Cellular mechanisms controlling EDRF/NO formation in endothelial cells.
We investigated the molecular mechanisms whereby Ca2+ enters the endothelial cytosol and regulates endothelial nitric oxide synthesis L-arginine-dependent nitric oxide synthesis by isolated endothelial cytosol as quantified by activation of a purified soluble guanylate cyclase was concentration-dependently enhanced by free Ca2+ (EC50 0.3 microM). The Ca(2+)-dependent activation was inhibited by the calmodulin antagonists mastoparan, melittin, and calcineurin (IC50 450, 350, and 60 nM, respectively) in a calmodulin-reversible manner. After removal of endogenous calmodulin the Ca(2+)-dependency of endothelial NO synthase was lost, but could be reconstituted with exogenous calmodulin. The results indicate that Ca(2+)-calmodulin directly activates the endothelial nitric oxide synthase, thereby transducing agonist-induced increases in intracellular free Ca2+ concentration to nitric oxide formation from L-arginine, K(+)-induced depolarization of the endothelial cells markedly inhibited the sustained, but not initial phase of the intracellular Ca2+ response to bradykinin, indicating that K(+)-induced depolarization depresses the transmembrane Ca2+ influx. On the contrary, the K+ channel activator Hoe 234 which elicits hyperpolarization of the endothelial cell membrane, augmented the sustained phase of the agonist-induced intracellular Ca2+ signal, but not the resting intracellular Ca2+ level. The effects of K+ and Hoe 234 on the agonist-induced Ca(2+)-response were reflected by corresponding changes in agonist-induced EDRF/NO release. From these data, we suggest that the endothelial membrane potential may play an important role for the extent of agonist-induced Ca2+ influx and, thereby, the endothelial EDRF/NO synthesis.
Busse
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R
Department of Applied Physiology, University of Freiburg, FRG.
Lückhoff
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Mülsch
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D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D000818:Animals; D001011:Aorta; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000494:pharmacology; D002417:Cattle; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology*; D004789:Enzyme Activation / Q000187:drug effects; D006162:Guanylate Cyclase / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase; D013552:Swine
D002147:Calmodulin; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D006162:Guanylate Cyclase; D001920:Bradykinin; D002118:Calcium