The Leading Role of Mitochondrial Depolarization in the Mechanism of Glutamate-Induced Disruptions in Ca2+ Homeostasis

Abstract

Data obtained in studies of the nature of the correlation which we have previously observed [10, 17] between mitochondrial depolarization and the level of disruption of Ca²⁺ homeostasis in cultivated brain neurons are summarized. Experiments were performed on cultured cerebellar granule cells loaded with Fura-2-AM or rhodamine 123 to measure changes in cytoplasmic Ca²⁺ and mitochondrial potential during pathogenic treatments of the cells. Prolonged exposure to 100 μM glutamate induced a reversible increase in [Ca²⁺]_i, which was accompanied by only a small degree of mitochondrial depolarization. A sharp increase in this mitochondrial depolarization, induced by addition of 3 mM NaCN or 300 μM dinitrophenol (DNP) to the glutamate-containing solution, resulted in further increase in [Ca²⁺]_i, due to blockade of electrophoretic mitochondrial Ca²⁺ uptake. Prolonged exposure to CN^– or DNP in the post-glutamate period maintained [Ca²⁺]_i at a high level until the metabolic inhibitors were removed. In most cells, this plateau was characterized by low sensitivity to removal of external Ca²⁺, demonstrating that the mechanisms of Ca²⁺ release from neurons were disrupted. Addition of oligomycin, a blocker of mitochondrial ATP synthase/ATPase, to the solution containing glutamate and CN^– or DNP eliminated the post-glutamate plateau. Parallel experiments with direct measurements of intracellular ATP levels ([ATP]) showed that profound mitochondrial depolarization induced by CN^– or DNP sharply enhanced the drop in ATP due to glutamate, while oligomycin significantly weakened this effect of the metabolic inhibitors. Analysis of these data led to the conclusion that blockade of mitochondrial Ca²⁺ uptake and inhibition of ATP synthesis resulted from mitochondrial depolarization and plays a key role in the mechanism disrupting [Ca²⁺]_i homeostasis after toxic exposure to glutamate.