Abstract

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 μm). When tested against kainate- (100 μm),N-methyl-d-aspartate- (30 μm), glutamate- (3 μm), or KCl- (50 mm) induced [Ca²⁺]_i transients (15 sec, in the presence of Na⁺ and Ca²⁺), KB-R7943 inhibited these transients with IC₅₀ values of 6.6, 8.2, 5.2, and 2.9 μm, respectively. [Ca²⁺]_iincreases caused by a higher concentration of glutamate (100 μm) also were inhibited by KB-R7943 (10 μm). 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 inhibitN-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.