Skip to main content
PMC home page
British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 1988 Nov;95(3):932–938. doi: 10.1111/j.1476-5381.1988.tb11723.x

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

D E Murphy 1, A J Hutchison 1, S D Hurt 1, M Williams 1, M A Sills 1
PMCID: PMC1854225  PMID: 2850065

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 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Full text

PDF
932

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Boast C. A., Gerhardt S. C., Pastor G., Lehmann J., Etienne P. E., Liebman J. M. The N-methyl-D-aspartate antagonists CGS 19755 and CPP reduce ischemic brain damage in gerbils. Brain Res. 1988 Mar 1;442(2):345–348. doi: 10.1016/0006-8993(88)91522-3. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Enna S. J., Snyder S. H. Influences ions, enzymes, and detergents on gamma-aminobutyric acid-receptor binding in synaptic membranes of rat brain. Mol Pharmacol. 1977 May;13(3):442–453. [PubMed] [Google Scholar]
  4. Fisher T. E., Tuchek J. M., Johnson D. D. A comparison of methods for removal of endogenous GABA from brain membranes prepared for binding assays. Neurochem Res. 1986 Jan;11(1):1–8. doi: 10.1007/BF00965160. [DOI] [PubMed] [Google Scholar]
  5. Foster A. C., Fagg G. E. Acidic amino acid binding sites in mammalian neuronal membranes: their characteristics and relationship to synaptic receptors. Brain Res. 1984 May;319(2):103–164. doi: 10.1016/0165-0173(84)90020-1. [DOI] [PubMed] [Google Scholar]
  6. Foster A. C., Fagg G. E. Comparison of L-[3H]glutamate, D-[3H]aspartate, DL-[3H]AP5 and [3H]NMDA as ligands for NMDA receptors in crude postsynaptic densities from rat brain. Eur J Pharmacol. 1987 Jan 20;133(3):291–300. doi: 10.1016/0014-2999(87)90025-2. [DOI] [PubMed] [Google Scholar]
  7. Lehmann J., Schneider J., McPherson S., Murphy D. E., Bernard P., Tsai C., Bennett D. A., Pastor G., Steel D. J., Boehm C. CPP, a selective N-methyl-D-aspartate (NMDA)-type receptor antagonist: characterization in vitro and in vivo. J Pharmacol Exp Ther. 1987 Mar;240(3):737–746. [PubMed] [Google Scholar]
  8. Murphy D. E., Schneider J., Boehm C., Lehmann J., Williams M. Binding of [3H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid to rat brain membranes: a selective, high-affinity ligand for N-methyl-D-aspartate receptors. J Pharmacol Exp Ther. 1987 Mar;240(3):778–784. [PubMed] [Google Scholar]
  9. Murphy D. E., Snowhill E. W., Williams M. Characterization of quisqualate recognition sites in rat brain tissue using DL-[3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and a filtration assay. Neurochem Res. 1987 Sep;12(9):775–781. doi: 10.1007/BF00971514. [DOI] [PubMed] [Google Scholar]
  10. Wong E. H., Kemp J. A., Priestley T., Knight A. R., Woodruff G. N., Iversen L. L. The anticonvulsant MK-801 is a potent N-methyl-D-aspartate antagonist. Proc Natl Acad Sci U S A. 1986 Sep;83(18):7104–7108. doi: 10.1073/pnas.83.18.7104. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from British Journal of Pharmacology are provided here courtesy of The British Pharmacological Society

RESOURCES