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.