Glutamate receptor-induced 45Ca2+ accumulation in cortical cell culture correlates with subsequent neuronal degeneration

Abstract

Murine neuronal and glial cell cultures exposed briefly to glutamate accumulated large amounts of 45Ca2+ from the extracellular medium during the exposure. Most of the accumulation likely reflected influx into neurons, as little accumulation was observed in similarly treated glial cultures. When the concentration of glutamate was varied between 10 and 1000 μM, or exposure duration was varied between 0 and 10 min, the amount of 45Ca2+ accumulation correlated closely with the amount of neuronal death 24 hr later. Both 45Ca2+ accumulation and cell death could be attenuated in a dose-dependent manner by the competitive NMDA antagonist D- aminophosphonovalerate or the noncompetitive antagonist dextrorphan, with IC50 values of approximately 100 μM and 15 μM, respectively. In contrast, neither 45Ca2+ accumulation nor cell death was blocked by the α-amino- 3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate antagonist 6- cyano-7-nitroquinoxaline-2,3-dione (CNQX) in the presence of high glycine. With brief exposure, high concentrations of AMPA, kainate, or K+ produced much less death or 45Ca2+ accumulation than produced by glutamate, especially if 10 μM MK-801 was included in the exposure medium to block NMDA receptor activation. Kainate- or AMPA-induced 45Ca2+ accumulation or neuronal cell death was blocked with CNQX. However, high K+-triggered 45Ca2+ accumulation was only partially blocked with CNQX plus MK-801, consistent with mediation by voltage-gated Ca2+ channels. In addition to measuring the accumulation of 45Ca2+ occurring during agonist exposure, we also assessed accumulation during the 30 min immediately following completion of a 3-5 min exposure to 500 μM NMDA. We found this late 45Ca2+ accumulation to be approximately half that accumulated during the initial exposure period. Delayed addition of MK-801 blocked this late accumulation and produced a corresponding partial reduction in cell death. Our results support the hypothesis that excess net Ca2+ influx through NMDA receptor-gated channels is a key step in triggering the neuronal death induced by brief, intense glutamate exposure.