Characterization of the binding of [3H]-CGS 19755: a novel N-methyl-D-aspartate antagonist with nanomolar affinity in...

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 Kd values of 9 and 200 nM, and corresponding Bmax values of 0.55 and 1.00 pmol mg-1 protein. In contrast, a single binding component with a Kd value of 24 nM and an apparent Bmax 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-alpha-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 (34-+/-)2-carboxypiperazin-4-yl)propyl-1-phosphonic acid), the corresponding analogue of 2-amino-7-phosphonoheptanoic acid (AP7).