REGIONALLY DIFFERENT N-METHYL-D-ASPARTATE RECEPTORS DISTINGUISHED BY LIGAND-BINDING AND QUANTITATIVE AUTORADIOGRAPHY OF [H-3]CGP-39653 IN RAT-BRAIN

1 Binding of (E)-2-amino-4-[H-3]-propyl-5-phosphono-3-pentenoic acid ( [H-3]-CGP 39653), a high affinity, selective antagonist at the glutama te site of the N-methyl-D-aspartate (NMDA) receptor, was investigated in rat brain by means of receptor binding and quantitative autoradiogr aphy techniques. 2 [H-3]-CGP 39653 interacted with striatal and cerebe llar membranes in a saturable manner and to a single binding site, wit h K-D values of 15.5 nM and 10.0 nM and receptor binding densities (B- max values) of 3.1 and 0.5 pmol mg(-1) protein, respectively. These K- D values were not significantly different from that previously reporte d in the cerebral cortex (10.7 nM). 3 Displacement analyses of [H-3]-C GP 39653 in striatum and cerebellum, performed with L-glutamic acid, ( (+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and glyci ne showed a pharmacological profile similar to that reported in the ce rebral cortex. L-Glutamic acid and CPP produced complete displacement of specific binding with K-i values not significantly different from t he cerebral cortex. Glycine inhibited [H-3]-CGP 39653 binding with sha llow, biphasic curves, characterized by a high and a low affinity comp onent. Furthermore, glycine discriminated between these regions (P<0.0 05, one-way ANOVA), since the apparent K-i of the high affinity compon ent of the glycine inhibition curve (K-iH) was significantly lower (Fi sher's protected LSD) in the striatum than the cortex (33 mM and 104 n M, respectively). 4 Regional binding of [H-3]-CGP 39653 to horizontal sections of rat brain revealed a heterogeneous distribution of binding sites, similar to that reported for other radiolabelled antagonists a t the NMDA site (D-2-[H-3]-amino-5-phosphonopentanoic acid ([H-3]-D-AP 5) and [H-3]-CPP). High values of binding were detected in the hippoca mpal formation, cerebral cortex and thalamus, with low levels in stria tum and cerebellum. 5 [H-3]-CGP 39653 binding was inhibited by increas ing concentrations of L-glutamic acid, CPP and glycine. L-Glutamic aci d and CPP completely displaced specific binding in all regions tested, with similar IC50 values throughout. Similarly, glycine was able to i nhibit the binding in all areas considered: 10 mu M and 1 mM glycine r educed the binding to 80% and 65% of control (average between areas) r espectively. The percentage of specific [H-3]-CGP 39653 binding inhibi ted by 1 mM glycine varied among regions (P<0.05, two-ways ANOVA). Mul tiple comparison, performed by Fisher's protected LSD method, showed t hat the inhibition was lower in striatum (72% of control), with respec t to cortex (66% of control) and hippocampal formation (58% of control ). 6 The inhibitory action of 10 mu M glycine was reversed by 100 mu M 7-chloro-kynurenic acid (7-CKA), a competitive antagonist of the glyc ine site of the NMDA receptor channel complex, in all areas tested. Mo reover, reversal by 7-CKA was not the same in all regions (P<0.05, two -ways ANOVA). In fact, in the presence of 10 mu M glycine and 100 mu M 7-KCA, specific [H-3]-CGP 39653 binding in the striatum was 131% of c ontrol, which was significantly greater (Fisher's protected LSD) than binding in the hippocampus and the thalamus (104% and 112% of control, respectively). 7 These results demonstrate that [H-3]-CGP 39653 bindi ng can be inhibited by glycine in rat brain regions containing NMDA re ceptors; moreover, they suggest the existence of regionally distinct N MDA receptor subtypes with a different allosteric mechanism of [H-3]-C GP 39653 binding modulation through the associated glycine site.