Research reportTransporter reversal as a mechanism of glutamate release from the ischemic rat cerebral cortex: studies with dl-threo-β-benzyloxyaspartate
Introduction
Glutamate is a major excitatory neurotransmitter in the brain, and at high concentrations it can function as an excitotoxin, inducing neuronal injury and death [40]. Extracellular brain concentrations of glutamate, and of the related amino acid aspartate, increase rapidly during ischemic episodes and have been proposed as a significant cause of the ensuing death of susceptible neurons [2], [3], [46]. There is, however, a controversy concerning the underlying mechanisms responsible for the ischemia-induced release of excitotoxic amino acids. Although calcium-dependent, exocytotic, release likely contributes to the initial release of these neurotransmitters, other mechanisms including the functioning of plasma membrane glutamate/aspartate transporters appear to be involved in the later, more pronounced, phase of increases in extracellular amino acid levels. Glial glutamate transporters have been shown to play an important role in maintaining extracellular glutamate concentrations below neurotoxic levels in the normoxic brain [41], [51]. A reversal of high-affinity, sodium-dependent, transporters, as a result of changes in the transmembrane concentrations of sodium, potassium and chloride, has been proposed as a important contributor to the ischemia-evoked increases in extracellular glutamate/aspartate levels [4], [18], [27], [38].
Four subtypes of cerebral Na+-dependent glutamate transporters have been identified to date [37], [38]. These are: EAAT 1 (GLAST) which is primarily expressed in glia; EAAT 2 (GLT-1) also expressed in glia; EAAT 3 (EAAC 1) which is localized only in neurons; EAAT 4 is present in the dendrites of Purkinje cells in the cerebellum and is present at very low levels in the forebrain. Selective inhibitors of these transporters are essential for the elucidation of the physiological roles of glutamate transporters, and thus a number of pharmacological agents have been developed. The majority of these act as competitive substrates, inducing a transport current and a substrate-dependent chloride flux. Blockers, such as dl-threo-hydroxy-aspartate (THA), dihydrokainate (DHK) and 4-methylglutamic acid, can also activate ionotropic glutamate receptors [1], [11], [25] potentially complicating the interpretation of their actions.
Recent studies on the role that reversal of the glutamate transporters may play in ischemia-evoked increases in glutamate levels have primarily utilized three different inhibitors. dl-threo-Hydroxyaspartate is a potent inhibitor of glutamate uptake by EAAT 1 (42–70 μM), EAAT 2 (6 μM) and EAAT 3 (6 μM) [16], [21], [45]. l-trans-Pyrrolidone-2,4-dicarboxylate (PDC) is somewhat selective for EAAT 2 in comparison to EAAT 1 and EAAT 3, whereas dihydrokainate is highly selective for EAAT 2 [38]. Both THA and PDC are transported into cells, and must compete with intracellular glutamate and aspartate for access to the transport site.
Using these inhibitors, investigators have obtained evidence that transporter reversal plays a role in the ischemia-evoked increases in extracellular glutamate/aspartate levels, although the magnitude of the reductions observed following inhibitor application have varied. In vitro experiments on cultured astrocytes [22] and hippocampal slices [39], in which PDC and THA reduced 3H-labeled d-aspartate efflux by >50% suggested that reversal of transport is a major contributor to excitotoxic amino acid release. As DHK did not inhibit ischemia-evoked release, Roettger and Lipton [39] proposed that the glial transporter EAAT 2 was not involved. In in vivo studies PDC and THA did not significantly attenuate ischemia-evoked increases in extracellular glutamate [12], [28], [34], although DHK did effectively attenuate such increases, indicating a reversed role for the glial transporter EAAT 2 in ischemia-evoked release [34], [44].
The recent development of dl-threo-β-benzyloxyaspartate (dl-TBOA), a very potent, non-transported, competitive inhibitor of the EAAT 1, EAAT 2 and EAAT 3 glutamate transporters [21], [45], presented a novel opportunity to further examine the contribution of reversed glutamate transport to the increases in extracellular glutamate/aspartate concentrations in the in vivo ischemic rat cerebral cortex. In the experiments described in this report amino acid, glucose and lactate levels were measured in rat cerebral cortical superfusates during ischemia/reperfusion in the absence or presence of dl-TBOA.
Section snippets
Materials and methods
Fourteen male Sprague–Dawley rats (Charles River, 275–325 g) were anesthetized with halothane, and after insertion of a tracheal cannula, were maintained with methoxyflurane (Penthrane). Body temperature was controlled at 37°C with a rectal probe and abdominal heating pad. One femoral artery was cannulated for measurement of arterial blood pressure and to obtain arterial blood samples for pH and blood gas measurements. Cerebral ischemia was induced by coagulating the vertebral arteries and
Physiological parameters
Significant differences in the mean arterial blood pressure, blood gas and pH values in arterial blood samples recorded either prior to ischemia or after 40 min of reperfusion were observed only in the lower pH level observed in the dl-TBOA group of animals (Table 1). In both groups the EEG became isoelectric within a few seconds of the onset of ischemia. A discernible recovery of EEG activity during reperfusion was evident in one of the control group and three of the dl-TBOA group of rats.
Amino acid efflux
Discussion
dl-TBOA, the novel transport inhibitor used in this study, is a potent competitive blocker of glial transporters EAAT 1 and EAAT 2 and the neuronal transporter, EAAT 3 [45]. It has only a weak affinity towards ionotropic glutamate receptors. In binding competition studies with [3H]CGS 19755 (an NMDA receptor antagonist), [3H]kainate and [3H]AMPA on rat brain synaptic membranes the IC50 values for dl-TBOA were 472±139 μM, 550±250 μM and >1 mM, respectively [45]. It did not show any agonist or
Acknowledgements
Supported by USPHS award NS 26912. The gift of dl-threo-β-benzyloxyaspartate by Dr. K. Shimamoto of the Suntory Institute of Bioorganic Research, Wakayamadai, Mishima-gun, Osaka, Japan is gratefully acknowledged.
References (53)
The high affinity uptake system for excitatory amino acids in the brain
Progr. Neurobiol.
(1994)- et al.
Effects of a glutamate uptake inhibitor on glutamate release induced by veratridine and ischemia
Neurochem. Int.
(1995) - et al.
Does the ‘mystery of extra glucose’ during CNS activation reflect glutamate synthesis?
Neurochem. Int.
(1999) Taurine efflux and cell volume regulation in cerebral cortical slices during chronic hypernatraemia
Neurosci. Lett.
(1995)- et al.
New β-hydroxyaspartate derivatives are competitive blockers for the bovine glutamate/aspartate transporter
J. Biol. Chem.
(1997) - et al.
An amperometric glucose-oxidase/poly(o-phenylenediamine) biosensor for monitoring brain extracellular glucose: in vivo characterization in the striatum of freely-moving rats
J. Neurosci. Methods
(1998) - et al.
The release and uptake of excitatory amino acids
Trends Pharmacol. Sci.
(1990) - et al.
Mechanisms of glutamate and aspartate release in the ischemic rat cerebral cortex
Brain Res.
(1996) - et al.
Hypoxia/hypotension evoked release of glutamate and aspartate from the rat cerebral cortex
Neurosci. Lett.
(1989) - et al.
Characterization of glutamate, aspartate and GABA release from ischemic rat cerebral cortex
Brain Res. Bull.
(1994)
Inhibition by anion channel blockers of ischemia-evoked release of excitotoxic and other amino acids from rat cerebral cortex
Brain Res.
Tamoxifen, a chloride channel blocker, reduces glutamate and aspartate release from the ischemic cerebral cortex
Brain Res.
Knockout of glutamate transporters reveals a major role for astroglial transport in excitotoxicity and clearance of glutamate
Neuron
Lactate and pH change in close correlation in the extracellular space of the rat brain during cortical spreading depression
Neurosci. Lett.
Does taurine act as an osmoregulatory substance in the rat brain?
Neurosci. Lett.
The structural specificity of high affinity uptake of l-glutamate and l-aspartate by rat brain slices
J. Neurochem.
The excitotoxin hypothesis in relation to cerebral ischemia
Cerebrovasc. Brain Metab. Rev.
Excitotoxic cell death
J. Neurobiol.
Stimulated release of lactate in freely moving rats is dependent on the uptake of glutamate
J. Physiol.
Effects of anion channel blockers on hyposmotically-induced amino acid release from the in vivo rat cerebral cortex
Neurochem. Res.
Extracellular brain glucose levels reflect local neuronal activity: a microdialysis study in awake, freely moving rats
J. Neurochem.
Physiological stimulation increases non-oxidative glucose metabolism in the brain of freely moving rat
J. Neurochem.
The mechanisms controlling physiologically stimulated changes in rat brain glucose and lactate: a microdialysis study
J. Physiol.
Extracellular glucose turnover in the striatum of unanaesthetized rats measured by quantitative microdialysis
J. Physiol.
Synthesis, resolution, and biological evaluation of four stereoisomers of 4-methylglutamic acid: selective probes for kainate receptors
J. Med. Chem.
Astrocytes: glutamate producers for neurons
J. Neurosci. Res.
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