Specific Gq protein involvement in muscarinic M3 receptor‐induced phosphatidylinositol hydrolysis and Ca2+ release in mouse duodenal myocytes

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. Atropine, 4‐diphenylacetoxy‐N‐methylpiperidine (4‐DAMP, a muscarinic M3 antagonist), pirenzepine (a muscarinic M1 antagonist), methoctramine and gallamine (muscarinic M2 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 M2 receptors with methoctramine during 4‐DAMP mustard alkylation of muscarinic M3 receptors provided no evidence for muscarinic M2 receptor‐activated [Ca2+]i increase. 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). 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‐αq/α11 antibody. An antisense oligonucleotide approach revealed that only the Gq protein was involved in acetylcholine‐induced Ca2+ release. Intracellular applications of either an anti‐βcom antibody or a peptide corresponding to the Gβγ binding domain of the β‐adrenoceptor kinase 1 had no effect on acetylcholine‐induced Ca2+ release. Our results show that, in mouse duodenal myocytes, acetylcholine‐induced release of Ca2+ from intracellular stores is mediated through activation of muscarinic M3 receptors which couple with a Gq protein to activate a phosphatidylinositol‐specific phospholipase C.