Riluzole enhances the activity of glutamate transporters GLAST, GLT1 and EAAC1

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

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Riluzole enhances the activity of glutamate transporters GLAST, GLT1 and EAAC1 Elena Fumagalli a,⁎, Marcella Funicello a , Thomas Rauen b , Marco Gobbi a , Tiziana Mennini a Istituto di Ricerche Farmacologiche “Mario Negri”, Via La Masa 19, 20156 Milan, Italy Universität Osnabrück, Fachbereich Biologie/Chemie, Abteilung Biophysik, Barbarastr. 13 D-49076 Osnabrück, Germany a

b

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

Abstract Riluzole exerts a neuroprotective effect through different mechanisms, including action on glutamatergic transmission. We investigated whether 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 the apparent affinity of glutamate for the transporters was significantly increased, with similar effects in the three cell lines. This may facilitate the buffering of excessive extracellular glutamate under pathological conditions suggesting that riluzole's neuroprotective action might be partly mediated 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 system is crucial because excessive and prolonged presence of glutamate in the extracellular space is associated with several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) (Heath and Shaw, 2002; Mennini et al., 2003). The extracellular glutamate concentration is controlled by a family of sodium-dependent carrier proteins, the excitatory amino acid transporters (EAATs) (Danbolt, 2001). The EAATs gene family comprises five members; the first three subtypes cloned were the glial GLAST and GLT1 subtypes, and the neuronal EAAC1. Based on sequence similarities, EAAT1 is the human homologue of GLAST, EAAT2 of GLT1, and EAAT3 of EAAC1. Two additional neuronal glutamate transporters were identified, EAAT4 from cerebellum and EAAT5 from retina. Differences between these five transporters in terms of distribution and role have been reported. The glial transporters EAAT2/GLT1 and EAAT1/GLAST play a major role in glutamate removal in vivo, while the neuronal transporter EAAT3/ ⁎ Corresponding author. Tel.: +390239014570; fax: +39023546277. E-mail address: [email protected] (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 neuronal uptake of cysteine, the principal substrate for biosynthesis of the antioxidant agent glutathione (Aoyama et al., 2006). Perturbation of glutamatergic transmission, particularly altered mechanisms of glutamate release and/or uptake, is an important mechanism in ALS (Heath and Shaw, 2002); ALS patients have reduced levels of EAAT2, with impaired glutamate re-uptake in diseased areas of brain and spinal cord (Rothstein et al., 1992). Riluzole is the only drug currently approved for ALS treatment (Lacomblez et al., 1996). It is a neuroprotective drug, with a complex mechanism of action, involving several effects: inhibition of voltage-dependent sodium channels (Urbani and Belluzzi, 2000; Zona et al., 1998), high-voltage activated calcium and potassium channels (Huang et al., 1997; Zona et al., 1998), and inhibition of protein kinase C, suggesting involvement in antioxidative processes (Noh et al., 2000). Another interesting property is its effect on glutamatergic transmission: riluzole inhibits glutamate release from presynaptic terminals through a mechanism linked to G-protein signalling (Wang et al., 2004). Riluzole also affects neurotransmission 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 rat spinal cord synaptosomes in vitro and after treatment in vivo (Azbill et al., 2000; Dunlop et al., 2003). However, no information is available on the possible action of riluzole on individual 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. Its effect on synaptosomal preparations from rat cortex was also evaluated to confirm previous results. New findings on the drug's mechanism of action on glutamate transporters may be useful to find new pharmacological tools for the treatment of neurodegenerative 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. 14 Luglio 1994) and international law and policies (EEC Council Directive 86/609, OJ L 358, 1, December 12, 1987; NIH Guide for the Care and Use of Laboratory Animals, US National Research Council 1996). Brain cortices from adult male CRL:CD(SD)BR rats were homogenized in 20 volumes of ice-chilled phosphate-buffered 0.32 M sucrose, pH 7.4, in a glass/Teflon homogenizer. The homogenate was centrifuged at 1000 ×g for 5 min at 4 °C, then the supernatant was centrifuged at 12,000 ×g for 20 min at 4 °C to yield the crude synaptosomal pellet (P2). The P2 was diluted to 3 mg wet weight tissue/mL in assay buffer containing (in mM): 10 Tris–acetate, 128 NaCl, 10 D-glucose, 5 KCl, 1.5 NaH2PO4, 1 MgSO4, 1 CaCl2, pH 7.4. Samples of synaptosomal preparation were preincubated for 7 min at 37 °C with or without riluzole (Sanofi-Aventis) in a concentration range of 0.1 to 1000 μM. Non-specific uptake was determined using Na+free buffer (NaCl was replaced by an equimolar concentration of choline chloride). Uptake was started by adding L-[3H]glutamate to a final concentration of 10 nM (GE Healthcare; 49 Ci/ mmol) and stopped after 5 min by adding 2 mL of ice-chilled assay buffer. Samples were filtered through cellulose mixed esther filters (0.65 μm pore size, Millipore Corporation) and washed with 2 mL of ice-chilled assay buffer. Radioactivity on filters was counted in a liquid scintillation counter (counting efficiency 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 described (Berry et al., 2005). Cells were grown in minimal essential medium (Gibco, BRL) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 μg/mL Penicillin and 50 U/mL streptomycin at 37 °C in a humidified atmosphere of 5% CO2, 95% air. For uptake experiments, confluent cultures 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 prewarmed assay buffer and preincubated for 12 min at 37 °C in assay buffer. Uptake was started by adding L-[3H]glutamate to a final concentration of 10 nM and stopped by removing the incubation buffer and washing cells twice with 0.5 mL/well of ice-chilled assay buffer. Cells were lysed with 0.2 mL/well of 1% SDS; radioactivity in the lysate was counted as above. The sodium-dependence of glutamate uptake and non-specific uptake were evaluated using Na+-free buffer. For initial timecourse experiments, cells were incubated with L-[3H]glutamate for 6, 12 or 18 min. To evaluate the Km and Vmax of glutamate uptake, 10 nM L-[3H]glutamate was incubated with unlabelled glutamate at concentrations from 0.1 to 1000 μM. Cell lines were characterised using three inhibitors of glutamate transporters: DL-threo-βhydroxy-aspartic acid, THA (Sigma), dihydrokainic acid, DHK (Tocris) and L-serine Osulfate, SOS (Sigma). Drugs were preincubated at different concentrations (300 μM for THA; 1 to 1000 μM for DHK and SOS) for 12 min before co-incubation with L-[3H]glutamate to establish the concentration inhibiting specific uptake by 50% (IC50). Riluzole was preincubated at concentrations from 0.1 to 1000 μM, before co-incubation with L-[3H]glutamate. It was first dissolved (10×) in assay buffer with 20% absolute ethanol, then diluted to working concentrations using assay buffer with 2% absolute ethanol. This dilution buffer did not impair uptake in cell lines. The effect of riluzole was evaluated on sodiumdependent and independent glutamate uptake (Na+-containing or 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 GraphPad Prism software version 4.00. Km and Vmax were calculated using the homologous displacement equation; curves with and without riluzole were compared using the F-test for non-linear curves. The IC50 for THA, DHK and SOS with corresponding 95% confidence intervals (CI) were calculated using the onesite competition equation. One-way ANOVA with Dunnet's multiple 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-dependent manner; there was a significant 16% increase with 100 μM riluzole. The higher concentration, 300 μM, had no real effect on glutamate uptake, possibly because of toxic effects; 1 mM riluzole markedly reduced (about 50%) both specific and non-specific glutamate uptake, suggesting loss of synaptosomal integrity (Fig. 1). In HEKGLT1, HEKGLAST or HEKEAAC1 cells, L-[3H]glutamate uptake was strongly Na+-dependent, being decreased by more than 90% in Na+-free buffer (Fig. 2A). The specific Na+-dependent uptake was linear between 6 and 18 min, so subsequent experiments were carried out with 12 min preincubation. Km values of L-[3H]glutamate uptake were 108 μM (CI: 91125) for HEKGLAST, 198 μM (CI: 138-261) for HEKGLT1 and

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Fig. 1. Effect of riluzole on specific glutamate uptake in rat cortical synaptosomes. Riluzole 100 μM significantly increased specific glutamate uptake by 16%. ⁎⁎p b 0.01, One-way ANOVA with Dunnet's multiple comparison test. Each value is the mean ± SD of six individual replicates in two different experiments.

80 μM (CI: 56-104) for HEKEAAC1. Glutamate uptake in the three cells lines was completely blocked by 300 μM THA, a nonselective inhibitor of glutamate transporters. DHK, a selective inhibitor of GLT1, was only active on HEKGLT1 (IC50: 387 μM; CI: 321-465), not on HEKGLAST and HEKEAAC1 (IC50 N 1 mM) while SOS, a preferential inhibitor for GLAST and EAAC1, was more active in HEKGLAST (IC50: 148 μM; CI: 106–204) and

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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-dependent manner, with significant effects at concentrations as low as 0.01–0.1 μM and the highest effect at 100 μM (Fig. 2B). The increase in glutamate uptake induced by 100 μM riluzole was similar in all cell lines (+ 27% in HEKGLAST, + 38% in HEKGLT1, + 39% in HEKEAAC1). Higher riluzole concentrations (300 and 1000 μM) were toxic, since at the end of experiments the majority of cells were floating (specific and non-specific glutamate uptake were reduced by more than 50%). Preincubation with riluzole for different times had different effects on glutamate uptake; after 7 min the drug showed less effect than after 12 min preincubation, with no further increase after 25 min (data not shown). Subsequent experiments were carried out with 12 min preincubation for riluzole. To evaluate whether riluzole affected the Km and Vmax of glutamate uptake, we measured these parameters with and without 100 μM riluzole. We found that riluzole significantly

Fig. 2. Panel A: effect of riluzole on Na+-dependent glutamate uptake in HEKGLT1, HEKGLAST and HEKEAAC1 cell lines. Total uptake was decreased by 90% without sodium. Riluzole selectively increased glutamate uptake in the three cell lines in the Na+-containing buffer. ⁎⁎ p b 0.01, One-way ANOVA. Panel B: effect of riluzole on specific glutamate uptake in HEKGLT1, HEKGLAST and HEKEAAC1 cell lines. Riluzole significantly increased uptake in all cell lines at concentrations from 0.1 to 100 μM. At 100 μM, glutamate uptake was increased by 27% in HEKGLAST, 38% in HEKGLT1, 39% in HEKEAAC1. ⁎p b 0.05 and ⁎⁎p b 0.01, One-way ANOVA with Dunnet's multiple comparison test. Each value is mean ± SD of six individual replicates in three different experiments. Panel C: Lineweaver-Burk plot to linearize substrate-velocity data shows the kinetic constant Kmax and Vmax of glutamate uptake in HEKGLT1, HEKGLAST and HEKEAAC1 cell lines. The Y intercept value is 1 / Vmax and the X intercept is - 1 / Km Dashed lines are fitted with 100 μM riluzole, solid lines without riluzole.

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Table 1 Km and Vmax values for L-[3H]glutamate uptake in HEK293 cell lines stably expressing rat glutamate transporters GLAST, GLT1 or EAAC1 HEKGLAST − Riluzole Km (μM) Vmax (pmol/min/well)

107.9 (CI 91–125) 693.1 (CI 659–727)

HEKGLT1 +Riluzole 100 μM b

70.4 (CI 53–88) 540.7a (CI 505–576)

− Riluzole 198.5 (CI 136–261) 1026 (CI 909–1144)

HEKEAAC1 +Riluzole 100 μM a

110.8 (CI 95–127) 936.2 (CI 894–978)

− Riluzole

+Riluzole 100 μM

80.2 (CI 56–104) 365.5 (CI 328–385)

45.4b (CI 31–60) 348.2 (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. ap b 0.05, bp b 0.01, F-test for non-linear curve fit comparison. Each value is the mean and 95% confidence interval (CI) of six individual replicates in three different experiments.

reduced Km values by about 50% for HEKEAAC1 and HEKGLT1 and 35% for HEKGLAST, indicating an increased affinity for glutamate (Table 1 and Fig. 2C). We also found that Vmax values were unchanged in HEKGLT1 and HEKEAAC1 while there was a small decrease (20%) in HEKGLAST (Table 1 and Fig. 2C). This latter and the decrease in Km might be responsible for the smaller increase in glutamate uptake in HEKGLAST cells. 4. Discussion Riluzole, the only drug currently used for the treatment of ALS (Bensimon et al., 1994; Lacomblez et al., 1996), exerts its neuroprotective effect at least in part by reducing the excitotoxic effects of high extracellular glutamate concentrations and hyperactivation of post-synaptic glutamate receptors. Several studies have shown that riluzole inhibits glutamate release from presynaptic terminals (Martin et al., 1993; Wang et al., 2004), but data regarding the possible effects on glutamate transportermediated uptake are still few. Effects of riluzole on rat spinal cord synaptosomes have been previously investigated: Azbill et al. reported that 0.1 and 1 μM riluzole significantly increased glutamate uptake in vitro (67 and 46% increases, respectively) and after in vivo treatment of animals (49%); Vmax for glutamate uptake increased by 31% and Km decreased by 21% (Azbill et al., 2000). Dunlop et al. found a 25–30% increase of glutamate uptake in rat spinal cord synaptosomes at higher riluzole concentrations (10 to 300 μM) (Dunlop et al., 2003). In rat cortical synaptosomes we confirmed that riluzole behaves as a facilitator of glutamate uptake. The significant effect of 100 μM riluzole (16%), however, was lower than has been reported elsewhere; the difference might be due to different experimental conditions or, more likely, to the fact that previous studies were done in rat spinal cord preparations, while we used rat brain (cortical) synaptosomes. There might be differences 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 mechanism of action of riluzole and the lack of information about its possible interaction with glutamate transporters make it difficult to understand whether there is a direct effect on glutamate transporters, or if increases in glutamate uptake in these preparations are due to a more indirect mechanism. An attempt to clarify this point was made using astrocyte cultures (Frizzo et al., 2004), also in the light of evidence that glial cells are the most important site of glutamate uptake in

vivo. A biphasic effect was reported, with significant increases in uptake at 1 and 10 μM riluzole (15%); higher concentrations were ineffective or even toxic for cultures. The authors suggested that neuroprotective effects of riluzole might be partly due to enhancement of glutamate uptake mediated by glial transporters. No additional information was provided about the mechanism leading to the increase in uptake. In this study, we investigated the effect of riluzole on glutamate uptake in HEK293 cells expressing rat GLAST, GLT1 or EAAC1. In our conditions high-affinity glutamate uptake was Na+-dependent, and was inhibited by glutamate transporter blockers; Km values for glutamate uptake were similar in the three cell lines. The Km in intact cells were higher than those reported in synaptosomal preparations (Bridges et al., 1999) and in other systems, such as membrane vesicle preparations (Rauen et al., 1992); these discrepancies are very likely due to the large volume of intact cells, because membrane vesicles and synaptosomes are smaller and fast-saturating structures. Comparisons between different systems are always difficult, but it can be assumed that intact cells are a useful model for studying effects on neurotransmitters uptake. Riluzole at concentrations from 0.01 to 100 μM increased specific glutamate uptake in HEKGLT1, HEKEAAC1 and HEKGLAST in a dose-dependent fashion; at the highest concentration the increase was about 30% in the three cell lines. We found no significant differences between the three glutamate transporters, suggesting there is no selective effect. Kinetic analysis showed that this uptake increase was due to a significant decrease in Km. It may be hypothesised that riluzole might induce some conformational changes in the transporter structure, increasing its affinity for substrate. This might be due to a direct interaction between riluzole and glutamate transporters, at the glutamate binding site or different sites, or to an indirect mechanism, although it is difficult to answer these questions with the data provided in this and other published studies. Our results suggest that riluzole acts by changing the relative affinity for glutamate rather than modifying the expression or the trafficking of transporters, as indicated by the lack of changes or the small changes in the maximal uptake rate in the different cell lines. Azbill suggested a regulation of glutamate uptake mechanism due to riluzole acting on Gi/Go proteins in rat spinal cord synaptosomes (Azbill et al., 2000). However, we cannot speculate on any similar mechanisms in our cell lines because of differences in the experimental systems.

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An alternative mechanism might be related to regulation of glutamate transporters by specific proteins; it was reported that the glutamate transporter-associated protein, GTRAP3-18, specifically acts as a negative modulator for EAAC1-mediated glutamate uptake, in vivo and in HEK293 cells (Lin et al., 2001). Increased levels of GTRAP3-18 dose-dependently inhibited glutamate uptake by reducing the transporter's affinity for glutamate. It is likely that factors or drugs regulating the expression or activity of these interacting proteins might finally influence glutamate uptake. Differences in Km among the glutamate transporter subtypes have been suggested to explain buffering properties (Grewer and Rauen, 2005); the transport process shows a phase of rapid glutamate buffering in a short time scale with low intrinsic affinity, and a subsequent slow phase dominated by translocation of tightly bound glutamate, with higher affinity. A plausible interpretation of the decrease in Km in the presence of riluzole may be a shift of glutamate transporters from a low-affinity system (weak binding of glutamate with higher Km) to a highaffinity system (tight binding of glutamate with lower Km), a condition that would have higher buffering properties (Grewer and Rauen, 2005; Mim et al., 2005). This mechanism seems to be important for GLT1 and EAAC1-expressing cells, where the increase in affinity of glutamate for the transporter was more marked than in GLAST-expressing cells. We also found a reduced rate of uptake for this subtype, which might be associated with either slower translocation or with a smaller number of transporters at the plasma membrane. Riluzole may possibly act on glutamate uptake mediated by GLAST with different efficacy, affecting both buffering and translocation properties, although the differences between the subtypes we tested were not so marked. The increase in affinity for glutamate might be relevant in pathological conditions, when more effective glutamate buffering and subsequent removal could lead to a protective effect. This effect of riluzole might compensate, to some extent, the reduced glutamate uptake due to selective loss in EAAT2 protein, found in post-mortem tissue of ALS patients (Rothstein et al., 1995). In the plasma of ALS patients under riluzole treatment and in healthy volunteers given the clinical dosage, the drug concentration was in the low micromolar range (Le Liboux et al., 1997; Wokke, 1996) and we found that these concentrations significantly increased glutamate uptake in our cell lines, although not in cortical synaptosomes. It is of course difficult to compare very different experimental conditions, but these concentrations might potentially also be effective in vivo. In conclusion, we found that riluzole significantly increased glutamate uptake mediated by GLAST, GLT1 and EAAC1. This effect was due to an increased affinity for glutamate in the presence 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 neuroprotection of riluzole, particularly as an anti-glutamatergic drug. Acknowledgements Authors wish to thank Mrs. Judith Baggott for English style revision of the text.

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