Effects of glial glutamate transporter inhibitors on intracellular Na+ in mouse astrocytes

Abstract

The effects of inhibitors of the glial Na+/glutamate co-transporter on the intracellular Na+ concentration ([Na+]i) were investigated in mouse cortical astrocytes. [Na+]i was monitored by fluorescence microscopy on single astrocytes using the Na+-sensitive probe sodium-binding benzofuran isophtalate. Application of the competitive inhibitors threo-β-hydroxyaspartate (THA) and trans-pyrrolidine-2,4-dicarboxylic acid (t-PDC) resulted in robust and reversible increases in [Na+]i that were comparable in shape to the response to glutamate but about twice lower in amplitude. As previously observed with glutamate, the amplitude of the [Na+]i response to these compounds was concentration-dependent with EC50 values of 11.1 μM (THA) and 7.6 μM (t-PDC), as was the initial rate of [Na+]i rise (EC50 values of 14.8 μM for THA and 11.5 μM for t-PDC). Both compounds diminished the response to subsequent glutamate applications, possibly because of an inhibitory effect of the intracellularly-accumulated compounds. In comparison, the newly-developed compound threo-β-benzyloxyaspartate (TBOA) alone did not cause any significant alteration of [Na+]i up to a concentration of 500 μM. TBOA inhibited the [Na+]i response evoked by 200 μM glutamate in a concentration-dependent manner with IC50 values of 114 and 63 μM, as measured on the amplitude and the initial rate, respectively. The maximum inhibition of glutamate-evoked [Na+]i increase by TBOA was ∼70%. The residual response persisted in the presence of a non-NMDA receptor antagonist or the inhibitor of the GLT-1 glutamate transporters, dihydrokainate (DHK). In view of the complete reversibility of its effects, TBOA represents a very useful pharmacological tool for studies of glutamate transporters. © 2001 Elsevier Science B.V.