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PubMed1993

Changes in NAD(P)H fluorescence and membrane current produced by glutamate uptake into salamander Müller cells.

1. Glutamate uptake into isolated, whole-cell patch-clamped glial cells was studied by monitoring the increase of cell fluorescence generated as glutamate and NAD(P) were converted into alpha-ketoglutarate and NAD(P)H by glutamate dehydrogenase. The current generated by the glutamate uptake carrier was recorded simultaneously. 2. L-Glutamate evoked an increase of cell fluorescence and an inward uptake current. L- and D-aspartate generated an uptake current but no fluorescence response, consistent with the amino acid specificity of glutamate dehydrogenase. 3. In the absence of external sodium the glutamate-evoked fluorescence response and uptake current were abolished, showing that there is no sodium-independent glutamate uptake across the cell membrane. 4. Varying the glutamate concentration altered both the fluorescence response and the uptake current. The fluorescence response saturated at a lower glutamate concentration than the uptake current, and depended in a Michaelis-Menten fashion on the uptake current. 5. The fluorescence response and the uptake current were reduced by membrane depolarization, and also by removal of intracellular potassium. 6. The dependence of the fluorescence response on uptake current when membrane potential was altered or intracellular potassium was removed was the same as that seen when the external glutamate concentration was altered. 7. These fluorescence studies show that glutamate uptake is inhibited by depolarization and by removal of intracellular potassium, consistent with the conclusion of earlier work in which uptake was monitored solely as a membrane current. The data are consistent with high-affinity electrogenic sodium- and potassium-dependent glutamate uptake with fixed stoichiometry being the only significant influx route for glutamate. Other possible interpretations of the data are also discussed.

Action Potentials↗

PubMed1997

Glutamate transport and metabolism in astrocytes.

Glutamate uptake and metabolism was studied in cerebral cortical astrocytes. The expression of the astrocytic glutamate transporter GLAST was found to be stimulated by extracellular glutamate through activation of kainate receptors on the astrocytes. Energy metabolism and ammonia homeostasis are two important aspects of glutamate handling in astrocytes. It is well known that glutamate transport into astrocytes and glutamine formation are energy consuming processes. Furthermore, ammonia is required for glutamine production. On the other hand, glutamate metabolism through the tricarboxylic acid cycle is an energy and ammonia producing pathway. In the present study it was shown that at an extracellular glutamate concentration of 0.5 mM, high energy phosphates were reduced, and more than 50% of the glutamate carbon skeleton entered the tricarboxylic acid cycle to yield products like lactate, aspartate, and additionally glutamate and glutamine derived from tricarboxylic acid cycle intermediates. Entry into the cycle was not affected by the transaminase inhibitor aminooxyacetic acid, indicating that deamination is the major route for 2-oxoglutarate formation from glutamate. Synthesis of glutamate from 2-oxoglutarate, however, proceeded via transamination. In an earlier study it was shown that at glutamate concentrations at and below 0.2 mM, glutamine appears to be the major product and entry of glutamate into the tricarboxylic acid cycle is decreased 70% by aminooxyacetic acid. In an attempt to unify the above mentioned results, it is suggested that availability of ammonia and energy demands are major factors determining the metabolic fate of glutamate in astrocytes.

ATP-Binding Cassette Transporters↗

PubMed2005

Glial restricted precursors protect against chronic glutamate neurotoxicity of motor neurons in vitro.

We have examined the expression of glutamate transporters in primary and immortalized glial precursors (GRIPs). We subsequently transduced these cells with the GLT1 glutamate transporter and examined the ability of these cells to protect motor neurons in an organotypic spinal cord culture. We show that glial restricted precursors and GRIP-derived astrocytes predominantly express glutamate transporters GLAST and GLT1. Oligodendrocyte differentiation of GRIPs results in downregulation of all glutamate transporter subtypes. Having identified these precursor cells as potential vectors for delivering glutamate transporters to regions of interest, we engineered a line of GRIPS that overexpress the glutamate transporter GLT1. These cells (G3 cells) have a nearly fourfold increase in glutamate transporter expression and at least a twofold increase in the V(max) for glutamate transport. To assess whether G3 seeding can protect motor neurons from chronic glutamate neurotoxicity, G3s were seeded onto rat organotypic spinal cord cultures. These cultures have previously been used extensively to understand pathways involved in chronic glutamate neurotoxicity of motor neurons. After G3 seeding, cells integrated into the culture slice and resulted in levels of glutamate transport sufficient to enhance total glutamate uptake. To test whether neuroprotection was related to glutamate transporter overexpression, we isolated GRIPS from the GLT1 null mouse to serve as controls. The seeding of G3s resulted in a reduction of motor neuron cell death. Hence, we believe that these cells may potentially play a role in cell-based neuroprotection from glutamate excitotoxicity.

Amino Acid Transport System X-AG↗

PubMed2001

Glutamate decreases pyruvate carboxylase activity and spares glucose as energy substrate in cultured cerebellar astrocytes.

The effects of glutamate on [U-(13)C]glucose metabolism were studied in cerebellar astrocytes using (13)C magnetic resonance spectroscopy. Labeled glutamate, glutamine, aspartate, lactate, and alanine were observed both in the cell extracts and in media, and, additionally, labeled glycogen was detected in the cell extracts. However, only labeled lactate and alanine were quantifiable in the medium in addition to [U-(13)C]glucose. In the presence of unlabeled glutamate, the amount of [U-(13)C]glucose removed from the medium was decreased, indicating that glutamate might spare glucose as an energy substrate and thus decrease the uptake of glucose. Labeled glycogen, [4,5-(13)C]glutamate, [3,4,5-(13)C]glutamate, [3,4-(13)C]aspartate, and [U-(13)C]alanine were increased in the presence of glutamate. However, the increase in the amount of [3,4,5-(13)C]glutamate from the second turn in the tricarboxylic acid (TCA) cycle was less pronounced than that of [4,5-(13)C]glutamate from the first turn in the TCA cycle. This indicates the dilution of label, probably resulting from the synthesis of unlabeled oxaloacetate from glutamate in the TCA cycle. Furthermore, exogenous glutamate had an inhibiting effect on pyruvate carboxylation, presumably by formation of oxaloacetate from 2-oxoglutarate derived from glutamate. It could be shown that glucose is a better substrate for energy production than glutamate; it is, however, less efficient in labeling amino acids than glutamate in cerebellar astrocytes.

Amino Acids↗

PubMed1999

Neurons depend on astrocytes in a coculture system for protection from glutamate toxicity.

Glutamate can be toxic to neurons although it is a neurotransmitter. Regulation of extracellular glutamate levels is essential for prevention of glutamate neurotoxicity. Astrocytes play a major role in clearance of glutamate released by neurons. A coculture system combining cerebellar cells and astrocytes was employed to investigate the astrocytic control of glutamate toxicity. Coculture of astrocytes with cerebellar neurons enhanced uptake of glutamate by astrocytes. Inhibition of glutamate uptake in a coculture system led to death of cerebellar cells. This toxicity could be inhibited by MK801. However, in the presence of the glutamate uptake inhibitor, there was no increase in glutamate in the cultures compared to when the neurons were not cocultured. This indicated that neurons become more susceptible to glutamate toxicity in the presence of astrocytes and thus become dependent on astrocytes for prevention of glutamate toxicity. Astrocytes treated with conditioned medium from cerebellar cells did not show an increase in glutamate uptake but medium from astrocytes exposed to neuron conditioned medium was toxic to cerebellar cells. This toxicity was due to glutamate present in the medium. This suggests that a soluble factor released by neurons signals to astrocytes that neurons are present and stimulates a signal back to neurons which causes an increased sensitivity to glutamate toxicity.

Animals↗

PubMed2005

Time-dependent changes in extracellular glutamate in the rat dorsolateral striatum following a single cocaine injection.

Acute cocaine administration has been shown to alter dorsal striatal plasticity [Proc Natl Acad Sci USA 87 (1990) 6912; Brain Res Bull 30 (1993) 173] and produce long-term neurochemical changes [Pharmacol Biochem Behav 27 (1987) 533]. To date, the effects of acute cocaine on extracellular glutamate and nerve terminal glutamate immunolabeling in the rat dorsolateral striatum have not been reported. To investigate cocaine-induced changes in extracellular glutamate, in vivo microdialysis was carried out in the dorsolateral striatum of rats 1-14 days after receiving a single injection of either vehicle or 15 mg/kg cocaine. There was an increase in the group injected with cocaine 1 day prior to measuring extracellular glutamate as compared with the control group. The group injected with cocaine 3 days prior to the microdialysis session had decreased extracellular glutamate levels. Furthermore, extracellular glutamate remained attenuated 14 days after acute cocaine treatment. Striatal glutamate decreased in the cocaine-treated rats after calcium removal, suggesting that cocaine-induced changes in extracellular glutamate were partially calcium-dependent. The density of nerve terminal glutamate immunolabeling was measured using immunogold electron microscopy in the contralateral striatum of the same rats that had been acutely treated with cocaine or vehicle. There were no changes in the density of glutamate immunolabeling within identified nerve terminals making an asymmetrical (excitatory) synaptic contact 1, 2, 3, or 14 days after acute cocaine exposure as compared with the control groups. Hence, these alterations in extracellular glutamate did not result from changes in glutamate immunolabeling within the synaptic vesicle pool. In addition, no changes in glutamate immunolabeling were found in rats that received cocaine 2 h previously or were withdrawn after 1 week of cocaine administration. The results demonstrate that a single injection of cocaine produces biphasic, time-dependent changes in extracellular glutamate in the rat dorsolateral striatum.

Animals↗

PubMed2003

The role of neuropeptides and capsaicin-sensitive fibres in glutamate-induced nociception and paw oedema in mice.

This study sought to establish whether sensory neuropeptides and the capsaicin-sensitive fibres are involved in the nociception and oedema formation caused by intraplantar (i.pl.) injection of glutamate into the mouse paw. The i.pl. co-injection of the selective neurokinin (NK) NK(2) (SR 48968, 0.05-0.5 nmol/paw), and to a lesser extent the selective NK(1) (FK 888, 0.25-1.0 nmol/paw) receptor antagonists, resulted in a significant inhibition of glutamate-induced nociception. The percentages of inhibition were 82 and 37%, respectively. In contrast, the selective NK(3) receptor antagonist (SR 142801, 0.25-1.0 nmol/paw) failed to significantly affect glutamate-induced nociception. SR 48968, but not FK 888 or SR 142801, significantly inhibited (36%) glutamate-induced paw oedema formation. The i.pl. injection of kinin B(1) receptor antagonist des-Arg(9)-[Leu(8)]-BK (0.2-0.8 nmol/paw), but not the B(2) receptor antagonist HOE 140 (1.0-4.0 nmol/paw), together with glutamate, also inhibited glutamate-induced nociception (53%) in a graded manner, without affecting glutamate-induced paw oedema. The i.pl. co-injection of the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP(8-37) (1 nmol/paw) failed to significantly inhibit glutamate-induced nociception or oedema. Finally, neonatal-capsaicin (50 mg/kg, s.c.) treatment inhibited glutamate-induced nociception by 69% and to a lesser extent glutamate-mediated oedema formation (30%). Collectively, the current results indicate that the nociception caused by i.pl. injection of glutamate in mice is clearly mediated by capsaicin-sensitive fibres and by release of neurokinins from sensory neurones that activate NK(2) receptors and to a lesser extent NK(1) receptors. Furthermore, kinins acting at B(1) (but not at B(2)) receptors also largely account for glutamate-mediated nociceptive behaviour response. In contrast, glutamate-induced paw oedema seems to be primarily mediated via activation of NK(2) receptors and stimulation of capsaicin-sensitive C-fibres. CGRP receptors do not seem to be involved in either of the glutamate responses.

Animals↗

PubMed2000

Glutamate receptors in the enteric nervous system: ionotropic or metabotropic?

Intracellular recording methods were used to investigate actions of glutamate on morphologically identified neurones in the myenteric and submucous plexuses of guinea-pig small intestine. Glutamate evoked a tetrodotoxin-resistant, slowly activating depolarizing response in most of the submucous neurones (86 of 125, 69%) and a smaller number of myenteric neurones (6 of 60, 10%). The depolarizing responses were restricted to S-type neurones with uniaxonal morphology. The group I metabotropic glutamate receptor (mGluRs) agonists quisqualate, 1S, 3R-ACPD and DHPG mimicked the depolarizing action of glutamate. A group I mGluRs antagonist, S-4-carboxyphenylglycine (S-4CPG), suppressed the glutamate responses with an IC50 of 357 microM at 30 microM glutamate. Group II or III mGluRs agonists did not produce depolarizing responses and group II or III mGluRs antagonists did not alter glutamate-evoked depolarization. The ionotropic glutamate receptor (iGluRs) agonists NMDA, AMPA, or kainate did not evoke depolarizing responses and glutamate-evoked depolarization was unaffected by the iGluRs antagonists D-APV, MK-801, or DNQX. No rapidly activating fast depolarizing responses reminiscent of fast excitatory postsynaptic potentials (EPSPs) were ever observed during application of glutamate or AMPA and stimulus-evoked fast EPSPs were unaffected by DNQX. The results suggest that the excitatory action of glutamate on enteric neurones is mediated by group I metabotropic glutamate receptors and that ionotropic glutamate receptors are not involved. The results also suggest that glutamate-mediated fast EPSPs may not be present in myenteric and submucous neurones in guinea-pig small bowel.

Animals↗

PubMed1998

Assessment of myocardial glutamate requirements early after coronary artery bypass surgery.

Glutamate is an important substrate for the intermediary metabolism of the heart, particularly in association with ischemia. Early after coronary artery bypass surgery (CABG) myocardial uptake of glutamate seems to be limited by substrate availability (arterial levels). However, glutamate is not an innocuous substrate. As arterial levels of glutamate are important both for myocardial uptake and adverse effects, an attempt was made to determine a minimum dose of glutamate sufficient to supply the needs of the heart after CABG. Ten patients received and infusion of 220-240 ml of 0.1 M L-glutamic acid solution at varying rates during two 30-min periods, starting 2 h after uncomplicated elective CABG. Intravenous glutamate infusion caused a dose-dependent linear increase in arterial glutamate and an increased myocardial uptake of glutamate. However, myocardial uptake of glutamate correlated with arterial levels only at lower infusion rates. Although maximal peak uptake in individual patients (6.6 +/- 1.1 mumol/min) occurred at an average increase of arterial whole blood glutamate of 172 +/- 34 mumol/L, the greatest impact on myocardial glutamate uptake was achieved by increasing arterial whole blood glutamate by less than 100 mumol/L. This implies that an infusion rate of 30-40 mg glutamate/kg BW/h could suffice to achieve a maximal or near maximal myocardial glutamate uptake in most patients after CABG. The adequacy of this dosage remains to be confirmed in high-risk patients.

Aged↗

PubMed2000

Compartmentation of brain glutamate metabolism in neurons and glia.

Intrasynaptic [glutamate] must be kept low in order to maximize the signal-to-noise ratio after the release of transmitter glutamate. This is accomplished by rapid uptake of glutamate into astrocytes, which convert glutamate into glutamine. The latter then is released to neurons, which, via mitochondrial glutaminase, form the glutamate that is used for neurotransmission. This pattern of metabolic compartmentation is the "glutamate-glutamine cycle." This model is subject to the following two important qualifications: 1) brain avidly oxidizes glutamate via aspartate aminotransferase; and 2) because almost no glutamate crosses from blood to brain, it must be synthesized in the central nervous system (CNS). The primary source of glutamate carbon is glucose, and a major source of glutamate nitrogen is the branched-chain amino acids, which are transported rapidly into the CNS. This arrangement accomplishes the following: 1) maintenance of low external [glutamate], thereby maximizing signal-to-noise ratio upon depolarization; 2) the replenishing of the neuronal glutamate pool; 3) the "trafficking" of glutamate through the extracellular fluid in a nonneuroactive form (glutamine); 4) the importation of amino groups from blood, thus maintaining brain nitrogen homeostasis; and 5) the oxidation of glutamate/glutamine, a process that confers an additional level of control in terms of the regulation of brain glutamate, aspartate and gamma-aminobutyric acid.

Animals↗

PubMed2000

Effects of mild versus deep hypothermia on a cloned human brain glutamate transporter (GLT-1) expressed in Chinese hamster ovary cells.

Glutamate transporters, widely distributed in the brain and spinal cord, maintain extracellular glutamate concentrations below neurotoxic levels. In cerebral ischemia/anoxia, the glutamate transporter runs in reverse and releases glutamate into the extracellular space, causing irreversible neuronal damage. Although hypothermia reduces the elevation of extracellular glutamate concentration during cerebral ischemia/anoxia, little is known about the effect of hypothermia on the glutamate transporter. A human glial glutamate transporter (hGLT-1) cDNA was isolated by screening a human cerebral cortical library, and cloned cDNA was stably transfected in Chinese hamster ovary (CHO) cells. Effects of deep hypothermia (22 to 23 degrees C) on uptake and release of L-glutamate via hGLT-1 were investigated by whole-cell patch-clamp. The control study was performed at 34 to 35 degrees C. The hGLT-1 transporter had the capacity to take up extracellular L-glutamate under essentially physiological ionic conditions, whereas this transporter promoted release of L-glutamate under a nonphysiological condition mimicking complete ischemia. Deep hypothermia decreased a) uptake and b) release of L-glutamate via hGLT-1 to a) 4.8+/-4.8% (P < .01, n = 7) and b) 19.0+/-4.5% (P < .01, n = 15) of control values, respectively. The results suggest that deep hypothermia is a potent inhibitor of glutamate uptake by intact glial cells as well as glutamate release from glial cells under certain pathophysiological circumstances. The balance between these antagonistic effects of hypothermia may attenuate the elevation of the extracellular glutamate concentration during ischemia/anoxia.

ATP-Binding Cassette Transporters↗

PubMed2005

Skeletal muscle glutamate metabolism in health and disease: state of the art.

PURPOSE OF REVIEW: Glutamate is an amino acid of interest because it participates in many metabolic pathways. However, there is evidence that skeletal muscle glutamate metabolism is disturbed in disease. This review presents current knowledge regarding the metabolic function and regulation of glutamate in skeletal muscle under physiological and pathophysiological circumstances. Furthermore, several options for modulating muscle glutamate concentration in order to improve glutamate metabolism are discussed. RECENT FINDINGS: The high correlation between muscle glutamate concentration and muscle glutathione concentration suggests that glutamate plays a determining role in the glutathione synthesis pathway. During exercise, glutamate plays a central role in energy provision because it participates in the tricarboxylic acid and the purine nucleotide cycles. However, a consistent finding in several diseases is reduced skeletal muscle glutamate. Remarkably, only few studies focused on modulation of muscle glutamate status either by exercise or by nutritional supplementation. There are several options for modulating glutamate metabolism, but the specific effects of the individual options require further elucidation. Nutritional supplementation of glutamate or its precursors glutamine, (ornithine) alpha-ketoglutarate, or the branched chain amino acids can influence muscle glutamate status. SUMMARY: Specific intervention studies must be conducted to investigate the effect of supplementation on skeletal muscle glutamate turnover and its related metabolic and functional consequences in healthy individuals and in patients with acute or chronic disease.

Dietary Supplements↗

PubMed1979

Further observations on the interaction between glutamate and aspartate on lobster muscle.

1 The ability of bath-applied L-glutamate to enhance subsequent depolarizations produced by bath-applied L-aspartate on lobster muscle was further investigated by means of intracellular recording techniques. 2. Increasing the conditioning glutamate concentration or exposure time produced a greater enhancement of aspartate responses. Enhancement was also dependent on the time interval between glutamate and aspartate doses and was not prevented by overnight storage of preparations in vitro. 3. The dose-depolarization curve for enhanced aspartate responses (measured at a fixed time following a given dose of glutamate) was displaced to the left along the abscissa scale relative to control, with no detectable change in limiting log-log slope. 4. Conditioning doses of kainate or domoate (but not quisqualate, aspartate, or KCl) also enhanced aspartate responses; however, their conditioning effect was little affected by increasing the concentration, exposure time, or time interval before applying aspartate. The rate of onset and decline of the enhanced aspartate response always resembled that of the previous conditioning agonist. 5. D and L-Aspartate were approximately equieffective depolarizing agents whereas D-glutamate was approximately 1/40 as potent as L-glutamate. After a conditioning dose of D or L-glutamate, responses to D or L-aspartate were enhanced. 6. In a Na+-free (Li+) medium, both the glutamate depolarization and the conditioning effect towards aspartate were largely abolished. With kainate however, Na+ was not apparently important either for evoking the kainate response or for producing the conditioning effect. 7. Bath-applied glutamate greatly enhanced and prolonged the time course of the iontophoretic aspartate potential with only a small effect on the glutamate potential; however, these effects were not maintained after washout of glutamate. In contrast, bath-application of aspartate depressed the aspartate potential while enhancing the glutamate potential. Some sites that were insensitive to iontophoretically-applied aspartate became clearly responsive to this agent during a bath-application of glutamate. 8. It is proposed that during conditioning with bath-applied glutamate, kainate or domoate, some agonist is trapped by extrajunctional sites and is subsequently displaced by bath-applied aspartate to produce the long-term enhancement effect.

Animals↗

PubMed1999

Sodium-dependent glutamate uptake by an alkaliphilic, thermophilic Bacillus strain, TA2.A1.

A strain of Bacillus designated TA2.A1, isolated from a thermal spring in Te Aroha, New Zealand, grew optimally at pH 9.2 and 70 degrees C. Bacillus strain TA2.A1 utilized glutamate as a sole carbon and energy source for growth, and sodium chloride (>5 mM) was an obligate requirement for growth. Growth on glutamate was inhibited by monensin and amiloride, both inhibitors that collapse the sodium gradient (DeltapNa) across the cell membrane. N, N-Dicyclohexylcarbodiimide inhibited the growth of Bacillus strain TA2.A1, suggesting that an F1F0-ATPase (H type) was being used to generate cellular ATP needed for anabolic reactions. Vanadate, an inhibitor of V-type ATPases, did not affect the growth of Bacillus strain TA2.A1. Glutamate transport by Bacillus strain TA2.A1 could be driven by an artificial membrane potential (DeltaPsi), but only when sodium was present. In the absence of sodium, the rate of DeltaPsi-driven glutamate uptake was fourfold lower. No glutamate transport was observed in the presence of DeltapNa alone (i.e., no DeltaPsi). Glutamate uptake was specifically inhibited by monensin, and the Km for sodium was 5.6 mM. The Hill plot had a slope of approximately 1, suggesting that sodium binding was noncooperative and that the glutamate transporter had a single binding site for sodium. Glutamate transport was not affected by the protonophore carbonyl cyanide m-chlorophenylhydrazone, suggesting that the transmembrane pH gradient was not required for glutamate transport. The rate of glutamate transport increased with increasing glutamate concentration; the Km for glutamate was 2.90 microM, and the Vmax was 0.7 nmol. min-1 mg of protein. Glutamate transport was specifically inhibited by glutamate analogues.

Amiloride↗

PubMed1966

Energy requirement for L-glutamate uptake and utilization by Hansenula subpelliculosa cells.

Shieh, K. Z. (Illinois Institute of Technology, Chicago), and L. R. Hedrick. Energy requirement for l-glutamate uptake and utilization by Hansenula subpelliculosa cells. J. Bacteriol. 92:1638-1644. 1966.-Cells of the yeast Hansenula subpelliculosa require an energy source for the uptake of glutamate. A lag period of 20 to 40 min was required after the addition of glucose to the cells before glutamate uptake was initiated. When cells were preincubated in glucose, and washed with distilled water prior to the addition of glutamate, there was no lag period. Preincubation in glucose and glutamate lowered both the rate and the total uptake of glutamate as compared with cells preincubated in glucose alone. This is attributed to the partial utilization of the glucose-metabolite by glutamate or to the partial saturation of binding sites by glutamate during the preincubation period. Transport of glutamate by these yeast cells appears to be via a carrier, where energy is required for the binding of the amino acid to nonspecific binding sites. In addition to total uptake, some aspects of the C(14)-glutamate utilization were measured. Of the total uptake, 58% was metabolized and converted to CO(2), 25.2% remained in the soluble pool, and 16.8% was incorporated into trichloroacetic acid-insoluble products. When the available energy source was depleted, the processes of uptake, metabolism, and incorporation ceased, even though there was an ample supply of glutamate present within the cells. Removal of cells from glutamate and addition of glucose reinitiated the incorporation of glutamate into proteins and other trichloroacetic acid-insoluble compounds. Therefore, an additional energy source is required with this species of yeast for glutamate uptake, for the priming of mechanisms required for its metabolism, and for its incorporation.

Ascomycota↗

PubMed1994

Intracellular pH changes produced by glutamate uptake in rat hippocampal slices.

1. The mean intracellular pH in area CA1 of rat hippocampal slices was monitored fluorescently after loading the cells with the dye BCECF-AM. 2. Including L-glutamate in the solution superfusing the slice led to the intracellular pH becoming more acid. This acidification had a roughly Michaelis-Menten dependence on the superfused glutamate concentration with a half-maximal dose around 200 microM: this value must overestimate the glutamate concentration at most of the cells, which will be reduced by uptake. 3. The glutamate-evoked acidification was not significantly reduced by blockers of glutamate-gated ion channels [6-cyano-7-nitroquinoxaline-2,3- dione (CNQX) and D-aminophosphonovalerate (APV)] nor by blockers of gamma-aminobutyric acid (GABA)- and glycine-gated channels (picrotoxin and strychnine), and so was not produced by H+ entry through alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) or N-methyl-D-aspartate (NMDA) receptor channels nor by HCO3- exit through the chloride channels controlled by GABA or glycine. 4. The glutamate-evoked acidification was not reduced by tetrodotoxin (TTX), ruling out the possibility of it being generated by action potentials. It was also unaffected by saturation of presynaptic L-amino-4-phosphonobutanoate (AP4) receptors with AP4. 5. In the presence of blockers of glutamate-, GABA-, and glycine-gated channels, the acidification showed the pharmacology of glutamate uptake and was reduced by a glutamate uptake blocker. 6. The glutamate-evoked acidification showed an ion dependence similar to that of glutamate uptake. It was abolished by removal of extracellular sodium and was reduced by raising the extracellular potassium concentration. It was unaffected by blockers of Na+/H+ exchange (amiloride) and Na+/HCO3- cotransport [4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)] and so was not produced by the Na+ influx accompanying glutamate uptake changing the activity of these carriers. 7. These data show that the glutamate uptake carrier acidifies hippocampal cells, possibly because it transports a pH-changing anion out of the cell as in salamander glial cells. Glutamate uptake may thus contribute to activity-induced pH changes in the nervous system.

Acid-Base Equilibrium↗

PubMed1997

Prolonged physiological entrapment of glutamate in the synaptic cleft of cerebellar unipolar brush cells.

The cellular mechanism underlying the genesis of the long-lasting alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-receptor-mediated excitatory postsynaptic currents (EPSCs) at the mossy fiber (MF)-unipolar brush cell (UBC) synapse in rat vestibular cerebellum was examined with the use of whole cell and excised patch-clamp recording methods in thin cerebellar slices. Activation of MFs evokes an all-or-none biphasic AMPA-receptor-mediated synaptic current with a late component that peaks at 100-800 ms, which has been proposed to originate from an entrapment of glutamate in the MF-UBC synaptic cleft and is generated by the steady-state activation of AMPA receptors. Bath application of cyclothiazide, which blocks desensitization of AMPA receptors, produced a dose-dependent enhancement of the amplitude of the synaptic current (median effective dose 30 microM) and slowing of the rise time of the fast EPSC. N-methyl-D-aspartate-receptor-mediated EPSCs in UBCs were not potentiated in amplitude or time course by cyclothiazide (100 microM). The dose-response relations for the steady-state current evoked by glutamate acting at AMPA receptors in excised outside-out patches from UBC and granule somatic membranes was biphasic, peaking at 50 microM and declining to 50-70% of this value at 1 mM glutamate. When glutamate was slowly washed from patches to simulate the gradual decline of glutamate in the synapse, a late hump in the transmembrane current was observed in patches from both cell types. The delivery of a second MF stimulus at the peak of the slow EPSC evoked a fast EPSC of reduced amplitude followed by an undershoot of the subsequent slow current, consistent with the hypothesis that the peak of the slow EPSC reflects the peak of the biphasic steady-state dose-response curve. Estimates of receptor occupancy and glutamate concentration derived from the ratio of fast EPSC amplitudes, and the amplitude and polarity of the initial steady-state current in paired-pulse experiments, predict a slow decline of glutamate with a time constant of 800 ms, declining to ineffective concentrations at 5.4 s. Manipulation of cleft glutamate concentration by lowered extracellular calcium or delivery of brief stimulus trains abolished the slow EPSC and restored the undershoot to paired stimuli, respectively, in a manner consistent with a prolonged lifetime of glutamate in the cleft. The slow component of the EPSC was prolonged in duration by the glutamate reuptake inhibitor L-trans-pyrrolidine-2,4-dicarboxylate, suggesting that glutamate transport contributes to the time course of the synaptic current in UBCs. The data support the notion that the MF-UBC synapse represents an ultrastructural specialization to effectively entrap glutamate for unusually prolonged periods of time following release from MF terminals. The properties of the postsynaptic receptors and constraints on diffusional escape of glutamate imposed by synaptic ultrastructure and glutamate transporters act in concert to sculpt the time course of the resulting slow EPSC. This in turn drives a long-lasting train of action potentials in response to single presynaptic stimuli.

Animals↗

PubMed2000

Evaluation of L-glutamate clearance capacity of cultured rat cortical astrocytes.

Astrocytes have a function in the uptake of excitatory neurotransmitter L-glutamate via the glutamate transporter. To evaluate the L-glutamate clearance capacity of astrocytes, we developed a colorimetric method for the determination of L-glutamate concentration and measured changes in extracellular L-glutamate concentration in rat cortical astrocyte cultures. When L-glutamate (50-200 microM) was added to astrocyte cultures and incubated for 1-8 h, the extracellular L-glutamate concentration declined with time. When L-glutamate was mixed with astrocyte culture supernatants only, no significant change in L-glutamate concentration was observed, ruling out the possibility that L-glutamate is spontaneously or enzymatically degraded in the extracellular space. Alternatively, the time-dependent decline of extracellular L-glutamate concentration was blocked by the presence of glutamate uptake inhibitors, indicating that the glutamate uptake system of astrocytes plays a major role in the clearance of extracellular L-glutamate.

Animals↗