atractyloside and 2-2--azobis(2-amidinopropane)

This study was designed to determine which enzyme activities were first impaired in mitochondria exposed to 2,2'-azobis-(2-amidinopropane) dihydrochloride (AAPH), a known radical initiator. EPR spin-trapping revealed generation of reactive oxygen species although malondialdehyde formation remained very low. With increasing AAPH concentrations, State-3 respiration was progressively depressed with unaltered ADP/O ratios. A top-down approach demonstrated that alterations were located at the phosphorylation level. As shown by inhibitor titrations, ATP/ADP translocase activity was unaffected in the range of AAPH concentrations used. In contrast, AAPH appeared to exert a deleterious effect at the level of F1F0-ATPase, comparable with dicyclohexylcarbodi-imide, which alters Fo proton channel. A comparison of ATP hydrolase activity in uncoupled and broken mitochondria reinforced this finding. In spite of its pro-oxidant properties, AAPH was shown to act as a dose-dependent inhibitor of cyclosporin-sensitive permeability transition initiated by Ca2+, probably as a consequence of its effect on F1F0-ATPase. Resveratrol, a potent antiperoxidant, completely failed to prevent the decrease in State-3 respiration caused by AAPH. The data suggest that AAPH, when used under mild conditions, acted as a radical initiator and was capable of damaging F1F0-ATPase, thereby slowing respiratory chain activity and reducing mitochondrial antioxidant defences.

Topics: Amidines; Animals; Atractyloside; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cyclic N-Oxides; Dicyclohexylcarbodiimide; Electron Spin Resonance Spectroscopy; Female; Free Radicals; Intracellular Membranes; Kinetics; Lipid Peroxidation; Malondialdehyde; Membrane Potentials; Mitochondria, Liver; Oxidative Phosphorylation; Oxygen Consumption; Proton-Translocating ATPases; Rats; Rats, Wistar; Spin Labels; Succinates