cgp-39653 and 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic-acid

Pretreatment with sulfhydryl-reactive agents, such as N-ethylmaleimide and p-chloromercuriphenylsulfonic acid, invariably resulted in marked inhibition of the binding of DL-(E)-2-amino-4-[3H]propyl-5-phosphono-3-pentenoic acid ([3H]CGP 39653), a competitive antagonist at an N-methyl-D-aspartate (NMDA)-sensitive subclass of central excitatory amino acid receptors, in brain synaptic membranes extensively washed and treated with Triton X-100, but did not significantly affect the binding of L-[3H]-glutamic acid ([3H]Glu), an endogenous agonist. The pretreatment was effective in reducing the binding of [3H]-CGP 39653 at equilibrium, without altering the initial association rate, and decreased the affinity for the ligand. Pretreatment with sulfhydryl-reactive agents also enhanced the potencies of NMDA agonists to displace [3H]-CGP 39653 binding and attenuated those of NMDA antagonists, but had little effect on the potencies of the agonists and antagonists to displace [3H]Glu binding. The binding of both [3H]CGP 39653 and [3H]Glu was similarly sensitive to pretreatment with four different proteases in Triton-treated membranes, whereas pretreatment with phospholipase A2 or C markedly inhibited [3H]CGP 39653 binding without altering [3H]Glu binding. Moreover, both phospholipases not only induced enhancement of the abilities of NMDA agonists to displace the binding of [3H]CGP 39653 and [3H]Glu, but also caused diminution of those of NMDA antagonists. These results suggest that both sulfhydryl-reactive agents and phospholipases may predominantly interfere with radiolabeling of the NMDA recognition domain in a state favorable to an antagonist by [3H]CGP 39653, with concomitant facilitation of that in an antagonist-preferring form by [3H]Glu. The possible presence of multiple forms of the NMDA recognition domain is further supported by these data.

Topics: 2-Amino-5-phosphonovalerate; 4-Chloromercuribenzenesulfonate; Animals; Brain; Dithionitrobenzoic Acid; Dizocilpine Maleate; Ethylmaleimide; Glutamates; Glutamic Acid; Kinetics; Male; N-Methylaspartate; Piperazines; Rats; Rats, Wistar; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate; Sulfhydryl Reagents; Synaptic Membranes

An approximately 54-kDa protein that has binding sites for the competitive N-methyl-D-aspartate (NMDA) receptor antagonists 3-((+-)-2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP) and (+-)-(E)-2-amino-4-propyl-5-phosphonopentenoic acid (CGP 39653) was purified from rat brain synaptic membranes. Polyclonal antibodies to this protein reacted specifically with an approximately 54-kDa protein in synaptic membranes and immunoextracted approximately 60% of [3H]CGP 39653 binding sites associated with solubilized membrane proteins. The antibodies also labeled antigenic sites in the perikaryon and apical and basilar dendrites of pyramidal neurons of the hippocampus and cerebral cortex.

Topics: 2-Amino-5-phosphonovalerate; Animals; Binding, Competitive; Chromatography, Affinity; Dendrites; Enzyme-Linked Immunosorbent Assay; Immunochemistry; Immunohistochemistry; In Vitro Techniques; Membrane Proteins; Piperazines; Precipitin Tests; Protein Binding; Rats; Receptors, Antigen; Receptors, N-Methyl-D-Aspartate; Synaptic Membranes

Treatment with a low concentration of Triton X-100 almost tripled the binding of [3H]D,L-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid (CGP 39653), a novel competitive antagonist at an N-methyl-D-aspartate (NMDA)-sensitive subclass of brain excitatory amino acid receptors, in synaptic membranes of the rat brain. The binding linearly increased with increasing protein concentrations of up to 0.4 mg/ml and also increased in proportion to incubation time with a plateau within 60 min after the initiation of incubation at 2 degrees C in Triton-treated membranes. Elevation of incubation temperature from 2 degrees C to 30 degrees C resulted in a marked decrease in the binding at equilibrium by 80%, and a maximal level was obtained within 1 min after the initiation of incubation at 30 degrees C with a gradual decline of up to 10 min. Bound [3H]CGP 39653 was rapidly dissociated by the addition of excess unlabeled L-glutamic acid (Glu), and the time required to attain complete dissociation was 60 min at 2 degrees C and 1 min at 30 degrees C, respectively. Among several agonists and antagonists tested, Glu was the most potent displacer of [3H]CGP 39653 binding with progressively less potent displacement by D-2-amino-5-phosphonovaleric, (+-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic (CPP), D-2-amino-7-phosphonoheptanoic, N-methyl-D-aspartic and N-methyl-L-aspartic acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 2-Amino-5-phosphonovalerate; Animals; Binding, Competitive; Brain Chemistry; Detergents; Glutamates; Glutamic Acid; In Vitro Techniques; Male; Octoxynol; Piperazines; Polyethylene Glycols; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Synaptic Membranes

D,L-(E)-2-Amino-4-propyl-5-phosphono-3-pentenoic acid (CGP 39653), a new, high-affinity, selective NMDA receptor antagonist, interacts with rat cortical membranes in a saturable way and apparently to a single binding site, with a KD of 10.7 nM and a receptor density of 2.6 pmol/mg of protein. Displacement analysis of [3H]CGP 39653 binding shows a pharmacological profile similar to that reported for another NMDA antagonist, 3-[(+/-)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid (CPP). Glycine, however, is able to discriminate between the two ligands; in fact, it does not affect [3H]CPP binding but inhibits [3H]CGP 39653 binding in a biphasic way. D-Serine, another agonist at the strychnine-insensitive glycine binding site of the NMDA receptor complex, inhibits [3H]CGP 39653 binding in the same way as glycine, with a potency that correlates with its binding affinity at the glycine site. In addition, 7-chlorokynurenic acid, an antagonist at the glycine site, is able to reverse the displacement of [3H]CGP 39653 by glycine in a dose-dependent manner. Furthermore, the dissociation rate constant of [3H]CGP 39653 is enhanced in the presence of glycine, whereas the presence of NMDA receptor ligands does not modify the rate of dissociation of [3H]CGP 39653 from the receptor. These results indicate that part of the binding of the NMDA antagonist CGP 39653 can be potently modified by glycine through an allosteric mechanism, and suggest the existence of two antagonist preferring NMDA receptor subtypes that are differentially modulated through the glycine binding site.

Topics: 2-Amino-5-phosphonovalerate; Animals; Binding, Competitive; Brain; Glycine; Kinetics; Kynurenic Acid; Male; Piperazines; Rats; Rats, Sprague-Dawley; Tritium