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 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.

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.

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