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 Ca²⁺-free medium. The changes in [Glu]_o in both directions were temperature-sensitive, and reversed at around 30mM 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-β-hydroxy-aspartate (THA), transportable substrates of glutamate transporters, increased the [Glu] _o in standard media. In contrast, β-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.