BINDING OF [H-3] 3-(2-CARBOXYPIPERAZIN-4-YL)PROPYL-1-PHOSPHONIC ACID TO RAT-BRAIN MEMBRANES - A SELECTIVE, HIGH-AFFINITY LIGAND FOR N-METHYL-D-ASPARTATE RECEPTORS

Abstract

3-(2-Carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP), a rigid analog of 2-amino-7-phosphonoheptanoic acid, has been reported as a selective N-methyl-D-aspartate (NMDA) antagonist. [3H]CPP bound with relatively high affinity (Kd = 201 nM) to Triton-treated rat brain crude synaptic membranes using a centrifugation assay. Binding was saturable, reversible, heat sensitive and dependent on protein concentration. Specific binding, which represented 75 to 85% of the total counts bound, was enriched in synaptosomal and microsomal fractions of rat brain, suggesting an involvement in events related to synaptic transmission. On a regional basis, binding was highest in hippocampus, followed by cortex > striatum > cerebellum = thalamus. No specific binding could be detected in pons medulla or in liver, kidney, heart, lung and adrenal tissue. [3H]CPP binding was stereoselective for the isomers of glutamate, 2-amino-5-phosphonopentanoic acid, homocysteic acid, .alpha.-aminoadipic acid and N-methyl-aspartate. The most potent compounds tested were L-glutamate and CPP, which were equiactive in displacing [3H]CPP. The order of activity of other excitatory amino acid receptor ligands was D-2-amino-5-phosphonopentanoic acid > L-homocysteic acid .gtoreq. DL-2-amino-7-phosphonoheptanoic acid = D-aspartate = L-aspartate > L-serine-O-sulfate = D-.alpha.-aminoadipic acid = ibotenate > NMDA > DL-2-amino-6-phosphonohexanoic acid > quisqualate > N-methyl-L-aspartate. The quisqualate- and kainate-type receptor agonists DL-.alpha.-amino-3-hydroxy-5-methylisoxazole-4-propionate and kainic acid, respectively, had negligible activity at 100 .mu.M. Binding was also unaffected by phencyclidine, dexoxadrol, DL-2-amino-4-phosphonobutyric acid, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate, .gamma.-D-glutamylaminomethyl-sulfonic acid, .gamma.-aminobutyric acid and balcofen. These results are consistent with [3H]CPP being a selective ligand for the NMDA excitatory amino acid receptor. The specificity and relatively high affinity of [3H]CPP, together with its high degree of specific binding make this the current ligand of choice to evaluate NMDA receptors in vitro.