FUNCTIONAL-ROLE OF M-2 AND M-3 MUSCARINIC RECEPTORS IN THE URINARY-BLADDER OF RATS IN-VITRO AND IN-VIVO

1 Urinary bladder smooth muscle is enriched with muscarinic receptors, the majority of which are of the M-2 subtype whereas the remaining mi nority belong to the M-3 subtype. The objective of the present study w as to assess the functional role of M-2 and M-3 receptors in the urina ry bladder of rat in vitro and in vivo by use of key discriminatory an tagonists. 2 In the isolated bladder of rat, (+)-cis-dioxolane produce d concentration-dependent contractions (pEC(50)=6.3) which were unaffe cted by tetrodotoxin (0.1 mu M). These contractions were antagonized b y muscarinic antagonists with the following rank order of affinity (pA (2)) estimates: atropine (9.1) > 4-diphenyl acetoxy-methyl piperidine methiodide (4-DAMP) (8.9) > darifenacin (8.5) > para fluoro hexahydros iladifenidol (p-F-HHSiD) (7.4) > pirenzepine (6.8) > methoctramine (5. 9). These pA(2) estimates correlated most favourably (r = 0.99, P < 0. 001) with the binding affinity (pK(i)) estimates of these compounds at human recombinant muscarinic m(3) receptors expressed in Chinese hams ter ovary cells, suggesting that the receptor mediating the direct con tractile responses to (+)-cis-dioxolane equates with the pharmacologic ally defined M-3 receptor. 3 As M-2 receptors in smooth muscle are neg atively 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 relaxa tion of KCl pre-contracted tissues). Muscarinic M-3 receptors were pre ferentially alkylated by exposing tissues to 4-DAMP mustard (40 nM, 1 h) in the presence of methoctramine (0.3 mu M) to protect M-2 receptor s. Under these conditions, (+)-cis-dioxolane produced concentration-de pendent reversal (re-contraction) of isoprenaline-induced relaxation ( pEC(50) = 5.8) but had marginal effects on pinacidil-induced, adenosin e 3':5'-cyclic monophosphate (cyclic AMP)-independent, relaxation. The re-contractions were antagonized by methoctramine and darifenacin, yi elding 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 s ubtypes. 4 In urethane-anaesthetized rats, the cholinergic component ( similar to 55%) of volume-induced bladder contractions was inhibited b y muscarinic antagonists with the following rank order of potency (ID3 5%inh, nmol kg(-1), i.v.): 4-DAMP (8.1) > atropine (20.7) > methoctram ine (119.9) > darifenacin (283.3) > pirenzepine (369.1) > p-F-HHSiD (1 053.8). These potency estimates correlated most favourably (r = 0.89, P = 0.04) with the pK(i) estimates of these compounds at human recombi nant muscarinic m(2) receptors. This is consistent with a major contri bution of M-2 receptors in the generation of volume-induced bladder co ntractions, although the modest potency of darifenacin does not exclud e a role of M-3 receptors. Pretreatment with propranolol (1 mg kg(-1), i.v.) increased the ID35%inh of methoctramine significantly from 95.9 to 404.5 nmol kg(-1) but had no significant effects on the inhibitory responses to darifenacin. These data suggest an obligatory role of be ta-adrenoceptors in M-2 receptor-mediated bladder contractions in vivo . 5 The findings of the present study suggest that both M-2 and M-3 re ceptors can cause contraction of the rat bladder in vitro and may also mediate reflex bladder contractions in vivo. It is proposed that musc arinic M3 receptor activation primarily causes direct contraction of t he detrusor whereas M-2 receptor activation can contract the bladder i ndirectly by reversing sympathetically (i.e. beta-adrenoceptor)-mediat ed relaxation. This dual mechanism may allow the parasympathetic nervo us system, which is activated during voiding, to cause more efficient and complete emptying of the bladder.