F1F0-ATPase, early target of the radical initiator 2,2'-azobis-(2-amidinopropane) dihydrochloride in rat liver mitochondria in vitro.
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.
Beauseigneur
F
F
EA DRED 1867, Dijon, France.
Goubern
M
M
Chapey
M F
MF
Gresti
J
J
Vergely
C
C
Tsoko
M
M
Demarquoy
J
J
Rochette
L
L
Clouet
P
P
D000578:Amidines / Q000494:pharmacology*; D000818:Animals; D001278:Atractyloside / Q000031:analogs & derivatives / Q000494:pharmacology; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D003497:Cyclic N-Oxides; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D005260:Female; D005609:Free Radicals / Q000378:metabolism; D007425:Intracellular Membranes / Q000187:drug effects / Q000502:physiology*; D007700:Kinetics; D015227:Lipid Peroxidation / Q000187:drug effects; D008315:Malondialdehyde / Q000032:analysis; D008564:Membrane Potentials / Q000502:physiology; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D010085:Oxidative Phosphorylation / Q000187:drug effects*; D010101:Oxygen Consumption / Q000187:drug effects; D006180:Proton-Translocating ATPases / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013113:Spin Labels; D013386:Succinates / Q000378:metabolism
D000578:Amidines; D003497:Cyclic N-Oxides; D005609:Free Radicals; D013113:Spin Labels; D013386:Succinates; D001278:Atractyloside; D008315:Malondialdehyde; D004024:Dicyclohexylcarbodiimide; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; C017245:5,5-dimethyl-1-pyrroline-1-oxide; C046728:2,2'-azobis(2-amidinopropane); D006180:Proton-Translocating ATPases; C003853:carboxyatractyloside