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-glutamate transport activity in the human U373 astrocytoma cell line. U373 cells exhibited a robust accumulation of L-glutamate which was predominantly (85%) extracellular Na(+)-dependent. Kinetic analysis of this transport activity 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 microM for K(m) and 0.13 +/- 0.02 nmol min-1 mg-1 protein for Vmax. A total of 14 known inhibitors of high-affinity L-glutamate transport were examined for their abilities to inhibit L-glutamate uptake by U373 cells. Three compounds, kainate (KA), dihydrokainate (DHK) and alpha-aminoadipic acid produced less than 30% inhibition at 1 mM. The lack of effect of both KA and DHK indicates that the predominant astroglial L-glutamate transporter EAAT2 (excitatory amino acid transporter 2) does not contribute to the uptake activity present in these cells. The rank order of inhibitory potency for the remaining 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 of 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 manner by the PKC inhibitor bisindolylmaleimide I. Expression of L-glutamate transporters by U373 cells was examined by reverse transcriptase polymerase chain reaction (RT-PCR) and Western analysis. Transcripts for both the EAAT1 and EAAT3 transporter subtypes were detected but not for EAATs 2, 4, and 5. Immunoblot analysis confirmed the presence of EAAT3 protein, however, we were unable to detect EAAT1 protein. In conclusion, the Na(+)-dependent high-affinity L-glutamate transport into human U373 astrocytoma cells appears to be mediated predominantly by the EAAT3 subtype.
Dunlop
J
J
CNS Disorders, Wyeth-Ayerst Research, Princeton, NJ 08543-8000, USA. dunlopj@war.wyeth.com
Lou
Z
Z
McIlvain
H B
HB
D018528:ATP-Binding Cassette Transporters / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D027322:Amino Acid Transport System X-AG; D000818:Animals; D001254:Astrocytoma; D001692:Biological Transport / Q000187:drug effects / Q000235:genetics; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics; D027341:Excitatory Amino Acid Transporter 1; D027342:Excitatory Amino Acid Transporter 2; D050580:Excitatory Amino Acid Transporter 3; D015972:Gene Expression Regulation, Neoplastic; D050491:Glutamate Plasma Membrane Transport Proteins; D018698:Glutamic Acid / Q000493:pharmacokinetics*; D006801:Humans; D051381:Rats; D017470:Receptors, Glutamate / Q000032:analysis / Q000235:genetics; D017981:Receptors, Neurotransmitter / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012964:Sodium / Q000494:pharmacology; D027981:Symporters; D014158:Transcription, Genetic / Q000502:physiology; D014316:Tritium / Q000493:pharmacokinetics; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D002352:Carrier Proteins; D027341:Excitatory Amino Acid Transporter 1; D027342:Excitatory Amino Acid Transporter 2; D050580:Excitatory Amino Acid Transporter 3; D050491:Glutamate Plasma Membrane Transport Proteins; D017470:Receptors, Glutamate; D017981:Receptors, Neurotransmitter; C493400:SLC1A1 protein, human; C493397:SLC1A2 protein, human; C493394:SLC1A3 protein, human; C493398:Slc1a1 protein, rat; C493395:Slc1a2 protein, rat; C493392:Slc1a3 protein, rat; D027981:Symporters; D014316:Tritium; D018698:Glutamic Acid; D012964:Sodium