In vitro uptake of glutamate in GLAST-and GLT-1-transfected mutant CHO-K1 cells is inhibited by the ethylmercury-containing preservative thimerosal

Thimerosal, also known as thimersal, Merthrolate, or sodiumethyl-mercurithiosalicylate, is an organic mercurial compound that is used in a variety of commercial as well as biomedical applications. As a preservative, it is used in a number of vaccines and pharmaceutical products. Its active ingredient is ethylmercury. Both inorganic and organic mercurials are known to interfere with glutamate homeostasis. Brain glutamate is removed mainly by astrocytes from the extracellular fluid via high-affinity astroglial Na⁺-dependent excitatory amino acid transporters, glutamate/ aspartats transporter (GLAST) and glutamate transporter-1 (GLT-1). The effects of thimerosal on glutamate homeostasis have yet to be determined. As a first step in this process, we examined the effects of thimerosal on the transport of [³H]-D-aspartate, a nonmetabolizable glutamate analog, in Chinese hamster ovary (CHO) cells transfected with two glutamate transporter subtypes, GLAST (EAAT1) and GLT-1 (EAAT2). Additionally, studies were undertaken to determine the effects of thimerosal on mRNA and protein levels of these transporters. The results indicate that thimerosal treatment caused significant but selective changes in both glutamate transporter mRNA and protein expression in CHO cells. Thimerosal-mediated inhibition of glutamate transport in the CHO-K1 cell line DdB7 was more pronounced in the GLT-1-transfected cells compared with the GLAST-transfected cells. These studies suggest that thimerosal accumulation in the central nervous system might contribute to dysregulation of glutamate homeostasis.     

Methylmercury alters the in vitro uptake of glutamate in GLAST- and GLT-1-transfected mutant CHO-K1 cells

In order to maintain normal functioning of the brain, glutamate homeostasis and extracellular levels of excitotoxic amino acids (EAA) must be tightly controlled. This is accomplished, in large measure, by the astroglial high-affinity Na⁺-dependent EAA transporters glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1). Methylmercury (MeHg) is a potent neurotoxicant. Astrocytes are known targets for MeHg toxicity, representing a site for mercury localization. Mehg is known to cause astrocytic swelling, EAA release, and uptake inhibition in astrocytes, leading to increased extracellular glutamate levels and ensuing neuronal excitotoxicity and degeneration. However, the mechanisms and contribution of specific glutamate transporters to MeHg-induced glutamate dyshomeostasis remain unknown. Accordingly, the present study was carried out to investigate the effects of MeHg on the transport of [d-2, 3-³H]-d-aspartate, a nonmetabolizable glutamate analog in Chinese hamster ovary cells (CHO) transfected with the glutamate transporter subtypes GLAST or GLT-1. Additional studies examined the effects of MeHg on mRNA and protein levels of these transporters. Our results indicate the following (1) MeHg selectively affects glutamate transporter mRNA expression. MeHg treatment (6 h) led to no discernible changes in GLAST mRNA expression; however, GLT-1 mRNA expression significantly (p<0.001) increased following treatments with 5 or 10 μM MeHg. (2) Selective changes in the expression of glutamate transporter protein levels were also noted. GLAST transporter protein levels significantly (p<0.001, both at 5 and 10 μM MeHg) increased and GLT-1 transporter protein levels significantly (p<0.001) decreased followign MeHg exposure (5 μM). (3) MeHg exposure led to significant inhibition (p<0.05) of glutamate uptake by GLAST (both 5 and 10 μM MeHg), whereas GLT-1 transporter activity was significantly (p<0.01) increased following exposure to 5 and 10 μM MeHg. These studies suggest that MeHg contributes to the dysregulation of glutamate homeostasis and that its effects are distinct for GLAST and GLT-1.     

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

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

The GLT-1 and GLAST glutamate transporters are expressed on morphologically distinct astrocytes and regulated by neuronal activity in primary hippocampal cocultures

The GLT-1 and GLAST astroglial transporters are the glutamate transporters mainly involved in maintaining physiological extracellular glutamate concentrations. Defects in neurotransmitter glutamate transport may represent an important component of glutamate-induced neurodegenerative disorders (such as amyotrophic lateral sclerosis) and CNS insults (ischemia and epilepsy). We characterized the protein expression of GLT-1 and GLAST in primary astrocyte-neuron cocultures derived from rat hippocampal tissues during neuron differentiation/maturation. GLT-1 and GLAST are expressed by morphologically distinct glial fibrillary acidic protein-positive astrocytes, and their expression correlates with the status of neuron differentiation/maturation and activity. Up-regulation of the transporters paralleled the content of the synaptophysin synaptic vesicle marker p38, and down-regulation was a consequence of glutamate-induced neuronal death or the reduction of synaptic activity. Finally, soluble factors in neuronal-conditioned media prevented the down-regulation of the GLT-1 and GLAST proteins. Although other mechanisms may participate in regulating GLT-1 and GLAST in the CNS, our data indicate that soluble factors dependent on neuronal activity play a major regulating role in hippocampal cocultures.     

Selective down-regulation of the astrocyte glutamate transporters GLT-1 and GLAST within the medial thalamus in experimental Wernicke's encephalopathy

Although earlier studies on thiamine deficiency have reported increases in extracellular glutamate concentration in the thalamus, a vulnerable region of the brain in this disorder, the mechanism by which this occurs has remained unresolved. Treatment with pyrithiamine, a central thiamine antagonist, resulted in a 71 and 55% decrease in protein levels of the astrocyte glutamate transporters GLT-1 and GLAST, respectively, by immunoblotting in the medial thalamus of day 14 symptomatic rats at loss of righting reflexes. These changes occurred prior to the onset of convulsions and pannecrosis. Loss of both GLT-1 and GLAST transporter sites was also confirmed in this region of the thalamus at the symptomatic stage using immunohistochemical methods. In contrast, no change in either transporter protein was detected in the non-vulnerable frontal parietal cortex. These effects are selective; protein levels of the astrocyte GABA transporter GAT-3 were unaffected in the medial thalamus. In addition, astrocyte-specific glial fibrillary acidic protein (GFAP) content was unchanged in this brain region, suggesting that astrocytes are spared in this disorder. Loss of GLT-1 or GLAST protein was not observed on day 12 of treatment, indicating that down-regulation of these transporters occurs within 48 h prior to loss of righting reflexes. Finally, GLT-1 content was positively correlated with levels of the neurofilament protein alpha-internexin, suggesting that early neuronal drop-out may contribute to the down-regulation of this glutamate transporter and subsequent pannecrosis. A selective, focal loss of GLT-1 and GLAST transporter proteins provides a rational explanation for the increase in interstitial glutamate levels, and may play a major role in the selective vulnerability of thalamic structures to thiamine deficiency-induced cell death.     

Regulation of glutamate transporter GLT-1 by MAGI-1

The sodium-dependent glutamate transporter, glutamate transporter subtype 1 (GLT-1) is one of the main glutamate transporters in the brain. GLT-1 contains a COOH-terminal sequence similar to one in an isoform of Slo1 K(+) channel protein previously shown to bind MAGI-1 (membrane-associated guanylate kinase with inverted orientation protein-1). MAGI-1 is a scaffold protein which allows the formation of complexes between certain transmembrane proteins, actin-binding proteins, and other regulatory proteins. The glutathione S-transferase pull-down assay demonstrated that MAGI-1 was a binding partner of GLT-1. The interaction between MAGI-1 and GLT-1 was confirmed by co-immunoprecipitation. Immunofluorescence of MAGI-1 and GLT-1 demonstrated that the distribution of MAGI-1 and GLT-1 overlapped in astrocytes. Co-expression of MAGI-1 with GLT-1 in C6 Glioma cells resulted in a significant reduction in the surface expression of GLT-1, as assessed by cell-surface biotinylation. On the other hand, partial knockdown of endogenous MAGI-1 expression by small interfering RNA in differentiated cultured astrocytes increased glutamate uptake and the surface expression of endogenous GLT-1. Knockdown of MAGI-1 increased dihydrokainate-sensitive, Na(+) -dependent glutamate uptake, indicating that MAGI-1 regulates GLT-1 mediated glutamate uptake. These data suggest that MAGI-1 regulates surface expression of GLT-1 and the level of glutamate in the hippocampus.     

Cerebral ischemic preconditioning reduces glutamate excitotoxicity by up-regulating the uptake activity of GLT-1 in rats

Our previous study has shown that cerebral ischemic preconditioning (CIP) can up-regulate the expression of glial glutamate transporter-1 (GLT-1) during the induction of brain ischemic tolerance in rats. The present study was undertaken to further explore the uptake activity of GLT-1 in the process by observing the changes in the concentration of extracellular glutamate with cerebral microdialysis and high-performance liquid chromatography. The results showed that a significant pulse of glutamate concentration reached the peak value of sevenfold of the basal level after lethal ischemic insult, which was associated with delayed neuronal death in the CA1 hippocampus. When the rats were pretreated 2 days before the lethal ischemic insult with CIP which protected the pyramidal neurons against delayed neuronal death, the peak value of glutamate concentration decreased to 3.9 fold of the basal level. Furthermore, pre-administration of dihydrokainate, an inhibitor of GLT-1, prevented the protective effect of CIP on ischemia-induced CA1 cell death. At the same time, compared with the CIP + Ischemia group, the peak value of glutamate concentration significantly increased and reached sixfold of the basal level. These results indicate that CIP induced brain ischemic tolerance via up-regulating GLT-1 uptake activity for glutamate and then decreasing the excitotoxicity of glutamate.     

The glutamate transporter GLAST is involved in spinal nociceptive processing

GLAST and GLT-1 are the most abundant glutamate transporters in the CNS and protect neurons from glutamate neurotoxicity. Here, we investigated the role of GLAST in spinal nociceptive processing. GLAST protein expression was not altered after treatment of rats with either formalin or zymosan. Surprisingly, knock-down of GLAST in the spinal cord using antisense-oligonucleotides decreased glutamate concentrations in cerebrospinal fluid (CSF) and reduced the nociceptive behaviour in the rat formalin assay. However, it did not influence thermal hyperalgesia in the zymosan-induced paw inflammation model indicating that GLAST is associated with spontaneous rather than inflammatory nociception. Mechanisms that might explain the decreased response in the formalin assay may include compensatory activation of other glutamate transporters, inhibition of glutamate release or disturbance of glutamate recycling. In conclusion, these data suggest that inhibition of GLAST expression in the spinal cord reduces excitatory synaptic activity and thereby spontaneous responses after nociceptive stimulation of the paw.     

The investigation of glutamate transporter 1 (GLT-1) degradation pathway in glioblastoma cells

Molecular Biology Reports - Glioblastoma multiform is a primary brain tumor derived from glial cells. The aim of this study is to investigate how glutamate metabolism is regulated by glutamate...     

Glutamate-induced glioma cell proliferation is prevented by functional expression of the glutamate transporter GLT-1

A tetracycline-dependent inducible system was used to achieve controlled expression of the glutamate transporter 1 (GLT-1) in C6 glioma cells. Non-induced cells show modest glutamate uptake and, in the presence of L-cystine, these cells tend to release substantial amounts of glutamate. Overnight exposure to doxycycline increased D-[3H]-aspartate uptake, reaching similar capacity as observed in cultured astrocytes. Efficient clearance of exogenously applied glutamate was evidenced in these cells, even in the presence of l-cystine. The addition of glutamate (100 microM) to the medium of non-induced cells significantly increased their proliferation rate, an effect that was blocked when the expression of GLT-1 was induced. This suggests that impaired glutamate uptake capacity in glioma cells indirectly contributes to their proliferation.     

The accessibility of a novel reentrant loop of the glutamate transporter GLT-1 is restricted by its substrate

The excitatory neurotransmitter glutamate is removed from the synaptic cleft by several related sodium- and potassium-coupled transporters. They thereby restrict the neurotoxicity of this transmitter. Based on the accessibility of single cysteines to the large sulfhydryl reagent 3-N-maleimidyl(propionyl)biocytin, we have proposed a topological model for the astroglial glutamate transporter GLT-1 (Grunewald, M., Bendahan, A. and Kanner, B. I. (1998) Neuron 21, 623-632). Because of several unexpected observations, we have investigated the topological disposition of 19 cysteine residues engineered into a loop proposed to be intracellular. We have probed the accessibility of these cysteines to small and large sulfhydryl reagents. The impermeant hydrophilic sulfhydryl reagent [(2-trimethylammonium)ethyl] methanethiosulfonate inhibits transport activity only at two of these positions, weakly at G365C and potently at A364C. Glutamate and its nontransportable analogue dihydrokainate markedly protect A364C transporters against this impermeant reagent. Using a biotinylated maleimide, we found that, among the 14 mutants tested with it, only A364C is accessible to it from the extracellular side. This, together with our previous observations, indicates that the loop-including amino acid residues 354, 359, 373, and 379-is largely intracellular, but a short region of it forms a reentrant pore-loop-like structure, the accessibility of which is dependent on the conformation of the transporter.     

Differentiation of human trophoblast cells in vitro is inhibited by dimethylsulfoxide

Dimethyl sulfoxide (DMSO) exerts a number of biological effects, the most frequently cited being induction of cell differentiation. The compound also increases invasiveness and metastatic potential. In contrast to the many reports of DMSO-induced cell differentiation, we report here that DMSO inhibits the morphological differentiation of human cytotrophoblast cells to syncytiotrophoblast, as revealed by immunofluorescence staining for desmosomal protein and nuclei. Cytotrophoblast cells treated with DMSO under differentiation-inducing conditions remained mononucleated with intense desmosomal staining. The effect was dose dependent, with a maximal effect seen at 1.5% DMSO. Concentrations of ≤0.5% had no effect and concentrations >2% were cytotoxic. In addition to these morphological changes, DMSO inhibited secretion of human chorionic gonadotropin in a dose-dependent manner. At a concentration of 1.5%, DMSO inhibited secretion by 70%. If cytotrophoblast cells were cultured in the presence of DMSO and then switched to DMSO-free medium, they proceeded to differentiate normally. While the precise mechanism of action remains unknown, judicious use of DMSO may be a useful tool for studying and manipulating the differentiation of human trophoblast cells in vitro. The findings also indicate that care should be used in interpreting results obtained using DMSO as a carrier in drug and inhibitor studies. J. Cell Biochem. 65:460–468. © 1997 Wiley-Liss Inc.     

Effect of topiramate and dBcAMP on expression of the glutamate transporters GLAST and GLT-1 in astrocytes cultured separately, or together with neurons

The mechanism of the antiepileptic drug topiramate is not fully understood, but interaction with the excitatory neurotransmission, e.g. glutamate receptors, is believed to be part of its anticonvulsant effect. The glutamate transporters GLAST and GLT-1 are responsible for the inactivation of glutamate as a neurotransmitter and it was therefore investigated if topiramate might affect the expression of GLAST and GLT-1 in astrocytes cultured separately or together with neurons. Since expression and membrane trafficking of glutamate transporters are affected by the protein kinase C system as well as by dBcAMP it was also investigated if these signalling pathways might play a role. In astrocyte cultures expressing mainly GLAST treatment with dBcAMP (0.25 mM) led to an increased expression of the total amount of GLAST as well as of its membrane association. The enhanced expression in the membrane was particularly pronounced for the oligomeric form of GLAST. No detectable effect on the expression of GLAST in astrocytes treated with topiramate in the presence and absence of protein kinase C activators or inhibitors was observed. Astrocytes co-cultured with neurons expressed both GLAST and GLT-1. In these cultures prolonged exposure to 30 muM topiramate (10 days) led to a statistically significant increase (P<0.025) in the membrane expression of GLAST. In case of GLT-1, culture in the presence of 30 microM topiramate for 1 and 10 days led to alterations in the total, cytoplamic and membrane expression of the oligomeric form of the transporter.     

Functional characterization of the NCC27 nuclear protein in stable transfected CHO-K1 cells.

NCC27 belongs to a family of small, highly conserved, organellar ion channel proteins. It is constitutively expressed by native CHO-K1 and dominantly localized to the nucleus and nuclear membrane. When CHO-K1 cells are transfected with NCC27-expressing constructs, synthesized proteins spill over into the cytoplasm and ion channel activity can then be detected on the plasma as well as nuclear membrane. This provided a unique opportunity to directly compare electrophysiological characteristics of the one cloned channel, both on the nuclear and cytoplasmic membranes. At the same time, as NCC27 is unusually small for an ion channel protein, we wished to directly determine whether it is a membrane-resident channel in its own right. In CHO-K1 cells transfected with epitope-tagged NCC27 constructs, we have demonstrated that the NCC27 conductance is chloride dependent and that the electrophysiological characteristics of the channels are essentially identical whether expressed on plasma or nuclear membranes. In addition, we show that a monoclonal antibody directed at an epitope tag added to NCC27 rapidly inhibits the ability of the expressed protein to conduct chloride, but only when the antibody has access to the tag epitope. By selectively tagging either the amino or carboxyl terminus of NCC27 and varying the side of the membrane from which we record channel activity, we have demonstrated conclusively that NCC27 is a transmembrane protein that directly forms part of the ion channel and, further, that the amino terminus projects outward and the carboxyl terminus inward. We conclude that despite its relatively small size, NCC27 must form an integral part of an ion channel complex.     

Selective induction of glial glutamate transporter GLT-1 by hypertonic stress in C6 glioma cells.

Glial glutamate transporter GLT-1 mRNA was selectively induced in C6 glioma cells exposed to hypertonic stress (HS), while the expression of two other subtypes, GLAST and EAAC1, was suppressed. HS increased phosphorylation of the MAPK family, ERK, p38 MAPK, and JNK. Treatment with a PKC inhibitor showed that phosphorylation of both p38 MAPK and JNK is PKC-dependent but ERK phosphorylation is independent. Inhibition of either ERK or p38 MAPK did not abolish GLT-1 mRNA induction. Inhibition of PKC also had no effect. These findings indicate that the induction of GLT-1 mRNA by HS is independent of the MAPK pathways. This is the first report that the expression of glial glutamate transporters is osmotically regulated.     

Zinc uptake into MCF-10A cells is inhibited by cholesterol depletion

The mechanism for cellular Zn uptake was investigated by depleting cell cholesterol levels, a treatment that disrupts lipid rafts/caveolae-dependent processes and inhibits coated-pit budding. Incubation of MCF-10A human breast epithelial cells with hydroxypropyl-beta-cyclodextrin significantly lowered cell cholesterol levels and significantly inhibited cellular zinc uptake measured at 10 min, but had no effect on 2-deoxyglucose uptake. Replacing potassium for sodium in the uptake buffer significantly stimulated Zn uptake by 20%. The effects of potassium depletion and chlorpromazine on Zn uptake were investigated to determine the contribution of coated-pit endocytosis. Potassium depletion following hypotonic shock significantly inhibited Zn uptake into MCF-10A cells approximately 15%. Chlorpromazine at 20 microg/ml inhibited uptake approximately 30%. The data support the hypothesis that Zn uptake into MCF-10A cells involves lipid rafts/caveolae. The relatively mild effects of potassium depletion and chlorpromazine suggest that a small portion of Zn uptake may require coated pit endocytosis.     

The in vitro activity of ADAM-10 is inhibited by TIMP-1 and TIMP-3

A recombinant soluble form of the catalytic domain of human ADAM-10 was expressed as an Fc fusion protein from myeloma cells. The ADAM-10 was catalytically active, cleaving myelin basic protein and peptides based on the previously described 'metallosheddase' cleavage sites of tumour necrosis factor alpha, CD40 ligand and amyloid precursor protein. The myelin basic protein degradation assay was used to demonstrate that hydroxamate inhibitors of matrix metalloproteinases (MMPs) were also inhibitors of ADAM-10. The natural MMP inhibitors, TIMP-2 and TIMP-4 were unable to inhibit ADAM-10, but TIMP-1 and TIMP-3 were inhibitory. Using a quenched fluorescent substrate assay and ADAM-10 we obtained approximate apparent inhibition constants of 0.1 nM (TIMP-1) and 0.9 nM (TIMP-3). The TIMP-1 inhibition of ADAM-10 could therefore prove useful in distinguishing its activity from that of TACE, which is only inhibited by TIMP-3, in cell based assays.     

The effects of wild-type and mutant HFE expression upon cellular iron uptake in transfected human embryonic kidney cells

In order to measure the effects of HFE (haemochromatosis) upon iron uptake, stable expression of wild-type and C282Y, H63D and S65C mutant HFE cDNA was established in HEK 293 cells. Control cells were transfected with empty vector. Expression of HFE mRNA and protein was detected in the cell lines transfected with HFE cDNA, but not in the control cell line. The ferritin concentration in wild-type cells cultured in 40 microM ferric ammonium citrate was 69% of that in control cells and 81% of that in C282Y cells. The ferritin concentration in H63D cells was intermediate between wild-type and C282Y and the ferritin concentration in S65C cells was similar to wild-type cells. Uptake of transferrin-iron in wild-type, C282Y and control cells was measured over 45 min. The Hill coefficients for transferrin-iron uptake were similar. The V(max) for transferrin-iron uptake in wild-type cells was 59.5% of control cells and 69.5% of C282Y cells. Estimates of K(m) were 232 nM for wild-type cells, 338 nM for C282Y cells and 570 nM for controls. Transferrin receptor levels were lowered, but not significantly, in the HFE transfected cells. The results show that HFE reduces transferrin-iron uptake, probably as an uncompetitive inhibitor.     

Differential expression of the glutamate transporter GLT-1 in pancreas

The glutamate uptake transporter GLT-1 is best understood for its critical role in preventing brain seizures. Increasing evidence argues that GLT-1 also modulates, and is modulated by, metabolic processes that influence glucose homeostasis. To investigate further the potential role of GLT-1 in these regards, the authors examined GLT-1 expression in pancreas and found that mature multimeric GLT-1 protein is stably expressed in the pancreas of wild-type, but not GLT-1 knockout, mice. There are three primary functional carboxyl-terminus GLT-1 splice variants, called GLT-1a, b, and c. Brain and liver express all three variants; however, the pancreas expresses GLT-1a and GLT-1b but not GLT-1c. Quantitative real time-PCR further revealed that while GLT-1a is the predominant GLT-1 splice variant in brain and liver, GLT-1b is the most abundant splice variant expressed in pancreas. Confocal microscopy and immunohistochemistry showed that GLT-1a and GLT-1b are expressed in both islet β- and α-cells. GLT-1b was also expressed in exocrine ductal domains. Finally, glutamine synthetase was coexpressed with GLT-1 in islets, which suggests that, as with liver and brain, one possible role of GLT-1 in the pancreas is to support glutamine synthesis.