Alkalinization prolongs recovery from glutamate-induced increases in intracellular Ca2+ concentration by enhancing Ca2+ efflux through the mitochondrial Na+/Ca2+ exchanger in cultured rat forebrain neurons.
Increasing extracellular pH from 7.4 to 8.5 caused a dramatic increase in the time required to recover from a glutamate (3 microM, for 15 s)-induced increase in intracellular Ca2+ concentration ([Ca2+]i) in indo-1-loaded cultured cortical neurons. Recovery time in pH 7.4 HEPES-buffered saline solution (HBSS) was 126 +/- 30 s, whereas recovery time was 216 +/- 19 s when the pH was increased to 8.5. Removal of extracellular Ca2+ did not inhibit the prolongation of recovery caused by increasing pH. Extracellular alkalinization caused rapid intracellular alkalinization following glutamate exposure, suggesting that pH 8.5 HBSS may delay Ca2+ recovery by affecting intraneuronal Ca2+ buffering mechanisms, rather than an exclusively extracellular effect. The effect of pH 8.5 HBSS on Ca2+ recovery was similar to the effect of the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxyphenyl)hydrazone (FCCP; 750 nM). However, pH 8.5 HBSS did not have a quantitative effect on mitochondrial membrane potential comparable to that of FCCP in neurons loaded with a potential-sensitive fluorescent indicator, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine++ + iodide (JC-1). We found that the effect of pH 8.5 HBSS on Ca2+ recovery was completely inhibited by the mitochondrial Na+/Ca2+ exchange inhibitor CGP-37157 (25 microM). This suggests that increased mitochondrial Ca2+ efflux via the mitochondrial Na+/Ca2+ exchanger is responsible for the prolongation of [Ca2+]i recovery caused by alkaline pH following glutamate exposure.
Department of Pharmacology, University of Pittsburgh School of Medicine, Pennsylvania 15261, USA.
K R
KR
Hoyt
I J
IJ
Reynolds
D000468:Alkalies / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002478:Cells, Cultured; D002998:Clonazepam / Q000031:analogs & derivatives / Q000494:pharmacology; D018698:Glutamic Acid / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D009474:Neurons / Q000378:metabolism; D009994:Osmolar Concentration; D016548:Prosencephalon / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013841:Thiazepines / Q000494:pharmacology; D014475:Uncoupling Agents / Q000494:pharmacology
D000468:Alkalies; D019831:Sodium-Calcium Exchanger; D013841:Thiazepines; D014475:Uncoupling Agents; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D018698:Glutamic Acid; D002998:Clonazepam; C079032:CGP 37157; D002118:Calcium