			Home About us Contact

Glutamate Concentration (glutamate + concentration)

Distribution by Scientific Domains

Life Sciences	60%
Medical Sciences	34%

Kinds of Glutamate Concentration

extracellular glutamate concentration

Selected Abstracts

Mechanisms of substrate transport-induced clustering of a glial glutamate transporter GLT-1 in astroglial,neuronal cultures

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2008
Takayuki Nakagawa
Abstract Glutamate uptake by the Na+ -dependent glutamate transporter GLT-1, which is predominantly expressed in astrocytes, is crucial for regulating glutamate concentration at the synaptic cleft and achieving proper excitatory neurotransmission. A body of evidence suggests that GLT-1 constitutively traffics between the plasma membrane and endosomes via an endocytosis/recycling pathway, and forms a cluster. Here, we report substrate transport via GLT-1-induced formation of GLT-1 cluster accompanied by intracellular trafficking in rat astroglial,neuronal cultures. We constructed a recombinant adenovirus expressing enhanced green fluorescence protein (EGFP)-tagged GLT-1. Adenoviral infection resulted in the expression of functional GLT-1,EGFP preferentially in astrocytes, partly as clusters. Treatment with glutamate, but not N -methyl-D-aspartate, dramatically increased the number of GLT-1 clusters within 1 h. The estimated EC50 value of glutamate was 240 ,m. In addition, glutamate decreased the cell surface expression and increased the intracellular expression of GLT-1. The GLT-1 clusters were found in early and recycling endosomes and partly in lysosomes, and were inhibited by blockade of endocytotic pathways. Ionotropic and metabotropic glutamate receptor antagonists had no effect on glutamate-induced GLT-1 clustering. The non-transportable glutamate uptake inhibitors (2S,3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate and dihydrokainate, as well as Na+ -free conditions, prevented the glutamate-induced GLT-1 clustering, whereas the competitive substrates, aspartate and L- trans -pyrrolidine-2,4-dicarboxylate, induced GLT-1 clustering. Furthermore, the Na+/K+ -ATPase inhibitor, ouabain, and the Na+ ionophores, gramicidin and monensin, produced GLT-1 clustering. Modulators of intracellular Ca2+signaling or membrane depolarization had no effect on GLT-1 clustering. Taken together, these results suggest that Na+ influx associated with GLT-1 substrate transport triggers the formation of GLT-1 clusters accompanied by intracellular trafficking via endocytotic pathways in astrocytes. [source]

Keratinocytes: a source of the transmitter l -glutamate in the epidermis

EXPERIMENTAL DERMATOLOGY, Issue 12 2009
Matthias Fischer
Abstract:, Various glutamate receptors have been described in both keratinocytes and melanocytes. l -Glutamate is the physiological agonist of the glutamate receptor family. The source of this transmitter had not yet been identified. In normal human epidermal keratinocytes (NHEK) and HaCaT-keratinocytes, cell supernatants were sampled in various stages of cell density and the l -glutamate content photometrically determined. The following examination time-points were defined: non-confluent (ca. 33%), subconfluent (ca. 70%) and confluent (90,100%). The l -glutamate concentration originally in the culture medium was 14.7 mg/l (0.1 mm/l). The l -glutamate concentration in the cell supernatant increased in NHEK with increasing cell density: non-confluent 39.9 ± 4 mg/l, subconfluent 60.6 ± 15.8 mg/l, confluent 100.7 ± 33.2 mg/l. A linear increase of l -glutamate concentration was also found for HaCaT cells. The investigations show that keratinocytes are capable of producing and releasing l -glutamate. Thus they are a source of l -glutamate which acts as a transmitter on epidermal glutamate receptors. [source]

Role of a serine residue (S278) in the pore-facing region of the housefly l -glutamate-gated chloride channel in determining sensitivity to noncompetitive antagonists

INSECT MOLECULAR BIOLOGY, Issue 4 2008
K. Hirata
Abstract ,-Hexachlorocyclohexane (,-HCH), fipronil and picrotoxinin are noncompetitive antagonists (NCAs) of l -glutamate-gated chloride channels (GluCls), yet their potencies are weaker than those on ,-aminobutyric acid receptors (GABARs). The A302S mutation of Drosophila RDL (resistant to dieldrin) GABAR confers NCA resistance, and housefly GluCls (MdGluCls) possess S278 as the residue corresponding to the A302. Thus, the effects of S278A mutation on the NCA actions on MdGluCls were investigated. The S278A mutation resulted in enhanced blocking by NCAs of the MdGluCl response to 30 µM l -glutamate. However, such actions of ,-HCH and picrotoxinin, but not of fipronil, on the S278A mutant were reduced with 200 µM l -glutamate. Further increases in the l -glutamate concentration led to potentiation by NCAs of the mutant response to l -glutamate. [source]

Use of L -[15N] glutamic acid and homoglutathione to determine both glutathione synthesis and concentration by gas chromatography-mass spectrometry (GCMS)

JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 7 2001
Bernard Humbert
Abstract A method for simultaneous measurement of both glutathione enrichment and concentration in a biological sample using gas chromatography mass spectrometry is described. The method is based on the preparation of N,S -ethoxycarbonylmethyl ester derivatives of glutathione, and the use of homoglutathione (glutamyl,cysteinyl,alanine) as an internal standard. A procedure for determination of glutamate concentration and enrichment is also reported. Both methods have within-day and day-to-day inter-assay coefficients of variation less than 5%, and recoveries of known added amounts of glutathione and glutamate are close to 100%. Taken together, these methods allowed determination of glutathione concentration and fractional synthesis rate in red blood cells using L -[15N] glutamic acid infusion. This approach was applied in vivo to investigate the effects of a 72 h fast, compared with a control overnight fast, on erythrocyte glutathione in a single dog. The 72 h fast was associated with a 39% decline in erythrocyte glutathione level, (2.9 ± 0.4 versus 4.7 ± 0.5 mmol l,1, fasting versus control) with no change in glutathione fractional synthesis (67.4 versus 71.3% d,1, fasting versus control). Copyright © 2001 John Wiley & Sons, Ltd. [source]

Glutamate levels and transport in cat (Felis catus) area 17 during cortical reorganization following binocular retinal lesions

JOURNAL OF NEUROCHEMISTRY, Issue 6 2003
Ann Massie
Abstract Glutamate is known to play a crucial role in the topographic reorganization of visual cortex after the induction of binocular central retinal lesions. In this study we investigated the possible involvement of the glial high-affinity Na+/K+ -dependent glutamate transporters in cortical plasticity using western blotting and intracortical microdialysis. Basal extracellular glutamate levels and the re-uptake activity for glutamate have been determined by comparing the extracellular glutamate concentration before and during the blockage of glutamate removal from the synaptic cleft with the potent transporter inhibitor l - trans -pyrrolidine-3,4-dicarboxylic acid. In cats with central retinal lesions we observed increased basal extracellular glutamate concentrations together with a decreased re-uptake activity in non-deprived, peripheral area 17, compared with the sensory-deprived, central cortex of the same animal as well as the topographically matching regions of area 17 in normal subjects. Western blotting experiments revealed a parallel decrease in the expression level of the glial glutamate transporter proteins GLT-1 and GLAST in non-deprived cortex compared with sensory-deprived cortex of lesion cats and the corresponding regions of area 17 of normal subjects. This study shows that partial sensory deprivation of the visual cortex affects the removal of glutamate from the synaptic cleft and implicates a role for glial,neuronal interactions in adult brain plasticity. [source]

Collapse of extracellular glutamate regulation during epileptogenesis: down-regulation and functional failure of glutamate transporter function in rats with chronic seizures induced by kainic acid

JOURNAL OF NEUROCHEMISTRY, Issue 3 2001
Yuto Ueda
We used northern and western blotting to measure the quantity of glutamate and GABA transporters mRNA and their proteins within the hippocampal tissue of rats with epileptogenesis. Chronic seizures were induced by amygdalar injection of kainic acid 60 days before death. We found that expression of the mRNA and protein of the glial glutamate transporters GLAST and GLT-1 were down-regulated in the kainic acid-administered group. In contrast, EAAC-1 and GAT-3 mRNA and their proteins were increased, while GAT-1 mRNA and protein were not changed. We performed in vivo microdialysis in the freely moving state. During the interictal state, the extracellular glutamate concentration was increased, whereas the GABA level was decreased in the kainic acid group. Following potassium-induced depolarization, glutamate overflow was higher and the recovery time to the basal release was prolonged in the kainic acid group relative to controls. Our data suggest that epileptogenesis in rats with kainic acid-induced chronic seizures is associated with the collapse of extracellular glutamate regulation caused by both molecular down-regulation and functional failure of glutamate transport. [source]

Inhibition of the Activity of Excitatory Amino Acid Transporter 4 Expressed in Xenopus Oocytes After Chronic Exposure to Ethanol

ALCOHOLISM, Issue 7 2008
Seung-Yeon Yoo
Background:, The extracellular glutamate concentration is tightly controlled by excitatory amino acid transporters (EAATs). EAAT4 is the predominant EAAT in the cerebellar Purkinje cells. Purkinje cells play a critical role in motor coordination and may be an important target for ethanol to cause motor impairments. We designed this study to determine the effects of chronic ethanol exposure on the activity of EAAT4 and evaluate the involvement of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) in these effects. Methods:, EAAT4 was expressed in Xenopus oocytes following injection of EAAT4 mRNA. Oocytes were incubated with ethanol-containing solution for 24 to 96 hours. Membrane currents induced by l -aspartate were recorded using 2-electrode voltage clamps. Responses were quantified by integration of the current trace and reported in microCoulombs (,C). Results:, Ethanol dose- and time-dependently reduced EAAT4 activity. EAAT4 activity after a 96-hour exposure was significantly decreased compared to the control values at all concentrations tested (10 to 100 mM). Ethanol (50 mM) significantly decreased the Vmax (2.2 ± 0.2 ,C for control vs. 1.6 ± 0.2 ,C for ethanol, n = 18, p < 0.05) of EAAT4 for l -aspartate. Preincubation of ethanol-treated (50 mM for 96 hours) oocytes with phorbol-12-myrisate-13-acetate (100 nM for 10 minutes) abolished the ethanol-induced decrease in EAAT4 activity. While staurosporine (2 ,M for 1 hour) or chelerythrine (100 ,M for 1 hour) significantly decreased EAAT4 activity, no difference was observed in EAAT4 activity among the staurosporine, ethanol, or ethanol plus staurosporine groups. Similarly, EAAT4 activity did not differ among the chelerythrine, ethanol, or ethanol plus chelerythrine groups. Pretreatment of the oocytes with wortmannin (1 ,M for 1 hour) also significantly decreased EAAT4 activity. However, no difference was observed in the wortmannin, ethanol, or ethanol plus wortmannin groups. Conclusions:, The results of this study suggest that chronic ethanol exposure decreases EAAT4 activity and that PKC and PI3K may be involved in these effects. These effects of ethanol on EAAT4 may cause an increase in peri-Purkinje cellular glutamate concentration, and may be involved in cerebellar dysfunction and motor impairment after chronic ethanol ingestion. [source]

Optimization of glutamate concentration and pH for H2 production from volatile fatty acids by Rhodopseudomonas capsulata

LETTERS IN APPLIED MICROBIOLOGY, Issue 6 2005
X.-Y. Shi
Abstract Aims:, This study attempted to employ response surface methodology (RSM) to evaluate the effects of glutamate concentration and pH on H2 production from volatile fatty acids by Rhodopseudomonas capsulata. Methods and Results:, A mixture of acetate, propionate and butyrate was used as a carbon source for the H2 production by R. capsulata. The H2 yield and H2 production rate were strongly affected by the glutamate concentration, pH and their interaction. The predicted maximum H2 yield of 0·534 was obtained when glutamate concentration and pH were 6·56 mmol l,1 and 7·29 respectively. On the contrary, the maximum H2 production rate of 18·72 ml l,1 h,1 was achieved at a glutamate concentration of 7·01 mmol l,1 and pH 7·31. Conclusions:, Taking H2 yield and H2 production rate together into account, a glutamate concentration of 6·56,7·01 mmol l,1 and pH of 7·29,7·31 should be selected for H2 production from a mixture of acetate, propionate and butyrate by R. capsulata. Significance and Impact of the Study:, The RSM was a useful tool for maximizing H2 production by photosynthetic bacteria (PSB). [source]

Molecular characterization, function and regulation of ammonium transporters (Amt) and ammonium-metabolizing enzymes (GS, NADP-GDH) in the ectomycorrhizal fungus Hebeloma cylindrosporum

MOLECULAR MICROBIOLOGY, Issue 2 2003
Arnaud Javelle
Summary External hyphae, which play a key role in nitrogen nutrition of trees, are considered as the absorbing structures of the ectomycorrhizal symbiosis. Here, we have cloned and characterized Hebeloma cylindrosporum AMT1, GLNA and GDHA genes, which encode a third ammonium transporter, a glutamine synthetase and an NADP-dependent glutamate dehydrogenase respectively. Amt1 can fully restore the pseudohyphal growth defect of a Saccharomyces cerevisiae mep2 mutant, and this is the first evidence that a heterologous member of the Mep/Amt family complements this dimorphic change defect. Dixon plots of the inhibition of methylamine uptake by ammonium indicate that Amt1 has a much higher affinity than the two previously characterized members (Amt2 and Amt3) of the Amt/Mep family in H. cylindrosporum. We also identified the intracellular nitrogen pool(s) responsible for the modulation of expression of AMT1, AMT2, AMT3, GDHA and GLNA. In response to exogenously supplied ammonium or glutamine, AMT1, AMT2 and GDHA were downregulated and, therefore, these genes are subjected to nitrogen repression in H. cylindrosporum. Exogenously supplied nitrate failed to induce a downregulation of the five mRNAs after transfer of mycelia from a N-starved condition. Our results demonstrate that glutamine is the main effector for AMT1 and AMT2 repression, whereas GDHA repression is controlled by intracellular ammonium, independently of the intracellular glutamine or glutamate concentration. Ammonium transport activity may be controlled by intracellular NH4+. AMT3 and GLNA are highly expressed but not highly regulated. A model for ammonium assimilation in H. cylindrosporum is presented. [source]

Excitatory amino acid transporters EAAT-1 and EAAT-2 in temporal lobe and hippocampus in intractable temporal lobe epilepsy

APMIS, Issue 4 2009
SINAN SARAC
Intractable temporal lobe epilepsy (TLE) is an invalidating disease and many patients are resistant to medical treatment. Increased glutamate concentration has been found in epileptogenic foci and may induce local over-excitation and cytotoxicity; one of the proposed mechanisms involves reduced extra-cellular clearance of glutamate by excitatory amino acid transporters (EAAT-1 to EAAT-5). EAAT-1 and EAAT-2 are mainly expressed on astroglial cells for the reuptake of glutamate from the extra-cellular space. We have studied the expression of EAAT-1 and EAAT-2 in the hippocampus and temporal lobe in 12 patients with TLE by immunhistochemistry and densitometry. The expression of EAAT-1 and EAAT-2 was reduced to approximately 40% and 25%, respectively, in CA1 of the hippocampus. In the same area, an increased expression of glial fibrillary acid protein (GFAP) at 90% reflected molecular rearrangements and upregulation of GFAP in the existing astrocytes as Ki-67 staining failed to demonstrate any signs of astrocytic proliferation. The aetiology of the reduced expression of EAAT-1 and EAAT-2 remains unclear. The downregulation of EAAT-1 and EAAT-2 may be an adaptive response to neuronal death or it may be a causative event contributing to neuronal death. Further studies of the EAATs and their function are needed to clarify the mechanisms and significance of EAAT-1 and EAAT-2 disappearance in TLE. [source]

In vitro neurotoxic properties and excitatory aminoacids concentration in the cerebrospinal fluid of amyotrophic lateral sclerosis patients.

ACTA NEUROLOGICA SCANDINAVICA, Issue 2 2010
Relationship with the degree of certainty of disease diagnoses
Fiszman ML, Ricart KC, Latini A, Rodríguez G, Sica REP. In vitro neurotoxic properties and excitatory aminoacids concentration in the cerebrospinal fluid of amyotrophic lateral sclerosis patients. Relationship with the degree of certainty of disease diagnoses. Acta Neurol Scand: 2010: 121: 120,126. © 2009 The Authors Journal compilation © 2009 Blackwell Munksgaard. Objective,,, To determine glutamate and aspartate levels in the cerebrospinal fluid (CSF) in patients with sporadic amyotrophic lateral sclerosis (SALS) grouped according to El Escorial diagnostic criteria, and to perform an in vitro assessment of the neurotoxicity of the CSF in murine cortical neurons. Methods,,, SALS patients were sorted according to El Escorial diagnostic criteria. Glutamate and aspartate were measured in the CSF using high performance liquid chromatography. Cultured cortical neuron viability was determined after exposure to CSF for 24 h. Results,,, Glutamate levels were elevated in 28 out of the 29 patients with definite, probable or possible SALS. There were no differences in glutamate concentrations when the three clinical forms of the disease were compared; neither there were significant variation across disease duration and clinical presentation. In agreement with previous reports, we concluded that CSF-SALS-induced in vitro neurotoxicity is mediated by ionotropic glutamate receptors. We found no relationship between the degree of in vitro neurotoxicity and glutamate concentration in the CSF. Conclusions,,, Glutamate but not aspartate CSF levels may contribute to ALS pathogenesis. However, glutamate levels may not influence the degree of diagnosis certainty or lesion extension. [source]

Increased Expression of the Neuronal Glutamate Transporter (EAAT3/EAAC1) in Hippocampal and Neocortical Epilepsy

EPILEPSIA, Issue 3 2002
Peter B. Crino
Summary: ,Purpose: To define the changes in gene and protein expression of the neuronal glutamate transporter (EAAT3/EAAC1) in a rat model of temporal lobe epilepsy as well as in human hippocampal and neocortical epilepsy. Methods: The expression of EAAT3/EAAC1 mRNA was measured by reverse Northern blotting in single dissociated hippocampal dentate granule cells from rats with pilocarpine-induced temporal lobe epilepsy (TLE) and age-matched controls, in dentate granule cells from hippocampal surgical specimens from patients with TLE, and in dysplastic neurons microdissected from human focal cortical dysplasia specimens. Immunolabeling of rat and human hippocampi and cortical dysplasia tissue with EAAT3/EAAC1 antibodies served to corroborate the mRNA expression analysis. Results: The expression of EAAT3/EAAC1 mRNA was increased by nearly threefold in dentate granule cells from rats with spontaneous seizures compared with dentate granule cells from control rats. EAAT3/EAAC1 mRNA levels also were high in human dentate granule cells from patients with TLE and were significantly elevated in dysplastic neurons in cortical dysplasia compared with nondysplastic neurons from postmortem control tissue. No difference in expression of another glutamate transporter, EAAT2/GLT-1, was observed. Immunolabeling demonstrated that EAAT3/EAAC1 protein expression was enhanced in dentate granule cells from both rats and humans with TLE as well as in dysplastic neurons from human cortical dysplasia tissue. Conclusions: Elevations of EAAT3/EAAC1 mRNA and protein levels are present in neurons from hippocampus and neocortex in both rats and humans with epilepsy. Upregulation of EAAT3/EAAC1 in hippocampal and neocortical epilepsy may be an important modulator of extracellular glutamate concentrations and may occur as a response to recurrent seizures in these cell types. [source]

Mu opioid receptor modulation of somatodendritic dopamine overflow: GABAergic and glutamatergic mechanisms

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 2 2009
V. I. Chefer
Abstract Mu opioid receptor (MOR) regulation of somatodendritic dopamine neurotransmission in the ventral tegmental area (VTA) was investigated using conventional microdialysis in freely moving rats and mice. Reverse dialysis of the MOR agonist DAMGO (50 and 100 ,m) into the VTA of rats produced a concentration-dependent increase in dialysate dopamine concentrations. Basal dopamine overflow in the VTA was unaltered in mice lacking the MOR gene. However, basal ,-aminobutyric acid (GABA) overflow in these animals was significantly increased, whereas glutamate overflow was decreased. Intra-VTA perfusion of DAMGO into wild-type (WT) mice increased dopamine overflow. GABA concentrations were decreased, whereas glutamate concentrations in the VTA were unaltered. Consistent with the loss of MOR, no effect of DAMGO was observed in MOR knockout (KO) mice. These data provide the first direct demonstration of tonically active MOR systems in the VTA that regulate basal glutamatergic and GABAergic neurotransmission in this region. We hypothesize that increased GABAergic neurotransmission following constitutive deletion of MOR is due to the elimination of a tonic inhibitory influence of MOR on GABAergic neurons in the VTA, whereas decreased glutamatergic neurotransmission in MOR KO mice is a consequence of intensified GABA tone on glutamatergic neurons and/or terminals. As a consequence, somatodendritic dopamine release is unaltered. Furthermore, MOR KO mice do not exhibit the positive correlation between basal dopamine levels and the glutamate/GABA ratio observed in WT mice. Together, our findings indicate a critical role of VTA MOR in maintaining an intricate balance between excitatory and inhibitory inputs to dopaminergic neurons. [source]

Mice with astrocyte-directed inactivation of connexin43 exhibit increased exploratory behaviour, impaired motor capacities, and changes in brain acetylcholine levels

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 8 2003
Christian Frisch
Abstract Gap junctions mediate communication between many cell types in the brain. Gap junction channels are composed of membrane-spanning connexin (Cx) proteins, allowing the cell-to-cell passage of small ions and metabolites. Cx43 is the main constituent of the brain-spanning astrocytic gap junctional network, controlling activity-related changes in ion and glutamate concentrations as well as metabolic processes. In astrocytes, deletion of Cx43-coding DNA led to attenuated gap junctional coupling and impaired propagation of calcium waves, known to influence neuronal activity. Investigation of the role of Cx43 in behaviour has been impossible so far, due to postnatal lethality of its general deletion. Recently, we have shown that deletion of Cx30, which is also expressed by astrocytes, affects exploration, emotionality, and neurochemistry in the mouse. In the present study, we investigated the effects of the astrocyte-directed inactivation of Cx43 on mouse behaviour and brain neurochemistry. Deletion of Cx43 in astrocytes increased exploratory activity without influencing habituation. In the open field, but not in the elevated plus-maze, an anxiolytic-like effect was observed. Rotarod performance was initially impaired, but reached control level after further training. In the water maze, Cx43 deficient mice showed a steeper learning course, although final performance was similar between groups. Cx43 inactivation in astrocytes increased acetylcholine content in the frontal cortex of water maze-trained animals. Results are discussed in terms of altered communication between astrocytes and neurons, possible compensation processes, and differential effects of Cx30- and astrocyte-specific Cx43 deletion. [source]

The medullary dorsal reticular nucleus enhances the responsiveness of spinal nociceptive neurons to peripheral stimulation in the rat

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2003
Christophe Dugast
Abstract Single-unit spinal recordings combined with application of glutamate into the medullary dorsal reticular nucleus were used to assess the action of this nucleus upon deep dorsal horn neurons in rats. Injection of high glutamate concentrations (10 and 100 mm) induced a dramatic and long-lasting increase of the responses of wide-dynamic range neurons to electrical stimulation of the sciatic nerve in the noxious range, without affecting ongoing discharges. Post-stimulus time histograms revealed that this increase concerned the post-discharge, but not A- or C-fibre-mediated responses, which remained unchanged independently of the stimulation frequency applied. The onset of the glutamate-induced response enhancement occurred with a concentration-dependent time delay and developed slowly until its maximum. These data indicate that the medullary dorsal reticular nucleus exerts a facilitating action upon deep dorsal horn wide-dynamic range neurons by enhancing their capacity to respond to peripheral stimulation through prolongation of their discharge. This action is accompanied by the strengthening of wind-up of deep dorsal horn wide-dynamic range neurons, hence providing a plausible substrate for chronic pain states. These results are in agreement with previous behavioural studies suggesting a pronociceptive role for the dorsal reticular nucleus [Almeida et al. (1996) Brain Res. Bull., 39, 7,15; Almeida et al. (1999) Eur. J. Neurosci., 11, 110,122], and support the involvement of a reverberating circuit, previously described in morphological studies [Almeida et al. (1993) Neuroscience, 55, 1093,1106; Almeida et al. (2000) Eur. J. Pain, 4, 373,387], which probably operates only at a certain threshold of activation. [source]

The effects of local perfusion of DAMGO on extracellular GABA and glutamate concentrations in the rostral ventromedial medulla

JOURNAL OF NEUROCHEMISTRY, Issue 3 2008
Raf Jan-Filip Schepers
Abstract Electrophysiological data suggest an involvement of rostral ventromedial medulla (RVM) GABA and glutamate (GLU) neurons in morphine analgesia. Direct evidence that extracellular concentrations of GABA or GLU are altered in response to mu opioid receptor (MOP-R) activation is, however, lacking. We used in vivo microdialysis to investigate this issue. Basal GABA overflow increased in response to intra-RVM perfusion of KCl (60 mmol/L). Reverse microdialysis of the MOP-R agonist d -Ala(2),NMePhe(4),Gly-ol(5)]enkephalin (DAMGO) (20,500 ,mol/L) produced a concentration-dependent decrease of RVM GABA overflow. Behavioral testing revealed that concentrations that decreased GABA levels increased thermal withdrawal thresholds. A lower agonist concentration that did not increase GABA failed to alter thermal thresholds. DAMGO did not alter GLU concentrations. However, KCl also failed to modify GLU release. Since rapid, transporter-mediated uptake may mask the detection of changes in GLU release, the selective excitatory amino acid transporter inhibitor pyrrolidine-2,4-dicarboxylic acid (tPDC, 0.6 mmol/L) was added to the perfusion medium for subsequent studies. tPDC increased GLU concentrations, confirming transport inhibition. KCl increased GLU dialysate levels in the presence of tPDC, demonstrating that transport inhibition permits detection of depolarization-evoked GLU overflow. In the presence of tPDC, DAMGO increased GLU overflow in a concentration-dependent manner. These data demonstrate that MOP-R activation decreases GABA and increases GLU release in the RVM. We hypothesize that the opposing effects of MOP-R on GLU and GABA transmission contribute to opiate antinociception. [source]

Glutamate levels and transport in cat (Felis catus) area 17 during cortical reorganization following binocular retinal lesions

Subunit-specific desensitization of heteromeric kainate receptors

THE JOURNAL OF PHYSIOLOGY, Issue 4 2010
David D. Mott
Kainate receptor subunits can form functional channels as homomers of GluK1, GluK2 or GluK3, or as heteromeric combinations with each other or incorporating GluK4 or GluK5 subunits. However, GluK4 and GluK5 cannot form functional channels by themselves. Incorporation of GluK4 or GluK5 into a heteromeric complex increases glutamate apparent affinity and also enables receptor activation by the agonist AMPA. Utilizing two-electrode voltage clamp of Xenopus oocytes injected with cRNA encoding kainate receptor subunits, we have observed that heteromeric channels composed of GluK2/GluK4 and GluK2/GluK5 have steady state concentration,response curves that were bell-shaped in response to either glutamate or AMPA. By contrast, homomeric GluK2 channels exhibited a monophasic steady state concentration,response curve that simply plateaued at high glutamate concentrations. By fitting several specific Markov models to GluK2/GluK4 heteromeric and GluK2 homomeric concentration,response data, we have determined that: (a) two strikingly different agonist binding affinities exist; (b) the high-affinity binding site leads to channel opening; and (c) the low-affinity agonist binding site leads to strong desensitization after agonist binding. Model parameters also approximate the onset and recovery kinetics of desensitization observed for macroscopic currents measured from HEK-293 cells expressing GluK2 and GluK4 subunits. The GluK2(E738D) mutation lowers the steady state apparent affinity for glutamate by 9000-fold in comparison to GluK2 homomeric wildtype receptors. When this mutant subunit was expressed with GluK4, the rising phase of the glutamate steady state concentration,response curve overlapped with the wildtype curve, whereas the declining phase was right-shifted toward lower affinity. Taken together, these data are consistent with a scheme whereby high-affinity agonist binding to a non-desensitizing GluK4 subunit opens the heteromeric channel, whereas low-affinity agonist binding to GluK2 desensitizes the whole channel complex. [source]

Glutamate levels and activity of the T cell voltage-gated potassium Kv1.3 channel in patients with systemic lupus erythematosus

ARTHRITIS & RHEUMATISM, Issue 5 2008
C. Poulopoulou
Objective Alterations in glutamate homeostasis and Kv1.3 voltage-gated potassium channel function have been independently associated with T cell dysfunction, whereas selective blockade of Kv1.3 channels inhibits T cell activation and improves T cell,mediated manifestations in animal models of autoimmunity. Because low extracellular glutamate concentrations enhance the activity of this channel in normal T cells ex vivo, we undertook this study to examine serum glutamate concentrations and Kv1.3 channel activity in patients with systemic lupus erythematosus (SLE). Methods We used high-performance liquid chromatography for glutamate measurements, and we used the whole-cell patch-clamp technique for electrophysiologic studies performed in freshly isolated, noncultured peripheral T cells. Results Mean ± SD serum concentrations of glutamate were lower in patients with either clinically quiescent SLE (77 ± 27 ,M [n = 18]) or active SLE (61 ± 36 ,M [n = 16]) than in healthy controls (166 ± 64 ,M [n = 24]) (both P < 0.0001). The intrinsic gating properties of the Kv1.3 channels in lupus T cells were found to be comparable with those in healthy control,derived T cells. Notably, electrophysiologic data from SLE patient,derived T cells exposed to extracellular glutamate concentrations similar to their respective serum levels (50 ,M) demonstrated Kv1.3 current responses enhanced by almost 20% (P < 0.01) compared with those subsequently obtained from the same cell in the presence of glutamate concentrations within control serum levels (200 ,M). Conclusion Based on the key role of Kv1.3 channel activity in lymphocyte physiology, an enhancing in vivo effect of low serum glutamate concentrations on the functional activity of this channel may contribute to lupus T cell hyperactivity. Studies to further elucidate Kv1.3 responses in SLE, as well as the possible pathogenetic role of this unsuspected metabolic abnormality, may have therapeutic implications for SLE patients. [source]

Production of indole diterpenes by Aspergillus alliaceus

BIOTECHNOLOGY & BIOENGINEERING, Issue 5 2006
B. Junker
Abstract Production of two related indole diterpenes (differing by a dimethyl leucine side chain) by Aspergillus alliaceus was improved through several pilot scale fermentations. Media were optimized through focus primarily on initial increases, as well as mid-cycle additions, of carbon and nitrogen sources. Fermentation conditions were improved by varying ventilation conditions using various combinations of air flowrate and back-pressure set points. Production improvements were quantified based on total indole diterpene concentration as well as the ratio of the major-to-minor by-product components. Those changes with a positive substantial impact primarily on total indole diterpene concentration included early cycle glycerol shots and enhanced ventilation conditions (high air flowrate, low back-pressure). Those changes with a significant impact primarily on ratio included higher initial cerelose, soybean oil, monosodium glutamate, tryptophan, or ammonium sulfate concentrations, higher broth pH, and enhanced ventilation conditions. A few changes (higher initial glycerol and monosodium glutamate concentrations) resulted in less notable and desirable titer or ratio changes when implemented individually, but they were adopted to more fully realize the impact of other improvements or to simplify processing. Overall, total indole diterpene titers were improved at the 600 L pilot scale from 125,175 mg/L with a ratio of about 2.1 to 200,260 mg/L with a ratio of about 3.3,4.5. Thus, the ability to optimize total indole diterpene titer and/or ratio readily exists for secondary metabolite production using Aspergillus cultures. © 2006 Wiley Periodicals, Inc. [source]