ABSTRACT: Binding of D,L-(E)-2-amino-4-[(3)H]-propyl-5-phosphono-3-pentenoic acid ([(3)H]-CGP39653), a selective antagonist at the glutamate site of the NMDA receptor, is modulated by glycine in rat brain tissue. We have further investigated this phenomenon in rodent and human brain by means of receptor binding and quantitative autoradiography techniques. In rat cerebral cortical membranes the glycine antagonist 3-[2-(Phenylaminocarbonyl)ethenyl]-4,6-dichloro-indole-2-carboxylic acid sodium salt (GV150526A) did not change basal [(3)H]-CGP39653 binding, but competitively reversed the high affinity component of [(3)H]-CGP39653 binding inhibition by glycine, with a pK(B) value of 8.38, in line with its affinity for the glycine site (pK(i)=8.49 vs. [(3)H]-glycine). Glycine (10 microM) significantly decreased [(3)H]-CGP39653 affinity for the NMDA receptor (with no change in the B(max)), whereas enhanced L-glutamate affinity (P<0.05, paired-samples Student's t-test). In rat brain sections the addition of GV150526A (30 microM) to the incubation medium increased [(3)H]-CGP39653 binding to 208% of control (average between areas), indicating the presence of endogenous glycine. The enhancement presented significant regional differences (P<0.05, two-way ANOVA), with striatum higher than cerebral cortex (282 and 187% of control, respectively; P<0.05, Fisher's LSD). On the contrary, there was not any significant variation in affinity values of [(3)H]-CGP39653, L-glutamate, glycine and GV150526A in striatal and cortical membranes. These results confirmed the existence of regionally distinct NMDA receptors subtypes with different glycine/glutamate allosteric modulation. Whole brain autoradiography revealed an uneven distribution of [(3)H]-CGP39653 binding sites in human brain. High levels of binding were determined in hippocampus and in cingulate, frontoparietal and insular cortex. Intermediate to low levels of binding were found in diencephalic nuclei and basal ganglia. [(3)H]-CGP39653 binding was increased to 216% of control (mean between areas) by 30 microM GV150526A. The enhancement, however, did not present significant regional differences. These results introduce GV150526A as a useful tool to identify NMDA receptor subtypes by means of receptor autoradiography; moreover, they demonstrate that the allosteric inhibition of [(3)H]-CGP39653 binding by glycine parallels an increase in receptor affinity to the endogenous ligand L-glutamate. Finally, this study provides the first detailed anatomical description of the regional distribution of [(3)H]-CGP39653 binding sites in human brain.
