Localization of neuronal and glial glutamate transporters

The cellular and subcellular distributions of the glutamate transporter subtypes EAAC1, GLT-1, and GLAST in the rat CNS were demonstrated using anti-peptide antibodies that recognize the C-terminal domains of each transporter. On immunoblots, the antibodies specifically recognize proteins of 65-73 kDa in total brain homogenates. Immunocytochemistry shows that glutamate transporter subtypes are distributed differentially within neurons and astroglia. EAAC1 is specific for certain neurons, such as large pyramidal cortical neurons and Purkinje cells, but does not appear to be selective for glutamatergic neurons. GLT-1 is localized only to astroglia. GLAST is found in both neurons and astroglia. The regional localizations are unique to each transporter subtype. EAAC1 is highly enriched in the cortex, hippocampus, and caudate-putamen and is confined to pre- and postsynaptic elements. GLT-1 is distributed in astrocytes throughout the brain and spinal cord. GLAST is most abundant in Bergmann glia in the cerebellar molecular layer brain, but is also present in the cortex, hippocampus, and deep cerebellar nuclei.     

Ibudilast prevents oligodendroglial excitotoxicity

Previously we have demonstrated that cells of oligodendroglial lineage express non-N-methyl-D-aspartate (NMDA) glutamate receptor (GluR) genes and are damaged by kainate induced Ca2+ influx via non NMDA GluR channels of the alpha-amino-3-hydroxy-5-methyl 4 isoxazole propionate (AMPA) type, representing oligodendroglial excitotoxicity. We here present the finding that ibudilast, which is used clinically for treat patients with asthma and cerebrovascular diseases, prevents oligodendroglia excitotoxicity. The oligodendrocyte like cells (OLC), differentiated from the CG-4 cell line established from rat oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells, were exposed to 2 mM kainate for 24 h and cell death was evaluated by measuring activity of lactate dehydrogenase (LDH) released into the culture medium. Kainate induced cell death was prevented by 10 to 100 microM ibudilast, which increased intracellular cAMP. A 45Ca2+ influx study revealed that ibudilast attenuated kainate-induced Ca2+ influx. Inhibition of kainate-induced Ca2+ influx by ibudilast was decreased by H-89, a protein kinase A (PKA) inhibitor, but increased by okadaic acid, an inhibitor of phosphatase 1 and 2A. Therefore, we concluded that ibudilast prevented oligodendroglial excitotoxicity by a PKA-dependent phosphorylation process resulting in decreased kainate-induced Ca2+ influx.     

Expression of high-affinity neuronal and glial glutamate transporters in the rat optic nerve

Recent studies have revealed that a dynamic axon-glial signaling occurs in the rat optic nerve, which is devoid of synapses. This interaction is postulated to be mediated by non-vesicular release of glutamate via a reversal of high-affinity glutamate transporters. Here we examined the expression of glial glutamate transporters (GLAST and GLT-1) and a neuronal transporter (EAAC1) in the rat optic nerve. RT-PCR analysis revealed the presence of mRNAs for GLT-1 and GLAST, but not EAAC1. RNase protection assays showed that of the two glial transporters, mRNA for GLAST was expressed at much higher level than was GLT-1. A similar expression pattern was found in primary astrocyte culture cells. GLAST mRNA level in the optic nerve was comparable to that in the cerebellum. Developmentally, GLAST mRNA level was highest at P2 and dropped slightly by adulthood. Nerve transection resulted in little or no change in mRNA levels for GLAST and GLT-1 assayed at 4 to 14 days post-transection, but GLAST mRNA level was decreased at 64 days. Western blot analysis revealed that the rat optic nerve showed immunoreactivity to antibodies against GLT-1, GLAST, and EAAC1. In conclusion, we suggest that glial and neuronal transporters are present in the rat optic nerve, where dynamic axon-glial interaction has been known to occur. In particular, the unusually high level of expression of GLAST in the optic nerve suggests a possible role for this glial transporter in protecting optic nerves from neurotoxicity during postnatal development.     

Mitochondria and neuronal glutamate excitotoxicity

The role of mitochondria in the control of glutamate excitotoxicity is investigated. The response of cultured cerebellar granule cells to continuous glutamate exposure is characterised by a transient elevation in cytoplasmic free calcium concentration followed by decay to a plateau as NMDA receptors partially inactivate. After a variable latent period, a secondary, irreversible increase in calcium occurs (delayed calcium deregulation, DCD) which precedes and predicts subsequent cell death. DCD is not controlled by mitochondrial ATP synthesis since it is unchanged in the presence of the ATP synthase inhibitor oligomycin in cells with active glycolysis. However, mitochondrial depolarisation (and hence inhibition of mitochondrial calcium accumulation) without parallel ATP depletion (oligomycin plus either rotenone or antimycin A) strongly protects the cells against DCD. Glutamate exposure is associated with an increase in the generation of superoxide anion by the cells, but superoxide generation in the absence of mitochondrial calcium accumulation is not neurotoxic. While it is concluded that mitochondrial calcium accumulation plays a critical role in the induction of DCD we can find no evidence for the involvement of the mitochondrial permeability transition.     

Regulation of the glial Na+-dependent glutamate transporters by cyclic AMP analogs and neurons

Sodium-dependent transport into astrocytes is critical for maintaining the extracellular concentrations of glutamate below toxic levels in the central nervous system. In this study, the expression of the glial glutamate transporters GLT-1 and GLAST was studied in primary cultures derived from cortical tissue. In primary astrocytes, GLAST protein levels were approximately one half of those observed in cortical tissue, but GLT-1 protein was present at very low levels compared with cortical tissue. Maintenance of these astrocytes in medium supplemented with dibutyryl-cAMP (dbcAMP) caused a dramatic change in cell morphology, increased GLT-1 and GLAST mRNA levels approximately 5-fold, increased GLAST protein approximately 2-fold, and increased GLT-1 protein >/=8-20-fold. These increases in protein expression were accompanied by 2-fold increases in the Vmax and Km values for Na+-dependent L-[3H]glutamate transport activity. Although GLT-1 is sensitive to inhibition by dihydrokainate in heterologous expression systems, no dihydrokainate sensitivity was observed in astrocyte cultures that expressed GLT-1. Biotinylation with a membrane-impermeant reagent, separation of the biotinylated/cell surface proteins, and subsequent Western blotting demonstrated that both GLT-1 and GLAST were present at the cell surface. Coculturing of astrocytes with neurons also induced expression of GLT-1, which colocalized with the glial specific marker, glial fibrillary acidic protein. Neurons induced a small increase in GLAST protein. Several studies were performed to examine the mechanism by which neurons regulate expression of the glial transporters. Three different protein kinase A (PKA) antagonists did not block the effect of neurons on glial expression of GLT-1 protein, but the addition of dbcAMP to mixed cultures of neurons and astrocytes did not cause GLT-1 protein to increase further. This suggests that neurons do not regulate GLT-1 by activation of PKA but that neurons and dbcAMP regulate GLT-1 protein through convergent pathways. As was observed with GLT-1, the increases in GLAST protein observed in cocultures were not blocked by PKA antagonists, but unlike GLT-1, the addition of dbcAMP to mixed cultures of neurons and astrocytes caused GLAST protein to increase approximately 2-fold. Neurons separated from astrocytes with a semipermeable membrane increased GLT-1 protein, indicating that the effect of neurons was mediated by a diffusible molecule. Treatment of cocultures with high concentrations of either N-methyl-D-aspartate or glutamate killed the neurons, caused GLT-1 protein to decrease, and caused GLAST protein to increase. These studies suggest that GLT-1 and GLAST protein are regulated independently in astrocyte cultures and that a diffusible molecule secreted by neurons induces expression of GLT-1 in astrocytes.     

Ifenprodil prevents glutamate cytotoxicity via polyamine modulatory sites of N-methyl-D-aspartate receptors in cultured cortical neurons

Neuroprotective effects of ifenprodil, a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, against glutamate cytotoxicity were examined in cultured rat cortical neurons. The viability of the cultures was markedly reduced by a 10-min exposure to glutamate followed by incubation with glutamate-free medium for 60 min. Ifenprodil and its derivative SL 82.0715 dose-dependently prevented cell death induced by glutamate. The NMDA antagonists MK-801 and 3-[(+/-)-2-carboxypiperazin-4-yl]propyl-1-phosphonic acid also prevented glutamate cytotoxicity with a potency similar to that of ifenprodil. Ifenprodil as well as MK-801 prevented NMDA-induced cytotoxicity, but did not affect kainate-induced cytotoxicity. Glutamate cytotoxicity was inhibited by removing extracellular Ca++ during and immediately after glutamate exposure. Ifenprodil and MK-801 reduced NMDA-induced Ca++ influx measured with rhod-2. Either spermidine, a polyamine modulatory site agonist, or glycine, a strychnine-insensitive glycine site agonist, potentiated NMDA- and glutamate-induced cytotoxicity. The protective effects of ifenprodil against NMDA- and glutamate-induced cytotoxicity were significantly reduced by spermidine, but not by glycine. These findings indicate that ifenprodil protects cortical neurons against glutamate cytotoxicity by selective antagonism of the polyamine modulatory site of the NMDA receptor complex.     

Synaptic laminin prevents glial entry into the synaptic cleft

Presynaptic and postsynaptic membranes directly oppose each other at chemical synapses, minimizing the delay in transmitting information across the synaptic cleft. Extrasynaptic neuronal surfaces, in contrast, are almost entirely covered by processes from glial cells. The exclusion of glial cells from the synaptic cleft, and the long-term stability of synapses, presumably result in large part from the tight adhesion between presynaptic and postsynaptic elements. Here we show that there is another requirement for synaptic maintenance: glial cells of the skeletal neuromuscular synapse, Schwann cells, are actively inhibited from entering the synaptic cleft between the motor nerve terminal and the muscle fibre. One inhibitory component is laminin 11, a heterotrimeric glycoprotein that is concentrated in the synaptic cleft. Regulation of an inhibitory interaction between glial cells and synaptic cleft components may contribute to synaptic rearrangements, and loss of this inhibition may underlie the loss of synapses that results from injury to the postsynaptic cell.     

The elusive transporters with a high affinity for glutamate

Removal of glutamate from the synaptic cleft is an essential component of the transmission process at glutamatergic synapses. This requirement is fulfilled by transporters that have a high affinity for glutamate and exhibit a unique coupling to Na+, K+ and OH- ions. Independently, three groups have succeeded in cloning cDNAs encoding high-affinity Na(+)-dependent glutamate transporters. These transporters are structurally distinct from previously characterized neurotransmitter transporters and show sequence identity with prokaryotic glutamate and dicarboxylate transporters. In addition, they exhibit significant differences in their structure, function and tissue distribution. This review compares and contrasts these differences, and incorporates into the existing body of knowledge these new breakthroughs.     

Monitoring glutamate release during LTP with glial transporter currents

Aminopeptidase (AP) A is a transmembrane type II molecule widely distributed in mammalian tissues. Since APA expression may be absent in renal cell carcinoma (RCC), it is possible that there is an altered regulation or other defect of APA upon malignant transformation of proximal tubular cells. However, investigations into the regulation of APA on tumour cells are rare. We report, for the first time, that both transforming growth factor-beta 1 (TGF-beta1) and tumour necrosis factor-alpha (TNF-alpha) down-regulate APA mRNA as well as protein expression in renal tubular epithelial cells and RCC cells in culture. In addition to this, both cytokines decrease dipeptidylpeptidase (DP) IV/CD26 mRNA, but not APN/CD13 mRNA expression. Otherwise, IL-4 and IL-13 increase CD13 as well as CD26 expression, but do not alter APA expression. Interferon-alpha (IFN-alpha), IFN-beta and IFN-gamma increase mRNA expression of all the three membrane ectopeptidases, whereas IL-1, IL-6, IL-7, IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been found to be without any significant effect. Treatment of cultured cells with cAMP-increasing agents, such as 8-bromo-cAMP or A23187, results in an increase in APA and DPIV/CD26, but no change in APN/CD13 mRNA expression or even a decrease in it. Furthermore, AP inhibitors can influence APA mRNA expression, since bestatin causes an increase in APA expression in a time- and dose-dependent manner, whereas bestatin does not change CD13 or CD26 expression. No difference could be found with respect to the modulation by different mediators between RCC cells and renal epithelial cells, though permanent tumour cell lines such as Caki-1 and Caki-2 may have lost some of the normally expressed peptidases.     

Phosphorylation and modulation of brain glutamate transporters by protein kinase C

High affinity sodium- and potassium-coupled L-glutamate transport into presynaptic nerve terminals and fine glial processes removes the neurotransmitter from the synaptic cleft, thereby terminating glutamergic transmission. This report describes that the purified L-glutamate transporter from pig brain is phosphorylated by protein kinase C, predominantly at serine residues. Upon exposure of C6 cells, a cell line of glial origin, to 12-O-tetradecanoylphorbol-13-acetate, about a 2-fold stimulation of L-glutamate transport is observed within 30 min. Concomitantly, the level of phosphorylation increases with similar kinetics. The phorbol ester also stimulates L-glutamate transport in HeLa cells infected with a recombinant vaccinia virus expressing T7 RNA polymerase and transfected with pT7-GLT-1. The latter is a recently cloned rat brain glutamate transporter of glial origin. Mutation of serine 113 to asparagine does not affect the levels of expressed transport but abolishes its stimulation by the phorbol ester. To our knowledge, this is the first direct demonstration of the regulation of a neurotransmitter transporter by phosphorylation.     

Localization and function of five glutamate transporters cloned from the salamander retina

Glutamate is the major excitatory neurotransmitter in the vertebrate retina. Native glutamate transporters have been well characterized in several retinal neurons, particularly from the salamander retina. We have cloned five distinct glutamate transporters from the salamander retina and examined their localization and functional properties: sEAAT1, sEEAAT2A, sEAAT2B, sEAAT5A and sEAAT5B. sEAAT1 is a homologue of the glutamate transporter EAAT1 (GLAST), sEAAT2A and sEAAT2B are homologues of EAAT2 (GLT-1) and sEAAT5A and sEAAT5B are homologues of the recently cloned human retinal glutamate transporter EAAT5. Localization was determined by immunocytochemical techniques using antibodies directed at portions of the highly divergent carboxy terminal. Glutamate transporters were found in glial, photoreceptor, bipolar, amacrine and ganglion cells. The pharmacology and ionic dependence were determined by two-electrode voltage clamp recordings from Xenopus laevis oocytes which had previously been injected with one of the glutamate transporter mRNAs. Each of the transporters behaved in a manner consistent with a glutamate transporter and there were some distinguishing characteristics which make it possible to link the function in native cells with the behavior of the cloned transporters in this study.     

BDNF prevents NO mediated glutamate cytotoxicity in cultured cortical neurons

The effects of brain-derived neurotrophic factor (BDNF) on glutamate-induced cytotoxicity were examined using primary cultures of rat cortical neurons. BDNF induced TrkB tyrosine phosphorylation in rat cultured cortical neurons. The cell viability was significantly reduced when cultures were briefly exposed to glutamate and incubated with normal medium for 24 h. Glutamate cytotoxicity was prevented by MK-801, which is a non-competitive blocker of N-methyl-D-aspartate and N(omega)-nitro-L-arginine, which is a blocker of nitric oxide synthetase. Delayed neurotoxicity was also induced by ionomycin, a calcium ionophore, and nitric oxide (NO) donors such as S-nitrosocysteine (SNOC) and 3-morpholinosydnonimine (SIN-1). Incubating cultures with BDNF for 10 min to 24 h protected cortical neurons against glutamate neurotoxicity. The protective effects of BDNF against glutamate cytotoxicity were dependent on both its concentrations and incubation time. BDNF also prevented the ionomycin-, SNOC-, and SIN-1 induced cytotoxicity. These results indicate that BDNF protects cultured cortical neurons from NMDA receptor-mediated glutamate neurotoxicity by reducing cytotoxic action of NO.     

Serotoninergic control of the activity and expression of glial GABA transporters in the rat cerebellum

OBJECTIVE: The purpose of the study was to compare staff versus patient perceptions of the causes and emotional impact of verbal and physical aggression on a psychiatric inpatient unit, and the corrective measures each group would endorse. METHODS: Fifty-four patients and 32 nursing staff members responded to similar questions about physical and verbal aggression. They also reported their emotional responses to aggression and steps they would endorse to reduce aggression at the medical center. Data was analyzed by chi-square tests for proportion comparisons between groups. RESULTS: "Verbal Abuse" was viewed an important contributor to physical aggression. Staff stressed patient substance abuse and violent lifestyles. Patients focused on the use of involuntary procedures and cultural differences between patients and staff. CONCLUSIONS: Patients endorsed more restrictive safety measures as long as the measures such as metal detectors and searches were applied to staff and visitors, as well as patients. Patients requested more input into decision-making processes through patient-staff workgroups.     

Antithrombin III prevents and rapidly reverses leukocyte recruitment in ischemia/reperfusion

BACKGROUND: P-selectin has recently been shown to be essential for leukocyte rolling after the reperfusion of ischemic mesentery. However, the mediators responsible for neutrophil rolling in ischemic microvessels remain entirely unclear. METHODS AND RESULTS: Intravital microscopy was used to examine leukocyte kinetics in a feline mesentery ischemia/reperfusion model. Sixty minutes of ischemia followed by reperfusion caused a profound increase in leukocyte rolling and adhesion. Pretreatment with the endogenous antithrombotic agent antithrombin III (ATIII) infused as a bolus (250 U/kg) reduced neutrophil rolling and adhesion to preischemic levels during reperfusion. No effect was seen with heat-inactive ATIII. Importantly, ATIII posttreatment also significantly reduced neutrophil rolling and adhesion during reperfusion, suggesting that ATIII can reverse the leukocyte recruitment response induced by ischemia/reperfusion. Vascular permeability was also reduced by 50% after ATIII administration. To determine whether ATIII could reverse thrombin-induced rolling directly, neutrophil rolling was performed on human endothelium in flow chambers. Indeed, thrombin-induced rolling, but not histamine-induced rolling, could be rapidly reversed with ATIII on endothelium, suggesting that ATIII affects thrombin rather than directly affecting neutrophils or the endothelium. CONCLUSIONS: This study demonstrates for the first time that thrombin plays an important role in ischemia-induced leukocyte rolling and adhesion and that ATIII can be used therapeutically postreperfusion to attenuate the leukocyte recruitment response in inflammation without the nonspecific effects associated with anti-adhesion molecule therapy.     

Identification of functional domains of the human glutamate transporters EAAT1 and EAAT2

Glutamate transporters serve the important function of mediating removal of glutamate released at excitatory synapses and maintaining extracellular concentrations below excitotoxic levels. Excitatory amino acid transporter subtypes EAAT1 and EAAT2 have a high degree of sequence homology and similar predicted topology and yet display a number of functional differences. Several recombinant chimeric transporters were generated to identify domains that contribute to functional differences between EAAT1 and EAAT2. Wild-type transporters and chimeric transporters were expressed in Xenopus laevis oocytes, and electrogenic transport was studied under voltage clamp conditions. The differential sensitivity of EAAT1 and EAAT2 to transport blockers, kainate, threo-3-methylglutamate, and (2S, 4R)-4-methylglutamate as well as L-serine-O-sulfate transport and chloride permeability were employed to characterize chimeric transporters. One particular region, transmembrane domains 9 and 10, plays an important role in defining these functional differences. The intracellular carboxyl-terminal region may also play a minor role in conferring an effect on chloride permeability. This study provides important insight into the identification of functional domains that determine differences among glutamate transporter subtypes.     

Plasma-induced changes in the physiology of mammalian retinal glial cells: role of glutamate

Plasma can leak into the nervous system when the vascular endothelial barrier is compromised. Although this occurs commonly, little is known about the effects of plasma on the function of cells in the central nervous system. In this study, we focused on the responses of glial cells, which, because they ensheathe the blood vessels, are the first cells exposed to leaking plasma. We used the perforated-patch configuration of the patch-clamp technique to assess the effects of plasma on freshly dissociated bovine and human Müller cells, the principal glia of the retina. To monitor the function of Müller cells in situ, we recorded electroretinograms from isolated retinas. We found that plasma activates an electrogenic glutamate transporter and inhibits inward-rectifying K+ channels, as well as a transient outward current. Glutamate, a normal constituent of the blood, mimicked these effects. Unlike our recent findings with serum, which contains molecules generated by the clotting process, plasma neither activated a nonspecific cation conductance nor inhibited the slow P(III) component of the electroretinogram, which is generated by Müller cells responding to light-evoked changes in the extracellular potassium concentration ([K+]o). Taken together, our observations indicate that a leakage of serum into the retina compromises the regulation of [K+]o by Müller cells; however, when plasma enters the retina at sites of a breakdown in the blood-retinal barrier, these glia can maintain K+ homeostasis while reducing the potentially neurotoxic levels of glutamate.     

Ethanol prevents the glutamate release induced by N-methyl-D-aspartate in the rat striatum

The administration of ethanol (2 g/kg, i.p.) or of the non-competitive antagonist(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloepten-5,1 0-imine maleate (MK-801; 1 mg/kg, i.p.) induced a decrease in the extracellular concentrations of glutamate, as studied by microdialysis in the striatum of awake rats. Moreover, ethanol and MK-801 completely prevented the increase in extraneuronal glutamate concentration induced by the focal application of N-methyl-D-aspartate (NMDA). The present results suggest that ethanol suppresses glutamate release through an inhibition of NMDA glutamate receptors in the rat striatum.     

Structural determinants of substrates and inhibitors: probing glutamate transporters with 2,4-methanopyrrolidine-2,4-dicarboxylate

Using an intramolecular [2 + 2] photocyclization, 2,4-methanopyrrolidine-2,4-dicarboxylate was prepared as a conformationally locked analogue of glutamate. This compound, in combination with two other pyrrolidine dicarboxylates, has been used to define the structural elements that differentiate substrate and nonsubstrate inhibitors of a high-affinity, sodium-dependent glutamate transporter.