Measurement of glutamate uptake and reversed transport by rat synaptosome transporters

To establish an assay system for evaluation of the uptake and reversed transport of glutamate, we examined the effects of Na(+)-concentration and pharmacological agents on the extracellular glutamate concentration ([Glu](o)) in rat cortical synaptosomes in vitro. There was a decrease and increase of the [Glu](o) at high and low Na(+) concentrations, respectively, in a Ca(2+)-free medium. The changes in [Glu](o) in both directions were temperature-sensitive, and reversed at around 30 mM of Na(+). Dihydrokainate (DHK), a non-transportable inhibitor selective for glial glutamate transporter GLT-1, suppressed the decrease in [Glu](o), and the reversal of [Glu](o) change was shifted to about 60 mM Na(+). There was no change in the maximum [Glu](o) at total Na(+) substitution. Further pharmacological analysis revealed that D-aspartate and DL-threo-beta-hydroxy-aspartate (THA), transportable substrates of glutamate transporters, increased the [Glu](o) in standard media. In contrast, beta-phenylglutamic acid, a structural analogue of glutamate, suppressed both the decrease in [Glu](o) in standard medium and the increase in [Glu](o) in low Na(+) medium. It is, thus, concluded that both the direction and the amount of [Glu](o) changes are determined by a balance of the uptake and reversed transport of glutamate, and that this assay system is suitable for evaluation of the effect of this on glutamate transporters.     

Glycine taken up through GLYT1 and GLYT2 heterotransporters into glutamatergic axon terminals of mouse spinal cord elicits release of glutamate by homotransporter reversal and through anion channels

Glycine concentration-dependently elicited [3H]D-aspartate ([3H]D-ASP) release from superfused mouse spinal cord synaptosomes. Glycine effect was insensitive to strychnine or 5,7-dichlorokynurenic acid, but was prevented by the glycine transporter blocker glycyldodecylamide. Glycine also evoked release of endogenous glutamate, which was sensitive to glycyldodecylamide and abolished in low-Na+ medium. Experiments with purified synaptosomes and gliasomes show that the glycine-evoked [3H]D-ASP release largely originates from glutamatergic nerve terminals. The glycine-evoked [3H]D-ASP release was halved by NFPS, a selective blocker of GLYT1 transporters, or by Org 25543, a selective GLYT2 blocker, and almost abolished by a mixture of the two, suggesting that activation of GLYT1 and GLYT2 present on glutamatergic terminals triggers the release of [3H]D-ASP. Accordingly, confocal microscopy experiments show localization of GLYT1 and GLYT2 in purified synaptosomes immuno-stained for the vesicular glutamate transporter vGLUT1. The glycine effect was independent of extra- and intraterminal Ca2+ ions. It was partly inhibited by the glutamate transporter blocker DL-TBOA and largely prevented by the anion channel blockers niflumic acid and NPPB. To conclude, transporters for glycine (GLYT1 or/and GLYT2) and for glutamate coexist on the same spinal cord glutamatergic terminals. Activation of glycine heterotransporters elicits glutamate release partly by homotransporter reversal and largely through anion channels.     

Chronic haloperidol treatment impairs glutamate transport in the rat striatum.

High-affinity, Na(+)-dependent transport of glutamate into neurons and glial cells maintains the extracellular concentration of this neurotransmitter at a sub-toxic level. Chronic blockade of dopamine D(2) receptors with haloperidol elevates extracellular glutamate levels in the striatum. The present study examines the effect of long-term haloperidol treatment on glutamate transporter activity using an assay based on measuring the uptake of D-[3H]aspartate in striatal synaptosomes prepared from male Wistar rats. The maximal rate of glutamate transport in the striatum is reduced by 63% following 27 weeks of haloperidol treatment. This impairment of glutamate transport may be important in chronic neuroleptic drug action.     

Enhancing glutamate transport: mechanism of action of Parawixin1, a neuroprotective compound from Parawixia bistriata spider venom

Previous studies have shown that a compound purified from the spider Parawixia bistriata venom stimulates the activity of glial glutamate transporters and can protect retinal tissue from ischemic damage. To understand the mechanism by which this compound enhances transport, we examined its effects on the functional properties of glutamate transporters after solubilization and reconstitution in liposomes and in transfected COS-7 cells. Here, we demonstrate in both systems that Parawixin1 promotes a direct and selective enhancement of glutamate influx by the EAAT2 transporter subtype through a mechanism that does not alter the apparent affinities for the cosubstrates glutamate or sodium. In liposomes, we observed maximal enhancement by Parawixin1 when extracellular sodium and intracellular potassium concentrations are within physiological ranges. Moreover, the compound does not enhance the reverse transport of glutamate under ionic conditions that favor efflux, when extracellular potassium is elevated and the sodium gradient is reduced, nor does it alter the exchange of glutamate in the absence of internal potassium. These observations suggest that Parawixin1 facilitates the reorientation of the potassium-bound transporter, the rate-limiting step in the transport cycle, a conclusion further supported by experiments showing that Parawixin1 does not stimulate uptake by an EAAT2 transport mutant (E405D) defective in the potassium-dependent reorientation step. Thus, Parawixin1 enhances transport through a novel mechanism targeting a step in the transport cycle distinct from substrate influx or efflux and provides a basis for the design of new drugs that act allosterically on transporters to increase glutamate clearance.     

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。     

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.     

谷氨酸转运体亚型谷氨酸转运体1与其调控药物的研究进展

胞外谷氨酸浓度的动态平衡是由谷氨酸转运体精确调控的,谷氨酸转运体功能或表达失调时导致胞外谷氨酸水平异常,引起一系列神经系统疾病。其中谷氨酸转运体1(GLT-1)起着"谷氨酸泵"作用,近年来还发现了仅在肽链C末端发生改变的GLT-1剪切变异体;其中GLT-1a、GLT-1b和GLT-1v发现与某些疾病具有相关性。药物调控谷氨酸转运体的表达或功能,维持胞外谷氨酸正常浓度,能有效改善病理状况。目前已有多种药物被报道对谷氨酸转运体具有激动或抑制作用,如能够上调GLT-1活性的药物有头孢曲松、苯环己哌啶、胞二磷胆碱、利鲁唑、凝血酶、蛋白激酶B等;下调GLT-1活性的药物有依托咪酯、氯氮平、天冬酰胺类衍生物、内皮素等。该文将调控谷氨酸转运体的药物做一总结,为药物开发和临床治疗提供新的思路。     

Reduction in the MK-801 binding sites of the NMDA sub-type of glutamate receptor in a mouse model of congenital hyperammonemia: prevention by acetyl-L-carnitine

Our earlier studies on the pharmacotherapeutic effects of acetyl-L-carnitine (ALCAR), in sparse-fur (spf) mutant mice with X linked ornithine transcarbamylase deficiency, have shown a restoration of cerebral ATP, depleted by congenital hyperammonemia and hyperglutaminemia. The reduced cortical glutamate and increased quinolinate may cause a down-regulation of the N-methyl-D-aspartate (NMDA) receptors, observed by us in adult spf mice. We have now studied the kinetics of [3H]-MK-801 binding to NMDA receptors in spf mice of different ages to see the effect of chronic hyperammonemia on the glutamate neurotransmission. We have also studied the Ca2+-dependent and independent (4-aminopyridine (AP) and veratridine-mediated) release of glutamate and the uptake of [3H]-glutamate in synaptosomes isolated from mutant spf mice and normal CD-1 controls. All these studies were done with and without ALCAR treatment (4 mmol/kg wt i.p. daily for 2 weeks), to see if its effect on ATP repletion could correct the glutamate neurotransmitter abnormalities. Our results indicate a normal MK-801 binding in 12-day-old spf mice but a significant reduction immediately after weaning (21 day), continuing into the adult stage. The Ca2+-independent release of endogenous glutamate from synaptosomes was significantly elevated at 35 days, while the uptake of glutamate into synaptosomes was significantly reduced in spf mice. ALCAR treatment significantly enhanced the MK-801 binding, neutralized the increased glutamate release and restored the glutamate uptake into synaptosomes of spf mice. These studies point out that: (a) the developmental abnormalities of the NMDA sub-type of glutamate receptor in spf mice could be due to the effect of sustained hyperammonemia, causing a persistent release of excess glutamate and inhibition of the ATP-dependent glutamate transport, (b) the modulatory effects of ALCAR on the NMDA binding sites could be through a repletion of ATP, required by the transporters to efficiently remove extracellular glutamate.     

Cellular mechanisms of acute decrease of glutamate release induced by raloxifene in rat cerebral cortex

Raloxifene, a selective estrogen receptor modulator, has been observed to offer a neuroprotective effect in several in?vitro models of neurotoxicity. An excessive release of glutamate is considered to be related to neuropathology of several neurological diseases. In this study, we investigated whether raloxifene could affect endogenous glutamate release in nerve terminals of rat cerebral cortex (synaptosomes) and explored the possible mechanism. Raloxifene exhibited a dose-dependent inhibition of 4-aminopyridine (4-AP)-evoked release of glutamate, and this effect was not blocked by the estrogen receptor antagonists. The effect of raloxifene on the evoked glutamate release was prevented by the chelating extracellular Ca(2+) ions, and by the vesicular transporter inhibitor bafilomycin A1, but was insensitive to the glutamate transporter inhibitor DL-TBOA. Raloxifene decreased the depolarization-induced increase in the cytosolic free Ca(2+) concentration ([Ca(2+)](C)), whereas it did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization. The effect of raloxifene on evoked glutamate release was prevented by blocking the Ca(v)2.2 (N-type) and Ca(v)2.1 (P/Q-type) channels, but not by blocking intracellular Ca(2+) release or Na(+)/Ca(2+) exchange. In addition, the inhibitory effect of raloxifene on evoked glutamate release was abolished by the mitogen-activated/extracellular signal-regulated kinase kinase (MEK) inhibitors, PD98059 and U0126. Furthermore, raloxifene significantly decreased the depolarization-induced phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase 1 and 2 (MAPK/ERK1/2) and synapsin I, the main presynaptic target of ERK. Thus, the effect of raloxifene on evoked glutamate release is linked to a decrease in [Ca(2+)](i) contributed by Ca(2+) entry through presynaptic voltage-dependent Ca(2+) channels and to the subsequent suppression of the ERK/synapsin I signaling cascade.     

The glutamate and neutral amino acid transporter family: physiological and pharmacological implications

The solute carrier family 1 (SLC1) is composed of five high affinity glutamate transporters, which exhibit the properties of the previously described system XAG-, as well as two Na+-dependent neutral amino acid transporters with characteristics of the so-called "ASC" (alanine, serine and cysteine). The SLC1 family members are structurally similar, with almost identical hydropathy profiles and predicted membrane topologies. The transporters have eight transmembrane domains and a structure reminiscent of a pore loop between the seventh and eighth domains [Neuron 21 (1998) 623]. However, each of these transporters exhibits distinct functional properties. Glutamate transporters mediate transport of L-Glu, L-Asp and D-Asp, accompanied by the cotransport of 3 Na+ and one 1 H+, and the countertransport of 1 K+, whereas ASC transporters mediate Na+-dependent exchange of small neutral amino acids such as Ala, Ser, Cys and Thr. Given the high concentrating capacity provided by the unique ion coupling pattern of glutamate transporters, they play crucial roles in protecting neurons against glutamate excitotoxicity in the central nervous system (CNS). The regulation and manipulation of their function is a critical issue in the pathogenesis and treatment of CNS disorders involving glutamate excitotoxicity. Loss of function of the glial glutamate transporter GLT1 (SLC1A2) has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), resulting in damage of adjacent motor neurons. The importance of glial glutamate transporters in protecting neurons from extracellular glutamate was further demonstrated in studies of the slc1A2 glutamate transporter knockout mouse. The findings suggest that therapeutic upregulation of GLT1 may be beneficial in a variety of pathological conditions. Selective inhibition of the neuronal glutamate transporter EAAC1 (SLC1A1) but not the glial glutamate transporters may be of therapeutic interest, allowing blockage of glutamate exit from neurons due to "reversed glutamate transport" of EAAC1, which will occur during pathological conditions, such as during ischemia after a stroke.     

Hispidulin inhibits the release of glutamate in rat cerebrocortical nerve terminals

Hispidulin, a naturally occurring flavone, has been reported to have an antiepileptic profile. An excessive release of glutamate is considered to be related to neuropathology of epilepsy. We investigated whether hispidulin affected endogenous glutamate release in rat cerebral cortex nerve terminals (synaptosomes) and explored the possible mechanism. Hispidulin inhibited the release of glutamate evoked by the K⁺ channel blocker 4-aminopyridine (4-AP). The effects of hispidulin on the evoked glutamate release were prevented by the chelation of extracellular Ca^2 + ions and the vesicular transporter inhibitor bafilomycin A1. However, the glutamate transporter inhibitor dl-threo-beta-benzyl-oxyaspartate did not have any effect on hispidulin action. Hispidulin reduced the depolarization-induced increase in cytosolic free Ca^2 + concentration ([Ca^2 +]_C), but did not alter 4-AP-mediated depolarization. Furthermore, the effect of hispidulin on evoked glutamate release was abolished by blocking the Ca_v2.2 (N-type) and Ca_v2.1 (P/Q-type) channels, but not by blocking ryanodine receptors or mitochondrial Na⁺/Ca^2 + exchange. Mitogen-activated protein kinase kinase (MEK) inhibition also prevented the inhibitory effect of hispidulin on evoked glutamate release. Western blot analyses showed that hispidulin decreased the 4-AP-induced phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) and synaptic vesicle-associated protein synapsin I, a major presynaptic substrate for ERK; this decrease was also blocked by the MEK inhibitor. Moreover, the inhibition of glutamate release by hispidulin was strongly attenuated in mice without synapsin I. These results show that hispidulin inhibits glutamate release from cortical synaptosomes in rats through the suppression of presynaptic voltage-dependent Ca^2 + entry and ERK/synapsin I signaling pathway.     

6-羟基多巴的细胞毒作用与谷氨酸转运的关系

目的探讨6-羟基多巴(6-OHDA)导致细胞毒性与谷氨酸(glutamate,Glu)递质水平和谷氨酸转运体的相关性。方法大鼠脑黑质内定位注射6-OHDA,制备帕金森病(Parkinson's disease,PD)动物模型;用在体微透析技术收集大鼠纹状体细胞外液;用高效液相色谱法测定PD大鼠纹状体和PC12细胞的细胞外液中Glu的水平;用流式细胞仪和酶标仪检测细胞凋亡率和细胞活性;通过测定L-［³H］-Glu的摄取能力确定谷氨酸转运体的功能。结果6-OHDA诱导PC12细胞和大鼠损毁侧纹状体释放Glu增加,使PC12细胞凋亡和活性降低,而PC12细胞和突触体上的谷氨酸转运体功能显著下降。结论6-OHDA引起的神经毒性与其增加Glu释放和降低谷氨酸转运体功能有关。     

Idebenone inhibition of glutamate release from rat cerebral cortex nerve endings by suppression of voltage-dependent calcium influx and protein kinase A

The present study was aimed at investigating the effect and the possible mechanism of idebenone on endogenous glutamate release in nerve terminals of rat cerebral cortex (synaptosomes). Idebenone inhibited the release of glutamate that was evoked by exposing synaptosomes to the K⁺ channel blocker 4-aminopyridine (4-AP), and this phenomenon was concentration dependent. Inhibition of glutamate release by idebenone was prevented by chelating extracellular Ca²⁺, or by the vesicular transporter inhibitor bafilomycin A1, but was insensitive to DL-threo-beta-benzyl-oxyaspartate, a glutamate transporter inhibitor. Idebenone decreased the depolarization-induced increase in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_C),whereas it did not alter the resting synaptosomal membrane potential or 4-AP-mediated depolarization. The inhibitory effect of idebenone on evoked glutamate release was prevented by blocking the Ca_v2.2 (N-type) and Ca_v2.1 (P/Q-type) channels, but not by blocking intracellular Ca²⁺ release or Na⁺/Ca²⁺ exchange. Furthermore, the idebenone effect on 4-AP-evoked Ca²⁺ influx and glutamate release was completely abolished by the protein kinase A (PKA) inhibitors, H89 and KT5720. On the basis of these results, it was concluded that idebenone inhibits glutamate release from rat cortical synaptosomes and this effect is linked to a decrease in [Ca²⁺]_C contributed by Ca²⁺ entry through presynaptic voltage-dependent Ca²⁺ channels and to the suppression of PKA signaling cascade.     

The role of astroglia in Pb-exposed adult rat brain with respect to glutamate toxicity

Astrocytes maintain neuronal homeostasis in brain and controlling of the released glutamate is one of the most important functions. Since it is suggested that glutamatergic component underlies lead-induced neurotoxic effects and simultaneously, astrocytes serve as a cellular lead (Pb) deposition site, it was of interest to investigate the functioning of astroglia in adult rat brain after short-term exposure to Pb. We examined the expression of main astrocytic glutamate/aspartate transporters--GLAST and GLT-1, which regulate extracellular glutamate concentration. Molecular evidence is provided which indicates overexpression of GLAST mRNA and protein. Simultaneously, decreased expression of GLT-1 mRNA and protein was observed, indicating that of the two glial transporters, GLT-1 is more susceptible to the toxic Pb effect. Protein expression of glutamine synthetase (GS), which converts toxic glutamate to non-toxic glutamine, was doubly enhanced. Moreover, Na+-dependent transport of radioactive glutamine to astroglia-derived fraction was affected in Pb-exposed rats. Both the rate of accumulation and the efflux of amino acid were diminished. Additionally, we observed enhanced expression of glutathione-protein complexes after Pb treatment what suggests activation of S-glutathionylation processes. The results of current studies indicate that lead toxicity in adult rat brain activates astrocytic processes connected with the controlling of glutamate homeostasis. The response of astroglia is rather of neuroprotective character however, downexpression of GLT-1 glutamate transporter and activation of S-glutathionylation processes lead to the question about their significance in Pb-induced neurotoxicity.     

Amphetamine increases the extracellular concentration of glutamate in striatum of the awake rat: involvement of high affinity transporter mechanisms.

Using microdialysis it was found that intracerebral infusions of amphetamine increase the extracellular concentration of glutamate, and also of dopamine, aspartate, GABA, and taurine. The increases in glutamate produced by amphetamine was independent of calcium in the perfusion medium but was significantly attenuated by specific blockers of the high affinity transporters of this neurotransmitter. Amphetamine infusions also produced a decrease in the extracellular concentration of Na+, an increase in the extracellular concentration of lactate, and a decrease in haemoglobin in the area of perfusion. All these data suggest that amphetamine increases the extracellular concentration of glutamate and other neurotransmitters through a hypoxic mediated process. This study also shows that an alpha-noradrenergic receptor antagonist is able to attenuate the effects of amphetamine on the release of glutamate, dopamine, GABA and taurine, which further suggests a vasoconstrictor effect of amphetamine as a result of which hypoxia could develop.     

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.     

Nontransportable inhibitors attenuate reversal of glutamate uptake in synaptosomes following a metabolic insult.

Na+-dependent, high-affinity glutamate transporters in the central nervous system are generally credited with regulating extracellular levels of L-glutamate and maintaining concentrations below those that would induce excitotoxic injury. Under pathological conditions, however, it has been suggested that these same transporters may contribute to excitotoxic injury by serving as sites of efflux for cellular L-glutamate. In this study, we examine the efflux of [3H]D-aspartate from synaptosomes in response to both alternative substrates (i.e., heteroexchange), such as L-glutamate, and a metabolic insult (5 mM potassium cyanide and 1 mM iodoacetate). Exposure of synaptosomes containing [3H]D-aspartate to either L-glutamate or metabolic inhibitors increased the efflux of the radiolabeled substrate to over 200% of control values. Two previously identified competitive transport inhibitors (L-trans-2, 3-pyrrolidine dicarboxylate and dihydrokainate) failed to stimulate [3H]D-aspartate efflux but did inhibit glutamate-mediated heteroexchange, consistent with the action of nontransportable inhibitors. These compounds also attenuated the efflux of [3H]D-aspartate from synaptosomes exposed to the metabolic inhibitors. These results add further strength to the model of central nervous system injury-induced efflux of L-glutamate through its high-affinity transporters and identify a novel strategy to attenuate this process.     

Glutamate-based therapeutic approaches: targeting the glutamate transport system

Although the ionotropic and metabotropic receptors for synaptically released glutamate have been extensively mined in the pursuit of novel therapeutic agents for a diverse array of central nervous system disorders, pursuit of the transport proteins--or excitatory amino acid transporters (EAATs)--toward a similar end has been a road much less travelled. Recent progress has seen the use of cloned EAAT subtypes to develop transporter inhibitors with improved subtype selectivity, providing important tools for elucidating the precise contribution of each transporter subtype to the regulation of extracellular glutamate homeostasis. In addition, momentum has been gained with the discovery of compounds capable of upregulating the activity of the predominant forebrain glutamate transporter, EAAT2.     

Regulation and dysregulation of glutamate transporters

Glutamate is the primary excitatory neurotransmitter in the central nervous system. During synaptic activity, glutamate is released into the synaptic cleft and binds to glutamate receptors on the pre- and postsynaptic membrane as well as on neighboring astrocytes in order to start a number of intracellular signaling cascades. To allow for an efficient signaling to occur, glutamate levels in the synaptic cleft have to be maintained at very low levels. This process is regulated by glutamate transporters, which remove excess extracellular glutamate via a sodium-potassium coupled uptake mechanism. When extracellular glutamate levels rise to about normal, glutamate overactivates glutamate receptors, triggering a multitude of intracellular events in the postsynaptic neuron, which ultimately results in neuronal cell death. This phenomenon is known as excitotoxicity and is the underlying mechanisms of a number of neurodegenerative diseases. A dysfunction of the glutamate transporter is thought to contribute to cell death during excitotoxicity. Therefore, efforts have been made to understand the regulation of glutamate transporter function. Transporter activity can be regulated in different ways, including through gene expression, transporter protein targeting and trafficking and through posttranslational modifications of the transporter protein. The identification of these mechanisms has helped to understand the role of glutamate transporters during pathology and will aid in the development of therapeutic strategies with the transporter as a desirable target.     

Ceramide-induced alterations in dopamine transporter function

The purpose of this study was to determine the effects of ceramide on dopamine and serotonin (5-HT, 5-hydroxytryptamine) transporters. Exposure of rat striatal synaptosomes to C2-ceramide caused a reversible, concentration-dependent decrease in plasmalemmal dopamine uptake. In contrast, ceramide exposure increased striatal 5-HT synaptosomal uptake. This increase did not appear to be due to an increased uptake by the 5-HT transporter. Rather, the increase appeared to result from an increase in 5-HT transport through the dopamine transporter, an assertion evidenced by findings that this increase: (1) does not occur in hippocampal synaptosomes (i.e., a preparation largely devoid of dopamine transporters), (2) occurs in striatal synaptosomes prepared from para-chloroamphetamine-treated rats (i.e., a preparation lacking 5-HT transporters), (3) is attenuated by pretreatment with methylphenidate (i.e., a relatively selective dopamine reuptake inhibitor) and (4) is inhibited by exposure to exogenous dopamine (i.e., which presumably competes for uptake with 5-HT). Taken together, these results reveal that ceramide is a novel modulator of monoamine transporter function, and may alter the affinity of dopamine transporters for its primary substrate.