REGULATION OF CA2+-DEPENDENT NITRIC-OXIDE SYNTHASE IN BOVINE AORTIC ENDOTHELIAL-CELLS

Vascular endothelium responds to Ca2+-mobilizing agonists by producing nitric oxide (NO), a potent vasodilator and inhibitor of platelet aggregation. Regulation of constitutively expressed endothelial NO synthase (eNOS) in intact cells is not well understood. We investigated the kinetics of NO formation in response to Ca2+-mobilizing agonists, the requirement for extracellular L-arginine, and the role of NO in regulating eNOS activity. When endothelial cells were stimulated with bradykinin and ATP in the presence of 100 mu M L-arginine, we observed a rapid and transient rise in intracellular Ca2+ concentration ([Ca2+](i)) from 50 +/- 8 nM to 698 +/- 74 and 637 +/- 53 nM, respectively, and a rapid and transient rise in NO production from a basal level of 37 pmol . min(-1) . mg protein(-1) to 256 and 275 pmol . min(-1) . mg protein(-1), respectively. When cells were stimulated with A-23187 or thapsigargin in the presence of 100 mu M L-arginine, we observed a sustained increase in [Ca2+](i) and a sustained increase in NO production. The rate of NO synthesis was linear over 30 min, rising above control levels of 7 pmol/min to 53 pmol/min for A-23187 and 62 pmol/min for thapsigargin. Thapsigargin stimulated NO production and [Ca2+](i) with 50% effective concentration values of 0.01 and 0.05 mu M, respectively. Ca2+-stimulated NO production was attenuated by the NO synthase inhibitor N-G-monomethyl-L-arginine, the removal of extracellular L-arginine, and the Ca2+-chelator ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid. When we exposed cells to NO gas (3.1 mM for 15 min) and S-nitrosoglutathione (10 mM for 1 h), thapsigargin-stimulated NO production was decreased by 50%. These studies in intact cells demonstrate that eNOS activity is tightly coupled to Ca2+, mirroring agonist-stimulated rises and falls in [Ca2+](i). In addition, eNOS requires supplementation with low levels of extracellular L-arginine to support a full response. Finally, although eNOS is inhibited by high doses of exogenous NO, it is not sensitive to feedback inhibition by endogenous NO.