cgp-39653 and 7-chlorokynurenic-acid

Antisense oligodeoxynucleotides were used to determine whether alterations in the expression of N-methyl-D-aspartate (NMDA) receptor subunit mRNA are responsible for developmental changes in the sensitivity of receptors to agonists and antagonists. Xenopus laevis oocytes were injected with mRNA prepared from neonatal and adult rat cerebral cortex, and the effects of agonists and antagonists were determined under voltage-clamp conditions. Glycine-site antagonists like 7-chlorokynurenate and glutamate-site antagonists like CGP-39653 were more potent at NMDA receptors expressed from mRNA from adult rat cerebral cortex than those expressed from mRNA from 1-day-old rat. NMDA receptors from 1-day-old rat cerebral cortex were more sensitive to activation by glycine than were receptors from adult rat cerebral cortex. 7-Chlorokynurenate and CGP-39653 were more potent inhibitors of responses seen with heteromeric NR1/NR2A receptors than with NR1/ NR2B receptors. Conversely, heteromeric NR1/NR2B receptors were more sensitive to activation by glycine than were NR1/NR2A receptors. We previously described a delay in the expression of the NR2A subunit in developing rat brain. Anti-sense oligodeoxynucleotides were used to determine whether the delayed expression of the NR2A subunit underlies changes in pharmacological properties observed during development. The properties of receptors seen when adult brain mRNA was coinjected with antisense oligodeoxynucleotides against the NR2A subunit were similar to those found in receptors from 1-day-old rat brain. These data suggest that changes in the sensitivity of NMDA receptors to antagonists and to glycine seen during development are a result of alterations in the expression of different species of NR2 subunit mRNA.

Topics: 2-Amino-5-phosphonovalerate; Aging; Animals; Animals, Newborn; Base Sequence; Cerebral Cortex; Excitatory Amino Acid Antagonists; Female; Gene Expression Regulation, Developmental; Glycine; Kynurenic Acid; Macromolecular Substances; Molecular Sequence Data; N-Methylaspartate; Oligonucleotides, Antisense; Oocytes; Rats; Rats, Sprague-Dawley; Receptors, Glycine; Receptors, N-Methyl-D-Aspartate; RNA, Messenger; Xenopus laevis

7-Chlorokynurenate, an antagonist at the glycine recognition site of the NMDA receptor complex, increases the binding of the competitive NMDA receptor antagonist [3H]CGP39653 ([3H]D,L-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid) to well washed rat brain membranes but only in the presence of 100 microM spermine. Conversely, spermine only increases [3H]CGP39653 binding in the presence of 10 microM 7-chlorokynurenate, through a mechanism insensitive to the putative polyamine antagonists ifenprodil, arcaine or putrescine. Thus, the effects of glycine antagonists and polyamines on the binding of competitive NMDA receptor antagonists may depend on the residual glycine and polyamine content of the membrane preparation or the state of the glycine recognition site. These data further attest to the complexity of interactions between spermine and the glycine and glutamate recognition sites of the NMDA receptor.

Topics: 2-Amino-5-phosphonovalerate; Allosteric Regulation; Animals; Biguanides; Binding Sites; Glycine; In Vitro Techniques; Kynurenic Acid; Male; Piperidines; Prosencephalon; Putrescine; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Spermine

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