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Sequestration of glutamate-induced Ca2+ loads by mitochondria in cultured rat hippocampal neurons.
J Neurophysiol. 1996 Sep; 76(3):1611-21.JN

Abstract

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.

Authors+Show Affiliations

Program in Neuroscience, University of Minnesota Medical School, Minneapolis 55455, USA.No affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, Non-P.H.S.
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8890280

Citation

Wang, G J., and S A. Thayer. "Sequestration of Glutamate-induced Ca2+ Loads By Mitochondria in Cultured Rat Hippocampal Neurons." Journal of Neurophysiology, vol. 76, no. 3, 1996, pp. 1611-21.
Wang GJ, Thayer SA. Sequestration of glutamate-induced Ca2+ loads by mitochondria in cultured rat hippocampal neurons. J Neurophysiol. 1996;76(3):1611-21.
Wang, G. J., & Thayer, S. A. (1996). Sequestration of glutamate-induced Ca2+ loads by mitochondria in cultured rat hippocampal neurons. Journal of Neurophysiology, 76(3), 1611-21.
Wang GJ, Thayer SA. Sequestration of Glutamate-induced Ca2+ Loads By Mitochondria in Cultured Rat Hippocampal Neurons. J Neurophysiol. 1996;76(3):1611-21. PubMed PMID: 8890280.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Sequestration of glutamate-induced Ca2+ loads by mitochondria in cultured rat hippocampal neurons. AU - Wang,G J, AU - Thayer,S A, PY - 1996/9/1/pubmed PY - 1996/9/1/medline PY - 1996/9/1/entrez SP - 1611 EP - 21 JF - Journal of neurophysiology JO - J Neurophysiol VL - 76 IS - 3 N2 - 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. SN - 0022-3077 UR - https://www.unboundmedicine.com/medline/citation/8890280/Sequestration_of_glutamate_induced_Ca2+_loads_by_mitochondria_in_cultured_rat_hippocampal_neurons_ L2 - https://journals.physiology.org/doi/10.1152/jn.1996.76.3.1611?url_ver=Z39.88-2003&rfr_id=ori:rid:crossref.org&rfr_dat=cr_pub=pubmed DB - PRIME DP - Unbound Medicine ER -