Abstract

The effects of pertussis toxin on muscarinic receptor-induced contractions of the isolated guinea pig ileum were investigated. In control tissues, isoproterenol (1 microM) only slightly inhibited contractions in response to oxotremorine-M; however, in pertussis toxin-treated tissues, isoproterenol exhibited an enhanced inhibition of the concentration-effect curve. Under basal conditions, pertussis toxin had no dampening effect on oxotremorine-M-induced contractions. When ilea were precontracted with histamine (1 microM) and then relaxed back to base line using forskolin (10 microM) before contractile responses to oxotremorine-M were measured, pertussis toxin shifted the concentration-effect curve to oxotremorine-M by a 6.1-fold increase in the EC50 value. Ilea were then incubated with [N-(2-chloroethyl)-4-piperidinyl diphenylacetate] (40 nM; 1 hr) in the presence of [[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11- dihydro-6H-pyrido[2,3b][1,4]benzodiazepine-6-one (1 microM) to selectively inactivate the M3 muscarinic receptors. Under these conditions, pertussis toxin blocked the concentration-effect curve to oxotremorine-M by a 30-fold increase in the EC50 value. Prior treatment of guinea pigs in vivo with pertussis toxin diminished the labeling of a 41-kDa protein in membranes suspensions of the longitudinal muscle incubated with [32P]nicotinamide adenine dinucleotide] and pertussis toxin. This ADP-ribosylation caused a functional uncoupling of the G protein from its receptor as indicated by radioligand-binding studies and second messenger assays. Our results verify that the M2 muscarinic acetylcholine receptor, like the M3 receptor, can elicit contractions of the guinea pig ileum and that the mechanism of this action involves inhibition of adenylate cyclase.