ABBREVIATIONS: NMDA, N-methyl-D-aspartate; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; DNQX, 6,7-dinitro-quinoxaline-2,3,-dione; TTX, tetro- dotoxin; CPP, 3-(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid; EGTA, ethylene glycol bis($-aminoethyl ether)-N,N,N’,N’-tetraacetic acid; HEPES, 4-2-hydroxyethyl)-1 -piperazineethanesulfonic acid. 565 0026-895X/89/050565-06$02.00/0 Copyright (#{231}) by The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 35:56.5-570 ACCELERA TED COMMUNICATION Interaction of 6-Cyano-7-nitroquinoxaline-2,3-dione with the N- Methyl-o-aspartate Receptor-Associated Glycine Binding Site ROBIN A. J. LESTER, MERRIT L. QUARUM, JOEL D. PARKER, ECKARD WEBER, and CRAIG E. JAHR Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland, Oregon 97201 Received December 7, 1988; Accepted February 14, 1989 SUMMARY The interaction of newly described antagonist of the non-NMDA glutamate receptor 6-cyano-7-nitroquinoxaline-2,3-dione(CNQX) with the glycine site of the NMDA receptor complex has been investigated. In whole-cell patch recordings from hippocampal neurons maintained in culture, currents induced by N-methyl-D- aspartate (NMDA) were dependent on extracellular glycine. Re- sponses to both NMDA (30 pM) and kainate(20 pM) were reduced by CNQX (1 0-30 pM). The antagonism by CNQX of NMDA, but not kainate, receptor-mediated responses could be reversed by increasing the concentration of glycine in the external medium. Glycine concentration-response curves constructed in the pres- ence of 30 cM NMDA were shifted to the right by CNQX, suggesting that CNQX was competing with glycinefor the glycine binding site. However, even at high concentrations of glycine (300 pM) the maximal NMDA current obtained in the presence of CNQX (1 0-30 pM) was not restored to control levels. Because CNQX had no effect on responses produced by supramaximal concentrations of NMDA (500 pM) and glycine (300 pM), it is suggested that CNQX also interacts with the NMDA recognition site. The antagonism of currents induced by NMDA was not dependent on the membrane potential, and the rapid onset and offset of the block suggested that there was little or no use dependence. Radioligand binding experiments were performed using [3H]glycine to label the strychnine-insensitive glycine rag- ulatory site of the NMDA receptor complex in guinea pig brain frontal cortex membranes. CNQX displaced [3H]glycine binding in a concentration-dependent manner (IC50 = 5.7 pM). Scatchard analysis of the inhibition showed a decrease in the affinity (in- crease in K0) of [3H]glycine binding, but no change in the number of binding sites (Bmax) in the presence of 5 pM CNQX, suggesting a competitive interaction. These data provide evidence that CNQX antagonizes NMDA receptor-mediated responses by competing with glycine for a modulatory site associated with the NMDA receptor complex. Furthermore, the results indicate that CNQX may not be as selective an antagonist for non-NMDA receptors as initially described, although its selectivity will depend on the concentration of the NMDA receptor ligand and may be enhanced by increasing the extracellular concentration of glycine. Receptors in the vertebrate central nervous system that mediate the actions of the excitatory amino acid transmitter L- glutamate have been broadly divided into two classes, NMDA and non-NMDA (1). Although selective NMDA receptor an- tagonists have been available for some time (2), only recently have specific antagonists become available for non-NMDA receptors (3). In the original report, two compounds, DNQX and CNQX, were observed to markedly depress the excitation of spinal cord neurons induced by the non-NMDA receptor agonists kainate and quisqualate, with little effect on responses produced by NMDA (3). In addition, these compounds showed a higher affinity for non-NMDA than for NMDA receptor binding sites (3). Electrophysiological (4, 5) and release (6) This work was supported by grants from the McKnight Foundation and the National Institutes of Health (NS21419) to C.E.J. and grants from the National Institute of Mental Health (MH40303 and MH42068) to E.W. studies utilizing in vitro preparations have since confirmed these findings and suggested that CNQX interacts competi- tively with non-NMDA receptors. However, there are reasons to believe that the quinoxaline- diones may be interacting with the NMDA receptor system in a more complex manner. In quantitative pharmacological stud- ies on neonatal rat spinal cord preparations, DNQX produced a shift to the right in the NMDA concentration-response curve, which was accompanied by a depression of the maximal re- sponse at high antagonist concentrations, suggesting a noncom- petitive inhibition (7). A similar noncompetitive block of NMDA receptor-induced responses has been observed with kynurenic acid (7-9) and 7-chiorokynurenic acid (10). Based on the observations that both these compounds could displace [3H]glycine from a strychnine-insensitive binding site (10, 11) and that an increase in the extracellular glycine concentration at BIN 868/Biomed Sci Lib(Wessex Biomedical) on January 31, 2012 molpharm.aspetjournals.org Downloaded from