Research reportRiluzole increases high-affinity glutamate uptake in rat spinal cord synaptosomes
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.
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