l-701324 and 7-chlorokynurenic-acid

NMDA receptors are glutamate-regulated ion channels that are of great importance for many physiological and pathophysiological conditions in the mammalian central nervous system. We have previously shown that, at low pH, glutamate decreases binding of the open-channel blocker [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten, 5,10-imine ([3H]MK-801) to NMDA receptors in the presence of 1 mM Mg2+ but not in Krebs buffer. Here, we investigated which cations that block the glutamate-induced decrease in Krebs buffer, using [3H]MK-801 binding assays in membrane preparations from the rat cerebral cortex. At pH 6.0, Na+, K+, and Ca2+ antagonized the glutamate-induced decrease with cross-over values, which is a measure of the antagonist potencies of the cations, of 81, 71, and 26 mM, respectively, in the absence of added glycine. Thus, in Krebs buffer only the concentration of Na+ (126 mM) is sufficiently high to block the glutamate-induced decrease observed at low pH. In the presence of 1 mM Mg2+ and 10 mM Ca2+ at pH 7.4, the cross-over values for Na+, K+, and Ca2+ were 264, 139, and 122 mM, respectively, in the absence of added glycine. This is the same rank order of potency as observed at pH 6.0, suggesting that the less H+-sensitive and the less Ca2+-sensitive, glutamate-induced decreases in [3H]MK-801 binding represent the same entity. The glycine site antagonists 7-chlorokynurenate (10 microM) and 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(H)-quinoline (L-701,324; 1 microM) antagonized the glutamate-induced decrease in [3H]MK-801 binding observed in presence of Mg2+ at pH 6.0, suggesting that glycine is required together with glutamate to induce the decrease observed at low pH. These results suggest that in addition to a previously described high-affinity binding site for H+ and Ca2+ there exist a low-affinity binding site for H+, Ca2+, Na+, and K+ on NMDA receptors. The latter site may under physiological conditions be blocked by Na+ or K+, depending on the extra/intracellular localization of the modulatory site. Both the high-affinity and low-affinity cation sites mediate antagonistic effects on the glutamate- and glycine-induced decrease of the affinity of the [3H]MK-801 binding site, which may correspond to similar changes in the affinity of the voltage-sensitive Mg2+-block site inside the NMDA receptor channel pore, which in turn may affect current and Ca2+ influx through activated NMDA receptor channels.

Topics: Animals; Calcium; Cations; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Glutamates; Glycine; Hydrogen-Ion Concentration; Kynurenic Acid; Magnesium; Male; Potassium; Protein Binding; Quinolones; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Sodium

The pharmacological effects of two novel N-methyl-D-aspartate (NMDA) receptor glycine site antagonists, L-701,324 and L-695,902 were examined on whole-cell voltage-clamped cells and compared to a prototypic antagonist, 7-chlorokynurenic acid. Both L-701,324 and L-695,902 non-competitively antagonised NMDA responses elicited in rat cultured cortical neurones, this was shown to be due to a competitive interaction at the glycine co-agonist site on the receptor complex (Kb values: 19 nM and 2.6 microM, respectively). Inhibition curves for the antagonism of responses to combined applications of NMDA and glycine showed that both antagonists were devoid of any intrinsic activity i.e. "full" antagonists and were, therefore, capable of completely abolishing inward currents. Despite this fact, both of these novel antagonists apparently modulated glutamate affinity for its recognition site-a property hitherto associated only with glycine site partial agonists. Human recombinant NMDA receptors comprising NR1a/NR2A and NR1a/NR2B subunits expressed in mouse fibroblast cells were also used to determine whether L-701,324 and L-695,902 were capable of discriminating between subtypes of NMDA receptor. Inhibition curves to each antagonist showed no difference in affinity for either of these subunit assemblies (mK1 values L-701,324 = 0.005 microM on both assemblies; L-695,902 = 4.37 and 3.7 microM on NR1a/NR2A and NR1a/NR2B, respectively). Kinetic analysis of the off-rates of antagonism with L-701,324 revealed that the high affinity of this compound compared to 7-chlorokynurenic acid were attributable to an exceptionally slow dissociation of the antagonist from the receptor.

Topics: Animals; Cells, Cultured; Cerebral Cortex; Electrophysiology; Excitatory Amino Acid Antagonists; Humans; Kinetics; Kynurenic Acid; Membrane Potentials; Mice; Neurons; Patch-Clamp Techniques; Quinolones; Rats; Receptors, Glycine; Receptors, N-Methyl-D-Aspartate