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PubMed1999

Glutamate receptors and circuits in the vertebrate retina.

We survey the evidence for L-glutamate's role as the primary excitatory neurotransmitter of vertebrate retinas. The physiological and molecular properties of glutamate receptors in the retina are reviewed in relation to what has been learned from studies of glutamate function in other brain areas and in expression systems. We have focused on (a) the evidence for the presence of L-glutamate in retinal neurons, (b) the processes by which glutamate is released, (c) the presence and function of ionotropic receptors for L-glutamate in retinal neurons, (d) the presence and function of metabotropic receptors for L-glutamate in retinal neurons, and (e) the variety and distribution of glutamate transporters in the vertebrate retina. Modulatory pathways which influence glutamate release and the behavior of its receptors are described. Emphasis has been placed on the cellular mechanisms of glutamate-mediated neurotransmission in relation to the encoding of visual information by retinal circuits.

Animals↗
PubMed1992

Positive feedback of glutamate exocytosis by metabotropic presynaptic receptor stimulation.

Glutamate is important in several forms of synaptic plasticity such as long-term potentiation, and in neuronal cell degeneration. Glutamate activates several types of receptors, including a metabotropic receptor that is sensitive to trans-1-amino-cyclopenthyl-1,3-dicarboxylate, coupled to G protein(s) and linked to inositol phospholipid metabolism. The activation of the metabotropic receptor in neurons generates inositol 1,4,5-trisphosphate, which causes the release of Ca2+ from intracellular stores and diacylglycerol, which activates protein kinase C. In nerve terminals, the activation of presynaptic protein kinase C with phorbol esters enhances glutamate release. But the presynaptic receptor involved in this protein kinase C-mediated increase in the release of glutamate has not yet been identified. Here we demonstrate the presence of a presynaptic glutamate receptor of the metabotropic type that mediates an enhancement of glutamate exocytosis in cerebrocortical nerve terminals. Interestingly, this potentiation of glutamate release is observed only in the presence of arachidonic acid, which may reflect that this positive feedback control of glutamate exocytosis operates in concert with other pre- or post-synaptic events of the glutamatergic neurotransmission that generate arachidonic acid. This presynaptic glutamate receptor may have a physiological role in the maintenance of long-term potentiation where there is an increase in glutamate release mediated by postsynaptically generated arachidonic acid.

4-Aminopyridine↗
PubMed2006

Secretion of L-glutamate from osteoclasts through transcytosis.

Osteoclasts are involved in the catabolism of the bone matrix and eliminate the resulting degradation products through transcytosis, but the molecular mechanism and regulation of transcytosis remain poorly understood. Upon differentiation, osteoclasts express vesicular glutamate transporter 1 (VGLUT1), which is essential for vesicular storage and subsequent exocytosis of glutamate in neurons. VGLUT1 is localized in transcytotic vesicles and accumulates L-glutamate. Osteoclasts secrete L-glutamate and the bone degradation products upon stimulation with KCl or ATP in a Ca2+-dependent manner. KCl- and ATP-dependent secretion of L-glutamate was absent in osteoclasts prepared from VGLUT1-/- knockout mice. Osteoclasts express mGluR8, a class III metabotropic glutamate receptor. Its stimulation by a specific agonist inhibits secretion of L-glutamate and bone degradation products, whereas its suppression by a specific antagonist stimulates bone resorption. Finally, it was found that VGLUT1-/- mice develop osteoporosis. Thus, in bone-resorbing osteoclasts, L-glutamate and bone degradation products are secreted through transcytosis and the released L-glutamate is involved in autoregulation of transcytosis. Glutamate signaling may play an important role in the bone homeostasis.

3T3 Cells↗
PubMed2006

Glutamate metabolism in HIV-infected macrophages: implications for the CNS.

Central nervous system disorders are still a common complication of human immunodeficiency virus (HIV) infection and can lead to dementia and death. They are mostly the consequences of an inflammatory macrophagic activation and relate to glutamate-mediated excitotoxicity. However, recent studies also suggest neuroprotective aspects of macrophage activation through the expression of glutamate transporters and glutamine synthetase. We thus aimed to study whether HIV infection or activation of macrophages could modulate glutamate metabolism in these cells. We assessed the effect of HIV infection on glutamate transporter expression as well as on glutamate uptake by macrophages and showed that glutamate transport was partially decreased in the course of virus replication, whereas excitatory amino acid transporter-2 (EAAT-2) gene expression was dramatically increased. The consequences of HIV infection on glutamine synthetase were also measured and for the first time we show the functional expression of this key enzyme in macrophages. This expression was repressed during virus production. We then quantified EAAT-1 and EAAT-2 gene expression as well as glutamate uptake in differentially activated macrophages and show that the effects of HIV are not directly related to pro- or anti-inflammatory mediators. Finally, this study shows that glutamate transport by macrophages is less affected than what has been described in astrocytes. Macrophages may thus play a role in neuroprotection against glutamate in the infected brain, through their expression of both EAATs and glutamine synthetase. Because glutamate metabolism by activated macrophages is sensitive to both HIV infection and inflammation, it may thus be of potential interest as a therapeutic target in HIV encephalitis.

Anti-Inflammatory Agents↗
PubMed1985

Effect of bicarbonate on glutamine and glutamate metabolism by rat kidney cortex mitochondria.

Isolated rat kidney cortex mitochondria were incubated at pH 7.4 in the presence or absence of a CO2/bicarbonate buffer (28 mM) to investigate the pH-independent role of bicarbonate on glutamine and glutamate metabolism. Changes in the concentration of key intermediates and products during the incubations were used to calculate metabolite flux rates through specific mitochondrial enzymes. With 1 mM glutamine and 2 mM glutamate as substrates, bicarbonate caused an inhibition of glutamate oxalacetate transaminase flux and a stimulation of glutamate deamination. The same effects were also produced with addition of either aminooxyacetate or malonate. These effects of bicarbonate were prevented when 0.2 mM malate was included as an additional substrate. Bicarbonate ion was identified as a potent competitive inhibitor of rat kidney cortex succinate dehydrogenase. These results indicate that aminooxyacetate, malonate, and bicarbonate all act to stimulate glutamate deamination through a suppression of glutamate transamination, and that the control by transamination of glutamate deamination is due to alterations in alpha-ketoglutarate metabolism. In contrast, in mitochondria incubated with glutamine in the absence of glutamate, bicarbonate was found to inhibit glutamate dehydrogenase flux. This effect was found to be due in part to the lower intramitochondrial pH observed in incubations with bicarbonate. These findings indicate that bicarbonate ion, independent of pH, may have an important regulatory role in renal glutamine and glutamate metabolism.

Aminooxyacetic Acid↗
PubMed1993

Quisqualate and ACPD are agonists for a glutamate-activated current in identified Aplysia neurons.

1. Glutamate is an important neurotransmitter in both vertebrates and invertebrates, yet the characterization of molluscan glutamate receptors and their relationships to vertebrate receptors is incomplete. This study uses two-electrode voltage clamp to characterize glutamate-evoked currents in cultured neurons from the marine gastropod mollusk Aplysia californica. 2. The identified buccal ganglion neurons, B1 and B2, display two pharmacologically distinct current responses to pressure-applied glutamate. One response is a desensitizing chloride current similar to that found in other large buccal ganglion neurons. The other is a nondesensitizing potassium current that is strongly outwardly rectifying. 3. The potassium current response has a higher sensitivity to glutamate than the chloride response. 4. Individual neurons, isolated in primary cell culture, exhibit different relative proportions of the two currents. 5. The glutamate agonists quisqualate and (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid specifically evoke the potassium response but not the chloride response. The glutamate agonist (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid does not evoke any appreciable response. Kainate and N-methyl-D-aspartate also have no effect on these cells. Another glutamate analogue, ibotenate, evokes the transient chloride current but not the potassium current. 6. These results indicate that the glutamate receptor mediating the outward potassium response does not conform in its pharmacological profile to any of the known vertebrate glutamate receptor types.

Animals↗
PubMed1989

Glutamate receptors of the N-methyl-D-aspartate subtype in the myenteric plexus of the guinea pig ileum.

The excitatory amino acids L-glutamate and N-methyl-D-aspartate (NMDA) produced contractions of the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum over the concentration range of 3 X 10(-6) to 10(-3) M. The contractile response to L-glutamate and NMDA, but not carbamyl choline, was blocked noncompetitively by 0.6 mM Mg++. In the absence of Mg++, concentration-dependent increases in contractile force also were produced by, in order of potency, L-aspartate, L-homocysteate and D-glutamate, but not by quisqualate, kainate or quinolinate. L-Glutamate was competitively antagonized by the selective NMDA receptor antagonists D-2-amino-5-phosphonovalerate and 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (3 X 10(-6)-3 X 10(-5) M), as well as by the nonselective excitatory amino acid antagonist gamma-D-glutamylglycine (3 X 10(-4) M). Glutamic acid diethyl ester (3 X 10(-4) M) noncompetitively antagonized L-glutamate. L-Glutamate was not blocked by gamma-D-glutamylaminomethyl sulphonate (3 X 10(-4) M), an antagonist which preferentially antagonizes kainate and quisqualate. In addition, the phencyclidine-like drugs etoxadrol (10(-7)-10(-5) M), dextromethorphan (10(-6)-10(-5) M) and 5-methyl-10,11-dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10-imine (10(-9)-10(-7) M) noncompetitively antagonized L-glutamate. The (+) isomer of 5-methyl-10, 11-dihydroxy-5H-dibenzo(a,d)cyclohepten-5,10-imine was approximately 10-fold more potent than the (-) isomer in antagonizing L-glutamate. The present results demonstrate that receptors for the excitatory amino acid L-glutamate are present in the guinea pig myenteric plexus and are of the NMDA subtype.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Characterization of a glutamic acid neurotransmitter binding site on neuroblastoma hybrid cells.

Glutamate is thought to be a major excitatory neurotransmitter in the central nervous system. To study the glutamate receptor and its regulation under carefully controlled conditions, the specific binding of [3H]glutamate was characterized in washed membranes isolated from a neuroblastoma X retina hybrid cell line, N18-RE-105. [3H]Glutamate bound in a saturable and reversible fashion with an apparent dissociation constant, KD, of 650 nM and a maximum binding capacity, Bmax, of 16 pmol/mg of protein. Pharmacologic characterization of the site indicates that it closely resembles the Na+-independent binding site for glutamate found on brain membranes and thought to be an excitatory amino acid neurotransmitter receptor. Thus, while kainate, N-methyl-DL-aspartate, and nonamino acid ligands did not displace [3H]glutamate, quisqualate and ibotenate were potent inhibitors of specific binding. Furthermore, this binding site is regulated by ions in a manner which resembles that described in the hippocampus (Baudry, M., and Lynch, G. (1979) Nature (Lond.) 282, 748-750). Calcium (10 mM) increased the number of binding sites 2.6-fold with no change in receptor-ligand affinity. Lanthanum (1 mM) was the only other cation added which enhanced (3-fold) the binding of [3H]glutamate. Monovalent cations resulted in a decrease in the number of glutamate binding sites. Incubation of membranes in the presence of chloride ions caused a marked increased in [3H] glutamate binding, an effect which was synergistic with that of calcium incubation. Thus, N18-RE-105 cells possess a binding site for [3H]glutamate pharmacologically similar to an excitatory neurotransmitter binding site in brain and which exhibits regulatory properties resembling those previously described in hippocampal membranes, providing an excellent model for mechanistic studies.

Animals↗
PubMed1984

Agonists and cations regulate the glutamic acid receptors on intact neuroblastoma hybrid cells.

Glutamate is thought to be a major excitatory neurotransmitter in the vertebrate brain. In the preceding paper (Malouf, A. T., Schnaar, R. L., and Coyle, J. T. (1984) J. Biol. Chem. 259, 12756-12762), we demonstrated specific binding of [3H]glutamate to membranes from a neuroblastoma hybrid cell line, N18-RE-105. These sites are pharmacologically and kinetically similar to those seen on rat brain membranes and are regulated by ions added to the isolated membranes. In the current paper, we describe an additional level of regulation for the glutamate receptor in this cell line. Long-term incubation (72 h) of intact N18-RE-105 cells with glutamate (10 mM) results in a 2- to 3-fold increase in [3H]glutamate binding. Scatchard analysis reveals that the increase in binding is due to an increase in the number of glutamate receptors without significant change in their affinity. The ability of glutamate analogs to induce such up-regulation mirrors their ability to compete for [3H]glutamate binding to isolated membranes, suggesting that up-regulation is receptor-mediated. Binding of [3H]glutamate to membranes isolated from cells grown in the presence of glutamate can be further up-regulated by brief exposure (10 min) of the isolated membranes to calcium ions. This suggests that agonist-induced and calcium-induced up-regulation occur via independent mechanisms. The short-term ion-induced up-regulation and the long-term agonist-induced up-regulation described in this paper may model two levels of synaptic potentiation reported to occur in the vertebrate hippocampus. The N18-RE-105 cell line may offer a homogeneous cell type in which to study the molecular mechanisms underlying these phenomena.

Amino Acids↗
PubMed1999

Structure and Bonding of Cu(II)-Glutamate Complexes at the gamma-Al(2)O(3)-Water Interface.

Thecomposition and mode of attachment of Cu(II) complexes at the gamma-Al(2)O(3)-water interface in suspensions containing a simple amino acid (glutamate) were characterized with EXAFS and FTIR spectroscopies. The spectroscopic results indicate that two types of Cu(II)-glutamate-alumina interactions are primarily responsible for Cu(II) and glutamate uptake between pH 4 and 9. In acidic suspensions of alumina, glutamate forms a bridge between Cu(II) ions and the (hydr)oxide surface (Type B complex). In this Type B surface complex, Cu(II) is bonded to amino acid headgroups (i.e., (+)H(3)NCHRCOO(-)) of two glutamate molecules. Spectroscopic and ionic strength dependent uptake results are combined to propose that the nonbonded side chain carboxylate groups of this complex are attracted to the oxide surface through long-range forces, leading to enhanced Cu(II) uptake relative to the glutamate-free system. In alkaline suspensions the relative amount of surface-bound Cu(II) complexed by glutamate decreases, and a direct Cu(II)-surface bond becomes the dominant mode of attachment (Type A complex). These surface complexes differ markedly from the species found in the alumina-free Cu(II)-glutamate aqueous system under similar solution conditions, where Cu(H(2)O)(2+)(6) and Cu(glutamate)(2-)(2) are the dominant species in acidic and alkaline solutions, respectively. Based on these spectroscopic results, surface complexation reactions are proposed for the Cu(II) and glutamate ternary interactions with the alumina surface in this system. Similarities between the results of this study and Cu(II) uptake behavior and complexation in the presence of natural organic material (NOM) indicate that Cu(II)-glutamate interactions mimic those in more complex Cu(II)-NOM-mineral-water systems. Copyright 1999 Academic Press.

Journal Article↗
PubMed2003

Regulation of glial glutamate transporter expression by growth factors.

Injuries to the brain result in the decline of glial glutamate transporter expression within hours and a recovery after several days. One consequence of this disturbed expression seems to consist in the temporary accumulation of toxic extracellular glutamate levels followed by secondary neuronal cell death. Whereas evidence exists that the decline in glutamate transporter expression results from a loss of neuronal PACAP influences on astroglia, the mechanism(s) inducing the reexpression of glial glutamate transporters is presently unknown. We now demonstrate that the injury-induced growth factors EGF, TGFalpha, FGF-2, and PDGF all promote the expression of the glutamate transporters GLT-1 and/or GLAST in cultured cortical astroglia. In contrast, similar stimulatory influences were absent with GDNF and BDNF, growth factors not affected by brain injuries. The effects of EGF, TGFalpha, FGF-2, and PDGF on glial glutamate transport were only partly redundant and involved distinctly different signaling pathways. Unlike EGF, TGFalpha, and FGF-2, PDGF promoted GLT-1, but not GLAST expression and further failed to increase the maximal velocity of sodium-dependent glutamate uptake. Moreover, FGF-2 only affected glial glutamate transport when the RAF-MEK-ERK signaling pathway was concomitantly inhibited with PD98059. Depending on the extracellular growth factor and glutamate transporter subtype, the observed stimulatory effects required the activation of PKA, PKC, and/or AKT. We suggest that after brain injury, reactive processes may limit secondary neuronal cell death by promoting glial glutamate transport. The detailed knowledge of these compensatory mechanisms will eventually allow us to therapeutically interfere with glutamate-associated neuronal cell death in the brain.

Amino Acid Transport System X-AG↗
PubMed1994

Characterization of the coenzyme-B12-dependent glutamate mutase from Clostridium cochlearium produced in Escherichia coli.

The glutamate mutase dependent on adenosylcobalamin (coenzyme B12) catalyzes the carbon skeleton rearrangement of (S)-glutamate to (2S,3S)-3-methylaspartate, the first step of the glutamate fermentation pathway of the anaerobic bacterium Clostridium cochlearium. The enzyme consists of two protein components, E, a dimer epsilon 2 (epsilon, 53.5 kDa) and S, a monomer (sigma, 14.8 kDa). The corresponding genes (glmE and glmS) were cloned, sequenced and over-expressed in Escherichia coli. The genes glmS and glmE are separated by glmL encoding a protein of unknown function. The deduced amino acid sequence of GlmL contains an ATP-binding motif which is common to chaperones of the HSP70-type, actin and procaryotic cell-cycle proteins. Both components of glutamate mutase were purified with excellent yields from cell-free extracts of E. coli carrying the corresponding genes. In contrast to component E, component S was shown to bind coenzyme B12. This observation strongly supports the idea that significant similarities of the amino acid sequences of component S and several other cobamide-dependent enzymes represent a common binding motif. Incubation of pure components E and S with coenzyme B12 resulted in the formation of a fully active glutamate mutase heterotetramer (epsilon 2 sigma 2) containing one molecule of coenzyme B12. EPR spectra of recombinant glutamate mutase, now available in sufficiency large amounts, were recorded after incubation of the enzyme with coenzyme B12 and (S)-glutamate. The EPR signals (gx,y approximately 2.1, gz = 1.985) were of much better resolution than observed earlier with the clostridial enzyme. Their typical hyperfine splitting is clearly derived from Co(II), which is involved in the formation of the paramagnetic species but is different from cob(II)alamin (gx,y = 2.25). The spin concentration was 34-50% of the concentration of the enzyme (epsilon 2 sigma 2) coenzyme complex. The competitive inhibitors (2S, 4S)-4-fluoroglutamate and 2-methyleneglutarate induced similar but not identical signals with spin concentrations of 134-148% of the enzyme concentration. Even (S)-[2,3,3,4,4-2H5]glutamate induced a signal significantly different to that of (S)-glutamate with an intensity of only 7%. These data suggest an involvement of the Co(II)-containing paramagnetic species in catalysis, the concentration of which reflects a steady state between its formation and decomposition. The large difference in the spin concentrations observed with (S)-glutamate as compared to the predeuterated glutamate is probably due to a kinetic isotope effect and indicates a cleavage of a C-H bond during formation of the paramagnetic species.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Isomerases↗
PubMed1990

Osmoregulation in Rhizobium meliloti: Production of Glutamic Acid in Response to Osmotic Stress.

Rhizobium meliloti, like many other bacteria, accumulates high levels of glutamic acid when osmotically stressed. The effect was found to be proportional to the osmolarity of the growth medium. NaCl, KCI, sucrose, and polyethylene glycol elicited this response. The intracellular levels of glutamate and K began to increase immediately when cells were shifted to high-osmolarity medium. Antibiotics that inhibit protein synthesis did not affect this increase in glutamate production. Cells growing in conventional media at any stage in the growth cycle could be suspended in medium causing osmotic stress and excess glutamate accumulated. The excess glutamate did not appear to be excreted, and the intracellular level eventually returned to normal when osmotically stressed cells were suspended in low-osmolarity medium. A glt mutant lacking glutamate synthase and auxotrophic for glutamate accumulated excess glutamate in response to osmotic stress. Addition of isoleucine, glutamine, proline, or arginine stimulated glutamate accumulation to wild-type levels when the mutant cells were suspended in minimal medium with NaCl to cause osmotic stress. In both wild-type and mutant cells, inhibitors of transaminase activity, including azaserine and aminooxyacetate, reduced glutamate levels. The results suggest that the excess glutamate made in response to osmotic stress is derived from degradation of amino acids and transamination of 2-ketoglutarate.

Journal Article↗
PubMed2005

Role of spinal cord glutamate transporter during normal sensory transmission and pathological pain states.

Glutamate is a neurotransmitter critical for spinal excitatory synaptic transmission and for generation and maintenance of spinal states of pain hypersensitivity via activation of glutamate receptors. Understanding the regulation of synaptically and non-synaptically released glutamate associated with pathological pain is important in exploring novel molecular mechanisms and developing therapeutic strategies of pathological pain. The glutamate transporter system is the primary mechanism for the inactivation of synaptically released glutamate and the maintenance of glutamate homeostasis. Recent studies demonstrated that spinal glutamate transporter inhibition relieved pathological pain, suggesting that the spinal glutamate transporter might serve as a therapeutic target for treatment of pathological pain. However, the exact function of glutamate transporter in pathological pain is not completely understood. This report will review the evidence for the role of the spinal glutamate transporter during normal sensory transmission and pathological pain conditions and discuss potential mechanisms by which spinal glutamate transporter is involved in pathological pain.

Amino Acid Transport System X-AG↗
PubMed1989

Effects of monensin and pH on the production and utilization of pyro-glutamate, a novel product of ruminal glutamine deamination.

Pure cultures of monensin-sensitive ruminal bacteria were involved in both the production and utilization of pyro-glutamate (pyrrolidone carboxylic acid), a novel product of glutamine fermentation, but the effect of monensin on pyro-glutamate accumulation in mixed cultures had not been assessed. Mixed ruminal bacteria (n = 3) fermented glutamine (40 mM) at a rapid rate (63 nmol/[mg protein-min]) and there was a transient increase in pyro-glutamate (3 mM). Monensin (5 ppm) decreased the rate of glutamine fermentation 70% and greatly enhanced the conversion of glutamine to pyro-glutamate (50 vs 7%). Glutamate was fermented at nearly the same rate as glutamine, and monensin eliminated glutamate fermentation. Glutamate was never converted to pyro-glutamate. Trypticase, a pancreatic digest of casein, contained pyro-glutamate, and a significant fraction (30%) of its N was never converted to ammonia. Pyro-glutamate utilization was reduced by monensin and low pH (less than 6.0). Based on these in vitro observations, pyro-glutamate could be a significant end-product of glutamine degradation when animals are fed monensin.

Animals↗
PubMed1999

Mature hippocampal astrocytes exhibit functional metabotropic and ionotropic glutamate receptors in situ.

Astrocytes closely contact neurons where they respond to neuronally released glutamate in immature brain slices. In previous studies, neither metabotropic nor ionotropic glutamate receptor-mediated responses were detected by imaging Ca2+ in astrocytes from mature (P21-P42) animals, suggesting astrocyte glutamate receptors only contribute to hippocampus physiology during development. In contrast to Ca2+ imaging, published electrophysiological experiments suggest P30-P35 astrocytes have alpha-amino-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. For this study, we imaged astrocytes in P31-P38 hippocampal slices to determine if metabotropic and ionotropic glutamate receptor activation elevates intracellular calcium in mature astrocytes. Drugs were perfused while [Ca2+]i was monitored (confocal imaging) in cells loaded with Calcium Green 1-AM. Imaged cells were subsequently identified as astrocytes by GFAP/S-100 immunostaining. Astrocytic Ca2+ increased after glutamate application in the presence of a glutamate uptake inhibitor. An agonist at group I/II metabotropic glutamate receptors, (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (t-ACPD), elicited Ca2+ increases as did group I agonist 3,5-dihydroxyphenylglycine (DHPG), suggesting that mature astrocytes respond to glutamate via metabotropic glutamate receptors. AMPA also elicited Ca2+ elevations that were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and occurred after treatment with omega-conotoxin MVIIC to block neurotransmitter release. These results demonstrate that astrocytes in mature hippocampus have functional ionotropic and metabotropic glutamate receptors that regulate astrocytic calcium levels. Glutamatergic regulation of astrocytic [Ca2+]i may be involved in synapse modeling, long-term potentiation, excitotoxicity and other events dependent on glutamatergic transmission in adult hippocampus.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1988

Glutamine and glutamate nitrogen exchangeable pools in cultured fibroblasts: a stable isotope study.

Glutamine's role as an energetic fuel has been extensively studied in the past using 14C- and 3H-labeled tracers in cultured human cells. Yet another prominent role of glutamine, that of a nitrogen shuttle, cannot be approached without an N-tracer. We therefore used 15N-labeled glutamine and glutamate to address the following questions: 1) is it possible to study the exchangeable pools of intracellular free glutamine and glutamate nitrogen with stable isotope methods? and 2) to what extent is intracellular glutamine pool regulated by extracellular glutamine? We observed that: 1) intracellular [15N]-glutamine enrichment reached a plateau at 80% within 20 min of incubation in a buffer containing 0.7 mM pure 15N-glutamine and no glutamate; in contrast, intracellular 15N-glutamate enrichment rose only to 40% after 4 hours of incubation in a buffer containing 0.5 mM pure 15N-glutamate and no glutamine; 2) the cell-free glutamine content was tightly dependent on extracellular glutamine level, while the cell-free glutamate remained steady irrespective of the extracellular glutamate level; 3) the cells took up glutamine and glutamate against a concentration gradient; the rate of glutamine uptake accounted for 90% of the cell glutamine turnover rate; and 4) when cells were confronted with a glutamine-free medium, only one fourth of intracellular glutamine was derived from the exchangeable glutamate. We conclude that: 1) The size and turnover rate of the intracellular pool of free glutamine nitrogen are measureable using stable isotope methodology; 2) glutamine uptake from the extracellular medium accounts for most of glutamine turnover rate in cultured fibroblasts; and 3) intracellular free glutamate is divided up between several pools in cultured human fibroblasts.

Body Fluids↗
PubMed1996

Differential abundance of glutamate transporter subtypes in amyotrophic lateral sclerosis (ALS)-vulnerable versus ALS-resistant brain stem motor cell groups.

Previous studies have suggested that defective high-affinity glutamate uptake, due mainly to a major loss of the astroglial-specific GLT-1 glutamate transporter, underlies the selective motoneuron degeneration observed in sporadic ALS (24, 28). If a defect in glutamate transport underlies the pathogenesis of sporadic ALS, the glutamate transporter subtype found to be lost in sporadic ALS should be present in abundance in the affected motor nuclei under normal conditions. To investigate this, we used immunohistochemical methods to analyze the localization of two subtypes of high-affinity glutamate transporters in the cranial motor nuclei of normal monkey brain stem: GLT-1, localized to astroglia; and EAAC1, localized to neurons. Our results indicated that all motor cell groups of monkey brain stem are rich in the GLT-1 glutamate transporter, which is localized to astroglial cells and processes that surround and envelop motoneuron cell bodies and dendrites. Image analysis indicated that the abundance of GLT-1 immunoreactive astroglial elements in ALS-vulnerable motor cell groups (i.e., the trigeminal, facial, and hypoglossal motor cell groups) is higher than in ALS-resistant motor cell groups (i.e., the oculomotor, trochlear, and abducens motor cell groups), and statistical analysis showed that this difference is significant. Our results also indicated that both ALS-vulnerable and ALS-resistant motor cell groups of monkey brain stem are relatively poor in EAAC1 immunoreactivity. Therefore, in the case of a loss in the GLT-1 glutamate transporter in sporadic ALS, glutamate may increase in the vicinity of motoneurons in all brain-stem motor cell groups, but especially in the ALS-vulnerable motor cell groups, which are normally richer in GLT-1. Increased extracellular glutamate could lead to excess entry of Ca2+ into motoneurons via glutamate-gated or voltage-activated Ca2+ channels and produce degeneration of those motoneurons unable to resist the insult. Since motoneurons in the ALS-resistant motor cell groups of the brain stem are enriched in the Ca2+ buffering protein parvalbumin, they should be better able to resist the damage than the majority of motoneurons in the ALS-vulnerable motor cell groups, which lack parvalbumin (20).

ATP-Binding Cassette Transporters↗