Direct and reversible inhibition of endothelial nitric oxide synthase by nitric oxide.
The objective of this study was to investigate the regulation of endothelial nitric oxide (NO) synthase by NO. Partially purified endothelial NO synthase was exposed to authentic NO (10-200 microM) and to the nitrovasodilators sodium nitroprusside (SNP; 10-1,000 microM) and S-nitroso-N-acetylpenicillamine (SNAP; 100-1,000 microM), and enzyme activity was assayed by measuring the conversion of L-[3H]arginine to L-[3H]citrulline in the presence of added cofactors. NO, SNP, and SNAP inhibited NO synthase activity in a dose-dependent manner, NO being the most potent inhibitor. The Michaelis constant for L-arginine was not altered (4.87 microM) by NO (50 microM), whereas the maximal velocity of the enzyme decreased from 784 to 633 pmol.mg-1.min-1. Oxyhemoglobin (10 microM) partially prevented the inhibition of NO synthase by NO (50 microM). The data also suggest that NO inhibits endothelial NO synthase activity by directly interacting with the NO synthase and not by an indirect mechanism such as limitation of cofactor or oxygen availability. Dialysis of NO synthase treated with NO (50 microM) partially restored the enzyme activity. This study demonstrates a direct and reversible inhibition of NO synthase by NO, suggesting a feedback mechanism in vivo.
Ravichandran
L V
LV
Department of Anesthesiology, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
Johns
R A
RA
Rengasamy
A
A
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D000704:Analysis of Variance; D000818:Animals; D001011:Aorta; D001120:Arginine / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D002956:Citrulline / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000201:enzymology*; D007700:Kinetics; D008163:Luminescent Measurements; D009569:Nitric Oxide / Q000494:pharmacology*; D019001:Nitric Oxide Synthase; D009599:Nitroprusside / Q000494:pharmacology; D010108:Oxyhemoglobins / Q000494:pharmacology; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D026423:S-Nitroso-N-Acetylpenicillamine; D014316:Tritium; D014665:Vasodilator Agents / Q000494:pharmacology
D010108:Oxyhemoglobins; D014665:Vasodilator Agents; D014316:Tritium; D009599:Nitroprusside; D002956:Citrulline; D009569:Nitric Oxide; D026423:S-Nitroso-N-Acetylpenicillamine; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D010396:Penicillamine; D004229:Dithiothreitol