piperidines and 5-7-dichlorokynurenic-acid

The present study investigated the role of the peripheral NR2 subunits of N-methyl-d-aspartatic acid (NMDA) receptors in inflammatory orofacial pain. Experiments were carried out using adult male Sprague-Dawley rats weighing 220 to 280 g. Formalin (5%, 50 μl) was applied subcutaneously to the vibrissa pad. For each animal, the number of noxious behavioral responses, including rubbing or scratching of the facial region proximal to the injection site, was recorded for 9 sequential 5 min intervals. NR2 subunit antagonists were injected subcutaneously at 20 min prior to formalin injection. The subcutaneous injection of 100 or 200 μg of memantine significantly suppressed the number of scratches in the second phase of the behavioral responses to formalin. The subcutaneous injection of 0.25, 2.5, or 25 μg of 5,7-dichlorokynurenic acid also produced significant antinociceptive effects in the second phase. The subcutaneous injection of AP-5 at high dose produced significant antinociceptive effects in the second phase. The subcutaneous injection of PPPA and Ro 25-6981 both significantly suppressed the number of scratches in the second phase. The antinociceptive doses of memantine (200 μg), 5,7-dichlorokynurenic acid (25 μg), AP-5 (20 μg), PPPA (2.5 μg), or Ro 25-6981 (50 μg) injected into the contralateral hind paw did not affect the number of scratches in both the first and second phases. Moreover, the peripheral administration of NR2 subunit antagonists, including other NMDA receptor blockers, did not produce any motor dysfunction. These results indicate that a targeted blockade of peripheral NR2 receptors is a potentially important new method of treating inflammatory pain in the orofacial area.

Topics: Animals; Behavior, Animal; Excitatory Amino Acid Antagonists; Facial Pain; Injections; Kynurenic Acid; Male; Memantine; Pain Measurement; Phenols; Piperidines; Postural Balance; Rats; Rats, Sprague-Dawley; Receptors, Glycine; Receptors, N-Methyl-D-Aspartate; Vibrissae

To examine the antinociceptive effects of N-Methyl-D-aspartate (NMDA) receptor NR2 subunit antagonists in a rat model of the facial formalin test.. Experiments were carried out on adult male Sprague-Dawley rats weighing 220 to 280 g. Anesthetized rats were individually mounted on a stereotaxic frame and a polyethylene tube was implanted for intracisternal injection and, 72 hours later, formalin tests were performed. NMDA receptor antagonists were administered intracisternally 10 minutes prior to subcutaneous injection of 5% formalin (50 MicroL) into the vibrissal pad.. The intracisternal administration of 25, 50, or 100 Microg of memantine, an antagonist that acts at the NMDA ion channel site, significantly suppressed the number of scratches in the second phase of the behavioral responses to formalin. Intracisternal administration of a range of doses of 5,7-dichlorokynurenic acid, a glycine site antagonist, or DL-2-amino-5-phosphonopentanoate (AP-5), a nonselective NMDA site antagonist, produced significant antinociceptive effects in the second phase. Intracisternal administration of 1, 2.5, or 5 Microg of (2R,4S)-4-(3 Phosphonopropyl)-2-piperidine_carboxylic acid (PPPA), a competitive NR2A antagonist, significantly suppressed the number of scratches in the second phase, while only the highest dose of PPPA (5 Microg) significantly suppressed the number of scratches in the first phase. The antinociceptive effects of intracisternal injection of (alphaR, betaS)-alpha-(4Hydroxyphenyl)-_ methyl-4-(phenylmethyl)-1-Piperidinepropanol maleate(Ro 25-6981), a selective NR2B antagonist, were similar to those of PPPA. Injection of memantine, AP-5, Ro 25-6981, or vehicle did not result in any motor dysfunction. A low dose of PPPA (1 microg) or 5,7-dichlorokynurenic acid (2.5 microg) did not affect motor function. However, higher doses of PPPA and 5,7-dichlorokynurenic acid produced motor dysfunction.. The present results suggest that central NR2 subunits play an important role in orofacial nociceptive transmission. Moreover, this data also indicate that targeted inhibition of the NMDA receptor NR2 subunit is a potentially important new treatment approach for inflammatory pain originating in the orofacial area.

Topics: Animals; Behavior, Animal; Cisterna Magna; Disease Models, Animal; Excitatory Amino Acid Antagonists; Facial Pain; Formaldehyde; Injections; Injections, Subcutaneous; Kynurenic Acid; Male; Memantine; Motor Activity; Nociceptors; Phenols; Piperazines; Piperidines; Pruritus; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Synaptic Transmission; Time Factors; Vibrissae

In our previous experiments, severe cellular damages and neuronal cell loss were observed following 24h of alcohol withdrawal in primary cultures of rat cortical neurones pre-treated with ethanol (50-200 mM) repeatedly for 3 days. Increased NMDA induced cytosolic calcium responses and excitotoxicity were also demonstrated in the ethanol pre-treated cultures. Thus, the enhancement in functions of NMDA receptors was supposed to be involved in the adaptive changes leading to the neurotoxic effect of alcohol-withdrawal. In this study, we investigated the effect of the 3-day repeated ethanol (100 mM) treatment on the function and subunit composition of the NMDA receptors. Here, we demonstrate that the maximal inhibitory effect of ethanol was significantly increased after ethanol pre-treatment. Similarly, the inhibitory activity of the NR2B subunit selective antagonists threo-ifenprodil, CP-101,606 and CI-1041 was also enhanced. On the contrary, the efficiency of the channel blocker agent MK-801 and the glycine-site selective antagonist 5,7-dichlorokynurenic acid was the same as in control cultures. According to these observations, a shift in subunit expression in favour for the NR2B subunit was suggested. Indeed, we provided evidence for increased expression of the NR2B and the C1 and C2' cassette containing splice variant forms of the NR1 subunit proteins in ethanol pre-treated cultures in further experiments using a flow cytometry based immunocytochemical method. These changes may constitute the basis of the increased NMDA receptor functions and subsequently the enhanced sensitivity of ethanol pre-treated cortical neurones to excitotoxic insults resulting in increased neuronal cell loss after ethanol withdrawal. Such alterations may play a role in the neuronal adaptation to ethanol as well as in the development of alcohol dependence, and might cause neuronal cell loss in certain areas of the brain during alcohol withdrawal.

Topics: Animals; Animals, Newborn; Cells, Cultured; Cerebral Cortex; Culture Media, Serum-Free; Dizocilpine Maleate; Ethanol; Excitatory Amino Acid Antagonists; Gene Expression Regulation; Hippocampus; Kynurenic Acid; N-Methylaspartate; Nerve Tissue Proteins; Neurons; Piperidines; Protein Subunits; Rats; Receptors, N-Methyl-D-Aspartate; RNA Splicing

The human NMDAR2D subunit was cloned, and the pharmacological properties of receptors resulting from injection of transcripts encoding human NMDAR1A and NMDAR2D subunits in Xenopus oocytes were characterized by profiling NMDA receptor agonists and antagonists. We found that glutamate, NMDA, glycine, and D-serine were significantly more potent on hNMDAR1A/2D than on hNMDAR1A/2A or hNMDAR1A/2B. Also, the potencies of NMDA and glycine were higher for hNMDAR1A/2D than for hNMDAR1A/2C. Ifenprodil was more potent at hNMDAR1A/2B than at hNMDAR1A/2D, whereas 5,7-dichlorokynurenate was more potent at hNMDAR1A/2A than at hNMDAR1A/2D. As measured in transiently transfected human embryonic kidney 293 cells, the maximal inward current in the presence of external Mg2 occurred at -40 mV, and full block was not observed at negative potentials. Kinetic measurements revealed that the higher affinity of hNMDAR1A/2D for both glutamate and glycine relative to hNMDAR1A/2A and hNMDA1A/2B can be explained by slower dissociation of each agonist from hNMDAR1A/2D. The hNMDAR1A/2D combination represents a pharmacologically and functionally distinct receptor subtype and may constitute a potentially important target for therapeutic agents active in the human CNS.

Topics: Animals; Calcium; Cells, Cultured; DNA, Complementary; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Fetus; Glutamic Acid; Glycine; Humans; Kidney; Kynurenic Acid; Membrane Potentials; Molecular Sequence Data; N-Methylaspartate; Oocytes; Patch-Clamp Techniques; Pipecolic Acids; Piperidines; Receptors, N-Methyl-D-Aspartate; Recombinant Proteins; Sequence Homology, Amino Acid; Serine; Xenopus laevis

Topics: 2-Amino-5-phosphonovalerate; Animals; Binding, Competitive; Dizocilpine Maleate; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; gamma-Aminobutyric Acid; In Vitro Techniques; Kainic Acid; Kynurenic Acid; Male; Piperidines; Radioligand Assay; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate

Activation of NMDA receptors in dissociated cerebellar granule cells reduced mitochondrial membrane potential (MMP), as measured by rhodamine 123 fluorescence in a flow cytometer. This effect was inhibited by several NMDA-receptor antagonists with the following rank order of potency: MK-801 > PCP > TCP > dextrorphan > dichlorokynurenic acid > D-AP5 > dextromethorphan. Neither spermine nor arcaine modified the NMDA-induced reduction in MMP, whereas ifenprodil and eliprodil inhibited this response in the micromolar range. The mechanism responsible for the alteration of MMP mediated by NMDA was studied. Mepacrine and dibucaine prevented the MMP reduction induced by NMDA, as did W13 (calmodulin antagonist). In contrast, this effect was not blocked by cyclooxygenase or lipooxygenase inhibitors, H7 (a protein kinase C inhibitor) or nitroarginine (nitric oxide synthase inhibitor). These data suggest a direct interaction between NMDA-receptor activation and arachidonic acid formation, and indicate that NMDA receptor-mediated effect on MMP could involve arachidonic acid.

Topics: 2-Amino-5-phosphonovalerate; Anesthetics, Local; Animals; Arachidonic Acid; Biomarkers; Cells, Cultured; Cerebellum; Dibucaine; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Flow Cytometry; Fluorescent Dyes; Glycine; Intracellular Membranes; Kynurenic Acid; Membrane Potentials; Mitochondria; N-Methylaspartate; Piperidines; Quinacrine; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Rhodamine 123; Rhodamines; Sulfonamides

We have recently demonstrated that a single administration of m-chlorophenylpiperazine (m-CPP, a preferential 5-HT2C receptor agonist) produces tolerance to its stimulatory effect on adrenocorticotropic hormone (ACTH) concentrations when challenged 24 h later with the same dose of m-CPP. In the present study, we studied the effects of pretreatment with various N-methyl-D-aspartate (NMDA) receptor antagonists on development of tolerance to m-CPP's stimulatory effect on ACTH concentrations. Pretreatment with various NMDA receptor antagonists such as 5.7-dichlorokynurenic acid (1.0 mg/kg), 3-amino-1-hydroxy 2-pyrrolidone (1.0 mg/kg), dizocilpine (0.1 mg/kg) and ifenprodil (1.0 mg/kg) injected 30 min before the first injection of m-CPP (2.5 mg/kg) blocked development of tolerance to m-CPP's stimulatory effect on ACTH concentrations in rats injected 24 h later with the same dose (2.5 mg/kg) of m-CPP. These findings suggest that tolerance to postsynaptic 5-HT2C receptor-mediated response is initiated though stimulation of NMDA receptor complex and, furthermore, demonstrate a functional interaction between the 5-HT and glutamate systems.

Topics: Adrenocorticotropic Hormone; Animals; Dimethyl Sulfoxide; Dizocilpine Maleate; Drug Tolerance; Kynurenic Acid; Male; Phencyclidine; Piperazines; Piperidines; Pyrrolidinones; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Serotonin Receptor Agonists

The cytotoxicity induced by the transient expression of functional N-methyl-D-aspartate (NMDA) receptors has been examined with the use of a luciferase reporter assay in Chinese hamster ovary cells. Various NMDA receptor antagonists, in a dose-dependent manner, prevented a loss of luciferase activity 24 to 48 hr post-transfection of either the NR1/NR2A or NR1/ NR2B subunit receptor configurations, likely correlating to the time required to express functionally these receptors. Both glutamate and NMDA were potently cytotoxic to transfected cells previously protected by antagonists. The novel ifenprodil analog (1S,2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (CP101,606-27) protected cells expressing NR1/NR2B, but not those cells expressing either NR1/NR2A or, putatively, NR1/NR2A/NR2B. Decreased cytotoxicity was observed when a mutated NR1 subunit (N616R) with reduced Ca++ permeability was used in coexpression studies with NR2A or NR2B. In contrast to our results with NR1/NR2A or NR1/NR2B, cells expressing NR1/NR2C did not perish. Our studies suggest that expression of functional NMDA receptors in non-neuronal cells leads to a form of excitotoxicity similar to that observed in mammalian neurons in vitro.

Topics: Animals; Calcium; Cell Survival; CHO Cells; Cricetinae; Dizocilpine Maleate; Kynurenic Acid; L-Lactate Dehydrogenase; Luciferases; Pipecolic Acids; Piperidines; Receptors, N-Methyl-D-Aspartate; Transfection

Functional N-methyl-D-aspartate (NMDA) antagonists including competitive antagonists, glycine partial agonists, and use-dependent channel blockers exhibit antidepressant-like actions in preclinical models. The present study examined the effects of eliprodil (SL-82.0715), an NMDA antagonist acting at polyamine sites, in behavioral and neurochemical tests predictive of antidepressant activity. In mice, eliprodil produced a dose-dependent reduction in immobility in the forced swim test, but was inactive in the tail suspension test. Chronic treatment with eliprodil produced both a significant downregulation of beta-adrenoceptors and a reduction in the potency of glycine to inhibit [3H]5,7-dichlorokynurenic acid binding to strychnine-insensitive glycine receptors in neocortical membranes. In toto, these data indicate that like other NMDA antagonists, eliprodil possesses antidepressant-like actions in preclinical tests predictive of clinical efficacy.

Topics: Adrenergic beta-Antagonists; Adrenergic Uptake Inhibitors; Animals; Antidepressive Agents; Behavior, Animal; Biogenic Polyamines; Brain Chemistry; Down-Regulation; Excitatory Amino Acid Antagonists; Glycine; Imipramine; Kynurenic Acid; Male; Mice; Mice, Inbred C57BL; Motor Activity; Piperidines; Receptors, Glycine

Addition of several polyamines, including spermidine and spermine, was effective in inhibiting binding of the antagonist ligand [3H]5,7-dichlorokynurenic acid ([3H]-DCKA) a Gly recognition domain on the N-methyl-D-aspartic acid (NMDA) receptor ionophore complex in rat brain synaptic membranes. In contrast, [3H]DCKA binding was significantly potentiated by addition of proposed polyamine antagonists, such as ifenprodil and (+/-)-alpha-(4-chlorophenyl)-4-[(4-fluorophenyl) methyl]-1-piperidine ethanol, with [3H]Gly binding being unchanged. The inhibition by spermidine was significantly prevented by inclusion of ifenprodil. In addition, spermidine significantly attenuated the abilities of four different antagonists at the Gly domain to displace [3H]DCKA binding virtually without affecting those of four different agonists. Phospholipases A2 and C and p-chloromercuribenzosulfonic acid were invariably effective in significantly inhibiting [3H]DCKA binding with [3H]Gly binding being unaltered. Moreover, the densities of [3H]DCKA binding were not significantly different from those of [3H]-Gly binding in the hippocampus and cerebral cortex, whereas the cerebellum had more than a fourfold higher density of [3H]Gly binding than of [3H]DCKA binding. These results suggest that the Gly domain may have at least two different forms based on the preference to agonists and antagonists in the rodent brain.

Topics: Animals; Binding Sites; Binding, Competitive; Brain; Glycine; Kinetics; Kynurenic Acid; Male; Piperidines; Radioligand Assay; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Synaptic Membranes; Tritium

Pre-weaning rat pups emit ultrasonic vocalizations when removed from the litter. These 'separation-induced vocalizations' (SIV) are suppressed by classical benzodiazepine anxiolytics and by non-benzodiazepine anxiolytics which lack muscle relaxant and sedative properties. The present study used the SIV model to assess potential anxiolytic properties of compounds which target different sites associated with the NMDA receptor complex. Comparison was made to drugs which affect benzodiazepine or serotonin (5-HT) receptors. Muscle relaxant potential was assessed using 'TIP' (time on an inclined plane), the amount of time a pup was able to retain its position on a steeply inclined surface. Mephenesin, a centrally acting muscle relaxant, significantly suppressed TIP but not SIV. The benzodiazepine agonist diazepam suppressed both SIV and TIP, whereas the 5-HT1A partial agonists, buspirone and MDL 73,005EF, suppressed SIV without affecting TIP. The 5-HT2 antagonist MDL 11,939 suppressed TIP but not SIV, whereas neither measure was affected by the 5-HT3 antagonist MDL 73,147EF. SIV was suppressed by NMDA antagonists including those acting at the glutamate recognition site (D,L-amino-phosphonovaleric acid (AP5) and MDL 100,453) or at the ion channel (MK-801), or by the strychnine-insensitive glycine antagonist 5,7-dichlorokynurenic acid (5,7-DCKA). TIP was suppressed even more potently by AP5, MDL 100,453 and MK-801, whereas 5,7-DCKA was inactive on this measure. Thus, antagonists acting at different sites present on the glutamate recognition site exhibit potential anxiolytic activity, but the glycine antagonist was unusual in its lack of prominent muscle relaxant side effects.

Topics: 2-Amino-5-phosphonovalerate; Animals; Anti-Anxiety Agents; Anxiety, Separation; Behavior, Animal; Buspirone; Diazepam; Dioxins; Dizocilpine Maleate; Dose-Response Relationship, Drug; Indoles; Kynurenic Acid; Maternal Deprivation; Mephenesin; Piperidines; Quinolizines; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate; Receptors, Serotonin; Serotonin Antagonists; Spiro Compounds; Valine; Vocalization, Animal