Delayed calcium dysregulation in neurons requires both the NMDA receptor and the reverse Na+/Ca2 + exchanger

Glutamate-induced delayed calcium dysregulation (DCD) is a causal factor leading to neuronal death. The mechanism of DCD is not clear but Ca2 + influx via N-methyl-d-aspartate receptors (NMDAR) and/or the reverse plasmalemmal Na+/Ca2 + exchanger (NCXrev) could be involved in DCD. However, the extent to which NMDAR and NCXrev contribute to glutamate-induced DCD is uncertain. Here, we show that both NMDAR and NCXrev are critical for DCD in neurons exposed to excitotoxic glutamate. In rat cultured hippocampal neurons, 25 μM glutamate produced DCD accompanied by sustained increase in cytosolic Na+ ([Na+]c) and plasma membrane depolarization. MK801 and memantine, noncompetitive NMDAR inhibitors, added shortly after glutamate, completely prevented DCD whereas AP-5, a competitive NMDAR inhibitor, failed to protect against DCD. None of the tested inhibitors lowered elevated [Na+]c or restored plasma membrane potential. In the experiments with NCX reversal by gramicidin, MK801 and memantine robustly inhibited NCXrev while AP-5 was much less efficacious. In electrophysiological patch-clamp experiments MK801 and memantine inhibited NCXrev-mediated ion currents whereas AP-5 failed. Thus, MK801 and memantine, in addition to NMDAR, inhibited NCXrev. Inhibition of NCXrev either with KB-R7943, or by collapsing Na+ gradient across the plasma membrane, or by inhibiting Na+/H+ exchanger with 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and thus preventing the increase in [Na+]c failed to preclude DCD. However, NCXrev inhibition combined with NMDAR blockade by AP-5 completely prevented DCD. Overall, our data suggest that both NMDAR and NCXrev are essential for DCD in glutamate-exposed neurons and inhibition of individual mechanism is not sufficient to prevent calcium dysregulation.

► AP-5 inhibits NMDA receptor but not reverse NCX in glutamate-treated neurons.
► MK801 and memantine inhibit both NMDAR and NCXrev in glutamate-exposed neurons.
► AP-5 does not prevent glutamate-induced delayed Ca dysregulation (DCD).
► MK801 and memantine do prevent glutamate-induced DCD.
► Both NMDAR and NCXrev inhibitions are necessary for preventing glutamate-induced DCD.