MITOCHONDRIAL PRODUCTION OF REACTIVE OXYGEN SPECIES IN CORTICAL-NEURONS FOLLOWING EXPOSURE TO N-METHYL-D-ASPARTATE

Increasing evidence suggests that glutamate neurotoxicity is partly me diated by reactive oxygen species, formed as a consequence of several processes, including arachidonic acid metabolism and nitric oxide prod uction. Here we used an oxidation-sensitive indicator, dihydrorhodamin e 123, in combination with confocal microscopy, to examine the hypothe sis that electron transport by neuronal mitochondria may be an importa nt 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, demons trated by oxidation of nonfluorescent dihydrorhodamine 123 to fluoresc ent rhodamine 123. Electron paramagnetic resonance spectroscopy studie s using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a radical-trapping agent, also showed production of ROS by cortical neurons after NMDA bu t not kainate exposure. NMDA-induced ROS production depended on extrac ellular Ca2+, and was not affected by inhibitors of nitric oxide synth ase or arachidonic acid metabolism. The increased production of ROS wa s was blocked by inhibitors of mitochondrial electron transport, roten one or antimycin, and mimicked by the electron transport uncoupler, ca rbonyl cyanide p-trifluoromethoxyphenylhydrazone. These data support t he possibility that NMDA receptor-mediated, Ca2+-dependent uncoupling of neuronal mitochondrial electron transport may contribute to the oxi dative stress initiated by glutamate exposure.