Distribution and function of muscarinic receptor subtypes in the ovine submandibular gland

1

Distribution and function of muscarinic receptor subtypes in

the ovine submandibular gland

By

G. Tobin*#, A.T. Ryberg*, S. Gentle

§ and the late A.V. Edwards

§

*Department of Pharmacology, the Sahlgrenska Academy at Göteborg University,

Medicinaregatan 15D, Goteborg 413 90, Sweden and §Physiological Laboratory,

University of Cambridge, Downing Street, Cambridge, CB2 3EG, UK

Running title: Muscarinic receptors in the ovine submandibular gland

Keywords: saliva, vasodilatation, muscarinic receptor

#Corresponding author: [email protected]

Articles in PresS. J Appl Physiol (December 1, 2005). doi:10.1152/japplphysiol.00779.2005

Copyright © 2005 by the American Physiological Society.

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The effects of muscarinic receptor antagonists on responses to electrical stimulation of the

chorda-lingual nerve were determined in pentobarbitone-anaesthetized sheep and

correlated to the morphology of tissue specimens. Stimulation at 2 Hz continuously, or in

bursts of 1 s at 20 Hz every 10 s, for 10 min induced similar submandibular fluid

responses (19±3 vs. 21±3 l.min-1

g gland-1

), while vasodilatation was greater during

stimulation in bursts (-52±4 vs. -43±5 %; P<0.01). Continuous stimulation at 8 Hz induced

substantially greater responses (66±9 l.min-1

.g gland-1

and -77±3 %). While atropine (0.5

mg.kg-1

I.V.) abolished the secretory response at 2 and 20 (1:10 s) Hz, a small response

persisted at 8 Hz (<5 %). The “M1-selective” antagonist pirenzepine (40 g.kg-1

I.V.)

reduced the fluid response at all frequencies tested (P < 0.05-0.01), most conspicuously at

2 Hz (reduced by 69 %). Methoctramine (“M2/M4-selective”; 100 g.kg-1

IV; n=5) had

no effect on fluid or the vascular responses but increased the protein output at 2 (+90 %, P

< 0.05) and 8 Hz (+45 %, P <0.05). The immunoblotting showed distinct bands for

muscarinic M1, M3, M4 and M5 receptors, and immunohistochemistry showed

muscarinic M1 and M3 receptors to occur in the parenchyma. Thus, muscarinic M1

receptors contribute to the secretory response to parasympathetic stimulation but have

little effect on the vasodilatation in the ovine submandibular gland. Increased transmitter

release caused by blockade of neuronal inhibitory receptors of the M4 subtype would

explain the increase in protein output.

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Introduction

The increase in salivary flow evoked by muscarinic agonists has generally been attributed

to activation of muscarinic receptors solely of the M3 subtype (Caulfield, 1993; Baum &

Wellner, 1999). This concept has been supported by findings obtained in studies using

subtype-specific antisera as well as by functional studies on rat parotid glands (Dai et al.,

1991). However, contradictory results have been found in the examination of other

salivary glands. Binding and molecular experiments on the rat submandibular gland have

indicated the expression of muscarinic M1 and M5 receptors in addition to the muscarinic

M3 receptors (Hammer et al., 1980; Buckley & Burnstock, 1986; Martos et al., 1987;

Vilaro et al., 1990; Flynn et al., 1997). Functional significance of muscarinic M1

receptors for the secretory response has been reported, in vivo as well as in vitro, in the

rabbit submandibular gland (Tobin, 1995) and in the rat sublingual (Culp et al., 1996;

Tobin et al., 2002), and of muscarinic M1 as well as M5 receptors in the rat

submandibular gland (Meloy et al., 2001; Tobin et al., 2002).

In salivary glands, parasympathetic nerve transmission may involve not only the classical

autonomic transmitter acetylcholine but also non-adrenergic, non-cholinergic transmitters

(Ekström, 1999). The parasympathetically nerve-evoked vasodilatation, which is evoked

by non-adrenergic, non-cholinergic transmitters, is particularly resistant to atropine

(Edwards, 1999). However, a secretory response also persists after full atropinization in

salivary glands of several species including the ovine submandibular gland (Edwards et

al., 2003), albeit much reduced and, preferentially, at high-frequency stimulation

(Ekström, 1999). In ovine salivary glands, the neuropeptides vasoactive intestinal peptide

and calcitonin gene-related peptide seem to mediate most of the atropine-resistant

parasympathetic responses (Edwards et al., 1988; Edwards et al., 2003). Nevertheless,

4

acetylcholine acting on muscarinic receptors is the principal stimulation for evoking fluid

responses in salivary glands (Cook et al., 1994), while the vasodilator response, although

markedly resistant to atropine, has a cholinergic component particularly at low-frequency

stimulation (Edwards, 1999; Edwards et al., 2003). The specific muscarinic receptor

mediating vasodilatation in salivary glands has not been characterized, although functional

studies in the rat parotid gland indicate that this response may be mediated, at least in part,

via muscarinic M3 receptors (Tobin, 1998).

Nerve transmission in the parasympathetic innervation of salivary glands may be

modulated by prejunctional muscarinic receptors (Tobin, 1995, 1998, 2002). In rat

salivary glands, muscarinic M1 receptors normally facilitate transmitter release during

short, intense nerve activity. At low frequencies, on the other hand, muscarinic M2

receptors, or possibly muscarinic M4 receptors, inhibit cholinergic as well as peptidergic

transmission, but only after some delay. Furthermore, it was first described in the feline

submandibular gland that stimulation of the parasympathetic innervation in a burst pattern

at high frequencies causes a conspicuous enhancement of vasodilatation and secretion in

comparison with continuous stimulation (Bloom & Edwards, 1979; Andersson et al.,

1982b). These observations have subsequently been confirmed in salivary glands of other

species including the ovine submandibular gland (Andersson et al., 1982a; Tobin et al.,

1990; Tobin, 1998, 2002). The phenomena have been attributed to the release of

neuropeptides, which preferentially occurs at high stimulation frequencies (Bloom &

Edwards, 1979; Andersson et al., 1982a), and to a short-lasting stimulation activating

facilitator and not inhibitory receptor mechanisms (Tobin, 1998, 2002).

In the present study, the contribution of muscarinic receptors other than the M3 subtype to

the in vivo responses in ovine salivary glands was determined using antagonists with

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different muscarinic profiles (pirenzepine, methoctramine, p-fluorohexahydro-sila-

diphenidol, atropine (Caulfield, 1993; Eglen & Nahorski, 2000)). The effects of these

antagonists were examined on responses evoked by electrical stimulation at varying

frequencies and patterns of the parasympathetic chorda tympanic nerve and morphological

correlates sought by employing Western blotting and immunohistochemistry. A

preliminary report of some of these results has been published previously (Tobin &

Edwards, 2004).

Methods

Animals

The experiments were carried out on 18 adult ewes of various breeds (35-72 kg body

weight) under the Animals Scientific Procedures Act (1986); Project Licence PPL

80/1316. Food but not water was withheld for 48 h prior to each experiment. Anaesthesia

was induced and maintained with sodium pentobarbitone (Sagatal, Rhône Mérieux Ltd.,

Harlow, U.K.; 15-30 mg kg-1

I.V. and then 0.1 – 0.3 mg min-1

kg –1

I.V. (adjusted to

maintain a stable blood pressure)). At the end of each experiment the animal was given a

lethal dose of barbiturate (Pentoject, Animalcare Ltd., York, U.K.; ca 15 ml 20% w/v) and

the ipsi- and contra-lateral submandibular glands dissected out and weighed (18±2 vs.

13±1 g; n=13).

Surgical and experimental procedures

The trachea was intubated and then exposed via a mid-line incision low in the neck. The

ipsilateral ascending cervical sympathetic nerve was identified and cut. An arterial

catheter was introduced into the abdominal aorta via a femoral artery and later employed

to monitor arterial blood pressure and heart rate; samples of arterial blood were also

6

collected periodically for measurements of packed cell volume (at start of experiment:

28±1 %; at the end: 26±1 %; n=13). The femoral vein was cannulated to provide a conduit

for the continuous infusion of sodium pentobarbitone. The chorda-lingual nerve was

exposed and cut and the submandibular duct was cannulated with the widest bore nylon

tubing practicable. The free end was then positioned above a photoelectric drop-counter.

A neighbouring length of the hypoglossal nerve was excised in order to minimise spread

of stimulus. Each of the tributaries of the ipsilateral linguofacial vein, except that draining

the submandibular gland, was ligated. The animal was heparinized (Mutiparin. CP

Pharmaceuticals Ltd., Wrexham, U.K.; 1000 i.u. kg –1

I.V.) and the linguofacial vein

cannulated with a short length of polythene tubing. The submandibular venous effluent

was thereby diverted through a second photoelectric drop-counter and returned to the

animal by a pump, via the ipsilateral jugular vein, in such a way as to match input to

output. Finally, a bipolar platinum stimulating electrode was placed under the duct and

chorda tympani close to the hilum of the gland. The protocol involved parasympathetic

stimulation at 2 and 8 Hz continuously for 10 min (20 V square-wave; 10 msec pulse-

width), and intermittent stimulation at 20 Hz given for 1 s at 10 s intervals for 10 min.

This thus resulted in the same total number of impulses as 2 Hz given continuously. The

stimulation was applied at the specified frequencies before and after administration of

antagonists at doses previously validated to be selective; examined in pilot experiments

(n=3) and compared with results from the literature (“M1-selective”; pirenzepine

dihydrochloride, 40 µg kg-1

I.V. (100 nmol kg-1

); “M2/M4-selective”; methoctramine

tetrahydrochloride, 100 g kg-1

IV (140 nmol kg-1

); “M3(M1/M5)-selective”; p-fluoro-

hexahydro-sila-diphenidol hydrochloride (pFHHSiD, 4 µg kg-1

I.V. (10 nmol kg-1

; all

from Sigma, St. Louis, US (Tobin, 1995; Tobin & Sjogren, 1995; Tobin, 1998, 2002;

Tobin et al., 2002)). The rates of flow of submandibular blood and saliva were recorded

photometrically drop by drop and also estimated gravimetrically. No spontaneous flow of

7

saliva occurred. During stimulation collection of samples was delayed for 2 min in order

to ensure complete evacuation of the submandibular dead space. The samples of blood

were weighed for gravimetric estimation of blood flow and then returned to the animal to

preserve the circulating blood volume. Aortic blood pressure and heart rate were

monitored continuously by means of a pressure transducer and amplifier. In three animals

the responses to nerve stimulation were also determined following the administration of

atropine (atropine sulphate, Sigma; 0.5 mg kg-1

, I.V. (2 µmol kg-1

).

Immunohistochemistry and immunoblotting

After administration of a lethal dose of anaesthetic, tissue from the contralateral

submandibular gland was dissected out from the animal for histological examinations; a

central, lower part of the gland was removed. The specimens were either fixed in

phosphate buffered 4 % paraformaldehyde (pH 7.0), and then embedded in paraffin, or

immediately placed in -80° C until prepared for Western immunoblotting.

For the immunohistochemical investigation of muscarinic receptor expression, transverse

sections of the different specimens were prepared in a cryostat at a thickness of 4 µm. The

sections were de-paraffinized by heating the slides to 60 °C for 15 minutes and then

subjecting them to two 30-minute changes in 100% xylene; the sections were then re-

hydrated by serial incubations in 100%, 95%, 85% and 70% ethanol, followed by tris-

buffered saline (TBS). Then the sections were immersed in 10 mM citrate buffer (pH 6.0)

and were microwaved for four cycles of 6 min. Endogenous peroxidase was blocked with

0.03% hydrogen peroxidase for 30 min. Non-specific protein binding was blocked with

5% bovine serum albumin (BSA) in TBS for 30 min. The sections were thereupon

incubated overnight at room temperature in a humidified chamber with polyclonal rabbit

anti-mAChR subtype specific antibodies (Research and Diagnostic Antibodies, Berkley,

8

US) diluted 100 x in TBS containing 1% BSA. The presence of the muscarinic receptors

was revealed using an avidin-biotin-complex immunoperoxidase method (ABC Staining

System, Santa Cruz Biotechnology, Santa Cruz, US; system used following the

manufacturer’s instructions) that uses 3,3P-diaminobenzidine (DAB) as a substrate. The

sections were counterstained using Mayer’s hematoxylin (Histolab, Göteborg, Sweden).

As a negative control, duplicate sections were immunostained without exposure to the

primary antibody, which resulted in no brown staining of the tissue. As an additional

control, the antibodies were pre-absorbed with the appropriate immunogen before

proceeding as described above.

Muscarinic receptor expression in the ovine submandibular gland was examined using

Western blotting. All tissues were homogenized in ice cold PE buffer (phosphate EDTA

buffer) containing 0.1 mM leupeptin (Sigma, St Louis, US), 0.1 mM pepstatin (Sigma),

1.5 mM aprotinin (Sigma), 4 mM Pefabloc SC (Fulka Chemie, Buchs, Swittzerland), 50

µM sodium fluoride (Sigma), 0.2 µM sodium orthovanadate and 5 mM CHAPS (Sigma).

The lysate was heated to 70 °C for 10 minutes in a reducing sample buffer. The proteins

were fractionated by reducing NuPAGE 4-12 % Bis-Tris gels (Invitrogen, Carlsbad, US)

and electroblotted onto PDVF membranes (Invitrogen), which when were incubated in

PBS-T (PBS-T: phosphate-buffered saline containing 0.3 % Tween 20 (Sigma))

containing 0.2 % I-Block (Tropix, Bedford, US) to block non-specific binding. The

membranes were then incubated overnight with polyclonal antibodies directed against

each of the five muscarinic receptors respectively (Research and Diagnostic Antibodies,

Berkley, US). The antibodies were used at the following dilutions: 1:500 (anti-M1, anti-

M3 and anti-M4), 1:2000 (anti-M2) and 1:2500 (anti-M5). The antibodies were validated

using positive controls. The binding was visualized with a chemilumniscent detection

system that utilizes enzyme-linked immunodetection, detected using enhanced

9

chemiluminescence, visualized by Flour-S (BioRad, Hercules, US) and analyzed using the

QuantityOne software (version 4.4.1, build 067; BioRad). For negative controls primary

antibodies were omitted in the procedure described above. As an additional control the

antibodies were pre-absorbed with the appropriate peptide immunogen as well, before

proceeding as described above.

Estimations

Submandibular vascular resistance (SVR) was estimated by dividing the perfusion

(arterial blood) pressure (mm Hg) by the submandibular blood flow ( l min-1 [g gland]-1

)

and expressed as the % changes from experimental time=0. Results are expressed as mean

values ± S.E.M. and were assessed statistically (Prism 4, GraphPad Software Inc., US) by

means of paired or unpaired Student’s t test or by repeated measures ANOVA followed by

a Bonferroni test as appropriate with n =number of animals. P values less than 0.05 are

considered to be statistically significant. All flows and outputs are expressed per unit

weight of the contralateral gland.

Results

Cardiovascular and secterory responses to stimulation of the parasympathetic innervation

Under resting conditions, in the absence of any stimulation, no fluid secretion occurred

from the submandibular gland. The mean glandular blood flow was 0.33±0.04 ml min-1

g

gland-1

(n=13) and did not vary significantly during the resting periods throughout the

experiments: After the three basal stimulations performed in the absence of any antagonist

(after 120 – 180 min), the blood flow was still 0.30±0.03 ml min-1

g gland-1

(n=13). Mean

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aortic blood pressure (92±5 vs. 88±4 mmHg) and heart rate (107±4 vs. 103±5 bpm) did

not differ significantly before and after stimulation.

Stimulation of the peripheral end of the chorda tympani at 2 and 8 Hz produced a

frequency-dependent increase in the flow of saliva and a fall in the submandibular

vascular resistance without affecting aortic blood pressure or heart rate (Fig. 1). Typical

responses from a single animal are illustrated in figure 2, which demonstrates the rapidity

of the onset of salivation and the associated vasodilatation in response to continuous

stimulation at 8 Hz. Both responses were well maintained for the duration of stimulation

and subsided gradually towards the initial values over a 5 – 10 min period after cessation

of stimulation. Stimulation at 8 Hz induced a mean flow of saliva over the 10 min

stimulation period that was approximately three times as large as that to 2 Hz (66±9 vs.

19±3 µl min-1

g gland-1

; P<0.001, n=13; Fig. 1). Also the mean decrease in SVR (-77±3

% vs. -43±5 %; P<0.001, n=13) was significantly greater at 8 Hz. This change in vascular

resistance corresponded to a mean blood flow of 0.53±0.05 ml min-1

g gland-1

at 2 Hz and

of 1.24±0.13 ml min-1

g gland-1

at 8 Hz. Stimulation at 20 Hz given in bursts for 1 s at 10 s

intervals (1:10), by so delivering precisely the same number of impulses to the chorda

tympani fibres over the 10 min test period as the continuous stimulation at 2 Hz, produced

instantaneous flow of saliva and fall in SVR similarly well-maintained during stimulation

and steadily subsiding thereafter (Fig. 1). The mean vasodilator response (mean blood

flow: 0.60±0.07 ml min-1

g gland-1

) during stimulation at 20 Hz given in bursts was

significantly greater than that during stimulation at 2 Hz continuously (-52±4 vs. -43±5 %;

P<0.01; n=13) but no such difference was recorded between the effects of the two patterns

of stimulation regarding the flow of saliva (21±3 and 19±3 µl min-1

g gland-1

at 20 Hz 1:10

and at 2 Hz, respectively). Continuous stimulation at 8 Hz was thus substantially and

significantly more effective than any of the other two stimulation patterns regarding flow

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of saliva and change in SVR, but also regarding saliva protein output (P<0.01-0.001). The

mean protein output over the stimulation period in response to 8 Hz was 100±27 µg min-1

g gland-1

, and to 2 Hz and 20 Hz 1:10 s, 18±5 and 25±7 µg min-1

g gland-1

, respectively.

Whereas the output at 8 Hz was significantly greater than any of the other stimulation

patterns (P<0.01-0.001; n=13), the mean output over the whole period did not differ

significantly between 2 Hz and 20 Hz 1:10. However, during the initial few minutes (2-4)

of the stimulation period, a significantly greater output in response to the intermittent

stimulation at 20 Hz occurred in comparison with that to 2 Hz given continuously (17±4

vs. 27±6 µg min-1

g gland-1

; P<0.05, n=13). Furthermore, the latency of the secretory

response was invariably reduced when the intermittent pattern of stimulation was

employed at this range (2 Hz: 34 ± 8 s; 20 Hz 1:10: 16 ± 2 s; P<0.05; n=13). Also

stimulation at 8 Hz evoked a more instantaneous secretory response (13 ± 1 s; P<0.05;

n=13) than stimulation at 2 Hz did, but the latency did not differ from that at 20 Hz 1:10.

Responses following the administration of muscarinic antagonists

Pre-treatment with atropine (0.5 mg kg-1

I.V.; n=3) effectively abolished the secretion of

saliva at 2 and 20 Hz 1:10, but a small atropine-resistant response was observed during

chorda tympani stimulation at the higher frequency of stimulation (Fig. 2). This small

response at continuous stimulation at 8 Hz was characterized by a long latency (90 – 180

s) and the maximal flow over the 10 min stimulation period amounted to 2±0.5 µl min-1

g

gland-1

. It was invariably associated with an intense vasodilatation in the gland and the rise

in blood flow preceded the onset of salivation. The fall in SVR at 8 Hz was closely similar

before and after administration of atropine, whereas the fall in SVR during stimulation at 2

Hz continuously and 20 Hz 1:10 were -37±6 % and -44±7 % in the absence of atropine but

only -15±5 % and -36±3 % in the presence, respectively.

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The intravenous administration of methoctramine 100 µg kg-1

(n=5) significantly and

consistently increased the heart rate (P<0.05-0.01), without affecting mean aortic blood

pressure (Fig. 4). Neither did methoctramine affect the flow of saliva or the change in

SVR at any of the stimulation patterns and frequencies when assessing the changes over

the whole 10 min stimulation period, albeit a tendency towards an inhibition of the fall in

SVR could be noted at 2 Hz (-39±8 vs. -31±5 %; Fig. 3). At the continuous stimulation at

2 and 8 Hz, the antagonist caused significant increases of the saliva protein output by 90

and 45 %, respectively (Fig. 3). The increase in the protein output at 2 Hz occurred

primarily during the initial few minutes (2-6) of stimulation (+170 %; P<0.01; Fig. 4).

Likewise methoctramine caused a significant increase during the first minutes (2-4) of

stimulation at 20 Hz 1:10 (+125%; P<0.05; not shown), but not of the output when

assessed for the whole period (Fig. 3). Administration of methoctramine was without

effect on the latency for the fluid response at 2 and 8 Hz, whereas the antagonist increased

the latency at 20 Hz 1:10 (15 ± 3 vs. 46 ± 6 s; P<0.001).

Pirenzepine (40 µg kg-1

I.V.; n=5) significantly (P<0.05-0.01) reduced the flow of saliva

at all frequencies examined, most conspicuously at 2 Hz given continuously, at which the

flow was reduced by 69 % (before: 22±4 µl min-1

g gland-1

; after: 7±3 µl min-1

g gland-1

;

P<0.01; Fig. 3). In addition to the reduction of the flow of saliva, pirenzepine consistently

delayed the onset of the flow of saliva, especially at the lower frequencies (latency at 2

Hz: 30±7 vs. 180±30 s, P<0.05; 20 Hz 1:10: 17±2 vs. 115 ± 32 s, P<0.05). At 8 Hz the

latency was changed from 11±1 s to 25±2 s (P<0.05). The inhibitory effect on the onset of

the secretory response could also be detected in the different shapes of the time-response

curves (flow and protein output; Fig. 5). Although pirenzepine did not significantly affect

the vascular responses a tendency towards a decrease could be noted at 2 Hz (-46±4 vs. -

35±3 %; n.s., multiple comparisons; Fig. 3). The additional administration of pFHHSiD (4

13

µg kg-1

I.V.; n=5) almost completely abolished the fluid response at 2 Hz (1±0.5 µl min-1

g gland-1

; P<0.05 in comparison with response in presence of pirenzepine), whereas the

antagonist had no or little effect on the pirenzepine-inhibited fluid response to 20 Hz 1:10

and 8 Hz (Fig. 3). Neither did pFHHSiD affect the vasodilator response, even though a

tendency towards a reduction could be noticed at 2 Hz (-29±3 %; n.s., Fig. 3 and 5).

However, the change in SVR at 2 Hz, in the presence of pirenzepine and pFHHSiD, was

significantly less than the response in the absence of any antagonist (-46±4 % vs. -29±3

%, P<0.05; Fig. 3). Whereas pirenzepine on its own did not affect the protein output at

any frequency or pattern, the addition of pFHHSiD significantly reduced the output at 2

Hz. The output was reduced at this frequency in spite of four-fold increase in the protein

concentration (1.1±0.3 vs. 4.1±1.0 µg µl-1

), much because of the markedly diminished

fluid response. Neither pirenzepine nor pFHHSiD affected heart rate or blood pressure

significantly, even though both tended to decrease during the course of the experiment

(Fig. 5).

Immunoblotting and immunohistochemistry

Using Western immunoblotting and specific antibodies against the receptor subtype

protein, distinct bands at levels that corresponded to the molecular size for muscarinic M1,

M3, M4 and M5 receptors could be detected in the glandular tissues (Fig. 6). No band

could be detected for the muscarinic M2 receptor. Semi-quantification of the

immunoblottings indicated relative constant signals of the specimens from the three

animals examined (muscarinic M1, M3, M4 and M5 receptors: 0.49±0.09, 0.41±0.03,

0.38±0.08 and 0.29±0.04 arbitrary units, respectively; n=3 in each case). Furthermore, the

addition of the respective blocking peptides suppressed or abolished the bands

corresponding to the estimated receptor molecular size for all muscarinic receptor

14

subtypes. The immunohistochemical examination of the localization of the muscarinic

receptors demonstrated a pronounced reactivity in the acinar and demilunar cells against

the muscarinic M1, M3, M4 and M5 receptor (M1R-IR – M5R-IR; Fig. 7). While M3R-IR

seemed to be primarily distributed in the demilunar cells, M1R-IR occurred more densely

on demilunar than on acinar cells and the M4R-IR and M5R-IR, showing resemblance in

their distributions, occurred close to or in demilunar cells, but also vaguely in acinar cells.

Muscarinic receptors were also detected in the stroma (M1R-IR, M4R-IR and M5R-IR).

Vague staining for the muscarinic M5 receptor could occasionally be detected in arterial

endothelium. Furthermore, the addition of the respective blocking peptides suppressed or

abolished the immunoreactivity for the muscarinic M1, M3, M4 and M5 receptors.

However, the staining for muscarinic M2 receptors caused a vague signal that could not be

suppressed by its immunogen and therefore the staining is not shown.

Discussion

This study shows that besides the muscarinic M3 receptors, a considerable portion of the

muscarinic receptor population within the ovine submandibular gland consists of

muscarinic M1, M4 and M5 receptors when using Western blotting. The muscarinic

receptors are widely distributed within the glandular parenchyma and particularly

muscarinic M1 and M4 receptors occur in the stroma. In the intraglandular vasculature

muscarinic M5 receptors were found in the arterial endothelium. Determination of the

functional significance of the receptors showed that muscarinic M1 receptors accounted

for a significant proportion of the cholinergic parasympathetic secretory response of the

ovine submandibular gland, particularly at low intensity of nerve activity. At low

intensities, the contribution of muscarinic M1 receptors to the fluid response was greater

than that of the muscarinic M3 receptors. In contrast to the pronounced secretory effect,

15

muscarinic M1 receptors do not seem to contribute to the parasympathetic nerve-evoked

vasodilatation; the cholinergic vasodilatation may involve muscarinic M5 receptors

mediated effects. Furthermore, the only muscarinic inhibitory receptor found in the ovine

submandibular gland is the M4 subtype, and its stromal localization make the occurrence

on parasympathetic nerve fibres possible. Consequently, the receptor may act as an

inhibitory autoreceptor and in the gland it modulates transmitter release at low stimulation

frequencies and, then, mainly peptidergic transmitters. The parasympathetic innervation

seems to exhibit muscarinic receptors, possibly of the M1 subtype, facilitating transmitter

release also.

In the present study, polyclonal antibodies were used in the Western blotting, which

caused some non-specific bands. Despite this, the bands that corresponded to the predicted

molecular masses of the muscarinic receptors were easily identified. Identification was

based partly on the molecular mass estimates for the muscarinic receptor subtypes

reported in other tissues (McLeskey & Wojcik, 1990; Ndoye et al., 1998; Preiksaitis et al.,

2000), and partly on the specificity of the band estimated by pre-incubation of the

antibody with its specific antigen. Even though a negative finding should be interpreted

with caution, it seems likely that no muscarinic M2 receptors seemed to occur within the

submandibular gland in view of the functional findings. Since muscarinic M1, M3 and M5

receptors are excitatory, the location of the M5 subtype in the intraglandular vasculature

can imply that this receptor exert indirect vascular effects. Nevertheless, the observations

that the ovine submandibular gland has several different muscarinic receptor subtypes is

consistent with the findings in the rat submandibular gland (Flynn et al., 1997).

The ovine submandibular (Edwards et al., 2003), and parotid glands (Reid & Titchen,

1988), share the characteristics of salivary glands of several other species in that

16

stimulation of the parasympathetic innervation at relatively high frequencies produces an

atropine-resistant fluid secretion mediated by peptidergic transmitters. In the current

experiments, an atropine-resistant fluid response appeared at 8 Hz, whereas stimulation at

2 Hz and 20 Hz in bursts was below the threshold frequency (totally atropine-sensitive).

Concerning the vasodilatation, on the other hand, it was almost totally resistant to atropine

at high frequencies and a significant reduction in the presence of muscarinic antagonists

occurred only at 2 Hz. Furthermore, the comparison of the responses to 2 and 20 Hz in

bursts, showed no significant difference in salivary flow, whereas the vasodilatation as

well as protein output were greater at 20 Hz in bursts. Since peptides, such as VIP and

CGRP, have been shown to be particularly potent in evoking vasodilatation and protein

output (Lundberg et al., 1982; Reid & Heywood, 1988; Ekström & Tobin, 1990; Buckle et

al., 1995), the present findings at 20 Hz in bursts fit well with the idea that the release of

peptides is increased at the high-frequency intermittent stimulation. A recent study on the

ovine submandibular gland showed that stimulation at 20 Hz efficiently released VIP from

the parasympathetic innervation and that intermittent stimulation was a more efficient

mode of stimulation than a continuous (Edwards et al., 2003). Even though an overt

atropine-resistant response is absent, peptidergic transmitters may anyway act in concert

with the classical transmitters, e.g. acetylcholine, and enlarge the amount or alter the

quality of the saliva (Lundberg et al., 1982; Ekström & Tobin, 1990).

The interpretation of the results from experiment, in which muscarinic antagonists with

different selectivity profiles was employed, is usually hampered by the narrow selectivity

window of the antagonists. However, it must be emphasized that pirenzepine and

methoctramine were used at doses previously validated in vivo for their selectivity for

muscarinic M1 and M2/M4 receptors, respectively (Tobin, 1995, 1998, 2002; Tobin et al.,

2002). Notably, methoctramine discriminates very poorly between muscarinic M2 and M4

17

receptors (2 times greater affinity for M2 over M4) in contrast to the more substantial

affinity difference for the inhibitory (muscarinic M2 and M4 receptors) over the excitatory

receptors (muscarinic M1, M3 and M5 receptors; (Caulfield & Birdsall, 1998; Eglen &

Nahorski, 2000)). In order to avoid an “unselective” effect by the “muscarinic M3 receptor

antagonist” pFHHSiD, an exceptionally low dose was used (4 µg kg-1

(10 nmol kg-1

) I.V.).

This dose is more than 10 times lower than the expected threshold dose for inhibiting

effects via the muscarinic M1 and M5 receptor (Tobin & Sjogren, 1995; Eglen &

Nahorski, 2000). Nevertheless, the current addition of the pFHHSiD to the pirenzepine

blockade served mainly to verify the selectivity of the pirenzepine antagonism. Overall,

the selectivity of the antagonist doses used in the current study is supported by the

following observations in the current experiments. First, methoctramine, and only

methoctramine, significantly raised the heart rate. Secondly, in general methoctramine had

opposite effects to the other two antagonists. Thirdly, pirenzepine had a substantially

greater inhibitory effect on secretion than on vasodilatation, and lastly, the extremely low

dose of pFHHSiD caused, in the presence of a larger pirenzepine dose, a significant

reduction in the parasympathetic nerve-evoked secretion; in pilot studies, when pFHHSiD

was given before pirenzepine, a small reduction occurred followed by a large pirenzepine

inhibitory effect. However, there appears to be one exception, even though not statistical

significant: pirenzepine showed strong tendencies towards increasing the protein output at

8 Hz. In this context, it is worth noting that the “muscarinic M1 receptor antagonist”

pirenzepine shows greater inhibitory potency on muscarinic M4 receptor-mediated effects

than on effects mediated by any of the other of the muscarinic M2-M5 receptor subtypes

(Eglen & Nahorski, 2000).

In the current study, the ovine submandibular gland was shown to belong to the glands in

which muscarinic M1 receptors evoke a flow of saliva. In previous studies, muscarinic M1

18

receptor-activated salivary flow has been demonstrated in the rabbit submandibular gland

(Tobin, 1995) and in the rat sublingual gland (Culp et al., 1996; Tobin et al., 2002). In

these glands, as well as the murine parotid gland (Watson et al., 1996), a co-expression of

muscarinic M1 and M3 receptors occurs, and in the sublingual gland of the rat a

simultaneous activation of both subtypes seems to be a prerequisite for evoking a maximal

fluid response (Luo et al., 2001). Because of the number of different ways positive

interactions could be exerted regarding the fluid response, it is hard to make any absolute

estimation of the relative contribution to the fluid response of muscarinic M1 and M3

receptors in the current study. Nevertheless, at low frequencies muscarinic M1 receptors

account for a large part, if not nearby the whole secretory response. Furthermore, in no

case was a maximal secretory response elicited under muscarinic M1 receptor blockade.

The contribution of other muscarinic receptor subtypes than M3 is supported by findings

in knockout mice also. Here, in muscarinic M1 receptor knockouts a decreased flow of

saliva occurs and in M1/M3 double knockouts a trace secretion seem to persists in

response to pilocarpine (Gautam et al., 2004). Regarding vasodilation, both pirenzepine

and pFHHSiD showed less inhibitory potency on the atropine-sensitive part than on

salivation; only when the two drugs were combined was statistical significance attained.

The obvious explanation is that muscarinic M1 receptors do not contribute to the

vasodilatation, but further, the lesser inhibitory potency of pFHHSiD could imply that a

receptor subtype other than the muscarinic M3 receptor is involved.

In the rat parotid and submandibular glands, pre-treatment with methoctramine may

double the parasympathetic nerve-evoked fluid secretion (Tobin, 1998, 2002). This effect

is attributable to a blockade of inhibitory muscarinic receptors on glandular nerve fibres,

and affects the neuronal release of acetylcholine as well as that of VIP (Tobin et al., 1991;

Tobin et al., 1994; Tobin, 1998). Also in sheep, the VIP submandibular output has been

19

shown to increase substantially by pre-treatment with atropine (Edwards et al., 2003).

However, it has not been established previously which particular type of muscarinic

receptor is responsible for presynaptic inhibition of VIP release in the sheep. The

morphological findings in the current study indicated the existence of only one subtype of

inhibitory muscarinic receptor in the submandibular gland, namely the M4 subtype located

within the stroma. In contrast to the findings in rats (Tobin, 1998, 2002) and rabbits

(Tobin, 1995), no enhancement of fluid secretion occurred in the presence of muscarinic

receptor blockade in the sheep. However, responses that could be attributed to the release

of VIP, i.e. protein output and vasodilatation were enhanced by methoctramine. In view of

the morphological findings, and the fact that methoctramine shows almost identical

affinity for muscarinic M2 and M4 receptors, it seems most plausible to ascribe the

increased responses to a blockade of muscarinic M4 receptors that enhanced release of

VIP. In salivary glands of rats and rabbits, facilitation of the nerve transmission occurs by

acetylcholine acting on muscarinic M1 receptors located prejunctionally (Tobin, 1995,

1998, 2002). A similar facilitator mechanism may occur in the ovine submandibular gland,

since pirenzepine invariably increased the latencies of both fluid and protein output (see

figure 5).

Thus, the ovine submandibular gland shows the same muscarinic receptor characteristics

as salivary glands in rabbits (Tobin, 1995) and rats (Culp et al., 1996; Luo et al., 2001;

Tobin et al., 2002) in that acetylcholine acting on muscarinic M1 receptors contribute

substantially to the secretory response. Other common features seem to be the occurrence

of prejunctional muscarinic receptors that inhibit or facilitate the release of transmitter

(Tobin, 1995, 1998, 2002). The cholinergic blood flow regulation in the ovine

submandibular gland may involve endothelial muscarinic M5 receptors, possibly via

20

nitric-oxide. Nonetheless, the present in vivo study provides compelling evidence for

muscarinic M1 receptors mediating a large part of the parasympathetic fluid response.

Ackowledgement

This study was supported by the Swedish Dental Society, Wilhelm and Martina Lundgrens

Foundation and Magn. Bergvall´s Foundation. We are indebted to professor Abigail

Fowden for valuable comments on the manuscript and to Histo-Center, Va Frolunda,

Sweden, for expert technical assistance (sectioning for immunohistochemistry).

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Figure legends

Figure 1. Comparison of the changes in submandibular flow of saliva (upper panel),

submandibular protein output (second panel from top), mean aortic blood pressure (middle

panel), mean heart rate (second panel from bottom) and submandibular vascular resistance

(bottom panel) in response to chorda tympani stimulation at 2 Hz continously ( ),

20 Hz in bursts (1:10 s; ) and 8 Hz continuously ( ) for 10 min in 13 anaesthetized

sheep. Vertical bars: S.E. M. value. Horizontal bar: duration of stimulation (CT stim).

Figure 2. Registrations in a single anaesthetized sheep before and after intravenous injection

of atropine (0.5 mg kg-1). Panels show changes in mean aortic blood pressure (upper panel),

in heart rate (next panel below), in submandibular blood flow (second panel from bottom)

and in submandibular flow of saliva (bottom panel). Middle registration shows the event

marker (stimulation at 8 Hz continuously for 10 minutes).

Figure 3. Comparison of the submandibular changes in mean salivary secretion (left

panels), in mean protein output (middle panels) and in mean vascular resistance (right

panels) over the 10 min stimulation period. The column doublets (upper panels) and

27

triplets (lower panels) in each panel show from left to right the mean responses to chorda

tympani stimulation at 2 Hz continuously, 20 Hz in bursts and 8 Hz continuously. The

upper panels show the mean responses in the absence ( ) and presence ( ) of

methoctramine (100 µg kg-1 I.V.) in five anaesthetized sheep and the lower panels show

the mean responses in the absence ( ) and presence of pirenzepine (40 µg kg-1 I.V.)

before ( ) and after administration pFHHSiD (4 g kg-1

I.V.) in five other anaesthetized

sheep.Vertical bars: S.E. M. *P<0.05, ** P<0.01.

Figure 4. Comparison of the changes in submandibular flow of saliva (upper panel),

submandibular protein output (second panel from top), mean aortic blood pressure (middle

panel), mean heart rate (second panel from bottom) and submandibular vascular resistance

(bottom panel) in response to chorda tympani stimulation at 2 Hz continously for 10 min in

the absence ( ) and in the presence of methoctramine (100 µg kg-1 I.V.; ) in five

anaesthetized sheep. Vertical bars: S.E. M. value. Horizontal bar: duration of stimulation (CT

stim).

Figure 5. Comparison of the changes in submandibular flow of saliva (upper panel),

submandibular protein output (second panel from top), mean aortic blood pressure (middle

panel), mean heart rate (second panel from bottom) and submandibular vascular resistance

(bottom panel) in response to chorda tympani stimulation at 2 Hz continously for 10 min in

the absence of antagonists ( ) and in the presence of pirenzepine (40 µg kg-1 I.V.; )

before and after administration of pFHHSiD (4 g kg-1

I.V.; ) in five anaesthetized sheep.

Vertical bars: S.E. M. value. Horizontal bar: duration of stimulation (CT stim).

Figure 6. Immunoblotting for muscarinic M1 and M3-M5 receptor subtypes in the ovine

submandibular gland. Bands corresponding to the predicted molecular mass of the

respective receptor subtype are indicated by arrows to the right of each image (M1 = 53

kDa, M3 = 67 kDa, M4 = 57 kDa, and M5 = 67 kDa; see Preiksaitis et al., 2000).

28

Immunoreactive protein bands corresponding to each receptor subtype were identified.

Immunoblotting in the absence (left lane) and presence (right lane) of antipeptide is shown in

each panel. Arrows indicate the MagicMark (40, 50, 60, 80 and 100 kDa; no band could be

detected for muscarinic M2 receptor; not shown).

Figure 7. Immunohistochemical labelling of ovine submandibular glands. Panels

demonstrate staining in absence of antibody (control); staining in the presence of

muscarinic M1, M3, M4 and M5 receptor antibodies (M1R-IR, M3R-IR, M4R-IR, M5R-

IR, respecetively; inserts in M1R-IR, M4R-IR and M5R-IR for demonstration of

appearances in stroma and endothelium). All sections are counterstained with

haematoxyline. Bar indicates 50 µm and the arrow close to the letters a, d, e and s indicate

acinar cells, demilunar cells, endothelial cells and stroma, respectively.

0

20

40

60

80

100

Sa

liva

(l m

in-1

g g

lan

d-1

)

Pro

tein

ou

tpu

t

(g

min

-1 g

gla

nd

-1)

0

40

80

120

160

200

Va

sc

ula

r re

sis

tan

ce

(% c

ha

ng

e)

-30

-90

10

-70

-50

-10

70

80

90

100

Blo

od

pre

ss

ure

(mm

Hg

)

80

100

120

He

art

ra

te

(bp

m)

Time (min)0 5 10 15-5

CT stim

1.0

0

10

0

15

5

0.5

125

50

150

100

Blo

od

pre

ssu

re

(mm

Hg

)

100

75

Hearr

rate

(bp

m)

Blo

od

flo

w

(ml m

in-1

)S

aliva

(ml m

in-1

)

Event marker (8 Hz)

Atropine 0.5 mg kg-1

*

*

***

*

Sa

liv

a (

l m

in-1

g g

lan

d-1

)S

ali

va

(l

min

-1 g

gla

nd

-1)

Pro

tein

(g

min

-1 g

gla

nd

-1)

Pro

tein

(g

min

-1 g

gla

nd

-1)

Va

sc

ula

r re

sis

tan

ce

(%

ch

an

ge

)

Va

sc

ula

r re

sis

tan

ce

(%

ch

an

ge

)

0

20

40

60

80

100

120

0

20

40

60

80

100

120

*

0

20

40

60

80

100

120

140

0

20

40

60

80

100

120

140

*-90

-80

-70

-60

-50

-40

-30

-20

0

-10

-80

-70

-60

-50

-40

-30

-20

0

-10

2 Hz 20 Hz

1:10

8 Hz 2 Hz 20 Hz

1:10

8 Hz 2 Hz 20 Hz

1:10

8 Hz

2 Hz 20 Hz

1:10

8 Hz 2 Hz 20 Hz

1:10

8 Hz 2 Hz 20 Hz

1:10

8 Hz

*

0

5

10

15

20

25

Sa

liva

(l m

in-1

g g

lan

d-1

)

Pro

tein

ou

tpu

t

(g

min

-1 g

gla

nd

-1)

0

20

40

60

80

100

Time (min)0 5 10 15-5

CT stim

70

80

90

100

Blo

od

pre

ss

ure

(mm

Hg

)

80

100

120

He

art

ra

te

(bp

m)

Va

sc

ula

r re

sis

tan

ce

(% c

ha

ng

e)

-30

10

-70

-50

-10

0

10

20

30

Sa

liva

(l m

in-1

g g

lan

d-1

)

Pro

tein

ou

tpu

t

(g

min

-1 g

gla

nd

-1)

0

5

10

15

20

70

80

90

100

Blo

od

pre

ss

ure

(mm

Hg

)

80

100

120

He

art

ra

te

(bp

m)

Va

sc

ula

r re

sis

tan

ce

(% c

ha

ng

e)

-30

10

-70

-50

-10

Time (min)0 5 10 15-5

CT stim

M1 M3 M4 M5100 kDa

40 kDa

60 kDa

M4R-IR

control M1R-IR M3R-IR

M5R-IR

a

d

ad

s

a

d

se

d

a

da