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Notes: Abstract: Glycine is the principal inhibitory neurotransmitter in posterior regions of the brain. In addition, glycine serves as an allosteric regulator of excitatory neurotransmission mediated by the N-methyl-D-aspartate (NMDA) acidic amino acid receptor subtype. The studies presented here characterize [3H]glycine binding to washed membranes prepared from rat spinal cord and cortex, areas enriched in glycine inhibitory and NMDA receptors, respectively, in an attempt to define the glycine recognition sites on the two classes of receptors. Specific binding for [3H]glycine was seen in both cortex and spinal cord. Saturation analyses in cortex were best fitted by a two-site model with respective equilibrium dissociation constants (KD values) of 0.24 and 5.6 μM and respective maximal binding constants (Bmax values) of 3.4 and 26.7 pmol/mg of protein. Similar analyses in spinal cord were best fitted by a one-site model with a KD of 5.8 μM and Bmax of 20.2 pmol/mg of protein. Na+ had no effect on [3H]glycine binding to cortical membranes but increased the binding to spinal cord membranes by 〈15-foId. This Na+-dependent binding may reflect glycine binding to the recognition site of the high-affinity, Na+-dependent glycine uptake system. Several short-chain, neutral amino acids displaced [3H]glycine binding from both cortical and spinal cord membranes. The most potent displacers of [3H]glycine binding to cortical membranes were D-serine and D-alanine, followed by the L-isomers of serine and alanine and β-alanine. In contrast. D-serine and D-alanine were similar in potency to L-serine in spinal cord membranes. Compounds active at receptors for the acidic amino acids had disparate effects on the binding of [3H]glycine. At 10 μM, NMDA resulted in a 25% increase, whereas D- and L-2-amino-5-phosphonovaleric acid at 100 μM resulted in a 30% decrease, in [3H]glycine binding to cortical membranes. Kynurenic acid was the most potent of the acidic amino acid-related compounds at displacing [3H]glycine binding. In cortical membranes, kynurenic acid displacement was resolved into a high- and a low-affinity component; the high-affinity component displaced the high-affinity component of [3H]glycine binding. Strychnine, 5,6,7,8-tetrahydro-4H-isoxazolo[4,5-d]azepin-3-ol, and 5,6,7,8-tetrahydro-4H-isoxazolo[3,4-d]azepin-3-ol, antagonists at the glycine postsynaptic inhibitory neurotransmitter receptor, displaced from 25 to 40% of f3H]glycine binding in spinal cord membranes but had no effect on the binding in cortical membranes, a result suggesting that this amount of [3H]gIycine binding in spinal cord membranes was to the inhibitory postsynaptic glycine receptor. The data suggest that [3H]glycine binding measures different receptor populations in cortical versus spinal cord membranes. The results are discussed in terms of binding to the glycine allosteric site on the NMDA receptor in cortical membranes and binding to the glycine recognition site on the glycine inhibitory postsynaptic receptor in spinal cord membranes.