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 (O-2(-).) in cultured bovine coronary endothelium, employing lucigenin (250 mu M)-elicited chemiluminescence (CL). In the homogenate from these cells, 100 mu M NADPH increased O-2(-) 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 mu M 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 O-2(-). superoxide dismutase (100 mu g/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 mu M) nor antimycin (10 mu M) + succinate (5 mM) had any significant effect on basal CL levels, thereby excluding xanthine oxidase and mitochondria, respectively, as a detectable sources of O-2(-). generation. The presence of NAD(+) (100 mu M) 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 O-2(-). production. Diphenyliodonium at 1 and 10 mu M inhibited both NADH-mediated CL in homogenate and lactate-mediated CL in intact endothelium by 50 and 33%, respectively. Nitric oxide synthase was not a detectable cellular source of O-2(-). since NG-nitro-L-arginine (0.1 mM) did not alter CL from intact endothelium. Exposure of cells or the homogenate to arachidonic acid (10 mu M) also caused minor increases in CL, originating from sources that remain to be identified. Thus NADH-oxidoreductase is the major source of O-2(-). detected by CL in bovine coronary endothelium.