title: Transporter reversal as a mechanism of glutamate release from the ischemic rat cerebral cortex: studies with DL-threo-beta-benzyloxyaspartate.
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-beta-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 microM; 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.
authors.affiliation: Department of Physiology, School of Medicine, 5374 Scott Hall, Wayne State University, 540 E. Canfield, Detroit, MI 48201, USA. jphillis@med.wayne.edu
authors.forename: J W
authors.initials: JW
authors.lastname: Phillis
authors.forename: J
authors.initials: J
authors.lastname: Ren
authors.forename: M H
authors.initials: MH
authors.lastname: O'Regan
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D027322:Amino Acid Transport System X-AG; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism / Q000494:pharmacology; D002545:Brain Ischemia / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism*; D018698:Glutamic Acid / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D019344:Lactic Acid / Q000378:metabolism; D008297:Male; D009498:Neurotoxins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000378:metabolism; D013654:Taurine / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D009498:Neurotoxins; C120673:benzyloxyaspartate; D013654:Taurine; D001224:Aspartic Acid; D019344:Lactic Acid; D018698:Glutamic Acid