[31P]/[1H] Nuclear Magnetic Resonance Study of Mitigating Effects of GYKI 52466 on Kainate-Induced Metabolic Impairment in Perfused Rat Cerebrocortical Slices

Summary: Purpose: Kainic acid (KA) has long been used in experimental animals to induce status epilepticus (SE). A mechanistic implication of this is the association between ex-citotoxicity and brain damage during or after SE. We evaluated KA-induced metabolic impairment and the potential mitigating effects of GYKI 52466 [1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine] in superfused rat cerebral cortical slices. Methods: Interleaved [³¹P]/[¹H] magnetic resonance spectroscopy (MRS) was used to assess energy metabolism, intra-cellular pH (pH_i), N-acetyl-L-aspartate (NAA) level, and lactate (Lac) formation before, during, and after a 56-min exposure to 4 mM KA in freshly oxygenated artificial cerebrospinal fluid (OXY-ACSF). Results: In the absence of GYKI 52466 and during the KA exposure, NAA, PCr, and ATP levels were decreased to 91.1 ± 0.8, 62.4 ± 3.9, and 59.1 ± 4.3% of the control, respectively; Lac was increased to 118.2 ± 2.1%, and pH, was reduced from 7.27 ± 0.02 to 7.13 2 0.02. During 4-h recovery with KA-free ACSF, pH_i rapidly and Lac gradually recovered, NAA decreased further to 85.5 ± 0.3%, and PCr and AW showed little recovery. Removal of Mg²⁺ from ACSF during KA exposure caused a more profound Lac increase (to 147.1 ± 4.0%) during KA exposure and a further NAA decrease (to 80.4 ± 0.5%) during reperfusion, but did not exacerbate PCr, ATP, and pH, changes. Inclusion of 100 μM GYKI 52466 during KA exposure significantly improved energy metabolism: the PCr and ATP levels were above 76.6 ± 2.1 and 82.0 & 2.9% of the control, respectively, during KA exposure and recovered to 101.4 ± 2.4 and 95.0 ± 2.4%, respectively, during reperfusion. NAA level remained at 99.8 ± 0.6% during exposure and decreased only slightly at a later stage of reperfusion. Conclusions: Our finding supports the notion that KA-induced SE causes metabolic disturbance and neuronal injury mainly by overexcitation through non-N-methy1-D-aspartate (NMDA) receptor functions.     

Molecular pathways mediating activation by kainate of mitogen-activated protein kinase in oligodendrocyte progenitors

Oligodendroglial cells express ionotropic glutamate receptors of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid hydrobromide (AMPA) and kainate (KA) subtypes. Recently, we reported that AMPA receptor agonists increased 45Ca2+ uptake and phospholipase C (PLC) activity. To further elucidate the intracellular signaling mechanisms, we examined the effects of AMPA and KA on mitogen-activated protein kinase (MAPK). KA caused a time- and concentration-dependent increase in MAPK activity (predominantly the p42mapk or ERK2) and the effect was blocked by 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), a competitive AMPA/KA receptor antagonist. Furthermore, the noncompetitive antagonists of AMPA receptor GYKI 52466 and LY 303070 prevented the actions of the agonists, indicating that the effect of KA on MAPK activation is mediated through AMPA receptors in oligodendrocyte progenitors. Chelation of extracellular Ca2+ by EDTA or inhibition of PLC with U73122 abolished MAPK activation by KA. In addition, KA-stimulated MAPK activation was reduced by the protein kinase C (PKC) inhibitors, H7 and bisindolylmaleimide, as well as downregulation of PKC by prolonged exposure to phorbol esters. The involvement of PKC in the signal transduction pathways was further supported by the ability of KA to induce translocation of PKC measured by [3H]PDBu binding. Interestingly, a wortmannin-sensitive phosphatidylinositol 3-kinase and a pertussis toxin (PTX)-sensitive G protein form part of the molecular pathways mediating MAPK activation by AMPA receptor. A specific inhibitor of MAPK kinase, PD 098059, blocked MAPK activation and reduced KA-induced c-fos gene expression. All together, these results indicate that MAPK is implicated in the transmission of AMPA signaling to the nucleus and requires extracellular Ca2+, and PLC/PKC activation.     

Short-term effects of kainic acid on CA1 hippocampal interneurons differentially vulnerable to excitotoxicity

PURPOSE: We sought to identify the inhibitory interneurons of the rat hippocampal CA1 region selectively vulnerable in the kainic acid (KA) model of temporal lobe epilepsy and to determine whether their selective vulnerability could be due to differential short-term KA effects. METHODS: We quantified vulnerable interneurons in stratum oriens-alveus (O/A) by using immunohistochemistry for glutamic acid decarboxylase (GAD), parvalbumin (PV), and somatostatin (SS) after KA injections in rats, and then compared in normal slices the effects of KA on interneurons either in O/A (vulnerable to KA) or in strata radiatum and lacunosum-moleculare (R/LM) (resistant to KA) by using whole-cell recording and calcium imaging. RESULTS: GAD-, PV- and SS-positive cells in O/A were decreased after KA treatment in P20 and P30 rats. Both short (1-min) and long (10-min) applications of KA produced similar tetrodotoxin (TTX)-insensitive membrane depolarization and decrease in input resistance in O/A and R/LM interneurons. KA responses were antagonized by CNQX and GYKI52466, suggesting AMPA receptor activation. KA also generated a similar increase in intracellular Ca2+ in O/A and R/LM interneurons, which was antagonized by CNQX and GYKI52466. CONCLUSIONS: The selective vulnerability of GAD-, PV-, and SS-immunopositive O/A interneurons in the KA model may not arise from cell-specific short-term membrane effects or calcium responses induced by KA, but from other glutamate receptor-mediated excitotoxic processes.     

Differential effects of NMDA and AMPA/kainate receptor antagonists on nitric oxide production in rat brain following intrahippocampal injection

Stimulation of glutamate receptors induces neuronal nitric oxide (NO) release, which in turn modulates glutamate transmission. The involvement of ionotropic glutamate NMDA and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainite (KA) receptors in the induction of NO production in the rat brain was examined after injection of kainate, non-NMDA receptor agonist, KA+6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), selective AMPA/KA receptor antagonist, or KA+2-amino-5-phosphonopentanoic acid (APV), selective NMDA receptor antagonist. Competitive glutamate receptor antagonists were injected with KA unilaterally into the CA3 region of the rat hippocampus. The accumulation of nitrite, the stable metabolite of NO, was measured by the Griess reaction at different times (5 min, 15 min, 2 h, 48 h and 7 days) in the ipsi- and contralateral hippocampus, forebrain cortex, striatum and cerebellum homogenates. The detected increase of NO production in distinct brain regions, which are functionally connected via afferents and efferents, suggests that these regions are affected by the injury. The effect of KA on nitrite production was blocked by the glutamate antagonists. Intrahippocampal KA+CNQX injection resulted in decrease of nitrite production, around control levels, in all tested brain structures. Significant decrease in nitrite levels was found only in comparison to KA-treated animals, i.e. the overall effect of selective AMPA/KA receptor antagonist was a decrease of KA-induced excitotoxicity. The accent effect of intrahippocampal KA+APV injection resulted, also, in decrease of nitrite production. However, this effect was detected after 5 min from the injection indicating the existence of an NMDA receptor-mediated component of basal nitrite production in physiological conditions and difference in mechanisms and time dynamics between CNQX and APV. The used antagonists showed same pattern in all tested brain structures. APV and CNQX both expressed sufficient neuroprotection in sense of reducing nitrite concentrations, but with differential effect in mechanisms and time dynamics. Our findings suggest that NMDA and AMPA/KA receptors are differentially involved in NO production.     

The anticonvulsant effect of the non-NMDA antagonists, NBQX and GYKI 52466, in mice.

The excitatory amino acid antagonists, NBQX (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(F)quinoxaline) and GYKI 52466 (1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine) that act on non-NMDA receptors, provide potent anticonvulsant protection against AMPA [RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)-induced seizures in Swiss mice and against sound-induced seizures in seizure-susceptible DBA/2 mice. Maximal anticonvulsant protection is observed 5-30 min after the i.p. administration of NBQX and 5-15 min after the i.p. administration of GYKI 52466 in DBA/2 mice. The ED50 values for the protection against AMPA-induced seizures by NBQX (30 min, i.p.) and GYKI 52466 (15 min, i.p.) are 23.6 (11.6-48.0) and 18.5 (11.5-29.5) mumol/kg, respectively. The ED50 values at 15 min for the protection against sound-induced seizures in DBA/2 mice are 31.3 (24.9-39.4) mumol/kg (NBQX, i.p.), 37.8 (21.2-67.4) mumol/kg (NBQX, i.v.) and 13.7 (11.5-16.5) mumol/kg (GYKI 52466, i.p.). In DBA/2 mice the therapeutic index (ratio of ED50 values for impaired rotarod performance and anticonvulsant action) is 6.6 for NBQX (15 and 30 min, i.p.) and 2.0 for GYKI 52466 (15 min, i.p.).     

Comparison of excitotoxic profiles of ATPA, AMPA, KA and NMDA in organotypic hippocampal slice cultures

The excitotoxic profiles of (RS)-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl)propionic acid (ATPA), (RS)-2-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainic acid (KA) and N-methyl-D-aspartate (NMDA) were evaluated using cellular uptake of propidium iodide (PI) as a measure for induced, concentration-dependent neuronal damage in hippocampal slice cultures. ATPA is in low concentrations a new selective agonist of the glutamate receptor subunit GluR5 confined to KA receptors and also in high concentrations an AMPA receptor agonist. The following rank order of estimated EC(50) values was found after 2 days of exposure: AMPA (3.7 mM)>NMDA (11 mM)=KA (13 mM)>ATPA (33 mM). Exposed to 30 microM ATPA, 3 microM AMPA and 10 microM NMDA, CA1 was the most susceptible subfield followed by fascia dentata and CA3. Using 8 microM KA, CA3 was the most susceptible subfield, followed by fascia dentata and CA1. In 100 microM concentrations, all four agonists induced the same, maximal PI uptake in all hippocampal subfields, corresponding to total neuronal degeneration. Using glutamate receptor antagonists, like GYKI 52466, NBQX and MK-801, inhibition data revealed that AMPA excitotoxicity was mediated primarily via AMPA receptors. Similar results were found for a high concentration of ATPA (30 microM). In low GluR5 selective concentrations (0.3-3 microM), ATPA did not induce an increase in PI uptake or a reduction in glutamic acid decarboxylase (GAD) activity of hippocampal interneurons. For KA, the excitotoxicity appeared to be mediated via both KA and AMPA receptors. NMDA receptors were not involved in AMPA-, ATPA- and KA-induced excitotoxicity, nor did NMDA-induced excitotoxicity require activation of AMPA and KA receptors. We conclude that hippocampal slice cultures constitute a feasible test system for evaluation of excitotoxic effects and mechanisms of new (ATPA) and classic (AMPA, KA and NMDA) glutamate receptor agonists. Comparison of concentration-response curves with calculation of EC(50) values for glutamate receptor agonists are possible, as well as comparison of inhibition data for glutamate receptor antagonists. The observation that the slice cultures respond with more in vivo-like patterns of excitotoxicity than primary neuronal cultures, suggests that slice cultures are the best model of choice for a number of glutamate agonist and antagonist studies.     

In vivo, the direct and seizure-induced neuronal cytotoxicity of kainate and AMPA is modified by the non-competitive antagonist, GYKI 52466

The 2,3-benzodiazepine GYKI 52466, administered intracerebrally or systemically, was assessed for its ability to protect against the neuronal death in the brain caused by intra-hippocampal injections of the non-N-methyl-D-aspartate (NMDA) receptor agonists, kainate and L-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). In contrast to a previous report, a low intra-hippocampal dose of GYKI 52466 (25 nmol) did not protect against kainate toxicity. In order to achieve higher doses of GYKI 52466, solubilization in 2-hydroxypropyl-beta-cyclodextrin was used, and limited protection against AMPA, but not kainate toxicity was found. There was a commensurate reduction in seizure-related neuronal loss in the limbic regions of the brain. When diazepam was used to prevent seizures, GYKI 52466 had no effect on hippocampal neuronal loss caused by the direct toxicity of AMPA and kainate on hippocampal neurons. Systemic administration of GYKI 52466 had only a minimal effect on preventing neuronal death caused by AMPA. In vivo, GYKI 52466 is only weakly effective as a neuroprotective agent.     

Diazoxide and cyclothiazide convert AMPA-induced dark cell degeneration of Purkinje cells to edematous damage in the cerebellar slice

Using the in vitro cerebellar slice preparation from young rats (8–12 days old), AMPA produced in addition to dark cell degeneration (DCD), an edematous type of toxicity (edematous damage; ED) in a minority (35%) of Purkinje cells. The intent of this study was to evaluate whether AMPA receptor desensitization is a primary factor that governs the type of toxicity induced by AMPA in the in vitro cerebellar slice preparation. Both the competitive and noncompetitive AMPA antagonists CNQX and GYKI 52466, respectively, blocked ED when given during the entire protocol (30 min AMPA exposure followed by 90 min of recovery). Cyclothiazide (100 μM) and diazoxide (500 μM), two antagonists of AMPA receptor desensitization reversed DCD and unveiled a fulminating ED. Lowering Na⁺ and lowering Cl⁻ proved effective in blocking ED, whereas removal of Ca²⁺ proved to be ineffective. Kainate (KA) (30–100 μM) produced only 50% as much ED as AMPA. This study indicates that AMPA can elicit more than one type of degeneration in the same neuronal population and that a primary governing factor in determining which toxicity is expressed seems to be the ability of AMPA receptors to desensitize.     

Involvement of AMPA Receptors in Trigeminal Post-synaptic Potentials Recorded in Rat Abducens Motoneurons In Vivo

The pharmacology of trigeminal excitatory postsynaptic potentials (EPSPs) evoked by electrical stimulation of the vibrissal pad was investigated in vivo in rat abducens motoneurons using intracellular recordings combined with microionophoretic applications of excitatory amino acid agonists [α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), NMDA, kainate] and a selective non-NMDA receptor antagonist (GYKI-52466). Intravenous applications of GYKI-52466 were also performed during synaptic and amino acid excitations. GYKI-52466, applied intravenously or microionophoretically, reversibly antagonized AMPA-induced depolarizations and trigeminal EPSPs in rat abducens motoneurons without affecting NMDA and kainate responses. The inhibition of AMPA-induced depolarizations was similar following i.v. and ionophoretic applications of GYKI-52466. Intravenous applications of GYKI-52466 (0.3–4 mg/kg) reversibly and dose-dependently reduced trigeminal EPSPs, which could be totally suppressed at the highest doses of GYKI-52466 (2–4 mg/kg). The antagonist effect, which developed very quickly, could last several minutes and recovered gradually. The effect of GYKI-52466 on the EPSPs and AMPA responses were compared in the same motoneurons. The partial inhibition of trigeminal EPSPs during microionophoretic applications of GYKI-52466 was probably due to the distribution of the synapses in the dendritic arborization of abducens motoneurons. Our results show that AMPA receptors are involved in the generation of trigeminal EPSPs in rat abducens motoneurons in vivo.     

Ionotropic glutamate receptors in isolated horizontal cells of the rabbit retina

With the use of the whole-cell voltage-clamp technique, we have recorded the currents induced by ionotropic glutamate receptor agonists on isolated axonless horizontal cells (HC) of rabbit retina. Bath application of the non-N-methyl-D-aspartate receptor agonists: kainate (KA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and L-glutamate (GLU) produced an increase in the conductance for non-selective cations. All the isolated horizontal cells responded to GLU, AMPA and KA. Responses elicited by GLU and AMPA but not KA exhibited a concentration-dependent desensitization. Application of N-methyl-D-aspartate (NMDA) evoked no responses. The rank order affinities of the agonists as estimated from EC50 values were AMPA > GLU > KA. Whereas KA had the lowest affinity of the agonists tested, it produced the largest currents. Hill coefficients of the concentration-response data were near 1 for AMPA, and 2 for KA and GLU. Coapplication of AMPA with cyclothiazide (CTZ) blocks AMPA receptor desensitization, and enhanced its effects on conductance. However, CTZ did not change the KA -induced conductances. In all cells tested, 6,7-dinitroquinoxaline (DNQX) completely and reversibly blocked the effects of KA and AMPA. The KA- and AMPA-induced currents were also completely blocked by 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466), a selective AMPA receptor antagonist. These results indicate that the responses to glutamate agonists in HC were mediated almost exclusively by AMPA receptors. Our study indicates that AMPA receptors play a fundamental role in mediating the synaptic input into rabbit horizontal cells.     

GYKI 52466 has positive modulatory effects on AMPA receptors

The 2,3-benzodiazepine derivative GYKI 52466 has been well characterized as a negative modulator of AMPA-type glutamate receptors. The present study re-examined the effects of GYKI 52466 on AMPA receptor-mediated currents in patches excised from pyramidal neurons in the hippocampal CA1 field and found that this drug has positive modulatory effects in addition to its receptor blocking action. A low concentration of GYKI 52466 (10 microM) reliably increased the steady-state current by about three-fold, while the peak current was reduced by 30% only. Higher drug concentrations produced parallel reductions in both the steady-state and peak currents. The increase in the steady-state current was not accompanied by a change in the deactivation time constant and thus, is more likely to result from a change in desensitization than a slowing of channel closing. The results indicate that GYKI 52466 modulates AMPA receptor-mediated currents in a complex manner, perhaps by acting through more than one binding site.     

AMPA receptor antagonists, GYKI 52466 and NBQX, do not block the induction of long-term potentiation at therapeutically relevant concentrations

The involvement of alpha-amino-3-hydroxy-5-methylizoxazole-4-propionic acid (AMPA) receptors in induction of long-term potentiation (LTP) was examined in rat hippocampal slice preparation. Using conventional extracellular recording, excitatory postsynaptic potentials (EPSPs) and population action potentials (PSs), evoked by low-frequency stimulation of the Schaffer collateral-commissural fibres, were recorded in the CA1 region. The effects of a competitive AMPA receptor antagonist, 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2, 3-dione (NBQX), and that of a non-competitive blocker, 1-(4-aminophenyl)-4-methyl-7,8-methylendioxy-5H-2,3-benzodiazepine (GYKI 52466) have been examined. 0.25-0.5 microM of NBQX and 20-40 microM of GYKI 52466 did not suppress the induction of LTP. LTP was attenuated only at the highest concentrations tested (1 microM NBQX or 80 microM GYKI 52466). These in vitro concentrations, however, exceed the brain levels needed for in vivo anticonvulsant action. Furthermore, even at the highest concentrations both compounds suppressed only the expression but not the induction of LTP. Namely after their washout LTP reappeared. Thus, at pharmacologically relevant concentrations these AMPA receptor antagonists apparently do not suppress LTP, a cellular mechanism underlying memory formation. These experiments suggest that in clinical practice AMPA receptor blockade may have some advantage over N-methyl-D-aspartate receptor antagonism, which is accompanied by severe memory impairment.     

Interactions of allosteric modulators of AMPA/kainate receptors on spreading depression in the chicken retina

The functional role of AMPA and kainate receptors in spreading depression (SD) was investigated in the isolated chicken retina. Competitive (NBQX) and non-competitive (GYKI 52466, GYKI 53405 and GYKI 53655) antagonists of the AMPA receptor inhibited AMPA-induced SD in a concentration-dependent manner. Concentrations of drugs caused 50% inhibition (IC(50) values) are 0.2, 16.6, 7.0 and 1.4 microM, respectively. AMPA receptor positive modulator cyclothiazide was more effective in the potentiation of SD evoked by AMPA than by kainate. Slight potentiation of either AMPA- or kainate-induced SD was observed only at high concentration (1 mg/ml) by the kainate receptor modulator concanavalin A. Compounds that positively modulate AMPA receptor function (cyclothiazide, IDRA-21, S 18986, 1-BCP and aniracetam) caused a concentration-dependent potentiation in SD. Concentrations of drugs that caused 50% potentiation (estimated EC(50) values) are 9, 135, 142, 450 and 1383 microM, respectively. Interaction between cyclothiazide, aniracetam or S 18986 administered with each other, or with GYKI 52466, respectively, was also investigated. When cyclothiazide and S 18986 were co-applied, their effects seemed to be additive. However, lack of additivity was obtained when S 18986 was added together with aniracetam. Positive modulators applied at equiactive concentrations reduced the inhibitory action of GYKI 52466 and differently shifted its concentration-response curve. In this respect, S 18986 was the most effective (IC(50) of GYKI 52466 changed from 16.6 to 51.9 microM). Our findings indicate the contribution of AMPA rather than kainate receptors in the mediation of retinal spreading depression. Our data further support the idea that multiple positive modulatory sites are present on the AMPA receptor complex in addition to a negative modulatory site.     

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 μM cyclothiazide (a specific AMPA receptor desensitization blocker) enhanced the glutamate-induced current by about 5 fold. In contrast, 300 μg ml⁻¹ ConA (a specific kainate receptor desensitization blocker), had no effect. GYKI 52466 (a specific AMPA receptor antagonist) at 30 μM almost completely suppressed the glutamate-induced inward current in HCs. Cyclothiazide at 100 μM 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 μg ml⁻¹ had no effect on either the dark potential or the light responses of the HC. GYKI 52466 (30 μM) 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.     

Mechanism of Inhibition of the GluA2 AMPA Receptor Channel Opening: the Role of 4-Methyl versus 4-Carbonyl Group on the Diazepine Ring of 2,3-Benzodiazepine Derivatives

2,3-Benzodiazepine derivatives are synthesized as drug candidates for a potential treatment of various neurodegenerative diseases involving the excessive activity of AMPA receptors. Here, we describe a rapid kinetic investigation of the mechanism of inhibition of the GluA2Q(flip) AMPA receptor channel opening by two 2,3-benzodiazepine derivatives, i.e. the prototypic 2,3-benzodiazepine compound GYKI 52466 [(1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine)] and 1-(4-aminophenyl)-3,5-dihydro-7,8-methylenedioxy-4H-2,3-benzodiazepin-4-one (BDZ-2). GYKI 52466 and BDZ-2 are structurally similar in that the 4-methyl group in the diazepine ring of GYKI 52466 is replaced by a carbonyl group, yielding BDZ-2. Using a laser-pulse photolysis technique with ～60 μs time resolution, we characterize the effect of the two compounds individually on the channel-opening process of the GluA2Q(flip) receptor expressed in HEK-293 cells. We find that BDZ-2 preferentially inhibits the open-channel state, whereas GYKI 52466 is more selective for the closed-channel state of the GluA2Q(flip) receptors. Each inhibitor binds independently to its own noncompetitive site, yet the two sites do not interact allosterically. The significance of these results in the context of both the structure-activity relationship and the properties of the GluA2Q(flip) receptor channels is presented.     

Activation of c-Jun NH2-terminal kinase 3 is mediated by the GluR6.PSD-95.MLK3 signaling module following cerebral ischemia in rat hippocampus

Kainate receptor glutamate receptor 6 (GluR6) binds to the postsynaptic density protein 95 (PSD-95), which in turn anchors mixed lineage kinase 3 (MLK3) via SH3 domain in rat brain tissue. MLK3 subsequently activates c-Jun NH(2)-terminal kinase (JNK) via MAP kinase kinases (MKKs). We investigated the association of PSD-95 with GluR6 and MLK3, MLK3 autophosphorylation, the interaction of MLK3 with JNK3, and JNK3 phosphorylation following cerebral ischemia in rat hippocampus. Our results indicate that the GluR6.PSD-95.MLK3 complex peaked at 6 h of reperfusion. Furthermore, MLK3 autophosphorylation and the interaction of MLK3 with JNK3 occurred with the alteration of GluR6.PSD-95.MLK3 signaling module. To further prove whether JNK3 activation in ischemic hippocampus is mediated by GluR6.PSD-95.MLK3 signaling pathway, the AMPA/KA receptor antagonist 6,7-dinitroquinoxaline-2, (1H, 4H)-dione (DNQX), the GluR6 antagonist 6,7,8,9-Tetrahydro-5-nitro-1H-benz[g]indole-2,3-dione-3-oxime (NS102), the AMPA receptor antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzo diazepine (GYKI52466), and the NMDA receptor antagonist ketamine were given to the rats 20 min prior to ischemia. Our findings indicate that both DNQX and NS102 significantly attenuated the association of PSD-95 with GluR6 and MLK3, MLK3 autophosphorylation, interaction of MLK3 with JNK3, and JNK3 phosphorylation, while GYKI52466 and ketamine had no effect. Moreover, administration of NS102 before cerebral ischemia significantly increased the number of the surviving hippocampal CA1 pyramidal cells at 5 days of reperfusion. Consequently, GluR6, one subunit of kainate receptor, plays a critical role in inducing JNK3 activation after ischemic injury.     

Effect of upregulation of AMPA glutamate receptors on cerebral O2 consumption and blood flow in rat

AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate) receptors, in cerebral cortex, underwent upregulation (35% increase) following chronic blockade with a non-competitive AMPA receptor antagonist, GYKI 52466 (1-(aminophenyl)-4-methyl-7, 8-methylenedioxy-5H-2,3-benzodiazepine). Such upregulation did not alter basal cerebrocortical blood flow or O(2) consumption. There was a much higher increase in blood flow and O(2) consumption in the upregulated, agonist (AMPA) stimulated cortices of anesthetized rats.