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 microM 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 microM) 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 > or = 300 microM. The discrepancy between the glutamate concentration required to evoke a maximal rise in [Ca2+]i and the higher concentration necessary elicit maximal Ca(2+)-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 microM, 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 A1. 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 microM, 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 A1, 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.
Program in Neuroscience, University of Minnesota Medical School, Minneapolis 55455, USA.
G J
GJ
Wang
S A
SA
Thayer
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002478:Cells, Cultured; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D018698:Glutamic Acid / Q000494:pharmacology*; D006019:Glycolysis / Q000187:drug effects; D006624:Hippocampus / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D007091:Image Processing, Computer-Assisted; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D010783:Photometry; D051381:Rats; D015222:Sodium Channels / Q000187:drug effects / Q000378:metabolism; D014475:Uncoupling Agents / Q000494:pharmacology
D018690:Excitatory Amino Acid Agonists; D015222:Sodium Channels; D014475:Uncoupling Agents; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D018698:Glutamic Acid; D016202:N-Methylaspartate; D007770:L-Lactate Dehydrogenase; D002118:Calcium