Glucose Regulates Glutamate-Evoked Arachidonic Acid Release from Cultured Striatal Neurons

Abstract: l -Glutamate stimulates the liberation of arachidonic acid from mouse striatal neurons via the activation of N -methyl- d -aspartic acid (NMDA) receptors and by the joint stimulation of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and metabotropic receptors. In this study, we investigated whether starving cultured mouse striatal neurons of glucose would modify glutamatergic receptor-mediated arachidonic acid release. Glucose deprivation for 30 min led to enhancement of the NMDA-evoked release of arachidonic acid, compared with that observed in the presence of glucose. This enhanced response depended on both the concentration of glucose and the length of time of glucose deprivation. The enhanced NMDA response appeared to result from both a release of glutamate and the subsequent additional release of arachidonic acid due to the activation of AMPA and metabotropic receptors. Indeed, the increased NMDA response was completely reversed when extracellular glutamate was enzymatically removed. Moreover, glucose deprivation potentiated the combined AMPA/metabotropic receptor-evoked release of arachidonic acid, even in the absence of extracellular glutamate. However, removing glucose did not improve the calcium rise induced by AMPA or NMDA. The ATP-evoked release of arachidonic acid from striatal astrocytes was not altered by glucose starvation. In summary, glucose deprivation affected two properties of striatal neurons: (a) it induced an NMDA-evoked release of glutamate from striatal neurons and (b) it selectively potentiated the AMPA/(1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylic acid-evoked release of [³H]arachidonic acid without altering the authentic NMDA-mediated response.     

Effects of Non-NMDA Receptor Modulators on [3H]Dopamine Release from Rat Mesencephalic Cells in Primary Culture

Abstract: The effects of AMPA and kainate on [³H]dopamine release from fetal (embryonic day 15) rat mesencephalic neurons in primary culture were enhanced markedly in a dose-dependent fashion by cyclothiazide, a recently described inhibitor of AMPA receptor desensitization. The EC₅₀ value for cyclothiazide was 2.2 ± 0.8 µ M . The release of [³H]dopamine induced by both AMPA (or kainic acid) and the combination of AMPA (or kainic acid) with cyclothiazide was antagonized by specific antagonists like 6-cyano-7-nitroquinoxaline-2,3-dione or the noncompetitive benzodiazepine GYKI 52466. Unlike cyclothiazide, the lectin concanavalin A did not stimulate [³H]dopamine release. These results established the involvement of AMPA-preferring receptors on [³H]dopamine release from rat mesencephalic neurons in primary culture and provided further evidence for the existence of regulatory allosteric sites on AMPA receptor subunits.     

Cyclothiazide Modulates AMPA Receptor-Mediated Increases in Intracellular Free Ca2+ and Mg2+ in Cultured Neurons from Rat Brain

Abstract: We investigated the modulation of (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced increases in intracellular free Ca²⁺ ([Ca²⁺]_i) and intracellular free Mg²⁺ ([Mg²⁺]_i) by cyclothiazide and GYKI 52466 using microspectrofluorimetry in single cultured rat brain neurons. AMPA-induced changes in [Ca²⁺]_i were increased by 0.3–100 µ M cyclothiazide, with an EC₅₀ value of 2.40 µ M and a maximum potentiation of 428% of control values. [Ca²⁺]_i responses to glutamate in the presence of N -methyl- d -aspartate (NMDA) receptor antagonists were also potentiated by 10 µ M cyclothiazide. The response to NMDA was not affected, demonstrating specificity of cyclothiazide for non-NMDA receptors. Almost all neurons responded with an increase in [Ca²⁺]_i to both kainate and AMPA in the absence of extracellular Na⁺, and these Na⁺-free responses were also potentiated by cyclothiazide. GYKI 52466 inhibited responses to AMPA with an IC₅₀ value of 12.0 µ M . Ten micromolar cyclothiazide significantly decreased the potency of GYKI 52466. However, the magnitude of this decrease in potency was not consistent with a competitive interaction between the two ligands. Cyclothiazide also potentiated AMPA- and glutamate-induced increases in [Mg²⁺]_i. These results are consistent with the ability of cyclothiazide to decrease desensitization of non-NMDA glutamate receptors and may provide the basis for the increase in non-NMDA receptor-mediated excitotoxicity produced by cyclothiazide.     

Ampakine CX546 bolsters energetic response of astrocytes: a novel target for cognitive-enhancing drugs acting as alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor modulators

Glutamate was previously shown to enhance aerobic glycolysis i.e. increase glucose utilization and lactate production with no change in oxygen levels, in mouse cortical astrocytes by a mechanism involving glutamate uptake. It is reported here that a similar response is produced in both hippocampal and cerebellar astrocytes. Application of the cognitive-enhancing drug CX546 promoted further enhancement of glucose utilization by astrocytes from each brain area following glutamate exposure. alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors represent the purported molecular target of cognitive-enhancing drugs such as CX546, and the presence of AMPA receptor subunits GluR1-4 was evidenced in astrocytes from all three regions by immunocytochemistry. AMPA itself did not stimulate aerobic glycolysis, but in the presence of CX546, a strong enhancement of glucose utilization and lactate production was obtained in cortical, hippocampal and cerebellar astrocytes. The effect of CX546 was concentration-dependent, with an EC(50) of 93.2 microm in cortical astrocytes. AMPA-induced glucose utilization in the presence of CX546 was prevented by the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the negative modulator GYKI 52466. In addition, the metabolic effect of CX546 in the presence of AMPA was mimicked by the AMPA receptor modulator cyclothiazide. Our data suggest that astrocyte energetics represents a novel target for cognitive-enhancing drugs acting as AMPA receptor modulators.     

Inherent desensitisation-preventing properties of a novel,subtype-selective AMPA receptor agonist, (S)-CPW 399, as a possible explanation for its excitotoxic action in cultured cerebellar granule cells

The synthesis and pharmacological characterisation of (S)-CPW 399 as a novel, potent and subtype-selective agonist of the AMPA receptor was recently reported. Studies have been extended to investigate its excitotoxic action in primary cultures of mouse cerebellar granule cells. (S)-CPW 399 induced neuronal cell death in a time- and concentration-dependent manner (EC(50) approximately 70 microM) at 24-h exposure. (S)-CPW-induced neuronal death could be prevented by co-administration with either of the AMPA/kainate selective receptor antagonists 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) and 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX) or by the 2,3-benzodiazepine, GYKI 53655 (a selective AMPA receptor antagonist); while no protection was afforded by either the NMDA receptor antagonist D,L(+/-)-2-amino-5-phosphonopentanoate (APV) or by nifedipine (an L-type calcium channel antagonist) when used alone or in combination. Cyclothiazide, which blocks AMPA receptor desensitisation, caused minimal potentiation of (S)-CPW 399-induced neuronal death, supporting accumulating evidence that (S)-CPW 399 is a full AMPA receptor agonist that markedly prevents a receptor desensitised conformation. (S)-AMPA, (S)-willardiine (a naturally-occurring heterocyclic excitatory amino acid) and its halogenated derivative, (S)-5-fluorowillardiine, had no deleterious effect on neuronal viability when used alone but each, in the presence of cyclothiazide, induced a concentration-dependent excitotoxic cell death with a rank order of potency (fluorowillardiine>AMPA=willardiine). (S)-CPW 399 stimulated an increase in intracellular free-calcium levels ([Ca(2+)](i)) in a concentration-dependent fashion (EC(50) approximately 5 microM) attaining a value of six-fold that of 'resting' cells at maximum stimulation; achieved at approximately 100 microM (S)-CPW 399. The (S)-CPW 399-stimulated increase in [Ca(2+)](i) was virtually abolished by GYKI 53655, NBQX, CNQX and by cobalt ions; markedly inhibited by nifedipine and marginally affected by D-APV. These results suggest that (S)-CPW 399 may be used as a pharmacological tool to aid in the investigation of the role of AMPA receptors in excitotoxicity and their molecular mechanisms of desensitisation.     

Excitatory Amino Acid Receptors in the Ventral Tegmental Area Regulate Dopamine Release in the Ventral Striatum

Abstract: The role of excitatory amino acid (EAA) receptors located in the ventral tegmental area (VTA) in tonic and phasic regulation of dopamine release in the ventral striatum was investigated. Microdialysis in conscious rats was used to assess dopamine release primarily from the nucleus accumbens shell region of the ventral striatum while applying EAA antagonists or agonists to the VTA. Infusion of the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (25 and 100 µ M ) into the VTA did not affect dopamine release in the ventral striatum. In contrast, intra-VTA infusion of the NMDA receptor antagonist 2-amino-5-phosphopentanoic acid (100 and 500 µ M ) dose-dependently decreased the striatal release of dopamine. Intra-VTA application of the ionotropic EAA receptor agonists NMDA and AMPA dose-dependently (10 and 100 µ M ) increased dopamine efflux in the ventral striatum. However, infusion of 50 or 500 µ M trans -(±)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD), a metabotropic EAA receptor agonist, did not significantly affect these levels. These data suggest that NMDA receptors in the VTA exert a tonic excitatory influence on dopamine release in the ventral striatum. Furthermore, dopamine neurotransmission in this region may be enhanced by activation of NMDA and AMPA receptors, but not ACPD-sensitive metabotropic receptors, located in the VTA. These data further suggest that EAA regulation of dopamine release primarily occurs in the VTA as opposed to presynaptically at the terminal level.     

Cyclothiazide Selectively Potentiates AMPA- and Kainate-Induced [3H]Norepinephrine Release from Rat Hippocampal Slices

Abstract: Activation of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) subtype of ionotropic glutamate receptors has been shown to result in a rapid desensitization of the receptor in the presence of certain agonists. One effect of AMPA receptor desensitization in the hippocampus may be to decrease the efficacy of AMPA receptor agonists at stimulating the release of norepinephrine from noradrenergic terminals. Recently, cyclothiazide was reported to inhibit AMPA receptor desensitization by acting at a distinct site on AMPA receptors. We have examined the effect of cyclothiazide on AMPA- and kainate (KA)-induced norepinephrine release from rat hippocampal slices to determine whether cyclothiazide would increase the efficacy of AMPA-induced [³H]norepinephrine release by inhibiting AMPA receptor desensitization. Cyclothiazide was observed to potentiate markedly both AMPA- and KA-induced [³H]norepinephrine release. This potentiation is selective for AMPA/KA receptors as cyclothiazide did not potentiate N -methyl- d -aspartate-induced [³H]norepinephrine release or release induced by the nonspecific depolarizing agents veratridine and 4-aminopyridine. These results demonstrate that AMPA receptor-mediated modulation of [³H]norepinephrine release from rat brain slices is a useful approach to studying the cyclothiazide modulatory site on the AMPA receptor complex.     

Characterization of the Glutamate Receptors Mediating Release of Somatostatin from Cultured Hippocampal Neurons

Abstract: l -Glutamate, NMDA, dl -α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and kainate (KA) increased the release of somatostatin-like immunoreactivity (SRIF-LI) from primary cultures of rat hippocampal neurons. In Mg²⁺-containing medium, the maximal effects (reached at ∼100 µ M ) amounted to 737% (KA), 722% (glutamate), 488% (NMDA), and 374% (AMPA); the apparent affinities were 22 µ M (AMPA), 39 µ M (glutamate), 41 µ M (KA), and 70 µ M (NMDA). The metabotropic receptor agonist trans -1-aminocyclopentane-1,3-dicarboxylate did not affect SRIF-LI release. The release evoked by glutamate (100 µ M ) was abolished by 10 µ M dizocilpine (MK-801) plus 30 µ M 1-aminophenyl-4-methyl-7,8-methylenedioxy-5 H -2,3-benzodiazepine (GYKI 52466). Moreover, the maximal effect of glutamate was mimicked by a mixture of NMDA + AMPA. The release elicited by NMDA was sensitive to MK-801 but insensitive to GYKI 52466. The AMPA- and KA-evoked releases were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX) or by GYKI 52466 but were insensitive to MK-801. The release of SRIF-LI elicited by all four agonists was Ca²⁺ dependent, whereas only the NMDA-evoked release was prevented by tetrodotoxin. Removal of Mg²⁺ caused increase of basal SRIF-LI release, an effect abolished by MK-801. Thus, glutamate can stimulate somatostatin release through ionotropic NMDA and AMPA/KA receptors. Receptors of the KA type (AMPA insensitive) or metabotropic receptors appear not to be involved.     

Two Pathways of Cyclic GMP Production Through Glutamate Receptor-Mediated Nitric Oxide Synthesis

The selective agonists for the metabotropic glutamate receptor and the ionotropic non-N-methyl-D-aspartate (NMDA) glutamate receptor, (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (ACPD) and (R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), respectively, increased the cyclic GMP (cGMP) content in cerebellar slices prepared from adult rats. The ACPD-induced rise in cGMP level was blocked by compounds known to antagonize metabotropic glutamate receptors, such as DL-2-amino-3-phosphonopropionic acid and L-2-amino-4-phosphonobutyric acid, but not by ionotropic glutamate receptor antagonists, D-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), whereas the AMPA-induced rise in cGMP level was suppressed by CNQX. Both rises in cGMP level involved nitric oxide synthase (NOS), because NG-methyl-L-arginine (NMLA), an inhibitor of NOS, blocked both cGMP level rises, and excess L-arginine reversed the effect of NMLA. After lithium chloride treatment, which could exhaust phosphatidylinositol phosphates, ACPD no longer increased cGMP levels, whereas AMPA was still effective. In a calcium-free medium, ACPD still induced a rise in cGMP level, whereas AMPA did not. When the molecular layer was isolated to determine the cGMP content separately from that in the rest of the cerebellar cortex, it was found that ACPD raised the cGMP level mainly in the molecular layer, whereas AMPA raised it in both sections.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMDA receptors in nucleus tractus solitarii are linked to soluble guanylate cyclase

We sought to test the hypothesis that cardiovascular responses to activation of ionotropic, but not metabotropic, glutamate receptors in the nucleus tractus solitarii (NTS) depend on soluble guanylate cyclase (sGC) and that inhibition of sGC would attenuate baroreflex responses to changes in arterial pressure. In adult male Sprague-Dawley rats anesthetized with chloralose, the ionotropic receptor agonists N-methyl-d-aspartate (NMDA) and dl-alpha-amino-3-hydroxy-5-methylisoxazole-propionic acid (AMPA) and the metabotropic receptor agonist trans-dl-amino-1,3-cyclopentane-dicarboxylic acid (ACPD) were microinjected into the NTS before and after microinjection of sGC inhibitors at the same site. Inhibition of sGC produced significant dose-dependent attenuation of cardiovascular responses to NMDA but did not alter responses produced by injection of AMPA or ACPD. Bilateral inhibition of sGC did not alter arterial pressure, nor did it attenuate baroreflex responses to pharmacologically induced changes in arterial pressure. This study links sGC with NMDA, but not AMPA or metabotropic, receptors in cardiovascular signal transduction through NTS.     

Differential Involvement of Metabotropic and p75 Neurotrophin Receptors in Effects of Nerve Growth Factor and Neurotrophin-3 on Cultured Purkinje Cell Survival

Abstract: We have examined the role of the p75 neurotrophin receptor in survival-promoting effects of nerve growth factor (NGF) and neurotrophin-3 (NT-3) on cultured Purkinje cells. Previously, we showed that NGF promotes Purkinje cell survival in conjunction with (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), an agonist of metabotropic excitatory amino acid receptors, whereas NT-3 by itself increases cell number. We now present evidence that p75 plays different roles in Purkinje cell responses to the two neurotrophins. A metabotropic receptor of the mGluR1 subtype may interact with p75 function, so as to regulate Purkinje cell responsiveness to neurotrophins. When cerebellar cultures were grown for 6 days in the presence of ACPD and a mutant form of NGF that does not bind to p75, no increase in Purkinje cell number was observed. Moreover, the survival-promoting effect of wild-type NGF and ACPD could be inhibited by a neutralizing antiserum to p75 or by a pyrazoloquinazolinone inhibitor of neurotrophin binding to p75. In contrast, the response to NT-3 was potentiated by anti-p75 treatment and by the quinazolinone. These data indicate the mediation of p75 in the trophic response to NGF-ACPD and a negative modulatory role of p75 in the action of NT-3. To probe the role of ACPD in the p75-dependent response to NGF, metabotropic receptor subtype-specific ligands were tested. The pattern of agonist specificity implicated the mGluR1 subtype, a receptor that is expressed at high levels by Purkinje cells and linked to activation of protein kinase C (PKC). Down-regulation or blockade of PKC abolished the response to NGF-ACPD. Consistent with the opposite roles of p75 in effects of the two neurotrophins, blockade of mGluR1 or PKC potentiated the survival response elicited by NT-3. In sum, our data suggest that afferent excitatory transmitters activate specific metabotropic receptors to elicit a p75-mediated action of NGF. NT-3 acts on Purkinje cells by a different mechanism that is not absolutely p75-dependent and that is reduced by neurotrophin access to p75 and metabotropic receptor activity.     

Pharmacological characterization of metabotropic glutamate receptors in cultured cerebellar granule cells

A detailed pharmacological characterization of metabotropic glutamate receptors (mGluR) was performed in primary cultures of cerebellar granule cells at 6 days in vitro (DIV). The rank order of agonists induced polyphosphoinositide (PPI) hydrolysis (after correcting for the ionotropic component in the response) was as follows: in terms of efficiency, Glu>quisqualate (quis)=ibotenate (ibo)>(1_S,3_R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD)>-methyl-amino-l-alanine (BMAA) and in terms of potency, quis>ACPD>Glu>ibo=BMAA. Ionotropic excitatory amino acid (EAA) receptor agonists, such as -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) were relatively inactive (in the presence of Mg²⁺). Quis and ACPD-induced PPI hydrolysis was unaffected by ionotropic Glu receptor antagonists, but was inhibited, in part by L-2-amino-3-phosphonopropionate (AP3). In contrast, Glu-or ibo- induced PPI hydrolysis was reduced, in part, by both AP3 and NMDA receptor antagonists. Characteristic interactions involving different transmitter receptors were noted. PPI hydrolysis evoked by quis and 1_S,3_R-ACPD was not additive. In contrast, PPI hydrolysis stimulated by quis/ACPD and carbamylcholine was additive (indicating different receptors/transduction pathways). In the presence of Mg²⁺, the metabotropic response to quis/AMPA and NMDA was synergistic (this being consistent with AMPA receptor-induced depolarization activating NMDA receptor). On the other hand, in Mg²⁺-free buffer the effects of quis and NMDA, at concentrations causing maximal PPI hydrolysis, were additive (indicating that PPI hydrolysis was effected by two different mechanisms). Thus, in cerebellar granule cells EAAs elicit PPI hydrolysis by acting at two distinct receptor types: (i) metabotropic Glu receptors (mGluR), with pharmacological characteristics suggesting the expression of a unique mGluR receptor that shows certain similarities to those observed for the mGluR1 subtype (Aramori and Nakanishi, 1992) and (ii) NMDA receptors. The physiological agonist, Glu, is able to stimulate both receptor classes.Abbreviations ACPD (1_S,3_R)-1-amino-cyclopentane-1,3-dicarboxylic acid - AMPA -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid - AP₃ L-2-amino-3-phosphono-propionate - AP₅ D-2-amino-5-phosphonopentenoate - BMAA -methyl-amino-L-alanine - DIV days in vitro - DNOX 6,7-dinitroouinoxoline-2,3-dione - EAA excitatory amino acids - Glu glutamate - InsP inositol monophosphate - mGluR metabotropic glutamate receptors - MK-801 (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohept-5,10-imine hydrogen maleate - NMDA N-methyl-D-aspartate - PPI polyphosphoinositide - quis quisqualate     

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.     

Differential modulation by AMPA of signals from red- and green-sensitive cones in carp retinal luminosity-type horizontal cells

Intracellular recordings were made from luminosity-type horizontal cells (LHCs) in the isolated superfused carp retina and the effect of AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid), a glutamate receptor agonist, on these cells was studied. AMPA suppressed the responses of LHCs driven by red-sensitive (R-) cones whereas it potentiated the responses driven by green-sensitive (G-) cones. The AMPA effect could be completely blocked by GYKI 53655, a specific AMPA receptor antagonist, indicating the exclusive involvement of AMPA-preferring receptors. The AMPA effect persisted in the presence of picrotoxin (PTX) or dihydrokainic acid (DHK), suggesting that the feedback from LHCs onto cones and glutamate transporters on cones may not be involved. It is suggested that there may exist different AMPA receptor subtypes with distinct characteristics on LHCs, which mediate signal transfer from R-and G-cones to LHCs, respectively.     

Differential modulation by AMPA of signals from red- and green-sensitive cones in carp retinal luminosity-type horizontal cells

Intracellular recordings were made from luminosity-type horizontal cells (LHCs) in the isolated superfused carp retina and the effect of AMPA (α-amino-3-hydroxy-5-methylisoxa-zole-4-propionic acid), a glutamate receptor agonist, on these cells was studied. AMPA suppressed the responses of LHCs driven by red-sensitive (R-) cones whereas it potentiated the responses driven by green-sensitive (G-) cones. The AMPA effect could be completely blocked by GYKI 53655, a specific AMPA receptor antagonist, indicating the exclusive involvement of AMPA-preferring receptors. The AMPA effect persisted in the presence of picrotoxin (PTX) or di-hydrokainic acid (DHK), suggesting that the feedback from LHCs onto cones and glutamate transporters on cones may not be involved. It is suggested that there may exist different AMPA receptor subtypes with distinct characteristics on LHCs, which mediate signal transfer from R- and G-cones to LHCs, respectively.     

Mitochondrial apoptotic cell death and moderate superoxide generation upon selective activation of non-desensitizing AMPA receptors in hippocampal cultures

In the present work we investigated the effect of selective stimulation of non-desensitizing alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in the intracellular processes leading to hippocampal neuronal death and production of reactive oxygen species (ROS). Activation of AMPA receptors in the presence of cyclothiazide (CYZ), a blocker of AMPA receptor desensitization, resulted in the death of approximately 25% of neurones, which was prevented by 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(f)quinoxaline (NBQX), an AMPA-preferring receptor antagonist. (+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801) protected the neurones from necrotic death induced by AMPA or NMDA receptor activation. Neurodegeneration caused by selective activation of non-desensitizing AMPA receptors, in the presence of AMPA, CYZ and MK-801, significantly decreased the number of Co2+-positive neurones, used as a cytochemical marker of Ca2+-permeable AMPA receptors, but maintained intracellular ATP/ADP. The AMPA-mediated apoptotic cell death involved mitochondrial cytochrome c release and the activation of caspases-1 and -3, which was prevented by NBQX. Interestingly, although selective activation of AMPA receptors was not associated with production of intracellular peroxides, a moderate increase in superoxide production was observed upon exposure to antimycin A (AA). Furthermore, increased activity of Mn- superoxide dismutase (SOD) was observed on selective activation of non-desensitizing AMPA receptors. Taken together, these data make important contributions to the elucidation of the downstream pathways activated in AMPA receptor-mediated excitotoxicity in cultured rat hippocampal neurones.     

Negative allosteric modulators of AMPA-preferring receptors inhibit [(3)H]GABA release in rat striatum

The effect of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), a selective glutamate receptor agonist, on the release of previously incorporated [(3)H]GABA was examined in superfused striatal slices of the rat. The slices were loaded with [(3)H]GABA in the presence of beta-alanine (1 mM) and superfused with Krebs-bicarbonate buffer containing nipecotic acid (0.1 mM) and aminooxyacetic acid (0.1 mM) to inhibit GABA uptake and metabolism. AMPA (0.01 to 3 mM) increased basal [(3)H]GABA outflow and nipecotic acid potentiated this effect. The [(3)H]GABA releasing effect of AMPA was an external Ca(2+)-dependent process in the absence but not in the presence of nipecotic acid. Cyclothiazide (0.03 mM), a positive modulator of AMPA receptors, failed to evoke [(3)H]GABA release by itself, but it dose-dependently potentiated the [(3)H]GABA releasing effect of AMPA. The AMPA (0.3 mM)-induced [(3)H]GABA release was antagonized by NBQX (0.01 mM) in a competitive fashion (pA(2) 5.08). The negative modulator of AMPA receptors, GYKI-53784 (0.01 mM) reversed the AMPA-induced [(3)H]GABA release by a non-competitive manner (pD'(2) 5.44). GYKI-53784 (0. 01-0.1 mM) also decreased striatal [(3)H]GABA outflow on its own right, this effect was stereoselective and was not influenced by concomitant administration of 0.03 mM cyclothiazide. GYKI-52466 (0. 03-0.3 mM), another negative modulator at AMPA receptors, also inhibited basal [(3)H]GABA efflux whereas NBQX (0.1 mM) by itself was ineffective in alteration of [(3)H]GABA outflow.The present data indicate that AMPA evokes GABA release from the vesicular pool in neostriatal GABAergic neurons. They also confirm that multiple interactions may exist between the agonist binding sites and the positive and negative modulatory sites but no such interaction was detected between the positive and negative allosteric modulators. Since GYKI-53784, but not NBQX, inhibited [(3)H]GABA release by itself, AMPA receptors located on striatal GABAergic neurons may be in sensitized state and phasically controlled by endogenous glutamate. It is also postulated that these AMPA receptors are located extrasynaptically on GABAergic striatal neurons.     

AMPA receptor-mediated toxicity in oligodendrocyte progenitors involves free radical generation and activation of JNK,calpain and caspase 3

The molecular mechanisms underlying AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) receptor-mediated excitotoxicity were characterized in rat oligodendrocyte progenitor cultures. Activation of AMPA receptors, in the presence of cyclothiazide to selectively block desensitization, produced a massive Ca(2+) influx and cytotoxicity which were blocked by the antagonists CNQX and GYKI 52466. A role for free radical generation in oligodendrocyte progenitor cell death was deduced from three observations: (i) treatment with AMPA agonists decreased intracellular glutathione; (ii) depletion of intracellular glutathione with buthionine sulfoximine potentiated cell death; and (iii) the antioxidant N -acetylcysteine replenished intracellular glutathione and protected cultures from AMPA receptor-mediated toxicity. Cell death displayed some characteristics of apoptosis, including DNA fragmentation, chromatin condensation and activation of caspase-3 and c-Jun N-terminal kinase (JNK). A substrate of calpain and caspase-3, alpha-spectrin, was cleaved into characteristic products following treatment with AMPA agonists. In contrast, inhibition of either caspase-3 by DEVD-CHO or calpain by PD 150606 protected cells from excitotoxicity. Our results indicate that overactivation of AMPA receptors causes apoptosis in oligodendrocyte progenitors through mechanisms involving Ca(2+) influx, depletion of glutathione, and activation of JNK, calpain, and caspase-3.     

Metabotropic Excitatory Amino Acid Receptor Activation Stimulates Phospholipase D in Hippocampal Slices

Metabotropic excitatory amino acid (EAA) receptors are coupled to effector systems through G proteins. Because various G protein-coupled receptors stimulate the hydrolysis of phosphatidylcholine by phospholipase D (PLD), we examined the possibility that metabotropic EAA receptors exist that are coupled to the activation of PLD. We found that the selective metabotropic glutamate receptor (mGluR) agonists 1S,3R-amino-1,3-cyclopentanedicarboxylic acid (ACPD) and 1S,3S-ACPD, but not the inactive isomer, 1R,3S-ACPD, induce a concentration-dependent increase in PLD activity in hippocampal slices. Selective ionotropic glutamate receptor (iGluR) antagonists did not block 1S,3R-ACPD-induced PLD stimulation. Furthermore, although selective iGluR agonists did not activate this response, the nonselective mGluR-iGluR agonists, ibotenate and quisqualate, caused significant increases in PLD activity (all in the presence of iGluR antagonists). L-2-Amino-3-phosphonopropionic acid, which blocks the mGluR that is coupled to phosphoinositide hydrolysis in various brain regions, activates PLD to the same extent as the active isomers of ACPD. These data suggest that metabotropic EAA receptors exist in hippocampus that are coupled to PLD activation and are pharmacologically distinct from phosphoinositide hydrolysis-coupled mGluRs.