Properties of excitatory amino acid transport in the human U373 astrocytoma cell line

In this study, we describe the presence of Na+-dependent high-affinity L-gl utamate transport activity in the human U373 astrocytoma cell Line. U373 ce lls exhibited a robust accumulation of L-glutamate which was predominantly (85%) extracellular Na+-dependent. Kinetic analysis of this transport activ ity revealed that the uptake followed first-order Michaelis-Menten kinetics and was high-affinity in nature. The kinetic parameters estimated by Eadie -Hofstee transformation of the saturable uptake were 37.3 +/- 5.1 mu M for K-m and 0.13 +/- 0.02 nmol min(-1) mg(-1) protein for V-max. A total of 14 known inhibitors of high-affinity L-gIutamate transport were examined for t heir abilities to inhibit L-glutamate uptake by U373 cells. Three compounds , kainate (KA), dihydrokainate (DHK) and alpha-aminoadipic acid produced le ss than 30% inhibition at 1 mM. The lack of effect of both KA and DHK indic ates that the predominant astroglial L-glutamate transporter EAAT2 (excitat ory amino acid transporter 2) does not contribute to the uptake activity pr esent in these cells. The rank order of inhibitory potency for the remainin g 11 compounds tested was L-cysteine sulphinate = L-CCG-III = L-cysteate = L-aspartate = threo-beta-hydroxyaspartate > trans-PDC > D-aspartate = MPDC > beta-glutamate > L-CCG-IV = L-aspartate-beta-hydroxamate. Pre-treatment o f U373 cells with phorbol ester for 30 min resulted in a 56% decrease in L- glutamate uptake and this effect was blocked in a concentration-dependent m anner by the PKC inhibitor bisindolylmaleimide I. Expression of L-glutamate transporters by U373 cells was examined by reverse transcriptase polymeras e chain reaction (RT-PCR) and Western analysis. Transcripts for both the EA AT1 and EAAT3 transporter subtypes were detected but not for EAATs 2, 4, an d 5. Immunoblot analysis confirmed the presence of EAAT3 protein, however, we were unable to detect EAAT1 protein. In conclusion, the Na+-dependent hi gh-affinity L-glutamate transport into human U373 astrocytoma cells appears to be mediated predominantly by the EAAT3 subtype. (C) 1999 Elsevier Scien ce B.V. All rights reserved.