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 μM 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 μM) 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 ≤300 μM. 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 μM, 5 min) greatly increased the amplitude of glummate-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 μM, 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).