SIGNAL-TRANSDUCTION MECHANISM(S) INVOLVED IN PROSTACYCLIN PRODUCTION ELICITED BY ACETYLCHOLINE IN CORONARY ENDOTHELIAL-CELLS OF RABBIT HEART

The purpose of this study was to elucidate the mechanism by which acet ylcholine (ACh) promotes prostacyclin (PGI(2)) production in cultured coronary endothelial cells (CEC) of the rabbit heart. ACh-induced prod uction of PGI(2), measured as immunoreactive 6-keto-PGF(1 alpha), was enhanced by increasing the extracellular calcium (Ca++) concentration and reduced by Ca++ depletion. The receptor-operated Ca++ channel bloc ker SK&F96365, but not the voltage-dependent Ca++ channel blockers ver apamil or nifedipine, attenuated ACh-induced 6-keto-PGF(1 alpha) produ ction and the associated rise in cytosolic Ca++. Thapsigargin, which d epleted Ca++ accumulation from the intracellular Ca++ store, did not p revent the ACh-induced rise in cytosolic Ca++. In the absence of extra cellular Ca++, ACh and ATP increased cytosolic Ca++ but did not alter 6-keto-PGF(1 alpha) production. In permeabilized CEC, guanosine 5'-O-( 3-thiotriphosphate) (GTP-gamma-S) but not ACh enhanced 6-keto-PGF(1 al pha) synthesis. ACh increased 6-keto-PGF(1 alpha) production in the pr esence of GTP-gamma-S. These effects of GTP-gamma-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 beta-escin and loaded with GTP-gamma-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 alpha) was inhibi ted by pertussis toxin. The phospholipase C inhibitor D609, which atte nuated ACh- and ATP-induced mobilization of intracellular Ca++, did no t alter 8-keto-PGF(1 alpha) production. The NO synthase inhibitor N-mo nomethyl-arginine also failed to alter ACh-induced 6-keto-PGF(1 alpha) synthesis. These data suggest that, in CEC of the rabbit heart, ACh s timulates 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.