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 μM measured in the absence of Mg2+. NMDA‐stimulated [Ca2+]j increases could be observed in buffer with Mg2+ when the cells were predepolarized with 15 mMKQ prior to NMDA addition. Under these predepolarized conditions, 100 mMethanol inhibited 25 μM 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 μM 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 μM NMDA responses was independent of the external Ca2+ or Mg2+ concentrations. Glycine (1, 10, and 100 μM) enhanced 25 μM NMDA‐induced increases in [Ca2+]i by approximately 50%. Glycine (1‐100 μM) prevented the 100 mM ethanol inhibition of NMDA‐stimulated [Ca2+]i observed in the absence of exogenous glycine. MK‐801 (25‐400 μM) inhibited 25 μM 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 μM, 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.