Sequestration of glutamate-induced Ca2+ loads by mitochondria in cultured rat hippocampal neurons

1. Buffering of glutamate-induced Ca2+ loads in single rat hippocampal neurons grown in primary culture was studied with ratiometric fluorescent Ca2+ indicators. The hypothesis that mitochondria buffer the large Ca2+ loads elicited by glutamate was tested. 2. The relationship between glutamate concentration and the resulting increase in the free intracellular Ca2+ concentration ([Ca2+](i)) reached an asymptote at 30 mu M glutamate. This apparent ceiling was not a result of saturation of the Ca2+ indicator, because these results were obtained with the low-affinity (dissociation constant = 7 mu M) Ca2+ indicator coumarin benzothiazole. 3. Five minutes of exposure to glutamate elicited concentration-dependent neuronal death detected 20-24 h later by the release of the cytosolic enzyme lactate dehydrogenase into the media. Maximal neurotoxicity was elicited at glutamate concentrations greater than or equal to 300 mu M. The discrepancy between the glutamate concentration required to evoke a maximal rise in [Ca2+](i) and the higher concentration necessary elicit maximal Ca2+-triggered cell death suggests that large neurotoxic Ca2+ loads are in part removed to a noncytoplasmic pool. 4. Treatment of hippocampal neurons with the protonophore carbonyl cyanide p-(trifluoro-methoxy) phenylhydrazone (FCCP; 1 mu M, 5 min) greatly increased the amplitude of glutamate-induced [Ca2+](i) transients, although it had little effect on basal [Ca2+](i). The effect of FCCP was more pronounced on responses elicited by stimuli that produced large Ca2+ loads. Similar results were obtained by inhibition of electron transport with antimycin Al. Neither agent, under the conditions described here, significantly depressed cellular ATP levels as indicated by luciferase-based ATP measurements, consistent with the robust anaerobic metabolism of cultured cells. Thus inhibition of mitochondrial function disrupted the buffering of glutamate-induced Ca2+ loads in a manner that was not related to changes in ATP. 5. Removal of extracellular Na+ for 20 min before exposure to N-methyl-D-aspartate (NMDA) (200 mu M, 3 min), presumably reducing intracellular Na+, evoked a prolonged plateau phase in the recovery of the [Ca2+](i) transient that resembled the mitochondrion-mediated [Ca2+](i) plateau previously observed in sensory neurons. Return of extracellular Na+ immediately after exposure to NMDA increased the height and shortened the duration of the plateau phase. Thus manipulation of extracellular Na+ altered the plateau in a manner consistent with plateau height being modulated by intracellular Na+ levels. 6. In neurons depleted of Na+ and challenged with NMDA, a plateau resulted; during the plateau, application of FCCP in the absence of extracellular Ca2+ produced a large increase in [Ca2+](i). In contrast, similar treatment of cells that were not depleted of Na+ failed to increase [Ca2+](i). Thus Na+ depletion traps Ca2+ within an FCCP-sensitive intracellular store. 7. Glutamate-induced Ca2+ loads are sequestered by an intracellular store that had a low affinity and a high capacity for Ca2+ was released by FCCP, was sensitive to antimycin Al, and was modulated by intracellular Na+ levels. We conclude that mitochondria sequester glutamate-induced Ca2+ loads and suggest that Ca2+ entry into mitochondria may account for the poor correlation between glutamate-induced neurotoxicity and glutamate-induced changes in [Ca2+](i).