N-methyl-d-aspartate Excitotoxicity: Relationships among Plasma Membrane Potential, Na+/Ca2+ Exchange, Mitochondrial Ca2+ Overload, and Cytoplasmic Concentrations of Ca2+, H+, and K+
- The Psychiatric Institute, Departments of Psychiatry and Pharmacology, College of Medicine, The University of Illinois at Chicago, Chicago, Illinois
Abstract
A high cytoplasmic Na+ concentration may contribute to N-methyl-d-aspartate (NMDA)-induced excitotoxicity by promoting Ca2+ influx via reverse operation of the Na+/Ca2+ exchanger (NaCaX), but may simultaneously decrease the electrochemical Ca2+driving force by depolarizing the plasma membrane (PM). Digital fluorescence microscopy was used to compare the effects of Na+ versus ions that do not support the NaCaX operation, i.e., N-methyl-d-glucamine+ or Li+, on: PM potential; cytoplasmic concentrations of Ca2+, H+, and K+; mitochondrial Ca2+ storage; and viability of primary cultures of cerebellar granule cells exposed to NMDA receptor agonists. In the presence of Na+ or Li+, NMDA depolarized the PM and decreased cytoplasmic pH (pHC); in the presence of Li+, Ca2+ influx was reduced, mitochondrial Ca2+ overload did not occur, and the cytoplasm became more acidified than in the presence of Na+. In the presence ofN-methyl-d-glucamine+, NMDA instantly hyperpolarized the PM, but further changes in PM potential and pHC were Ca-dependent. In the absence of Ca2+, hyperpolarization persisted, pHC was decreasing very slowly, K+ was retained in the cytoplasm, and cerebellar granule cells survived the challenge; in the presence of Ca2+, pHC dropped rapidly, the K+concentration gradient across the PM began to collapse as the PM began to depolarize, and Ca2+ influx and excitotoxicity greatly increased. These results indicate that the dominant, very likely excitotoxic, component of NMDA-induced Ca2+ influx is mediated by reverse NaCaX and that direct Ca2+ influx via NMDA channels is curtailed by Na-dependent PM depolarization.
Footnotes
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Send reprint requests to: Lech Kiedrowski, Ph.D., The Psychiatric Institute, 1601 W. Taylor St., Chicago, IL 60612. E-mail:lkiedr{at}psych.uic.edu
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This work was supported in part by National Institutes of Health Grant NS 37390 and presented in part in abstract form, Society of Neuroscience Abstracts 300.5, 1998.
- Abbreviations:
- [Na+]C
- [K+]C, and [Ca2+]C, cytoplasmic concentrations of Na+, K+, and Ca2+, respectively
- BCECF
- 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein
- CaDF
- electrochemical force for Ca2+ influx
- CGCs
- cerebellar granule cells
- CM
- conditioned medium
- DiBAC4(3)
- bis(1,3-dibutylbarbituric acid)trimethine oxonol
- Em
- plasma membrane potential
- [K+]E
- extracellular concentration of K+
- Li-L
- NMG-L, Cs-L, Na-free Locke’s buffers in which Na+ was substituted with Li+, NMG+, or Cs+, respectively
- MDC
- mitochondria-depolarizing cocktail
- NaCaX and NaHX
- Na+/Ca2+ and Na+/H+exchanger, respectively
- Na-L
- standard Locke’s buffer containing physiological Na+ concentrations
- nF488
- normalized fluorescence emitted by DiBAC4(3) after excitation at 488 nm
- NMDA
- N-methyl-d-aspartate
- NMG+
- N-methyl-d-glucamine+
- PBFI
- K+-binding benzofuran isophthalate
- pHC
- cytoplasmic pH
- PM
- plasma membrane
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- Received December 7, 1998.
- Accepted June 9, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
