6-cyano-7-nitroquinoxaline-2-3-dione and 1-hydroxy-3-amino-2-pyrrolidone

Previous research has demonstrated that intrathecal i.t. morphine in a dose of 60.0 nmol into the spinal subarachnoid space of mice can evoke nociceptive behavioral responses consisting of a severe hindlimb scratching directed toward the flank followed by biting/licking of the hindpaw. The present study was undertaken to examine the involvement of spinal N-methyl-D-aspartate (NMDA) and opioid receptors on the behavioral responses evoked by high-dose i.t. morphine. Pretreatment with naloxone, an opioid receptor antagonist (1.0 and 4.0 mg/kg, s.c.), failed to reverse the morphine-evoked behavioral response, suggesting that the morphine effect is not mediated through the opioid receptors in the spinal cord. The morphine-induced behavior was dose-dependently inhibited by i.t. co-administration of the competitive NMDA receptor antagonists, D(-)-2-amino-5-phosphonovaleric acid (D-APV) (6.25-50.0 pmol) and 3-((+)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) (3.125-25.0 pmol). The characteristic behavior was also reduced by co-administration of (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5,10-imine maleate (MK-801) (74.1-250 pmol), an NMDA ion-channel blocker. Ifenprodil, a competitive antagonist of the polyamine recognition site of NMDA receptor ion channel complex, produced a dose-related inhibitory effect on the behavioral response to i.t. morphine with less potency than the competitive and non-competitive antagonists examined. High doses of (+)-HA-966, a glycine/NMDA antagonist, induced a dose-dependent inhibition of morphine-induced response. The effective dose of i.t. 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist, needed to reduce the morphine-induced response, was approximately 10-fold greater than that of D-APV. These results suggest that spinal NMDA receptors, but not non-NMDA receptors, may be largely involved in elicitation of the behavioral episode following i.t. injection of morphine in mice.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Binding, Competitive; Dizocilpine Maleate; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Hindlimb; Hyperalgesia; Injections, Spinal; Male; Mice; Mice, Mutant Strains; Morphine; Naloxone; Narcotic Antagonists; Nerve Tissue Proteins; Pain Measurement; Piperazines; Piperidines; Pyrrolidinones; Reaction Time; Receptors, N-Methyl-D-Aspartate; Receptors, Opioid; Spinal Cord; Subarachnoid Space

The interactions between the glycine and glutamate binding sites of the NMDA receptor have been studied in outside-out patches and synapses from hippocampal neurons in culture using rapid drug application techniques. Desensitization of NMDA receptor-mediated currents elicited by glutamate in newly excised outside-out patches was reduced in the presence of saturating concentrations of glycine. This suggests that the glutamate and glycine binding sites of the NMDA receptor are allosterically coupled as has been reported in whole-cell preparations. A glycine-insensitive form of desensitization increased rapidly over the first few minutes of recording and largely occluded the glycine concentration-sensitive desensitization in outside-out patches. However, even in old patches that displayed no glycine-sensitive desensitization, the unbinding rate of glycine was increased fourfold by the presence of glutamate, suggesting that the two binding sites were still allosterically coupled. These data suggest the existence of two forms of NMDA receptor desensitization in outside-out patches, only one of which is dependent on the concentration of glycine. In the presence of saturating levels of glycine, activation of NMDA receptors by synaptic stimulation or by exogenous glutamate resulted in currents that relaxed biexponentially. Addition of the partial glycine-site agonist 1-hydroxy-3-aminopyrrolid-2-one (HA-966) increased the rate of decay of both synaptic and patch currents. This suggests that HA-966 increases the dissociation rate of glutamate from NMDA receptors. These results support the hypothesis that the glutamate and glycine binding sites of NMDA receptors interact allosterically; ligand binding at both types of sites can affect the affinity of the other type for its agonist.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Binding Sites; Cells, Cultured; Glutamates; Glutamic Acid; Glycine; Hippocampus; Kinetics; Neurons; Pyrrolidinones; Quinoxalines; Rats; Receptors, N-Methyl-D-Aspartate; Synapses; Time Factors

N-methyl-D-aspartic acid (NMDA) and non-NMDA ionotropic receptors mediating increase of norepinephrine (NE) release coexist on NE rat hippocampus axon terminals. Activation of non-NMDA receptors permits activation of NMDA receptors also in presence of Mg++ ions and induces important changes in the NMDA receptor recognition site and in its intrinsic ion channel. We have now studied the effects of this receptor-receptor interaction on the glycine site of the NMDA receptor by using two antagonists, 7-chloro-kynurenic acid and (+-)-3-amino-1-hydroxy-2-pyrrolidone (HA-966). Both the [3H]NE releases induced from rat hippocampus synaptosomes by NMDA (no Mg++ added) and by NMDA+quisqualic acid (QA), in the presence of 1.2 mM Mg++, were prevented by 7-chloro-kynurenic acid with almost identical potency (IC50 values: 0.19 and 0.39 microM, respectively). In contrast, HA-966, up to 1000 microM, was ineffective toward NMDA (no Mg++), but it blocked the effect of NMDA + QA in presence of Mg++ (IC50 = 1.1 microM). HA-966 also antagonized NMDA + QA in Mg(++)-free medium. Thus coactivation of non-NMDA and NMDA receptors seems to permit the antagonistic activity of HA-966. In the presence of Mg++, L-glutamic acid (L-Glu) enhanced [3H]NE release. The sensitivity of the L-Glu effect to various antagonists was similar to that of the effect of NMDA + QA, indicating that the NMDA receptor complex activated either by NMDA + QA or by the physiological transmitter L-Glu in presence of Mg++ ions undergoes dramatic conformational changes at the recognition site, at the ion channel as well as at the glycine site.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Cells, Cultured; Dizocilpine Maleate; Hippocampus; Magnesium; Male; N-Methylaspartate; Norepinephrine; Pyrrolidinones; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate; Synaptosomes

Central activation of excitatory amino acid receptors has been implicated in neuropathic pain following nerve injury. In a rat model of painful peripheral mononeuropathy, we compared the effects of non-competitive NMDA receptor antagonists (MK 801 and HA966) and a non-NMDA receptor antagonist (CNQX) on induction and maintenance of thermal hyperalgesia induced by chronic constrictive injury (CCI) of the rat common sciatic nerve. Thermal hyperalgesia to radiant heat was assessed by using a foot-withdrawal test and NMDA/non-NMDA receptor antagonists were administered intrathecally onto the lumbar spinal cord before and after nerve injury. Four daily single treatments with 20 nmol HA966 or CNQX beginning 15 min prior to nerve ligation (pre-injury treatment), reliably reduced thermal hyperalgesia in CCI rats on days 3, 5, 7 and 10 after nerve ligation. Thermal hyperalgesia was also reduced in CCI rats receiving a single post-injury treatment with HA966 (20 or 80 nmol) or MK 801 (5 or 20 nmol) on day 3 after nerve ligation when thermal hyperalgesia was well developed. In contrast, a single post-injury CNQX (20 or 80 nmol) treatment failed to reduce thermal hyperalgesia or to potentiate effects of HA966 or MK 801 (5 or 20 nmol) on thermal hyperalgesia in CCI rats. Moreover, multiple post-injury CNQX treatments utilizing the same dose regime as employed for the pre-injury treatment attenuated thermal hyperalgesia but only when the treatment began 1 or 24 h (but not 72 h) after nerve ligation.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Dizocilpine Maleate; Hot Temperature; Hyperalgesia; Injections, Spinal; Male; Pain; Peripheral Nervous System Diseases; Pyrrolidinones; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Sciatic Nerve

Direct intracerebellar administration of quisqualate resulted in marked increases in levels of cGMP in the cerebellum of the mouse, with a Hill number of 2.0. Quinoxalinediones, DNQX (6,7-dinitroquinoxaline-2,3-dione) and CNQX (6-nitro,7-cyanoquinoxaline-2,3-dione) attenuated the quisqualate-induced response. 6,7-Dinitroquinoxaline-2,3-dione also attenuated the D-serine-induced increases in levels of cGMP in a competitive manner. Intracerebellar injection of DNQX also antagonized the response to parenterally-administered harmaline. Similar results were also obtained with CNQX. These results indicate that these quinoxalinediones can attenuate the responses, mediated through the NMDA-associated glycine recognition sites, as well as the NMDA receptor complex. However, the glycine antagonist HA-966 (3-amino-1-hydroxypyrrolidone-2), at doses which completely reversed the increases induced by D-serine, failed to alter the response to quisqualate, indicating a lack of effect of glycine antagonists on quisqualate-mediated synaptic events. These results further support the interaction of the quinoxalinediones, DNQX and CNQX, with the NMDA receptor complex as established in receptor binding and electrophysiological studies.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Cerebellum; Cyclic AMP; Glycine; Harmaline; Kinetics; Male; Mice; N-Methylaspartate; Pyrrolidinones; Quinoxalines; Quisqualic Acid; Serine