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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...
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...
1 To characterize the muscarinic receptors on human pulmonary veins associated with the acetylcholine (ACh)-induced relaxation, isolated venous and arterial preparations were precontracted with noradrenaline (10 mM) and were subsequently challenged with ACh in the absence or presence of selective muscarinic antagonists. 2 ACh relaxed venous preparations derived from human lung with a pD 2 value of 5.82+0.09 (n=16). In venous preparations where the endothelium had been removed, the ACh relaxations were abolished (n=4). ACh relaxed arterial preparations with a pD 2 value of 7.06+0.14 (n=5). 3 Atropine (1 mM), the non selective antagonist for muscarinic receptors, inhibited ACh-induced relaxations in human pulmonary veins. The a nity value (pK B value) for atropine was: 8.64+0.10 (n=5). The selective muscarinic antagonists (darifenacin (M 3 ), himbacine (M 2 ,M 4 ), methoctramine (M 2 ) and pFHHSiD (M 1 ,M 3 )) also inhibited ACh-induced relaxations in venous preparations. The pK B values obtained for these antagonists were not those predicted for the involvement of M 2 ± 5 receptors in the ACh-induced relaxation in human pulmonary veins. 4 The pK B value for darifenacin (1 mM) was signi®cantly greater in human pulmonary arterial (8.63+0.14) than in venous (7.41+0.20) preparations derived from three lung samples. 5 In human pulmonary veins, the pK B values for pirenzepine (0.5 and 1 mM), a selective antagonist for M 1 receptors, were: 7.89+0.24 (n=7) and 8.18+0.22 (n=5), respectively. In the venous preparations, the pK B values derived from the functional studies with all the di erent muscarinic antagonists used were correlated (r=0.89; P=0.04; slope=0.78) with the a nity values (pK i values) previously published for human cloned m1 receptors in CHO cells. 6 These results suggest that the relaxations induced by ACh are due to the activation of M 1 receptors on endothelial cells in isolated human pulmonary veins.
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