Increase of [Ca2+]i and release of arachidonic acid via activation of M2 receptor coupled to Gi and Rho proteins in oesophageal muscle

We have previously shown that acetylcholine-induced contraction of oesophag eal circular muscle depends on activation of phosphatidylcholine selective phospholipase C and D, which result in formation of diacylglycerol, and of phospholipase 2 which produces arachidonic acid. Diacylglycerol and arachid onic acid interact synergistically to activate protein kinase C. We have th erefore investigated the relationship between cytosolic Ca2+ and activation of phospholipase A, in response to acetylcholine-induced stimulation, by m easuring the intracellular free Ca2+ ([Ca2+](i)), muscle tension, and [H-3] arachidonic acid release. Acetylcholine-induced contraction was associated with increased [Ca2+](i) and arachidonic acid release in a dose-dependent manner. In Ca2+-free medium, acetylcholine did not produce contraction, [Ca 2+](i) increase, and arachidonic acid release. In contrast, after depletion of Ca2+ stores by thapsigargin (3 mu M), acetylcholine caused a normal con traction, [Ca2+](i) increase and arachidonic acid release. The increase in [Ca2+](i) and arachidonic acid release were attenuated by the M2 receptor a ntagonist methoctramine, but not by the M3 receptor antagonist p-fluoro-hex ahydro siladifenidol. Increase in [Ca2+](i) and arachidonic acid release by acetylcholine were inhibited by pertussis toxin and C3 toxin. These findin gs indicate that contraction and arachidonic acid release are mediated thro ugh muscarinic M2 coupled to Gi or rho protein activation and Ca2+ influx. Acetylcholine-induced contraction and the associated increase in [Ca2+](i) and release of arachidonic acid were completely reduced by the combination treatment with a phospholipase A(2) inhibitor dimethyleicosadienoic acid an d a phospholipase D inhibitor pCMB, They increased by the action of the inh ibitor of diacylglycerol kinase R59949, whereas they decreased by a protein kinase C inhibitor chelerythrine. These data suggest that in oesophageal c ircular muscle acetylcholine-induced [Ca2+](i) increase and arachidonic aci d release are mediated through activation of M2 receptor coupled to Gi or r ho protein, resulting in the activation of phospholipase A(2) and phospholi pase D to activate protein kinase C. (C) 2000 Elsevier Science Inc. All rig hts reserved.