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Characterization of the binding of [ 3 H]‐CGS 19755: a novel N‐methyl‐ d ‐aspartate antagonist with nanomolar affinity in rat brain
Author(s) -
Murphy Deborah E.,
Hutchison Alan J.,
Hurt Steven D.,
Williams Michael,
Sills Matthew A.
Publication year - 1988
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1988.tb11723.x
Subject(s) - nmda receptor , phencyclidine , ampa receptor , stereochemistry , chemistry , glutamate receptor , competitive antagonist , binding site , antagonist , receptor , biochemistry
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 [ 3 H]‐CGS 19755 bound with high affinity and selectivity to the NMDA‐type excitatory amino acid receptor. 2 [ 3 H]‐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 [ 3 H]‐CGS 19755 selectively labels the NMDA receptor. The most active inhibitors of [ 3 H]‐CGS 19755 binding were l ‐glutamate and CGS 19755 (IC 50 values = 100 n m ). 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 [ 3 H]‐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 [ 3 H]‐CPP (3‐(±)−(2‐carboxypiperazin‐4‐yl)propyl‐l‐phosphonic acid), the corresponding analogue of 2‐amino‐7‐phosphonoheptanoic acid (AP7).