Perturbation of intracellular calcium and hydrogen ion regulation in cultured mouse hippocampal neurons by reduction of the sodium ion concentration gradient.
Na(+)-Ca2+ exchange has been identified as a mechanism for regulation of intracellular Ca ion concentration ([Ca2+]i) in neurons of invertebrates and vertebrates, but for mammalian central neurons its role in restoration of resting [Ca2+]i after transient increases induced by stimulation has been less clear. We have examined the recovery of [Ca2+]i following K+ depolarization and glutamate receptor activation of cultured mouse hippocampal neurons using the Ca(2+)-sensitive dye Fura-2. Reduction of the transmembrane Na+ gradient by removal of external Na+ slowed the recovery of neurons from imposed Ca2+ loads. We observed that [Ca2+]i regulation was disrupted more severely when N-methyl-D-glucamine (N-MG), Tris, or choline rather than Li+ replaced external Na+. Additional disruption of intracellular pH regulation by substitutes other than Li+ may account for this difference. Measurement of [Ca2+]i and [H+]i (using the H(+)-sensitive dye BCECF) during glutamate receptor activation indicated that Ca2+ influx resulted in production of intracellular H+, and that Li+ but not N-MG could prevent cytoplasmic acidification on removal of external Na+. We also observed that intracellular acidification alone was sufficient to slow recovery from Ca2+ load. We conclude, therefore, that Na(+)-Ca2+ exchange contributes to recovery of [Ca2+]i after stimulation leading to Ca2+ entry into hippocampal neurons, and that Na(+)-H+ exchange limits the acidification (and secondary increase in [Ca2+]i) that accompanies Ca2+ influx. We suggest that because both Na(+)-Ca2+ and Na(+)-H+ exchangers will be compromised during ischemia and hypoglycemia, increased intracellular H+ may synergize with cytoplasmic Ca2+ to potentiate excitotoxic neuronal death.
Koch
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Division of Neurosciences, Beckman Research Institute of the City of Hope, Duarte, California 91010.
Barish
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D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004594:Electrophysiology / Q000379:methods; D005260:Female; D005452:Fluoresceins; D005456:Fluorescent Dyes; D005971:Glutamates / Q000494:pharmacology; D018698:Glutamic Acid; D006624:Hippocampus / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D011188:Potassium / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism* / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D005452:Fluoresceins; D005456:Fluorescent Dyes; D005971:Glutamates; D018698:Glutamic Acid; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium; D011188:Potassium; D002118:Calcium