concanavalin-a and 2-3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline

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

Studies demonstrated that intrathecal 1,2,3,4-tetrahydro-6-nitro-2, 3-dioxo[f]quinoxaline-7-sulfonamide disodium (NBQX), an antagonist of AMPA/kainate receptors, induced antinociception in the spinal cord of rats. The present study demonstrated that the NBQX-induced increases in hindpaw withdrawal latencies (HWLs) were dose-dependently attenuated by intrathecal pretreatment of the AMPA receptor desensitization inhibitor, diazoxide. The effect was unrelated to the opening of K+ channels by diazoxide. On the other hand, intrathecal pretreatment of concanavalin A, which selectively inhibits the desensitization of kainate receptor, produced no significant influence on the NBQX-induced antinociception. The results suggest that the NBQX-induced antinociception was mediated by AMPA receptors, not by kainate receptors, in the spinal cord of rats.

Topics: Analgesics; Animals; Concanavalin A; Diazoxide; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Injections, Spinal; Male; Pain Measurement; Potassium Channels; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Receptors, Kainic Acid; Spinal Cord

Responses of cultured rat cortical and spinal cord neurones to kainate (KA) have been related to the expression of KA receptor subunits revealed by single-cell reverse transcription polymerase chain reaction (RT-PCR). Semi-rapid application of KA evoked non-desensitizing responses with EC(50) values of 82 microM for cortical and 67 microM for spinal cord neurones. In the presence of concanavalin A, GYKI 53655 (100 microM) reduced responses of both types of neurone to KA by about 80% to leave a KA receptor-mediated response with an EC(50) of 4 microM on spinal cord neurones and 27 microM (P<0.001) on cortical neurones. Ultra-fast application of KA to outside-out patches of cortical neurones evoked a non-decaying response which was reduced by about 30% by GYKI 53655 to reveal a transient response that desensitized by 92.5% with a time constant (tau(des)) of 26.2 ms. Responses of spinal cord patches decayed by 47.8%. GYKI 53655 reduced the peak response by 8.3% and the residual response desensitized by 75.8%, with a tau(des) of 17.3 ms, all values being significantly smaller than for cortical neurones. Single-cell RT-PCR showed relative abundances of mRNAs for the KA receptors, GluR5, GluR6 and GluR7, of 12, 33 and 54% for cortical neurones and 38, 10 and 54% for spinal cord neurones, respectively. The relative abundances of KA1 and KA2 were 12 and 88% for cortical neurones, and 19 and 79% for spinal cord neurones, respectively. The most likely expression patterns of functional KA receptors is GluR6/KA2 for cortical neurones and GluR5/KA2 for spinal cord neurones.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Benzodiazepines; Cells, Cultured; Cerebral Cortex; Concanavalin A; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Gene Expression; Kainic Acid; Membrane Potentials; Neurons; Patch-Clamp Techniques; Quinoxalines; Rats; Rats, Wistar; Receptors, AMPA; Receptors, Kainic Acid; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spinal Cord

The developmental expression of calcium (Ca2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors in cultured neocortical neurons was evaluated by using cobalt uptake, a histochemical method that identifies cells expressing Ca2+-permeable, non-N-methyl-D-aspartate (non-NMDA) receptors. At a concentration of 500 microM, AMPA was found to stimulate cobalt uptake only late in development, resulting in staining of 2.7%+/-0.3% of the neurons maintained in culture for 12 days in vitro (DIV). When AMPA receptor desensitization was blocked with 50 microM cyclothiazide, the developmental profile of cobalt uptake mediated by 25 microM AMPA changed dramatically. The cobalt staining now appeared in young cultures (5 DIV), and the percentage of stained cells increased from 3.4%+/-0.2% at 5 DIV to 21.7%+/-1.6% at 12 DIV. The effect of 200 microM kainate was similar to that seen with 25 microM AMPA plus 50 microM cyclothiazide, resulting in 17.7%+/-0.3% stained neurons at 12 DIV. The cobalt uptake was specific to AMPA and kainate receptors because NMDA receptors and voltage-gated calcium channels were found not to mediate any cobalt staining. In addition, 10 microM 6-nitro-7-sulphamoylbenzo-[f]-quinoxaline-2,3-dione (NBQX) was able to prevent all staining at 5 and 8 DIV and most of the staining at 12 DIV, indicating that the non-NMDA ionotropic glutamate receptors are involved in cobalt uptake into the neurons. The AMPA receptor-selective antagonist GYKI 53655 was used to differentiate between cobalt influx through AMPA- or kainate-preferring receptors. After pretreatment with concanavalin A (con A), an inhibitor of kainate receptor desensitization, cobalt uptake was assessed after stimulation by 200 microM kainate in the presence of 25 microM GYKI 53655. No cobalt staining was observed under these conditions, indicating that most if not all of the cobalt influx induced by kainate was mediated through AMPA receptor channels.

Topics: Animals; Calcium; Cells, Cultured; Cobalt; Concanavalin A; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Mice; Neocortex; Neurons; Permeability; Quinoxalines; Receptors, AMPA; Receptors, Kainic Acid; Staining and Labeling

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

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

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

1. The shift in d.c. potential in dorsal roots (EC50 8.0 microM +/- 0.9 s.e. mean, n = 5) or depression of the C elevation of the compound action potential (EC50 3.0 microM +/- 0.3, n = 7) have been used to measure the depolarizing action of kainate on dorsal root C fibres of immature (3 to 5 day old) rats. Depolarization of motoneurones was measured from the shift in d.c. potential in ventral roots. 2. 6-Cyano-7-nitroquinoxaline,2-3,dione (CNQX) (pA2 5.78 +/- 0.06, n = 8) and 6-nitro-7-suplhamobenzo(f)quinoxaline-2,3-dione (NBQX) (pA2 5.75 +/- 0.04, n = 7) had similar potencies as antagonists of kainate at dorsal root fibres. The potency of NBQX as a kainate antagonist was similar also at motoneurones (pA2 5.72 +/- 0.07, n = 3). At motoneurones, NBQX was less potent as an antagonist of domoate (pA2 5.29 +/- 0.05) and more potent as an antagonist of S-alpha-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (pA2 6.80 +/- 0.09) than as an antagonist of kainate. 3. Application of L-glutamate, quisqualate and RS-AMPA to dorsal roots produced only short lasting depolarizations but kainate concentration-effect plots were shifted to the right in the presence of these three agonists (pA2 5.08 +/- 0.08, (n = 3), 5.59 +/- 0.04, (n = 4) and 4.46 +/- 0.04 (n = 4) respectively). Slopes of dose-ratio against concentration were significantly less than one for the latter antagonism. 4. The amplitude of depolarizations induced by L-glutamate, AMPA and quisqualate were increased up to ten fold and those induced by kainate up to two fold following treatment of dorsal roots with concanavalin A. The duration of the responses was increased also by the latter treatment. Folowing 85 s applications of glutamate, quisqualate, AMPA and kainate the mean respective times (s +/- s.e.mean (n))taken for responses to decay to half the peak amplitude were increased from 63 +/- 7 (10), 86 +/- 17 (4),95 +/- 19 (4) and 135 +/- 3 (12) to 202 +/- 49 (10), 147 +/- 7 (4), 160 +/- 13 (6) and 163 +/- 10 (10). Under similar conditions the mean decay time of y-aminobutyric acid-induced responses was 145 +/- 7 (10). This was not significantly altered by concanavalin A treatment.5. Application to dorsal roots of L-aspartate at concentrations up to 5 mm (with or without concanavalin A treatment), the selective metabotropic agonist 1S,3R-trans-1-aminocyclopentane-1,3-dicarboxylate (1 mM,) and D-serine (20 pM) in the presence or absence of N-methyl-D-aspartate (NMDA,500 pM) neither depolarized the prep

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Action Potentials; Animals; Concanavalin A; In Vitro Techniques; Kainic Acid; Motor Neurons; Nerve Fibers; Quinoxalines; Rats; Receptors, Amino Acid; Spinal Cord