concanavalin-a and cyclothiazide
concanavalin-a has been researched along with cyclothiazide* in 27 studies
Other Studies
27 other study(ies) available for concanavalin-a and cyclothiazide
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Functional characterisation of homomeric ionotropic glutamate receptors GluR1-GluR6 in a fluorescence-based high throughput screening assay.
We have constructed stable HEK293 cell lines expressing the rat ionotropic glutamate receptor subtypes GluR1(i), GluR2Q(i), GluR3(i), GluR4(i), GluR5Q and GluR6Q and characterised the pharmacological profiles of the six homomeric receptors in a fluorescence-based high throughput screening assay using Fluo-4/AM as a fluorescent Ca2+ indicator. In this assay, the pharmacological properties of nine standard GluR ligands correlated nicely with those previously observed in electrophysiology studies of GluRs expressed in Xenopus oocytes or mammalian cells. The potencies and efficacies displayed by the agonists (S)-glutamate, (S)-quisqualate, kainate, (RS)-AMPA, (RS)-ATPA, (RS)-ACPA] and (S)-4-AHCP at the six GluRs were in concordance with electrophysiological studies. Furthermore, the Ki values exhibited by the competitive antagonists NBQX and (RS)-ATPO were also in agreement with findings of previous studies. Finally, the effects of various concentrations of Ca2+ in the assay buffer and of the allosteric modulators cyclothiazide and concanavalin A on GluR signalling were examined. This study represents the most elaborate functional characterisation of multiple AMPA and KA receptor subtypes in the same assay reported to date. We propose that high throughput screening of compound libraries at the six GluR-HEK293 cell lines could be helpful in the search for structurally and pharmacologically novel ligands acting at the receptors. Topics: Aniline Compounds; Benzothiadiazines; Calcium; Cell Line; Combinatorial Chemistry Techniques; Concanavalin A; Drug Evaluation, Preclinical; Electrophysiology; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Fluorescence; Glutamic Acid; Humans; Kainic Acid; Quinoxalines; Receptors, AMPA; Receptors, Glutamate; Signal Transduction; Xanthenes | 2006 |
Nootropic agents enhance the recruitment of fast GABAA inhibition in rat neocortex.
It is widely believed that nootropic (cognition-enhancing) agents produce their therapeutic effects by augmenting excitatory synaptic transmission in cortical circuits, primarily through positive modulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptors (AMPARs). However, GABA-mediated inhibition is also critical for cognition, and enhanced GABA function may be likewise therapeutic for cognitive disorders. Could nootropics act through such a mechanism as well? To address this question, we examined the effects of nootropic agents on excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) recorded from layer V pyramidal cells in acute slices of somatosensory cortex. Aniracetam, a positive modulator of AMPA/kainate receptors, increased the peak amplitude of evoked EPSCs and the amplitude and duration of polysynaptic fast IPSCs, manifested as a greater total charge carried by IPSCs. As a result, the EPSC/IPSC ratio of total charge was decreased, representing a shift in the excitation-inhibition balance that favors inhibition. Aniracetam did not affect the magnitude of either monosynaptic IPSCs (mono-IPSCs) recorded in the presence of excitatory amino acid receptor antagonists, or miniature IPSCs (mIPSCs) recorded in the presence of tetrodotoxin. However, the duration of both mono-IPSCs and mIPSCs was prolonged, suggesting that aniracetam also directly modulates GABAergic transmission. Cyclothiazide, a preferential modulator of AMPAR function, enhanced the magnitude and duration of polysynaptic IPSCs, similar to aniracetam, but did not affect mono-IPSCs. Concanavalin A, a kainate receptor modulator, had little effect on EPSCs or IPSCs, suggesting there was no contribution from kainate receptor activity. These findings indicate that AMPAR modulators strengthen inhibition in neocortical pyramidal cells, most likely by altering the kinetics of AMPARs on synaptically connected interneurons and possibly by modulating GABA(A) receptor responses in pyramidal cells. This suggests that the therapeutic actions of nootropic agents may be partly mediated through enhanced cortical GABAergic inhibition, and not solely through the direct modification of excitation, as previously thought. Topics: Animals; Antihypertensive Agents; Benzothiadiazines; Cognition; Concanavalin A; Evoked Potentials; Neocortex; Neural Inhibition; Nootropic Agents; Pyramidal Cells; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Receptors, GABA-A | 2005 |
Spontaneous synchronized calcium oscillations in neocortical neurons in the presence of physiological [Mg(2+)]: involvement of AMPA/kainate and metabotropic glutamate receptors.
Primary cultures of neocortical neurons exhibit spontaneous Ca(2+) oscillations under zero or low extracellular [Mg(2+)] conditions. We find that mature murine neocortical neurons cultured for 9 days also produce spontaneous Ca(2+) oscillations in the presence of physiological [Mg(2+)]. These Ca(2+) oscillations were action potential mediated inasmuch as tetrodotoxin eliminated their occurrence. AMPA receptors were found to regulate the frequency of Ca(2+) oscillations. In contrast, Ca(2+) oscillations were independent of activation of L-type Ca(2+) channels, and NMDA receptors provided only a minor contribution. Release of intracellular Ca(2+) stores was involved in the oscillatory activity since thapsigargin reduced the amplitude and frequency of the oscillations. S-4-carboxyphenylglycine (S)-4CPG), an antagonist of group I metabotropic glutamate receptor (mGluR), also reduced the amplitude of oscillations. In addition, 1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD), a group I mGluR agonist, increased the oscillation frequency, suggesting a critical role for mGluR in the generation of Ca(2+) oscillations. The mGluR-mediated release of intracellular Ca(2+) stores appeared to be mediated by phospholipase C (PLC) since the PLC inhibitor U73122 eliminated the Ca(2+) oscillations. These results indicate that Ca(2+) oscillations in neocortical cultures in the presence of physiologic [Mg(2+)] are primarily initiated by excitatory input from AMPA receptors and involve mobilization of intracellular Ca(2+) stores following activation of mGluR. Topics: Anesthetics, Local; Aniline Compounds; Animals; Antihypertensive Agents; Benzothiadiazines; Calcium Channel Blockers; Calcium Signaling; Cells, Cultured; Concanavalin A; Dose-Response Relationship, Drug; Embryo, Mammalian; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Magnesium; Mice; Neocortex; Neurons; Nifedipine; Receptors, Glutamate; Tetrodotoxin; Thapsigargin; Veratridine; Xanthenes | 2004 |
Both N-methyl-D-aspartate (NMDA) and non-NMDA receptors mediate glutamate-induced cleavage of the cyclin-dependent kinase 5 (cdk5) activator p35 in cultured rat hippocampal neurons.
Cyclin-dependent kinase 5 (cdk5) regulates crucial neurobiological events, and deregulation of cdk5 has been implicated in several neurodegenerative disorders. The deregulation is suggested to occur due to cleavage of the cdk5 activator protein p35 to a smaller p25 fragment by the calcium-activated protease calpain. Here we have elucidated the role of different calcium-permeable ionotropic glutamate receptors in the induction of p35 cleavage in cultured rat hippocampal neurons. The glutamate receptor agonists glutamic acid, N-methyl-D-aspartate (NMDA), kainic acid, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) were all able to induce p35 cleavage, in a manner depending on extracellular calcium. The effect of glutamate was mediated by NMDA receptors, as it was prevented by the NMDA antagonist MK-801. Cyclothiazide (CTZ), an inhibitor of AMPA receptor desensitization, enhanced glutamate-induced p35 cleavage. In immature 6-day-old cultures the non-NMDA agonist kainic acid provoked p35 cleavage, whereas glutamate and NMDA were ineffective. The data suggest that both NMDA and non-NMDA receptors are able to induce p35 cleavage. Different factors, such as maturation state of neurons or desensitization properties of non-NMDA receptors, may determine which receptor predominantly mediates the effect of glutamate on p35 cleavage. Topics: Animals; Benzothiadiazines; Blotting, Western; Calcium Channel Blockers; Cells, Cultured; Concanavalin A; Cysteine Proteinase Inhibitors; Dipeptides; Dose-Response Relationship, Drug; Drug Interactions; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Hippocampus; Nerve Tissue Proteins; Neurons; Nifedipine; Rats; Rats, Wistar; Receptors, Glutamate; Time Factors | 2004 |
Functional characteristics of non-NMDA-type ionotropic glutamate receptor channels in AII amacrine cells in rat retina.
The properties of non-NMDA glutamate receptor channels in AII amacrine cells were studied by patch-clamp recording from rat retinal slices. Application of AMPA or kainate to intact cells evoked currents with no apparent desensitization (EC50 of 118 microM for AMPA and 169 microM for kainate). Application of AMPA to patches evoked desensitizing responses with an EC50 of 217 and 88 microM for the peak and steady-state responses, respectively. Kainate-evoked responses of patches displayed no desensitization (EC50 = 162 microM). Cyclothiazide strongly potentiated AMPA-evoked responses and the AMPA-receptor antagonist GYKI 53655 inhibited both AMPA- and kainate-evoked responses (IC50 = 0.5-1.7 microM). Pre-equilibration with GYKI 53655 completely blocked the response to kainate and pretreatment with concanavalin A did not unmask a response mediated by kainate receptors. AMPA- and kainate-evoked currents reversed close to 0 mV. AMPA-evoked peak and steady-state response components in patches displayed linear and outwardly rectifying I-V relations with an RI (ratio of the slope conductances at +40 mV and -60 mV) of 0.96 +/- 0.11 and 5.6 +/- 1.3, respectively. AMPA-evoked currents displayed a voltage-dependent relaxation after steps to positive or negative membrane potentials, indicating that the outward rectification of the steady-state response is caused by a voltage-dependent kinetic parameter of channel gating. Under bi-ionic conditions ([Ca2+](out) = 30 mM, [Cs+)(in) = 171 mM), the reversal potentials of AMPA- and kainate-evoked currents indicated channels with significant Ca2+ permeability (P(Ca)/P(Cs) = 1.9-2.1). Stationary noise analysis indicated that kainate activated channels with an apparent chord conductance of approximately 9 pS. Non-stationary noise analysis indicated that AMPA and glutamate activated channels with apparent chord conductances of approximately 9, approximately 15, approximately 23 and approximately 38 pS. Discrete single-channel gating corresponding to chord conductances of approximately 23 pS could be directly observed in some responses. Thus, our results indicate expression of high-affinity, voltage-sensitive AMPA receptors with significant Ca2+ permeability and relatively large single-channel chord conductances in AII amacrine cells. Topics: Algorithms; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Benzodiazepines; Benzothiadiazines; Calcium; Concanavalin A; Electric Stimulation; Electrophysiology; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; In Vitro Techniques; Kainic Acid; Membrane Potentials; Patch-Clamp Techniques; Rats; Receptors, AMPA; Receptors, Glutamate; Retina; Sodium | 2002 |
AMPA-kainate subtypes of glutamate receptor in rat cerebral microglia.
Microglial cells were isolated from rat cerebral cortex, and kainate (KA)-induced inward current was measured at a holding potential of -40 or -60 mV. 6-Cyano-7-nitroquinoxaline-2, 3-dione-sensitive KA-induced currents increased with increasing KA concentration. The half-activation concentration and Hill coefficient were 3.3 x 10(-4) M and 1.4, respectively. Although glutamate (Glu) and AMPA-induced currents were much smaller than that induced by KA, all KA-, Glu-, and AMPA-induced currents were greatly and consistently enhanced in the presence of cyclothiazide (CTZ). On the other hand, KA-induced currents were much less sensitive to potentiation by concanavain A, suggesting that the KA-induced response in rat microglia is predominantly mediated by AMPA-preferring receptors (subunits GluR1-GluR4). The current-voltage relationships of KA- and AMPA-CTZ-induced currents were almost linear or slightly outward rectifying. The reversal potential of KA-induced current shifted to negative potentials (from +4 to -40 mV) on switching from high Na(+) to high Ca(2+) external solution, indicating the low Ca(2+) permeability through the AMPA-KA receptor channel complexes. AMPA-KA receptor expression was studied with immunohistochemistry and reverse transcription-PCR, from which GluR2, GluR3, GluR4, and GluR5 were identified. Lower levels of mRNAs for GluR7 and KA-1-KA-2 were also indicated. Finally, activation of these receptors with KA or Glu significantly enhanced the production of tumor necrosis factor-alpha. These results suggest that primary cultured rat microglia possesses functional Glu receptor, which may mediate neuron to microglia communication in the physiological and pathological states. Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Antihypertensive Agents; Benzothiadiazines; Calcium; Cells, Cultured; Cerebral Cortex; Concanavalin A; Excitatory Amino Acid Agonists; Glutamic Acid; Kainic Acid; Membrane Potentials; Microglia; Patch-Clamp Techniques; Rats; Rats, Wistar; Receptors, AMPA; Receptors, Kainic Acid; Reverse Transcriptase Polymerase Chain Reaction; Stimulation, Chemical; Tumor Necrosis Factor-alpha | 2000 |
Glutamate and GABA activate different receptors and Cl(-) conductances in crab peptide-secretory neurons.
Responses to rapid application of glutamic acid (Glu) and gamma-aminobutyric acid (GABA), 0.01-3 mM, were recorded by whole-cell patch clamp of cultured crab (Cardisoma carnifex) X-organ neurons. Responses peaked within 200 ms. Both Glu and GABA currents had reversal potentials that followed the Nernst Cl(-) potential when [Cl(-)](i) was varied. A Boltzmann fit to the normalized, averaged dose-response curve for Glu indicated an EC(50) of 0.15 mM and a Hill coefficient of 1.05. Rapid (t(1/2) approximately 1 s) desensitization occurred during Glu but not GABA application that required >2 min for recovery. Desensitization was unaffected by concanavalin A or cyclothiazide. N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, quisqualate, and kainate (to 1 mM) were ineffective, nor were Glu responses influenced by glycine (1 microM) or Mg(2+) (0-26 mM). Glu effects were imitated by ibotenic acid (0.1 mM). The following support the conclusion that Glu and GABA act on different receptors: 1) responses sum; 2) desensitization to Glu or ibotenic acid did not diminish GABA responses; 3) the Cl(-)-channel blockers picrotoxin and niflumic acid (0.5 mM) inhibited Glu responses by approximately 90 and 80% but GABA responses by approximately 50 and 20%; and 4) polyvinylpyrrolydone-25 (2 mM in normal crab saline) eliminated Glu responses but left GABA responses unaltered. Thus crab secretory neurons have separate receptors responsive to Glu and to GABA, both probably ionotropic, and mediating Cl(-) conductance increases. In its responses and pharmacology, this crustacean Glu receptor resembles Cl(-)-permeable Glu receptors previously described in invertebrates and differs from cation-permeable Glu receptors of vertebrates and invertebrates. Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Benzothiadiazines; Brachyura; Cells, Cultured; Chloride Channels; Chlorides; Concanavalin A; gamma-Aminobutyric Acid; Glutamic Acid; Glycine; Kainic Acid; N-Methylaspartate; Neurons; Patch-Clamp Techniques; Quisqualic Acid; Receptors, AMPA; Receptors, GABA; Receptors, Glutamate | 2000 |
Kainate receptor-mediated activation of the AP-1 transcription factor complex in cultured rat cerebellar granule cells.
The sequence-specific DNA-binding activity of the AP-1 transcription factor complex was measured in cultured rat cerebellar granule cells by electrophoretic mobility shift assay. A low concentration of kainate (KA; 10 microM), but not alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA; 10 microM), enhanced DNA-binding of the AP-1 transcription factor in cultures pretreated with Concanavalin A (Con A), to prevent KA receptor desensitization. In the presence of cyclothiazide (an inhibitor of AMPA receptor desensitization), KA (10 microM) caused only a slight increase of AP-1 DNA-binding, in contrast to the threefold enhancement produced by AMPA (10 or 30 microM) or by a higher concentration of KA (30 microM), suggesting that the effect of KA, in the presence of Con A, is mediated by activation of putative KA receptors. To confirm this, the effects of the AMPA receptor-selective, non-competitive antagonist, 1-(4-aminophenyl)-3-methylcarbamoyl-4-methyl-3,4-dihydro-7, 8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 53655; 50 microM), the mixed AMPA/KA receptor competitive antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 50 microM), and the AMPA and GluR5 KA receptor competitive antagonist, (-)(3S,4aR,6R, 8aR)-6-[2-(1(2)H-tetrazole-5-yl)ethyl]-1,2,3,4,4a,5,6,7,8, 8a-decahydroisoquinoline-3-carboxylic acid monohydrate (LY 326325; 100 microM), were examined on AMPA- and KA-induced AP-1 activation, respectively. Our results suggest that stimulation of native KA receptors is responsible for the observed KA-specific activation of the AP-1 transcription factor complex. Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Benzodiazepines; Benzothiadiazines; Cells, Cultured; Cerebellum; Concanavalin A; Dimerization; Excitatory Amino Acid Antagonists; Isoquinolines; Kainic Acid; Neurons; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, Kainic Acid; Tetrazoles; Transcription Factor AP-1 | 2000 |
Excitatory amino acid responses in relay neurons of the rat lateral geniculate nucleus.
Responses to glutamate receptor agonists were recorded from identified relay neurons in the dorsal lateral geniculate nucleus of the rat, using the nystatin-perforated patch-clamp technique. Rapid application of glutamate, N-methyl-D-aspartate, (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate induced inward currents at a holding potential of -44 mV. The responses to low concentrations of each agonist were composed only of steady-state currents, but the responses to high concentrations were additionally composed of a rapid transient peak component except in the kainate-induced current. The currents induced by 10(-3)M N-methyl-D-aspartate in the external solution containing 0 mM Mg2+ and 10(-6)M glycine were reduced in amplitude when the external solution contained 1 mM Mg2+, and were abolished when the solution contained no glycine. The currents induced by a neurotransmitter candidate at retinogeniculate synapses, N-acetyl-aspartyl-glutamate, were markedly reduced in amplitude when the solution contained 1 mM Mg2+ or 10(-4)M DL-2-amino-5-phosphonovaleric acid. The current abolished in the Mg2+-containing, glycine-free solution (N-methyl-D-aspartate component) and the current remaining in the same solution (non-N-methyl-D-aspartate component) of the N-acetyl-aspartyl-glutamate response were both increased in a concentration-dependent manner, as the N-acetyl-aspartyl-glutamate concentration was increased. The current-voltage relationship of the currents induced by N-methyl-D-aspartate and N-acetyl-aspartyl-glutamate was characterized by Mg2+-dependent block at hyperpolarized potentials. The inward currents induced by 3 x 10(-4)M AMPA and 3 x 10(-4)M glutamate were markedly potentiated by 10(-4)M cyclothiazide, but the currents induced by 3 x 10(-4)M kainate and 10(-3)M N-acetyl-aspartyl-glutamate (non-N-methyl-D-aspartate component) were little affected. The currents induced by any agonist were not affected by 3 x 10(-4)g/ml concanavalin A. The current induced by 10(-4)M kainate was markedly suppressed by pretreatment with 10(-4)M AMPA or 10(-4)M glutamate, but only weakly by 10(-3)M N-acetyl-aspartylglutamate. The Ca2+ permeability (PCa/PCs) of the N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors was 9.57 and 0.16, respectively. These results suggest that dorsal lateral geniculate nucleus relay neurons of the rat possessed both Ca2+-permeable N-methyl-D-aspartate receptors and less permeable non-N-methyl-D-aspartate ( Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Antihypertensive Agents; Benzothiadiazines; Concanavalin A; Dipeptides; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Geniculate Bodies; Glutamic Acid; Glycine; Ionophores; Kainic Acid; Membrane Potentials; N-Methylaspartate; Neurons; Neuroprotective Agents; Nystatin; Patch-Clamp Techniques; Rats; Rats, Wistar; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Synaptic Transmission | 1999 |
NS-257, a novel competitive AMPA receptor antagonist, interacts with kainate and NMDA receptors.
In this study, we examined the effects of a novel, water-soluble, putative competitive AMPA receptor antagonist, 1,2,3,6,7, 8-hexahydro-3-(hydroxyimino)-N,N,7-trimethyl-2-oxobenzo[2,1- b:3, 4-c']dipyrrole-5-sulfonamide (NS-257) on AMPA, kainate and NMDA receptors using the two-electrode voltage-clamp technique in Xenopus oocytes. All glutamate receptor subtypes were inhibited by NS-257 in a voltage-independent way. When kainate was applied to oocytes injected with total mouse brain mRNA, mainly AMPA receptors were activated. The antagonistic effects of NS-257 on these kainate-induced currents were concentration-dependent and competitive. In the same way, NS-257 blocked kainate-induced currents recorded from oocytes expressing homomeric GluR-1 receptors. In our experiments higher concentrations (>1 microM) of NS-257 also produced inhibitory effects on kainate and to a lesser extent on NMDA receptor function as indicated by recordings from GluR-6 or NR-1b/2A cRNA injected oocytes. While NMDA receptor function was inhibited in a competitive fashion, kainate responses recorded from homomeric GluR-6 receptors were blocked in a mixed competitive-noncompetitive manner. This mixed antagonistic action of NS-257 might have been caused by preincubating oocytes with concanavalin A, which blocks desensitization of kainate receptors. Although NS-257 appeared to be a less potent AMPA receptor antagonist then other known antagonists like NBQX, its main advantage over all other reported compounds so far is its higher aqueous solubility which still represents the major weakness of the other AMPA receptor antagonists, especially for clinical use. Topics: Animals; Antihypertensive Agents; Benzothiadiazines; Concanavalin A; Dose-Response Relationship, Drug; Electrophysiology; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Indoles; Kainic Acid; Membrane Potentials; Mice; Mice, Inbred C57BL; Microinjections; N-Methylaspartate; Oocytes; Patch-Clamp Techniques; Receptors, AMPA; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; RNA, Complementary; RNA, Messenger; Sulfonamides; Xenopus | 1999 |
Modulation of desensitization at glutamate receptors in isolated crucian carp horizontal cells by concanavalin A, cyclothiazide, aniracetam and PEPA.
In horizontal cells freshly dissociated from crucian carp (Carassius auratus) retina, we examined the effects of modulators of glutamate receptor desensitization, concanavalin A, cyclothiazide, aniracetam and 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyacetam ide (PEPA), on responses to rapid application of glutamate and kainate, using whole-cell voltage-clamp techniques. Incubation of concanavalin A suppressed the peak response but weakly potentiated the equilibrium response of horizontal cells to glutamate. Cyclothiazide blocked glutamate-induced desensitization in a dose-dependent manner, which resulted in a steady increase of the equilibrium current. The concentration of cyclothiazide causing a half-maximal potentiation for the equilibrium response was 85 microM. Furthermore, cyclothiazide shifted the dose-response relationship of the equilibrium current to the right, but slightly suppressed the kainate-induced sustained current. These effects of concanavalin A and cyclothiazide are consistent with the supposition that glutamate receptors of carp horizontal cells may be an alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-preferring subtype. In order to further characterize the AMPA receptors of horizontal cells, modulation by aniracetam and PEPA of glutamate- and kainate-induced currents was studied. Aniracetam, a preferential modulator of flop variants of AMPA receptors, considerably blocked desensitization of glutamate-induced currents, but only slightly potentiated kainate-induced currents. It was further found that PEPA, a flop-preferring allosteric modulator of AMPA receptor desensitization, slightly suppressed the peak current, while it dramatically potentiated the equilibrium current induced by glutamate in a dose-dependent manner. PEPA was much potent than aniracetam at these receptors and showed the effect on glutamate-induced desensitization even at a concentration as low as 3 microM. PEPA also potentiated non-desensitizing currents induced by kainate, but with much less extent. These modulatory effects of concanavalin A, cyclothiazide, aniracetam and PEPA on AMPA receptors in carp horizontal cells were rather similar to those obtained at AMPA receptors assembled from flop variants expressed in Xenopus oocyte and HEK cell. Consequently, we speculate that the AMPA receptor on carp horizontal cells may predominantly carry the flop splice variants. Topics: Animals; Aspartic Acid Endopeptidases; Benzothiadiazines; Concanavalin A; Goldfish; Nootropic Agents; Patch-Clamp Techniques; Pyrrolidinones; Receptors, AMPA; Receptors, Glutamate; Receptors, Kainic Acid | 1999 |
Characterisation of kainate receptor mediated whole-cell currents in rat cultured cerebellar granule cells.
Whole-cell voltage clamp recordings have been used to identify and characterise inward currents mediated by native kainate receptors in rat cultured cerebellar granule cells. While the selective AMPA receptor antagonist GYKI 53655 (50 microM) completely abolished inward currents evoked by AMPA (10-100 microM) in the presence of cyclothiazide (100 microM), kainate evoked currents in cells pretreated with concanavalin A (Con A) always showed a component (35-140 pA, n = 13) resistant to blockade. The majority (73+/-7%, n = 5) of GYKI 53655-resistant kainate-evoked inward currents remained in the presence of 100 microM AMPA. However, these currents were reversibly blocked by the competitive AMPA/kainate receptor antagonist NBQX (100 microM). (2S, 4R)-4-methylglutamate (SYM 2081, 10 microM) evoked inward currents in Con A treated cells (15-60 pA, n = 7), which were resistant to complete blockade by GYKI 53655 (50 microM) but antagonised by NBQX (100 microM). Kainate-evoked responses in the presence of GYKI 53655 (50 microM) had linear or slightly outwardly rectifying current-voltage (I-V) relationships in all cells examined (n = 5) and were resistant to blockade by Joro spider toxin (JsTx, 1 microM; n = 5). These results provide evidence that rat cultured cerebellar granule cells express functional kainate receptors made up of subunits which are edited at the Q/R site, and that SYM 2081 is an agonist at these native kainate receptors with a greater selectivity than kainate itself. Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Benzodiazepines; Benzothiadiazines; Cells, Cultured; Concanavalin A; Evoked Potentials; Excitatory Amino Acid Antagonists; Glutamates; Hippocampus; Kinetics; Neurons; Neurotoxins; Patch-Clamp Techniques; Rats; Receptors, Kainic Acid; Spider Venoms | 1999 |
AMPA-preferring glutamate receptors in cochlear physiology of adult guinea-pig.
1. The present study was designed to determine which glutamate (Glu) receptors are involved in excitatory neurotransmission at the first auditory synapse between the inner hair cells and the spiral ganglion neurons. 2. The Glu receptors present at the membrane level were investigated on isolated spiral ganglion neuron somata from guinea-pigs by whole-cell voltage-clamp measurements. Glu and AMPA induced a fast onset inward current that was rapidly desensitized, while kainate induced only a non-desensitizing, steady-state current. NMDA induced no detectable current. 3. To further discriminate between the AMPA and kainate receptors present, we used the receptor-specific desensitization blockers, cyclothiazide and concanavalin A. While no effect was observed with concanavalin A, cyclothiazide greatly enhanced the Glu-, AMPA- and kainate-induced steady-state currents and potentiated Glu-induced membrane depolarization. 4. To extrapolate the results obtained from the somata to the events occurring in situ at the dendrites, the effects of these drugs were evaluated in vivo. Cyclothiazide reversibly increased spontaneous activity of single auditory nerve fibres, while concanavalin A had no effect, suggesting that the functional Glu receptors on the somata may be the same as those at the dendrites. 5. The combination of a moderate-level sound together with cyclothiazide increased and subsequently abolished the spontaneous and the sound-evoked activity of the auditory nerve fibres. Histological examination revealed destruction of the dendrites, suggesting that cyclothiazide potentiates sound-induced Glu excitotoxicity via AMPA receptors. 6. Our results reveal that fast synaptic transmission in the cochlea is mainly mediated by desensitizing AMPA receptors. Topics: Animals; Benzothiadiazines; Cochlea; Concanavalin A; Dendrites; Electric Stimulation; Electrophysiology; Guinea Pigs; Hair Cells, Auditory, Inner; In Vitro Techniques; Membrane Potentials; Nerve Fibers; Organ of Corti; Patch-Clamp Techniques; Receptors, AMPA; Receptors, Kainic Acid; Spiral Ganglion; Synapses; Synaptic Transmission; Vestibulocochlear Nerve | 1999 |
Ca2+-permeable non-NMDA glutamate receptors in rat magnocellular basal forebrain neurones.
1. Ionotropic glutamate receptor-mediated responses were recorded from rat magnocellular basal forebrain neurones under voltage clamp from a somatically located patch-clamp pipette. Currents were recorded from both acutely dissociated neurones and neurones maintained in culture for up to 6 weeks. 2. Non-NMDA and NMDA receptor-mediated events could be distinguished pharmacologically using the selective agonists (S)-alpha-amino-3-hydroxy-5-methyl-isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartate (NMDA), and antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D(-)-2-amino-5-phosphonopentanoic acid (AP5). 3. Responses to rapid application of AMPA displayed pronounced and rapid desensitization. Responses to kainate showed no desensitization. Steady-state EC50 values for AMPA and kainate were 2.7 +/- 0.4 microM (n = 5) and 138 +/- 25 microM (n = 10), respectively. Cyclothiazide markedly increased current amplitude of responses to both agonists, whereas concanavalin A had no clear effect on either response. The selective AMPA receptor antagonist GYKI 53655 inhibited responses to kainate with an IC50 of 1.2 +/- 0.08 microM (n = 5) at -70 mV. These data strongly suggest that AMPA receptors are the predominant non-NMDA receptors expressed by basal forebrain neurones. 4. At -70 mV, approximately 6 % of control current amplitude remained, at a maximally effective concentration of GYKI 53655. This residual response displayed desensitization, was insensitive to cyclothiazide and was potentiated by concanavalin A, suggesting that it was mediated by a kainate receptor. 5. Current-voltage relationships for non-NMDA receptor-mediated currents were obtained from both nucleated patches pulled from neurones in culture and from acutely dissociated neurones. With 30 microM spermine in the recording pipette, currents frequently displayed double-rectification characteristic of non-NMDA receptors with high Ca2+ permeabilities. Ca2+ permeability, relative to Na+ and Cs+, was investigated using constant field theory. The measured Ca2+ to Na+ permeability coefficient ratio was 0.26-3.6; median, 1.27 (n = 15). 6. Current flow through non-NMDA receptors was inhibited by Ca2+, Cd2+ and Co2+ ions. At a holding potential of -70 mV, a maximally effective concentration of Cd2+ (> 30 mM) reduced current amplitude by approximately 90 %, with an IC50 of 44 microM. In six out of seven cells tested, block by Cd2+ was voltage sensitive. 7. Ca2+ permeability of many of the n Topics: Animals; Basal Ganglia; Benzodiazepines; Benzothiadiazines; Calcium; Cations, Divalent; Cells, Cultured; Concanavalin A; Diuretics; Electrophysiology; Excitatory Amino Acid Antagonists; Membrane Potentials; Neurons; Patch-Clamp Techniques; Prosencephalon; Rats; Receptors, AMPA; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Sodium Chloride Symporter Inhibitors | 1998 |
Postsynaptic responses of horizontal cells in the tiger salamander retina are mediated by AMPA-preferring receptors.
The postsynaptic responses of sign-preserving second-order retinal neurons (horizontal cells (HCs) and off-bipolar cells) are mediated by CNQX-sensitive AMPA/KA glutamate receptors. In this study we used receptor-specific allosteric regulators of desensitization and selected antagonists to determine the glutamate receptor subtypes in tiger salamander horizontal cells. Two approaches were employed in this study. The first was to measure postsynaptic currents induced by exogenously applied glutamate under voltage clamp conditions in living retinal slices; and the second was to record voltage responses controlled by endogenous glutamate released from photoreceptors in whole retinas. Application of 100 microM cyclothiazide (a specific AMPA receptor desensitization blocker) enhanced the glutamate-induced current by about 5 fold. In contrast, 300 microgram ml-1 Co nA (a specific kainate receptor desensitization blocker), had no effect. GYKI 52466 (a specific AMPA receptor antagonist) at 30 microM almost completely suppressed the glutamate-induced inward current in HCs. Cyclothiazide at 100 microM depolarized the HC dark membrane potential by about 5 mV and reduced the amplitudes of the voltage responses to dim lights, but enhanced the voltage responses to bright lights. Cyclothiazide had no effect on either the dark potential or the light responses of rods and cones. Con A at 300 microgram ml-1 had no effect on either the dark potential or the light responses of the HC. GYKI 52466 (30 microM) hyperpolarized the HC dark membrane potential by about 55 mV and almost completely suppressed the light responses. We conclude from these results that the postsynaptic glutamate- and light-induced responses in the tiger salamander retinal horizontal cells are mediated by AMPA-preferring, and not kainate-preferring glutamate receptors. The functional roles of AMPA receptors and their desensitization kinetics in visual information processing are discussed. Topics: Ambystoma; Animals; Anti-Anxiety Agents; Antihypertensive Agents; Benzodiazepines; Benzothiadiazines; Concanavalin A; Dark Adaptation; Electrophysiology; Excitatory Amino Acid Antagonists; Fluorescent Dyes; Glutamic Acid; Isoquinolines; Membrane Potentials; Patch-Clamp Techniques; Photic Stimulation; Photoreceptor Cells; Receptors, AMPA; Synapses | 1998 |
Functional diversity of synaptic AMPA/KA receptors from rat as revealed by subtype-specific antagonists.
Subtype-specific pharmacological compounds represent important tools to identify the molecular components of synaptically activated glutamate receptors in central neurones. Here, we utilized a collection of subtype-specific antagonists and modulators to investigate the functional profile of glutamate receptors in identified synapses in thin slices of the cerebellum, hippocampus and brain stem. During whole-cell patch-clamp recordings alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate (AMPA/KA) receptor-mediated synaptic currents (EPSCs) in cerebellar Purkinje cells were (i) prolonged by 100 microM cyclothiazide, (ii) not significantly changed after preincubation in 10 microM concanavalin A, (iii) not affected by 1 microM Evans Blue or polyamine toxin analogue N-(4-hydroxyphenylpropanolyl)-spermine (NHPPS), but (iv) significantly reduced by high (> or = 100 microM) concentrations of Evans Blue. These pharmacological properties were distinct from those observed in hippocampal granule cells and brain stem interneurones and markedly different from those of recombinant glutamate receptor channels GluR1-GluR6 previously investigated in heterologous expression systems. Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Antihypertensive Agents; Benzothiadiazines; Brain Stem; Cerebellum; Coloring Agents; Concanavalin A; Evans Blue; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Hippocampus; Interneurons; Patch-Clamp Techniques; Polyamines; Rats; Rats, Wistar; Receptors, AMPA; Receptors, Kainic Acid; Spermine; Synapses; Tetrodotoxin | 1998 |
NMDA and AMPA receptors evoke transmitter release from noradrenergic axon terminals in the rat spinal cord.
N-methyl-D-aspartate (NMDA) stimulated release of [3H]noradrenaline (NA) from prelabelled rat spinal cord slices. The release was partially insensitive to tetrodotoxin (TTX) and was inhibited by the NMDA antagonist MK-801. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) also evoked release of [3H]NA, which was enhanced by blocking AMPA receptor desensitization with cyclothiazide. AMPA-evoked release was inhibited by the non-NMDA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)-quinoxaline (NBQX) but was not affected by TTX. NMDA and AMPA showed synergistic effects, indicating co-existence of NMDA and AMPA receptors on noradrenergic terminals. Kainate evoked [3H]NA release only at high concentrations and the release was not potentiated by blocking kainate receptor desensitization with concanavalin A. Thus, the results indicate that there are stimulatory presynaptic NMDA and AMPA receptors on noradrenergic axon terminals in the spinal cord and that they interact synergistically to evoke release of [3H]NA. Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Benzothiadiazines; Concanavalin A; Dizocilpine Maleate; In Vitro Techniques; Kainic Acid; Male; N-Methylaspartate; Norepinephrine; Presynaptic Terminals; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Spinal Cord; Tetrodotoxin | 1998 |
Role of non-NMDA receptors in osmotic and glutamate stimulation of vasopressin release: effect of rapid receptor desensitization.
Previous studies demonstrated that the increase in vasopressin (VP) release and induction of VPmRNA content by osmotic stimulation was blocked by kynurenic acid, a non-specific antagonist of excitatory amino acid (EAA) receptors. In order to identify the type of EAA receptor involved, perifused explants of the hypothalamo-neurohypophyseal system (HNS) were exposed to a ramp increase in osmolality (40 mOsm over 6 h achieved by increasing NaCl) in the presence and absence of 10 microM 6,7-dinitroquinoxaline-2,3-dione (DNQX), an antagonist of non-n-methyl-d-aspartate (NMDA) excitatory amino acid receptors. Vasopressin release and VP mRNA content were significantly increased by exposure to the osmotic stimulus. 6,7-dinitroquinoxaline-2,3-dione inhibited osmotically stimulated VP release (F=16.65, P=0.0008) without significantly reducing basal release. It also prevented the osmotically stimulated increase in VP mRNA content (P <0.05). Although these results implicated glutamate, the primary endogenous ligand for EAA receptors, in the regulation of VP, exogenous glutamate was ineffective in stimulating VP release from HNS explants in either low-Mg2+ or Mg2+-replete medium. However, blockade of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor desensitization with cyclothiazide (100 microM) caused a marked increase in VP release in response to 100 microM glutamate, and blockade of kainate receptor desensitization with concanavalin A resulted in a small, but significant increase in VP release in response to 1 mM glutamate. These results support a role for non-NMDA receptor activation in osmotic regulation of VP release. Topics: Animals; Benzothiadiazines; Concanavalin A; Down-Regulation; Excitatory Amino Acid Antagonists; Glutamic Acid; Hypothalamo-Hypophyseal System; In Vitro Techniques; Magnesium; Male; Osmolar Concentration; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, Glutamate; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; RNA, Messenger; Sodium Chloride; Vasopressins | 1998 |
Glutamate receptor ion channel properties predict vulnerability to cytotoxicity in a transfected nonneuronal cell line.
Excessive activation of glutamate receptors is thought to play a critical role in neuronal excitotoxicity. To compare the cytotoxic potential of different glutamate receptor subtypes and correlate receptor biophysical properties with cytotoxicity, we have expressed recombinant receptors in human embryonic kidney 293 (HEK-293) cells. Survival of transfected cells was analyzed under conditions of defined agonist concentration and exposure time. For HEK-293 cells transfected with N-methyl-D-aspartate (NMDA) receptors, the EC50 for NMDA-induced cytotoxicity was 300 microM. Experiments using ion substitution, or cells expressing mutant NMDA receptors with low calcium permeability, suggested that both calcium and sodium influx through NMDA receptors contributed to cytotoxicity. In contrast, cytotoxicity was not observed in cells transfected with calcium permeable alpha-amino 3-hydroxy-5-methyl-4-isoxazole propionate- or kainate-type glutamate receptors even at saturating agonist concentrations, unless inhibitors of agonist-dependent desensitization were included. These results directly demonstrate that calcium permeability and desensitization kinetics play important roles in determining the excitotoxic potential of different glutamate receptor subtypes. Topics: 2-Amino-5-phosphonovalerate; Animals; Base Sequence; Benzothiadiazines; Cell Line; Cell Survival; Concanavalin A; Dizocilpine Maleate; DNA Primers; Evoked Potentials; Glutamic Acid; Humans; Ion Channels; Kainic Acid; Kidney; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; N-Methylaspartate; Neurotoxins; Patch-Clamp Techniques; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate; Transfection | 1996 |
Fast desensitizing kainate-gated current resolved in whole-cell experiments on isolated rat hippocampal neurons.
In whole-cell concentration-jump experiments on dissociated rat hippocampal neurons the existence of a fast desensitizing component of a kainate-gated current was demonstrated. More than 90% of all neurons tested possessed such a receptor. Electrophysiological and pharmacological data suggest an association of this fast kainate-gated current with that mediated through a combination of subunits forming a low-affinity "(RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolopropionic acid (AMPA)-preferring' receptor. Kinetic data suggest the existence of a compact spatial location of fast desensitizing "AMPA-preferring' receptors which are naturally expressed on the surface of the neurons. An AMPA-insensitive current component mediated through fast desensitizing high-affinity kainate receptors was not observed. Topics: Animals; Benzothiadiazines; Cells, Cultured; Concanavalin A; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Hippocampus; Kainic Acid; Male; Patch-Clamp Techniques; Rats | 1996 |
Kainate responses of leech Retzius neurons in situ and in vitro.
Responses to the ionotropic glutamate receptor agonist kainate were measured in Retzius cells (RCs) of intact segmental ganglia (in situ), acutely isolated RCs, and cultured RCs (in vitro) of the leech Hirudo medicinalis. RCs in intact ganglia responded to kainate (5-20 microM) with depolarizations up to 30 mV or with an inward current under voltage-clamp that reversed near -10 mV. The membrane conductance increased by a factor of 2.5 at a holding potential of -70 mV in the presence of 20 microM kainate. In RCs in situ the membrane responses to 5 microM kainate increased when applied repeatedly 3-5 times. After this potentiation, the amplitude and time course of the membrane responses to 5 microM kainate were similar to the membrane response to 20 microM kainate. In current-clamp experiments kainate evoked an increase in intracellular calcium concentrations ([Ca2+]i) only when the membrane depolarized beyond -40 mV. In voltage-clamped RCs at a holding potential of -70 mV, kainate caused no significant rise in [Ca2+]i, indicating that the Ca2+ permeability of these kainate-gated ion channels appears to be negligible. The potentiation of the kainate-induced responses in RCs in situ was also present in voltage-clamped cells, where no or only small changes in [Ca2+]i occurred, suggesting that the underlying mechanism seemed to be independent of intracellular Ca2+ changes. In addition, the potentiation of the kainate-induced membrane responses was unaffected by cyclothiazide (100 microM), concanavalin A (0.5 mg/mL), and in the presence of extracellular low-Ca2+ and high-Mg2+ concentrations to suppress synaptic transmission in the ganglion. During whole-cell patch-clamp recordings (up to 50 min) potentiation remained the same indicating that small intracellular messenger molecules, which would be expected to dissipate, were not likely to be involved in mediating this potentiation. In acutely isolated RCs kainate induced no or only very small voltage responses. A potentiation of the kainate response was never observed in acutely isolated RCs. In cultured RCs (2-7 days in vitro) kainate evoked membrane responses with no apparent potentiation. Cultured RCs also responded with Ca2+ transients only when depolarized beyond -40 mV. The results show that RCs respond differently to kainate when kept isolated in culture compared to RCs in intact ganglia. The mechanism underlying the potentiation of the kainate response of RCs in situ, however, could not yet be identified Topics: Animals; Benzothiadiazines; Calcium; Calcium Channels; Cell Membrane Permeability; Cells, Cultured; Concanavalin A; Drug Synergism; Excitatory Amino Acid Agonists; Ganglia, Invertebrate; Kainic Acid; Leeches; Neurons | 1996 |
Three types of postsynaptic glutamatergic receptors are activated in DMNX neurons upon stimulation of NTS.
While it is widely accepted that parasympathetic activity plays a significant role in cardiovascular, bronchomotor, and gastrointestinal function, little is known about the synaptic control of parasympathetic vagal neurons. In this study, we identified the neurotransmitter(s) and postsynaptic responses in dorsal motor nucleus of the vagus (DMNX) neurons upon stimulation of the nucleus of the solitary tract (NTS). Neurons were visualized in rat brain stem slices, and perforated patch-clamp techniques were used to record postsynaptic currents. NTS stimulation activated glutamatergic currents in DMNX that were separated into N-methyl-D-aspartate (NMDA) and non-NMDA components using D-2-amino-5-phosphonovalerate and 6-cyano-7-nitroquinoxaline-2,3-dione, respectively. The non-NMDA component was further characterized using cyclothiazide and concanavalin A to block desensitization of DL-alpha-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA) and kainate receptors, respectively. Cyclothiazide increased the postsynaptic amplitude, whereas concanavalin A augmented duration, suggesting kainate, but not AMPA, currents are curtailed by desensitization. High frequency stimulations did not alter synaptic efficacy. In conclusion, this study demonstrates the existence of a monosynaptic glutamatergic pathway from NTS that activates NMDA, kainate, and AMPA postsynaptic receptors in DMNX neurons. Topics: 2-Amino-5-phosphonovalerate; 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Benzothiadiazines; Brain; Concanavalin A; Electric Stimulation; Electrophysiology; Excitatory Amino Acid Antagonists; Neurons; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, Glutamate; Solitary Nucleus; Synapses; Vagus Nerve | 1996 |
Properties of AMPA receptors expressed in rat cerebellar granule cell cultures: Ca2+ influx studies.
Cultured cerebellar granule cells become vulnerable to excitatory amino acids, especially to NMDA and kainate, by 9 days in vitro. In the same time, the sensitivity of cells to (RS)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA), in terms of AMPA-induced toxicity or 45Ca2+ uptake, was very low. The low AMPA responsiveness was due to receptor desensitization, because agents known to block desensitization, cyclothiazide and the lectins concanavalin A and wheat germ agglutinin, rendered granule cells vulnerable to AMPA and produced a pronounced stimulation of 45Ca2+ accumulation. 45Ca2+ influx was induced specifically by AMPA-receptor stimulation, because it was blocked virtually completely by 2,3-dihydroxy-6-nitro-7-sulfamoylbenzoquinoxaline (NBQX) and the benzodiazepine GYKI 52466 (selective non-NMDA receptor antagonists). Nevertheless, indirect routes activated by cellular responses to AMPA-receptor stimulation contributed significantly to the overall 45Ca2+ influx. These included Ca2+ uptake through NMDA-receptor channels, voltage-sensitive Ca2+ channels, and via Na+/Ca2+ exchange. However, nearly one-fifth of the total 45Ca2+ influx remained unaccounted for and this estimate was similar to 45Ca2+ influx observed under Na(+)-free conditions. This observation suggested that a significant proportion of the Ca2+ flux passes through the AMPA-receptor channel proper, a view supported by Co2+ uptake into nearly all granule cells on exposure to AMPA in the presence of cyclothiazide. Results are discussed in light of the reported AMPA receptor-subunit composition of cerebellar granule cells in vitro. Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Anti-Anxiety Agents; Benzodiazepines; Benzothiadiazines; Calcium; Calcium Radioisotopes; Cell Survival; Cells, Cultured; Cerebellum; Cobalt; Concanavalin A; Excitatory Amino Acids; Kainic Acid; N-Methylaspartate; Quinoxalines; Rats; Receptors, AMPA; Wheat Germ Agglutinins | 1995 |
AMPA-type glutamate receptors in glial precursor cells of the rat corpus callosum: ionic and pharmacological properties.
Glial cells in fiber tracts express various functional transmitter receptors, e.g., got glutamate. However, little is known about their biophysical and pharmacological profile. Using the in situ patch-clamp technique, kainate- and AMPA-induced conductance changes of glial precursor cells in the rat corpus callosum were investigated to study these aspects. Precursor cells were identified by their voltage-gated currents and were easily discernable from astrocytes and oligodendrocytes. Kainate induced two overlying effects in these cells: the activation of a cationic current and the block of potassium conductances. Cesium in the pipette solution blocked potassium conductances nearly completely and the ionic profile of the kainate-induced cationic current could be studied in detail. Full replacement of the sodium in the bath by calcium resulted only in a small kainate-induced (calcium) inward current flow, but the kainate-induced outward current carried by Cs+ was less affected reflecting a weak calcium permeability. The kainate response could be blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX) and millimolar zinc concentrations. Co-application of micromolar concentrations of zinc slightly enhanced the kainate-induced current, while Evans Blue was without any significant effect. Cyclothiazide increased the kainate response by a factor of x6 while concanavalin A did not enlarge it. The AMPA-induced current was amplified by a factor of x39 by cyclothiazide. The present data suggested the expression of weakly calcium-permeable AMPA receptors on glial precursor cells in the rat corpus callosum. Only a small fraction of the agonist-induced current could be seen without the appropriate blockers of receptor desensitization. An additional expression of kainate-preferring glutamate receptors could not be shown. Topics: Animals; Benzothiadiazines; Calcium; Chlorides; Concanavalin A; Corpus Callosum; Diuretics; Evans Blue; Kainic Acid; Neuroglia; Patch-Clamp Techniques; Quinoxalines; Rats; Receptors, AMPA; Receptors, Kainic Acid; Sodium Chloride Symporter Inhibitors; Zinc Compounds | 1995 |
Differential antagonism of alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid-preferring and kainate-preferring receptors by 2,3-benzodiazepines.
Whole-cell recordings were used to study the antagonism of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-preferring and kainate-preferring receptors by 2,3-benzodiazepines. Current through kainate-preferring receptors was recorded in rat dorsal root ganglion (DRG) neuron-s, whereas AMPA receptor current was measured in cultured neurons from rat cerebral cortex. In both cell types 2,3-benzodiazepines produced noncompetitive inhibition; however, antagonist potency was much higher against AMPA-preferring receptors than against kainate receptors. The most potent compound, 1-(4-aminophenyl)-3-methylcarbamyl-4-methyl-7,8- methylenedioxy-3,4-dihydro-5H-2,3-benzodiazepine (GYKI 53655), blocked AMPA receptor currents with an IC50 of approximately 1 microM. A second benzodiazepine, 1-(4-aminophenyl)-4-methyl-7,8- methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466), was about 20-fold less potent at AMPA receptors (IC50 = 18 microM). Both drugs were markedly weaker against kainate currents in DRG neurons. At 200 microM, the highest concentration tested, GYKI 53655 and GYKI 52466 produced only 30-40% inhibition in DRG cells, suggesting that for both compounds the IC50 against kainate receptors is > 200 microM. Our study suggests that GYKI 53655, at a concentration of approximately 10 microM, should produce > 90% block of AMPA-preferring receptors but < 5% inhibition of kainate-preferring receptors. Because the antagonism by this drug is noncompetitive, its effectiveness should not be influenced by phasic changes in transmitter concentration, making it an ideal compound for functional studies of the role of kainate and AMPA receptors in synaptic transmission. Topics: Animals; Anti-Anxiety Agents; Benzodiazepines; Benzothiadiazines; Binding, Competitive; Cerebral Cortex; Concanavalin A; Electrophysiology; Ganglia, Spinal; Kainic Acid; Membrane Potentials; Neurons; Rats; Rats, Inbred Strains; Receptors, AMPA; Receptors, Kainic Acid; Substrate Specificity | 1995 |
Selective modulation of desensitization at AMPA versus kainate receptors by cyclothiazide and concanavalin A.
Potentiation by cyclothiazide of recombinant glutamate receptor responses in Xenopus oocytes showed absolute selectivity for AMPA versus kainate receptors. In contrast, concanavalin A strongly potentiated responses at kainate but not AMPA receptors. Rapid desensitization in HEK 293 cells transfected with AMPA receptors was blocked by cyclothiazide, but only weakly attenuated by concanavalin A. Desensitization at kainate receptors was blocked by concanavalin A but unaffected by cyclothiazide. Selective effects of these modulators following coexpression of subunits from different families suggest independent assembly of functional AMPA and kainate receptors. Northern blot analysis of mRNA for dorsal root ganglia revealed a predominant expression of GluR5, indicating that modulation of desensitization by concanavalin A but not cyclothiazide in sensory neurons accurately predicts subunit expression for native glutamate receptors. Topics: Animals; Benzothiadiazines; Blotting, Northern; Cell Line; Cerebellum; Cloning, Molecular; Concanavalin A; Female; Ganglia, Spinal; Gene Expression; Kinetics; Macromolecular Substances; Membrane Potentials; Neurons; Oocytes; Prosencephalon; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, Glutamate; Receptors, Kainic Acid; Recombinant Proteins; RNA; Transfection; Xenopus laevis | 1993 |
Differential modulation by cyclothiazide and concanavalin A of desensitization at native alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid- and kainate-preferring glutamate receptors.
Concanavalin A, cyclothiazide, and aniracetam, ligands that modulate desensitization at glutamate receptors, were tested for their actions on responses at kainate-preferring receptors in dorsal root ganglion (DRG) neurons and at alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-preferring receptors in hippocampal neurons. In DRG neurons concanavalin A blocked desensitization produced by either kainate or 5-chlorowillardiine and strongly potentiated the peak amplitude of responses to both agonists. However, in hippocampal neurons concanavalin A produced only weak potentiation of responses to kainate and 5-chlorowillardiine, and after treatment with lectin responses to 5-chlorowillardiine remained strongly desensitizing. In contrast, cyclothiazide completely blocked desensitization produced by 5-chlorowillardiine in hippocampal neurons and strongly potentiated responses to kainate; the action of aniracetam was similar but much weaker. In DRG neurons cyclothiazide and aniracetam had no effect on desensitization and instead produced weak inhibition of responses to kainate. The different sensitivities of native AMPA- and kainate-preferring glutamate receptors to cyclothiazide and concanavalin A should prove useful for the differentiation of glutamate receptor subtypes in other areas of the central nervous system. Topics: Alanine; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Benzothiadiazines; Concanavalin A; Ganglia, Spinal; Hippocampus; Ibotenic Acid; In Vitro Techniques; Kainic Acid; Neurons; Pyrrolidinones; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, Glutamate; Receptors, Kainic Acid | 1993 |