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

The contractile roles of the M-2 and M-3 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 mu M) 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 M-3 receptors with 4-DAMP mustard, the contractile response can be mediated by the pertussis toxin-sensitive M-2 receptor. After pertussis toxin treatment, the kinetics of alkylation of muscarinic receptors in the colon were consistent with a single, 4-DAMP mustard-sensitive, M-3 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 mu M) and either forskolin (10 mu M) or isoproterenol (0.60 mu M), the contractile responses to oxotremorine-M were pertussis toxin-sensitive and potently antagonized by the M-2 selective antagonist, AF-DX 116. Collectively, our results indicate that the M-2 receptor elicits contraction through two mechanisms, a direct contraction and an indirect contraction by preventing the relaxant effects of cAMP-generating agents.