Substrate-induced modulation of glutamate uptake in human platelets

In the central nervous system (CNS), glutamate rapidly upregulates the activities of different excitatory amino-acid transporter subtypes (EAATs) in order to help protect neurons from excitotoxicity. Since human platelets display a specific sodium-dependent glutamate uptake activity, and express the three major glutamate transporters, which may be affected in neurological disorders, we investigated whether platelets are subject to substrate-induced modulation as described for CNS. A time- and dose-dependent upregulation of [3H]-glutamate uptake (up to two-fold) was observed in platelets preincubated with glutamate. There was an increase in maximal velocity rate without affinity changes. Glutamate receptor agonists and antagonists did not modulate this upregulation and preincubation with glutamate analogues failed to mimic the glutamate effect. Only aspartate preincubation increased the uptake, albeit approximately 35% less with respect to glutamate. The effect of glutamate preincubation on the expression of the three major transporters was studied by Western blotting, showing an increase of approximately 70% in EAAT1 immunoreactivity that was completely blocked by cycloheximide (CEM). However, L-serine-O-sulphate, at a concentration (200 microM) known to block EAAT1/3 selectively, did not completely inhibit the effect of glutamate stimulation, indicating the possible involvement of EAAT2. In fact, glutamate stimulation was completely abolished only when, following CEM pre-incubation, the experiment was run in the presence of the selective EAAT2 inhibitor dihydrokainic acid. Since surface biotinylation experiments failed to show evidence of EAAT2 translocation, our results suggest the existence of a different way of regulating EAAT2 activity. These findings indicate that human platelets display a substrate-dependent modulation of glutamate uptake mediated by different molecular mechanisms and confirm that ex vivo platelets are a reliable model to investigate the dysfunction of glutamate uptake regulation in patients affected by neurological disorders.     

谷氨酸转运体靶点药物的抗脑缺血作用研究进展

兴奋性氨基酸毒性是脑缺血损伤的主要机制之一。缺血期间谷氨酸的大量累积会导致神经元细胞、星形胶质细胞等神经细胞发生兴奋性毒性损伤,因此对缺血期间谷氨酸水平的调控一直是脑缺血防治药物研究的重点。近年来研究表明,通过上调星形胶质细胞上谷氨酸转运体GLAST(EAAT1)和GLT-1(EAAT2)的表达或活性,增加缺血时谷氨酸的摄取,维持突触间隙内谷氨酸的正常浓度,从而降低兴奋性毒性,减轻缺血性脑损伤。一些化合物如β-内酰胺类抗生素、尿酸、甲状腺激素、雌激素、山楂酸等已在体内或体外实验中被证实对谷氨酸转运体的调节作用,对抗谷氨酸毒性,发挥神经保护作用。研究和开发以星形胶质细胞谷氨酸转运体为作用靶点的药物,为缺血性脑损伤的预防和治疗提供了一条新的途径。     

Bromocriptine, an ergot alkaloid, inhibits excitatory amino acid release mediated by glutamate transporter reversal

Bromocriptine, a dopamine D₂ receptor agonist, has widely been used for patients with Parkinson's disease. The aim of the present study was to investigate the effect of bromocriptine on glutamate transporter. Since the astroglial glutamate transporter GLT-1 (EAAT2) is the predominant isoform in the forebrain, we generated EAAT2-expressing human embryonic kidney cells and immortalized mouse astrocytes. In the present studies, we observed a GLT-1-immunoreactive band and significant Na⁺-dependent d-[³H] aspartate uptake. Furthermore, the glutamate transporter inhibitors, dl-threo-β-benzyloxyaspartic acid (TBOA) and dihydrokainate (DHK), displayed a dose-dependent reduction of d-[³H] aspartate uptake in both types of cells. In contrast, cells exposed to either chemical anoxia or high KCl elicited a marked release of d-[³H] aspartate, and the release was inhibited by TBOA and DHK, implying the contribution of glutamate transporter reversal. Interestingly, we found that bromocriptine dose-dependently inhibits d-[³H] aspartate release elicited by chemical anoxia or high KCl, while no changes occurred in the uptake. The inhibitory action of bromocriptine was not affected by sulpiride, a dopamine D₂ receptor antagonist. On the other hand, bromocriptine had no effect on swelling-induced d-[³H] aspartate release, which is mediated by volume-regulated anion channels. In vivo studies revealed that bromocriptine suppresses the excessive elevation of glutamate levels in gerbils subjected to transient forebrain ischemia in a manner similar to DHK. Taken together, these results provide evidence that bromocriptine inhibits excitatory amino acid release via reversed operation of GLT-1 without altering forward transport.     

Excitatory amino acid transporters as emerging targets for central nervous system therapeutics

In the mammalian central nervous system (CNS) the excitatory amino acid transporter (EAAT) family of proteins are responsible for the high-affinity sodium-dependent uptake of glutamate into both astroglial cells and neurones. Normal EAAT function is required both for the efficient termination of glutamatergic neurotransmission and for the maintenance of low extracellular glutamate concentrations, thereby preventing glutamate excitotoxicity. It is widely believed that a dysfunction of glutamate transmission participates in the aetiology of a number of neurodegenerative and neuropsychiatric disorders and diseases. This review introduces the EAATs as a new family of emerging therapeutic targets for CNS disorders by virtue of their central role in maintaining glutamate homeostasis. We examine recent findings on the modulation and regulation of EAATs and review the changes in both EAAT function and expression which have been described in a number of neuropathological conditions.     

Etomidate reduces glutamate uptake in rat cultured glial cells: involvement of PKA

Background and purpose:

Glutamate is the main excitatory neurotransmitter in the vertebrate CNS. Removal of the transmitter from the synaptic cleft by glial and neuronal glutamate transporters (GLTs) has an important function in terminating glutamatergic neurotransmission and neurological disorders. Five distinct excitatory amino-acid transporters have been characterized, among which the glial transporters excitatory amino-acid transporter 1 (EAAT1) (glutamate aspartate transporter) and EAAT2 (GLT1) are most important for the removal of extracellular glutamate. The purpose of this study was to describe the effect of the commonly used anaesthetic etomidate on glutamate uptake in cultures of glial cells.

Experimental approach:

The activity of the transporters was determined electrophysiologically using the whole cell configuration of the patch-clamp recording technique.

Key results:

Glutamate uptake was suppressed by etomidate (3–100 μM) in a time- and concentration-dependent manner with a half-maximum effect occurring at 2.4±0.6 μM. Maximum inhibition was approximately 50% with respect to the control. Etomidate led to a significant decrease of V_max whereas the K_m of the transporter was unaffected. In all cases, suppression of glutamate uptake was reversible within a few minutes upon washout. Furthermore, both GF 109203X, a nonselective inhibitor of PKs, and H89, a selective blocker of PKA, completely abolished the inhibitory effect of etomidate.

Conclusion and implications:

Inhibition of glutamate uptake by etomidate at clinically relevant concentrations may affect glutamatergic neurotransmission by increasing the glutamate concentration in the synaptic cleft and may compromise patients suffering from acute or chronic neurological disorders such as CNS trauma or epilepsy.     

β-cypermethrin-induced acute neurotoxicity in the cerebral cortex of mice

A Type II pyrethroid pesticide β-cypermethrin is widely used in agriculture and domestic applications for pest control. However, the effect of β-cypermethrin on the glutamate neurotransmitter has not been well-documented. In the current study, mice were treated with 20, 40, or 80?mg/kg β-cypermethrin by a single oral gavage, with corn oil as a vehicle control. Four hours after treatment, we investigated glutamate levels and glutamate-metabolizing enzyme (phosphate-activated glutaminase, PAG; glutamine synthetase, GS) activities in the cerebral cortex of mice, using a HPLC system with ultraviolet detectors and a colorimetric assay. Glutamate uptake levels in the synaptosomes of cerebral cortex and mRNA expression levels of PAG, GS, and glutamate transporter-1 (GLT-1) in the cerebral cortex were detected by a radioactive labeling method and qRT-PCR, respectively. Toxic symptoms were observed in mice treated with 40 or 80?mg/kg β-cypermethrin. Compared with the control, significant decreases in glutamate level and GS activity, and an obvious increase in synaptosomal glutamate uptake, were found in the cerebral cortex of mice treated with 80?mg/kg β-cypermethrin. No significant changes were found among groups in PAG activity or PAG, GS, and GLT-1 mRNA expression levels. These results suggest that β-cypermethrin treatment may reduce the glutamate level in the mouse cerebral cortex, which is associated with decreased GS activity and increased synaptosomal glutamate uptake. Our findings provide a partial explanation for the neurotoxic effects of synthetic β-cypermethrin insecticides.     

Aroclor 1254 selectively inhibits expression of glial GLT-1 glutamate transporter in the forebrain of chronically exposed adult rat

Aroclor 1254, a commercially produced mixture of polychlorinated biphenyls, is known to cause many adverse conditions, including neurotoxicity. It has been recently postulated that upregulation of N-methyl-d-aspartate receptors (NMDARs) and enhanced glutamate signalling which leads to excitotoxicity, is the mechanism of Aroclor-induced neurotoxicity. To obtain insights into the mechanisms underlying glutamatergic overstimulation, we investigated the function and expression of sodium-dependent glutamate transporters which are known to regulate extracellular glutamate concentrations in the brain. Exposure to Aroclor 1254 was found to significantly lower the uptake of radioactive glutamate into gliosomal fractions obtained from adult rat brains. It also markedly decreased the expression of both protein and mRNA of GLT-1, the main glial glutamate transporter. This indicates that downregulation of GLT-1 may potentially lead to disturbances in glutamate clearance. The expression of GLAST, another astroglial glutamate transporter, was unchanged under conditions of Aroclor toxicity. Conversely, we observed enhanced glutamate uptake into nerve-endings fractions paralleled by increased EAAC1 protein expression. This may reflect the induction of protective mechanisms.     

Inducible expression and pharmacology of the human excitatory amino acid transporter 2 subtype of L-glutamate transporter

1. In this study we have examined the use of the ecdysone-inducible mammalian expression system (Invitrogen) for the regulation of expression of the predominant L-glutamate transporter EAAT2 (Excitatory Amino Acid Transporter) in HEK 293 cells. 2. HEK 293 cells which were stably transformed with the regulatory vector pVgRXR (EcR 293 cells) were used for transfection of the human EAAT2 cDNA using the inducible vector pIND and a clone designated HEK/EAAT2 was selected for further characterization. 3. Na+-dependent L-glutamate uptake activity (3.2 pmol min-1 mg-1) was observed in EcR 293 cells and this was increased approximately 2 fold in the uninduced HEK/EAAT2 cells, indicating a low level of basal EAAT2 activity in the absence of exogenous inducing agent. Exposure of HEK/EAAT2 cells to the ecdysone analogue Ponasterone A (10 microM for 24 h) resulted in a > or = 10 fold increase in the Na+-dependent activity. 4. L-glutamate uptake into induced HEK/EAAT2 cells followed first-order Michaelis-Menten kinetics and Eadie-Hofstee transformation of the saturable uptake data produced estimates of kinetic parameters as follows; Km 52.7+/-7.5 microM, Vmax 3.8+/-0.9 nmol min-1 mg-1 protein. 5. The pharmacological profile of the EAAT2 subtype was characterized using a series of L-glutamate transport inhibitors and the rank order of inhibitory potency was similar to that described previously for the rat homologue GLT-1 and in synaptosomal preparations from rat cortex. 6. Addition of the EAAT2 modulator arachidonic acid resulted in an enhancement (155+/-5% control in the presence of 30 microM) of the L-glutamate transport capacity in the induced HEK/EAAT2 cells. 7. This study demonstrates that the expression of EAAT2 can be regulated in a mammalian cell line using the ecdysone-inducible mammalian expression system.     

Parawixin1: a spider toxin opening new avenues for glutamate transporter pharmacology

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system. After release from glutamatergic nerve terminals, glial and neuronal glutamate transporters remove glutamate from the synaptic cleft to terminate synaptic transmission and to prevent neuronal damage by excessive glutamate receptor activation. In this issue of Molecular Pharmacology, Fontana et al. (p. 1228) report on the action of a venom compound, Parawixin1, on excitatory amino acid transporters (EAATs). They demonstrate that this agent selectively affects a glial glutamate transporter, EAAT2, by specifically increasing one particular step of the glutamate uptake cycle. Disturbed glutamate homeostasis seems to be a pathogenetic factor in several neurodegenerative disorders. Because EAAT2 is a key player in determining the extracellular glutamate concentration in the mammalian brain, drugs targeting this protein could prevent glutamate excitotoxicity without blocking glutamatergic transmission. Its specificity and selectivity makes Parawixin1 a perfect starting point to design small molecules for the treatment of pathological conditions caused by alterations of glutamate homeostasis.     

The Rho kinase inhibitor Fasudil up-regulates astrocytic glutamate transport subsequent to actin remodelling in murine cultured astrocytes

BACKGROUND AND PURPOSE

Glutamate transporters play a major role in maintaining brain homeostasis and the astrocytic transporters, EAAT1 and EAAT2, are functionally dominant. Astrocytic excitatory amino acid transporters (EAATs) play important roles in various neuropathologies wherein astrocytes undergo cytoskeletal changes. Astrocytic plasticity is well documented, but the interface between EAAT function, actin and the astrocytic cytoskeleton is poorly understood. Because Rho kinase (ROCK) is a key determinant of actin polymerization, we investigated the effects of ROCK inhibitors on EAAT activity and astrocytic morphology.

EXPERIMENTAL APPROACH

The functional activity of glutamate transport was determined in murine cultured astrocytes after exposure to the ROCK inhibitors Fasudil (HA-1077) and Y27632 using biochemical, molecular and morphological approaches. Cytochemical analyses assessed changes in astrocytic morphology, F-/G-actin, and localizations of EAAT1/2.

RESULTS

Fasudil and Y27632 increased [³H]-d-aspartate (d-Asp) uptake into astrocytes, and the action of Fasudil was time-dependent and concentration-related. The rapid stellation of astrocytes (glial fibrillary acidic protein immunocytochemistry) induced by Fasudil was accompanied by reduced phalloidin staining of F-actin and increased V_max for [³H]-d-Asp uptake. Immunoblotting after biotinylation demonstrated that Fasudil increased the expression of EAAT1 and EAAT2 on the cell surface. Immunocytochemistry indicated that Fasudil induced prominent labelling of astrocytic processes by EAAT1/2.

CONCLUSION AND IMPLICATIONS

These data show for the first time that ROCK plays a major role in determining the cell surface expression of EAAT1/2, providing new evidence for an association between transporter function and astrocytic phenotype. ROCK inhibitors, via the actin cytoskeleton, effect a consequent elevation of glutamate transporter function – this activity profile may contribute to their beneficial actions in neuropathologies.     

WAY-855 (3-amino-tricyclo[2.2.1.02.6]heptane-1,3-dicarboxylic acid): a novel, EAAT2-preferring, nonsubstrate inhibitor of high-affinity glutamate uptake

The pharmacological profile of a novel glutamate transport inhibitor, WAY-855 (3-amino-tricyclo[2.2.1.0(2.6)]heptane-1,3-dicarboxylic acid), on the activity of the human forebrain glutamate transporters EAAT1, EAAT2 and EAAT3 expressed in stable mammalian cell lines and in Xenopus laevis oocytes is presented. WAY-855 inhibited glutamate uptake mediated by all three subtypes in a concentration-dependent manner, with preferential inhibition of the CNS-predominant EAAT2 subtype in both cells and oocytes. IC50 values for EAAT2 and EAAT3 inhibition in cells were 2.2 and 24.5 microM, respectively, while EAAT1 activity was inhibited by 50% at 100 microM (IC50 values determined in oocytes were 1.3 microM (EAAT2), 52.5 microM (EAAT3) and 125.9 microM (EAAT1)). Application of WAY-855 to EAAT-expressing oocytes failed to induce a transporter current, and the compound failed to exchange with accumulated [3H]d-aspartate in synaptosomes consistent with a nonsubstrate inhibitor. WAY-855 inhibited d-aspartate uptake into cortical synaptosomes by a competitive mechanism, and with similar potency to that observed for the cloned EAAT2. WAY-855 failed to agonise or antagonise ionotropic glutamate receptors in cultured hippocampal neurones, or the human metabotropic glutamate receptor subtype 4 expressed in a stable cell line. WAY-855 represents a novel structure in glutamate transporter pharmacology, and exploration of this structure might provide insights into the discrimination between EAAT2 and other EAAT subtypes.     

Rapamycin prevents the mutant huntingtin-suppressed GLT-1 expression in cultured astrocytes

Aim:

To investigate the effects of rapamycin on glutamate uptake in cultured rat astrocytes expressing N-terminal 552 residues of mutant huntingtin (Htt-552).

Methods:

Methods: Primary astrocyte cultures were prepared from the cortex of postnatal rat pups. An astrocytes model of Huntington''s disease was established using the astrocytes infected with adenovirus carrying coden gene of N-terminal 552 residues of Huntingtin. The protein levels of glutamate transporters GLT-1 and GLAST, the autophagic marker microtubule-associated protein 1A/1B-light chain 3 (LC3) and the autophagy substrate p62 in the astrocytes were examined using Western blotting. The mRNA expression levels of GLT-1 and GLAST in the astrocytes were determined using Real-time PCR. ³H]glutamate uptake by the astrocytes was measured with liquid scintillation counting.

Results:

The expression of mutant Htt-552 in the astrocytes significantly decreased both the mRNA and protein levels of GLT-1 but not those of GLAST. Furthermore, Htt-552 significantly reduced ³H]glutamate uptake by the astrocytes. Treatment with the autophagy inhibitor 3-MA (10 mmol/L) significantly increased the accumulation of mutant Htt-552, and reduced the expression of GLT-1 and ³H]glutamate uptake in the astrocytes. Treatment with the autophagy stimulator rapamycin (0.2 mg/mL) significantly reduced the accumulation of mutant Htt-552, and reversed the changes in GLT-1 expression and ³H]glutamate uptake in the astrocytes.

Conclusion:

Rapamcin, an autophagy stimulator, can prevent the suppression of GLT-1 expression and glutamate uptake by mutant Htt-552 in cultured astrocytes.     

Identification of glutamate transporters and receptors in mouse testis

INTRODUCTION Glutamate is the predominant excitatory neu-rotransmitter in the central nervous system (CNS). Itsneurotransmission can be mediated by various ligand-gated ion channels, of which there are three subtypes.These subtypes, which are classified on the basis ofsequence homologies and agonist affinities, are N-methyl-D-aspartate (NMDA) receptors (NR1 andNR2A-D), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors (GluR1-4), and kainate(KA) receptors (GluR5-7…     

Isoflurane induces a protein kinase C alpha-dependent increase in cell-surface protein level and activity of glutamate transporter type 3

Glutamate transporters regulate extracellular concentrations of glutamate, an excitatory neurotransmitter in the central nervous system. We have shown that the commonly used anesthetic isoflurane increased the activity of glutamate transporter type 3 (excitatory amino acid transporter 3, EAAT3) possibly via a protein kinase C (PKC)-dependent pathway. In this study, we showed that isoflurane induced a time- and concentration-dependent redistribution of EAAT3 to the cell membrane in C6 glioma cells. This redistribution was inhibited by staurosporine, a pan PKC inhibitor, or by 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Go6976) at a concentration that selectively inhibits conventional PKC isozymes (PKC alpha, -beta, and -gamma). This isoflurane-induced EAAT3 redistribution was also blocked when the expression of PKC alpha but not PKC beta proteins was down-regulated by the respective antisense oligonucleotides. The isoflurane-induced increase of glutamate uptake by EAAT3 was abolished by the down-regulation of PKC alpha expression. Immunoprecipitation with an anti-EAAT3 antibody pulled down more PKC alpha in cells exposed to isoflurane than in control cells. Isoflurane also increased the phosphorylated EAAT3 and the redistribution of PKC alpha to the particulate fraction of cells. Consistent with the results in C6 cells, isoflurane also increased EAAT3 cell-surface expression and enhanced the association of PKC alpha with EAAT3 in rat hippocampal synaptosomes. Our results suggest that the isoflurane-induced increase in EAAT3 activity requires an increased amount of EAAT3 protein in the plasma membrane. These effects are PKC alpha-dependent and may rely on the formation of an EAAT3-PKC alpha complex. Together, these results suggest an important mechanism for the regulation of glutamate transporter functions and expand our understanding of isoflurane pharmacology at cellular and molecular levels.     

The neuroprotective agent MS-153 stimulates glutamate uptake

We investigated the effect of (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153), a novel neuroprotective agent, on L-[3H]glutamate uptake through GLT-1, a Na(+)/K(+)-dependent glial glutamate transporter, expressed in COS-7 cells. MS-153 (1-100 microM) accelerated the L-[3H]glutamate uptake through GLT-1 in a concentration-dependent and time-dependent manner. Eadie-Hofstee analysis revealed that MS-153 significantly decreased the K(m) of the glutamate uptake by COS-7 cells expressing GLT-1. In contrast, [3H]gamma-aminobutyric acid (GABA) uptake through a glial GABA transporter was not affected. In addition, MS-153 increased Na(+) currents through GLT-1 expressed in Xenopus oocytes. We also investigated the effect of MS-153 on amino acid efflux from rat hippocampal slices. The increase in glutamate efflux induced by 50 mM KCl was significantly attenuated by the treatment with MS-153 at 10 microM, while MS-153 had no significant effect on the K(+)-evoked efflux of GABA. Furthermore, the increase in glutamate efflux by ischemia (hypoxia/aglycemia) was partially, but significantly inhibited by MS-153. These results suggest that the cerebroprotective effect of MS-153 in this ischemic model in vivo is due to the specific reduction of the glutamate concentration in the extracellular space, which can probably be attributed to the acceleration of glutamate uptake by the indirect modulation of the glutamate transporter activity.