Contractile roles of the M2 and M3 muscarinic receptors in the guinea pig colon.

The contractile roles of the M2 and M3 muscarinic receptors were investigated in guinea pig longitudinal colonic smooth muscle. Prior treatment of the colon with N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard) (40 nM) in combination with [[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11- dihydro-6H-pyrido[2,3b][1,4]benzodiazepine-6-one (AF-DX 116) (1.0 microM) caused a subsequent, irreversible inhibition of oxotremorine-M-induced contractions when measured after extensive washing. The estimate of the degree of receptor inactivation after 2 hr (97%) was not much greater than that measured after 1 hr (95%), which suggests that both 4-DAMP mustard-sensitive and -insensitive muscarinic subtypes contribute to the contractile response. Pertussis toxin treatment had no significant inhibitory effect on the control contractile response to oxotremorine-M, but caused an 8.8-fold increase in the EC50 value measured after a 2-hr treatment with 4-DAMP mustard. These results suggest that, after elimination of most of the M3 receptors with 4-DAMP mustard, the contractile response can be mediated by the pertussis toxin-sensitive M2 receptor. After pertussis toxin treatment, the kinetics of alkylation of muscarinic receptors in the colon were consistent with a single, 4-DAMP mustard-sensitive, M3 receptor subtype mediating the contractile response. When measured after a 2-hr treatment with 4-DAMP mustard and in the presence of histamine (0.30 microM) and either forskolin (10 microM) or isoproterenol (0.60 microM), the contractile responses to oxotremorine-M were pertussis toxin-sensitive and potently antagonized by the M2 selective antagonist, AF-DX 116. Collectively, our results indicate that the M2 receptor elicits contraction through two mechanisms, a direct contraction and an indirect contraction by preventing the relaxant effects of cAMP-generating agents.