SITES OF SUPEROXIDE ANION PRODUCTION DETECTED BY LUCIGENIN IN CALF PULMONARY-ARTERY SMOOTH-MUSCLE

Sources of superoxide anion (O-2(-).) production in calf pulmonary artery smooth muscle homogenate and subcellular fractions were examined in this study by measurement of the chemiluminescence produced by the reaction of O-2(-). with 50 mu M lucigenin, because recent evidence suggests that endogenously produced reactive O-2 species appear to mediate certain vascular responses. In the homogenate fraction, an NADH (0.1 mM)-dependent oxidoreductase activity was the major detected source of chemiluminescence. NADPH (0.1 mM) produced only 3% of the O-2(-). observed with NADH. Quantitation of certain other potential sources of O-2(-). (under optimized conditions), including xanthine oxidase (0.1 mM hypoxanthine), mitochondria (5 mM succinate + 30 mu M antimycin), cyclooxygenase/lipoxygenase (1 mu M arachidonic acid + 0.1 mM NADPH), or autooxidation (0.1 mg/ml superoxide dismutase), resulted in the detection of minimal amounts (<3% of NADH) of chemiluminescence. Estimation of mitochondrial O-2(-). production from tissue respiration rates suggests that lucigenin is a poor detector of intramitochondrial O-2(-).. These observations were confirmed by examination of chemiluminescence produced by subcellular fractions, where the major activity detected was an NADH oxidoreductase, which fractionated in a manner closely matching the activity of the microsomal marker enzyme rotenone-insensitive NADH-cytochrome c reductase. Because this NADH oxidoreductase appears to be a major vascular smooth muscle-derived source of O-2(-). production, this system has the potential to be an important endogenous source for the generation of vasoactive reactive O-2 species.