Specific G(q) protein involvement in muscarinic M(3) receptor-induced phosphatidylinositol hydrolysis and Ca(2+) release in mouse duodenal myocytes

1. Cytosolic Ca(2+) concentration ([Ca(2+)](i)) during exposure to acetylcholine or caffeine was measured in mouse duodenal myocytes loaded with fura-2. Acetylcholine evoked a transient increase in [Ca(2+)](i) followed by a sustained rise which was rapidly terminated after drug removal. Although L-type Ca(2+) currents participated in the global Ca(2+) response induced by acetylcholine, the initial peak in [Ca(2+)](i) was mainly due to release of Ca(2+) 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 Ca(2+) 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 [Ca(2+)](i) increase. 3. Acetylcholine-induced Ca(2+) 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 Ca(2+) 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)/α(11) antibody. An antisense oligonucleotide approach revealed that only the G(q) protein was involved in acetylcholine-induced Ca(2+) 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 Ca(2+) release. 6. Our results show that, in mouse duodenal myocytes, acetylcholine-induced release of Ca(2+) 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