N-methyl-D-aspartate Excitotoxicity: Relationships among Plasma Membrane Potential, Na/Ca Exchange, Mitochondrial Ca Overload, and Cytoplasmic Concentrations

A high cytoplasmic Na concentration may contribute to Nmethyl-D-aspartate (NMDA)-induced excitotoxicity by promoting Ca influx via reverse operation of the Na/Ca exchanger (NaCaX), but may simultaneously decrease the electrochemical Ca driving force by depolarizing the plasma membrane (PM). Digital fluorescence microscopy was used to compare the effects of Na versus ions that do not support the NaCaX operation, i.e., N-methyl-D-glucamine or Li, on: PM potential; cytoplasmic concentrations of Ca, H, and K; mitochondrial Ca storage; and viability of primary cultures of cerebellar granule cells exposed to NMDA receptor agonists. In the presence of Na or Li, NMDA depolarized the PM and decreased cytoplasmic pH (pHC); in the presence of Li , Ca influx was reduced, mitochondrial Ca overload did not occur, and the cytoplasm became more acidified than in the presence of Na. In the presence of N-methyl-D-glucamine, NMDA instantly hyperpolarized the PM, but further changes in PM potential and pHC were Ca-dependent. In the absence of Ca , hyperpolarization persisted, pHC was decreasing very slowly, K was retained in the cytoplasm, and cerebellar granule cells survived the challenge; in the presence of Ca, pHC dropped rapidly, the K concentration gradient across the PM began to collapse as the PM began to depolarize, and Ca influx and excitotoxicity greatly increased. These results indicate that the dominant, very likely excitotoxic, component of NMDA-induced Ca influx is mediated by reverse NaCaX and that direct Ca influx via NMDA channels is curtailed by Na-dependent PM depolarization. Excitotoxicity has been causally linked with glutamateelicited Ca influx (Choi, 1987; Hartley et al., 1993; Eimerl and Schramm, 1994) and with Ca accumulation in mitochondria (Kiedrowski and Costa, 1995; White and Reynolds, 1996; Schinder et al., 1996; Stout et al., 1998). Although exposure to glutamate also greatly elevates cytoplasmic Na concentrations ([Na]C) in cultured neurons (Kiedrowski et al., 1994; Pinelis et al., 1994), it is yet unclear what role the increase in [Na]C plays in excitotoxicity. Although excessive swelling caused by the influx of Na, Cl, and water leads to neuronal death (Rothman, 1985; Choi, 1987), such a mechanism characterizes kainaterather than glutamateor NMDA-induced excitotoxicity (Kiedrowski, 1998). Earlier studies have shown that high [Na]C contributes to the NMDA-induced Ca influx by activating the reverse operation of the plasma membrane Na/Ca exchanger (NaCaX) (Kiedrowski et al., 1994; Hoyt et al., 1998). The activation of the reverse NaCaX also requires that cytoplasmic Ca be raised to micromolar levels (Hilgemann et al., 1992). This makes the channels that are permeable for both Na and Ca, such as NMDA receptor channels (Mayer and Westbrook, 1987), perfect triggers of the reverse NaCaX activa-