Monitoring of brain spiking activity and autoantibodies to N-terminus domain of GluR1 subunit of AMPA receptors in blood serum of rats with cobalt-induced epilepsy

We have monitored EEG spontaneous spiking activity and analyzed serum from rats with cobalt-induced epilepsy for the presence of autoreactive antibodies to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) glutamate receptor subunits. The presence and the level of autoantibodies were assessed using immunoblot and ELISA with synthetic peptide specific to the N-terminus domain of the GluR1 subunit of the AMPA receptor. Rats with cobalt-induced epilepsy exhibited strong GluR1 immunoreactivity at the end of the first week after surgery compared with vehicle-treated rats. We showed that GluR1 autoantibodies in blood serum of rats with cobalt-induced epilepsy preceded the spiking activity maximum in the EEG. Levels of autoantibodies to GluR1 detected in blood of these rats remained elevated when EEG spiking activity was significantly reduced and seizures disappeared. The EEG monitoring of spiking activity showed a correlation with accumulation of GluR1 autoantibodies in blood serum of rats with cobalt-induced epilepsy.     

SAP97 is associated with the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR1 subunit

Rapid glutamatergic synaptic transmission is mediated by ionotropic glutamate receptors and depends on their precise localization at postsynaptic membranes opposing the presynaptic neurotransmitter release sites. Postsynaptic localization of N-methyl-D-aspartate-type glutamate receptors may be mediated by the synapse-associated proteins (SAPs) SAP90, SAP102, and chapsyn-110. SAPs contain three PDZ domains that can interact with the C termini of proteins such as N-methyl-D-aspartate receptor subunits that carry a serine or threonine at the -2 position and a valine, isoleucine, or leucine at the very C terminus (position 0). We now show that SAP97, a SAP whose function at the synapse has been unclear, is associated with alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors. AMPA receptors are probably tetramers and are formed by two or more of the four AMPA receptor subunits GluR1-4. GluR1 possesses a C-terminal consensus sequence for interactions with PDZ domains of SAPs. SAP97 was present in AMPA receptor complexes immunoprecipitated from detergent extracts of rat brain. After treatment of rat brain membrane fractions with the cross-linker dithiobis(succinimidylpropionate) and solubilization with sodium dodecylsulfate, SAP97 was associated with GluR1 but not GluR2 or GluR3. In vitro experiments with recombinant proteins indicate that SAP97 specifically associates with the C terminus of GluR1 but not other AMPA receptor subunits. Our findings suggest that SAP97 may be involved in localizing AMPA receptors at postsynaptic sites through its interaction with the GluR1 subunit.     

AMPA glutamate receptor subunits are differentially distributed in rat brain

To demonstrate the regional, cellular and subcellular distributions of non-N-methyl-D-aspartate glutamate receptors in rat brain, we generated antipeptide antibodies that recognize the C-terminal domains of individual subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-preferring glutamate receptors (i.e. GluR1, GluR4, and a region highly conserved in GluR2, GluR3 and GluR4c). On immunoblots, antibodies detect distinct proteins with mol. wts ranging from 102,000 to 108,000 in homogenates of rat brain. Immunocytochemistry shows that glutamate receptor subunits are distributed abundantly and differentially within neuronal cell bodies and processes in cerebral cortex, basal ganglia, limbic system, thalamus, cerebellum and brainstem. The precise patterns and cellular localizations of glutamate receptor subunit immunoreactivities are unique for each antibody. In neocortex and hippocampus, pyramidal neurons express GluR1 and GluR2/3/4c immunoreactivities; many non-pyramidal, calcium-binding, protein-enriched neurons in cerebral cortex are selectively immunoreactive for GluR1. In striatum, the cellular localizations of GluR1, GluR2/3/4c and GluR4 immunoreactivities are different; in this region, GluR1 co-localizes with many cholinergic neurons but is only present in a minor proportion of nicotinamide adenine dinucleotide phosphate diaphorase-positive striatal neurons. GluR1 co-localizes with most dopaminergic neurons within the substantia nigra. In several brain regions, astrocytes show GluR4 immunoreactivity. Within the cerebellar cortex, cell bodies and processes of Bergmann glia express intense GluR4 and GluR1 immunoreactivities; perikarya and dendrites of Purkinje cells show GluR2/3/4c immunoreactivity but no evidence of GluR1 or GluR4. Ultrastructurally, GluR subunit immunoreactivities are localized within cell bodies, dendrites and dendritic spines of specific subsets of neurons and, in the case of GluR1 and GluR4, in some populations of astrocytes. This investigation demonstrates that individual AMPA-preferring glutamate receptor subunits are distributed differentially in the brain and suggests that specific neurons and glial cells selectively express glutamate receptors composed of different subunit combinations. Thus, the co-expression of all AMPA receptor subunits within individual cells may not be obligatory for the functions of this glutamate receptor in vivo.     

The differential expression patterns of messenger RNAs encoding non-N-methyl-D-aspartate glutamate receptor subunits (GluR1-4) in the rat brain

The messenger RNA expression of non-N-methyl-D-aspartate glutamate receptor subunits (GluR1-4), considered alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid type, was investigated in rat brain by in situ hybridization histochemistry using oligonucleotide probes specific to each subunit sequence. GluR1-4 subunit messenger RNAs were expressed widely and abundantly throughout the CNS. However, the combination of expression pattern varied notably according to location. GluR2 messenger RNA was expressed most strongly and widely, with most areas except the Bergmann glia containing this messenger RNA. GluR4 messenger RNA was also present widely, although the expression level was low. However, we observed many areas which lacked or expressed very little GluR1 messenger RNA, such as some nuclei in the general motor system and auditory system. In addition, some nuclei in the hypothalamus and general somatosensory system lacked or expressed very little GluR3 messenger RNA. These results suggest that in the rat CNS non-N-methyl-D-aspartate receptors varied their composition according to the area where they were expressed, and that the combination pattern might be related to the functional role of neurons.     

Seasonal changes in growth and energy status in the Third World

Two-color immunofluorescence histochemistry and immunohistochemistry in combination with retrograde tract-tracing techniques were used to examine the relationship of alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA)-selective glutamate receptor subunits (GluR1, GluR2/3/4c and GluR4) to identified populations of striatal projection neurons and interneurons. The majority of striatonigral and striatopallidal neurons were double-labeled for GluR2/3/4c. These findings were confirmed using calbindin to label matrix projection neurons. In contrast, immunostaining of the GluR1 subunit was not observed to co-localize with any striatal projection neurons. Striatal interneurons immunostained for parvalbumin were also labeled by antibodies directed against the GluR1 subunit. Approximately 50% of parvalbumin neurons also contained GluR2/3/4c. Somatostatin immunoreactivity did not co-localize with either the GluR1 or GluR2/3/4c subunits. GluR4-immunoreactive neurons were not observed in striatum. This study demonstrates that AMPA-selective glutamate receptors are differentially localized on subpopulations of striatal neurons and interneurons. These findings suggest that discrete striatal neuron populations may express different AMPA receptor subunit combinations which may account for their functional specificity.     

Cellular localizations of AMPA glutamate receptors within the basal forebrain magnocellular complex of rat and monkey

The cellular distributions of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors within the rodent and nonhuman primate basal forebrain magnocellular complex (BFMC) were demonstrated immunocytochemically using anti-peptide antibodies that recognize glutamate receptor (GluR) subunit proteins (i.e., GluR1, GluR4, and a conserved region of GluR2, GluR3, and GluR4c). In both species, many large GluR1-positive neuronal perikarya and aspiny dendrites are present within the medial septal nucleus, the nucleus of the diagonal band of Broca, and the nucleus basalis of Meynert. In this population of neurons in rat and monkey, GluR2/3/4c and GluR4 immunoreactivities are less abundant than GluR1 immunoreactivity. In rat, GluR1 does not colocalize with ChAT, but, within many neurons, GluR1 does colocalize with GABA, glutamic acid decarboxylase (GAD), and parvalbumin immunoreactivities. GluR1- and GABA/GAD-positive neurons intermingle extensively with ChAT-positive neurons. In monkey, however, most GluR1-immunoreactive neurons express ChAT and calbindin-D28 immunoreactivities. The results reveal that noncholinergic GABAergic neurons, within the BFMC of rat, express AMPA receptors, whereas cholinergic neurons in the BFMC of monkey express AMPA receptors. Thus, the cellular localizations of the AMPA subtype of GluR are different within the BFMC of rat and monkey, suggesting that excitatory synaptic regulation of distinct subsets of BFMC neurons may differ among species. We conclude that, in the rodent, BFMC GABAergic neurons receive glutamatergic inputs, whereas cholinergic neurons either do not receive glutamatergic synapses or utilize GluR subtypes other than AMPA receptors. In contrast, in primate, basal forebrain cholinergic neurons are innervated directly by glutamatergic afferents and utilize AMPA receptors.     

Pharmacological properties of homomeric and heteromeric GluR1o and GluR3o receptors

Homomeric and heteromeric alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits GluR1o and GluR3o were expressed in Spodoptera frugiperda (Sf9) insect cells. Membranes containing the recombinant receptors showed a doublet of bands of the expected size (99-109 kDa) after western immunoblotting which was shifted to a single band upon deglycosylation. In (R,S)-[3H]AMPA binding experiments, high expression was seen (Bmax = 0.8-3.8 pmol/mg protein) along with high affinity binding to a single site (Kd, nM+/-S.D.): GluR1o, 32.5+/-2.7; GluR3o, 23.7+/-2.4; GluR1o + GluR3o, 18.1+/-2.9. The pharmacological profiles of these receptors resembled that of native rat brain AMPA receptors: AMPA analogues > L-glutamate > quinoxaline-2,3-diones > kainate. In the Xenopus oocyte expression system we had previously shown that the agonist (R,S)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionate (ACPA) exhibited an 11-fold selectivity for GluR3o vs. GluR1o. In this study, it was found that ACPA has 3-fold higher affinity at homomeric GluR3o and heteromeric receptors than at homomeric GluR1o, suggesting that its efficacy and/or desensitisation properties are different at GluR1o vs. GluR3o.     

In-vitro characterization of YM872, a selective, potent and highly water-soluble alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor antagonist

The in-vitro pharmacological properties of (2,3-dioxo-7-(1H-imidazol-1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxal inyl)-acetic acid monohydrate, YM872, a novel and highly water-soluble alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-receptor antagonist were investigated. YM872 is highly water soluble (83 mg mL(-1) in Britton-Robinson buffer) compared with 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX), 6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione hydrochloride (YM90K) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). YM872 potently inhibits [3H]AMPA binding with a Ki (apparent equilibrium dissociation constant) value of 0.096 +/- 0.0024 microM. However, YM872 had very low affinity for other ionotropic glutamate receptors, as measured by competition with [3H]kainate (high-affinity kainate binding site, concentration resulting in half the maximum inhibition (IC50) = 4.6 +/- 0.14 microM), [3H]glutamate (N-methyl-D-aspartate (NMDA) receptor glutamate binding site, IC50 > 100 microM) and [3H]glycine (NMDA receptor glycine-binding site, IC50 > 100 microM). YM872 competitively antagonized kainate-induced currents in Xenopus laevis oocytes which express rat AMPA receptors, with a pA2 value of 6.97 +/- 0.01. In rat hippocampal primary cultures, YM872 blocked a 20-microM AMPA-induced increase of intracellular Ca2+ concentration with an IC50 value of 0.82 +/- 0.031 microM, and blocked 300-microM kainate-induced neurotoxicity with an IC50 value of 1.02 microM. These results show that YM872 is a potent and highly water-soluble AMPA antagonist with great potential for treatment of neurodegenerative disorders such as stroke.     

Determination of degradation rates of transfer and ribosomal ribonucleic acids in cultured rat hepatocytes by measuring N6-threoninocarbonyladenosine, dihydrouridine, and pseudouridine in medium using high-performance liquid chromatography

The cause of the selective degeneration of motor neurons in amyotrophic lateral sclerosis (ALS) remains unexplained. One potential pathogenetic mechanism is chronic toxicity due to disturbances of the glutamatergic neurotransmitter system, mediated via alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-sensitive glutamate receptors. Functional AMPA receptors consist of various combinations of four subunits (designated GluR1-4). The GluR2 subunit is functionally dominant and renders AMPA receptors impermeable to calcium. Most native AMPA receptors in the mammalian central nervous system (CNS) contain the GluR2 subunit and are calcium impermeable. We have investigated the composition of AMPA receptors expressed on normal human spinal motor neurons by in situ hybridization to determine their likely subunit stoichiometry. Highly significant levels of mRNA were detected for the GluR1, GluR3, and GluR4 subunits. However, GluR2 subunit mRNA was not detectable in this cell group. The absence of detectable GluR2 mRNA in normal human spinal motor neurons predicts that they express calcium-permeable AMPA receptors unlike most neuronal groups in the human CNS. Expression of atypical calcium-permeable AMPA receptors by human motor neurons provides a possible mechanism whereby disturbances of glutamate neurotransmission in ALS may selectively injure this cell group.     

M3 muscarinic receptor-mediated enhancement of NMDA-evoked adenosine release in rat cortical slices in vitro

Acetylcholine plays an important role in cortical arousal. Adenosine is released during increased metabolism and has been suggested to be a sleep-promoting factor. To understand the interaction of acetylcholine and adenosine in regulating cortical excitability, we examined the effect of carbachol on NMDA-evoked adenosine release and identified the muscarinic receptor subtype that mediated this effect in adult rat cortical slices in vitro. Carbachol (to 300 microM) alone did not affect the basal release of adenosine. However, carbachol (100 microM) induced a 253% increase in NMDA (20 microM)-evoked adenosine release in the presence of Mg2+. In the absence of Mg2+, carbachol's potentiating effect was less (60% increase). The nonselective muscarinic antagonist atropine (1.5 microM) blocked the facilitatory effect of carbachol on NMDA-evoked adenosine release, and this was mimicked by the M3-selective antagonist 4-diphenylacetoxy-N-methylpiperidine (1 microM). Neither an M1-selective dose of pirenzepine (50 nM) nor the M2-selective antagonist methoctramine (1 microM) affected carbachol's action on NMDA-evoked adenosine release. Carbachol had no effect on adenosine release evoked by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA). These results suggest that acetylcholine does not affect basal adenosine release but enhances NMDA receptor-mediated evoked adenosine release by acting at M3 receptors in the cortex. This interaction may have a role in regulating cortical neuronal excitability on a long-term basis.     

The regulation of AMPA receptor-binding sites

A wide variety of mechanisms have been identified that can regulate the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-receptor complex. Modulation has been shown to occur at the nucleic acid level via RNA editing and alternative splicing. At the posttranslational level, processes such as phosphorylation, glycosylation, chemical modification of reactive groups on the receptor proteins, interaction with a putative receptor-associated modulatory protein, and changes in the lipid environment have been reported to regulate receptor binding and function. In this review, we discuss general aspects of the cell biology, pharmacology, and function of AMPA receptors. In particular, we focus on some factors shown to modulate agonist binding and discuss possible molecular mechanisms underlying the regulation observed.     

AMPA receptors in the rat and primate hippocampus: a possible absence of GluR2/3 subunits in most interneurons

Amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors are assembled from the four subunits GluR1, 2, 3, 4 (or GluRA, B, C, D). AMPA channels that do not contain the GluR2 subunit are permeable to calcium. Recent studies indicate that excitotoxic as well as epileptic and ischemic cell damage may be mediated not only by N-methyl-Daspartate receptors, but also by AMPA receptors. The majority of interneurons in the hippocampus are resistant, but subsets of interneurons are consistently damaged in different disease states. Single immunolabeling using antibodies against AMPA receptor subunits, together with double immunolabeling for calcium-binding proteins (parvalbumin, calbindin and calretinin) and the neuropeptide somatostatin, were performed to study GluR1-4 immunoreactivity in interneuronal populations and principal cells. The ultrastructure of GluR1-4 labeled neurons was also examined using electron microscopy. With the exception of calbindin-positive interneurons, GluR2/3 was absent from hippocampal interneurons in both rat and monkey. In the rat, interneurons were more strongly immunoreactive against GluR1 than principal cells. In the monkey, immunoreactivity for GluR4 in interneurons was stronger than for GluR1. All GluR subunits were confined to spines, dendritic membrane and cytoplasm surrounding the nucleus but absent from axons and presynaptic terminals. Our findings suggest that hippocampal principal cells and interneurons express different complements of AMPA receptor subunits. Furthermore, the absence of GluR2 and/or GluR3 in both vulnerable and resistant interneurons subtypes indicates that knowledge of receptor subunit composition is not sufficient to predict neuronal vulnerability.     

Selective reduction of GluR2 protein in adult hippocampal CA3 neurons following status epilepticus but prior to cell loss

Kainic acid (KA) induces status epilepticus and delayed neurodegeneration of CA3 hippocampal neurons. Downregulation of glutamate receptor 2 (GluR2) subunit mRNA [the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) subunit that limits Ca2+ permeability] is thought to a play role in this neurodegeneration, possibly by increased formation of Ca2+ permeable AMPA receptors. The present study examined early hippocampal decreases in GluR2 mRNA and protein following kainate-induced status epilepticus and correlated expression changes with the appearance of dead or dying cells by several histological procedures. At 12 h, in situ hybridization followed by emulsion dipping showed nonuniform decreases in GluR2 mRNA hybridization grains overlying morphologically healthy-appearing CA3 neurons. GluR1 and N-methyl-D-aspartate receptor mRNAs were unchanged. At 12-16 h, when little argyrophilia or cells with some features of apoptosis were detected by silver impregnation or electron microscopy, single immunohistochemistry with GluR2 and GluR2/3 subunit-specific antibodies demonstrated a pattern of decreased GluR2 receptor protein within CA3 neurons that appeared to predict a pattern of damage, similar to the mRNA observations. Double immunolabeling showed that GluR2 immunofluorescence was depleted and that GluR1 immunofluorescence was sustained in clusters of the same CA3 neurons. Quantitation of Western blots showed increased GluR1:GluR2 ratios in CA3 but not in CA1 or dentate gyrus subfields. Findings indicate that the GluR1:GluR2 protein ratio is increased in a population of CA3 neurons prior to significant cell loss. Data are consistent with the "GluR2 hypothesis" that reduced expression of GluR2 subunits will increase formation of AMPA receptors permeable to Ca2+ and predict vulnerability to a particular subset of pyramidal neurons following status epilepticus.     

Non-plaque dystrophic dendrites in Alzheimer hippocampus: a new pathological structure revealed by glutamate receptor immunocytochemistry

Alzheimer's disease is a progressive dementia characterized by a pronounced neurodegeneration in the entorhinal cortex, hippocampal CA1, and subiculum. Excitatory amino acid receptor-mediated excitotoxicity is postulated to play a role in the neurodegeneration in Alzheimer's disease. The present study investigated immunocytochemical localization of excitatory amino acid receptor subunits in the hippocampus of twelve Alzheimer's disease and eleven controls, matched for age, sex and post mortem interval. Immunocytochemistry with antibodies specific for glutamate receptors GluR1, GluR2(4) (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid), GluR5/6/7 (kainate) and NR1 (N-methyl-D-aspartate) receptor subunits demonstrated that virtually all projection neurons in all subfields contained subunits from each receptor class. However, regional differences in immunoreactivity were apparent in Alzheimer's disease vs normal human brain. In the vulnerable regions (i.e. CA1) immunolabelling of GluR1, GluR2(4), GluR5/6/7 and NR1 was reduced, presumably due to cell loss. In contrast, GluR2(4) immunolabelling appeared to be increased in the inner portion of the molecular layer of the dentate gyrus. In addition to cellular labelling, GluR1, GluR2(4) and NR1 immunolabelling revealed a novel pathological structure in 12 of 12 Alzheimer's disease, but none of the control brains. The lesions were juxtacellular clusters of granular immunoreactivity in the neuropil of the pyramidal cell layer. Ultrastructural analysis revealed these to be cellular processes containing dense vesicles and flocculent material with immunolabelling localized to plasma and vesicular membranes. They were not specifically associated with amyloid fibrils and did not contain paired helical filaments and they were also distinct from granulovacuolar degeneration. Several structures contained Hirano body filaments indicating that the dystrophic processes were most likely dendritic in origin. Additional studies are needed to determine the pathogenesis of these lesions, which could provide an additional index of dendritic deterioration in Alzheimer's disease.     

Relative concentrations and seizure-induced changes in mRNAs encoding three AMPA receptor subunits in hippocampus and cortex

In situ hybridization was used to determine 1) the relative concentrations of mRNAs encoding different subunits of the alpha-amino 3-hydroxy-5-methyl-4- isoxazolepropionate receptor family in select regions of rat forebrain and 2) whether limbic seizures alter the balances of the subunit mRNAs. GluR1 and GluR2 mRNA levels were about equal and were much greater than GluR3 mRNA levels in the principal neurons of each hippocampal subdivision. Probable interneurons in hippocampal molecular layers had much higher levels of GluR1 mRNA than of either GluR2 or GluR3 mRNA. Pyramidal cell layers in neo- and paleocortex had a balance of mRNAs that was significantly different from the balance in hippocampus: GluR1 mRNA and GluR3 mRNA levels were about equal and were substantially lower than those of GluR2 mRNA. Lesion-induced limbic seizures caused transient changes in mRNA levels that were differentiated with regard to subunit and brain region. All three mRNAs were decreased in the pyramidal layers of cortex, and changes in hippocampal pyramidal cells were smaller. Seizure-induced changes in granule cells of the dentate gyrus differed from all other regions examined: GluR1 mRNA was reduced to a greater degree than GluR2 mRNA, whereas GluR3 mRNA content was markedly increased. These data strongly suggest that the subunit composition of alpha-amino 3-hydroxy-5-methyl-4-isoxazolepropionate receptors differs significantly between areas of the cortical telencephalon. Furthermore, the data indicate that aberrant patterns of physiological activity differentially influence the expression of subunit mRNAs in a region-specific and/or cell-type-specific manner.     

Disulfide bonding and cysteine accessibility in the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor subunit GluRD. Implications for redox modulation of glutamate receptors

Redox agents elicit a wide variety of effects on the ligand affinity and channel properties of ionotropic glutamate receptors and have been proposed as potential therapeutic agents for neuropathological processes. One such effect is the dithiothreitol (DTT)-induced increase in agonist affinity of certain ionotropic glutamate receptors (GluRs), presumably due to reduction of a disulfide bridge formed between cysteine residues conserved among all GluRs. Using biochemical techniques, this disulfide is shown to exist in the ligand-binding domain of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit GluRD, although GluRD homomeric receptors are not modulated by DTT. The disulfide is inaccessible to DTT, explaining the insensitivity of the intact receptor. Single mutants C260S and C315S show a 2-3-fold higher ligand affinity than wild-type, as observed for several intact GluRs, indicating that the affinity switch is completely contained within the ligand-binding domain. Also, mutants lacking the native disulfide show non-native oligomerization and dramatically reduced specific activity. These facts suggest that the disulfide bridge is required for the stability of the ligand-binding domain, explaining its conservation. A third cysteine residue in the ligand-binding domain exists as a free thiol, partially sequestered in a hydrophobic environment. These results provide a framework for interpreting a variety of GluR redox modulatory phenomena.     

Hypoxic cell death in human NT2-N neurons: involvement of NMDA and non-NMDA glutamate receptors

Human NTera2 teratocarcinoma cells were differentiated into postmitotic NT2-N neurons and exposed to hypoxia for 6 h. The cultures were evaluated microscopically, and percent lactate dehydrogenase (LDH) release after 24 and 48 h was used as an assay for cell death. After 48 h LDH release was 24.3 +/- 5.6% versus 13.8 +/- 3.7% in controls (p < 0.001). Cell death was greatly diminished by MK-801 pretreatment (15.4 +/- 5.1%, p < 0.001). If glutamine was omitted from the medium, glutamate levels after 6 h of hypoxia were reduced from 101 +/- 63 to 2.3 +/- 0.3 microM, and cell death at 48 h was also markedly reduced (15.4 +/- 4.5%, p < 0.001). The alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (18.7 +/- 5.1%, p < 0.001) and mild hypothermia (33.5-34 degrees C) during hypoxia (19.5 +/- 2.7%, p < 0.05) were moderately protective. Basic fibroblast growth factor (24.1 +/- 3.2%), the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (22.8 +/- 8.1%), the antioxidant N-tert-butyl-o-phenyinitrone (18.9 +/- 5.9%), and the 21-aminosteroid U74389G (24.0 +/- 3.4%) did not protect the cells. N-Acetyl-L-cysteine even tended to increase cell death (30.1 +/- 2.5%, p = 0.06). Treatment with MK-801 at the end of hypoxia did not reduce cell death (23.3 +/- 2.3%). In separate experiments, a 15-min exposure to 1 mM glutamate without hypoxia did not result in significant cell death (14.7 +/- 2.4 vs. 12.2 +/- 2.1%, p = 0.07). We conclude that, although somewhat resistant to glutamate toxicity when normoxic, NT2-N neurons die via an ionotropic glutamate receptor-mediated mechanism when exposed to hypoxia in the presence of glutamate. As far as we know, this is the first reported analysis of the mechanism of hypoxic cell death in cultured human neuronlike cells.