Mechanism of Inhibition of N‐Methyl‐d‐Aspartate‐Stimulated Increases in Free Intracellular Ca2+ Concentration by Ethanol

Abstract

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.