PERTUSSIS TOXIN BLOCKS M(2) MUSCARINIC RECEPTOR-MEDIATED EFFECTS ON CONTRACTION AND CYCLIC-AMP IN THE GUINEA-PIG ILEUM, BUT NOT M(3)-MEDIATED CONTRACTIONS AND PHOSPHOINOSITIDE HYDROLYSIS

The effects of pertussis toxin on muscarinic receptor-induced contract ions of the isolated guinea pig ileum were investigated. In control ti ssues, isoproterenol (1 mu M) only slightly inhibited contractions in response to oxotremorine-Rn; however, in pertussis toxin-treated tissu es, isoproterenol exhibited an enhanced inhibition of the concentratio n-effect curve. Under basal conditions, pertussis toxin had no dampeni ng effect on oxotremorine-M-induced contractions. When ilea were preco ntracted with histamine (1 mu M) and then relaxed back to base line us ing forskolin (10 mu M) 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 EC(50) value. Ilea we re then incubated with [N-(2-chloroethyl)-4-piperidinyl diphenylacetat e] (40 nM; 1 hr) in the presence of 1-dihydro-6H-pyrido[2,3b][1,4]benz odiazepine-6-one (1 mu M) to selectively inactivate the M(3) muscarini c receptors. Under these conditions, pertussis toxin blocked the conce ntration-effect curve to oxotremorine-M by a 30-fold increase in the E C(50) value. Prior treatment of guinea pigs in vivo with pertussis tox in diminished the labeling of a 41-kDa protein in membranes suspension s of the longitudinal muscle incubated with [P-32]nicotinamide adenine dinucleotide] and pertussis toxin. This ADP-ribosylation caused a fun ctional uncoupling of the G protein from its receptor as indicated by radioligand-binding studies and second messenger assays. Our results v erify that the M(2) muscarinic acetylcholine receptor, like the M(3) r eceptor, can elicit contractions of the guinea pig ileum and that the mechanism of this action involves inhibition of adenylate cyclase.