Calcium-dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin.
We investigated whether calmodulin mediates the stimulating effect of Ca2+ on nitric oxide synthase in the cytosol of porcine aortic endothelial cells. Nitric oxide was quantified by activation of a purified soluble guanylate cyclase. The Ca2(+)-sensitivity of nitric oxide synthase was lost after anion exchange chromatography of the endothelial cytosol and could only be reconstituted by addition of calmodulin or heat-denatured endothelial cytosol. The Ca2(+)-dependent activation of nitric oxide synthase in the cytosol was inhibited by the calmodulin-binding peptides/proteins melittin, mastoparan, and calcineurin (IC50 450, 350 and 60 nM, respectively), but not by the calmodulin antagonist, calmidazolium. In contrast, Ca2(+)-calmodulin-reconstituted nitric oxide synthase was inhibited with similar potency by melittin and calmidazolium. The results suggest that the Ca2(+)-dependent activation of nitric oxide synthase in endothelial cells is mediated by calmodulin.
Department of Applied Physiology, University of Freiburg, FRG.
R
R
Busse
A
A
Mülsch
D000594:Amino Acid Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D019703:Calcineurin; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000494:pharmacology* / Q000502:physiology; D002148:Calmodulin-Binding Proteins / Q000494:pharmacology; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D004789:Enzyme Activation; D006162:Guanylate Cyclase / Q000378:metabolism; D007093:Imidazoles / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D008555:Melitten / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase; D010455:Peptides; D010749:Phosphoprotein Phosphatases / Q000494:pharmacology; D013552:Swine; D014862:Wasp Venoms / Q000494:pharmacology
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D007093:Imidazoles; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D014862:Wasp Venoms; D008555:Melitten; D009569:Nitric Oxide; C031938:calmidazolium; C037500:mastoparan; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D006162:Guanylate Cyclase; D002118:Calcium