Elsevier

Brain Research

Volume 868, Issue 1, 16 June 2000, Pages 105-112
Brain Research

Research report
Transporter reversal as a mechanism of glutamate release from the ischemic rat cerebral cortex: studies with dl-threo-β-benzyloxyaspartate

https://doi.org/10.1016/S0006-8993(00)02303-9Get rights and content

Abstract

Elevated levels of the excitotoxic amino acids, glutamate and aspartate, have been implicated in the pathogenesis of neuronal injury and death induced by cerebral ischemia. This study evaluated the contribution of reversed high-affinity, Na+-dependent, glutamate transport to the ischemia-evoked release of glutamate and aspartate using dl-threo-β-benzyloxyaspartate (dl-TBOA), a newly developed competitive, non-transported blocker of the EAAT 1–3 transporters. Changes in the extracellular levels of these and other amino acids, and of glucose and lactate in cerebral cortical superfusates during four-vessel occlusion-elicited global cerebral ischemia were examined using a cortical window technique. Basal and ischemia-evoked amino acid, glucose and lactate efflux were compared in control versus dl-TBOA (100 μM; applied topically for 35 min prior to ischemia) animals. Twenty minutes of ischemia caused large increases in aspartate, glutamate, GABA and taurine effluxes into cortical superfusates, with non-significant effects on the efflux of glycine, glutamine, alanine and serine. Application of dl-TBOA caused a 2-fold increase in basal, preischemic, extracellular glutamate levels, but did not affect those of the other compounds. In the presence of dl-TBOA, ischemia-evoked release of aspartate, glutamate, taurine and glutamine was significantly reduced; that of the other amino acids was not affected. The ischemia-evoked declines in glucose were significantly attenuated, and lactate release was enhanced above that in control animals. The amino acid data are interpreted as indicating that aspartate and glutamate releases were reduced as a consequence of dl-TBOA inhibition of reversed transport by high-affinity, Na-dependent carriers, predominantly involving the glial EAAT 2 transporter. The reduction in ischemia-evoked taurine release is interpreted as being due to a decrease in cell swelling prior to and during the initial phase of ischemia due to reduced entry of the Na+, and other ions, associated with a decreased glutamate uptake. Glucose-sparing and availability for lactate formation would also result from a reduced glutamate/Na+ uptake. These results indicate that reversed transport, primarily from glial cells by the EAAT 2 carrier, is responsible for a substantial (42 and 56%) portion of the ischemia-evoked increase in extracellular glutamate and aspartate levels, respectively. As a potent, competitive, non-transported blocker of high-affinity, Na+-dependent, glutamate transporters, dl-TBOA promises to be a valuable new compound for the study of glutamatergic mechanisms.

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.

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