Vol. 282, Issue 1, 113-122, 1997

Signal Transduction Mechanism(s) Involved in Prostacyclin Production Elicited by Acetylcholine in Coronary Endothelial Cells of Rabbit Heart¹

H. Kan², Y. Ruan³ and K. U. Malik

Department of Pharmacology, College of Medicine, The University of Tennessee Center for the Health Sciences, Memphis, Tennessee

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-PGF₁, 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-PGF₁ 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-PGF₁ production. In permeabilized CEC, guanosine 5-O-(3-thiotriphosphate) (GTP--S) but not ACh enhanced 6-keto-PGF₁ synthesis. ACh increased 6-keto-PGF₁ production in the presence of GTP--S. These effects of GTP--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 -escin and loaded with GTP--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-PGF₁ 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-PGF₁ production. The NO synthase inhibitor N-monomethyl-arginine also failed to alter ACh-induced 6-keto-PGF₁ 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.