FUNCTIONAL-ROLE OF THE M(2)-MUSCARINIC RECEPTOR IN SMOOTH-MUSCLE OF GUINEA-PIG ILEUM

A functional role for the M2 muscarinic receptor in smooth muscle contraction was investigated in isolated guinea pig ileum. Contractile responses to the muscarinic agonist oxotremorine-M (oxo-M) were measured in isolated ilea that had been pretreated with histamine (0.32 muM) and isoproterenol (0.64 muM) to achieve conditions of elevated cAMP. The resulting concentration-effect curve was biphasic, consisting of high (0-50 nm) and low (> 50 nm) potency components. The reversible M2-selective antagonist AF-DX 116 ([[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3b][1,4]benzodiazepine-6-one) (1 and 10 muM) shifted this curve in a manner that was inconsistent with competitive antagonism at a single receptor site; the high affinity component was significantly blocked, whereas there was little effect on the low aff inity portion of the curve. To inactivate the M3 muscarinic receptors selectively, ilea were incubated with the irreversible M1/M3-selective muscarinic antagonist 4-DAMP mustard [N-(2-chloroethyl)-4-piperidinyldiphenylacetate] (40 nm) for 1 hr in the presence of AF-DX 116 (1 muM) and were then washed extensively. Under these conditions, the contractile responses to oxo-M, in the presence of histamine and isoproterenol or forskolin, were antagonized by AF-DX 116 (1 muM) in a manner consistent with that mediated by an M2 receptor. AF-DX 116 caused 6.6- and 11-fold increases in the EC50 value for oxo-M for ilea pretreated with isoproterenol and forskolin, respectively, and a significant increase in the Hill coefficient in both cases. Under basal conditions, AF-DX 116 caused only a 1.34-fold increase in the EC50 value and no change in the Hill coefficient. In addition, under basal conditions 4-DAMP mustard treatment shifted the oxo-M contractile response curve to the right approximately 20-fold. However, when histamine was present in combination with isoproterenol or forskolin 4-DAMP mustard treatment shifted the concentration-effect curves for oxo-M to the right only about 3.5-fold. Oxo-M produced an M3-mediated stimulation of phosphoinositide hydrolysis in the longitudinal muscle of rat ileum with an EC50 value of 30 muM. 4-DAMP mustard (10 nM; 1 hr) prevented this response, resulting in a 6.6-fold increase in the EC50 value with a 65% reduction of the maximal response. In contrast, this treatment blocked M2-Mediated inhibition of isoproterenol-stimulated adenylate cyclase with only a 2-fold increase in EC50, without affecting maximum inhibition. A more selective blockade of the M3-mediated response was achieved by incubation of the longitudinal muscle slices with 4-DAMP mustard (40 nm) for 1 hr in the presence of AF-DX 116 (1 muM), followed by extensive washing. Under these conditions, the EC50 value for phosphoinositide hydrolysis was shifted 2.5-fold, with an 80% reduction in the maximum response. However, no change was observed in the ability of oxo-M to inhibit adenylate cyclase with this treatment. Our results demonstrate that 4-DAMP mustard can be used to inactivate M3-mediated responses selectively and that contractile responses of guinea pig ileum can be elicited not only by activation of phosphoinositide-coupled M3 receptors but also through activation of the more abundant cAMP-inhibitory M2 receptors.