Functional role of M₂ and M₃ muscarinic receptors in the urinary bladder of rats in vitro and in vivo

Abstract

1 Urinary bladder smooth muscle is enriched with muscarinic receptors, the majority of which are of the M 2 subtype whereas the remaining minority belong to the M 3 subtype. The objective of the present study was to assess the functional role of M 2 and M 3 receptors in the urinary bladder of rat in vitro and in vivo by use of key discriminatory antagonists. 2 In the isolated bladder of rat, (+)-cis-dioxolane produced concentration-dependent contractions (pEC 50 =6.3) which were unaected by tetrodotoxin (0.1 mM). These contractions were antagonized by muscarinic antagonists with the following rank order of anity (pA 2 ) estimates: atropine (9.1) 4 4-diphenyl acetoxy-methyl piperidine methiodide (4-DAMP) (8.9) 4 darifenacin (8.5) 4 para¯uoro hexahydrosiladifenidol (p-F-HHSiD) (7.4) 4 pirenzepine (6.8) 4 methoctramine (5.9). These pA 2 estimates correlated most favourably (r=0.99, P50.001) with the binding anity (pK i ) estimates of these compounds at human recombinant muscarinic m 3 receptors expressed in Chinese hamster ovary cells, suggesting that the receptor mediating the direct contractile responses to (+)-cis-dioxolane equates with the pharmacologically de®ned M 3 receptor. 3 As M 2 receptors in smooth muscle are negatively coupled to adenylyl cyclase, we sought to determine whether a functional role of M 2 receptors could be unmasked under conditions of elevated adenylyl cyclase activity (i.e., isoprenaline-induced relaxation of KCl pre-contracted tissues). Muscarinic M 3 receptors were preferentially alkylated by exposing tissues to 4-DAMP mustard (40 nM, 1 h) in the presence of methoctramine (0.3 mM) to protect M 2 receptors. Under these conditions, (+)-cis-dioxolane produced concentration-dependent reversal (re-contraction) of isoprenaline-induced relaxation (pEC 50 =5.8) but had marginal eects on pinacidil-induced, adenosine 3':5'-cyclic monophosphate (cyclic AMP)-independent, relaxation. The re-contractions were antagonized by methoctramine and darifenacin, yielding pA 2 estimates of 6.8 and 7.6, respectively. These values are intermediate between those expected for these compounds at M 2 and M 3 receptors and were consistent with the involvement of both of these subtypes. 4 In urethane-anaesthetized rats, the cholinergic component (*55%) of volume-induced bladder contractions was inhibited by muscarinic antagonists with the following rank order of potency (ID 35%inh , nmol kg 71 , i.v.): 4-DAMP (8.1) 4 atropine (20.7) 4 methoctramine (119.9) 4 darifenacin (283.3) 4 pirenzepine (369.1) 4 p-F-HHSiD (1053.8). These potency estimates correlated most favourably (r=0.89, P=0.04) with the pK i estimates of these compounds at human recombinant muscarinic m 2 receptors. This is consistent with a major contribution of M 2 receptors in the generation of volumeinduced bladder contractions, although the modest potency of darifenacin does not exclude a role of M 3 receptors. Pretreatment with propranolol (1 mg kg 71 , i.v.) increased the ID 35%inh of methoctramine signi®cantly from 95.9 to 404.5 nmol kg 71 but had...

M3 muscarinic receptors mediate contraction of human urinary bladder

“…These data demonstrate that contraction of human bladder in response to carbachol is mediated predominantly if not exclusively by the M 3 muscarinic receptor. This situation mimics closely the previously reported ®ndings in rats (Choppin et al, 1998;Hegde et al, 1997;Longhurst et al, 1995;Longhurst & Levendusky, 2000;Tong et al, 1997), mice (Choppin & Eglen, 2001b), pigs (Yamanishi et al, 2000) and dogs (Choppin & Eglen, 2001a). These data suggest that a drug for the e ective treatment of overactive bladder should have high a nity for M 3 receptors.…”

“…Nevertheless analysis of the shifts of the carbachol concentration response curve yielded slopes close to unity for all four antagonists. The rank order of the antagonist potencies (atropine5darifenacin4pirenzepine4methoctramine) as well as the absolute potencies were in good agreement with those at cloned M 3 receptors and those determined functionally for antagonist of contraction in rat, mouse and dog bladder (Choppin & Eglen, 2001a, b;Hegde et al, 1997). These data demonstrate that contraction of human bladder in response to carbachol is mediated predominantly if not exclusively by the M 3 muscarinic receptor.…”

“…A similar situation is found in the bladder of rats, rabbits, guinea-pigs (Wang et al, 1995) and pigs (Goepel et al, 1998;Yamanishi et al, 2000). The contractile response to the exogenous muscarinic agonist carbachol and to endogenous agonist released by ®eld stimulation, however, occurs predominantly if not exclusively via M 3 receptors in rats (Choppin et al, 1998;Hegde et al, 1997;Longhurst et al, 1995;Longhurst & Levendusky, 2000;Tong et al, 1997), mice (Choppin & Eglen, 2001b), pigs (Yamanishi et al, 2000) and dogs (Choppin & Eglen, 2001a). Moreover, M 3 (but not M 2 ) receptor knock-out mice exhibit bladder distension and develop urinary retention (Matsui et al, 2000).…”

“…While atropine, pirenzepine and methoctramine did not signi®cantly a ect the maximum response to carbachol, darifenacin signi®cantly reduced the maximum carbachol responses. Similar unsurmountable darifenacin e ects on bladder contraction have previously been described in rat (Hegde et al, 1997) and canine bladder (Choppin & Eglen, 2001a), but the underlying reasons are unknown. 2nd curve 97+3 9 7 +3 101+3 9 4 +3 9 2 +5 3rd curve 96+2 8 6 +4 9 8 +5 9 1 +6 7 7 +11* 4th curve 90+3 7 6 +6 9 7 +7 8 7 +10 65+10* 5th curve 85+3 6 7 +9 7 4 +8 7 4 +12 53+9* E max is expressed as % of the e ect in the ®rst curve within each preparation which was always performed in the absence of any antagonist; subsequent curves were generated in the presence of increasing antagonist concentrations (see Figure 1).…”

3-iodothyronamine (T1AM), a novel antagonist of muscarinic receptors

“…In this preparation, all muscarinic subtypes are expressed and their activation evokes micturition. Consensus evidence indicates that the pharmacological effects mediated by the different subtypes are hard to distinguish (Hegde et al, 1997) and that, in preparations from normal bladders with an intact urothelium, studies indicate that carbachol contraction is mediated by a homogenous muscarinic type 3 population (Hegde et al, 1997). In this tissue exposed to carbachol, mimicking increased cholinergic tone, we demonstrated that the addition of T1AM reduced contraction calculating an IC50M perfectly overlapping with pKi values measured from binding experiments.…”