Mitochondrial production of reactive oxygen species in cortical neurons following exposure to N-methyl-D-aspartate.
Increasing evidence suggests that glutamate neurotoxicity is partly mediated by reactive oxygen species, formed as a consequence of several processes, including arachidonic acid metabolism and nitric oxide production. Here we used an oxidation-sensitive indicator, dihydrorhodamine 123, in combination with confocal microscopy, to examine the hypothesis that electron transport by neuronal mitochondria may be an important source of glutamate-induced reactive oxygen species (ROS). Exposure to NMDA, but not kainate, ionomycin, or elevated potassium stimulated oxygen radical production in cultured murine cortical neurons, demonstrated by oxidation of nonfluorescent dihydrorhodamine 123 to fluorescent rhodamine 123. Electron paramagnetic resonance spectroscopy studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a radical-trapping agent, also showed production of ROS by cortical neurons after NMDA but not kainate exposure. NMDA-induced ROS production depended on extracellular Ca2+, and was not affected by inhibitors of nitric oxide synthase or arachidonic acid metabolism. The increased production of ROS was blocked by inhibitors of mitochondrial electron transport, rotenone or antimycin, and mimicked by the electron transport uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. These data support the possibility that NMDA receptor-mediated, Ca(2+)-dependent uncoupling of neuronal mitochondrial electron transport may contribute to the oxidative stress initiated by glutamate exposure.
D000818:Animals; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D051379:Mice; D008815:Mice, Inbred Strains; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D016202:N-Methylaspartate / Q000494:pharmacology*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism*; D012235:Rhodamines / Q000378:metabolism
D017382:Reactive Oxygen Species; D012235:Rhodamines; C058319:dihydrorhodamine 123; D016202:N-Methylaspartate