Glutamate-induced calcium increase mediates magnesium release from mitochondria in rat hippocampal neurons.

Excess administration of glutamate is known to induce Ca(2+) overload in neurons, which is the first step in excitotoxicity. Although some reports have suggested a role for Mg(2+) in the excitotoxicity, little is known about its actual contribution. To investigate the role of Mg(2+) in the excitotoxicity, we simultaneously measured intracellular Ca(2+) and Mg(2+), using fluorescent dyes, Fura red, a fluorescent Ca(2+) probe, and KMG-104, a highly selective fluorescent Mg(2+) probe developed by our group, respectively. Administration of 100 μM glutamate supplemented with 10 μM glycine to rat hippocampal neurons induced an increase in intracellular Mg(2+) concentration (Mg(2+)). Extracellular Mg(2+) was not required for this glutamate-induced increase in Mg(2+), and no increase in intracellular Ca(2+) concentration (Ca(2+)) or Mg(2+) was observed in neurons in nominally Ca(2+)-free medium. Application of 5 μM carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), an uncoupler of mitochondrial inner membrane potential, also elicited increases in Ca(2+) and Mg(2+). Subsequent administration of glutamate and glycine following FCCP treatment did not induce a further increase in Mg(2+) but did induce an additive increase in Ca(2+). Moreover, the glutamate-induced increase in Mg(2+) was observed only in mitochondria localized areas. These results support the idea that glutamate is able to induced Mg(2+) efflux from mitochondria to the cytosol. Furthermore, pretreatment with Ru360, an inhibitor of the mitochondrial Ca(2+) uniporter, prevented this Mg(2+) increase. These results indicate that glutamate-induced increases in Mg(2+) result from the Mg(2+) release from mitochondria and that Ca(2+) accumulation in the mitochondria is required for this Mg(2+) release.