Nitric oxide synthesis in endothelial cytosol: evidence for a calcium-dependent and a calcium-independent mechanism

Abstract

Release of nitric oxide (NO) from endothelial cells critically depends on a sustained increase in intracellular free calcium maintained by a transmembrane calcium influx into the cells. Therefore, we studied whether the free cytosolic calcium concentration directly affects the activity of the NO-forming enzyme(s) present in the cytosol from freshly harvested porcine aortic endothelial cells. NO was quantified by activation of a purified soluble guanylate cyclase co-incubated with the cytosol. In the presence of 1 mM L-arginine, 0.1 mM NADPH and 0.1 mM EGTA, endothelial cytosol (0.2 mg of cytosolic protein per ml) stimulated the activity of guanylate cyclase 5.0 +/- 0.5-fold (from 31 +/- 9 to 153 +/- 15 nmol cyclic GMP formed per min per mg guanylate cyclase). Calcium chloride increased this stimulation further in a concentration-dependent fashion by up to 136 +/- 15% (with 2 microM free calcium; EC50 0.3 microM). The calcium-dependent and -independent activation of guanylate cyclase was enhanced by superoxide dismutase (0.3 microM) and was inhibited by the stereospecifically acting inhibitor of L-arginine-dependent NO formation NG-nitro-L-arginine (1 mM) and by LY 83583 (1 microM), a generator of superoxide anions. Our findings suggest a calcium-dependent and -independent synthesis of NO from L-arginine by native porcine aortic endothelial cells.