Measurement of glutamate uptake and reversed transport by rat synaptosome transporters

To establish an assay system for evaluation of the uptake and reversed transport of glutamate, we examined the effects of Na(+)-concentration and pharmacological agents on the extracellular glutamate concentration (Glu](o)) in rat cortical synaptosomes in vitro. There was a decrease and increase of the Glu](o) at high and low Na(+) concentrations, respectively, in a Ca(2+)-free medium. The changes in Glu](o) in both directions were temperature-sensitive, and reversed at around 30 mM of Na(+). Dihydrokainate (DHK), a non-transportable inhibitor selective for glial glutamate transporter GLT-1, suppressed the decrease in Glu](o), and the reversal of Glu](o) change was shifted to about 60 mM Na(+). There was no change in the maximum Glu](o) at total Na(+) substitution. Further pharmacological analysis revealed that D-aspartate and DL-threo-beta-hydroxy-aspartate (THA), transportable substrates of glutamate transporters, increased the Glu](o) in standard media. In contrast, beta-phenylglutamic acid, a structural analogue of glutamate, suppressed both the decrease in Glu](o) in standard medium and the increase in Glu](o) in low Na(+) medium. It is, thus, concluded that both the direction and the amount of Glu](o) changes are determined by a balance of the uptake and reversed transport of glutamate, and that this assay system is suitable for evaluation of the effect of this on glutamate transporters.