| 15228597 |
Gyulkhandanyan AV, Pennefather PS: Shift in the localization of sites of hydrogen peroxide production in brain mitochondria by mitochondrial stress. J Neurochem. 2004 Jul;90(2):405-21.
We have determined the underlying sites of H (2) O (2) generation by isolated rat brain mitochondria and how these can shift depending on the presence of respiratory substrates, electron transport chain modulators and exposure to stressors. H (2) O (2) production was determined using the fluorogenic Amplex red and peroxidase system. H (2) O (2) production was higher when succinate was used as a respiratory substrate than with another FAD-dependent substrate, alpha-glycerophosphate, or with the NAD-dependent substrates, glutamate/malate. Depolarization by the uncoupler p-trifluoromethoxyphenylhydrazone decreased H (2) O (2) production stimulated by all respiratory substrates. H (2) O (2) production supported by succinate during reverse transfer of electrons was decreased by inhibitors of complex I (rotenone and diphenyleneiodonium) whereas in glutamate/malate-oxidizing mitochondria diphenyleneiodonium decreased while rotenone increased H (2) O (2) generation. The complex III inhibitors antimycin and myxothiazol decreased succinate-induced H (2) O (2) production but stimulated H (2) O (2) production in glutamate/malate-oxidizing mitochondria. Antimycin and myxothiazol also increased H (2) O (2) production in mitochondria using alpha-glycerophosphate as a respiratory substrate. In substrate/inhibitor experiments maximal stimulation of H (2) O (2) production by complex I was observed with the alpha-glycerophosphate/antimycin combination. In addition, three forms of in vitro mitochondrial stress were studied: Ca (2+) overload, cold storage for more than 24 h and cytochrome c depletion. In each case we observed (i) a decrease in succinate-supported H (2) O (2) production by complex I and an increase in succinate-supported H (2) O (2) production by complex III, (ii) increased glutamate/malate-induced H (2) O (2) generation by complex I and (iii) increased alpha-glycerophosphate-supported H (2) O (2) generation by complex III. Our results suggest that all three forms of mitochondrial stress resulted in similar shifts in the localization of sites of H (2) O (2) generation and that, in both normal and stressed states, the level and location of H (2) O (2) production depend on the predominant energetic substrate. |
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