Abstract

The purpose of this study was to elucidate the mechanism by which acetylcholine (ACh) promotes prostacyclin (PGI₂) production in cultured coronary endothelial cells (CEC) of the rabbit heart. ACh-induced production of PGI₂, measured as immunoreactive 6-keto-PGF_{1 α}, was enhanced by increasing the extracellular calcium (Ca⁺⁺) concentration and reduced by Ca⁺⁺ depletion. The receptor-operated Ca⁺⁺channel blocker SK&F96365, but not the voltage-dependent Ca⁺⁺ channel blockers verapamil or nifedipine, attenuated ACh-induced 6-keto-PGF_{1 α} production and the associated rise in cytosolic Ca⁺⁺. Thapsigargin, which depleted Ca⁺⁺ accumulation from the intracellular Ca⁺⁺ store, did not prevent the ACh-induced rise in cytosolic Ca⁺⁺. In the absence of extracellular Ca⁺⁺, ACh and ATP increased cytosolic Ca⁺⁺ but did not alter 6-keto-PGF_{1 α} production. In permeabilized CEC, guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-S) but not ACh enhanced 6-keto-PGF_{1 α} synthesis. ACh increased 6-keto-PGF_{1 α} production in the presence of GTP-γ-S. These effects of GTP-γ-S were attenuated by guanosine 5′-O-(2-thiotriphosphate). In the absence of extracellular Ca⁺⁺, ACh or ATP increased cytosolic Ca⁺⁺ in cells permeabilized with β-escin and loaded with GTP-γ-S; this effect was attenuated by guanosine 5′-O-(2-thiotriphosphate). The effect of ATP but not ACh to mobilize intracellular Ca⁺⁺ or increase 6-keto-PGF_{1 α} was inhibited by pertussis toxin. The phospholipase C inhibitor D609, which attenuated ACh- and ATP-induced mobilization of intracellular Ca⁺⁺, did not alter 6-keto-PGF_{1 α} production. The NO synthase inhibitor N-monomethyl-arginine also failed to alter ACh-induced 6-keto-PGF_{1 α} synthesis. These data suggest that, in CEC of the rabbit heart, ACh stimulates prostacyclin production via a pertussis toxin-insensitive G protein and by increasing the influx of extracellular Ca⁺⁺ through a G protein-independent receptor-operated Ca⁺⁺ channel.