Meeting Report for: The 7th International Symposium on Subtypes of Muscarinic Receptors

The muscarinic acetylcholine receptors are monomeric proteins with seven hydrophobic, membrane spanning helices, and share a common evolutionary origin with the other members of the superfamily of membrane proteins known as seven-helix receptors. The amino acid sequences of five different muscarinic acetylcholine receptors, called m1, m2, m3, m4 and m5 have been determined. The five subtypes are expressed to different extent in different tissues. A large number of low molecular ligands for muscarinic receptors are known, but they bind to all five subtypes of receptors and only a few of them have a slightly higher (five-six fold) affinity for one of the subtypes, e.g. pirenzepine for M1 (1) and tripitramine for M2 receptors (2). Several neurotoxins have been isolated from snake venoms and used as pharmacological tools. Mambas, African snakes of genus Dendroaspis, have toxins that recognize muscrinic receptors and some of these muscarinic toxins are the most selective ligands for M1 and M4 receptors known to date.

The treatment of airway obstructive disease may be improved by antimuscarinic agents which selectively block M1 and M3 receptors but do not inhibit prejunctional cholinergic autoreceptors which limit release of acetylcholine. Revatropate is a novel antimuscarinic agent which shows some 50-fold selectivity for M1 and M3 receptors in guinea pig trachea and rabbit vas deferens over the M2 subtype in atria. This selectivity profile was seen in vivo in anaesthetised guinea pigs and conscious dogs where bronchodilator activity was produced in the absence of any effect on heart rate. Revatropate, in contrast to the non-selective agent ipratropium, did not potentiate bronchoconstrictor responses induced by vagal nerve stimulation, indicating that inhibitory autoreceptors were still functional. Early clinical studies in COAD patients showed that inhaled revatropate was an effective bronchodilator which was well tolerated. Darifenacin differs from revatropate by showing selectivity for M3 receptors relative to both M2 and M1 subtypes. [3H] darifenacin had 5-fold higher affinity for the human m3 relative to m1 receptors while there was significantly reduced binding to m2, m4 and m5 receptors. The degree of selectivity in functional tissue preparations was even greater, with darifenacin showing 100-fold selectivity for the ileum M3 receptors over M2 receptors in atria and 30-fold over M1 receptors in rabbit vas deferens. Darifenacin was able to differentiate between M3 receptors in different tissues; although darifenacin was equipotent with atropine in the ileum and bladder, it was some 10-fold and 6-fold less potent at inhibiting muscarinic responses in the trachea and submandibular salivary gland respectively, relative to atropine. Studies in anaesthetised dogs confirmed this selectivity profile. Thus darifenacin inhibited responses of the gut and bladder to cholinergic stimulation without affecting heart rate. Salivary gland responses were inhibited at doses some 6-10 fold higher than those required to inhibit gut and bladder responses. Clinical studies are ongoing in urge incontinence and functional bowel disease which may confirm this selectivity profile.

The molecular mechanisms involved in the regulation of muscarinic receptor gene expression are poorly understood. In an effort to gain a better understanding of the regulation of M2 receptors, we have investigated homologous and heterologous regulation of M2 muscarinic receptor protein and gene expression in human embryonic lung fibroblasts (HEL 299 cells). HEL 299 cells constitutively express m2 receptors, with no evidence of other muscarinic receptor subtypes. We have shown that M2 receptors in these cells can be down-regulated by muscarinic and beta2-adrenergic receptor agonists. Unlike the down-regulation mediated by muscarinic and beta-adrenergic stimulation, activation of PKC with PDBu was mediated through changes in m2 muscarinic receptor mRNA through reduced gene transcription. Because of the inflammatory nature of asthma, we have focused on delineating the interactions between cytokines and M2 receptors in an attempt to define potential endogenous modulators of M2 receptor expression. We have shown that the multi-functional cytokine, transforming growth factor beta1 (TGF-beta1), which is involved in several inflammatory conditions induces desensitization and down-regulation of M2 muscarinic receptor protein and gene expression that was mediated through a reduction in the rate of m2 receptor gene transcription. Other cytokines of interest are tumor necrosis factor alpha (TNF-alpha) and interleukin 1beta (IL-1beta) which are elevated in asthma. We have demonstrated that TNF-alpha and IL-1beta synergise to induce down-regulation of M2 muscarinic receptor protein and mRNA which was associated with functional desensitization of the receptor protein. The M2 receptor mRNA down-regulation appeared to be mediated through a reduction in the rate of m2 receptor gene transcription which may be dependent on the transcription and translation of unknown protein factor(s). Moreover, a role of PKA and ceramide pathways in M2 receptor regulation is suggested. Collectively, our work provides a mechanistic explanation of previous reports indicating altered function of M2 receptors in asthma. Ours results also suggest that the expression of this receptor subtype may be under the control of a cytokine network at the airways.

The ternary allosteric model predicts the possibility of discovering molecules with novel and highly subtype-selective modes of action. This approach has been applied to muscarinic receptors. The alkaloid brucine is capable of selectively enhancing by an allosteric mechanism the effects of low but not high concentrations of acetylcholine at only the m1 subtype of muscarinic receptors. A simple derivative of brucine, N-chloromethylbrucine, enhances acetylcholine actions selectively at only m3 receptors. In addition it binds to, but does not affect, the properties of m4 receptors, thereby demonstrating neutral cooperativity and an 'absolute' selectivity of action at m3 receptors over m4 receptors. Brucine N-oxide enhances acetylcholine binding at m3 and m4 receptors and is neutral at m1 and m5 receptors. These findings allow the possibility of developing muscarinic agents that have a novel and highly targeted mode of action; they may act only on a single muscarinic receptor subtype which is functioning sub-optimally and therefore be of use therapeutically in the early stages of Alzheimer's Disease.

In longitudinal muscle of guinea-pig ileum, activation of muscarinic receptors causes contraction antagonised by M3 receptor subtype antagonists despite a preponderance of M2 receptor subtype binding sites. Experiments on single smooth muscle cells under voltage-clamp described here show that the cationic current evoked by carbachol which normally causes depolarization of the muscle is inhibited competitively by M2 antagonists with affinities typical of antagonism at a M2 receptor. However, M3 antagonists strongly reduced the maximum cationic current which could be evoked by carbachol in a non-competitive manner with affinities typical for an action at M3 receptors. Thus cation channels are gated by M2 receptor activation but strongly modulated by activation of M3 receptors.

In some cell systems muscarinic receptor stimulation can induce proliferation or transformation. This phenomenon is subtype-specific (only m1 and m3 receptors are effective) and cell type dependent. In 1321N1 astrocytoma cells activation of m3 receptors stimulates phospholipase C, but does not induce DNA synthesis. In contrast the thrombin receptor, which also couples to phospholipase C, is strongly mitogenic and induces AP-1-dependent gene expression. Various experimental findings indicate that this discrepancy is not due to muscarinic receptor desensitization or blockade of growth stimulatory pathways. Muscarinic receptor number may be limiting, in particular for receptor coupling to the pertussis toxin-insensitive G-protein G12. This G-protein is required for thrombin-induced mitogenesis in 1321N1 cells and may couple selectively to the thrombin versus muscarinic receptor. In cardiomyocytes hypertrophic cell growth is induced by heterologously expressed m1 or m3 receptors but not by the endogenous m2 receptors. Studies using chimeric receptors confirm that induction of hypertrophy requires signalling through phospholipase C, but indicate that additional signals are needed to induce the morphological features of this response. We suggest that small G-proteins of the Rho subfamily, in addition to G12, mediate growth responses to G-protein-coupled receptors.

The receptor subtype and transduction mechanisms involved in the regulation of various neuronal ionic currents are reviewed, with some recent observations on sympathetic neurons, hippocampal cell membranes and basal forebrain cells.

As therapeutic agents, M1 agonists in the short-term may palliate symptoms of AD and improve memory function. In the long-term, M1 agonists have the potential to modify the underlying pathophysiology of AD, and thereby prevent or retard the course of dementia.

m1 and m2 receptors are traditionally linked to tissue specific functions performed by fully differentiated cells. However, these receptors have been also implicated in growth stimulation. The mechanisms whereby these receptors regulate proliferative signaling pathways are still poorly understood. Furthermore, pharmacological evidence suggest that many growth promoting agents act on Gi coupled receptors, but there is no formal proof that induction of DNA-synthesis results from decreased intracellular levels of cAMP. In our laboratory, we have used the expression of ml and m2 receptors as a model for studying proliferative signaling through G protein-coupled receptors. Currently available evidence suggest that these receptors signal to distinct members of the MAP kinase superfamily, MAP kinase and JNK, through betagamma subunits of heterotrimeric G proteins acting, respectively, on a Ras and Rac1 dependent pathway.

Alzheimer's disease involves progressive degeneration of the cortex and the limbic system. Loss of afferent forebrain neurochemical modulatory systems is also seen, most significantly of the basal forebrain cholinergic system. Drug discovery programmes have pursued enhancement of forebrain muscarinic function as a therapeutic target. The most promising muscarinic agonists described achieve functional selectivity as agonists as the M1/M3 receptors in the CNS and M2 antagonists. These compounds have fewer cardiac and other cholinergic side effects. In rodent and monkey models of reference and working memory, these compounds reverse the cognitive impairment induced with plopolamine. Acetylcholinesterase inhibitors are even more efficacious in these models.

One of the most interesting recent developments in the pharmacology of muscarinic receptors has been the finding of small proteins in the venoms of mamba snakes that bind with high affinity and selectivity to different subtypes of muscarinic receptors. In the workshop on muscarinic toxins, the practicalities of isolating, characterising and using these toxins as tools in the study of muscarinic receptors were discussed.

The prostate gland from several animal species contains variable levels of muscarinic subtypes, but only the human prostate expresses significant levels of the m1 subtype. We studied muscarinic receptor activity in human benign prostatic hypertrophy (BPH) as well as several cell lines derived from prostate cancer. The BPH we studied expresses approximately 75% of the m1 receptor and undetectable levels of the other receptor subtypes whereas PC3 cells express only the m3 receptor subtype. DU145 and LnCaP cells express approximately equal levels of m1 and m3 receptor subtypes. Only the PC3 cells responded to carbachol with an increase in turnover of polyphosphoinositides, and none of the cell lines responded with effects on cAMP metabolism. Co-precipitation of receptors with heterotrimeric guanine nucleotide-binding regulatory proteins demonstrated interactions of the m1 receptors with Gi, Gq and G16 in BPH tissue and of the m1 and m3 receptors with Gi, Gq and G12 in PC3 and DU145 cells. Mitogen activated protein kinase (ERK) activity was seen in response to carbachol in PC3 and DU145 but not LnCaP cells. Finally, carbachol promoted cell proliferation in all three cell lines. Thus, there appears to be no consistent relationship between ERK activity, cell proliferation, and the subtype mediating the proliferative response, amongst these prostate cancer cell lines.

We have investigated the molecular and cellular basis for the regulation of expression and function of the muscarinic acetylcholine receptors. Treatment of cultured chick cardiac cells with the agonist carbachol results in decreased levels of mRNA encoding the m2 and m4 receptors. Treatment of chick embryos in ovo with carbachol results in decreased levels of mRNA encoding the potassium channel subunits GIRK1 and GIRK4 as well as the m2 receptor. There are thus multiple pathways for the regulation of mAChR responsiveness by long-term agonist exposure. Immunoblot, immunoprecipitation, and solution hybridization analyses have been used to quantitate the regulation of mAChR expression in chick retina during embryonic development. The m4 receptor is the predominant subtype expressed early in development, while the expression of the m3 and m2 receptors increases later in development. A cAMP-regulated luciferase reporter gene has been used to demonstrate that the m2 and m4 receptors have distinct specificities for coupling to G-protein subtypes to mediate inhibition of adenylyl cyclase. This system has also been used to demonstrate that beta-arrestin1 and beta-adrenergic receptor kinase-1 act synergistically to promote receptor desensitization. We have isolated the promoter region for the chick m2 receptor gene, identified regions of the promoter required to drive high level expression in cardiac and neural cells, and have identified a region which confers sensitivity of gene expression to neurally active cytokines. Finally, in order to determine the role of individual receptor subtypes in muscarinic-mediated responses in vivo, we have used the method of targeted gene disruption by homologous recombination to generate mice deficient in the m1 receptor.

Muscarinic agonists regulate the L-type calcium current in isolated cardiac myocytes. The second messengers pathways involved in this regulation are discussed briefly, with particular emphasis on the involvement of cAMP and cGMP pathways.

Amyloid deposits in Alzheimer's disease are composed of amyloid beta-peptides (A beta) that are derived from the larger amyloid precursor protein (APP). Proteolytic APP processing is activity-dependent, and it can be regulated by muscarinic acetylcholine receptors. In particular, muscarinic m1 receptor subtypes increase cleavage within the A beta domain, followed by the release of the soluble APP ectodomain (APPs). In this study, we show that the m1-selective agonist talsaclidine concentration-dependently increased APPs release from both transfected human astrocytoma cell lines and rat brain slices. This increase was blocked by atropine. In contrast, the M2 antagonist BIBN 99 failed to increase APPs release, and decreased it at higher concentrations. These results show that talsaclidine can effectively modulate alpha-secretase processing of APP in human cell lines and in brain tissue. The data suggest that talsaclidine may be a useful candidate drug to modulate APP processing in Alzheimer's disease.

Current concepts regarding the regulation and coupling of muscarinic m3 receptors to G-proteins and various effectors are discussed. The last few years have provided much evidence that although muscarinic m1, m3 and m5 subtypes couple predominantly via pertussis toxin-insensitive G-proteins (Gq/11) to activate phosphoinositidase C (PIC), interactions with other G-proteins (Gi, Go, Gs) can be readily observed in cells expressing recombinant muscarinic receptors even at relatively low levels. The significance of this diversity and the potential for agonist "trafficking" could open up opportunities for novel approaches to selective agonist action. Finally, mechanisms underlying the regulation of muscarinic m3 coupling through Gq/11 to PIC are discussed. In particular, our recent studies on precursor lipid depletion, whether regulation is receptor or cell specific and the identification and role of receptor kinases are briefly reviewed.

Tolterodine is a new, potent and competitive muscarinic receptor antagonist in clinical development for the treatment of urge incontinence and other symptoms of unstable bladder. Tolterodine has a high affinity and specificity for muscarinic receptors in vitro and it exhibits a selectivity for the urinary bladder over salivary glands in vivo. A major active metabolite, (PNU-200577) the 5-hydroxymethyl derivative of tolterodine, has a similar pharmacological profile. Based on pharmacological and pharmacokinetic data, it has been concluded that this metabolite contributes significantly to the therapeutic effect of tolterodine. The bladder selectivity demonstrated by tolterodine and PNU-200577 in vivo cannot be attributed to selectivity for a single muscarinic receptor subtype. Moreover, this favourable tissue-selectivity seems to occur also in humans. Tolterodine is well tolerated and it exerts a marked effect on bladder function in healthy volunteers. Phase II data indicate that tolterodine is an efficacious and safe treatment for patients with idiopathic detrusor instability or detrusor hyperreflexia. An optimal efficacy/side-effect profile is obtained with tolterodine, at a dosage of 1 or 2 mg twice daily, which seems to have less propensity to cause dry mouth than the currently available antimuscarinic drugs.

By comparison to the other subtypes of muscarinic receptors, very little is known about the binding properties, locations, mechanisms and physiological functions of the M5 (m5)* receptor subtype. Studies of the m5 receptor have been hampered by the lack of m5-selective ligands or antibodies and a source that endogenously expresses predominantly the m5 receptor subtype. We have developed a pharmacological labeling strategy using the non-selective muscarinic antagonist [3H]NMS, in the presence of muscarinic antagonists and toxins in green mamba venom to occlude the m1-m4 receptor subtypes, to selectively label the m5 receptor subtype. This m5-selective labeling approach, along with those developed for the other four receptor subtypes, has permitted for the first time a comparison of the relative expression levels and anatomical localizations of the five muscarinic receptor subtypes in the brain. The distribution profile of the m5 receptor is distinct from the other four subtypes and is enriched in the outer layers of the cortex, specific subfields of the hippocampus, caudate putamen, olfactory tubercle and nucleus accumbens. These studies have also demonstrated that the levels of m5 receptor protein expression are apparently higher and more widespread than anticipated from previous in situ hybridization and immunoprecipitation studies. Taken together, the results suggest a unique and potentially physiologically important role for the m5 receptor subtype in modulating the actions of acetylcholine in the brain.

The muscarinic acetylcholine receptor (mAChR) molecular subtype, m2, has been postulated to be the presynaptic cholinergic autoreceptor in many brain regions. However, due to a lack of subtype-specific pharmacological agents, conclusive evidence for m2 as an autoreceptor remains elusive. The development of subtype-specific antibodies has enabled extensive characterization of the synaptic localization of the m2 subtype. Specifically, double-labeling immunocytochemistry with m2 antibodies and antibodies to the vesicular acetylcholine transporter (VAChT), a novel specific marker of cholinergic terminals, in the striatum has allowed the first direct anatomical evidence of m2 localization in cholinergic terminals. Additionally, other anatomical studies in striatum and the septohippocampal pathway have revealed that this subtype is also expressed presynaptically in non-cholinergic terminals, and is postsynaptically expressed in both cholinergic and non-cholinergic neurons. The implications of these data for understanding the functional roles of this subtype are discussed.

The role of small molecular weight guanine nucleotide-binding proteins (G proteins) of the Rho family in muscarinic acetylcholine receptor (mAChR) signaling to phospholipase C (PLC) and phospholipase D (PLD) was studied in human embryonic kidney (HEK) cells, stably expressing the human m3 receptor subtype. Evidence for the involvement of Rho proteins in m3 mAChR signaling to both phospholipases is based on findings obtained with Clostridium (C.) difficile toxin B and C. botulinum C3 exoenzyme, both of which specifically, although by different mechanisms, inactivate Rho family G proteins. Toxin B potently inhibited both the mAChR-stimulated PLC and PLD activities in intact cells as well as the stimulation of both phospholipases by the stable GTP analog GTPgammaS in permeabilized cells, the latter effect being mimicked by C3 exoenzyme. In contrast, PLC and PLD activities, measured in the presence of exogenous phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], a substrate and cofactor for PLC and PLD, respectively, were not altered. These data suggested that the Rho-inactivating toxins inhibit stimulation of PLC and PLD by reducing the cellular level of PtdIns(4,5)P2, which was indeed found with both toxin B and C3 exoenzyme. In agreement with a crucial role of cellular PtdIns(4,5)P2 supply for PLC signaling, we observed that short-term agonist (carbachol) treatment of HEK cells caused a long-lasting increase in PtdIns(4,5)P2 level, accompanied by a potentiation of receptor- and G protein-stimulated inositol phosphate formation. Finally, studies with tyrosine kinase and tyrosine phosphatase inhibitors strongly suggest that PtdIns(4,5)P2 synthesis and mAChR-stimulated PLD activity in HEK cells apparently also involve a tyrosine phosphorylation-dependent mechanism(s). Thus, m3 mAChR signaling to PLC and PLD in HEK cells requires the concerted action of various intracellular components, most notably the complex regulation of PtdIns(4,5)P2 synthesis.

The cholinergic septohippocampal system has been associated with learning and memory, as evidenced by the severe loss of these functions in lesioned animals as well as in senile demented patients. In an attempt to comprehend the physiological basis of the cholinergic innervation for hippocampal functions, numerous studies employed the in-vitro hippocampal slice preparation and analyzed the consequences of exposing the cells to cholinergic ligands. Many effects of activating a cholinergic receptor in the hippocampus were thus described, including blockade of several types of potassium conductances, yet few of these effects are intuitively related to the involvement of the cholinergic system in hippocampal plasticity. An alternative approach involves focusing on the possible effect of low concentration of cholinergic ligands on reactivity of the hippocampus to afferent stimulation. We found two new actions of acetylcholine (ACh); The first one is a fast onset, short lived increase in cellular responses to activation of the N-methyl-D-aspartate (NMDA) receptor, and the second one is a slow onset, long lasting increase in reactivity to afferent stimulation, resembling that produced by a tetanic stimulation, which we called muscarinic long term potentiation (LTPm). The latter effect is mediated by a postsynaptic M2 receptor, and it shares several properties with the more familiar tetanic LTP. In addition, LTPm involves a rise of intracellular calcium concentration and an activation of both a tyrosine kinase and a serine/threonine kinase. Intuitively, LTPm is better related to hippocampal plasticity than the other reported effects of ACh in the hippocampus. Indeed, aged rats, which are cognitively impaired, lack LTPm while they do express other muscarinic actions. It is proposed that LTPm is an important link between the cholinergic action and function in the hippocampus.

Butylthio[2.2.2] (LY297802 / NNC11-1053) is a mixed muscarinic cholinergic receptor agonist/antagonist that produces antinociception in mice and rats. As such, butylthio[2.2.2] may have therapeutic utility in the treatment of pain. Butylthio[2.2.2] was fully efficacious in the mouse grid shock, writhing, tail-flick and hot plate tests with ED50 values ranging from 1.5 to 12.2 mg/kg after oral administration. In contrast, the ED50 values for morphine ranged from 7.3 to 72 mg/kg after oral administration. Scopolamine was a competitive antagonist of the antinociceptive effects of butylthio[2.2.2]. Butylthio[2.2.2] did not produce either salivation or tremor at therapeutic doses; rather, there was a 50- to >100-fold separation between therapeutic doses and doses which produced side-effects. Butylthio[2.2.2] had high affinity for muscarinic receptors, but little if any affinity for other neurotransmitter receptors or uptake sites. In isolated tissues, butylthio[2.2.2] was an agonist with high affinity at M1 receptors in rabbit vas deferens, an antagonist at M2 receptors in guinea pig atria as well as an antagonist at M3 receptors in guinea pig urinary bladder. Although it has been suggested that M1 receptors mediate the antinociceptive effects of muscarinic agonists, M1 efficacy is not a requirement for antinociception, and, in vivo, the antinociceptive effects of muscarinic agonists are blocked by the intrathecal administration of pertussis toxin, indicating the involvement of m2 or m4 receptors. Since butylthio[2.2.2] is an M2 antagonist, antinociception is therefore most likely mediated by m4 receptors. Butylthio[2.2.2] is currently undergoing clinical development as a novel analgesic.

Five muscarinic acetylcholine receptor (mAChR) subtypes, m1-m5, have been cloned and sequenced to date. The question as to which mAChR subtypes exist in mammalian heart has been studied extensively and is still under considerable debate. We used the reverse transcriptase-polymerase chain reaction to amplify mRNA from adult rat ventricular myocytes, and found that these cells express mRNA for m1 and m2 mAChRs. Immunocytochemical analysis confirmed that m1 and m2, but not m3, mAChR proteins are present on the surface of these cells. Finally, the functional significance of these receptors was examined. Administration of the m1 mAChR antagonist pirenzepine inhibited the stimulatory effect of the muscarinic agonist carbachol on Ca transients. These findings are consistent with the presence of at least two mAChR subtypes in mammalian heart, m1 and m2, and suggest that activation of m1 mAChRs is involved in the stimulatory effects of muscarinic agonists in mammalian heart.

The aim of this investigation in anaesthetized dogs was to provide direct evidence for an activation of the sympathetic nervous system by the muscarinic agonist talsaclidine (WAL 2014 FU). Intravenous infusion at a rate of 1 mg/kg/min increased plasma catecholamines and in particular epinephrine, thus indicating a predominant stimulation of the adrenals. Sympathetic activation was also indicated by increases in renal vascular resistance, an effect which was sensitive to alpha-adrenolysis. It is concluded that the sympathetic activation by talsaclidine is due to full agonism at the M1-receptor and the ability to cross the blood-brain barrier. As talsaclidine is less potent and only a partial agonist at M2- and M3-receptors many peripheral actions mediated by these receptor subtypes are functionally antagonized by the concomitant sympathetic activation.

A study was undertaken to assess the receptor binding characteristics of [3H]4-benzylpempidine to an allosteric site on calf brain membranes associated with nicotinic cholinergic receptors and to compare the binding affinity of novel arylpempidine analogs with their ability to antagonize the behavioral effects of nicotine in mice. Scatchard analysis of the binding yielded a K(d) of 20 nM and a B(max) of 330 fmols/mg membrane protein. [3H]4-benzylpempidine appears to be a more satisfactory ligand than [3H]mecamylamine, since it possessed a 50-fold greater affinity and its binding was far less sensitive to inorganic ions and Tris. Among the arylpempidine analogs 4-m-chlorobenzylidenepempidine and 4-benzylidenepempidine had the lowest K(i) values (1.4 nM and 5.0 nM, respectively) and were the most potent in antagonizing nicotine-induced seizures in mice. Although the K(i) values for pempidine and mecamylamine were 1-2 orders of magnitude greater than any of the arylpempidines, the dose required to antagonize nicotine-induced seizures in mice was comparable to the arylpempidines. One explanation for this apparent discrepancy in the correlation of binding affinity and nicotine antagonism is the lower brain penetration of arylpempidines compared to mecamylamine, following their systemic administration to mice.

Different muscarinic acetylcholine receptor subtypes were used as model systems to study the structural basis of receptor/G protein coupling selectivity. Extensive mutagenesis studies have previously led to the identification of single amino acids on the m3 muscarinic receptor protein (located in the second intracellular loop (i2) and at the N- and C-terminus of the third intracellular loop (i3)) that dictate selective recognition of Gq/11 proteins by this receptor subtype. Based on these results, we proposed a model of the intracellular m3 receptor surface in which the functionally critical residues project into the interior of the transmembrane receptor core. To identify specific regions on the G protein(s) that are contacted by these different, functionally critical receptor sites, we recently employed a novel experimental strategy involving the coexpression of hybrid m2/m3 muscarinic receptors with hybrid G alpha-subunits. Using this approach, we could demonstrate that the C-terminus of G protein alpha i/o-subunits is recognized by a short sequence element in the m2 muscarinic receptor ("VTIL") that is located at the junction between the sixth transmembrane domain (TM VI) and the i3 loop. We could show that this interaction is critically involved in determining coupling selectivity and triggering G protein activation. By using a similar strategy (coexpression of mutant muscarinic receptors with hybrid G alpha-subunits), other major receptor/G protein contact sites are currently being identified. These studies, complemented by biochemical and biophysical approaches, should eventually lead to a detailed structural model of the ligand-receptor-G protein complex.

Muscarinic M1, M2, and M3 receptor subtypes have been shown to be involved in the pre- and postjunctional control of airway diameter of various species, including man. In a guinea pig model of allergic asthma, the prejunctional M2 receptor was shown to become dysfunctional already during the early allergic reaction, thereby contributing to exaggerated vagal reflex activity and airway hyperreactivity. Moreover, a deficiency of endogenous nitric oxide was observed after allergen provocation, which may also contribute to an enhanced postjunctional M3 receptor-mediated cholinergic response. Both in human and in animal airway preparations it was shown that enhanced cholinergic contractions are relatively resistent to beta-adrenoceptor-mediated relaxation. The reduced beta-adrenoceptor function may primarily be due to transductional cross-talk between PI metabolism and adenylyl cyclase, including protein kinase C-induced uncoupling of the beta-adrenoceptor from the effector system. Cross-talk between postjunctional M2 receptor-mediated inhibition and beta-adrenoceptor-induced activation of adenylyl cyclase appears to be of minor functional importance, but could be enhanced in allergic asthma due to increased expression of the inhibitory G protein as induced by cytokines.