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.

Cytosolic calcium ([Ca 2ϩ ] i ) is an important mediator of neuronal signal transduction, participating in diverse biochemical reactions that elicit changes in synaptic efficacy, metabolic rate, and gene transcription. Excessive [Ca 2ϩ ] i also has been implicated as a cause of acute neuronal injury, although measurement of [Ca 2ϩ (Rothman and Olney, 1986;Choi, 1992). Studies using primary neuronal culture systems have shown that cultured neurons are selectively vulnerable to brief applications of glutamate (Rothman, 1985; or to selective agonists of the NMDA class of glutamate receptors (Rothman, 1985;Choi et al., 1988;Hartley and Choi, 1989). In contrast, prolonged activation of the AM PA or kainate receptors is required to produce neurotoxicity (Koh et al., 1990).Schanne and colleagues (1979) originally described the dependence of extracellular calcium in hepatocyte toxicity, which provided a potential mechanism for neuronal toxicity with the subsequent discovery that glutamate receptor activation produced neuronal calcium influx (Connor et al., 1987(Connor et al., , 1988Murphy et al., 1987). Additional studies by other investigators led to the hypothesis that calcium entry was responsible for excitotoxic neuronal injury (Choi, 1987) (for review, see Dubinsky, 1993a). A correlation between intracellular calcium levels and glutamate toxicity was suggested by the dependence of the latter on extracellular calcium Rothman et al., 1987 influx during nonlethal AMPA treatment (Marcoux et al., 1988;Eimerl and Schramm, 1994; Lu et al., 1996). The study by Hartley and coworkers (1993) suggested that a direct relationship existed between calcium accumulation and subsequent death in neurons exposed to NMDA. Although previous studies using fluorescent intracellular calcium indicators have noted that lethal excitotoxic insults may be followed by prolonged or delayed [Ca 2ϩ ] i elevation (Ogura et al., 1988;de Erausquin et al., 1990;Randall and Thayer, 1992;Dubinsky, 1993b; Tymianski et al., 1993a;Limbrick et al., 1995), such indicators have failed to demonstrate a consistent difference between intracellular free calcium concentration ([Ca 2ϩ ] i ) in cells during lethal and nonlethal challenges (de Erausquin et al., 1990;Michaels and Rothman, 1990; Tymianski et al., 1993a).One potential interpretation of these results is that there is not a direct relationship between the magnitude of acute [Ca 2ϩ ] i elevation and the extent of subsequent neuronal death. Another possibility is that current methods of [Ca 2ϩ ] i measurement fail to distinguish toxic levels of [Ca 2ϩ ] i . In the present study we examine the hypothesis that conventional, high-affinity calcium indicators (e.g., fura-2) are unable to measure accurately the [Ca 2ϩ ] i in the micromolar range and therefore fail to detect differences in [Ca 2ϩ ] i between lethal and nonlethal excitotoxic exposures. We Received April 18, 1997; revised June 2, 1997; accepted June 12, 1997. This work was supported by National Institutes o...

Patients with several inherited human encephalomyopathies exhibit systemic and neurological symptoms in association with specific mitochondrial mutations. The mechanisms by which these mitochondrial mutations result in cellular injury have not been elucidated. One potential cause of neuronal vulnerability is an inability to effectively buffer intracellular calcium. We report that fibroblasts from patients with one specific inherited encephalomyopathy, MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) syndrome, have elevated levels of ionized calcium and cannot normally sequester calcium influxes. Quantitative fluorescence imaging demonstrated that this abnormality was associated with a relative decrease in mitochondrial membrane potential compared to control fibroblasts. This documentation of pathological calcium homeostasis in a genetic neurological disease extends the calcium hypothesis of toxic cell injury to human mitochondrial encephalomyopathies.

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