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 cont raction was investigated in isolated guinea pig ileum. Contractile res ponses to the muscarinic agonist oxotremorine-M (oxo-M) were measured in isolated ilea that had been pretreated with histamine (0.32 muM) an d 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-sel ective antagonist AF-DX 116 -dihydro-6H-pyrido[2,3b][1,4]benzodiazepin e-6-one) (1 and 10 muM) shifted this curve in a manner that was incons istent with competitive antagonism at a single receptor site; the high affinity component was significantly blocked, whereas there was littl e effect on the low aff inity portion of the curve. To inactivate the M3 muscarinic receptors selectively, ilea were incubated with the irre versible M1/M3-selective muscarinic antagonist 4-DAMP mustard [N-(2-ch loroethyl)-4-piperidinyldiphenylacetate] (40 nm) for 1 hr in the prese nce of AF-DX 116 (1 muM) and were then washed extensively. Under these conditions, the contractile responses to oxo-M, in the presence of hi stamine and isoproterenol or forskolin, were antagonized by AF-DX 116 (1 muM) in a manner consistent with that mediated by an M2 receptor. A F-DX 116 caused 6.6- and 11-fold increases in the EC50 value for oxo-M for ilea pretreated with isoproterenol and forskolin, respectively, a nd 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 E C50 value and no change in the Hill coefficient. In addition, under ba sal conditions 4-DAMP mustard treatment shifted the oxo-M contractile response curve to the right approximately 20-fold. However, when hista mine was present in combination with isoproterenol or forskolin 4-DAMP mustard treatment shifted the concentration-effect curves for oxo-M t o the right only about 3.5-fold. Oxo-M produced an M3-mediated stimula tion of phosphoinositide hydrolysis in the longitudinal muscle of rat ileum with an EC50 value of 30 muM. 4-DAMP mustard (10 nM; 1 hr) preve nted this response, resulting in a 6.6-fold increase in the EC50 value with a 65% reduction of the maximal response. In contrast, this treat ment blocked M2-Mediated inhibition of isoproterenol-stimulated adenyl ate cyclase with only a 2-fold increase in EC50, without affecting max imum inhibition. A more selective blockade of the M3-mediated response was achieved by incubation of the longitudinal muscle slices with 4-D AMP mustard (40 nm) for 1 hr in the presence of AF-DX 116 (1 muM), fol lowed by extensive washing. Under these conditions, the EC50 value for phosphoinositide hydrolysis was shifted 2.5-fold, with an 80% reducti on in the maximum response. However, no change was observed in the abi lity of oxo-M to inhibit adenylate cyclase with this treatment. Our re sults demonstrate that 4-DAMP mustard can be used to inactivate M3-med iated responses selectively and that contractile responses of guinea p ig ileum can be elicited not only by activation of phosphoinositide-co upled M3 receptors but also through activation of the more abundant cA MP-inhibitory M2 receptors.