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Hydrogen peroxide removal and glutathione mixed disulfide formation during metabolic inhibition in mesencephalic cultures
Author(s) -
Ehrhart Julie,
Zeevalk Gail D.
Publication year - 2001
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.2001.00355.x
Subject(s) - malonate , buthionine sulfoximine , glutathione , oxidative stress , catalase , reactive oxygen species , chemistry , biochemistry , glutathione disulfide , toxicity , aminooxyacetic acid , hydrogen peroxide , pharmacology , biology , enzyme , organic chemistry
Compromised mitochondrial energy metabolism and oxidative stress have been associated with the pathophysiology of Parkinson's disease. Our previous experiments exemplified the importance of GSH in the protection of neurons exposed to malonate, a reversible inhibitor of mitochondrial succinate dehydrogenase/complex II. This study further defines the role of oxidative stress during energy inhibition and begins to unravel the mechanisms by which GSH and other antioxidants may contribute to cell survival. Treatment of mesencephalic cultures with 10 µ m buthionine sulfoximine for 24 h depleted total GSH by 60%, whereas 3 h exposure to 5 m m 3‐amino‐1,2,4‐triazole irreversibly inactivated catalase activity by 90%. Treatment of GSH‐depleted cells with malonate (40 m m ) for 6, 12 or 24 h both potentiated and accelerated the time course of malonate toxicity, however, inhibition of catalase had no effect. In contrast, concomitant treatment with buthionine sulfoximine plus 3‐amino‐1,2,4‐triazole in the presence of malonate significantly potentiated toxicity over that observed with malonate plus either inhibitor alone. Consistent with these findings, GSH depletion enhanced malonate‐induced reactive oxygen species generation prior to the onset of toxicity. These findings demonstrate that early generation of reactive oxygen species during mitochondrial inhibition contributes to cell damage and that GSH serves as a first line of defense in its removal. Pre‐treatment of cultures with 400 µ m ascorbate protected completely against malonate toxicity (50 m m , 12 h), whereas treatment with 1 m m Trolox provided partial protection. Protein–GSH mixed disulfide formation during oxidative stress has been suggested to either protect vulnerable protein thiols or conversely to contribute to toxicity. Malonate exposure (50 m m ) for 12 h resulted in a modest increase in mixed disulfide formation. However, exposure to the protective combination of ascorbate plus malonate increased membrane bound protein–GSH mixed disulfides three‐fold. Mixed disulfide levels returned to baseline by 72 h of recovery indicating the reversible nature of this formation. These results demonstrate an early role for oxidative events during mitochondrial impairment and stress the importance of the glutathione system for removal of reactive oxygen species. Catalase may serve as a secondary defense as the glutathione system becomes limiting. These findings also suggest that protein–GSH mixed disulfide formation under these circumstances may play a protective role.