Nitric oxide synthesis in endothelial cytosol: evidence for a calcium-dependent and a calcium-independent mechanism.
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.
Mülsch
A
A
Department of Applied Physiology, University of Freiburg, Federal Republic of Germany.
Bassenge
E
E
Busse
R
R
D000634:Aminoquinolines / Q000494:pharmacology; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D002122:Calcium Chloride / Q000494:pharmacology; D002849:Chromatography, Gas; D003600:Cytosol / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000378:metabolism*; D006162:Guanylate Cyclase / Q000378:metabolism; D066298:In Vitro Techniques; D009249:NADP / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D019335:Nitroarginine; D013552:Swine
D000634:Aminoquinolines; D019335:Nitroarginine; D009569:Nitric Oxide; D009249:NADP; C041715:6-anilino-5,8-quinolinedione; D001120:Arginine; D006162:Guanylate Cyclase; D002122:Calcium Chloride; D002118:Calcium