2-methyl-6-(4-methoxyphenyl)-3-7-dihydroimidazo(1-2-alpha)pyrazin-3-one and 10-10--dimethyl-9-9--biacridinium

Lucigenin-dependent chemiluminescence together with 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H tetrazolium monosodium salt (WST-1) reduction can be detected following addition of NADH to many cell types, including human sperm suspensions. Although many reports suggest that such a phenomenon is due to reactive oxygen species production, other oxygen detecting metabolite probes, such as MCLA and luminol, do not produce a chemiluminescent signal in this model system. The enzyme responsible for NADH-dependent lucigenin chemiluminescence was purified and identified as cytochrome-b5 reductase. In support of this concept, COS-7 cells overexpressing cytochrome-b5 reductase displayed at least a 3-fold increase in the previously mentioned activity compared with mock-transfected cells. Fractions containing cytochrome-b5 reductase were capable of inducing both lucigenin-dependent chemiluminescence and WST-1 reduction. Oxygen radicals clearly did not mediate the cytochrome b5-mediated activation of these probes in vitro since neither luminol nor MCLA gave a chemiluminescence response in the presence of the enzyme and the cofactor NADH. These results emphasize the importance of the direct NADH-dependent reduction of these putative superoxide-sensitive probes by cytochrome-b5 reductase even though this enzyme does not, on its own accord, produce reactive oxygen species.

Topics: Acridines; Animals; Blotting, Western; Chlorocebus aethiops; Coloring Agents; COS Cells; Cytochrome-B(5) Reductase; Humans; Imidazoles; Luminescent Measurements; Male; NAD; Oxidation-Reduction; Pyrazines; Reverse Transcriptase Polymerase Chain Reaction; RNA; Spectrometry, Fluorescence; Spermatozoa; Tetrazolium Salts; Transfection

Since an increased endothelial superoxide formation plays an important role in the pathogenesis of endothelial dysfunction its specific detection is of particular interest. The widely used superoxide probe lucigenin, however, has been reported to induce superoxide under certain conditions, especially in the presence of NADH. This raises questions as to the conclusion of a NAD(P)H oxidase as the major source of endothelial superoxide. Using independent methods, we showed that lucigenin in the presence of NADH leads to the production of substantial amount of superoxide (approximately 15-fold of control) in endothelial cell homogenates. On the other hand, these independent methods revealed that endothelial cells without lucigenin still produce superoxide in a NAD(P)H-dependent manner. This was blocked by inhibitors of the neutrophil NADPH oxidase diphenyleniodonium and phenylarsine oxide. Our results demonstrate that a NAD(P)H-dependent oxidase is an important source for endothelial superoxide but the latter, however, cannot be measured reliably by lucigenin.

Topics: Acridines; Arsenicals; Cells, Cultured; Cytochrome c Group; Imidazoles; Luminescent Measurements; NAD; NADH, NADPH Oxidoreductases; NADP; NADPH Oxidases; Neutrophils; Onium Compounds; Pyrazines; Superoxides; Umbilical Veins; Xanthine; Xanthine Oxidase