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PubMed1998

[U-13C]glutamate metabolism in astrocytes during hypoglycemia and hypoxia.

The ability of cultured astrocytes to metabolize [U-13C]glutamate in the absence of glucose was investigated by utilizing 13C nuclear magnetic resonance spectroscopy to identify 13C-labeled metabolites. Control cultures (3 mM glucose), hypoglycemic cultures (glucose-deprived), severe hypoglycemic cultures (glucose-deprived, 0.5 mM iodoacetate as an inhibitor of glycolysis), hypoglycemic/hypoxic cultures, and cultures deprived of all additional substrates were incubated for 2 hr in medium containing 0.5 mM glutamate (50% [U-13C]glutamate). Glucose deprivation alone had little effect on removal of glutamate from the culture medium, but the presence of iodoacetate or incubating cultures in a low-oxygen atmosphere decreased glutamate clearance. Only the withdrawal of all substrates other than glutamate decreased glutamine synthesis. Metabolism of glutamate through the tricarboxylic acid (TCA) cycle was evident by the appearance of [1,2,3-13C]glutamate and [U-13C]aspartate in cell extracts and [U-13C]lactate in cell media. Lactate derived from TCA cycle intermediates was significantly reduced after glucose deprivation and even more so after severe hypoglycemia. Release of glutamate from astrocytes was observed under all incubation conditions. [U-13C]Aspartate was not detected in control media but was released from glucose-deprived cells when oxygen was available. Increased release was observed in the presence of iodoacetate. After withdrawal of all substrates other than glutamate, [U-13C]aspartate was the only metabolite observed intracellularly, whereas aspartate, glutamine, and 5-oxoproline were detected in the incubation medium. The present results indicate that glutamate-to-aspartate conversion is preferentially utilized by astrocytes when oxygen is available but glycolysis is impaired.

Animals↗
PubMed1999

Noradrenaline inhibits glutamate release in the rat bed nucleus of the stria terminalis: in vivo microdialysis studies.

The microdialysis technique was used to simultaneously study the in vivo extracellular levels of noradrenaline, glutamate, and gamma aminobutyric acid (GABA) in the bed nucleus of the stria terminalis in order to assess the regulation that noradrenaline may exert upon the release of amino acid neurotransmitters. Perfusion through the probe with UK14304, a selective alpha2-adrenergic agonist, produced a significant decrease of noradrenaline and glutamate extracellular levels. Perfusion through the probe with RX821002, a selective alpha2-adrenergic antagonist, produced a significant increase of noradrenaline and glutamate basal extracellular levels. Perfusion with prazosine, a selective alpha1-adrenergic antagonist, produced a significant decrease of noradrenaline basal extracellular levels without affecting glutamate levels. Under the same conditions, GABA basal extracellular levels were not changed in the presence of any of the alpha-adrenergic ligands studied. The perfusion of high potassium through the probe induced a significant Ca++dependent release of the three neurotransmitters; however, extracellular noradrenaline returned to normal levels even though potassium was still present. In addition, it was observed that alpha-adrenergic receptor ligands exerted differential effects upon K+-induced release of noradrenaline and glutamate. Perfusion with the nonselective alpha-adrenergic antagonist, phenoxybenzamine, presented a biphasic effect upon K+-induced release of noradrenaline; a significant decrease during the first 5 min of stimulation followed by a significant increase in the next 5 min of stimulation. Perfusion with RX821002 produced a significant increase in K+-induced release of noradrenaline that returned to normal basal values before the end of the stimulation period. In contrast, local perfusion with prazosine caused a significant decrease of K+-induced noradrenaline release. In the case of glutamate, perfusion through the probe with phenoxybenzamine produced a significant increase in K+-induced release of glutamate. In addition, RX821002 and prazosine produced a significant increase in K+-induced release of glutamate. Perfusion through the probe with UK14304 produced a significant decrease of both noradrenaline and glutamate K+-induced release. The present results show that noradrenaline in the bed nucleus of stria terminalis exerts a significant inhibition over its own release through alpha2-adrenergic receptors and over glutamate release mainly through alpha2-adrenergic receptors. Thus, the results suggest that noradrenaline in the bed nucleus of the stria terminalis maintains an inhibitory tone over the information flow mediated by glutamate.

Adrenergic Agents↗
PubMed1998

4-hydroxynonenal, a lipid peroxidation product, impairs glutamate transport in cortical astrocytes.

Astrocytes possess plasma membrane glutamate transporters that rapidly remove glutamate from the extracellular milieu and thereby prevent excitotoxic injury to neurons. Cellular oxidative stress is increased in neural tissues in a variety of acute and chronic neurodegenerative conditions. Recent findings suggest that oxidative stress increases neuronal vulnerability to excitotoxicity and that membrane lipid peroxidation plays a key role in this process. We now report that 4-hydroxynonenal (HNE), an aldehydic product of membrane lipid peroxidation, impairs glutamate transport in cultured cortical astrocytes. Impairment of glutamate transport occurred within 1-3 h of exposure to HNE; FeSO4, an inducer of membrane lipid peroxidation, also impaired glutamate transport. Vitamin E prevented impairment of glutamate transport induced by FeSO4, but not that induced by HNE, consistent with HNE acting as an effector of lipid peroxidation-induced impairment of glutamate transport. Glutathione, which binds and thereby detoxifies HNE, prevented HNE from impairing glutamate transport. Western blot, immunoprecipitation, and immunocytochemical analyses using an antibody against HNE-protein conjugates provided evidence that HNE covalently binds to many different astrocytic proteins including the glutamate transporter GLT-1. Data further suggest that HNE promotes intermolecular cross-linking of GLT-1 monomers to form dimers. HNE also induced mitochondrial dysfunction and accumulation of peroxides in astrocytes. Impairment of glutamate transport and mitochondrial function occurred with sublethal concentrations of HNE, concentrations known to be generated in cells exposed to various oxidative insults. Collectively, our data suggest that HNE may be an important mediator of oxidative stress-induced impairment of astrocytic glutamate transport and may thereby play a role in promoting neuronal excitotoxicity.

Aldehydes↗
PubMed1998

Astrocytes protect neurons from neurotoxic injury by serum glutamate.

Serum is used widely for culturing neurons and glial cells, and is thought to provide essential, albeit undefined, factors such as hormones, growth factors, and trace elements that promote the growth of cells in vitro. Moreover, serum can have profound effects on cell proliferation, differentiation, and cell morphology, and may even influence cell fate decisions. Despite the overall growth-promoting influence of serum on cell culture, frequent media changes have been shown to be detrimental to neuronal cultures, significantly reducing the yield of viable neurons. The reason for this loss of neurons by frequent media changes has been puzzling. We demonstrate that bovine and horse sera, the most popular serum complements for CNS cell culture, are a significant source for glutamate, supplying glutamate at concentrations sufficient to kill primary cultured hippocampal neurons. By using the bioluminescence detection method, we determined the glutamate concentration [Glu] in several batches of fetal bovine (calf) sera (FBS) to be close to 1 mM, and that of horse sera to be approximately 0.3 mM. Thus 10% serum supplement to culture media results in [Glu] of 30-100 microM due to serum alone. We subsequently produced glutamate depleted media (GDM) by using primary cultures of hippocampal astrocytes to absorb glutamate from media containing 10% FBS. Within 3 h, astrocytes reduced the [Glu] in the medium from approximately 90 microM to less than 1 microM. Sister cultures of hippocampal neuron that underwent frequent media changes with GDM or GDM + partial untreated media demonstrated that GDM significantly increase neuronal survival (10-fold at 21 DIV). Subsequent exposure to glutamate provided by either untreated serum or by equivalent doses of exogenous glutamate added to GDM led to dose-dependent neuronal cell death. The relative sensitivity of hippocampal neurons to glutamate increased with increasing culture age from initial ED50 values of > 100 microM (< 6 DIV) to approximately 6 microM in cultures maintained for 3 weeks or longer. The relative sensitivity to exogenous glutamate was at least 2-fold higher in neurons cultured in GDM than in sister cultures maintained in media containing untreated serum. The death of neurons exposed to untreated media was blocked by the NMDA receptor antagonist MK-801. These experiments suggest that the vulnerability of neurons to media changes can be solely explained by excitotoxicity resulting from serum-borne glutamate. Moreover, we propose that use of GDM may be advantageous for culturing hippocampal neurons and may eliminate the possible selection for glutamate resistant neurons. The use of GDM could be particularly important for studies of excitotoxicity; our study predicts that the ED50 for neuronal culture with regular serum will be artificially high and may not adequately reflect the in vivo state.

Animals↗
PubMed2000

Nicotine stimulation of extracellular glutamate levels in the nucleus accumbens: neuropharmacological characterization.

In the present study, we have characterized the neuropharmacological regulation of nicotine-induced increases in extracellular nucleus accumbens glutamate levels. Sprague-Dawley rats were stereotaxically implanted with 2 mm microdialysis probes in the nucleus accumbens and on the following day in vivo microdialysis experiments were performed in awake, freely moving animals. An acute dose of nicotine (0.3-0.6 mg/kg, s.c.) produced an increase in nucleus accumbens glutamate levels with a maximal increase of approximately 50% following the higher dose. No changes in nucleus accumbens aspartate levels were found. The increase in glutamate levels following nicotine (0.3 mg/kg, s.c.) was blocked by mecamylamine (1 mg/kg, i.p. ) but not by haloperidol (0.2 mg/kg, i.p.) pretreatment. Local perfusion of artificial cerebrospinal fluid (CSF) without calcium did not alter nicotine (0.3 mg/kg, s.c.) stimulation of glutamate levels. Local perfusion with a selective blocker for the GLT-1 glutamate transporter, dihydrokainic acid (DHKA) (10(-4) M), had no effect, while local perfusion with a nonselective glutamate transporter blocker, L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC) (10(-4) M), blocked nicotine (0.3 mg/kg, s.c.) stimulation of glutamate levels. In animals previously dopamine denervated by local injections of 6-hydroxydopamine (6-OHDA) into the nucleus accumbens, nicotine (0.3 mg/kg, s.c.) stimulation of glutamate levels was enhanced vs. sham-lesioned animals. These findings demonstrate a novel form of nucleus accumbens glutamate release that is dopamine- and calcium-independent. The ability of PDC to block the effects of nicotine suggest that a glutamate transporter may be involved in mediating the stimulation of glutamate release.

3,4-Dihydroxyphenylacetic Acid↗
PubMed2003

Molecular mechanisms of glutamate neurotoxicity in mixed cultures of NT2-derived neurons and astrocytes: protective effects of coenzyme Q10.

Although glutamate excitotoxicity has long been implicated in neuronal cell death associated with a variety of neurological disorders, the molecular mechanisms underlying this process are not yet fully understood. In part, this is due to the lack of relevant experimental cell systems recapitulating the in vivo neuronal environment, mainly neuronal-glial interactions. To explore these mechanisms, we have analyzed the cytotoxic effects of glutamate on mixed cultures of NT2/N neurons and NT2/A astrocytes derived from human NT2/D1 cells. In these cultures, the neurons were resistant to glutamate alone (up to 2 mM for 24-48 hr), but they responded to a simultaneous exposure to 0.5 mM glutamate and 6 hr of hypoxia. Neuronal cell death occurred during subsequent periods of reoxygenation (>30% within 24 hr). This was associated with a marked decrease of intracellular ATP, a significant increase in reactive oxygen species (ROS) and downregulation of glutamate uptake by astrocytes. Thus, under energy failure and high levels of ROS production, only the neurons from these mixed cultures succumbed to glutamate neurotoxicity; the astrocytic cells remained unaffected by the treatment. Taken together, our data suggested that glutamate excitotoxicity might be due to the energy failure and oxidative stress affecting the properties of the NMDA glutamate receptors and causing impairment of glutamate transporters. Cells pretreated for 72 hr with 10 microg/ml of coenzyme Q(10) (functions both as a ROS scavenger and co-factor of mitochondrial electron transport), were protected, suggesting a useful role for coenzyme Q(10) in treatments of neurological diseases associated with glutamate excitotoxicity. A model of the complex interactions between neurons and astrocytes in regulating glutamate metabolism is presented.

Astrocytes↗
PubMed2004

C(a2+)-dependent glutamate release involves two classes of endoplasmic reticulum Ca(2+) stores in astrocytes.

Astrocytes can modulate synaptic transmission by releasing glutamate in a Ca(2+)-dependent manner. Although the internal Ca(2+) stores have been implicated as the predominant source of Ca(2+) necessary for this glutamate release, the contribution of different classes of these stores is still not well defined. To address this issue, we cultured purified solitary cortical astrocytes and monitored changes in their internal Ca(2+) levels and glutamate release into the extracellular space. Ca(2+) levels were monitored by using the Ca(2+) indicator fluo-3 and quantitative fluorescence microscopy. Glutamate release was monitored by an L-glutamate dehydrogenase-linked detection system. Astrocytes were mechanically stimulated with a glass pipette, which reliably caused an increase in internal Ca(2+) levels and glutamate release into the extracellular space. Although we find that the presence of extracellular Cd(2+), a Ca(2+) channel blocker, significantly reduces mechanically induced glutamate release from astrocytes, we confirm that internal Ca(2+) stores are the predominant source of Ca(2+) necessary for this glutamate release. To test the involvement of different classes of internal Ca(2+) stores, we used a pharmacological approach. We found that diphenylboric acid 2-aminoethyl ester, a cell-permeable inositol 1,4,5-trisphosphate (IP(3)) receptor antagonist, greatly reduced mechanically induced glutamate release. Additionally, the preincubation of astrocytes with caffeine or ryanodine also reduced glutamate release. Taken together, our data are consistent with dual IP(3)- and caffeine/ryanodine-sensitive Ca(2+) stores functioning in the control of glutamate release from astrocytes.

Aniline Compounds↗
PubMed1994

Poly(ADP-ribose) polymerase: early involvement in glutamate-induced neurotoxicity in cultured cerebellar granule cells.

Glutamate neurotoxicity is correlated with an increase of cytosolic free Ca2+. In some cell systems, activation of Ca2+ dependent endonucleases or formation of free radicals can damage DNA and activate the chromatin bound enzyme poly(ADP-ribose) polymerase (pADPRP). We have investigated whether pADPRP may be involved in glutamate neurotoxicity in vitro. Cerebellar granule cells at 12 days in culture when treated with a toxic dose of glutamate (100 microM) showed a rapid and transient increase of polyADP-ribose immunoreactivity. Cellular immunostaining was heterogeneous and returned to control levels after washout of glutamate. In the same cell preparations glutamate elicited a marked increase in enzyme protein immunoreactivity which persisted at later times. Non-toxic doses of glutamate did not affect immunostaining. In another set of experiments, pADPRP mRNA was increased 30 min after glutamate. In order to investigate the role of pADPRP in glutamate-mediated neurotoxicity, structurally different inhibitors of pADPRP (3-aminobenzamide, benzamide,3-aminophthalhydrazide) and their inactive analogues (benzoic acid and phthalimide) were tested in this model. Addition of the inhibitors to cultures 60 min before and during the 30 min of glutamate treatment prevented neuronal death by 60-100%, assessed 24 hr later. Glutamate-induced Ca2+ influx was not affected. Inactive analogues failed to afford neuroprotection. These data indicate that not only is pADPRP activated by the early, possibly Ca(2+)-mediated mechanisms initiated by glutamate, but that it might also actively contribute to the subsequent neuronal death.

Animals↗
PubMed1995

Neurotoxicity of acute glutamate transport blockade depends on coactivation of both NMDA and AMPA/Kainate receptors in organotypic hippocampal cultures.

Excessive activation of glutamate receptors is neurotoxic, contributing to brain injury caused by cerebral ischemia. The pharmacology of glutamate neurotoxicity is difficult to study in animals because it is efficiently cleared from the extracellular space by a family of glutamate transporters. We have investigated the receptor specificity of endogenous glutamate's toxic effects in organotypic cultures of the hippocampus by acute blockade of these transporters. The organotypic cultures used in these transporters. The organotypic cultures used in these experiments preserve the intrinsic connections and regional differentiation of the hippocampus in long term culture and may more closely reproduce the pharmacology of the mature brain region. Membrane injury was measured with digital fluorescence imaging of the vital dye, propidium iodide, 24 h after a 30-min exposure to glutamate receptor agonists or to antagonists of glutamate transport. Confirming our previous results, bath-applied, exogenous glutamate caused dose-dependent neuronal injury. Glutamate was less potent than the selective agonists NMDA, AMPA, and quisqualate. Blockade of glutamate transport with the selective antagonists threo-hydroxy-aspartate and pyrrolidine-dicarboxylic acid also caused dose-dependent neuronal injury. Endogenous or exogenous glutamate toxicity was caused by a coactivation of both NMDA and AMPA/kainate receptors; blockade of either was sufficient to substantially prevent neuronal injury. Protective effects of combined application of antagonists were generally less than additive. We conclude that AMPA/kainate receptors play a more prominent role in glutamate neurotoxicity in organotypic cultures than in dissociated cortical or hippocampal cultures, acting together with NMDA receptors to cause neuronal injury.

ATP-Binding Cassette Transporters↗
PubMed2002

BMP-7 and excess glutamate: opposing effects on dendrite growth from cerebral cortical neurons in vitro.

Glutamate is an important regulator of dendrite development. During cerebral ischemia, however, there is massive release of glutamate reaching millimolar concentrations in the extracellular space. An early consequence of this excess glutamate is reduced dendrite growth. Bone morphogenetic protein-7 (BMP-7) a member of the transforming growth factor-beta (TGF-beta) superfamily has been demonstrated to enhance dendrite output from cerebral cortical and hippocampal neurons in vitro. However, it is not known whether BMP-7can prevent the reduced dendrite growth associated with excess glutamate or enhance dendrite growth after glutamate exposure. Therefore we quantified axon and primary, secondary, and total dendrite growth from embryonic mouse cortical neurons (E18) grown at low density in vitro in a chemically defined medium and exposed to glutamate (1 or 2 mM) for 48 h. Morphology and double immunolabeling (MAP2, NF-H) were used to identify cortical dendrites and axons after 3 DIV. In these short-term cultures, glutamate did not influence neuron survival. The addition of glutamate to cortical neurons, however, significantly attenuated dendrite output. This effect was mimicked by the addition of NMDA but not AMPA agonists and inhibited by the specific NMDA receptor antagonist MK-801. The reduction in dendrite growth mediated by excess glutamate was ameliorated by the administration of 30 or 100 ng/ml of BMP-7. In addition, when administered in a delayed fashion between 1 and 24 h after the initial glutamate exposure, BMP-7 was able to enhance dendrite growth, including primary dendrite number, primary dendrite length, and secondary dendritic branching. These findings demonstrate that BMP-7 can ameliorate reduced dendrite growth from cerebral cortical neurons associated with excess glutamate in vitro and are important because they may help explain why BMP-7 administration is associated with enhanced functional recovery in models of cerebral ischemia.

Animals↗
PubMed1990

Synthesis of transmitter glutamate and the glial-neuron interrelationship.

Glutamate in glutamatergic neurons exists in a cytosolic pool, as well as a transmitter pool, which is assumed to be localized in synaptic vesicles. Transmitter glutamate released from glutamatergic neurons is taken up by both neurons and glial cells, giving rise to a flux of glutamate from neurons to astrocytes. In astrocytes, glutamine is formed from glutamate by the glial-specific enzyme glutamine synthetase (EC 6.3.1.2). Glutamine diffuses back to neurons, where glutamate is formed by phosphate-activated glutaminase (EC 3.5.1.2). However, this cycle is not stoichiometric, and glutamine obtained from glial cells cannot replenish all transmitter glutamate lost from neurons. 2-Oxoglutarate is another putative precursor for transmitter glutamate. Net synthesis of citric acid cycle intermediates is dependent on carbon dioxide fixation to pyruvate, catalyzed by pyruvate carboxylase (EC 6.4.1.1). Since this enzyme is exclusively glial, a net flow of citric acid cycle intermediates from glial cells to neurons probably exists. The quantitative contribution of each transmitter precursor may not be the same in different regions of the brain and may vary with the metabolic state of the neuron. The pool of transmitter glutamate is most likely regulated by the activity of glutamate-forming enzymes in the nerve terminal, and/or by uptake/release of glutamate and glutamate precursors through the synaptosomal plasma membrane.

Animals↗
PubMed2001

Electrically evoked release of glutamate in rat hippocampal slices: effects of various drugs and fimbria-fornix lesions.

A model of electrically evoked release of glutamate from rat hippocampus was developed and used to detect possible changes induced by lesions of hippocampal afferences. Neuronal glutamate in hippocampal slices was labelled by preincubation with [3H]glutamine. The slices were then superfused with physiological medium in the presence of the glutamate uptake inhibitor L-transpyrrolidine-2,4-dicarboxylic acid (100 microM or 3 microM) and stimulated twice electrically (S1, S2: 240 pulses, 3 Hz, 2 ms, 26-30 mA); various drugs were added before S2. In order to determine the basal and evoked outflow of [3H]glutamate only, the mixture of 3H-labelled compounds (glutamine, glutamate and GABA) was separated by ion exchange chromatography in superfusate fractions and slices. The electrically evoked overflow of [3H]glutamate was largely Ca2+-dependent and tetrodotoxin-sensitive and hence represented action potential-induced exocytotic release of [3H]glutamate. Evoked [3H]glutamate release was significantly increased by the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 0.1 microM), suggesting the presence of endogenous inhibitory adenosine, and reduced by the A1 receptor agonist N6-cyclopentyladenosine (1 microM, antagonized by DPCPX, 0.1 microM). There was no evidence for a cholinergic, serotonergic, or adrenergic modulation of the evoked release of [3H]glutamate: the corresponding selective agonists (or antagonists) were ineffective. After aspirative lesions of the septohippocampal pathways the hippocampal noradrenaline content was markedly increased, whereas cholinergic and serotonergic markers were reduced. The evoked release of [3H]glutamate in hippocampal slices of lesioned rats was significantly increased by a mechanism which still has to be determined, but which is not related to alterations in A1 receptor function. It is concluded that the present model was able to detect lesion-induced differences in electrically evoked release of [3H]glutamate, but the relationship of these differences to changes of noradrenergic, cholinergic or serotonergic hippocampal innervations remains to be established.

Action Potentials↗
PubMed2004

Glutamate activates cation currents in the plasma membrane of Arabidopsis root cells.

The effect of glutamate on plant plasma membrane cation transport was studied in roots of Arabidopsis thaliana (L.) Heynh. Patch-clamp experiments using root protoplasts, (22)Na(+) unidirectional fluxes into intact roots and measurements of cytosolic Ca(2+) activity using plants expressing cytosolically-targeted aequorin in specific cell types were carried out. It was demonstrated that low-millimolar concentrations of glutamate activate within seconds both Na(+) and Ca(2+) currents in patch-clamped protoplasts derived from roots. The probability of observing glutamate-activated currents increased with increasing glutamate concentration (up to 29% at 3 mM); half-maximal activation was seen at 0.2-0.5 mM glutamate. Glutamate-activated currents were voltage-insensitive, 'instantaneous' (completely activated within 2-3 ms of a change in voltage) and non-selective for monovalent cations (Na(+), Cs(+) and K(+)). They also allowed the permeation of Ca(2+). Half-maximal Na(+) currents occurred at 20-30 mM Na(+). Glutamate-activated currents were sensitive to non-specific blockers of cation channels (quinine, La(3+), Gd(3+)). Although low-millimolar concentrations of glutamate did not usually stimulate unidirectional influx of (22)Na(+) into intact roots, they reliably caused an increase in cytosolic Ca(2+) activity in protoplasts isolated from the roots of aequorin-transformed Arabidopsis plants. The response of cytosolic Ca(2+) activity revealed a two-phase development, with a rapid large transient increase (lasting minutes) and a prolonged subsequent stage (lasting hours). Use of plants expressing aequorin in specific cell types within the root suggested that the cell types most sensitive to glutamate were in the mature epidermis and cortex. The functional significance of these glutamate-activated currents for both cation uptake into plants and cell signaling remains the subject of speculation, requiring more knowledge about the dynamics of apoplastic glutamate in plants.

Aequorin↗
PubMed1975

Purification and properties of glutamate binding protein from the periplasmic space of Escherichia coli K-12.

Glutamate binding protein released from the periplasmic space of Escherichia coli K-12 by lysozyme-EDTA treatment was purified to homogeneity and its physical and chemical properties were studied. It is a basic protein with a pI of 9.1. Its molecular weight, determined in an analytical ultracentrifuge, and by gel filtration on Sephadex G-100 and dodecylsulphate acrylamide is 29 700, 27 800 and 32 000, respectively. The KD value for glutamate was 6.7 - 10- minus 6 M. L-Aspartate, reduced glutathione, G-glutamate-gamma-benzylester and L-glutamate-gamma-ethylester competitively inhibited glutamate binding with K-i; values of 7.8 - 10- minus 5, 1.1 - 10- minus 5, 1.0 - 10- minus 5 and 1.0 - 10- minus 5 M, respectively. Spheroplasts retained 40% of glutamate transport as compared to intact cells. The glutamate binding activity of a glutamate-utilizing strain (CS7), was 1.6 times as high as that of the glutamate non-utilizing parent strain (CS101). Similarly, the glutamate binding activity of a temperature conditional glutamate-utilizing mutant (CS2-TC) was 1.9 times higher when grown at the permissive temperature (42 degrees C) than when grown at the restrictive temperature (30 degrees C).

Amino Acids↗
PubMed1990

Metabolism of glutamine and glutamate in human lenses.

Glutamate is important to lenses as a central intermediate in amino acid metabolism, as well as for synthesis of proteins and glutathione. In rat and calf lenses, the principal source of lenticular glutamate is glutamine, which enters the lens and is deamidated to form glutamate. In contrast, monkey lenses use external glutamate more readily than glutamine. Amino acid metabolism was studied in human lenses by incubating them with amino-labeled [15N]glutamine or [15N]glutamate. The lenticular free amino acids were then isolated and analysed by gas chromatography-mass spectrometry to determine 15N-labeled products. The results were compared with those of similar experiments with lenses from other species. Label was measured in aspartate, alanine, serine and proline, as well as lenticular glutamine and glutamate. Glutamine enters lenses more readily than glutamate in all the species examined. Nevertheless, aspartate, alanine and serine were more rapidly labeled by incubating human lenses in [15N]glutamate than in [15N]glutamine. This observation is similar to reports of experiments with monkey lenses, which unlike rat lenses, preferentially utilize glutamate rather than glutamine. In contrast, human lens proline was more rapidly labeled by incubating lenses with [15N]glutamine than with [15N]glutamate. In human lenses, the relatively slow utilization of glutamine for the transamination reactions which form aspartate, alanine and serine appears to result from slow deamidation. The relative preference for glutamine over glutamate as a precursor for proline synthesis in human lenses may be related to the mitochondrial location of the enzymes involved.

Adolescent↗
PubMed1995

Glutamate and glutamine metabolism in cultured GABAergic neurons studied by 13C NMR spectroscopy may indicate compartmentation and mitochondrial heterogeneity.

Primary cultures of mouse cerebral cortical neurons were incubated for 3 h with 250 microM [U-13C]glutamate or 250 microM [U-13C]glutamine and 6 mM glucose. 13C NMR spectra of cell extracts exhibited distinct multiplets for glutamate, aspartate and GABA. Incorporation of label into aspartate can only occur through the tricarboxylic acid (TCA) cycle, but as demonstrated by formation of the 3,4-13C2-isotopomer of GABA and the 1,2,3-13C3-isotopomer of glutamate, these amino acids were also to some extent derived from this pathway. Formation of the 1,2-13C2-(1J1,2 = 50 Hz) and 3,4-13C2-isotopomer (1J3,4 = 50.9 Hz) in aspartate occurs exclusively when oxaloacetate (containing 12C) is derived from the second turn of the TCA cycle. From [U-13C]glutamine, the 12C containing isotopomers in aspartate accounted for 27% of the total label and from [U-13C]glutamate, it was less than 10%. When [U-13C]glutamine was the precursor, 36% of the labeled glutamate and 52% of the labeled GABA contained 12C incorporated during the first turn of the TCA cycle. These numbers decreased to 15 and 30%, respectively when [U-13C]glutamate was used. Since glutamine must be converted to glutamate before it can enter the TCA cycle as 2-oxoglutarate, appearance of 12C incorporation in aspartate should be identical when [U-13C]glutamate and [U-13C]glutamine was used as substrates. This was, however, not observed which may be indicative of (1) compartmentation of mitochondrial glutamate metabolism or (2) differences in the amount of intramitochondrial glutamate derived from external glutamate or glutamine.

Animals↗
PubMed1988

A comparison of the localization of choline acetyltransferase and glutamate decarboxylase immunoreactivity in rat cerebral cortex.

The neurotransmitter-synthesizing enzymes choline acetyltransferase and glutamate decarboxylase were localized immunocytochemically at the light microscopic level. Their respective laminar distributions were compared in 17 different cytoarchitectural areas, comprising limbic and neocortical regions of rat cerebral cortex. The immunoreactive intensities within these areas were measured with an image analysis system and dark-field optics. Choline acetyltransferase and glutamate decarboxylase immunoreactivity displayed distinctive distribution patterns throughout the cerebrum. In general, limbic cortex showed greater intensity of both choline acetyltransferase and glutamate decarboxylase immunoreactivity than neocortex. For example, choline acetyltransferase immunoreactivity in pyriform and retrosplenial cortex was 54% and 29% greater, respectively, than in neocortex, and glutamate decarboxylase immunoreactivity in the same cortical areas was 5% and 17% greater, respectively. In addition to these regional differences, the marked variations of choline acetyltransferase and glutamate decarboxylase immunostaining were characterized as either coincidental or complementary when comparing their laminar distributions. The laminar pattern and relative intensities of choline acetyltransferase and glutamate decarboxylase immunostaining were coincident in some layers of all cortical regions. For example, both choline acetyltransferase and glutamate decarboxylase immunoreactive intensities were high in cellular layers II and IV of the entorhinal cortex. In contrast, examples of complementary choline acetyltransferase and glutamate decarboxylase immunoreactive patterns were observed in retrosplenial cortex and neocortex. In neocortex, layers III and part of V were intensely glutamate decarboxylase-positive, whereas these same layers were less intensely choline acetyltransferase immunoreactive than the intervening layer IV and upper part of V. Quantitatively, choline acetyltransferase immunoreactivity in layers IV and upper V was 27-37% greater than adjacent layers II and deep V. The glutamate decarboxylase immunostaining pattern was complementary in that layer IV was 19-23% less intensely stained than adjacent layers III and V. Our results demonstrate that terminals immunoreactive for choline acetyltransferase and glutamate decarboxylase, and presumably the synaptic terminals that respectively use acetylcholine or gamma-aminobutyric acid as their neurotransmitters, are distributed in distinct laminar patterns that are strategically situated for modulating either afferent information in the case of cholinergic terminals or efferent transmission for GABAergic endings.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

The involvement of multiple calcium channel sub-types in glutamate release from cerebellar granule cells and its modulation by GABAB receptor activation.

In this study, we have examined both the ability of various Ca2+ channel sub-types to support the release of [3H]glutamate from cerebellar granule neurons and the mechanism of action involved in the modulation of glutamate release by the GABAB receptor agonist, (-)-baclofen. Cerebellar granule neurons were stimulated to release newly synthesized [3H]glutamate by K(+)-evoked depolarization. Stimulated release was entirely calcium-dependent and abolished by the presence of 200 microM cadmium. Release of glutamate was not affected by either tetrodotoxin or 5-aminophosphonovaleric acid but was potentiated by dihydrokainate and inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione. Stimulated glutamate release was partially inhibited by both the L-type calcium channel blocker, nicardipine, and the N-type calcium channel blocker, omega-conotoxin GVIA; however, the P/Q-type calcium channel blocker omega-agatoxin IVA inhibited release of glutamate only after pre-incubation of cells with omega-conotoxin GVIA. K(+)-stimulated release of glutamate was observed when stimulated either in the presence of Ca2+ or of Ba2+ and similar inhibition of release by (-)-baclofen was seen under both conditions. In contrast to these results, ionomycin-evoked glutamate release was greatly reduced as compared to K(+)-evoked release and was not modulated by (-)-baclofen. In the presence of omega-conotoxin GVIA alone, inhibition of release by (-)-baclofen was attenuated but not abolished. Following block of nicardipine-sensitive channels, inhibition of release by (-)-baclofen was still present, and after prior block of omega-conotoxin GVIA-sensitive channels the presence of nicardipine restored the ability of (-)-baclofen to inhibit residual release of glutamate. Modulation of glutamate release by (-)-baclofen was unaffected by the presence of omega-agatoxin IVA alone; however, after block of both omega-conotoxin GVIA- and omega-agatoxin IVA-sensitive channels, inhibition of release by (-)-baclofen was completely abolished. These results indicate that multiple sub-types of voltage-dependent calcium channels are present on the presynaptic terminals of cerebellar granule neurons and support K(+)-stimulated release of [3H]glutamate. Modulation of release by GABAB receptor activation appears to be dependent upon interaction of this receptor with a number of voltage-sensitive calcium channels, including omega-conotoxin GVIA-sensitive and omega-agatoxin IVA-sensitive channels.

Animals↗