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 (NCX_rev) could be involved in DCD. However, the extent to which NMDAR and NCX_rev contribute to glutamate-induced DCD is uncertain. Here, we show that both NMDAR and NCX_rev are critical for DCD in neurons exposed to excitotoxic glutamate. In rat cultured hippocampal neurons, 25μM 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 NCX_rev while AP-5 was much less efficacious. In electrophysiological patch-clamp experiments MK801 and memantine inhibited NCX_rev-mediated ion currents whereas AP-5 failed. Thus, MK801 and memantine, in addition to NMDAR, inhibited NCX_rev. Inhibition of NCX_rev 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, NCX_rev inhibition combined with NMDAR blockade by AP-5 completely prevented DCD. Overall, our data suggest that both NMDAR and NCX_rev are essential for DCD in glutamate-exposed neurons and inhibition of individual mechanism is not sufficient to prevent calcium dysregulation.