SEARCH · Search PubMed

Results for “GLUTAMATES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,153 records · Page 64Linked to original sources

PubMed1970

Relation of renal cortical gluconeogenesis, glutamate content, and production of ammonia.

Glutamate is an inhibitor of phosphate dependent glutaminase (PDG), and renal cortical glutamate is decreased in metabolic acidosis. It has been postulated previously that the rise in renal production of ammonia from glutamine in metabolic acidosis is due primarily to activation of cortical PDG as a consequence of the fall in glutamate. The decrease in cortical glutamate has been attributed to the increase in the capacity of cortex to convert glutamate to glucose in acidosis. In the present study, administration of ammonium chloride to rats in an amount inadequate to decrease cortical glutamate increased the capacity of cortex to produce ammonia from glutamine in vitro and increased cortical PDG. Similarly, cortex from potassium-depleted rats had an increased capacity to produce ammonia and an increase in PDG, but glutamate content was normal. The glutamate content of cortical slices incubated at pH 7.1 was decreased, and that at 7.7 was increased, compared to slices incubated at 7.4, yet ammonia production was the same at all three pH levels. These observations suggest that cortical glutamate concentration is not the major determinant of ammonia production. In potassium-depleted rats there was a 90% increase in the capacity of cortex to convert glutamate to glucose, yet cortical glutamate was not decreased. In vitro, calcium more than doubled conversion of glutamate to glucose by cortical slices without affecting the glutamate content of the slices, and theophylline suppressed conversion of glutamate to glucose yet decreased glutamate content. These observations indicate that the rate of cortical gluconeogenesis is not the sole determinant of cortical glutamate concentration. The increase in cortical gluconeogenesis in acidosis and potassium depletion probably is not the primary cause of the increase in ammonia production in these states, but the rise in gluconeogenesis may contribute importantly to the maintenance of increased ammoniagenesis by accelerating removal of the products of glutamine degradation.

Acid-Base Equilibrium↗

PubMed2006

Different mechanisms exist for the plasticity of glutamate reuptake during early long-term potentiation (LTP) and late LTP.

Regulation of glutamate reuptake occurs along with several forms of synaptic plasticity. These associations led to the hypothesis that regulation of glutamate uptake is a general component of plasticity at glutamatergic synapses. We tested this hypothesis by determining whether glutamate uptake is regulated during both the early phases (E-LTP) and late phases (L-LTP) of long-term potentiation (LTP). We found that glutamate uptake was rapidly increased within minutes after induction of LTP and that the increase in glutamate uptake persisted for at least 3 h in CA1 of the hippocampus. NMDA receptor activation and Na+-dependent high-affinity glutamate transporters were responsible for the regulation of glutamate uptake during all phases of LTP. However, different mechanisms appear to be responsible for the increase in glutamate uptake during E-LTP and L-LTP. The increase in glutamate uptake observed during E-LTP did not require new protein synthesis, was mediated by PKC but not cAMP, and as previously shown was attributable to EAAC1 (excitatory amino acid carrier-1), a neuronal glutamate transporter. On the other hand, the increase in glutamate uptake during L-LTP required new protein synthesis and was mediated by the cAMP-PKA (protein kinase A) pathway, and it involved a different glutamate transporter, GLT1a (glutamate transporter subtype 1a). The switch in mechanisms regulating glutamate uptake between E-LTP and L-LTP paralleled the differences in the mechanisms responsible for the induction of E-LTP and L-LTP. Moreover, the differences in signaling pathways and transporters involved in regulating glutamate uptake during E-LTP and L-LTP indicate that different functions and/or sites may exist for the changes in glutamate uptake during E-LTP and L-LTP.

Animals↗

PubMed1996

Glutamate immunoreactivity in the tiger salamander retina differentiates between GABA-immunoreactive and glycine-immunoreactive amacrine cells.

A higher proportion of retinal amacrine cells, largely thought to be inhibitory, in tiger salamander retina are immunoreactive (+) for the excitatory amino acid glutamate than for either of the inhibitory amino acids, GABA and glycine. The colocalizations of glutamate immunoreactivity, GABA immunoreactivity and glycine immunoreactivity were studied in the proximal retina of tiger salamander using postembedding immunocytochemical methods in consecutive semithin sections. A considerable degree of colocalization was observed between glutamate immunoreactivity and GABA immunoreactivity and to a lesser extent with glycine immunoreactivity. No glutamate-immunoreactive cells were single labelled in INL3 (proximal inner nuclear), 12% were single labelled in INL2 (mid INL; these are bipolar cells), 62% were singled labelled in the ganglion cell layer (GCL) and likely to be ganglion cells. Single-labelled glycine-immunoreactive cells comprised 16% in INL3, 2%; INL2 and GCL, 0%). Glutamate immunoreactivity co-localized four times as often with GABA immunoreactivity than glycine immunoreactivity in INL3 (77% versus 19%), comparably in INL2 (37% and 32%) and six times as often in the GCL (29% versus 5%). A computerized image analysis of the relative intensities of glutamate immunoreactivity in GABA-immunoreactive/glutamate-immunoreactive and glycine-immunoreactive/glutamate-immunoreactive double labelled amacrine cells in INL3 revealed that all glycine-immunoreactive cells contained low intensities of glutamate immunoreactivity; whereas 35% of GABA-positive cells had medium to high intensities of glutamate immunoreactivity. There was a significant correlation (r = 0.44; p < 0.01) between the intensities of glutamate immunoreactivity and GABA immunoreactivity in GABA-immunoreactive/glutamate-immunoreactive amacrine cells. The common colocalization of glutamate immunoreactivity with GABA immunoreactivity and the positive correlation of their relative intensities suggests that glutamate is likely to serve as a precursor for GABA synthesis. However, intense glutamate immunoreactivity in 5% of the amacrine cells suggests a transmitter function. Glycine-immunoreactive amacrine cells contained little or no glutamate immunoreactivity, suggesting a minimal role for glutamate as a neurotransmitter in these cells. The major distribution of intense glutamate immunoreactivity is in bipolar cells and ganglion cells, consistent with data suggesting that glutamate is the transmitter in the retinal through pathway.

Animals↗

PubMed1985

Glial and neuronal glutamate transport following glutamine synthetase inhibition.

Glutamate transport into striatal tissue preparations was studied following inhibition of glutamine synthetase with methionine sulfoximine (MSO). Glutamate uptake in striatal tissue prisms was elevated for up to 7 days following an intraventricular (i.c.v.) injection of MSO. Kinetic analysis of glutamate uptake revealed that a high- and a low-affinity carrier system mediated the transport of glutamate into tissue slices. MSO altered the transport of glutamate via the high-affinity carrier without changing the characteristics of low-affinity glutamate transport. MSO increased the Km for glutamate and the Vmax at the high-affinity uptake site. The changes in the Km and the Vmax for glutamate uptake were maximal 24 hr after administration of MSO, but the transport system returned to normal by 14 days after injection. In addition, MSO increased high-affinity aspartate uptake into tissue slices, but it was without effect on leucine uptake. Glutamate uptake into striatal synaptosomes and bulk-isolated glial cells or neurons was, in all cases, mediated by a low- and high-affinity carrier. The Km and Vmax values for high-affinity glial-glutamate uptake were increased 24 hr after i.c.v. injection of MSO, while the low-affinity kinetic parameters for glial glutamate uptake were not altered by MSO. Neither high-affinity nor low-affinity glutamate uptake into bulk-isolated neurons or synaptosomes was altered by MSO 24 hr after injection. These results suggest that MSO induced alterations in glutamate transport within striatal slices may be due to changes in glial glutamate transport arising from the disruption of glutamate metabolism.

Animals↗

PubMed1965

EXCHANGE TRANSAMINATION AND THE METABOLISM OF GLUTAMATE IN BRAIN.

1. Experiments were performed to throw light on why the incorporation of (14)C from labelled carbohydrate precursors into glutamate has been found to be more marked in brain than in other tissues. 2. Rapid isotope exchange between labelled glutamate and unlabelled alpha-oxoglutarate was demonstrated in brain and liver mitochondrial preparations. In the presence but not in the absence of alpha-oxoglutarate the yield of (14)CO(2) from [1-(14)C]glutamate exceeded the net glutamate removal, and the final relative specific activities of the two substrates indicated that complete isotopic equilibration had occurred. Also, when in a brain preparation net glutamate removal was inhibited by malonate, isotope exchange between [1-(14)C]glutamate and alpha-oxoglutarate and the formation of (14)CO(2) were unaffected. 3. The time-course of isotope exchange between labelled glutamate and unlabelled alpha-oxoglutarate was followed in uncoupled brain and liver mitochondrial fractions, and the rate of exchange calculated by a computer was found to be 3-8 times more rapid than the maximal rate of utilization of the two substrates. 4. The physiological situation was imitated by the continuous infusion of small amounts of alpha-oxo[1-(14)C]glutarate into brain homogenate containing added glutamate. The fraction of (14)C infused that was retained in the glutamate pool depended on the size of the latter, and the final relative specific activities of the two substrates indicated almost complete isotope exchange. Isotopic equilibration also occurred when alpha-oxoglutarate was generated from pyruvate through the tricarboxylic acid cycle in a brain mitochondrial preparation containing [1-(14)C]glutamate. 5. The differences in the incorporation of (14)C from labelled glucose into the glutamate of brain and liver are discussed in terms of the rates of isotope exchange, the glutamate pool sizes and the rates of formation of labelled alpha-oxoglutarate in the two tissues. It is concluded that the differences between tissues in the incorporation of glucose carbon into glutamate reflect features of their metabolism largely unrelated to that of glutamate.

Amino Acids↗

PubMed1991

An electron microscopic description of glutamate-like immunoreactive axon terminals in the rat principal sensory and spinal trigeminal nuclei.

The spinal and principal sensory trigeminal nuclei relay noxious and nonnoxious stimuli from the orofacial region to the thalamus. Physiological studies have implicated glutamate as an important neurotransmitter in this region. Despite its importance as a potential transmitter, few studies have examined the anatomical distribution of glutamate within these nuclei. We therefore chose to use a monoclonal antibody raised against glutamate conjugated to a carrier protein to identify and describe glutamate-like immunoreactive processes at the electron microscopic level. Glutamate-like immunoreactive axon terminals were identified throughout the spinal trigeminal and principal sensory trigeminal nucleus. In subnucleus caudalis glutamate-like immunoreactive terminals occurred frequently in all laminae and were morphologically heterogeneous. In lamina I, glutamate-like immunoreactive terminals were primarily ovoid, contained spherical synaptic vesicles, and participated in synaptic complexes with both dendritic and axonal profiles. In laminae II and III many glutamate-like immunoreactive axon terminals were identified as the central element in synaptic glomeruli. Within discrete patches of lamina II, large numbers of glutamate-like immunoreactive terminals contained dense core vesicles. The majority of glutamate-like immunoreactive terminals in subnucleus interpolaris, subnucleus oralis, and principal sensory trigeminal nucleus were similar in morphology and synaptic interaction to the glutamate-like immunoreactive terminals found in subnucleus caudalis. Glutamate-like immunoreactive terminals that were the central presynaptic element in glomerular complexes were seen in all subnuclei. In sections from subnucleus interpolaris and subnucleus oralis central glutamate-like immunoreactive terminations within glomerular complexes had much smoother profiles, and in subnucleus interpolaris participated primarily in axodendritic synaptic junctions. In the principal sensory trigeminal nucleus central glutamate-like immunoreactive terminations were highly scalloped and participated in numerous axoaxonic synaptic junctions. The above observations are consistent with the hypothesis that glutamate-like immunoreactivity is present in some primary afferent terminations and functions as an important excitatory transmitter involved in the relay of sensory information to the spinal trigeminal and principal sensory trigeminal nucleus.

Animals↗

PubMed2005

Glutamate transport by retinal Muller cells in glutamate/aspartate transporter-knockout mice.

Glutamate transporters are involved in maintaining extracellular glutamate at a low level to ensure a high signal-to-noise ratio for glutamatergic neurotransmission and to protect neurons from excitotoxic damage. The mammalian retina is known to express the excitatory amino acid transporters, EAAT1-5; however, their specific role in glutamate homeostasis is poorly understood. To examine the role of the glial glutamate/aspartate transporter (GLAST) in the retina, we have studied glutamate transport by Muller cells in GLAST-/- mice, using biochemical, electrophysiological, and immunocytochemical techniques. Glutamate uptake assays indicated that the Km value for glutamate uptake was similar in wild-type and GLAST-/- mouse retinas, but the Vmax was approximately 50% lower in the mutant. In Na+-free medium, the Vmax was further reduced by 40%. In patch-clamp recordings of dissociated Muller cells from GLAST-/- mice, application of 0.1 mM glutamate evoked no current showing that the cells lacked functional electrogenic glutamate transporters. The result also indicated that there was no compensatory upregulation of EAATs in Muller cells. [3H]D-Aspartate uptake autoradiography, however, showed that Na+-dependent, high-affinity transporters account for most of the glutamate uptake by Muller cells, and that Na+-independent glutamate transport is negligible. Additional experiments showed that the residual glutamate uptake in Muller cells in the GLAST-/- mouse retina is not due to known glutamate transporters-cystine-glutamate exchanger, ASCT-1, AGT-1, or other heteroexchangers. The present study shows that while several known glutamate transporters are expressed by mammalian Muller cells, new Na+-dependent, high-affinity glutamate transporters remain to be identified.

Amino Acid Transport System ASC↗

PubMed1993

Quantitative mapping of glutamate presynaptic terminals in the supraoptic nucleus and surrounding hypothalamus.

Although the hypothalamus is generally regarded to have low levels of glutamate receptors, anatomical and physiological studies have provided consistent evidence implicating glutamate as a potential transmitter for the control of neuroendocrine cell activity. To clarify the extent of the contribution of synapses utilizing glutamate for control of vasopressin/oxytocin neuroendocrine cells, we mapped the density and location of glutamate immunoreactive terminals in the supraoptic nucleus and surrounding hypothalamus. Colloidal gold particle densities in presynaptic terminals were measured from electron micrographs of: (1) the magnocellular neuroendocrine cell perikarya (main body of the supraoptic nucleus), (2) the dendritic field of the magnocellular neuroendocrine cells (ventral dendritic neuropil) and (3) the hypothalamic perinuclear zone dorsal to the supraoptic nucleus. In addition, serial sections were stained, alternatively, for glutamate or GABA to determine glutamate staining in GABA cells. Terminals with high glutamate immunoreactivity were clearly distinguished from the glutamate precursor staining found in GABA terminals and were abundant at all rostral-caudal levels within each region. The number of glutamate terminals identified in each region was similar but represented a very high proportion of all terminals in the ventral dendritic neuropil (38%) vs. the main body of the supraoptic nucleus and the perinuclear zone (20-22%). The regional variation in the relative proportion of glutamate terminals was determined largely by differences in the number of non-glutamate terminals within each region. Glutamate and GABA terminals together accounted for over two-thirds of the innervation of vasopressin/oxytocin neuroendocrine cells. No systematic relationship was observed between excitatory and inhibitory inputs on the same cell. These results suggest that glutamate is the predominant excitatory transmitter used for control of vasopressin/oxytocin cells. The relative contribution of glutamate neurotransmission to a particular region will depend, in part, on the number and type of competing non-glutamate terminals.

Animals↗

PubMedDate not supplied

Involvement of glutamate in ischemic neurodegeneration in isolated retina.

Retinal ischemia, a major cause of visual loss, is believed to result from overexcitation of glutamate receptors. However, under euglycemic and normoxic conditions, exogenously applied glutamate is not neurotoxic in the retina. Under such conditions, exogenous glutamate typically causes glia swelling and requires very high concentrations to produce neurotoxicity. To determine whether ischemic conditions enhance the neurotoxicity of endogenous and exogenous glutamate, we examined the effects of simulated ischemia (deprivation of both glucose and oxygen) on retinal morphology and lactate dehydrogenase (LDH) release. In an ex vivo rat retinal preparation, glutamate was administered during simulated ischemia in the presence of riluzole, an inhibitor of glutamate release. Deprivation of both glucose and oxygen for 60 min at 30 degrees C produced severe acute neurodegeneration. This neurodegeneration, characterized by bull's eye formation in the inner nuclear layer and spongy appearance in the inner plexiform layer, was prevented by the combination of MK-801 and DNQX, antagonists of N-methyl-D-aspartate (NMDA) and non-NMDA receptors, indicating that the damage results from activation of both glutamate receptors. We also found that administration of glutamate pyruvate transaminase (alanine aminotransaminase) with pyruvate diminished the neurodegeneration during simulated ischemia. Furthermore, riluzole, an inhibitor of glutamate release, attenuated the neurodegeneration, suggesting the importance of endogenous glutamate in ischemic damage. In the presence of riluzole and simulated ischemia, exogenously applied glutamate failed to cause Müller cell swelling but was extremely neurotoxic. These results suggest that simulated ischemia enhances glutamate-mediated neurotoxicity in part by depressing glutamate uptake. When glutamate transport is impaired, sub-millimolar glutamate concentrations become profoundly neurotoxic.

Adenosine Triphosphate↗

PubMed2002

Prevention of ammonia and glutamate neurotoxicity by carnitine: molecular mechanisms.

Carnitine has beneficial effects in different pathologies and prevents acute ammonia toxicity (ammonia-induced death of animals). Acute ammonia toxicity is mediated by excessive activation of the NMDA-type of glutamate receptors, which mediates glutamate neurotoxicity. We showed that carnitine prevents glutamate neurotoxicity in primary cultures of cerebellar neurons. This supports the idea that the protective effect of carnitine against ammonia toxicity is due to the protective effect against glutamate neurotoxicity. We are studying the mechanism by which carnitine protects against glutamate neurotoxicity. Carnitine increases the binding affinity of glutamate for metabotropic glutamate receptors. The protective effect of carnitine is lost if metabotropic glutamate receptors are blocked with specific antagonists. Moreover, activation of metabotropic glutamate receptors by specific agonists also prevents glutamate neurotoxicity. This indicates that the protective effect of carnitine against glutamate neurotoxicity is mediated by activation of metabotropic glutamate receptors. The molecule of carnitine has a trimethylamine group. Different compounds containing a trimethylamine group (carbachol, betaine, etc.) also prevent ammonia-induced animal death and glutamate-induced neuronal death. Moreover, metabotropic glutamate receptor antagonists also prevent the protective effect of most of these compounds. We summarize here some studies aimed to identify the mechanism and the molecular target that are responsible for the protective effect of carnitine against ammonia and glutamate neurotoxicity. Finally it is also shown that carnitine inhibits the hydrolysis of inositol phospholipids induced by activation of different types of metabotropic receptors, but this effect seems not responsible for its protective effects.

Ammonia↗

PubMed2000

Calcium sequestering ability of mitochondria modulates influx of calcium through glutamate receptor channel.

The excitotoxicity of glutamate is believed to be mediated by sustained increase in the cytosolic Ca2+ concentration. Mitochondria play a vital role in buffering the cytosolic calcium overload in stimulated neurons. Here we have studied the glutamate induced Ca2+ signals in cortical brain slices under physiological conditions and the conditions that modify the mitochondrial functions. Exposure of slices to glutamate caused a rapid increase in [Ca2+]i followed by a slow and persistently rising phase. The rapid increase in [Ca2+]i was mainly due to influx of Ca2+ through the N-methyl-D-aspartate (NMDA) receptor channels. Glutamate stimulation in the absence of Ca2+ in the extracellular medium elicited a small transient rise in [Ca2+]i which can be attributed to the mobilization of Ca2+ from IP3 sensitive endoplasmic reticulum pools consequent to activation of metabotropic glutamate receptors. The glutamate induced Ca2+ influx was accompanied by depolarization of the mitochondrial membrane, which was inhibited by ruthenium red, the blocker of mitochondrial Ca2+ uniporter. These results imply that mitochondria sequester the Ca2+ loaded into the cytosol by glutamate stimulation. Persistent depolarization of mitochondrial membrane observed in presence of extracellular Ca2+ caused permeability transition and released the sequestered Ca2+ which is manifested as slow rise in [Ca2+]i. Protonophore carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) depolarized the mitochondrial membrane and enhanced the glutamate induced [Ca2+]i response. Contrary to this, treatment of slices with mitochondrial inhibitor oligomycin or ruthenium red markedly reduced the [Ca2+]i response. Combined treatment with oligomycin and rotenone further diminished the [Ca2+]i response and also abolished the CCCP mediated rise in [Ca2+]i. However, rotenone alone had no effect on glutamate induced [Ca2+]i response. These changes in glutamate-induced [Ca2+]i response could not be explained on the basis of deficient mitochondrial Ca2+ sequestration or ATP dependent Ca2+ buffering. The mitochondrial inhibitors reduced the cellular ATP/ADP ratio, however, this would have restrained the ATP dependent Ca2+ buffering processes leading to elevation of [Ca2+]i. In contrast our results showed repression of Ca2+ signal except in case of CCCP which drastically reduced the ATP/ADP ratio. It was inferred that, under the conditions that hamper the Ca2+ sequestering ability of mitochondria, the glutamate induced Ca2+ influx could be impeded. To validate this, influx of Mn2+ through ionotropic glutamate receptor channel was monitored by measuring the quenching of Fura-2 fluorescence. Treatment of slices with oligomycin and rotenone prior to glutamate exposure conspicuously reduced the rate of glutamate induced fluorescence quenching as compared to untreated slices. Thus our data establish that the functional status of mitochondria can modify the activity of ionotropic glutamate receptor and suggest that blockade of mitochondrial Ca2+ sequestration may desensitize the NMDA receptor operated channel.

Adenosine Diphosphate↗

PubMed1999

Inhibition of uptake unmasks rapid extracellular turnover of glutamate of nonvesicular origin.

Maintaining glutamate at low extracellular concentrations in the central nervous system is necessary to protect neurons from excitotoxic injury and to ensure a high signal-to-noise ratio for glutamatergic synaptic transmission. We have used DL-threo-beta-benzyloxyaspartate (TBOA), an inhibitor of glutamate uptake, to determine the role of glutamate transporters in the regulation of extracellular glutamate concentration. By using the N-methyl-D-aspartate receptors of patched CA3 hippocampal neurons as "glutamate sensors," we observed that application of TBOA onto organotypic hippocampal slices led to a rapid increase in extracellular glutamate concentration. This increase was Ca(2+)-independent and was observed in the presence of tetrodotoxin. Moreover, prevention of vesicular glutamate release with clostridial toxins did not affect the accumulation of glutamate when uptake was inhibited. Inhibition of glutamine synthase, however, increased the rate of accumulation of extracellular glutamate, indicating that glial glutamate stores can serve as a source in this process. TBOA blocked synaptically evoked transporter currents in astrocytes without inducing a current mediated by the glutamate transporter. This indicates that this inhibitor is not transportable and does not release glutamate by heteroexchange. These results show that under basal conditions, the activity of glutamate transporters compensates for the continuous, nonvesicular release of glutamate from the intracellular compartment. As a consequence, acute disruption of transporter activity immediately results in significant accumulation of extracellular glutamate.

ATP-Binding Cassette Transporters↗

PubMed2000

Herniated lumbar disc material as a source of free glutamate available to affect pain signals through the dorsal root ganglion.

STUDY DESIGN: Combined prospective human cohort and prospective controlled animal model. OBJECTIVES: To determine whether free glutamate is available in herniated disc material in concentrations sufficient to diffuse to glutamate receptors and affect the activity of neurons in the dorsal root ganglion that may transmit pain information. SUMMARY OF BACKGROUND DATA: The severity of lumbar radicular pain cannot be fully explained by physical pressure on nerve roots or ganglions. In experimental models, inflammatory processes are relatively modest under conditions of disc herniation. The hypothesis for the current study was that the proteoglycan link and core proteins, which contain high fractions of acidic amino acids, may be a source of glutamate when enzymatically degraded in an environment without glutamate reuptake systems. Glutamate would be free to diffuse to the dorsal root ganglion to affect glutamate receptors. METHODS: Disc material was harvested during surgery from herniated and nonherniated portions in patients undergoing elective lumbar disc surgery and subjected to immunohistochemistry and high-performance liquid chromatography for assessment of the presence of extracellular disc matrix glutamate. Miniosmotic pumps with differing concentrations of radiolabeled glutamate based on human data were implanted in the rat epidural space for 72 hours and dorsal root ganglion (DRG) in the region were harvested. RESULTS: Densitometry of disc matrix demonstrated immunohistochemical evidence for significant extracellular glutamate (P < 0.002). High performance liquid chromatography showed significant concentrations of glutamate in disc material and significantly more in herniated than in nonherniated disc material (P < 0.05). Significant radiolabeling of the dorsal root ganglion after epidural glutamate infusion was found at concentrations two orders of magnitude below measured disc glutamate levels. Autoradiography demonstrated radiolabeling of adjacent DRG. CONCLUSIONS: Glutamate originating from degenerated disc proteoglycan may diffuse to the dorsal root ganglion and effect glutamate receptors. Consideration may be given to treating disc radiculopathy with epidural glutamate receptor antagonists.

Adult↗

PubMed1985

Glutamate dehydrogenase (NADP-dependent) mRNA in relation to enzyme synthesis in Euglena gracilis. Evidence for post-transcriptional control.

Cells of Euglena gracilis Klebs strain z Pringsheim had high NADP-dependent glutamate dehydrogenase activity when grown on glutamate as nitrogen source but activity was completely repressed in cells grown on ammonium (NH4+). A 120-fold purification of NADPH-glutamate dehydrogenase (subunit Mr = 45 000) was achieved from glutamate-grown cells by affinity chromatography on blue Sepharose CL-6B. Antisera raised against the homogeneously pure protein were used to demonstrate that increase in NADPH-glutamate dehydrogenase activity on transfer from NH4+ to glutamate medium resulted from an increase in the amount of protein. Glutamate NH4+-grown cells were labelled with L-[35S]methionine and anti-(NADPH-glutamate dehydrogenase) used to immunoprecipitate the dehydrogenase from cell extracts. NADPH-glutamate dehydrogenase protein was detected in glutamate-grown but not NH4+-grown cells. Anti-(NADPH-glutamate dehydrogenase) was used to detect NADPH-glutamate dehydrogenase resulting from the translation of total polyadenylated RNA from Euglena in a cell-free rabbit reticulocyte lysate system. NADPH-glutamate dehydrogenase mRNA was present in glutamate NH4+-grown cells, there being no apparent difference in mRNA abundance between cells showing a tenfold difference in NADPH-glutamate dehydrogenase specific activity. These results indicate that the synthesis of this dehydrogenase is regulated primarily at the post-transcriptional level.

Culture Media↗

PubMed1985

Induced release of gamma-aminobutyric acid by a carrier-mediated, high-affinity uptake of L-glutamate in cultured chick retina cells.

[3H]gamma-aminobutyric acid (GABA) was taken up by cultured embryonic retina cells during the initial stages of cell differentiation. The accumulated GABA was released in the bathing medium and a transient increase in the efflux of GABA was observed when cultures were pulse-stimulated (2 min) with 0.1 mM L-glutamate but not with D-glutamate. The EC50 for L-glutamate to evoke [3H]GABA release was approximately 15 microM. This value is close to the Km for high-affinity uptake of L-glutamate by retina cells. When Na+ ions were replaced by Li+ ions, L-glutamate-induced release of GABA was abolished. Moreover, L-[14C]glutamate uptake by retina cells was significantly reduced when NaCl was replaced by LiCl in the incubation medium. L-Glutamate elicited release of GABA was Ca2+ independent, and was observed when Ca2+ was replaced by Co2+ or when Mg2+ ions were increased to 10 mM concentration. D-Aspartate, which is taken up by the same high-affinity uptake mechanism as L-glutamate, induced an increase in [3H]GABA efflux comparable to L-glutamate. The addition of unlabeled GABA to the medium also promoted the release of accumulated [3H]GABA. However, GABA was twofold less effective than L-glutamate in eliciting [3H]GABA release. The addition of both GABA and L-glutamate to the incubation medium indicated that [3H]GABA efflux due to L-glutamate and GABA was additive. L-Aspartate also promoted an increase in the efflux of [3H]GABA accumulated by retina cells. However, L-aspartate effect was significantly decreased in the absence of Ca2+ or when Na+ ions were replaced by Li+. Our results indicate that at least three releasable pools of GABA are present in the chick embryo retina cells: (a) a GABA-promoted GABA release-homoexchange, (b) a Ca2+-dependent L-aspartate-promoted release, and (c) a Ca2+-independent, Na+-dependent L-glutamate-evoked release. In addition, our data strongly suggest that the L-glutamate-promoted GABA release is due to a process of exchange of L-glutamate with GABA, which may play a fundamental role in the fine control of the excitability of local circuits in the retina.

Animals↗

PubMed1986

Binding of L-[3H]glutamate to fresh or frozen synaptic membrane and postsynaptic density fractions isolated from cerebral cortex and cerebellum of fresh or frozen canine brain.

Synaptic membrane (SPM) and postsynaptic density (PSD) fractions isolated from cerebral cortex (CTX) and cerebellum (CL) of canine brain, either fresh or frozen and isolated from either fresh or frozen tissue, were found to contain L-[3H]glutamate binding sites. It was found that there was a concentration of L-glutamate binding sites in CTX-PSD and CL-PSD over the respective membrane fractions, and the Bmax value of CL-PSD (92.0 pmol/mg protein) was about three times that of CTX-PSD (28.9 pmol/mg). The results, together with those of others, suggest that the thin CL-PSD are probably derived from the excitatory synapses in the molecular layer. The ion dependency of L-glutamate binding to canine CTX-SPM fraction was found to be similar to that reported for a rat brain SPM fraction: (a) Cl- increased the number of L-glutamate binding sites and the effect was enhanced by Ca2+; Ca2+ alone had no significant effect; (b) the Cl-/Ca2+-sensitive binding sites were abolished by 2-amino-4-phosphonobutyrate (APB) or freezing and thawing; (c) the effect of Na+ ion was biphasic; low concentration of Na+ (less than 5 mM) decreased Cl-/Ca2+-dependent L-glutamate binding sites, whereas at higher concentrations of Na+ the binding of glutamate was found to increase either in the presence or absence of Ca2+ and Cl-. In addition, the K+ ion (50 mM) was found to decrease the Na+-independent and Cl-/Ca2+-independent binding of L-glutamate to fresh CTX-SPM by 18%, but it decreased the Na+-dependent and Cl-/Ca2+-independent L-glutamate binding by 93%; in the presence of Cl-/Ca2+, the K+ ion decreased the Na+-dependent binding by 78%. Freezing and thawing of CTX-SPM resulted in a 50% loss of the Na+-dependent L-glutamate binding sites assayed in the absence of Ca2+ and Cl-. The CL-SPM fraction showed similar ion dependency of L-glutamate binding except for the absence of Na+-dependent glutamate binding sites. The CTX-PSD fraction contained neither Na+-dependent nor APB (or Cl-/Ca2+)-sensitive L-glutamate binding sites and its L-glutamate binding was unaffected by freezing and thawing, in agreement with the reported findings using rat brain PSD preparation. L-Glutamate binding to CTX-SPM or CTX-PSD fraction was not affected by pretreatment with 10 mM L-glutamate, nor by simultaneous incubations with calmodulin.(ABSTRACT TRUNCATED AT 400 WORDS)

Aminobutyrates↗

PubMed1983

Kinetics of glutamate efflux in rat liver mitochondria.

The transport of glutamate was studied in isolated rat liver mitochondria preloaded with glutamate in the presence of respiratory inhibitors. Glutamate efflux was initiated by dilution of the loaded mitochondria into a glutamate-free medium, and the rate of transport was measured by following the disappearance of glutamate from the mitochondrial matrix following rapid centrifugation through silicone oil. Glutamate efflux was inhibited extensively by bromcresol purple and partially by N-ethylmaleimide, compounds which are both known to inhibit mitochondrial glutamate uptake. The efflux process was stereospecific for L-glutamate and exhibited an activation energy of 19.2 kcal/mol. The rate of glutamate efflux was not affected by changes in the mitochondrial membrane potential. However, a good correlation was observed between the rate of glutamate efflux and the matrix pH, the efflux rate being stimulated by a decrease in matrix pH in the range from 8.0 to 7.2. In contrast, acidification of the incubation medium in the pH range 7.4 to 6.5 inhibited the rate of glutamate efflux. A kinetic analysis was made of the efflux reaction by a computer curve-fitting procedure which fits the experimental data to an integrated rate equation (Williamson, J.R., and Viale, R.O. (1979) Methods Enzymol. 56, 252-278). The results indicated that a fall in the matrix pH primarily caused a decrease in the K'm for matrix glutamate, with little change in V'max. In contrast, a low external pH had an effect on the V'max but not on the K'm for intramitochondrial glutamate. The results are in agreement with a symmetrical sequential model of glutamate transport where the glutamate anion binds to the protonated carrier.

Animals↗

PubMed2000

Glutamate transport and metabolism in dopaminergic neurons of substantia nigra: implications for the pathogenesis of Parkinson's disease.

Parkinson's disease (PD) is associated with degeneration of the pigmented dopaminergic neurons located in the ventral mesencephalon. Although the mechanisms by which these neurons degenerate in PD are poorly understood, indirect evidence suggests involvement of glutamatergic mechanisms in the pathogenesis of this disorder. Glutamate, the major excitatory transmitter in the mammalian central nervous system, is known to be neurotoxic when present in excess at the synapses. Two major mechanisms protect neurons from glutamate-induced toxicity: (a) removal of synaptic glutamate via a high affinity uptake carried out by cytoplasmic membrane proteins known as excitatory amino acid transporters (EAAT); and (b) metabolism and recycling of glutamate by synaptic astrocytes via glutamine synthetase, an ATP-requiring reaction. However, when extra-cellular glutamate levels are high (0.5-1.0 mM), glutamate metabolism may be shifted toward the ATP-generating oxidative deamination (glutamate dehydrogenase)-TCA cycle pathway. We have cloned and characterized two human glutamate dehydrogenases (GDH), one of which is nerve tissue specific. This isoenzyme requires ADP for its activity and it may become functional when cellular energy charge is low. We have also cloned three human glutamate transporters. One of these (EAAT3) is neuron specific. In situ hybridization studies using human brain revealed that the pigmented dopaminergic neurons, which degenerate in PD, express EAAT3 at high levels. Primary nerve tissue cultures derived from rat ventral mesencephalon were established and studied for their ability to metabolize glutamate. Results showed that mature cultures expressing high levels of GDH activity were capable of rapidly utilizing glutamate added to the medium at high concentrations (1-1.2 mM). This was associated with little release of aspartate and alanine into the medium. In contrast, immature cultures expressing low GDH activity utilized glutamate at lower rates while releasing substantial amounts of aspartate and alanine into the medium. These data suggest that immature mesencephalic cells metabolize a substantial fraction of the glutamate they take up from the medium via the transamination pathway, compared to mature mesencephalic cultures. Immunocytochemical studies on these cultures revealed that dopaminergic neurons (identified by their tyrosine hydroxylase content) showed intense staining for GDH. Furthermore, inhibition of GDH expression by antisense oligonucleotides was toxic to cultured mesencephalic neurons, with dopaminergic neurons being affected at the early stages of this inhibition. Hence, the dense expression by dopaminergic neurons of proteins involved in the transport and metabolism of glutamate may serve particular biological needs intrinsic to these cells. Further studies are required to test whether these properties render these neurons vulnerable to excitotoxic mechanisms or to abnormalities of glutamate metabolism.

ATP-Binding Cassette Transporters↗