Reverse Na+/Ca2+ exchange contributes to glutamate-induced intracellular Ca2+ concentration increases in cultured rat forebrain neurons

Activation of ionotropic glutamate receptors causes increases in intracellular Ca2+ concentration ([Ca2+](i)) and intracellular Na+ concentration in neurons. It has been suggested that reversal of the plasma membrane Na+/Ca2+ exchanger (NCE) may account in part for the rise in [Ca2+](i). Recently, KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate) was reported to selectively inhibit the reverse mode of the NCE in non-neuronal cells. We investigated the effects of KB-R7943 on glutamate-stimulated increases in [Ca2+](i). In cultured rat forebrain neurons loaded with indo-1 acetoxymethyl ester, KB-R7943 inhibited the reverse mode of NCE (IC50 = 0.7 mu M). When tested against kainate- (100 mu M), N-methyl-D-aspartate- (30 mu M), glutamate- (3 mu M), or KCl- (50 mM) induced [Ca2+](i) transients (15 sec, in the presence of Na+ and Ca2+)(i) KB-R7943 inhibited these transients with IC50 values of 6.6, 8.2, 5.2, and 2.9 mu M, respectively. [Ca2+](i) increases caused by a higher concentration of glutamate (100 mu M) also were inhibited by KB-R7943 (10 mu M). However, KB-R7943 had no effect on peak [Ca2+](i) changes caused by prolonged application of glutamate and did not inhibit glutamate-induced neuronal injury. KB-R7943 did not inhibit N-methyl-D-aspartate- or kainate-induced whole-cell currents, nor did it substantially inhibit voltage-sensitive Ca2+ currents, excluding a direct inhibition of these ion channels. These results suggest that reverse NCE contributes to the immediate rise in [Ca2+](i) resulting from glutamate receptor activation. However, reverse NCE becomes less important as the stimulus time is increased, and Ca2+ entry by this route is not critical for the expression of excitotoxic injury.