Specific G(q) protein involvement in muscarinic M3 receptor-induced phosphatidylinositol hydrolysis and Ca2+ release in mouse duodenal myocytes

Abstract

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 M3 antagonist), pirenzepine (a muscarinic M1 antagonist), methoctramine and gallamine (muscarinic M1 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 M1 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-α(q)/α11 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-β(com) antibody or a peptide corresponding to the Gβγ binding domain of the β-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 M3 receptors which couple with a G(q) protein to activate a phosphatidylinositol-specific phospholipase C.