Smooth Muscle Muscarinic Receptors Research Articles

PD24-10 NICOTINIC RECEPTORS ON NERVE TERMINALS INDUCE ACETYLCHOLINE RELEASE IN CANINE BLADDER

INTRODUCTION AND OBJECTIVE: Carbachol, a mixed muscarinic and nicotinic agonist similar to acetylcholine, is often used for in-vitro bladder contraction with the implicit assumption that it causes contraction by only activating bladder smooth muscle muscarinic receptors. We sought to determine whether nicotinic receptors may also be involved in canine detrusor muscle contractions in- vitro. METHODS: Mucosa denuded canine bladder muscle strips from sham operated animals were used from a larger study of nerve transfer for bladder reinnervation. Strips were fixed between force transducers and positioners and suspended in Tyrode's solution bubbled with 95% O2/5% CO2 at 37 C. After stretching to a length of optimal force production, maximal responses to 120 mM KCl were determined then various agents were added for 20 minutes before inducing contraction with the nicotinic agonists epibatidine and nicotine. RESULTS: Epibatidine induced contractions that were approximately 40% of the maximal response to 120 mM KCl whereas nicotine only induced contractions that were 20% of KCl. The muscarinic receptor antagonist atropine (10 uM) completely blocked 10 uM epibatidine or 1 mM nicotine induced contractions but desensitization of purinergic receptors with 10 uM α,β methylene ATP only blocked these contractions by 40%. Blocking sodium channels with 1 uM tetrodotoxin (TTX) had no statistically significant inhibitory effect on epibatidine or nicotine induced contractions. The α7 nAChR-selective agonist AR-R17779 had no effect and the α7 selective antagonist MLA had no effect at α7 selective concentrations on epidbatidine induced contractions. The α4β2 selective agonist TC2559 also had no effect whereas the α3β4 selective agonist NS3861 and the non-selective ganglionic receptor agonist DMPP induced small contractions. Also, the α3β4 selective antagonist SR-16584 (1-3 uM) blocked eipibatidine induced contractions by 50% . At relatively high concentrations, the skeletal muscle neuromuscular junction nicotinic receptor antagonists atracurium besylate (5 uM) and tubocurarine (0.1 uM) blocked epibatidine induced contractions. At concentrations reported to be selective for ganglionic nicotinic receptors, the neuronal nicotinic receptor antagonists hexamethonium (100 uM) and mecamylamine (10uM) blocked epibatidine induced contractions. CONCLUSIONS: Because of atropine blockade but only minor blockade by α,β methylene ATP desensitization, the nicotinic agonists induce bladder contractions indirectly by releasing predominately acetylcholine from intramural nerve terminals. Because TTX was ineffective, these nicotinic receptors do not need to induce action potentials and thus are likely located near the neuromuscular junction. Based on the pharmacological data, the α3β4 receptor subtype is likely involved in contraction of the canine bladder. Source of Funding: NINDS R01NS070267

Migrating Motor Complex in Colectomized Ileo Stoma Patients

In colectomized patients with ileo stoma, the reflex modulation of small intestinal functions is disturbed, resulting in high enteric stoma outputs and malabsorption. Serotonin has a pivotal role in initiating motor and secretory reflexes involving activation of neuronal 5-HT(3) and smooth muscle muscarinic receptors. We aimed to evaluate the effect of 5-hydroxytryptamine (5-HT), ondansetron and atropine on fasting and stimulated antro-duodeno-jejunal migrating motor complex (MMC) in colectomized patients with ileo stoma compared with healthy subjects. Manometric recordings were obtained in a blinded, age- and gender-matched design. The effects of either standard meal or intravenous 5-HT (10 nmol/kg/min.) treatment with pre-treatment of saline (placebo) or ondansetron (250 μg/kg) or atropine (10 μg/kg) were compared. Adverse effects, blood pressure, heart rate and electrocardiographic data were also evaluated. 5-HT increased the frequency (threefold) and migration velocity (twofold) of MMC phase III in both experimental groups. Ondansetron reduced 5-HT-induced frequency of MMC phase III in patients (p < 0.05) but not in healthy subjects. Atropine reduced 5-HT-induced frequency of MMC phase III in healthy subjects (p < 0.05). Ondansetron did not alter fasting or postprandial MMC in either experimental group (p > 0.05). Atropine did not change fasting MMC in healthy subjects (p > 0.05). We conclude that 5-HT is a stimulator of MMC phase III and that ondansetron reduces the 5-HT-induced MMC phase III without affecting fasting or postprandial properties in colectomized patients with ileo stoma. Similar effects were observed for 5-HT and atropine in healthy subjects.

Tissue selective localization of oral cimetropium bromide to muscarinic receptors in colonic smooth muscle

Abstract The time-related distribution and muscarinic receptor binding of orally administered cimetropium, a quaternary ammonium antimuscarinic drug, in longitudinal smooth muscle of intestinal segments and other bodily tissues of the rat was investigated in parallel with atropine in an in-vivo binding study combined with autoradiograpic imaging. Unlike the binding of the quaternary drug in duodenum, jejunum and ileum, which decreased with time, the binding of the drug to colonic smooth muscle increased with time over a 15-h period. Binding was absent in the right atrium and minor and temporary in submandibular glands. The tissue distribution of (3H)cimetropium 24 h after oral administration mirrored the binding data. Micro-autoradiographic imaging performed 24 h after oral administration of (14C)cimetropium showed silver grains to be distributed in highest density in colonic tissues, i.e. the smooth muscles, blood vessels (mainly venulesj and the deep mucosal glands; and in hepatocytes. Negligible grain densities were seen in heart, exocrine glands and other bodily tissues. The results indicate that oral cimetropium, differently from atropine, accumulates selectively in colon–and binds specifically there to muscarinic receptors in smooth muscle–by a mechanism more reflective of transit along the enterai tract, and local uptake and elimination, than absorption in the upper gastrointestinal region.

Interaction of agonists and selective antagonists with gastric smooth muscle muscarinic receptors

The interaction of cholinergic agonists and antagonists with smooth muscle muscarinic receptors has been investigated by measurement of displacement of the muscarinic antagonist [3H]QNB (quinuclidinyl benzilate) in membranes prepared from toad stomach. The binding of [3H]QNB was saturable, reversible and of high affinity (KD = 423 pM). The muscarinic receptor subtypes present in gastric smooth muscle were classified by determining the relative affinities for the selective antagonists pirenzepine (M1), AF-DX 116 (M2) and 4-DAMP (M3). The results from these studies indicate the presence of a heterogeneous population of muscarinic receptor subtypes, with a majority (88%) exhibiting characteristics of M3 receptors and a much smaller population (12%) exhibiting characteristics of M2 receptors. The binding curve for the displacement of [3H]QNB binding by the agonist oxotremorine was complex and was consistent with presence of two affinity states: 24% of the receptors had a high affinity (KD = 4.7 nM) for oxotremorine and 76% displayed nearly a 1,000-fold lower affinity (KD = 4.4 microM). When oxotremorine displacement of [3H]QNB binding was determined in the presence GTP gamma S, high affinity binding was abolished, indicating that high affinity agonist binding may represent receptors coupled to G proteins. Moreover, pertussis toxin pretreatment of membranes also abolished high affinity agonist binding, indicating that the muscarinic receptors are coupled to pertussis toxin-sensitive G proteins. Reaction of smooth muscle membranes with pertussis toxin in the presence [32P]NAD caused the [32P]-labelling of a 40 dD protein that may represent the alpha subunit(s) of G proteins that are known to by NAD-ribosylated by the toxin.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential signalling by muscarinic receptors in smooth muscle: m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and m3-mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway.

Signalling via m3 and m2 receptors in smooth muscles involved activation of two G-protein-dependent pathways by each receptor. m2 receptors were coupled via Gbetagammai3 with activation of phospholipase C-beta3, phosphoinositide 3-kinase and Cdc42/Rac1 (where Cdc stands for cell division cycle) and p21-activated kinase 1 (PAK1), resulting in phosphorylation and inactivation of myosin light chain kinase (MLCK). Each step was inhibited by methoctramine and pertussis toxin. PAK1 activity was abolished in cells expressing both Cdc42-DN (where DN stands for dominant negative) and Rac1-DN. MLCK phosphorylation was inhibited by PAK1 antibody, and in cells expressing Cdc42-DN and Rac1-DN. m3 receptors were coupled via Galpha(q/11) with activation of phospholipase C-beta1 and via RhoA with activation of Rho-associated kinase (Rho kinase), phospholipase D and protein kinase C (PKC). Rho kinase and phospholipase D activities were inhibited by C3 exoenzyme and in cells expressing RhoA-DN. PKC activity was inhibited by bisindolylmaleimide, and in cells expressing RhoA-DN; PKC activity was also inhibited partly by Y27632 (44+/-5%). PKC-induced phosphorylation of PKC-activated 17 kDa inhibitor protein of type 1 phosphatase (CPI-17) at Thr38 was abolished by bisindolylmaleimide and inhibited partly by Y27632 (28+/-3%). Rho-kinase-induced phosphorylation of myosin phosphatase targeting subunit (MYPT1) and was abolished by Y27632. Sustained phosphorylation of 20 kDa regulatory light chain of myosin II (MLC20) and contraction were abolished by bisindolylmaleimide Y27632 and C3 exoenzyme and in cells expressing RhoA-DN. The results suggest that Rho-kinase-dependent phosphorylation of MYPT1 and PKC-dependent phosphorylation and enhancement of CPI-17 binding to the catalytic subunit of MLC phosphatase (MLCP) act co-operatively to inhibit MLCP activity, leading to sustained stimulation of MLC20 phosphorylation and contraction. Because Y27632 inhibited both Rho kinase and PKC activities, it could not be used to ascertain the contribution of MYPT1 to inhibition of MLCP activity. m2-dependent phosphorylation and inactivation of MLCK precluded its involvement in sustained MLC20 phosphorylation and contraction.

Pharmacological Analysis of the Contractile Role of M 2 and M 3 Muscarinic Receptors in Smooth Muscle

Muscarinic receptors expressed on smooth muscle cells are primarily of the M 2 and M 3 subtypes. The M 3 subtype triggers contraction through an interaction with G q proteins to stimulate phosphoinositide hydrolysis and mobilize Ca 2+ . In contrast, activation of M 2 receptors modulates contraction by preventing relaxation or by potentiating M 3 receptor-mediated contractions, which enhances heterologous desensitization. These effects can be explained by the coupling of M 2 receptors to G i proteins that mediate an inhibition of adenylyl cyclase and calcium-activated potassium channels. The pharmacological antagonism of a response mediated through an interaction between M 2 and M 3 receptors has been shown to resemble the profile of the directly acting receptor (M 3 ), primarily, and not that of the conditional receptor (M 2 ). Evidence for a contractile role of the M 2 receptor has been obtained by inactivating its signaling pathway with pertussis toxin or by measuring contractile effects of muscarinic agonists after M 3 receptors have been covalently inactivated. Under these conditions, M 2 receptors have been shown to mediate an inhibition of the relaxant effects of agents, like isoproterenol, on the contractile effects of nonmuscarinic spasmogens. Muscarinic M 2 and M 3 receptor knockout mice are useful tools for exploring interactions between these receptors in smooth muscle.

Smooth muscle cholinergic denervation hypersensitivity in diverticular disease

Evidence from clinical and laboratory investigations into the causes of diverticular disease suggests that disturbances in cholinergic activity are important, the effector mechanisms of which have yet to be established. We aimed to investigate the role of smooth muscle and neural cholinergic activity in the pathogenesis of this disease. Two investigators independently did a blinded immunohistochemical image analysis of localising antibodies to choline acetyltransferase, co-localised with protein gene product (PGP)--a marker of general neural tissue-and smooth muscle muscarinic M3 receptors, on three histological sections of sigmoid colons from ten patients with diverticular disease and ten controls, after resections for rectal tumours. We also did isotonic organ bath experiments to assess muscle strip sensitivities to exogenous acetylcholine. In circular muscle, activity of choline acetyltransferase was lower in patients with diverticular disease than in controls: median percentage surface area of choline acetyltransferase over PGP was 17.5% (range 10.0-37.0) in patients with diverticular disease and 47.0% (29.0-54.0) in controls (p<0.0001). M3 receptors were upregulated in patients with diverticular disease compared with controls: the median surface area was 13.2% (6.0-23.3) in patients with diverticular disease and 2.5% (1.6-3.7) in controls (p<0.0001). The sensitivity to exogenous acetylcholine was increased in patients with diverticular disease (mean -log EC(50) 5.6 [SD 0.3]) compared with controls (4.9 [0.5]; difference 0.7 [95% CI 0.3-1.1], p=0.006). In longitudinal muscle, choline acetyltransferase activity was lower in patients with diverticular disease (median 19.5%, range 12.0-30.0) than in controls (47.0%, 35.0-60.0; p<0.0001), with upregulation of M3 receptors in diverticular disease (diverticular disease 7.8% [1.9-20.4], controls 1.7% [0.8-3.0]; p<0.0001). However, sensitivity to exogenous acetylcholine did not differ between the two groups (diverticular disease mean 5.6% [SD 0.3], controls 5.2% [0.4]; difference 0.4% [95% CI -0.02-0.7], p=0.06). Our results suggest that cholinergic denervation hypersensitivity can affect smooth muscle. Upregulation of smooth muscle M3 receptors might account for specific clinical, physiological, and pharmacological abnormalities associated with diverticular disease.

Pharmacological Analysis of the Contractile Role of M 2 and M 3 Muscarinic Receptors in Smooth Muscle

Muscarinic receptors expressed on smooth muscle cells are primarily of the M(2) and M(3) subtypes. The M(3) subtype triggers contraction through an interaction with G(q) proteins to stimulate phosphoinositide hydrolysis and mobilize Ca(2+). In contrast, activation of M(2) receptors modulates contraction by preventing relaxation or by potentiating M(3) receptor-mediated contractions, which enhances heterologous desensitization. These effects can be explained by the coupling of M(2) receptors to G(i) proteins that mediate an inhibition of adenylyl cyclase and calcium-activated potassium channels. The pharmacological antagonism of a response mediated through an interaction between M(2) and M(3) receptors has been shown to resemble the profile of the directly acting receptor (M(3)), primarily, and not that of the conditional receptor (M(2)). Evidence for a contractile role of the M(2) receptor has been obtained by inactivating its signaling pathway with pertussis toxin or by measuring contractile effects of muscarinic agonists after M(3) receptors have been covalently inactivated. Under these conditions, M(2) receptors have been shown to mediate an inhibition of the relaxant effects of agents, like isoproterenol, on the contractile effects of nonmuscarinic spasmogens. Muscarinic M(2) and M(3) receptor knockout mice are useful tools for exploring interactions between these receptors in smooth muscle.

The initial inositol 1,4,5-trisphosphate response induced by histamine is strongly amplified by Ca2+ release from internal stores in smooth muscle

We have studied the Ca2+-dependence and wortmannin-sensitivity of the initial inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) response induced by activation of either histamine or muscarinic receptors in smooth muscle from guinea pig urinary bladder. Activation of H1 receptors with histamine (100μM) produced a significant elevation in Ins(1,4,5)P3 levels with only 5s stimulation and in the presence of external Ca2+. However, this response was abolished fully by either the prolonged absence of external Ca2+ or the depletion of internal Ca2+ stores with thapsigargin (100nM) or ryanodine (10μM). In contrast, the same conditions only slightly reduced the initial Ins(1,4,5)P3 response induced by carbachol. The prolonged incubation of smooth muscle in 10μM wortmannin to inhibit type III PI 4-kinase abolished both the early histamine-evoked Ins(1,4,5)P3 and Ca2+ responses. Conversely, wortmannin did not alter Ca2+ release induced by carbachol, despite a partial reduction of its Ins(1,4,5)P3 response. Collectively, these data indicate that the detectable histamine-induced increase in Ins(1,4,5)P3 is more the consequence of Ca2+ release from internal stores than a direct activation of phospholipase C by H1 receptors. In addition, the effect of wortmannin implies the existence of a Ca2+-dependent amplification loop for the histamine-induced Ins(1,4,5)P3 response in smooth muscle.

Influence of nitric oxide synthase inhibition and endothelin-1 receptor blockade on acetylcholine-induced coronary artery contraction in vitro in dilated and ischemic cardiomyopathies.

The normal dilatory response to acetylcholine (ACH) is reduced in coronary vessels from patients with dilated cardiomyopathy (DCM) and reversed to a contraction in patients with coronary artery disease (CAD) and ischemic cardiomyopathy (ICM). This study investigated the influence of nitric oxide synthase inhibition and endothelin (ET)-1 receptor blockade on the reactivity to ACH of coronary arteries isolated from patients with end-stage congestive heart failure (CHF) associated or not with CAD. Small (approximately 400 microm) epicardial right coronary arteries were isolated from explanted hearts of patients undergoing transplantation for DCM or ICM. Segments were mounted on a wire myograph to record changes in isometric tension. ACH (1 microM) dilated pre-contracted vessels from DCM hearts but contracted pre-contracted vessels from ICM hearts. In the absence of pre-contraction, ACH (10(-9)-3 x 10(-5) M) induced a small contraction of rings from DCM hearts and a larger contraction (p < 0.05) of rings from ICM hearts. N(omega)-nitro-L-arginine (L-NNA, 100 microM), a NO synthase inhibitor, increased (p < 0.05) sensitivity and maximal response to ACH of vessels from DCM hearts only. In the presence of L-NNA, blockade of ET(A) with BQ123 (1 microM) prevented the effects of L-NNA in DCM, whereas blockade of ET(A/B) receptors with bosentan (10 microM) only reduced vascular sensitivity to ACH without significantly reducing the maximal contraction to ACH in DCM. The antagonists had no effects in vessels from ICM hearts. ACH, however, induced similar contractions of vessels without endothelium in DCM and ICM. These results suggest that ACH induces a contraction by stimulating smooth muscle muscarinic receptors. In coronary arteries isolated from DCM hearts, the contraction is regulated by NO and ET-1, whereas these factors seem to have little influence in ICM. This suggests that endothelial muscarinic receptors are either not expressed or uncoupled in ICM hearts.

New leads for asthma therapy

The role of tryptic enzymes, such as epithelial trypsin and mast-cell tryptase, in airway function has long been an enigma in lung physiology. Stimulation of protease-activated receptors (PARs) on airway smooth muscle cells can lead to bronchoconstriction, and it has been assumed that mast-cell proteases contribute to allergy-mediated asthma symptoms. Now, Cocks et al.1xA protective role for protease-activated receptors in the airways. Cocks, T.M. et al. Nature. 1999; 398: 156–160Crossref | PubMed | Scopus (295)See all References present compelling evidence for protease-mediated bronchorelaxation via stimulated PAR2 receptors on airway epithelial cells. PAR2 is expressed on endothelial or epithelial cells, and its levels are particularly high in the gut epithelium where it protects against digestive proteolytic enzymes. It is remarkable that a similar mechanism has been adopted to protect the airways against protease-mediated bronchoconstriction. Activation of mouse airway epithelial cell PAR2 by trypsin or by the PAR2 tethered-ligand sequence peptide, SLIGRL-NH2 (which mimics the protease-activated PAR2) relaxed bronchi that had been constricted with 5-hydroxytryptamine (5HT) or with carbachol, bronchoconstrictors that act via airway smooth muscle cell serotonergic and muscarinic receptors, respectively. Relaxation was lost following epithelial cell removal, indicating that the relevant PAR2 were located there. PAR2-induced relaxation of the bronchi was blocked by the cyclooxygenase inhibitor indomethacin, suggesting that relaxation was mediated by prostaglandin E2 (PGE2), the most abundant airway epithelium prostanoid. Indeed, PGE2 is a potent bronchial relaxing agent1xA protective role for protease-activated receptors in the airways. Cocks, T.M. et al. Nature. 1999; 398: 156–160Crossref | PubMed | Scopus (295)See all References. The bronchorelaxing effects of inhaled SLIGRL-NH2 aerosol were evident in vivo: it inhibited 5HT-induced changes in airway resistance in anaesthetized rats, suggesting that similar mechanisms might regulate normal airway tone1xA protective role for protease-activated receptors in the airways. Cocks, T.M. et al. Nature. 1999; 398: 156–160Crossref | PubMed | Scopus (295)See all References.Another new study showed that the arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) relaxed rabbit bronchi that had been contracted with histamine2xAirway synthesis of 20-hydroxyeicosatetraenoic acid: metabolism by cyclooxygenase to a bronchodilator. Jacobs, E.R. et al. Am. J. Physiol. 1999; 276: L280–L288PubMedSee all References. Similar to the PAR2 study, 20-HETE-mediated relaxation was blocked by indomethacin or by epithelium removal, implicating its conversion to a bronchial relaxant, probably PGE2, by epithelial cyclooxygenase.Enjoying the salty seaside air or venturing into deep salt mines can also relieve asthma symptoms. The third study addressed this phenomenon by demonstrating that intraluminally applied hyperosmotic NaCl or KCl solutions relaxed bronchi that had been contracted by muscarinic or electric-field stimulation. Relaxation was again epithelium-dependent and sensitive to Na+ and Cl− channel blockers3xOsmotic regulation of airway reactivity by epithelium. Fedan, J.S. et al. J. Pharmacol. Exp. Ther. 1999; 289: 901–910PubMedSee all References. However, this intriguing type of airway relaxation was not sensitive to indomethacin but to inhibitors of nitric oxide synthase. Thus several distinct mechanisms operate in orchestrating airway relaxation by epithelial cells.Research into the mechanisms of PAR2, 20-HETE and NaCl-induced bronchorelaxation should foster new leads for effective asthma drug targets. For example, inhaled proteases or PAR2 tethered-ligand mimetic peptides, as well as inhaled prostanoid precursors, such as 20-HETE, could promote epithelial PAR2-mediated airway relaxation. Aerosols of hyperosmotic NaCl might similarly mediate airway relaxation, possibly in synergy with PAR2 or prostanoid-operated pathways.

Subtypes of the Muscarinic Receptor in Smooth Muscle

Subtypes of the Muscarinic Receptor in Smooth Muscle

Subtypes of the Muscarinic Receptor in Smooth Muscle

Muscarinic receptors are expressed in smooth muscle throughout the body. In most instances, the muscarinic receptor population in smooth muscle is composed of mainly the M2 and M3 subtypes in an 80% to 20% mixture. The M3 subtype mediates phosphoinositide hydrolysis and calcium mobilization, whereas the M2 subtype mediates an inhibition of cAMP accumulation. In addition, a variety of ionic conductances are elicited by muscarinic receptors. Muscarinic agonists stimulate a nonselective cation conductance that is pertussis toxin-sensitive and dependent on calcium. The pertussis toxin-sensitivity of this response suggests that it is mediated by M2 receptors. Following agonist induced depolarization of smooth muscle, voltage dependent calcium channels are activated to enable an influx of calcium. In some instances, muscarinic agonists enhance this conductance through a mechanism involving protein kinase C, whereas in other instances, muscarinic agonists suppress this calcium conductance. Smooth muscle often contains calcium activated potassium channels that tend to repolarize the membrane following calcium influx. Activation of muscarinic receptors suppresses this potassium conductance in some smooth muscles. Under standard conditions, muscarinic agonists elicit pertussis toxin-insensitive contractions through activation of the M3 receptor. When most of the M3 receptors are inactivated, it is possible to measure a pertussis toxin-sensitive contractile response to muscarinic agonists that is most likely mediated through M2 receptors. M2 receptors also cause an indirect contraction by inhibiting the relaxant effects of agents that increase cAMP (e.g., forskolin and isoproterenol).

The use of irreversible ligands to inactivate receptor subtypes: 4-DAMP mustard and muscarinic receptors in smooth muscle

Irreversible ligands are useful tools for investigating the function of receptor subtypes in various physiological processes. The mechanism for alkylation involves the formation of a reversible receptor complex followed by a covalent reaction. The extent of receptor alkylation is determined by the dissociation constant of the reversible complex and the rate constant for conversion to the covalent complex. Selectivity can be achieved if the irreversible ligand exhibits a difference in its dissociation constants for receptor subtypes. Selective alkylation can also be achieved using a selective competitive inhibitor to protect the desired receptor subtype. By using the non-M 2-selective irreversible antagonist, 4-DAMP mustard, in combination with the competitive M 2-selective antagonist, AF-DX 116, it has been possible to achieve a highly selective inactivation of all non-M 2 subtypes of the muscarinic receptors in smooth muscle and has enabled the discovery of the functional role of M 2 receptors in smooth muscle.

Inflammatory modulation of muscarinic receptor activation in canine ileal circular muscle cells

BACKGROUND & AIMS: Spontaneous in vivo phasic contractions are suppressed during ileal inflammation. The aim of this study was to test the hypothesis that modulation of the contractile response to muscarinic receptor activation contributes to this inhibition.METHODS: Muscarine, methacholine, and arecaidine propargyl ester were infused close-intra-arterially in the normal and inflamed ileum of conscious dogs. Concurrent studies were performed on in vitro muscle strips.RESULTS: Prior close-intra-arterial infusions of tetrodotoxin or hexamethonium had no significant effect on the contractile response to any agonist, indicating that they acted primarily on smooth muscle muscarinic receptors. The contractile response to each agonist was suppressed significantly during inflammation. The median inhibitory dose of 4-diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP) was 57-244-fold smaller than that of pirenzepine, methoctramine, and tropicamide to inhibit the contractile response to muscarine. Inflammation induced a significant leftward shift in the inhibitory dose-response curve to methoctramine but not to 4-DAMP, pirenzepine, or tropicamide. The data from in vitro muscle strips were similar to those in conscious dogs.CONCLUSIONS: Inflammation suppresses the phasic contractile response to muscarinic receptor activation in circular smooth muscle cells. The spontaneous and muscarinic agonist-stimulated in vivo phasic contractions of ileal circular smooth muscle cells are mediated primarily by M3 receptors.(Gastroenterology 1997 Mar;112(3):864-74)

The supine venous pump function test: normal values and results in patients with the post-thrombotic syndrome: J.A.H.R. Claassen 1, M.C.H. Janssen 1, W.N.J.C. van Asten 3, H. Wollersheim 1, M.J.M. de Rooij 2, Th. Thien 1. Department of 1 Medicine, 2 Dermatology and 3 Clinical Vascular Laboratory, University Hospital, Nijmegen, Netherlands

The muscarinic receptor mediating vasolidation of resistance vessels in the rat isolated, constant-pressure perfused kidney (preconstriction by 10−7 M cirazoline) was characterized by subtype-preferring agonists and selective antagonists. The agonists produced vasolidation with the following rank order of potency: arecaidine propargyl ester (APE) > 5-methylfurtrethonium = methacholine = oxotremorine > (R)-aceclidine > arecaidine 2-butyne-1,4-diyl bisester > 4-Cl-McN-A-343 = (R)-nipecotic acid ethyl ester = N-ethyl-guvacine propargyl ester (R)-aceclidine = (S)-nipocotic acid ethyl ester > McN-A-343. Agonist-induced vasodilation disappeared after destruction of the endothelium with detergent. Highly significant correlations of agonist potencies for vasodilation were found between rat kidney and guinea-pig ileum submucosal arterioles as well as agonist potencies at smooth muscle muscarinic M3 receptors of the guinea-pig ileum. The rank order of antagonist potencies (4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) > (R)-hexahydro-difenidol ∼ hexahydro-sila-difenol > pirenzepine ∼ p-fluorohexahydro-sila difenol ∼ himbacine ∼ AF-DX 384 ∼ AQ-RA 741 > (S)-hexahydro-difenidol) to attenuate vasodilation to APE in rat kidney, correlated significantly with affinities at M3 receptors in submucosal arterioles and in smooth muscle of the guinea-pig ileum, but differed from those at M1 and M2 receptors in rabbit vas deferens. The agonist and antagonist potencies suggest that vasodilation elicited by muscarinic stimuli in endothelium-intact rat renal vasculature is mediated by functional muscarinic M3 receptors.

MS-5-5 - Desensitization of muscarinic receptors in smooth muscle.

MS-5-5 - Desensitization of muscarinic receptors in smooth muscle.

Characterization of the airway smooth muscle muscarinic receptor in vivo

We examined the bronchodilator activity of eight subtype-selective and non-selective muscarinic antagonists in anesthetized, ventilated guinea pigs bronchoconstricted by carbachol aerosols. Relative bronchodilator potencies were consistent with M 3 receptor antagonism and correlated with inhibition of bladder smooth muscle contraction in vivo. We conclude that the airway smooth muscle muscarinic receptor can be functionally characterized in vivo as M 3 and that it is of the same subtype as the muscarinic receptor in bladder smooth muscle.

Hexocyclium derivatives with a high selectivity for smooth muscle muscarinic receptors

1. The affinity of a number of derivatives of the muscarinic antagonist, hexocyclium, containing an amidine cationic head, for guinea-pig cardiac and ileal receptors was investigated. 2. All the compounds studied displayed a greater affinity for muscular than for cardiac muscarinic receptors. 3. The 5 fold ileal selectivity of hexocyclium was increased by a number of chemical substitutions. The largest discrimination between receptors (about 200 fold) was found for the formamidine derivative. 4. The selectivity displayed by the hexocyclium derivatives stemmed from a greater decrease in affinity towards cardiac as compared to ileal receptors.

Differential effects of pancuronium bromide on cardiopulmonary function in the neonatal lamb

The effect of pancuronium bromide (Panc Br) on resting cardiopulmonary function and cardiopulmonary responses to intravenous injection of acetylcholine (Ach) and histamine (H) was evaluated in neonatal lambs. The animals were mechanically ventilated and managed to maintain physiologic gas exchange and acid-base conditions. A proximal segment of the cervical trachea was bypassed; the developed pressure response of this segment (P cervical trach) was used as a direct indication of airway smooth muscle contraction and bronchoconstriction. Pulmonary resistance (Rp) and functional residual capacity were determined. The change in Rp from resting values was used as a functional indicator of central and peripheral airway bronchoconstriction. Cardiovascular function and responses were evaluated from changes in mean arterial pressure and heart rate. Following Panc Br, there was a significant reduction in Ach-induced P cervical trach (-50 +/- 9.2% SE) and Rp (-46 +/- 2.4% SE). In contrast, Panc Br did not significantly change Ach-induced bradycardia and hypotension, cardiopulmonary responses to H, and resting cardiopulmonary function. The differential effects of Panc Br on cardiopulmonary function appear to be related to regional differences between cardiovascular and airway smooth muscle muscarinic receptors in the neonate. The results of this study elucidate a mechanism which may explain previously reported variability in the effect of Panc Br on neonatal cardiopulmonary function. Furthermore, the Panc Br-related attenuation of airway smooth muscle responses suggests that this form of neuromuscular blockade affects the regulation of airway tone and may influence the susceptibility of the neonate to airway deformation consequent to mechanical ventilation.