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

Life Sciences 74 (2003) 355 – 366 www.elsevier.com/locate/lifescie

Contractile role of M2 and M3 muscarinic receptors in gastrointestinal, airway and urinary bladder smooth muscle Frederick J. Ehlert * Department of Pharmacology, College of Medicine, University of California, Irvine, Irvine, CA 92697-4625, USA

Abstract Both M2 and M3 muscarinic receptors are expressed in smooth muscle and influence contraction through distinct signaling pathways. M3 receptors interact with Gq to trigger phosphoinositide hydrolysis, Ca2 + mobilization and a direct contractile response. In contrast, M2 receptors interact with Gi and Go to inhibit adenylyl cyclase and Ca2 +-activated K+ channels and to potentiate a Ca2 +-dependent, nonselective cation conductance. Ultimately, these mechanisms lead to the prediction that the influence of the M2 receptor on contraction should be conditional upon mobilization of Ca2 + by another receptor such as the M3. Mathematical modeling studies of these mechanisms show that the competitive antagonism of a muscarinic response mediated through activation of both M2 and M3 receptors should resemble the profile of the directly acting receptor (i.e., the M3) and not that of the conditionally acting receptor (i.e., the M2). Using a combination of pharmacological and genetic approaches, we have identified two mechanisms for the M2 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 M3 receptor. The latter mechanism may be involved in muscarinic agonist-mediated heterologous desensitization of smooth muscle, which requires activation of both M2 and M3 receptors. D 2003 Elsevier Inc. All rights reserved. Keywords: Muscarinic receptors; Knockout mice; Smooth muscle; Ileum; Trachea; Urinary bladder

Introduction Muscarinic agonists are known to elicit contraction in numerous types of smooth muscle from the gastrointestinal (GI), respiratory and urinary tracts (Eglen et al., 1996a, 1997). This contractile action is * Tel.: +1-949-824-6208; fax: +1-949-824-4855. E-mail address: [email protected] (F.J. Ehlert). 0024-3205/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2003.09.023

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mediated through muscarinic receptors located on the muscle itself. In some of these tissues, activation of muscarinic receptors causes relaxation; however, this effect can be attributed to the release or synthesis of chemical mediators. For example, activation of muscarinic receptors on the endothelium of many peripheral blood vessels stimulates the synthesis of nitric oxide, which diffuses to the muscle to cause relaxation (Knispel et al., 1992; Saenz de Tejada et al., 1988). In the lower esophageal sphincter, agonists activate muscarinic receptors on neurons, which release an inhibitory neurotransmitter that relaxes the muscle (Goyal and Rattan, 1978). Finally, activation of muscarinic receptors in smooth muscle sometimes stimulates the production of arachidonic acid metabolites that relax the muscle (Tachado et al., 1994). It has long been thought that it is primarily the M3 muscarinic receptor that elicits contraction in smooth muscle (see reviews by Eglen et al. (1996a) and Ehlert et al. (1997)). The initial basis for this conclusion rests on the findings of several investigators who have shown that the potencies of subtype selective muscarinic antagonists for blocking contraction agree best with their respective binding affinities at M3 muscarinic receptors, but not at other subtypes. An example of this relationship for antagonists can be found in a review by Ehlert et al. (1997) where the dissociation constants determined by antagonism of the contractile response (KB values) are compared the with binding affinities (KD values) measured at recombinant M1 –M5 muscarinic receptors. It was shown that the KB values of antagonists agree best with the binding affinity at M3 muscarinic receptors. This result is consistent with the postulate that M3 receptors mediate the direct contractile response in many smooth muscle types. These pharmacological data are consistent with more recent data on mutant mice lacking M3 muscarinic receptors (M3 muscarinic receptor knockout mice; M3 KO mice). It has been shown that the muscarinic contractile response is greatly inhibited in ileum and urinary bladder of M3 KO mice (Matsui et al., 2000), whereas a much smaller decrement in contractile function was noted in M2 muscarinic receptor knockout mice (M2 KO mice) (Stengel et al., 2000). In mice lacking both M2 and M3 muscarinic receptors (M2/M3 KO mice), the muscarinic contractile response is nearly completely eliminated in the ileum and urinary bladder (Matsui et al., 2002).

Signaling mechanisms of muscarinic receptors in smooth muscle The contractile role of the M3 muscarinic receptor in smooth muscle is consistent with its signaling mechanism. The M3 receptor is known to interact with Gq to elicit phosphoinositide hydrolysis in smooth muscle (Candell et al., 1990; Noronha-Blob et al., 1989; Roffel et al., 1990; Yang, 1991; Zhang and Buxton, 1991) as well as in cell lines in which the receptor has been heterologously expressed (Peralta et al., 1988). Activation of phospholipase-Ch in smooth muscle leads to Ca2 + mobilization and contraction. However, even before the signaling mechanisms of muscarinic receptor subtypes were resolved, an enigma began to appear in the pharmacological literature in the late 1980’s when it was shown that a variety of smooth muscle types express a majority of M2 muscarinic receptors (Candell et al., 1990; Choo et al., 1985; Choo and Mitchelson, 1988; Giraldo et al., 1987; Lazareno and Roberts, 1989; Michel and Whiting, 1987, 1988; Roffel et al., 1988; Yang, 1990; Zhang et al., 1991). This finding was surprising because it was expected that the receptor thought to elicit contraction (i.e., the M3) would be the most abundant receptor expressed in smooth muscle. The M2 receptor was shown to interact with Gi and Go to elicit pertussis toxin-sensitive responses in smooth muscle (Ostrom and Ehlert, 1999; Shehnaz et al., 2001; Thomas and Ehlert, 1994) as well as in cell lines transfected with the receptor (Lai

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et al., 1991; Peralta et al., 1988). Thus, pertussis toxin is a powerful tool for discriminating between M2 and M3 responses in smooth muscle. M2 muscarinic receptors are known to mediate an inhibition of adenylyl cyclase in cell lines (Lai et al., 1991; Peralta et al., 1988) and in smooth muscle (Candell et al., 1990; Yang et al., 1991; Zhang and Buxton, 1991). This action results in a decrease in cAMP accumulation elicited by h-adrenoceptors in GI and tracheal smooth muscle (Griffin and Ehlert, 1992; Ostrom and Ehlert, 1998; Thomas et al., 1993). Stimulation of muscarinic receptors in smooth muscle causes an activation of a nonselective cation conductance (Icat) (Bolton, 1979; Inoue, 1991; Inoue and Isenberg, 1990a). This effect is pertussis toxin-sensitive and dependent on Ca2 + mobilization (Inoue and Isenberg, 1990b; Pacaud and Bolton, 1991; Unno et al., 1995). Its sensitivity to pertussis toxin as well as M2 selective antagonists (Bolton and Zholos, 1997) indicates that the conductance is mediated by M2 receptor activation. However, the dependence of Icat on Ca2 + indicates that M2 receptor-mediated Icat is contingent upon Ca2 + mobilization by another receptor, like the M3. When measured using whole cell recording electrodes, it has been demonstrated that muscarinic stimulation of smooth muscle causes an efflux of K+ through Ca2 + dependent K+ channels (BKCa) (Carl et al., 1995; Cole et al., 1989; Heppner et al., 1997; Kume et al., 1992; Wade and Sims, 1993). This effect is thought to be mediated by the increase in Ca2 + elicited by M3 receptor stimulation. This K+ conductance acts as an inhibitory feedback mechanism to hyperpolarize the cell following stimulation by any Ca2 + mobilizing receptor. In isolated patches of tracheal smooth muscle, muscarinic agonists have been shown to cause a direct inhibition of BKCa channels (Cole et al., 1989; Kume et al., 1992; Wade and Sims, 1993). This effect is pertussis toxin-sensitive (Kotlikoff et al., 1992), indicating that M2 receptors mediate an inhibition of BKCa.

Interactions between M2 and M3 receptors in smooth muscle The literature summarized in the preceding paragraph is consistent with the postulate that the M3 receptor elicits a direct contractile response through Ca2 + mobilization, whereas the M2 receptor is capable of mediating effects that potentiate the contractile response of the M3 receptor or other Ca2 + mobilizing receptors. Moreover, the train of events elicited by the M2 receptor is not necessarily capable of eliciting a direct contraction by itself because these mechanisms are dependent on Ca2 + mobilization by another receptor. For example, there is no obvious mechanism whereby M2 receptor-mediated inhibition of cAMP accumulation could lead to calcium mobilization and a direct contractile response. Nevertheless, this effect could inhibit h-adrenoceptor mediated stimulation in cAMP accumulation, which normally inhibits smooth muscle contraction. Thus, the M2 receptor could mediate a disinhibition of contraction by inhibiting the relaxant effects of h-adrenoceptor activation. According to this mechanism, one would expect that the simultaneous activation of both M2 and M3 receptors would elicit a greater contractile stimulus in the presence of a h-agonist as compared to that generated under similar conditions when only M3 receptors are activated. A similar type of conclusion can be made for the effects of the M2 receptor on Icat and BKCa. Activation of M2 receptors by themselves would not appear to trigger contraction through these channels. In resting smooth muscle, the Ca2 + concentration is low, and thus, M2 receptor activation would be expected to have little effect on Icat, because this conductance is dependent on Ca2 +. Similarly, M2 receptor activation would be expected to have little affect contraction through BKCa channels under resting conditions because these channels are inactive at rest and a further inhibition would have little effect. However, if M3 receptors are activated, the resulting Ca2 + mobilization would satisfy the Ca2 + requirement of Icat, and

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thus, the M2 receptor-activation might potentiate M3 receptor-mediated contractions through stimulation of Icat. Similarly, the Ca2 + mobilization by the M3 receptor would trigger K+ efflux through BKCa channels, which tend to inhibit contraction. However, M2 receptor activation under these conditions would inhibit BKCa channels, and hence, potentiate M3 receptor mediated contractions. Thus, it can be seen that signaling pathways of M2 receptors that have been described in smooth muscle have the potential to enhance M3 receptor-mediated contractions, but might be ineffective by themselves. Thus, we have designated the contractile action of M2 receptors a conditional response, because it depends on Ca2 + mobilization by another receptor. So far, we have identified two conditional contractile responses to M2 receptor activation in smooth muscle: 1) a high potency inhibition of the relaxant action of agents that increase cAMP and 2) a low potency potentiation of the contractile response to M3 receptor activation. The evidence for these two responses is described below. The idea that the M2 receptor can only elicit conditional responses might seem to conflict with data on M3 KO mice in which direct contractions to muscarinic stimulation, albeit small, have been noted in ileum, trachea and urinary bladder (Matsui et al., 2000; Stengel et al., 2002). No evidence for this type of contraction has been observed in smooth muscle from guinea pigs in which most of the M3 receptors have been inactivated with the irreversible antagonist, N-2-chloroethyl-4-piperidinyl diphenylacetate (4DAMP mustard) (Sawyer and Ehlert, 1998; Thomas et al., 1993). It is possible that the M2 receptor is capable of a weak direct contractile response in mice and that this direct contraction is potentiated by M2 receptor signaling through the conditional mechanisms described above. It is also possible that there is a compensatory increase in the contractile function of the M2 receptor in M3 KO mice because of the lack of expression of M3 receptors. In guinea pig smooth muscle, it is possible that a 4-DAMP mustardalkylated M3 receptor may behave like a dominant negative mutant to prevent direct contractile signaling by the M2 receptor.

Competitive antagonism of responses mediated through an interaction between M2 and M3 muscarinic receptors It has been frequently stated that the functional role of the M2 receptor in contraction is questionable, because the potencies of subtype-selective muscarinic antagonists for blocking contraction are consistent with an M3 mechanism, but not that of an M2. It seems implicit in this rationale, that if the M2 receptor contributes to contraction, then M2 selective antagonists ought to block contraction with greater potency than that expected for a pure M3 response. However, this conclusion does not take into account the differences in the signaling mechanisms of muscarinic receptors. As described above, the M3 receptor mediates a direct contractile response, whereas the contribution of the M2 receptor to contraction is conditional upon M3 receptor activation. Thus, it is less than obvious what the antagonistic profile of such an M2/M3 interactive response should be. To address this question, we carried out modeling studies to determine the antagonistic profile of such a response. The results of our analysis showed that the competitive antagonism of such an interaction has a tendency to resemble the pharmacological profile of the directly acting receptor (i.e., the M3) and not that of the conditionally acting receptor (i.e., the M2) (Ehlert, 2003; Ehlert et al., 1999; Sawyer and Ehlert, 1999). This analysis explains why the competitive antagonism of muscarinic agonist induced contractions of smooth muscle exhibits an M3 profile even though muscarinic agonists activate both M2 and M3 receptors in smooth muscle and both receptors probably contribute to contraction.

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An example of this phenomenon can be gleaned from the data in Fig. 1. The upper left panel shows a concentration-response curve for the contractile effect of the muscarinic agonist, oxotremorine-M, in guinea pig ileum. It can be seen that the M2 selective antagonist, [[2-[(diethylamino)-methyl]-1piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3b][1,4]-benzodiazepine-6-one (AF-DX 116) (1 AM), has little inhibitory effect on the concentration response curve. The small two-fold increase in the EC50 value of oxotremorine-M is consistent with an M3 contractile mechanism. Similarly, the upper right panel shows that pertussis toxin-treatment has no inhibitory effect on contraction; in fact, a small potentiating effect is observed with pertussis toxin-treatment. These experiments were repeated in the presence of isoproterenol (see Fig. 1, lower panels). In the presence of isoproterenol (1 AM), one might expect that both M2 and M3 receptors contribute to contraction, because activation of M2 receptors should inhibit the relaxant effect of isoproterenol on M3 receptor-mediated contractions. However, the lower left panel shows that AF-DX 116 still has little competitive effect on muscarinic agonist induced contractions in the presence of isoproterenol. Thus, this M2 selective antagonist provides no evidence for a role of the M2 receptor in contraction. However, in the presence of isoproterenol, pertussis toxin-

Fig. 1. Effects of AF-DX 116 and pertussis toxin (PTX) on the contractile response to oxotremorine-M (Oxo-M) in guinea pig ileum in the absence (a and b) and presence (c and d) of isoproterenol (1 AM). a: The contractile response to Oxo-M was measured in the absence (o) and presence of AF-DX 116 (1.0 AM) (E). b: The contractile response to Oxo-M was measured in ilea from control guinea pigs (o) and guinea pigs treated with pertuss toxin (E). c: The contractile response to Oxo-M was measured in the presence of isoproterenol (o) and isoproterenol plus AF-DX 116 (E). d. The contractile response to Oxo-M was measured in the presence of isoproterenol in ilea from control guinea pigs (o) and guinea pigs that had been treated with pertussis toxin (E). The data are from Thomas et al. (1993) and Ehlert et al. (2001).

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treatment causes an inhibition of oxotremorine-M-induced contractions (see lower right panel). These results show that the M2 receptor is involved in contraction under these conditions. Our modeling studies have shown that competitive M2 selective antagonists are unable to reveal the role of the M2 receptors in contraction under conditions of a simultaneous activation of both M2 and M3 receptors. Nevertheless, pertussis toxin is a useful reagent to dissect out the role of the M2 receptor under these conditions (Ehlert, in 2003; Ehlert et al., 1999; Sawyer and Ehlert, 1999).

M2 muscarinic receptors mediate an inhibition of the relaxant effect of agents that increase cAMP in smooth muscle To determine whether muscarinic M2 receptors participate in contraction indirectly by inhibiting the relaxant effects of agents that increase cAMP, we developed a novel experiment (Thomas et al., 1993). This procedure eliminates the problems described above related to the detection of a conditional potentiation of contraction. Muscarinic M3 receptors in isolated smooth muscle preparations are first inactivated with 4-DAMP mustard-treatment, and the tissue is washed extensively. Then contractile responses to the muscarinic agonist, oxotremorine-M, are measured in the presence of both histamine and a cAMP-stimulating relaxant agent, like isoproterenol. When present together, histamine and the relaxant agent (i.e., isoproterenol) have no net contractile effect because the relaxant agent prevents the contraction to histamine. Nevertheless, under these conditions in GI smooth muscle, muscarinic agonists elicit a highly potent contractile response that is pertussis toxin-sensitive and exhibits a pharmacological profile for competitive antagonism consistent with an M2 receptor mechanism (Ehlert and Thomas, 1995; Ehlert et al., 1997). Presumably, the mechanism involves an M2 receptor-mediated inhibition of the relaxant effect on histamine-induced contractions. Using variations of this protocol we and others have identified a role for the M2 receptor in mediating contraction in the ileum (Ostrom and Ehlert, 1999; Reddy et al., 1995; Thomas et al., 1993; Thomas and Ehlert, 1994), colon (Sawyer and Ehlert, 1998, 1999), esophagus (Eglen et al., 1996b), trachea (Ostrom and Ehlert, 1998, 1999; Thomas and Ehlert, 1996) and urinary bladder (Hegde et al., 1997). The results in the trachea are particularly interesting. We showed that M2 receptors inhibit the relaxant effects of forskolin, but not isoproterenol, on H1 histamine and M3 muscarinic receptor induced contractions (Ostrom and Ehlert, 1998, 1999; Thomas and Ehlert, 1996). Other investigators also found a lack of effect of the M2 receptor in opposing isoproterenol-induced relaxation (Roffel et al., 1993, 1995; Watson et al., 1995). These results leave open the question of the physiological role of postjunctional M2 receptors in tracheal smooth muscle. It is possible that activation of M2 receptors may enhance M3 receptor-mediated contractions and those of inflammatory mediators in asthma. If M2 receptors mediate an inhibition of the relaxant action of cAMP stimulating agents on M3 receptor-mediated contractions, one would expect that the relaxant activity of forskolin and isoproterenol might be enhanced in smooth muscle from M2 KO mice. We found that forskolin exhibited greater relaxant activity against oxotremorine-M-induced contractions in ileum, trachea and urinary bladder form M2 KO mice (Matsui et al., 2003). We also found that the relaxant action of isoproterenol against oxotremorine-M-induced contractions was increased in the ileum and urinary bladder, but not in the trachea from M2 KO mice (Matsui et al., 2003). These results demonstrate that the M2 receptor functions to inhibit the relaxant action of agents that increase cAMP in smooth muscle. The results also suggest that isoproterenol elicits relaxation through a non-cAMP dependent mechanisms in the trachea,

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which explains why its relaxant action was unaffected by M2 receptor activation. We have previously obtained evidence for a non-cAMP-dependent mechanism of relaxation for isoproterenol in bovine trachea (Ostrom and Ehlert, 1998). Our results cannot be explained by a general increase in the sensitivity of smooth muscle from M2 KO mice to relaxant agents because there was no increase in the relaxant action of forskolin and isoproterenol against KCl-induced contractions. Also, there was no significant change in the sensitivity of smooth muscle to the relaxant effects of the potassium channel activator, pinacidil.

M2 receptors mediate a low potency enhancement of M3 receptor-mediated contractions in GI smooth muscle Following inactivation of the majority of M3 receptors with 4-DAMP mustard in guinea pig colon, the muscarinic agonist, oxotremorine-M, is still capable of eliciting contraction, but does so with reduced potency (Sawyer and Ehlert, 1998, 1999). Under these conditions as well as without 4-DAMP mustardtreatment, the muscarinic contractile response exhibits an M3 profile for pharmacological antagonism, which would seem to indicate that residual M3 receptors elicit contraction after 4-DAMP mustard treatment. However, following 4-DAMP mustard-treatment, the contractile response to oxotremorine-M in the colon is pertussis toxin-sensitive, suggesting an M2 mechanism. We previously showed that these enigmatic data (i.e., M3 antagonistic profile, yet pertussis toxin sensitive) can be explained by a model whereby the M3 receptor elicits a direct contractile response that is potentiated by M2 receptor activation (Ehlert et al., 1999; Sawyer and Ehlert, 1999). However, the M2 receptor is unable to elicit contraction by itself. Thus, we designate the mechanism of the M2 receptor as conditional upon M3 receptor activation. We postulate that this mechanism could involve M2 receptor mediated inhibition of BKCa or stimulation of Icat. We also postulate that this conditional M2 mechanism is less potent than the direct contractile mechanism of the M3 receptor. This latter postulate rests on the observation that, under control conditions, pertussis toxin-treatment has no inhibitory effect on contraction (Ehlert et al., 2001; Sawyer and Ehlert, 1999; Thomas and Ehlert, 1994). GI smooth muscle in the guinea pig typically exhibits a large receptor reserve for the contractile effects of a highly efficacious muscarinic agonist, like oxotremorine-M. This situation implies that the more potent M3 contractile mechanism can trigger a maximal contraction at agonist concentrations that are too low to activate the conditional M2 mechanism. However, after the sensitivity of the M3 pathway has been reduced by partial inactivation of M3 receptors with 4-DAMP mustard, then the sensitivity of the M2 conditional mechanism should greater relative to the residual M3 mechanism. Activation of M2 receptors with oxotremorine-M under these conditions should increase the contractile response elicited by the M3 receptor, and this potentiation should be sensitive to pertussis toxin. This rational can explain why the muscarinic contractile response is sensitive to pertussis toxin after 4-DAMP mustard treatment, but not before. Moreover, as described above, our modeling studies showed that the contractile response after 4-DAMP mustard treatment should exhibit an M3 profile for competitive antagonism. Since this conditional M2 potentiation of M3 receptor mediated contractions is only observed in isolated tissue bath experiments after partial inactivation of M3 receptors, one wonders about its physiological significance. It is possible that it plays a role in the dynamics of neurotransmission at smooth muscle on a rapid time scale that is not detected under standard conditions that are usually optimized for equilibrium measurements and not rapidly changing kinetic measurements. Also, it is

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possible that a subset of postjunctional sites undergo this M2/M3 interaction that is not detected when a global activation of muscarinic receptors occurs after introduction of muscarinic agonist to the organ bath.

Both M2 and M3 muscarinic receptors mediate heterologous desensitization in GI smooth muscle Although the physiological role of the conditional M2 mechanism described above is unclear because of its low potency, we hypothesized that it might play a role in heterologous desensitization because desensitization usually occurs after treatment of isolated smooth muscle with high concentrations of muscarinic agonist. Short term, heterologous desensitization is traditionally viewed as uninteresting because the mechanism involves distal elements in the signaling pathway such as depletion of stored Ca2 + (Hishinuma et al., 1992; Hishinuma and Uchida, 1988, 1989). In the isolated ileum, excessive muscarinic stimulation causes a subsequent decrease in contractile sensitivity to a variety of spasmogens (Cantoni and Eastman, 1946; Dale, 1958; Paton, 1961). This generalized subsensitivity is not surprising, given the metabolic costs required to maintain muscle contraction for several minutes. Thus, the receptors that mediate contraction probably also cause heterologous desensitization. For this reason, we are interested in heterologous desensitization, because if provides an indirect look at the receptors involved in contraction. We have shown that pertussis toxin-treatment prevents short-term (20 min) ACh-mediated desensitization of the contractile response to both histamine and the muscarinic agonist oxotremorine-M (Ehlert et al., 2001; Shehnaz et al., 2001). These results show that a G protein of the Gi family is involved in mediating desensitization, which strongly suggests a role for the M2 receptor. We also found that selective inactivation of M3 receptors with 4-DAMP mustard prevented ACh-mediated desensitization of histamine-induced contractions (Shehnaz et al., 2001). Collectively, the results suggest that both M2 and M3 receptors are involved in mediating desensitization, and that activation of either receptor by itself is insufficient to cause desensitization. The mechanism for the subsensitivity is likely to be downstream from receptor activation as described above, because ACh-treatment had no effect on histamine- or oxotremorine-M-stimulated phosphoinositide hydrolysis. The requirement of both M2 and M3 muscarinic receptors for heterologous desensitization is consistent with the conditional role of the M2 receptor in contraction. It is conceivable that the cellular stimulation elicited by M3 receptors is insufficient by itself to elicit much heterologous desensitization, perhaps because of K+ efflux through BKCa channels. Similarly, activation of the M2 receptor by itself would not be expected to cause desensitization because, in the guinea pig, the M2 receptor appears incapable of eliciting a direct contractile response. Nevertheless, if M2 receptor activation potentiates the contractile response to M3 receptors, then this mechanism could explain why heterologous desensitization requires activation of both receptor subtypes. The mechanism could involve an M2 receptormediated inhibition of BKCa channels, which normally put a brake on the contractile response to M3 receptor stimulation. The involvement of both M2 and M3 receptors in heterologous desensitization raises the question of how the competitive antagonism of a response contingent upon activation of two types of receptors should behave. We have used receptor modeling techniques to show that the competitive antagonism of such a response should obey the pharmacological profile of the least sensitive receptor signaling pathway, which we would predict to be that of the M2 receptor as described above (Ehlert et al.,

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1999). In preliminary data, we have found that the competitive antagonism of heterologous desensitization is most consistent with an M2 profile (Griffin et al., 2003). Thus it is interesting to note that activation of both M2 and M3 receptors initiates a train of events in smooth muscle, and depending upon which event one examines in competitive antagonism studies, empirical data for either an M2 response (e.g., desensitization) or an M3 response (e.g., contraction) can be obtained. These seemingly opposite results for responses triggered by the same two receptors can be rationalized by the nature of the dependence of the response on the interaction between M2 and M3 receptors. Acknowledgements The authors’ work described in this review was supported by N.I.H. Grants NS 26511 and NS 30882.

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