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+ ] i ). Extracellular Mg 2+ was not required for this glutamate‐induced increase in [Mg 2+ ] i , and no increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) or [Mg 2+ ] i 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+ ] i and [Mg 2+ ] i . Subsequent administration of glutamate and glycine following FCCP treatment did not induce a further increase in [Mg 2+ ] i but did induce an additive increase in [Ca 2+ ] i . Moreover, the glutamate‐induced increase in [Mg 2+ ] i 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+ ] i increase. These results indicate that glutamate‐induced increases in [Mg 2+ ] i result from the Mg 2+ release from mitochondria and that Ca 2+ accumulation in the mitochondria is required for this Mg 2+ release. © 2010 Wiley‐Liss, Inc.