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: email@example.com.
APStracts 3:0080N, 1996.
SUMMARY AND CONCLUSIONS
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