Elsevier

Brain Research

Volume 871, Issue 2, 21 July 2000, Pages 175-180
Brain Research

Research report
Riluzole increases high-affinity glutamate uptake in rat spinal cord synaptosomes

https://doi.org/10.1016/S0006-8993(00)02430-6Get rights and content

Abstract

The purpose of this study was to examine the effect of the anti-convulsant agent, riluzole, on high-affinity glutamate uptake as measured in rat spinal cord synaptosomes. The rate of glutamate uptake was significantly increased in the presence of 0.1 μM and 1.0 μM riluzole, but not at the higher concentrations examined. Kinetics analysis demonstrated that riluzole (0.1 μM) decreased the apparent Km by 21% and increased the Vmax by 31%. Glutamate uptake also was significantly increased in spinal cord synaptosomes obtained from rats treated with 8 mg/kg (i.p.) of riluzole and sacrificed 4 h later. The increase in glutamate uptake in vitro was not affected by pretreatment either with H7, an inhibitor of PKA and PKC, or with the PKC activating phorbol ester, 12-O-tetradecanoylphorbol 13-acetate. Previous studies have shown that some of the actions of riluzole are mediated by G proteins sensitive to pertussis toxin. Surprisingly, treatment of synaptosomes with pertussis toxin alone increased the rate of glutamate uptake, while having no effect on uptake in the presence of riluzole. However, pretreatment with cholera toxin was found to completely block the effects of riluzole on glutamate uptake. These results reveal an additional mechanism by which riluzole can affect glutamatergic neurotransmission, and provides further support that riluzole may prove beneficial in the treatment of traumatic central nervous system injuries involving the excitotoxic actions of glutamate.

Introduction

Glutamate is one of the most abundant and widespread excitatory amino acid neurotransmitters in the central nervous system (CNS), and its actions are mediated by a number of receptor subtypes located on both neurons and glia. Following release, the concentrations of glutamate in the extracellular space are highly regulated and controlled, primarily by a sodium-dependent uptake mechanism involving several transporter proteins. The major glutamate transporter proteins found in the CNS include GLAST-1, GLT-1, and EAAC1, with GLT-1 being the most predominantly expressed form [8], [15], [19]. In addition, these transporters are differentially expressed in specific cell types, with GLAST-1 and GLT-1 being found primarily in glial cells, and EAAC1 being localized in neurons. The physiological events regulating the activity of the glutamate transporters are not well understood, although there is evidence that phosphorylation of the transporters by protein kinases may differentially affect glutamate uptake [6], [7], [11], [25].

The ability of the glutamate transporters to remove glutamate from the extrasynaptic space is a critical step in regulating the potential excitotoxic actions of this excitatory amino acid. The extracellular levels of glutamate have been shown to rise to excitotoxic levels within minutes following traumatic or ischemic CNS injury, and there is evidence that the function of the glutamate transporters becomes impaired under these excitotoxic conditions [16], [21], [33], [34]. Glutamate mediated excitotoxicity has also been implicated in certain neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, and Huntington’s disease [10]. For example, it has been shown that the ability of the glutamate transporters to remove glutamate from the extracellular space is significantly impaired in CNS regions that exhibit neuronal degeneration in ALS [28], [29].

Given the potential role of glutamate in CNS injury and neurodegenerative diseases, several treatment strategies have been implemented to reduce glutamate-mediated excitotoxicity. One approach involves the use of riluzole, an anticonvulsant that has been shown to be neuroprotective in animal models of Parkinson’s disease, ischemia, and traumatic CNS injury [1], [2], [22], [24], [26], [31], [35], [36], [38]. In addition, recent clinical trials in patients with ALS have suggested that riluzole may have beneficial effects in this neurodegenerative disorder [4]. The neuroprotective effect of riluzole is thought to be related, in part, to its ability to inhibit glutamate release in vivo through G proteins sensitive to pertussis toxin [9], [14], [23]. However, not all of the effects of riluzole can be explained by the presynaptic modulation of glutamate release [13], [37].

To address an alternative action of riluzole, we examined the ability of this compound to affect glutamate uptake in vitro and in vivo in rat spinal cord synaptosomes. There is clear evidence that traumatic injury to brain or spinal cord results in down-regulation of glial glutamate transporters [20], [27], [30], [32]. In addition, we examined whether riluzole affects glutamate uptake through the activation of protein kinases (PKA and PKC), or involves signaling mechanisms dependent upon G protein activation. The results of this study demonstrate that riluzole significantly increases glutamate uptake in a dose-dependent fashion, and involve a G protein signaling mechanism sensitive to cholera toxin.

Section snippets

Animal treatments and synaptosomal preparation

Female Long–Evans rats (200–250 g) were used in all studies. All procedures used followed the guidelines established in the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals, and the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and were approved by the University of Kentucky Institutional Animal Care and Use Committee. For the in vitro studies, animals were anesthetized with sodium pentobarbital (40 mg/kg) and sacrificed by

Results

The first set of studies was performed to determine the effects of different concentrations of riluzole on sodium-dependent glutamate uptake. Glutamate uptake was significantly increased (P<0.01) in the presence of 1 μM (46%) and 0.1 μM (67%) riluzole, but was ineffective at the higher concentrations examined (Fig. 1A). The effect of riluzole (0.1 μM) was completely abolished in the absence of sodium or in the presence of PDC (100 μM), a specific inhibitor of glutamate uptake (data not shown).

Discussion

The results of this study demonstrate that riluzole is a potent facilitator of high-affinity glutamate uptake in vitro and in vivo. Riluzole was found to increase the affinity (Km) and rate (Vmax) of glutamate uptake, did not involve the phosphorylation events mediated by PKA or PKC, and may depend on signaling mechanisms involving CTX-sensitive G proteins. The observation that riluzole was not effective in facilitating glutamate uptake at relatively higher concentrations suggests that the

Acknowledgements

This work was supported by grants NS-30248 from the National Institutes of Health and SA-9502-K3 from the Kentucky Spinal Cord and Head Injury Research Trust to J.E.S.

References (38)

  • J. Pratt et al.

    Neuroprotective actions of riluzole in rodent models of global and focal cerebral ischaemia

    Neurosci. Lett.

    (1992)
  • F. Wahl et al.

    Effect of riluzole on focal cerebral ischemia in rats

    Eur. J. Pharmacol.

    (1993)
  • F. Bareyre et al.

    Time course of cerebral edema after traumatic brain injury in rats: effects of riluzole and mannitol

    J. Neurotrauma

    (1997)
  • E. Benoit et al.

    Riluzole specifically blocks inactivated Na channels in myelinated nerve fibre

    Pflugers Arch.

    (1991)
  • G. Bensimon et al.

    A controlled trial of Riluzole in amyotrophic lateral sclerosis

    N. Engl. J. Med.

    (1994)
  • R.J. Bridges et al.

    Conformationally defined neurotransmitter analogues. Selective inhibition of glutamate uptake by one pyrrolidine-2,4-dicarboxylate diastereomer

    J. Med. Chem.

    (1991)
  • M. Casado et al.

    Activation of high-affinity uptake of glutamate by phorbol esters in primary glial cell cultures, J Neurochem

    (1991)
  • M. Conradt et al.

    Inhibition of the high-affinity brain glutamate transporter GLAST-1 via direct phosphorylation

    J. Neurochem.

    (1997)
  • A. Doble

    The pharmacology and mechanism of action of riluzole

    Neurology

    (1996)
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