SC-46275: a potent and highly selective agonist at the EP3 receptor.

The agonist properties of SC-46275 have been investigated in EP receptor subtype-specific smooth muscle assays. In the isolated guinea pig vas deferens (GPVD), prostaglandin E2 (PGE2), via the EP3 receptor, potently inhibited electrically induced contractions with an EC50 of 5.4 +/- 1.1 nM. Sulprostone and misoprostol were both potent relaxers of the GPVD yielding EC50s of 1.6 +/- 0.4 nM and 4.3 +/- 0.9 nM, respectively, while butaprost (10,000 nM) was inactive. SC-46275 was by far the most potent agonist in the GPVD exhibiting an EC50 of 0.04 +/- 0.02 nM. PGE2, via the EP1 receptor, stimulates contractions in the longitudinal muscle layer of the guinea pig ileum (GPIL) with an EC50 of 74.4 +/- 10.6 nM. SC-46275 was extremely weak in this preparation, generating only 33% of the maximal PGE2 effect at 30,000 nM. The circular muscle layer of guinea pig ileum (GPIC) is responsive to inhibition of electrically stimulated contractions by PGE2 (EC50 = 179.6 +/- 20.8 nM) via the EP2 receptor. SC-46275 (up to 10,000 nM) was completely inactive in this preparation. We conclude from these findings that SC-46275 is a very potent and highly selective EP3 receptor agonist. SC-46275 should prove to be an extremely valuable tool in probing the physiological significance of EP3 receptors. The high potency of SC-46275 at the EP3 receptor may account for its antisecretory and cytoprotective actions, while its lack of activity at the EP1 or EP2 sites may explain its very weak diarrheagenic potential.

SC-49518 enhances gastric emptying of solid and liquid meals and stimulates gastrointestinal motility in dogs by a 5-hydroxytryptamine4 receptor mechanism.

SC-49518 (N-[exo-(hexahydro-1H-pyrrolizine-1-yl)methyl]-2-methoxy-4- amino-5-chlorobenzamide HCl), a new benzamide gastrointestinal prokinetic compound, was investigated to determine its ability to stimulate gastrointestinal motility in vivo and whether these actions could be mediated by agonist activity at the putative 5-hydroxytryptamine (5-HT)4 receptor. In conscious fasted dogs with strain gauge transducers and myoelectrodes, SC-49518 disrupted gastric and small intestinal migrating motility complex cycling for more than 3.5 hr. It stimulated gastric antral contractile and intestinal myoelectric spike burst activities during the normally quiescent Phase I of the migrating motility complex at doses as low as 0.01 and 0.03 mg/kg i.v., respectively. In a canine model of gastroparesis, SC-49518 reversed completely alpha-2 adrenergically delayed gastric emptying of a solid meal with an ED50 value of 0.1 mg/kg intragastrically and partially reversed delayed emptying of a liquid meal. SC-49518, like 5-HT, cisapride and renzapride, acted as an agonist (EC50 = 6.6 +/- 1.1 x 10(-8) M) at the putative 5-HT4 receptor in rat esophageal tunica muscularis mucosae by relaxing carbachol-induced contractions. SC-49518 was a partial agonist at 5-HT4 receptors, but also blocked high affinity (5-HT4-mediated) responses to 5-HT (10(-9) M to 3 x 10(-7) M) in guinea pig ileum with a pA2 value of 8.39.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidant-evoked release of acetylcholine from enteric neurons of the rat colon.

In the presence of halides, granulocytes generate hypochlorous acid and, subsequently, chlorinated amines (chloramines). These lipophilic, potent reactive oxygen metabolites may contribute to the mucosal pathophysiology associated with inflammatory bowel disease. A common symptom of inflammatory bowel disease is mucosal secretion of fluid and electrolytes, leading to diarrhea. Because acetylcholine (Ach) can stimulate colonic fluid secretion, we determined the effect of monochloramine (NH2Cl) on Ach release by mucosal/submucosal nerves. Mucosa from the rat colon was separated from outer muscle layers and minced before incubation with [14C]choline to label stores of Ach in cholinergic neurons. Release of [14C]Ach was evoked with NH2Cl in the absence and presence of 5-aminosalicylic acid, glutathione, nordihydroguaiarectic acid or the cyclooxygenase inhibitor piroxicam. NH2Cl produced concentration-related increases in [14C] Ach release into the medium; greater than 100% over base line was observed at 0.5 mM. Glutathione inhibited the NH2Cl-evoked release in a concentration-dependent fashion. Release induced by 0.1 mM NH2Cl was abolished by 5-aminosalicylic acid and significantly inhibited by nordihydroguaiarectic acid. Piroxicam also prevented the effect of NH2Cl on release of [14C] Ach. None of these agents alone had any effect on base line [14C]Ach release. Tetrodotoxin (5 microM) did not significantly inhibit the NH2Cl-evoked transmitter release. We conclude that NH2Cl, at concentrations believed to exist in inflamed tissue, causes the release of Ach from mucosal/submucosal nerves primarily through nonspecific neural membrane injury. Endogenous prostaglandins, possibly liberated as a consequence of the injury, may be involved in the Ach release process.

Endothelin-1 stimulates contraction and ion transport in the rat colon: different mechanisms of action.

Endothelin-like immunoreactivity has been detected in all regions of the rat gastrointestinal tract. In the present study, we studied the effect of endothelin-1 (ET-1) on muscle contraction and ion transport in the rat colon. Isometric tension was recorded in colonic muscle strips oriented along their longitudinal axis. The effect of ET-1 on ion transport was investigated by assessing changes in short-circuit current in segments of muscle-stripped rat colon in Ussing chambers. ET-1 induced concentration-dependent contraction of the colon (EC50, 3 nM). The concentration-response curve to ET-1 was not modified by the neuronal blocker tetrodotoxin (0.1 microM) or by atropine (1 microM). Pretreatment of colon muscle strips with the calcium channel blockers diltiazem (0.1 microM) or nicardipine (1 microM) had no effect on the contractile response to ET-1. Furthermore, the response was not affected by removal of extracellular calcium. In the ion transport studies, serosal addition of ET-1 produced a transient, bumetanide (chloride secretion inhibitor) -sensitive, increase in transepithelial short-circuit current. The maximal increase was 107 +/- 13 microA/sq. cm, with an EC50 of 2.5 nM. The increase in short-circuit current evoked by ET-1 was not significantly affected by 1 microM atropine, but was reduced by 50% (P less than .05) by 1 microM tetrodotoxin, or removal of extracellular calcium. We conclude that ET-1 stimulates smooth muscle directly, whereas its effect on epithelial chloride secretion is mediated in part via the enteric nerves. Moreover, the effect of ET-1 in these two systems can be differentiated on the basis of sensitivity to extracellular calcium.

5-HT4 receptor activation induces relaxation and associated cAMP generation in rat esophagus.

Changes in mechanical events and intracellular levels of cAMP induced by the activation of the 5-HT4 receptor were investigated in the rat esophagus tunica muscularis mucosae preparation. Serotonin (5-HT) and 5 methoxytryptamine (5-MOT; 5-HT4 agonist) caused concentration-related relaxation responses, while 5-carboxamidotryptamine (5-CT; 5-HT1 agonist), 1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane (DOI; 5-HT2 agonist) and 2-methyl-serotonin (2-methyl-5-HT; 5-HT3 agonist) were less active. The prokinetic agents, cisapride and renzapride also induced concentration-dependent relaxation of rat esophagus which was intermediate to 5-HT and 5-MOT in potency. The relaxation was not due to activity at receptors other than the 5-HT4 since methysergide (5-HT1 and 5-HT2 antagonist) and granisetron (5-HT3 antagonist) did not block the relaxant response to 5-HT while ICS 205930 (5-HT4 antagonist) antagonized this response (pA2 = 6.45). Serotonin also caused concentration-related increases in tunica muscularis mucosae cAMP with the rank order of efficacy of 5-HT agonists in raising tissue cAMP levels reflecting their relaxant activities (5HT greater than or equal to 5-MOT greater than 5-CT greater than DOI = 2-methyl-5-HT = control). Enhancement of cAMP concentrations was also observed following renzapride treatment. This cAMP relaxation response was specific for 5-HT4 receptor activation as demonstrated by the lack of ICS 205930 inhibition of rat esophagus relaxation caused by isoproterenol, 16,16-dimethyl-prostaglandin E2 and forskolin.(ABSTRACT TRUNCATED AT 250 WORDS)

Li+ increases accumulation of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate in cholinergically stimulated brain cortex slices in guinea pig, mouse and rat. The increases require inositol supplementation in mouse and rat but not in guinea pig.

Li+, beginning at a concentration as low as 1 mM, produced a time- and dose-dependent increase in accumulation of [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4,5)P4 in acetylcholine (ACh)-stimulated guinea-pig brain cortex slices prelabelled with [3H]inositol and containing 1 mM-inositol in the final incubation period. Similar results were obtained by mass measurement of samples incubated with 10 mM-Li+ by using a receptor-binding assay, although the percentage stimulation of Ins(1,4,5)P3 accumulation by Li+ was somewhat less by this assay. The increase in accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 by Li+ was absolutely dependent on the presence of ACh. In the absence of added inositol, 1-5 mM-Li+ produced smaller increases in Ins(1,4,5)P3, but the Li(+)-dependent increase in Ins(1,3,4,5)P4 was not as affected by inositol omission. In previous studies with cholinergically stimulated rat and mouse brain cortex slices, Li+ inhibited accumulation of Ins(1,4,5)P3 in rat and inhibited Ins(1,3,4,5)P4 accumulation in rat and mouse [Batty & Nahorski (1987) Biochem. J. 247, 797-800; Whitworth & Kendall (1988) J. Neurochem. 51, 258-265]. We found that Li+ inhibited both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 accumulation in these species, but we could reverse this inhibition by adding 10-30 mM-inositol; we then observed a Li(+)-induced increase in Ins(1,4,5)P3 and Ins(1,3,4,5)P4. The species differences observed in the absence of supplemented inositol were explained by the fact that a much higher concentration of inositol was required to bring the Li(+)-elevated levels of CDP-diacylglycerol (CDPDG) down to baseline in the rat and mouse. These data suggest that inositol is more rate-limiting for phosphatidylinositol synthesis in the presence of Li+ in rat and mouse, which can account for the previous reports of inhibition of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 accumulation by this ion in these species. Thus, in all species examined. Li+ could be shown to increase accumulation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 in cholinergically stimulated brain cortex slices if the slices were supplemented with sufficient inositol to bring the Li(+)-elevated level of CDPDG down to near baseline, as seen in the absence of Li+. In guinea-pig brain cortex slices, increases in Ins(1,4,5)P3 and Ins(1,3,4,5)P4 accumulation could then be seen at Li+ concentrations as low as 1 mM, which falls within the therapeutic range of plasma concentrations in the treatment of manic-depressive disorders. These observations may have therapeutic implications.

Effect of endothelin-1 on guinea pig gallbladder smooth muscle in vitro.

The purpose of this study was to investigate the pharmacological activity of endothelin-1 (ET-1) on guinea pig gallbladder smooth muscle. Guinea pig gallbladder muscle strips were mounted in 10-ml siliconized organ baths containing Krebs' solution. After 1 hr of equilibration, ET-1 was added cumulatively. ET-1 induced slow-developing and long-duration contractile responses. The EC50 was approximately 10 nM. ET-1 was 5 times less potent than cholecystokinin (EC50, 2 nM), but 20 and 40 times more potent than carbachol (EC50, 200 nM) and histamine (EC50, 400 nM), respectively. The concentration-response curve to ET-1 was not affected by tetrodotoxin (0.1 microM) or by the muscarinic antagonist, atropine (10 microM). The neuronal N-type calcium channel blocker, omega-conotoxin (0.1 microM), had no significant effect on the ET-1 concentration-response curve. In contrast, the contractile effect to ET-1 was reduced markedly by removal of extracellular calcium or by the voltage-dependent calcium channel blockers nicardipine and diltiazem. Substitution of strontium (an inhibitor of intracellular calcium release) for Ca++ significantly reduced the response to ET-1. The cyclooxygenase inhibitor indomethacin had no significant effect on the contractile activity of ET-1. These finding suggest that ET-1 is a potent contractile stimulant of guinea pig gallbladder and that it acts directly on the smooth muscle. The activity depends on extracellular Ca++, suggesting involvement of Ca++ influx via the voltage-dependent Ca++ channel and on intracellular calcium.

Monochloramine induces contraction of guinea pig gallbladder via two different pathways.

The purpose of the present investigation was to examine the effect and mechanism of action of the reactive oxygen metabolites monochloramine (NH2Cl), hypochlorous acid (HOCl), and hydrogen peroxide (H2O2) on gallbladder smooth muscle contractility. All oxidants caused concentration-dependent increases in resting tension of gallbladder muscle; the rank order of potencies (half-maximal concentration) was NH2Cl (30 microM) greater than HOCl (70 microM) greater than H2O2 (100 microM). The oxidant concentrations employed are those found to exist in inflamed tissue. Tetrodotoxin (0.5 microM) had no effect on gallbladder muscle contraction caused by the oxidants, suggesting a direct, nonneural action. The maximal response induced by NH2Cl, HOCl, or H2O2 was significantly (P less than 0.05) inhibited by 5 microM indomethacin and 5 microM piroxicam. The calcium channel blocker verapamil partially inhibited the contractile effect of NH2Cl but had no effect on the contraction induced by exogenous cyclooxygenase products. Monochloramine induced significant prostaglandin E2 release from the gallbladder, which was blocked by indomethacin. Furthermore, the effect of NH2Cl on the smooth muscle was blocked by 5-aminosalicylic acid (1 mM). We conclude that reactive oxygen metabolites induce contraction of gallbladder smooth muscle by a direct action. The effect is mediated via cyclooxygenase metabolites and activation of calcium influx.

CGRP and ANF cause relaxation of opossum internal anal sphincter via different mechanisms.

This investigation examined and compared the role of cyclic nucleotides in the mediation of internal anal sphincter (IAS) relaxation caused by the addition of neuropeptide calcitonin gene-related peptide (CGRP) and atrial natriuretic factor (ANF). The studies were performed in vitro on smooth muscle strips of opossum IAS. The relaxation produced by CGRP and ANF was examined before and after the addition of tetrodotoxin (TTX) (1 x 10(-6)M). At this concentration, TTX did not have any significant effect on the relaxation produced by either CGRP or ANF, suggesting that these peptides act directly on the smooth muscle. Addition of CGRP (3 x 10(-6) M) produced the maximal relaxation and significantly increased cAMP content without changing cGMP. On the other hand, addition of ANF (3 x 10(-6) M) caused a similar fall in IAS tension that was accompanied by a significant elevation in cGMP without any change in cAMP content. The rises in the levels of cyclic nucleotides preceded the onset of fall in the resting tension of IAS. Our results demonstrate that CGRP and ANF relax isolated strips of opossum IAS by their action directly at the smooth muscle and that this relaxation is associated with an increase in cAMP and cGMP, respectively. The studies suggest the presence of both cAMP and cGMP pathways in the IAS and that the relaxation of IAS smooth muscle in response to different peptides may occur via a specific intracellular biochemical pathway.

Neutrophil-derived oxidants modify CCK-OP-stimulated guinea pig gallbladder contraction in vitro.

Neutrophil-derived oxidants such as hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and monochloramine (NH2Cl) may contribute to gallbladder inflammation in cholecystitis. We examined the influence of oxidants on the biological activity of different agonists and antagonists of gallbladder smooth muscle function. The concentration-response curves for cholecystokinin-octapeptide (CCK-OP) and carbachol were examined before and after incubation of the tissues with NH2Cl (30 microM). The 50% effective concentration of CCK-OP was shifted from 0.5 +/- 0.09 nM (control) to 4 +/- 1.2 nM in the presence of NH2Cl. The effect of carbachol was not affected by NH2Cl. The contractile effect of CCK-OP (3 nM) was abolished by prior exposure to HOCl or NH2Cl. These actions were prevented by 60 microM glutathione. Oxidant-induced degradation of CCK-OP was confirmed by high-performance liquid chromatography and thin-layer chromatography. NH2Cl also significantly reduced the contractile response to neurokinin A, bradykinin, leukotriene D4, and phorbol 12,13-dibutyrate and the relaxant response to isoproterenol. Prior exposure of acetylcholine, histamine, serotonin, prostaglandin E2, vasoactive intestinal polypeptide, or calcitonin gene-related peptide to NH2Cl had no effect on their activity. The results indicate that NH2Cl generated during inflammation may decrease the biological activities of different agonists and antagonists of smooth muscle function.

Differential effects of reactive oxygen metabolites on neurally stimulated and nonstimulated guinea pig ileum.

Large numbers of polymorphonuclear leukocytes which generate reactive oxygen metabolites are found in mucosa and submucosa of the intestinal wall of subjects suffering from inflammatory bowel disease. We have, therefore, examined the relative influences of hydrogen peroxide (H2O2), hypochlorous acid (HOCl) and N-chloramines such as NH2Cl, on the neurally stimulated and nonstimulated guinea pig ileum. In separate experiments the oxidants were tested in the presence and absence of the cyclooxygenase inhibitor piroxicam and the antioxidant glutathione. All three oxidants, in concentrations produced by activated neutrophils, increased the muscle tone (concentration-dependent, peak at 0.3 mM for NH2Cl and H2O2 and 1 mM for HOCl). Tetrodotoxin (0.5 microM) inhibited the NH2Cl and H2O2 effects by 50% and 70%, respectively. Piroxicam (5 microM) partially blocked maximal contractions induced by all three oxidants. The contractile response to carbachol (10 microM) was blocked by 0.3 mM NH2Cl, but not by H2O2 and HOCl. In electrically stimulated ileum the oxidants produced a concentration-dependent biphasic response (transient enhancement of neurally mediated twitch contraction followed by marked inhibition). This response was not modified by piroxicam, hexamethonium, atropine and pyrilamine. The inhibition of twitch contraction was irreversible for NH2Cl and HOCl, in contrast to H2O2, which was reversed by repeated washing. Neither the contractile effect nor the effects on nerve stimulation-induced contraction were affected by preincubation of the tissue with glutathione, whereas prior combination of NH2Cl with glutathione prevented the effects of NH2Cl. Oxidant-induced contraction of guinea pig ileum appears to be via release of prostaglandins and one or more neurotransmitters. High concentrations of reactive oxygen metabolites may alter receptor function.(ABSTRACT TRUNCATED AT 250 WORDS)

Gastrointestinal hormone receptors on isolated smooth muscle cells from gastric antrum of the rabbit.

The regulation by gastrointestinal polypeptide hormones of contraction and relaxation of functionally isolated smooth muscle cells from gastric antrum of the rabbit has been investigated. Gastrin, cholecystokinin (CCK-8) and motilin induced a rapid contraction of isolated cells: significant response occurred within a 5-sec incubation with these peptides and maximal response (40% decrease in cell length) after 30 sec. A higher sensitivity of smooth muscle cells to gastrin and CCK-8 than to motilin stimulations was demonstrated (EC50 = 10 pM for both gastrin and CCK-8 and EC50 = 1 nM for motilin). The minimal gastrin fragment required to get full contraction was the C-terminal pentapeptide amide common to gastrin and CCK. Proglumide inhibited gastrin- or CCK-8- but not motilin-induced contractions with an IC50 of 50 microM. contraction induced by gastrin and motilin required normal levels of extracellular calcium, whereas that due to CCK-8 seemed to be independent of extracellular calcium. Vasoactive intestinal polypeptide (VIP) caused a relaxation of smooth muscle cells maximally contracted by carbachol or CCK-8 or gastrin (EC50 = 2.2 nM) with a parallel increase in intracellular cAMP content.

Influence of stimulators and inhibitors of cyclic nucleotides on lower esophageal sphincter.

These studies were performed in vitro to investigate the nature of the second messenger for lower esophageal sphincter (LES) smooth muscle relaxation in response to electrical field stimulation (EFS) and vasoactive intestinal polypeptide (VIP). It was seen that VIP, permeant derivatives of the cyclic nucleotide 8-bromo cyclic GMP (BrcGMP) and 8-bromo cyclic AMP (8-BrcAMP), the guanylate cyclase stimulant sodium nitroprusside (SNP), the adenylate cyclase stimulant forskolin, M&B 22,948 (cGMP phosphodiesterase inhibitor) and SK&F 94,120 (cAMP phosphodiesterase inhibitor) caused dose-dependent and tetrocotoxin resistant fall in LES tension. Guanylate cyclase inhibitor methylene blue (MB) (3 x 10(-5) M), caused significant antagonism of fall in LES tension by SNP without modifying the inhibitory response of forskolin. The possible adenylate cyclase inhibitor N-ethylmaleimide (NEM) (1 x 10(-4) M), on the other hand, caused significant antagonism of fall in LES tension by forskolin without any effect on that caused by SNP. The inhibitory responses of 8-BrcGMP and 8-BrcAMP were not modified by MB or NEM. NEM (1 x 10(-4) M) and MB (3 x 10(-5) M) caused significant inhibition of the fall in LES tension with EFS. NEM also caused inhibition of fall in LES tension by VIP. Furthermore, SK&F 94,120 and not M&B 22,948 caused significant potentiation of fall in LES tension by EFS. From these results we conclude that: 1) cAMP and cGMP may act as second messengers for LES relaxation with EFS and VIP, and 2) VIP may act primarily via cAMP system and remains a strong possibility for one of the inhibitory neurotransmitters in the LES.

Effect of methylene blue and N-ethylmaleimide on internal anal sphincter relaxation.

The present studies were performed in vitro to define the participation of regulatory cyclic nucleotides in the relaxation of internal anal sphincter (IAS) smooth muscle in response to neural stimulation by electrical field stimulation (EFS) vs. exogenous vasoactive intestinal peptide (VIP). EFS and VIP both caused relaxation of the resting tone in the opossum-isolated IAS smooth muscle strips. The addition of permeant cyclic nucleotide derivatives, the guanylate cyclase stimulant sodium nitroprusside (SNP), and the adenylate cyclase stimulant forskolin caused a dose-dependent fall in the resting tension of IAS smooth muscle. The inhibitory effect of the agonists on the IAS smooth muscle was not modified by tetrodotoxin (TTX), a neurotoxin. TTX almost abolished the IAS responses to EFS. The effects of SNP and forskolin were selectively blocked by the putative inhibitors of corresponding enzyme systems, i.e., methylene blue (MB) (3 X 10(-5) M) for guanylate cyclase and N-ethylmaleimide (NEM) (10(-4) M) in the case of adenylate cyclase. NEM and not MB caused significant antagonism of the fall in IAS tension in response to both EFS and VIP during the control experiments. Such data suggest a common biochemical link (adenosine 3',5'-cyclic monophosphate as second messenger system) between the IAS smooth muscle relaxations with neural stimulation and VIP. In addition, a part of the IAS smooth muscle relaxation in response to EFS also involves the mediation of guanosine 5'-cyclic monophosphate.

Muscarinic receptors in isolated smooth muscle cells from gastric antrum.

Smooth muscle cells from the gastric antrum of the rabbit were isolated using collagenase and pronase. We examined the characteristics of muscarinic receptors that control contraction of the muscle cell: kinetics, stoichiometry and specificity of both contractile response to muscarinic agents and binding of labeled N-methyl-scopolamine. Cells contracted in the presence of muscarinic agents after a short time (30 sec) while binding of (3H)-NMS reached a plateau after 10 min exposure. Specific binding was saturable and Scatchard analysis revealed a single class of high-affinity binding sites (Kd: 0.5 nM). Oxotremorine was the most potent agonist with an ED50 of 0.6 pM; acetylcholine and carbachol were 10 times less potent. Muscarinic antagonists competed with (3H)-NMS for binding with IC50 values in the same range (nanomolar or less) than those obtained for inhibition of acetylcholine-induced contractions. Pirenzepine antagonized contractile effect of muscarinic agonists with EC50 in a micromolar range. Intracellular levels of cyclic AMP were lowered by muscarinic agonists. Monoclonal anti-muscarinic receptor antibodies M-35 displayed agonist-like activities triggering contraction and lowering cyclic AMP levels of the cells. However, although the antagonist inhibits M-35-induced contractions and cAMP decrease, M-35 had no effect on binding of the antagonist to the muscarinic receptor. These data revealed the presence of an M2-muscarinic receptor subtype involved in the contractile response of the isolated smooth muscle cell.