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 mu M, for 15 s)-induced increase in intracellular Ca2+ concentration ([Ca2+](i)) in indo-1-loaded cultured cortical neurons. Recovery time in pH 7.4 HERES-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-I). 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 mu M). This suggests that increased mitochondrial Ca2+ afflux via the mitochondrial Na+/Ca2+ exchanger is responsible for the prolongation of [Ca2+](i) recovery caused by alkaline pH following glutamate exposure.