PMID- 10519046
OWN - NLM
STAT- MEDLINE
DCOM- 19991117
LR  - 20190614
IS  - 0006-8993 (Print)
IS  - 0006-8993 (Linking)
VI  - 839
IP  - 2
DP  - 1999 Aug 28
TI  - Properties of excitatory amino acid transport in the human U373 astrocytoma cell 
      line.
PG  - 235-42
AB  - 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.
FAU - Dunlop, J
AU  - Dunlop J
AD  - CNS Disorders, Wyeth-Ayerst Research, Princeton, NJ 08543-8000, USA. 
      dunlopj@war.wyeth.com
FAU - Lou, Z
AU  - Lou Z
FAU - McIlvain, H B
AU  - McIlvain HB
LA  - eng
PT  - Journal Article
PL  - Netherlands
TA  - Brain Res
JT  - Brain research
JID - 0045503
RN  - 0 (ATP-Binding Cassette Transporters)
RN  - 0 (Amino Acid Transport System X-AG)
RN  - 0 (Carrier Proteins)
RN  - 0 (Excitatory Amino Acid Transporter 1)
RN  - 0 (Excitatory Amino Acid Transporter 2)
RN  - 0 (Excitatory Amino Acid Transporter 3)
RN  - 0 (Glutamate Plasma Membrane Transport Proteins)
RN  - 0 (Receptors, Glutamate)
RN  - 0 (Receptors, Neurotransmitter)
RN  - 0 (SLC1A1 protein, human)
RN  - 0 (SLC1A2 protein, human)
RN  - 0 (SLC1A3 protein, human)
RN  - 0 (Slc1a1 protein, rat)
RN  - 0 (Slc1a2 protein, rat)
RN  - 0 (Slc1a3 protein, rat)
RN  - 0 (Symporters)
RN  - 10028-17-8 (Tritium)
RN  - 3KX376GY7L (Glutamic Acid)
RN  - 9NEZ333N27 (Sodium)
SB  - IM
MH  - ATP-Binding Cassette Transporters/analysis/*genetics/*metabolism
MH  - Amino Acid Transport System X-AG
MH  - Animals
MH  - *Astrocytoma
MH  - Biological Transport/drug effects/genetics
MH  - Carrier Proteins/analysis/genetics
MH  - Excitatory Amino Acid Transporter 1
MH  - Excitatory Amino Acid Transporter 2
MH  - Excitatory Amino Acid Transporter 3
MH  - Gene Expression Regulation, Neoplastic
MH  - Glutamate Plasma Membrane Transport Proteins
MH  - Glutamic Acid/*pharmacokinetics
MH  - Humans
MH  - Rats
MH  - Receptors, Glutamate/analysis/genetics
MH  - Receptors, Neurotransmitter/analysis/genetics
MH  - Reverse Transcriptase Polymerase Chain Reaction
MH  - Sodium/pharmacology
MH  - *Symporters
MH  - Transcription, Genetic/physiology
MH  - Tritium/pharmacokinetics
MH  - Tumor Cells, Cultured/chemistry/metabolism
EDAT- 1999/10/16 00:00
MHDA- 1999/10/16 00:01
CRDT- 1999/10/16 00:00
PHST- 1999/10/16 00:00 [pubmed]
PHST- 1999/10/16 00:01 [medline]
PHST- 1999/10/16 00:00 [entrez]
AID - S0006-8993(99)01714-X [pii]
AID - 10.1016/s0006-8993(99)01714-x [doi]
PST - ppublish
SO  - Brain Res. 1999 Aug 28;839(2):235-42. doi: 10.1016/s0006-8993(99)01714-x.