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

Sources of superoxide anion (O-2(-).) production in calf pulmonary art ery smooth muscle homogenate and subcellular fractions were examined i n this study by measurement of the chemiluminescence produced by the r eaction of O-2(-). with 50 mu M lucigenin, because recent evidence sug gests that endogenously produced reactive O-2 species appear to mediat e certain vascular responses. In the homogenate fraction, an NADH (0.1 mM)-dependent oxidoreductase activity was the major detected source o f chemiluminescence. NADPH (0.1 mM) produced only 3% of the O-2(-). ob served with NADH. Quantitation of certain other potential sources of O -2(-). (under optimized conditions), including xanthine oxidase (0.1 m M hypoxanthine), mitochondria (5 mM succinate + 30 mu M antimycin), cy clooxygenase/lipoxygenase (1 mu M arachidonic acid + 0.1 mM NADPH), or autooxidation (0.1 mg/ml superoxide dismutase), resulted in the detec tion of minimal amounts (<3% of NADH) of chemiluminescence. Estimation of mitochondrial O-2(-). production from tissue respiration rates sug gests 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 mat ching the activity of the microsomal marker enzyme rotenone-insensitiv e NADH-cytochrome c reductase. Because this NADH oxidoreductase appear s to be a major vascular smooth muscle-derived source of O-2(-). produ ction, this system has the potential to be an important endogenous sou rce for the generation of vasoactive reactive O-2 species.