NADH oxidoreductase is a major source of superoxide anion in bovine coronary artery endothelium.
In this study we examined the intracellular sources of superoxide anion (O2-.) in cultured bovine coronary endothelium, employing lucigenin (250 microM)-elicited chemiluminescence (CL). In the homogenate from these cells, 100 microM NADPH increased O2-. by 81% from 8.9 +/- 1.5 to 16.0 +/- 1.5 x 10(5) cpm/mg protein (P < 0.01, n = 8). In the presence of 100 microM NADH, however, CL increased by 458% from 8.9 +/- 1.6 to 49.6 +/- 12.0 x 10(5) cpm/mg protein (P < 0.01, n = 8). Scavengers of O2-., superoxide dismutase (100 micrograms/ml), or 4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt (Tiron, 10 mM) inhibited NADH-mediated CL by 70 and 83%, respectively. Neither hypoxanthine (100 microM) nor antimycin (10 microM)+succinate (5 mM) had any significant effect on basal CL levels, thereby excluding xanthine oxidase and mitochondria, respectively, as a detectable sources of O2-. generation. The presence of NAD+ (100 microM) and lactate (1 mM) increased CL by 88% (n = 8, P < 0.01). In the intact cells, basal production of CL was increased by 205% (P < 0.01) by 5 mM lactate, but not by 5 mM pyruvate, and CL was inhibited by 10 mM Tiron, suggesting the reduction of cytosolic NAD by lactate dehydrogenase stimulates O2-. production. Diphenyliodonium at 1 and 10 microM inhibited both NADH-mediated CL in homogenate and lactate-mediated CL in intact endothelium by 50 and 33%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Mohazzab
K M
KM
Department of Physiology, New York Medical College, Valhalla 10595.
Kaminski
P M
PM
Wolin
M S
MS
D000166:Acridines; D000818:Animals; D000838:Anions / Q000378:metabolism; D016718:Arachidonic Acid / Q000378:metabolism; D002417:Cattle; D002478:Cells, Cultured; D003331:Coronary Vessels / Q000166:cytology / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D007773:Lactates / Q000494:pharmacology; D019344:Lactic Acid; D008163:Luminescent Measurements; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D014969:Xanthine Oxidase / Q000378:metabolism
D000166:Acridines; D000838:Anions; D007773:Lactates; D017382:Reactive Oxygen Species; D013481:Superoxides; C033472:10,10'-dimethyl-9,9'-biacridinium; D016718:Arachidonic Acid; D019344:Lactic Acid; D014969:Xanthine Oxidase; D009247:NADH, NADPH Oxidoreductases