Prostacyclin (PGI2) Synthesi in ths e Vascular Wall of ...hyper.ahajournals.org/content/hypertensionaha/5/6_Pt_3/V38.full.pdf · Prostacyclin (PGI2) Synthesi in ths e Vascular Wall

Prostacyclin (PGI2) Synthesis in the Vascular Wallof Rats with Bilateral Renal Artery Stenosis

SILVIA MORERA, PH.D. , FEDERICO M. SANTORO, M . S C , MARfA 1. ROS6N, M . S C ,

AND IGNACIO J. DE LA RlVA, M . D .

SUMMARY The ability of vessels (rings of arteries and vena cava) to synthesize prostacyclin (PGIJ"in vitro" was analyzed in the initial (6-day) and chronic (6-week) phase of two-kidney, two cliphypertension. Male Wistar CHbb THOM rats were used. Tissues were incubated for two hours inKrebs solution containing l4C-arachidonk acid as exogenous substrate. Specimens (in benzene-eth-anol 4:1 vol/vol) and the unlabeled standard solutions of arachidonic acid, 6-keto PGF,n, PGf^, andPGE2, were spotted on silica gel-G plates for thin layer chromatography. Conversion of l4C-arachi-donic acid to stable metabolite 6-keto PGF,a was used as an index of PGI2 synthesis. Results shown: 1)PGI2 is the major PG synthesized by the rat artery wall; 2) PGI2 synthesis was increased 2.4 times iqthe initial 6-day period of development of rendvascular hypertension (RH); 3) no changes in PGI2production were observed in arteries during the chronic 6-week period of RH; 4) abdominal vena cavahas little or no capacity to produce PGI2. As PGI2 is a potent vasodilator, higher production by arteriesduring the 6-day period suggests that prostacyclin could play a modulator role on peripheral resis-tance during the initial phase of renal hypertension. (Hypertension 5 (supp V): V-38-V-42, 1983)

KEY WORDS • prostaglandins • blood vessels • renovascular hypertension

STRUCTURAL changes of the vessel wall cer-tainly contribute to increased peripheral vascu-lar resistance (PVR) in hypertensive disease,1

but vasoconstriction must be present for high bloodpressure (BP) levels to develop. Thus, in the hyperten-sive process, factors that regulate contraction of vascu-lar smooth muscle are of particular interest. Prosta-glandin I2 (PGI2) has been recognized as a potentvasodilator and inhibitor of platelet aggregation2'3 andis the principal metabolite of the arachidonic acid orprostaglandin endoperoxides in a number of blood ves-sels of several animal species and humans.3"" Theenzyme that synthesizes PGI2 is almost exclusivelyconcentrated in the endothelial lining of the vessels,7'l2

but it has been reported to appear, irk small proportion,in other vascular layers.7' l3~13 Like other prostaglan-dins, PGI2 synthesized intramurally can modify vascu-

From the Instituto de Investigaciones Cardiologicas, Facultad deMedicina, Universidad de Buenos Aires, Buenos Aires, Argentina.

Supported in part by the ConSejo Nacionai de Investigaciones Cientf-ficas y Tecnicas. Dr. de la Riva is an Established Investigator and Drs.Morera and Santoro are Fellows of the Consejo Nacionai de Investiga-ciones Cientlficas y Tecnicas.

Address for reprints: IgnacioJ. de la Riva, M.D. .Instituto de Investi-gaciones Cardiologicas, Facultad de. Medicina, M. T. de Alvear 2270,(1122) Buenos Aires, Argentina.

lar smooth muscle tension directly'6"18 or indirectly,altering either the response to diverse vasoactive sub-stances19"21 or the adrenergic neurotransmitter releasefrom sympathetic nerve endings.22 This paper dealswith the ability of vessels (arteries and vena cava)obtained from rats with bilateral renal artery stenosis tosynthesize PGI2 "in vitro." As far as we know, there isno previous information on this subject in this particu-lar model of renovascular hypertensive animals. Both,the,initial (6-day) and chronic (6-week) periods of hy-pertension were analyzed. Present results show higherPGI2 synthesis only in arteries after 6 days of increas-ing BP; the fact suggests that prostacyclin could act asa modulator of PVR in the initial phase of renovascularhypertension (RH); furthermore, because of the ab-sence of demonstrable changes in vessels of 6-weekhypertensive rats, the possible role of PGI2 during thechronic period remains undefined.

MethodsThe experiments were carried out on male Wistar

CHbb THOM rats. High BP was elicited by bilateralstenosis of the renal arteries. Both the acute phase (6-day period) and the chronic phase (6-week period) ofthe hypertensive process were analyzed. In each study,

V-38

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the animals were divided into three groups: Intacts,Shams, and Clips. Intacts were rats without any ex-perimental maneuver that were matched in weight;there were 24 rats weighing 250 g that were studied fora 6-day period and 15 rats weighing 370 g studied for a6-week period. Shams had clips placed on the renalarteries but immediately removed; there were 33 ratsfor the 6-day period and 14 rats for the 6-week period.Clips had one solid silver clip 0.25 mm of lumenplaced on each renal artery; there were 22 rats for the 6-day period and 20 rats for the 6-week period. BP wasindirectly recorded by a tail pulse pneumatic trans-ducer connected to an oscilloscope. On the followingday, the animals were sacrificed by decapitation, andblood samples were obtained for creatinine measure-ments (cinetic-colorimetric method). The aorta (thearch excluded) plus iliac arteries and the abdominalvena cava were removed and cleaned of periadventitialtissue. The vascular segments were sliced to 1 mmthickness, and the vessels from several rats were usual-ly pooled (two or three in the case of arteries and threeor four in the case of veins). The tissues were preparedin cold temperature (0° to 4°C) and then kept wet in 1.5ml of cold Krebs solution until incubation took place.After 30 minutes at 37°C, the vascular segments weretransferred to other tubes containing 0.2 /xCi l4C-ara-chidonic acid (New England Nuclear Corporation,Boston, Massachusetts, specific activity = 54.5 mCi/mmol) in 1.5 ml of Krebs solution. Incubation wascarried out for 2 hours, with shaking at 37°C. Thisperiod of time seems to be adequate, as has beenshown by Limas et al.10 The reaction was stopped byacidifying to pH 2.5 to 3 with 0.5 M citric acid. Thereaction medium was extracted twice with 4 ml ethylacetate, and the combined extracts were dried undernitrogen. Then they were taken up in 100 (JL\ of ben-zene:ethanol (4:1 vol/vol) for thin layer chromatog-raphy on silica gel (G type 60, Merck Sharp & Dohme)plates. Standard solutions of arachidonic acid, 6-ketoPGFla, PGF^, and PGE^ were spotted on the sameplate of specimens. The chromatographic solvent wasthe upper phase of ethyl acetate:isooctane:acetic acid:water (110:50:20:100, by volume), and the plates weredeveloped to 15 cm from origin. After drying, thestandards were visualized with phosphomolibdic acid(ethanolic solution, 15% p/v), and the correspondingradioactive spots were identified by autoradiographyand scrapped off for radioactivity measurement. Con-version of l4C-arachidonic acid to 6-keto PGFla, thestable metabolite of PGI2, was used as an index ofvascular PGI2 synthesis. To test the inhibition of PGI2

synthesis, tranylcypromine (Sigma Chemical Com-pany, St. Louis, Missouri) 500, 1000, and 1500 /ug/mlwas added to the incubation medium of three samplesfrom nine intact rats, 5 minutes before MC-arachidonicacid was incorporated. Proteins were measured by theLowry method.

Data were expressed as means ± SE. Statistical sig-nificance of the differences were determined either bythe two-tailed Student's t test or by one-way analysisof variance and the Newman-Keuls test.

ResultsDevelopment of Hypertension

Mean BP values in Shams were: in the 6-day group,107 ± 2 mm Hg (range 90 to 115) and, in the 6-weekgroup, 121 ± 3 mm Hg (range 100 to 135). Clip rats inwhich the BP was 10 mm Hg above the highest valueof the Sham group were considered hypertensive.Therefore, two Clip rats of the 6-day group (BP under125 mm Hg) and two of the 6-week group (BP under145 mm Hg) were discarded. Mean BP values in Cliprats were: in the 6-day group, 147 ± 4 mm Hg (range125 to 180) and in the 6-week group, 178 rt 5 mm Hg(range 145 to 200). Differences between Shams andClips were statistically significant both in the 6-dayperiod (r test, p < 0.001) and in the 6-week period (ttest, p < 0.001).

Synthesis of Prostaglandins by ArteriesSignificant amounts of PGI2 (quantified as 6-keto-

PGFla metabolite) were always present in samples ofartery ring incubates (fig. 1). As pointed out in theMethods section, arteries from two to three rats werepooled per sample to be incubated. The BP valueswere individually recorded, however. This experimen-tal design accounts for the apparent discrepancies inthe number of cases in the Sham and Clip groupsexpressed in Methods and figure 1. In the 6-day peri-od, arteries from hypertensive rats synthesize 2.4times more PGI2 than arteries from Sham rats (p <0.01). In the 6-week period, there were no significantdifferences among the groups. Shams of the 6-weekperiod were statistically different from Shams of the 6-day period (p < 0.02).

800

600

400

200

nU

6-day group

pmol»*/100 prottint

800

T 600

^ ^ ^ | 400T ^^M

(101

nttcts Sram* Clip*

6-w»«k group6KF|#<

pmt>(»s/IOO mg prottlns

T

16)

InUd*

*

SKum Clip*

FIGURE 1. Biosynthesis ofPGI2 is expressed as 6-keto PGFla

(6KF,a in the figure). Columns represent mean values, and barsrepresent the standard errors. In parentheses are the number ofexperimental cases. In each sample, vessels from two or threerats were incubated. In the 6-day period, data were analyzed byone-way analysis of variance and the Newman-Keuls test. Aster-isk (*) indicates a significant difference: Clips vs Intacts, p <0.05, Clips vs Shams, p < 0.01. In the 6-week period, theasterisk (*) indicates the statistical significance of the differ-ence determined by Student's t test: Shams (6-weeks) vs Shams(6-days) p < 0.02.

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6- day Group

ARTERY VENA CAVA

6-w*ek Group

ARTERY ARTERY

AAo

AAo

I

6KF,a

• - • - J L USHAM CUP SHAM CUP

:i

FIGURE 2. Autoradiographs of thin layerchromatography plates from vessel wall in-cubates. The positions in which standardsrun in this system are indicated by the cir-cles; spots of 6-keto-PGF,a (6KF,a in thefigure) were always clearly present in thecase of arteries. In addition to PGI2, sig-nificant amounts ofPGF2a and PGE2 werepresent in some samples (see text). PGI2synthesis was not visualized in vena cavaincubates.

SIMM CLIP SHAW a i P

Identification of the compound as 6-keto-PGFla wasobtained from the following lines of evidences: 1) thecompound migrated as unlabeled standards of 6-keto-PGF,a on thin layer chromatography. Moreover, thissubstance was shown to be more polar than PGF^ andPGEj, as has been reported by other investigators23 u

for 6-keto-PGF,a. (In our results, Rf = 0.23 as com-pared with 0.29 for PGF^ and 0.35 for PGE2.) 2)Quantitatively, this compound was the principal prod-uct of the PG cascade obtained by incubation of theartery wall, as stated in previous reports.7 8 1223 3)Preincubation of the samples with tranylcyprominelargely decreased radioactivity (50% to 55% of con-trol) in the spots ascribed to be 6-keto-PGFla, evenwhen the lowest dose (500 /xg/ml) was used.

In addition to the spot of 6-keto-PGF]a, the presenceof other less polar substances was observed in autora-diographs of thin layer chromatography plates of somesamples (figure 2). Two of the substances migratedalmost similarly to the nonlabeled PGF^ and PGE2

standards; when clearly identified, the correspondentspots were separately scrapped off and quantified. Re-sults (in pmol/100 mg protein) were: in the 6-day peri-od, Intacts (n = 3) PGF^ 149 ± 47, PGF^ 252 ± 100;PGF^ and PGE2 in Shams and Clips were indetectable.In the 6-week period, Intacts (n = 6)PGF2a241 ± 25,PGE2 268 ± 31; Shams (n = OPGF^ HOjPGE, 131;Clips (n = 2) PGF^ 148; PGEj 170.

Synthesis of Prostaglandins by VeinsRings of abdominal vena cava from either Intact,

Sham, or Clip rats of the 6-day period had a clear lowcapacity to synthesize PGI2; this assertion is based onthe fact that 6-keto-PGF, a was not visualized in theautoradiographs in our experiments (fig. 2). Similarly,PGF^ was not observed. Instead, PGF^ was quantifiedin the six samples from the vena cava of Sham rats (480

± 125, pmol/100 mg protein) and in three of sevensamples from the vessels of clip rats (344 ± 78 pmol/100 mg protein). Further research is needed for reach-ing conclusive results regarding PGE; synthesis in theveins of the 6-day period. In the 6-week period, PGI2

synthesis was intended to be enforced by includingmore tissue per incubation sample (up to 11 veins werepooled in one case). Unfortunately, the determinationshad a bad resolution with thin layer chromatography(possibly due to an excess of adventitial lipids) and,consequently, results of PGI2 synthesis by veins in thechronic groups were unclear and are not included inthis paper.

Plasma CreatininePlasma creatinine was determined in some animals

to test renal function. Data, expressed as mg/100 ml,were: 6-day period, Intacts (n = 9) 0.59 ± 0.02;Shams (n = 6) 0.58 ± 0.03; Clips (n = 10) 0.70 ±0.04; 6-week period, Intacts (n = 20) 0.58 ± 0.02;Shams (n = 4) 0.61 ± 0.04; Clips (n = 6) 0.75 ±0.05. Differences among groups were not statisticallysignificant (one-way analysis of variance). We havepreviously reported23 plasma creatinine values at the1st, 2nd, 4th, and 10th week in two-kidney, two cliphypertensive rats when no significant differences be-tween hypertensive and control animals were ob-served.

DiscussionIn this study, 6-keto-PGFla was actually the major

labeled product separated from the artery incubationmedium. This finding is in keeping with a series ofrecent papers that analyzed prostaglandin (PG) synthe-sis by the entire vessel wall.8' ">24 The authors haveshown that the stable nonenzymatic metabolite of PGI2

may quantitatively reflect its intramural synthesis, at

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least under experimental "in vitro" conditions. So far,similar results have been reported in cultured vascularendothelial cells.7- M

In addition to prostacyclin, other PGs and metabo-lites have been reported formed when homogenatesinstead of intact vascular tissue were investigated.8-24"26

Nevertheless, it must be noted that other substancesless polar than 6-keto-PGFla were also observed insome of our samples (13 of 57 analyzed) after incuba-tion of the entire artery wall and separation of theproducts by thin layer chromatography; similar resultswere reported by Limas et al.10 in different strains.Remarkably, arteries from intact rats, in particular,showed that tendency (see Results). When the radioac-tive spots were clearly distinguished in the autoradio-graphs, two of such substances were quantified asPGF^ and PGE^ since they had migrated similarly tononlabeled standards in the chromatography plates. Itis likely that they represent individual differences inthe capacity of the artery wall to generate PGs.

On the other hand, 6-keto-PGFla was not visualizedin autoradiographs from thin layer chromatographyplates of vena cava incubates; thus, its presence, ifany, would have been insignificant. Furthermore, be-cause of interference from the background radioactiv-ity in the runs, its quantization from the plates wasconsidered unreliable. These results are in harmonywith those of Skidgel and Printz,27 also in rats, usinghomogenates of the same vessel; the authors concludedthat the abdominal segment is even less active than thethoracic segment to produce PGI2. Wong et al.23-26

have proposed that veins would contain more 15-hy-droxyprostaglandin dehidrogenase than arteries andthis enzyme could form 6-15-diketo-PGFla in additionto the nonenzymatic metabolite 6-keto-PGFla. How-ever, because of the absence of distinguishable radio-labeled 6-keto-PGFla in vena cava incubates, we thinkPGI2 synthesis was clearly insignificant in our experi-ments. In this regard, an unequal capacity to synthetizeprostacyclin of endothelial cells from different originscould be postulated; as a matter of fact, Herman et al.28

have observed that the amount of 6-keto-PGFla formedby the peritoneum after incubation with labeled pros-taglandin endoperoxide H2 was about three times high-er than the amount formed by sections of the aorta.Nonsignificant synthesis of PGI2 by the wall of theabdominal vena cava suggests that production of intra-mural prostacyclin is not fundamental to regulateblood dynamics in great veins of rats either under nor-mal conditions or early hypertension. This conclusionmust not be considered to apply to all veins or speciesin general since human endothelial cells from umbili-cal cord veins,12-29 walls of bovine umbilical veins,30

and colic and gastric human veins6 have been shown toproduce PGI2.

In summary, results of the present experimentsclearly show that rings of arteries from rats after a shortperiod of RH (6-days) synthesized double the amountof PGI2 produced in tissues of Sham rats incubatedwith l4C-arachidonic acid substrate. As far as datafrom conductive arteries apply to small resistance ves-

sels, the fact suggests that intramural prostacyclin mayparticipate in regulation of peripheral vascular toneduring the initial phase of RH. We have not foundprevious information on such brief period of hyperten-sion but similar enhancement of PG production hasbeen reported for vessels from other long-term hyper-tensive models such as genetic salt-induced hyperten-sion10 or spontaneous hypertension;8- 3I~33 in thesecases, it has been postulated that the increment in bio-synthesis could represent a compensatory mechanismby which the increase in BP would be attenuated.10-32

Nevertheless, our results from 6-week hypertensiverats showed that stable high BP levels were developedwithout demonstrable changes in artery PGI2 synthe-sis. Thus, the adaptive response of blood vessels medi-ated by a PG mechanism after prolonged RH seems tobe ruled out. However, a modulatory role of PGI2 inthe chronic 6-week period could not be completelydisregarded since differences in sensitivity to otherPGs (PGE2, F^ , A2, B2, D2) have been described forhypertensive vessels.32-M In this connection, the possi-bility of changes in vascular responses to local PGI2,circulating PGI2

33 or even some enzymic metabolitefrom plasma36 has to be kept in mind.

Finally, we must add that the actual "in vivo" sig-nificance of the present observations remains unclear.As usual, extrapolations from "in vitro" experiments,as intended in the course of this discussion, must berecognized merely as thought-provoking suggestions.

AcknowledgmentsThe authors especially thank Drs. Alicia and Norberto Terragno

for encouraging this study. The contributions of Dr. Juan a Pasquiniand Dr. Angela Ubios and the technical assistance of Mireya T. deFernandez are also acknowledged.

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S Morera, F M Santoro, M I Rosón and I J de la RivaProstacyclin (PGI2) synthesis in the vascular wall of rats with bilateral renal artery stenosis.

Print ISSN: 0194-911X. Online ISSN: 1524-4563 Copyright © 1983 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Hypertension doi: 10.1161/01.HYP.5.6_Pt_3.V38

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