Sites of superoxide anion production detected by lucigenin in calf pulmonary artery smooth muscle

Sources of superoxide anion (O2-.) 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 O2-. with 50 microM lucigenin, because recent evidence suggests that endogenously produced reactive O2 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 O2-. observed with NADH. Quantitation of certain other potential sources of O2-. (under optimized conditions), including xanthine oxidase (0.1 mM hypoxanthine), mitochondria (5 mM succinate + 30 microM antimycin), cyclooxygenase/lipoxygenase (1 microM 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 O2-. production from tissue respiration rates suggests that lucigenin is a poor detector of intramitochondrial O2-.. 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 O2-. production, this system has the potential to be an important endogenous source for the generation of vasoactive reactive O2 species.