Regulation of intracellular calcium in N1E-115 neuroblastoma cells:: the role of Na+/Ca2+ exchange

In fura 2-loaded N1E-115 cells, regulation of intracellular Ca2+ concentration ([Ca2+](i)) following a Ca2+ load induced by 1 muM thapsigargin and 10 muM carbonylcyanide p-trifluoromethyoxyphenylhydrazone (FCCP) was Na+ dependent and inhibited by 5 mM Ni2+. In cells with normal intracellular Na+ concentration ([ Na+](i)), removal of bath Na+, which should result in reversal of Na+/Ca2+ exchange, did not increase [Ca2+](i) unless cell Ca2+ buffer capacity was reduced. When N1E-115 cells were Na+ loaded using 100 muM veratridine and 4 mug/ml scorpion venom, the rate of the reverse mode of the Na+/Ca2+ exchanger was apparently enhanced, since an similar to4- to 6-fold increase in [Ca2+](i) occurred despite normal cell Ca2+ buffering. In SBFI-loaded cells, we were able to demonstrate forward operation of the Na+/Ca2+ exchanger (net efflux of Ca2+) by observing increases (similar to 6 mM) in [Na+](i). These Ni2+ (5 mM)-inhibited increases in [Na+](i) could only be observed when a continuous ionomycin-induced influx of Ca2+ occurred. The voltage-sensitive dye bis-(1,3-diethylthiobarbituric acid) trimethine oxonol was used to measure changes in membrane potential. Ionomycin (1 muM) depolarized N1E-115 cells (similar to25 mV). This depolarization was Na+ dependent and blocked by 5 mM Ni2+ and 250-500 muM benzamil. These data provide evidence for the presence of an electrogenic Na+/Ca2+ exchanger that is capable of regulating [Ca2+](i) after release of Ca2+ from cell stores.