evidence implicating the natriuretic peptide system in the ...

EVIDENCE IMPLICATING THE NATRIURETIC PEPTIDE SYSTEM IN THE ANTIHYPERTENSIVE

EFFECT OF MODERATE ETHANOL CONSUlMPTION

Pascal Guillaume

Department of Physiology McGill University, Montréal, Canada

A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfilment of the requirements

for the degree of Doctor of Philosop&

Q 1997 by Pascal Guillaume

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ABSTRACT

Chronic moderate ethanol (ETOH) consumption prevents the development of the age-

dependent increase m blood pressure (BP) in both humans and experimental animals. In the

present midies we proposed that the natriuretic peptide f a d y may be partially responsible

for this antihypenensive effect of ETOH. The natnuretic -stem consists of the atrial

natriuretic peptide (ANP), the brain natriuretic peptide (BNP). the C-type natriuretic peptide

(CNP) and the natriuretic peptide receptors (NPRs). The major function of the natriuretic

-stem is to decrease BP. Thus. the main objective of the present midies was to investigate

the interactions of acute and chronic ETOH administration with various components of the

natriuretic systern Acute studies: The injection of 1 and 2 g of ETOWkg B. W. in Sprague-

Dawley rats and 0.25 and 0.50 g of ETOWkg B.W. in hurnans resulted in a rapid transient

increase of circulating ANP levels. in the rats, this increase in plasma ANP levels was

associated with a rapid decrease of atrial ANP content and with a delayed increase in

ventricular ANP levels. Chronic studies: A 20°h v/v solution of alcohol was -+en to

spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats for 8 months. In both SHR

and WKY rats. chonic ETOH treatment decreased the BP. This lower BP in ETOH-treated

animals was associated with lower circulatmg ANP and BNP levels. whereas ETOH treatment

increased atrial ANP and BNP tissue contents. but not ANP and BNP mRNA. Furthemore.

chronic ETOH treatment reduced heart ventncular ANP content and ANP rnRNA while it

increased ventricular BNP content and BNP mRNA in SHR, but not in WKY rats. Chronic

ETOH reduced total natnuretic peptide binding sites (NPR-A and NPR-C) in the renal

elornerdi. Quantification of the receptor mbtypes demonstrated that this decrease was due Ci

to the dom-regdation of NPR-C. In the renal papilla. chronic ETOH treatrnent increased

natnuretic peptide binding sites (NPR-A). In addition to its effects on the penpheral

natnuretic synem chronic ETOH treatment altered the activity of the natnuretic system at

the level of the brain. Thus. chronic ETOH increased ANP and CNP levels in the

hypothalamus, pons and medulla of SHR rats. In WKY rats, ETOH had no effect on brain

ANP levels. but enhanced the concentration of CNP in the hypothalamus and medulla.

Chronic ETOH treatment also decreased the a&ty of NPR-C in some of the

circumventrîcular organs ofthe brain. in the nibfomical organ and cboroid plexus. but not in

the area postrerna.

These results demonstrate that both acute and chronic moderate ETOH administration

ahers the a c t ~ t y of vanous components of the natriuretic system Therefore. these ETOH-

induced modifications in cardiac. renal and brain natriuretic peptides and receptors may

contnbute to the antihypertensive effect of chronic moderate ETOH drinking.

RÉsUMÉ

La consommation continue d'éthanol (ETOH) en quantité modérée previent

l'augmentation de la pression sanguine liée à Fige autant chez l'animal que chez Iliumam.

Cette présente série d'études avançe l'hypothèse que la famiue des peptides natriurétiques

pourrait être en partie responsable de cet effet antihypertenseur de l'ETOK Le système

natriUrétique est composé du peptide natriurénque auriculaire (ANP), du peptide natriurétique

cérébral (BNP), du peptide natriurétique de type C (Cm) et des réce?teurs natriurétiques

(NPRs). Le rôle principal de ce système est de diminuer la pression sanguine. L'objectif

premier de la présente séries d'études est donc l'analyse des effets aigues et chroniques de

llalcool sur les différentes composantes du système natriurétique. Études a'gües: I L'injection

de 1 et 2 g d'ETOWkg de poid corporel chez le rat Sprague-Dawley et l'ingestion de 0.25 et

0.50 g d'ETOH/kg de poid corporei chez 1'buma.h provoque une rapide mais coune

augmentation des niveaux plasmatiques &ANP. Chez le rat, cette augmentation est associée

à une rapide diminution du contenu ANP de l'oreilette et à une plus lente augmentation du

contenu ANP du ventricule. Études chroniques: Une solution &ETOH à 20% viv est

administrée pendant 8 mois a des rats spontanément hypertendus ( S m ) et Wistar-Kyoto

(WKY). Chez les deux groupes de rats, la consommation contmue d'alcool diminue la

pression sanguine. Cette diminution de la pression sanguine se traduit par une diminution des

niveaux plasmatiques d'ANP et de BNP. Cependant, le contenu ANP et BNP de l'oreilette est

augmenté, bien que les niveaux auriculaires d'ARNrn ne soient pas modifiés. De plus, la

consommation continue d'ETOH réduit les niveaux ANP et ANP ARNm et augmente les

niveaux BNP et BNP ARNm du ventricule chez les rats SHR seulement. L'administration

d'alcool est également associée a une réduction du nombre total de récepteurs natriurétiques

(NPR-A et NPR-C) dans les glomérules rénaux. La quantification sélective des types de

récepteurs démontre que cette diminution est due principalement à la régulation négative des

récepteurs NPR-C. Dans la papille rénale, l'administration d'ETOH augmente le nombre total

de récepteurs natriurétiques (NPR-A). En plus de ces effets sur le système natriurétique

périphérique, la consommation contmue GETOH modifie également l ' a c t ~ t é du syçtème

natriurétique au niveau du cerveau. En effet, l'administration d'alcool augmente les niveaw

ANP et CNP de l!hypothalamus. du pons et de la médulla des rats SHR Chez les rats WKY.

la consommation d'ETOH n'a pas d'effet sur les niveaux ANP du cerveau mais augmente les

niveaux CNP de lliypothalarnus et de la rnédulla. Enfm. I'aftinité des récepteurs NPR-C des

organes circurnventriculaires du cerveau . t'organe subfomical et le choroid pexus mais non

la région postréma. est diminuée à la suite d'une consommation continue d'ETOH.

Ces résultats démontrent que l'administration aigue et chronique de quantitées modérées

d'alcool modifie les composantes principales du système natriurétique. De plus. la direction

de ces altérations produites par I'ETOH air les peptides et récepteurs natnurétiques du coeur.

du rem et du cerveau indique qu'elles peuvent contniuer à l'effet antihypertenseur des doses

modérées et chroniques d'alcool.

TABLE OF CONTENTS

Preface

Acknowledgements

Publications

Original contributions to knowledge

List of figures

List of tables

List of abbreviations

CHAPTER 1: GENERAL INTRODUCTION

1.1. ALCOHOL (ETOH)

1.1.1. HISTORICAL PERSPECTIVES

1.1.2. PHARMACOLOGY

1.1.3. ABSORPTION AND DlSTRIBUTION

1.1.4. EXCRETION AND METABOLISM

1.1.3.1. ETOH to acetaldehyde

a) .-l lcohol dehirdrogertnse (ADHI

b) Mcrosoninl ETOH-oxrdizrtlg -'stem ~~/EOS)

cl Cntnlclse

d) Noti-oxidative p a t h v q s

1.1 -3 -2. Acetaldehyde to acetate

a) A ldehyde dehydroger iase (A L DH)

1.1.5. MAJOR BIOLOGICAL ACTIONS OF ETOH

1.1 S. 1. Effects of ETOH on the central nervous sysrem

n) y-amfiiobii^;ric ocid (GA BA) receptors

b) iV-niethyl-D-aspartate ( N A D A ) receptors

j Oproidr

1.1.5 2. Effects of ETOH on the endocrine system

a) rlrgir wie vmopressiri /A 1 F)

b) Fivpothalamic-pi tir itaty-adred a i s

C) Nypothulani ic-p i hr 2 t a - g o a i s

e) Catecholumitre

J Etidothelitrni- a~rd platelet-derived wsoactive

factors

gl Others

1.1.5 -3. Effects of ETOH on the kidney

1.1.5.4. Effects of ETOH on the h e r

1.1.5.5. Effects of ETOH on the cardiovascular system

1.2. ALCOHOL AND BLOOD PRESSüRE

1.2.1. ACUTE ETOH C O N S W T I O N

1 . L l . l . Human studies

a) ETOH-iridirced decrecrse iri BP

b) E TOH-irldziced irrcrecrse irr BP

C) NO effect of ETOH oti BP

d) Factors irrflzmrci~rg the restrlts observed itr

htrnrntl stttdies

r ) Szmtmq-

1.2.1.2. Animal studies

a) E TOH-irrdtrced decrease ir r BP

b) No effect of ETOH or1 BP

C) Factors itrflireticitrg the results observed irl

nrrintnl st~rdies

d) Szrnrniary

1.2.1.3. Mechanisms

6) C'mocorzstrictiorz

C ) E TOH-irdziced itlcreme i ~ z HR

1.2.2. CHRONIC ETOH CONSUMPTION

1.2.2.1. Hirman studies

a) H e q ETOH cotrszimptzoti

b) Light a d nioderate ETOH cotrszinptzoti

C) Factors itifliieticing the resiilts observed iti

epidemiologicai stiidies

d) Szrnrnq

1 -2.2.2. Animal studies

CI) ETOH-itidziced decrease in BP

b) E TOH-it i h ced itrcreme itr BP

c) No effecct of ETOH ut1 BP

d) Factors itifliierici~ig the residts observeci U r

atrinial stzïdies

r ) Szmirmn-

1.2.2.3. Meclianisms

a) .\/cchnn~cms rc.rponrib/c_t;w rhc h ~ p ~ r r c n r I ~ L ' -!/l'cf r!t'

chronrc hc*crr-* E7'0tI consrtmprlon

b) .\ lcchtrnnms r"sponr h i c f i w rhc unrrb pcrrenr rrc qfl2cr r!/ '

chrrmrc / i g h ~ crnJ moricrcrrc. I!TOH conrrtmprion

1.3. NATRIURETIC PEPTIDE FAMILY

1.3.1. A-TYPE NATRIURETIC PEPTIDES

1.3.1.1. Atrial natriuretic peptide

a) Historical perspectives

b) Biuchemisrp atid release

C) Tissue distribiitiori

4 Ilfetcrbolisni

1.3.1.2. Urodiatin

1.3.1.3. Kterminal fragments of proANP

1.3.2. B-TYPE NATRIURETIC PEPTIDE

1 -3.2.1. Brain natriuretic peptide

0) Biocliemistg arld release

b) Tisszte disrrib~rtior~

C) .bfernbolisnt

1.3.3. C-TYPE NATRlURETIC PEPTIDE

1.32.1. C-type natriuretic peptide

a) Biochenzistn md refeme

C) hfetabolisnt

1.3.4. NATRIURETIC PEPTIDE RECEPTORS

1.3.4.1. Guanylate-cyclase receptors

1.3.4.2. Clearance receptor

1.3-4.3- Receptors for N-terminal fia_gnents of proANP

1.3 .-W. Tissue distribution

1.3.5. PHYSIOLOGICAL ACTIONS OF THE NATRIURETIC

PEPTIDES

1.3.5.1. Renal actions

1.3.5.2. Cardiovascular actions

1.3-5.3. Adrenal actions

1.3.5.1. Central newous system actions

1.3.5.5. Hormonal actions

1.3.5 -6. Pulmonary actions

1.4. ALCOHOL, BLOOD PRESSURE AND THE NATIUURETIC

PEPTIDE F.4MtLY

1 -4.1. ETOH AND NATMURETIC PEPTIDE FAMILY

1.4.2. WOREUNG HYPOTHESIS AND DWISION OF THE

THESIS

CHAPTER 2: ACUTE ETOH STUDIES

2.1, INCREASED PLASMA ATRIAL NATRIURETIC PEWIDE

.M'TER ACUTE iNJECTION OF ALCOAOL IN RATS

2.1.1. ABSTRACT

2.1.2. rNTRODUCTION

2.1.3. PdATERiALS AND METHODS

2.1.3.1. Animal treatments

2.1.3.2. Blood alcohol content

2.1.3.3. Tissue extraction

2 - 1 3 -4. Plasma preparation for hormonal measurements

2.1.2-5. Cornparison between direct and extracted assays

2. 1.3 - 6 . Radioirnrnunoassay procedures

2.1 -3.7. StatisticaI analysis

2.1.4. RESULTS

2.1.4.1. Eqeriment 1

2.1.1.2. Experiment 2

2.1.4.3. Evperirnent 3

2.1.3.4. Correlations

2.1 S. DISCUSSION

2.1.6. ACKNOWLEDGEMENTS

2.2. INCREASED PLASMA ATRIAL NATRIURETIC PEPTiDE

M'TER INGESTION OF LOW DOSES OF ETOH IN HUMANS

2.2.1. ABSTRACT

2-2.2. INTRODUCTION

2.2.3. MATERIALS AND METHODS

2.2.3.1 Subjects

2.2.3.2. Experimental design

2.2.3 -3. Estimation of blood aIcohol content

2.2.3 -4. Estimation of plasma ANP. AVP and cortisol

2.2.3.5. Statistical analysis

2.2.4. RESULTS

2.2.5. DISCUSSION

2.2.6. ACKNOWLEDGMENTS

CHAPTER 3: CKRONIC ETOH STUDIES

3.1. EFFECT OF CIERONIC ETOEI CONSUMITION ON THE

ATRLAL NATRlURETIC SYSTEM OF S m RATS

3.1.1. ABSTRACT

3.1.2. INTRODUCTION


3.1.3.1. Treatments

3.1.3.2. Estimation of bIood ETOH

3.1.3.3- heparation of blood and tissue extracts for RiAs

3.1.3.4. RiAs

3.1.3.5. RNA extraction and hybridization

3.1.3.6. RT-l'CR

3.1.3-7. Statistical analysis

3.1.4. RESULTS

3.1.4.1. Effect of ETOH on BP and HR

3.1.1.2. .Effect of ETOH on the heart ANP system

3.1.4.3- Effect of ETOH on circulating hormone levels

3.1.5. DISCUSSION


3.2. EFFECT OF CEtRONIC ETOH CONSUMPTION ON EIEART

BRAIN NATRWRETIC PEPTIDE

3.2.1. ABSTRACT 166

3 2.2. INTRODUCTION 166

3.2.3. MATERlALS AND METHODS 167

3.2.4. RESULTS 171

3.2.4.1. Effect of age and ETOH on body weight. BP. HR 17 1

and total protein content in atrial and ventricdar

tissues

3.2.1.2. Effect of age on BNP

3.2.4.3. Effect of ETOH on BNP

3 - 2 - 4 4 Ventricular BNP mRNA by RT-PCR

3.2.5. DISCUSSION


3.3. RENAL ALTEUTIONS OF ATRIAL NATRiURETIC

PEPTIDE RECEPTOUS BY CFIRONIC MODERATE ETOH

TREATMENT

3.3.1. ABSTRACT

3.3.2. INTRODUCTION

3.3.3. METHODS

3.3.3.1. Animal treatments 1 1 -

J .J .J -2. Urine collection

3.3.3.3. Urinary cGMP excretion

3.3.3.4. ANP RIA

3.3.3.5. Blood aIcohol content

3 Z3.6. Preparation of membranes for cornpetitive binding

assay

3-3-3.7. Cornpetitive binding assay

3.3.3.8. Autoradiographic studies

3 -3.3.9. Statistics

3.3.4. RESULTS

3.3.4.1. BP and plasma ANP levels

3 -3.4-2. Urine analysis

3.3.4.3. Competitive binding studies

3 3.4.4. Autoradiography

3.3.4.5. Urinary cGMP excretion

3.3.5. DISCUSSION


3 A .QLTERATIONS IN B W LEVELS OF ATRIAL AND C-TYPE

NATRIURETIC PEPTIDES AFTER CEiRONIC MODER4TE

ETOH CONSUMPTION IN SHR RATS

3-41. ABSTRACT

3.4.2. INTRODUCTION

3-4.3. MATERIALS AND METHODS


3.4.3.3. Tissue extraction

3.4.3.3. RIAS


3-44. RESULTS

3.4.4.1. Effect of age

3.4.4.2. Effect of strain

3.4.4.3. Effect of ETOH

3.4.5. DISCUSSION

3-46. ACKNOWLEDGEMENTS

3.5. CIRCUMVENTRICULAR ORGAN NATRZURETIC PEPTIDE

RECEPTORS FOLLOWING CEiROMC MODERATE ETOH

CONSUMPTION IN SBR AND WKY RATS

3.5.1. ABSTRACT

3.5.2. INTRODUCTION

3.5 -3. MATERIALS AND METHODS


3.5.3.2. Autoradiographic studies


3.5.4. RESULTS

3.5.4.1. Subfomical organ and choroid plexus

3.5.4.2. Area postrema

3.5.5. DISCUSSION


CELAPTER 4: GENERAL DISCUSSION

4.1. EFFECT OF ETOH ON TEE BLOOD PRESSURE

4.2. EFFEC'ï OF ETOH ON PLASMA AND =ART

NATRIURETIC PEPTIDES

4.2.1. PLASMA

4.2.2. HEART ATRiA

3.2.3. HEART VENTRICLES

4.3. EFFECT OF ETOH ON RENAL NATRllTRETIC RECEPTORS

4.4. EFFECT OF ETOH ON BRAIN NATIUURETIC PEPTIDES

AND RECEPTORS

4.5. GENERAL SUMMARY AND FUTURE STUDIES

4.5.1. KEART AND VASCULATURE

4.5.2. KIDNJSY

4.5.3. BRArN

APPENDTX

REFERENCES

PREFACE

The present thesis consihg of four chapters. describes the effects of acute and chronic

moderate ETOH consurnption on the major aspects of the natriuretic peptide system. n i e

ETOH-induced alterations are fùrther analyzed as pan of the mechanism by which moderate

ETOH treatment prevents the age-dependent increase in BP. Chapter 1 includes a

comprehensive review of the Iiterature on ETOH on the effect of ETOH on BP. on the

natriuretic peptide family and on the interactions between natnuretic peptides and ETOH.

Chapters 2 and 3 comprise scientSc papers published. accepted or submitted for publication

on the general alterations in the natriuretic peptide system produced by acute and chronic

ETOH drinking. respectively. in cornpliance with the guidelines for thesis preparation

provided by the Faculty of Graduate Studies and Research. the following te.xt is reproduced

below:

'Candidates have the option of including, as part of the thesis, the text of a paper(s) submitted

or to be submitted for publication, or the clearly-duplicated text of a published paper(s). These

texts must be bound as an integral part of the thesis.

If this opfion is chosen, connecimg texts that provide logical bridges between the different papers

are mandatory. The thesis must be written in such a way that it is more than a mere collection of

manuscripts; in other words, results of a series of papers must be integrated.

The thesis must sfill con fm to dl other requirements of the 'Guidelines for Thesis Preparation'.

The thesis must indude: A Table of contents, an abstract in English and French, an introduction

which clearly States the rationale and objectives of the study, a comprehensive review of the

literature, a final conclusion and summary, and a thorough bibliography or reference k t .

Additional material must be provided where appropriate (e.g. in appendices) and in sufficient

detail to dlow a dear and precise judgement to be made of the importance and originality of the

research reported in the thesis.

In the case of manuscripts CO-authored by the candidate and others, the candidate is required

to make an expliat statement in the thesis as to who contributed to such work and to what extent.

Supervisors must attest to the accuracy of such statements at the doctoral oral defense. Since

the task of the exam'ners is made more difficult in these cases, it is in the candidate's interest to

make perfectly dear the responsibiliti es of al1 the authors of the CO-authored papers. Under no

circumstances can a co-author of any component of such a thesis serve as an examiner for that

thesis."

Chapter 2. Acute ETOH experiments. describes the effects of a single administration

of a low and moderate ETOH dose on cardiac and circulating ANP levels in rats (Section

2.1. ) and human volunteers (Section 2.2. ). The demonstration of a stimulatory effect of acute

ETOH treatment on plasma ANP levels in both human and animal studies indicated that the

naniuretic peptide system was sensitive to ETOH exposure. Furthermore. the direction of the

ETOH-induced changes in plasma ANP levels suggested the possibility of a depressor effect

of this hormonal syaem in ETOH-treated ind~duals . Therefore. we sou& to analyze the

long-terrn modifications in the major components of the natriuretic peptide synem during

regular rnoderate ETOH drinking. in order to investigate the role of this hormonal family in

mediating moderate ETOH's antiliypertensive effect.

in Chapter 3. Chronic ETOH experiments. the long-term administration of moderate

ETOH levels was found io prevent the age-dependent increase in BP in both spontaneously

hypertensive ( SHR) and Wistar-Kyoto (W) rats (Section 3.1. ). This antihyp ertensive effect

of ETOH was associated with specific alterations in cardiac and circulating ANP and BNP

levels and in cardiac ANP and BNP mRNA levels (Sections 3.1. and 3.2.). The ETOH

treatment also modified the natriuretic peptide binding sites in the liidney. thus affecting botli

ends of the endocrine synem (Section 3.3.). Furthermore. the contribution of the brain

natriuretic system was evaiuated following chronic ETOH treatment via natnuretic peptide

measurements in the major brain areas (Section 3.4. ) and natriuretic peptide binding sites in

the circumventncular organs ( Section 3 . 5 . ).

Thus. the major parameters of the natriuretic peptide system were analyzed followüq

chronic moderate ETOH treatment. Interestingly, all of these parameters were specifically

altered by ETOH m a direction supporthg a general role for this hormonal system in ETOHs

antihypertensive effect.

Fmaily. Chapter 4 contains a detailed discussion of the various ETOH-induced aiterations

m the natrhiretic peptide syaem and of the mechanisms by which these alterations are Lilcely

to contribute to the antihypertensive effect o f moderate ETOH drinkmg. A descrÏption of

future directions for studies investigating ETOH and the natriuretic peptide f a d y is also

included.

1 am particularly indebted to Dr. Chriaina Gianoulakis for her patience and guidance

throughout the course of my audies. The iittle 1 know about scieutifk writmg and thinking

is certainly due in large parts to her exceptional generosity. For her t h e &en in countless

scientific and general discussions. for her days spent correcthg and improving my

manuscnpts. for her enthusiasm and Irindness. 1 am extremely grateful.

1 am also thankfÙl to Dr. Jolanta Gutkowska for her advices and excellent knowledge of

science. for her support and encouragements throughout my doctoral midies. Her sugeestions

and criticisms were greatly appreciated.

1 wish to thank past and present -dents in the labs for their fnendship and their help:

Jean-Pascal De Waele and Neil Jamenski m Douglas Hospital: Salima Menari. Eric Morin. and

especially Sheila Emest (Pour l'amitié et les sentils coups de téléphone. pour l'opéra aussi ...)

in the Hôtel-Dieu.

1 am also appreciative of the excellent technical eqertise and assistance of Céline

Coderre and Nathalie Charron (Merci beaucoup!!). of Dr. Suheyla Mukaddam-Daher. Dr.

Marek Jankowski. Dr. Tham-Vinh Dam Sylvie Larocque and Diane Beaudry .

1 wish to express my gratitude to Ricardo Claudio for his assistance and patience with

the animals. His fnendship is certainly the gea t ea accomplishment of these studies (Merci

a toi aussi Sylvie ... ).

Fmaily. I am especially grateful to my family: My rnother. Monique. my father. Roland.

and my brother. Emmanuel. for their constant and unrelenting encouragements and supports

(Sans vous tous. je n'y serais peut-ètre pas amvé..!).

1 should also thank - with a toast - these daily glasses of wine -never more than two - wbich ailowed me to survive stress and maintained rny blood pressure to a relatively normal

level.. .

PUBLICATIONS

Guillaume P, Gutkowska J, Gianoulakis C ( 1994) Increased plasma atrial natriuretic peptide d e r acute injection of alcohol m rats. J P h m a c o l E r - nter 27 1 ; 1656- 1665.

Gianoulakis C, Guillaume P. De Waele JP, Angelogianni P ( 1995) Effect of stress and alcohol on the proopiornelanocortin/ Bendorphin system bz: Stress, gender, and alcohol-seekmg behaviour, Research rnonograph 29, Hunt WA. Zakhari S (ed.), NIH NIAAA. Bethesda. p. 145- 165.

GuiIIaume P. Jankowski M, Gutkowska J, Gianoulakis C ( 1996) Effect of chronic moderate ethanol consumption on heart brah natriuretic peptide. Eur J Phamtacol 3 16: 49-58.

Guillaume P, Jankowski M, Gianoulakis C, Gutkowska J ( 1996) Effect of chronic etbanol consumption on the atnal natriuretic system of spontaneously hypertensive rats (SHR). AIcoholExp Clin Res 20; 1653-1661.

Guillaume P, Dam TV, Gianoulakis C, Gutkowska J ( 1997) Renal alterations of atrial natriuretic peptide receptors by chronic moderate ethanol treatment. Am J Physioi272 (Rerial Physiof 41); F107-F116.

Gianodakis C, Guillaume P, Thavundayd J, Gutkowska J ( 1997) Increased plasma atriai natriuretic peptide afkr mgestion of low doses of ethanol in human. AIcohol C h Exp Res (in press).

Guillaume P. Gutkowska J, Gianoulakis C ( 1997) Alterations in brain levels of atrial and C- type natriuretic peptides after chronic moderate ethanol consumption in spontaneously hypenensive rats. Ezir J Phannocol (m press).

Guiilaume P, Gianoulakis C, Gudcowska J ( 1997) Circmentricular organ natriuretic peptide receptors following chronic moderate ethanol consumption in SHR and WKY rats. (manuscript in preparation).

ABSTRACTS-COMMUNICATIONS

mP, Gianoulakis C (1992) Alcohol-mduced secretion of atrial natriuretic factor (ANP) m rats: Possible implication of 8-endorphin. 2~~idAnrzuo~ meetirig of the Sociev for Neurmcierzce, Anaheim, USA.

urne P, Gutkowska J, Gianoulakis C ( 1993) Plasma atrial natriuretic factor after acute injection of alcohol in rats. 2e Jounzée de la Recherche de l'Hôtel-Dieu de Montréal. Montréal, Canada.

Guillaume P, Gianoulakis C ( 1993 ) Effect of iti vivo ethanol administration on the HPA-ais and pituitary D-endorphin. Xth R S ' sczetitrfic cot,feretice. Washington. USA.

Guillaume P. Gutkowska J. Gianoulakis C (1993) Acute and chronic effect of moderate ethanol consumption on the atrial natriuretic syaem Sernorar, 3th Pqchiatry Research dqi: Dozlglas Hospital Research Centre, Verdun. Canada.

Guillaume P. Gutkowska J. Gianoulakis C ( 1994) Plasma atrial natriuretic factor following acute mjection of ethano 1 in rat S. MIth it~teniatzoiral cotigress of Pharniacologr.. Mont réal. Canada.

Guillaume P, Gutkowska J. Gianoulakis C ( 1994) increased plasma atrial natriuretic peptide after acute injection of alcohol in rats. Firsr prce. Sémitraire de Recherche. Hôtel-Dieu de hfot~tréal, Montréal, Canada.

Guillaume P, Gutkowska J. Gianoulakis C ( 1994) Acute and chronic effects of ethanol on the atrial natriuretic peptide system. Sentitinr. Dcnrglas Hospital Research Ceirtre. Verdun. Canada.

G u i l l a e , Jankowski M. Gutkowska J. Gianoulakis C ( 1994) Effect of chronic moderate ethanol consumption on the atnal natriuretic peptide syaem in SHR and WKY rats. 3e Jozirtlée de la Recherche de l'Hôtel-Dieu de Motitréal. Montréal, Canada.

Guillaume P. Jankowski M. Gianoulakis C. Gutkowska J ( 1995) Effect of chronic ethanol consurnption on the atriai natriuretic system of spontaneously hypertensive rats (SHR). 6r Coitgrès de in Sucies rli<ébécoise d'&perret~;ioti artérielle. Québec. Canada.

Guillaume P, Gutkowska J. Gianoulakis C ( 1995) Acute and chronic effects of ethanol consumption on blood pressure and ANP syaem in S H R .rith Scret1tzfic rneetitigfor the Itlter-Atnerrcnti socrey of khpertet~sioti. Montréal. Canada.

Guillaume P. Gianoulakis C. lankowslii M. Gutkowska J ( 1995 ) Acute and chronic effects of ethanol connimption on blood pressure and ANP system in spontaneously hypertensiie rat S. 7th Eirropeati nzeetitig or1 Hypertetisiori. Milan. Italy .

Guillaume P, Dam TV. Trernblay J. Gianoulakis C. Gutkowska J ( 1995) Renal alterations of ANP receptors by chronic ethanol treatment in SHR and WKY rats. First Ezrropem cotferetice or1 Pharmacoiogy, Milan. Italy.

Guillaume P. Jankowski M. Gutkowska J. Gianoulakis C ( 1995) Effect of acute and chronic ethanol treatment on the heart ANP and BNP system First Atunial Physioiogy Resenrch dq, McGilI University. Montréal. Canada.

Guillaume P, Dam TV. Tremblay J. Gianoulakis C. Gutkowska J ( 1995) Renal alterations of atrial nahuretic peptide receptors by chronic moderate ethanol treatment. -le Jmtniée de la Recherche de l'hrotel-Dieu de Montréal. Montréal. Canada.

Guillaume P. Gutkowslia J. Gianoulakis C ( 1996) Effect of chronic ethanol consumption on brain levels of ANP. Sem imr. Doziglas Hospital Research Ceritre. Verdun. Canada.

Guillaume P. Jankowski M. Gutkowska J. Gianoulakis C ( 1996) Effect of chronic ethanol consump tion on heart brain natriuretic peptide. Semimr, bui A wual Physiologv Research d q McGill University. Canada.

Gutkowska J. Guillaume P. Jankowski M. Gianoulakis C ( 1996) Effect of alcohol on blood pressure and atrial natriuretic peptide system in spoataneously hypertensive rats. 16th Scietifrjic nreetirig of the hrtenzatiorral Sotie+* of Hyertemiori. Glasgow. Scotland.

vii

ORIGINAL CONTRIBUTION TO KNOWLEDGE

L in order to mvestigate the role of the natriuretic peptide family m the prevention of the age-

dependent increase in the blood pressure (BP) by moderate ethanol (ETOH) consumption.

the effect of acute and chronic ETOH treatment on the major components of the natriuretic

peptide syaems and their receptors are inveaigated in the heart and circulation. kidney and

brain. The chronic administration of moderate levels of ETOH resulted in a Iower BP and HR

m the ETOKtreated rats when compared to the water-treated animals. These results confirm

and ergand previous reports and are nipponing the existence of a unknown antihypertensive

mechanism associated with the alcohol consumption.

CI. Acute ingestion of moderate ETOH levels in rats and humans resulted in increased plasma

ANP levels. In the rats. this mcrease is associated with a rapid decrease in atnal ANP content

and a delayed increase in ventncular ANP content. suggeaing the release of ANP fiom the

heart. These observations indicate that the heart natriuretic peptide system is stimulated by

the ETOH exposure.

[IL Chronic administration of moderate ETOH levels resulted in Iower plasma ANP and BNP

levels. Nevertheless. atrial and ventricular ANP and BNP mRNA activities are not reduced

by ETOH. despite the lower BP. Rather. increased atrial ANP and BNP contents and

increased ventncular BNP levels are observed in ETOH-treated rats. Moreover. the chronic

moderate ETOH treatment prevented the development of ventricular hypertrophy.

Considering the pattern of food and Buid intake during the iight and dark phases of the daily

cycle. these results indicate that the heart natnuretic system in the ETOH-treated rats is

mamtamed at an higher potential actMty by a dinerent rnechanism than in the water-treated

rats, suggeaing the possibility of a chronic stimulation of the heart oatnuretic -stem by

ETOH.

W . Chronic admirisration of moderate ETOH levels resulted in a reduction of the clearance

receptors (NPR-C ) m the renal glomeruli. Furthemore. chronic ETOH treatment mcreased

natriuretic peptide binding sites in the renal papiua. Excretory cGMP levels were either

elevated or unfhaaged foIlowing the ETOH administration despite the Iower BP. suggesting

a firnctional effect of the ETOH-induced natriuretic receptor alterations. These observations

suggest enhanced renal a c t ~ t y of the natriuretic system in ETOH-treated rats.

V. Cbronic administration of moderate ETOH levels resulted m increased or unchanged ANP

and CNP levels in the hypothalamus. pons and medulla. despite the lower BP. Moreover.

there was a reduction in the afEin.ity of NPR-C in the subfornical organ and choroid plexus.

but not in the area postrema following the alcohol treatment. These results suggen some

stimulations of the brain natriuretic peptides by the ETOH administration.

VI. In general. the direction of these cardiac. renal and brain alterations in the natriuretic

peptide system foster the hypothesis that the natnuretic peptides and receptors may mediate.

at least in part. the antihypenensive effect of moderate ETOH consumption.

LIST OF FIGURES

Chapter I

Figure 1.1.1. Schematic representation of gastric and hepatic alcohol metabolimi during acute and chronic ETOH consumption

F i e 3 1 Schematic representation of the transcription. translation and processing of the natriuretic peptide f a d y

Figure 1.3.2. Schematic representation of the structure of the natriuretic peptide farnily in rats

Figure 1 -3.3. Diagram of the natnuretic peptide receptors (NPR)

F e 1 3 4 Physiological actions of the natriuretic peptides

Figure 1 3 . 5 . Schematic diagram s u r n r n a ~ n g the ( 1 ) tubular. (2) hemodynamic. and ( 3 ) hormonal effects of the natnuretic peptide family on the kidney

Figure 1.3.6. Schematic diagram summarin'ng the effects o f the natriuretic peptide family on the heart and the vasculature

Figure 1.3.7. Schematic diagram m r i z i n g the effects of the natriuretic peptide family on the adrenal gland

Figure 1.3.8. Schematic diagram nimmarizuig the effiects of the natnuretic peptide famiIy on the brain

Chapter 2

Figure 2.1.1. Effect of the i.p. injection of morphine on the circulating ANP levels using either extracted (A) or unextracted (B) plasma for the RLA

Figure 2.1.2. Changes in blood alcohol content (BAC) with tirne after the i.p. injection of 1 or 2 g of ETOWkg B.W. (40% v/v solution)

Figure 2.1.3. Changes m plasma ANP (A). fi-endorphin (B) and corticosterone (C) content with t h e after the i.p. injection o f 1 or 2 g of ETOWlrg B.W. (JO0/0 v/v solution) or the equivalent amount of saline

Figure 2.1.4.

Figure 2.1.5.

Figure 2.1.6.

Figure 2.1.7.

Figure 2.2.1.

Figure 2.2.2.

Figure 2.2.3.

Figure 2.2.4.

Figure 2.2.5.

Figure 3.1.1.

Figure 3.1.2.

Figure 3.1.;.

Variations of ANP fiom basal levels in the plasrna (A). nght atrium (B). left atrium (C) and ventricles (D) after the i-p. injection of 2 g of ETOWkg B.W. (40% v/V solution) or the equivalent amount of saline

Correlation between the ETOH-induced changes in the plasma ANP and O-endorphin (i3-EP) contents

Correlations between the blood alcohol content (BAC) and the plasma ANP (A) and O-endorphin (B ) contents

Correlations between the increase in plasma ANP levels and the corresponding decrease in right atrial (A) and left atrial (B) ANP content at 1 5 min post-ETOH

Changes in blood alcohol content following ingestion of 0.25 and 0.50 g ETOH/kg B.W.

Hean rate foUowing (A) the placebo, (B) the 0.25 and (C) the 0.50 g ETOWkg B.W. drmks. Systolic and diastolic BP following (D) the placebo. (E) the 0.25 and (F) the 0.50 g ETOWkg B.W. drinks

Effect of(A) the placebo. (B) the 0.25 and (C) the 0.50 g ETOWkg B. W. drinks on the plasma ANP content

Effea of (A) the placebo. (B) the 0.25 and (C) the 0.50 g ETOHkg B.W. drinlis on the plasma content of vasopressin

Effect of(A) the placebo. ( B ) the 0.25 and (C) the 0.50 g ETOHkg B.W. drinks on the plasma cortisol content

Chapter 3

Progression of blood pressure dunng 8 months of water or 20% viv ETOH consurnption in WKY rats and SHR

Progression of heart rate during 8 months of water or 20°/o v/v ETOH consumption in WKY rats and SHR

Effect of ETOH treatment on the right atrial ANP system (content. concentration and mRNA) in WKY rats and SHR

Figure 3.1.4. Effect of ETOH treatment on the left atrial ANP system (content. 153 concentration and mRNA) m WKY rats and SHR

Figure 3.1.5.

Figure 3.1.6.

Figure 3.1.7.

Figure 3.2.1.

Figure 3.2.2.

Figure 3.2.3.

Figure 3.2.4.

Figure 3.2.5.

Figure 3.2.6.

Figure 3.2.7.

Figure 3.2.8.

Figure 3.3.1.

Effect of ETOH treatment on the ventncular ANP system (content. concentration and mRNA) in WKY rats and SHR

Autoradiograms of a representative Northem blot for atrial (A) and ventricular (B ) ANP mRNA m WKY and SHR rats after 8 months of water (H) or 20% vlv ETOH treatment (E)

Relative quantification of ANP mRNA in rat ventricles by RT-PCR

Circulating BNP levels in WKY rats and SHR at 7 weeks of age and afier 8 rnonths of water or 20% vlv ETOH consumption

Effect of age on nght atrial (A-B), lefi atnal ( G D ) and ventricular (E-F) heart BNP content and concentration

Effect of water or chronic ETOH treatment on the right atrial BNP system (total content (A). concentration (B) and mRNA (C)) in WKY rats and SHR

Effect of water or chronic ETOH treatment on the left atrial BNP system (total content (A), concentration (B) and mRNA (C)) in WKY rats and SKR

Autoradiogram of a representative Northem blot of atrial BNP mRNA in WKY and SHR rats after 8 months of water (H) or 20% vlv ETOH treatment (E)

Effect of water or chronic ETOH treatment on the ventricular BNP system (total content (A), concentration (B) and mRNA (C)) in WKY rats and SHR

Autoradiogram of a representative Northem blot of ventricular BNP mRNA in WKY and SHR rats after 8 months of water (H) or 20°h V/V ETOH treatment (E)

Ventncular BNP mRNA by RT-PCR in adult SHR and WKY rats

Urine volume and osmolanty in SHR and WKY rats d e r 7% months of ETOH or water treatment

Figure 3.3.2.

Figure 3.3.3.

Figure 5.3.5.

Figure 3.3-6.

Figure 3.41.

Figure 3.42.

Figure 3.43.

Figure 3.4.4.

Urine sodium concentration (A), sodium excretion (B). potassium concentration (C) and potassium excretion (D) m SHR and WKY rats afier 7% months of ETOH or water treatment

Cornpetitive inhiôition of '"EANP binding by increasing concentrations of unlabelled ANP and cANF to the glomemlar membranes in SHR and WKY rats at age 7 weeks (before ETOH treatment) and at age 38 weeks (afler 8 months of ETOH or water treatment )

Competitve inhibition of '"1-ANP binding by increasing concentrations of unlabeiied ANP to the papillary (CM) membranes in SHR and WKY rats at age 7 weeks (before ETOH treatment) and at age 38 weeks (afier 8 rnonths of ETOH or water treatment)

Autoradiographs of bmdmg of 50 pM '%ANP m the kidneys of adult SHR and WKY rats, afier 8 months of ETOH or water treatment

Quantification by densitometry of the displacement of total "'1-ANP bmding by 10" M unlabeiied cANF in the kidneys of adult SHR and WKY rats. after 8 months of ETOH or water treatment

Unnary excretion of cGMP (nM1day) measured from the urine collected during the iight phase of the daily cycle following 7 ' 2

months of water or ETOH treatment in SHR and WKY rats

Standard RIA curve of CNP

ANP content (ng) and concentration (@mg protein) in the hypothalamus (HYPO). pons and meduiia (MED) of 7 and 38 week- old SHR and WKY rats

CNP content (ng) and concentration (@mg protein) in the hypothalamus (HYPO). pons and medulla (MED) of 7 and 38 week- old SHR and WKY rats

ANP content (ng ) and concentration (ng/mg protein) after 8 months of water or ETOH (20% v/v) treatment m the hypothalamus (HYPO). pons and medulla (MED) of 7 and 38 week-old SHR and WKY rats

Figure 3 - 4 5

Figure 3.5.1.

Figure 3.5.2.

Figure 4.2.1.

Figure 4.2.2.

Figure 4.3.1.

Figure 4.3.2.

Figure 4.41.

Figure 4.4.2.

Figure 4.5.1.

CNP content (ng) and concentration (ng/mg protem) after 8 months of water or ETOH (20% v h ) treatment m the hypothalamus (HYPO). pons and medulla (MED) of 7 and 38 week-old SHR and WKY rats

Average cornpetition bbding curves of "1-ANP in adult (38 weeks- old) SHR and WKY rats subfomical organ (SFO). after 8 months of water o r ETOH (20% VI\;) treatment

Average cornpetition binding c w e s of ' 2 5 ~ - A N P in adult (38 weeks- old) SHR and WKY rats choroid plexus (CP). afier 8 months of water o r ETOH (20% v h ) treatment

Average cornpetition binding curves of "'EANP in adult (38 weeks- old) SHR and WKY rats area postrerna (AP). after 8 months of water or ETOH (20% v/v) treatment

Chapter 4

Schernatic representation of the natriuretic peptide syaem ( ANP and BNP) in the heart

Schematic representation of the possible ETOH-induced changes in circulating and heart natriuretic peptides during the Light and dark phases of the daily cycle

Schematic representation of the renal natriuretic system

Schematic representation of the ETOH-induced changes in renal natnuretic peptide receptors

Schematic representation of the natriuretic peptide system in the central nervous system (CNS)

Schematic representation of the ETOH-induced changes in brain natriuretic peptides and receptors

Schematic diagram s u m m a ~ n g the effects of chronic moderate ETOH consumption on the natriuretic peptide system and the mechanisms by which these modifications may mediate part of alcohol's antihypertensive effect

LIST OF TABLES

Chapter 1

Table 1.1.1

Table 1.1 .2.

Table 1.2.1.

Table 1.2.2.

Table 1.2.3.

Table 1.2.4.

Table 2.1.1.

Table 2.1.2.

Table 2.1.3.

Table 2.2.1.

Table 3.1.1.

Table 3.1 .2.

Table 3.2.1.

Table 3.2.2.

ETOKinduced alterations in the a c t ~ t y of the central nervous system (CNS)

ETOH-induced alterations in the activity of the endocrine system

Human studies on the acute effect of ETOH on the BP and the E-R

Animal studies on the acute effect of ETOH on the BP and the HR

Hurnan studies on the chronic effect of ETOH on the BP

Animal studies on the chronic eEect of ETOH on the BP

Chapter 2

Effeas of ETOH (2 &kg B.W.) on the plasma levels of B-endorphin. corticosterone. ACTH, aldosterone and AVP

Effects of ETOH (2 @hg B.W.) on blood pressure and heart rate

Correlations between the various parameters

Urine output and urine sodium and potassium excretion (rnean = SEM) following ingestion of O. 0.25 and 0.50 g ETOH/kg B.W.

Chapter 3

Blood alcohol, ANP. AVP. corticosterone and aldosterone in SHR and WKY rats at age 7 weeks (before ETOH treatment) and at age 38 weeks (afier 8 months of ETOH or water treatment)

Rotein levels in the cardiac compartments of WKY and SHR rats

Body weight and da* liquid consumption in WKY and SHR rats during the 8 months of treatment

Systolic blood pressure and heart rate in WKY and SHR rats during the 8 months of treatment

Table 3.2.3. Protein levels in the cardiac compartments of WKY and SHR rats 1 74

Table 3.3.1. Blood pressure (BP). body weight (BW). liquid connimption (LC) 198 and circulating ANP levels in SHR and WKY rats at age 7 weeks (before ETOH treatment) and at age 38 weeks (afier 8 months of ETOH or water treatment)

Table 3 -3.2. Kinetic parameters for glomerular natriuretic receptors in SHR and 202 WKY rats at age 7 weeks (before ETOH treatment) and at age 3s weeks (afier 8 months of ETOH or water treatment)

Table 3.3.3- Kinetic parameters for papillary (IM) oatnuretic receptors in SHR 706 and WKY rats at age 7 weeks (before ETOH treatment) and at age 38 weeks (afler 8 months of ETOH or water treatment)

Table 3.4.1. Blood pressure (BP), body weight (BW) and daily liquid connimption 227 (LC) m SHR and WKY rats at age 7 weeks (before ETOH treatment) and at age 38 weeks (afier 8 months of ETOH or water treatment)

Table 3.5.1. Natnuretic receptor cliaracteristics (B,, and K.J in the SFO. CP and 21 1 AP afler 8 months ofwater or ETOH treatment in WKY and SHR rats

AD: angiotensin II

AC: adenylate cyclase

ACTH: adrenocorticotropic hormone

ADH: alcohol dehydrogenase

ALD: alcoholic liver disease

ALDH: aldehyde dehydrogenase

AMPA: a-amino-3-hydroxy- 5-rnethyl-4-isoxazole p roionic acid

ANOVA: analysis of variance

ANP: atrial natnuretic peptide

ANP,,.: long acting sodium stimulator

.&NP,,,,: vesse1 dilator

.&NP,,-,,: kahuretic stimulator

AP: area postrema

-4V3V: anteroventral third ventricie

AVP: arginine vasopressin

BAC: blood alcohol content

bFGF: basic fibrobast growth factor

BNP: brain natnuretic peptide

BP: blood pressure

B.W.: body weight

CAMP: 3'. 5' -cyclic adenosine monophosphate

cANF: des-[GlnHh. Seri". Gly"". Leu"'. Gly""]ANP,,2.,,,

cGMP: Y. 5' -cyclic guanosine moaophosphate

CCIF: congestive heart failure

CNP: C-type natriuretic peptide

CNS: central nervous system

CO: cardiac output

CP: choroid ple.nis

CSF: cerebrospmal fluid

EAA: excitatory aminoacid

EC F: extracellular fluid

EDRF: endothelium-derived relaxhg factor

ETOH: ethanol

GABA: y-arninobutync acid

GC: guanylate cyclase

GFR: glomedar filtration rate

G 1: gastrointestinal

HPA: hypothalamic-pituitary-adrenal

APG: hypothalamic-pituitary-gonadal

HPT: hypothalamic-pituitary-thyroid

i.c.v.: intracerebroventricular

IMC D: inner medullary collecting duct

i.p.: intrap entoneal

LHRH: luteinking hormone releasing hormone

mRNA: messenger ribonucleic acid

M EOS: microsomaI ethanol-o'cidizing system

NAD: nicotinamide-adenine dinucleotide

NMDA: N-methyl-D-aspartate

NO: nitric oxide

NPR: natriuretic peptide receptor

OVLT: organum vasculosum of the laminae terminalis

PGE,: prostaglandin EI

PNS: penpberal nervous system

POMC: proopiomelanocortin

RAAS: renin-angiot ensin-aldosterone system

RIA: radioimmunoassay

RT-PCR: reverse transcriptase polymerase chah reaction

SFO: subfornical organ

SHR: spontaneously hypertenske rats

SMC: smooth muscle celIs

T, : triiodothyronine

T,: thyroxine

TGF-II: transfonning growth factor-D

TNF-a : tumor necrosis factor- a

UD: urodilatin

VR: venous return

WKY: Wistar-Kyoto rats

CEIAPTER 1

GENERAL INTRODUCTION

1.1. ALCOHOL (ETOA)

1 .l. 1. RISTORICAL PERSPECTIVES

The discovery of alcohol dates back from the early steps of history. when coUected h i t s

and plants were lefl unattended in the sun. This natural fermentation of fi-uits and plants

produced a liquid (beer. wine) whose taste was pleasant and whose consumption promoted

leisure and conversation. It soon became part of the daily Life of early civilizations. in rituals.

m bonding. in politics. in medicine. Later. the Arabs discovered that they codd increase the

alcohol concentration of their liquids through alcohol distillation (liquor). intereaingly. the

word "alcohol' is also of Arabic origin. meaning "he ly divided spirit".

Yet. its detrimental effects when consumed in excess were also rapidly noticed since the

oldest code of laws. the code of Harnrnurabi of Babylooia. aiready regimented its

consumption. With the industrialization and the mass production of the XiXth century.

alcoliolic beverages became increasingly commercialized and the problems associated with

excessive drinking started to be considered seriously.

There is therefore a delicate balance between a positive. therapeutic or social use of

alcohol and a aegative. excessive or unhealthy alcohol consumption. As a consequence of this

dual action of alcohol. the cultures of the XXth century have oscillated between two

extremes. fiom the complete prohibition of alcohol m the United-States to the complete lasity

in alcohol consumption in France or Italy. A better approach. in my opinion. would be to

recognized both the beneficial effects of light alcohol drinking and the detnmental

consequences of heavy alcohol consumption.

1.1.2. PAARMACOLOGY

The chemical structure of alcohols consists of an hydroxyl group (OH) attached to a

chah of saturated carbons of vanous lengths. Although different types of alcohol e'cist. ethyl

alcohol or ethanol (ETOH) is the intoxïcating agent present in au alcoholic beverages. It is

a relatively simple molecule formed by a two carbon chah: CH,-CH,-OH. The molecular

composition of ETOH is both hydrophillic and hydrophobic. The polar hydroql group

renders ETOH misci'ble m all proportions with water. On the other hand. the non-polar CH,-

C H - portion makes ETOH soluble in lipid-dissoking fluids. Nevertheless. ETOH is poorly

soluble m Lipids themselves since its pamtion coefficient between the lipid and aqueous phases

is around 0.1 (molkg membrane)/(molll water). meaning that its concentration in tissue

lipids is always 10°/o of its concentration in body waters (Seeman et al.. 1972).

This characteristic of ETOH suggeas a rather weak dmg. Due to its strong preference

for the aqueous phase. the ETOH concentration in the blood must be high enough to force

ETOH into the ce11 membranes in order to achieve any effect on the various tissues of the

body (Goldstem. 1992). Paradoxically. this low potency of ETOH as a dmg is the main cause

of the ETOH-related health problems. associated with chronic heaw alcohol consumption.

n i e physical mtercalation of ETOH mto the Lipid membrane fits the Meyer-Overton concept.

which correlates the potency of an anesthetic with its iipid solubility (Meyer. 1937: Seeman

et al.. 1972: McCreely and Hunt. 19%). Non-saturable ETOH binding to ce11 membranes has

also been demonstrated. suggeaing the absence of specific receptors (Seeman et al.. 1972).

The nature of the ETOH interaction with the ce11 membrane is therefore considered similar

to that of a weak anesthetic. ETOH dissohies physically into the phospholipid membrane to

produce expansion and disordering. associated with elevated fluidity (Seeman et al.. 1974:

Chm and Goldstem. 1977; Taraschi and Rubin. 1985; Hunt. 1993 ). Most of the p hysiological

actions oT ETOH result from these non-specific interactions and from the secondary

modifications m ionic channels (Messing et al.. 1986). honnonal/neurotransminer systems and

their receptors ( V a k e ~ s et al.. 1989: White et al.. 1990). G-proteins and second messengers

(Stenstrom et al.. 1986: Diamond et al.. 1987: Chamess et al., 1988). and gene evpression

characteristics (Wilke et ai., 1994).

The blood alcohol content (BAC) depends on the dose administered. the route of alcohol

into the vascular system and the rate of its metabolism and excretion.

1.1.3. ABSORPTION AND DISTRIBUTION

The major route by whicli ETOH reaches the circulation is through the gastrointestinal

(CI) tract. In rare occasions ETOH may also be absorbed through pulmonary tissues (Leaer

and Greenberg. 195 1). The skin however is a structure quite impermeable to allow ETOH

a s i o n (Bowers er al., 1942).

Within the GI tract. ETOH is absorbed by passive diffusion from the small intestine and.

to a d e r extent fiom the stomch (Li 1980: Brick et al.. 1988). Several factors may affect

the rate of absorption. The diffusion of ETOH into the blood is directly related to its

concentration. Stornach emptying tirne is also important because of the high proportion of

ETOH abçorbed fiom mteainal tissues. The passive difision of ETOH fiom the GI tract to

the vaxulature is also regulated by the diffusion gradient. modified by the blood flow or the

BAC (Berggren and Goldberg. 1940). Another major factor is the presence of food in the

stomach known to delay both ETOH diffusion and stomach emptying (Melanby er a/.. 19 19:

Kalant et al.. 197 1). It is of importanc.z to note that longer ETOH transit tirnes in the nomach

and slower absorption rates are associated with lower final BAC values. because of the

significant contribution of the gastric and hepatic fira-pass ETOH metabolism (see section

1.1.4. ) (Goldstein, 1992).

Once into the cuculation. ETOH is distributed throughout the aqueous compartments

of the body. Because of their rich blood supply. the brain. liver. kidneys and lungs equilibrate

faster with BAC than less vascularized tissues (Pohorecky and Brick. 1988).

1.1 .4. EXCRETION .AND METABOLISM

A d l fiaction of mgested ETOH is eliminated mtact through the kidneys. the lungs and

the skin. The rea is metabolized. first to acetaldehyde and then to acetate.

1.1.4.1. ET0 H to acetaldehvde

al d kohol dehydrogetiase

CH,CH,OH + NAD' =s CH,CHO + NADH + W tETOI1) ( ci~litrildCh'.dL')

The major enzymatic pathway for the oxidation of ETOH to acetaldehyde involves the

enzyme alcohol dehydrogenase (ADH). ADH is a cytosolic zinc-containing enzyme which

requires nicotinamide-adenine dinucleotide (NAD) as a coenzyme to activate the reaction

(Von Wartburg et al.. 1964; Ehrig et al.. 1990). ADH is expressed m the liver and on the

gastnc walls (Smith et al., 1972; Ehrig et al., 1990) and is characterized by its Iow Michaelis

constants a, which suggest an actMty even with very low ETOH concentrations (Lieber,

1994).

Even though the ADH m o l d e s mediate most of ETOH oxidation, the rat e-limiting nep

m the reaction is usu* the regaieration of NAD' f?om NADH During alcohol mtoxication.

the hepatocytes use the majority of their available dehydrogenases to reoxidize NADY often

m spite of theh mability to maintah redox homeostasis. This results in the altered metabolism

of various other compounds, such as Eits, steroids and carbohydrates (Lundquist er ai-. 1962:

Forsander, 1966).

During acute alcohol consumption, ETOH is metabolized by gastric and hepatic ADH

(figure 1.1.1. ). FM-pas metabolisn reduces the bioavailability of ETON both directly fiorn

the stomach and during its first passage through the liver (JuIhunen et al.. 1985; Lim er al..

1993). During chronic heavy alcohol consumption or in alcoholics, the gaaric first-pass

metabolism is reduced due to the decreased activity of the gaanc ADH (Di Padova et al..

1987). in contrast to gastric ADH, the a c t ~ t y of the h e r ADH system is not sigdicantly

altered by chronic ETOH consumption, until the appearance of serious h e r disease.

6) Micrmonrai etha~rol-oxidizi~rg system MEOS)

CH,CH,OH + NADPH + H* + 0, =, CH,CHO + NADP+ + 2H,O (ETOH) (acddebyde)

A second enzymatic pathway for the oxidation of ETOH, independent of ADH, has been

identified in liver microsornes (Lieber and De Carli, 1968, 1970). It involves a family of

ETOH-inducible P-450 enzymes (P-450 2E 1 is the major one induced by ETOH) and the

presence of oxygen and NADPH (Ohnishi and Lieber, 1977). The Y, of MEOS for ETOH

is about one order of magnitude higher than the Y, of ADH, meaning that at low ETOH

concentrations the hepatic ADH pathway is responsible for most of ETOH metabolism (figure

1.1.1 .). However, during ETOH intoxication, a significant portion of ETOH may be

catabolized by MEOS.

Similarly, t6e long-term use of alcohol in both experimental animais and alcoholics bas

ALCOHOL '7

Acetaldehyde

Acetaldehyde

ACUTE 7 1 ALCOHOL 1 ALCOHOL 1

Acetaldehyde

Acetate CH RONlC ALCOHOL

ALCOHOL ,

f

Aceta dehyde + ?

Acetate

F ig u fe 1 .1.1. Schematic representation of gastric and hepatic alcohol metabolism during acute and chronic ETOH consumption (ADH: Alcohol dehydrogenase, ALDH: Aldehyde dehydrogenase, MEOS: Microsomal ethanoi~xid~ing system)

been associated with a sigdicant induction of P-450 2E 1 and with elevated levels of P-150

2E 1 transnipts (Tnitsumi et a/. . 1989: Diehl et ai.. 199 1 a: Takahashi et ai.. 1 993 ). Therefore.

during chronic ETOH connimptioa. a subaantial portion of ETOH metabolism is achieved

through the MEOS (figure 1.1.1. ).

CH3CH20H + H20Z * CHICHO + 2Hr0 (ErOEI) c r i ~ ~ & h ' . & )

A third minor enzymatic pathway has been described for the oxidation of ETOH. Hepatic

catalase has been s h o w to metabolize ETOH iti vitro in the presence of hydrogen peroxide

(H201) (Keilin and Hartree. 1945). However. this pathway is unlikely to contniute much to

ETOH metabolism since the rate-lirniting step is the formation of HZ02 . hdeed. in the liver

HZOZ is generated m significantly lower levels than expected based on the content of hepatic

catalase (Bovens et al.. 1972). Furthemore. indirect estimations of the Y, for ETOH by the

catalase reaction has reponed high values. m the same range as those reported for the MEOS

(Oshino et al.. 1 973 ).

d) iVor1-oxïdative patlnvqs

The production of fatty acid ethyl esters fiom ETOH by the action of the enzyme fatty

acid ethyl ester synthase has been reported (Mogelson and Lange. 1984). This non-oxidative

metabolism of ETOH is present in ogans lacking the traditional ox ida t~e ETOH pathways.

such as the brain. the pancreas and the heart. and has been implicated in alcohol-related

mjuries (Laposata and Lange. 1986: Bora and Lange. 1993 ). Interestingiy. the actkity of the

brain fatty acid ethyl ester synthase is increased in alcoholics (Laposata et ni.. 1987).

1.1.4.2 Acetaldehvde to acetate

CH,CHO + NAD+ + H,O =s CH3COOH + NADH + W ( ~~xtdd&> dC) ( ~ L X ~ L . )

The major consequence of the oxidation of ETOH to acetaldehyde is to convert a rather

weak drug to a toxic one. Acetaldehyde has been s h o w to form protein adducts with liver

microsomal proteins such as collagen (Baraona et ai.. 1993) and with cuculating protems

such as hemogiobin (Stevens er ai.. 198 1 ) and Lipoproteins (Wehr et al.. 1993). resulting in

antibody production. alterations in protein release and modifications in enzymatic activities

(Hoemer et al.. 1986: Solomon 1987: Tuma et al.. 1990). Acetaldehyde has also been s h o w

to promote lipid peroxidation and f?ee radical-mediated to'ricity (Müller and Sies. 1982).

Furthemore. acetaldehyde accumulation is associated with autonomic and cardiovascular

effects such as the "flush syndrome". hyperventilation and nausea. Therefore. under normal

conditioos. the rapid oxidation of acetaldehyde to acetate by hepatic ALDK in the presence

of NAD'. maintains acetaldehyde levels in very low concentrations (figure 1.1.1. ).

Long-term ETOH consumption renilts in the significant reduction of ALDH actMty

(figure 1.1.1. ) (Hasumura er ai.. 1975). This lower capacity of acetaldehyde oxidation.

associated with the enhanced production of acetaldehyde fiom MEOS. leads to bcreased

circulatmg and t h e acetaldehyde levels and the possibility of chronic toxicity (Pikkarainen

er nl., 198 1 : Nomura and Lieber. 198 1 ).

t .1.5. iMAJOR BIOLOGICAL ACTIONS OF ETOH

The incorporation of alcohol into the cell membrane of the cells of vanous tissues

produces a cohon of physiological effects. Among the physiological systems greatly affected

by alcohol are: the central nervous system (CNS). the endocrine system the kidney. the liver

and the cardiovascular qaem The majority of the behavioral effects of ETOH are rnediated

through ETOH-induced alterations in the a c t ~ t y of the CNS. Various components of the

cardiovascular system nich as the contractile properties of the heart or the blood pressure.

are also xnodulated by alcohol consumption. Because of its importance in ETOH metabolisrn

the h e r is greatly affected by ETOH. especially during long-tem consumption. The diuretic

effects of alcohol are mediated in pan through the kidney. Finally. a ournber of hormooal

syaerns. such as arginine vasopressin (AVP), prolactin. testosterone or the hypothalamic-

pituitary-adrenal (HPA) a i s , are also rnodified by the presence of ETOH.

1.1.5.1. Effects of ETOA on the central nervous svstem lCNSl

ETOH is classified as a general neurodepressant dmg. Nevertheless. low levels of alcohol

have been found to produce excitation. suggesting a biphasic effect (Hunt. 1993). At low

BAC (below 50 mgdl). the cognitive h c t i o n s of the cerebral cortex are mostly afTected.

resuitmg in reduced mbibition. decreased tension and general euphona. Higher BAC (between

50 and 150 m m ) impair motor coordination of cerebellar ori_gin. whereas BACS above 200

mg/dl greatly depress consciousneçs. respiration and cardiovascular regulations. With chronic

heavy ETOH consumption. tolerance to these effects may appear. Furthemore. when long-

tenn ETOH drinking is abruptly aopped. a state of general hyperexcitability called "alcohol

withdrawal syndrome" may occur indicating addiction and physical dependence and

characterized by convulsions. seinire. tremulousness and delirium tremens. These various

effects of alcohol are mediated by ETOH-induced aiterations in the actMty of a number of

neurotranmitter systems such a s y-amhobutyric acid (GABA). glutamate or opioids. A brief

description of these alterations and their physiological si_guificance is outlined (table 1 . 1 . 1 . ).

a) y -aminobzc~*rrc acrd (GA BA) receptors

GABA is the major Uihibitory neurotransrnitter in the brain. Its actions are mediated via

two classes of receptors. GABA, and GABA,. GABA, is responsible for mon of the

inhibitory effect of GABA through the activation of the chloride channel associated with the

receptor, leadmg to an ~ U Y of Cl- ions which hyperpolarizes the affected neuronal cells and

therefore decreases the e'rcitability of the neurons (Olsen and Tobin. 1990).

Acute ETOH consumption has been impiicated with the potentiation of GABA, activity

and with the facilitation ofthe association between GABA and its major receptor (Suzdac et

al., 1986: Deitrich et a/. , 1989; Leidenheimer and Hams. 1992; Hunt, 1993). The resulting

neuronal hyperpolarization and lower firing rate in those tissues rnay evlain in part the

intoxicating and depressant eEects associated with high BAC.

In contrast. long-term ETOH exposure is associated with a reduction of the ETOH-

mediated enhancement of GABA, actMty. suggesting tolerance to its acute effect ( M a n and

Harris, 1987: Morrow er al. 1988). Aiterations in the gene expression of GABA, have been

Table 1.1.1. ETOH-irrduced alteratiorzs irr the actzvitv o f the central rrervous systern (C-ï.%

Acute ETOE consum~tion Chronic ETOB coosum~tion

GABA receptors

NMDA receptors

Ca" channels

Adenylate cyclase (AC)

Opioids

Potentiation of GABA, activity (Depressant CNS effec)

lnhr'bition of NMDA activity (Depressant CNS effect) {Short-term amrresia)

inhibition of Ca" curent Oepressarrt C M e ffec t)

Potentiation of AC activity {Grearer p b s iological response fir

neurorransmirrers using rhrs parhway)

Potentiation of DA a c t ~ t y lReirrfocirig effect of ETOH)

Potentiation of 5-HT actMty Reirforcirzg eflect of ETOH)

inhibition of NE a c t ~ t y (Depressarit CNS effect)

Potentiation of opioid activity

Inhibition of GABA, actMty ETOH withdrawal: seizzue arui arzxiev)

Upregulation of NMDA receptors (ETOH withdrmval: h3perexcitability a d seizztre)

Upregulation of L-type Ca2'channels ETOH withdrmvai: hyperexcitabiiity arui seizztre)

Inhibition of AC activÏty (Tolermce to the amte eflecr of ETOH)

lnhiibition of DA actMty meit florcirzg e ffect of E TOH)

inhibition of 5-HT a c t ~ t y (Reirforcirrg effecr of ETOH)

Potentiation of NE activity ETOH rvithdmvai)

Luhibition of opioid activity

postuiated (Morrow et al.. 199 1 ). To sorne extent, these chronic m o ~ c a t i o n s m GABAergic

actMty may provide an explanation for the seizures and anxiety observed during ETOH

withdrawal (Hunt. 1993 ).

b) iV-nie thvl- D-asparta t e N34DA) receptors

Glutamate is one of the major excitatory neurotransmitters in the brain and has been

show to interact maHily with 3 types of receptors: NMDA kainate and a-amino-3-hydroxy-

5-methyl-4-isoxazole proionic acid (AMPA). Like GABA,. these receptors are ionotropic

since the): are coupled to an ion channel permeable to various cations. Kainate and AMPA

receptors are believed to mediate fast neuronal excitation through Na'. whereas the NMDA

receptor is mvoived m slower excitatory responses through Ca". NMDA receptor actMty and

inward calcium flunes have been implicated with newotransminer release. learning and

memory (Collingridge and Leiter. 1989: Cotman et al.. 1989).

Acute intoxicating levels of ETOH are reported to depress NMDA receptor functions

and to mhibit NMDA-activated Ca2+ currents (Lovinger et al.. 1989: H o b et al.. 1989:

White et al.. 1990). The iower intracellular Ca2+ IeveIs decrease neuronal activity and may

account for the sedative effect of alcohol. Similarly. the ETOKmediated inhibition of NMDA

activity in hippocampal cells may explain the "short-term amnesia" observed ofien afier

excessive drinking (Diamond and Messing. 1994). In contrast. lower levels of ETOH have

been associated with enhanced NMDA receptor functions. suggesting a biphasic effect of

alcohol ( Lima-Landman and Albuquerque. 1989).

Long-term ETOH treatment induces the upregulation of NMDA receptors. in order to

cornpensate for the chronic reduction of CaL' infiuxes by the continuous presence of alcohol

(Michaelis et al.. 1978. I W O : lorio et al.. 1992). This overexpression of NMDA receptors

produces a state of excessive glutamate and excitatory amino acids (EAA) activation.

conmbutsig to the hyperexcitabdity and seipire production during alcohol withdrawal (Grant

et al., 1990: Nutt and Peters, 1994).

cl C bllage-dependent calcitin~ (Ca'-) charnels

ETOH does not only disrupt receptor-mediated ion channels but also voltage-gated ion

channels. 'The L-type ~ a " channel is particularly sensitive to ETOH. Much &e for NMDA

receptor functions acute ETOH administration mhiits Ca2+ current through this channel

(Leslie et a/.. 1983: Treistman el ni.. 199 1). resulting in reduced byaptic release of

neurotransmitters. whereas adaptive compensations occur with chronic alcohol consumption.

producing the upregulation of L-type Ca" channels. This upregulation of L-type Ca'+

channels contniutes to a number of ETOHs withdrawal syndromes, such as seizures and

overexcitability (Messing et al.. 1 986: Little. 1 99 1 ).

d) S e c o d nzesserrger gstenrr (Adeqvlate cydase)

The incorporation of ETOH into neuronal ceil membranes has been implicated with

alterations of the adenosine 3'. 5'-cyclic monophosphate (CAMP) second messenger system

(Hofban and TabakoE 1990). Acute ETOH exposure stimulates adenylate cyclase (AC) Ma

the enhanced activity of stimulatory G-proteins. resulting in greater physiological responses

to neurotransmitters using this pathway. such as monoamines (Rabin and Molinoff. 198 1 :

Leithm and TabakofE 1984). In contraa. chronic ETOH consumption appears to reduce the

efficiency of the CAMP system and therefore of the translation between the neurotranmitter

signal and the biological effect. probably via heterologous desensitization (Israel et al.. 1972:

Gordon er al.. 1986). This is an example of tolerance to ETOH following its prolonged

exposure.

e) h./otrmtirres

n i e major monoamine neurotransmitters are dopamine. serotonin and norepinephnne.

Acute ETOH consumption is associated with elevated dopamine levels in the nucleus

accumbens, probably because of the ETOH-mediated mcreased activity of serotonin receptors

(Di Chiara and Imperato. 1988: Lovinger. 199 1 ). In contrast. lower brain levels of dopamine

and serotonin have been obsewed following chronic ETOH conaimption, suggesting adaptive

compensations (Wu et al.. 1986: Wang et al.. 1993). The implication of serotonin and

dopamine in the reinforcing effect of alcohol is suggeaed by lower brain concentrations of

both monoamines and by elevated serotonin receptor numbers in ETOH-prefening arains of

rats. predisposing these animals to alcohol drinking (Murphy et al.. 1987: McBride et al..

IWO: Hunt. 1993).

Acute ETOH consumption is associated with lower brain levels of norepmephrine (Alan

et a/.. 1987: Patel and Pohorecky. 1989). During chronic ETOH drlliking. norepinephnne

release is increased in the brain. possibly because of the dom-regdation of the inhibitory

presynaptic a*-adrenoceptors located on norepinephric neurons (Nutt et al.. 1988: Wang et

O!.. 1993). This overac t~ ty of the CNS norepinephnne system has been implicated with the

alcohol withdrawal syndrome (Hawley et al.. 1985 ).

Opioidî

Alcohol preference may also depend on the a c t ~ t y of the opioid system (Gianoulaliis.

1993). Various opioid receptor antagonids have been reported to reduce voluntary ETOH

htake (Hyytia et al.. 1993 ). Moreover. although acute ETOH consumption increases plasma

and CSF Bendorphin leveis (Borg et al-. 1982: Gianoulakis and Barcomb. 1987: Gianoulakis

et ai.. 1996). chronic ETOH exposure, such as in alcohol addicts, is associated with reduced

central 0-endorphin levels. suggesting a desensitization of the opioid syaem and the

possibility of opioid-dependent ETOH addiction and dependence (Gienazzani et al.. 1982:

Hutchison et d.. (988).

Although most short-terrn and moderate ETOH consumptions produce only reversible

changes m neurophysiological functions. prolonged heavy ETOH drinlimg has been implicated

with various more permanent brain Iesions. such as Wernicke's encephalopathy and

Korsakoff s psychosis (Tuk. 1 992: Chames. 1993 ). The former is characterized by confiision.

ataxia and oculornotor abnonnalities. The latter is a sequelae of Wemicke's encephalopathy

and consists of short-term memory anmesia. n i e exact role of alcohol in the etiolow of those

diseases is unclear. However. chronic ETOH consumption-associated malnutrition and

especiaiiy thiamine deficiency have been positively h k e d with the development of these

neurological disorders (Blass et ai.. 1977: Victor er al. . 1989).

1 . l S . Z . Effects of ETOH on the endocrine system

A number of ETOH-mediated manifestations such as diwesis, tolerance. withdrawal and

even liver abnormalities. have been associated with specific ETOH-induced alterations in the

a c t ~ t y of some endocrine hormonal systems (table 1.1.2.).

a) ArgirNrie vasopressir~ (A 17')

Acute ETOH consumption induces diuresis (Murray. 1932). This effect has been

attnbuted. at leaa in part. to the ETOH-mediated inhibition of AVP release from the

posterior pituitary gland (Eisenhofer and Johnson. 1982: Leppaluoto er ai.. 1992). More

specificaUy. AVP suppression by ETOH appears to be limited to the ascending dope of BAC.

whereas an increase in plasma AVP content is observed during the descending BAC levels

(Linkola et al.. 1978: Eisenhofer and Johnson. 1982). This biphasic AVP response may be

due to a secondary increase in osmolarity. suppressing the direct ETOH inhibition of AVP.

In contrast. stimulations of AVP release have been observed with chronic ETOH

connunption to cornpensate for the continuous presence of alcohol. redting in water

retention during alcohol withdrawal (Eisenhofer et ni.. 198% Emsley et al.. 1987: Taivainen

er al.. 1995). interestingly. many peptides of the brain. including AVP. are important for the

acquisition and retention of certain learned behaviours and for the etiology of convulsive

disorders (Kasting et al. . 1980: Cicero. 198 1 ). The dernonaration that both the tolerance to

the hypothermie effects of alcohol and the duration of the ETOH withdrawal seinires are

prolonged in ACT-treated rats suggests that elevated AVP levels may also participate in the

developrnent of tolerance and physical dependence (HotEnau et al.. 1978: Rigter et al. . 1 980).

b) Hypothaianric-piti~iraq-adred IHPA) ais

Acute ETOH drinking increases circulahg cortisol (humans) / corticosterone (rodents)

levels (Ellis et al.. 1966: Ienkins and Connelly. 1968). This elevatioo appears to be under

hypothalamic and pituitaiy controL smce plasma ACTH levels are also increased after ETOH

Acute ETOB Chronic ETOH consumption

AVP

I-IPA axis

HPG a i s

HPT a i s

Catecholamines

ml

Endothelin- 1

Prolactin

GH

inhibition of AVP release (Ditiretic effect of ETOH)

Stimulation of cortisol/ corticosterone and ACTH release

Reduction m plasma testosterone levels

Reduction in plasma T, levels

Elevation in plasma cat ectiolamine levels 0 'asocorzstrictiorl)

Stimulation of PGI, release f i 'mdilatiorr)

Stimulation o f NO release flrasodilutiorl)

lnhiition of TXAl release ,~'asodilutiott)

Stimulation of endothelin- 1 release flhsocortstrictio?l)

Elevated plasma prolactin levels

Inhidition of GH release

Stimulation of AVP release (ETOH withdrawal: wuter reterrtiorl arld se izure) (Fzir~ctiorral toferance to ETOH)

Stimulation of cortisoUcorticosterone and ACTH release ETOH tvithdrawal: seiztrre) (Ftrrzctiorral tolerurlce to ETOH)

Reduction m plasma testost erone tevels flesrictdar arrophy) (Disruptiorr of the rner~striral cycle) Metabofic toferailce to E TOH)

Reduction in plasma T, levels (ETOH hepatitis and cirrhosis)

Elevation in plasma catecholamine levels (heavy E TOH-iridiiced hypertemiort)

inhibition of PGI, release fieavy ETOH-iruiuced hyperterzsiorz P))

Stimulation of NO release (Fwlctiorraf tolerurtce to the vasocorntrictive effect of ETOH)

Unchanged or stimulated TXA: release (heavy E TOH- irzdziced hijoertemioti PI/

Stimulation of endothelin- 1 release k e m y ETOH-induced hyperterrsiori PI)

Elevated plasma prolactin levels (ETOH withdrawal)

consumption (Rivier et al.. 1984: Redei et al.. 1986).

Excessive long-term use of ETOH is also associated with high plasma levels of cortisol

(humans) / corticosterone (rodents). leadmg to various clinical features descnied as "pseudo-

Cushing's syndrome" (Frajria and Angeli. 1977: Van ThieL 1983). hcreased

CoibsoVcorticosterone levels may also be related to the development of phytical dependence

to ETOH. since adrenalectomy markedly decreased the severity of the alcohol withdrawaI

seinires m rats (Sze et al.. 1974). The HPA a i s may also participate in functional tolerance

to ETOH since the administration of corticosterone antagonists attenuated the development

of the tolerance to the hypothermie effect of chronic ETOH drinking in rats (Sze. 1977:

Tabakoff and Yanai. 1979).

Both acute and chronic ETOH administration reduce circulating testosterone levels

(Gordon et al.. 1976; Cicero and Badger. 1977: Lester and Van =el. 1977). Elevated

hepatic catabolism of testosterone (Rubm er a/.. 1976). increased arornatase a c t ~ t y (Gordon

et al.. 1979a). decreased testosterone biosynthesis by the testis (Gordon et al.. 1980: Cicero

and B e l 1980) and diininished lutemiMg hormone releasing hormone (LHRH) release from

the hypothalamus (Chapm et al.. 1980: Cicero. 198 1 ) have ail been implicated with this effect.

Longtemi ETOH drinking is associated with testicular atrophy in males and disruption

ofthe menstrual cycle in females (Van Thiel et al.. 1977: Cobbs et al.. 1978). The increased

conversion of androgens to estrogens also produces " feminizationt' in some long-tenn

alcoholics (Gordon et ni.. 1979b). Fiiially. ETOH-mediated hypogonadism Las been

implicated m the metabolic tolerance to alcohol. indeed. the levels of hepatic ADH are under

the negative control of testosterone. so that with the suppression of testosterone synthesis by

ETOH. there is a reduction in this inhibitory control and a si&cant increase in the

subsequent rnetabolism of alcohol. leading to metabolic tolerance (Rachamin et al.. 1980:

Cicero. 1982).

d) &pothalamic-pinrita~~thyroid (Wu mis

Cûculating triiodothyronine (T,) levels are often lower with acute and chronic ETOH

constmptioa especially in alcoholics with h e r diseases (Bexmudez et al., 1975; Israel er al.,

1979). The most ~ e l y mechanism invohred appears to be a direct effect of ETOH on the

conversion of thyroxine (T,) to T, in the liver (Israel er al.. 1979). Long-term ETOH

alterations of the thyroid hormone may play a role in the etioloa of alcobolic hepatitis and

cirrhosis since the hypermetabolic state induced in the h e r by chronic h e a y ETOH

administration is eliminated in thyroidectomized animals (Bernstein et al., 1975 ).

e) Cntechoiantirle

Plasma catecholamine levels are generaily elevated following acute and chroaic ETOH

consumption (Guaza and Borrell. 1983 : Ireland et al.. 1984: Howes and Reid. 1985 : Howes

et ni.. 1986). interestingly. these ETOH-induced alterations in cuculating catecholamine

levek appear to remit prirnarily fiom reductions m epinephrine and norepinephrine clearance.

rather than fiom mcreased catecholamine release (Howe and Reid 1985 : Howes et a/. . 1986).

The substantial pressor effects of catecholamine may also explain in part the hypertension

associated with chronic heavy ETOH consumption (see section 1.2.2.3.).

f) Etldotheliirni- arrd plorelet-cierived vnsoactive factors

Tlie production and release of prostacyclin (PGI,). a local vasodepressor agent Iocated

m endothelial celis. is increased foilowing acute ETOH administration (Karanian et ni.. 1985:

Mehta er al.. 1987: Karanian and Salem, 1988). in contrast. its release is significantly

decreased following chronic Iieaky ETOH consumption or in alcoholics (Foraermann and

Feuerstain. 1987: Karanian and Salem, 1988).

The secretion of nitric oade (NO). another vasodepressor factor produced by endothelial

cells. is also increased afier acute alcohol drinking in several vascular beds. such as in

pulmonary arieries (Greenberg et al.. 1993 ). However. the ETOH-induced contraction

observed in certain vessels. such as in the aorta. has been s h o w to r e d t fiom the partial

suppression of endothelium-dependent vasorelaxation, çuggesting an inhibitory effect of

ETOH on NO release in these vessels (Hatake et al.. 1989: Wang and Fang. 1993: Hatake

et al., 1993). Nevertheles a reduction of the ETOH-induced contraction has been obsewed

m these vessels following chronic ETOH consumption. mediated by a secondary increase in

NO production and release fiom endothelial ceils and suggesting functional tolerance to the

vasoactive effects of ETOH (Knych. 1992: Hatake et al.. 1994).

Acute ETOH consumption sijpficantly lowers the production and release of

thromboxane A, (TXAZ). a vasoconstnctor agent produced by the platelet (Miai l idis et 01..

1983: Mehta er al.. 1987: Karanian and Salem 1988). TXAl is unchanged or slightly

increased following chronic ETOH treatment (Forstermano and Feuerstain. 1987: Karanian

and Salem, 1988).

Both acute and chronic ETOH connimption have been associated with elevated levels

of endothelin- 1. a local vasoconstrictor factor located in endothelial cells (Kawano et al..

199 1 : Nanji er al.. 1 994).

The balance of these opposhg effects of ETOH on the various vascular beds may explain

the presence of either vasodilation and vasoconstriction following acute and chronic ETOH

consumption (see section 1.2.1.3. and 1.2.2.3. )

g) Others

Although conflictmg, aiterations m the actMty of additional hormonal systerns by ETOH

have been observed. Elevated plasma prolactin levels have been described in alcoholics

(Linclholm et al.. 1978). The negative correlation between prolactin levels and the severity

of alcohol withdrawal rnay suggest a role for prolactin in the withdrawal symptoms

(Pohorecki and Brick 1988). The release of growth hormone (GH) is reduced both by acute

and chronic ETOH consumption (Gandai et al.. 1978: Redmond. 198 1).

1.1.5.3. Effects of ETOH on the kidnev

The modifications m water and electrolytes balance by alcohol drinking are also mediated

by direct effects of ETOH on the kidneys. Acute ETOH consumption produces diuresis. ai

least during the ascending portion of the BAC (Jones. 1990). Sodium excretion is initially

unaffected but is later iocreased to restore plasma ion levels altered by the initial water

exmetion (Ponticelli and Montagnino. 1979). Althou& the diuretic effect of ETOH is due in

part to the inhibition of AVP release. direct modifications m renal hemodynamics may also

occur. such as elevated giornedar filtration rate (GFR) (Kmck and Krecke. 1965). The

delayed natriuretic effect may also depend on GFR but appears to r e d t mainly fiom reduced

Na' reabsorption by the nephron. probably because of the inhibition of basolateral N a * K -

ATPase punip activity and the reduced gradient for Na+ reabmrption (Israel and Kalant. 1963:

Parenti et al.. 199 1 : Rothman et al.. 1992).

Chronic ETOH consumption is associated with adaptive alterations to compensate for

the repentnie acute effect of aicohol driuking. Tbe water retention and s i w c a n t antidiuresis

observed in alcoholics and chronic ETOH animal models may result 6om the elevation in

AVP release (Taivamen et al., 1995). Sodium retention is generally noted probably because

of the secondary mcreased activity of Na+/K+-ATPase with chronic ETOH. resulting in lower

mtracellular Na+ concentrations and elevated gradients for Na' reabsorption by the nephron

(Beard and Knon. 1968: Ponticelli and Montagnho. 1979: Rodrigo et al.. 199 1 ).

Hypokalemia is sometimes associated with alcoholism mainly because of deficient nutrition

(Wadstein and Ohlin. 1979). The general stimulation of Na*/K'-ATPase actMty rnay also

increase intracellular K' and hyperpolarize the cells. leading to lower circulating K- levels.

Hypokalemia lias been implicated with the development of deiirium tremens (Wadstein and

Ohlin. 1979). Plasma rnagnesium levels are also decreased by chronic ETOH consumption

and hypomagnesemia may be important for withdrawal symptoms such as seinire (Nielsen.

1965).

Renal dysfunctions are usually reversible with cessation of druiliing. although renal

papillan, necrosis rnay develop in alcoholics with liver diseases (Edmondson er al.. 1966: De

Marchi et al., 1993).

ï.l.S.4. Effects of ETOA on the liver

The central role of the h e r in the metabolism of ETOH predisposes this organ to the

toxic effects of oxidative enzymes and ETOH metabolites. However, long-term heavy ETOH

intoxication is needed for the appearance of hepatotoxicity since both hurnan and animal

studies have s h o w that chronic moderate ETOH connunption is not associated with any

manifestation of alcoholic h e r diseases (ALD) (Van de Wiel et al.. 1990: Savolainen er al..

1993). ALD is characterized by three specific histopathologic stages: fatty liver. hepatitis and

Gbrosis/cirrhosis (Crabb. 1993). Malnutrition is a conmbuting factor. but ALD rnay ail1 occur

in the absence of nutritional deficiencies (Lieber, 1994).

The oxidations of ETOH and acetaldehyde by ADH and ALDH use NAD+ as a cofactor.

With the chronic presence of large amounts of ETOH elevated levels of reducing agents such

as NADH are produced by the liver. overwheiming the hepatic capacity for NAD'

regeoeration (redo't mechanism) and leadmg to various metabolic disorders. lncreased

NADWNAD' ratio is associated with trigiyceride accumulation in the liver and the

development of fàtty liver and hypoglycemia (Salaspuro et ai.. 198 1 : Jauhonen er ai.. 1982:

Lieber. 1994). The increased MEOS actMty in alcoholics not only accelerates the oxidation

of ETOH. but also the metabolism of vanous other microsoma1 substrates. generating on

occasion metabolites more toxic than the precursors. nich as superoxide radicals (Dai et ai..

1993).

Acetaldehyde is responsiile for several hepatotoxic effects of ETOH. Lipid pero'adation

caused by acetaldehyde may damage hepatic tissue (Müller and Sies. 1982). Acetaldehyde-

protein adducts may also delay protein secretion into the plasma. resulting in hepatocyte

enlargement (Baraona et al.. 1977). modify enzymatic a c t ~ t y (Solomon. 1 987) and produce

immune reactions (Hoemer et ol.. 1986: Niemela et al.. 1987). Finally. acetaldehyde rnay

stimulate the collagen accumulation observed during the various stages of ALD (Mann et al..

1979; Moskage et al., IWO).

1.1.5.5. Effects of ETOH on the cardiovascular svstem

The two major components of the cardiovascular syaem the blood vessels and the heart

itself are both greatly affected by ETOH consumption. Because of the controversial nature

of the data obtained fiom studies mvestigating the interaction of alcohol with the vasculature.

m tenns of both blood pressure and vascular disorders. and because of their importance for

the description of the thesis hypothesk, the ETOH-blood vessels interactions wili be discussed

extensively m the section of Alcohol and Blood Pressure (section 1.2.). Consequently, the

present section wiil review only the modifications mduced by acute and chronic ETOH

drinking on the cardiac tissue.

The acute exposure of the heart tissue to ETOH reduces cardiac contractility (Wong,

1973; HoMlitz and Atkins, 1974; Urbano-Marquez et al.. 1989; Pagala et al., 1995). The

strength of the myocardium contraction is a weil defined process depending on the electrical

excitation of the cellular membrane and the subsequent activation of the mtracellular

contractile protems. Briefly, the openhg of the fast Na' channels leads to mtracellular

depolarization and to the openhg of the voltagedependent Ca" channels fiom the

cytoplasmic and sarcoplasmic reticulum membranes. Extracellular and aored Ca" rushes into

the cardiac cells, giving nse to a transient mcrease in cytosoiic Ca2+ levels. This, in tum,

activates rnyonlament contractioq via the bmdmg of Ca2+ to troponin C. The activation of the

contractile protem is terminated by the active transport of Ca2' out of the cytosol through

ATP-dependent pumps and Na+/Ca2' exchangers (Thomas et al.. 1994). The physical

intercalation of ETOH into cardiac cell membranes reduces the peak amplitude of the

cytosolic Ca" mcrease, probably via several modifications in the excitation-contraction

process (Thomas et aL. 1989). Reductions m the CaL' currents 60m the cytoplasmic and

sarcoplasmic reticulum membranes (Mongo et al.. 1990) and alterations in ionic pump

activity, such as the inhibition of the Na', K'-ATPase a c t ~ t y (Williams et ai.. 1975), have

been iniplicated with the lower cardiac contractiiity. Lower Ca2+ norage m the sarcoplasmic

retic* probably by the reduction m the activity of the Ca" pump (Thomas et ai.. 1994).

and a decreased sensitivity of the myofilaments to CaL' (Damiger et al., 199 1; Rozanski et

al.. 1992) may dso contniute to the lower contractility induced by acute ETOH.

Chronic ETOH exposure is associateci with adaptive modi6cations in cardiac morphology

to compensate for the cardiodepressant action of repeated alcohol intoxication. Gradua1

mcrease m total heart mass and dilation of the ventricles are noted (Capasso et al., 1992). The

Can channel den* is Hicreased, refiecting m n d a r y changes and tolerance to the inhiibitory

effects of acute ETOH exposure (Littleton, 1988). Components of the alcohol degradation

produas, such as acetaldehyde and fatty acid ethyl esters, which are elevated after chroaic

ETOH consumption, may dso depress cardiac contractility (Williams et al.. 1975: Lange,

1991). Eventuaily m humans, after at least a decade of heavy ETOH consumption, permanent

damages in the har t tissue may occur. Cardiomyopathy, the moa common long-tem ETOH-

related disorder, is characterized by cardiomegaly, dilation of the left ventricle and low-output

congestive heait fadure (Sheehy, 1992; Rubm and Urbano-Marquez, 1994; Reedy and

Richardson, 1994). It is a Sgdicant cause of cardiac morbidity and mortaiity. Chronic heavy

ETOH consumption is also associated with arrhythmias and in certain instances with sudden

death (Regan, 1990).

1.2. ALCOHOL AND BLOOD PRESSURE

1.2.1. ACLJTE ETOEI CONSUMPTION

1.2.1.1. Auman studies

In the last 25 years, a number of studies have mvestigated the acute hemodynarnic effects

of ETOH m human subjects (Table 1.2.1 .). However, a consensus regarding the direction. if

any, of the ETOH-induced change in BP has been difncult to obtain.

a) ETOH-ir2duced decreme irl BP

A majority of studies particuiarly in recent years, have reported a depressor effect of

acute ETOH connimption (in 38% of the midies mvestigating acute ETOH administration

and BP) (see table 1.2.1.. part A). in generai, these studies demonstrated a slow but long-

lasting hypotensive effect of acute ETOH administration fiom 30 minutes to 8 hours

following the alcohol ingestion. Furthemore, the BP r e a c t ~ t y to various stimuli mch as

exercise, was also sigiuficantly reduced in ETOH-treated mdividuals. However, HR values

were mcreased in most studies.

Source ETOH ingestion BAC (max) Results

Table 1.2.1, Hzintari studies on the mute effecr of ETOH on the BP artd the HR

B

1

(A) Studies demonstrating a decrease in BP

Conway 1968

Kupari 1983

Eisenhofer et al. 1984

Howes and Reid 1985


Adesso et al. 1990

Kawano et al. 1992

Kojima et al. 1993

Abe et al. 1994

lg/kg in 60 min 25 mmoVL

lg/kgBWmlO-15 BACnot min measured

0.9 g/kg BW in 10 125 mg/d min

1 g/kg BW in 15 min BAC not measured

l g k g B W i n 5 r n i n BACnot rneasured

1 g k g BW with food BAC not measured

1 g/kg BW with food BAC not measured

1 g k g BW with food BAC not measured

1 g/kg BW/day (4 BAC not d a ~ s ) measured

(B) Studies demonstrating an increase in BP

Orlando et al. 2 and 5 oz of whiskey 39.3 and 103 1976 in 12 min mg/dl

Ireland et al. 0.5 g/kg BW in 20 20 mmol/L 1984 min

Potter et al. 0.75 g/kg BW 84 mg/dl 1986

4 BP (30 min) no change HR

uBP(12O to 180 min) h HR(l5 to 180 min)

4 BP r e a c t ~ t y (to NE and methoxamine)

4 BP (4 hrs) h HR (4 to 7 hrs)

4 BP reactMty (to exercice and methoxarnine)

4 BP (20 min) no HR

4 BP ( 1 to 8 hrs) h HR ( 1 to 8 hrs)

4 BP (2 hrs) h KR (2 hrs)

4 BP ( 1 to 8 hrs) fi HR ( 1 to 8 hrs)

4 BP r e a c t ~ t y (to exercice)

fi BP(10 min) fi HR ( 10 min)

h BP ( 10 and 20 min) h HR (20 min)

h BP (30 to 60 min) fi HR (4 to 5 hrs)

Maheswaran et al. -1 dday 199 1

( C ) Siudies demonstrating no change in BP

R S e r al. 1969

Gould et al. 1971

Zostér and SeIlers 1977

Giles et al. 1982


Howes and Reid 1985

Potter et al. 1986

Stott et al. 1987

Made et al. 1993

Koskinen et ai. 1994

O'Caiiaghan et ai. 1995

6 oz of whiskey in 10 min

2 oz of whiskey in 10 min

0.3 and 0.6 g/kg B W in 10 min

180 cc of volka in 5- 15 min

0.5 and 1 g/kg B W in 10-15 min

Igkg in 60 min

0.9 g/kg BW in 10 min



1 g/kg BW in 30 min

BAC not measured

100 mg/d

BAC not measured

36.6 and 85.3 mg/dl

130 mg/dl

50 and 80 mg/a

25 mmol/L

125 mg/dl

84 mg/dl

99 rng/dl

62 mg/dl

18.9 mg/dl (breath)

BAC not davs) measured

no change BP fi HR (30 to 90 min)

no change BP no HR

no change BP 11 HR ( 15 and 45 min)

no change BP lt HR (30 to 240 min)

no change BP r e a c t ~ t y (to NE)

no change BP ( 15 to 90 min) 'IIHR(15 to 180 min)

no change BP ( I O min to 3 hrs) 'II HR (4 to 7 hrs)

no change BP ( 1 to 5 hrs) ft HR (4 to 5 hrs)

no change BP ( 1 hr to 2 days) R HR(1 to 9 hrs)

no change BP ft HR (25 and 40 min)

no change BP (3 hrs) 1 HR variability

no change BP

b) ETOH-irzcizrced irzcrease in BP

hcreased BP values after acute ETOH consumption have been reponed in a few studies

(m 16% of the studies mvestigating acute ETOH adtninistration and BP) (see table 1.7.1. pan

B). In general . these studies observed a rapid ETOH-induced hypertensive effect in the fira

30 minutes following the alcohol mgestion. Furthemore. short-lasting pressor effects of acute

ETOH administration are also noticed in heavy alcoholics. HR values were also elevated in

these studies.

C) No eDct of ETOH orz BP

Several experiments. particularly the older ones. were unable to find any m o ~ c a t i o n s

in the BP values d e r ETOH administration (m -16% of the studies investigating acute ETOH

administration and BP) (see table 1.2.1.. part C). In generaL the doses of ETOH adrninistered

in these studies were lower than m the other experiments investigating acute ETOH

conçumption and BP. Nevertheless. ETOH-mediated increases in HR va lus were noticed in

most studies.

d) Factors rrzflzrertcrng the restr lts obsen'ed nt htrmm stzrdies (A cute ETON)

A nurnber of factors have prevented a straight-fonvard comparative analysis between

studies and lirnited the value of the proposed final eEect of acute ETOH on the BP.

1 ) Previous ETOH consumption : One of the major limitation in human e~~erirnents is the

impossibility to control previous habits of alcohol consumption. Early nudies did not even

report the average ETOH consurnption of their subjects (Conway. 1968). Others simply

mixed ail the drinkmg habits of the patients together. observùig the combined effect of ETOH

m excessive. rnoderate and non-drinkers (Ra et al.. 1969: Gould et nl., 197 1 ; Ireland et al..

1984: Howes and Reid. 1985: Potter et ai., 1986: Kawano et n i . 1992: Kojima et al.. 1993:

Abe er ai.. 1994). Only a few studies screened specifically for low or moderate alcohol

consumption (Zsotér and SeIIers. 1977; Giles et al.. 1982; Kupari. 1983; Maule et al.. 1993:

Koskmen et al.. 1994: OCallaghan er al.. 1995).

2 ) Dose of ETOH: Another difncdty is the alcoholic beverage itself. The quantity. volume

and concentration of ETOH are dBerent in almost every experiment. The BAC may be

considered a relatively good parameter in order to standardize most of the results: its absence

in about a third of the experiments is therefore unfortunate.

3 ) hiration of ETOH drinkmg: The duration of the alcohol consumption may also affect the

way ETOH mteracts with the BP. This parameter varied from 5 minutes in the experiment of

Adesso et al. ( 1990) to 60 minutes in the experiment of Kupari ( 1983).

4) Resence of food: The administration of ETOH with food (ffiwano et al.. 1992) or in a

faaing state (Orlando et al.. 1976) may also affect the outcorne. since the presence of food

in the stomach delay ETOH diffusion and stomach emptying. increasing the first-pass ETOH

metabo lism (Kalant et ai., 1 97 1 ).

5 ) PathophysioloPical conditions: Various clinical conditions with potentially detrimental

consequences on BP sensdMty to ETOH. such as hypertension (Kojima er al.. 1993: Abe et

al.. 1994). heart disease (Conway. 1968: Gould et aL. 197 1 ) or angina pectoris (Orlando er

al.. 1976) have been evaluated without proper controls.

5) Aee of the subiects: Alcohol phannacokinetics are altered by the biological changes

associated wati a& (Pohorecky and Brick. 1988). This may alter the effect of acute ETOH

mgestion on the BP in the vanous studies. since the average age of the subjects ranged fiom

20 years (Adesso et a/.. 1990) to 57 years (Conway. 1968).

6) Time-course of BP measurements: Some e~~er imen t s measured the BP values minutes

afier the ETOH administration (Conway. 1968: Zsotér and Sellers. 1977: treland et al..

1984), whereas others descnied the BP alterations m terms of hours (Howes and Reid. 1985:

Kawano et al.. 1992: Koskinen et al.. 1994) and even days (Stott et ai-. 1987: Abe et al..

1994). Therefore. the initial a d o r delayed effect of ETOH administration on the BP may be

missed in some of these studies.

7) Office versus ambulatorv BP: The stress and the discodort of office BP may have been

responsble for some of the short-laaing pressor effects of ETOH. but have been elirninated

in recent years with the development of ambulatory BP (Kawano et al.. 1992: Abe et al..

1994; 0'Callagha.n er ai., 1995).

8) Isovolumetic versus isocaloric conditions: The type of control used in each audy may also

affect the r e d t . Isovolumetri~ conditions are used m most of the experiments (Zsotér and

Stellers. 1977: Ireland et aL. 1984: Adesso et al.. 1990: Koskinen et al.. 1994). although the

calonc component of alcohol is best accounted for with isocaloric controls (Stott et al.. 1987:

Made et al., 1993 : Abe et ai., 1994).

e) Srrmman

Because of the dinerent methodological procedures and the numerous factors that rnay

influence the resuits observed in human studies. it is dBcult to conclude on the exact effect

of acute ETOH consumption on the BP. Nevertheless. a tentative general description of the

effect of ETOH m human aibjeas is possible. indeed. rnoderate ETOH consumption appears

to produce longlasting reductions in the BP. Such reductions rnay be seen a s early as 20 to

30 minutes &er the ETOH administration (Conway, 1968: Adesso et ni.. 1990). but are most

probably significantly difEerent around the first hour and up to 8 hours following the alcohol

mgestion (Kupari. 1983: Howes and Reid. 1985: Kawano et ai.. 1992: Kojima et ai.. 1993:

Abe et a(. . 1994). hterestingly. a rapid and short-lasting pressor effect of ETOH rnay exist

m the firn minutes following alcohol consumption (Orlando et al.. 1976: Lreland et ai-. 1984:

Potter et ai.. 1986). The absence of this rapid BP increase durinp ambulatory BP

meawements rnay suggest an effect of stress and discodort rather than alcohol itself (Abe

et ai. 1994: O'Callaghan et al.. 1995). The caloric load of ETOH rnay also eqlain tliis fast

mcrease m BP m experiments using isovolumetric controls (Ireland et ai.. 1984: Potter et ni..

1986). The possibility of a secondary withdrawal reaction rnay also exist. especially in audies

where the subjects are regular ETOH drinkers (3 and more drinkdday) (Potter et al.. 1986).

Lower quantities or longer time of ETOH administration are not associated wkh sipificant

modifications on the BP (Zostér and Sellers. 1977; Stott et ai.. 1987; Made et al., 1993 ). In

contrast. almost al1 experiments have reported a significant increase in the E-R lasting from

a few minutes to a few hours following the ETOH ingestion.


There have been comparatively few animals studies on the acute effect of ETOH on the

BP (Table 1.2.2.). Surprisingly. the direction of the ETOH-induced change is Iess

controversial.

CI) E TOH-irrdztced decrease itt BP

Moa of the animal audies have demonstrated a depressor effect of acute ETOH

connimption on the BP (in 6706 of the studies investigating acute ETOH administration and

BP) (see table 1.2.2.. part A). in generaL the ETOH-induced hypotensive effect was noticed

m studies using gastric mtubation as their route of alcohol administration. Furtherrnore. these

studies demonstrated a slow but long-lasting hypotensive effect of ETOH from 30 minutes

to 4 hours. The BP reactivity to vanous stimuli. nich as stress. was also reduced in ETOH-

treated animals. However, the HR values were elevated in most studies.

6) No effect of E TOH mi BP

A few experiments reported no significant variation in the BP following ETOH or

acetaldehyde administration (in 33% of the nudies investigating acute ETOH administration

and BP) (see table 1.2.2.. part B). in general. these studies used i.p. administration of low

levels of ETOH. Nevertheless. the IiR values were elevated in certain studies.

C) Factors itrjltretrcitrg the reszdts obsened itr mimai stirdies (Acirte ETOH)

I ) Previous ETOH exposure: The main advantage of animal versus human studies is the

coqlete control over the conditions. pan and present. of the e'cperimental subject S. ETOH-

naive animals enable to audy the initial acute effects of alcohol. without the possibility of

secondary modifications produced by years of regular alco bol consumption.

2 ) Dose of ETOH: Nevertheless. the concentration and volume of the ETOH &&en in any

study may ail1 result in dserent BAC levels and therefore in different effects on the BP

( Roine et ai.. 1993).

3) Route of ETOH administration: Comparative analysis is further complicated by the route

of ETOH administration in animal studies. either i.p. (Sparrow et al.. 1987). i. t: (Gulati er

ai.. 1989; Tabuzchi and Pang, 1992) or through gastric intubation (Malinowska et al.. 1989:

Source Species and ETOH BAC Results

Table 1.2.2. Animal studies on the amte eflect of ETOH ori the BP ard the HR

I

1

A) Studies demonstrating a decrease in BP

Guiati et al. 1989

Tabrizchi and Pang 1992

Sparrow et al. 1987

Malinowska et al. 1989

Thompson and Adams 1994

Brackett et al. 1994

and 90%) (Cats)

2.4 and 4.8 i-v. mg/min/kg BW ( Sprague-Dawley rats)

[bl Z.P. admirlistmtior

1.p. ( Sprague-Dawley rats)

2 g k g BW gmtric iritztbatiotl

(Wistar rats)

5 gfkg BW gastric itltn batzoti (Sprague- Dawley rats)

2,4 and 6 g/kg BW gmtric itrtu batioti ( Sprague- Dawley rats)

BAC not measured

BAC not measured

BAC not measured

BAC not measured

BAC not rneasured

63, IO3 and 22 1

mg/ dl

4 BP (30 min) 8 HR (30 min)

6 BP (12 min) (4.8 m a l 3 BW) no change HR (12 min)

4 BP reactMty (to strtss) fi H R ( 5 to 120 min)

8 BP (30 to 60 min) fi HR(l5 to 60 min)

8 BP (60 to 230 min) h HR (60 to 240 min)

4 BP (60 to 120 min) (4 and6 g/kg BW) f i HR (for 4 hrs)

B) Studies demonstrating no change in BP

[a 1 i. p. adnzirlstratiorz

Sparrow et al. 0.08 to 6.3 g/kg BW BAC not 1987 i-p. measured

( Sprague-Dawley rats)

Pawlak et al. 3.6 and 12 mgkg BAC not 1993 B W i. p. acetaldehyde meanired

( Wistar rats)

[b 1 i. v. adnz fizistrarioit

Abdel- Rahman 0.1.0.5andlg/kg 71.8 1994 BW i.v. ( SHR. WKY mg/dl(O.5

and Sprague-Dawley g/kg BW)

no change BP ( 5 to 130 mm) h HR ( 5 to 120 min)

no change BP ( 15 to 60 mm) no change HR ( 15 to 60 min )

no change BP (90 min) fi' HR

I rats)

Brackett et ai., 1994).

4) Different specieh: Inter-species variability and strah differences in the cardiovascular

response to acute ETOH consumption may also produce confusion Hi the direction and

magnitude of the BP m o ~ c a t i o n s .

5) Human versus animal studies: Although comparative analysis between animal and human

studies are possible. a direct extrapolation of results obtained on laboratory animals to human

physiology and pathophysiology is hazardous. For example. alcohol metabolism is faner in

rats than in humans (40 vernis 18 mgdl per hour). reducing the effect of a particular BAC

level in rats compared to the effect observed in humans (York 1982: GiIl et al.. 1986).

Nevertheless. animal experiments remain useful models of ETOH consumption and

alcoholism by allowing the nudy of parameters not accessible on human subjects.

d) S z i m n i q

in generai these studies support a slow and long-lasting depressor effect of acute ETOH

consumption in animal experiments. The short-Iasting mcrease in the BP observed

immediately afier ETOH administration in human studies is absent fiom evperiments

performed on rats and cats. The direct recording of BP through a catheter in several studies

may prevent the stress and discornfort caused by the irnmobilition or manipulation of the

animals (Sparrow et al.. 1987: Mahowska et al.. 1989: Thoinpson and Adams. 1994).

Smaller ETOH doses are not associated with any modification in BP values (Tabrizchi and

Pang. 1992: Abdel-Rahman. 1994). In contrast. the HR is elevated in most experiments.

1.2.1.3. Mechanisms

Because of the short-term duration of the acute ETOH effect on the BP. vascular flow

and resistance rather thm the renal characteristics have been investigated. Non-specific

association of ETOH with vascular smootli muscle cells and secondary modifications in ion

cliannels and pumps. as well as ETOH-induced modifications in local and endocrine

vasoactive factors have been postulated. Depending on the vascular bed and the doses of

ETOH @en, vasodilation or vasoconsûiction have been O bserved ( Altura and Altura. 1 9 82).

Low ETOH concentration increases the blood flow in mesenteric. splanchnic. coronary

and portal vessek (Fewings et al., 1966: Carmichael et al.. 1988: Maule et ai.. 1 993 ; Pirwitz

et al.. 1995). Si@cant reductions in total peripheral resistance (TPR) during the

hypotensive phase of acute ETOH administration have also suggested a general peripheral

vasodilation (Kupan. 1983: Kawano et al.. 1992: Abe et ai.. 1994).

1 ) Direct vasodilatory effect of ETOH: A portion of the ETOH-mediated relaxation of

precapillary çphrinters. artenoies and muscdar venules is proposed to result fiom a direct

action of alcohol on smooth muscle cells (Altura and Altura. 1982).

2) Ca2'levels: Lower mtraceilular levels of C a . probabiy via the reduction of intracellular Na-

levels. have been demonstrated with acute ETOH administration and may decrease the

contractile a c t ~ t y of smooth muscle ceiis (Blauaein, 1977: Turlapaty et al.. 1979: Kojirna

et al.. 1993 ).

3 ) Inhibition of ~ressor hormonal vasoconstriction: Moderate amounts of ETOH have also

been shown to reduce the vasoconariction produced by hormonal factors. such as

catecholarnine or AVP (Edgarian and Altura. 1976: Altura and Altura. 1 983 ). Similarly. the

production of local vasoconstrictor agents. such as thromboxane A, (TXA) fiom the

platelets. is reduced foUovhg light ETOH consumption (Mikhailidis et al.. 1983 ). In contrast.

the ETOH-induced mhiition of AVP release has been demonstrated not to contribute much

to the initial decrease m BP d e r ETOH consumption. but may be imponant in sustainin_o the

hypotensive effect by srirnulating diuresis (Cowley and Liard. 1988: Kawano et ni.. 1 992).

4) Stimulation of hormonal vasodilation: The release of local vasodepressor agents. such a s

prostacyclin (PGL) and nitric oxide (NO) fiom the endothelial celis. is increased foilowing

acute consumption of low levels of ETOH (Karanian et al.. 1985: Greenberg et al.. 1993 ).

6) C'mocor lstrictiorr

ETOH is associated with direct vasoconstriction in cerebral. intrapulrnonary and renal

arteries (Altura and Altura. 1982: Toda et al.. 1983).

1 ) Inhibition of hormonal vasodilation: There is the possibility that elevated levels of ETOH

reduce the vasodilation produced by acetylchohe and ATP. although low ETOH

concentrations may potentiate the vasorela?tation caused by these agents (Criscione et ai..

1989). The suppression by hi& ETOH concentration of endothelium-dependent

vasorelaxation. possibly via NO. has also been posnilated in rat aorta (Hatake et ai.. 1989:

Wang and Panp 1993: Hatake et al.. 1993).

2) Stimulation ofhomonal vasoconstriction: The potentiation by heavy ETOH consumption

of the pressor effects of vasoconstrictor agents such as catecliolamines may also occur

(Edganan and Altura. 1976: Talesnik et al., 1980). In several studies. there is an

augmentation in circulating catecholamine levels following acute ETOH administration.

probabiy due to an mcreased release from the adrenal medulla and sympathetic nerves as weli

as to a reduction in catecholarnine clearance (Eisenhofer et al., 1983; Ireland et al., 1981:

Howes and Reid 198% Grassi et ai.. 1989). However. this increase may be secondary to the

hypotensive effect of ETOH since an inverse relationship has been found between the a c t ~ t y

of catecholamine and the extent of the BP reduction (Kawano et a/., 1992). Sirnilarly. the

actMty of the RAAS appears to be unchanged following a single exposure to ETOH. or only

slightly mcreased as a compensatory mechanism to the depressor effect of alcobol (Porter et

ai., 1986: Stott et al., 1987: Kawano et al.. 1992).

3 ) Ca"leve1s: n i e ETOH-induced vasoconstriction rnay ultimately depend on intracellular

Ca" levels. There is some reports that suggest that Ca" levels are increased in certain

vascular beds following acute ETOH admmistration through the inhibition of Na'.K'-ATPase

a c t ~ t y ( Arkwright et ai.. 1984).

C) E TOH-ir zdzt ced irrcrease irr HR

The ETOH-mduced tachycardia obsewed m most studies rnay represent a reflex response

to compensate for the reduction in BP. both fiom initial vasodilation and fiorn delayed

diuresis, thus explainhg the prolonged effect of ETOH on HR a c t ~ t y (Stott et ai.. 1987:

Malinowslia et al.. 1989; Kawano et al.. 1992). It may also be secondary to the decreased

myocardial contractility ( Urbano-Marquez er al.. 1989).

1.2.2. CBRONIC ETOB CONSUMPTION

1.2.2.1. Human studie~

There have been 60 or so epidemiological and experimental studies investigating the

relationship between chronic alcohol consumption and BP since 19 15 (Table 1.2.3. ).

Remarkably. this issue is stiU very controversial. Most of the experiments rneasured the

average systolic and diastolic BP for several speci6c groups of ETOH drinkers. ranghg fiom

occasional to heaw ETOH coonimption. ûne ofthe early problem was the various and often

misleadmg ways the alcohol consurnption was reported in these specific groups. either as o r

ml. g or ?h of ETOH. complicating comparative analysis (Turner. 1990). Therefore. for

standardkation of this chapter. the levels of alcohol consumption m humans are aIi expressed

m standard druiks per day. As a d e . 1 drink is dehed as the equivalent of log of ETOH. or

about 1 glas of wine. one c m of beer or a single measure of spirits. Thus low. moderate and

heavy ETOH con~uniption usuaiiy refer to less than 1 drink/day. between 2 and 3 drinkslday

and a bove 4 drinlidday respectively.

O) Heap E TOH cotuirnlptiori

With the exception of two eqeriments (Baghurt and Dwyer. 198 1 : Coates et al.. 1985 ).

heaw ETOH consumption has always been associated with elevated BP and hypertension

(Table 1.2.3.). The £ira evidence of a pressor efFect of large quantities of alcohol was

published by Camille Lian m 19 15 wbo reported a linear mcrease in systolic BP among French

s e ~ c e m a i with mcreasing amounts of ETOH consumed daily. interestingiy. the group with

the lowea average consumption of alcohol. descnbed as "sobres" (sober). still managed to

dnnk 1 liter of wine/day! Subsequently. from the late 1960s to 1995. a great number of

epidemiological midies observed significant elevations in BP following heacy ETOH

consumption. such as Gyntelberg and Meyer ( 1974) ( 2 5 drinksiday). Klatsky et al. ( 1977)

( r 5 drinkskiay) or Trevisan er al.. ( 1987) ( r 7 drinkdday). Sirnilarly, experimental studies on

heavy alcoholics revealed sigruficant mcreases in their BP compared to moderate drinkers and

non-drinkers (Saunders et al.. 198 1 : Ibsen et ni.. 198 1 : Potter and Beevers. 1984: Puddey et

al.. 1985: Seppa et al.. 1994).

Table 1.2.3. Hzrmari snrdies or1 the chrotiic effecrs of ETOH oti the BP (the range of the

Source Number of Age Results

(A) Studies demonstrating a U- or J-shaped curve

[a J Laver dme of ETON measwed: < I dril~Wdav

Harburg et al. 1980

Criqui et al. 1981

Gordon and Kannel 1983

Harlan et al. 1984

Klastsky et al. 1986

Weissfeld et al. 1988

Marmot et al. 1994

GiUrnan et al. 1995

MacMahon et al. 1984

[b] Laoer dase ETOH rneaszrreci: f drirzWdqv

5550 25-64 J-shaped curve m males ( 1 to >3 drinkdday ) U-shaped cuve in females

doses of daiiy ETOH corrrumptior2 memured h i each stuc& is irrdicated iri parenthesis).

I m

1

1

J-shaped curve in males (0.04 to 7.3 drinkdda y) U-shaped curve in females

J-shaped c w e in males (< 1 to >3 drinksiday ) U-shaped curve in females

J-shaped curve in males (< 1 to >3 drinksiday ) U-shaped c w e in females

U-shaped curve in males (< 1 to >3 drinkdda y ) U-shaped curve in females

S-shaped curve in females ( < 1 to >6 drinkdday )

I-shaped and threshold curves in males ( < O S to >2 drinkdday) U- and J-shaped curves in fernales

J-shaped curve in fernales (< 1 to > 10 drinkdday )

J-shaped c w e in males (< l to >3 drinkdd a y) J-shaped curve in females

Gruchow er al. 9553 18-74 Vproportionofhypertentionintight 1985 and moderate ETOH drinkers

Jackson et al. 1985

Lang et ai. 1987

Moore er al. 1990

Kagan et ai. 1981

Shaper er al. 1988

Keil et al. 1989

1429 35-64 J-shaped curve m m i e s ( 1 to >3 drinkdda y) U-shaped curve in females

6632 18-60 J-shaped curve in fernaIes ( 1 to >8 drinkdday )

2500 ZO+ J-shaped curve in males ( 1 to >2 drinks/day)

[c 1 Laver dose of E TOH rnemzired: < 2 drirzks/dai:

83 947 15-79 J-shaped curve in females (2 to >6 drinkdday )

8006 46-68 J-shaped curve in males (<2 to >4 drinkdday)

773 5 - J-shaped c w e in males

3 100 30-69 J-shaped curve in males (<2 to >4 drinkdda y)

II (B) Studies detnonstrating a threshold eurve (for 11 BP)

[a 1 Lower dose cf ETOH nreaswed: < 1 dritiWdav

Reed et ai. 8000 46-65 threshold curve in males (< 1 to >3 1982 drinkdda y )

Gordon and Doyle 1910 3 8-5 5 threshold curve in maIes (< 1 to >6 1986 driokslday )

DeFrank et al. 4 16 26-49 threshold curve in males (<l to >4 1987 drinkdday )

Dyer et al. 1990

503 1 18-30 threshold c w e in males (< 1 to >7 drinkdday ) threshold curve in females II ~;-lpatz et al. 1784 25-50 threshold cuve in females (<O. 1 to > 1 drinkdday )

Marmot et ai. 1994

Gyntelberg and Meyer 1974

Mitchell et al. 1980

Milon er al. 1982

Cooke et al. 1982

Fortmann et al. 1983

Lang et al. 1987

Klatsky et al. 1977

Cairns et al. 1984

Savdie et al. 1984

ïrevisan et ni. 1987

Keil er al. 1989

Keil et al. i991

Kondo and Ebihara 1984

968 1 20-59 threshold curve in males (< 1 to > 10 drinkdday )

[b] Laver dose qf ETOH rneaîured: I dritik/d-

5249 40-59 threshold c w e in males ( 1 to >5 drinkdda y )

85 -36 threshold curve in males ( 1 to >8 drinkdda y)

1134 20-59 threshold curve in males ( 1 to >5 drinkdday )

20 920 18-70 threshold c u v e in females ( 1 to >4 drinkdday )

1842 20-74 threshold curve in females ( 1 to >3 drinkdday )

6632 18-60 threshold curve in males ( 1 to >8 drinkdda y)

[c 1 Lower dase of ETOH measztred 52 dritcksh"d

83 947 15-79 threshold curve in males (2 to >6 drinkdda y)

3198 30-69 threshold cuve in males (<2 to >8 drinkdda y) threshold curve in females

11 O00 -43 threshold curve in females (<2 to >9 drinkdda y j

6699 20-59 threshold curve in males (<2 to > 1 O drinkdday ) threshold curve in females

3 100 30-69 threshold cuve in females (<2 to >4 dnnkdday)

53 12 25-64 threshold curve in females (<2 to >8 drinkdda y)

[d 1 Lwer dose of ETON nlemwed: 53 + dritzksldaÿ

3083 - 5 3 threshold curve in females (>3 drinks/day)

Mishima et al. 200 1990

Klag et al.

5 0- 54 threshold cuve m males (c4.3 to >4.3 drinkdday) threshold curve in females

15-89 threshold cuve in males (<3 to >7 1 9 6 drinkdda y )

(C) Studies demonstrating a Iinear curve

[a 1 Luwer dose of E TOH rneasztred: < i drir&dày

Strogatz et al. 199 1

Cooke et al. 1982

Fortmann et al. 1983

Arkwright et al. 1982a

Savdie et ni. 1984

Paulin et al. 1985

Keil et al. 199 1

Wakabayashi et al. 1994

66 5 10 - linear curve in males ( 4 to drinkdda y)

1784 25-50 linear curve in males ( < O 2 to > 1 drinkdday ) threshold curve in females

[b 1 Lmver dose of ETOH nieasured: I drirzWd~v

20 920 18-70 linear curve for males ( 1 to >4 drinkdday )

1842 20-74 linear curve in males ( 1 to >3 drinkdday )

[c 1 Laver dose of ETOH nzeasured: 57 dfitrks/da

49 1 20-45 linear cuve in males (2 to >4 drinkdda y)

1 1 O00 -43 h e a r curve in males (<2 to >9 drinkdda y)

90 1 19+ h e a r curve in males (>2 to >4 drinkdday )

53 12 25-64 Linear curve in males (<3 to >8 drinkdday )

2439 48-56 Linear curve in males (<2 to >6 drinkdda y)

[ d 1 Lower dose of E TOH memureci: 53 + dri>ih/dm

1 O0 2 1 + linear curve in males (>5 to 1 5 drinkdda y)

Clark et al. 1967

Peu and D'Alonzo 1968

Dyer et al. 1977

Kozararevic et al. 1980

Saunders et al. 1981

Ibsen et al. 1981

Salonen et al. 1983

UeshUna et al. 1984

Kondo and Ebihara 1984

Krornhout et al. 1985

17-64 T BP with heaky ETOH conçumption

40-55 linear cume in males (>3 drinkdday)

35-62 h BP with fF ETOH (males)

19-71 l inearcweinmales( lOto34 drinkslda y)

44 h BP in high ETOH male drinkers (>5 drinkdday ) compared to low ETOH male drinkers (2.8 drinks/day )

30-64 ft BP with h ETOH

40-69 ünear curve in males (<3 to '8 drinkdday )

-53 Linear c w e in males (>3 drinkdday)

(D) Studies demonstrating no association between ETOR and the BP

Bagburst and m e r 3 50 -23 no association 1981

Coates et al. 1418 20+ no association 1985

Paul i et al. 90 1 19+ no association in females ( l to >4 1985 drinkdday )

b) Light and nzoderate E ?'OH corrsurnprio~

The controversy lies with the effect of Light and moderate alcohol consumption on the

BP. This area of the ETOH-B P relationship is important to consider since it represents the

alcohol consumption of the ovenvhelmmg majority ofthe population (Kaplan. 199 1 : Harburg

et al.. 1994). Vanous patterns of BP progression with increasing ETOH connimption have

been descnied (Gleibemian and Harburg 1986: Criqui et al.. 1986: MacMahon et al.. 1 987).

1 ) U- or J-shaped curve: In quite a few number of nudies. the existence of a U- or J-shaped

cuve has been reported. suggeshng that light and moderate ETOH drinkers have sigmficantly

lower BP than both heavy and non-drinkers (see table 1.2.3.. part A). in the experirnent of

Harburg et ai. ( 1980) for example. male and female subjects with m~des t ETOH consumption

ranging from 0.04 to 1 and fiom 0.04 to 3.2 drllilidday respectively were associated with

reduced systolic BP when compared to non-drinkers. Heavy ETOH consumption (27

drinkdday) was still associated with hypertension. interestingly. the range and extent of the

ETOH-induced hypotensive etfect has been consistently geater in female than in male

subjects.

2) Threshold-sha~ed curve: There are also several studies where ETOH consumption below

a certain level has no sigdicant effect on the BP. suggesting a threshold-shaped curve with

increasing quantity of alcohol. in these studies. although there is no significant hypo- or

hypeitensive effect of light and moderate ETOH consumption. heavy alcohol drinlcing above

a certain quantity is associated with hypertension (see table 1 .LX. part B). For esample.

Trevisan et ai. (1987) noted no significant effect of ETOH on the BP below alcohol

consumption ofabout 5 W d a y . Above these levels. elevations in BP were reported. The

lefi-end portion of the threshold-shaped curve has been consiaently longer in female

compared to male subjects.

3 ) Lmear-shaped cuve: A smaller number of experiments have proposed a linear association

between ETOH and the BP. suggesting a significant BP increase with the lowest quantity of

reported ETOH (see table 1.2.3.. part C). In the epidemiological study of Ueshirna et a/.

( 1984) for example. a linear increase in systolic BP is noted from i3 to 28 drinkdday.

c) Factors irifz<erici~ig the restrlts observed iri epidemiological shrdies

There are a number of factors to consider when investigating the comparative value of

these audies. particularly when lookuig at the low end of the ETOH-BP relationship.

1 ) Low spectrum of ETOH consumotion: T O some extent. the shape of this relationship

depends primady on the number and values of the lowest categories of ETOH consumptions

&en in each experiments. Indeed. most of the Linear-shaped curves are reponed in studies C

where the lowest group of aicohol drinkers consumed 52 drinkdday (Savdie et al.. 1984: Keil

et al.. 199 1 : Wakabayashi et al.. 1994) or even s3 drinkdday (Lian. 19 15: Dyer et al.. 1977:

Sauoders et al.. 198 1 : Arkwright et al.. 1982a: Ueshima et al.. 1984). Any effect of light and

moderate ETOH consumption is loa in such e'rperiments. In fact. only a handfùl

epidemiological studies specifically recorded more than 1 dose of alcohol consumption below

2 drinkdday (Harburg et al.. 1980: Criqui et al.. 198 1 : Gordon and KanneL 1983: Harlan et

al.. 1984: Weissfeld et al.. 1988: Strogatz et al.. 199 1 : Marmot et al.. 1994: Gillman et a/..

1995). Ofthese experiments only one failed to observe a .J- or U-shaped relationship for the

BP (Strogatz et al.. 1991).

2) Non-drinker catepory: Nwertheless the scientific community has been reluctant to accept

the possibility of an hypotensive effect of chronic light and moderate ETOH consumption.

One of the major concem is the possible heterogeneity of the non-drinkers categoiy. It has

been suggeaed that previously heavy ETOH drinkers and ex-drinkers who were forced to

stop drinkmg because of developmg health problems are also present in this category. so that

the average BP is higher than it should have been if Me-long non-drinkers were the only

members of this eroup (Shaper. 1990: Beilin and Puddey. 1992). Likewise. it has been

postulated that socioeconomical factors could explain the lower BP with light ETOH

consumption. since wine drinkers who represent the majority of light and moderate alcohol

drinkers tend to be fiom higher social class than non-druikers (Colslier and Wallace. 1989:

Burke et al.. 1995). However. J-shaped cuwes were still observed in epidemiological nudies

with life-long non-drinkers. Similarly. the exclusion of ETOH abstainers because of poor

health or the consideration of the social level throughout the ETOH consumption spectrum

did not modify the pattern of the BP alterations, suggesting a distinct effect of the lower

ETOH levels on the BP (Marmot and Bninner. 199 1 : Cullen, 1993; Kem 1993 ).

3 ) Self-reporting: There is a tendency to underestimate the quantity of ETOH consumed in

epidemiological self-reports particularly m the lower leveis of ETOH consumption (Redman

et al.. 1987). This tendency may increase the range of the depressor action of alcohol and

limit M e r the value of a number of linear- and threshold-shaped experiments with hi@

initial ETOH consumption.

4) Contnîuting factors: The age of the subjects is likely to mod@ the relationship between

ETOH and the BP (Lang et al.. 1987: Weissfeld et ai., 1988: Keil et al.. 199 1: Gillman et al.,

1995). Likewise. the lipid content (Salonen et al.. 1983: Gruchow et d. 1985: Minishima

et al.. 1990). the race (Klatsky et aL. 1986: Dyer et al.. 1990). the smoking habits (Savdie

et al.. 1984: Keil et al.. 199 1). the physical actMty (Reed et aL. 1982: Keil et al.. 199 I ) or

the presence of ilInesses such as hypertension (Potter and Beevers. 1984) have al1 been

demonarated to contribute differently to the s ens i t~ ty of the ETOH-BP curve. Sex-

dependent modifications in this relationship have also been noticed in almoçt every

eqerirnent. Indeed there has been a longer and deeper hypotensive portion of the ETOKBP

spectmm in female cornpared to male subjects (Harburg et ni.. 1980: Weissfeld et al.. 1988).

5) Pattern of ETOH connimption: The pattern of the ETOH consumption rnay influence the

BP. since binge rather than replar drinking is associated with elevated BP ( Seppa et O/. . 1994).

6) Time-course of ETOH measurement: The time from the last aicoholic drink may also

~ e a t l y m o d e the BP values. since the experirnent may measure either the depressor = component of the repeated acute ETOH consumption or a secondary withdrawai reaction

(Howes et al., 1986: Abe et al.. 1994).

7) Type of alcoholic beveraq: distinct components of the various ETOH beverages. such as

wine. beer or spirits. may daerently alter the ETOKBP relationship.

Interestingly. the existence of a U- or J-shaped cuwe has been achowledged for the

effect of alcohol on coronary heart disease and mortality (Veenstra. 199 1 : Renaud and De

LorgeriL 1992; Coate. 1993: Gronbaek et a/.. 1994). It is therefore iikely that the effect of

ETOH on cardiovascular components such as the BP is also biphasic in nature. with hypo-

and hypertensive actions of low and heavy ETOH consumption. respectively.


Much like for the acute studies. there have been fewer animal experiments on the chronic

effects of ETOH on the BP (Table 1.2.4. ). The issue remams controversial.

a) ETOH-iridztced decrease itl BP

Vanous studies have demonstrated an antihypertensive effect of chronic moderate ETOH

consumption (m 50% ofthe çtudies mvestigating chronic ETOH administration and BP) (see

table 1.2.1.. part A). in general. these studies reponed a slow and progressive

antihypertensive effect of ETOH in spontaneously hypertensive ( S m ) and Wistar-Kyoto

(WKY) rats that needed at least 12 weeks to reach çignificance. Most studies used ETOH

rnixed in water in moderate concentration (20% v/v).

b) ETOH-i~rd~tced increase iti BP

Despite the experirnental evidence. acceptance of the possibility of a depressor effect of

chronic moderate ETOH consumption by the scientific comrnunity has been diicult. This is

probably due to several reports suggeaing a pressor effect of chronic ETOH administration

(m 42% ofthe snidies investigating chronic ETOH administration and BP) (see table 1.2.4..

part B). In general. these studies reported a fast hypertensive effect of ETOH in Sprague-

Dawley and Wistar rats that need between 1 to 4 weeks to reach sigtüficance. Al1 studies used

ETOH mixed in water in low to moderate concentrations (5 to 20% vk) .

c,i !Vo effect of ETOH oti BP

In one study. the administration of a low concentration of ETOH ( 10% v/v) for 1 1 weeks was

not associated with significant modifications in the BP of SHR and WKY rats (see table

1.2.4.. part C).

tource Species Treatments R e d t s

A) Studies demonstrating a decrease in BP

[al ETOH iri water

vlames and Aldinger Spague- 25% v/v for 30 8 BP

$anderson et ai. 1983

loues et al. 1988

Kowe et al. 1989

Beilin et al. I99Z

Karanian et ai. 1986

Hatton er al. 1992

Dawley weeks rats

SHR and 5 and 20% v/v Wistar for 16 weeks rats

SHRand 20%v/vfor24 Wistar weeks rats

SHRSP. 20% v/v for 28 SKR and weeks WKY rats

SHRand 20°/0v/vfor12 WKY rats weeks

[bl ETOH i ~ i vapor

Sprague- 25 mg/L in Dawley vapor for 8 rats days

Wistar Lieber- De rats Carli diet

(36%) for 13 weeks

8 BP with 20% vlv (Sm) no change BP (Wistar)

8 BP (SHR) no change BP (Wistar)

4 BP (fiom week 16) (SHRSP) 4 BP (fkom week 16) (SHR) & BP (fiom week 16) (WKY)

8 BP (SHR) 8 BP (WKY)

8 BP with h BAC

6 BP (fiom week 3)

(B) Studies demonstrating an increase in BP

[a ( ETOH iri water

Chan and Sutter 1983

Chan et al. 1985

Abdel-Rahman and Wooles 1987

Strickland and Wooles 1989

Hsieh et al. 1992

Vasdev et a/. 1993

Wist ar rats

W ist ar rats

Sp rague- Dawley and W ist ar rats

Sp rague- Dawley rats

Wistar rats

WKY rats

20% v/v for 12 T BP (Eom week 1) weeks

20% v/v for 12 f l BP (6om week 4) weeks (stressed and unstressed rats)

20% vlv for 12 h BP (fiom week 6) weeks

20% vlv for 22 ft BP (Eom week 4) weeks

15% v/v for 4 f i BP (fiorn week 1 ) weeks

5% vlv for 14 fi BP (fiom week 1 ) weeks

(C) Studies demonstrating no change in BP

[a 1 E TOH iti ivater

Khetarpal and SHR and 10% v/v for 1 1 no change in BP (Sm) Volicer WKYrats weeks no change in BP (WKY) 1979

d) Factors Nzfrz~etzcitzg the reszihs obserced in attinzai shcdies (Chrotzic E TUH)

1 ) Dehvdration: It has been argued that the antihypertensive effect of chronic moderate

ETOH consumption may not be the r e d t of alcohol per se but of secondary dehydration

because of the rat aversion to ETOH. Smail but significant reductions in fluid consumption

have been observed m a few studies (Khetarpal and Volicer. 1979: Howe et ai.. 1989: Beilin

et ai.. 1992). However. the administration of water for 1 dayiweek to offset any dehydration

caused by ETOH stiil resuited in the prevention of the age-dependent hypertension (Jones et

al., 1988). Moreover. the hematocrit was unaltered in ETOH-treated animals. suggesting no

modiications in plasma volume (Sanderson et ni.. 1983: Beilin et al.. 1992).

2) Nutritional deficit: The presence of nutritional deficit is unlü<eiy since most studies reported

no change in the body weight of the ETOH-treated animals (Khetarpal and Volicer. 1979:

Sanderson et ai.. 1983: Jones et d.. 1988: Howe et al.. 1989: Hanon et a!.. 1992). Sirnilarly.

cardiac mass was unaKected by the ETOH treatment ( Howe et al.. 1 989). Finally. in a study

where nutritional deficits were specifically avoided by the administration of a Lieber-De Cadi

ETOH diet. a significant antihypertensive effect of alcohol was still observed (Hatton et ai..

1992).

3 ) Stress: The surprisingly rapid and extremely significant increase in the BP afier less than

1 (Hsieh et ai., 1992; Vasdev et al.. 1993) or 4 (Chan and Sutter. 1983: Chan et ai.. 1985:

Strickland and Wooles. 1989) weeks of ETOH administration suggen a stress response to

the novel liquid mixture rather than a direct pressor effect of ETOH. The result of Vasdev et

al. ( 1993) with 5% viv ETOH is questionable, particularly the huge 20 (week 1 ) to 80 (week

I I ) mm HG BP increase. considering the low level of ETOH &en (tested with no success

by Khetarpal and Volicer in 1979) and the slow and subtle modikations in BP reported in

every other experiment.

4) Time-course of BP measurement: The timing of the BP measurement may be critical for

the pressor and depressor effect of chronic ETOH consumption. much like for repeated acute

ETOH administration when the initial hypotensive response is foliowed by a secondary

hypertensive reaction (Abe et aL. 1994). Similarly. Stricklmd and Wooles ( 1989) reported

that afiemoon BP calculation is associated with some hypotensive eEects. whereas moming

recordmg is hypertensive. suggesting a direct relationshrp between BAC levels and BP values.

5) Genetics: Genetic predispositions may also e.uplain some of the pressor actions of chronic

ETOH drinking. The antihypertensive effect of chronic ETOH consumption is stronger in

SHR rats (Jones et al., 1988; Howe et ai.. 1989: Beilin et al., 1992). in contrast, the

hypertensive or the lack ofhypotenske effect of chronic ETOH consumption is noted almost

esclusively in Wistar rats (Chan and Sutter. 1983: Sanderson et al.. 1983: Chan et al.. 1985:

Jones et al.. 1988: Hsieh et al.. 1992). uiterestingly. even the group of Chan casts doubts on

the possibility to produce a pressor effect with a difrent ntain of rats (Chan et al.. 1985).

e) Sunimary

A progressive general antihypertensive action of chronic moderate ETOH consumption

has been demonstrated in rats. although some pressor response to ETOH may e i a .

depending on the timing of the BP recording and the strain of animal used.

1.2.2.3. iVlechanisms

A few hypotheses have been proposed to explain both the pressor and depressor

components of chronic ETOH consumption. but the exact mechanisms remain unclear.

a) Mecht iisnis resporrrible for rhe bpertetsive eefjecr of chrotiic h e m ~ E TO ff cotrrmprioii

1 ) Withdrawal : The well documented pressor effect of replar large amounts of ETOH may

be the result of a secondary withdrawal reaction (Clark and Friedman. 1985: King er al..

199 1 ). It is possible that in regular heaw drinkers the repeated hypotensive effect observed

d e r each acute alcohol administration is followed by a compensatory hypertensivc reaction.

probably due to secondary alterations in vascular membrane charactenstics. In the long m.

wch a repetition of "intemittent withdrawal reactions" may produce a sustained state of

hypertension in alcoholics (Melville. 198 1 ). Partial validation of this hypothesis has been

obtahed recently from the group of Abe in short-term chronic experiments. Indeed. a single

administration of moderate ETOH m human subjects produced ody a direct depressor effect

measured by ambulatory BP. In contrast. repeated alcohol administration for a weeli

produced a secondaq pressor response. aiggesting the initiation of a short-term withdrawal

reaction (Abe et ai.. 1994). in most cases. lowering the alcohol consumption would reduce

the BP m alcoholic patients. demonstrating the reversibility of ETOH-induced hypertension

(Maheswaran et al., 1992: Ueshima et ai.. 1993).

2) Calcium: Cellular modifications in Ca" content have been W e d with alcohol-induced

hypertension (Somlyo and Souûyo. 1994). indirect evidence has shown that the pressor effect

of acute (Potter and Beevers 1984) and chronic (Arkwright et ai.. i982b: Hsieh et al.. 1992)

alcohol consumption may be due to the increased accumulation of Ca" in smooth muscle

ceils which causes an increase in the vascular tone. Exactly how this shift of Ca" from

extracelhdar to mtracellular space is achieved is uncertain. However. a nurnber of mechanisms

have been proposed mcluding (a) the inhibition of Ca2' ATPase activity (Sun. 1979). (b) the

mcreased Na' permeability . (c) the inhibition of NaT/K'-ATPase a c t ~ t y (Blaustein. 1977:

Arkwight et al.. 1984: Coca et ni.. 1992) and (d) the upregulation of voltage-activated Ca2'

channels (Littleton. 1988) foiiowing ETOH mtake and the intercalation of ETOH hto cellular

membranes.

3 ) Pressor hormones: Several pressor hormones. nich as catecholamine. cortisol and renin.

have been suggested to contribute to the high BP associated with chronic heavy ETOH

consumption. It has been proposed that ETOH could mcrease both theù levels and potentiate

dieu pressor action on the vasculature. Of particular interest is the hypothesis proposing that

the repetitive ETOH-mediated increases in circulating norepinephrine levels and

norepinephrine-dependent vasoconstriction may lead to a slow pressor effect of catecholamine

in long-terni alcohol drinkers (Aitura and Ahna. 1982; Ireland et ai.. 1984: Chan et al.. 1985:

Criscione et ai.. 1989). n i e elevation in norepinephnne niay also reflect enhanced actMty of

the sympathetic nervous system by ETOH exposure (Malhotra et al.. 1985: Beilin and

Puddey, 1992: R a n h et al., 1995). However. nich increases in plasma catecholamine levels

and sympathetic a c t ~ t y have been absent f?om other studies investigating chronic ETOH

consumption (Ibsen et ai.. 198 1 : Arkwright er al.. I98Za: Poaer and Beevers. 1 984: Hatton

et ai,. 1992). Similarly. inconsistent results have been obtained for cortisol and the renin-

angiotensin-aldosterone system (RAAS), so that the sigdicance of these modifications. if

any. seems linnted (Arkwright et ai.. 1982b: Potter and Beevers. 1984: Vasdev et ai.. 1993 ).

4) Others: A genetic predisposition to hi& BP and hi& alcohol consumption may esia in

certain i n d ~ d u a l s (Myrhed. 1974). The salt present in alcoholic beverages. particularly in

beers. has also been proposed as a mediator of the alcohol-induced hypertension (Puddey et

al.. 1986). ETOH may also be indirectly related to high BP in some cases since psychological

factors. such as stress, may predispose an i n d ~ d u a i to hypertension and alcohol drinliing

(Harburg et ai.. 1973: McQueen and Cellentano. 1982). In nich cases. alcohol is a way to

cope with detrimental psychological conditions rather than the cause of elevated BP.

b) Mechariisnts respomi b le for the arttihypertercsive e ffecr of chrorttc light a d moderute

ET0H coratrrnptiort

I ) Lipoproteins: The major hypothesis for the beneficial effect of low and moderate ETOH

consumption on BP and cardiovascular diseases involves modifications by ETOH of the

various Lipoprotein subfiactions such as hi&-density lipoprotein (HDL). low-density

lipoprotein (LDL) and very low-density lipoprotein (VLDL). HDL is imp licated in "reverse

cholesterol transport" fiom the vascular walls to the liver and is important in preventing

cholesteroi-derived v a d r damage (Gordon et al.. 1977: Gordon et al.. 1989). Ln contrast.

LDL has been positivety associated with hypertension and coronary heart disease (Castelli and

Anderson. 1986; Sachinidis et al.. 1990).

Early epidemiological and experimental audies have observed significant increases in

HDL levels after chronic alcohol connimption. even in low doses (CasteIli et ai.. 1977:

Barboriak et ni.. 1979; AUen and Adena. 1985: Langer et al.. 1992). Interesiingly. lower

threshold levels have been found in female subjects. explainhg perhaps to some estent the

greater incidence of U-shaped curves in women compared to men (Weidner et al.. 199 1 ).

However. the analysis of the two HDL nibfiactions. HDL, and D L , . have indicated that

modest ETOH consumption mcreases mody the levels of HDL-,, whereas HDL, is augmented

prirnady following chronic heavy ETOH consumption or in alcoholics (Haskeli er al.. 1981:

Moore and Pearson, 1986: Diehl et ai.. 1988). This is of importance since HDL was initially

believed to produce moa of the HDL beneficial effects on BP and cardiovascular disease

(Miller et aL. 198 1 : Ballantnie et a[. 1982). Fortwiately. in recent years. HDL, has also been

impiicated m these beneficial eEects ( H a 6 e r et al.. 1985: Langer et al. 1992). Furthemore.

various recent experiments have reported elevations in both HDL, and HDL, levels with light

and moderate alcohol consumption. confirming the hypothesis that HDL may indeed explain

parts of the hypotensive action of iow to moderate alcohol consumption (Miller et ai.. 1 988:

Hojnacki et ai.. 1988: Stampfer et al.. 199 1: Razai et al.. 1992: Rossouw et al.. 1992:

Gaziano et al.. 1993). A relative reduction in the LDUHDL ratio has also been found with

moderate. but not heavy. ETOH consumption in monkeys (Hojnacki et ai.. 1988) and with

the phenotic compounds of red wines (Frankel et al.. 1993).

2) Hemostasis and Thrombosis: Antithrombotic effects of alcohol have also been linked vliith

the beneficial actions of chronic moderate ETOH consumption and especially with the

reduction of coronary artery disease. Long-term consumption of modest amounts of alcohol

have been associated with lower plasma fibrinogen levels and with increased fibrinolytic

a c t ~ t y . assessed by endogenous tissue-type plasminogen activator (t-PA) (Maede et al..

1 979: Veenstra et ai.. 1990: Ridker et al.. 1 994). A reduction in platelet aggregation has also

been demonstrated with chronic ETOH drinking (Elmer et al.. 1984: Pikaar et al.. 1987:

Rubin and Rand. 1991). The hypothesis tliat alcohol interferes with hemostasis and

thrombosis is funher supported by studies indicatmg that acute and short-term chronic alcohol

coosumption decreased TXAz release fiom platelets and increased PGI, synthesis f?om

endothelial cells (Mikhailidis et al., 1983: Landolfi and Steiner. 1984: James and Walsh.

1985).

3) Others: The non-specifk association of ETOH with the cellular membranes produces

widespread modifications m basic cell physiology and therefore m the communication between

cells and between organs. Not-surpnsingly (section 1.1.5.2, ). most aspects of endocrine

physiology. such as hormone production. release. receptor Ievels or second messenger

systems. are altered by alcohol and the various homeostatic systems regulated by these

endocrine agents. such as the BP. are likely to be modified accordingly. Several very general

homonal systems with vascular. rend and central componeats nich as RAAS, catecholamine

or AVP, have been associated with pressor responses and positive regdation of BP

homeostasis. Accordin& experimental studies have tried to link such systems with the hi@

ETOH-induced hypertension (section 1.2.2.3. pan 3). The absence of a seneral and

widespread hormonal family with hypotensive actions and a negative regulation of BP

homeostasis prevented early investigations on the humoral contribution to the beneficial

effects of light and moderate ETOH connimption. Nevertheless. a few reports have noted the

attenuation of the pressor effect of acute ETOH in certain vascular beds foilowing chronic

ETOH chking, mediated by the increased actMty of local vasodilating factors. such as NO

and PGIz(Karanian et aL. 1985: Knych. 1992: Hatake er aL. 1994). However. in the lan 15

years the discovery of an expanding family of natriuretic and vasorelaxant peptides opened

a new avenue to investigate the mechanisms responsible for the hypotensive effects of low

ETOH consumption. Much like the hypothesis of a slow pressor effect of epinephrine

following heavy ETOH consumption. a slow hypotensive action of "natnuretic peptides"

mediated by vascular. central and renal modifications may exia in chronic moderate ETOH

drinkers. leading to a sustain antihypertensive effect. The report by Colantonio et al. ( 1 99 1 )

of a significant mcrease in circulating atrial natriuretic peptide (ANP) levels. the fin member

of this natriuretic peptide family. after acute administration of modest amounts of ETOH

further demonstrated the need of designing eqeriments to survey the possible implication of

the natriuretic peptide system in the cardiovascular effects of acute and chronic ETOH

consumption.

1.3. NATFUURETIC PEPTLDE FAMILY

During the paa 15 years. a new hormonal system for the negative regulation of total

body fluid and BP homeostasis has been identified and characterized. Three major members

have been isolated. The first two. the A- and B-type natriuretic peptides (ANP and BNP) are

located mainly m the heart tissue. whereas the third one, the C-type natriuretic peptide (CNP).

is found mainly in the CNS. Their major biological activities include natriuresis. diuresis.

vasorelaxation and the mhiiition of several pressor hormones. These physiological effects are

mediated by the association of the natriuretic peptides with three specific natriuretic peptide

receptors. Interactions of the natriuretic peptides with the &st two natriuretic peptide

receptors (NPR-A and NPR-B) stimuiate the production and accumulation of guanosine Y.5'-

monophosphate (cGMP). cGMP appears to be the second messenger system of the natriuretic

peptide farnily. Interactions of the natriuretic peptides with the third natriuretic peptide

receptor (NPR-C) does not stimulate the production of cGMP. Therefore. NPR-C is moaiy

considered to be a clearance receptor.

1.3.1. A-TYPE NATRIURETIC PEPTIDES

1.3.1.1. Atrial natriuretic peptide

a) Historrcal perspecrives

Modifications in body fluid volumes and therefore in BP have long been known to

control the secretion of sodium and water by the kidney (Starling. 1909). However. the

humoral aspect of this "pressure-natriuresis" rnechanisrn (Guyton. 1980) escaped investigation

until relatively recently. in 1956. Kisch noted the presence of granules in atrial cardiocytes.

an obsenration that wodd open a new concept. the heart as an endocrine organ (Braunward.

1964). These granules were later better described by Jarnieson and Palade ( 1964). who

suggested for them a secretory role. In the next decade. more specific expenments were

performed. These studies indicated that atrial distention produced diuresis. whereas it

decreased circulatory renin and AVP levels (Brennan et al.. 197 1 ). In 1976. Marie and

colleagues obsewed that atnal granulanty was increased by salt and water load. This

"suggested a relationship between atnal specific granules and the replation of water-

electrolyte balance ..." (De Bolci, 1979). The crincal last step was made in 198 1 when De Bold

et al. ( 198 1 ) demonstrated that the mjection of atnal extracts in aneçthetized rats led to rapid

natrkesis (30-fold mcrease) and diuresis ( IO-fold increase) and to a sigruficant fa11 in the BP.

The atrial natriuretic peptide (ANP) was found.

b) Biochemisby and release

The ANP gene consists of 3 exons (codmg regions) and 2 mirons (interverhg sequences)

(Greenbug et al., 1984; Nemer et al.. 1984; Gardner et ai.. 1988). In bumaas and rats, the

first exon encodes for the 24 aminoacid signal peptide and the £ira 20 aminoacid of the

prohormone. The second exon encodes for the remainder of proANP, with the exception of

the laa (in humans) or three laa (in rats) residues, coded by the third exon (figure 1.3.1.).

Translation of ANP mRNA produces the aaual 152 aminoacid preproANP precursor (Cwie

et al., 1983; Oikawa et al. , 1984). The removal of the 24 aminoacid signal sequence and the

two carboxyl (C)tterminal ar@e ( k g ) residues fomd in rats, but not in humans, @es nse

to the 126 aminoacid proANP (Flynn et al.. 1983; Atlas et al.. 1984). The prohomone was

iater found to be the major storage form of the peptide m atrial granules (Bloch et al.. 1985:

Thiiauit et al., 1987), m contrast to most other hormones which are stored m their active

fom. It is believed that membrane-bound enzymes cleave the prohormone either upon or

immediately afier secretion (Thiibault et al., 1986; Sei et al., 1992). The final proteolytic

processing is made behiveen arginine (Arg), and serine (Ser), forming the mature C-terminal

28 aminoacid ANPw931, identified in the circulation (Gutkowska er al.. 1984; Tanaka et al..

1984; Theiss et al.. 1987) (figure 1.3.2.). Even though ANPw-12, is the main C-terminal

peptide f o m processed fiom proANP, cleavages of proANP at other atypical sites have been

detected in other tissues. For example, amino (Nkelongated forms of ANP are found in the

kidney (ANP,,,,) (see section 1.3.1.2.) (Schulz-happe et al.. 1988) and the testis (ANP,,

2 6 ) (Pandey and Orgebm-Cria . 199 1 ), while shorter processed forms of ANP have been

identzed in the brain ( A N P l o ~ l x and ANPI,-,,) (Shiono et al.. 1986; Ueda el al., 1987).

A common characteristic of the C-terminal natriuretic peptides is the presence of a

-hide bridge, which is essential for biological aaMty (Misono et al.. 1984). Interestmgly,

the structural similarity between most of the natriuretic peptides resides withm the 17

aminoacid ring structure formed by the disulphide bridge. in fact, among the various known

natriuretic homones (ANP, BNP and CNP), 1 1 aminoacid of the ring structure are identical

(Koller and Goeddel, 1992). Among species, the ANP amhoacid sequence is also remarkably

well conserved. Betweeo humans and rats for example, mature ANP is identical, with the

5ut

ANP mRNA

PreproANP

fragment

Exon 1 Exon 2 Exon 3

BNP gene I Sut

BNP mRNA V

PrepraBNP ! I -26

ProBNP

N-terminal fragment B N P-45

CNP gene

CNP

Exon 1 Exon 2 I

mRNA Y

PreproCNP 1 I -23

ProCNP I

N-terminal fragment 1 1 O3

CNP-53

F ig ure 1.3.1 . Schematic representation of the transcription, translation and processing of the natriuretic peptide family

A-type natriuretic

B-type natriuretic

F ig ure 1 3.2. Schematic representation of the stmcture of the natriuretic peptide famiîy in rats

exception of one substitution at position 1 10. fkom isoleucine (Ile) (humans) to methionine

(Met) (rats) (Kangawa and Matsuo, 1984).

The major stimulus for the atrial release of ANP appears to be atnal stretch (Haass et al..

1987: Greenwald et al.. 1989: Ruskoaho. 1992: Espmer. 1994). Therefore. factors promotmg

atnal distention by volume expansion. such as head-out water immersion (Katsube et al..

1985: Anderson et al.. 1986). changes in body posture (Solomon er al.. 1986). increased

heart rate (Schielbmger and Lmden, 1986) or elevated dietary sodium intake (Richards et al..

1986) increase circulating ANP levels. Similarly. plasma ANP levels are also elevated in

pathophysiological conditions associated with increased atrial filling pressure nich as

congestive hean failure (Cm) (Burnett et al.. 1986: Tsunoda et al.. 1986) or hypertension

(Gutkowska et al.. I986a: Morü et al.. 1986a: Sagneila et al.. 1986: Hollister and Luagami.

199 1 ; Naruse et al.. 1994). Other stimuli have been associated with cardiac ANP release.

indeed, vasoconstrictor hormones such as AVP (Itoh et al., 1987: houe et ni.. 1988 ).

angiotensin II (Diets 1988 ) or endotheiin (Schielbinger and Greening, 1992) are found to

affect ANP production and release. Glucocorticoids (Gardner et al.. l986a). testosterone

(Deng and Kaufkaa 1993 ) and thyroid hormone (T,) (Argentin et al.. 1987) accelerate ANP

secretion. Opioids (such as O-endorphin) are also possibly involved in the regulation of

cardiac ANP release (Voilmar et al., 1987: Widera er al.. 1992).

C) Tissue distribzrtiorr

As implied by its name. ANP was first isolated fiom atrial tissue (Flynn et al.. 1983).

Early radioimmunoassays demonstrated higher total ANP content in the right compared to

the left atrium of rats (Gutkowska et al.. 1984; Gutkowska et al,. l987a). As was previously

discussed. ANP is stored m the atrial granules in its higher molecular weight form (proANP)

and is released upon atrial aretch. Most of the ANP in circulation originates fiom the hean

atria. However. ANP immunoreact~ty and transcripts have also been found although in

srnailer quantities, in several extra-atrial tissues (Gutkowska and Nemer. 1989).

Heart ventricular tissue has been reported to contain ANP mRNA and proANP (Nemer et

a[. 1986). Morphological (absence of granules) and secretory differences between the atria

and the ventricles have however suggested that contrary to the atria. m the ventricles ANP

is not stored in granules but it is released in a constitutive rnanner (Bloch et al., 1986). In

view of the relative large size of the ventricles and the conthuous secretion of ventricular

ANP. the ventricles contniute substantially to circulatmg ANP levels. This contribution is

particularly important in pathophysiological conditions associated with ventricular

hyperirophy. such as congestive heart failure (Cantin et ai., 1988) or hypertension ( Arai rr

ai.. 1988: Ogawa et al.. 199 1 ). in such cases. the ventricular tissue is considered as the major

source of plasma ANP levels.

ANP. proANP and ANP mRNA are also present in the lungs (Sakamoto et ai.. 1986:

Gutkowska et al.. 1987~: Gutkowka et al.. 1989). Within the respiratory syaem ANP has

been localized by various techniques in the respiratory epithelium (Gutkowska and Nemer.

1989), pleural fluid (Vesely et al.. 1989) and pulmonary vems ( SpringalI et al.. 1 988). Marked

elevations in plasma ANP levels and in lung ANP content and transcnpt are observed in

cardiomyopathie hamsters. which exhibit decreased atrial ANP production (Currie et ai-.

1987: Gutkowska et al.. 1989). These observations suggest that as was observed for the heart

ventricles. under certain pathophysiological conditions. the lmg may enhance the production

and secretion of ANP. contributhg to the elevated plasma ANP levels in the presence of

diminished a c t ~ t y of the heart ANP system. Furthemore. a paracrine role for ANP in

pulmonary homeostasis has been proposed (Perreault and Gutkowska. 1995).

Extensive localization of ANP immunoreact~ty has been identified in the central (CNS)

and peripheral (PNS) newous systems (Kawata et al.. 1985: Standaert et al.. 1986). n i e

highest concentrations of ANP (Morii et ai., 1985) and ANP mRNA (Gundlach and Knobe.

1992) are found in the hypothalamus. and particularly in areas involved in the replation of

fluids and BP. such as the anteroventral third ventricle region (AV3V) (Ku and Zhanp 1994).

Other brain areas where immunoreactive ANP has been detected include the midbrain.

thalams. septum, spmal cord oi£àctory bulbs and mucosa. pons and medda (Kawata et a/-.

1985 ; Gutkowska and Marcinkiewicz, 1989; Gutkowska et al., 199 1 ). ANP is also seen in

the cerebrospmal fluid (Manuno er al.. 1987). Atypical processing of proANP in the CNS is

suggested by the isolation of shorter 24 and 25 aminoacid ANPs (Ueda er al.. 1987). In the

PNS. ANP has been detected in the autonomie system, panicularly in the m a t h e t i c and

parasympathetic ganglia (Debinski et al.. 1986).

Both ANP and ANP mRNA are found in the antenor pituitary (Gardner er ai.. 1986b:

Gutkowska and Cantïn. 1988). Local modulation of pituitary hormones by ANP has been

posnilated. in contraa. only mature ANP is present m the posterior puititary. suggesting an

exogenous production. possibly fiom the hypothalamus (Gutkowska et al.. 1987b).

The chromaffm ceüs ofthe adrenal meduila have been reported to possess both proANP

and ANP (McKenzie er al.. 1985: ûng et al.. 1987). The possibility of local ANP rynthesis

is also hypothesized in the vasculanire. notably in the aortic arch (Gardner et al.. 1987). the

gonads (Gutkowska et al.. 1993) and the GI tract (Vuolteenaho et ai., 1988). because of the

presence of the hi@ (proANP) and low (ANP) molecular weight forms.

Small quantities of ANP (Figueroa et al.. 1990) and ANP mRNA (Greenward et al..

1992) are found throughout the renal nephron. The added presence of an elongated N-

terminal form of ANP (ANP,,,) (see section 1.3.1-2.) in the kidney indicates a local role for

the A-type natriuretic peptides in this organ (Goetz 199 1 ).

d) Me rabolisnt

ANP has a shon half-life in plasma, ranging fiom Iess than a minute in rats (Katsube er

ai.. 1986: Krieter and Trapain. 1989) to less than 5 minutes in humans (Gnadinger et ol..

i 986: Biollaz et of.. 1987). The rapid removal of ANP fiom the circulation has been shown

to occur primarily in the kidney (Needleman et al.. 1989) with lesser contributions of otlier

ogans. such as the her. lungs and mtestine (Crozier et al.. 1986: Krieter and Trapain. 1989:

Gerbes and Vollmar, 1990). Two major processes. one proteolytic and the other receptor-

mediated have been reported in ANP clearance. Proteolytic degradation is rnoaly the result

of peptide cleavage wittiin the ring of ANP by endopeptidase 21.1 1. yielding inactive products

(Stephenson and Kenny. 1987: Seymour et al.. 1991 : Kanazawa et ai.. 1992). Endopeptidase

24.1 1 is a membrane-bound enzyme disaiiuted primady in rend glornenili and on the luminal

side of the proximal tubules (Olins et ai.. 1987: Erdos and SkidgeL 1989; Landry et al..

1993). It is also eqressed in vascuiar tissues (Johnson et al.. 1990: Dussaule et ab. 1993 ).

Receptor-mediated clearance of ANP is beiieved to be through NPR-C (see section

1.3.1.4.3.). the so-called "clearance receptor" (Maack et ai.. 1987). Upon ANP binding. the

receptor- ligand complex is mtemalized and ANP is degraded by lysosornal hydrolysis

(Nussenzveig et ai.. 1990: Maack et ai.. 1993 ). The abundance of NPR-C in the vasculature.

kidney. b r a h lungs heart and adrenals M e r supports for a specific role of this receptor in

ANP metaboiism (Nakao et ai.. 1992: Maack et ai.. 1993 ).

1.3.1.2. Urodilatin (UD)

Lately. the atypical processing of proANP in the kidney has attracted attention. Schulz-

Knappe et al. m 1988. isolated from human urine a 32 aminoacid peptide who tumed out to

be a 4 aminoacid N-terminal extension of mature ANP. ANP,,_,,, - .. - or urodilatin (UD) as it

is now called - is absent Ciom the circulation ( D r u m e r et ai.. 199 1 ). It has been postulated

that the distal tubular nephron is the major. if not the only. site of synthesis for UD in the

body (Goetz 199 1 : Forssmann. 1995). Low levels of gene expression (Greenward et ai..

1992) and proANP (Ritter et ai.. 1992) have been identified in this tissue. suggeaing local

synthesis. Therefore. UD may be released fi-om the cells of the distal tubule and interact with

the luminally-situated natriuretic receptors (see section 1.2.3.1.) of the inner medulla

collecting ducts (IMCD) (Gunning and Brenner. 1992: Valentin et al.. 1993). Aiternatively.

the lridneys may represent only a distinct processing site for exogenous proANP. although the

absence ofthe ANP precursor m the circulation renders this hypothesis unlikely (Gutkowslia

et al.. 1984; Forssmann. 1995). Experimental evidence has demonstrated Iittle or no storage

space in renal tubular cells. suggeaing a constitutive rather than regulated mode of UD

release (Ritter et al.. 199 1 ). The regulation of UD secretion is still unclear. Initially. a neural

control was pormlated (Emmeluth et ni.. 1992). However. the demonstration of unaltered UD

release in denervated kidneys suggested the presence of an humoral regulation as well

(Emmeluth et al.. 1993 ). The clearance of UD is done mostly through urine excretion. In

contraa to ANP. üD appears to be quite resistant to proteolytic degradation by

endopepidase 24.1 1 (Gagelmann et al.. 1988). Recently, a paracrine role has been attributed

to CID for the tubular diuretic and natriuretic effects previously ascribed solely to circulating

1.3.1 3 . N-terminal frarmen ts of proANP

It is a common occurrence m endocrinology that several different hormones may

originate ficm a single prohormone. As a example of this, proopiomelanocortm (POMC) is

the precursor of at Ieast 4 known peptides, includhg adrenocorticotropic hormone (ACTH)

and B-endorphin (Mains et al.. 1977; Eipper and Mains, 1980). Recent aidence has

suggested a nmilar muhihormonal role for the ANP precursor (Vesely, 1995). Indeed 3

distinct N-terminal peptides are derived from the first 98 aminoacid of proANP, nameiy 1 )

ANPI-, or "long acting sodium stimulator", 2) ANP,,,, or "vesse1 dilator" and 3) ANP,,,

or "kaliuretic stimulator" (Vesely et al.. 1987; Martin et al.. 1990). The 3 new peptides lack

the characteristic 17 aminoacid ring structure associated with other natriuretic peptides

(YandIe, 1 994). Nevertheless, N-teminal ANP molecules have been found t O produce similar

natriuretic, diuretic and BP lowering effects as C-terminal ANP (Martm et al., 1990: Vesely

et al.. 1994). Whereas the hal maturation step for ANP is believed to occur on or just afier

Rs release fiom hem atria, for N-terminal ANP molecuies a M e r proteolytic process may

occur within the circuiation to form the 3 N-terminal peptides (Vesely, 1995). Specific RIAS

toward various portions of N-terminal ANP molecules have demonstrated that ANP,.,,,I

ANP,,, and ANP,,, indeed circulate as distinct peptides (Winters et al.. 1988: Gower et

al., 1994). The release ofthe N-terminal ANPs is reguiated Ma the same stimuli as ANP. such

as atrial stretch and rapid heart beat (Ngo et al., 1989; Dietz et al.. 199 1). The tissue

distnibution of N-terminal ANPs is dl under investigation but should parallel to some exqent

the tissue distribution of ANP. Thus, the atrial tissue is certainiy their major site of spthens

(Vesely et al., 1992). ANP,,, and ANP,,,, have been localized in the kidney, particuiarly in

the p r o d tubule @mirez et al.. 1992; Saba et al.. 1993). Immunofluorescent studies have

also demonstrated the presence of the same two peptides m cholinergie neurons of the CNS

(Veseiy et al., 1992). in the circulation, ANP,,, and AN&,,, are found m concentrations 10

to 24 times higher than that of ANP, whereas plasma Ievels are approxirnatively 3

times higher than those of ANP (Winters et al.. 1988; Vesely et al.. 1994). Ln addition, the

vanous half-hes cf the N-terminal peptides are about 15 times longer than the half-live of

ANP (Achemian et ai.. 1992), a situation accounting in part for their much geater

concentration in circulation than that of the classical natrîuretic peptides (ANP. BNP and

CNP) (Vesely. 1995). The exact clearance mechanism is not clear. although it appears to be

independent corn clearance receptors (Vesely. 1995). Much aiil has to be leamed fiom N-

terminal natriuretic peptides. but their elevated plasma levels and longer half-lives may

suggea prolonged effects. in contrast to the acute nature of the C-terminal natriuretic

peptides responses.

1 .3.t. B-TYPE NATRrURETIC PEPTIDE

1.3.3.1. Brain natriuretic ~ept ide

a) Biochentistp artd releuse

The BNP gene consists of 3 exons and 2 introns (Roy and Flynn, 1990: Steinhelper.

1993). Like ANP. moa of mature BNP is encoded by the second exon (figure 1.3.1.)

(Seilhamer et al.. 1989). In addition. the BNP gene contains an ATTTA motif in the 3'-

untranslated region. a motif absent from the ANP gene. This sequence has been implicated

wRh mRNA instability and suggests a differential transcriptional reepiation between the ANP

and BNP genes (Davidson and Struthers. 1994: Ogawa et al.. . 1995). Post-transcriptional

dserences have also been noted between the two natriuretic peptides. There is a lower

structural homology for BNP and proBNP between species than for ANP and ANP

precursors. indicating interspecies diversity (Lang et al.. 1992: Datidson and Struthers.

1994). In human cardiac tissue. BNP mRNA produces a 134 aminoacid precursor which

becomes. after the removal of the 26 aminoacid signal peptide. the 108 aminoacid proBNP

(Sudoh et al.. 1989: Seilhamer e t al.. 1989). A shorter coding sequence for the second exon

of rat BNP gene gives rise to a 95 aminoacid BNP precursor for this specie (Roy et aL.

1990). A final proteot)ac cleavage imrnediateiy after an Arg-X-X-Arg sequence produces the

C-terminal mature BNP fom with the characteristic 17 aminoacid ring structure of natriuretic

peptides (Steinhelper et al.. 1993). Interestingiy. there is a greater sequence h o m o l o ~

between ANP and BNP than between the BNPs of various species (Yandle. 1994).

Consequently. a 32 aminoacid f o m of BNP circulates in humans (Sudoh et al . 1989). a 45

aminoacid form m rats (Aburaya et al.. 1989a) (figure 1.3.2. ) and a 26 aminoacid form in pigs

(Aburaya et ai.. 1989b). To date. no experiments have investigated the proBNP portion of

the peptide. although its greater interspecies sequence bomology than the C-teminal BNP

(Stemhelper er ai.. 1993) and the elevated plasma lwels of the N-terminal proBNP (Tateyama

et al.. 1992: Yandle et ai.. 1993 ) may suggest same biological fùnctions. much like for the

newly identified proANPs.

BNP was originaily isolated fiom the porcine brain (Sudoh ft al.. 1988). However.

subsequent experiments have indicated that the major site of BNP synthesis. in hurnans. pigs

and rats is the heart rather than the brain (Minamino et al., 1988: Saito et al.. 1989:

Mukoyama er al.. 199 1 ). Withui the heart itself. the ventricles rather than the atria appear to

be the main site ofBNP production. Although about 100-fold higher concentrations of BNP

are found in atrial tissue. 6096 of the total BNP secretion is still observed after the

experimental removal of the atna. comparatively to onIy 50h for ANP (Ogawa et ai.. 199 1 ).

Pathophysiological conditions associated with hypertrophied ventricles tùrther accelerate the

synthesis and secretion of BNP fiom this tissue (Kohno et ai.. 1 992a: Dagnino er ai.. 1992:

Yokota et ai.. 1993). The scarcity of ventricular granules suggeas a constitutive mechanism

of secretion for BM? much like for ventricular ANP (Lang et al.. 1992). in the cardiac

atrium BNP is colocalized with ANP in granules (Lida et al.. 1990: Hasegawa et al.. 199 1 :

Thibault et al.. 1992). However. the storape foms of BNP in atrial tissue Vary amongst

species. fiom mature BNP in rats (Thibault et ai.. 1992) to a misture of BNP and proBNP

in humans (Takeyama et ai.. 1990) or mainly proBNP in pigs (Minamino et d.. 1988).

Because of the ventricular l o c ~ t i o n of BNP within the heart. its mechanisrn of release

depends more on left ventricular end-diastolic pressure than on atrial stretch (Nakamura et

ai.. 1992: Richards et al.. 1993 ). Thus. stimuli favouring elevated BP (Lang et ai.. 199 1 :

Yokota et al.. 1993) or supraventricular tachycardia (Kohno et al.. 1992b) have been

demonstrated to increase heart BNP release. Humoral factors such as endothelin- 1. but not

AVP or angiotensin iI . have also been shown to modulate BNP secretion (Hirata et ai., 1990;

Horio et al.. 1992). Nevertheles the avial portion of BNP colocalized with ANP m granules

is still repiated by atrial distension (Kohno et al.. 1992b).

6) Tisstre distribzrtiotr

As previously discussed the cardiac tissue and particularly the heart ventricles appear to

be the most prominent site of BNP synthesis and release in the body (Mukoyama et al. .

199 1 ). Whereas BNP is present in the porcine brai.. mainly in the bramstem the spinal cord

and the hypothalamus (Ueda et al.. 1988), ody smail amounts of this natnuretic peptide have

been detected in the rat and human brain. with the exception of the spinal cord where

si@cant levels are found (Ogawa et al.. 1990: Nakao et al.. 1990: Hira et al.. 199 1 ). BNP

and proBNP (also termed "aldosterone secretion inhiiitory factor or ASIF") have been

localized m adrenal chromaffin cells and proposed to locally modulate aldosterone secretion

(Nguyen et ai.. 1989a.b). The lung and the aorta have also been found to contain the BNP

gene (Dagnino et al.. 199 1 ). ûther peripheral tissues (e.g. GI tract) have been shown to

possess BNP imrnunoreacthdy. but local biosynthesis is still uncenain (Sharkey et ai.. 199 1 ).

in the circulation. BNP levels are usually about 50h of those of ANP (Takeyama et al.. 1992).

although they may equal or surpass ANP levels in pathophysiological conditions such as heart

failure. largely because of ventncular hypenrophy (Mukoyama et al.. 199 1 ).

C) Metabolisnt

Although the structural similanty between the A- and B-type natnuretic peptides suggest

similar metabolic pathways. a longer haü-live for BNP than for ANP has been reported iii

plasma (Holmes et a/.. 1993). Resumably. this ditference is the result of the lower affuiity of

BNP to the clearance receptors (Mukoyama et ai.. 199 1: Suga et al.. l992a). It may also

resuit fiom a slower hydrolysis of BNP tban ANP by the enzymatic pathway of endopeptidase

24.1 1 (Kenny et al.. 1993).

1-3.3. C-TYPE NATRILJRETIC PEPTIDE

1.3.3.1. C - t y ~ e natriuretic peptide

a) Biochernktry and releare

The CNP gene is composed of at lean 2 exons and 1 intron (figure 1.3.1 .) (Tawaragi et

01.. 1990. 199 1 : Ogawa et ai.. 1992). Exon 1 codes for a 23 aminoacid signal peptide and for

the hst 7 aminoacid of proCNP, the rest of which is encoded by evon 2 (Yandle. 1994). As

for BNP. several ATTTA motifs have been identified fiom the 3'-untranslated region of rat

cDNA. suggesting mRNA denabilkation (Kojima et al.. IWO). The cleavage of the signal

peptide fiom the 126 aminoacid preproCNP results in the production of the 1 O3 aminoacid

proCNP. interehgly, CNP precursors have the highest interspecies sequence homology of

al1 natriuretic peptides (above 9 6 O i o h o m o l o ~ between rat. pig and human proCNPs) (Kojima

et ni.. IWO: Tawaragi et al.. 199 1 ). As for the A- and B-type natriuretic peptides. the mature

fonns of CNP. a 22 aminoacid (CNP-22) and a 53 aminoacid form (CNP-53). are contained

in the C-terminal end of the prohormone. CNP-53 appears to be the predominant form in

mon tissues (Yandle. 1993). The ha1 cleavage is believed to occur immediately afler an Are-

X-X-Arg sequace 8 proCNP for CNP- 5 3 and on a dibasic Lys-Lys sequence. direct- f?om

proCNP or from CNP-53. for the CNP-22 (Yandle. 1994). Both biologicaily active forms of

CNP exhibit the characteristic 17 aminoacid ring structure and disulphide bridge. but lack the

C-terminal Mig extension present in ANP and BNP (figure 1.3.2.) (Sudoh et nl.. 1990). As

for BNP. the production of additional biologically active peptides fiom the N-terminal portion

of proCNP awaits investigation.

CNP was originally discovered in the porcine brain (Sudob er al.. 1990). in contrast to

ANP and BNP which are located prùnarily within the heart. CNP is virtually absent fiom the

cardiac tissue and located almost exclusively in the CNS (Komatsu et al.. 199 1: Minamino

et ai.. 199 1 ). A role for CNP as a neurotransmitter has been postulated. The regdation of

CNP synthesis and release, particularly in the CNS, remains unclear (Espiner. 1994).

b) Tissue distributiorl

CNP is the most prevalent of the 3 natriuretic peptides in the brain ( M m a e o et ai..

199 1 : Togashi et al.. 199 1 ). Within the CNS. its regional distribution cliffers From that of

ANP. Species variations have also been observed. in the rat. CNP immunoreact~ty is the

highest in the cerebellum foilowed by the hypothalamus. thalamus. septum and olfactov

buibs (Komatsu et al.. 199 1 ). whereas in the human brain the highea CNP concentration is

found in the hypothalamus. followed by the rnidbrain and the thalamus (Komatsu et al..

199 1 ). CNP mRNA has also been detected in the CNS. notably in the anteroventral preoptic

area (AV3V) and the arcuate nucleus ofthe hypothalamus (Herman et a/.. 1993). Some CNP

immunoreact~ty has also been detected m other areas of the nervous system such as the

spinal cord (Ueda er al.. 199 1 ). the anterior and posterior pituitary gland (Komatsu er al..

199 1 ) and the CSF (Kaneko er CIL, 1993).

CNP immunoreact~ty (Suga et al.. 1 992b: Stingo et al.. 1992: Heublein et al.. 1992)

and gene expression (Komatsu et al.. 1992a) have been reponed in vascular endothelial cells.

providing the concept of a "vascular natriuretic peptide system". The modulation of CNP

secretion by V ~ ~ O U S growth factors and cytokines. nich as transfonning growtth factor4

(TGF-B). tumor necrosis factor-a (TNF-a). basic fibroblast growth factor (bFGF).

mterteukin- 1 and thrombin. suggests a role for the CNP-mediated vasorelalration and growth

inhibition in vascular remodelling ( Suga et al., 1 992b. 1 993 ).

Smaller concentrations of CNP have been found in the kidney (Komatsu et al.. 199 1 :

Suzuki et al.. 1993). The recent identification of CNP trmscript in various nephron segments

by PCR experiments suggests the possibility of a local role for the C-type natriuretic peptide

in this tissue (Terada et al., 1994).

The presence of CNP within the heart itself remains controversial. The low

concentrations of atrial and ventricular immunoreactive CNP detected by early eqeriments

have been shown to be due to the cross-react~ty with the high cardiac concentrations of

ANP and BNP (Minamho et al.. 199 1 ). However. the recent demonstration of CNP mRNA

gene expression m the hean atria and ventricles may indicate a small paracrine role for CNP

m the heart (Vollmar et ai.. 1993). particularly in pathophysiological conditions (Wei et al..

i 993 ).

CNP and CNP mRNA have also been detected m the adrenal chromaffin cells (Babinski

eral., 1991).

c) ~letuboiisnz

Very low CNP levels are present in the plasma. in agreement with its central rather than

peripheral localization m the body (Mmamino et ni.. 199 1 : Stingo et al.. 1992). Nevertheless.

it has been s h o w that of the 3 natriuretic peptides. CNP is the best substrate for

endopeptidase 24.1 1 (Kenny et ai.. 1993). Moreover. the binding atfinity of CNP to the

clearance receptor. although lower than that of ANP. is higher than the binding affinity of

BNP (Suga et al.. 1992a).

1.3.4. NATRHJRETIC PEPTIDE RECEPTORS

1.3.41. Guanvlate-cvclase (CC1 receotors (NPR-A and NPR-B)

The guanylate-cyclase (GC) receptors consist of an extracellular igand-binding region.

a single traosmembrane domain and two mtraceliular regioos. a kinase homology domain and

a GC catalytic domain (figure 1.3.3.) ( KoUer and Goeddel, 1992: Nakao et ni.. 1992: Maack

er ai.. 1993). The gene coding for the GC receptors contain 22 exons and 2 1 introns in the

rat (Yamaguchi et al.. 1990). The fùn 6 exons encode for the extracellular portion of the

receptors. Exoo 7 represents the membrane-spanning region. The intracellular portion of the

receptors is separated in two. with exons 8 to 15 encoding for a protein-kinase like domain

and exons 16 to 22 for the GC domain. The transcription of the GC receptor genes results in

the production of two distinct giycoprotein receptors structurally M a r . a 1057 aminoacid

NPR-A and a 1047 aminoacid NPR-B (Schulz et al.. 1989; Chang et al.. 1989: Chinkers c f

al., 1989). The second messager guanilate Y. 5'-monophosphate (cGMP) is produced upon

peptide bmding to the extracellular region by the activation of the GC catalytic domain (Kuno

et al.. 1986: Shmg et ni., 1988). Most of the pbysiological actMties of the natriuretic peptides

are believed to occur via the production and cytoplasrnic accumulation of cGMP. The kinase

kinase homology domain

g uanylyl cyclase domain

N PR-A N PR-B NPR-C

44% homology

a-D

88% homology

0-D

30% homology

a-P 33%

homology

-I>

extracellular

cell membrane

Figure 1.3.3. Diagramofthenatriureticpeptide receptors (NPR)

homology domain may be important in transducing binding information to cGMP production

(Chinicers and Garbers. 1989: Koller et al., 1992). A negative regulation of cGMP by this

spec%c area has been postulated. since the deletion of the kinase homolou domam results

in constitutive production of the second messenger (Larose et al.. 199 1 : Chinliers et al..

1991).

The aminoacid sequence between the two receptors presents a 44% homolou in the

extracellular region. 63% homology in the kinase homology domain and 880h homolog in

the GC region (Chang et ai., 1989). The GC receptors also have about a 30% sequence

homology with the extraceMar peptide-buidhg domain of NPR-C. the third and stnicturally

different receptor of the natriuretic peptides (Fuller et al.. 1 988).

The relative selectkity of the GC receptors to the various natriuretic peptides lias been

assessed ni ce11 membrane preparations fiom various tissues (Suga et al.. 1992a). The NPR-A

receptor bmds most effectively ANP and BNP. However. BNP is usually 1 O-times kss potent

than ANP m stimulahg cGMP production by this receptor (Schulz et ai.. 1989). In contrast.

CNP bas Little effect on the cGMP production by the NPR-A receptor. suggesting the

following rank order of selectivity: ANPzBNPKNP (Suga er al.. 1992a). The NPR-B

receptor recognizes especially CNP. suggesting CNP>ANPzBNP as a rank order of

selectivity (Koller et ai.. 199 1 ). Thus. ANP and CNP are postulated to be the major ligands

for NPR-A and NPR-B respectively. As yet. no specific receptor for BNP has been identified.

1.3.4.2. Clearance receotor (NPR-Cl

The mature NPR-C receptor. structuraily vety cliffereut fiom the GC NPR-A and NPR-B

receptors. exists predomioantly as a homodimer and consists of an extracellular peptide-

bmdmg domaIn. a single transmembrane area and a small(37 aminoacid) intracellular region

(figure 1 - 3 2 . ) (Fuller et al., 1988: Uchida et al.. 1989). The NPR-C gene contains 8 e'rons

and 7 introns (Lowe et ai., 1990; Porter et ai., 1990; Sahelci et al.. 199 1). Exons 1 to 6

encode for the extracellular domain, whereas exons 7 and 8 code for the transmembrane-

spanning region and the intracellukir area respectively. The translation of the mRNA gives rise

to a 537 aminoacid NPR-C precursor in bovine tissue (Fuller er al., 1988). In contrast to

NPR-A and NPR-B. this receptor lacks the cytoplasmic GC and kinase homology domams.

Eariy experiments faiied to noticed a si@cant association between peptide binding to this

receptor and any physiologicd effect of the natriuretic peptides (Schenk et ai.. 198% Leitman

et ai.. 1986). Furthemore. the demonstration of NPR-C intemaikation upon ligand binding

and the subseguent lysosomal degradation of the ligand itself (Nussemeig et ai.. 1990:

Maack et al.. 1993). led Maack et ai.. m 1987. to propose a role for NPR-C in natriuretic

peptide clearance. This so-called "clearance receptor" then may function as a b d e r system

to prevent sudden variations in natnuretic peptide levels and BP. However. other Imes of

evidence indicate a biological role for the NPR-C receptor (Anand-Srivastava and Trachte.

1993 : Smyth and Keenan. 1994). Indeed recent experimeots suggest that NPR-C may inhibit

adenylase cyclase (AC)-mediated adenosine 3'. 5'-monophosphate (CAMP) production

( Anand-Scrivastava et al.. 1990: Tseng et cri.. 1990: Drewett er ai.. 1992). activate the

phosphomositol pathway (Hirata et al.. 1989) and mediate parts of the antiproliferative action

of the natnuretic peptides (Levin. 1993 ).

In contrast to both GC NPR-A and NPR-B receptors. NPR-C does not have stringent

requirements for peptide binding. It exhibits hi& a f i i t y for ANP. BNP. CNP and even for

vanous tnmcated. ring-deleted and linear biologically inactive ANP analogs (Leitman et al..

1986: Maack et ai.. 1987: Olins et nf.. 1988: Bovy et oi., 1989). Its rank order of specificity

for the major natriuretic peptides is ANP>CNP>BNP (Suga et al.. 1992a).

1.3.4.3. R e c e ~ tors for N-terminal fra~ments of D~oANP

Recent experiments have postulated the existence of separate and distinct receptors for

the newly identified peptides produced fiom the processing of the N-terminal fragments of

the proANP molecule (see section 1.3.1.3) (Vesely et ai.. 1990: Gwining et al.. 1992: Zeidel.

1995). However. the characterization or the molecdar cloning of these receptors have not

yet been reported.

1.3.1.4. Tissue distribution

Both GC (NPR-A and NPR-B) and clearance (WR-C) receptors are present in renal

tissues (Napier er al.. 1984: De Léan et al.. 1985: Brown and Zuo. 1992). Within the

nephron. the glomenilus and huer medda collectmg ducts (LMCD) have the hi&ea

concentration of natriuretic peptide binding sites (Mattin er al.. 1989: Brown and Zuo. 1992).

The quantification of the receptor subtypes with des-(Glnl". Ser"'. Gl y "". Leu"'.

Gly"" ]ANF,u2-,2, (or cANF). a tmca ted and ring deleted ANP analog with NPR-C

s p d c i t y (Maack et al.. 1987). demonsh-ated a &ed receptor population in the glomenilus

with 50 to 80% of the receptors being of the NPR-C type. and a homogenous GC receptor

population (NPR-A or NPR-B) in the iMCD (Martin er al.. 1989: Nugiozeh er al.. 1990).

Although the messages for NPR-A NPR-B and NPR-C have been detected throughout the

kidney (TaUenco-Melnyk er al., 1992: Greenwald et al.. 1992: Canaan-Kuhl et ai.. 1992:

Teada et al.. 1994). no expression of NPR-B mRNA bas been reponed yet. suggesting the

presence of mature NPR-A and NPR-C. but not NPR-B. in renal tissues (Brown and Zuo.

1992: Luk et al.. 1994). Kidney vascular segments. such as the vasa recta. and renal

meduilary interstitial cells have also been s h o w to possess nattiuretic peptide receptors

(Bianchi er al., 1987; Fontoura et al., 1990).

The vascular tissues primanly express the NPR-C receptor (Leitman et al.. 1986: Cahill

et al.. 1990). Nevertheless. the message for both GC and clearance receptors have been

detected in vascular cells. although the subtype of GC receptor varies with the cell type. since

only NPR-A or NPR-B are present in aortic endothelial and smooth muscle cells. respectively

(Suga er al.. 1992b: KatAchi et ai.. 1992). The NPR-C receptor is also localized in platelets

(Anand-Scrivastava et al., 199 1 ; Schif in et ai.. 199 1 ).

NPR-A. NPR-B and NPR-C mRNAs are expressed in cardiac tissues (Nunez er ai..

1992; Rutherford et al.. 1992). The expression ofthe NPR-C receptor appears to be the mon

abundant. whereas the prevalence of NPR-B mRNA seems lower than that of the other two

receptors (Nunez er al., 1992).

In the CNS. the NPRs are mainly localized in the circumventricular organs. such as the

subfomical organ (SFO). area postrema (AP). choroid plexus (CP) and organum vasculosum

of the lamina terminalis (OVLT) (Quuion et al.. 1986: Mantyh er al., 1987: Brown and

Czaniecki 1990a). The quantification of the receptor subtypes has revealed the presence of

on& GC receptors in the SFO and AP (NPR-A ancilor NPR-B) (Himeno et al.. 1992: Konrad

et al.. 1992a.b; Brown and Zuo. 1993). but of the mixture of GC and clearance receptors in

the CP (Himeno et al.. 1992: Zorad et al.. 1993 ). Lower NPRs concentrations are expressed

within the brain. but Sumners and Tang. in 1992. postulated that NPR-A and NPR-B

predorninate in fibroblan and neuronal cells. respectively. NPRs have also been described in

the olfactory bulb (Gutkowska et al.. 1991), spinal cord (Sirnonnet et al.. 1989). cerebral

microvessels (Whitson et al., 1991) and on the blood side of the BBB (Ettnish, 1992).

Similady. natriuretic peptide binding sites have been demonstrated in the anterior. but not

posterior. pituitary (Von Schroeder et al.. 1985: Pang et al.. 199 1 ).

in the adrenal gland, NPRs are present in the glomenilosa layer of the cortex (Meloche

et al.. 1986: Takayanagi et ai.. 1987). Both NPR-A and NPR-C are expressed in the adrenal

cottes. aithough the proportion of each receptor subtype varies between species (Schiffrin er

al., 1985; Meloche et al., 1986; Ohashi et al., 1988).

Various experiments have demonstrated the preponderance of NPR-C receptors in lune

membranes. in similar proportion than those found in the glornerulus and vasculature

(Leitman et al., 1987: Uchida et ai.. 1989: Abe et al., 1995). Other organs have also shown

natriuretic peptide bhdmg sites. such as the thyroid gland (Tseng et al.. 1990). testis (Pandey

et al.. 1986). liver (TalIerico-Melnyk et al.. 1992) and uterus (Itoh et al.. 1994: Dos Reis er

al., 1995).

1.3.5. PAYSIOLOGICAL ACTIONS OF THE NATRIURETIC PEPTiDES

It is generally accepted that the natriuretic peptide family plays a sipficant role in 1 )

body fluid and 2) cardiovascular homeostasis (figure 1.3.4 ). The p hysiological regulation and

control of these two major functions by the natriuretic hotmones involves the interaction of

the natriuretic peptides with various organs and hormonal systems throughout the body.

1.3.5.1. Renal actions

The natxiuretic and diuretic properties of the natriuretic peptides have been reco+pked

a) Diuresis b) Natriuresis

a) l nhibition of aldoste rone release Zona qlomerulosa

aldos te rone u ADRENAL GLAND

H E A R T and VASCULATURE 0

i - ty~od~alarnus Medulla

AVP release I

a) Inhibition of salt and m t e r intake b) Stimulation of salt and w t e r excretion

CENTRAL NERVOUS S Y S T E M (CNS) Barorecepnrs

Figure 1.3.4. Major s i s s ofaction and physiological effeco; ofnatriuretic peptides: (1) Kidney induces diuresis and naaiuresis,

(2) Heanand vasculahire induce vasorelaxanon, (3) Adrenal gland i n h i b i ~ aldosterone secretion and (4) Cenaal

netvous system inhibits (a) saltand viater intake, (b) saltand vm&r excretion and (c) AVP release.

early (De Bold et al.. 198 1 : Maack et ai.. 1984; Weidmann et al.. 1986: Zeidel and Brenner.

1987: Hoimes et ni.. 1993). in addition. elevations in potassium magnesium chloride and

phosphate excretion have also been observed followkg ANP administration (Maack et ai-.

1984: Cody et al., 1986). Altliough not completely elucidated. tubular. hemodynamic and

hormonal contriiutions have been postdated to explam the natriuretic and diuretic properties

of the natriuretic peptides (figure 1.3.5. ).

( 1 ) A direct effect of ANP. as well as of BNP and UD. on tubular sodium reabsorption

has been reported (Murray et ab. 1985: Blaine. 1990). ln the IMCD. the C-terminal

oaaiuretic peptides have been shown to inhibit sodium reuptake by regdathg the amiloride-

semitnie sodium channels. via NPR-A receptors and cGMP production (Sonnenberg et al..

1986: Zeidel et al.. 1988; Zeidel 1993 ). interestiogly. the N-terminal ANP molecules have

also been show to produce natriuresis. but mdependently From cGMP production. This effect

is mediated by the mhibition of the Na+/K+-ATPase actMty- probably through enhanced PGE,

actMty (Gunning et al. , 1 992; Zeidel- 1995 ).

(2) A modest mcrease of the glomemlar filtration rate (GFR) has been demonstrated by

mcreases of both plasma ANP and BNP levels (Atlas er al.. 1984: Maack et al.. 1 983). This

effect is most likely due to an eievation in the gIomemlar capillary hydraulic pressure induced

by the relaxation and constriction of the afferent and efferent anenoles. respectively (Marin-

Grez et al.. 1986: Veldkamp et al.. 1988: Kimura et ai.. 1990: Lanese et ai.. 199 1 ). The

relaxation of the glomerdar mesangial cells by ANP may also esplain. at leaçt in part. the

effect of the natriuretic peptides on the G R by expanding the capilIary surface area available

for filtration and thus mcreasing the filtration coefficient (Singhal et ai.. 1989). However. the

correlatioii between GFR and natriuresis is ail1 incompletely understood. since some

expedents have demoostrated natriuretic effects of ANP without sigdicant modications

in the GFR (Salazar er ai., 1986: Blaine. IWO). Another intrarenal hemodparnic process

possibly important in natriuresis consias of the natriuretic peptide-mediated increases in

medullary blood flow. particularly Ma the vasa recta (Kiberd et ai.. 1987). This vasodilation

is believed to rnodiSi the medullary tonicity and pressure gradients between the vasa recta and

coilechg ducts. leading to a reduced passive reabsorption of sodium and wat er by the tubular

INP

I, m 2 u

Efierentvasoconsmction

) ANP receptors

O receptors

2+ (Mg , CI-, ~ 0 ~ -

Figure 1.3.5. Schematic diagram summarizing the (1) tubular, (2) hemodynamic, and (3) hormonal effects of the natriuretic peptide farnily on the kidney. (G FR : glomerular filtration rate, AI 1: angiotensin I 1, AVP: arginine vasopressin)

structures and to sodium secretion tiom the mterstitnim into the tubular fluid (renal rnedullary

washout) (Takezawa et ai.. 1987: Davis and Briggs, 1987: Zeidel et al.. 1993).

(3) Intrarenal interactions between the natnuretic peptide family and other hormonal

systems implicated in body fluid homeostasis have also been noted. Thus. in the cortical

collecting ducts. ANP. BNP and üD have an inhibitory action on the antidiuretic effects of

AVP (Zeidel et al.. 1987: Espmer. 1994). In addition. inhibition of the antinatnuretic effects

of angiotensin II at the level of the proximal rubules (Harris and Skinner. 1990) and of

aldosterone in the kidney cytosol (Honuchi et ai.. 1 989) by ANP have been demonstrated.

E.utrarenal interactions between the natnuretic peptides and vanous hormonal systems have

also been observed and are discussed m the foliowing sections (sections 1-3-53. to 1.3.5-5.).

1.3.5.2. Cardiovascular actions

The significant BP reducing effects of the natriuretic peptides have been documented in

both experimental a-1s and hurnans (figure 1.5.6.) (Maack et al., 1984: Volpe et al.. 1981:

Richards et ai., 1985: Tikkanen et al., 1985: Weidmann et al., 1986).

( 1 ) hi vitro studies iiidicated that this hypotensive effect may be due to a direct effect of

ANP or BNP on the vascular smooth muscle cells. producing relaxation (Cume et al.. 1983:

Garcia et al.. 1985: Bolli et al.. 1987). The vasorelaxant effects of the natriuretic peptides are

usually more pronounced in larger arteries than in smaIIer vessels. and appear to be associated

with cGMP accumulation (Winquia et al., 1984: Faison er al.. 1985). In recent years. the

identification of CNP in endotheliai cells and NPR-B in smooth muscle cells have also

suggested the existence of a local "vascuiar natriuretic peptide system" with vasodilatory and

grow-th inhibitory properties (Komatsu et nL. 1 W2a: Ogawa, 1995 ).

(2) Acute in vivo experiments have associated the hypotensive response to the natriuretic

peptides with a reduction in the cardiac output (CO). which may be the result of a Iower

venous retum (VR) (Breuhaus et ai.. 1985: k I I e et ai.. 1992). The reduced VR is probably

due to the ANP-mediated decrease m blood volume, secondary to the shift in the extracellular

fluid (ECF) fiom the vascular to the interstitial space (Almeida et al.. 1986; Trippodo and

Barbee. 1987: Groban et al.. 1990) and to the increased resistance to the VR (Chien et al..

BNP

ANP

Atriu m

Vasorelaxation

Figure 1.3.6. Schematic diagram summarizing the effects of the natriuretic peptide family on the heart and the vasculature. (1) vasorelaxation, and (2) reduction in CO, TPR and VR. (CO: cardiac output, TPR : total peripheral resistance, VR : venous return)

1987; Christensen, 1993). With long-terni ANP administration, the initial decrease in CO is

no longer observed, but the continued hypotensive response appears to result fiom a

secondary decrease in total peripheral resistance (TPR) (Parkes et al., 1988; Charles et al..

1993). The exact mechanisms by wfüch chronic ANP Uifiision lower TPR are unclear, but

ANP-induced inhibition of angiotensin II or endothelin-dependent vasoconstriction has been

suggested (Kohno et al.. 199 1).

An action ofthe natriuretic peptides on baroreceptors has also been postulated because

of the absence of reflex tachycardia after the lower BP and CO resultmg from ANP infùsions

(Schulz et al., 1988).

1.3.5.3. Adrenal actions

( 1 ) Natriuretic peptides have been s h o w to inhibit aldosterone release (figure 1.3.7.)

(Maack et al.. 1984; Jennhgs et al.. 1990). Although this inhi'bition is mediated in part by the

ANP-induced suppression of the action of various aldosterone secretagogues nich as

angiotensin II (Cuneo et al., 1987), potassium (Clark er al.. 1992) or ACTH (Agdera.

1987), a direct effect on the adrenal gland has also been demonstrated (Chartier et al.. 1984:

Higuchi et al., 1986; Rosenberg et al.. 1 989). The mhiitioa of the adrenal zona glomedosa

aldosterone production by ANP appears to be exerted primarily at the early aeps of

steroidogenesis, duriog the formation of pregnenolone (Racz et al.. 1985: ElIiott and

ûoodfiiend, 1986) and also to some extent during the final conversion of corticosterone to

aldosterone (Campbell et al., 1985). In contrast to most other effects of the natriuretic

peptides, the exact mechanism of action for this inhibition remains controversial. None of

cGMP analogs affect sigiilficantly the aldosterone production (Matsuoka er al.. 1987;

Gan& er al., 1989; Gangdy, 1992). Therefore. a biological role has been suggested for the

NPR-C in the adrenal corte- even though some reports aill argue for a contniution of the

GC recepton (Bahr et ai., 1993). The possiiility of a local "adrenal natriuretic peptide

systern" bas also been suggested foUowing the demonstration of ANP, BNP and CNP

transcripts m adrenal chromfi celk (Ong et al., 1987; Nguyen et al., 1989a; Babinski et al.,

199 1).

zona alornerulosa

1 BNP Aldosderone

Angiotensin II

Renin

O

Figure 1.3.7. Schematic diagram summarizing the effects of the natriuretic peptide family on the adrenal gland. (1 1 l nhibition of aldosterone, and (2) of renin.

(2) ANP and BNP also inhibit renin release fiom the renal juxtaglomerular cells (Maacli

et al.. 1984: K u t z et al.. 1986). The renin suppression by the natriuretic peptides seems to

be cGMP dependent (Kurtz et al.. 1986).

1.3.5.4. Central nervous svstem KNS) actions

Recently. a major role for the central component of the natriuretic peptide system in

cardiovascular and k i d homeostasis has beai postulated ( h u r a et al.. 1992). The penpheral

stimuli received by the bram natriuretic system such as plasma ANP binding to the

circumventncular organ receptors or the neuronal signais fiom the baroreceptors. are believed

to produce a cohort of CNS responses that rnay include three components: the modulation

of £luid and salt intake. the modulation of fluid and salt excretion and the modulation of other

honnonal syaems implicated in fluid and salt homeostasis (figure 13.8.).

( 1 ) Intracerebroventricular (i.c. v. ) ANP injections have been shown to inhibit water

mtake during angiotensin Il -induced drinking or in water-deprived rats (Antues-Rodrigues

er al.. 1985: Nakamura et al.. 1985: Katsuura et al.. 1986: Lappe er ai.. 1986). Similarly. an

inhibition of salt appetite is reported after central administration of ANP in rats. rabbits and

sheeps ( Antunes-Rodrigues et al.. 1986: Ta jan et al.. 1988: Weisinger et al.. 1992). The

exact location for these antidipsogenic and antiadipsianic actions of natriuretic peptides is

uriclear. but it may mvofve the AV3 V area. an hypothalamic structure important in body fluid

homeostasis (Buggy and Bealer. 1987: Ku and B a n g 1994). The implication o f the SFO has

also been dernonstrated. since ANP microinjections into the subfomical organ attenuate

angiotensin 11 -induced drinking (Ehrlich and Fitts 1990) and inhibit the actkity of

angiotensùi II -sensitive neurons projecting to the AV3V area (Hattori et ai.. 1988: Shibata

et al.. 1992).

(2) Central mfùsions of ANP have been associated with diuresis and. in some instances.

natriuresis (Fhs er al.. 198% Israel and Barbeila. 1986: Shoji er al.. 1987). Although part of

the water excretion appears to result Eom the ANP-mediated modulation of central AVP

(Standaert et al.. 1987). a direct effect of the brain natriuretic system on renal diuresis has

also been suggested (Shoji et al., 1987). Again. the AV3V area seems important in the central

HYPOTHALAMUS ME DULLA

NPR

NPR

NPR

CNP

SON

PVN I

I C Diuresis and nalriuresis

Figure 1.3.8.

NTS

I I Ba roreceptors

Schematic diagram summarizing the effects of the natriuretic peptide family on the brain. (1 ) Inhibition of -ter and salt intake, (2) Stimulation of m t e r and salt excretion, and (3) inhibition of AVP release. (AP:area postrema, AV3V: a nteroventrai third ventricle area, AVP: arginine vasopressin, NTS: nucleus tractus solitarii, PVN: paraventricular nucleus, S FO: subfornical organ, SON: supra-optic nucleus)

control of water and sodium handling by the kidney (Imura et al.. 1992). Some reports have

demonstrated a depressor action of i.c. v. ANP mjections. especially during central angiotensin

[I -mediated hypertension. but the issue remains controversial (Itoh et al.. 1986a: Castro et

al.. 1987). The hypothalamus may be the site of action for this central hypotensive effect of

natriuretic peptides. In addition. the meduila oblongata. particularly around the NTS and the

nucleus arnbiguous. may be important. since ANP microinjections in these areas have

produced significant reductions in BP (McKitnck and Calaresu. 1988: Levin et al.. 1989:

Ermirio et a', 199 1).

(3) Several hormonal and neurotransmitter systems are modulated by the central

administration of ANP. The release of AVP fiom the supraoptic and paraventricular nuclei

m mhibited by central infusion of natriuretic peptides (Crandall and Gregg, 1986: Samson et

al.. 1987: Standaert et al.. 1987). Basal AVP release 6om the posterior pituitary is also

reduced foilowing I.C. v. ANP administration (Obana et al.. 1985: Januszewicz et al.. 1985).

An inhibitory action of centrally-administrated ANP on dopaminergic (Nakao et al.. 1986).

noradrenergic (Vatta et al.. 1992) and angiotensin II -sensitive neurons (Hattori et al.. 1985)

has also been demonstrated. In the cases where appropriate studies were perfomed. these

central effects ofthe natriuretic peptides appeared to be mediated by cGMP (Giridhar et al..

1992).

A role for ANP m CSF formation. particularly in the choroid plexus. lias been suggested

(Tsutsurni et al.. 1987: Steardo and Nathanson. 1987).

1.3.5.5. )iormonal actions

Enhanced aaivay of the natriuretic peptide system has been associated with the inhibition

of the secretion of ACTH (King and Baertschi 1989: Dayanilhi and Antoni 1990: Fink et al..

199 1 ), prolactin (Samson and Bianchi 1 988) and luteinking hormone secretion (Samson et

al., 1988: Franci et al.. 1990; Samson et al.. 1993 ), but not of thyroid-stirnulating hormone

(Franci et al.. 1992). in contrast. acute exposure to ANP has been shown to enhance

testosterone secretion (Mukhopadhyay et al.. 1986) and to inhibit progesterone production

(Pandey et al., 1985).

The naaniretic peptides have been shown to inhibit most of the angiotensm U actions ( at

the levels of the vasculature. brain. kidney and adrenals). suggesting the concept of

physiological antagonism between the angiotensin and natriuretic peptide systems

(Mendelsohn et ai., 1987: Espiner. 1994).

1.3.5.6. Pulmonarv actions

Bronchorelaxation (Potvin and Vanna. 1989). regdation of lung pemeability and thus

prevention of pulmonary edema (Lofton et ai.. 1990) and a role m &actant production (Ishii

et ai.. 1989) have been suggested for ANP in pulmonary tissues. The mechanim, of action of

the natriuretic peptides at the level of the lungs is still unclear. There is some evidence that

production of cGMP is needed.

1.4. ALCOHOL (ETOH), BLOOD PRESSURE (BP) AND THE

NATRKRETIC PEPTIDE FAMILY

1 .-LI, ETHANOL (ETOH) .AND THE NATRCZTRETIC PEPTIDE FAMILY

in 199 1. when this project was initiated. there was only one published report on the

mteractions between alcohol and the natriuretic peptide system. The group of Colantonio er

al. (1991) had investigated the effect of a single ETOH dnnk ( 1 mVkg B.W.) on the

circulahg ANP levels m healthy human males. They noticed a rapid increase in plasma ANP

levels following the ETOH consumption. lasting one hour. in quasi-isovolurnetric conditions

(0.1 L of urine on average). They concluded on a possible role of ANP on the ETOH-induced

diuresis. probably through the Hihibition of AVP release as wggested by the opposite changes

in circulating ANP and AVP levels.

Since then. four other publications with divergent methodologies have proposed various

and often different associations behhieen acute ETOH consumption and ANP. comp licating

the issue. Leppiiluoto et al. ( 1992) observed a significant decrease in senun ANP levels 2

hours d e r a 1.5 g/kg B.W. ETOH drink in seven healthy males. However, the lack of blood

analysis early afier the ETOH consumption may renilt in missing the rapid and short-lived

ANP increasr noted by Colantonio et al. ( 199 1). Moreover. the rapid diuresis obsewed

during the fust hour (0.9 L on average) and the corresponding lower plasma volume should

rapidly counteract any initial stimulating effect of ETOH on the ANP system. Intereaingly.

the lower ANP levels are associated with a secondary increase in plasma AVP contents.

confirming the possibility of a link between the two hormonal syaems. The same year.

Hpynen er al. ( 1992) reported the absence of any eBect of alcohol ( 1 b@kg B.W.) on the

circulating ANP levels of ten healthy male volunteers. However. the water loading of the

subjects prior to the ETOH experiment and the corresponding increase in plasma volume

mua have sigdïcantly augmented plasma ANP content and limited the possibility or the

e.xtent of a further ANP increase by ETOH. Variations in circulating AVP levels were also

absent fiom this study (Hynynen et al.. 1992). In addition. the siguikant diuresis produced

even before the alcohol was administered could limit any subsequent potentiation of the ANP

system by ETOH. In 1994. Ekman and colleagues noticed the inhibition of the noctumal

increase Ui plasma ANP levels more than 8 hours afier the administration of a IgAg B.W.

ETOH drink in nme heaithy subjects. However. in this study the firn blood sample was taken

two hours afier ingestion of the ETOH drink lirniting the value of the experiment in

mvestigating the early rapid changes on the release of ANP. However. they noticed that the

content of the proANP molecules was not affected by the ETOH treatrnent. suggestin_e an

mcreased peripheral elunination of ANP rather than the inhibition of ANP release following

the ETOH drink to explain the observed result.

While our studies were in progress. the first midy on the interaction of ETOH with the

ANP system using experirnental animals was published (Wigle et al.. 1993a. 1995). Animal

experiments are important since they allow an accurate control of a number of parameters.

such as previous alcohol habits. heterogeneity of the subjects. food and water/ETOH intake.

etc. that may affect the outcome of the mteraction between ETOH and the natriuretic peptide

system and that cannot be controlled in human studies. Acute heavy ETOH consumption ( 5

e/kg B.W.) was administered through gastric gavage. Because of the hi& volumes of water C

@en to control rats, plasma ANP levels progressively increased during the experiment. In

contran. no modification was observed in circulatmg ANP levels after the ETOH

administration. Both atrial and ventricular ANP contents foilowed the same progressive

changes m the two groups of rats. This may be the result of the mhibition of gastnc emptying

and of the presence of secondary gastnc irritation. by such swere alcohol intoxication, rather

than of a direct eEect of ETOH itselfon the ANP system hdeed, the extremely slow and

progressive mcrease m BAC suggests a partial suppression of the normal rate of ETOH

absorption by the GI tract. Lower doses of alcohol should be used in fiiture experiments in

order to mimic the BAC c w e observed in human subjects after acute ETOH consumption.

The effects of chronic ETOH exposure on the natriuretic peptide system are even less

clear. This issue was investigated for the fïrst and ody tirne m 1993 following a 6 weeks

administration of a 20% vlv ETOH solution, using the Sprague-Dawley rats (Wigle et al..

1993b,c). No modification m the BP was noted, which is not surprishg considering the slow

and progressive nature of the antihypertensive effect of moderate ETOH consumption in rats

(see section 1.2.2.2.). Plasma and atrial ANP levels were unaffected by the ETOH treatment.

but ventricular ANP content was significantly elevated in ETOH-treated rats (Wigle er al..

1993b). Intereçtingiy, circulahg BNP Ievels were also augmented in these animais (Wigle et

al., 1993~). in order to really analyze the possible contribution of the natnuretic peptide

system in the prevention of the age-dependent increase m BP by chronic rnoderate ETOH

drinking, it may be necessary to investigate this systern after the antihypertensive effect of

ETOH has been established (after at least 6 months) and not only during the early stages of

the treatment (after 6 weeks). Furthemore, since this prevention of the age-dependent

increase m BP is more evident in the hypertensive strains of rats, such as S m experiments

should be designed to compare the effects of ETOH m the hypertensive rats versus the

normot ensive controls.

In recent years, ANP has also been implicated with the attenuation of delirium tremens

and convulsions during ETOH withdrawal (Bezzegh et al.. 199 1; Kovacs. 1993).

1.4.2. WORKLNG EiYPOTKESIS AND DlVISION OF TEE TaESlS

in the previous sections, the evidence for the antihypertensive ef5ects of acute and

chronic moderate ETOH consumption has been rwiewed. A few mechanisms, nich as

lipoproteins or hemostatic processes, have been descnbed to explain this effect of alcohoL but

a conviucing hypothesk is stin lachg. It has been suggested that ETOH may alter the actMty

of a number of physiological systems which rnay then mediate its antihypertensive effects. A

new and expandmg fandy of natriuretic peptides with widespread BP lowering and

vasorelaxant effects has also been discussed. Recent evidence of some alterations m the

a+ of this system by ETOH just opened the possiibility of a new mediator for several of

the ETOH-mduced effects on the cardiovascular system

InteresMgly, alcohol and the naviuretic peptide f d y have several nmilar effects on

the kictney, the cardiovascular system and the brah. They both have some depressor effects

on various brain functions. They both produce transient antihypertensive effects and lower

cardiac contractiiity. They both mduce diuresis and natriuresis. Since a number of studies have

demonstratecl that ETOH alters the activity of a number of hormonal syaems such as AVP.

it is possible that the activity of various componaits of the natriuretic peptide system may also

be affected by alcohoL It is surprising then that the implication of the natriuretic peptide

family on some of the effects of alcohol had not been investigated pnor to 1991. For

example, it has been postdated that the dimesis caused by ETOH is mediated by the inhibition

of AVP (Eisenhofer and Johnson, 1982; Leppaluoto et al., 1 992). Interestmgly, AVP release

kom the hypothalamus is also suppressed by AM> (Januszewicz et al., 1986a; Crandall and

Gregg, 1986; Standaen et al., 1987). Thus, it is possible that the inhiiitory effect of ETOH

on AVP release is mediated by a aimulatory effect of ETOH on ANP secretion. Then. some

of the cardiovascular and rend effects of ETOH previously attnbuted to AM> may be

mediated rather by the natriuretic peptide family. Similady, such modifications in the

natriuretic peptide system may produce, during long-tenu moderate ETOH administration.

permanent antihypertensive effects that may explain, at leaa m part, the U-shaped BP c w e

and the prevention of the age-dependent mcrease in BP observed m human subjects and

experirneatal animals following chronic moderate ETOH consumption.

Therefore, the present series of experiments were undertaken to mvestigate the following

hyp othesis:

Acute and chronic alcohol exposure alters the activity of distinct

components of the natriuretic system (natriuretic peptides and natriuretic

receptors). These alterations may mediate some of the effects of ETOH on the

cardiovascular system.

(1) ACUTE ETOH STUDiES: Chapter 2 describes our audies investigating the eEects

of acute low and moderate ETOH consutnption on cardiac and circulating ANP levels in rats

(Section 2.1. ) and human volunteers (Section 2.2.).

(a) A n h l studies (Section 2.1.): Male LongEvans rats receive a 1 or 2 g of ETOWkg

BW through i-p. injection. Circulating and cardiac ANP levels are measured at various time

points foiiowing the ETOH injection.

(b) Human studies (Section 2.2.): Normotensive i n d ~ d u a l s receive a 0.25 or 0.50 g of

ETOWkg BW in unsweeteaed orange juice. Plasma ANP levels are measured for 3 hours

following the ETOH ingestion.

These studies should d o w the determination of any acute and/or short-lasting activation

of the natriuretic peptide syaem. The demonstration of a stimulatory effect of acute ETOH

treatment on plasma ANP levels should indicate whether the natnuretic peptide syaem is

sensitive to alcohol exposure. in addition. any stimulatory effect of ETOH on the circulating

ANP levels may suggen the possiiility of a sipficant antihypertensive effect of this hormonal

system during chronic moderate ETOH drinking.

(LI) CHROMC ETOH STUDIES: Chapter 3 describes our studies surveying the effects

of chronic moderate ETOH consumption on the a c t ~ t y of distinct components of the

naaiuretic system (natrituetic peptides and natriuretic receptors). Male Wistar- Kyoto ( WKY )

and spontaneously hypertensive ( S m ) rats receive a 20% vlv solution of ETOH for 8

months The effect of chronic moderate ETOH consumption on the age-dependent increase

m the BP is descnied in section 3.1. The studies of Chapter 3 are performed on some of the

major sites of natriuretic peptides synthesis and action. such as the heart. liidney and brain.

following long-term moderate alcohol consumption.

(a) Cardiac natriuretic -stem (Sections 3.1. and XZ.1 : The heart is the central

component of the ANP and BNP systems. It may directly alter the BP. both fiom

modifications in cardiac contractility or hormonal release. It is also sensitive to ETOH.

Circulating ANP (chapter 3.1) and BNP (chapter 3.2) levels are measured following chronic

moderate ETOH consumption. Modifications in atrial and ventricular ANP. BNP. ANP

mRNA and BNP mRNA are also mvestigated following the alcohol treatment.

(b) Renal natnuretic svstem (Section 3.3.): The kidney is associated with the

dwelopment of hypertension. It mediates some of the major effects of the natriuretic peptides

through specific receptors. Its functions are altered by ETOH exposure.

Modifkations m glomenilar and papillary natriuretic peptide receptor characteristics are

investigated following chronic moderate ETOH consumption.

(c) Brain natriuretic wstem (Sections 3.1. and 3.5.): The brain is the main target for

several of alcohol-induced effects. In recent years. its importance in the natriuretic peptide

family physiology has been demonstrated. It also controls hdirectly the BP via hormonal

sensors in circumventncular organs and through neuronal inputs via baroreceptors.

Modifications in ANP and CNP levels in the major brain areas (chapter 3.4.) and

alterations in natriuretic peptide receptor characteristics in the circumventncular organs

(chapter 3.5.) are investigated following the chrooic administration of moderate amounts of

ETOH.

Thus, the present studies describe the general modifications in the actMty of the

natriuretic peptide system following chronic moderate ETOH consumption. This work

repreçents the kst s w e y on the existence and direction of such alterations and proposes the

hypothesis that this homonai system may mediate parts of the antihypertensive effect usually

observed following clironic moderate ETOH consumption.

CHAPTER 2

ACUTE ETOH STUDIES

Section 2.1.

INCREASED PLASMA ATRIAL NATRIURETIC PEPTIDE

AFTER ACUTE INJECTION OF ALCOHOL IN RATS

P. GuiUaume, J. Gutkowska and C. Gianoulakis

The Journal of Pharmacology and Experimentd Therapeutics

(J Pharmacol Exp Ther 27 1 : 1656- 1665. 19941

Contribution by CO-authors: Dr. J. Gutkowska and Dr. C . Gianoulakis were my CO-

supenisors.

Acknowledeements: R Claudio was the animal technician responsible for the weCbeing of

the mimals. D. Beaudry performed some of the O-endorph iodinations.

2.1. INCREASED PLASMA ATRIAL NATRIURETIC PEPTIDE

AFTER ACUTE INJECTION OF ALCOHOL IN RATS

2.1.1. ABSTRACT

A number of mechanisms may be involved in the protective effect of low ethanol

(ETOH) consumption on the development of the age-dependent hypertension in both

human and experimental animals. I t was the objective of the present studies to test the

hypothesis that acute administration of low doses of ETOH would increase the plasma

content of Atrial Natriuretic Peptide (ANP). an hormone known to decrease blood

pressure. Plasma ANP levels were signiticantly increased within 15 minutes following the

i .p. injection of 1 or Zg ETOH/ kg B.W.. The increase in plasma ANP was more

pronounced and longer lasting following the i.p. injection of 2 than ig ETOHkg

B. W. .This increase in plasma A N P level was associated with a rapid decrease of atrial

ANP. but not of ventricular A N P which on the contrary was significantly elevated at 120

minutes post-injection. It has been suggested that opioids could play a significant role in

controlling ANP release. In fact. circulating levels of Il-endorphin were also rapidly

increased following the ETOH injection. with a timecourse pattern similar to that of ANP.

Furthermore. a highl y positive correlation was found be tween the ET0 H- induced changes

of plasma ANP and R-endorphin contents. S ignificant increases in plasma corticosterone

and adrenoconicotropic hormone. but not aldosterone contents. were observed follow ing

the i.p. injection of Zg ETOH/kg B.W.. while plasma arginine vasopressin levels were

significantly decreased at 15 but not at 170 minutes post-ethanol. There was no significant

elevation in blood pressure during the 120 minutes experimental period. although a small

tachycardia did develop in the ETOH-treated animals. Thus. acute in v i w administration

of ETOH increased plasma ANP content in a dose-dependent manner and rnay play a role

in the "protective" effect of low ETOH consumption in the development of the age-

dependent hypertension.

2.1.2. INTRODUCTION

A number of epidemiological studies have revealed a positive association between

h igh ethanol ( ETOH) consurnption and hypertension (Gleiberman and Harburg. 1986).

However. the effect on blood pressure of low ETOH consumption is less clear

( MacMahon. 1987). Indeed. several investigations have reported a significant decrease in

blood pressure with low regular consumption of alcoholic beverages ( 1 to 3 drinks per

day). while others reported no significant changes in blood pressure ( MacMahon. 1987).

Moreover. animal s tud ies have demonstrated the prevention or delay in the development

of the age dependent hypertension with chronic low ETOH ingestion (Howe et ai.. 1989).

The mechanisms by which low ETOH consurnption rnay prevent the age dependent

increase in blood pressure are still not clear. One such mechanism may involve the Atrial

Naniuretic Peptide (ANP). a 28 arnino acids peptide synthesized and released mostly from

the hem atria (De Bold eral., 1981. Needleman ef al., 1989. Nakao et al.. 1993). This

hormone has a definite role in body tluid homeostasis (Blaine. 1990). I t s major effect is

to lower b i d pressure through increased diuresis. natriuresis and vascular relaxation. and

a decrease in the secretion of several pressor hormones (Inagami et al.. 1987). Among the

mechanisms modulating the release of ANP are changes in atrial stretch (due to changes

in blood volume). in blood pressure. in osmolarity. or in the concentration of opioid

peptides (Blaine. 1990. Pesonen et al., 1990).

Recently. several studies have been published on the effects of acute ETOH on the

A N P system in man. However. the interactions between the two are still unclear since

either an increase (Colantonio et ai.. 199 1 ). or a decrease (Leppaluoto et al.. 1992) or no

change (Hynynen et al., 1992) in the plasma ANP levels were found following acute

ETOH administration. This variability of the acute effects of ETOH on the A N P system

in human could be attributed to various factors, such as dose of ETOH used, nutritional

status. previous ETOH exposure. genetic factors. etc. (Beilin et Puddey. 1992). Therefore.

it would be interesting to study the effects of ETOH on the ANP systern using an

experimental animal mode1 such as the rat. where al1 the confounding factors (nutritional

status. previous ETOH exposure. genetic factors. etc.) could be thoroughly controlled.

Acute administration of ETOH has been reported to decrease plasma levels of

arginine vasopressin (AVP) (Pohorecky and Brick. 1988). Interestingly. ANP has also

been shown to suppress AVP release from the supra-optic nucleus (SON) (Clark et al..

1991). Similady. acute ETOH is known to increase plasma concentrations of

catecholamine (Zgombick et al.. 1986). corticosterone (Ellis. 1966). and 8-endorphin. a

potent endogenous opioid peptide (Gianoulakis. 1989). 1 ntzrestingl y. it has been reported

that p-opioid receptor antagonists decrease ANP secretion (Gutkowska er al.. l986b.

Widera et al.. 1992). while p-opioid receptor agonists increase ANP secretion (Vollmar

et al.. 1987). One might therefore hypothesize that one of the rnechanisrns by which

ETOH rnay enhance the release of ANP is through irs effects on the release of endogenous

opioid peptides. such as 8-endorphin. which binds with about equal aftïnity to both p and

8 recep tors (Charness. 1989). Additional mechanisms could include changes in plasma

osmolarity and release of other hormones. such as epinephrine. by ETOH intake

( MacMahon. 198% Colantonio et al., 199 1 )

Stress has been reported to increase the release of ANP (Horky et al.. 1985).

Therefore. it is important to distinguish between the stress mediated and ETOH specific

ef'fects on the activity of the ANP system. Since both ETOH and stress have been shown

to increase the release of O-endorphin. adrenocorticotropic hormone (ACTH ) and

conicosterone. changes in the plasma content of these hormones would indicate both the

effectiveness of the ETOH dose administered in the present studies. as well as the presence

or absence of stress due to the necessary handling and manipulations of the animals during

the course of the experiment. Furthermore. estimation of the plasma 8-endorphin levels

(a potent endogenous opioid peptide which could interact with the p-opioid receptors and

increase the release of ANP) would allow us to investigate the possibility that the ETOH-

induced changes in the plasma B-endorphin content may mediate. at least in part. the

effects of ETOH on the release of ANP. by estimating the correlation between the ETOH-

induced changes in plasma A N P and R-endorphin contents.

The aim of the present studies was to test the hypothesis that acute administration of

a low to moderate dose of ETOH in the rat would induce a rapid increase in plasma ANP

levels. A positive correlation between moderate ETOH intake and increased activity of the

A N P system might help to explain. at least in part. the prevention or delay of the age

dependent increase in blood pressure by chronic low ETOH consumption observed both

in human and experimental animals (Howe et al., 1989). The content of ANP in the hean

aria and ventricles was also rneasured and correlated with the changes in the plasma ANP

content. In addition to ANP. the changes in plasma O-endorphin. corticosterone. ACTH.

aldosterone and AVP were rneasured.


2.1 .3.1. -al Treatrnents

Male Long-Evans rats (purchased from Charles River Breeding laboratories. S t-

Constant. Québec. Canada) weighing 100-300 grams were used in this study . A seven days

period was allowed for acclimatization of the animals before the initiation of the

experirnents. Al1 animals were handled twice daily to reduce stress levels during the

experiment.

EXPERlMENT 1 : The objective of the studies in experiment 1 was to perform a time

course study on the effect of ETOH on plasma ANP content. Rats were lightly

anaesthetized with "Metofane" ( Methoxytlurane. inhalation anaesthetic. MTC Canberra

Packard). and a siiastic catheter (0.035 in 1. D.. Dow Corning) was inserted into the letl

jugular vein. The catheter was tunnelled subcutaneously to the back of the neck and the

disral end was plugged by a süiinless steel pin after being tïlled with heparinized saline (70

pllml). The animals were then placed in individual cages and allowed at least a two days

post-surgery rest. On the day of the experiment. at time O. an initial blood sample (0.3 ml)

was withdrawn through the catheter to calculate initial hormone levels. prior to any

treatrnent. Then. the animals were randoml y assig ned to the alcohol treatment groups or

to the saline treatrnent. control group. The animals in the ETOH groups were injected

intraperitoneally (i.p.) with I or Ig ETOH/kg B.W. (40% vlv solution in saline). The

animals in the control group were injected i.p. with the equivalent volume of saline.

Additional blood samples. of 0.4 ml each. were withdrawn through the catheter at 15. 30.

60 and 170 minutes for estimation of the blood alcohol content and levels of ANP. fi-

endorphin and corticosterone. Following each withdrawal of 0.4 ml blood. an equal

volume of 0.9% saline was injected through the catheter to replace the withdrawn blood.

EXPERIMENT 2: The objective of the studies in experiment ? was to investigate the

effects of stress and of the temporary changes in blood volume due to serial withdrawal

of blood samples in experiment 1 (although an equivalent amount of sa1 ine was injected).

Thus. in experiment 2. animals did not have a catheter. but they were sacri ficed at 15 and

120 minutes following the administration of Zg ETOH/kg B.W.. the dose which as

demonstrated in experiment 1 induced a pronounced and long lasting increase of plasma

ANP levels. or the equivalent volume of saline. Thus. 35 male Long-Evans rats were

randomly assigned to 5 treatment groups (5 animals per group). Animals in group 1 and

group 7 were injected i.p. with 2g ETOH/kg B.W. and were sacrificed at 15 and 120

minutes post-ETOH injection respectively . Animals in group 3 and group 4 were injecred

i.p. with the equivalent volume of saline. and were sacrificed at 15 and 110 minutes

respectively. To estimate the hormone levels at tirne O (prior to administration of the

ETOH or saline solution), animals in group 5 received no injection and were sacrificed

within 70 seconds following their removal from the home cage. Immediatel y following

sacrifice, tmnk blood was collected for estimation of the blood alcohol content (BAC) and

the concentration of ANP. fi-endorphin. corticosterone. ACTH. aldosterone and A V P in

the plasma. In addition, the atria and ventricles were dissected to study the effects of

ETOH on the tissue ANP content.

EXPERIMENT 3: The objective of the studies of experiment 3 was to investigate the

e&ct of ETOH on blood pressure (BP) and heart rate (HR) . Thus. rats were placed in a

ratrainer and the BP and HR were recorded regularly on a polygraph (Grass Instruments.

Quincey, MASS) prior to and at specific intervals following the i.p. injection of either Ig

ETOH/kg B. W. or the equivalent volume of saline (for a 2 hours period). using the tail-

cuff method (Pfeffer et al.. 197 1 ). The dose of 2g ETOHIkg B. W. was chosen because.

as demonstrated in experiment 1. it induced a pronounced and long lasting increase in

plasma ANP levels.

2.1.3.23lood Alcohol C o n t a

50 pl of whole blood from each blood sample was deproteinized with 450 pl of ice-

cold trichloroacetic acid (6.15% w/v. Sigma) for estimation of the blooci ETOH levels

using the dehydrogenase enzymatic method (Hawkins et al.. 1966).

2.1.3.3. m u e Extraction

To estimate the content of ANP. ventricles as well as the left and right atria were

carefully removed and placed separately in ice-cold 0.1 N HCl. Tissues were rapidly

boiled for 5 minutes and then cooled on ice. Atria were homogenized with a

microultrasonic cell disrupter (Kondes. Vineland. N. J. ). whereas ventricles were

homogenized using a Polyuon. An aliquot was taken to measure the protein content using

the Bradford's method (Bradford. 1976). and the remaining homogenate was centrifuged

for 15 minutes at 4°C and supernatants were stored frozen at -75°C for estimation of the

ANP content using a specific RIA (Gutkowska et al.. 1984).

2.1.3.4. ~ r m o n for homoml memuremen&

Blood samples were collected in chilled eppendorf tubes (experiment 1 ) or 15 ml

conical centrifuge tubes (experiment 2) and proteases inhibitors were added to a final

concentration of ethylenediamine-retra-acetate (EDTA) ( 1 mglml), phenylmethylsulfonyl

tluoride (PMSF) (40 pl ImMIml) and Pepstatin A (20 pl 0.5 mM1ml). and were

immediately centrifuged at 4°C. The plasma was stored frozen at -75°C for subsequent

RIAS. for estimation of the ANP. AVP.B-endorphin. ACTH. corticosterone and

aldosterone content.

2.1.3.5. Cornparison between direct and e-ed ANP

Due to the scarcity of blood in each time interval of experiment 1. unextracted plasma

was used for estimation of the plasma ANP content (direct ANP RIA). Therefore. in order

to evaluate the sensitivity of the direct ANP assay. we injected i.p. 1 groups of rats (4 in

each group) with either saline or morphine. a known stimulus of ANP release (Gutkowska

et al.. 1986b) and sacrificed the animals 15 minutes following the injection. Trunk blood

was collected. and both direct and extracted ANP RIA were performed. As it is clearly

shown in Figure 1.1.1 .. though the absolute values of plasma ANP content are different.

both methods indicated a similar change in plasma ANP levels following morphine

injection. Thus. the direct assay is suitable to investigate the changes in plasma ANP

content following ETOH administration.

2.1.3.6. Radioimmunoassav (RIA) ~rocedura . . Determination of atrial (final dilution. 1: 15000). ventricular ( 1 : 1000) and plasma (50

or 100 ANP content was done using a direct second-antibody RIA, developed from che

method of Gutkowska et al.. 1984. The antiserum (produced by the immunization

procedure of Gutk~wska et al.. 1984. final dilution 1:30000) showed a good cross-

reactivity with other fragments of the pro-peptide. but less than 5% cross-reactivity with

oxidized ANP. The iodination of the peptide. using lactoperoxydase. was done as

described elsewhere (Gutkowska et al.. 1 %Va). 0.75 pghube was the minimum quantity

of ANP detecrable with this RIA. The intra- and inter-assay coefficients of variation were

3 .2 and 8.5% respectively.

Estimation of plasma content of B-endorphin-like irnmunoreactivity was performed

as described previously (Bourassa et al.. 1978) using an antiserum (final dilution. 1 :3OOOO)

specific to the C-terminal portion of B-endorphin( 1-3 1 ). This antiserum shows a 100 %

cross-reactivity with 8-1 ipotropin and w ith the non-acetylated and a-N-acetyl forms of O-

endorphin(1-3 1). 70% cross-reactivity with Bzndorphin(1-27). but no cross-reactivity with

ACTH. a and fi-melanotropin and a and gamma-endorphin (Gianoulakis. 1987).

Iodination of standard B-endorphin(1-31) to be used as tracer was donc using the

1 Saline Morphine

NON-EXTRACTE D

Figure 2.1.1. Effectofthei.p.injectionof morphineonthecirculatingANP levels using either extracted (A) or unextracted (B) plasma for the RIA. Values are expressed as Means *SEM (n=4). Levels of signlicance refer to the drfference between the treatments; ""P<O.OOI.

chloramine T method (Bourassa et al.. 1978). 10 pg/tube was the smallest quantity of R-

endorphin detectable with this RIA. Intra- and inter-assay coefficients of variation were

2.8 and 8.3 % respectively.

Plasma conicosterone levels were measured in ETOH extracted plasma samples. as

previousl y described (Krey et al.. 1975). using a specific antiserum (83- 163. Endocrine

Science. Tarzana. CA) which showed a small cross-reactivity with desoxycorticosterone

( ~ 4 % ) but not with cortisol. ['H 1-corticosterone was purchased from New Eng land Nuclear

(101 Cihnmol). The minimum level of detection with this assay was 10 pg/tube. The intra-

and inter-assay coefficients of variation were 10.4 and 14.4 % respectively .

Plasma ACTH was rneasured with a sensitive RIA (Orth, 1979). The antiserum

(Peninsula laboratories. final dilution. 1 :3OOûO) cross-reacted 1 0 % with ACTH( 1-39) and

ACTH(1-24) but less than 1 % with O-endorphin, a and 8-melanotropin. and a and B-

lipotropin. Iodinated ACTH( 1-34) was purchased from Incstar (Steel Water. MIN). The

minimal detectable level of plasma ACTH was about 0.3 pg/tube. Intra- and inter-assay

coefficients of variation were 5.4 and 10.7 % respectively.

Plasma aldosterone was determine in ETOH extracted samples using a sensitive RIA

previously described (Mayes er al., l97O). This antiserum (A3-375. Endocrine Science.

Tarzana. CA) shows a 0.04% cross-reactivity with adrenosterone and a 0.01% cross-

reactivity with corticosterone. ['H I-aldosterone was purchased from New England Nuclear

(100 Cilmmol). 10 pg/tube was the lowest amount of the steroid detectable using this

assay. The intra- and inter-assay coefficients of variation were 11.2 and 15.1 %

respect ive1 y.

Estimation of plasma AVP levels was performed in I ml of acetone extracted plasma

using a second-antibody RIA (Stowsky et al.. 1974). Iodination of the peptide was done

by the chloramine T method. The antiserum (Peninsula Laboratories. final dilution

1 :500ûû) showed a 3 % cross-reactivity with Lysn-vasopressin but no cross-reactivity w ith

ANP. 0.2 pglml was the smallest quantity of AVP detectable with this RIA. Intra- and

inter-assay coefficients of variation were 4 and 1 1 % respectively.

. . 2.1.3.7. Statacal

The data are presented as the mean S . E. M. The significance of difference among

the various groups was evaluated by a two-way analysis of variance (ANOVA). with time

as the first independent variable and trestment as the second independent variable.

followed by the Neuman-Keuls multiple cornparison test. Correlat ions were calculated

through the Pearson's r test. A P value of 50.05 was considered significant.

2.1.4. RESULTS

2.1.4.1. Ex~eriment 1

Injection of ETOH as a 40 % vlv solution resulted in a rapid increase in the blood

alcohol content (BAC). reaching maximum levels within l5 minutes following the i.p.

injection of Ig ETOH/kg B. W. (95.7 + 2.6 mgldl) and within 30 minutes following the

i.p. injection of 3g ETOHkg B. W. (705 + 1 1 mgldl) (Figure 2.1.2. ). The i.p. injection

of ETOH. but not of saline. induced a fast increase in the plasma ANP content. reaching

maximum concentrations within 15 minutes post-injection for both lg ETOHIkg B. W.

(43.4 + 3.6 pg1100 pl) and 2g ETOHIkg B.W. (63.0 f 3.7 pgllûû p l ) (Figure 2.1.3A.).

Following this initial increase. the plasma ANP content started to decl ine slowly toward

the basal levels. At 120 minutes post-injection. the ANP concentration in the plasma of the

rats injected with the Ig ETOH dose was still significantly higher than the plasma ANP

content prior to the ETOH injection. as well as the ANP content in the plasma of the

saline-injected animals (Figure 1.1.3A.). However. the ANP concentration in the plasma

of the rats injected with the lg ETOH dose was not significantly different frorn tliat in the

saline-injected animals at 30. 60 and 120 minutes post-injection. A two-way ANOVA with

time as the first independent variable and treatment as the second independent variable

indicated a significant effect of time (F,.,, =7 1-09; PrO.O 1) and treatment (F1.,, =MAO:

P~0 .0 1). and a significant interaction of time and treatment (F,,,=9.47: PcO.0 1). The i.p.

injection of both 1 and Zg ETOHIkg B.W.. but not of saline. induced an increase in the

plasma content of Bendophin (Figure 2.1.3 B. ) and corticosterone (Figure 1.1.3C. ). For

B-endorphin. significant differences due to time (F,.,, = 2 2 2 1 : P5O.O 1) . treatment

- -- - 1 1gofETOHlkgB.W.

++ 2 g of ETOHlkg B.W.

O 30 60 90 120 Time (min)

Figure 2.1.2. Changes in blood alcohol content (BAC) with time after the i.p. injection of 1 or 2 g ETOHkg B.W. (40% vfv solut ion). Values shown are Means I SEM (n=5).

+ Saline

- 1 1gofETOHlkgB.W.

+ 2 g of ETOHlkg B.W.

o 1 I 1 I i 1 1 I

1

O 30 60 90 120 Time (min)

Figure 2.1 3. Changes in plasma ANP (A), Rendorphin (B) and corticosterone (C) content with time after the i.p. injection of 1 or 2 g ETOHlkg B.W. (40% vhr solution) or the equivalent amount of saline. Values shown are Means i SEM (n=5). Significantly drfferent from the corresponding value of the saline- treated animals; "P~0.00I1 "P<0.01, 'Pc0.05. Significantiydifferent from the corresponding value of the 1 g ETOHkg B.W.-treated animals; ~ ~ 0 . 0 0 1 , tp<O.OI, p<0.05.

(F,., =48.76: Pc0.01) and time by treatment interaction (F,.,=5.03: Pdl.01) were

observed. Similarly. for corticosterone. significant effects of time (F,.,, = 19.04: P4.0 1).

ueatrnent (F:.,, =76.O 1 : Pg0.0 1 ) and time by treatment interaction (F,., =6.56: PcO.0 1 )

were present.

2.1.4.2. -riment 2

Figure 4 presents the % change from basal levels (time O) in the concentration of

ANP in the plasma (Figure 7.1.4A.). the right (Figure 2-1-48.) and left (Figure 1 . 1 AC. )

atria and in the ventricles (Figure 2.1.4D.) of animals sacrificed at 15 and 120 minutes

following the i.p. injection of Zg ETOHIkg B.W. or saline. In agreement with the results

of experiment 1. the i.p. injection of 2g ETOHIkg B. W. induced an increase in the plasma

ANP content at 15 and 120 minutes post-injection. while i.p. injection of saline did not

induce statistically signiiïcant changes in the plasma ANP content either at 15 or at 120

minutes post-injection. The increase in the plasma ANP content was associated with a

decrease in the ANP content in the right and the lefi atria at both 15 and 120 minutes post-

ETOH injection. In the right atria. the ANP content changed from 6.90 + 0.45 pglrng

protein prior to ETOH to 4.41 + 0.33 and 5.61 f 0.5 1 pglmg protein at 15 and 120

minutes post-ETOH respective1 y. In the left atria. the i.p. ETOH injection decreased the

ANP content tiom 4.92 f 0.2 1 pglmg protein prior to the ETOH injection to 3.00 f 0.2 1

pglmg protein at 15 minutes and 3.27 f 0.15 pglmg protein at 120 minutes post-ETOH.

The i.p. injection of the equivalent volume of saline did not alter significantly the content

of A N P either in the right atria (6.90 + 0.45 pglmg protein at O minutes. 6.17 f 0.15

pglmg protein at 15 minutes and 6.42 + 0.24 pglmg protein at 120 minutes) or the left

atria (4.92 f 0.2 1 pglmg protein at O minutes. 4.74 0.30 pglmg protein at 15 minutes

and 4.1 1 + 0.42 pglmg protein at 120 minutes). Contrary to the atria. i.p. injection of

ETOH induced a slow increase in the tissue ANP content in the ventricles. from 306.8 f

15.5 ng/mg protein at time O (prior to the ETOH injection) to 355.6 + 3 1 .O nglmg protein

at 15 minutes and 651.4 & 19.9 nglmg protein at 120 minutes post-injection. The i.p.

injection of saline did not induce a significant change in the ventricular ANP content

Ventricles

VARIATIONS (% DIFF.) FROM BASAL VALUES OF THE ANP CONTENTS IN

Left atrium Rig ht atrium Plasma

(370.6 + 14.2 ng/mg protein at 15 minutes and 184.6 + 41.1 nglmg protein at 120

minutes post-saline injection).

Table 2.1.1. shows the concentration of B-endorphin. corticosterone. ACTH.

aldosterone and AVP in the plasma of animals sacrificed without any injection (tirne 0) and

of animals sacrificed at 15 and 120 minutes following the i.p. injection of Ig ETOHikg

B.W.. or the equivalent volume of saline. ETOH induced an initial 3 fold increase in

plasma B-endorphin content at 15 minutes post-injection. At 2 hours post-ETOH. the

plasma O-endorphin levels were still 2 times higher than the basal levels. No significant

change in plasma B-endorphin content was observed following the i.p. injection of saline.

Although plasma ACTH levels presented a 6 fold increase over basal at 15 minutes and

only a 3 fold increase at 120 minutes post-ETOH injection. plasma corticosterone

presented a significant increase at 15 minutes (4 times higher than basal) and at 120

minutes post-injection (7 times higher than basal). As shown for R-endorphin. sa1 ine

treatment did not significantl y al ter the plasma ACTH and corricosterone content. In

contrat to ANP. fi-endorphin. ACTH and corticosterone. the plasma aldosterone levels

remained unaffected by either ETOH or saline treatments. at both 15 and 120 minutes

post-injection. Finally. plasma AVP levels were significantly decreased in the ETOH-

treated rats at 15 minutes cornpareci to both conuol and saline-treated animals. while at 170

minutes post-injection the plasma AVP levels were back to control values.

2.1.4.3.

The effects of the i.p. injection of Zg ETOHlkg B.W. or saline on the blood pressure

and hem rate are shown on Table 2.1 2. No significant variation in the blood pressure was

observed. However. a significant difference did developed in the heart rate between the

2 treatrnent groups. Within 15 minutes. heart rate values were significantly higher in the

ETOH-treated animals. and remained elevated for the remainder of the experiment .

2.1.4.4.

Since similar patterns of changes in the plasma R-endorphin and ANP levels were

Table 2.1.1. Effects of ETOH [2g/kg B. W. (40% v/v solutzoti)] ori p l m a Ievels 00-eruiorphirz, cortzcmterone. A CTH. ido os ter or te ami A C.'P II Hormone

-

Treatment Time post-injection

Il-endorp hin (pg/ 1 O0 p 1) Saline

ETOH

Corticosterone (ng/ 1 O0 pl) Saline

ETOH

ACTH (pg/mU Saline

ETOH

Aldosterone (pg1100 pl) Saline

ETOH

AVP (pg/ml) Saline

ETOH 0.372 0.035*** 0.63 1 = 0.039 Values s h o w are Means * S.E.M. Levels of sigdicance refer to the ciifference between treatrnents: ***PsO.OO 1 : **Ps0.0 1 ; *Ps0.05. n = number of animals in each group.

Table 2.1.2. Efiects ofETOH [&/kg B. W. (40% v/v sol~~tiotJJ ot, bloodpessrrtv otid hemt torite

Treatriieiit Tiiiie post-iiijectioii

ll~lood pressure (iniii Hg) Saline 106.7 * 4.0 I l 1.3 * 1.4 110.0 4.3 102.0 * 2.7 102.5 * 3.4

ll~eart rate (beatslniiii) Saline 338 k 6 3 5 5 * 7 348 * 5 354 11: 7 351 k 5

ETOH 349 i 5 402 k 14** 398 k 1 l * 388 k IO* 390k 13* Values show are Meaiis -t S.E. M. Levels of sigiiificaiice refer to the differeiice betweeii treatineiits: **P 4.0 1 ; *P d.05. ti = riumber of atiinials iii eacli group.

observed following the i.p. injection of ETOH. the presence of a positive correlation

between the ETOH-induced changes in the plasma 0-endorphin and ANP contents was

investigated. A positive and highly significant correlation (P<0.001) was found between

the 3 hormones (Figure 2.1.5.). Sirnilarly. positive correlations were also found between

ANP and BAC (Pc0.001) and between &-endorphin and BAC (P~0.01) respectively

(Figure 2.1.6A. and 2.1.68.). Moreover. a signitïcant inverse correlation (Figure 2.1.7. )

was observed between the decrease in atrial ANP content and the increase in plasma ANP

levels ( P ~ 0 . 0 1 ) at 15 minutes post-ETOH administration. Table 2.1.3. presents the

possible correlations between the parameters measured in the present studies. Interest ing l y.

an inverse correlation was also found between plasma ANP and plasma AVP levels. as

well as between BAC and plasma AVP.

2.1.5. DISCUSSION

The present investigations examined the acute in vivo e k t of the i.p. injection of

a moderate dose of ETOH on the circulating ANP levels as well as on the atrial and

ventricular ANP content. I t is important to note that due to the small arnount of blood

removed in the tirne-course study. a direct RIA for ANP was performed. using unextracted

plasma. This explains the somewhat higher plasma levels of ANP in the present study

compared to other publications. However. the major objective of this investigation was to

study the relative differences in the plasma ANP levels between the ETOH and saline

treated groups of animals. The direct plasma assay has been used previously in several

studies with reliable resulü. showing good correlations between the direct and the

extracted plasma assays (Steinhelper et al.. 1 990. B id mon et al., 199 1 ). Moreover . in the

present studies. the suitability of the direct assay was evaluated by estimating the plasma

A N P levels following the i.p. injection of morphine. which is known to increase ANP

release (Gutkowska et al., l986b). us ing unextracted (direct assay) and ex tracted plasma.

Indeed. the percent increase of plasma A N P levels using the direct assay was similar to

that obtained using a plasma extraction step prior to the RIA (Figure 2.1. L . ) . supporting

O 20 40 60 80 Plasma ANP (pg1100 pl)

Figure 2.1.5. Correlation between the ethanokinduced changes in the plasma ANP and Bendorphin (R-EP) contents. Values correspond to individual animais (n=75).

O 50 100 150 200 BAC (mgldl)

O 50 1 O0 150 200 BAC (mgldl)

Figure 2.1.6. Correlations between the blood alcohol content (BAC) and the plasma ANP (A) and Rendorphin (B) contents. Values are expressed as Means I SEM (n=9).

Plasma (pg1100 pl)

Plasma (pg1100 pl)

Figure 2.1.7. Correlations between the increase in plasma ANP levels and the corresponding decrease in right atrial (A) and left atrial (B) ANP content at 15 minutes post-ETOH. Values correspond to individual animais (n=20).

Table 2.1.3. Correlations betweerr the variais riarameters

Coefficient of Level o f sigdïcance

Plasma ANP vs. right atnal ANP ( ~ 2 0 )

Plasma ANP vs. left atnal ANP (n=20)

Plasma ANP vs. ventricular ANP (n=20)

Plasma ANP vs. plasma B-endorphin (n=75)

Plasma ANP vs. plasma corticosterone (n=75)

Plasma ANP vs. plasma AVP (n= 1 1 )

BAC vs. plasma ANP (n=9)

BAC vs. pIasma B-endorphin (n=9)

BAC vs. plasma ACTH (n=5)

BAC vs. corticosterone (n=9)

BAC vs. plasma AVP (n= 1 1 )

BAC vs. nght atnal ANP (n=5)

BAC vs. lefi atnal ANP (n=5)

BAC vs. ventncular ANP (n=5) 0.507 n. S.

n = number of points: BAC = blood alcohol content: n.s. = not significant.

the suitability of the direct plasma RIA for comparing changes in plasma ANP

concentrations following drug treatmenü or physiological conditions.

Results indicated that i.p. injection of 1 or 2g ETOHlkg B. W. induced an increase

in the circulating ANP levels in a dose-dependent manner. Funhermore. the ETOH-

mediated increase in plasma ANP levels was longer lasting following the 2 than the Ig

ETOH/kg B. W. dose. Since rats are rnetabolizing alcohol more quickly than humans

(Roine et al.. 1991). the high BAC obtained i n the present studies does not have the same

et'fect at the level of consciousness in the rat as it would have in humans. hdeed. BACs

that may produce coma or stupor in most humans subjects. in the rats produce some motor

incoordination but the animals remain fully conscious. as was observed in the present

studies and was reported by others investigators (Sparrow et al.. 1987). Therefore. in

many studies using experimental anirnals. doses up to 2g ETOHlkg B.W. (and BACs up

to 200 mg/dl) are considered moderate with respect to the effects of ETOH on

coordination and other behavioral responses (Subramanian et al.. 1990. Kettunen et al..

1993).

Acute ETOH has also been shown to increase plasma corticosterone (Ellis. 1966) and

ACTH (Rivier et al.. 1984) content. although this effect seems to depend on the ETOH

dose used and the time post-injection. Stress due to manipulation of the anirnals did not

seem to account for the observed increase in the plasma ANP levels. Indeed. the stable

baseline in plasma ACTH and corticosterone levels of the saline-treated anirnals was a

good indication that there was no activation of the hypothalam ic-h ypop hyseal-adrenal axis.

due to stress induced by the handling of the experirnental anirnals.

The increare in plasma ANP levels at both 15 and 120 minutes following the 2g ETOH/kg

B.W. dose was associated with a decrease in the atrial ANP content. in the atria. ANP is

known to be stored in its higher molecular weight form in the ce11 granules (Lang er al..

1992). The decreased atrial ANP content is therefore consistent with an increased

secretion, at a tirne when the increase in the rate of ANP biosynthesis is not yet

contributing to the tissue ANP content. Moreover. a significant inverse correlation was

observed between the changes in atrial ANP content and the changes in the circulating

ANP levels. In the ventricles. opposite to the atria. ANP is released in a constitutive

rnanner (Lang et al.. 1992) and there is no. or very little storage of ANP. Thus. an

increase in the rate of ANP biosynthesis would be associated with increased tissue content.

as was observed at 2 hours post-ETOH administration in the present studies and could

indicate an increased contribution of ventricular ANP in the plasma. Alternatively. the

increased ANP content in the ventricles could be due to an inhibitory effect of ETOH on

the ANP release by the ventricles. Thus. further studies estimating changes in the ANP

mRNA levels in this tissue at various intervals post-ETOH administration are needed to

understand better the effects of ETOH on the ventricular ANP system.

The ETOH-induced increase in plasma ANP levels is consistent with the tïndings of

Colantonio et al. (1991) in humans. where a drink of 1 g ETOHIkg B.W. induced a

significant increase in the plasma ANP content for the 2 hours of the study. In contrasr.

Leppaluoto et al. ( 1992) administering 1.5 g ETOHIkg B. W. and Hynynen et al. ( 1992)

administering 1 g ETOHIkg B.W. reported either a decrease. or no effect of ETOH. on

the plasma A N P content. The hypothesis put forward to explain the ETOH-ANP

interaction States that alcohol rapidly increases plasma osmolarity . which in turn increases

the release of ANP from the hem atria (Kurnik et al., 1991). Subsequently. the slower

ETOH-mediated dehydration and diuresis. induced at least in part by the ETOH-mediated

inhibition of AVP release (Pohorecky and Brick. 1988 and this report) decreases plasma

volume and atrial stretch. thus lowering A N P release. Any effect preventing the

hyperosmolarity observed following ETOH administration m ight prevent the increase in

ANP release. It is possible then that the initial volume-loading (prior to ETOH

administration) of the patients in the experiment of H ynynen et al. ( 1992) elevared plasma

ANP levels and masked any effect of the subsequent ETOH administration on further

release of the natriuretic peptide. Similarly. higher doses of ETOH. like those used in the

studies of Leppaluoto et al. (1992). might lead to a more rapid and more consistent

diuresis. an effect which will rapidly override the initial ETOH-induced hyperosmolarity.

Thus. lower doses of ETOH and initial isovolumetric conditions may be necessary to

demonstrate the ETOH-induced increase in plasma ANP levels.

Recently. Wigle et al. ( l993a). in the only reponed study on the effects of acute

ETOH administration on ANP using an animal model (Sprague-Dawley rats). reported a

lack of effect of ETOH on plasma ANP levels for 2 hours following the administration of

a high ETOH dose (5g/kg B. W.) through gastric gavage. while animals receiving an equal

volume of water presented a signifiant increase in plasma ANP levels. Interestingly. both

ETOH and water treated animals presented a decrease in the ventricular ANP content and

an increase in the atriai ANP content. A possible explanation for these changes could be

that the high amount of liquid administered in this particular study resulted in a water-

loading effecr. with a rapid decrease in the ventricular ANP content and a slower increase

in the atrial ANP content of both the ETOH and water treated animals. indicating

increased ANP release and availability to cope with this water-loading situation (Wigle et

al.. l993a). Knowing that circulating ANP is mostly derived from the h e m (Nakao et al..

1993). it is surprising that despite the similar changes in heart ANP contents. the ETOH-

given rats exhibited lower plasma ANP levels than their controls. However. it is possible

that the tremendous diuresis usually associated witli a very high dose of ETOH (and

therefore decreased plasma volume) may explain the decrease or rather. the absence of

increase. in plasma ANP levels. Moreover. such high doses of ETOH and the route of

administration may cause gastric irritation and increase sympathetic activi ty. therefore

interfering with the interactions of the drug with various physiological systems.

Furthermore. some investigators (Colantonio et al.. 199 1 ) have proposed an indirect

effect of ETOH on the activity of the ANP system. mediated by its effects on various

hormonal and neurotransmitter systems. among which could be the endogenous opioid

peptide systems. Experimental evidence has indicated that opioids may play a significant

role in controlling ANP release (Gutkowska et al.. 1986b. Widera et al.. 1997). Indeed.

p-opioid receptor agonists. like fentanyl. have been shown to increase ANP release

(Vollrnar et al.. 1987. Pesonen et al.. 1990). while naloxone (an opioid receptor

antagonist) has been reported to significantiy suppress the increase in plasma ANP levels

follow ing water-immersion in human (W idera n al., 1992). Moreover. a'-adrenerg ic

agonists. such as clonidine. also stimulate ANP, via activation of central opioid receptors

(Pan and Gutkowska. 1988). Interestingly. naloxone also suppressed the water immersion-

induced decrease in blood pressure. suggesting a definite role of the endogenous opioid

system in the conuol of ANP and its subsequent effect on blood pressure. Since ETOH has

long been shown to induce a rapid increase in plasma O-endorphin in both humans and

experimental animals (Gianoulakis. 1989). it was interesting to determine whether a

correlation between the ETOH-induced changes of plasma ANP and fi-endorphin could be

detected in the present study. Indeed. a strong. positive association (r=0.707. Pc0.001)

was found between the effects of ETOH on plasma ANP and O-endorphin levels.

Therefore. the increased plasma levels of A N P following injection of a low doses of

ETOH may represent the additive effect of the ETOH-induced increase in both osmolarity

and plasma levels of endogenous opioid peptides. such as O-endorphin. The presence of

an indirect effect of ETOH on the heart A N P system is further supported by the

observation that exposure of cultured myocytes to ETOH did not alter the rate of ANP

release (Wigle et al.. 1993a).

The effect of acute ETOH on the blood pressure is still not fully understood. Several

studies (Eisenhofer et ai.. 1984. Howes et al.. 1985. Eisenhofer et al.. 1987. Sparrow et

ai.. i987. Maiinowska et al.. 1989. Adesso et al.. 1990. Kawano et al., 1992) have

reported a decrease. wh ile others (Ireland et al.. 1984. Howes et ai.. 1986. Potter et al..

1986. Maheswaran et ai.. 1991) have shown an increase or no change (Stott et al.. 1987)

in blood pressure following ETOH ingestion. The renin-ang iotensin-aldosterone system

is not believed to play an important role in the acute effects of ETOH on blood pressure.

and any changes observed in the activity of the renin-ang iotensin-aldosterone sys tem may

be secondary to the variations in sympathetic activity and blood pressure (Stott et al..

1987). The absence of ETOH-induced changes in the plasma levels of aldosterone. which

was also seen in the present studies. provide further support to this hypothesis. The

elevated h a r t rate. the only consistent observation in the various investigations (also seen

in the present studies). may be due to : (a) a reflex mechanism to maintain blood pressure

(Kawano et al.. 1992): (b) a decrease in blood volume (Stott et al.. 1987): ( c ) ETOH

metabolites. such as acetaldehyde and their mediated secondary increase in catecholarn ine

(Potter et al., 1986); and ( d ) CNS-mediated effects (Malinowska et al,, 1989).

Several hypothesis have been put forward to explain the effects of ETOH on the

blood pressure. Various confounding factors. like the dose of ETOH. its duration of

exposure. genetic predispositions. nutritional status. timing of measurement fol low ing the

ETOH administration. or the route of ETOH administration rnay play an important role

and must be taken into account when considering the general effect of ETOH on various

physiological systems (Beilin et Puddey. 1992). At the vascular level. ETOH could cause

vasoconstriction by decreasing the clearance of Norepinephrine (Howes et al.. 1986). by

a translocation of Ca" from plasma to smooth muscle cells (Potter et al.. 1986) or by

decreasing the vasorelaxation induced by Acetylcholine and thus affecting the release of

Endothelium-Derived Relaxing Factor (EDRF) from the endothelial cells (Hatake et al..

1993. Criscione el al.. 1989). On the other hand. ETOH may also cause vasodilation by

a direct effect on arterioles. capillaries and venules (Altura and Altura. 1981) or by an

inhibitory eftèct on the a-adrenoceptors induced vasoconstriction ( Eisenhofer et al. 1984).

I t has also been proposed that the rapid increase in blood pressure. seen in sorne studies

following acute ETOH exposure. could be simply due to the caloric load of ETOH. similar

to chat observed after a glucose load (Stott et al.. 1987). More recently. Kawano et al.

(1992) suggested a biphasic effect of ETOH in man. with a transient increase in blood

pressure observed within the first hour. followed by a sustained hypotension due CO

peripheral vasod i lation (associated w ith increased Cardiac Output and decreased Total

Peripheral Raistance) for up to 8 hours. In this model. sympathetic activity is initiated by

the vasodilatory effects of ETOH. and prevents either directly. or through an action on

renin, or by increasing the heart rate. the development of a more severe hypotension.

From our study. it seems that ANP could be added to chis growing number of hormonal

factors implicated in the complex action of ETOH on the cardiovascular system. Consistent

with the mode1 of Kawano et al.. (1992). an acute elevation of circulating ANP could help

modulate the effects of ETOH on the vasculature and the kidneys. and could prevent the

initial activation of counterbalancing systems by iü inhibition of sympathetic activity and

its suppression of renin and aldosterone release. The body could therefore achieve a

s igni ficant decrease in blood pressure. before the stimulus for compensatory actions is

strong enough to override the initial hypotensive effect of ANP. Again. the elevated heart

rate, observed in the present studies. is seen as the result of those compensatory

rnechanisms . In addition. extra airial ANP systems may also play an important role in the ETOH

induced diuresis and blood pressure changes. For example. ANP is known to suppress the

release of AVP from the supra-optic nucleus (SON) (Clark et al.. 1991) and an increase

in central ANP levels could be responsible. at least in part. for the ETOH mediated

decrease in AVP release reported previousl y (Gutkowska and Nemer. 1989) and observed

in the present studies. Whether the decrease in circulating AVP levels following the ETOH

administration is the result of a direct effect of ETOH or is mediated by the increase in

central and peripheral ANP activity rernains to be evaluated.

H igh ETOH drinking is associated with an increased prevalence of hypertension

(MacMahon. 1987). On the other hand. low doses of ETOH are associated with

signi tkant lower blood pressures as indicated by a number of epidemiological studies

demonstrating a non4 inear association between increased ETOH consump t ion and blood

pressure (the J or U shaped curves) (MacMahon. 1987). Interestingly. a similar non-linear

association is now well documented between alcohol consumption and cardiovascular

diseases (Marmot and Brunner. 199 1 ). Variations in Lipoproteins (HDL and LDL)

(Criqui. 1986. Hojnacki et al. 1988) or an attenuation of the vasoconstrictor responses to

Norepinephrine (Criscione et al., 1989) with low doses of ETOH. are a few proposed

rnechanisms for this protective effect of low ETOH consumption on hypertension and

cardiovascular diseases. The present investigations suggest that ANP could be an additional

rnechanism involved in the protective effects of chronic low ETOH consumption on the

blood pressure. Chronic studies are presently under way to study this hypothesis.

In conclusion. acute ETOH has been shown to significantly and rapidly increase

plasma ANP levels, associated with a decrease of the atrial A N P content. This increase in

circulating ANP levels could be mediated in part by the ETOH-induced increase in the

plasma content of endogenous opioid peptides, such as fi-endorphin. S tudies of chronic

ethanol treatment as well as evaluation of the effects of ETOH on the extraatrial natriuretic

peptide system. are necessary before any conclusions could be drawn on the possible

importance of ANP in the prevention of the age-dependent hypertension observed with

regular low alcohol consumption.


The authors wish to ihank Mr Ricardo Claudio and Mme Diane Beaudry for technical

assistance. as well as Dr Dominique Walker for the generous donation of the ACTH

tracer. This work was supported by a grant of MRC of Canada (MT-10337). by the

Canadian Heart and Stroke Foundation (J.G.) and by the Alcohol Research Program ar

Douglas Hospital ( C G . ) . P.G. is a recipient of a scholarship from the "Fonds pour la

formation de Chercheurs et ['Aide à la Recherche" (FCAR).

Section 2.2.

INCREASED PLASMA ATRIAL NATRIURETIC PEPTIDE

AFTER INGESTION OF LOW DOSES OF ETHANOL IN HUMANS

C. Gianodakis, P. Guillaume, J. Thavundayil and J. Gutkowska

Alcoholism: Clinicd and Experimental Rerearch

Wcohol Clin Grp Res, in press, 1997)

Contribution by CO-authors: 1 was in charge of the AVP radioimmunoassay, the urine volume

and urine electrolytes measurements. I took an active part in the writing and editmg of the

manuscript.

2.2. INCREASED PLASMA ATRIAL NATRIURETIC PEPTIDE

AFIXR INGESTION OF LOW DOSES OF ETHANOL IN

HUMANS

2.2.1. ABSTRACT

Previous studies have demonstrated that in human, acute consurnption of high doses

of ethanol (ETOH). administered in a large quantity of fluid. with or without volume-

loading. induced either a decrease or an increase in the plasma content of atrial natriuretic

peptide (ANP). a substance which has a hypotensive effect. The objective of the present

investigations was <O examine the effect of low doses of ETOH (0: 0.75: and 0.50 g

ETOH per kg. B. W.) administered to 6 normotensive individuals. in a small volume of

fluid without prior volume-loading. Prior to and at various intervals following

administration of the placebo or ETOH drinks the heart rate and blood pressure (BP) were

measured and blood samples were taken for estimation of the plasma ANP. arginine

vasopressin (AVP) and cortisol contents. Results indicated small changes in BP and heart

rate following ingestion of either the placebo or ETOH drinks. On the other hand a

signifiant increase in the plasma ANP content was observed at 15 min following ingestion

of both the 0.15 and 0.50 g ETOH per kg B.W. doses. but not following the placebo

drink. The plasma ANP levels were still elevated at 45 min post ETOH intake. bur had

returned to basal levels at 120 min after the ETOH drink. Interestingly. it was noticed that

the higher dose of ETOH (0.50 g) did not induce a higher plasma ANP concentration than

the Iower dose (0.25 g) of ETOH. however the plasma ANP content remained elevated for

a longer period. Furthermore. the increase in plasma ANP content was not due to ETOH

or stress induced increases in the plasma AVP and cortisol contents. since the plasma

concentration of these hormones remained either at basal or below basal levels for the

duration of the experiment. In conclusion. ingestion of low amounts of ETOH equivalent

to 1 or 2 standard drinks induced an increase in plasma A N P content.

2.2.2. INTRODUCTION

A number of epidemiological studies have indicated that though high (more than 1

dr i n ks per day ) ethanol ( ETOH) consump t ion induces hypertension. low to moderate

ETOH consumption ( 1-3 drinks per day) not only does not cause hypertension. but it

seems to prevent or delay the development of the age dependent increase in blood pressure

(BP) (Klatsky et al.. 1977: Gleiberman and Harburg. 1986: MacMahon. 1987). Thus. it

was observed that the BP of non-drinkers was higher than the BP of low ETOH drinkers

matched forage. sex. obesity etc (MacMahon. 1987). Since ETOH does not have specitïc

receptors to interact with. the mechanism by which it induces its antihypertensive effect

is not known. In general. it is accepted that ETOH alters the activity of a number of

neurotransmitter and hormonal systems which in turn mediate a number of its effects. I t

is reasonable then to suggest that low ETOH intake inhibits the activity of substances

having a pressor effect and/or stimulates the activity of substances having antihypertensive

effect. The mechanisms or physiolog ical systems mediating this antihypertensive effect of

ETOH are not known. One such mechanism may be the inhibition of the vasoconstrictor

effect of norepinephrine by small but not by high concentrations of ETOH (Criscione et

al.. 1989). Another suggested mechanism may be associated with the effect of ETOH on

the concentration of plasma Very Low Density Lipoproteins (VLDLs) and High Density

Lipoproteins (HDLs) (Criqui. 1986). A trial Natriuretic Peptide ( ANP) is a 28-am ino acid

peptide which has been shown to have a hypotensive effect (Cantin and Genest. 1987).

Though ANP was originaily isolated from the heart atria (De Bold et al.. 1981). its

presence has also been demonstrated in the heart ventricles. and in a nurnber of neuronal

and endocrine tissues such as the brain. pituitary gland. ganglia of the autonornic nervous

system and the lungs (Samson. 1985: Cantin and Genest. 1987: Gurkowska et al.. 1982:

Gutkowska and Nemer. 1989). ANP has been shown to decrease the BP by inducing

vasorelaxation, by increasing diuresis and natriuresis and decreasing the activity of a

number of pressor hormones and /or neurotransmitters such as vasopressin. aldosterone.

angiotensin I I . renin and norepinephrine (Cantin and Genest. 1987: Gutkowska and

Nemer. 1989). Thus, it has been hypothesized that part of the antihypertensive effect of

low ETOH consumption may be mediated by the activation of the ANP system (Colantonio

et al., 1991). It is possible that following low ETOH consumption ANP is released into

the circulation inducing a transient hypotensive effect. In contrat. when the ETOH

consumption is high. in addition to ANP various pressor systems are activated. such as

catecholarnines. which evennially may lead to the development of hypertension in chronic

alcoholics. However, the published reports on the effect of acute ETOH ingestion on the

ANP system in human have indicated controversial results. since an increase (Colantonio

et al.. 199 1 ). a decrease (Leppaluoto et al.. 1992) or no change (Hynynen ef al.. 1991)

in plasma ANP levels have been reported following ETOH intake. These different effects

of ETOH on plasma ANP content rnay be due to differences in the experirnental conditions

between the various studies. such as differences in the dose of ETOH administered. the

volume in which die ETOH was administered. previous loading of the subjects. time when

the study was performed and the nutritional state of the subjects.

Interestingly. al1 the studies investigating the effect of ETOH on plasma ANP content

have used doses of ETOH. ranging from 0.5 to 1.5 g ETOH / kg B.W. . adrninistered in

large volumes of fluid (from 500 to 750 ml). wirh (Hynynen et al.. 1992) or without

(Colantonio et al.. 1991 : Leppaluoto et al.. 1992: Ekman et al.. 1994) previous volume-

loading of the subjects. Volume loading could itself trigger ANP release (Lang et al..

1985). In fact presently. there are no published studies investigating the effect of low doses

of ETOH administered in smdl volumes of tluid. Thus. it was the objective of the present

investigations to examine the effect of low doses of ETOH (0. 0.15. and 0.50 g ETOH per

kg B.W.) administered in a mal1 volume of tluid. on the circulating ANP levels at various

intervals post-drink. in 6 normotensive individuals. These doses of ETOH were chosen

because when taken by a 60-70 kg B.W. individual. they correspond to one (0.15 g

ETOH) and two (0.50 g ETOH) standard drinks. which the epidemiological studies have

shown to have a protective effect on the age dependent development of hypertension. A

standard drink is equivalent to approximately 14 g of ETOH. In addition to plasma ANP.

the plasma content of vasopressin (AVP) and cortisol were estimated. Furthermore. the

BP. heart rate and urine excretion were recorded during the 3 hrs of the experimental

per iod .


2.2.3.1.

Six (4 males and 2 fernales) Caucasian normotensive individuals. 15-30 years old

with body weight between 59 to 73 kg (65 + 2) participated in the study. All subjects

were tested for excess alcohol consumption using the Michigan Alcohol ism Screening Test

(MAST) (Selzer. 197 1 ), and a drinking behaviour interview (Shelton et al.. 1969). The

subjects consumed alcohol socially. but none of the subjects was an alcoholic at the time

of testing. In addition. al1 subjects underwent a complete rnedical examination. lndividuals

under chronic medication suffering from diabetes or hypertension. or having liver. brain.

kidney. or other chronic diseases as well as subjects with high ETOH consumption were

excluded from the study. Furthermore. the subjecü were not taking any over the counter

med icat ion or steroidal antiintlammatory drugs. The fernale subjecü were not on birtli

control medication and were tested during the fol1 icular phase of the reproductive cycle.

The investigations were approved by the Douglas Hospital H urnan Research Comm ittee.

and were conducted in accordance with the guidelines proposed in the declaration of

Helsinki. Informed consent was obtained from al1 subjects.

2.2.3.2. merimental Design

Al1 subjects abstained from alcohol for 48 hr prior to testing. Subjects participated

in three testing sessions. In the tïrst testing session subjects received a placebo drink

consisting of unsweetened orange juice and tonic water (2 parts orange juice 1 part tonic

water). The g las was dipped in ETOH to provide the smell and taste of ETOH. On the

second and third testing sessions. the subjects were randomly given either 0.25 g ETOH

per kg B. W. (as a mixture of 1 part 95 % ETOH 5 parts unsweetened orange juice). or

0.50 g ETOH per kg B. W.. (as a mixture of 1 part 95 % ETOH 2 parts unsweetened

orange juice). The total volume of each drink was between 1 16- 145 ml. The exact volume

depended on the subject's body weight. However. for the same subject the same volume

of tluid was ingested in ail 3 testing sessions. There was a minimum of one week interval

between testing sessions.

On the day of testing. subjects arrived at the research unit early in the morning

without having breakfast. A catheter was placed in the lefi arrn vein at 8 a m . The subjects

were allowed to rest for 1 hour. To avoid the feeling of nausea. often occurring following

ETOH ingestion with an empty stomach. which couid act as a stressor and release

hormones known to influence the ANP secretion (cortisol. epinephrine. norepinephrine).

following placement of the catheter the subjects were given a light breakfast consisting of

a toast without butter or marmalade. They were allowed to have 100 ml of water with their

breakfast, but not tea or coffee and were not allowed to smoke for the duration of the

testing session. 60 min afier placing the i.v. catheter. a 10 ml blood sample was drawn and

BP and hem rate were measured. Then. the subjects were given the placebo or the

appropriate ETOH drink. which they were requested to drink within 5 min. Additional 10

ml blood samples were drawn at 15. 45. 110 and 180 min post drink. These time intervals

were chosen based on the blood ETOH content following ETOH ingestion which rises

sharply within 15 min. reaches maximum within 45 to 60 min and then decreases

gradually. so that at 3 hr post drink the alcohol in blood is low but still measurable.

Following the drawing of the blood samples. BP and heart rate were measured while the

subject was lying in bed. During the intervals between blood sampling. the subjects were

allowed to get up. sit in the living room of the research unit, watch television. read and

converse with research unit personnel. The subjects collected the urine for estimation of

the total volume excreted during the 3 hrs of the experirnental period. as well as Na' and

K' excretion. The content of Na' and K' in urine was measured direcily by a tlame

p hotometer (Instrumentation Laboratory Autoanalyser. 1 L943).

2.2.3.3. Estimation of Blood Alcohol Content

To estirnate the blood alcohol content, 750 pl of whole blood were rnixed with 1 .O

ml of 6 % ice cold perchloric acid and centrifuged at 4°C. The supernatant was kept

frozen at -75°C till the time of estimation of the blood alcohol content using the alcohol

dehydrogenase enzymatic method (Hawkins et al.. 1966).

2.2.3.4. Estimation of Plasmê ANP. AVP and Cortisol Contents

10 ml of blood were drawn and placed in ice-chilled EDTA vacutainer tubes

containing 100 ui of 10 mM pepstatin A (Sigma Chernical Co. St. Louis MO. USA. No

P-4265) to prevent degradation of ANP and AVP. Blood samples were centrifuged at 4°C

and the plasma was stored at -75 "C until the tirne of the assay.

The ANP content in the plasma was estimated using a sensitive RIA as described

previously (Gutkowska et al.. 1985). The antibody for ANP was raised in the laboratory

of Dr. Gutkowska (Gutkowska et al.. 1985). Prior to RIA. ANP peptides were extracted

from 1 ml of plasma using C-18 Sep-Pak Cartridges (Waters Scientific Mississauga

Ontario. Canada). The iodination of ANP, .:, was perforined using the lactoperoxidase

rnethod (Gutkowska et ai.. 1987a). The minimum quantity of ANP detectable with this

RIA was 0.12 fmoles/tube. AI1 samples were analyzed in the sarne assay to avoid the

between assays variation. The intra-assay coefficient of variation was 6.8 % .

Estimation of plasma AVP content was performed in 1 ml of acetone-extracted

plasma (Skowsky er al.. 1974). The antisemm was purchased from Peninsula Laboratories

(Belmont CA. USA) and was used at a final dilution of 1:50000. I r presented a 3 % cross-

reactivity with Lys,-vasopressin and no cross-reactivity with ANP. AVP was iodinated

using the chloramine T method. The sensitivity of the antiserum was 0.2 pg/ml. Al1

samples were analyzed in the same assay to avoid the between the assays variation. The

intra-assay coefficient of variation was 4 % .

For estimation of the plasma cortisol levels. plasma was extracted in absolute ETOH

and cortisol was determined by RIA according to previously pub1 ished procedure (Krey

et al., 1975). using a cortisol antiserurn (F3-3 14: Endocrine Sciences. Tarzana. CA. USA)

at a final dilution of 1 : 1000. The intra-assay coefficient of variation was 9 % . Of the

physiologically occurring steroids tested for cross-reactivity only cortisone and

desoxycortisol presented a signifiant cross-reactivity with the antibody. 4.7 % and 7.5 %

respect ive1 y. The contribution of these cross-reactions in normal human plasma

determinations is small due to the ordinarily low concentrations of desoxycortisol and

cortisone relative to cortisol. Total cortisol (bound plus free) was determined by this

method.

. . 2.2.3.5. Statistical A-

Statistical evaluation of hormone content and other physiological measures was done

by 2 way analysis of variance (ANOVA) for repeated measures across time and dose with

the various sample times as the B n t variable and treatment (placebo. 0.15 g and 0.50 g

ETOHlkg B.W.) as the second variable. The ANOVA was followed by the Neuman-Keuls

multiple comparison test to determine the significance of difference between the different

treatments and time intervals of testing. A value of p < 0.05 was considered signitïcant.

2.2.4. RESULTS

in Figure L I . 1.. the changes in blood alcohol content with time after ingestion of the

ETOH drinks are shown. The concentration of ETOH in the circulation increased sharpl y

within the first 15 min and was still high at 45 and 120 min. The ETOH was cleared from

the blood slowly so that it was still detectable at 180 min after the ETOH drink (Fig.

7.7.1.). In Figure 2.2.2.. the changes in heart rate (Fig. 2.2.2A. B and C) and BP (Fig.

1.1.2D. E and F) following the placebo and the alcohol drinks are shown. There was no

significant change in the heart rate following the placebo as well as the 0 . 3 and 0.50 g

ETOHIkg B.W. drinks. Small changes in both systolic and diastolic pressure were

observed during the three hour experimental period. Statistical analysis (two way ANOVA)

indicated a significant effect of time on both systolic [F4.,,,=6.32: p < 0.001 1 and diastolic

[F,,,= 10.28: p < 0.01 1 BP. Thus. systolic BP was decreased following ingestion of the

placebo, 0.25g and OSOg ETOH/kg B.W. drinks. Post-hoc analysis using the Neuman-

Keul's multiple comparison test indicated a significantly lower systolic BP at 60 min

following the placebo drink and at 120 and 180 min following the 0.15 g ETOH drink

.+ 0.25 g ETOHIkg B.W.

+ 0.50 g ETOHIkg B.W.

Time of drink (min)

Figure 2.2.1. Changes in blood alcohol content folloning ingestion of 0.25 and 0.50 g ETOHIkg B.W.

Pta ce bo 0.25 g E TOH 0.50 g ETOH

O a 60 90 120 150 180

Time (min)

I /

I I I 1 60 0 O 30 60 90 120 150 180 O 30 60 90 120 150 180

Time (min) Time (min)

Figure 2.2.2. Heart rate follodng (A) the placebo, (B) the 0.25 and (C) the 0.50 g ETOHkg B.W. drinks. Systolic and diastolic BP folloming (D) the placebo, (E) the 0.25 and (F) the 0.50 g ETOHIkg B.W. drinks.

cornparecf to the corresponding systolic BP at time O min. Following ingestion of the 0.50

g ETOH drink a gradual decrease in systolic BP was also observed which however did not

reach a level of significance at any of the time points that it was cested. The diastolic BP

also presented a small decrease with time following the placebo and the 0.15 g ETOH/kg

B.W. drinks. At 180 min following the placebo drink the diastolic BP was signitïcantly

lower than at time O min. Following the 0.15 g ETOH drink the gradual decrease in

diastolic BP did not reach a level of significance. Interestingly. at 15 min following the

0.5 g ETOH drink there was a short lasting. but statistically significant increase in the

diastolic BP.

The ef'fect of ETOH on the plasma ANP content is shown in Figure 2.7.3A.B and

C. Both 0.25 and 0.50 g ETOH per kg B.W. induced an increase in plasma ANP content.

As shown on Figure 2 . L 3 . . the plasma ANP content was elevated at 15 and 45 min but

had returned to basal levels by 120 min post-ETOH ingestion. Statistical analysis (two way

ANOVA for repeated measures) indicated a signiticant effect of time [F,.,, = 8: p < 0.00 1 1.

However. there was no significant effect of treatment and no significant interaction

between treatment and time factors. Post-hoc analysis using the Neuman-Keul's multiple

comparison test indicated that at 15 min following the 0.75 g alcohol drink and at 15 and

45 min following the 0.5 g alcohol drink the plasma ANP content was significantly higher

than the basal ANP levels ( levels at time O) (p <0.01]. This increase in the concentration

of plasma ANP with time was a specific effect of ETOH since no increase was observed

following the placebo drink. In fact. a small decrease with time was observed in the

plasma ANP content following the placebo drink, which however did not reach a level of

significance. Furthermore. it was also noticed that the high dose of ETOH (0.5 g

ETOHfkg B.W.) did not induce a higher plasma ANP concentration than the low dose of

ETOH (0.25 g ETOH/kg B.W.) even though blood alcohol content was about 2 times

higher. However. the plasma ANP levels remained elevated for a longer period follow ing

the high ETOH dose.

Figure 2.2.4A. B and C demonstrate the effect of ETOH ingestion on the plasma

AVP content. Statistical analysis indicated a significant effect of time [F,.,(, = 2.5:

Placebo

r

3 30 60 90 120 1 5 0 180

Time (min)

l I ! I ! I

O 30 60 90 120 150 180

Time (min)

O 30 Hl 90 120 150 180

Time (min)

Figure 2.2.3. Effect of (A) the placebo, (B) the 0.25 and (C) the 0.50 g ETOHlkg B.W. drinks on the plasma ANP content.

Placebo

O 30 60 90 120 150 180

Time (min)

O 30 60 90 120 150 180 O 30 60 90 120 150 180

Time (min) Time (min)

Figure 2.2.4. Effect of (A) the placebo, (BI the 0.25 and (C) the 0.50 g ETOH/kg B.W. drinks on the plasma content of vasopressin.

p <0.05]. Post-hoc analysis using the Neuman-Keul's multiple comparison test indicated

that following either the placebo or the 0.25 g ETOH drinks there was no significant

change in the plasma AVP levels. while following the 0.5 g ETOH dose there was a

gradual decrease in the plasma AVP content. In fact the plasma AVP levels at 110 and 180

min post-drink were significantly different from those at O (prior to the drink) and at 15

min post-drink (p < 0.05. Neuman-Keuls multiple comparison test). As expected. this

decrease in plasma AVP content following the G.5 g ETOH dose was associated with a

higher urine excretion to 365 f 52 ml. compared to 230 + 17 ml and 265 + 38 ml

following the placebo drink and the 0.25 g ETOH dose respectively. Estimation of Na+

and K' contents in the urine collected during the 3 hr of the experimental period indicated

a signiticant increase of NaT excretion following the 0.25 g ETOH drink (p < 0.05) but

not following the 0.50 g ETOH drink . On the other hand. a small but significant

(p < 0.05) decrease in the excretion of K+ in the urine was observed following the 0.50 g

but not the 0.25 g ETOH drink (Table 2.2.1.).

Figure 3.1.5A. B and C indicate that ETOH ingestion did not induce an increase

above the basal levels in the plasma content of immunoreactive (ir)-cortisol . Statistical

analysis indicated a significant effect of time on the plasma cortisol content [F4.,,=?.81:

p < 0.051. A gradual decrease with time was observed as expected from the circadian

rhythm of circulating cortisol in humans. Post-hoc analysis using the Neuman-Keul's

multiple comparison test indicated that the plasma cortisol content at time O (prior to

administration of the drinks) was significantly higher than the plasma cortisol content at

60. 110 and 180 min following the placebo drink (p<0.05) and 110 and 180 minutes

following the 0.35 g ETOH drink (p < 0.05). while following the 0.50 g ETOH drink the

time dependent decrease in the plasma cortisol content did not reach a level of

significance. Thus. ingestion of the 0.5 g ETOH/kg B.W. drink did not induce an increase

in the plasma cortisol levels. However. it prevented the decrease in the plasma cortisol

levels that was observed with time following ingestion of either the placebo. or the 0.25

g ETOHkg B. W.

Placebo

O 30 60 90 120 150 180

Time (min)

O 30 60 90 120 150 180

Time (min) O 30 60 90 120 150 180

Time (min)

Figure 2.2.5. Effect of (A) the placebo, (B) the 0.25 and (C) the 0.50 g ETOHIkg B.W. drinks on the plasma cortisol content.

Table 2.2.1. Uritie output orid urzrze sodilirn and potassium excretiori heari * SEhl)

f o l h i ~ i g irrgestion of O. 0.25 und 0.50 g ETOWkg B. W.

Treatment Urine volume Na' (mmoVhr) K' (rnmoh)

[[ 0.50 2 ETOH 365 k 52* 4.4 1 + 1.36 2.10 * 0.61* 11 * Significant clifference fiom the placebo ( O g ETOH); p 50.05.

** Sigpficant Merence fkom the 0.50 g ETOH dose; p 50.05.

Neurnan-Keuls multiple cornparison test.

2.2.5. DISCUSSION

Both experimental (Howe et al.. 1989) and epidemiological (MacMahon. 1987)

smdies have shown that low to moderace ETOH consumption ( 1 to 2 drinks per day) delays

or prevents die age dependent increase in BP. The mechanism by which ETOH exerts this

antihypertensive effect is not clear .

The present studies indicated a small transient increase of diastolic but not systol ic

BP within 15 min following ingestion of the 0.50 g ETOH dose. A dissociation between

the effect of aicohol on the systolic and diastolic BP has been previously reponed (Criqui.

1986). It was proposed that ETOH's effect on systolic and diastolic BP may provide a clue

to the underl y ing pressor (or depressor) mechanism b y wh ic h alcohol modifies

cardiovascular homeostasis. Indeed. elevation in systolic BP may imply a greater effect of

ETOH on the cardiac output or tluid volume. whereas elevations in diastolic BP may

suggest moditkations in vascular resistance (Ireland et al.. 1984: Puddey et al.. 1985).

Thus. the initial and rapid increase in diastolic BP following the higher dose of alcohol

may represent sorne short-lived vasoconstriction produced by ETOH. Previous studies

investigating the acute eiiects of ETOH on BP have yielded conflicting results. Thus

following high ETOH intake (intoxicating doses) both a decrease (Conway. 1968) and no

change (Riff et al.. 1969) in BP have been reponed. Likewise. ingestion of ETOH at doses

below the intoxicating levels induced no change in BP (Gould et al., 1971). increase in BP

(Orlando et al.. 1976) or immediate increase followed by a decrease in BP (Ireland et al..

1984). In another study the contribution of (a) expectancy. (b) tluid volume and ( c )

intoxicating doses of ETOH on the BP changes following an ETOH drink were

investigated (Adesso et ai.. 1990). Surprisingly. results indicated that neither expectancy

nor tluid volume had a signitkant effect on BP. On the other hand. ETOH induced an

initial decrease in the BP followed by an increase toward the basal levels during the

detoxification period. This increase in BP during the detoxification period may be

attributed to ETOH withdrawal.

Estimation of the blood alcohol content following ETOH ingestion indicated that i t

remained well below the intoxicating levels. Furthermore, it was noticed that the dose of

0.5 g ETOH/kg B. W.. did not induce a larger increase in ANP content than the dose of

0.25 g ETOH/kg B.W.. indicating that large doses of ETOH may not be more effective

in stimulating the release of ANP than srnall doses of ETOH. However. following the 0.50

g dose of ETOH the plasma ANP content remained elevated for a longer period post-drink

than following the 0.25 g ETOH dose. suggesting a longer lasting effect of the higher

dose. A similar increase in plasma ANP content has been reported following

administration of 1 .O ml ETOH /kg B. W. in a total volume of 750 ml (Colantonio et al..

1991). Interestingly. data from this study demonstrated that the maximum increase in

plasma ANP content was obtained within the tïrst IO minutes post-ETOH when the blood

alcohol content was about 40 mg/dl. As blood ETOH content increased to intoxicating

values the plasma ANP content started to decrease. clearly indicating that maximum

response of the ANP system to ETOH is obtained with low concentrations of ETOH. in

agreement with the resulu of the present study . In addition. administration of the placebo

drink. consisting of two parts orange juice one part tonic water at a final volume equal to

that of the alcohol drinks did not alter the plasma ANP content. indicating that (a) the

volume of the fluid ingested was not high enough to induce sufficient increase in plasma

volume and suetching of the atria to stimulate the release of ANP and (b) the increase in

plasma ANP concentration following the ETOH drink was a specific effect of ETOH. and

was nor due to orange juice.

However. studies by other investigators have failed to observe an ETOH-induced

increase in plasma ANP content (Leppiiluoto et ai.. 1992: Hynynen et al.. 1992: Ekman

et ai., 1994). For example in a recent study when doses of 0. 0.50 and 1 g ETOH per kg

/ B.W. were administered in 500 ml of tluid at 18:OO there was no increase in plasma

ANP content. while a dose dependent inhibition of the nocturnal peak (occurring at

midnight) of plasma ANP was observed (Ekman et ai., 1994). In these studies the earliest

measurement of plasma ANP was at 2 hr after initiation of drinking. Since both the present

and previous (Colantonio et al.. l99 1 ) studies indicate that the increase in plasma ANP

content occun fast following drinking and is short lasting, returning to basal levels withi n

2-3 hrs post-drink. even if there was an initial increase in plasma ANP content. it could

not have been detected in the snidies by Ekman et al ( 1994). In another study ( Leppaluoto

er al.. 1992) a decrease in plasma ANP content was observed at 30 min and was

maintained for at least 2 hr, following ingestion of 1. 5 g ETOHfkg B. W. administered

in 500 ml tluid at 18:M) hr. On the other hand. there was no effect on plasma A N P content

following administration of 1 g ETOH /kg B.W. in volumes ranging from 415-560 ml at

18:00 hr to subjects who were slightly volume-loaded prior to ETOH administration

(Hynynen et al.. 1992). The differences in the results of those studies frorn those of the

present studies and of Colantonio et al (1991) rnay be attributed to a number of factors

such as the rime of day when the ETOH was administered. sarnpling time intervals post

ETOH ingestion and prior volume-loading of the subjects. For exarnple, volume-loading

may stimulate the heart ANP system prior to the administration of ETOH. so that a

subsequent stimulation by ETOH rnay not induce a further increase in ANP release. In

addition. rapid urine excretion usuall y associated wi th volume-loading and the

corresponding reduction in plasma volume. as was observed in the study by Leppaluoto

et al. (1992). may also counteract any stimulatory effect of ETOH on ANP release.

The present studies also indicated that the 0.50 g ETOH dose induced a gradua1

decrease in plasma AVP content and increased diuresis. This decrease in the plasma AVP

content and increased diuresis observed following the 0.50 g ETOH drink. can not be due

to die volume of the tluid ingested since it was not observed following either the placebo

or the lower dose of ETOH (0.25 g ETOH / kg B.W.) which were administered in the

saine volume of fluid. Indeed. the three drinks given to each subject were isovolumetric

but not isoosmotic. In fact. ETOH ingestion increases plasma osmolality. Interestingly.

it has been shown that increase in plasma osmolality stimulates the release of A N P and

increases plasma ANP content (Arjamaa and Vuolteenaho. 1985). Thus. one of the

mechanisms by which ETOH increases ANP release could be the increase in plasma

osmolality with increasing alcohol concentration in the blood. The absence of a decrease

in AVP secretion following the low dose of ETOH may be explained by the low

concentration of ETOH in circulation achieved with the 0.25 g dose. as was previously

reported (Ekman et al., i 994). Most of the diuretic effect associated with acute ETOH

consumption is attributed to the inhibition of AVP release from the posterior pituitary

(Eisenhofer and Johnson. 1982: Goldsmith and Dodge. 1985). Interestingl y, ANP has also

been reponed to suppress hypothalamic AVP (Januszewicz et al.. 1986: Colantonio et al..

1991: Ekman et al.. 1994). in the present studies. the increase in circulating ANP levels

by the 0.5 g ETOH drink is associated with a decrease in plasma AVP levels and increased

diuresis. It is conceivable therefore. that the inhibition of AVP release following ETOH

administration is mediated by the observed stimulatory effect of ETOH on ANP release.

The decrease in AVP and increase in ANP release would lead to increased diuresis as was

observed in the present (table 2.2.1 .) and other studies (Potter et al., 1986: Colantonio ef

al.. 1991: Hynynen et al.. 1991: Ekman et al.. 1994). Estimation of the content of

electrolytes (Na' and K') excreted in the urine during the three hour of experimental

period indicated a small increase in Na' excretion following ingestion of the 0.75 but not

of the 0.50 g ETOH / kg B.W. drink (table 2.2.1.). Other studies using ETOH doses

higher than 0.50 g ETOH also did not observe signitïcant changes in urine Na' and K-

concentrations (Potter et al.. 1986: Colantonio et al.. 199 1 : Hynynen et al.. 1992: Ekman

et al.. 1994). The use of specific ANP and AVP antagonists would help further detïne the

possible associations between these two hormonal systems in the ET0 H- i nduced di ures is . although a major problem for studies using ANP antagonists is the low potency and

specificity of the currently known ANP antagonists (Anand-Srivastava and Trachte. 1993).

Among the factors known to increase the release of A N P is stress (Horky er al..

1985). Therefore. it is possible that activation of the Hypothalamic-Pituitary- Adrenal

(HPA) mis. either by stress associated with the experimental procedure or by the ETOH

administration. could induce an increase in the ANP release. However. it seems that this

was not the case in the present studies since estimation of plasma cortisol content indicated

no increase in plasma cortisol levels following either the placebo or ETOH drinks and thus

no activation of the HPA-axis. confirrning the absence of stress associated with the

experimental procedure. Interestingl y. a number of studies have demonstrated activation

of the HPA axis associated with an increase in plasma cortisol levels following

administration of moderate to high doses of ETOH (Mendelson and Stein. 1966:

Mendelson et al.. 197 1 : Valimaki et al., 1984: Schuckit et al.. 1987. 1988) but not of

low doses of ETOH (Schuckit et al.. 1987. 1988) to humans. Since in the present studies

the doses of ETOH used are considered low (equivalent to one or two standard drinks).

the absence of a stimulating effect of ETOH on the HPA axis is in agreement with the

pub 1 is hed reports.

A number of mechanisms have been proposed to explain the antihypertensive effect

of low to rnoderate ETOH consumption. These include ETOH-induced increase of the

HDL content in circulation (Criqui. 1986) and the inhibitory effect of ETOH on the

activity of substances known to have a pressor effect. For example. low but not high

concentrations of ETOH induce an inhibitory effect on the vasoconstrictor activity of

norepinephrine (Kalsmer. 1970: Altura et al.. 1983: Criscione et al.. 1989). ETOH

consumption has also been shown to increase the secret ion of nitric oxide (NO) in several

vascular beds. such as in the pulmonary arteries (Greenberg et al.. 1993). NO is a

vasodilator produced by the endothelial cells.

The present studies indicated that an additional mechanisrn may be implicated in the

antihypertensive effect of low ETOH consumption. involving the A N P systern. ANP. a

hormone released from the hem atria. decreases BP by inducing vasorelaxation. increas i ng

diuresis. and decreasing the release and/or activity of a number of pressor hormones

(Chartier et al.. 1984: Yasujima et al.. 1985. 1986: lnagami et al.. 1987). lntravenous

administration of excess ANP antibodies induced a decrease in diuresis and natriuresis. an

increase in plasma renin but had no effect on plasma aldosterone and BP (Chartier et al..

1984). Furthermore. infusion of low doses of A N P suppressed the hyperrensive effect of

norepinephrine. epinephrine and angiotensin I I (Yasujima et al.. 1986: Inagami et al..

1987). Thus. it seems that ANP rnay act as a buffering system that protects the

cardiovascular system against a sudden increase of BP induced by sudden activation of the

noradrenergic or the renin-angiotensin systems (Inagami et al.. 1987). Therefore. it is

reasonable to propose that agents stimulating the release of ANP may provide a protection

against the activation of systems known to exert a hypertensive effect.

It is possible then that the protective effect of low and moderate doses of ETOH on

the age-dependent increase in BP. is partially due to its ability to activate the hem ANP

system. In addition. ETOH rnay alter the activity of other renal and /or cardiovascular

systems. such as AVP. and thus modi@ diuresis and BP. Supporting the stimulatory effect

of acute ETOH exposure on the hem ANP system are animal studies demonstrating a dose

dependent increase in plasma ANP content (section 2.1 .). This increase in plasma ANP

content is fast. reaches maximum levels within 15 min following an i.p. injection of

ETOH (as observed in the present studies) and is associated with a decrease in the ANP

content in the heart atria.

Therefore. it rnay be hypothesized that frequent activation of the heart A N P system

by low daily alcohol ingestion ( 1 to 2 drinks per day) will stimulate the release of ANP

and eventually rnay increase the rate of ANP synthesis and the content of specific ANP

mRNA. Conrrary to the beneficial effect of chronic low to moderate ETOH consumption

on BP. following chronic heavy use of ETOH the increased activity of the heart ANP rnay

not be sufficient to counteract the hypertensive effects of ETOH. such as increased

sympathetic activity , increased sensitivity of blood vessels to pressor substances and

intermittent ETOH withdrawal reactions and eventually hypertension will develop. in this

case. it rnay be expected that the ETOH-induced hypertension will be associated with high

circulating ANP levels as has been reported for other conditions of hypertension (Imada

er al.. 1985: Nozuri et al., 1986). Therefore. ETOH administration rnay induce both

h ypertensive and antihypertensive effects. At low ETOH concentrations. the net impact

of the antihypertensive effects rnay be dominant. while at high ETOH concentrations rhe

net impact of hypertensive effects rnay predom inate.

In summary. the present studies indicated that ingestion of low amounts of ETOH.

equivalent to I or 3 standard drinks. but not of a placebo drink increased the plasma ANP

content in norrnotensive subjects. This increase in the release of ANP by low doses of

ETOH rnay be partially responsible for the antihypertensive effects of low to moderate

daily ETOH consumption reported in a number of epidemioiogical (human) and

experimental (animal) studies.


The authors wish to thank the nursing staff of the research unit at Douglas Hospital

Research Centre. This study was supported by gram MT- 10337 and MT- 1 1674 from the

MRCC and grants from the Kidney Foundation and the Canadian Heart and Stroke

Foundation to Gutkowska J. and by the Alcohol Research Program at Douglas Hospital.

Guillaume P. is a recipient of a scholarship from the "Fond pour la Formation de

Chercheurs et l'Aide a la Recherche (FCAR) du Québec"

CBAPTER 3

CHRONIC ETOH STUDIES

Section 3.1.

EFFECT OF CHRONIC ETHANOL CONSUMPTION ON THE ATRIAL

NATRIURETIC SYSTEM OF SPONTANEOUSLY HYPERTENSIVE

RATS

P. Guiilaume, M. Jankowski, C. Gianoulakis and J. Gutkowska

Alcohoüsm: C h c d and Experimentai Research

Wcohol Clin Eirp Res 20: 1653- 166 1, 1996)

Contribution bv CO-authors: Dr. M. Jankowski introduced me to the technical aspects of

Northem blottmg and was responsible for the RT-PCR midies. Dr. C. Gianoulakis and Dr.

J. Gutkowska were my CO-supervisors.

Achowled~ements: - R Claudio was the animal technician. C. Coderre. N. Charron and S.

Laroque performed some of the ANP or AVP iodinations.

3.1. EFFECT OF CHRONIC ETHANOL CONSUMPTION ON THE

ATRIAL NATRIURETIC SYSTEM OF SPONTANEOUSLY

HYPERTENSIVE RATS ( S H R )

3.1.1. ABSTRACT

There is a lot of discussion on the effects of ethanol (ETOH) on the blood pressure

(BP). It has been suggested that chronic moderate ETOH consumption prevents the

development of agedependent hypertension in humans and spontaneously hypertensive rats

(SHR). However. the mechanism mediating this effect is unknown. In the present studies.

we hypothesized the implication of Atrial Natriuretic Peptide (ANP). a BP-lowering

hormone. on the antihypertensive effect of moderate ETOH consumption. A 10% viv

solution of alcohol was given as drinking iluid to SHR and normotensive Wistar-Kyoto

(WKY) rats for up to 32 weeks. This treatment prevented. at least in part. the age-

dependent increase of BP in SHR and WKY rats. The lower BP was associated with

s igni ficantl y lower levels of circulating atrial natriuretic peptide ( ANP) in both groups.

After chronic ETOH administration. total ANP content and concentration were higher in

the left and right atria of SHR and WKY rats than in water-treated controls. Despite the

ETOH- induced increase in atrial A N P content. there was no signitïcant change in atrial

ANP mRNA . suggesting decreased atrial release. Chronic ETOH treatment significantl y

reduced ANP mRNA in the ventricles of S H R but not of WKY rats. Correspondingly.

ventricular ANP content and concentration were lowered by ETOH in SHR only. Chronic

ETOH administration induced a signifiant increase of plasma arginine vasopress in ( AVP)

and a signitkant decrease of plasma aldosterone in S H R but not in WKY rats.

Thus. chronic ETOH treatment prevented the age-dependent elevation of BP in both

SHR and WKY rats. and altered the activity of heart ANP as well as of the aldosterone

and AVP systems.

3.1.2. INTRODUCTION

A positive correlation between chronic high ethanol (ETOH) consumption and

hypertension has long been recognized in humans (Lian. 1915). However. it appears that

aithough this relationship is linear for 3 or more standard drinks per day ( 1 standard drink

corresponds to 8-10 grams of ETOH). a different kind of association is seen with low or

moderate ETOH consumption ( MacMahon. 1987). Thus. a number of epidemiological

studies have reponed a U- or J-shaped curve for blood pressure (BP) with increased ETOH

drinking. suggesting that individuals consuming 1-3 drinks per day had lower or similar

BP as abstinent individuals and significantly lower BP than heavy (5 or more drinkdday )

drinkers (Gleiberman and Harburg. 1986: MacMahon. 1987). Interestingly. a similar

effect has been seen in animal studies where chronic moderate ETOH administration

prevented or delayed the development of age-dependent hypertension. particularly in rat

populations known to become hypertensive (SHR and SHRSP) (Sanderson et al.. 1983:

Jones et ai., 1988: Howe et al., f 989: Beil in er ai., I992).

The mechanisms by which chronic moderace ETOH administration prevents the

development of hypertension are still not known. One such mechanism could involve the

atrial natriuretic peptide (ANP).

Atrial natriuretic peptide (ANP). a 18-amino acid peptide synthesized mostly in heart

atria (De Bold et al.. 198 1 ) . is known to lower BP through several mechanisrns. including

natr iuresis. diuresis. vasodilation. and by decreasing the secretion of various pressor

hormones. such as renin-angiotensin I I . aldosterone. norepinephrine and arginine

vasopressin (AVP) (Needleman er al.. 1989: Blaine. 1990: Nakao et al.. 1993). Earlier

(Section 2.1.). we demonstrated that acute injection of moderate ETOH doses in rats

induced a rapid increase in plasma ANP levels. and this elevation was associated with a

reduction of right and left atrial ANP content. In contras. the acute ingestion of a large

dose of ETOH in Sprague-Dawley rats has been associated with no increase in circulating

ANP levels (Wigle er al.. 1993a). Recently. a short-term chronic ETOH study (6 weeks)

using Sprague-Dawley rats was also published by Wigle et al. ( 1993b. 1993c) who noted

that. although there were no changes in plasma ANP levels. plasma brain natriuretic

peptide (BNP) and ventricular ANP contents were significantly elevated. After 6 weeks

of ETOH administration however. BP values in the ETOH-treated and control animals

were not significantly diffèrent (Howe et al.. 1989). Therefore. it would be interesting to

examine the effect of chronic treatment with moderate amounts of ETOH for a duration

sufficient to produce a significant difference in BP between both groups.

Using Wistar-Kyoto (WKY) rats and SHR. the present studies investigated the effect

of chronic moderate ETOH consumption for 31 weeks (a duration suficient to demonstrate

prevention of the age-dependent increase in BP) (Howe et al.. 1989) on circulating ANP

levels, as well as on ANP content and ANP mRNA in h a r t atria and ventricles. BP. heart

rate (HR). body weight (BW) and fluid consumption were evaluated on a regular bais.

In addition to ANP. the plasma leveis of AVP. corticosterone and aidosterone were also

estimated to provide an indication on the implication of other hormonal systems in the

ancihypertensive effect of chronic moderate ETOH consumption and to contribute to a better

understanding of the interactions between the various endocrine systerns.


3 . 1 . 3 . 1 . T r e e

Male WKY rats and SHR. approximately 6 weeks old. were purchased from Charles

River Breeding Farms (St-Constant. Québec. Canada). Upon reception. the animals were

randomly assigned to 1 of 4 groups (Group 1 : WKY-H,O ( n = 15): Group 2: WKY-ETOH

( n = 15): Group 3: SHR-H,O ( n = 15) and Group 4: SHR-ETOH ( n = 15)). After a 7-day

acclimatization period. ETOH was gradualiy added to the drinking water of Groups 2 and

4. up io a 30% v/v solution in 15 days (5 % v/v for 5 days. 10% v/v for 5 days. 15 % v/v

for 5 days and 20% vlv subsequently. as described by Howe et al.. 1989). AI1 animals had

free access to pelleted rat chow (Purina. Richmond. VA) and tluids (water or ETOH

solution) for the duration of 8 months.

BP and HR were rneasured every 1 weeks and BW every week. Liquid consumprion

was recorded daily. BP and HU were evaluated by the tail-cuff method (Pfeffer er al..

1%' 1). After 8 months, a11 rats were killed by decapitation. Trunk blood was collected.

and atria and ventricles were removed carefully for subsequent estimation of A N P and

ANP mRNA content. To investigate the effect of age. a third group of animals ( 1 1 WKY.

II SHR) was sacrificed prior to the initiation of ETOH treatment. at about 7 weeks of age.

3.1.3.2, Estimation of blood ETOH

From fresh blood. 50-pl samples were deproteinized with 450 pl of ice-cold

trichloroacetic acid (6.35 w/v) for the measurement of blood ETOH levels at the tirne of

death. using the dehydrogenase enzymatic method (Hawkins et ai.. 1966).

. . 3.1-3.3. Prepgration of blood and ti.ssue e x t w for R a d i o i m m u n ~ v s (RIAS)

Trunk blood was collected in chilled 15 ml conical centrifuge tubes containing

protease inhibitors to the following final concentrations: 1 mg/rnl ethylenediamine-tetra-

acetate (EDTA). 10 mM phenylmethylsulfonyl fluoride (PMSF. P7626. Sigma Chemical

Co.. St-Louis. MO) and 5 m M pepstatin A (P4265. Sigma) . The tubes were imrnediately

centrifuged at 4°C and the plasma stored at -75°C for subsequent RIAS.

The ventricles as well as the left and right heart atria were excised and placed in

separate tubes containing ice-cold 0.1 N HCI. They were rapidl y boiled for 5 minutes and

then cooled on ice. The tissues were homogenized subsequently in a Polytron. and an

aliquot was taken for the estimation of protein content using Bradford's method (Bradford.

1976). The remaining homogenates were centrifuged for 15 minutes at 4°C and the

supernatants stored frozen at -75°C for the measurernent of ANP content.

3.1.3.4. RIAs

Atrial (final dilution. 1: 15 000). ventricular (final dilution. 1: 1 000) and plasma ( 100

p l ) ANP content was quantified by a direct second-antibody RIA (Gutkowska et al.. 1984:

Gutkowska et al., l987a). The val idity of the direct assay for the investigation of the

relative differences between various treatments has been previously demonstrated (Bidmon

et ai., 1991 and Section 2.1 .). AVP levels were estimated in acetone-extracted plasma

samples. also using a second-antibody RIA (Skowsky et al.. 1974). Corticosterone (Krey

et al.. 1975) and aldosterone (Mayes et al.. 1970) levels were measured by sensitive RI As

in ETOH-extracted plasma samples.

* * . 3.1.3.5. RNA extraction and hvbnd-

For estimation of ANP mRNA. the atria and ventricles tiom 5 animals per group

were careful [y dissected. frozen irnmed iatel y in isopentane. and stored singl y at -75 OC

until total R N A extraction.

RNA from cardiac tissues was isolated according to the acid guanidinium-thiocyanate-

phenol-chloroforrn methoci. as described elsewhere (Chomczynski and Sacchi. 1987). The

RNA concentration in each sample was determined by UV absorbance at 260 nm. Ten

pg of ventricular or two pg of atrial RNA were subjected to electrophoresis through 1.5 %

agarose gels containing 0.22 M formaldehyde and transfered ont0 nylon membranes

(Hybond N + . Amersham International PLC. UK) by capillary blotting. Immobilized RNA

samples were hybridized with randoml y primed a3'P-cDNA probes corresponding to ANP

and a-tubulin mRNA sequences. A random priming kit (Gibco. BRL. Bethesda. MD) and

a3'P-dCTP (3000 Ci/mmol. Amersham. Ar1 ington Heights. 1 L) were used for label1 ing

the probes. The Pst 1-digested 660 bp fragment from plasmid clone PN- 1-1 lserved as the

ANP probe (gift from Dr. Mona Nemer. Institut de Recherche Clinique de Montréal). A

550-bp fragment of a-tubulin cDNA probe. generated by reverse transcription (RT) of rat

atrial RNA and amplified by polymerase chain reaction (PCR). was used to monitor the

amount of RNA in each sarnple (Dagnino et al.. 1992). The blots were prehybridized in

buffer containing 120 mM Tris. 8 mM EDTA. 0.6 M NaCI. 0.01% sodium

pyrophosphate. 0.02% SDS, and 0.01 % heparin (H7005. Sigma). pH 7.4. Hybridization

was carried out in the same buffer for 16 hours with heparin content increased to 0.03 %

and supplemented with 10% of dextran sulfate as well as 10" dpm/ml of the labelled probe.

After hybridization. the membranes were washed in 2 X SSC/ 1 % SDS ( 10 minutes at

room temperature) and exposed on a Phosphorimager. Radioactive bands were measured

with Image-Quant software (Molecular Dynamics, Sunnyvale. CA). The tissue

concentrations of ANP mRNA reported in the text were normalized to the a-tubulin

rnRNA concentration in each sample to correct for differences in the RNA content applied.

3.1.3.6- RT-PCR

a) First strand cDNA qwthesis and quant~fication reaction product

Total RNA sarnples were treated with RQI DNAse (Promega. Fisher Scientific.

Canada) to eliminate any contamination with genomic DNA. Then. total RNA (2 pg) was

annealed with 0.5 pg of random hexamer and reverse transcribed in 50 mM Tris-HCI

buffer (pH 8.3). 7 rnM KCI. 3 rnM MgCl,. 10 mM DTT. 0.5 mM dNTP. 40 units

RNAsin (Pharmacia) and 200 units Moloney muiine leukemia virus reverse transcriptase

(BRL Laboratories, Gaithersburg. MD. USA). The reaction was left to incubate for 1 hour

at 37°C. A 3-pl aliquot of the RT reaction product was denaturated at 95 O C for 5 minutes

and then electrophoresed on 1 % agarose gel. After gel dry ing on autoradiographic films.

counts (over 400 bp in length) were integrated for each sample using Image-Quant

software. Input of cDNA sarnples in each PCR was normalized according to Irnage-Quant

rneasurements of the radioactive RT reaction product (Kolls et al.. 1993).

b) PCR ampiifkarion of ANP transcript

PCRs were conducted according to previously described procedures ( Dagnino er al..

1991: Dagnino et al.. 1992: Jankowski et al.. 1996). The number of cycles for the

exponential amplification of PCRs and the concentration of genomic DNA were

standarized in separate experirnents (Jankowski et al.. 1996). The following prirners were

used for PCR amplification: ANP forward. 5'-CAGCATGGGCTCCTTCTCCA-3'. and

ANP reverse. 5'-GTCAATCCTACCCCCGAAGCAGCT-3'. cDNA (and DNAse-treated

RNA samples used as negative control) was subjected to PCR amplification of 25 cycles

for 1 minute, at 94°C. 1 minute at 6 1 "C and 3 minutes at 73 O C . After separation on 1.5 %

agarose gels. products arising from arnplitication of cDNA (lower band) or genomic DNA

(upper band) yielded 430 bp or 541 bp fragments respectively. The bands from each

sarnple were measured by 1 mage-Quant software. and the cDNA am pl ificat ion products

were normalized to the arnount of genomic fragments (100%). as described previously

(Jankowski et al., 1996).

. . 3.1.3.7. -cal Audysis

The data are presented as Mean f SEM. The significance of differences among the

various groups was evaluated by two-way analysis of variance (ANOVA). with time as

the first independent variable and treatment as the second independent variable. fol lowed

by the Neuman-Keuls multiple comparison test. A P value of L 0.05 was considered to be

signit-cant.

3.1.4. RESULTS

3.1.4.1. Effect of ethanol on the hlpod m u r e and heart rate

Variations in the BP of SHR and WKY rats with age and treatment are shown in Fig.

3.1.1. Two-way ANOVA with time as the first independent variable and treatment as the

second independent variable indicated a significant effect of rime (F,.,, = D . S 6 . Pr0.0 1 )

and treatment (F,.,,=336.66. P~0.01) with a significant interaction of time and treatrnent

(F,,,,, =37.63. Pi0.0 1) in SHR. Similarly. a significant effect of time (F,.,, = 18.23.

P~0 .0 1 ) and treatment (F,.,,, =?O9.X. Pr0.01) with time-treatment interaction

(F, . - ,,,=23.36. P50.01) was noted in WKY rats. A rapid BP increase in both water- and

ETOH-ueated SHR was observed during the First half of the experimenr ( = 17 weeks) but

subsequently levelled off at around 200 mm Hg ( l? to 31" week). Meanwhile. WKY rats

experienced a slow and gradua1 elevation of BP values with age. The effect of ETOH

treatment in both SHR and WKY was seen as a slower rate of increase in BP compared

to corresponding water-treated controls. Thus. the BP rise in rats drinking ETOH was

s igni ficantly lower than in their H,O-treated control counterparts. In fact. a highl y

significant difference in BP between SHR-ETOH and SHR-H20 was observed from the

32"' week onward (P~O.ûû1). while in WKY rats. this significant difference was apparent

only from the 25" week of treatment (P50.05). After 32 weeks. the BP average was 189

+ 4 and 162 + 3 mm Hg ( n = 15) (p10.001) in SHR-H,O and SHR-ETOH (Groups 3 and

4) respectively. and 131 + 2 and 112 + 2 mm Hg ( n = 15) ( ~ ~ 0 . 0 5 ) in WKY-H,O and

WKY-ETOH rats (Groups 1 and 2) respectively.

A similar association was demonstratecl for HR (Fig. 3.1 2.). Again. H R values were

+ WKY water + SHR water

+ WKY ETOH + SHR ETOH

I 5 9 13 17 21 25 29 33 Time (weeks)

F Ïg ure 3.1.1. Progression of blood pressure during 8 months of water or 20% v/v ETOH consumption in WKY rats and SHR. Values are presented as means I SEM (n=15). *p<0.05, "pc0.001, ETOH versus water.

- - WKY water . SHR water

+ WKY ETOH + SHR ETOH

1 5 9 13 17 21 25 29 33 Time (weeks)

F igure 3.1.2. Progression of heart rate during 8 months of \Rater or 20% v h ETOH consumption in WKY rats and SHR. Values are presented as means *SEM (n =IO). 4.05, ETOH versus -ter.

significantly higher in SHR than in WKY rats. In both strains. HR declined gradually

during the fint 15 weeks of treatment. followed by a slow increase to initial values by the

3 P week. The effect of ETOH in both SHR and WKY was again a slower HR increase

so that by the end of this study there was a significant difference in HR between ETOH-

and H20-treated SHR. Thus. by the end of the experimental period. HR values were 441

+ 7 and 404 + 9 bears/min (n= 10) (ps0.05) in SHR-H,O and SHR-ETOH (Groups 3 and

4) respectively. and 379 + 8 and 363 + 7 beats/min ( n = 10) in WKY-H,O and WKY-

ETOH rats (Groups 1 and 2) respectively.

No signitïcant BW difference w u observed between ETOH- and water-treated

animals of both strains. Similarly. no siginificant variation between the liquid consumption

of ETOH- and water-treated SHR and WKY rats was noted. The average volume of tluids

ingested was around 50 ml/day. ETOH-treated animals consumed around 7.5 g of

ETOH/day .

3.1.4.2. Effect of ethanol on the heart ANP svstem

Plasma ANP levels were significantly lower in ETOH-treated rats than in their age-

matched controls (Table 3.1.1 .). Interestingly. there was no significant difference in

plasma A N P values between ETOH-treated adults and young 7-weeb-old animals

(p =0.795 and p =0.70 1 for WKY and SHR rats. respectively). Again. as observed for BP.

the variations in circulating ANP levels between the H,O- and ETOH-treated groups were

greater in SHR than in WKY rats. SHR-ETOH rats presented a 50 % decrease compared

to their water-treated counterparts wh ile WKY-ETOH showed a 34 % reduction (Table

3.1.1.).

The next 3 figures show corresponding tissue A N P variations in the right (Fig.

3.1.3.) and left (Fig. 3.1.4.) atria and ventricles (Fig. 3.1 S.) in terms of total ANP

content. concentration (pg/mg proteins) and m RNA levels. Au toradiograms of

representative Northern blots for atrial and ventricular ANP mRNA are shown on Fig.

3.1 A. Both ETOH- and water-treated SHR and WKY rats presented d a r levels of total

protein content in the atna (Table 3.1.2.). Since in most cases the changes in total protein

Table 3.1.1. Blood alcohol, ANP, A VP, corticosterone and aldosterone in SHR and WK Y

rats ut age 7 weeks (before ET0H treatment) and at age 38 weeks (Mer 8 months of

ETOH or H20 treatment).

WKY SHR

4.F 7 38 weeks 38 weeks 7 week. 38 weeks 38 weeks weeks Water ETOH Water ETOH

Blod alcohol content (me/dI)

12.41" 8.33 5 1.52 5 1.71 ( n = 10) (II= IO)

59.0'$' 94.0 + 16.1 & 8.7 (n = I O ) (n= IO)

307 8'''.3$ -. i: 17.7 (n= 15)

0.343'$ 4 0.037 in=4)

1 5.95" - - 2.16 In= 10)

1 1 1.6'."

I 10.2 cri= IO)

39-23 t - 18.97 (n=6)

151.9"' 5 14.9 tn= 15)

0.506'* * 0.033 (n=4)

10.88 - - 1-27 cn= I O )

633* 2 10.5 (n= I O )

Values shown are + SEM (n=number in each group). Effect of

treatment (water versus ETOH): ***P~0.00 1 . **P.0.0 1. *P<0.05. Effect of strain (WKY t-L= . . --

versus SHR): ' ''Pr0.001. ' 'PrO.01. 'P~0.05. Effect of age (7 weeks versus 38 weeks): . . . --- . . -- - "'P<O.OO 1. "P<O.O 1. 'P~0.05.

SHR WKY

water (38 weeks)

ETOH (38 weeks)

SHR

F 3.1.3. Effect of ETOH treatment on the right atrial ANP system (content, concentration and mRNA) in W rats and SHR. Values are presented as means I SEM (n=10). Effect of treatment (ETOH venus water): *p<0.05, "p<0.001. Effect of strain (SHR versus W): ttp<O.OOl.

SHR WKY SHR

1 water (38 weeks)

SHR

F ig Urê 3.1.4. Effect of ETOH treatment on the left atrial ANP system (content, concentration and mRNA) in WKY rats and SHR. Values are presented as means i SEM (n=10). Effect of treatment (ETOH verçus water): Pc0.05, "pc0.01, -p<0.001. Effect of strain (SHR versus W): m<0.001.

SHR I I

WKY SHR

SHR

water (38 weeks)

ETOH (38 weeks)

Figure 3.1.5. Effect of ETOH treatment on the ventricular ANP system (content, concentration and mRNA) in WKY rats and SHR. Values are presented as means I SEM (n=10). Effect of treatment (ETOH venus water): *p<0.05, "pc0.001. Effect of strain (SHR venus W): *<0.05, m<0.001.

Left atrium Right atrium

WKY SHR WKY SHR

ANP mRNA

Tubulin mRNA

Vent ric les WKY SHR

ANP mRNA

Tubulin mRNA

FIGURE 3.1.6. Automdiogmms of a representative Nodhern blot foratnal (A) and ventricular(6) ANP mRNA in WKY and SHR mts after 8 rnonths of water(H) or 20% ETOH treatment (E). Tubulin mRNA is used as interna1 contiol. The ANP-tub UR ratios are p resented on Fig. 3.1.6C, 3.1.7C and 3.1.8C.

Table 3.1 -2. Protein levek in the cardiuc compartmenîs of WK Y and SHR rats.

II Protein levels (mghotal tissue)

Right Atria Left Atria Ventricles

II WKY -water 7.14 + 0.21 6.17 t 0.47 8.43 + 1.05

1 S H R-ethanol 6.80 * 0.3 1 6.14 & 0.3 1 8.01 & l X * Values shown are means & S.E.M. (n= 10).

* Effect of treatment (ETOH versus water): pc0.05.

Effect of main (WKY versus SHR): 'p0.05.

content represent the changes m the total mas of the tissue and are usually proportional to

the changes in tissue weight (Deshaies et al.. 1975: Szabo et nf., 1979), this result suggens

no modification by ETOH on the atrial weight. Chronic ETOH administration resulted in

significantly higher ANP concentrations in the right and left atria of SHR and WKY

animals. Correspondingly. total ANP content was significantly higher in ETOH- versus

H,O-treated SHR and WKY rats . Northern bloc analysis revealed that ETOH had little

effect on the atrial ANP mRNA content of either WKY or SHR (Fig. 3.1.3C.. 3.1 .K.

and 3.1.6A.). although ANP mRNA was significantly higher in SHR compared to WKY

rats.

The ventricles of the SHR presented significantly higher total protein content than those

of WKY rats mdicahg ventricular hypertrophy (Table 3.1.2. ). However. ETOH prevented

the development of this hypertrophy in SHR as indicated by the reduction of the total

ventricular protein content in SHR-ETOH rats to levels observed for the total ventncuiar

protein content of WKY rats (Table 3.1.2.). Accordingly . the ventricles (Fig. 3.1 -5. ) of

H,O-treated SHR presented significantly higher total ANP content and ANP concentration

than those of water-treated WKY rats. This tïnding was further supported by a significant

elevation of ANP mRNA in hypertensive compared to normotensive animals. [n S H R .

chronic ETOH treatrnent resulted in a significant decrease of total A N P content. but not

of ventricular ANP concentration (Fig. 3. MA. and 3.1.58.). There was no sigr'f rcant

difference in the toal content and concentration of venuicular ANP between the water- and

ETOH-treated WKY rats. Correspondingly . in Northtrn Blot studies. ventricular ANP

mRNA was significantly decreased by long-term ETOH administration in SHR but not

in WKY rats (Fig. 3.1.5C. and 3.1.68.). In fact. ETOH-treated SHR presented total

ventricular ANP content and ANP mRNA values which were similar to those of water-

and ETOH-treated WKY. RT-PCR analysis was performed on ventricular tissue as an

additional control of the northern blot results (Fig. 3.1.7.). PCR amplification of cDNA

and genomic DNA gave products predicted from gene sequences. No PC R amplitïcation

was obtained when cDNA was replaced by total RNA samples. The abundance of the A N P

transcript was estimated by cornparing the hybridization signal of the cDNA PC R products

13 water (38 weeks) ETOH (38 weeks)

WKY SHR

Figure 3.1.7. Relative quantification of ANP mRNA in rat ventricles by RT-PCR The upper panel represents the mean * SEM of 3 mdependent RNA preparations. Values

were calculated as mRNA band/genomic band. A fixed amount of cDNA ( 10 ng of normalized input) was PCR amplified by the presence of 100 ng of rat gaiornic DNA acting as a cornpetitive intemal standard. The lower panel show representative phosphoimager d e n e bands of PCR products after agarose gel electrophoresis. The upper band of 534 bp originated nom genomic DNA. and the lower band of 430 bp resulted fiom the amplincation of mRNk Effect of treatment (ETOH versus water): ***p 50.00 1. Effect of strain (SHR versus WKY):Wp 50.0 1.

to the signal arising from the genornic template ( %). RT-PCR studies revealed a similar

significant decrease of ANP mRNA for SHR-ETOH only. without any modification in

WKY-ETOH rats (Fig. 3.1.7.). Again. the ANP mRNA level in SHR-ETOH was similar

to that in WKY-H.0 and WKY-ETOH.

3.1.4.3. Effect o f e t b o l on c i r m hormone leveh

Table 3.1.1. shows the blood alcohol . ANP. AVP, corticosterone and aldosterone

contenrs in the animals of the two strains. both before and after the 8 months experiment.

The animals were sacritïced during the light phase of the daily cycle (between 9 AM and

12) at a time when they are not active and are not consuming ETOH. Accordingly. the

blood alcohol content in ETOH-treated rats was generally low. although large variations

occurred. Plasma AVP levels were signitïcantly higher in the SHR-ETOH than in the

SHR-H,O group (pr0.01). but not in WKY-ETOH compared to the WKY-H,O group.

Circulating AVP levels were significantly decreased with age (psO.01) in both S H R and

WKY rats but. interestingly. significantly lower plasma AVP levels (pr0.01) were found

in young 7 weeks old S H R animais compared to age-matched WKY rats. Plasma

corticosterone levels had a tendency to be lower with ETOH but this never reached

significance. In contrat. plasma aldosterone levels were signitïcantl y lower in the S H R-

ETOH compared to the SHR-HIO group (pc0.05). but not between the ETOH and water

WKY groups.

3.1 S. DISCUSSION

The present studies demonstrated that chronic moderate ETOH consumption

prevented the age-dependent increase of BP in both hypertensive (SHR) and normotensive

(WKY) rats. The ETOH treatment was also associated with significant alterations in the

activity of the ANP system.

Since the pioneering work of Lian in 19 15. it has been well-documented that there

is a direct association between high ETOH consumption and hypertension. However. in

the last 15 years. with the publication of more extensive epidemiological studies. this

association has become less clearcut. especially with low and rnoderate ETOH

consumption (Gleiberman and Harburg. 1986: MacMahon. 1987). Indeed. it has been

repetitively reported that subjecn with moderate ETOH consumption (berween 1 and 3

standard drinks per day) had lower systolic blood pressure than both heavy drinkers and

non-drinkers (known as the U- or J-shaped curve) (MacMahon. 1987). Although some

investigators (Beilin and Puddey, 1992) still view this non-linear relationship as an artifact

(incorrect screening of subjects. etc.). a similar association between ETOH consumption

and cardiovascular diseases is now recognized (Marmot and Brunner . 1 99 1 ).

In animal models. where various confounding factors such as previous ETOH

exposure or nutritional status are easier to control. it was initially thought that ETOH at

moderate doses would either augment (Chan and Sutter. 1983) or have no significant effect

on BP (Khetarpal and Volicer. 1979). However. recent studies have shown that ETOH

in concentrations up to 70 % vlv may protect against the age-dependent increase in BP.

especially in hypertensive animals such as S H R and SHRSP (Sanderson et al.. 1983:

Jones et al.. 1988: Howe et al., 1989: Beilin et al.. 1992). Indeed. the present report

demonstrates a protective effect of ETOH on the age-dependent elevation of BP in both

hypertensive (SHR) and normotensive (WKY) rats. confirming previous reports

(Sanderson et al., 1983: Jones et al.. 1988: Howe et al., 1989: Beilin et al., 1992).

Interestingly. in WKY rats. ETOH appeared to completely prevent any age-dependent rise

in BP. Thus. this beneficiai effect of moderate alcohol consumption could be important not

only for hypertensive but also for normotensive individuals. However. i t is noteworth y that

the protective action of rnoderate ETOH consumption on the development of hypertension

is a slow process. requiring 23 weeks in SHR and 25 weeks in WKY rats to reach

statistical signi ticance.

The ETOH-induced decrease in BP could be due to dehydration and reduced blood

volume. However. this is unlikely since: a) the animals consumed equal volumes of water

and ETOH solution. b) no growth retardation was evident in ETOH-treated rats. as would

be expected if they suffered from chronic dehydration. c) plasma volumes have been

reporteci to remain unaltered w ith chronic administration of moderate amounts of alcohol

(Beilin m al.. 1992). and d) in studies where once a week water replaced ETOH solution

as the drinking fluid. to offset chronic dehydration. the BP-lowering effect of chronic

ETOH was not modified (Jones et al., 1988).

The mechanisms of this s d l e d "protective" action of alcohol are not known. In the

present experiments. we investigated the hypothesis that the A N P system may be

responsible. at least in part. for the antihypertensive action of ETOH. Indeed. A N P is

known to decrease BP (Needleman et ai., 1989: Nakao et al.. 19930. and in our

preliminary experiments we have observed that in humm. plasma ANP levels are elevated

by acute administration of low and moderate ETOH doses and that it thus may counteract

some of ETOH's pressor effects (Section 2 . 2 . ) . This was further affirmed in rats which

presented a rapid transient rise in plasma ANP levels following acute ETOH rreatment

(Section 2.1. ). 1 t thus appears important to investigate the impact of chronic ETOH

administration on ANP at a time when its antihypertensive effect is well-developed. The

previously pub1 ished chronic ETOH s tud ies have investigated the variations of natr iuret ic

peptides in normotensive (Sprague-Dawley ) rats follow ing a 6-week period of ETOH

ueatment (Wigle et al.. 1993b.c). at which time the protective effect of ETOH on BP has

not been developed yer.

Plasma ANP levels have been reported to be elevated in various models of

hypertension. in SHR (Morii et al., 1985: Gutkowska et al.. 1986a: Ruskoaho and

Leppaluoto. 1988). DOCA-sa1 t (Itoh et al.. 199 1 : Kohno et al.. 1992a) and one-kidney .

one-clip ( 1 K IC) hypertension (Garcia et al.. 1987). In the present studies. we have also

found significantly higher plasma ANP values in hypertensive animals. Circulating A N P

levels were measured by direct RIA. as described previously (Bidmon et al.. 1991 and

Section 2.1 .). This elevation appears to be secondary to the rise in BP and is therefore

believed to be. in both animal models and in humans (Hollister and Inagami. 1991). a

compensatory mechanism to prevent a furrher increase of BP. Similarly. in the present

studies. the significantly lower circulating ANP values in ETOH-treated rats (which

showed lower BP levels) could be the resul t of a diminished demand for counterbalancing

mechanisms.

In the present experirnents. moderate ETOH consumption elevated ANP

concentration and content of both the left and right atria. without altering ANP mRNA.

It can therefore be argued that the observed lower circulating ANP levels are due to

decreased ANP release From both atria. The increased ANP accumulation in atria and the

finding that atrial ANP mRNA is not decreased. but maintained at the sarne level in

ETOH-treated animais suggest also that the system is not down-regulated by long-term

ETOH use. Interestingly. higher atrial ANP mRNA and total ANP contents have been

observed in ETOH-treated (6 weeks of 20% v/v alcohol) Sprague-Dawley rats during high

BAC levels (Wigle et al.. 1993b). This rnay explain the absence of lower atrial ANP

mRNA levels during low BAC levels. despite the lower BP. Ir is possible also that when

an i mals treated chronical ly wi th moderate amounts of alcohol are exposed to stressful

situations or conditions which would increase BP (i.e.. via heightened sympathetic

activity). the atria rapidly release the larger quantities of stored ANP. thus counteracting

the hypertensive effects and preventing the development of hypertension. If this is true.

decreased plasma ANP concentrations could be expected under the normal non-stress

conditions and low BAC levels of the present studies as direct consequence of the ETOH-

induced fa11 in BP. Further experiments are needed to test this hypothesis.

Initially. it was thought that ANP was localized exclusively in heart atria (De Bold

et al.. 198 1). However. scientists have now demonstrated that ventricles synthesize and

release natriuretic hormones such as ANP and BNP. especiall y under pathophys iological

conditions (Arai et al.. 1988: Gutkowska and Nerner. 1989: Ogawa et al.. 199 1 ). 1 ndeed.

it appears that a shift in ANP synthesis and release from the atria to the ventricles takes

place with hypertension. leading to a significant increase in the proportion of circulating

ANP derived boom hypertrophied ventricular tissue (Ogawa et al. , 199 1 : Dagnino et al. .

1992). The same effect was seen in the present studies where adult S H R showed higher

ventricular ANP content and ANP mRNA than adult WKY. Interestingly. chronic ETOH

treatment prevented the development of the ventricular hypertrophy in SHR. as indicated

by the significant decrease in total ventricular protein content. total ventricular ANP

content and ventricular ANP mRNA compared to water-treated SHR, Since the BP

elevations in these animals are markedly reduced. bringing them closer to normotensives.

the shift in ANP production from atria to ventricles is thus likely to occur to a lesser

extent. This marked difference between WKY and S H R at the level of ventricular ANP

is likely to explain the greater decrease in circulating ANP observed in S HR compared

to WKY following ETOH treatment. Whereas the mild. albeit signitïcant decl ine of plasma

A N P in WKY-ETOH rats appears to be the result of atrial alterations only (decreased

release). a funher mechanism (reduced activity of the ventricular ANP system) seems to

be present in SHR. This would explain the greater effect of ETOH treatment on the ANP

system in SHR. Knowing that the ventricles have little or no storage space (Lang et al..

1992). in contrast to the atria. and therefore that ANP is released constitutively . a 3-fold

decrease of ANP mRNA in SHR will be reflected directly in plasma ANP levels. Of

course. in such a scherne. prevention of the age-dependent progression of hypertension by

ETOH is still the cause of the different modifications in the cardiac ANP systern of S H R

and WKY rats.

In fact. the important question is whether this general decrease in plasma and

ventricular ANP observed after chronic moderate ETOH consumption is secondary to the

effect of ETOH on BP? It is indeed possible that initially (as seen in acute studies for

example. Chapter 1). the activity of the ANP system is elevated by ETOH and this

increase is sufficient to mediate some of the long-term actions of ETOH on BP. The

observed results may therefore be compensatory to the repeated stimulatory effect of acute

ETOH consumption on the ANP system. such that its sensitivity is elevated in ETOH-

treated rats. Sirnilar compensatory reactions to a repeated stimulation by ETOH have been

demonstrated for several other physiolog ical systems. such as AVP. N M D A receptors.

GABA, receptors. calcium channels or Na'/K+-ATPase activity (Diamond and Messing.

1994; Nua and Peters. 1994). On the other hand. the reported ETOH effect on the heart

A N P system may also simply be secondary to iü impact on BP.

Acute ETOH consurnption mduces diuresis through the inhibition of AVP release

(Eisenhofer and Johnson, 1982). In contrast, chronic ETOH consumption is associated with

some stimulations of the AVP systern to compensate for the continuous presence of a1cohoL

resuiting m the possibility of some water retention (Eisenhofer et al.. 1985: Taivainen et a/. . 1995). Furthemore. ANP is known to suppress AVP release from the supra-optic nucleus

(Januszewicz et al.. 198613: Clark et ai.. 199 1) . Therefore. the significant elevation of

circulating AVP in the SHR-ETOH group may indicate a heightened activity of this

hormone with chronic ETOH treatment because of its lower inhibition by the significant

fail in plasma ANP. More studies are needed on the effects of ETOH on the interactions

between these two hormonal systems and on the role of these interactions in cardiovascular

regulation. Circulating aldosterone Ievels were also significantly lower in the SHR-ETOH

group. indicating a decreased activity of the renin-angiotensin-aldosterone system (RAAS)

in hypertensive anirnals chronically consuming alcohol. A similar lowering of plasma

aldosterone levels was observed in normotensive Sprague-Dawley rats by Wigle er al.

(1993b). but not in the WKY controls used in the present studies. Genetic differences

could explain this discrepancy. Indeed. there are genetic differences in the renin gene of

WKY and SHR (Kreutz er al.. 1993). I t would be interesting to Further investigate the

activity of the RAAS system in S H R as a possible rnechanism underlying the protective

effect of moderate ETOH administration.

In conclusion. the present investigations have demonstrated that the prevention of the

age-dependent increase in BP by chronic moderate ETOH consumption is associated with

significant aiterations of the heart ANP system. Indeed. circulating ANP levels are lower

fol low ing long-term chronic ETOH treatment and the development of ETOH-induced

protection against elevated BP. Atrial ANP concentration is slightly augmented without

significant variation of atrial ANP mRNA. which could suggest decreased atrial A N P

release. Ventricular ANP content and ANP mRNA are significantly lower in the SHR-

ETOH group. suggesting a possible reduction of ANP release from the ventricles.

Further experiments are needed during ETOH intoxication and with other components of

the natriuretic peptide system. such as B N P levels (Wigle et al.. 1993~). renal ANP

receptor density and central ANP and C-type natriuretic peptide. to understand the

interactions between ETOH and this system and to ascertain its possible importance in the

so-cal led " protective " effect of chronic rnoderate alcohol consumption observed in both

humans and experimental animals.


The authors thank Mr. Ricardo Claudio whose help in the care of animais made this

work so much easier. Special thanks are also given to Céline Coderre. Nathalie Charron

and Sylvie Laroque for their excellent technical assistance. This work was supported by

gram tiom Medical Research Council of Canada (MT- 10337 and MT- 1 1674). Canadian

Heart and Stroke Foundation. Caiiadian Kidney Foundation (J.G.) and by the Alcohol

Research program at Douglas Hospital (C.G.). P.G. is recipient of a studentship from the

"Fonds pour la formation de chercheurs et l'aide à la recherche" (FCAR).

Section 3.2.

EFFEXT OF CHRONIC MODERATE ETHANOL CONSWPTION ON

HEART BRAIN NATRIURETIC PEPTIDE

P. Guillaume, M. Jankowski, J. Gutkowska and C. Gianouiakis

European Journal of Pharmacology

(Eur ] Pharmacol, 3 16: 49-58, 1996)

Contniution bv CO-authors: Dr. M. Jankowski introduced me to the technical aspects of

Northem blotthg and was responsile for the RT-PCR studies. Dr. J. Gutkowska and Dr. C.

Gianouiakis were my CO-supervisors.

Acknowledgements: R Claudio was the animal technician. C. Coderre and N. Charron

performed the BNP iodinations.

3.2. EFFECI' OF cRRONIC MODERATE ETHANOL CONSUMPTION

ON HEART BRAIN NATFUURETIC PEPTIDE

3.2.1. ABSTRACT

There is experimental evidence indicating that chronic moderate ethanol consumption

delays the age-dependent mcrease m blood pressure. Smce the brain natriuretic peptide (BNP)

is a potent hypotensive hormone. the effect of chronic ethanol treatment on the heart BNP

systern was mvestigated ushg spontaneously hypertensive ( S m ) and Wistar- Kyoto ( WKY )

rats. Chronic moderate ethanol consumption resulted in significantly lower circulating BNP

IeveIs for both SHR (206.9 * 18.5 versus 306.9 i 28.1 pg/rni, n= 12. Ps0.05) and WKY rats

( 13 1.3 * 20.7 versus 220.6 * 25.0 p g / d n= 12. Ps0.05). Lefi and right atrial BNP content

and concentration in WKY rats and tefi atrial BNP content and concentration m SHR rats

were aupented by the ethanol treatment. but not atrial BNP mRNA. in ventricular tissue.

alcohol had no effect on total BNP content of either SHR or WKY rats. but it induced a

significant elevation in ventncular BNP concentration (pg/mg protein) and BNP rnRNA in

SHR but not WKY rats. Thus. chronic ethanol treatment resulted in specsc alterations in

the a c t ~ t y of the heart BNP systern.

3.2.2. INTRODUCTION

Brain natriuretic peptide (BNP). originaliy discovered in porcine brain (Sudoh et al..

1988) is now considered the second member of the natriuretic peptide family. the k a being

atrial natriuretic peptide (ANP) (Davidson and Struthers. 1994. Nakao et al.. 1993 ). BNP is

an NH2 elongated version of ANP with hi& sequence homology in its ring anicture (Lang

et ni.. 1992). However. in contraa to other natriuretic hormones. its aminoacid sequence

displays important interspecies diversity (Davidson and Struthers. 1994). In the rat. the -15

aminoacid BNP molecule is believed to be as potent as ANP in its ability to lower blood

pressure and to cause natriuresis, diuresis and vasorelaxation (Holmes et al.. 1993: Lang et

aL. 1992). Ahhough BNP is colocalized with ANP in atrial granules (Thibadt et al., 1992).

the major site of BNP synthesis and secretion in the body is the ventncle (Kohno et al..

l9Wa: Ogawa et al.. 199 1 ). Moreover. it appears that changes in the a c t ~ t y of ventricular

BNP correlate better with the progression of hypertension as well as other pathophysiologicd

conditions, such as congestive heart failure. than changes in the a a ~ t y of the ANP syaem

(Naruse et al.. 1994: Lang et al.. 1992: Kohno et al.. 1992~).

It has been reported by Howe et ni. ( 1989) and confirmed by us (Section 3.1.) that

chronic moderate ethanol consumption prevents or delays the age-dependent increase in blood

pressure in both nonnotensive Wktar-Kyoto ( WKY) and spontaneously hypertensive ( SHR)

rats. The mechanimis underlying this effect are ni1 unknown but the activation of one or

several uiembers of the natriuretic peptide family by chronic ethanol is an attractive

hypothesis. Furthermore. circulating B NP levels were recent ly found t O be elevat ed following

a short chronic ethanol treatmeot (six weeks) (Wigle et al.. 1993~). However. since the

prevention of the age-dependent increase in blood pressure by chronic moderate ethanol

consumption is a progressive effect. observed only afier 22 weeks of alcohol administration

(Section 3.1 .), it was the objective of the present studies to investigate the changes of the

plasma and heart BNP levels foliowing chroaic ethanol treatment for a period sufncient to

demonstrate significantky lower blood pressure values in the ethanol-treated compared to the

ethanol-naive rats. Circdating, atrial and ventricular BNP levels were measured by

radioimmunoassay before and after the chronic administration of ethanol. Contents of BNP

rnRNA were also evaiuated in the atria and ventricles of SHR and WKY rats.


A total of 72. 6 weeks-old. rats (36 male SHR and 36 male WKY rats) were purchased

fiom Charles River Breeding Laboratones (St-Constant, Québec. Canada). Following one

week acclirnatization period, the animals from each strain were randomly assigned to one of

the following 3 groups: 1 ) the initial age control group; 2) the water-treated control poup

and 3) the ethanol-treated group. in the initial age control group. 12 (7 weeks-old) SHR and

WKY rats were sacrificed to d o w estimation of the eEect of age on the BNP system. In the

water-treated control group, 12 SHR (SHR-water) and 12 WKY (WKY-water) rats were

given fiee access to drinking water for the 8 months of the experimental period. In the

ethanol-treated group, 12 SHR ( SHR-ethanol) and 1 2 WKY ( WKY-ethanol) rats were @en

6ee access to an ethanol solution that was gradudy increased to 20% viv in 15 days (5% viv

for 5 days 10% v/v for 5 days. 15% viv for 5 days and 20% v/v subsequently) (Howe et ai..

1989). The ethanol treatment laaed 8 months. AU animals were maintamed on a 12 h dark

and 12 h Li& cycle (lights on at 6:00 a.m) and _&en fiee access to pellet chow (Purina.

Richmond. Va) during the experirnental penod. The blood pressure and the heart rate were

measured monthly by the tail-cuff method (ffeffer et ai.. 197 1 ). At the end of the

experirnental penod the animals were sacrificeci. between 9:00 to 1 200 a. m.. during the light

cycle.

T h blood was collected in chilled 15 ml conical centrifùge tubes containing 1 mglm1

ethylenediaminetetraacetate (EDTA). 10 mM phenylmethylnilfonyl fluoride ( Sigma Chemical

Co.. P-7626) and 5 mM pepstatm A (Sigma Chemical Co.. P-4265). A 50 pl aliquot of whole

blood was placed m 450 pl of ice-cold trichloroacetic acid (6.25Y0 w/v. Sigma) for estimation

of the blood aicohol content at the tirne of death using the dehydrogenase enzymatic rnethod

(Hawkins et al.. 1966). The remaining blood was centrifuged and the plasma was stored

frozen at -75°C until assayed for BNP content. From each group. 6 animals were sacrificed.

the ventricles. lefi and nght atna were dissected and placed in separate tubes containing ice

cold 0.1 N HCI. The tissues were boiled for 5 min and homogenized using a Polytron

(Kmernatica GMBK Luzem S h r l a n d ) . Protems were estimated by the Bradford's method

(Bradford. 1976). The atria and ventricles fiom the remaining 6 animais per goups were

dissected. immediately fiozen in isopentane and aored at -75°C for estimation of BNP

rnRNA.

Plasma BNP was extracted fiom 2 ml plasma using C,, Sep-Pali cartridges (Millipore.

Milford, Ma) and was measured using a second-antibody radioimrnunoassay (RLA) ( Itoh er

ai.. 1989). Bnefly. the iodination of BNP was doue by the lactoperoxidase method. as

demibed elsewhere for ANP (Gutkowska et ai.. l987a). The standard curve was prepared

by serial dilution of synthetic rat BNP (fiom 12.2 to 3 125 pg/ml). The standards and

unknowns were incubated for 48 h at 4°C with an antibody specific for the rat BNP

(Peuinsula Laboratories, h a 1 dilution 1 :8000). This antibody reacted 100% with rat BNP-( 1-

45 ) and cross-reacted 100% with rat BNP-( 1-32), but had no cross-reactivity with rat or

human ANP. porcine or human BNP. or with arginine vasopressin. angiotensin II and

endothelin-1. %BNP (6000 cpm) was then added and incubated for 24 h at 4OC. To

separate bound fiom 6ee. the second-antiiody (GARGG. 1 5 0 dilution) and the n o m l rabbit

serum (NRS. 135 dilution) were added and lef€ to mcubate for 4 h at room temperature.

FinaUy. 6.25% polyethylengiycol was added prior to a 20 min (3000 g) centrifugation. the

supematants were aspirateci and the pellets counted in a gammacounter (Canberra Packard).

The sensitMty of this assay is 24 pdtube. The &a- and interassay coefficients of variation

were 5.8 and 9.5%. respectively. Resdts were expressed as pgml plasma.

Atrial and venaicular homogenates were ccentrifuged, the supematants were appropnately

diluted and the BNP content was measured directly using the same RIA procedure descnbed

for estimation of plasma BNP levels. Results were expressed a s pg/tissue (total content) and

@mg protein (concentration).

The RNA extraction Iiom the atrial and ventricular tissues was done by the acid

_müinidinium thiocyanate phenol chlorofonn method (Chomczynski and Sacchi. 1987). Total

RNA ( 10 yg) was separated by electrophoresis ( 1 SOh agarose gel containing 0.22 M

formaldehyde) and blot transfemed to a nylon membrane. B lots were su bsequently h ybndized

with random primed [a-"Pl-labelled cDNA probes corresponding to BNP and a-tubulin

mRNA sequences. Random priming kit (Gibco. BRL. Bethesda. MD) and [a-"PI-~CTP

(3000 Ciimmol) (Amersham. Arhgton Heights. IL) were used for labelling the probes. As

BNP probe, a 347 bp fiagment covering sequences in the 5'- untranslated region of the rat

BNP cDNA and in the 3'- sequence of the second exon of BNP gene was used (Dagnino et

al.. 1992). This fragment was generated by reverse transcription of rat ventncular RNA. As

a-tubulin probe. a 550 bp fragment of a-tubulin cDNA was generated by reverse

transcription of rat atrial RNA and amplified by the polymerase chah reaction (PCR)

(Dapino er al.. 1992). Membranes were hybrichd in b a e r containing 120 rnM Tns. 8 rnM

EDTA, 0.6 M NaCL 0.0 1% Na pyrophosphate. 0.02% sodium dodecyl sulfate (SDS). 0.03%

heparin (Sigma Chernical Co, St-Louis, MO. H7005), pH 7.4 and supplemented with 10%

dextran sulphate and IO6 dpdrnl of radioactive labelled probe. Washing was doue in 2 X

sodium chloride-sodium citrate buffer (SSC)/ 1% SDS ( I O min at room temperature). 0.1 X

SSC1O.O 1 % SDS ( 15-30 min at 65OC) and 0.1 X SSC ( 10 min at room temperature). The

inteosity of the hybridization signals were subsequently quantitated by densitomet~y using the

PhospliorIrnager (Molecular Dpamics. Sunnyvale. CA). In preliminary midies. we used both

a-tubulin and GAPDH probes to control for the hybridization signal. Our results indicated

that there was no sigiilficant effect of ethanol on either a-tubulin or GAPDH mRNAs. Thus.

to correct for ditferences in the amount of total mRNA applied on the electrophoresis geL a-

tubulin was used as intemal control and the results are expressed as the ratio of BNP/a-

tubulin rnRNAs.

RT-PCR was conducted according to previously described procedures (Dagnino et ai-.

1992). Normalized input of cDNA with 60 ng of liver @NA. 0.2 rnM dNTPs. 10 pCi "P

(ha1 concentration) and 40 pmol of fonvard and reverse primers correspondhg to sequences

in the 1 and 2 exon ofthe BNP gene. were added for amplification with 2.5 units of Thernzzrs

aqziuticzcs (taq) DNA polymerase ( Perkin-Elmer-Cetus. Nonvalk CT. USA). The following

primers were used for the PCR ampiifkation reaction: BNP fonvard. 5'-

CCATCGCAGCTGCCTGGCCCATCACTTCTG-3'. BNP reverse. 5'-

GACTGCGCCGATCCGGTC-3'. PCR amplification was camed out with 25 cycles of 1 min

at 94'C. 1 min at 6 I°C and 3 min at 72°C. Amplification of the cDNA and the genomic DNA

templates yielded respectively a 347-bp and a 546-bp fragment. M e r separation on 1.5

agarose gels BNP cDNA (lower band) and genomic cDNA (upper band) were measured on

Phosphorlrnager (Molecular Dynamics. Sunnyvale. CA) and BNP cDNA amplification

products were normaliied to the amount of genomic fragments.

Data are presented as means 5 S.E.M. The significance of difference among the various

groups was calculated by a two-way analysis of variance (ANOVA). This analysis was

followed by the Neuman-Keuls multiple cornparison test. A P value of sO.05 was considered

si+Pnificant.

33.4.1. Effert of age and ethanol on bodv weight. blood oressure. heart rate and total

grotein content in atrial and ventricular tissues.

SHR and WKY rats received a 20% v/v solution of ethanol for 8 months (32 weeks).

Controls of both grains had fiee access to normal water. Table 3.2.1. shows the changes in

body weight and daily fluid intake at various steps during the experirnental period. No

sipificant Merences were observed between the body weights of the water- vernis the

ethanol-treated animals. or between the daily fluid mtake of the water- versus the ethanol-

treated rats. For each strain of rats. a two-way ANOVA with the treatment as the fira

independent variable and time as the second independent variable was perfomed for the

blood pressure and heart rate (Table 3.2.2.). For the blood pressure. s i ~ c a n t effect of

treatment (F( l.l62)= 17-92. Ps0.00 1 ), time (F(8.162)=34.50. Ps0.00 1 ) and a significant

interaction oftreatment with time (F(8.162p4.95. Ps0.00 1 ) are demonstrated for SHR rats.

Sirnilarly. significant effects of treatment (F( l.l62)=J.O 1. Ps0.05). time (F(8.162)=4.77.

Pr0.00 1 ) and treatment by time interaction (F(8.162)=3.53. PiO.0 1 ) are present for WKY

animals. hdeed. chronic moderate ethanol consumption resulted in significantly lower BP

values from the 6' (in SKR) and 7' (in WKY) month oftreatment onward. as compared to

age-matched water-treated controls (Table 3.2.2.). For the heart rate. the two-way ANOVA

indicates a significant effect of treatment (F( J.l62)= 19.8 1. Ps0.00 1 ) and time

(F(8.162)=8.64. Ps0.00 1 ). but no significant interaction between treatment and time

(F(8.162)= 1.85. P=0.08) for SKR rats. A significant effect of treatment (F( 1.162)=4.50.

PsO.05) and time (F(8.162)=19.67. Pi0.00 1). but no significant interaction of treatment by

time (F(8,162)= 1 .O4. P=OA l ) is also observed in WKY animals. Chronic moderate ethanol

consumption significantly lowered beart rate values during the 7m month of treatment in WKY

rats and during the 8" month of treatment in SHR animals (Table 3 2 . 2 . ).

The total protein content in the atria and ventricles of SHR and WKY rats is s h o w on

Table 3-22 , in most cases. changes in total protein content represent changes in the total

mass of the tissue and are usually proportional to changes m tissue weight ( Szabo et al.. 1979:

Deshaies et al.. 1975). With age, both SHR and WKY rats presented similar increases in the

Table 3.2.2. Systolic blood pressrire attd heart rote iti WKY attd SHR rots diirittg the 8 mnths of treametrt.

BlotKi pressure (iiiiii Hg)

Hran rate (beats/iiiiii)

- -

I

1

1

4 .

I 1

7

(

l

Values shown are means f S . E . M . ( n = 10). C h The levels of significaiice refer to the differeiice betweeii treatiiieiits (water versus ethaiiol): P 4.00 1. 9 4.0 1 , P iU.05.

WKY-water 3 X 4 f I 2 3 8 0 f 1 7 3 7 0 3 3 3 9 6 k 1 1 3 4 6 1 9 3 4 8 f 1 3 3 6 8 1 1 4 3 7 3 f 3 397 f 5

WKY -eilüiiiol 3 H 6 f l I 3 9 5 f 9 3 7 2 i : 9 3 8 2 1 1 2 3 2 2 1 1 0 3 2 X f 9 344 f 5 346 I Sh 380 f 9

SHR-water 4 4 0 1 8 4 3 2 f 1 1 4 4 6 f I 6 4 2 8 k 1 2 4 2 4 k 5 448 f 13 418 1: 6 434 k 9 475 k 5

SHR-etli;iriol 4 3 O f I I 4 2 2 1 7 4 4 0 k 5 4 1 S f I S 4 0 7 k 1 1 33H f 6 382 f X 404 f 12 446 f Xh

The blood pressure and heari rate of eacli aiiiinal represent the average of tliree consecutive tail-cuff ineasuremeiits ai the end of eacli inontli.

Table 3 .LS. Protein fevels in the cardiac compartments of WKY and SHR rats.

Protein levels (mgltotal tissue)

Right Atria Left Atria Ventricles

WKY-ini tial 1.28 & O. 17 1.00 f 0.19 4.40 0.40

WKY-water 7.08 I 0. lga 6.19 I 0 . 5 8 ~ 8.54 + 1.12 b

WK Y-ethanol 7.12 + 0.42 5.24 f 0.17 8.19 _+ 1.26

SHR-initial 1.50 & 0.13 0.86 + 0.07 4.49 & 0.38

SHR-water 6.51 O. 19" 6.36 + O. 17" 14. 13 f 1 .82"~

S H R-ethanol 6.66 0.27 6.10 f 0.34 7.88 + 1.40'

Values shown are means & S.E.M. (n=6) . a h Effect of age (initial versus water): Pi0.001. C ~ r ~ . ~ l . Pc0.05. d Effect of strain (WKY versus SHR): P50.05.

Effect of treatment (water versus erhanol): ' ~ 1 0 . 0 5 .

total protem content m the atria, suggesting similar mcrease m the atrial weight. However.

even though at 7 weeks of age the total protem content m the ventricles of SHR and WKY

rats is Smilar, at 38 weeks of age the ventricles of the SHR rats presented significantly higher

total protein content compared to age-mtched WKY rats, mdicating that agkg mduced a

larger increase in the ventricular tissue weight of the SHR animals. Chronic moderate ethanol

consumption had no signifïcant effect on the total protem content m the atria and ventncles

of the WKY rats and in the atria of SHR animais. However, ethanol prevented the age-

induced mcrease in ventricuiar total protein content in SHR rats, suggestmg the prevention

of the ventricular hypertrophy usually observed with age m this strain.

3.2.4.2. Effect of age on BNP

Young (7 weebold) and aduh (38 weeks-old) SHR rats have sigiilficantly higher plasma

BNP values than WKY animals of the same age (3 32.3 p g h l 16.9 versus 194.9 pg/ml*

25.5, n= 12, Ps0.0 1, for 7 weeks-old anirnals and 306.9 p g / d 28.1 versus 220.6 pg/ml*

25.0, n=12, PsO.05, for 38 weeks-old rats) (Fig. 3.2.1.). Total BNP content (pg) is

significantly increased with age in both the nght and the lefi atria of SHR and WKY rats (Fig.

3.2.2A. and C.), while the concentration (&mg protem) is significantly decreased (Fig.

3.2.2B. and D.). As observed for the atria, total ventricular BNP content is increased with age

in both strains of animais (Fig. 3.2.28.). However, contrary to the atna, ventricular BNP

concentration is also augmented in the adult compared to young h l s (Fig. 3.2.2F. ).

Companson of the heart BNP content between the SHR and WKY rats of the same age

mdicated: (a) 7 week-old SHR rats presented a higher total BNP content in the nght atria. but

not in the lef€ atria and the ventncles, compared to 7 week-old WKY rats. No significant

difference is obsewed in the concentration of BNP in the right and left atna or in the

ventricles between the 7 week-old SHR and WKY rats. (b) At 38 weeks of age, the total BNP

content is significantiy higher m the right and lefi atria and in the ventricles of SHR compared

to WKY rats. However, this significant Merence in the total BNP content is not associated

with a Pgnificant Merence m the BNP concentration (&mg protein) in either the nght and

left atria or the ventricles of the 38 week-old SHR versus WKY rats.

WKY SHR

F ig urê 3 .2.1. Circulating BNP levels in WKY rats and SHR at 7 weeks of age (diagonal lines bars) and after 8 months of water (plain bars) or 20% v/v ethanol consurnption (horizontal lines bars). Values are presented as means i S.E.M. (n=12). Significant difTerence between ethanol- and water-treated animals of the same strain: * PcO.05. Significant difference between SHR and WKY rats of the same age and treatment: P<0.05, W<O.O1.

a wKY SHR SHR WKY

F igure 3.2.2. Effect of age on right atrial (A-B), left atrial (C-Dl and ventricular (E-F) heart BNP content and concentration. Diagonal lines bars represent 7 wek-o ld animals and plain bars represent 38 wek -o l d rats. Values are presented as means +S .E .M. (n =6). Significant difference be-en the 7 w e k s and 38 w e k s (nater) animals of the same strain: +(P 4.01, 4.001. Significant difference b e t w e n the SHR and WKY rats of the same age: ttP 4.01. ttP 4.001.

3.2.4.3. Effect of ethanol on BNP

At the time of sacrifice, the blood alcohol content detected was very low for both strains

(29.97 * 11.24 and 38.27 =: 17.82 mg/dL n=6. in WKY-ethanol and SHR-ethanoL

respectively). This low blood alcohol content is due to the fact that the animals are sacrificed

d u ~ g the light cycle (between 9:00 and 12:OO a m ) wben they are not actively consmhg

ethanoL Fig. 3.2.1. demonstrates that the circulating BNP levels are significantly lower in the

ethanol-treated rats of both strains. Plasma BNP is estimated to be 306.9 * 28.1 and 206.9

= 18.5 p g / d (n= 12. Pi0.05) in SHR-water and SHR-ethanol respectively. a 33% decrease.

and 220.6 * 25.0 and 13 1.3 * 20.7 p g M ( 0 4 2 . Ps0.05) in WKY-water and WKY-ethanol.

a 30% decrease.

The ethanol-induced changes m the total content (pg) and concentration (pumg protein)

of BNP. as well as BNP mRNA in the hean right and lefi atria and ventricles are s h o w on

the figures 3.2.3. to 3.2.7. WKY rats have increased right atrial BNP content and

concentration following chronic moderate ethanol consumption (Fig. 3.2.3. ). whereas right

atrial BNP levels in SHR are sirnilar following the 8 rnonths of either water or ethanol

administration. In lefi atnal tissue. SHR and WKY rats have sigruficantly elevated BNP levels.

both m ternis oftotal content (pg) and concentration (@mg protein) (Fig. 3.2.1.). However.

the contents of BNP mRNA m both atria are unchanged by the ethanol treatment (Fig. 3.2.5. ).

The ethanol treatment prevented the development of ventncular hypertrophy in SHR rats. as

mdicaîed by the Iower total protein content in the ventricles of ethanol- compared to water-

treated SHR animals (Table 3.2.3.). The chronic ethanol consumption also significantly

increased both ventricular BNP concentration (n=6, P=0.05) and ventricular BNP mRNA

(n=6. Ps0.05) in SHR rats (Fig. 3.2.6. and 3 -2.7. ). In contrast, total ventricular BNP content

and concentration and ventricular BNP mRNA were not affected by the ethanol treatment in

normotensive WKY animals (Fig. 3.2.6. and 3-2.7.)-

3.2.44. Ventricular BNP mRNA bv RT-PCR

To confirm the ethanol-induced increase in ventricular BNP mRNA content in the SHR

but not WKY rats, which was demonstrated by Northem blot analysis, the content of BNP

i

SHR

F igufê 3.2.3. Effect of water (plain bars) or chronic ethanol treatment (horizontal lines bars) on the right atrial BNP system (total content (A), concentration (B) and mRNA (C)) in WKY rats and SHR. Values are presented as means r S.E.M. (n=6). *P<0.01, " P<0.001, ethanol versus water.

SHR

F igure 3.2.4. Effect of water (plain bars) or chronic ethanol treatment (horizontal Iines ban) on the left atnal BNP system (total content (A), concentration (6) and mRNA (C)) in WKY rats and SHR. Values are presented as means k SEM. (n=6). * Pc0.05, ethanol versus water.

Left atrium Right atrium WKY SHR WKY SHR nnnn H E H E H E H E

BNP mRNA

Tubulin mRNA

FIGURE 3.2.5. Autoradiogram of a representative Northem blot of atrial BNP mRNA in WKY and SHR rats after 8 months of water (H) or 20% ETOH treatment (E). Tubulin mRNA is used as interna1 control. The BNP-tubulin ratios are presented on Fig. 3.2.3C and 3.2.4C.

SHR

F igufe 3.2.6. Effect of water (plain bars) or chronic ethanol treatment (horizontal lines bars) on the ventricular BNP system (total content (A), concentration (B) and mRNA (C)) in WKY rats and SHR. Values are presented as means I S.E.M. (n=6). +Pc0.05, ethanol venus water.

WKY SHR

BNP mRNA

Tubulin mRNA

FIGURE 3.2.7. Autoradiogram of a representative Northem blot of ventricular BNP mRNA in WKY and SHR rats after 8 months of water (H) or 20% ETOH treatment (E). Tubulin mRNA is used as intemal control. The BNP-tubulin ratios are presented on Fig. 3.2.6C.

mRNA in the ventricies of water- and ethanol-treated SHR and WKY rats was also estimated

by RT-PCR Results Eom the RT-PCR analysis demonstrated that long-term ethanol

treatment induced a sigdicant hcrease in the BNP mRNA content in the ventricles of SHR

rats, a 9 1% mcrease fkom the SHR-water group (Eg. 3.2.8. ). In agreement with the Nonhem

blot analysis, chronic ethanol treatment failed to mduce a signifïcant mcrease of the BNP

mRNA content m the ventricles of WKY rats.

3.2.5 DISCUSSION

Aithough the hypertensbe effect of the long-term consumption of hi& quantities of

alcohol has been weil documenteci (Riddey et ai.. 1985; Lian, 1915), the effect of chronic low

to moderate ethanol consumption remams controversial (MacMahon, 1987). indeed it has

been previousty reported by Howe et al. (1989) and confirmed by us that chronic

administration of moderate quantities of ethano1 delays or even prevents the age-dependent

increase in blood pressure not only in hypenensive animals, but in nonnotensive animals as

weil.

Since BNP has hypotensive properties, the objective of the present studies was to

mvestigate the effect of chronic moderate ethano1 consumption on the heart BNP system The

present audies demonstrated that chronic ethanol treatment induced a decrease in circulating

BNP levels of both SHR and WKY rats. The low plasma BNP levels are associated with

elevated aaial BNP content and concentration, without any alterations in atrial BNP mRNA.

In the ventricles, the BNP concentration and the BNP mRNA content are s iwcant ly

increased by the ethanol treatment m SHR, but not WKY rats.

Chronic moderate ethanol conçumption significantiy decreased plasma BNP levels and

significantly increased the content and concentration of BNP in the right and lefi atna of

WKY animals and in the lefi atria of SHR rats. It has been demonstrated that BNP is

cosecreted with ANP by the atrial tissue (Thibault et al . 1992; Iida et al.. 1990; Aburaya et

al.. 1989~). Indeed, two types of atnal granules have been reported , type 1 with ANP alone.

and type II with both ANP and BNP (Hasegawa et ai., 199 1). This may explain the similar

variations of ANP (Section 3.1. ) and BNP m the atrial tissue following chronic

SHR

1 Gencrric DNA Pcnd

BNP M?NA bnd

Figure 3.2.8. Venaicular BNP mRNA by RT-PCR in adult WKY and SHR rats. Values are calculated as BNP mRNA band/genornic DNA band (n=3). Siguficant difference between water (plain bars) and ETOH treatment (horizontal lines bars): *p ~0.05.

ethanol treatment. A differential regulation of lefi and right rat atrial cardiocytes has also been

reported (Marie et al.. 1976). It is therefore possible that ethanol may have a different effect

on the right than lefl atrial cardiocytes. as observed in SHR rats where lefi atnal BNP content

is increased by ethanol while right atrial BNP levels remain unchanged. The reason for this

strain ciifference in right atrial BNP content is unclear. but since numerous genetic and

hormonal deviations f?om normal rats have been observed in SHR anirnals (Kreutz et al.,

1992: Lang et a!.. 198 1 : Nagaoka and Lovenbeg. 1977). it may be possible that the BNP

system and its s e n s i t ~ t y to ethanol are also dif5erent in the hypertensive strain. The lower

circulating BNP levels rnay be secondary to the hemodynamic changes induced by the ethanol

treatment. However, &ce atrial BNP mRNA contents in ethanol-treated rats remained at the

same lwek as those of the water-treated controls. which displayed sigdicantly higher blood

pressure and plasma BNP levels. it may be suggested that the ethanol treatment maintained

the atnal BNP synthesis at the same level as that of the water-treated rats by a different

rnechanism than secondary hemodynamic changes due to reduced blood pressure. This

rnechanism may be either a direct or indirect effect of ethanol. Thus. it seems that in water-

treated controls the BNP a c t ~ t y is stimulated by the higher blood pressure. whereas in

ethanol-treated rats the BNP activity is stimulated by ethanol. It is possible then that when the

blood alcohol content is hi& (immediately afier drinkhg) there is an increase in atnal BNP

release. whereas in the absence or with tow levels of aIcohol, the release of BNP is not

stimuiated and BNP accumulation m the atria occurs. Indeed at the time of sacrifice (between

9:00 to 12:00 a.m), the blood alcohol content was very low for both strains of rats.

In contrast to ANP, in the heart the major site of BNP synthesis and release is the

ventricle (Lang et al.. 1992: Ogawa et a/.. 1991). Therefore. the BNP concentration in

circulation depends more on the ventncular BNP release which is controlled by the blood

pressure and the ventricular load than on atnal distention pressure (Richards et al.. 1993).

Ventncular granules are also practicaiiy nonexistent. forcing BNP to be released in a

constitutive rnanner (Davidson and Struthers. 1994). Moreover. it appears that in

pathophysiological conditions, Wte congestive heart failure or hypertension. a shifl occurs in

the production ofnatriuretic peptides (BNP, but also ANP) fiom atria to ventricles (Lang et

al., 1992). In consequence, the release of BNP (and ANP) fkom the hypertrophied ventricles

of SHR rats is PgniScantly higher than f?om the ventricles of WKY rats (Kohno et ai., 1994;

Yokota et al.. 1993; Kinnunen et ai., 1993; Roy et al., 1992; Kohno et al., 1992a; Yokota

et al., 1990; and this section).

In the present studies, chronic ethanol consumption augmented significantly BNP

concentration and BNP mRNA m the ventricular tissue of Sm but not WKY rats,

suggesting an mcrease in the rate of BNP synthesis by the ventricles. Interestingly, chronic

moderate ethanol connunption prevented ventricular hypertrophy m the SHR rats. Smce

ventricular hypertrophy is produced by high blood pressure and cardiac overload (Kinnunen

et ai., 1993; Kohno et al.. 1992a), the absence of elevated blood pressure in ethanol-treated

SHR rats may have prevented the development of the hypertrophy. Therefore, the elevated

ventricuiar BNP mRNA levels and BNP concentration, but not totaI BNP content, following

chronic alcohol treatment suggeas a direct mmulatory effect of ethanol on the ventricular

BNP n/stem of SHR rats, despite a lower ventricular mass and thus a lower basal ventricular

total BNP content than the water-treated SHR rats. Again, genetic differences between SHR

and WKY rats may explain this variation in ventricdar BNP s e n s i t ~ t y to chronic ethanol

consumption. Although the effects of acute moderate ethanol administration on the BNP

system are dl unhown, a recent study reported a significant mcrease in circulating BNP

levek (with no variations m ANP or heart BNP content and BNP mRNA) follovhg a short-

term (6 weeks) chronic ethanol treatment in Sprague-Dawley rats (Wigle er al., 1993~).

mdicathg an mcreased actiMty of the BNP system as was also suggested in the p resent midies

by the mcreased content of ventricular BNP mRNA in SHR rats. However, even though both

venûicular BNP mRNA and BNP concentration are increased in SHR rats following ethanol

treatment, plasna BNP content is decreased. The reason for the discrepancy between plasma

BNP Iwels and venaicular BNP concentration imd mRNA is unclear. It may be expected that

the lower blood pressure in ethanol-treated rats should have led to a decrease in ventricular

BNP synthesis and release and eventually to lower ventricular BNP mRNA content. inaead.

chronic ethanol treatment prevented the expected decrease in ventricular BNP a c t ~ t y in

WKY rats and even enhanced the potential BNP activity m SHR rats. Therefore, it is possible

that a transient mcrease in plasma BNP levels is produced after each ethanol drink during the

period of elevated blood alcohol content. so that the activity of the ventricular BNP system

is maintained (WKY rats) or even enhanced (SHR rats). Aiternatively. it has been proposed

that BNP mRNA is more unstable than ANP mRNA requiring lager amounts to maintain

basal tissue levels and secretion by the ventncles (Lang et al.. 1992). Thus. one would need

to ver@ (by pulse-chase experiments) whether this increase in ventricular BNP mRNA in

ethanol-treated SHR rats reflects substmtial elevations m BNP synthesis and release.

interestin&. acute and short-tem chronic ethanol experiment s have rep oned significant

increases in plasma BNP and ANP levels (Chapter 2 and Wigle et al.. 19932). In chronic

ethmol experiments, the blood pressure and circulating ANP (Section 3.1.) and BNP levels

are decreased. Nevertheless. the potential biosynthetic a c t ~ t y of the atrial and ventricular

natriuretic BNP system appears to be maintained or even enhanced cornpared to the potential

biosynthetic actMty of the correspondig water-treated controls. despite the decrease in

blood pressure observed following moderate ethanol consumption. This maintenance of the

potential biosynthetic BNP a c t ~ t y in the atria and ventncles of the ethanol-treated rats may

be due to the repetitive stimulatory effects of the daily ethanol consumption. Future audies

estimating plasma BNP levels following acute ethanol administration in rats chronically

exposed to moderate amounts of alcohol or the determination of circulating BNP levels at

various mtervals during the chronic ethanol treatment should provide a better insight on the

exact pattern of changes in the activity of the hean BNP s y a e a its possible correlation with

the changes m the blood pressure and the possible contribution of the natriuretic peptides in

the prevention of the age-dependent hypertension by moderate ethanol consumption.

Conversely. it is possible that these ethanol-induced increases in plasma BNP and ANP levels

following acute and short-term chronic ethanol exposure (Cliapter 2 and Wigle et a/. . 19%)

rnay have no long-term effects on the blood pressure. in such a case. the lower plasma levels

of BNP and ANP in rats treated chronically with ethanol couid represent only a seconda-

response to the ethanol-induced "hypotensivet' effect, which could have been mediated by

other physiological systerns, such as the ethanol-induced changes in lipoproteins (Hojnaclii

et al., 1988).

In sumrnary. the present experiments have demonstrated that chronic ethanol exposure

for 8 months alters significantly the heart %NP levels in both normotensive and hypertensÏve

rats. Circulatmg BNP levels are lowered by ethanol in both strains. Moreover. BNP mRNA

leveis are signrficant~ elevated in the ventricles but not atna of ethanol-treated SHR but not

WKY rats. Further studies should be performed to determine the significance of the ethanol-

induced changes in the a c t ~ t y of the heart BNP system on the prevention of the age-

dependent increase in blood pressure by low to moderate chronic ethanol coosumption.


The authors wish to express their appreciation to the excellent technical assistance of Ms

Céline Coderre and Nathalie Charron. Many thanks are also due to Mr Ricardo Claudio for

the excellent care of the animals. P. G. is a recipient of a "Fond pour la formation de

chercheurs et l'aide à la recherche (FCAR)" scholarship. This work was also supported by

gants fiom MRC CANADA (MT 10337 and MT 1 1674). by the Canadian Heart and Stroke

Foundation (J.G.) and by the Alcohol Research Program at Douglas Hospital (C.G. )

Section 3.3.

RENAL, ALTERATIONS OF ATRIAL NGTRIURETIC PEPTIDE

RECEPTORS BY CHRONIC MODERATE ETHANOL CONSUMPTION

P. Guillaume, T.V. Dam, C. Gianodakis and J. Gutkowska

Arnerican Journal of Physiology

(Am Physiol272: Rend Physiol. 4 1 : F107-F116, 1997)

Contribution bv CO-authors: Dr. T.V. Dam mtroduced me to the technical aspects of

autoradiography. Dr. C . Gianoulakis and Dr. J. Gutkowska were my CO-supervisors.

Acknowledgements: R Claudio was the animal technician. C. Coderre and N. Charron

perfomed the ANP and cGMP iodinations.

3.3. RENAL ALTERATIONS OF ATRIAL NATRIüRETIC PEPTIDE

RECEPTORS BY CHRONIC MODERATE ETHANOL TREATMENT

3.3.1. ABSTRACT

Previous studies have shown that chronic moderate ethanol (ETOH) consumption

prevents the age-dependent increase in blood pressure. However. the physiological syaems

mediating ETOHs antihypertensive effects are not knom. The objective of the present

studies was to investigate the effects of chronic (8 rnonths) moderate ETOH consumption on

renal natriuretic receptors of hypertensive (SHR) and normotensive (WKY) rats. using

cornpetitive binding assay and autoradiographic techniques. In the renal giomeruli. the

manmal bmdmg capacity ( B a of the heterogeneous ANP receptor population (NPR-A and

NPR-C) was significantly lower in ETOH-treated SHR and WKY rats compared to water-

treated controls. Quantification of receptor subtypes showed that this decrease was pnrnarily

the result of NPR-C down-regdation. The apparent dissociation constant (&) was also

decreased by the ETOH treatment. In the rend papilla. the B,, of the homogeneous receptor

population (NPR-A) was significantly elevated by long-terrn ETOH consumption in both

strains compared to water-treated controls. However. the Y, was unaltered by the ETOH

administration. Thus. ETOH treatment induced specific alteratioos in renal natriuretic

receptors which may play a role in the "protective" effect of moderate ETOH consumption

on the age-dependent increase in blood pressure.

3.3.2. [NTRO DUCTION

Chronic low to moderate ethanol (ETOH) consumption has been reported to prevent or

delay the age-dependent increase in the blood pressure (BP) in hypertensive (SHR) and

normotensive (WKY) rats (Howe et al., 1989 and Section 5 .1 .), and to lower systolic blood

pressure (BP) in h u m s (MacMahon, 1987). However, the mechanisrns mediating this

"beneficial" e f f i of low to moderate ETOH consumption on BP remah unknown. Following

the demonstration of elevated circulating levels of atnal natriuretic peptide (ANP) with acute

moderate ETOH administration. the natriuretic peptide family has been proposed as one of

the possible mediators of this effect (Section 2.1 .). ANP is part of a new farnily of hormonal

peptides implicated m the regdation of water and sodium homeostasis. and in the control of

BP (Gunning and Brenner. 1992). Specificaify. ANP. a 28 ammoacid peptide of cardiac

ori* has been s h o w to decrease BP by vasorelaxation. diuresis and natriuresis (Gunning

and Brenner. 1992). Additional natriuretic peptides have recently been characterized: brain

aatriuretic peptide (BNP) and C-type nafriuretic peptide (CNP) (Gunninp and Brenner. 1992).

ANP and BNP are mainly synthesized and release in the circulation by the hean. whereas

C N P is moaly found in the braio.

The physiological actions of the natriuretic peptides are mediated by 3 specific natriuretic

receptors (Koller and GoeddeL 1992). The ka two natriuretic peptide receptors (NPR-A and

NPR-B) are single transmembrane proteins with a guanylyl cyclase (GC) region in the

intraceiiular domain. producing cGMP as their second messager. ANP preferably binds to

NPR-A. whiie NPR-B appears to be the natural receptor for the C-type natriuretic peptide

(CNP) (Koller and GoeddeL 1992). The third natriuretic receptor. NPR-C. is an homodimer

protein with a very short intraceiluiar domain. Its abundance and its lower specificity to the

various natriuretic peptides prompted scientists to consider this receptor as part of a buffer

system to clear natriuretic hormones from plasma (Maack er al.. 1987). Yet. recent

association of NPR-C with CAMP has raised doubts about its sole function as a clearance

receptor (Anand-Srivastava and Trachte. 1993 ).

The presence of both NPR-A and NPR-C has been demonstrated on the epithelial

membrane of rend glomenrli, whereas only NPR-A was reported in the renal papilla (Manin

et al., 1989: Brown and Zuo. 1992). The presence of both NPR-A and NPR-C receptors in

the kidney confirms its importance m mediating the diuretic. natriuretic and hypotensive effect

of ANP. Furthemore. ETOH. a weak diuretic. is h o w n to alter the fluidity of renal

membranes and to mod* the a c t ~ t y of the Na'K+-ATPase pump in kidney tubules

(Goldstein. 1987). It is therefore possible that chronic ETOH conntmption is also causing

modifications in renal natriuretic receptor characteristics. These alterations rnay explain. at

least m part. the prevention ofthe age-dependent increase in BP by chronic moderate ETOH

conçumption.

The ami of the present studies was to investigate the effect of moderate chronic ETOH

consumption for 8 months on the natriuretic receptors in the renal glomenili and inner

meduila of spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. using

competitive bmding assays and autoradiographic techniques. In addition. the effects of ETOH

treatment on urine volume. osmolarity and Na' and K* content were measured at the end of

the experimental penod. The duration of the ETOH treatment was based on previous studies

demonstrating the presence of a ~ ~ c a n t antihypenensive effect of ETOH after 6 (Sm)

and 7 (WKY) months of alcohol consumption.

3.3.3. METHODS

3.3.3.1. Animai tre-

48 male SHR and 48 male WKY rars. aged 6 weeks. were used in the present studies

(purchased from Charles River Breeding Laboratories. St-Constant. Québec. Canada).

After a 7 days acclimatization period. the animals from each strain were randomly

assigned to one of the following 3 groups: Grou? 1 : 18 animals from each strain were

given drinking water containing moderate amounts of ETOH (WKY-ETOH and SHR-

ETOH). ETOH was gradually added ro the drinking water. as previously described by

Howe et al. ( 1989). Brie tly . rats received 5 % v/v for 5 days. 10 % vlv for 5 days. 15 %

vlv for 5 days and then 20 % v/v for the remaining of the 8 rnonth experimental period.

During that time. access to pellet chow (Purina. Richmond. VA) and liquid was ad

libitum. Grou? 2: 18 animals from each strain were given free access to food and water

(WKY -H,O and SHR-H,O). . G r o u : To characterize the renal natriuretic receptors of

SHR and WKY rats at the beginning of the study. 12 rats from each strain were sacrified

at 7 weeks of age.

Body weight and blood pressure were measured at the beginning of the study (7

weeks-old) and every month during the 8 months experimental period. The tail-cuff

method was used to record BP.

3.3.3.2.

7% months into the experiment. 6 rats per group were randomly selected to be placed

in metabolic cages. Urine collection was monitored on a 12 hours basis corresponding to

the light and dark cycle of the room. Animals were given free access to ETOH or water

throughout the 3 days of the experiment. while food was given only during the dark cycle

to prevent any contamination of the urine during the light cycle. Urine volumes were

measured directl y and the urine samples collected during the 1 ig ht cycle were subsequentl y

kept frozen for estimation of the Na+/K + concentration and osmolaliry. Na' and K ' were

meassured directl y by an Instrumentation Laboratory autoanalyser. Osmolality was

est imated using 50 pl-frozen samples in an osmorneter (W ide-Range Osmometer.

Advanced Instruments. Needham heights. MA).

3.3.3.3. Ilrinw cGMP excretion

Excreted cGMP levels in the urine samples collected during the light cycle were

determined by radioirnmunoassay as previously described (Tremblay er al.. 1993).

. . 3.3.3.4. ANP rad]-av (RI&

Trunk blood was collected in chilled 15 ml conical centrifuge tubes containing

protease inhibitors to the following final concentrations: 5 mM pepstatin A (P4265. Sigma

chemical CO.. St-Louis. MO), 10 m M phenylmethylsuifonyl tluoride (PMSF) (P7626.

Sigma) and 1 mg/ml ethylened iam ine-tetra-acetate (EDTA). The blood samples were

immediately centrifuged at 4°C and the plasma stored at -75°C for ANP measurements.

Plasma ANP levels were measured by a direct RIA (Gutkowska et al.. 1984). The

validity of the direct assay for the investigation of the relative differences between various

treatments has been previously demonstrated (Section 2. I .).

3.3.3.5.Jllood &oh01 content (BAC)

A 50 pl aliquot of whole blood was placed in 450 pl of ice-cold trichloroacetic acid

(6.25 % w/v, Sigma) for estimation of the BAC at the time of death. using the

dehydrogenase enzymatic method (Hawkins et al.. 1966).

. . . 3.3.3.6. Pre-n of membranes for competitive bind-

After 8 months of treatment. rats were sacrificed by decapitation and their kidneys

were rapidly excised and decapsulated. The kidneys of 12 rats/group were randomly

chosen to be analyzed by competitive binding assay. while the kidneys of the rernaining

6 rats/group were left for autoradiographic studies. The kidneys of 12 young (7 weeks-

old). ETOH-naive animals were also prepared for competitive binding assays. Each kidney

was cut longitudinaily and the inner medulla (IM) was removed and kept frozen ar -75°C

until assayed. The ourer medulla was discarded and the remaining cortical tissue was

minced in a tissue grinder. Subsequently . glomeruli were isolated according to the graded

sieving method of Misra (1972). slightly modified. Brietly. the cortical mesh was

suspended in 0.9% saline and filtered through a 100-pm nylon sieve. Then. the collected

material was passed successively through a 50-pm and a 100-pm sieves. Finally. a last

sieve of 150 -pm was used to collect the glorneruli preparation. This preparation was then

washed in 0.05 M Tris-HCI buffer. pH 7.4 (washing buffer). before being centrifugated

( 180 rpm for 3 minutes) and resuspended in the same buffer. The solution was kept at -

75°C until used.

The glornerular solutions were homogenized with a Polytron (Kinematica GMBH.

Luzern. Switzerland). The homogenates were centrifuged at l5OOOg (4°C) for 30 minutes

and were then washed twice with 0.05 M Tris-HCI buffer. pH 7.4 (washing buffer). The

pellets were then resuspended in 1 ml of the same buffer. Protein concentrations were

measured using a modified version of the Bradford's method. using bovine serurn albumin

(BSA) as standard. Specitïc binding of ANP increased linearly with increasing protein

concentrations between 25pg and 50 pg . Accordingl y. the membrane preparat ions were

diluted to a 35 pg150 pl concentration (well within the linear range) with the competitive

binding assay buffer which consisted of 0.05 M Tris-HCI. pH 7.4. 0.1 % Bacitracine

(Sigma chemical Co., B-0125). 0.5 mM phenylmethylsulfonyl fluoride (PMSF) (Sigma

chemical Co., P-7626). 5 mM MgCl, (Fisher, Fair Lawn. N1. M-33), 0.493 % MnCI,

(Sigma chemical Co.. M-3634). 0.037% EDTA and 0.4% BSA.

Renal inner medullary tissues were homogenized in washing buffer with a Polytron

(Kinematica GMBH. Luzern, Switzerland). Homogenates were centrifuged at 4000g (4°C)

for 70 minutes. Supernatants were subsequently centrifuged at 14000g (4°C) for 45

minutes. Pellers were resuspended in 2 ml of washing buffer. homogenized and centrifuged

again at 14000g (4°C) for 45 minutes. Pellets were finally resuspended in 500 ml of the

sarne buffer and an aliquot was taken for measurements of proteins. As observed for the

glomerular membranes. specific binding of ANP increased linearl y with increasing protein

concentration between 25pg and 50 pg. Aceordingly, the membrane preparations were

diluted to a 30 pg/50 pl concentration with the membrane buffer.

. . . . 3.3.3.7. -ive bind-

The competitive binding assays were carried out according to the method described

by Gutkowska et al. (1988). Binding as a function of time (room temperature) showed

bind ing equil ibrium after I hours. Displacement studies were therefore performed in

duplicates at room temperature for 3 hours with the following components added in

succession on ice: competitive binding assay buffer. standards ( 100 pl). '?-AN P (70000

cpm/50 pl. specific activity: 1000 Ci/mmol) and glomerular or IM membranes (50 pl ).

The standards were ANP,,.,,, ( 10"' M to M) and des-[CiIn"" Seri''. Gly"". Leu"'.

GlylYANPl,,2.,21 (or cANF) ( IO-" M to IO-' M). a ring-deleted ANP analog which binds

specifical l y to NPR-C receptors (Peninsula Laboratories) (Maack et al.. 1987).

Displacement with these unlabelled natriuretic peptides al lowed the d ifferentiation between

the receptor subtypes.

After the 3 hours incubation period. the reactions were stopped by dilution with 3 ml of

cold 0.05 M Tris-HCI buffer and filtered through 1 % polyethylenimine (PEI) (Sigma

chemical Co., P-3 143)-treated Whatman GF/C filters. Filters were washed twice with 3

ml of 0.05 M Tris-HCI buffer. allowed to dry and counted in a gammacounter (Canberra

Packard) .

3.3.3.8. AutorridiograDhi-

The kidneys of 6 ratslgroup were rapidly decapsulated. frozen intact in isopentane

and kept at -75°C until used. 20-pm-thick cryostat sections were prepared and thaw-

mounted ont0 poiy-L-lycine treated slides for autoradiography as described elsewhere

(Brown and Zuo. 1992: Mukaddarn-Daher et al.. 1995). Briefly. kidney sections were

preincubated for 15 minutes at 25°C in 50 m M Tris-HCI buffer, pH 7.4. containing O. 1 %

pol yethylenimine to reduce the non-specific binding of 1'51-ANP. Sections were then

incubated at 25°C with 50 m M HCL buffer. pH 7.4. containing 150 mM NaCI. 10 m M

MgCl?, 40 pglml Bacitracin. 0.5% BSA and 50 pM "'1-ANP. The cornpetitive inhibition

of total l5I-ANP binding to the membranes by unlabelled peptides was examined for each

animal by coincubating with various concentrations of unlabelled cANF ( 10-". IO-' . 10.'

and 10h M) at room ternperature for one hour. The binding observed in the presence of 10-

W was considered the non-specific binding (NSB). The incubation was stopped by

washing the slides in 4 successive dishes containing 50 mM Tris-HCl. pH 7.1 (4°C).

followed by a final dip in water ro wash the salts. Sections were left to dry overnight and

then cxposed for 5 days on Phosphor-sensitive screens. The intensity of the signals were

subsequently quantitated by densitometry using a Phosphor Imager (Molecular Dynamics.

Sunnyvale. CA). Relevant kidney sections were transferred to Maclntosh and processed

there for better quality of the irnaging.

3.3.3.9. Statisîia

For each group. the maximum binding capacities (BmJ and apparent dissociation

constants (K3) were calculated from the cornpetitive binding assays in both glornerul i and

IM using the LIGAND iterative model-fitting computer program. Results are presented

as Mean + SEM. Comparisons between groups were done by two-way ANOVAs with the

strain (WKY or SHR) as the first independent variable and the treatrnent (HLO or ETOH)

or the age (7 weeks-old or 38 weeks-old) as the second independent variable. followed by

the Neurnan-Keuls multiple cornparison test. A p value of 50.05 was considered

significant.

3.3.4. RESULTS

3.3.4.1. BP and ~ l a s m a ANP l e v e l ~

The chronic moderate ETOH consumption si@cantly prevented (m WKY rats) or

delayed (in SHR rats) the age-dependent increase in BP (Table 3.3.1. ). A two-way ANOVA

with the strain and the treatment as the two mdependent variables indicated a significant effect

of the strain (F,,,=307.80, ps0.001) and the treatment (F1,,=49.25, ps0.001), but no

significant strain by treatment mteraction (F1.,,=O. 10, p=0.75) on the BP.

The age-dependent increase in circulating ANP levels was also prevented by the ETOH

treatment m both SHR and WKY rats. Indeed, the plasma ANP levels were 54 and 28%

lower m the ETOH- versus the Hetreated SHR and WKY rats, respectively (Table 3 2.1. ).

At the tMe of sacrifice (between 9:00 and 12:OO AM), the blood alcohol content (BAC) was

28.9 * 12.2 and 39.2 * 18.9 mg/dl in WKY-ETOH and SHR-ETOH, respectively. This low

BAC is explained by the fàct that the animais were sacrificed during the light cycle, when they

are not actively eating and drinking and thus not consurning ETOH.

3.3.4.2. Urine analvsis

On the 36& week of age, urine was collected for 3 consecutive days fkom six animals of

each experimental group. Long-term alcohol drinkmg induced a sigmficant decrease in the

amount of urine coUected (Figure 3 -3.1 A. ). Urine volumes are measured as 8.4 * 0.6 and 4.6

* 0.6 d d a y (n=6, p 50.00 1) in WKY-H,O and WKY-ETOH respectively, and as 6.8 * 0.6

and 4.6 * 0.6 (n=6, poO.05) in SHR-H,O and SHR-ETOH respectively. No si&cant

dinereoce between strains is noted. Urine of chronicaily ETOH-fed animals is also more

concentrated, as demonstrated by the signiticantly elevated osmolarity values (Figure

3.3.1 B. ). Nevertheless, the daily fluid intake and the body weight (Table 3.3.1. ) are not

difTerent between the water- and ETOH-treated rats of both arains, suggeaing no

dehydration or volume depletion m alcohol-fed animals.

Urine sodium concentration is sigdicantly increased in ETOH-treated animals of both

strains (40.2 * 6.4 versus 8 1.1 * 8.3 mM, n=6, p sO.0 1, m WKY rats, and 35.0 5.1 vernis

55.1 k 6.2 mM, n=6, ps0.05, in SHR rats) (Figure 3.3.2AJ. Interestingly. the sodium

Table 3.3.1. Blood pressure (BP). body weight (B W). liquid consumption (ZC) and

circulnrng ANP levels in SHR and MY nus ut age 7 weekr &@ore ETOH trearmenr) and

at age 38 weeks (Mer 8 m n i h of ETOH or H,O treatment).

7 weeks BP (mm Hg) 1 1 3 2 4 133 * 4" BW (g) 288 i= 3 286 * 4

LC ( d d a y ) 48*4 49 I 4

PlasmaANP(pg/lOOyl) 104.0k5.1 144.4 * 5.9'

38 weeks BP (mm Hg) 132 k 2::j 186 * 4w.$$:

(water) BW(g) 462 k ît:: 45 1 * 8$z$

LC (mVday) 81 * St:: 82 i 6:::

PlasmaANP(pK/lOOpl) 147.9k7.0:: 259.5 k 1 8Srn*:::

Xweeks BP(rnmHg) 109 * 1 * * 165 * 4***

(ETOH) BW(g) 434 5 I I 460 i 6

LC ( d d a y ) 77 i 6 79 i 6

PlasmaANP(pg/lOOy1) 106.9*5.1" 120.4 * 12.0*** . . - -

Significant difference between strains (WKY versus SHR): 'P~0.05. "Pc0.01. . . . --- * * * P50.001.

. . . S ignificant difference between age (7 versus 38 week-old): ' i ~ c ~ . ~ 1. "'Pc0.00 1.

La **a

Significant difference between treatments (ETOH versus H20): Pc0.01. Pc0.001.

Values are expressed as means * S.E. M. (n= 12).

WKY SHR

WKY SHR

1 water 1

F igure 3.3.1. Urine volume and osmolarity in SHR and W rats after 7% months of ETOH or water treatment (n=6, mean of 3 consecutNe days). ETOH versus H20: *p<0.05, pc0 .01 , -p<0.001.

1 water UI] ETOH

I

SHR SHR WKY

T

WKY SHR SHR

F igure 3.3.2. Urine sodium concentration (A), sodium excretion (8). potassium concentration (C) and potassium excretion (D) in SHR and WKY rats after 7Xmonths of ETOH or meter treatment (n 4, mean of 3 consecutive days). ETOH versus H20: P 4.05, 4.01, -p 4.001. WKY versus SHR: mQ.01.

excretion is also increased by ETOH, even with the reduction m total urine volume (Figure

3 -3 -2B.). In contrast, urine potassium concentration is not different between the water and

ETOH groups of the same grain (Figure 3.3.2C.). However, K' concentration is higher in

ETOH-naive SHR (90.6 * 2.3 m . , n=6) compared to ETOH-naive WKY rats (75.1 * 2.6

mM, n=6) (po0.01). Because of the reduced urine volume, the potassium excretion is lower

in ETOH-treated rats (Figure 3.3.2D. ).

3.3.4.3. Comaetitive bindine studies

The density and afEnity of natriuretic receptors are evaluated by cornpetitive inhibition

of "1-ANP binding to the membrane receptors by mcreasing concentrations of unlabelled

ANP or CM.

a) Glomemlur nutriirretic receptors:

The kmetic parameters obtasied from the cornpetitive binding curves are s h o w on Table

3.3.2. To ailow the differentiation between guanylyl cyclase and "clearance" receptors.

IabeUed ANP is displaced by increasing concentrations of cold CM. This displacement

represents between 50 and 90% of the total glomedar natriuretic binding sites in WKY and

SHR rats, so that the majority of the natriuretic receptors in renal glornerular membranes are

of the NPR-C subtype.

Strain effect :

Both young (7 week-old) and adult (38 week-old) ETOH-naive SHR rats exhibit lower

total glomerular natriuretic bmding sites when compared to ETOKnaive WKY rats of the

same age, as observed by the lower displacement of '"1-ANP by unlabelled ANP (Figure

3.3.3A. and C.). The B, for glomerular NPR-C receptors obtained from the inhibition of

'%WP binding by cANF is also sigiuncantly lower m SHR compared to WKY rats of the

same age (Table 3.3.2.). In contrast, the density of the guanylyl cyclase receptor subtype

@PR-A), obtained by subtracting NPR-C receptor number fiom total glomerular binding, is

not Sgiuficantly different between the two strains of rats (Table 3.3.2. ) (Kollenda et aL . 1 990 :

Mukaddam-Daher et al., 1995).

Table 33.2. Kittetic parameters for giornemlor mtriuretic receptors in SHR a d WKY rats ut age

7 weeks @e@e ETOH treatrnent) and ai age 38 w e e b (after 8 month of ETOH or water

treatment) .

WKY SHR

Bm, Bm, Ed

7 weeks Total 1367 * 141 0.56 * 0.04 700 * 193* 0.32 * 0 . 0 4 ~

NPR-C 71 1 * 42 4.8 * 0.7 330 * 21m 10.3 = 1.0"

NPR-A 656*91 370 i 94

%NPR-C 52.0 47.1 +++

38weeks Total 4834*3lgtt: 1.4 1 * 0 . 0 4 ~ ~ ~ 2740 * 193".:: 1 .O5 i 0.04 +++

(water) NPR-C 4434 i 6.4 * 0.8 1970*274w0zz 25.3+2.5"*:

NPR-A 400 * 178 770 s 233

%R-C 91-7 71.9

38weeks Total 1966 * 188" 0.48 * 0.04~" 1142 168" 0.65 r 0.06"

(ETOH) NPR-C 1327 198"' 1.4 * 0.6 556 * 42" 16.3 1 1.1

NPR-A 539 155 586 * 82

%NPR-C 67.5 48.7 ..- ---

ignificant difference between ~trains (WKY versus SHR): 'Pr0.05. "PgO.01. ' "Pc0.001. . . . 7 - Y

Significant difference between age (7 versus 38 week-old): ' ~ i 0 . 0 5 . '*P.-0.0 1. "'PdLOû 1. . iL a+*

Signifiant difference between treatments (ETOH venus H,O): ' ~50 .05 . Pc0.0 1. Ps0.00 1.

Values are expressed as means S.E.M. of two separate bindmg experiments doue in tnplicate. Six

rats per group were used for each experiment.

7 weeks 38 wee ks

-log ANP (M) -log ANP (M)

O -12 -11 -10 -9 -8 -7 -6

-log cANF (M) -log cANF (M) - WKY water - SHR water

---I)--- WKY ETOH --a--- SHR ETOH

Figure 3.3.3. Cornpetitive inhibition of 1251-ANP binding by increasing concentrations of unlabelled ANP and cANF to the glomenilar membranes of SHR and W rats at age 7 weeks (before ETOH treatment) and at age 38 weeks (after 8 months of ETOH or water treatment). Each point is presented as %BR30 to show variations in Kd, where B and Bo are the binding with

- and without displacing peptides, respectiveiy, expressed as the mean (I S.E.M.) of two separate binding experiments done in tripkate. Six rats per group were used for each experiment. (Representative competition binding cums expressed as BTT to show variations in Bmax, where T is the total binding, are presented as inserts).

203

The dissociation constant (&) for total glomedar ANP bindmg sites is lower in SHR

compared to WKY rats of the same age. However. the affinity of glornedar NPR-C

receptors is lower m both young and adult SHR compared to age-rnatched WKY rats (Table

3.3.2.).

Age effect:

The total number of gIomedar natriuretic binding sites is increased with age for both

WKY and SHR rats (Table 3.3.2.). This increase is mady due to the augmentation of NPR-C

receptor numbers. The dissociation constant (KJ for the giomedar ANP binding sites is also

increased signifïcantly fiom the 7- to the 38-week-old animals of both strains.

ETOH eEect:

The displacement of ' 3 ~ - ~ by unlabelled ANP produces a significant reduction in the

total glomerular natriuretic bmding sites in chronically ETOH-treated animals of both strains

compared to controls (Figure 3 . 3 . K . ) . Similarly. the displacernent of labelled ANP by cold

cANF produces a sipificant decrease in the glomerular NPR-C receptor binding sites in

ETOH- compared to H20-treated rats (Figure 3.3.3D.). This result mggens a sensitive

discrimination between the two populations of receptors with chroaic ETOH treatment.

Although more specinc agonins of NPR-A are needed. the direct extrapolation of NPR-A

receptor numbers fiom the calculated glomemlar total ANP binding sites minus the NPR-C

receptor density (Kolienda et ai.. 1990: Muhddam-Daher et al., 1995 ) suggests a sipificant

decrease of clearance receptors. d o u t any significant variation in NPR-A receptor numbers

(Table 3.3.2.). Indeed the long-tem ETOH administration decreases the proportion of NPR-

C in glomedar membranes. as calculated by displacement studies with cold ANP and cold

cANF (fiom 71.9 to 48.7% and fiom 9 1.7 to 67.5% oftotal receptors number in SHR and

WKY rats. respectively ).

The dissociation constant (4) for the total glomerular natriuretic receptors and for the

NPR-C receptor bindiig sites are decreased with ETOH in both strains of animals (Table

3.3.2.).

6) Irrrrer meduifury (IM) mttriuretzc receptors:

The hetic parameters obtained fiom the cornpetitive bmdiog curves are shown on Table

3 -3.3. The rend papilla is beiieved to possess mostly NPR-A receptors. Nevertheless. the

possible presence of other natriuretic receptors is investigated in the present experiments by

the displacement of '"I-ANP with cold CANE As expected. mcreashg concentrations of

unlabeued CAM? are not progressively inhibithg the '"1-ANP binding. except at its highest

concentration ( IOn M) where it displaced the labelled peptide by ody 14?h (WKY) and 6%

(SHR). dernonaratkg the absence of NPR-C receptors (figure not show).

Strain effect:

in contraa to the glomedar natriuretic receptors, both young (7 week-old) and adult

(38 week-old) ETOH-naive SHR rats exhibit higher total b e r medullary ANP receptors

compared to age-rnatched ETOH-naive WKY aaimals. as demonstrated by the grearer

disp lacement of "'EANP by unlabelled ANP (Figure 3.3 -4 and Table 3.3.3. ).

The dissociation constant (b) for the total papillary ANP binding sites is not different

between the SHR and WKY rats of the same age (Table 3 - 3 3 . ).

Age effect:

The total inner medullary natriuretic receptors are decreased with age in both strauts of

rats (Table 3.3.3. ). However. the dissociation constant (KJ is unaltered between the 7- and

the 38-week-old SKR and WKY rats.

ETOH effect:

The displacement of "'1-ANP by unlabelled ANP demonstrates a significant

augmentation in the total b e r meddary ANP binding sites in chronkally ETOH-treated

animals of both strains compared to controls (Figure 3 34B. and Table 3 -3.3. ).

The a E t y for the total papillary natriuretic receptors is not altered by the ETOH

treatment in both SHR and WKY rats (Table 3.3.3.) .

3.3.4.4. Autoradio~ra

To furtber illustrate the differential regulation of the glomedar ANP receptor subtypes

by ETOH admhistration in adult SHR and WKY rats, the autoradiography of whole liidney

Table 3 3 3 . Kimtic praameters for papillary rzatriuretic receptors irt SHR arid WK Y rats

at age 7 weekr fiefore ETOH trentmertt) midut age 38 iveeh m e r 8 rnor~thr of ETOH or water

II SHR

7 weeks Total 429 * L 1 0.46 * 0.02 727 * 37" 0.38 = 0.0 1

38 weeks Total 345 * 14:: 0.42 * 0.04 105 * 25':tt 0.32 = 0.0 I

(water)

38 weeks Total 49 1 * 44* 0.4 1 * 0.02 717 i. 61" 0.35 - 0.06 11 (ETOH) Significant difference between strains (WKY versus SHR): '~~0.05. "~<0.01.

Significant difference between age (7 versus 38 week-old): ''~~0.01. " i ~ c O . O O 1 . - Significant difference between treatments (ETOH versus H,O): 'Pr0.05. P50.01.

Vahes are expressed as means * S.E.M. of two separate binding experiments done in tnplicate.

Six rats per group were used for each experiment.

7 weeks

A)

38 weeks

-log ANP (M) -log ANP (M)

- SHR water

--+--- WKY ETOH --a--- SHR ETOH

F ig urê 3 3.4. Cornpetitive inhibition of 1251-ANP binding by increasing concentrations of unlabelled ANP to the papillary (lM) membranes in SHR and W rats at age 7 weeks (before ETOH treatment) and at age 38 weeks (after 8 rnonths of ETOH or water treatment). Each point is presented as %B/Bo to show variations in Kd, where B and Bo are the binding with and wÏthout dispbcing peptides, respectkiy, expressed as the mean (i S.E.M.) of two separate binding experiments done in triplicate. Six rats per group were used for each experiment. (Representative cornpetlion binding cuwes expressed as B K to show variations in Brnax, where T is the total binding, are presented as inserts).

sections ("'1-ANP) displaced by unlabelled cANF is also perfomed.

The displacement of '151-ANP by cold cANF is shown on Figure 3 - 3 5 Because this

synthetic ANP analog binds specifically to NPR-C. it ailows the differentiation between the

"clearance" and guanylyl cyciaç- receptors. 80 to 90% of the total cortical bmding is inhibled

by IO4 M CM, coafirming that the majority of the namuretic receptors in the cortical tissue

is of the NPR-C abtype. In contrast, total inner meddary bindmg is ody partially inhibited

by the highest concentration of unlabelled cANF ( 1on M). suggesting the preponderance of

guanylyl cyclase receptors in the [M (Figure 3.3.5E. and F. ). L

ETOH effect:

Quantitative densitometric analysis shows a lower displacement of total glomerular '"I-

ANP binding by unlabelled cANF ( l ~ - ' and I O - ~ M) m ETOH-treated SHR and WKY rats than

in H@-treated controls (Figure 3.3.5. and 3.3.6A.). This result suggests that in the ETOH-

treated rats. the relative proportion of NPR-C receptors is lower than in the water-treated

controls. supporting the conclusion of the radioreceptor audies.

Although the s e n s i t ~ t y of the autoradiographic technique does not ailow the

visualization of ETOH-induced changes in the total inner medullary (LM) ANP receptors

(Figure 3.3.6C. ). a lower displacement of total outer meduiiary (OM) "'1-ANP binding by

cold cANF is observed for both grains of ETOH- compared to water-treated rats (Figure

3.3.6B.).

3.3.4.5. Urinarv cGMP excretion

in order to link the glomeruiar and papillary ETOKinduced alterations in renal

natriuretic receptors with the possibility of elevated renal effects of the natnuretic peptides.

the urinary excretion of cGMP is examined following 7% months of water or ETOH

treatment.

Chronic ETOH consurnption is associated with a significant increase (in SHR rats) and

with no signifiant change (in WKY rats) in the daily excretion of cGMP when compared to

water-treated controls, despite the lower plasma ANP levels in ETOH- compared to water-

treated animals (Figure 3.3.7.). This result suggests that the ETOH-induced glomerular and

SHR 10-7 M cANF

WKY 10-7 M cANF

106 M cANF

FIGURE 33.5. Autoradiograp hs of b M i n g of 50 p M labeled ANP in the kidneys of adult SHR and WKY rats, after 8 months of €iDH or water treatment. B inding is shown in presence of 10-7 (A to D) and 10-6 cANF (E and F).

1 O water ETOH 1

SHR

F igure 3.3.6. Quantification by densitometry of the displacement of total 1251- ANP binding by 10-7 M unlabelled cANF in the kidneys of adutt SHR and WKY rats, after 8 months of ETOH or water treatment (n=6). Resutts are expressed as: % of total 1251-ANP binding, in (A) glornenili, (B) outer medulla (OM), and (C) inner medulla (IM). ETOH versus H20: p<O.Ol , "p<0.001.

SHR

a water

ETOH

F ig ure 3.3.7. Unnary excretion of cGMP (nMfday) measured from the urine collected during the light phase of the daily cycle following 7% months of water or ETOH treatment in SHR and WKY rats (n=6. mean of 3 consecutive days). ETOH versus H20: *pc0.05.

papillary modifications in renal natriuretic receptors may produce potentially greater cGMP-

mediated renal effects. demonsrrating a fùnctional significance of these specific receptor

alterations.

3.3.5. DISCUSSION

The present experiments have: (a) demonstrated that chronic moderate ETOH

consumption &ers rend ANP receptors. mducing a decrease in total glomenilar ANP binding

sites (mostly NPR-C receptors) and an mcrease m total papillary ANP receptor density in both

SHR and WKY rats; (b) s h o w a lower number of total glomeruiar ANP bindmg sites and a

higher number of total papilIary ANP receptor bmding sites in both young (7 weeks-old) and

aduh (38 weeks-old) SHR compared to WKY animals: and (c) characterized the presence of

an heterogeneous ANP receptor population (NPR-A and NPR-C) in rat renal glomemlar

membranes and of a . homogeneous natriuretic receptor population (NPR-A) in the rat renal

papilla.

Alcoliol has two very difEerent effects (acute and chronic) on the body. Acute

administration of ETOH mcreases the disorder (fluidking effect) of the renal lipid membranes.

and is known to inhibit the a c t ~ t y of the Na'K'ATPase purnp throughout the nephron. thus

affecting the reabsorption of Na' (Rothman et al.. 1992). Water and salt excretion is generally

increased (Wadstein and ohlin. 1979). As tolerance develops. the membranes nSen

(Goldstein, 1987). the activity ofthe Na'K'ATPase enzymes is increased. and Na* and water

retention may occur duruig chroaic alcoholism (Ray et al.. 1992).

The present audies showed that chronic moderate ETOH consumption significantly

prevented the age-dependent increase of the BP in both WKY and SHR rats. This is in

agreement with earlier reports of an hypotensive effect of chronic low and moderate ETOH

administration (Howe er al.. 1989). Dehydration of the ETOH-treated animals is unliliely. as

indicated by the absence of a body weight or fluid intake modïcation fiom water-treated

controls.

a) Glomenr lar natrizïretzc receptors:

The present snidies confbmed earlier reports demonstrating the presence of NPR-A and

NPR-C receptors m renal glomedar membranes. with the "clearance" receptor in excess

amounts (50 to 80% of the total number) (Martin et al.. 1989: Brown and Zuo. 1992). The

two d i h a glomerular ANP receptor populations are mainiy located on the epithelial cells.

with smaller aumbers on the renal microvessels and mesangial cells (Bianchi er ai.. 1986:

Chansel er al., 1990).

Age and strain effects:

Decreased total glornerular ANP binding sites have been demonstrated in various models

of adult hypertensive aniu.uk. such as in the SHR (Ogura er ai.. 1989). the lKlC rat

(Bonhomme and Garcia. 1993). the Lyon hypertensive rat (Gauquelin et al. . 1 993 ) or m the

DOCA-salt treated rat (Nugiozeh et al.. 1990). The present report has extended this result

for young (7 week-old) SHR rats. suggesting the existence of lower total glomerular ANP

receptors during the pre-hypertens~e stage. This strain-induced modification in renal ANP

receptors may be secondary to the elevated circulatmg ANP levels noticed in 7 week-old SHR

compared to 7 week-old WKY rats.

ETOH effect:

In the present experiments. results have s h o w a significant decrease in total glornemlar

natriuretic binding sites in ETOH- compared to water-treated animals of both strains.

calculated fiorn the displacement of '"1-ANP by unlabelled ANP. Furthemore. the

displacement of IabeUed ANP by cold cANF lias suggested that this decrease may be due to

the dom-replation of NPR-C only. A differential regulation of glomedar ANP receptor

subtypes by extemal agents has already been described. indeed. salt load was reponed to

decrease significantly glomerular NPR-C receptors without altering the density of NPR-A

receptors. allowing a greater glomenilar effect of circulating ANP levels in order to remove

the excess salts (Kollenda er al.. 1 990; Fraenkel et ai.. 1994). Similady. the observed

differential regulation ofthe various classes of glomerular ANP receptors by ETOH may have

a funaional significance since a decrease m NPR-C receptors only would mean that a geater

proportion of circulatiag ANP b e l s would bind to the active receptors (NPR-A). Therefore.

the increased proportion of NPR-A receptors in the glomenili of ETOH-treated rats may

increase the glornerular effect of circulating natriuretic peptides. nich as diuresis and

natriuresis. so that it rnay represent one of the mechanisms for the beneficial effect of

moderate ETOH consumption on BP. It is also possîile that this reduction in glomerular

NPR-C binding sites by ETOH is secondary to the decrease in cüculating ANP levels

observed after chronic moderate ETOH consumption in both SHR and WKY rats. increasing

the relative NPR-A a c t ~ t y . To c o h these hypotheses. the exact role of the so-called

"clearance" receptor needs fùrther clarification ( Anand-Srivaçtava and Trachte. 1993). Until

it is h o w n whether the NPR-C receptor has additional fùnctions than ANP buffering. we may

onty speculate on the present dmease of total glomerular ANP bmding sites in ETOH-treated

SKR and WKY animals.

6) lmer medziliary mtrizrretzc receptors:

The present studies confirmed earlier reports dernonarathg the presence of NPR-A

receptors in renal inner medullary tissue (Bianchi er al.. 1987). The IM tissue preparations

used in the present experiments did not allow the distinction between the various ce11 types

in the papilla. Previous studies have dernonstrated that NPR-A alone is present on inner

medda collecting ducts (IMCD) (Bianchi er ai.. 1987) and interstitial cells (Fontoura et al..

1990). Thin loops of Hede and IM capillaries have also been shown to possess natnuretic

peptide receptors (Bianchi et al.. 1987). Furthemore. natnuretic peptide receptors are also

present in the outer medulla. mainly in the vasa recta (Bianchi er ai.. 1987). These papillary

natriuretic peptide receptors have been reported to influence Na* handling and medullary

hypenonicity ( C 3 g and Brenner. 1 992).

Age and strain effects;

Various animal models of hypertension possess higher numbers of natriuretic receptors

m the medulla (Nuglozeh et ai., 1990: Bonhomme and Garcia. 1993). Nugiozeh et al. ( 1990)

reported a significant increase in the density of ANP receptors in the papilla of DOCA-salt

rats, with similar a f i t y . Similarly, Bonhomme and Garcia ( 1993) found a modest

augmentation m the deisity of papillary ANP receptors in l K 1 C rats. Again. the affinity was

mchanged. The present experiments extended this fmding to young (7 week-old) SHR rats.

Several reasons may explain the effect of high BP on papillary ANP receptors. It may be

compensatory to the mcreased BP. The increased number of inner medullary NPR-A

receptors in SHR rats may also be caused by the hypertrophy of the kidneys (Nuglozeh et al. . 1990). However. values calculated as hoVrng protein and not only as hoVpapiUa d l

showed sipifkant elevations in the B,,. Similarly. the assumption that increased plasma

ANP levels must be associated with renal hypertrophy and therefore with mcreased B,, in

the IM is somewhat misleadmg. considering that lower densities of NPR-A were found in the

papilla of rats with congestive heart failure (CHF), a pathophysiological condition where

circulating ANP levels are also signtficantly increased (Yechieli et ai.. 1993 ).

ETOH effect:

The present studies reported increased total papillary ANP bmding sites afier long-tenn

ETOH administration m both WKY and SHR rats. Such an elwatioa depending on the actual

receptor occupancy and renal level of natriuretic peptides. may resdt in enhanced natriuresis

of tlie ETOH-fed rats. Interestingly. the present experiments have shown increased Na-

excretion in ETOH-treated animals. With chronic ETOH consurnption. these alterations in

natriuresis and papillary NPR-A receptors may explain. at least in part. the reduced BP of

ETOH-treated rats. The weak antidiuresis associated with the natnuretic effect of chronic

moderate ETOH connimption is puzzling, although previous experiments have demonstrat ed

the possibility of a dissociation between water and salt excretion followuig both ETOH or

ANP administration (Wadstein and ohlin. 1979: Singer et al., 1987).

Some reports have suggeaed that the hypotensive and natriuretic effects of circulating

ANP levels rnay be independent i?om papuary receptor interactions. because of the necessity

for natriuretic peptides to cross the endopeptidase 24.1 1 "barrier" (main enzyme for ANP

metabolism) of the proximal tubules in order to reach the luminal-situated receptors of the

medda (Goetz. 1991; Gunning and Brenner, 1992). Despite this. tlie discovery of an

intrarenal mernber of the natriuretic peptide family. the NH1-elongated Urodilatin (UD)

(Goetr 199 1 ), synthesized in the diaal tubules, released on the luminal side and binciing to

the same receptors as ANP with equal or greater afnnity (GumÎng and B r e ~ e r . 1992). has

open again the discussion for a role of inner meduilary NPR-A. not only in subtle Na'

variation. but also for BP control. Furthemore. ANP and ANP mRNA are present m the

glomeruli, distal tubules and cortical collecting ducts of rat kidneys (Gunning and Brenner.

1992). Interestingly. CNP and NPR-B mRNAs are also found throughout the nephron

(Terada et al.. 1994). although no expression of these mRNAs have been detected thus far

in the kidney (Mukaddam- Daher et al.. 1995 ). Therefore. the possibility of intrarenal

paracrine effects on papiliary ANP receptors. by UD. by ANP or even by CNP. suggen a

contniution of the IM and its natriuretic receptors for Na' and BP regulation during long-

terni moderate ETOH consumption. Howwer. one would need to determine whether chronic

alcohol treatment rnay increase the synthesis and expression of natriuretic peptides of renal

ongin.

c) U r i i z a ~ cGMP excretiot>

The urinary Ievels of cGMP have been examined in both SHR and WKY rats in order to

link the observed natriuretic receptor modifications of glornenilar and papillary membranes

witb the second messenger syaem of the natriuretic peptides and thus with the possibility of

chronic renal functiooal aherations. nich as mcreased natriuretic a c t ~ t y . that rnay rnodify BP

regdation. It is noteworthy that the cGMP levels are measured from urine s a q l e s collected

d u h g the light phase of the dady cycle. at a time when the animals are not active and are not

consurning ethanol. Accordingly. the blood alcohol levels (BAC) are relatively low and

plasma ANP levels are reduced in ethanol- compared to water-treated animals because of

secondary hemodynarnic effects associated with the lower blood pressure. Nevertheless.

excretory cGMP levels are elevated in ETOH-treated SHR rats whereas they remained

unchanged in ETOH-treated WKY rats. despite their lower plasma ANP levels. This result

directly supports the hypothesis that the natriuretic receptors modifications produced by

ETOH mcrease the rend effects of the natriuretic peptides and may possibly coninbute to the

antihypertensive effect of long-term moderate alcohol consumption.

in addition to the natriuretic receptors. long-term alcohol treatrnent rnay also alter the

activity of other systems implicated in the regdation of water and electrolytes. i.e. AVP or

the renin-angiotensin-aldosterone system. Angiotensin II receptors for example are present

on mesangial membranes of the g l o m d (Sexton et al.. 1992) and possible modifications in

th& deoZty or characteristics by long-term ETOH administration may be responsiile. at least

in part. for the ETOH-mediated modifications in BP.

In summary, the present midies have dernonstrated alterations of renal ANP receptors

with chrooic ETOH treatment and suggested a possible role for the renal natriuretic receptors

m the long-tenn effects of moderate alcohol conaimption on Na' handling and B P regdation.

Specificaliy, increased density of NPR-A in the papilla and decreased quantity of ANP

receptors (mostiy NPR-C) in the glomeruli with chronic moderate ETOH were found by

radioreceptor and autoradiographic studies. for both SHR and WKY rats. Whether these

alterations are responsible for the protective effect of long-term moderate ETOH

consurnption on BP remains to be confirmed.


The authors wish to e.xpress their gratitude to Mr Ricardo Claudio for his excellent worli

m the handling of the animals. Special thmks are also addressed to Mrs Céline Coderre and

Ms Nathalie Charron for their techoical assistance. P. G. is a recipient of a scholarship from

"Fonds pour la formation de Chercheurs et l'Aide a la Recherche" (FCAR). This work was

supported by gants fiom Canadian medical research council (MT- 10337 and MT- 1 1671).

the Heart and Stroke Foundation. the Canadian Kidney Foundation (J.G.) and the Alcohol

research program at Douglas hospital ( C G . ).

Section 3.4.

ALTERATIONS IN BRAIN LEVELS OF ATRIAL AND C-TYPE

NATRIURETIC PEPTIDES AFTER CHRONIC MODERATE

ETHANOL CONSUMPTION IN SPONTANEOUSLY HYPERTENSIVE

RATS

P. Guillaume, J. Gutkowska and C. Gianoulakis

European Journal of Pharmacology

(Eur Pharmacol, in press, 1997)

Contribution bv CO-authors: Dr. I. Gutkowska and Dr. C. Gianoulakis were my CO-

sup exvisors.

Acknowled~ements: R Claudio was the animal technician. C. Coderre and N. Charron

performed the ANP and CNP iodinations.

3.4. ALTERATIONS lN BRAIN LEVELS OF ATRIAL AND C-TYPE

NATRIURETIC PEPTIDES AFTER CHRONIC MODERATE

ETHANOL CONSUMPTION iN SPONTANEOUSLY

BkTERTENSIVE RATS

3.4.1. ABSTRACT

Atrial (ANP) and C-type (CNP) natriuretic peptides have been found in brain regions

associated with fluid homeostasis and blood pressure. Since chronic moderate ethanol

consumption has been show to prevent the age-dependent increase in blood pressure in

experimental animals the objective of the present studies was to investigate the effect of

ethano1 (20 O/O v h for 8 months) on the total content and concentration of ANP and CNP in

the brain of spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. Ethanol

mcreased the content and concentration ofboth AM> and CNP m the hypothalamus. pons and

medulla of SHR rats. in contrast. in the WKY rats ethanol had no effect on the levels of ANP

in any of the brain regions studied. but enhanced the concentration of CNP in the

hypothalamus and medulla. Thus. ethanol induced changes in the content of natriuretic

peptides in distinct brain regions associated with control of cardiovascular a c t ~ t y . Such

changes may be partialiy responsible for the effect of chronic moderate ethanol consumption

on blood pressure.

3.4.2. INTRODUCTION

The brain is one of the main targets for a number of ethanol-mediated effects (Nut t

and Peters. 1994. Diamond and Messing. 1994). Indeed. ethanol readily crosses the blood-

brain-barrier to modify electrical and chernical properties of the brain and allers

neuroendocrine, behavioral and cardiovascular responses. Ethanol is also associated with

changes in blood pressure (MacMahon. 1987). In fact, chronic heavy alcohol consumption

has been associated with the developrnent of hypertension (Lian. 19 15. MacMahon. 1987).

whereas low and moderate ethanol consurnption have been found to prevent the age-

dependent increase in blood pressure in both human (MacMahon. 1987) and experimental

animals (Howe et al.. 1989 and Section 3.1 .). This effect is more pronounced in

hypertensive animals. such as in spontaneously hypertensive rats (SHR) (Section 3.1.).

The mechanisms mediating alcohol's effects on blood pressure are still unknown. A

number of brain regions. such as the hypothalamus. pons and medulla have been associated

with the control of cardiovascular activity and blood pressure. It is possible then that

alterations in the activity of some of these brain regions by ethanol may be responsible.

at least in part. for the effects of alcohol on blood pressure.

Atriai natriuretic peptide (ANP). the first rnember of a natriuretic peptide famil y with

potent hypotensive properties. was discovered by De Bold and CO-workers in 198 1. S ince

then, brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) have been

isolated frorn the porcine brain (Sudoh et al.. 1988. Sudoh et al.. 1990). These natriuretic

peptides produce hypotension by vasorelaxation. diuresis and nauiures is. and b y inh ib i t ing

the release and activity of several pressor hormones. such as arginine vasopressin ( AVP).

angiotensin II (AH) and aldosterone (Blaine. 1990. Jamison et al.. 1991). In recent years.

the implication of the natriuretic peptides in the central control of blood pressure has been

demonstrated ([mura er al.. 1997). Indeed. immunohistochemical staining has

dernonstrated that ANP is mainly localized in the hypothalamus in the adult rat brain

(Kawata er ai.. 1985. Standaert et al.. 1986. Morii et al.. 1986b). and especially in the

anteroventral third ventricle region (AV3V) ( K u and Zhang. 1994). an area believed to be

important in the regulation of fluid homeostasis and blood pressure (Buggy and Bealer.

1987). ANP is also found throughout the pons. the medulla (Kawata et al.. 1985) and the

olfactory bulbs (OB) (Gutkowska et al.. 1991). Sirnilarly. the presence of CNP has been

demonstrated in the rat hypothalamus, pons. medulla and cerebellum (Kornatsu er al..

199 t . Herman et al., 1993). In contrast, BNP is absent from the rat brain.

Our initial studies have demonstrated that both acute and chronic ethanol exposure

1 alter the activity of the hem ANP and BNP systems (Section

the strong hypotensive effects of natriuretic peptides. it

2.1. and 3.1 .). Considering

was hypothesized that the

natriuretic system (peptides and recepton) may mediate. at least in part. the effects of low

to moderate ethanol consumption on blood pressure. Ethanol may also alter the activity of

the brain natriuretic peptide systems (ANP and CNP). which may play a role in rnediating

ethanol's effects on the cardiovascular system. There is no previous data on the subject.

Thus. the objective of the present studies was to investigate the effects of chronic rnoderate

ethanol consumption on the brai n natriure tic system of spontaneousl y h ypertensive ( S H R)

and Wistar-Kyoto rats (WKY). by estimating the total content and concentration (ng/mg

protein) of ANP and CNP in the hypothalamus. pons and medulla. brain regions important

in the regulation of fiuid homeostasis and cardiovascular responses.

3.4.3. MATERLALS AND METHODS

3.4.3.1. Animal T r e m m

6 week-old male Wistar-Kyoto (WKY) and spontaneously hypertensive rats t SHR)

were used in the presenr studies (Charles River Breeding Laboratories. St-Constant.

Québec. Canada). The animals were randomly separated in 4 different groups: WKY -H,O

( n = 12), WKY-ethano1 ( n = 12). SHR-H20 ( n = 12) and SHR-ethanol ( n = 12). After a week

acclimatization period. alcohol was gradually added to the drinking water of the ethanol

groups. as described previously (Howe et al.. 1989). Brietly . ethanol-treated rats ( WKY -

ethanol and SHR-ethanol) had free access to an ethanol solution of 5 % v/v for 5 days.

10% v/v for 5 days. 15 % v/v for 5 days and 20% v/v for the remaining of the 8 months

experimental period. The corresponding control rats (WKY-H,O and SHR-H,O) had free

access to tap water. Al1 animals had free access to pellet chow (Purina. Richmond. VA) .

Furthermore. from each strain of rats. an additional 10 animals were sacritïced at 7 weeks

of age to estimate the total content and concentration (ng/mg protein) of brain A N P and

CNP prior to the age-dependent increase in blood pressure and ethanol administration.

Blood pressure was measured via the tail-cuff method (Pfeffer et al.. 1971) at the

beginning of the study (7 weeks-old) and at the end of the 8 month experimental period.

In addition, body weight was recorded every 15 days and tluid intake (alcohol solution or

water) was recorded daily.

3.4.3.2. m u e E-

After decapitation. the hypothalami. pons and medulla were removed from the brain.

Each brain region was placed in separate tubes containing ice-cold 0.1 N HCI and protease

inhibitors at final concentrations of 1 mg/ml ethylenediamine-tetra-acetate (EDTA). 10

mM phenylrnethylsuifonylfluoride (PMSF) (Sigma Chemical Co.. P-7626) and 5 rnM

pepstatin A (Sigma Chemical Co.. P-4265). The tissues were rapidl y boiled for 5 min and

cooled on ice. The brain regions were then homogenized with a microultrasonic ce11

disrupter (Kondes. Vineland. NJ). Proteins were rneasured by a modification of the

Bradford rnethod (Bradford. 1976). The remaining homogenates were centrifuged for 15

min at 4°C and the supernatants stored frozen at -75°C until assayed for ANP and CNP.

3.4.3.3. m - a v s (B1Bsl .

Hypothalamic. pontine and medullary extracts were appropriatel y diluted and the

content of ANP and CNP was directly quantified by using second-antibody RIA

procedures for both ANP and CNP. The ANP RIA has been described elsewhere

(Gutkowska et al.. 1987a). The antibody (produced by the immunization procedure of

Gutkowska et al.. (1984). final dilution 1:30000) showed a 100% cross-reactivity with the

126 aminoacid prohorrnoiie and the circulating ANP. but less than 5 % cross-reactivity with

oxidized ANP. It also showed less than 0.001 % cross-reactivity with CNP. Iodination of

ANP was done by the lactoperoxidase method. The lowest quantity of ANP rneasurable

with this assay was 0.75 pgltube. The intra- and inter-assay coefficients of variation were

4.3 and 8.3% respectively.

The CNP RIA was performed by a RIA similar to that for ANP which we developed

in Our laboratory. A typical standard curve generated with "'I-CNP and cold CNP is

presented at Fig. 3.4.1. 12SI-CNP displacement by serial dilutions of rat hypothalamic

homogenates paralleled the dose-response standard curve of rat CNP. indicating that the

peptide rneasured by the RIA was immunologically identical with synthetic CNP (Fig.

3.4.1.). Similar parallelisms to the RIA standard curve are demonstrated for pontine and

medullary extracts. The antibody (Peninsula Laboratories. final dilution 1 : 8000) showed

- CNP standards

* Hypothalamic extracts

01 I I 1 I

1 10 100 1000 1 O000 Log [CNP] (pgltube)

Figure 3.4.1. Standard RIA cuwe of CNP. The serial dilutions of rat hypothaiarnic homogenates show a good parallelism to the RIA standard curve.

a 100% cross-reactivity with CNP. but only a 0.015% cross-reactivity with ANP. The

iodination of CNP was also done by the lactoperoxidase method. The minimal quantity of

CNP detectable with this RIA was 12 pgltube. The intra- and inter-assay coefficients of

variation were 4.8 and 9.7 % respective1 y.

. . 3.4.3.4. -1 A m

Results are presented as mean I S.E.M.. Statistical analysis was done by a two-way

analysis of variance (ANOVA). with the treatment or the age as the first independent

variable and the strain as the second independent variable. This analysis was followed by

the Neuman-Keuls multiple cornparison test. A p value of 0.05 or lower was considered

signiticant.

3.4.4. RESULTS

3.4.4.1. J3ffect of a s

There is a significant decrease with age ( p ~ 0 . 0 0 1 ) in hypothalamic. pontine and

medullary ANP levels of both SHR and WKY rats (Fig. 3 - 4 2 . ) .

Total CNP content in the hypothalamus is augmented with age (pc0.001). whereas

its concentration is decreased (pé0.001) (Fig. 3.4.3.). Pontine and medullary CNP content

and concentration are decreased with age in both strains of rats (~50.00 1).

3.4.4.2. Effect of strah

Both the content (pc0.05) and the concentration (ps0.05) of hypothalamic A N P are

higher in young SHRs cornpared to age-matched WKY rats (Fig. 3.4.2.). In adult anirnals.

the total hypothalamic ANP content is higher in the SHR compared to WKY rats (pc0.05).

but the hypothalarnic ANP concentration is sirnilar between the two strains of rats. Pontine

ANP content (pc0.001) and concentration ( p ~ 0 . 0 1 ) are lower in young SHRs compared

to young W K Y rats. However. adult SHRs have significantly higher pontine ANP content

(pc0.01) and A N P concentration ( p ~ 0 . 0 5 ) than age-matched WKY animals. There is no

difference between the medullary ANP levels of 7 week-old SHR and WKY rats. In

1 7weeks 0 38 weekç (water) 1

WKY rats

SHR rats

HYPO PONS MED

I I I

HYPO PONS MED

Figure 3.4.2. ANP content (ng) and concentration (nglmg proten) in the hypothahmus (HYPO), pons and medulia (MED) of 7 (diagonal line bars) and 38 week-old (plain bars) SHR and WKY rats (n=12). Signlicant difference from the 7 week-old animais: *p<0.01, "p~0.001. Significant difference between SHR and W rats of the same age: Wc0.05, m<O.O1, ttpc0.001.

I 7 weeks 0 38 weeks (water) 1

WKY rats

SHR rats

HYPO PONS MED HYPO PONS MED

Figure 3.4.3. CNP content (ng) and concentration (nglmg protein) in the hypothalamus (HYPO), pons and medulla (MED) of 7 (diagonal line bars) and 38 weekold (plain bars) SHR and WKY rats (n=12). Significant difference from the 7 weekold anirnals: -p<0.001.

contrast. both the total content (pi0.01) and concentration (pr0.05) of medullary ANP

are higher in the adult SHRs compared to adult WKY rats.

There is no significant difference in the hypothalamic. pontine and medullary CNP

levels (content and concentration) between young or adult SHR versus young or adult

WKY rats. respectively (Fig. 3.4.3 .).

3.4.4.3. Effect of e t h a d

After 8 monrhs of moderate ethanol consumption. both SHR and WKY rats had

significantly lower blood pressure than water-treated animals of the same strain (table

3.4.1. ). The body weight and the daily fluid consumption are not different between the

ethanol- and water-mted rats (shown for the last week of treatment only) (table 3.4.1. ).

There is a signitïcant increase in the hypothalamic ANP content of SHR. but not

WKY rats. after chronic ethanol consumption (Fig. 3 A.4.). Indeed. total hypothalamic

ANP content is raised from 0.58 f 0.02 ng in SHR-H,O to 1.16 f 0.06 ng (p~O.00 1 ) in

the SHRethanol group. Likewise. a two fold increase in hypothalamic ANP concentration

is observed following the chronic ethanol treatment (from 0.17 t 0.02 ng/mg protein in

SHR-H,O to 0.51 & 0.05 ng/mg protein. p-0.001 in SHR-ethanol). Chronic moderate

ethanol consumption induced an increase in the pontine ANP concentration in SHR. but

not in WKY rats (~50.05). Medullary ANP content (pc0.05) and concentration (pd.00 1 )

were also higher after long-term ethanol consumption in SHR. but not W K Y rats.

Hypothalamic CNP content (~50.01) and concentration ( ~ ~ 0 . 0 5 ) are eievated after

chronic ethanol treatment in both SHR and WKY rats (Fig. 3.4.5.). Pontine CNP

concentration. but not CNP content. is also signitïcantl y increased (ps0.0 1 ) by the erhanol

treatment in the SHR. but not WKY rats. Medullary CNP content and concentration are

increased by the ethanol treatrnent in the SHRs. However. in the rnedulla of WKY rats.

chronic ethanol treatment increased the concentration but not the total content of CNP.

3.4.5. DISCUSSION

The present experiments have demonstrated age, strain and ethanol-induced changes

Table 3.4.1. Blood pressure (BP), bodv weight (BW) and dai& Iiquid conrumption (LC) in

SHR and WKY rats at age 7 weeks (befiore ethanol treatrnent) and at age 38 weekr fafier 8

months of ethanol or water treatment) .

WKY S H R II 7 38 weeks 38 weeks 7 weeks 38 weeks 38 weeks weeks Water ETOH Water ETOH

BP 113 1 3~~ 1 0 9 ~ 133' 186"** 165' (mm Hg) - + 4 * 2 I 1 + 4 t 4 & 4

IL Values shown are means f S.E.M. ( n = t2).

a: Significant difference frorn 7 week-old animals: P4L00 1 . c. d: Signitïcant difference between SHR and WKY rats of the same age: c: Pc0.0 1 .

b. e: Signiticant difference between the ethanol- and water-treated animals of the same strain

and age: b: P ~ 0 . 0 1, e: P~0.00 1.

water

WKY rats

HYPO PONS

SHR rats

*

MED HYPO PONS MED

F igure 3.4.4. ANP content (ng) and concentration (ngimg protein) after 8 months of water (plain bars) or ethanol(20% vlv) (horizontal line bars) treatment in the hypothalamus (HYPO), pons and medulla (MED) of SHR and WKY rats (n=12). Significant difference behveen the ethanol- and water-treated animals of the same strain and age: '~~0.05, "p<0.001.

I 0 water ETOH I

WKY rats

SHR rats

HYPO PONS MED HYPO PONS

F ig u re 3.4.5. CNP content (ng) and concentration (nglmg

*

f MED

protein) after 8 months of water (plain bars) or ethanol(20% vlv) (horizontal line bars) treatment in the hypothalamus (HYPO), pons and medulla (MED) of SHR and WKY rats (n=12). Significant difference between the ethanol- and water-treated animais of the same strain and age: *p<0.05, wp<O.O1, "p<0.001.

in natriuretic peptide levels in brain regions involved in cardiovascular homeostasis. ( 1 )

There is a signifiant decrease with age in both ANP and CNP levels in the hypothalamus.

pons and medulla of SHR and WKY rats. (2) There is significantly higher brain ANP, but

not CNP, levels in SHR compared to WKY rats. (3) There is significantly higher brain

ANP levels following chronic moderate ethanol consumption in SHR but not WKY rats.

In contrast, brain CNP levels are higher in both SHR and WKY rats following the chronic

ethanol ueatment.

The importance of the brain in mediating a number of alcohol effects is indicated by

the nurnerous ethanol-induced behavioral effects (Nutt and Peters. 1994). 1 t has been

hypotheskd that some of the ethanol-induced effects on biood pressure may be mediated

through specific modifications in the activity of relevant neurohormonal and

neurommitter systerns of the brain, such as the natriuretic or renin-ang iotensin systerns

(Imura et al.. 1992, Steckelings. 1992). or through secondary modifications in the activity

of peripheral pressor and depressor agents by ethanol-induced changes in hypothalamic and

pituitary hormones (Cicero. 198 1). Ethanol. an amphiphilic dmg, rapidly crosses the

blood-brain-barrier to physically dissolve into brain ce11 membranes (Diamond and

Messing , 1994). This property of alcohol modifies membrane fluidity (Nutt and Peters.

1994) and inhibits brain Na'K'ATP ase activity (Sun, 1976, Nhamburo et al.. 1986.

Swann, 1990). leading to various alterations in the chernical and electrical properties of

brain cells. Interestingly , centrally administered ANP significantly enhanced the release

of Na'K'ATP ase inhibitors (Crabos et al., 1988). It is possible then that elevated brain

ANP levels in ethanol-treated rats serve as a mediator for the ethanol-induced inhibition

of N a X ' ATP ase activity. Further work is needed to elucidate this observation. Acute

ethanol also decreases the sensitivity of N-methyl-D- Aspartate (NMDA) receptors for

excitatory aminoacids like glutamate and potentiates y-aminobutyric acid (GABA)-A

recepton, explaining in part its sedative actions (Lovinger, L989, Wafford et al., 1991).

Ethanol also affects the activity of several neurohormonal and neurotransrnitter s ystems

such as AVP or angiotensin II, some of which are of critical importance in the long-term

control of blood pressure (Cicero. 1 98 1 ) . The eiTect of ethanol on blood pressure is biphasic. Indeed. although high ethanol

consumption has long been shown to produce hypertension (Lian. 1915). low and/or

moderate ethanol consumption has been s hown to prevent the age-dependent increase in

blood pressure ( MacMahon. 1987). Thus. a nurnber of epidemiological studies have

reported lower systolic blood pressure in light and moderate human drinkers than in both

heavy and non-drinkers (Harburg et al.. 1980. Gillman et al.. 1995). Similarly. animal

experiments have demonstrated the prevention of the age-dependent increase in blood

pressure with moderate ethanol consumption. especial l y in the hypertensive strains of rats

(Howe et al.. 1989 and Section 3.1.). The mechanism(s) by which ethanol induces this

antihypertensive effect remain unclear. Some studies have indicated that this ethanol-

mediated protection from this age-dependent increase in blood pressure could be due. at

least in part. to lipoproteins which are differently regulated by low and high ethanol

consumption (Hojnacki et al., 1988). Recently. the natriuretic peptide famil y was added

to the list of systems which centrally regulate blood pressure and fluid homeostasis ( Imura

et al.. 1992). Therefore. alterations in the activity of the brain A N P system may be

partially responsible for ethanol's effect on blood pressure.

In the adult brain. the highest A N P concentration is found in the hypothalamus

(Kawata et al.. 1985. Standaert et al.. 1986). Within this tissue. reg ions involved in the

regulation of fluid homeostasis and blood pressure. such as the AV3V area. m i n strongly

for ANP immunoreactivity (Kawata et ab. 1985). Pro-ANP mRNA is also present in high

quantities within the hypothalamic areas involved in cardiovascular regulation (Gundlach

and Knobe. 1992). Likewise. CNP (Komatsu et al., 1991) and CNP rnRNA (Herman et

al.. 1993) are found in high quantities in the hypothalamus. In addition to the

hypothalamus. natriuretic peptides are also present in the pons. mainly in the dorsal

tegmental area. and in the medulla. in close association with regions involved in

cardiovascular control (Kawata et al.. 1985).

I t has been reported that S H R rats exhibit higher levels of hypothalamic A N P

compared to WKY rats (Morii et al.. 1986b. Debinski et al.. 1989. Jin et al., 199 1 . Chen

et al.. 1997. Komatsu et al.. 1992b). The present studies confirmed this finding.

Furthermore. in contrast to the finding of lmada and collegues ( 1985). who reported no

significant change in hypothalamic A N P levels with age. the present stud ies demonstrated

a reduction in hypothalamic ANP levels with age in both strains of rats. This discrepancy

could be explained by the different ages of the animais used in the two studies (38 weeks-

old in the present study versus 15 weeks-old in that of Imada). Significantly higher content

of pontine and medullary A N P in adult compared to young SHRs was observed in the

present study. confirming previous reports (Imada et al.. 1985. Komatsu et al., 1992b).

Chronic ethanol consumption increases ANP levels in the hypothalamus. pons and

rnedulla of hypertensive. but not normotensive rats. The presence of elevated hypothalamic

ANP levels in SHR rats following chronic ethanol treatment may enhance the contribution

of central natriuretic peptides to cardiovascular homeostasis. I ndeed.

intracerebroventricular (i.c.v. ) injection of ANP (third and lateral ventricles) produces

dose-dependent inhibition of water intake and angiotensin-induced drinking in overniglit-

dehydrated rats (Nakamura et al.. 1985. Antunes-Rodrigues et al.. 1985. Katsuura er al..

1986). S imilarl y. centrally administered ANP (lateral ventricles) suppresses sait appetite

in salt-depleted rats and in strains known to have increased sa1 t preference. such as S H R

(Fitts et al., 1985. ltoh et al.. 1986b). Furthermore. i.c.v. injection of ANP (lateral

ventricles) is associated with diuresis (Israel and Barbella. 1986. Shoji et al., 1987. Lee

et al., 1989). Since i.c.v. injection of A N P also signitkantly blunts the A V P increase

induced by i.c.v. injection of angiotensin I I or by osmotic stimulation. parts of the diuretic

effect of central A N P administration rnay be explained by its effect on the A V P system

(Yamada et al., 1986. litake et al., 1986. Inoue et al.. 1990). Interestingly. the diuretic

effecr of ethanol is also anributed in part to the inhibition of AVP release (Eisenhofer and

Johnson. 1982). Therefore. the elevated brain A N P levels following chronic ethanol

consumption may produce a greater inhibit ion of vasopressinergic neurons and thus

elevated diuresis in these animals compared to water-treated rats. Final ly . central l y

administered ANP inhibits the pressor effects of i.c.v. injection of angiotensin I I in

conscious rats (Itoh et al., 1986a. Shimizu et al.. 1986. Castro et al., 1987). The exact

site of action of ANP injected within the hypothalamus is not clear. but recent evidence

suggests that ir rnay be the AV3V area (Ku and Zhang. 1994). The subfornical organ rnay

also be important since microinjections of ANP into this region blocks angiotensin II-

induced neuronal stimulation (Hanori et al.. 1988. Ehrlish and Fitts. 1990). I nterestingl y.

it was previously demonstrated that hypothalamic ANP concentration was signitïcantly

reduced in S H R rats fol low ing antihypertensive treatment. suggesting that the increased

hypothalamic ANP levels represented a homeostatic response to the elevated blood

pressure in order to control the increase in arter ial pressure (Rus koaho and Leppaluoto.

1988). In contrast. the fact that the hypotensive effect of chronic moderate ethanol

consumption in SHR rats is associated with significant increases in brain ANP leveis

suggests a specific effect of alcohol in central ANP regulation rather than a secondary

response to lower blood pressure. Thus. alcohol either directly or indirectly . through its

effects on other systems known to regulate the activity of hypothalamic ANP. such as the

cholinergie and norepinephrine systems (Nissen et al.. 1989. Baldissera et al.. 1989). rnay

modify the synthesis and release of ANP by the hypothalamus. Therefore. the increased

hypothalamic ANP concentrations associated with the possibil ity of chronic enhancement

of centrally-mediated ANP hypotensive effects rnay represent one mechanism by which

the prevention of the age-dependent increase in blood pressure is explained.

The elevated ANP levels found in the medulla of SHR rats following chronic ethanol

treatment rnay also rnodify ANP-med iated cardiovascular homeostasis. The ANP-rich

region of the nucleus tractus solitarius is the sire of termination of baroreceptor afferent

fibres. Therefore. ANP rnay be involved in lowering blood pressure by interfering with

the baroreceptor reflex. Indeed. microinjections of ANP into the nucleus tractus solitarius

or into adjacent areas such as the ventrolateral medulla caused significant decreases in

b l d pressure (McKitrick and Calaresu. 1988. Bergagl io and Calaresu. 1990). S im ilarl y.

the administration of A N P into the fourth ventricle produced a profound decrease in

arterial blood pressure (Levin et al.. 1989). Therefore. it is possible that elevated

medullary ANP levels in ethanol-neated SHR rats are associated with greater modifications

in the transmission of the baroreceptor information within the nucleus tractus solitarus and

medullary regions. leading to a lowering in blood pressure that may explain in part the

prevention of the age-dependent increase in blood pressure observed following chronic

moderate ethanol consumption.

There was no strain-specific difference in brain CNP levels between WKY and SHR

rats. In contrast. hypothalarnic CNP content was significantly increased with age in both

suains of rats. whereas pontine and medullary CNP levels were decreased during the same

period. To our knowledge. this is the first report of strain (WKY versus SHR) or age (7

versus 38 week-old animals) modifications in brain CNP levels.

C hronic ethanol consumption increases CNP levels in the hypothalamus. pons and

medulla of hypertensive rats. and hypothalamic and medullary CNP levels in WKY rats.

In contrast to ANP. the exact role of CNP in brain tissues and its possible contribution to

cardiovascular regulation is still unclear (Imada et al.. 1992). It is therefore difficult to

decide what the significance of the modifications in the activity of the brain CNP system

by chronic ethanol consumption is and what are its effects on blood pressure.

The reason for the different sensitivity of the central natriuretic system toward ethanol

between SHR and W K Y rats is unclear but may retlect genetic differences between the two

strains of rats. Indeed. grafting embryonic hypothalamic SHR cells into the hypothalamus

of adult WKY rats resulted in rapid elevations of the systolic blood pressure and heart rate.

suggesting profound differences in the brain characteristics between these two strains of

rats (Eilam et al., 199 I ). Other endocrine and neuroendocrine deviations from

normotensive rats have been observed in SHR rats. such as differences in renin, arginine

vasopressin (AVP) or vasoactive intestinal polypeptide (VI P) mRNAs (Lang et al.. 198 1 .

Avidor et al., 1989, Kreutz et al., 1993). Furthermore, alterations in the ANP and B N P

mRNAs of the heart ventricles by ethanol have also been demonstrated in SHR. but not

WKY rats (Sections 3.1. and 3.2.). However, other parts of the natriuretic peptide system

have been similarly modified by chronic moderate ethanol administration in both strains

of rats. such as atrial ANP and BNP contents (Sections 3.1 and 3.2.) . Considering the

genetic differences between the two strains and the more profound ant ih ypertensive e ffects

of moderate ethanol consumption in SHR compared to WKY rats (tabie 3 -4.1. ). a different

activation of the major components of the natriuretic peptide system in the brain. the hem

and the kidney may exist between the two strains of rats. producing faster and greater

effects on the blood pressure of ethanoi-treated SHR than ethanol-treated WKY rats.

In summary. during pathophysiological conditions such as hypertension. the elevated

ANP levels seen in the hypothalamus. pons and medulla are considered compensatory to

the higher blood pressure. On the other hand. the increase in brain ANP and CNP levels

of SHRs observed during chronic moderate ethanol administration. in spite of the

considerably lower blood pressure measured in these animais compared to water-treated

SHRs. suggests a direct or indirect stimulatory effect of ethanol on the activity of the A N P

system which is independent of the hemodynamic control. Such stimulation in the activity

of the brain A N P systern may play an important role in the antihypertensive effect of

chronic moderate ethanol consumption.


The authors wish to thank Ricardo Claudio for his excellent care of the animals.

Special thanks are also given ro Céline Coderre and Nathalie Charron for their technical

assis tance.

P.G. is the recipient of a doctoral scholarship from the "Fonds pour la formation de

Chercheurs et I 'Aide à la Recherche" (FCAR). This work was supported by gram from

the Medical Council of Canada (MRC) (MT- 10337 and MT- I 1674) (J.G.) and by the

alcohol research program at Douglas hospital (C.G.).

Section 3.5.

CIRCUMNENTRICUIAR ORGAN NATRIURETIC PEPTIDE

RECEPTORS FOLLOWING CHRONIC MODERATE ETHANOL

CONSUMPTION IN SHR AND WKY RATS

P. Guillaume, C. Gianoulakis and J. Gutkowska

Manuscript in preparcrüon

Contribution bv CO-authors: Dr. C. Gianoulakis and Dr. J. Gutkowska were my CO-

s u p e ~ s o r s .

Acknowled~ements: R Claudio was the animal technician. C. Coderre and N. Charron

performed the ANP iodinations.

3.5. CIRCUMVENTRICULAR ORGAN NATRWRETIC PEITIDE

RECEPTORS FOLLOWING CHRONIC MODERArlTE ETHANOL

CONSUMPTION IN SHR AND WKY RATS

3.5.1. ABSTRACT

Chronic moderate ethanol (ETOH) consumption is associated with the prevention of

the agedependent increase in the blood pressure of experimental animais. The rnechanisms

mediating this antihypertensive effect of ETOH are unclear, but the natriuretic peptide

system has been hypothetized to contribute to this effect. In the present experiments.

ETOH- induced modifications in the characteristics (B, and Ka) of the natriuretic

receptors are investigated in the subfornical organ (SFO), choroid plexus (CP) and area

postrema (AP) of spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. Both

strains received either water or a 20% v/v ETOH solution for 8 months. A significantly

lower number of total natriuretic recepton is observed in the water-treated SHR compared

to the water-treated WKY rats in the SFO and CP, but not in the AP. This lower B, in

the SFO and CP of hypertensive compared to normotensive animals is associated with

elevated proportions of NPR-C receptors. Furthermore, it was observed that chronic

moderate ETOH consumption significantly decreased the affinity of the NPR-C receptors

in the SFO and CP. In contrast, ETOH does not alter the characteristics (Bu and KJ of

the NPR-A receptors in the SFO, CP or AP of either SHR or WKY rats.

Thus, development of age-dependent hypertension seems to be associated w ith

changes in the total density as well as in the relative proportion of the natriuretic recepror

subtypes in the SFO and CP. Furthermore. the ETOH treatment is associated with

alterations in the circumventricular organ ANP receptors K, that may mediate. at least in

part. the antih ypertens ive effect of moderate alcohol consumption.

INTRODUCTION

Three distinct receptors have been described for the natriuretic peptides fam ily

( Anand-Srivastava and Trachte. 1 993). The natriuretic peptide receptor (NPR)-A and

NPR-B are both bel ieved to be active receptors, consisting of a single transmembrane

domain, with cGMP as their second messenger. Atrial natriuretic peptide (ANP) appears

to bind preferably to NPR-A while C-type natriuretic peptide (CNP) binds preferentially

to NPR-B (Suga et al., 1992a). The NPR-C. on the other hand. is less selective. binding

various ANP analogs, and seerns to be part of a buffer system which major function is the

clearance of excess amounts of natriuretic peptides frorn the circulation (Maack et al.,

1987). In the brain, the natriuretic receptors are found rnostly in the circurnventricular

organs such as the subfornical organ (SFO). the choroid plexus (CP) and the area postrema

(AP) (Quirion et al., 1986, Brown et Czamecki, 1990a), on brain microvessel endothelial

cells (Whitson et al., 1991) and on the blood side of the BBB (Ermish, 1992).

Long-term ethanol (ETOH) drinking has been associated with some antihypertensive

effects in animal experimenü, particularly in hypertensive strains of rats (Howe et al.,

1989 and Section 3.1 .). Likewise. lower systolic blood pressure (BP) has been found in

light and moderate human ETOH drinkers than in both heavy and non-drinkers. suggesting

the existence of some depressor effects of low and moderate alcohol consurnption

(MacMahon. 1987). It has been hypothetized that the diuretic and antihypertensive effects

of low and moderate ETOH consumption may be mediated, at least in part. by the

natriuretic peptide family (Colantonio et al.. 1991 and Section 2.1.). Modifications in

natriuretic receptors have alread y been noticed in the kidneys fol low ing chronic mode rate

ETOH drinking, implicating this system in the antihypertensive effects of alcohol (Section

3 -3 .). Considering the importance of the communication and feedback mechanisms

between the brain and the circulation in the control of long-term BP. it was interesting to

investigare also the possibility of ETOH-induced alterations in :he binding characteristics

of the natriuretic receptors in the circumventricular organs. Structures such as the SFO.

CP and AP lack a blood-brain-barrier (BBB), a unique situation that enables a direct link

between hormonal inputs from the periphery and neural syscems in the brain. For example.

the binding of circulating ANP to natriuretic receptors in the SFO has been shown to

inhibit AII-sensitive neurons projecting to the AV3V area (Hattori et al., 1988, Shibata

er al.. 1992) and therefore to block stimuli for AVP release and tluid intake. supporting

an inhibitory action of ANP on the hypothalamus and AVP system. Modifications in

c ircumventricular natriuretic recep tor characteristics have already been demonstrated in

various conditions. such as hypertension. and have been suggested to be responsibie to

some extent for the development of hypertension (Saavedra. 1986. McCarty et al.. 1986).

Li kewise, ETOH-induced al terations of the circumventricular natriuretic recep tors may

help to explain the prevention of the age-dependent hypertension in experimental animals

such as SHR and WKY by chronic Iow to moderate alcohol consumption.

Thus. in the present stud ies. the natriuretic receptor characteristics (B,, et K,) were

investigated in the circumventricular organs (SFO. CP and AP) following the chronic

administration of 20 % v/v ETOH in hypertensive (SHR) and normotensive (WKY) rats.


3.5.3.1. Animal Treatmem

6 weeks-old male Wistar-Kyoto (WKY) and spontaneously hypenensive rats (SHR)

were used in the present studies (purchased fiom Charles River Breeding Laboratories. St-

Constant. Québec. Canada). The animais were randomly separated in 4 different groups:

WKY-H,O (n=6). WKY-ETOH (n=6). SHR-H,O (n=6) and SHR-ETOH (n=6). Afrer

a week acclimatization period. alcohol was gradually added to the drinking water of the

ETOH groups. as described previously by us (Section 3.1. ) and by Howe ef al.. ( 1989).

Brietly, rats received 5 % v/v for 5 days. 10% v/v for 5 days. 15 % v/v for 5 days and

20 % v/v for the remaining of the 8 months experimental period. During that time. rats had

free access to their respective water or ETOH solutions and to pellet chow (Purina.

Richmond. VA).

3.5.3.2. Autoradiomhic Studie

The brains were frozen intact in isopentane and kept at -75 OC until used. Frozen. 20-

pm-thick coronal brain sections were cut in a cryostat at - 15 OC through the SFO-CP and

through the AP regions. These tissue sections were thaw-mounted ont0 poly-L-lysine-

treated siides and placed overnight into a desiccator under vacuum at 4°C.

The brain sections were washed for 15 minutes in a preincubation buffer consisting

of O. 1 % PEI in 50 m M Tris-HCI. pH 7.4. and subsequently incubated with 50 pM 131-

ANP (specific activity. Zûûû Ci/mmol) for 1 hour at room temperature. The incubation

buffer consisted of 50 mM HCI, pH 7.4. 150 m M NaCI. 5 rnM MnCl, (Sigma Chemicai

Co.. M-3634). 40 g/ml bacitracin (Sigma Chernical Co.. B-OL25) and 0.5 % bovine serum

albumin (BSA). The competitive inhibition of "'1-ANP binding to the membranes by

unlabeled peptides was examined for each animal by coincubating the membranes with

various concentrations of cold ANP (10"' to l ( r M) and des-(Glnllb. Seri''. GlyllX. Leu1''.

Gly'31ANF,,,.,,l (or cANF) (IO-"'. IO-". [O-' M). cANF is a ring deleted ANP analog

which binds specifically to the clearance receptors (NPR-C) (Maack et al.. 1987).

The highest concentration of cold peptides ( 10'" M) was considered to exhibit non-

specific binding (NSB). After incubation. the sl ides were washed 4 times in 50 mM Tris-

HCl. pH 7.4 (4°C). rinced quickly in distilled water (4°C) and left to dry overnight. The

tissue sections were then exposed on Phosporlmager cassettes for 5 days. The intensity of

the signals were subsequently quantitated by densitometry using a Phosporlmager

( Molecular Dynamics. Sunnyvale. CA). Relative quantities between experiments were

correlated by known quantities of I3I-ANP simultaneously exposed ont0 Phosphorlmager

cassettes. The average of at least 4 values per concentration per rat were calculated for

each individual brain SFO. CP and AP and their optical densities converted to radioactive

concentratioiis (cpm). based on the cornparison with the "'1-ANP standards.

3.5.3.3. Statistical t .

The maximum binding capacity (B-J and the apparent dissociation constant (K,)

were calculated when possible by the LIGAND iterative model-tïtting computer program

(Munson et al.. 1980).

3.5.4. RESULTS

Natriuretic receptors characteristics (B,, and Kd) are calculated by displacing "'1-

ANP with increasing concentrations of unlabeled A N P ( 10-l0 to IO-" M) and cANF ( IO?

IO-". 10-" M) in SFO. CP and AP of water- and ETOH-treated S HR and WKY rats. The

LIGAND -calculated B, and K, values for the three circumventricular organs are

presented on Table 3.5.1.

3.5.4.1. S u b f o ~ c a l orgao (SFO) and choroid plexus (CF')

The average (n=6) displacernent of labeled ANP by cold ANP is presented on Figure

3.5.1 A for SFO and Figure 3.5.2A for CP. As calculated from these values. signitkantly

lower (n =6. p~0.05) total ANP receptor densities are found in hypertensive compared to

normotensive anirnals (Table 3.5.1 .). In contrat. a significant decrease in K, is reported

in the CP only (n=6. ~ ~ 0 . 0 5 ) . When I3I-ANP is incubated with increasing concentrations

of unlabeled cANF (Figures 3.5. I B. and 3.5.2B.). another difference is visible between

SHR and WKY rats. cANF has no (in SFO) or little ( in CP) capacity to displace labeled

ANP in normotensive animals. However. a significant portion of '"1-ANP is displaced by

cANF in hypertensive rats. 1 ndeed. the B,, values for "'1- ANPlcANF displacement

represent 64.8 and 36.4% of maximal binding density calculated with 1251-ANPIANP in

the SFO of SHR-water and SHR-ETOH rats. respectively (Figure 3.5.1B). Likewise. the

B,,, values for 1251-ANP/cANF displacement represent 78.0 and 63.1 % of the ma.. imal

binding density for l'SI-ANP/ANP in the CP of SHR-water and SHR-ETOH animals

respectively. whereas they are calculated as only 28.0 and 35.1 % in WKY-water and

WKY-ETOH rats (Figure 3.5 -18). The availability of complete and specific antagonists

toward the various natriuretic receptors are needed to confirm this result. but these

calculations suggest that the lower numbers of total natriuretic receptors in hypertensive

anirnals are also of a different nature than those of normotensive controls. consisting of an

elevated proportion of NPR-C (or clearance) receptors.

No significant variation is seen between the total ANP receptor characteristics (B,,

and KJ of water- versus ETOH-treated anirnals (Table 3.5.1 .). Similady. the quantity of

NPR-C receptors. evaluated by the displacement of labeled ANP by cold cANF. is not

different between treatments. However, there is a significant increase in the K, measured

Table 3.5.1. Nott*iitrrtic tecepot* cliot*cicterrstics (B , , , o t d KA in the SFO, CP ritid A P nfret* 8 tirotrtlu. ojiinter or ETOH ttaenttnetit

itr WK Y n id SHR rats. "'1-A NP displc~erl by cold A NP ('"1-ANP~A NP) repesetrts totnl trotriirtatic peptide ivceptors. "%A NP

displnced bjt cold CA NF ('"1-A NP/cA NF) rel>t*esetrts the clenrntice (NPR-C) receptotrr.

WKY- 80.1 29.1 128.7 168.0 ~t 120.0 57.8 174.7 81.1

4 0 k 9.0 5.0 * 68.0 36.3 * 23.7 * 27.1 L 22.4 16.4

WKY- 106.5 43.4 250.7 89.0 88. O 257.8 212.2 136.1

ETOH I 10.2 * 8.2 * 47.4 k17.2 k l 7 . 2 i 45.2* k 28.5 21.0

SHR- 47.5 55.8 30.8 15.3 27.7 32.7 21.6 14.3 264.7 1 15.0

H,O * 7.1t 1 0 1 *9.2 i 9.8 k6.17t k9.l-t i4.81. 7.9 * 35.8 ;t 19.9

SHR- 37.1 46.9 13.5 74.7 35.9 25.3 22.3 124.0 160.7 125.5

ETOH i : l . l t t k9.4 i: 6.1 i: 15.0* * 5.977 i 7 3 * 2.1t *23,8* k4Z.l k 22.2

Values sliowii are inean * S.E.M. (ii=6). Tlie B,,,, are iiieasured iii fiiioViiig proteiii. The Y, are iiieesiired in pM.

'p 4.05, "p 4 . 0 1, SI-IR versus WKY; 'p 4.05, ETOH versus HIO.

l I I 1 -10 -9 -8 -7 -6

-log ANP (M)

T ?

-10 -9 -8 -7 -6

-log cANF (M)

I WKY water - SHR water

- -C - WKY ETOH - 4 - SHR ETOH

F iguie 3.5.1. Average (n 4) cornpetition binding curves of ' L 3 1 - ~ ~ ~ in adult (38 weks-old) SHR and WKY rats subfornical organ (S FOI, after 8 months of w t e r or ETOH (20% v/v) treatment. Autoradiographic sections are incubated \hith increasing concentrations of unlabeled ANP (A) and cANF (B).

CL 7 e - 40

1

in' CV T L.

20

O I T l I 1 O

-IO -9 -8 -7 -6

-log ANP (M)

1 I 1 I

-10 -9 -8 -7 -6 -log cANF (M)

-- WKY water - SHR water

-4- WKY ETOH 4 SHR ETOH

F igure 3.5.2. Average (n 4) cornpetition binding curves of ' 2 5 1 - ~ ~ ~ in adult (38 m e ks-old) S HR and WKY rats choroid plexus (CP), after 8 months of m t e r or E TOH (20% v/v) treatment. Autoradiographic sections are incu bated mith increasing concentrations of unlabeled ANP (A) and cANF (6).

for the NPR-C receptors of the ETOH-treated animals in the SFO (for SHR rats) and CP

(for both WKY and SHR rats) (Table 3.5.1. ). This result suggests a lower affinity of the

clearance receptors for the circulating natriuretic peptides and the possibility of a greater

proportion of the total plasma natriuretic peptides binding to the active NPR-A. B

receptors .

3.5.4.2.Area (AP)

Mean (n=6) displacement of "51-ANP by cold A N P and cANF (Figure 3.5.3.)

confirms the absence of B, or K, variation between strains (Table 3 S. 1. ). cANF has no

effect in displacing E51-ANP. suggesting the absence of NPR-C receptors in this tissue.

No significant variation is seen between the total ANP receptor characteristics (B,,

and K,) of water- versus ETOH-treated animals (Table 3.5.1 .).

3.5.5. DISCUSSION

The present experiments 1 ) demonstrated significantl y lower natriuretic peptide

receptor binding sites in SFO and CP of S H R cornpared to WKY rats. Furthermore.

rnoditications in the relative proportion of the different ANP receptor subtypes were noted

beween hypenensive and normotensive animals in the two circumventricular organs: and

1) showed a significant reduction by ETOH treatrnent in the affinity of the NPR-C

receptors located in the SFO and CP.

In contrast to the natriuretic peptides found mainly within the brain. most of the

natriuretic receptors in the central nervous system (CNS) are located in circurnventricular

organs. particularly in SFO. CP and AP (Quirion et ai.. 1986. Brown and Czarnecki.

1990a.b). These structures Iack a BBB. a unique situation in the CNS that enables them

to transduce directly hormonal signals from the circulation (for SFO and AP) and the

cerebrospinal tluid (CSF) (for CP) to the neural circuits within the brain. Thus. they

display high concentrations of receptors for the hormones involved in the regulation of

cardiovascular system, such as ANP and angiotensin 11 (AH) (Saavedra et al.. 1987).

S imilarl y. numerous nerve fibers are found connecting these structures to hypothalamic

I I I 1 i 1 I

-10 -9 -8 -7 -6

-log ANP (M)

I I 1

-10 -9 -8 -7 -6

-log cANF (M)

I WKY water

- 4- WKY ETOH

.L SHR water

- -8- SHR ETOH

F igure 3.5.3. Average (n 4) cornpetition binding curves of ' 2 5 1 - ~ ~ ~ in adult (38 w e ks-old) S HR and WKY rats area postrema (AP), after 8 months of w t e r or ETOH (20% vM treatment. Autoradiographic sections are incubated increasing concentrations of unlabeled ANP (A) and cANF (BI.

245

or rnedullary cardiovascular centres. such as the AV3V a r a or the NTS-baroretlex region.

For example. Paikovits et al. ( 1992) reported ANP-sensitive nerve fibers and terminals in

the SFO arising from deep into the hypothalamus. Likewise. AH-sens itive neurons have

been shown to connect SFO with the AV3V area (Buggy and Bealer. 1987).

The current hypothesis for the contribution of the brain natriuretic peptides farnily to

BP regulation involves two aspects: the natriuretic receptors present in circumventricuiar

organs and therefore m f e r i n g information from the periphery to the brain. and the ANP

and CNP stores which are located within the cardiovascular centres of the CNS. Brietly,

from the circulation. ANP may bind to specific receptors (in the SFO for example) and

inhibit AH-sensitive cells fibres to the AV3V region (Hattori et al.. 1988). This inhibition

would result in lower stimuli for tluid intake or for AVP secretion from the supra-optic

nucleus (SON). promoting BP lowering (Mangiapane. 1987). Likewise. A N P and CNP

from the hypothalamus rnay directly mediate a depressor action of the AV3V area on BP

(Ku et al.. 1994) and suppress in parts A V P release from SON (Standaert et al., 1987).

Al terations in natriuretic receptor numbers and affinity have been described in

pathophysiological conditions such as hypertension. SHR rats have been reported to exhibit

lower B, for the total natriuretic peptide receptors cornpared to WK Y anirnals in SFO

and CP (Saavedra. 1986. McCarty et ai.. 1986. Brown and Czarnecki. 1990a). but not in

AP (Saavedra et al.. 1987). These results are confirrned by the present studies. Recently.

different natriuretic receptor populations have been suggested to exist in the

circumventricular organs. Konrad et al. ( l992a) described an heterogeneous population of

active nauiuretic receptors (NPR-A and NPR-B). devoid of clearance receptors. in the AP.

This finding is confirmed by the present studies. The SFO has been reported to produce

an hornogeneous population of NPR-A (Himeno er al.. 1992. Brown and Zuo. 1993.

Zorad et ai.. 1993) or NPR-B (Konrad et al.. 1992b). In contrat, the presence of NPR-C

in association with active natriuretic receptors (NPR-A or NPR-B) in various proportions

has been described in the CP (Himeno et al., 1992, Brown and Zuo. 1993. Zorad et al.,

1993). These studies were all perforrned on normotensive anirnals and are in agreement

with the result of the present experiments for WKY rats. However. there are no published

snidies investigating the quantity of the various natriuretic receptor subtypes in conditions

such as hypertension or ETOH intoxication. The results of the present experiments suggest

the existence of a subpopulation of NPR-C receptors in the SFO and an elevated

proportion of NPR-C in the CP of SHR compared to WKY rats. I t is possible that with

high BP and high circulating ANP levels (Gutkowska et al., 1986a). secondary decreases

in natriuretic receptor numbers are seen, but also augmentations in NPR-C receptors to

remove excess ANP from the circulation. It could also be a genetic difference between

SHR and WKY rats, leading to a smaller depressor effect of peripheral ANP to central BP

regulation, and therefore to elevated BP in SHR anirnals.

Long-term moderate ETOH consumption had no effect on the total natriuretic

receptor characteristics (B, and KJ of the three circumventricular organ investigated.

However. the afinity for the NPR-C recepton. evaluated by the displacement of '"1-ANP

by cold cANF. was signiticantly lower in the SFO and CP of ETOH- compared to water-

treated rats. Therefore. it appears that a greater proportion of peripheral ANP rnay bind

to the active NPR-A,B receptors following chronic moderate alcohol consumption. leading

possibly to enhanced signal transduction of plasma (and/or cerebrosp inal fluid) ANP to the

brain cardiovascular centres. Such an effect rnay stimulate ANP-sensitive tïbers and inhibit

AH-sensitive neurons comecting the SFO (andlor the CP) to the AV3V area and produce

some depressor. diuretic, natriuretic and antidipsogenic effects that rnay mediate parts of

the antihypertensive effect of moderate ETOH treacment.

In summary . the significant decrease in natr iuretic recep tor dens ities in hypertens ive

animals rnay be considered secondary to the increased circulating ANP levels. However.

since this reduction in the B, is already present in Young, pre-hypertensive rats

(Saavedra. 86. McCarty et ai., 1986, Saavedra er ai., 1987), it rnay also represent part of

the reason for the elevated BP in SHR rats. The augmented proportion of clearance

receptors in the SFO and CP of hypertensive compared to normotensive rats reported in

the present studies would further diminish any effect of peripheral ANP on the CNS.

Conversely, this rnay also represent a compensatory mechanism to remove from the

circulation the increased plasma ANP levels in SHR rats.

Funhermore. the signifiant decrease in the afi-nity of the clearance receptors located

in the SFO and CP following long-term ETOH administration may contribute. at least in

part. to the prevention of the age-dependent increase in the BP following chronic alcohol

treatment.


The authors wish to thank Ricardo Claudio for his friendship and excellent care of

the animals. Special thanks are also given to Céline Coderre and Nathalie Charron for their

technical assistance.

P.G. is the recipient of a doctoral scholarship from the " Fonds pour la formation de

Chercheurs et l'Aide à la Recherche" (FCAR). This work was supponed by grants from

the Medical Council of Canada (MRC) (MT- 10337 and MT- 1 1674) (J.G.) and by the

alcohol research program at Douglas hospital ( C . G . ) .

GENERAL DISCUSSION

The present work descnbes the effects of acute and chronic moderate ETOH

consumption on the major components of the natriuretic peptide system (ANP. BNP. CNP.

NPR-A, NPR-B and NPR-C) in the heart, the kidney and the bram. It also coaârms the

existence of a lowering effkt on the BP of long-term moderate alcohol consumption in both

hypertensive and normotensive stragis of rats. Furthemore, the present studies explore a

specific mode1 for the ETOH-mduced preveution of the age-dependent mcrease in BP

observed m both rat and human populations. It thus provides msights into the mteractions of

ETOH with the natriuretic peptide syaem, which mteractions may contniute to the

antihypertensive effect produced by chronic moderate ETOH drinking.

4.1. EFFECT OF ETOH ON THE BLOOD PRESSURE

There is a progressive mcrease with age m BP. particularly in strams of rats with genetic

predisposition to hypertension nich as SHR rats (Howe et al.. 1989). Even though acute

eqosure to moderate concentrations of ETOH did not produce tigdïcant changes in the BP

of either rats (Section 2.1.) or humans (Section 2 - 2 4 the administration of moderate

quantities of ETOH (20% v/v) for 8 months resulted in the partial suppression of the age-

dependent increase in BP in both SHR and WKY rats (Section 3.1. ).

The absence of significant Merences either m body weight mcrease or m liquid

consumption between the water- and ETOH-treated groups indicated that the effect of

chronic moderate ETOH consumption on the BP was not due to secondary d d dehydration.

Thus, the ETOH-induced alterations m BP observed in the present studies agreed with the

majority of the human and animal midies in which an antihypertensive effect of chronic

moderate ETOH consumption has been reported (Section 1.2.2.). Currently, the mechanisms

mediahg this antihypertensive effect of ETOH are not weli understood. The present audies

suggest that the natriuretic peptide f d y may mediate some of the effects of ETOH on the

cardiovascular system.

4.2. EFFECT OF ETOH ON PLASMA A N D HEART NATRIURETIC

PEPTIDES

4.2.1. PLASMA

The administration of a single moderate dose of ETOH ( 1 and 2 g ETOWkg B.W.) by

[.p. mjection resuhed in a rapid but short-lasting mcrease in plasma ANP levels ( Section 2.1 . ).

Circulating ANP lwels were also sigmficantly elevated foilowing the ingestion of a 0.25 and

a 0.50 g/kg B. W. ETOH drink in human volunteers (Section 2.2.).

Ln contrast. the administration of a moderate 20% v/v ETOH solution for 8 months in

WKY and SHR rats was associated with significantly lower iirculating ANP (Section 3.1. )

and BNP (Section 3.2.) Ievels compared to water-treated controls.

Based on experirnental evidence dernonstrating (a ) that acute ETOH exposure induces

a transient increase m the release of natriuretic peptides fiom the heart and (B) that following

mcreases or decreases m BP there are hemodynamic modifications which would respectively

increase or decrease the release of cardiac natnuretic peptides. two hypotheses may br

proposed to explain the low content of natnuretic peptides in the plasma of rats treated

chronically with moderate amounts of alcohol:

( 1 ) According to the fïrst hypothesis. the low levels of circulating natriuretic peptides in rats

treated chronicaNy with ETOH are a direct consequence of the significant decrease in systolic

B P and the associated bemodynarnic modifications reflecting lower requirements for

circulating ANP and BNP levels and thus !ower rates of cardiac release.

(2) According to the second hypothesis. the low levels of circulating natriuretic peptides are

due to either the absence or low levels of ETOH in the circulation during the light phase of

the dady cycle d e n the estimation of plasma naaiuretic peptides were perforrned. During the

Light phase of the daily cycle. rats are not eating and drinking actively. thus the BAC is low

and there is no direct stimulation of the cardiac tissue by ETOH to increase the release of the

natnuretic peptides. in the absence of a direct stimulatory effect of ETOH on the cardiac

tissue. the hemodynamic modifications produced by the significantly lower BP predominate

and. as a consequence, there is a lower rate of ANP and BNP release and lower plasma ANP

and BNP Iwels. It is possible that during the dark phase of the daily cycle when the animals

are drinkmg and eating actively and when the BAC is hi&. the stimulatory effect of ETOH

on the cardiac tissue may predonmiate leadmg to enhanced release of natriuretic peptides and

higher contents of ANP and BNP m the circulation. The absence of down-regulation of the

atrial and ventncular ANP and BNP systems. as a logical consequence of the prolonged

decrease in BP. is in support of this hypothesis.

4.2.2. HE ART ATRL4

A schematic diagram of the heart ANP and BNP systems is presented on figure 4.2.1.

The translation of ANP and BNP mRNAs in the atnal tissue produces ANP and BNP

prohomones which are then stored in specific granules. The ANP contained in atrial gantdes

is the main contributor to cuculating ANP levels (Flynn et al.. 1983). Unlike ANP. BNP is

mostly produced by the heart ventncles (Ogawa et al.. 199 1). Nevertheless. BNP is also

colocalized with ANP m certam aûial granules (Thibault et al.. 1992). With the development

of high BP. such as in SHR rats. a t d ANP mRNA and total BNP content are increased.

resulting in higher plasma levels of natriuretic peptides (Sections 3.1. and 3.2.).

The administration of a single moderate dose of ETOH ( 1 and 2 g ETOWkg B.W.) by

i -p . injection resulted in a rapid decrease in lefi and right atrial content (Section 2.1.). In

contrast. following 8 months of continuous ETOH (20°h v/v) administration. there was a

sign5cant increase in both lefi and rigbt atnal ANP content and concentration in WKY and

SHR rats (Section 3.1 .). Similarly, ETOH also increased atnal BNP levels in WKY rats and

lefi atrial BNP content and concentration m SHR rats (Section 3.2.). There was no signifïcant

daerence in atrial ANP or BNP mRNA levels between the water- and the ETOH-treated

animals of both strains.

Based on our nudies demonstrating that acute. i j i vivo. exposure to ETOH induces a

transient mcrease m plasma ANP levels (retuming to basal levels within 2 hours post-ETOH

admbhration) as a consequence of a short-lasting increase in ANP release. and considering

the eating and drinking pattern of rats during the daily cycle (the eating and drinking occur

during the dark phase of the daiiy cycle), the a c t ~ t y of the atrial natriuretic peptide system

Ca rdiac natriuretic system

Ve nvicles ANP

4- mRNA ANP BNP

0 granule

F igu re 4.2.1 . Schematic representa tion of the natriuretic peptide system (ANP and BNP) in the heart. Atria: The translation of ANP (and BNPi mR NAs produces ANP (and BNP) prohormones. The prohormones are then stored in atrial granules. ANP (and BNP) are released in the circulation upon a trial stretch. Ventricles: The translation of BNP (and ANP) mRNAs produces BNP (and ANP). Due to the absence of ventricular granules, B NP (and ANP) are released constitutively in the circulation.

may be descnïed as outlined in Figure 4.2.2.:

( 1 ) During the dark phase of the daily cycle. the animals are active and thus eating and

conaiming sigaificant amounts of ETOH. Accordingly, BAC levels are hi&. The release of

ANP and BNP rnay be stirnulated by alcohol. leacüng to transient increases in plasma

natriuretic peptide levels and stimulating peptide and mRNA çynthesis in the atria. The only

other published study investigatmg the effect of chronic (6 weeks) moderate ETOH

consumption on the hem ANP system has reported a high BAC ( 172.0 * 13.9 mg/dl) (Wigle

et al.. 1993b). Interestingly. this hi& BAC level was associated with a si@cant increase

in atrial ANP mRNA levels.

(2) hiring the light phase of the daily cycle. the animals are inactive and thus are not eating

or consuming sigdicant amounts of ETOH. Accordmgly. BAC levels are low (28.92 c 12.25

and 39.23 * 18.97 mg/dl for the WKY and SHR rats of the present studies). Therefore. there

is no direct stimulation of ANP and BNP release fiom the atrial tissue by ETOH. so that the

release of the two natriuretic peptides is controlled mainly by the hemodynamic mechanisms.

Since the BP is lower in the ETOH- compared to the water-treated animals. the release of

ANP and BNP is lower. resulting in lower plasma levels and the increase in the accumulation

of ANP and BNP peptides in the avial granules. Iadeed. the higher accumulation of ANP and

BNP in the atria of the ETOH-treated animals compared to the water-treated controls. and

the finding that the contents of atrial ANP and BNP rnRNA are similar in the atria of the

ETOK and water-treated rats indicates that despite the low BP the atnal ANP and BNP

systems are not down-regulated by the chronic moderate ETOH e.xposure. It may then be

proposed that in the ETOH-treated animals when the BAC is high (usually during the dark

phase of the daily cycle), alcohol either directly or indirectly through its effects on vanous

hormonal and neurotransmitter systems. stimulates the release and synthesis of atrial ANP and

BNP (as seen by Wigle et al.. 1993b) and thus maintams the content of ANP and BNP mRNA

in the atria to the same levels as those in the atria of the water-treated animals which evhibit

a significantly higher BP. It rnay be suggeaed that in the water-treated rats. ANP and BNP

synthesis and release are higher probably because of the high BP. whereas in the ETOH-

treated rats. the atrial ANP and BNP systems are stirnulated to a higher level of a c t ~ t y by

@ Dark phase of the daily cycle (High BAC)

ANP B N P + + a Light phase of the daily cycle (Low BAC)

-

Ve nmcle s I- ANP WKY - BNP

p --- --.-=-O f-b BNP

+ hig he r leve Is ANP BNP

-: lover levels - -

Fig ure 4.2.2. Schemuc representation of the possible ETOH-induced changes in circulating and heart natnurenc pepbdes dunng the Iight

and dark phases of the daily cycle.

(1 ) Atria: The high BAC m y sumlate amal ANP (and BNP) synthesis and increase the release of ANP (and BNPI fromlhe

atnal granules.

Vent r ic les : The high BAC may st imlate BNP (and ANP?] synthesis and release from the ventncles.

12) Atria : Because of the low BAC. there is no direct stimulation of ANP (and BNPi release from the atnal granules by ETOH,

so that the release of ANP (and BNP) is controlled mainly by the hemdynamc mechanism. Therefore. ANP (and BNPI

release are decreased in ETOH- conpared to water-îreated rats as a direct consequence of the lower BP and ANP (and

BNPi accumilate in the amal granules. Neverttieless, ANPlBNP mRNAs are maintained a t the same levels as those of

water-mated rats, despite significandy lower BP, probabty because of the direct stimlaaon by ETOH dunng the dark phase.

Vent r ic les : During low BAC, BNPlANP M N A s are rmintained at the s a m levels in ETOH- and water-treated WKY rats,

despite significandy lower BP, probably because of the sumlation of BNPIANP synthesis by ETOH during the dark phase.

Oudng low BAC, BNP mR N A is higher in ETOH- conpared to water-ireated SHR rats, despite signrficandy lower BP.

probably because of the stin-ulation of BNP synthesis by ETOH during the dark phase. In conûast ANP rrClNA 1s lower in

ETOH- conpared to water-treated SHR rats.

255

a different mechanism than hemodynarnic changes, probably by ETOH. Thus, it may be

hypothesized that this enhanced potential actinty of both atrial ANP and BNP systems is

produced by the repetitive stimdatory effects of the daily ETOH consumption.

4.2.3. aEART VENTRICLES

The schernatic diagram of the ventricdar ANP and BNP systems is presented on figure

4.2.1. Due to the absence of grandes, the translation of ventncular ANP and BNP &As

produces mature natriuretic peptides that are released constitutively in the circulation. in

contrast to atrial ANP and BNP release (Lang et al.. 1992). The m m site of ANP production

in the body is the heart atria and 95% of the circulatmg ANP is produced by the atnal tissue

uuder normal conditions (Ogawa et al.. 199 1 ). However, with the development of hi& BP

and venaicular hypertmphy m the SHR rats. this contribution decreases to 60%. In contrast.

the ventrkular tissue contniutes to more than 60% of total circulatmg BNP levels under

normal conditions (Ogawa et al.. 199 1 ). This contnbution is fùrther increased m certain

pathophysiological conditions, such as hypertension (Yokota et al., 1993).

The admini_ctratiotl of a single moderate dose of ETOH ( 1 and 2 g ETOHkg B. W.) by

i.p. mjection resulted m a dekyed increase m ventficular ANP levels (Section 2.1 .). Following

chronic ETOH treatment, total ventncular ANP content and ventricular ANP mRNA were

sigaificantly lower in SHR, but not WKY rats (Section 3.1.). in contrast, ventricular BNP

conceniration and BNP mRNA levels were significantly increased in S m but not in WKY

rats (Section 3.2.).

Based on our studies demonstratmg that acute ETOH consumption induces a transient

hcrease m plasna ANP levels and a delayed mcrease in ventncular ANP content, and taking

into consideration the eating and drslkmg pattern of rats during the daily cycle, the mode1

proposed for the effect of ETOH on the atrial natriuretic peptide systems may be expanded

to include the ventncular natriuretic peptide systems (figure 4.2.2. ):

(A) WKY rat : The chronic ETOH treatment produced no aherations in ANP and BNP

contents or in ANP and BNP mRNA lwels, even though the BP was significantly lower in

the ETOH-compareci to the water-treated animals for at least a month pnor to sacrifice. The

fàct that a decrease m the act-ivity of the ventricular BNP and ANP qstems was not observed

in the ETOH-treated WKY rats mdicates that chronic ETOH treatment maintained the

potential actMty of the ventricuiar natriuretic peptide systems to the same levels as those of

water-treated WKY animals exhibithg higher BP. Thus, t may be hypothesized that during

the dark phase of the daily cycle, each ETOH drmking episode produces an mcrease m

ventricular ANP and BNP release, leadhg to an mcrease m ventricular ANP and BNP mKNA

b e l s and ANP and BNP biosynthesis. During the light phase of the cycle, the hemodynamic

fàctors predominate and the ventricular natriuretic peptides are not stimulated. In agreement

with this model, the study of Wigle and colleagues ( 1993b), performed in conditions of high

BAC, demonstrated Sgnificant elevations in both ventricular ANP mRNA and ANP content

following the administration of moderate ETOH b e l s for 6 weeks.

(B) SHR rats: The chronic ETOH consumption prevented the development of ventricular

hypertrophy in SHR rats. Indeed, the total ventricular protein content is sigiilficantly lower

in ETOH-compared to water-treated SHR rats, mdicatmg the absence of ventricular

hypertrophy. Thus, the significant increase in ventricular %NP &A levels suggests a

specific effect of ETOH mice the Iower BP of ETOH-treated SHR rats prevents the

amibution of this effect to compensatory mechanisms due to elevated BP. Therefore, it may

be hypothesized that during the dark phase of the daily cycle, the ETOH consumption

Stimulates venhicular BNP activity and thus BNP mRNA levels, producing transient increases

in circulating BNP levels. Interestingly, elevated plasma BNP levels have already been

observed following short-term chronic ETOH drinking, when the animals were sacrifïced

during high BAC conditions (Wigle et al, 1993~). Chrooic stimulation of the ventricular BNP

system by ETOH may then maintain higher basal levels of ventricular BNP mRNA in ETOH-

treated SHR rats as suggeaed by the higher BNP mRNAs, compared to water-treated SHR

rats which have higher BP. In contrast to BNP, the contniution of ventricular ANP content

to the plasma ANP levels must be minimal in ETOH-treated SHR rats. The sigdcant

decrease in ventricular ANP content and ANP mRNA levels shouid reflect the lower

ventncular mass due to ETOH. Therefore, any possible stimulatory effect of ETOH on the

ventricular ANP system of SHR rats may be masked by the sigdicant decrease in ventricular

4.3. EFFECT OF ETOH ON RENAL NATRKTRETIC RECEPTORS

The natriuretic and diuretic effects of circulating (ANP and BNP) and intrarenal (UD)

natriuretic peptides are mediated by receptors located in the glomenili (NPR-A and NPR-C)

and the coilecting duct of the b e r medda (NPR-A) (Figure 4.3.1. ):

(A) in the glomenili NPR-A receptors are considered effective mediators of the physiological

effects of natriuretic peptides through the activation of the secondary rnessenger. cGMP

(Kolier and Goeddel, 1992). Natriuretic peptide binding to tbese so-called active glomemlar

aatriuretic receptors (NPR-A) has been açsociated with the relaxation of giomemlar mesangial

ceils and with the expansion of the capillary surface area for filtration. producing a modest

increase in GFR (Singhal et al.. 1989). The relaxation of glomedar affereat anerioles and

the constriction of giomerular efferent anerioles produced by the binding of natriuretic

peptides to active natriuretic receptors also increase GFR Ma enhanced glomemlar capillary

hydraulic pressure (Marïn-Grey et ai.. 1986: Kumira et al.. 1990). in contraa. the exact role

of the NPR-C receptors is unclear. Any fuoction beside the weil-defined "clearance" role

remaius highly speculative (Maack et ai.. 1993).

( B ) An homogeneous NPR-A receptor population exias in the CM (Martin et al.. 1989). The

binding of natriuretic peptides to this receptor has been associated with the suppression of

sodium reuptake via amiloride-sensitive sodium channels (Sonnenberg et al.. 1986: Zeidel et

al., 1988) and with the inhibition of the antidiuretic effect of AVP (Zeidel et al.. 1987).

Interestingly, significant increases in IM natriuretic peptide binding sites have been obsewed

in hypertensive animais. suggesting increased NPR-A a c t ~ t y to compensate for the higher

BP (Nuglozeh et al.. 1990; Bonhomme and Garcia. 1993).

Chronic moderate ETOH drinking is associated with a significant reduction in total

glomedar natriuretic peptide bmding sites (Section 3.3. ). Furthemore. quantification of the

various receptor subtypes suggeaed a significant decrease in glomerular NPR-C (or

Plasma Glomerulus

NPR-C k NPR-C

""-A F lncrease in GFR

NEPHRON

Distal tubule Collecb'ng duct

O $ k eceptor-second messenger cornpiex (NPR -A)

NPR-A

O R eceptor (NPR-C)

G F R : glomerular filtration rate

Inhibi~on ofNa \eabsorption

Inhibibon o f H 2 0 reabsoipbon

Figure 4.3.1. Schematic representation of the renal natriuretic system. (A) The binding of plasma ANP and BNP to the NPR-A receptors of the glomeruli produces an increase in G FR. (5) The binding of ANP and BNP, as vudl as intrarenal UD, to the NPR -A receptors of the inner medulla produces the inhibition of sodium and w t e r reabsorption.

clearance) receptors, but not m glomenilar NPR-A receptors. Chronic ETOH consumption

is also associated with a significant mcrease m b e r medullary (IM) natriuretic peptide

binding sites (NPR-A) ( Section 3.3. ).

Based on our snidies demonstrating ETOH-induced changes in renal natriuretic peptide

receptors (NPR-A and NPR-C), a mode1 illustrating a possible role of the renal natriuretic

system m the antihypertaisive ef5ect of moderate alcohol connunption is proposed on Figure

43.2:

( 1 ) in the glomeruk the specific dom-regdation of NPR-C receptors suggests that a greater

proportion of circulating ANP and BNP levels may bind to the active receptors (NPR-A).

aUowing a greater glomerular effect at any given concentration of circulating natriuretic

peptides. It has been lmown that various stimuli such as salt Ioad or hypertension specifically

dom-regulate NPR-C receptors in glomenilar tissue in order to reduce the plasma ANP and

BNP butTering by NPR-C and to allow an enhanced renal diuretic and natriuretic effect

through the NPR-A receptors (Ogura et al.. 1989: Kollenda et al.. 1990). However. the

demonstration of a selective dom-replation of glomerular NPR-C receptors by ETOH.

despite the lower BP m these animals compared to water-treated rats. niggests that this effect

is not due to secondary hemodynarnic characteristics. Thus, the physiological consequence

of the effect of ETOH on the glomerular tissue may be enhanced GFR and as consequence

some antihypertensive effects.

(2) The demonstration of higher IM natriuretic peptide binding sites in ETOH-treated rats.

despite the sipificantly lower BP. suggests a specific effect of ETOH in renal tissue. Thus.

circulating ANP and BNP reaching the IM (as weU as intrarenal C I D ) have the possibility to

produce enhanced inhibition of sodium reabsorption by the collecting ducts in ETOH-

compared to water-treated rats.

The ETOH-induced modifications in the kidney. such as the increased NPR-A (LM) and

decreased NPR-C (glomedi), suggest enhanced diuretic and natriuretic effects. which may

lead to a long-lasting decrease of the BP. However, to assig. any functional significance on

the ETOH-induced changes in the renal natriuretic peptide receptors, the increase in the

relative proportion (glomeruli) and number (IM) of NPR-A must be associated with increased

Plasma Glomenilus Disal tubule Cokcbng duct

Increase in GFR

+

NEPHRON

7

NPR-A

NPR-A +

( Inhibi~foon o f H20 rea bsorp don

+

X: decrease in receptor quantity +: increase in receptor quantity

Figure 4.3.2. Schematic representation of the ETOH-induced changes in

renal natriuretic peptide receptors. (1) The reduction in glomerular NPR -C quantity ma y enhance the contribution of NPR-A, leading to a greater increase in the GFR. (2) The increase in IM NPR-A quantity may enhance the inhibition of sodium and vlater rea bsorption.

urinary excretion of cGMP in the ETOH-treated mimals. hterestmgly. there is either no

change (WKY) or an mcrease (SHR) in the h a r y excretion of cGMP in ETOH- compared

to water-treated rats. despite the reduced plasma levels of ANP and BNP observed during the

light phase of the daily cycle as a consequence to the secondary bernodynamic effects

associated with the lower BP (Section 4.2.). This suggests that a sirnilar level of circulating

natriuretic peptide would have a greater effect in ethanol- compared to water-treated SHR

rats at the level of the kidney. because of the ethanol-mediateci modifications of the natnuretic

peptide receptors in the glomenili and the huer medulla. In SHR rats. these receptor

alterations are sdicient to produce higher levels of urinary cGMP during the light cycle. at

a time when the plasma ANP levels are lower. In WKY rats. despite the Iower circulating

ANP and BNP levels, the urinary cGMP levels of the ETOH-treated rats are similar to those

of the water-treated WKY rats which exhibit higher circulating ANP and BNP levels.

indicatmg a functional significance of the ETOH-induced changes of the renal NPR-A

receptors. These r e d s support the theory that the ETOKinduced modifications in the renal

natriuretic receptors may contribute to the antihypertensive effect of long-term moderate

alcohol consumption. The potential signficance ofthe ETOH-induced changes in the renal

natriuretic peptide receptors rnay be even more important during the dark phase of the daily

cycle. when as a consequence of the higher BAC the plasma ANP and BNP levels rnay be

elevated. inducing a further increase in cGMP production and natriuresis. and a tùrther

decrease in BP.

4.4. EFFECT OF ETOH ON BRADJ NATMURETIC PEPTIDES AND

RECEPTORS

Under normal conditions, the basal a c t ~ t y of the brain natriuretic peptides syaem is

detennined by the BP. Two dinerent pathways have been postulated to influence central

natriuretic peptide levels and may serve as sensors of the BP (Figure 4.4.1.):

(A) Natriuretic peptide receptors located in the subfomical organ (SFO) and area postrerna

HYPOTHALAMUS MEDULLA

CP (CSFI

NPR-A

NPR-C

NPR-A

Plasma ANP

AVP

AVP

I l Ba roreceptors 0

NTS : nucleus tractus solita rus NL: neural lobe of the pituitary gland AP: area postrema ME : median eminence LC: locus coeruleus CP: choroid plexus AV3V: anteroventral third nucleus area CS F : cerebrospinal fluid S FO: subfornical organ NE n: norepinephrine neuron S ON: supraoptic nucleus All n: angiotensin II neuron PVN: paraventricular nucleus ANPn: ANP neuron

- I Inhibition

Figure 4.4.1. Schemaîic represeniation ofthe naüiurebc peptide farnily in the central neivous

sysem. (A) The naüiuretic peptide recepcrs located in the SFO and AP aansduce

hormonal inbrmation fiom bie plasma a, the brain cardiovascular centres in the

hypothalamus and medulla. (B) The barorecepmrs tansduce neuronal information

fiom the peripheryto the brain atihe level ofthe medulla.

The ANP and CNP fibers located inthe AV3V area suppress AVP release from the

SON and PVN,inhibitwterand saltintake and produce some depressoreffects.

Hypoihalamic ANP and CNP fibers mayalso conûibute î~ plasma ANP levels through

ANP release from lhe ME ordwough the release ofother facmrs fiom the NL siimulabng

heanANP release.

263

( AP), two circumventncular organs lackmg a blood-brain-bamer ( BBB ). transduce diuect ly

hormonal information fiom the circulation to the cardiovascular centres in the hypothalamic

AV3V area and meduliary nucleus tractus solitarü (NTS). respect~ely.

(B) Neuronal Somation ftom the baroreceptors in the nght atria. carotid and aortic suiuses

and in the kidneys are also reachhg the brah through the medulla and the NTS. The impulses

produced by the NTS fiom baroreceptor- and AP-mediated informations activate the locus

c o d e u s (LC) which m tm sends fibers of noradrenergic neurons (NEn) to the AV3V area

of the hypothalamus.

Interestin&. the NTS area is nch m ANP neurons (Kawata et al., 1985). Micromjections

of ANP into that region or into adjacent areas such as the ventrolateral medulIa have also

been associated with important decreases in BP ( McKitrick and Calaresu. 1 988: Bergaglio

and Calaresu, 1990). It seems then that medullary ANP may be involved in BP lowering by

interfering with the noradrenergic neurons and baroreceptor reflex since afferent

baroreceptor fibres terminate in the NTS.

Both the neuronal inputs fiom the medulla and the hormonal information fiom the SFO

(and the choroid plexus) terminate in the A V W area of the hypothalamus. This region is

central to the control of fluid intake and fluid excretion (Buggy and Bealer. 1987).

interestingly. this area bas also the highest concentration of ANP fibres in the brain (Kawata

et ai.. 1985). Intracerebroventncular (i.c.v.) injections of ANP have produced various

antidrpsogenic. dituetic and depressor effects which are believed to be mediated by the A V W

area. as suggeaed by a number of studies such as: (a) Centrally administered ANP

significantly inhibited the water intake d u ~ g water deprivation and NI-induced drinking

(Nakamura et al.. 1985: Antunes-Rodngues e t d . 1985; Katsuura et al.. 1986). ( b ) The i.c.v.

injection of ANP also reduced sigrilficantly the salt appetite during salt deprivation or in

mains usually know to have elevated salt appetite. such as SHR rats (Fitts et ni.. 1986: Itoh

el al.. 1986b). (c) Microinjections of ANP around the hypothalamus produced significant

increases in water excretion. possibly through the inhibitory effect of ANP on AVP release

f?om the supra-optic (SON) and paraventricular (PVN) nuclei (Israel and Barbella. 1986: Lee

et aL, 1989; houe et al., 1990). (d) 1.c.v. mjected ANP also signiticantly inhibited the pressor

action of ic.v. mjected An m conscious. unrestrained rats (Itoh et ai.. 1986a: Shimini er al..

1986; Castro et al., 1987).

Severai hypothalamic ANP neurons ( A m ) terminate in the median emhence (ME) or

the neural lobe of the pituitary gland (NL). probably leadmg to the release of natriuretic

peptides into the vasculature drainhg these two organs. The contribution of hypothalamic

ANP to circulating ANP levels is certaidy minimal. considering the low brain ANP levels

compared to those found in the heart atria. However. it is possible that the activation of

hypothalamic efferent ANP neurons to ME and NL produces the release of other factors fiom

the pmiitary gland. such as oxytocin (Haanwiuckel et al.. 1995), a d o r the activation of other

neuronal pathways to the heart. leading to the synthesis and release of ANP and BNP fiom

the heart atria and ventricles.

Following chronic moderate ETOH connimption. there is a significant increase in

hypothalamic. pontine and medullary ANP levels in SHR. but not WKY rats (Section 3.4. ).

Brain CNP levels are also elevated in SHR-ETOH rats and in the hypothalamus and medulla

of WKY-ETOH animals. Furthemore. chronic ETOH treatment significantly increased the

& of NPR-C receptors in the SFO (for the SHR rats) and CP (for both WKY and SHR rats).

but not in the AP (Section 3.5. ).

( 1) It rnay be speculated that elevated meduilary ANP levels produced by chronic moderate

ETOH consumption in SHR rats d o w a greater and prolonged inhibition of the baroreceptor

reflex. so that the reaing BP is calibrated to a lower level (Figure 4.1.2. ). it is also possible

that the increased concentration of medullary ANP levels in ETOH-treated rats enhances the

impulses of noradrenergic neurons to the hypothalamic ANP areas of the AV3V area.

promoting a greater antidipsogenic. diuretic and depressor effect.

(2) The significantly higher ANP content and concentration in the liypothalamus of SHR rats

following chronic moderate ETOH consumption may contribute to the ETOH-mediated

prevention of the age-dependent hypertension by enhancing the hhibitory effect on water and

sait mtake and the stimulatory effect on water and salt excretion by the ANP released in the

A V W area. Higher brah ANP levels following ETOH may also enhance the impulses of

hypothalamic efferent ANP neurons to the neurohypophysis and the median eminence.

HYPOTHALAMUS MEDULLA

SFO (plasmi

CP (CSFI

NPR-A

O N P R - C

NPR-A

AVP

AVP

+: higher levels Plasma ANP

-: i o w r levels

NPR-A

NPR-A

- I I - I I I 1 1 Barorecepmrs

I I

Figure 4.4.2. Schematic represenation o f h ETOH-induced changes in brain natriureic pepldes and

recepmrs.

(1) The increased medullary ANP and CNP IeveIs ma y produce a greaaer inhibibon ofthe

barorecepmrreflexand increase itie impulses ofNEn lo the hypothalamic AV3V area.

(2 The increased hypothalamic ANP and CNP levels may produce a g r e a ~ r inhibition of

AVP release fiom the SON and PVN,a greaorsuppressionofwoerand saltinmke and

enhance the depressor efkcts.

(3) The reduced affmityofihe NPR-Crecepmrs inthe SFO and CP may enhance the

conûibuiion ofttie NPR-A recepmrs, leading m a greaber inhibition ofthe pressor,

antidiuretic and dipsogenic e f k c ~ ofAlIn.

promoting higher basal levels of heart ANP and BNP synthesis and mRNA levels. despite the

lower BP (Figure 4.2.2.).

(3) The significant decrease m the affinrty of the NPR-C receptors m the SFO and CP

foUowing long-term alcohol consumption may slow a greater proportion of the natriuretic

peptides fiom the circulation (and/or the cerebrospinal fluid) to bind to the "active" NPR-AB

receptors. leading possibly to enhanced inhiibitioo of the pressor. antidiuretic and dipsogenic

effects mediated by the AI1 fibers (Figure 42.2.).

The ETOH-induced changes in brain CNP levels may also mediate some of the

antihypertensive effects of rnoderate ETOH consumption. Interestingly. CNP and CNP

mRNA have been isolated from the hypothalamus, particularly fiom the AV3V area ( H e m

er al.. 1993). However. hi& quantities of CNP have also been found in areas unrelated to

cardiovascular controL such as the cerebelium (Komatsu et al.. 199 1 ). Consequently. the

siflcance of the CNP changes in the mediation of the antihypertensive effect of ETOH are

difficult to assess until it is known to which extent central CNP may contribute to the

cardiovascular regdation.

4.5. GENERAL S U M M M Y AND FUTURE STUDIES

Based on our results for the ETOH-induced alterations in circulating, cardiac. brain and

renal natriuretic peptides and receptors. a tentative general pathway for the lowering of the

BP by alcohol may be outlined (Figure 4.5.1 .). The direction of most of the modifications in

the natriuretic peptide system foster the hypothesis that this hormonal family may mediate.

at least in part. the antihypertensive effect of chronic moderate ETOH consurnption.

( 1 ) Acute moderate ETOH consumption may increase circulating ANP and BNP levels.

During long-term alcohol treatment. this condition may occur during the dark phase of the

daily cycle, associated with high BAC.

(2) The elevated plasma natriuretic peptide levels results probably £?om the release of ANP

and BNP fiom the cardiac tissue. The greater ANP content accumulated in the atnal granules

Heart

B lood vesse1

1 1 B ra in (hvpothalamus. medu l la l

Kidne y

pressor efkct

vieterand saltinmke

barorecepbr reflex

a ntidiuresis

d iure s is

naaiuresis

3' -- O 4 reducedlevels / decrease

increased levels / increase

Figure 4.5.1. Schemaoc d iag ramsumnz ing the effects of chronic moderate ETOH consunpnon on the namureoc syçtemand the

m c h a n i s n s by which these modificauons rnay rnediate part of alcohol's antihypertensive effect.

During conditions of long-term moderate ETOH treaûmnt, acute E T O H consunption m y increase plasma ANP (and BNP)

levels (1). This effect results m s r likely from the release of accumilated ANP fmm heart aûial granules and from che

elevated BNP producaon from hean venmcular nssue (2). The elevated ANP (and CNP) contents in the CNS may also

contribute slighdy to the increased namureoc pepude levels in the circulation by ttre stinulauon of ANP release through ME

and by the activanon of horrmnal and neuronal pathways sunxilating heart ANP iand BNP) release through NL (3).

The elevated plasma ANP (and BNP) levels f o t l o ~ n g acute ETOH admnismuon may then lower BP by several

mechanism. They may produce vasorelaxaiion in the major blood vessels (4). They rnay enhance renal diuresis and

namuresis by binding to elevated NPR-AiNPR-C in the glomeruli and NPR-A in the IM (5 ) . They r m y reduce the Alln çignal

to rhe pressorcentres of the brain by binding to elevated NPR-AINPR-C in the SFO (6) . This effect associated w th the

elevated ANP iand CNP) contents in the CNS, m y reduce the antihypertensive effect of AVP, the dipsogenic and pressor

effects of All and che threshold forthe baroreceptor reflex (7).

of ETOH-treated animals during low BAC may represent the major source of elevated plasma

ANP levels following each acute alcohol exposure. Likewise. the enhanced ventricular BNP

mRNA and BNP content suggest elevated BNP production fkom the ventricular tissue and

qeater BNP release mto the circulation.

(3) Ahhough the major source of plasma ANP and BNP variations is the heart. the effect of

ETOH on the central ANP and CNP systems may also contniute slightly to the elevated

natriuretic peptide leveis. indeed the ETOH-mduced mcreases in hypothalamic and medullary

ANP and CNP contents may produce a greater activation of the ANP neurons (ANPn)

terminahg m the median eninence (ME) and neural lobe of the pituitary gland (NL). leading

to the release of natriuretic peptides mto the vasculature draining the ME or to the release of

other hormonal or neuronal pathways invohed in the stimulation of ANP and/or BNP eom

the heart.

The elevated plasma naaiuretic peptide levels foUowing each acute ETOH consumption

(or during hi& BAC) m chronicaUy ETOH-treated anirnals may then lower the BP by several

mechanisms.

(4) nie higher circulating ANP and BNP levels may produce some vasorelaxation in the

major blood vessels.

(5) The higher circulating ANP and BNP levels may cause elevated diuresis and natriuresis.

This effect is enhanced by the higher proportion of active NPR-A (dom-regdation of NPR-

C) in the renal glomeruli and by the increased quantity of NPR-A in the renal inner medulla

(IM) of ETOH-treated rats.

(6) The higher circulating ANP and BNP levels may reduce the pressor sipals to the brain

cardiovascular centres mediated by AI1 neurons ( A h ) . This effect is enhanced by the lower

a££ïnity of the clearance NPR-C in the SFO (and CP) of ETOH-treated rats.

(7) The effect of higher circulating ANP and BNP levels to the CNS is further enhanced by

elevated ANP and CNP contents in the hypothalamus and meduila of ETOH-treated rats.

These natriuretic peptide levels may reduce the antidiuretic effect of AVP. the pressor and

salt/water mtake produced by An and recalibrate the baroreceptor reflex to a lower BP value.

The objective of the present work was to mvestigate the implication of the natriuretic

peptide system in the prwention of the agedependent increase in BP by chronic moderate

ETOH consumption. In general terms, both the major sites of production for this hormonal

family, such as the heart, and the main effector areas, such as the kidneys and the bram.

presented ETOH-mduced modifications that may explain, at least in part, the effect of

moderate ETOH connimption on the BP. Therefore, both components of this endocrine

system, the peptides (ANP. BNP and CM) and the receptors (NPR-A NPR-B and NPR-C)

have undergone specific ETOH-mediated alterations, suggeamg widespread modifications

in the general characteristics of the syaem and ailowhg the possibility of a role in ETOHs

antihypertensive effect.

Smce the present studies are among the few experiments investigatmg the mteractions

of ETOH with the natriuretic peptide system, they are of an exploratory nature. Now that a

significant interaction between ETOH and the various cornponents of the natriuretic syaern

have been demonstrated, W e r studies should be perfonned focused on distinct components

ofthe natriuretic £àmily to evaluate the exact mechanisms mvolved and better defhe the role

ofthe natriuretic system on the lowering effect of chronic moderate ETOH consumption on

B P.

4.5.1. HEART A N D VASCULATURE

(a) The possiiility of an mdirect effect of ETOH on the cardiac natriuretic peptide system

is niggested by the observation that heart myocytes exposed to alcohol iti vitro present

no changes in ANP release (Wigle er al.. 1993b). interestingly, there is also a arong

positive correlation between the changes of plasma ANP and circulating O-endorphin

levels following acute ETOH administration (Section 2.1 .). Since ETOH consumption

rapidly increases plasma Il-endorphin levels (Gianoulakis et al., 1989) and p opioid

receptor agonists, such as fentanyl have been associated with the release of ANP fkom

the heart (Vollmar et al., 1987; Gutkowska et al., 1993), it may be hypothesized that the

ETOH-induced increase on circdating levels of û-endorphin peptides may mediate the

ETOH-induced mcrease in plasma ANP levels. Therefore, the effect of naloxone (an

opioid receptor antagooist) on the cirnilating ANP (and BNP) levels during acute ETOH

adminktmtion should illustrate whether the endogenous opioid system is mediahg the

ETORmduced mcrease in the plasma ANP levels. The use of opioid receptor

antagonists that do not cross the BBB nich as methyhaltrexone may M e r illustrate

whether the e f f i of alcohol on tûe nahiuretic peptides is produced in the heart or CNS.

(b) From the data obtained following acute and chronic ETOH exposure. we have

proposed that ETOH may increase the potential activity of the healt ANP and BNP

systems (section 4.2.). However, lower plasma ANP and BNP levels were observed m

animals chronicaily treated with alcohol, because of the hemodynamic modifications

produced by the lower BP. Yet. it was proposed that the ETOH-specific alterations in

cardiac ANP and BNP levels may enhance the ability of the heart natriuretic peptide

systems to respond to various physiological andlor pharmacological conditions (aich as

high BAC) by a more pronounced expression and release of the natnuretic peptides.

Thus. the effect of an acute ETOH administration in animals chronically treated with

ETOH should be mvestigated. The demonstration of an increased release of ANP andlor

BNP foilowhg an acute ETOH challenge would support the hypothesis of the tight and

dark phases of the daily cycle on ETOH consumption and its effect on the release of the

natriuretic peptides (section 4.2.).

(c) The wahration of the circulating and cardiac ANP and BNP levels at various intervals

during the 8 months of the experimental period should determine the progressive changes

in natriuretic peptide levels and allow their association with the development of the

antihypertensive effect of alcohol. The respective contribution of ETOH and of the

secondary hemodynamic changes to the actual b e l s of both ANP and BNP activity may

then become more clear.

(d) Puise and pulse-chase midies could determine the rate of biosynthesis of ANP and

BNP in the various components of the cardiac tissue and allow a direct correlation

between the effect of ETOH on the mRNA levels and the peptide biosynthesis. Such

experirnents are especiaily important for the BNP system seice the BNP gene contains

an ATlTA motif in the 3'-untranslateci region niggesting mRNA dest abilization ( Ogawa

et al., 1995).

(e) The circulating natriuretic peptide levels may also be modifled by ETOH-induced

alterations in NPR-C receptors either directly at the site of release (Nunez er al.. 1993 )

or m the blood aream nich as in the platelets. Changes in vascular and cardiac NPR-C

receptors should then be detennined following chronic ETOH treatment to see whether

ETOH may alter either their number or their a£Enity for ANP and BNP.

1.5.2. KIDNEY

(a) Chrooic ETOH treatment was associated with the increase in papillary NPR-A

bmding sites (Section 3 2.). However. the effect of circutating ANP and BNP levels on

these receptors is likely to be minimal. considering the important layer of endopeptidase

24.1 1 enrymes located in the proximal tubule of the nephron (Gunning and Brenner.

1992). hstead intrarenal UD. ANP and even CNP may bind to the NPR-A receptors in

the iM, producing paracrine natriuretic and depressor effects (Figueroa et al.. 1 990:

Goetz 199 1 : Terada et al.. 1991). Therefore. experirnents should be performed on the

expression and release of natnuretic peptides of renal ongui to evaluate whether ETOH

modifies their levels and whether nich ETOH-induced modifications parallel the changes

in papillary NPR-A receptors.

(b) The status of the proximal tubule endopeptidase 24.11 enzymes should also be

investigated following chronic moderate ETOH treatrnent to evaluate whether ETOH

alters the acthity of endopeptidase 24.11 and thus aUows a greater proportion of

circulating ANP and BNP levels to reach the natnuretic receptors in the IM and affect

directly the natriuresis of ETOH-treated rats.

(c) The modifications produced by ETOH on both the gIomedar and papillary

natriuretic peptide receptors suggest the possibility of an enhanced diuretic and

nahret ic e f f i ofnatriuretic peptides (Section 3.3.). It may be useful to inject specifïc

concentrations of ANP (and BNP) i v. mto ETOH- and water-treat ed animals t O evaluat e

whether natriuresis and diuresis are greater in the ETOH- than in water-treated rats.

Animais should also be placed in highly accurate metabolic cages to observe the exact

modifications in cGMP production. natriuresis and diuresis during both light and dark

phases of the daily cycle.

4s.3, BRAIN

(a) Chronic ETOH treatment produced significant mcreases in hypothalamic and

medullary ANP and CNP levels in SHR rats (Section 3.4.). ANP immunoreact~ty is

mamly l o c k d m the cardiovascular centres of the brain. nich as the AVW area or the

NTS (Kawata et al.. 1985). in contrast. CNP has been found in additional brah regions

which are unrelated to fluid or BP homeostasis (Komatsu et al.. 199 1). Future

experiments may inveaigate. through Ni sitic hybridization or immunohistocherniary.

which s p e d c nuclei present elevated ANP and CNP peptide levels as well as ANP and

CNP mRNA levels. allowing the exact mapping of the ETOH-induced changes in the

cardiovascular centres of the CNS.

(b) ANP (and C M ) may also be injected ;.C.V. into the third (hypothalamus) or fourth

(meduiia) ventricles of the brain to investigate whether the antidipsogenic. loss of sait

appetite. diuretic. natriuretic and depressor effects of ANP are modifîed in the brain of

ETOH-treated animais compared to the brain of water-treated rats.

(c) Chronic ETOH treatment has been associated with some specifk changes in the

uatrIuretic peptide receptor characteristics m the circumventricular organs (Section 3.5. ).

It is possible that natriuretic peptide receptors located inside the brain are also modZed

following ETOH consumption and that these modifications parallel those of the brain

ANP and CNP peptides. Future experiments may then explore NPR mRNA levels in

various hypothalamic and rneduhq nuclei and correlate the ETOH-induced changes on

NPR levels with the ETOH-induced changes on the ANP and CNP contents and their

specific mRNAs.

In conclusion this work dowed the identification of an hormonal system the natriuretic

peptide system that may be implicated m the prevention of the age-dependent increase in BP

by chronic moderate ETOH consurnption. The data obtained indicated that the general

aspects of this endocrine system such as the organs of production and the major bindmg sites.

have presented ETOH-induced modifications that may mediate ETOHs antihypertensive

effect at least in part. Therefore. the exact and specific role of this natriuretic peptide family

in mediatmg alcohoi's effects on the BP clearly ments further investigation. Future

experiments should provide new msights regarding the exact contribution of each component

of this endocrine system on the antihypertensive effect of moderate ETOH consumption

observed in both human and animal studies.

METHODOLOGIES

a) Estimation of the BP andor atrial pressure

In the present studies, the systolic BP was evaiuated by the non-mvasive mdirect tail-cuff

method using a photoelectnc sensor attached to a polygraph (Grass Instruments, Quincey.

MASS) (Pfeffer et al.. 1971). In general, the tail-cdfmethod mvolves the occlusion of the

arterial blood flow to the tail through an idated cuffand the subsequent detection of the

exact pressure, during the cddeflation, at which the fira arterial pulsation reappears. The

vddity of this technique ultimately dep ends on the sensitMty of the sensor used t O record this

initial pulsation. uiitialiy, anesthetized animals and extemai preheating of the tail were

necessas, to produce çome vasorelaxation and ailow the recording of artenal pulsations (Borg

and Viberg, 1980). However, these conditions have bem show to mod* the measured BP.

For example, preheating may by itself'elevate the BP m SHR rats because of their greater

sensitMty to thermal stress than WKY animals (Yen et a!., 1978). Modem taiI-cuffmethods

have bypassed these technicd düEculties by usmg better sensors, such as photoelectnc rather

than ultrasonic sensors, thus dowing repeated and accurate recordmgs of the BP in unheated

non-anesthetized rats (Bunag and Buttefield, 1982; ikeda et al.. 199 1 ; Spanos et al.. 199 1 ).

in fact, syaolic BP estimations using the mdirect method have shown an aimoa perfect

correlation with direct syaolic BP measurements using an indwehg arterial catheter

(r=0.970 m the experiment of Ikeda et al., 199 1 ). Furthemore, the mdirect technique proved

to be a superior means of BP measmement in long-term or chronic midies, because this non-

invasive method prevented the significant appetite suppression and growth impairment

associated with the chronic arterial catheterization (O'Neill and Kaufinan, 1990).

ANP is released pnmady fkom the cardiac atria m response to elevated atrial pressure and

the concomitant stretch of the atrial walis (Nose et al.. 1994; Javeshghani et ah, 1995).

interestingly, lefi atrial pressure has been shown to be twice as hi& m SHR compared to

WKY rats, explainhg in part the elevated levels of circulating ANP in the hypertensive

animals (Noresson et al., 1979). This effect appears to be secondq and conipensatoly to the

ventricular hypertmphy observeci with elevated BP m SHR rats (Ricksten et al., 1980). ui the

present sudies, chronic moderate ETOH coflsumption prevented the development of the age-

dependent hypertension in SHR rats and thus the ventricular hypemophy. Accordingiy, atrial

pressure should also be reduced in these animals compared to the water-treated SHR rats.

Although catheters could have been placed m both atria, it seems likely that these surgical

procedures would have caused stressfùl reactions of the animals during the chronic studies

and modined the relationships between ETOH and BP and between ETOH and ANP.

Furthemore, previous studies have mdicated that ANP release f?om the atria upon ETOH

stimulation is indirect and mdependent of water-loading and increased atrial stretch

(Colantonio et al.. 199 1 ; Wigle et al.. 1993a).

b) Route of ETOE injection in acute studies (rats)

in the present studies, 1 and 2 g of ETOWkg B.W. (as a 40% v/v solution) were injected

irztraperitor~eal& (i-p. ) in Long-Evans rats. Although btîraverzous (i-v.) injection or

irzîragutric (i.g.) administration couid have been used as well, the i.p. injection was chosen

for the following reasons:

I.D. hieetion conpareci to i. v. iniection: (a) Concentrated injections of ETOH in the blood

are IethaL AccordBigly, 1 and 2 g of ETOWkg B. W. çhould be mjected i. v. in rats as a diluted

solution, increasing greatly the final volume of the injection. In contraa, higher concentrations

of ETOH (up to 40% vh) may be çafely mjected i . p . reducing the chance of a water-Ioading

effect on the BP or the natriuretic peptide system (Roine et al., 199 1). (b) The BAC is

maximum immediately after the i. v. injection. However, because of the gradua1 diffusion of

ETOH mto the blood vessels after the i.p. injection, the BAC is maximum 30 minutes post-

injection and thus better mimics the BAC curve following oral administration of alcohol.

[.o. iniectiorz comoared to i.p admDiisiratzo~z: (a) intragastnc mtubation may stress the

animais. Furthemore, this route of administration may produce elevated levels of ETOH in

the stomach, causing gastric imitation and the possibhty of sympathetic activation. Both

effècts may modiS, the interaction between alcohol and the natriuretic peptide system (b) The

use of i-p. rather than i.g. admhktration prevents the possibility of different BAC levels £iom

the same i-g. ETOH administration m different animals because of the presence of different

amounts of food m the stomach and thus dserent Iweis of gashic first-pas metabolism

(Rome et al., 199 1).

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