Stimulation of N-methyl-D-aspartate receptor-mediated calcium entry into dissociated neurons by reduced and oxidized glutathione

The effects of GSH (γ-glutamylcysteinylglycine) and GSSG on intracellular calcium levels ([Ca²⁺]_i) were investigated using fura-2-loaded dissociated brain cells from newborn rat pups. Both produced concentration-dependent increases in [Ca²⁺] (EC₅₀ values of 914.3 ± 190.5 and 583.0 ± 97.2 μM for GSH and GSSG, respectively), similar to that observed with N-methyl-D-aspartate (NMDA) and other agonists at the NMDA receptor. Maximum response (expressed as percentage change in [Ca²⁺]_i relative to basal) was significantly greater for GSSG (37.5 ± 1.6%) than for GSH (25.3 ± 1.6%). The response to both agents was prevented or reversed by competitive (100 μM) (-)-2-amino-5-phosphonovalerate and noncompetitive (400 nM) MK-801 or 1.0 mM Mg²⁺ antagonists of NMDA receptor-mediated calcium entry, even at concentrations of GSH and GSSG normally producing maximal response. The idea that these effects are mediated, at least in part, by interaction with the NMDA receptor was supported by the effects of GSH and GSSG on the binding of the NMDA receptor ligand [³H]CGP-39653 to membranes isolated from hippocampal and cortical homogenates. Both GSH and GSSG displaced bound [³H]CGP-39653, with IC₅₀ values of 0.93 ± 0.18 and 11.02 ± 1.22 μM, respectively, and produced an increase in the apparent K_d of binding (control, 8.92 ± 0.83 nM, and GSH, 13.31 ± 1.19 nM; control, 11.59 ± 0.35 nM, and GSSG, 18.73 ± 0.66 nM). However, both also produced modest reductions in B_max (control, 1265 ± 69 fmol/mg of protein, and GSH, 901 ± 73 fmol/mg of protein; control, 1068 ± 30 fmol/mg of protein, and GSSG, 730 ± 18 fmol/mg of protein) and Hill slopes (GSH, 0.66 ± 0.02; GSSG, 0.62 ± 0.04). This suggests complex kinetics for the interaction of GSH and GSSG with the NMDA receptor. Taken together, the results suggest the potential for modulation of the NMDA receptor complex by GSH and GSSG.