Specific G(q) protein involvement in muscarinic M-3 receptor-induced phosphatidylinositol hydrolysis and Ca2+ release in mouse duodenal myocytes

1 Cytosolic Ca2+ concentration ([Ca2+](i)) during exposure to acetylcholine or caffeine was measured in mouse duodenal myocytes loaded with fura-2. Acetylcholine evoked a transient increase in [Ca2+](i) followed by a sustained rise which was rapidly terminated after drug removal. Although L-type Ca2+ currents participated in the global Ca2+ response induced by acetylcholine, the initial peak in [Ca2+](i) was mainly due to release of Ca2+ from intracellular stores. 2 Atropine, 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, a muscarinic M-3 antagonist), pirenzepine (a muscarinic M-1 antagonist), methoctramine and gallamine (muscarinic M-2 antagonists) inhibited the acetylcholine-induced Ca2+ release, with a high affinity for 4-DAMP and atropine and a low affinity for the other antagonists. Selective protection of muscarinic M-2 receptors with methoctramine during 4-DAMP mustard alkylation of muscarinic M-3 receptors provided no evidence for muscarinic M-2 receptor-activated [Ca2+](i) increase. 3 Acetylcholine-induced Ca2+ release was blocked by intracellular dialysis with a patch pipette containing either heparin or an anti-phosphatidylinositol antibody and by external application of U73122 (a phospholipase C inhibitor). 4 Acetylcholine-induced Ca2+ release was insensitive to external pretreatment with pertussis toxin, but concentration-dependently inhibited by intracellular dialysis with a patch pipette solution containing an anti-alpha(q)/alpha(11) antibody. An antisense oligonucleotide approach revealed that only the G(q) protein was involved in acetylcholine-induced Ca2+ release. 5 Intracellular applications of either an anti-beta(com) antibody or a peptide corresponding to the G beta gamma binding domain of the beta-adrenoceptor kinase 1 had no effect on acetylcholine-induced Ca2+ release. 6 Our results show that, in mouse duodenal myocytes, acetylcholine-induced release of Ca2+ from intracellular stores is mediated through activation of muscarinic M-3 receptors which couple with a G(q) protein to activate a phosphatidylinositol-specific phospholipase C.