A Two-Translocator Model for the Transport of 2-Oxoglutarate and Glutamate in Chloroplasts during Ammonia Assimilation in the Light

This study examines the transport of 2-oxoglutarate (2-OG) and other dicarboxylates during ammonia assimilation in illuminated spinach chloroplasts. The transport of all dicarboxylates examined was strongly inhibited by NH₄Cl preincubation in the light. Treatment with NH₄Cl caused a rapid depletion of the endogenous glutamate pool and a corresponding increase in endogenous glutamine content. The inhibition of transport activity by NH₄Cl was apparently linked to its metabolism in the light because inhibition of glutamine synthetase activity by the addition of l-methionine sulfoximine or carbonylcyanide-m-chlorophenylhydrazone abolished this affect. Measurements of endogenous metabolite pools showed that malate was most rapidly exchanged during the uptake of all exogenous dicarboxylates examined. Depending on the exogenous substrates used, the apparent half-times of efflux measured for endogenous malate, aspartate and glutamate were 10, 10 to 30, and 15 to 240 seconds, respectively. The transport of 2-OG was also inhibited by malate. But chloroplasts preincubated with malate in the presence or absence of NH₄Cl were found to have high transport activity similar to untreated chloroplasts. A two-translocator model is proposed to explain the stimulation of 2-OG transport as well as the stimulation of (NH₃, 2-OG)-dependent O₂ evolution by malate (KC Woo, CB Osmond 1982 Plant Physiol 69: 591-596) in isolated chloroplasts. In this model the transport of 2-OG on the 2-OG translocator and glutamate on the dicarboxylate translocator is coupled to malate counter-exchange in a cascade-like manner. This results in a net 2-OG/glutamate exchange with no net malate transport. Thus, during NH₃ assimilation the transport of 2-OG into and the export of glutamate out of the chloroplast occurs via the 2-OG and the dicarboxylate translocators, respectively.     

Amyloid-β Decreases Nitric Oxide Production in Cultured Retinal Neurons: A Possible Mechanism for Synaptic Dysfunction in Alzheimer’s Disease?

The neurotoxicity of the amyloid-β peptide (Aβ) appears to be, at least in part, related to pathological activation of glutamate receptors by Aβ aggregates. However, the downstream signaling pathways leading to neurodegeneration are still incompletely understood. Hyperactivation of nitric oxide synthase (NOS) and increased nitric oxide (NO) production have been implicated in excitotoxic neuronal damage caused by overactivation of glutamate receptors, and it has been suggested that increased NO levels might also play a role in neurotoxicity in Alzheimer’s disease. We have examined the effect of blockade of NO production on the neurotoxicity instigated by Aβ₄₂ and by elevated concentrations of glutamate in chick embryo retinal neurons in culture. Results showed that l-nitroarginine methyl ester, a potent inhibitor of all NOS isoforms, had no protective effect against neuronal death induced by either Aβ₄₂ (20 μM) or glutamate (1 mM). Surprisingly, at short incubation times both Aβ and glutamate decreased NO production in retinal neuronal cultures in the absence of neuronal death. Thus, excitotoxic insults induced by Aβ and glutamate cause inhibition rather than activation of NO synthase in retinal neurons, suggesting that cell death induced by Aβ or glutamate is not related to increased NO production. On the other hand, considering the role of NO in long term potentiation and synaptic plasticity, the decrease in NO levels instigated by Aβ and glutamate suggests a possible mechanism leading to synaptic failure in AD.     

Hypoxia suppresses astrocyte glutamate transport independently of amyloid formation

Sustained hypoxia alters the expression of numerous proteins and predisposes individuals to Alzheimer’s disease (AD). We have previously shown that hypoxia in vitro alters Ca²⁺ homeostasis in astrocytes and promotes increased production of amyloid β peptides (Aβ) of AD. Indeed, alteration of Ca²⁺ homeostasis requires amyloid formation. Here, we show that electrogenic glutamate uptake by astrocytes is suppressed by hypoxia (1% O₂, 24 h) in a manner that is independent of amyloid β peptide formation. Thus, hypoxic suppression of glutamate uptake and expression levels of glutamate transporter proteins EAAT1 and EAAT2 were not mimicked by exogenous application of amyloid β peptide, or by prevention of endogenous amyloid peptide formation (using inhibitors of either β or γ secretase). Thus, dysfunction in glutamate homeostasis in hypoxic conditions is independent of Aβ production, but will likely contribute to neuronal damage and death associated with AD following hypoxic events.     

Magnesium Sulfate Protects Against the Bioenergetic Consequences of Chronic Glutamate Receptor Stimulation

Extracellular glutamate is elevated following brain ischemia or trauma and contributes to neuronal injury. We tested the hypothesis that magnesium sulfate (MgSO₄, 3 mM) protects against metabolic failure caused by excitotoxic glutamate exposure. Rat cortical neuron preparations treated in medium already containing a physiological concentration of Mg²⁺ (1 mM) could be segregated based on their response to glutamate (100 µM). Type I preparations responded with a decrease or small transient increase in oxygen consumption rate (OCR). Type II neurons responded with >50% stimulation in OCR, indicating a robust response to increased energy demand without immediate toxicity. Pre-treatment with MgSO₄ improved the initial bioenergetic response to glutamate and ameliorated subsequent loss of spare respiratory capacity, measured following addition of the uncoupler FCCP, in Type I but not Type II neurons. Spare respiratory capacity in Type I neurons was also improved by incubation with MgSO₄ or NMDA receptor antagonist MK801 in the absence of glutamate treatment. This finding indicates that the major difference between Type I and Type II preparations is the amount of endogenous glutamate receptor activity. Incubation of Type II neurons with 5 µM glutamate prior to excitotoxic (100 µM) glutamate exposure recapitulated a Type I phenotype. MgSO₄ protected against an excitotoxic glutamate-induced drop in neuronal ATP both with and without prior 5 µM glutamate exposure. Results indicate that MgSO₄ protects against chronic moderate glutamate receptor stimulation and preserves cellular ATP following treatment with excitotoxic glutamate.     

Activation of microglia with zymosan promotes excitatory amino acid release via volume-regulated anion channels: the role of NADPH oxidases

Microglia are the resident immune cells of the CNS, which are important for preserving neural tissue functions, but may also contribute to neurodegeneration. Activation of these cells in infection, inflammation, or trauma leads to the release of various toxic molecules, including reactive oxygen species (ROS) and the excitatory amino acid glutamate. In this study, we used an electrophysiologic approach and a d‐[ ³ H] aspartate (glutamate) release assay to explore the ROS‐dependent regulation of glutamate‐permeable volume‐regulated anion channels (VRACs). Exposure of rat microglia to hypo‐osmotic media stimulated Cl^? currents and d ‐[³H]aspartate release, both of which were inhibited by the selective VRAC blocker, DCPIB. Exogenously applied H₂O₂ potently increased swelling‐activated glutamate release. Stimulation of microglia with zymosan triggered production of endogenous ROS and strongly enhanced glutamate release via VRAC in swollen cells. The effects of zymosan were attenuated by the ROS scavenger, MnTMPyP, and by two inhibitors of NADPH oxidase (NOX), diphenyliodonium and thioridazine. However, zymosan‐stimulated glutamate release was insensitive to other NOX blockers, apocynin and HEBSF. This pharmacologic profile pointed to the potential involvement of apocynin‐insensitive NOX4. Using RT‐PCR we confirmed that NOX4 is expressed in rat microglial cells along with NOX1 and NOX2. To check for potential involvement of phagocytic NOX2, we stimulated this isoform using protein kinase C (PKC) activator, phorbol 12‐myristate 13‐acetate or inhibited it with the broad spectrum PKC blocker, Gö6983. Both agents potently modulated endogenous ROS production by NOX2 but not VRAC activity. Taken together, these data suggest that the anion channel VRAC may contribute to microglial glutamate release and that its activity is regulated by endogenous ROS originating from NOX4.     

Chronic administration of valproic acid induces a decrease in rat striatal glutamate and taurine levels

Summary The effect of acute and chronic (10 days) administration of 200 mg/kg (i.p.) of valproic acid (VPA) on endogenous levels of aspartate, glutamate, alanine, glycine and taurine in the cerebral frontal cortex and corpus striatum of rats was studied. Quantification of the amino acid levels was performed by HPLC.Valproic acid (VPA) did not either induce changes on these neurotransmitters contents in corpus striatum after acute treatment. After chronic administration we found a decrease on the endogenous levels of glutamic acid (24%, p < 0.05) which was related to an increase (250%, p < 0.02) of the in vitro KCl evoked release of glutamate. We found decrements in taurine endogenous levels (22%, p < 0.05) which was not associated with an increase of its release.In cerebral frontal cortex there was not found any change neither under the acute nor under the chronic condition.Thus, it may be conclude that chronic treatment with VPA produces decreases on the endogenous levels of glutamate and taurine. However the relevance of this effect concerning it therapeutic action remains unclear.     

Effects of spinal transection on presynaptic markers for glutamatergic neurons in the rat

To evaluate the hypothesis that glutamic acid may be the neurotransmitter of descending, excitatory supraspinal pathways, the uptake and release ofl-[³H] glutamate and the levels of endogenous glutamate were measured in preparations from rat lumbar spinal cord following complete mid-thoracic transection. Following transection, the activity of the synaptosomal high-affinty glutamate uptake process was increased in both dorsal and ventral halves of lumbar cord between 1 and 14 days after transection and returned to control levels by 21 days posttransection. At 7 days, the increased activity of the uptake process forl-[³H] glutamate resulted in elevation ofV _max with no significant alteration inK _t as compared to age-matched controls. Depolarization-induced release ofl-[³H]glutamate from prelabeled slices did not differ significantly from control in the lesioned rat except at 21 days after lesion when the amount of tritium release was significantly greater in the transected preparations than in control. Amino acid analysis of the lumbar cord from control and transected rats indicated only a 10% decrease in the level of endogenous glutamate and no alterations in the concentration of GABA and glycine 7 days after lesion. These findings do not support the hypothesis that glutamate serves as a major excitatory neurotransmitter in supraspinal pathways innervating the lumbar cord of the rat.     

EFFECTS OF TETRODOTOXIN, CALCIUM AND MAGNESIUM ON THE RELEASE OF AMINO ACIDS FROM SLICES OF GUINEA-PIG CEREBRAL CORTEX

Abstract— Tetrodotoxin, Ca²⁺-deprivation and high-Mg²⁺ were used in an effort to identify the portion of the evoked release of endogenous amino acids, labelled via metabolism of [¹⁴C]-glucose, and several exogenous labelled amino acids, that came from nerve terminals when slices of guinea pig cerebral cortex were superfused with glucose-free solutions and stimulated electrically. With some exceptions, spontaneous release of labelled amino acids was decreased by 2 μm -tetrodotoxin but increased in Ca²⁺-free medium and in solutions containing an extra 24 mm -MgCl₂. Tetrodotoxin suppressed 85–90% of the stimulated release of almost all labelled amino acids, but had a smaller effect on the release of endogenous ¹⁴C-labelled threonine-serine-glutamine (unseparated). In Ca²⁺-free solution, the stimulated release of endogenous ¹⁴C-labelled glutamate, aspartate and GABA was suppressed by 80–90%, but that of endogenous ¹⁴C-labelled threonine-serine-glutamine was unaffected as was most of the release of the other labelled amino acids. In medium containing an extra 24mM-MgCl₂, the stimulated release of endogenous ¹⁴C-labelled glutamate, aspartate and GABA was suppressed by 75-85%, that of exogenous labelled aspartate and GABA by 50–65%, but the release of the other labelled amino acids was unaffected. The control stimulated releases of endogenous ¹⁴C-labelled glutamate, aspartate and GABA were much larger than those of other labelled amino acids but were reduced by tetrodotoxin, Ca²⁺-deprivation and high-Mg²⁺ to a level similar to that of the control stimulated releases of the other labelled amino acids. These results suggest that almost all of the stimulated release of endogenous ¹⁴C-labelled glutamate, aspartate and GABA came from nerve terminals while those of the other labelled amino acids came from other tissue elements. In addition, they are in accord with a transmitter role for glutamate, aspartate and GABA in cerebral cortex.     

Free amino acid production during tomato fruit ripening: a focus on l-glutamate

In tomato, free amino acids increase dramatically during fruit ripening and their abundance changed differentially. More evident is l-glutamate which gives the characteristic “umami” flavor. Glutamate is the principal free amino acid of ripe fruits of cultivated varieties. In this paper, we examined the capacity of tomato fruits to process endogenous as well as exogenous polypeptides during the ripening transition, in order to analyze their contribution to the free amino acid pool. In addition, the activity of some enzymes involved in glutamate metabolism such as γ-glutamyl transpeptidase (γ-GTase), glutamate dehydrogenase (GDH), α-ketoglutarate-dependent γ-aminobutyrate transaminase (GABA-T), alanine and aspartate aminotransferases was evaluated. Results showed that peptidases were very active in ripening fruits, and they were able to release free amino acids from endogenous proteins and glutamate from exogenously added glutamate-containing peptides. In addition, red fruit contained enough γ-GTase activity to sustain glutamate liberation from endogenous substrates such as glutathione. From all the glutamate metabolizing enzymes, GDH and GABA-T showed the higher increase in activities when the ripening process starts. In summary, tomato fruits increase free amino acid content during ripening, most probably due to the raise of different peptidase activities. However, glutamate level of ripe fruit seems to be mostly related to GDH and GABA-T activities that could contribute to increase l-glutamate level during the ripening transition.     

Vulnerability to glutamate toxicity of dopaminergic neurons is dependent on endogenous dopamine and MAPK activation

Dopaminergic neurons are more vulnerable than other types of neurons in cases of Parkinson disease and ischemic brain disease. An increasing amount of evidence suggests that endogenous dopamine plays a role in the vulnerability of dopaminergic neurons. Although glutamate toxicity contributes to the pathogenesis of these disorders, the sensitivity of dopaminergic neurons to glutamate toxicity has not been clarified. In this study, we demonstrated that dopaminergic neurons were preferentially affected by glutamate toxicity in rat mesencephalic cultures. Glutamate toxicity in dopaminergic neurons was blocked by inhibiting extracellular signal-regulated kinase (ERK), c- jun N-terminal kinase, and p38 MAPK. Furthermore, depletion of dopamine by α-methyl- dl - p -tyrosine methyl ester (α-MT), an inhibitor of tyrosine hydroxylase (TH), protected dopaminergic neurons from the neurotoxicity. Exposure to glutamate facilitated phosphoryration of TH at Ser31 by ERK, which contributes to the increased TH activity. Inhibition of ERK had no additive effect on the protection offered by α-MT, whereas α-MT and c- jun N-terminal kinase or p38 MAPK inhibitors had additive effects and yielded full protection. These data suggest that endogenous dopamine is responsible for the vulnerability to glutamate toxicity of dopaminergic neurons and one of the mechanisms may be an enhancement of dopamine synthesis mediated by ERK.     

Development of a primary microglia screening assay and its use to characterize inhibition of system xc- by erastin and its analogs

The inflammatory response in the central nervous system involves activated microglia. Under normal conditions they remove damaged neurons by phagocytosis. On the other hand, neurodegenerative diseases are thought to involve chronic microglia activation resulting in release of excess glutamate, proinflammatory cytokines and reactive oxygen species, leading to neuronal death. System x_C^- cystine/glutamate antiporter (SXC), a sodium independent heterodimeric transporter found in microglia and astrocytes in the CNS, imports cystine into the cell and exports glutamate. SXC has been shown to be upregulated in neurodegenerative diseases including multiple sclerosis, ALS, neuroAIDS Parkinson's disease and Alzheimer's disease. Consequently, SXC inhibitors could be of use in the treatment of diseases characterized by neuroinflammation and glutamate excitotoxicity. We report on the optimization of a primary microglia-based assay to screen for SXC inhibitors. Rat primary microglia were activated using lipopolysaccharides (LPS) and glutamate release and cystine uptake were monitored by fluorescence and radioactivity respectively. LPS-induced glutamate release increased with increasing cell density, time of incubation and LPS concentration. Conditions to screen for SXC inhibitors were optimized in 96-well format and subsequently used to evaluate SXC inhibitors. Known SXC inhibitors sulfasalazine, S-4CPG and erastin blocked glutamate release and cystine uptake while R-4CPG, the inactive enantiomer of S-4CPG, failed to inhibit glutamate release or cystine transport. In addition, several erastin analogs were evaluated using primary microglia and found to have EC₅₀ values in agreement with previous studies using established cell lines.     

Developmental change of endogenous glutamate and gamma-glutamyl transferase in cultured cerebral cortical interneurons and cerebellar granule cells,and in mouse cerebral cortex and cerebellum in vivo

The developmental change of endogenous glutamate, as correlated to that of gamma-glutamyl transferase and other glutamate metabolizing enzymes such as phosphate activated glutaminase, glutamate dehydrogenase and aspartate, GABA and ornithine aminotransferases, has been investigated in cultured cerebral cortex interneurons and cerebellar granule cells. These cells are considered to be GABAergic and glutamatergic, respectively. Similar studies have also been performed in cerebral cortex and cerebellum in vivo. The developmental profiles of endogenous glutamate in cultured cerebral cortex interneurons and cerebellar granule cells corresponded rather closely with that of gamma-glutamyl transferase and not with other glutamate metabolizing enzymes. In cerebral cortex and cerebellum in vivo the developmental profiles of endogenous glutamate, gamma-glutamyl transferase and phosphate activated glutaminase corresponded with each other during the first 14 days in cerebellum, but this correspondence was less good in cerebral cortex. During the time period from 14 to 28 days post partum the endogenous glutamate concentration showed no close correspondence with any particular enzyme. It is suggested that gamma-glutamyltransferase regulates the endogenous glutamate concentration in culture neurons. The enzyme may also be important for regulation of endogenous glutamate in brain in vivo and particularly in cerebellum during the first 14 days post partum. Gamma-glutamyl transferase in cultured neurons and brain tissue in vivo appears to be devoid of maleate activated glutaminase.Abbreviations used Asp-T aspartate aminotransferase (EC 2.6.1.1) - GABA-T GABA aminotransferase (EC 2.6.1.19) - GAD glutamate decarboxylase (EC 4.1.1.15) - gamma-GT gamma-glutamyl transferase (gamma-glutamyl transpeptidase) (EC. 2.3.2.2) - Glu glutamate - GDH glutamate dehydrogenase (EC 1.4.1.3) - GS glutamine synthetase (EC 6.3.1.2) - MAG maleate activated glutaminase - Orn-T ornithine aminotransferase (EC 2.6.1.13) - PAG phosphate activated glutaminase (EC 3.5.1.1)     

C-NMR spectroscopic evaluation of the citric acid cycle flux in conditions of high aspartate transaminase activity in glucose-perfused rat hearts

A new mathematical model, based on the observation of ¹³C-NMR spectra of two principal metabolites (glutamate and aspartate), was constructed to determine the citric acid cycle flux in the case of high aspartate transaminase activity leading to the formation of large amounts of labeled aspartate and glutamate. In this model, the labeling of glutamate and aspartate carbons by chemical and isotopic exchange with the citric acid cycle are considered to be interdependent. With [U-¹³C]Glc or [1,2-¹³C]acetate as a substrate, all glutamate and aspartate carbons can be labeled. The isotopic transformations of 32 glutamate isotopomers into 16 aspartate isotopomers or vice versa were studied using matrix operations; the results were compiled in two matrices. We showed how the flux constants of the citric acid cycle and the ¹³C-enrichment of acetyl-CoA can be deduced from ¹³C-NMR spectra of glutamate and/or aspartate. The citric acid cycle flux in beating Wistar rat hearts, aerobically perfused with [U-¹³C]glucose in the absence of insulin, was investigated by ¹³C-NMR spectroscopy. Surprisingly, aspartate instead of glutamate was found to be the most abundantly-labeled metabolite, indicating that aspartate transaminase (which catalyses the reversible reaction: (glutamate + oxaloacetate ↔ 2-oxoglutarate + aspartate) is highly active in the absence of insulin. The amount of aspartate was about two times larger than glutamate. The quantities of glutamate (G_o) or aspartate (A_O) were approximately the same for all hearts and remained constant during perfusion: G₀ = (0.74 ±0.03) μmol/g; A₀ = (1.49±0.05) μmol/g. The flux constants, i.e., the fraction of glutamate and aspartate in exchange with the citric acid cycle, were about 1.45 min⁻¹ and 0.72 min⁻¹, respectively; the flux of this cycle is about (1.07±0.02) μmol min^-1 g^-1. Excellent agreement between the computed and experimental data was obtained, showing that: i) in the absence of insulin, only 41% of acetyl-CoA is formed from glucose while the rest is derived from endogenous substrates; and ii) the exchange between aspartate and oxaloacetate or between glutamate and 2-oxoglutarate is fast in comparison with the biological transformation of intermediate compounds by the citric acid cycle.     

Crayfish sensory terminals and motor neurones exhibit two distinct types of GABA receptors

Motor neurones of the crayfish walking system display inhibitory responses evoked either by γ-amino butyric acid (GABA) or glutamate, possibly involving the same receptor (Pearlstein et al. 1994). In order to test if this sensibility to both GABA and glutamate was a specific property of crayfish GABA receptors, pharmacological characteristics of GABA-evoked responses in both sensory terminals from CB chordotonal organ and motor neurones of the walking system have been compared. Both receptors are GABA-gated Cl⁻ channels activated by specific GABA_A (muscimol, isoguvacine), GABA_B (3-aminopropyl phosphinic acid), and GABA_C (cis-4-amino crotonic acid) agonists, and blocked by competitive (β-guanidino propionic acid) and non-competitive (picrotoxin) antagonists. They were insensitive to specific GABA_A (bicuculline, SR-95531) and GABA_B (phaclofen) antagonists. Furthermore, in both cases, nipecotic acid and the modulatory drug diazepam had no effect. However, our results demonstrate that GABA receptors of sensory terminals are different from those of motor neurones. GABA-induced desensitisation only occurred in sensory terminals. Moreover, glutamate was shown to activate GABA-gated Cl⁻ channels in motor neurones, but not in sensory terminals. Therefore, GABA is likely to be the endogenous neurotransmitter of presynaptic inhibition in sensory terminals, whereas inhibition between antagonistic motor neurones would be achieved by glutamate. Accepted: 10 July 1996     

Gamma-Aminobutyric Acid (GABA) Modulates Nitrate Concentrations and Metabolism in the Leaves of Pakchoi (<Emphasis Type="Italic">Brassica campestris</Emphasis> ssp. <Emphasis Type="Italic">chinensis</Emphasis> Makino) Treated with a Nitrogen-Rich Solution

Pakchoi plants were grown in 32 mM NO₃^? nutrient solution with or without 2.5 mM γ-aminobutyric acid (GABA) to investigate metabolite changes, gene and protein expression levels, and the activities of key enzymes related to nitrate metabolism in the leaves over a period of 0–12 days. High-nitrogen treatment enhanced plant growth and the NO₃^?, NO₂^?, NH₄⁺, Gln, and Glu contents in the leaves; promoted the gene and protein expression of nitrate reductase (NR) and glutamate decarboxylase (GAD); and increased the activities of NR, nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT), and GAD. The endogenous GABA concentration in the leaves was enhanced in parallel with the increase in GAD activity. The GABA-treated leaves displayed the greatest increases in the gene and protein expression levels of NR and GAD and in the activities of NR, NiR, GS, GOGAT, and GAD. In addition, accelerated rates of nitrate reduction and assimilation were detected, and these changes occurred concurrently with the observed increases in gene or protein expression and enzyme activity. As a result, the concentrations of NH₄⁺, Gln, Glu, and endogenous GABA were significantly elevated, and the NO₃^? and NO₂^? contents were significantly decreased, in GABA-treated leaves compared with plants exposed to nitrogen-rich conditions. Our results reveal a potential positive that GABA may act as a nitrogen source to improve the plant growth and the most prominent effect of decreasing nitrate contents by accelerating NO₃^? reduction and assimilation. Exogenous GABA plays an important role in reducing the NO₃^? content of leaves, and thereby improves the ability to harvest leafy vegetables containing higher levels of endogenous GABA.     

Glutamate decarboxylase activity in striatal slices: characterization of the increase following depolarization.

Abstract— The activity of glutamate decarboxylase in rat striatal slices was estimated following preincubation in either a non-depolarizing medium or in a depolarizing medium. GAD activity was significantly increased following preincubation in normal Krebs-Ringer-phosphate medium as compared to activity in slices which were not preincubated. GAD activity in slices which were depolarized in high potassium Krebs-Ringer-phosphate medium was further increased when compared to activity in slices which were preincubated in normal medium. The depolarization-induced increase in GAD activity was graded in response to the time of depolarization, and both the increase following preincubation in normal medium and the increase following preincubation in high potassium displayed a relative requirement for calcium. In addition, net GABA formation from endogenous glutamate was increased in slices preincubated in high potassium medium as compared to net GABA formation from endogenous glutamate in slices preincubated in non-depolarizing medium. In support of the use of [¹⁴C]CO₂ trapping as an estimate of GAD activity in slices, preincubation of slices in the presence of the GAD inhibitor glutamic acid γ-hydrazide caused a concentration-related inhibition of [¹⁴C]CO₂ evolution.     

Mercuric chloride inhibits the in vitro uptake of glutamate in GLAST- and GLT-1-transfected mutant CHO-K1 cells

Glutamate is removed mainly by astrocytes from the extracellular fluid via high-affinity astroglial Na⁺-dependent excitatory amino acid transporters, glutamate/aspartate transporter (GLAST), and glutamate transporter-1 (GLT-1). Mercuric chloride (HgCl₂) is a highly toxic compound that inhibits glutamate uptake in astrocytes, resulting in excessive extracellular glutamate accumulation, leading to excitotoxicity and neuronal cell death. The mechanisms associated with the inhibitory effects of HgCl₂ on glutamate uptake are unknown. This study examines the effects of HgCl₂ on the transport of ³H-d-aspartate, a nonmetabolizable glutamate analog, using Chinese hamster ovary cells (CHO) transfected with two glutamate transporter subtypes, GLAST (EAAT1) and GLT-1 (EAAT2), as a model system. Additionally, studies were undertaken to determine the effects of HgCl₂ on mRNA and protein levels of these transporters. The results indicate that (1) HgCl₂ leads to significant (p<0.001) inhibition of glutamate uptake via both transporters, but is a more potent inhibitor of glutamate transport via GLAST and (2) the effect of HgCl₂ on inhibition of glutamate uptake in transfected CHO cells is not associated with changes in transporter protein levels despite a significant decrease in mRNA expression; thus, (3) HgCl₂ inhibition is most likely related to its direct binding to the functional thiol groups of the transporters and interference with their uptake function.     

Glutamate triggers intracellular Ca2+ oscillations and nitric oxide release by inducing NAADP- and InsP3-dependent Ca2+ release in mouse brain endothelial cells

The neurotransmitter glutamate increases cerebral blood flow by activating postsynaptic neurons and presynaptic glial cells within the neurovascular unit. Glutamate does so by causing an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in the target cells, which activates the Ca²⁺/Calmodulin-dependent nitric oxide (NO) synthase to release NO. It is unclear whether brain endothelial cells also sense glutamate through an elevation in [Ca²⁺]_i and NO production. The current study assessed whether and how glutamate drives Ca²⁺-dependent NO release in bEND5 cells, an established model of brain endothelial cells. We found that glutamate induced a dose-dependent oscillatory increase in [Ca²⁺]_i, which was maximally activated at 200 μM and inhibited by α-methyl-4-carboxyphenylglycine, a selective blocker of Group 1 metabotropic glutamate receptors. Glutamate-induced intracellular Ca²⁺ oscillations were triggered by rhythmic endogenous Ca²⁺ mobilization and maintained over time by extracellular Ca²⁺ entry. Pharmacological manipulation revealed that glutamate-induced endogenous Ca²⁺ release was mediated by InsP₃-sensitive receptors and nicotinic acid adenine dinucleotide phosphate (NAADP) gated two-pore channel 1. Constitutive store-operated Ca²⁺ entry mediated Ca²⁺ entry during ongoing Ca²⁺ oscillations. Finally, glutamate evoked a robust, although delayed increase in NO levels, which was blocked by pharmacologically inhibition of the accompanying intracellular Ca²⁺ signals. Of note, glutamate induced Ca²⁺-dependent NO release also in hCMEC/D3 cells, an established model of human brain microvascular endothelial cells. This investigation demonstrates for the first time that metabotropic glutamate-induced intracellular Ca²⁺ oscillations and NO release have the potential to impact on neurovascular coupling in the brain.     

Berberine Inhibits the Release of Glutamate in Nerve Terminals from Rat Cerebral Cortex

Berberine, an isoquinoline plant alkaloid, protects neurons against neurotoxicity. An excessive release of glutamate is considered to be one of the molecular mechanisms of neuronal damage in several neurological diseases. In this study, we investigated whether berberine could affect endogenous glutamate release in nerve terminals of rat cerebral cortex (synaptosomes) and explored the possible mechanism. Berberine inhibited the release of glutamate evoked by the K⁺ channel blocker 4-aminopyridine (4-AP), and this phenomenon was prevented by the chelating extracellular Ca²⁺ ions and the vesicular transporter inhibitor bafilomycin A1, but was insensitive to the glutamate transporter inhibitor DL-threo-beta-benzyl-oxyaspartate. Inhibition of glutamate release by berberine was not due to it decreasing synaptosomal excitability, because berberine did not alter 4-AP-mediated depolarization. The inhibitory effect of berberine on glutamate release was associated with a reduction in the depolarization-induced increase in cytosolic free Ca²⁺ concentration. Involvement of the Ca_v2.1 (P/Q-type) channels in the berberine action was confirmed by blockade of the berberine-mediated inhibition of glutamate release by the Ca_v2.1 (P/Q-type) channel blocker ω-agatoxin IVA. In addition, the inhibitory effect of berberine on evoked glutamate release was prevented by the mitogen-activated/extracellular signal-regulated kinase kinase (MEK) inhibitors. Berberine decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and synapsin I, the main presynaptic target of ERK; this decrease was also blocked by the MEK inhibition. Moreover, the inhibitory effect of berberine on evoked glutamate release was prevented in nerve terminals from mice lacking synapsin I. Together, these results indicated that berberine inhibits glutamate release from rats cortical synaptosomes, through the suppression of presynaptic Cav2.1 channels and ERK/synapsin I signaling cascade. This finding may provide further understanding of the mode of berberine action in the brain and highlights the therapeutic potential of this compound in the treatment of a wide range of neurological disorders.     

Glutamate as a Putative Transmitter in the Cerebellum: Stimulation by GABA of Glutamic Acid Release from Specific Pools

The aim of the present paper was to determine whether the release of glutamate from putative "glutamergic" terminals in the cerebellum is influenced by gamma-aminobutyric acid (GABA). In a group of preliminary experiments, we present biochemical evidence in favour of a neurotransmitter role of glutamate in the cerebellum: (1) endogenous glutamate was released from depolarized cerebellar synaptosomal preparations in a Ca2+-dependent away; (2) [14C]glutamate was synthesized from [14C]glutamine in cerebellar synaptosomes, and the newly synthesized [14C]glutamate was released released in a Ca2+-dependent way; (3) the elevation of cyclic GMP elicited by depolarization of cerebellar slices in the presence of Ca2+ was partly reversed by the glutamate antagonist glutamic acid diethyl ester, which probably prevented the interaction of endogenously released glutamate with postsynaptic receptors. GABA and muscimol at low concentrations (2--20 micrometers) potentiated the depolarization-induced release of D-[3H]aspartate (a glutamate analogue which labels the glutamate "reuptake pool") from cerebellar synaptosomes. The effect was concentration dependent and was largely prevented by two GABA antagonists, bicuculline and picrotoxin. The stimulation of D-[3H]aspartate release evoked by muscimol was linearly related to the logarithm of K+ concentration in the depolarizing medium. GABA did not affect the overall release of endogenous glutamate, but potentiated, in a picrotoxin-sensitive manner, the depolarization-evoked release of [14C]glutamate previously synthesized from [14C]glutamine. Since nerve endings are the major site of glutamate synthesis from glutamine, GABA and muscimol appear to exert their stimulatory effect at the level of "glutamergic" nerve terminals, probably after interacting with presynaptic GABA receptors. The possible functional significance of these findings is briefly discussed.