Characterization of the binding of [3H]‐CGS 19755: a novel N‐methyl‐d‐aspartate antagonist with nanomolar affinity in rat brain

Abstract

1 CGS 19755 (cis-4-phosphonomethyl-2-piperidine carboxylic acid), a rigid analogue of 2-amino-5-phosphonopentanoic acid (AP5), is one of the most potent competitive N-methyl-d-aspartate (NMDA) antagonists described. Using Triton-treated crude synaptic membranes from rat brain, binding studies indicated that [³H]-CGS 19755 bound with high affinity and selectivity to the NMDA-type excitatory amino acid receptor. 2 [³H]-CGS 19755 binding was saturable, reversible, heat-labile, pH-dependent and linear with protein concentration. Specific binding represented 80–85% of the total amount bound. 3 Using a centrifugation assay, saturation experiments revealed two distinct binding components with K_d values of 9 and 200 nM, and corresponding B_max values of 0.55 and l.00 pmol mg⁻¹ protein. In contrast, a single binding component with a K_d value of 24 nM and an apparent value of 0.74 pmol mg⁻¹ protein was observed with a filtration assay. 4 Competition experiments in which both assay techniques were used, showed that [³H]-CGS 19755 selectively labels the NMDA receptor. The most active inhibitors of [³H]-CGS 19755 binding were l-glutamate and CGS 19755 (IC₅₀ values = 100 nm). 5 In the centrifugation assay, a number of excitatory amino acids were found to generate shallow inhibition curves, and computer analysis indicated the presence of two binding components. The quisqualate receptor ligand AMPA (D,L-α-amino-3-hydroxy-5-methylisoxazole-4-propionate), kainic acid and the non-competitive NMDA antagonists, such as phencyclidine, tiletamine and MK-801, were without activity. 6 The high affinity binding obtained with [³H]-CGS 19755 by use of filtration techniques thus permits the more rapid evaluation of compounds as potential NMDA antagonists and agonists. Therefore, this rigid analogue of AP5 is a more suitable radioligand for NMDA receptors than [³H]-CPP (3-(±)−(2-carboxypiperazin-4-yl)propyl-l-phosphonic acid), the corresponding analogue of 2-amino-7-phosphonoheptanoic acid (AP7).