strychnine and 5-7-dichlorokynurenic-acid

Retinal ganglion cells exhibit fast and slow inhibitory synaptic glycine currents that can be selectively inhibited by strychnine and 5,7-dichlorokynurenic acid (DCKA), respectively. In this study we examined whether strychnine and DCKA selectivity correlated with the subunit composition of the glycine receptor. Homomeric alpha1, alpha2 or alpha2* glycine subunits were in vitro expressed in human embryonic kidney cells (HEK 293). In cells expressing the alpha1 subunit, responses to 200 microm glycine were blocked by 1 microm strychnine but not by 500 microm DCKA. In cells expressing the alpha2 subunit, both 1 microm strychnine and 500 microm DCKA were effective antagonists of 200 microm glycine. In cells expressing alpha2* subunits, which are much less glycine-sensitive, 10 mm glycine was inhibited by 500 microm DCKA but not by 1 microm strychnine. A single amino acid mutation in the alpha1 subunit (R196G), converted this subunit from DCKA-insensitive to DCKA-sensitive. In conclusion, the comparative effectiveness of strychnine and DCKA can be used to distinguish between the alpha1, alpha2 and alpha2* receptor responses. Furthermore, a single amino acid near the glycine receptor's putative agonist binding site may account for differences in DCKA sensitivity amongst the alpha subunits.

Topics: Animals; Arginine; Cell Line; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Glycine; Glycine Agents; Humans; Kynurenic Acid; Mutation; Osmolar Concentration; Rats; Receptors, Glycine; Strychnine

The feeding behaviour of the freshwater polyp Hydra vulgaris (Cnidaria, Hydrozoa) is modulated by a number of molecules acting as neurotransmitters in other nervous systems. Here we present biochemical and functional evidence of the occurrence of putative NMDA receptors in Hydra tissues. Saturation experiments showed the presence of one population of binding sites with nanomolar affinity and low capacity for [3H]MK-801. Before equilibrium, [3H]MK-801 binding was increased by the agonists glutamate and glycine as well as by reduced glutathione (GSH). In vivo the glutamate receptor agonist NMDA markedly decreased the duration of the response to GSH. This effect was linearly related to ligand doses in the nanomolar concentration range and was counteracted by either the NMDAR-specific antagonist D-AP5 or by the d-serine antagonist DCKA. When NMDA concentration was increased to 10 or 100 microm, duration of the response to GSH was no longer affected unless the lectin concanavalin A, which prevents receptor desensitization in other systems, was added to the test medium. Simultaneous administration of ineffective doses of NMDA and strychnine, glycine or d-serine, an agonist at the glycine binding site of the NMDA receptor in vertebrate CNS, resulted in a strong reduction of response duration. Both D-AP5 and DCKA suppressed this effect. These results, together with the decrease in response duration produced by d-serine, support the hypothesis that NMDA-like glutamate receptors may occur in Hydra tissues where they are involved in modulation of the response to GSH with opposite actions to those of GABA and glycine.

Topics: 2-Amino-5-phosphonovalerate; Animals; Behavior, Animal; Binding Sites; Cell Membrane; Concanavalin A; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Interactions; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Feeding Behavior; Glutamic Acid; Glutathione; Glycine; Hydra; Kynurenic Acid; N-Methylaspartate; Receptors, N-Methyl-D-Aspartate; Serine; Strychnine

Chronic treatment with 1-aminocyclopropanecarboxylic acid (ACPC) but not with dizocilpine or imipramine produces desensitization to the behavioral response in ACPC challenge in the forced swim test (forced swim test). The mechanism by which ACPC produces this effect is unclear and may depend upon either its functional antagonist or its agonist properties at the NMDA receptor. We now report that chronic treatment with glycine or ACPC desensitizes the behavioral effect of challenge with ACPC in the forced swim test. The desensitization of the acute effects of ACPC cannot be explained by the presence of residual glycine because 24 h after the last of 14 daily glycine injections (i.e. the time of forced swim test) cortical and hippocampal glycine concentrations were unchanged. Likewise, the affinity of glycine to displace specific [3H]5,7-DCKA binding to glycine sites of the NMDA receptor complex was unchanged by chronic glycine administration. These results support the hypothesis that antidepressants produce adaptation of the NMDA receptor complex by mechanisms other than simply increasing synaptic glycine concentrations. Moreover, these results indicate that the behavioral adaptation in the forced swim test induced by chronic treatment with ACPC results from its agonist properties.

Topics: Amino Acids; Amino Acids, Cyclic; Animals; Behavior, Animal; Binding Sites; Cerebral Cortex; Glycine; Hippocampus; Kynurenic Acid; Male; Mice; Psychomotor Performance; Radioligand Assay; Receptors, N-Methyl-D-Aspartate; Strychnine; Tritium

1. Protein kinase modulation of glycine-activated currents was examined in acutely dissociated ganglion cells from tiger salamander retina using whole-cell voltage-clamp techniques. 2. Glycine (100 microM) induced an outward chloride current in cells clamped at 0 mV. Co-application of 50 microM forskolin made the glycine-induced current more transient. The combination of forskolin and glycine reduced the later portion of current response without changing the initial peak amplitude. 3. 3-Isobutyl-1-methylxanthine (IBMX) or 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) produced effects similar to those of forskolin. H-89, a protein kinase A (PKA) inhibitor, blocked the effect of forskolin. 4. A protein kinase C (PKC) activator, OAG (1-oleoyl-2-acetyl-sn-glycerol), also made the glycine response more transient. Unlike PKA analogues, OAG enhanced the glycine peak response without changing the glycine late response. OAG effects were blocked by 1 microM GF-109203X, a PKC inhibitor. 5. Nanomolar concentrations of strychnine selectively blocked the fast phase of the glycine current and reversed the effect of OAG, but not that of forskolin. Conversely, forskolin occluded the effect of 5,7-dichlorokynurenic acid, which selectively suppresses the late phase of the glycine current. The action of OAG was not blocked by 5,7-dichlorokynurenic acid. 6. Thus, through a differential modulation, both protein kinase A and C shorten the decay time of the glycine current. PKA suppresses the slow component, while PKC potentiates the fast component.

Topics: 1-Methyl-3-isobutylxanthine; 8-Bromo Cyclic Adenosine Monophosphate; Ambystoma; Animals; Colforsin; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Glycine; Glycine Agents; Isoquinolines; Kinetics; Kynurenic Acid; Membrane Potentials; Patch-Clamp Techniques; Phosphodiesterase Inhibitors; Protein Kinase C; Retinal Ganglion Cells; Strychnine; Sulfonamides

Physiological and pharmacological properties of possible subtypes of the native glycine receptor were investigated in retinal neurons using whole-cell voltage-clamp techniques. Two discrete inhibitory glycine responses were identified in ganglion cells. The responses could be distinguished pharmacologically: one was sensitive to strychnine and the other to 5,7-dichlorokynurenic acid. The two responses had different kinetics: the former had a fast onset and fast desensitization, whereas the latter had a slower onset and was much more sustained. The physiological and pharmacological distinctions suggest that the responses are mediated by different receptors. These receptors transduce glycinergic synaptic signals to ganglion cells, where they serve as low- and high-pass filters, respectively, of EPSPs.

Topics: Alanine; Ambystoma; Animals; Electrophysiology; Excitatory Amino Acid Antagonists; Glycine; Glycine Agents; Kynurenic Acid; Receptors, Glycine; Retinal Ganglion Cells; Serine; Strychnine; Synaptic Transmission; Taurine

We have previously reported that enhanced glycine release is produced by epidural spinal cord stimulation, a clinical method for treating neuropathic pain. Our current hypothesis is that glycine administered intrathecally reduces neuropathic pain as measured by the Randall-Selitto method. Neuropathic rats created by unilateral partial ligation of the sciatic nerve were treated with intrathecal infusion of glycine, strychnine, MK-801, or 5,7-DKA at 0.1 mumol, or artificial CSF for 2 hours at a rate of 10 microliters/min. Force required to produce the pain response was significantly increased after glycine administration and reduced using strychnine, a specific glycine receptor (Gly l) antagonist. Strychnine blocked the response to glycine when infused together. Administration of the non-specific NMDA receptor MK-801 antagonist and 5,7-DKA, a specific glycine-NMDA receptor (Gly 2) antagonist, however, failed to block the response to glycine. Our results provide evidence for the use of glycine and related compounds to treat neuropathic pain.

Topics: Animals; Cerebrospinal Fluid; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Glycine; Glycine Agents; Injections, Spinal; Kynurenic Acid; Male; Mechanoreceptors; Neuralgia; Nociceptors; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Strychnine; Transcutaneous Electric Nerve Stimulation

The effect of felbamate on excitatory amino acid-induced biochemical changes was investigated in cultured cortical neurons. Felbamate inhibited NMDA- and glutamate-induced neuronal injury in a dose-dependent manner, but it did not rescue cells from kainate-induced neurotoxicity. The neuroprotective effect was accompanied by a decrease in NMDA- and glutamate-induced neuronal calcium (Ca2+) influx. Exogenous addition of glycine failed to modulate the effect of felbamate on NMDA-induced neurotoxicity or Ca2+ influx, although corresponding changes induced by the strychnine-insensitive glycine antagonist, 5,7-dichlorokynurenic acid could be modulated with glycine. Taken together, these results suggest that felbamate acts through a site on the NMDA receptor that is distinct from the strychinine-insensitive site, and that the effect of the drug on neuronal Ca2+ may be pivotal to its neuroprotective mechanism.

Topics: Animals; Anticonvulsants; Binding Sites; Calcium; Cell Death; Cells, Cultured; Cerebral Cortex; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Felbamate; Glycine; Kainic Acid; Kynurenic Acid; N-Methylaspartate; Neurons; Neuroprotective Agents; Neurotoxins; Phenylcarbamates; Propylene Glycols; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Strychnine

[3H]5,7 Dichlorokynurenic acid ([3H]DCKA) was used to define conditions for obtaining selective binding to strychnine-insensitive glycine receptors. The parameters were established in sections of human brain prior to localizing the receptors sites by autoradiography. The binding of [3H]DCKA was of high affinity (Kd = 14.5 nM), readily reversible (K-1 = 0.216 min-1), and specific (60% specific binding determined by inhibition with 100 microM glycine or D-serine). High levels of strychnine-insensitive glycine receptors were identified in several brain areas including portions of the cerebral cortex (Bmax in middle temporal gyrus: 174.0 fmol/mg tissue), basal ganglia, hippocampal formation, and midbrain. These results identify regions where glycine receptors may be involved in modulating NMDA-mediated channel activity.

Topics: Autoradiography; Basal Ganglia; Brain; Cerebral Cortex; Hippocampus; Humans; Kynurenic Acid; Mesencephalon; Radioligand Assay; Receptors, Glycine; Receptors, N-Methyl-D-Aspartate; Strychnine; Tritium

The dicarbamate felbamate has been shown to be capable of competing for the binding of 5,7-[3H]dichlorokynurenic acid ([3H]DCKA) to strychnine-insensitive glycine receptors in sections of human postmortem brain. The IC50 for this interaction was 305.8 microM and the inhibition was complete at 1 mM. Autoradiographic localization of [3H]DCKA binding revealed many regions of human brain in which strychnine-insensitive glycine receptors are manifest. The specific binding in most of these areas was markedly reduced in the presence of 625 microM felbamate. In many regions, [3H]DCKA binding was reduced to background in the presence of felbamate, but some areas retained binding by as much as 41% (i.e., the CA2 region of the hippocampus). This is in contrast to the binding of [3H]DCKA in the presence of carbamazepine, phenytoin, or valproic acid. The binding of the glycine receptor antagonist was not affected by any of these latter agents to the same degree as felbamate. Strychnine-insensitive glycine receptors represent a site of action of felbamate in the human brain.

Topics: Adult; Aged; Anticonvulsants; Autopsy; Autoradiography; Binding, Competitive; Brain; Felbamate; Female; Glycine; Hippocampus; Humans; Kinetics; Kynurenic Acid; Male; Mesencephalon; Middle Aged; Neurons; Organ Specificity; Phenylcarbamates; Propylene Glycols; Radioligand Assay; Receptors, Glycine; Strychnine; Tritium

Topics: Adult; Aged; Anticonvulsants; Autoradiography; Cerebral Cortex; Felbamate; Glycine; Humans; Kynurenic Acid; Male; Middle Aged; Phenylcarbamates; Propylene Glycols; Receptors, Glycine; Strychnine

Chronic (14 day) but not acute (1 day) treatment of mice with clinically active antidepressants produces a significant (approximately 1.8-4.3 fold) reduction in the potency of glycine to inhibit [3H]-5,7-dichlorkynurenic acid (5,7-DCKA) binding to strychnine-insensitive glycine receptors in neocortical membranes. Moreover, these effects were not observed following chronic treatment with a variety of nonantidepressant drugs such as D-deprenyl, chlorpromazine, salbutamol, scopolamine and chlordiazepoxide. The time course and dose-response relationships for this effect were examined after treatment with two representative antidepressant drugs (imipramine and citalopram) and electriconvulsive shock (ECS). Increases in the IC50 of glycine to inhibit [3H]-5,7-DCKA binding were observed after treatment for 7 days with ECS, 10 days with citalopram and 14 days with imipramine, respectively, and were no longer apparent by the 10th day after cessation of treatment. These findings indicate that the antidepressant-induced reduction in the IC50 of glycine to inhibit [3H]-5,7-DCKA binding is: 1) a slowly developing, adaptive phenomenon; 2) remarkably persistent after cessation of treatment; and 3) a significantly better predictor of antidepressant activity (22 of 23 drugs) than either beta adrenoceptor down-regulation (15 of 23 drugs) or efficacy in the forced swim test (13 of 23 drugs) [P < .01 vs. each measure, Fisher's Exact Test]. The ability of antidepressants drawn from every principal therapeutic class to effect adaptive changes in the N-methyl-D-aspartate receptor complex is consistent with the hypothesis that this ligand-gated ion channel serves as a final common pathway of antidepressant action and indicates that glutamatergic pathways may be involved in the pathophysiology of depression.

Topics: Adaptation, Physiological; Animals; Antidepressive Agents; Citalopram; Dose-Response Relationship, Drug; Electroshock; Glycine; Imipramine; Kynurenic Acid; Male; Mice; Receptors, N-Methyl-D-Aspartate; Sensitivity and Specificity; Strychnine; Time Factors; Tritium

Felbamate (2-phenyl-1,3-propanediol dicarbamate) is a novel agent effective against maximal electroshock, pentylenetetrazol and other chemically induced seizures in mice and rats. Felbamate has been proposed as a novel anticonvulsant for the treatment of generalized tonic-clonic and complex partial seizures. In addition, felbamate has been shown to have neuroprotectant effects (in vitro and in vivo) in neonate models of cerebral ischemia. However, few existing studies have contributed to the elucidation of the mechanism of anticonvulsant and neuroprotectant action of felbamate. Because glycinergic mechanisms have been demonstrated to be involved with seizure disorders and neuroprotection, we investigated the binding interaction of felbamate with strychnine-insensitive glycine receptors and compared these findings with brain and plasma levels of felbamate after drug treatment. Inhibition of [3H]5,7-dichlorokynurenic acid (a high-affinity glycine receptor antagonist) binding by felbamate (IC50 = 374 microM) corresponded well with peak felbamate concentrations found in brain (683 and 759 microM) and plasma (679 and 807 microM) 8 hr after 300 (i.p.) or 500 mg/kg (p.o.) doses, respectively. Chemically diverse anticonvulsants tested and MK 801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cyclohepten-5,10-imine maleate] did not modulate [3H]5,7-dichlorokynurenic acid binding. Additional studies have shown that felbamate does not interact with other sites associated with the N-methyl-D-aspartate receptor complex. Thus, the data presented in this report strongly indicate a mechanism of action for felbamate through strychnine-insensitive glycine receptor interaction.

Topics: Animals; Anticonvulsants; Brain; Dizocilpine Maleate; Felbamate; Female; In Vitro Techniques; Kynurenic Acid; Male; Neurons; Phenylcarbamates; Propylene Glycols; Rats; Rats, Sprague-Dawley; Receptors, Glycine; Receptors, Neurotransmitter; Strychnine

A strychnine-insensitive glycine binding site is located on the N-methyl-D-aspartate (NMDA)-preferring glutamate receptor complex. Kynurenic acid analogs are antagonists at this binding site. A derivative of kynurenic acid, 5,7-dichlorokynurenic acid (5,7-DCKA) was radiolabeled with 3H and used to study antagonist binding to the glycine recognition site. This ligand ( [3H]5,7-DCKA) showed high affinity (Kd = 69 nM), saturable (Bmax = 14.5 pmol/mg protein) binding to rat brain membranes. A variety of agonists and antagonists inhibited the binding of [3H]5,7-DCKA and [3H]glycine in a similar fashion (r = 0.93). In addition, glutamate site agonists and antagonists exerted opposite allosteric effects on [3H]5,7-DCKA binding suggesting that [3H]5,7-DCKA preferentially binds to the agonist-activated conformation of the receptor.

Topics: Allosteric Regulation; Animals; Binding Sites; Brain; Glycine; In Vitro Techniques; Kynurenic Acid; Ligands; Male; Polyamines; Radioligand Assay; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate; Strychnine; Synaptosomes