title: Ca2+-independent permeabilization of the inner mitochondrial membrane by peroxynitrite is mediated by membrane protein thiol cross-linking and lipid peroxidation.
abstract: Peroxynitrite anion, the reaction product of superoxide and nitric oxide, is a potent biological oxidant, which inactivates mammalian heart mitochondrial NADH-coenzyme Q reductase (complex I), succinate dehydrogenase (complex II), and ATPase, without affecting cytochrome c oxidase (complex IV). In this paper, we evaluated the effect of peroxynitrite on mitochondrial membrane integrity and permeability under low calcium concentration. Phosphate buffer was used in most of our experiments since Hepes, Tris, mannitol, and sucrose were found to inhibit the oxidative chemistry of peroxynitrite. Peroxynitrite (0.1-1.0 mM) caused a dose-dependent decrease in the ability of mitochondria to build up a membrane potential when N,N,N',N'-tetramethyl-p-phenylenediamine/ascorbate were used as substrate. Elimination of the membrane potential was accompanied by penetration of the osmotic support (KCl/NaCl) into the matrix as judged by the parallel occurrence of mitochondrial swelling. This swelling was partially inhibited by dithiothreitol (DTT) or butylated hydroxytoluene (BHT) and was insensitive to ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, ADP, and cyclosporin A. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins indicated that alterations in membrane permeability were associated with the production of protein aggregates due to membrane protein thiol cross-linking. The protective effect of DTT on both mitochondrial swelling and protein polymerization suggests the involvement of disulfide bonds in the membrane permeabilization process. In addition, the increase in thiobarbituric acid-reactive substances and the partial inhibitory effect of BHT indicate the occurrence of lipid peroxidation. These results support the idea that under our experimental conditions peroxynitrite causes mitochondrial structural and functional alterations by Ca2+-independent mechanisms through lipid peroxidation and protein sulfhydryl oxidation.
authors.affiliation: Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas, SP, Brazil.
authors.forename: F R
authors.initials: FR
authors.lastname: Gadelha
authors.forename: L
authors.initials: L
authors.lastname: Thomson
authors.forename: M M
authors.initials: MM
authors.lastname: Fagian
authors.forename: A D
authors.initials: AD
authors.lastname: Costa
authors.forename: R
authors.initials: R
authors.lastname: Radi
authors.forename: A E
authors.initials: AE
authors.lastname: Vercesi
mesh_terms: D000818:Animals; D002118:Calcium / Q000494:pharmacology; D003432:Cross-Linking Reagents; D005260:Female; D007425:Intracellular Membranes / Q000187:drug effects* / Q000378:metabolism; D015227:Lipid Peroxidation; D008564:Membrane Potentials / Q000187:drug effects; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000378:metabolism; D009566:Nitrates / Q000494:pharmacology*; D016877:Oxidants / Q000494:pharmacology; D010539:Permeability / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D013438:Sulfhydryl Compounds; D017392:Thiobarbituric Acid Reactive Substances / Q000032:analysis
chemical_list: D003432:Cross-Linking Reagents; D009566:Nitrates; D016877:Oxidants; D013438:Sulfhydryl Compounds; D017392:Thiobarbituric Acid Reactive Substances; C039642:peroxynitric acid; D002118:Calcium