6-cyano-7-nitroquinoxaline-2-3-dione and domoic-acid

Ionotropic glutamate receptors (iGluRs) are responsible for fast excitatory neurotransmission in the mammalian brain, and are critical regulators of neuronal activity and synaptic plasticity. The three main types of iGluRs (AMPA, NMDA, and kainate receptors) are composed of distinct subunit populations. The tetrameric kainate receptors can be assembled from a combination of five different types of subunits (GluK1-GluK5). GluK1-3 subunits are able to produce functional homomeric receptors, while GluK4-5 are obligate heteromers, and must assemble with a GluK1-3 subunit. The neurotoxin domoate is widely used as an agonist at kainate-type receptors because it produces a less desensitizing response compared to glutamate. We have identified an additional, subunit-dependent action of domoate at recombinant kainate receptors. When applied to heteromeric GluK2/K5 receptors, domoate generates a small, long-lasting, tonic current. In addition, brief exposure to domoate inhibits the GluK5 subunit, preventing its activation by other agonists for several minutes. These characteristics are not associated with the GluK1, K2, or K4 subunits and can be prevented by a mutation in GluK5 that reduces agonist binding affinity. The results also show that the domoate-bound, GluK2/K5 heteromeric receptors can be fully activated by agonists acting through the GluK2 subunit, suggesting that the subunits within the tetramer can function independently to open the ion channel, and that the domoate-bound state is not a desensitized or blocked conformation. This study describes new properties associated with domoate action at kainate receptors, and further characterizes the distinct roles played by different subunits in heteromeric receptors.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Binding Sites; Excitatory Amino Acid Antagonists; GluK2 Kainate Receptor; Glutamic Acid; HEK293 Cells; Humans; Kainic Acid; Membrane Potentials; Mutation; Neural Inhibition; Neurotoxins; Patch-Clamp Techniques; Receptors, Kainic Acid; Transfection

Neurite outgrowth is a fundamental step in establishing proper neuronal connections in the developing central nervous system. Dynamic control of outgrowth has been attributed to changes in growth cone Ca2+ levels in response to extracellular cues. Here we have investigated a possible role for Ca2+ permeable kainate (KA) receptors in regulating neurite outgrowth of nociceptive-like dorsal root ganglion (DRG) neurons. To identify KA receptor subunits likely to be involved, we used quantitative RT-PCR on acutely dissociated DRG and dorsal horn neurons. DRG neurons expressed more GluK1, particularly the GluK1b spice variant, than dorsal horn neurons. Conversely, dorsal horn neurons expressed more GluK2, particularly GluK2a, than DRG neurons. Further, an RNA editing assay indicated that the majority of GluK1 and GluK2 mRNA transcripts in DRG were unedited. Imaging Ca2+ transients following application of a KA receptor agonist to DRG and dorsal horn co-cultures revealed increases in intracellular Ca2+ in the growth cones of DRG neurons. In the majority of cases, this increase in Ca2+ was partly or completely blocked by Joro spider toxin (JSTX), an antagonist for Ca2+-permeable AMPA and KA receptors. Treatment of DRG/dorsal horn co-cultures with KA for 18 hours suppressed neurite outgrowth while application of the rapidly desensitizing KA receptor agonist SYM 2081, the competitive AMPA/KA receptor antagonist, CNQX, and JSTX or philanthotoxin enhanced neurite outgrowth and prevented KA effects on neurite outgrowth. Thus, Ca2+ entry through KA receptors at the growth cone of DRG neurons may be an important regulator of neurite outgrowth.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Adenosine Deaminase; Analysis of Variance; Animals; Calcium; Cells, Cultured; Dose-Response Relationship, Drug; Embryo, Mammalian; Excitatory Amino Acid Antagonists; Ganglia, Spinal; GAP-43 Protein; Gene Expression Regulation; GluK2 Kainate Receptor; Glutamates; Growth Cones; Kainic Acid; Membrane Proteins; Neurites; Neuromuscular Depolarizing Agents; Rats; Rats, Sprague-Dawley; Receptors, Kainic Acid; RNA Editing; RNA, Messenger; Sensory Receptor Cells; Spider Venoms; Subcellular Fractions

We have studied the effects of terfenadine on neurotoxicity and elevation of free cytoplasmic Ca2+ levels upon stimulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors in cultured cerebellar neurons. Pre-exposure to terfenadine (5 microM, 5 h) significantly increased neuronal death following specific stimulation of receptors by 100 microM AMPA or by subtoxic concentrations of domoate (8 microM), stimuli that are non-toxic when applied to terfenadine-untreated sister cultures. Terfenadine potentiation was prevented by the transcription inhibitor actinomycin D and was significantly ameliorated by histamine (1 mM). In terfenadine-treated neurons, AMPA increased [Ca2+](i) by approximately five fold, while AMPA induced no significant increase in [Ca2+](i) in the absence of terfenadine. Terfenadine reduced neuronal steady-state concentrations of [Ca2+](i) by approximately 75%. Our results suggest a role for histamine H1 receptors and intracellular calcium in the modulation of the excitotoxic response via AMPA receptors.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Analysis of Variance; Aniline Compounds; Animals; Animals, Newborn; Calcium; Cell Survival; Cells, Cultured; Cerebellum; Dactinomycin; Dizocilpine Maleate; Drug Interactions; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Histamine; Histamine H1 Antagonists; Kainic Acid; Neurons; Protein Synthesis Inhibitors; Rats; Receptors, AMPA; RNA; Terfenadine; Xanthenes

The effects of a novel vasopressin fragment analog NC-1900 (pGlu-Asn-Ser-Pro-Arg-Gly-NH2 acetate) were studied on learning and/or memory impairment in passive avoidance task and on cell damage of cultured cerebro-cortical neurocytes induced by glutamic acid. A small dose of NC-1900 (1 ng/kg, s.c.) ameliorated impairments of learning and/or memory induced by intracisternal injection of 467.6 micrograms of 10 microliters glutamic acid. NC-1900 also ameliorated the impairments induced by intracisternal NMDA, AMPA-antagonist CNQX and by metabotropic receptor (mGluR1) agonist 3,5-dihydroxyphenylglycine but not by kainate agonist domoic acid nor MK-801 in mice. NC-1900 (100 pM, 1nM) ameliorated the cell damage of cultured rat cerebro-cortical neurocytes induced by 100 and 1000 microM of glutamic acid. These results suggest that NC-1900 may serve as a remedies in various patients with certain brain disorders induced by excess glutamic acid.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Arginine Vasopressin; Avoidance Learning; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Glutamic Acid; Glycine; Kainic Acid; Learning Disabilities; Male; Memory Disorders; Mice; Mice, Inbred Strains; N-Methylaspartate; Neuromuscular Depolarizing Agents; Oligopeptides; Pyrrolidonecarboxylic Acid; Rats; Rats, Sprague-Dawley; Resorcinols

AMPA and NMDA receptor channels are closely related molecules, yet they respond to glutamate with distinct kinetics, attributable to differences in ligand binding and channel gating steps (for review, see Edmonds et al., 1995). We used two complementary approaches to investigate the number of functional binding sites on AMPA channels on outside-out patches from cultured hippocampal neurons. The activation kinetics of agonist binding were measured during rapid steps into low concentrations of selective AMPA receptor agonists and during steps from a competitive AMPA receptor antagonist, 6-cyano-7-nitro-quinoxaline-2,3-dione, into a saturating concentration of agonist. Both approaches revealed sigmoidal kinetics, which suggests that multiple agonist binding steps or antagonist unbinding steps are needed for channel activation. A kinetic model with two independent binding sites gave a better fit to the activation phase than models with one or three independent sites. A more refined analysis incorporating cooperative interaction between the two binding sites significantly improved the fits to the responses. The affinity of the first binding step was two to three times higher than the second step. These results demonstrate that binding of two agonist molecules are needed to activate AMPA receptors, but the two binding sites are not identical and independent. Because NMDA receptors require four ligand molecules for activation (two glycine and two glutamate; Benveniste and Mayer, 1991; Clements and Westbrook, 1991), it may be that some binding sites on AMPA receptors are functionally silent.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; Antihypertensive Agents; Benzothiadiazines; Binding Sites; Cells, Cultured; Enzyme Activation; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Hippocampus; Ion Channel Gating; Kainic Acid; Kinetics; Neuromuscular Depolarizing Agents; Neurons; Patch-Clamp Techniques; Quisqualic Acid; Rats; Rats, Sprague-Dawley; Receptors, AMPA

Domoic acid (50 nM) elevates cytosolic free calcium ([Ca2+]i) levels in 49% of the hippocampal pyramidal neurons isolated from postnatal day one (PND1) rats. This effect was prevented by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; an antagonist of non-N-methyl-D-asparate (NMDA) receptors, but not 2-amino-5-phosphonovaleric acid (AP-5; an antagonist of NMDA receptors). Domoic acid given at 5 microM also elevated [Ca2+]i levels in a second population (36%) of neurons in which the effect was only partially inhibited by 100 microM CNQX. Nimodipine given at 300 nM prevented the elevation in [Ca2+]i caused by 50 nM and 5 microM domoic acid, indicating that domoic acid induced Ca2+ entry through type L voltage dependent calcium channels. These results provide evidence for at least two domoic acid-sensitive non-NMDA receptor subtypes in primary cultures of neonatal hippocampal pyramidal cells and indicate that voltage-dependent calcium channels are a primary calcium entry mechanism for domoic acid action.

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Calcium; Calcium Channel Blockers; Cells, Cultured; Cytosol; Excitatory Amino Acid Antagonists; Kainic Acid; Neurotoxins; Nimodipine; Pyramidal Cells; Rats; Receptors, N-Methyl-D-Aspartate

We investigated the role of kainate receptor activation in modulating the influx of Ca2+ coupled to the exocytotic release of glutamate in rat hippocampal synaptosomal fractions (P2). In whole hippocampus synaptosomes stimulation with domoic acid increased the intracellular free Ca2+ concentration ([Ca2+]i) in a dose-dependent manner with an EC50 of 0.16 microM, whereas the EC50 for kainate was 0.86 microM and for (+/-)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) was 43.04 microM. Stimulation of the synaptosomes with 10 microM domoic acid induced Ca(2+)-dependent release of endogenous glutamate. Also, in synaptosomes isolated from the CA3 sub-region of the hippocampus the domoic acid-induced release of glutamate was higher than that from the dentate gyrus (221.3%), from the CA1 (188.1%) or from the whole hippocampal synaptosomes (131.5%). These results support the existence of a presynaptic kainate receptor which may control the exocytotic release of glutamate in the CA3 sub-region of the rat hippocampus.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Excitatory Amino Acid Antagonists; Glutamic Acid; Hippocampus; In Vitro Techniques; Kainic Acid; Male; Rats; Rats, Wistar; Receptors, Kainic Acid; Receptors, Presynaptic; Synaptosomes

The binding of [3H]NS 257 (1,2,3,6,7,8-hexahydro-3-(hydroxyimino)-N,N-[3H]dimethyl-7-methyl- 2- oxobenzo[2,1-b:3,4-c']dipyrrole-5-sulfonamide) to rat cortical membranes was characterized in the absence and presence of thiocyanate. Specific [3H]NS 257 binding was saturable and reversible, and the stimulating effect of thiocyanate on binding was optimal at 100 mM. In the presence of thiocyanate [3H]NS 257 bound to a single population of binding sites with an affinity of 225 +/- 8 nM and a binding site density of 0.61 +/- 0.04 pmol/mg of original tissue. Thiocyanate increased the affinity of the binding site labeled by [3H]NS 257 for both alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and L-glutamate by a factor of 20 and 5, respectively. However, the affinity of the agonist domoate and the antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)-quinoxaline (NBQX) was decreased in the presence of thiocyanate. Apparently, the affinities of antagonists as well as agonists for the AMPA receptor can be either increased or decreased by thiocyanate. The rank order of potency of the putative agonists quisqualate > AMPA > L-glutamate > domoate > kainate and of the antagonists NBQX > CNQX is consistent with the labeling of AMPA receptors. Autoradiographic studies showed that the distribution of [3H]NS 257 binding sites in rat brain was similar to that of [3H]AMPA binding sites. NS 257 is the first AMPA antagonist to be described showing an increased affinity for the AMPA receptor in the presence of thiocyanate.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Autoradiography; Binding, Competitive; Cell Membrane; Cerebral Cortex; Hydrogen-Ion Concentration; Indoles; Kainic Acid; Male; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Sulfonamides; Thiocyanates; Tritium

When rabbit retinas are exposed in vitro to specific excitatory amino acid receptor agonists certain GABAergic amacrine cells are activated to cause a release of GABA. The GABA that is not released can be detected by immunohistochemistry. Exposure of tissues to kainate or NMDA each caused a characteristic change in the GABA immunoreactivity. CNQX antagonised the kainate effect specifically while MK-801 counteracted the influence of NMDA. The effect produced by kainate was mimicked by domoic acid while the influence of homocysteic acid was identical with NMDA. Flupirtine alone did not influence the nature of the GABA immunoreactivity and so did not act as a kainate or NMDA agonist. However, flupirtine counteracted the influence produced by NMDA and homocysteic acid but had no effect on the kainate and domoic acid responses. Thus in this system flupirtine acts as an NMDA antagonist.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Aminopyridines; Animals; Dizocilpine Maleate; gamma-Aminobutyric Acid; Homocysteine; Immunohistochemistry; Kainic Acid; Rabbits; Receptors, N-Methyl-D-Aspartate; Retina

The effect of the neurotoxin domoic acid (DOM), a structural analogue of kainic acid, on the release of [3H]gamma-aminobutyric acid (GABA) and on the [Ca2+]i was studied in cultured chick retina cells. DOM stimulated dose-dependently the release of [3H]GABA with an EC50 of 2.5 microM. In Ca(2+)-containing medium (1 mM), DOM (5 microM) increased the [Ca2+]i by about 190 nM and evoked the release of 11.8 +/- 1.3% of the intracellular [3H]GABA, while in the absence of extracellular Ca2+ DOM induced the release of only 7.9 +/- 1.4% of the accumulated [3H]GABA. The Ca(2+)-independent release of [3H]GABA was blocked by the non-competitive inhibitor of the GABA carrier 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-py ridine- carboxylic acid hydrochloride (NNC-711), but a component of Ca(2+)-dependent release remains. DOM evoked Ca(2+)-independent release of [3H]GABA was significantly depressed in the absence of external Na+ and completely blocked by the non-selective antagonist of the non-NMDA glutamate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Similarly, CNQX decreased the [Ca2+]i response to DOM, whereas L(+)-2-amino-3-phosphonopropionic acid (L-AP3), an antagonist of the metabotropic glutamate receptors, was without effect. MK-801 did not affect the release of [3H]GABA stimulated by DOM. Taken together our results indicate that DOM evokes both Ca(2+)-dependent and Ca(2+)-independent release of [3H]GABA, most likely by activating kainate receptors.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Calcium; Cells, Cultured; Chick Embryo; Excitatory Amino Acid Antagonists; GABA Antagonists; gamma-Aminobutyric Acid; Kainic Acid; Neurotoxins; Nicotinic Acids; Nipecotic Acids; Oximes; Quinoxalines; Retina

Glutamate mediates fast synaptic transmission at the majority of excitatory synapses throughout the central nervous system by interacting with different types of receptor channels. Cloning of glutamate receptors has provided evidence for the existence of several structurally related subunit families, each composed of several members. It has been proposed that KA1 and KA2 and GluR-5, GluR-6, and GluR-7 families represent subunit classes of high-affinity kainate receptors and that in vivo different kainate receptor subtypes might be constructed from these subunits in heteromeric assembly. However, despite some indications from autoradiographic studies and binding data in brain membranes, no functional pure kainate receptors have so far been detected in brain cells. We have found that early after culturing, a high percentage of rat hippocampal neurons express functional, kainate-selective glutamate receptors. These kainate receptors show pronounced desensitization with fast onset and very slow recovery and are also activated by quisqualate and domoate, but not by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate. Our results provide evidence for the existence of functional glutamate receptors of the kainate type in nerve cells, which are likely to be native homomeric GluR-6 receptors.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Calcium; Cell Membrane Permeability; Cells, Cultured; Embryo, Mammalian; Hippocampus; Kainic Acid; Membrane Potentials; N-Methylaspartate; Neurons; Neurotoxins; Quinoxalines; Quisqualic Acid; Rats; Receptors, AMPA; Receptors, Glutamate

As excitatory amino acid receptors have been implicated in nociceptive sensory transmission, the principal objective of the present study was to investigate the effects of various excitatory amino acid antagonists on naturally evoked responses in spinal dorsal horn neurons. Extracellular single unit activity was recorded from functionally identified, spinal dorsal horn neurons in unanesthetized, decerebrated cats and in alpha-chloralose-anesthetized cats. The tests included iontophoretic application of the N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovaleric acid (APV), the non-N-methyl-D-aspartate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and kynurenate, and also the intravenous administration of the N-methyl-D-aspartate receptor antagonist, ketamine. In addition, attempts were made to determine the effects on these neurons of iontophoretic application of the excitatory amino acid agonists, L-glutamate, N-methyl-D-aspartate, quisqualate, (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and domoate. Marked differences were noted in the actions of agonists and antagonists between the responses observed in the unanesthetized, decerebrated and the anesthetized animals. In decerebrated cats, responses to hair afferent stimulation were blocked by kynurenate, 6-cyano-7-nitroquinoxaline-2,3-dione and 2-amino-5-phosphonovaleric acid. Responses to noxious thermal stimulation were attenuated by 2-amino-5-phosphonovaleric acid and in one unit also by ketamine. Neither 6-cyano-7-nitroquinoxaline-2,3-dione nor kynurenate affected the responses to noxious thermal stimulation. The proportion of cells responding to the agonists were: N-methyl-D-aspartate 24/27 (89%), quisqualate 12/13 (92%) and domoate 6/7 (86%). In chloralose-anesthetized cats, responses to hair afferent stimulation were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione and kynurenate but not by 2-amino-5-phosphonovaleric acid. Responses to noxious thermal stimulation were not affected by any of these antagonists, while the response to non-noxious thermal stimulation was blocked by 2-amino-5-phosphonovaleric acid, ketamine and kynurenate in the one neuron studied. The proportion of cells excited by the agonists differed from those observed in decerebrated cats: N-methyl-D-aspartate 9/32 (28%), quisqualate 50/54 (93%), (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate 19/23 (83%) and domoate 17/38 (45%). Application of the putative endoge

Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Cats; Decerebrate State; Evoked Potentials; Glutamates; Glutamic Acid; Ibotenic Acid; Kainic Acid; Ketamine; Kynurenic Acid; N-Methylaspartate; Neurons; Quinoxalines; Quisqualic Acid; Receptors, Amino Acid; Receptors, N-Methyl-D-Aspartate; Spinal Cord; Synapses

We have characterized a high-affinity kainate binding site in in vitro living rat neocortical slices using [3H]kainate. [3H]Kainate labelled at least two binding sites, the higher affinity site with a Kd of 7.1 nM and a Bmax of 71.2 fmol/mg protein. This high-affinity binding site showed a pharmacology consistent with a kainate receptor with competition by kainate and domoic acid, as well as the (RS)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate antagonist 6-cyano-2,3-dihydroxy-7-nitroquinoxaline. Increases in cellular depolarization induced by 2-h preincubations in veratridine and glutamate led to a significant 55% average decrease in [3H]kainate binding in adult cortex. Similarly, preincubation in kainate led to a significant average 26% decrease in binding. In both instances, Eadie-Hofstee analysis of saturation binding data revealed that the decreased binding reflected changes in receptor number. At different postnatal ages, increases in cellular depolarization significantly decreased binding (< 20 days postnatal age, -86%; > 60 days, -48%). Kainate treatment also significantly decreased binding at all ages (-64% at < 20 days; > 60 days, -18%), with significant differences noted between ages. These age-dependent effects are unlike those previously described for either N-methyl-D-aspartate [Lanius and Shaw (1992) Anat. Rec. 232, 54(A)] or (RS)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate high affinity receptors [Shaw and Lanius (1992) Devl Brain Res. 68, 225-233].(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Age Factors; Animals; Binding, Competitive; Cerebral Cortex; Glutamates; Glutamic Acid; Kainic Acid; Kinetics; Male; N-Methylaspartate; Nerve Tissue Proteins; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, Glutamate; Receptors, Kainic Acid; Veratridine

A detailed pharmacological characterization of the quisqualate (QA) receptor coupled to phospholipase C (Qp) was performed in striatal neurons. The experiments were carried out in the presence of the ionotropic antagonists MK-801 (1 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (30 microM), concentrations that block N-methyl-D-aspartate (NMDA) or alpha-amino-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in these cells. QA, ibotenate and trans-1-aminocyclopentyl-1,3-dicarboxylate (ACPD) evoked dose-dependent inositol phosphate formations with EC50 values of 0.3, 6.7 and 29 microM, respectively. QA and ibotenate had the same maximal effect (295.7 +/- 17.9% of basal, n = 6) whereas the efficacy of ACPD was somewhat lower (70.2 +/- 8.9% of the maximal quisqualate effect, n = 4). The QA-, ibotenate- and ACPD-induced maximal effects were not additive, and the inositol phosphate formations induced by high concentrations of L-aspartate (L-ASP), AMPA, kainate (KA) and domoate (DO) (100 microM or higher) were also not additive. The inositol phosphate responses induced by all these agonists were totally blocked by the phorbol ester phorbol 12,13-dibutyrate (PdBu), but not by atropine or prazosin suggesting that all these substances were able to stimulate the Qp excitatory amino acid receptor in striatal neurons. Of the excitatory amino acid receptor antagonists tested, only D,L-2-amino-3-phosphonopropionate (D,L-AP3) inhibited QA-induced InsP formation in a competitive manner (mean pKi = 4.45 +/- 0.43, n = 4). However, this drug was also a partial agonist of the Qp receptor since it stimulated the inositol phosphate formation. We found that D,L-AP3 also inhibited NMDA-induced calcium increase, in a competitive manner (mean pIC50 = 4.34 +/- 0.22, n = 8, and mean pKi = 3.7 +/- 0.11, n = 5). The Qp excitatory amino acid receptor in striatal neurons therefore closely resembles Qp receptors with high potency for agonists as described in striatal and retinal slices and synaptoneurosomes, and has several pharmacological differences compared to the Qp receptors which have low potency for agonists described in hippocampal and cortical slices, cerebellar granule cells, astrocytes and rat brain mRNA-injected oocytes.

Topics: 2-Aminoadipic Acid; 6-Cyano-7-nitroquinoxaline-2,3-dione; Alanine; Aminobutyrates; Animals; Cells, Cultured; Corpus Striatum; Dizocilpine Maleate; Fura-2; Ibotenic Acid; Inositol Phosphates; Kainic Acid; Mice; Neurons; Phorbol 12,13-Dibutyrate; Phosphoserine; Quinoxalines; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Receptors, Neurotransmitter; Type C Phospholipases

The presynaptic effects of domoic acid (Dom) on hippocampal mossy fiber synaptic transmission were examined using a subcellular fraction enriched in mossy fiber synaptosomes. Domoic acid significantly increased the K(+)-evoked release of endogenous glutamate from superfused guinea pig mossy fiber synaptosomes. The presynaptic facilitation produced by Dom was dose-dependent and was antagonized by the prior application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). At a concentration of 30 microM, both domoic acid and kainic acid significantly increased the extent to which membrane depolarization augmented the availability of cytosolic free calcium in mossy fiber synaptosomes. These results are consistent with the suggestion that domoic acid enhances the release of mossy fiber neurotransmitters in the guinea pig hippocampus through the activation of a CNQX-sensitive presynaptic receptor.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Calcium; Drug Synergism; Glutamates; Glutamic Acid; Guinea Pigs; Hippocampus; Kainic Acid; Male; Nerve Fibers; Potassium; Quinoxalines; Synaptosomes

Recent evidence implicates excitatory amino acids (EAAs), acting as excitotoxic agents, in the pathogenesis of neurological disorders involving the spinal cord. In this study, we used the chick embryo spinal cord as an in vitro model for studying the sensitivity of spinal neurons to the excitotoxic effects of EAA agonists. Compounds tested include the prototypic receptor-specific agonists, N-methyl-D-aspartate (NMDA), quisqualic acid (Quis), and kainic acid (KA), and the plant-derived excitotoxic food poisons, beta-N-oxalylamino-L-alanine, beta-N-methylamino-L-alanine, and domoic acid. Each agonist induced concentration-dependent acute degeneration of neurons distributed throughout the spinal cord. These cytopathological changes consisted of acute edematous degeneration of dendrosomal structures in the dorsal horn and intermediate zone, and dark cell changes with intracytoplasmic vacuolization of motor neurons; this damage is identical to that induced by excitotoxin agonists in other regions of the central nervous system. The NMDA receptor-specific antagonist MK-801 completely blocked toxicity of NMDA, and the nonNMDA antagonist CNQX preferentially blocked the toxicity of Quis- and KA-type agonists in the spinal cord. Our findings suggest that (1) the majority of spinal neurons have all three subtypes of EAA receptors, making them acutely vulnerable to excitotoxin exposure; and (2) EAA antagonists are effective in preventing excitotoxin-induced damage of the spinal cord.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Amino Acids, Diamino; Animals; beta-Alanine; Chick Embryo; Cyanobacteria Toxins; Dizocilpine Maleate; Glutamates; Glutamic Acid; Ibotenic Acid; Kainic Acid; Motor Neurons; N-Methylaspartate; Nerve Degeneration; Neurotoxins; Quinoxalines; Quisqualic Acid; Spinal Cord

1. Neuropharmacological actions of several kainate derivatives (kainoids) were examined for electrophysiological effects in the isolated spinal cord and the dorsal root fibre of the newborn rat. 2. Some kainoids caused depolarization of the motoneurone much more effectively than kainic acid or domoic acid and others were weaker. The rank order of the depolarizing activities of the kainoids tested here is as follows: 4-(2-methoxyphenyl)-2-carboxy-3-pyrrolidineacetic acid (MFPA) greater than acromelic acid A greater than domoic acid greater than or equal to 4-(2-hydroxyphenyl)-2-carboxy-3-pyrrolidineacetic acid (HFPA) greater than or equal to acromelic acid B greater than kainic acid. 3. In the isolated dorsal root fibre, domoic acid caused the most significant depolarization. There were distinct differences with regard to the rank order of the depolarizing activity between the motoneurone and the dorsal root fibre. The rank order in the dorsal root fibre is domoic acid greater than acromelic acid B greater than 5-bromowillardiine greater than or equal to MFPA greater than acromelic acid A greater than HFPA greater than kainic acid. 4. Significant desensitization of kainate receptors was observed in the isolated dorsal root fibre during prolonged application of L-glutamate, kainate and its derivatives. Cross desensitization was also observed among these excitatory amino acids. Receptors desensitized by kainate did not respond to MFPA, HFPA and acromelic acids, suggesting that these kainate derivatives activated common kainate receptors in the dorsal root fibre.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Animals, Newborn; In Vitro Techniques; Kainic Acid; Motor Neurons; Neuromuscular Depolarizing Agents; Piperazines; Quinoxalines; Rats; Rats, Inbred Strains; Receptors, N-Methyl-D-Aspartate; Spinal Cord; Structure-Activity Relationship