Sequestration of Glutamate-Induced Ca2+ Loads by Mitochondria in Cultured Rat Hippocampal Neurons. Wang, Guang Jian and Stanley A. Thayer. Program in Neuroscience and Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455, Phone: (612) 626-7049; FAX: (612) 625-8408; EM:
APStracts 3:0080N, 1996.
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 mM glutamate. This apparent ceiling was not a result of saturation of the Ca2+ indicator because these results were obtained with the low affinity (Kd=7 ÁM) Ca2+ indicator, coumarin benzothiazole (BTC). 3. 5 min exposure to glutamate elicited concentration- dependent neuronal death detected 20-24 hrs later by the release of the cytosolic enzyme, lactate dehydrogenase, into the media. Maximal neurotoxicity was elicited at glutamate concentrations equal to and greater than 300 mM. 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 non-cytoplasmic pool. 4. Treatment of hippocampal neurons with the protonophore carbonyl cyanide p-(trifluoro-methoxy) phenylhydrazone (FCCP; 1 mM, 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 prior to exposure to NMDA (200 mM, 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 (Werth and Thayer 1994). 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 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.

Received 29 March 1995; accepted in final form 1 April 1996.
APS Manuscript Number J208-5.
Article publication pending J. Neurophysiol.
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 8 May 96