The pharmacological profile of L-glutamate transport in human NT2 neurones is consistent with excitatory amino acid transporter 2.
The human teratocarcinoma cell line NTera2/D1 can be differentiated to produce post-mitotic neurones (NT2-N cells) by prolonged (> 3 week) exposure to retinoic acid. In this study, we describe the characterisation of high-affinity Na+-dependent L-glutamate transport activity in post-mitotic differentiated NT2-N cells. NT2-N cells, but not the undifferentiated precursor cells, transported L-glutamate in a Na+-dependent manner, as determined by equimolar replacement of Na+ with choline. L-glutamate uptake was saturable and Eadie-Hofstee transformation of the saturation data revealed a Km of 10.6+/-0.8 microM, and a maximum transport capacity (Vmax) of 100.3+/-12.3 pmol min(-1) mg(-1) protein. Pharmacological characterisation of the transport activity in NT2-N cells produced a rank order of inhibitory activity which was identical to that determined for the human excitatory amino acid transporter 2 which we have analysed in a stable mammalian cell line (Madin Darby Canine Kidney (MDCK) cells). Of particular note, L-glutamate transport by NT2-N cells was sensitive to both dihydrokainate and kainate. The expression of human excitatory amino acid transporter mRNAs was studied using reverse transcriptase polymerase chain reaction. NT2-N cells expressed transcripts for excitatory amino acid transporters 2 and 3, but not for the subtypes 1, 4 and 5. We conclude that although the mRNA expression studies suggest the presence of transcripts for both excitatory amino acid transporter 2 and 3 in NT2-N cells, the sensitivity to dihydrokainate and kainate determined in the pharmacological analysis indicates that, of the known transporter subtypes, excitatory amino acid transporter 2 contributes to the bulk of the L-glutamate transport activity present in these cells.
Dunlop
J
J
CNS Disorders, Wyeth Ayerst Research, Princeton, NJ 08543-8000, USA. dunlopj@war.wyeth.com
Beal McIlvain
H
H
Lou
Z
Z
Franco
R
R
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D004285:Dogs; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D027342:Excitatory Amino Acid Transporter 2; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007608:Kainic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D017981:Receptors, Neurotransmitter / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013237:Stereoisomerism; D014407:Tumor Cells, Cultured
D018690:Excitatory Amino Acid Agonists; D027342:Excitatory Amino Acid Transporter 2; D017981:Receptors, Neurotransmitter; D018698:Glutamic Acid; C020889:dihydrokainic acid; D007608:Kainic Acid