Mitochondria, Calcium Regulation, and Acute Glutamate Excitotoxicity in Cultured Cerebellar Granule Cells

Abstract

Abstract: Exposure of cultured cerebellar granule cells to 100 µ M glutamate plus glycine in the absence of Mg 2+ causes calcium loading of the in situ mitochondria and is excitotoxic, as demonstrated by a collapse of the cellular ATP/ADP ratio, cytoplasmic Ca 2+ deregulation (the failure of the cell to maintain a stable cytoplasmic free Ca 2+ concentration), and extensive cell death. Glutamate‐evoked Ca 2+ deregulation is exacerbated by the mitochondrial respiratory chain inhibitor rotenone. Cells maintained by glycolytic ATP, i.e., in the presence of the mitochondrial ATP synthase inhibitor oligomycin, remain viable for several hours but are still susceptible to glutamate; thus, disruption of mitochondrial ATP synthesis is not a necessary step in glutamate excitotoxicity. In contrast, the combination of rotenone (or antimycin A) plus oligomycin, which collapses the mitochondrial membrane potential, therefore preventing mitochondrial Ca 2+ transport, allows glutamate‐exposed cells to maintain a high ATP/ADP ratio while accumulating little 45 Ca 2+ and maintaining a low bulk cytoplasmic free Ca 2+ concentration determined by fura‐2. It is concluded that mitochondrial Ca 2+ accumulation is a necessary intermediate in glutamate excitotoxicity, whereas the decreased Ca 2+ flux into cells with depolarized mitochondria may reflect a feedback inhibition of the NMDA receptor mediated by localized Ca 2+ accumulation in a microdomain accessible to the mitochondria.