title: Mitochondrial complex I, aconitase, and succinate dehydrogenase during hypoxia-reoxygenation: modulation of enzyme activities by MnSOD.
abstract: Both NADH dehydrogenase (complex I) and aconitase are inactivated partially in vitro by superoxide (O2-.) and other oxidants that cause loss of iron from enzyme cubane (4Fe-4S) centers. We tested whether hypoxia-reoxygenation (H-R) by itself would decrease lung epithelial cell NADH dehydrogenase, aconitase, and succinate dehydrogenase (SDH) activities and whether transfection with adenoviral vectors expressing MnSOD (Ad.MnSOD) would inhibit oxidative enzyme inactivation and thus confirm a mechanism involving O2-. Human lung carcinoma cells with alveolar epithelial cell characteristics (A549 cells) were exposed to <1% O2-5% CO2 (hypoxia) for 24 h followed by air-5% CO2 for 24 h (reoxygenation). NADH dehydrogenase activity was assayed in submitochondrial particles; aconitase and SDH activities were measured in cell lysates. H-R significantly decreased NADH dehydrogenase, aconitase, and SDH activities. Ad.MnSOD increased mitochondrial MnSOD substantially and prevented the inhibitory effects of H-R on enzyme activities. Addition of alpha-ketoglutarate plus aspartate, but not succinate, to medium prevented cytotoxicity due to 2,3-dimethoxy-1,4-naphthoquinone. After hypoxia, cells displayed significantly increased dihydrorhodamine fluorescence, indicating increased mitochondrial oxidant production. Inhibition of NADH dehydrogenase, aconitase, and SDH activities during reoxygenation are due to excess O2-. produced in mitochondria, because enzyme inactivation can be prevented by overexpression of MnSOD.
authors.affiliation: Birmingham Department of Veterans Affairs Medical Center, Birmingham, AL 35233, USA.
authors.forename: Charles S
authors.initials: CS
authors.lastname: Powell
authors.forename: Robert M
authors.initials: RM
authors.lastname: Jackson
mesh_terms: D000154:Aconitate Hydratase / Q000378:metabolism; D000231:Adenocarcinoma, Papillary; D000256:Adenoviridae / Q000235:genetics; D001224:Aspartic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D042967:Electron Transport Complex I; D004789:Enzyme Activation / Q000187:drug effects / Q000528:radiation effects; D004847:Epithelial Cells / Q000166:cytology / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic; D005822:Genetic Vectors; D006801:Humans; D007656:Ketoglutaric Acids / Q000494:pharmacology; D008027:Light; D008928:Mitochondria / Q000201:enzymology*; D009245:NADH Dehydrogenase / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D010100:Oxygen / Q000494:pharmacology; D011837:Radiation-Protective Agents / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D015427:Reperfusion Injury / Q000378:metabolism*; D020545:Respiratory Mucosa / Q000166:cytology / Q000201:enzymology*; D012235:Rhodamines; D013385:Succinate Dehydrogenase / Q000378:metabolism; D019802:Succinic Acid / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D007656:Ketoglutaric Acids; D011837:Radiation-Protective Agents; D017382:Reactive Oxygen Species; D012235:Rhodamines; C098097:dihydrorhodamine 6G; D001224:Aspartic Acid; D019802:Succinic Acid; D013482:Superoxide Dismutase; D013385:Succinate Dehydrogenase; D009247:NADH, NADPH Oxidoreductases; D009245:NADH Dehydrogenase; D000154:Aconitate Hydratase; D042967:Electron Transport Complex I; D010100:Oxygen; D004229:Dithiothreitol