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European Journal of Pharmacology 578 (2008) 171–176www.elsevier.com/locate/ejphar

Short communication

Riluzole enhances the activity of glutamate transportersGLAST, GLT1 and EAAC1

Elena Fumagalli a,⁎, Marcella Funicello a, Thomas Rauen b, Marco Gobbi a, Tiziana Mennini a

a Istituto di Ricerche Farmacologiche “Mario Negri”, Via La Masa 19, 20156 Milan, Italyb Universität Osnabrück, Fachbereich Biologie/Chemie, Abteilung Biophysik, Barbarastr. 13 D-49076 Osnabrück, Germany

Received 3 July 2007; received in revised form 11 October 2007; accepted 16 October 2007Available online 25 October 2007

Abstract

Riluzole exerts a neuroprotective effect through different mechanisms, including action on glutamatergic transmission. We investigatedwhether this drug affects glutamate transporter-mediated uptake, using clonal cell lines stably expressing the rat glutamate transporters GLAST,GLT1 or EAAC1. We found that riluzole significantly increased glutamate uptake in a dose-dependent manner; kinetic analysis indicated that theapparent affinity of glutamate for the transporters was significantly increased, with similar effects in the three cell lines. This may facilitate thebuffering of excessive extracellular glutamate under pathological conditions suggesting that riluzole's neuroprotective action might be partlymediated by its activating effect on glutamate uptake.© 2007 Elsevier B.V. All rights reserved.

Keywords: Riluzole; Glutamate transporter; Uptake; Cell lines; Synaptosomes

1. Introduction

The homeostasis of glutamate in the central nervous systemis crucial because excessive and prolonged presence of gluta-mate in the extracellular space is associated with severalneurodegenerative diseases, including amyotrophic lateral scle-rosis (ALS) (Heath and Shaw, 2002; Mennini et al., 2003).

The extracellular glutamate concentration is controlled by afamily of sodium-dependent carrier proteins, the excitatoryamino acid transporters (EAATs) (Danbolt, 2001). The EAATsgene family comprises five members; the first three subtypescloned were the glial GLAST and GLT1 subtypes, and theneuronal EAAC1. Based on sequence similarities, EAAT1 is thehuman homologue of GLAST, EAAT2 of GLT1, and EAAT3 ofEAAC1. Two additional neuronal glutamate transporters wereidentified, EAAT4 from cerebellum and EAAT5 from retina.

Differences between these five transporters in terms ofdistribution and role have been reported. The glial transportersEAAT2/GLT1 and EAAT1/GLAST play a major role in gluta-mate removal in vivo, while the neuronal transporter EAAT3/

⁎ Corresponding author. Tel.: +390239014570; fax: +39023546277.E-mail address: fumagalli@marionegri.it (E. Fumagalli).

0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2007.10.023

EAAC1 has a less important role in glutamate uptake (Danbolt,2001). A primary role for EAAC1 has been suggested in neu-ronal uptake of cysteine, the principal substrate for biosynthesisof the antioxidant agent glutathione (Aoyama et al., 2006).

Perturbation of glutamatergic transmission, particularlyaltered mechanisms of glutamate release and/or uptake, is animportant mechanism in ALS (Heath and Shaw, 2002); ALSpatients have reduced levels of EAAT2, with impaired gluta-mate re-uptake in diseased areas of brain and spinal cord(Rothstein et al., 1992).

Riluzole is the only drug currently approved for ALS treat-ment (Lacomblez et al., 1996). It is a neuroprotective drug, witha complex mechanism of action, involving several effects:inhibition of voltage-dependent sodium channels (Urbani andBelluzzi, 2000; Zona et al., 1998), high-voltage activated cal-cium and potassium channels (Huang et al., 1997; Zona et al.,1998), and inhibition of protein kinase C, suggesting involve-ment in antioxidative processes (Noh et al., 2000).

Another interesting property is its effect on glutamatergictransmission: riluzole inhibits glutamate release from presyn-aptic terminals through a mechanism linked to G-proteinsignalling (Wang et al., 2004). Riluzole also affects neurotrans-mission mediated by AMPA/kainate receptors and reduces

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NMDA-evoked responses (Albo et al., 2004; De Sarro et al.,2000). Finally, it enhances high-affinity glutamate uptake in ratspinal cord synaptosomes in vitro and after treatment in vivo(Azbill et al., 2000; Dunlop et al., 2003). However, no in-formation is available on the possible action of riluzole onindividual glutamate transporter subtypes.

The aim of the present study was to investigate the effect(s)of riluzole on the three main glutamate transporters GLAST,GLT1 and EAAC1, individually expressed in HEK293 cells. Itseffect on synaptosomal preparations from rat cortex was alsoevaluated to confirm previous results. New findings on thedrug's mechanism of action on glutamate transporters may beuseful to find new pharmacological tools for the treatment ofneurodegenerative disorders.

2. Materials and methods

2.1. [3H]glutamate uptake in rat cortical synaptosomes

Experiments were conducted according to national (D.L. no.116, G.U. suppl. 40, 18 Febbraio 1992, circolare no.8, G.U. 14Luglio 1994) and international law and policies (EEC CouncilDirective 86/609, OJ L 358, 1, December 12, 1987; NIH Guidefor the Care and Use of Laboratory Animals, US NationalResearch Council 1996).

Brain cortices from adult male CRL:CD(SD)BR rats werehomogenized in 20 volumes of ice-chilled phosphate-buffered0.32 M sucrose, pH 7.4, in a glass/Teflon homogenizer. Thehomogenate was centrifuged at 1000 ×g for 5 min at 4 °C, thenthe supernatant was centrifuged at 12,000 ×g for 20 min at 4 °Cto yield the crude synaptosomal pellet (P2). The P2 was dilutedto 3 mg wet weight tissue/mL in assay buffer containing (inmM): 10 Tris–acetate, 128 NaCl, 10 D-glucose, 5 KCl, 1.5NaH2PO4, 1 MgSO4, 1 CaCl2, pH 7.4. Samples of synapto-somal preparation were preincubated for 7 min at 37 °C with orwithout riluzole (Sanofi-Aventis) in a concentration range of 0.1to 1000 μM. Non-specific uptake was determined using Na+-free buffer (NaCl was replaced by an equimolar concentrationof choline chloride). Uptake was started by adding L-[3H]glu-tamate to a final concentration of 10 nM (GE Healthcare; 49 Ci/mmol) and stopped after 5 min by adding 2 mL of ice-chilledassay buffer. Samples were filtered through cellulose mixedesther filters (0.65 μm pore size, Millipore Corporation) andwashed with 2 mL of ice-chilled assay buffer. Radioactivity onfilters was counted in a liquid scintillation counter (countingefficiency about 50%).

2.2. [3H]glutamate uptake in cell lines

Stable cell lines for GLT1, GLAST or EAAC1 (HEKGLT1,HEKGLAST or HEKEAAC1) were developed as previously de-scribed (Berry et al., 2005). Cells were grown in minimalessential medium (Gibco, BRL) supplemented with 10% fetalbovine serum, 2 mM L-glutamine, 50 μg/mL Penicillin and50 U/mL streptomycin at 37 °C in a humidified atmosphereof 5% CO2, 95% air. For uptake experiments, confluent cul-tures were separated by trypsinization and cells were seeded

into 48-well culture plates at the density of 200,000 cells/well,then maintained until they reached confluence (2–3 days).Confluent cells were washed twice with 0.5 mL/well of pre-warmed assay buffer and preincubated for 12 min at 37 °C inassay buffer. Uptake was started by adding L-[3H]glutamate to afinal concentration of 10 nM and stopped by removing theincubation buffer and washing cells twice with 0.5 mL/well ofice-chilled assay buffer. Cells were lysed with 0.2 mL/well of1% SDS; radioactivity in the lysate was counted as above. Thesodium-dependence of glutamate uptake and non-specificuptake were evaluated using Na+-free buffer. For initial time-course experiments, cells were incubated with L-[3H]glutamatefor 6, 12 or 18 min. To evaluate the Km and Vmax of glutamateuptake, 10 nM L-[3H]glutamate was incubated with unlabelledglutamate at concentrations from 0.1 to 1000 μM.

Cell lines were characterised using three inhibitors ofglutamate transporters: DL-threo-βhydroxy-aspartic acid, THA(Sigma), dihydrokainic acid, DHK (Tocris) and L-serine O-sulfate, SOS (Sigma). Drugs were preincubated at differentconcentrations (300 μM for THA; 1 to 1000 μM for DHK andSOS) for 12 min before co-incubation with L-[3H]glutamate toestablish the concentration inhibiting specific uptake by 50%(IC50). Riluzole was preincubated at concentrations from 0.1 to1000 μM, before co-incubation with L-[3H]glutamate. It wasfirst dissolved (10×) in assay buffer with 20% absolute ethanol,then diluted to working concentrations using assay buffer with2% absolute ethanol. This dilution buffer did not impair uptakein cell lines. The effect of riluzole was evaluated on sodium-dependent and independent glutamate uptake (Na+-containingor Na+-free buffer); the effect of different pre-incubation times(7, 12 and 25 min) was also evaluated.

All fittings and data analyses were done using the GraphPadPrism software version 4.00. Km and Vmax were calculatedusing the homologous displacement equation; curves with andwithout riluzole were compared using the F-test for non-linearcurves. The IC50 for THA, DHK and SOS with corresponding95% confidence intervals (CI) were calculated using the one-site competition equation. One-way ANOVA with Dunnet'smultiple comparison test was used for statistical analysis.

3. Results

Riluzole (0.1–100 μM) increased Na+-dependent L-[3H]glutamate uptake in rat cortical synaptosomes in a dose-de-pendent manner; there was a significant 16% increase with100 μM riluzole. The higher concentration, 300 μM, had no realeffect on glutamate uptake, possibly because of toxic effects;1 mM riluzole markedly reduced (about 50%) both specific andnon-specific glutamate uptake, suggesting loss of synaptosomalintegrity (Fig. 1).

In HEKGLT1, HEKGLAST or HEKEAAC1 cells, L-[3H]gluta-mate uptake was strongly Na+-dependent, being decreasedby more than 90% in Na+-free buffer (Fig. 2A). The specificNa+-dependent uptake was linear between 6 and 18 min, sosubsequent experiments were carried out with 12 min preincuba-tion. Km values of L-[3H]glutamate uptake were 108 μM (CI: 91-125) for HEKGLAST, 198 μM (CI: 138-261) for HEKGLT1 and

Fig. 1. Effect of riluzole on specific glutamate uptake in rat corticalsynaptosomes. Riluzole 100 μM significantly increased specific glutamateuptake by 16%. ⁎⁎pb0.01, One-way ANOVA with Dunnet's multiplecomparison test. Each value is the mean±SD of six individual replicates intwo different experiments.

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80 μM (CI: 56-104) for HEKEAAC1. Glutamate uptake in thethree cells lines was completely blocked by 300 μMTHA, a non-selective inhibitor of glutamate transporters. DHK, a selectiveinhibitor of GLT1, was only active on HEKGLT1 (IC50: 387 μM;CI: 321-465), not on HEKGLAST and HEKEAAC1 (IC50N1 mM)while SOS, a preferential inhibitor for GLAST and EAAC1, wasmore active in HEKGLAST (IC50: 148 μM; CI: 106–204) and

Fig. 2. Panel A: effect of riluzole on Na+-dependent glutamate uptake in HEKGLT1, Hsodium. Riluzole selectively increased glutamate uptake in the three cell lines in the Non specific glutamate uptake in HEKGLT1, HEKGLAST and HEKEAAC1 cell lines. Rilu100 μM. At 100 μM, glutamate uptake was increased by 27% in HEKGLAST, 38% inDunnet's multiple comparison test. Each value is mean±SD of six individual replicsubstrate-velocity data shows the kinetic constant Kmax and Vmax of glutamate uptakeVmax and the X intercept is - 1 /Km Dashed lines are fitted with 100 μM riluzole, so

HEKEAAC1 cells (IC50: 60 μM; CI: 44–82), than in HEKGLT1

(IC50: 670 μM; CI: 482–933).We tested the effect of riluzole on glutamate uptake with and

without Na+ and found that it increased uptake only with Na+,without effects on non-specific glutamate uptake (Fig. 2A).

Riluzole increased Na+-dependent uptake in a dose-depen-dent manner, with significant effects at concentrations as lowas 0.01–0.1 μM and the highest effect at 100 μM (Fig. 2B).The increase in glutamate uptake induced by 100 μM riluzolewas similar in all cell lines (+27% in HEKGLAST, +38% inHEKGLT1, +39% in HEKEAAC1). Higher riluzole concentrations(300 and 1000 μM) were toxic, since at the end of experimentsthe majority of cells were floating (specific and non-specificglutamate uptake were reduced by more than 50%).

Preincubation with riluzole for different times had differenteffects on glutamate uptake; after 7 min the drug showed lesseffect than after 12 min preincubation, with no further increaseafter 25 min (data not shown). Subsequent experiments werecarried out with 12 min preincubation for riluzole.

To evaluate whether riluzole affected the Km and Vmax

of glutamate uptake, we measured these parameters with andwithout 100 μM riluzole. We found that riluzole significantly

EKGLAST and HEKEAAC1 cell lines. Total uptake was decreased by 90% withouta+-containing buffer. ⁎⁎ pb0.01, One-way ANOVA. Panel B: effect of riluzolezole significantly increased uptake in all cell lines at concentrations from 0.1 toHEKGLT1, 39% in HEKEAAC1. ⁎pb0.05 and ⁎⁎pb0.01, One-way ANOVAwithates in three different experiments. Panel C: Lineweaver-Burk plot to linearizein HEKGLT1, HEKGLAST and HEKEAAC1 cell lines. The Y intercept value is 1 /

lid lines without riluzole.

Table 1Km and Vmax values for L-[3H]glutamate uptake in HEK293 cell lines stably expressing rat glutamate transporters GLAST, GLT1 or EAAC1

HEKGLAST HEKGLT1 HEKEAAC1

−Riluzole +Riluzole 100 μM −Riluzole +Riluzole 100 μM −Riluzole +Riluzole 100 μM

Km (μM) 107.9 70.4b 198.5 110.8a 80.2 45.4b

(CI 91–125) (CI 53–88) (CI 136–261) (CI 95–127) (CI 56–104) (CI 31–60)Vmax (pmol/min/well) 693.1 540.7a 1026 936.2 365.5 348.2

(CI 659–727) (CI 505–576) (CI 909–1144) (CI 894–978) (CI 328–385) (CI 321–375)

Km and Vmax for glutamate uptake were evaluated with and without 100 μM riluzole. Km values significantly decreased with 100 μM riluzole in all three cell lines;Vmax were unchanged in cell lines expressing GLT1 or EAAC1 and decreased in cell line expressing GLAST. apb0.05, bpb0.01, F-test for non-linear curve fitcomparison. Each value is the mean and 95% confidence interval (CI) of six individual replicates in three different experiments.

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reduced Km values by about 50% for HEKEAAC1 and HEKGLT1

and 35% for HEKGLAST, indicating an increased affinity forglutamate (Table 1 and Fig. 2C). We also found that Vmax valueswere unchanged in HEKGLT1 and HEKEAAC1 while there was asmall decrease (20%) in HEKGLAST (Table 1 and Fig. 2C). Thislatter and the decrease in Km might be responsible for thesmaller increase in glutamate uptake in HEKGLAST cells.

4. Discussion

Riluzole, the only drug currently used for the treatment ofALS (Bensimon et al., 1994; Lacomblez et al., 1996), exerts itsneuroprotective effect at least in part by reducing the excitotoxiceffects of high extracellular glutamate concentrations andhyperactivation of post-synaptic glutamate receptors. Severalstudies have shown that riluzole inhibits glutamate release frompresynaptic terminals (Martin et al., 1993; Wang et al., 2004),but data regarding the possible effects on glutamate transporter-mediated uptake are still few.

Effects of riluzole on rat spinal cord synaptosomes have beenpreviously investigated: Azbill et al. reported that 0.1 and 1 μMriluzole significantly increased glutamate uptake in vitro (67 and46% increases, respectively) and after in vivo treatment ofanimals (49%); Vmax for glutamate uptake increased by 31% andKm decreased by 21% (Azbill et al., 2000). Dunlop et al. found a25–30% increase of glutamate uptake in rat spinal cordsynaptosomes at higher riluzole concentrations (10 to 300 μM)(Dunlop et al., 2003). In rat cortical synaptosomes we confirmedthat riluzole behaves as a facilitator of glutamate uptake. Thesignificant effect of 100 μM riluzole (16%), however, was lowerthan has been reported elsewhere; the difference might be due todifferent experimental conditions or, more likely, to the fact thatprevious studies were done in rat spinal cord preparations, whilewe used rat brain (cortical) synaptosomes. There might bedifferences in various areas of the CNS in the characteristics and/or abundance of the different glutamate transporter subtypes(Manzoni and Mennini, 1997). However, the complex mecha-nism of action of riluzole and the lack of information about itspossible interaction with glutamate transporters make it difficultto understand whether there is a direct effect on glutamatetransporters, or if increases in glutamate uptake in these prep-arations are due to a more indirect mechanism.

An attempt to clarify this point was made using astrocytecultures (Frizzo et al., 2004), also in the light of evidence thatglial cells are the most important site of glutamate uptake in

vivo. A biphasic effect was reported, with significant increasesin uptake at 1 and 10 μM riluzole (15%); higher concentrationswere ineffective or even toxic for cultures. The authorssuggested that neuroprotective effects of riluzole might bepartly due to enhancement of glutamate uptake mediated byglial transporters. No additional information was providedabout the mechanism leading to the increase in uptake.

In this study, we investigated the effect of riluzole onglutamate uptake in HEK293 cells expressing rat GLAST,GLT1 or EAAC1. In our conditions high-affinity glutamateuptake was Na+-dependent, and was inhibited by glutamatetransporter blockers; Km values for glutamate uptake weresimilar in the three cell lines. The Km in intact cells were higherthan those reported in synaptosomal preparations (Bridges et al.,1999) and in other systems, such as membrane vesiclepreparations (Rauen et al., 1992); these discrepancies are verylikely due to the large volume of intact cells, because membranevesicles and synaptosomes are smaller and fast-saturatingstructures. Comparisons between different systems are alwaysdifficult, but it can be assumed that intact cells are a usefulmodel for studying effects on neurotransmitters uptake.

Riluzole at concentrations from 0.01 to 100 μM increased spe-cific glutamate uptake in HEKGLT1, HEKEAAC1 and HEKGLAST

in a dose-dependent fashion; at the highest concentration theincrease was about 30% in the three cell lines. We found nosignificant differences between the three glutamate transpor-ters, suggesting there is no selective effect. Kinetic analysisshowed that this uptake increase was due to a significantdecrease in Km. It may be hypothesised that riluzole mightinduce some conformational changes in the transporterstructure, increasing its affinity for substrate. This might bedue to a direct interaction between riluzole and glutamatetransporters, at the glutamate binding site or different sites, orto an indirect mechanism, although it is difficult to answerthese questions with the data provided in this and otherpublished studies.

Our results suggest that riluzole acts by changing the relativeaffinity for glutamate rather than modifying the expression orthe trafficking of transporters, as indicated by the lack ofchanges or the small changes in the maximal uptake rate in thedifferent cell lines. Azbill suggested a regulation of glutamateuptake mechanism due to riluzole acting on Gi/Go proteins inrat spinal cord synaptosomes (Azbill et al., 2000). However, wecannot speculate on any similar mechanisms in our cell linesbecause of differences in the experimental systems.

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An alternative mechanism might be related to regulation ofglutamate transporters by specific proteins; it was reported thatthe glutamate transporter-associated protein, GTRAP3-18,specifically acts as a negative modulator for EAAC1-mediatedglutamate uptake, in vivo and in HEK293 cells (Lin et al.,2001). Increased levels of GTRAP3-18 dose-dependently in-hibited glutamate uptake by reducing the transporter's affinityfor glutamate. It is likely that factors or drugs regulating theexpression or activity of these interacting proteins might finallyinfluence glutamate uptake.

Differences in Km among the glutamate transporter subtypeshave been suggested to explain buffering properties (Grewer andRauen, 2005); the transport process shows a phase of rapidglutamate buffering in a short time scale with low intrinsic af-finity, and a subsequent slow phase dominated by translocationof tightly bound glutamate, with higher affinity. A plausibleinterpretation of the decrease in Km in the presence of riluzolemay be a shift of glutamate transporters from a low-affinitysystem (weak binding of glutamate with higher Km) to a high-affinity system (tight binding of glutamate with lower Km), acondition that would have higher buffering properties (Grewerand Rauen, 2005; Mim et al., 2005). This mechanism seems to beimportant for GLT1 and EAAC1-expressing cells, where theincrease in affinity of glutamate for the transporter was moremarked than inGLAST-expressing cells.We also found a reducedrate of uptake for this subtype, which might be associated witheither slower translocation or with a smaller number of trans-porters at the plasma membrane. Riluzole may possibly act onglutamate uptake mediated by GLAST with different efficacy,affecting both buffering and translocation properties, although thedifferences between the subtypes we tested were not so marked.

The increase in affinity for glutamate might be relevant inpathological conditions, when more effective glutamate buffer-ing and subsequent removal could lead to a protective effect.This effect of riluzole might compensate, to some extent, thereduced glutamate uptake due to selective loss in EAAT2protein, found in post-mortem tissue of ALS patients (Rothsteinet al., 1995). In the plasma of ALS patients under riluzoletreatment and in healthy volunteers given the clinical dosage,the drug concentration was in the low micromolar range (LeLiboux et al., 1997; Wokke, 1996) and we found that theseconcentrations significantly increased glutamate uptake in ourcell lines, although not in cortical synaptosomes. It is of coursedifficult to compare very different experimental conditions, butthese concentrations might potentially also be effective in vivo.

In conclusion, we found that riluzole significantly increasedglutamate uptake mediated by GLAST, GLT1 and EAAC1. Thiseffect was due to an increased affinity for glutamate in thepresence of riluzole and was similar on the three main EAATs,since no selective effect was seen on anyone of the subtypes.These results may help elucidate the mechanism of neuropro-tection of riluzole, particularly as an anti-glutamatergic drug.

Acknowledgements

Authors wish to thank Mrs. Judith Baggott for English stylerevision of the text.

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