antimycin and 10-10--dimethyl-9-9--biacridinium

To determine if 30 min of hypoxia (PO2 8-10 Torr) affects basal, mitochondrial, or xanthine oxidase-derived lucigenin-detectable superoxide anion (O2.-) production by intact isolated bovine coronary microvessels and myocytes.. O2.- was quantitated by lucigenin-elicited chemiluminescence. Antimycin A (10 microM) and hypoxanthine (0.1 mM) were employed to increase O2.- from mitochondria and xanthine oxidase, respectively.. Chemiluminescence from microvessels and myocytes was enhanced (approximately twofold, P < 0.05, n = 8-10) by inhibition of Cu,Zn-SOD via pretreatment with diethyldithiocarbamate (10 mM, 30 min) and was decreased (P < 0.05, n = 8-10) by an intracellular scavenger of O2.- (10 mM Tiron), but not by added SOD (3 microM, n = 8-10). In the presence of SOD inhibition, hypoxia produced a hypoxanthine-dependent (n = 8-10) twofold increase in chemiluminescence (P < 0.05, n = 10) in microvessels but not in myocytes. Other combinations of hypoxia, antimycin, or hypoxanthine did not significantly alter chemiluminescence.. Lucigenin appears to detect a basal intracellular source of O2.- in both microvessels and myocytes that is not derived from mitochondria or xanthine oxidase. Exposure to hypoxia does not appreciably increase basal O2.- in vessels or myocytes, but if exogenous hypoxanthine is supplied, microvessels show an increase in O2.- production presumably derived from xanthine oxidase.

Topics: 1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; Acridines; Animals; Antimycin A; Capillaries; Cattle; Coronary Circulation; Estradiol; Hypoxanthine; Hypoxanthines; In Vitro Techniques; Luminescent Measurements; Myocardium; Oxygen; Superoxide Dismutase; Superoxides; Xanthine Oxidase

We find spontaneous light emission from isolated Krebs-Henseleit-perfused rabbit lungs when the light-emitting super-oxide trap lucigenin is added to the perfusate. Lucigenin light emission appears to be specific for superoxide anion, because light emission from the lung caused by a superoxide-generating system is abolished by superoxide dismutase but not by catalase or dimethylthiourea. We also studied the relative sensitivity of lucigenin photoemission to superoxide and to H2O2 in vitro. Lucigenin photoemission is three to four orders of magnitude more sensitive to superoxide than to H2O2 and probably cannot detect H2O2 in concentrations thought to occur in biological systems. Basal lucigenin photoemission by the lung is oxygen dependent, because severe hypoxia completely inhibits light emission. Superoxide dismutase reduces basal photoemission by 50%, and administration of the low-molecular-weight superoxide scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron) inhibits basal photoemission by approximately 90%. These observations suggest that endogenous superoxide production is primarily intracellular and that approximately half of the superoxide reaches the extracellular space. Superoxide transport may involve anion channels, because the anion channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid increases photoemission, suggesting intracellular accumulation of superoxide. A cytochrome P-450 inhibitor, SKF 525A, or the mitochondrial transport inhibitor antimycin decreased basal photoemission by approximately 50%, suggesting that cytochrome P-450-mediated reactions and perhaps mitochondrial function contribute to basal superoxide production in the isolated perfused lung. Endogenous superoxide production may be important in regulation of pulmonary vascular reactivity and may contribute to the pathogenesis of lung reperfusion injury.

Topics: 1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Acridines; Animals; Antimycin A; Cyanides; Hypoxia; In Vitro Techniques; Luminescent Measurements; Lung; Perfusion; Proadifen; Rabbits; Superoxide Dismutase; Superoxides