Pre-clinical and clinical pharmacology of selective muscarinic M3 receptor antagonists

Muscarinic M3 receptor antagonists have therapeutic potential for the treatment of disorders associated with altered smooth muscle contractility or tone. These include irritable bowel syndrome (IBS), chronic obstructive airways disease (COAD) and urinary incontinence. Zamifenacin is a potent muscarinic receptor antagonist on the guinea pig ileum (pA2 value 9.27) with selectivity over M2 receptors in the atria (135-fold) and M1/M4 receptors in the rabbit vas deferens (78-fold). In addition, zamifenacin had lower affinity for the M3 receptor in the salivary gland (pKi 7.97). In animals, zamifenacin potently inhibited gut motility in the absence of cardiovascular effects and with selectivity over inhibition of salivary secretion. In healthy volunteers, zamifenacin inhibited small and large bowel motility and increased the rate of gastric emptying over a dose range which was associated with minimal anticholinergic side effects. These data show that zamifenacin, a selective muscarinic M3 receptor antagonist, was well tolerated in man and was efficacious as an inhibitor of gut motility. Further studies in patients are required with muscarinic M3 receptor antagonists to confirm efficacy against symptoms in diseases associated with altered smooth muscle contractility.     

Comparison of in vitro selectivity profiles of solifenacin succinate (YM905) and current antimuscarinic drugs in bladder and salivary glands: a Ca2+ mobilization study in monkey cells

We investigated the effects of the new muscarinic receptor antagonist solifenacin succinate [YM905; (+)-(1S,3'R)-quinuclidin-3'-yl 1-phenyl-1,2,3,4-tetrahydroisoquinoline-2-carboxylate monosuccinate] and the current antimuscarinic drugs for the treatment of overactive bladder (oxybutynin, tolterodine and darifenacin) on intracellular Ca(2+) mobilization in response to M(3) muscarinic receptor activation in bladder smooth muscle and submandibular gland cells isolated from Cynomolgus monkeys. Solifenacin concentration-dependently inhibited carbachol-induced Ca(2+) mobilization, with affinity constant values (pKi) of 8.5 +/- 0.053 in bladder smooth muscle cells and 8.2 +/- 0.051 in submandibular gland cells (n = 5). The pKi value of solifenacin was almost equivalent to the values of oxybutynin, tolterodine and darifenacin in bladder smooth muscle cells (8.7, 8.5 and 8.4, respectively), while being lower than those in submandibular gland cells (9.0, 8.7 and 8.8, respectively). The bladder-selectivity index (Ki ratio: submandibular gland/bladder) for solifenacin (2.1) was statistically higher, moreover, than those for oxybutynin, tolterodine and darifenacin (0.51, 0.65 and 0.46, respectively). These findings consequently indicate solifenacin's unique profile in terms of its selectivity for bladder smooth muscle cells over salivary gland cells in non-human primates, relative to oxybutynin, tolterodine and darifenacin. Solifenacin may, therefore, confer a promising therapeutic advantage for reducing adverse effects, such as dry mouth, exhibited by current antimuscarinic therapy for overactive bladder.     

In vivo demonstration of M3 muscarinic receptor subtype selectivity of darifenacin in mice

A novel muscarinic receptor antagonist, darifenacin, inhibited specific binding of [N-methyl-(3)H]scopolamine ([(3)H]NMS) in the mouse bladder, submaxillary gland and heart in a concentration-dependent manner. The inhibitory effect was most potent in the submaxillary gland, followed by the bladder and heart. In addition, darifenacin inhibited specific [(3)H]NMS binding in the membranes of CHO-K1 cell lines expressing muscarinic M(2) and M(3) receptor subtypes, and the potency was significantly (22-fold) greater at the M(3) than at the M(2) subtype. At 0.5 to 12 h after oral administration of darifenacin, a significant increase in K(d) values for specific [(3)H]NMS binding was seen in the bladder, submaxillary gland and lung of mice, compared with control values. Also, there was a sustained decrease in the B(max) values in the submaxillary gland. These data suggest that muscarinic receptor binding of oral darifenacin is rapid in onset and of a long duration. On the other hand, oral darifenacin exerted only temporary or little binding of muscarinic receptors in the heart and colon. Pilocarpine-induced salivary secretion in mice was continuously suppressed by oral darifenacin. The time-course of suppression coincided well with that for the muscarinic receptor binding in the submaxillary gland. The antagonistic effect of darifenacin against the dose-response curves for pilocarpine appeared to be insurmountable. In conclusion, the present study has shown that oral darifenacin may exert a pronounced and long-lasting binding of muscarinic receptors in tissues expressing the M(3) subtype.     

Muscarinic receptors and gastrointestinal tract smooth muscle function

Over the last decade, several lines of evidence have shown that both muscarinic M2 and M3 receptors are postjunctionally expressed in many smooth muscles, including the gastrointestinal tract. Although in vitro data suggests that both receptors are functional in that they inhibit adenylate cyclase activity and activate non-selective cation channels, few studies support a role in vivo. Thus, data from procedures that ablate the signaling pathway of the muscarinic M2 receptor, including receptor antagonism, pertussis toxin pretreatment reveal little effect on gastrointestinal smooth muscle responsiveness to muscarinic agonists. Recently, information from knockout mice, lacking either M2 or M3 receptor, indicate reveal a role for both subtypes. However, the contribution of the M2 receptor appears greater in the ileum than in the urinary bladder. Therapeutically, non-selective, as well as selective M3 receptor antagonists are being clinically studied, although it remains to be shown which is the optimal approach to disorders of smooth muscle motility.     

Cardioselective profile of AF-DX 116, a muscarine M2 receptor antagonist

AF-DX 116 (see chemical name below) is a competitive antagonist of muscarine receptors in peripheral organs. In contrast to pirenzepine, its behaviour in functional experiments indicates selectivity for the M2 muscarinic subtype. In pithed rats AF-DX 116 inhibits vagally-induced bradycardia, an M2 response, (ED50 32 micrograms/kg i.v.) in preference to the M1-mediated pressor response to McN-A-343 (ED50 211 micrograms/kg i.v.). AF-DX 116 further discriminates among M2 receptors, showing a high affinity for the cardiac muscarine receptors. In isolated preparations, AF-DX 116 has a tenfold higher affinity for the muscarine receptors of the heart (pA2 7.33) than for those in smooth muscles (pA2 6.39-6.44). The same profile appears from animal studies, where the compound is a more potent antagonist of either endogenously or exogenously activated cardiac muscarine responses as compared to vascular, smooth muscle or secretory responses. In general, the ratios of potencies (ED50) observed in cardiac vs. other muscarine mediated functions ranged between 30 and 50. Atropine showed no discrimination, inhibiting all muscarine responses in the same range of doses. In the conscious dog intravenous AF-DX 116 increased basal heart rate, and completely reversed the reflex bradycardia induced by clonidine. Tachycardia was dose-related (ED50 79 micrograms/kg i.v.), and occurred independently of background sympathetic tone. AF-DX 116 clearly distinguishes between M1- and M2-mediated responses; it also emphasizes the long-recognized heterogeneity among the peripheral M2 subtypes. AF-DX 116, for its pronounced cardioselectivity, may have a therapeutic potential in the treatment of sinus bradycardia.     

The M2 muscarinic receptor mediates in vitro bladder contractions from patients with neurogenic bladder dysfunction

Bladder muscle specimens from seven patients with neurogenic bladder dysfunction were analyzed to determine whether the muscarinic receptor subtype mediating contraction shifts from M(3) to the M(2) subtype as found in the denervated, hypertrophied rat bladder. Seven bladder specimens were analyzed from six female and one male patients. Six of the patients had traumatic cervical spinal cord injuries (C(4)-C(7)), and the other patient had an L(1) congenital myelomeningocele. This was compared with results from bladder specimens obtained from eight organ transplant donors. The affinities of three subtype-selective muscarinic receptor antagonists for inhibition of carbachol-induced contractions were determined. The affinity of the M(3) selective antagonists darifenacin or p-fluoro-hexahydrosiladifenadol (p-F-HHSiD) was determined in six of the seven spinal injury patient specimens. The affinity was consistent with M(2)-mediated contractions in four of these six specimens, intermediate between M(2) and M(3) in one specimen, and within the M(3) range in one specimen. The other specimen, tested only with the M(2) selective antagonist methoctramine, showed an M(3) affinity. In the organ donors, the affinity of p-F-HHSiD was within the M(2) range for six of seven specimens, whereas the affinity of darifenacin was within the M(3) range for five of six and intermediate between M(2) and M(3) for the other specimen tested. The affinity of methoctramine in both organ donor specimens tested was within the M(3) range. Whereas normal detrusor contractions are mediated by the M(3) receptor subtype, in patients with neurogenic bladder dysfunction as well as certain organ transplant donors, contractions can be mediated by the M(2) muscarinic receptor subtype.     

Reserpine-induced post-receptor reduction in muscarinic-mediated airway smooth muscle contraction

Radioligand binding was conducted on airways of the rat and human, surgically subdivided into trachea, lung airways, and parenchyma. 3H-QNB bound uniformly to receptors in separate sections of the rat and human airway. Receptor densities generally were ranked: lung airways greater than trachea greater than parenchyma. Receptor subtypes were identified mostly by pirenzepine displacement of bound 3H-QNB. The rat trachea, and rat and human lung airways had a uniformly low affinity for pirenzepine while rat and human parenchyma demonstrated both high and low affinity pirenzepine binding. Inhibition of methacholine-stimulated smooth muscle contraction by the M1 receptor antagonist, pirenzepine, and M2 receptor antagonist, gallamine, was studied in rat trachea and bronchus in vitro. Schild plot pA2 values were compatible with low potency antagonism, thereby favoring the presence of M3 receptors at these smooth muscle sites. Reserpine treatment of rats (0.5 mg kg-1 day-1 for 7 days) produced a decrease in peak tension in response to methacholine without changing the muscarinic receptor character (Kd 3H-QNB), population density (Bmax in fmol mg-1 protein), or function (methacholine EC50). These results indicate that muscarinic receptor heterogeneity exists in the airway of both laboratory rat and man. While the muscarinic receptor subserving airway smooth muscle contraction appears to be the M3 subtype, decreased contractile responses to methacholine by trachea and bronchus from reserpine-treated rats were receptor independent.     

Neuronal soma-dendritic and prejunctional M1-M4 receptors in gastrointestinal and genitourinary smooth muscle

A variety of neurons in gastrointestinal and genitourinary smooth muscle express muscarinic auto- as well as heteroreceptors. These receptors are found on the soma and dendrites of many cholinergic, sympathetic and NANC neurons and on axon terminals. A given neuron may contain both excitatory and inhibitory presynaptic muscarinic receptors. The subtypes involved are species- and tissue-dependent, and neuronal M1 to M4 receptors have been shown to be expressed in smooth muscle tissues. In this study, the ability of several selective muscarinic receptor antagonists to inhibit the effect of arecaidine propargyl ester (APE) on prejunctional muscarinic receptors on sympathetic nerve endings in the rabbit anococcygeus muscle (RAM) was investigated to characterise the receptor subtype involved. Electrical field stimulation (EFS) resulted in a release of noradrenaline (NA) eliciting monophasic contractions due to stimulation of postjunctional alpha1-adrenoceptors. The selective muscarinic agonist APE did not reduce contractions to exogenous NA, but caused a concentration-related and N-methylatropine-sensitive inhibition of neurogenic responses. All muscarinic antagonists investigated failed to affect the EFS-induced contractions, but shifted the concentration-response curve of APE to the right in a parallel and surmountable fashion. Schild analysis yielded regression lines of unit slope, indicating competitive antagonism. The following rank order of antagonist potencies (pA2 values) was found: tripitramine (9.10) > AQ-RA 741 (8.26) > or = himbacine (8.04) > or = (S)-dimethindene (7.69) > pirenzepine (6.46) > or = p-F-HHSiD (6.27). A comparison of the pA2 values determined in the present study with literature binding and functional affinities obtained at native or recombinant M1 to M5 receptors strongly suggests that NA release from sympathetic nerve endings in RAM is inhibited by activation of prejunctional muscarinic M2 receptors.     

Biochemical studies on muscarinic receptors

Muscarinic receptors have been characterized in smooth muscle and brain by the binding of reversible (e.g. atropine, quinuclidinylbenzylate) or irreversible (benzilylcholine or propylbenzilylcholine mustards) ligands. There is a close correlation between affinity constants derived from binding experiments and the affinities of muscarinic ligands for these sites obtained in pharmacological experiments on smooth muscle. Whereas atropine shows a single high affinity binding component (in subcellular preparations) several other ligands (QNB, ACh, oxotremorine) show multiple affinity binding. This indicated the existence of several types of binding sides which show selectivity toward certain cholinergic effectors. Most detergents inhibit the binding of ligands to the receptor site and therefore cannot be used to solubilize the receptor protein from the membrane. Treatment of brain subcellular membrane preparations with high salt concentrations (2M NaI) solubilize proteins which possess the muscarinic ligand binding properties observed in the membrane preparation. The affinities for muscarinic antagonists however are decreased, which suggests that a conformational change occurs in the protein upon solubilization.     

Role of M2 muscarinic receptors in airway smooth muscle contraction

Airway smooth muscle expresses both M2 and M3 muscarinic receptors with the majority of the receptors of the M2 subtype. Activation of M3 receptors, which couple to Gq, initiates contraction of airway smooth muscle while activation of M2 receptors, which couple to Gi, inhibits beta-adrenergic mediated relaxation. Increased sensitivity to intracellular Ca2+ is an important mechanism for agonist-induced contraction of airway smooth muscle but the signal transduction pathways involved are uncertain. We studied Ca2+ sensitization by acetylcholine (ACh) and endothelin-1 (ET-1) in porcine tracheal smooth muscle by measuring contractions at constant [Ca2+] in strips permeabilized with Staphylococcal alpha-toxin. Both ACh and ET-1 contracted airway smooth muscle at constant [Ca2+]. Pretreatment with pertussis toxin for 18-20 hours reduced ACh contractions, but had no effect on those of ET-1 or GTPgammaS. We conclude that the M2 muscarinic receptor contributes to airway smooth muscle contraction at constant [Ca2+] via the heterotrimeric G-protein Gi.     

Muscarinic receptors on nerves and muscles in opossum esophagus muscularis mucosa

The muscarinic receptors of muscularis mucosa have some recognition properties that suggest they resemble receptors of the M1 subtype. The nerves of these tissues also contain muscarinic receptors which inhibit tonic contractions caused by release of a substance-P-like material by field stimulation. These receptors also appear to be M1 in type as they are maximally activated by McNeil A343 as well as by carbachol (pD2, 5.5 and 7.5, respectively). They are also inhibited by pirenzepine, as well as by atropine (negative logarithms of the required dose for 50% inhibition or potentiation, 6.6-6.7 compared with 8.2-8.3). Hexahydrosiladifenidol, an antagonist selective or M2 receptors of guinea pig ileum, had a low (approximately 7.1) pA2 value for antagonism of both agonists in smooth muscle in this tissue. However, it was closer to atropine in potency with respect to potentiating tonic responses to field stimulation or to inhibiting phasic responses to field stimulation than it was to antagonizing smooth muscle contractions. Thus, atropine was about 40 times more potent than pirenzepine and 2-5 times more potent than hexahydrosilafenidol. There were some quantitative differences in the effectiveness of these three antagonists in blocking the phasic (acetylcholine-mediated) response to field stimulation. Atropine was 70-100 times more potent than pirenzepine and 8-25 times more potent than hexahydrosiladifenidol. This greater potency difference for inhibition of phasic contractions compared with potentiation of tonic contractions was discussed. This tissue appears to be one of the first smooth muscles in which both nerves and muscles contain muscarinic receptors with some recognition properties resembling those of the M1 subtype.     

Contractile role of M2 and M3 muscarinic receptors in gastrointestinal, airway and urinary bladder smooth muscle

Both M(2) and M(3) muscarinic receptors are expressed in smooth muscle and influence contraction through distinct signaling pathways. M(3) receptors interact with G(q) to trigger phosphoinositide hydrolysis, Ca(2+) mobilization and a direct contractile response. In contrast, M(2) receptors interact with G(i) and G(o) to inhibit adenylyl cyclase and Ca(2+)-activated K(+) channels and to potentiate a Ca(2+)-dependent, nonselective cation conductance. Ultimately, these mechanisms lead to the prediction that the influence of the M(2) receptor on contraction should be conditional upon mobilization of Ca(2+) by another receptor such as the M(3). Mathematical modeling studies of these mechanisms show that the competitive antagonism of a muscarinic response mediated through activation of both M(2) and M(3) receptors should resemble the profile of the directly acting receptor (i.e., the M(3)) and not that of the conditionally acting receptor (i.e., the M(2)). Using a combination of pharmacological and genetic approaches, we have identified two mechanisms for the M(2) receptor in contraction: 1) a high potency inhibition of the relaxation elicited by agents that increase cytosolic cAMP and 2) a low potency potentiation of contractions elicited by the M(3) receptor. The latter mechanism may be involved in muscarinic agonist-mediated heterologous desensitization of smooth muscle, which requires activation of both M(2) and M(3) receptors.     

Characterization of muscarinic receptor subtypes in human tissues

The affinities of selective, pirenzepine and AF-DX 116, and classical, N-methylscopolamine and atropine, muscarinic cholinergic receptor antagonists were investigated in displacement binding experiments with [3H]Pirenzepine and [3H]N-methylscopolamine in membranes from human autoptic tissues (forebrain, cerebellum, atria, ventricle and submaxillary salivary glands). Affinity estimates of N-methylscopolamine and atropine indicated a non-selective profile. Pirenzepine showed differentiation between the M1 neuronal receptor of the forebrain and the receptors in other tissues while AF-DX 116 clearly discriminated between muscarinic receptors of heart and glands. The results in human tissues confirm the previously described selectivity profiles of pirenzepine and AF-DX 116 in rat tissues. These findings thus reveal the presence also in man of three distinct muscarinic receptor subtypes: the neuronal M1, the cardiac M2 and the glandular M3.     

The changing role of eosinophils in long-term hyperreactivity following a single ozone exposure

Ozone hyperreactivity over 24 h is mediated by blockade of inhibitory M(2) muscarinic autoreceptors by eosinophil major basic protein. Because eosinophil populations in the lungs fluctuate following ozone, the contribution of eosinophils to M(2) dysfunction and airway hyperreactivity was measured over several days. After one exposure to ozone, M(2) function, vagal reactivity, smooth muscle responsiveness, and inflammation were measured in anesthetized guinea pigs. Ozone-induced hyperreactivity to vagal stimulation persisted over 3 days. Although hyperreactivity one day after ozone is mediated by eosinophils, AbVLA-4 did not inhibit either eosinophil accumulation in the lungs or around the nerves or prevent hyperreactivity at this time point. Two days after ozone, eosinophils in BAL, around airway nerves and in lungs, were decreased, and neuronal M(2) receptor function was normal, although animals were still hyperreactive to vagal stimulation. Depleting eosinophils with AbIL-5 prevented hyperreactivity, thus eosinophils contribute to vagal hyperreactivity by mechanisms separate from M(2) receptor blockade. Three days after ozone, vagal hyperreactivity persisted, eosinophils were again elevated in BAL in lungs and around nerves, and M(2) receptors were again dysfunctional. At this point, airway smooth muscle was also hyperresponsive to methacholine. Eosinophil depletion with AbIL-5, AbVLA-4, or cyclophosphamide protected M(2) function 3 days after ozone and prevented smooth muscle hyperreactivity. However, vagal hyperreactivity was significantly potentiated by eosinophil depletion. The site of hyperreactivity, muscle or nerve, changes over 3 days after a single exposure to ozone. Additionally, the role of eosinophils is complex; they mediate hyperreactivity acutely while chronically may be involved in repair.     

Tiotropium bromide (Ba 679 BR), a novel long-acting muscarinic antagonist for the treatment of obstructive airways disease

Tiotropium bromide (Ba 679 BR) is a novel potent and long-lasting muscarinic antagonist that has been developed for the treatment of chronic obstructive airways disease (COPD). Binding studies with [3H]tiotropium bromide in human lung have confirmed that this is a potent muscarinic antagonist with equal affinity for M1-, M2- and M3-receptors and is approximately 10-fold more potent than ipratropium bromide. Tiotropium bromide dissociates very slowly from lung muscarinic receptors compared with ipratropium bromide. In vitro tiotropium bromide has a potent inhibitory effect against cholinergic nerve-induced contraction of guinea-pig and human airways, that has a slower onset than atropine or ipratropium bromide. After washout, however, tiotropium bromide dissociates extremely slowly compared with the dissociation of atropine and ipratropium bromide. Measurement of acetylcholine (ACh) release from guinea-pig trachea shows that tiotropium bromide, ipratropium bromide and atropine all increase ACh release on neural stimulation and that this effect is washed out equally quickly for the three antagonists. This confirms binding studies to transfected human muscarinic receptors which suggested that tiotropium bromide dissociates slowly from M3-receptors (on airway smooth muscle) but rapidly from M2 autoreceptors (on cholinergic nerve terminals). Clinical studies with inhaled tiotropium bromide confirm that it is a potent and long-lasting bronchodilator in COPD and asthma. Furthermore, it protects against cholinergic bronchoconstriction for > 24 h. This suggests that tiotropium bromide will be a useful bronchodilator, particularly in patients with COPD, and may be suitable for daily dosing. The selectivity for M3- over M2-receptors may also confer a clinical advantage.     

Stereoselectivity at muscarinic receptor subtypes: observations with the enantiomers of phenglutarimide

The affinity of the enantiomers of phenglutarimide at three muscarinic receptor subtypes was examined in vitro using field-stimulated rabbit vas deferens (M1 receptors) and guinea pig atria (M2 alpha receptors) and ileum (M2 beta receptors). Extremely high stereoselectivity was observed and higher affinities (up to 6000-fold) were found for the (+)-S-enantiomer. The stereoselectivity ratios were different at the three subtypes, and the stereochemical demands made by the muscarinic receptors were most stringent at M1 receptors. (+)-(S)-Phenglutarimide was found to be a potent M1-selective antagonist (pA2 at M1 = 8.53). Its receptor selectivity profile is qualitatively similar to that of pirenzepine. (-)-(R)-Phenglutarimide showed no comparable discriminatory properties.     

Muscarinic receptor subtypes modulating smooth muscle contractility in the urinary bladder

Normal physiological voiding as well as generation of abnormal bladder contractions in diseased states is critically dependent on acetylcholine-induced stimulation of contractile muscarinic receptors on the smooth muscle (detrusor) of the urinary bladder. Muscarinic receptor antagonists are efficacious in treating the symptoms of bladder hyperactivity, such as urge incontinence, although the usefulness of available drugs is limited by undesirable side-effects. Detrusor smooth muscle is endowed principally with M2 and M3 muscarinic receptors with the former predominating in number. M3 muscarinic receptors, coupled to stimulation of phosphoinositide turnover, mediate the direct contractile effects of acetylcholine in the detrusor. Emerging evidence suggests that M2 muscarinic receptors, via inhibition of adenylyl cyclase, cause smooth muscle contraction indirectly by inhibiting sympathetically (beta-adrenoceptor)-mediated relaxation. In certain diseased states, M2 receptors may also contribute to direct smooth muscle contraction. Other contractile mechanisms involving M2 muscarinic receptors, such as activation of a non-specific cationic channel and inactivation of potassium channels, may also be operative in the bladder and requires further investigation. From a therapeutic standpoint, combined blockade of M2 and M3 muscarinic receptors would seem to be ideal since this approach would evoke complete inhibition of cholinergically-evoked smooth muscle contractions. However, if either the M2 or M3 receptor assumes a greater pathophysiological role in disease states, then selective antagonism of only one of the two receptors may be the more rational approach. The ultimate therapeutic strategy is also influenced by the extent to which pre-junctional M1 facilitatory and M2 inhibitory muscarinic receptors regulate acetylcholine release and also which subtypes mediate the undesirable effects of muscarinic receptor blockade such as dry mouth. Finally, the consequence of muscarinic receptor blockade in the central nervous system on the micturition reflex, an issue which is poorly studied and seldom taken into consideration, should not be ignored.     

The design of novel methoctramine-related tetraamines as muscarinic receptor subtype selective antagonists

Several novel methoctramine-related tetraamines were designed, and their biological profiles at muscarinic receptor subtypes were assessed by functional experiments in isolated guinea pig and rat atria (M2) and smooth muscle (ileum and trachea, M3) and by binding assays in rat cortex (M1), heart (M2), and submaxillary gland (M3) homogenates and NG 108-15 cells (M4). Tripitramine, a nonsymmetrical tetraamine, resulted in the most potent and the most selective muscarinic M2 receptor antagonist of the series (pA2 = 9.14-9.85; pKi = 9.54). Spirotramine (FC 15-94), a symmetrical tetraamine, was able to differentiate between muscarinic M1 receptors (pKi = 7.88) and the other subtypes (M2, pKi = 6.20; M3, pKi = 5.81; M4, pKi = 6.27). Thus, tripitramine and spirotramine could be valuable tools for the pharmacological classification and characterization of muscarinic receptor subtypes.     

G protein-mediated dysfunction of excitation-contraction coupling in ileal inflammation

Inflammation impairs the circular muscle contractile response to muscarinic (M) receptor activation. The aim of this study was to investigate whether the expression of muscarinic receptors, their binding affinity, and the expression and activation of receptor-coupled G proteins contribute to the suppression of contractility in inflammation. The studies were performed on freshly dissociated single smooth muscle cells from normal and inflamed canine ileum. Northern blotting indicated the presence of only M(2) and M(3) receptors on canine ileal circular muscle cells. Inflammation did not alter the mRNA or protein expression of M(2) and M(3) receptors. The maximal binding and K(d) values also did not differ between normal and inflamed cells. However, the contractile response to ACh in M(3) receptor-protected cells was suppressed, whereas that in M(2) receptor-protected cells was enhanced. Further experiments indicated that the expression and binding activity of G alpha(q/11) protein, which couples to M(3) receptors, were downregulated, whereas those of G alpha(i3), which couples to M(2) receptors, were upregulated in inflamed cells. We concluded that inflammation depresses M(3) receptor function, but it enhances M(2) receptor function in ileum. These effects are mediated by the differentially altered expression and binding activity of their respective coupled G alpha(q/11) and G alpha(i3) proteins.     

1,5-Benzodioxepin derivatives as a novel class of muscarinic M3 receptor antagonists

The structure-activity relationships of novel 1,5-benzodioxepin derivatives as muscarinic M(1)-M(3) receptor antagonists are reported. Some of these compounds were found to possess high binding affinity for the muscarinic M(3) receptor and potent effect on rhythmic increase in bladder pressure in unanesthetized rats following oral administration. These compounds displayed selectivity for the bladder over the salivary gland.