Superoxide production from paraquat evoked by exogenous NADPH in pulmonary endothelial cells

Superoxide production from paraquat in a pulmonary microvascular endothelial cell (PMEC) suspension was demonstrated using 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyraz in-3-one (MCLA), a chemiluminescence probe, to detect superoxide anions. Increased rates of superoxide production from paraquat, which were sensitive to superoxide dismutase (SOD), required the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) in the reaction medium, and occurred instantaneously after the addition of NADPH, which is impermeable to cell membranes. NADH as an electron donor was not as effective, and xanthine or succinate had no influence. Paraquat was anaerobically reduced in the presence of NADPH and PMECs to yield a one-electron reduced radical, and the reduction was inhibited by NADP+. Diphenyleneiodonium, an inhibitor of flavoprotein reductases, also markedly inhibited both paraquat reduction and superoxide production. These results indicate that NADPH-dependent superoxide production from paraquat probably occurs by a flavoprotein with NADPH-dependent reductase activity in cell membranes. NADPH-dependent superoxide production from paraquat was also reproduced using adherent PMECs on wells. Under these conditions, superoxide production was enhanced with agonists, including interleukin-1beta, A23187, and phorbol 12-myristate 13-acetate. The effect of the former two was blocked with staurosporine, while the latter's effect was suppressed with calyculin A.