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J Appl Physiol 100: 1215-1223, 2006. First published December 1, 2005; doi:10.1152/japplphysiol.00779.2005
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Distribution and function of muscarinic receptor subtypes in the ovine submandibular gland

G. Tobin,1 A. T. Ryberg,1 S. Gentle,2 and A. V. Edwards 2,{dagger}

1Department of Pharmacology, Sahlgrenska Academy at Göteborg University, Goteborg, Sweden; and 2Physiological Laboratory, University of Cambridge, Cambridge, United Kingdom

Submitted 1 July 2005 ; accepted in final form 29 November 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
The effects of muscarinic receptor antagonists on responses to electrical stimulation of the chorda-lingual nerve were determined in pentobarbitone-anesthetized 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 µl·min–1·g gland–1), whereas 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 µl·min–1·g gland–1 and –77 ± 3%). While atropine (0.5 mg/kg iv) abolished the secretory response at 2 and 20 Hz (1:10 s), a small response persisted at 8 Hz (<5%). The "M1-selective" antagonist pirenzepine (40 µg/kg iv) 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 µg/kg 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.

saliva; vasodilatation

THE INCREASE IN SALIVARY FLOW evoked by muscarinic agonists has generally been attributed to activation of muscarinic receptors solely of the M3 subtype (3, 7). This concept has been supported by findings obtained in studies by using subtype-specific antisera, as well as by functional studies on rat parotid glands (11). 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 (6, 18, 20, 23, 39). 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 (30) and in the rat sublingual gland (10, 37), and of muscarinic M1 as well as M5 receptors in the rat submandibular gland (25, 37).

In salivary glands, parasympathetic nerve transmission may involve not only the classical autonomic transmitter acetylcholine but also nonadrenergic, noncholinergic transmitters (16). The parasympathetically nerve-evoked vasodilatation, which is evoked by nonadrenergic, noncholinergic transmitters, is particularly resistant to atropine (12). However, a secretory response also persists after full atropinization in salivary glands of several species, including the ovine submandibular gland (14), albeit much reduced and, preferentially, at high-frequency stimulation (16). In ovine salivary glands, the neuropeptide vasoactive intestinal peptide (VIP) and CGRP seem to mediate most of the atropine-resistant parasympathetic responses (13, 14). Nevertheless, acetylcholine acting on muscarinic receptors is the principal stimulation for evoking fluid responses in salivary glands (9), whereas the vasodilator response, although markedly resistant to atropine, has a cholinergic component, particularly at low-frequency stimulation (12, 14). 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 (31).

Nerve transmission in the parasympathetic innervation of salivary glands may be modulated by prejunctional muscarinic receptors (30–32). 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 compared with continuous stimulation (2, 4). These observations have subsequently been confirmed in salivary glands of other species, including the ovine submandibular gland (1, 31, 32, 35). The phenomena have been attributed to the release of neuropeptides, which preferentially occurs at high-stimulation frequencies (1, 4), and to a short-lasting stimulation activating facilitator and not inhibitory receptor mechanisms (31, 32).

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 by using antagonists with different muscarinic profiles [pirenzepine, methoctramine, p-fluorohexahydro-sila-diphenidol (pFHHSiD), atropine (7, 15)]. 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 (33).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animals.   The experiments were carried out on 18 adult ewes of various breeds (35–72 kg body wt) under the Animals Scientific Procedures Act (1986); Project Licence PPL 80/1316. The ethics committee of the respective university approved the study design. Food, but not water, was withheld for 48 h before each experiment. Anesthesia was induced and maintained with pentobarbitone sodium [Sagatal, Rhône Mérieux, Harlow, UK; 15–30 mg/kg iv and then 0.1–0.3 mg·min–1·kg–1 iv (adjusted to maintain a stable blood pressure)]. At the end of each experiment, the animal was given a lethal dose of barbiturate (Pentoject, Animalcare, York, UK; ca. 15 ml, 20% wt/vol), and the ipsi- and contralateral submandibular glands were 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 midline 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 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 pentobarbitone sodium. 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 neighboring length of the hypoglossal nerve was excised to minimize 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, Wrexham, UK; 1,000 IU/kg iv), and the linguofacial vein was 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 helium of the gland. The protocol involved parasympathetic stimulation at 2 and 8 Hz continuously for 10 min (20-V square wave; 10-ms pulse width), and intermittent stimulation at 20 Hz given for 1 s at 10-s intervals (1:10) 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 iv, 100 nmol/kg; "M2/M4-selective": methoctramine tetrahydrochloride, 100 µg/kg iv, 140 nmol/kg; "M3(M1/M5)-selective": pFHHSiD, 4 µg/kg iv, 10 nmol/kg; all from Sigma, St. Louis, MO (30–32, 37, 38)]. The rates of flow of submandibular blood and saliva were recorded photometrically drop by drop and also estimated gravimetrically. No spontaneous flow of saliva occurred. During stimulation, collection of samples was delayed for 2 min 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 iv, 2 µmol/kg).

Immunohistochemistry and immunoblotting.   After administration of a lethal dose of anesthetic, 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 they were 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 deparaffinized by heating the slides to 60°C for 15 min and then subjecting them to two 30-min changes in 100% xylene; the sections were then rehydrated 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. Nonspecific protein binding was blocked with 5% BSA in TBS for 30 min. The sections were thereupon incubated overnight at room temperature in a humidified chamber with polyclonal rabbit anti-muscarinic acetylcholine receptor subtype-specific antibodies (Research and Diagnostic Antibodies, Berkley, CA) diluted 100x 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, CA; system used following the manufacturer's instructions) that uses 3,3'-diaminobenzidine 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 preabsorbed with the appropriate immunogen before proceeding as described above.

Muscarinic receptor expression in the ovine submandibular gland was examined by using Western blotting. All tissues were homogenized in ice-cold phosphate EDTA buffer containing 0.1 mM leupeptin (Sigma), 0.1 mM pepstatin (Sigma), 1.5 mM aprotinin (Sigma), 4 mM Pefabloc SC (Fulka Chemie, Buchs, Switzerland), 50 µM sodium fluoride (Sigma), 0.2 µM sodium orthovanadate, and 5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Sigma). The lysate was heated to 70°C for 10 min in a reducing sample buffer. The proteins were fractionated by reducing NuPAGE 4–12% Bis-Tris gels (Invitrogen, Carlsbad, CA) and electroblotted onto polyvinylidene difluoride membranes (Invitrogen), which, then were incubated in phosphate-buffered saline-0.3% Tween 20 (Sigma), containing 0.2% I-Block (Tropix, Bedford, MA) to block nonspecific binding. The membranes were then incubated overnight with polyclonal antibodies directed against each of the five muscarinic receptors respectively (Research and Diagnostic Antibodies). The antibodies were used at the following dilutions: 1:500 (anti-M1, anti-M3, and anti-M4), 1:2,000 (anti-M2), and 1:2,500 (anti-M5). The antibodies were validated using positive controls. The binding was visualized with a chemiluminescent detection system that utilizes enzyme-linked immunodetection, detected using enhanced chemiluminescence, visualized by Flour-S (Bio-Rad, Hercules, CA), and analyzed using the QuantityOne software (version 4.4.1, build 067; Bio-Rad). For negative controls, primary antibodies were omitted in the procedure described above. As an additional control, the antibodies were preabsorbed 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 (mmHg) by the submandibular blood flow (µl·min–1·g gland–1) and expressed as the percent changes from experimental time = 0. Results are expressed as mean values ± SE and were assessed statistically (Prism 4, GraphPad Software) 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 <0.05 are considered to be statistically significant. All flows and outputs are expressed per unit weight of the contralateral gland.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Cardiovascular and secretory 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 aortic blood pressure (92 ± 5 vs. 88 ± 4 mmHg) and heart rate (107 ± 4 vs. 103 ± 5 beats/min) 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 SVR without affecting aortic blood pressure or heart rate (Fig. 1). Typical responses from a single animal are illustrated in Fig. 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 toward the initial values over a 5- to 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 1:10, by so delivering precisely the same number of impulses to the chorda tympani fibers over the 10-min test period as the continuous stimulation at 2 Hz, produced instantaneous flow of saliva and fall in SVR that were 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 not only regarding flow 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, it was 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 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 compared 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.


Figure 1
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  		Fig. 1. Comparison of the changes in submandibular flow of saliva (first panel), submandibular protein output (second panel), mean aortic blood pressure (third panel), mean heart rate (fourth panel) and submandibular vascular resistance (fifth panel) in response to chorda tympani (CT) stimulation at 2 Hz continuously ({blacktriangleup}), 20 Hz in bursts [bursts for 1 s at 10-s intervals (1:10); bullet], and 8 Hz continuously ({blacksquare}) for 10 min in 13 anesthetized sheep. Values are means ± SE. Horizontal bar: duration of stimulation (CT stim). bpm. Beats/min.

 

Figure 2
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  		Fig. 2. Registrations in a single anesthetized sheep before and after intravenous injection of atropine (0.5 mg/kg). Panels show changes in mean aortic blood pressure (first panel), heart rate (second panel), submandibular blood flow (third panel), and submandibular flow of saliva (fourth panel). Middle registration shows the event marker (stimulation at 8 Hz continuously for 10 min).

 
Responses following the administration of muscarinic antagonists.   Pretreatment with atropine (0.5 mg/kg iv; 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.

The intravenous administration of 100 µg/kg methoctramine (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 the changes over the whole 10-min stimulation period were assessed, albeit a tendency toward 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 min) of stimulation (+170%; P < 0.01; Fig. 4). Similarly, methoctramine caused a significant increase during the first minutes (2–4 min) 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).


Figure 4
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  		Fig. 4. Comparison of the changes in submandibular flow of saliva (first panel), submandibular protein output (second panel), mean aortic blood pressure (third panel), mean heart rate (fourth panel), and submandibular vascular resistance (fifth panel) in response to CT stimulation at 2 Hz continuously for 10 min in the absence ({square}) and in the presence ({blacksquare}) of methoctramine (100 µg/kg iv) in 5 anesthetized sheep. Values are means ± SE. Horizontal bar: duration of stimulation.

 

Figure 3
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  		Fig. 3. Comparison of the submandibular changes in mean salivary secretion (left), mean protein output (middle), and mean vascular resistance (right) over the 10-min stimulation period. The column doublets (top) and triplets (bottom) in each panel show, from left to right, the mean responses to CT stimulation at 2 Hz continuously, 20 Hz in bursts, and 8 Hz continuously. Top: mean responses in the absence (open bars) and presence (solid bars) of methoctramine (100 µg/kg iv) in 5 anesthetized sheep. Bottom: mean responses in the absence (open bars) and presence of pirenzepine (40 µg/kg iv) before (hatched bars) and after (solid bars) administration of p-fluorohexahydro-sila-diphenidol (pFHHSiD; 4 µg/kg iv) in 5 other anesthetized sheep. Values are means ± SE. *P < 0.05, **P < 0.01.

 
Pirenzepine (40 µg/kg iv; 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 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 toward a decrease could be noted at 2 Hz (–46 ± 4 vs. –35 ± 3%; not significant, multiple comparisons; Fig. 3). The additional administration of pFHHSiD (4 µg/kg iv; n = 5) almost completely abolished the fluid response at 2 Hz (1 ± 0.5 µl·min–1·g gland–1; P < 0.05 compared 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 toward a reduction could be noticed at 2 Hz (–29 ± 3%; not significant, Figs. 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, despite a fourfold increase in the protein concentration (1.1 ± 0.3 vs. 4.1 ± 1.0 µg/µl), 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).


Figure 5
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  		Fig. 5. Comparison of the changes in submandibular flow of saliva (first panel), submandibular protein output (second panel), mean aortic blood pressure (third panel), mean heart rate (fourth panel), and submandibular vascular resistance (fifth panel) in response to CT stimulation at 2 Hz continuously for 10 min in the absence of antagonists ({square}) and in the presence of pirenzepine (40 µg/kg iv; {blacksquare}) before and after administration of pFHHSiD (4 µg/kg iv; {blacktriangleup}) in 5 anesthetized sheep. Values are means ± SE. Horizontal bar: duration of stimulation.

 
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. Semiquantification 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 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-immunoreactivity (IR) to 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.


Figure 6
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  		Fig. 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 Ref. 27). 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
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  		Fig. 7. Immunohistochemical labeling 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-immunoreactivity (IR), M3R-IR, M4R-IR, M5R-IR, respectively]. Insets in M1R-IR, M4R-IR, and M5R-IR are for demonstration of appearances in stroma and endothelium. All sections are counterstained with hematoxylin. Bar indicates 50 µm, and the arrows close to the letters a, d, e, and s indicate acinar cells, demilunar cells, endothelial cells, and stroma, respectively.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
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, muscarinic M1 receptors do not seem to contribute to the parasympathetic nerve-evoked vasodilatation; the cholinergic vasodilatation may involve muscarinic M5 receptor-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 fibers 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 nonspecific 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 (24, 26, 27) and partly on the specificity of the band estimated by preincubation 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 exerts 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 (18).

The ovine submandibular (14) and parotid glands (29) share the characteristics of salivary glands of several other species in that stimulation of the parasympathetic innervation at relatively high frequencies produces an atropine-resistant fluid secretion mediated by peptidergic transmitters. In the present experiments, an atropine-resistant fluid response appeared at 8 Hz, whereas stimulation at 2 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. Because peptides such as VIP and CGRP have been shown to be particularly potent in evoking vasodilatation and protein output (5, 17, 21, 28), 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 one (14). 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 (17, 21).

The interpretation of the results from experiment, in which muscarinic antagonists with different selectivity profiles were 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 (30–32, 37). Notably, methoctramine discriminates very poorly between muscarinic M2 and M4 receptors (two 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; (8, 15)]. To avoid an "unselective" effect by the "muscarinic M3 receptor antagonist" pFHHSiD, an exceptionally low dose was used (4 µg/kg, 10 nmol/kg iv). This dose is more than 10 times lower than the expected threshold dose for inhibiting effects via the muscarinic M1 and M5 receptor (15, 38). 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 present study is supported by the following observations in the present experiments. First, methoctramine, and only methoctramine, significantly raised the heart rate. Second, in general, methoctramine had opposite effects as the other two antagonists. Third, pirenzepine had a substantially greater inhibitory effect on secretion than on vasodilatation, and last, 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 it is not statistically significant: pirenzepine showed strong tendencies toward 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 muscarinic M2-M5 receptor subtypes (15).

In the present 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 receptor-activated salivary flow has been demonstrated in the rabbit submandibular gland (30) and in the rat sublingual gland (10, 37). In these glands, as well as the murine parotid gland (40), a coexpression 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 (22). 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 present study. Nevertheless, at low frequencies, muscarinic M1 receptors account for a large part of, if not nearly 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 seems to persist in response to pilocarpine (19). 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, furthermore, 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, pretreatment with methoctramine may double the parasympathetic nerve-evoked fluid secretion (31, 32). This effect is attributable to a blockade of inhibitory muscarinic receptors on glandular nerve fibers and affects the neuronal release of acetylcholine as well as that of VIP (31, 34, 36). Also in sheep, the VIP submandibular output has been shown to increase substantially by pretreatment with atropine (14). 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 present 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 (31, 32) and rabbits (30), 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 (30–32). A similar facilitator mechanism may occur in the ovine submandibular gland, since pirenzepine invariably increased the latencies of both fluid and protein output (see Fig. 5).

Thus the ovine submandibular gland shows the same muscarinic receptor characteristics as salivary glands in rabbits (30) and rats (10, 22, 37) in that acetylcholine acting on muscarinic M1 receptors contributes 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 (30–32). The cholinergic blood flow regulation in the ovine submandibular gland may involve endothelial muscarinic M5 receptors, possibly via nitric oxide. Nonetheless, the present in vivo study provides compelling evidence for muscarinic M1 receptors mediating a large part of the parasympathetic fluid response.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This study was supported by the Swedish Dental Society, Wilhelm and Martina Lundgrens Foundation, and Magn. Bergvall's Foundation.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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 ACKNOWLEDGMENTS
REFERENCES
 
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).


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Tobin, Dept. of Pharmacology, the Sahlgrenska Academy at Göteborg Univ., Medicinaregatan 15D, Göteborg 413 90, Sweden (e-mail: gunnar.tobin{at}pharm.gu.se)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

{dagger} Deceased. Back


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 ACKNOWLEDGMENTS
 REFERENCES
 

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