MECHANISM OF INHIBITION OF N-METHYL-D-ASPARTATE-STIMULATED INCREASES IN FREE INTRACELLULAR CA2+ CONCENTRATION BY ETHANOL

Dissociated brain cells were isolated from newborn rat pups and loaded with fura-2. These cells were sensitive to low N-methyl-D-aspartate (NMDA) concentrations with EC50 values for NMDA-induced intracellular Ca2+ concentration ([Ca2+])i) increases of approximately 7-16-mu-M measured in the absence of Mg2+. NMDA-stimulated [Ca2+]i increases could be observed in buffer with Mg2+ when the cells were predepolarized with 15 mM KCl prior to NMDA addition. Under these predepolarized conditions, 100 mM ethanol inhibited 25-mu-M NMDA responses by approximately 50%, which was similar to the ethanol inhibition observed in buffer without added Mg2+. Ethanol did not alter [Ca2+]i prior to NMDA addition. In the absence of Mg2+, 50 and 100 mM ethanol did not significantly alter the EC50 value for NMDA, but did inhibit NMDA-induced increases in [Ca2+]i in a concentration-dependent manner at 4, 16, 64, and 256-mu-M NMDA. Whereas NMDA-induced increases in [Ca2+]i were dependent on extracellular Ca2+ and were inhibited by Mg2+, the ability of 100 mM ethanol to inhibit 25 mu-M NMDA responses was independent of the external Ca2+ or Mg2+ concentrations. Glycine (1, 10, and 100-mu-M) enhanced 25-mu-M NMDA-induced increases in [Ca2+]i by approximately 50%. Glycine (1-100-mu-M) prevented the 100 mM ethanol inhibition of NMDA-stimulated [Ca2+]i observed in the absence of exogenous glycine. MK-801 (25-400 nM) inhibited 25-mu-M NMDA-stimulated rises in [Ca2+]i in a concentration-dependent manner. Unlike the additive inhibition observed with Mg2+ plus ethanol, as the concentration of MK-801 increased above 50 nM, ethanol (at 100 mM) did not produce further inhibition of NMDA responses compared with MK-801 alone. These results suggest that ethanol may produce a noncompetitive inhibition of NMDA-stimulated Ca2+ influx in dissociated brain cells, with interactions at the glycine and possibly the phencyclidine site on the NMDA-receptor complex.