Recent evidence suggests that a strychnine-insensitive glycine modulatory site is associated with the N-methyl-d-aspartate receptor-channel complex. A quantitative autoradiographic method was used to characterize the pharmacological specificity and anatomical distribution of strychnine-insensitive [3H]glycine binding sites in rat brain. [3H]Glycine binding was specific, saturable, reversible, pH and temperature-sensitive and of high affinity. [3H]Glycine interacted with a single population of sites having a KD of approximately 200 nM and a maximum density of 6.2 pmol/mg protein (stratum radiatum, CA1). Binding exhibited a pharmacological profile similar to the physiologically defined strychnine-insensitive glycine modulatory site. Binding was stereoselective; the rank order of potency of simple amino acids as displacers of binding was: glycine > d-serine > d-alanine > l-serine > l-alanine > l-valine > d-valine. Binding was not altered by the inhibitory glycine receptor ligand, strychnine, by the glutamate agonists, quisqualate and kainate, or by GABA receptor selective ligands. Most competitive agonists or antagonists of the N-methyl-d-aspartate recognition site were ineffective displacers of glycine binding. The exceptions were the aminophosphono series of antagonists, d-alpha-aminoadipate, gamma-d-glutamylglycine and beta-d-aspartylaminomethylphosphonic acid. However, the inhibition of [3H]glycine binding produced by the aminophosphono compounds could be accounted for by the level of glycine contamination present in these compounds. The non-competitive NMDA receptor-channel blockers, phencyclidine, its thienyl derivative, and MK-801 did not alter glycine binding. Kynurenate, glycine methylester, l-serine-O-sulfate, l-homocysteic acid, and several glycine-containing dipeptides were effective displacers of glycine binding. Structure-activity relations of agonists and antagonists of the strychnine-insensitive glycine binding site are discussed. The distribution of strychnine-insensitive [3H]glycine binding was heterogeneous with the following rank order of binding densities: hippocampus > cerebral cortex > caudate-putamen ≥ thalamus > cerebellum > brain stem. This distribution of binding was correlated with N-methyl-d-aspartate-sensitive [3H]glutamate binding (r2 = 0.77; P< 0.001; Pearson product-moment) and [3H]thienylcyclohexylpiperidine binding (r2 = 0.72; P < 0.001). These observations are consistent with the hypothesis that the strychnine-insensitive glycine binding site is closely associated with the N-methyl-d-aspartate receptor-channel complex. These data also suggest that the stoichiometry between these binding sites is approximately 1:3:4 (thienylcyclohexylpiperidine: glycine:glutamate) in stratum radiatum of area CA1. However, in other brain regions, there were more glycine than glutamate binding sites suggesting that there may be heterogeneity in the N-methyl-d-aspartate receptor-channel complex.
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