Increased agonist and antagonist sensitivity of N-methyl- d-aspartate stimulated calcium flux in cultured neurons following chronic ethanol exposure

Abstract

Cortical cultures of rat brain neurons were exposed to ethanol (100 mM) for 4 days in order to examine whether the pharmacological characteristics of N-methyl- d-aspartate (NMDA) receptors expressed by these neurons were altered by this treatment. In fura-2 loaded control neurons, NMDA (plus 10μM glycine) stimulated a dose-dependent increase in intracellular calcium concentrations with an estimated EC 50 value of 6.8 μM. NMDA-stimulated increases in intracellular calcium reached a plateau at approximately 30 μM with no further increases observed at 100 μM. The EC 50 value for NMDA in ethanol-exposed neurons was reduced to 1.8 μM with no alteration in the maximal response. Similarly, the EC 50 value for glycine (tested with 100 μM NMDA) was reduced from 2.3μM in control cultures to 0.67 μM in ethanol-treated cultures. Ifenprodil inhibited NMDA-stimulated increases in intracellular calcium in control cultures only at concentrations of 3 μM and above, with 100 μM producing approximately a 58% inhibition. In ethanol-treated cultures, 0.3 μM ifenprodil inhibited the NMDA response by approximately 60% with 100 μM ifenprodil producing a 72% inhibition. Over the concentration range of ifenprodil tested, half-maximal inhibition occurred at 1.4 μM and 0.18 μM, respectively, for control and ethanol-treated neurons. Although chronic ethanol treatment appeared to alter the sensitivity of neurons to NMDA agonists and antagonists, the inhibitory effects of 50 mM ethanol on NMDA-stimulated increases in intracellular calcium were not different between control (28% inhibition) and ethanol-treated neurons (27% inhibition). Finally, the changes in NMDA receptor sensitivity observed in ethanol-treated neurons were accompanied by an enhanced sensitivity to the neurotoxic effects of NMDA as measured by propidium iodide staining. These results suggest that chronic exposure of neurons to ethanol may result in an altered expression of agonist-sensitive/ifenprodil selective NMDA receptor subunits.