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

Abstract

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. In the isolated bladder of rat, (+)‐cis‐dioxolane produced concentration‐dependent contractions (pEC 50=6.3) which were unaffected by tetrodotoxin (0.1 μ m). These contractions were antagonized by 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 hexahydrosiladifenidol (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 (p Ki) 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 defined M 3 receptor. 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 n m, 1 h) in the presence of methoctramine (0.3 μ m) 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 effects 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. 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−1, i.v.): 4‐DAMP (8.1) > atropine (20.7) > methoctramine (119.9) > darifenacin (283.3) > pirenzepine (369.1) > p‐F‐HHSiD (1053.8). These potency estimates correlated most favourably (r=0.89, P=0.04) with the p Ki 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 volume‐induced bladder contractions, although the modest potency of darifenacin does not exclude a role of M 3 receptors. Pretreatment with propranolol (1 mg kg−1, i.v.) increased the ID 35%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 β‐adrenoceptors in M 2 receptor‐mediated bladder contractions in vivo. The findings of the present study suggest that both M 2 and M 3 receptors can cause contraction of the rat bladder in vitro and may also mediate reflex bladder contractions in vivo. It is proposed that muscarinic M 3 receptor activation primarily causes direct contraction of the detrusor whereas M 2 receptor activation can contract the bladder indirectly by reversing sympathetically (i.e. β‐adrenoceptor)‐mediated relaxation. This dual mechanism may allow the parasympathetic nervous system, which is activated during voiding, to cause more efficient and complete emptying of the bladder. British Journal of Pharmacology (1997) 120, 1409–1418; doi:10.1038/sj.bjp.0701048