title: Induction of xCT gene expression and L-cystine transport activity by diethyl maleate at the inner blood-retinal barrier.
abstract: OBJECTIVE
  In this study, the expression and regulation of the L-cystine transporter, system x(-)(c), at the inner blood-retinal barrier (inner BRB) was investigated using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) as an in vitro model.
  
  
  METHODS
  For the uptake study, TR-iBRB2 cells were cultured at 33C in the presence or absence of diethyl maleate (DEM), and the uptake rate of [(14)C]L-cystine was measured at 37C. The mRNA levels of system x(-)(c), which consists of xCT and 4F2hc, were determined by quantitative real-time RT-PCR analysis with specific primers.
  
  
  RESULTS
  The xCT and 4F2hc mRNAs were expressed in TR-iBRB2 cells. The [(14)C]L-cystine uptake by TR-iBRB2 cells appeared to be mediated through a saturable Na(+)-independent process. The corresponding Michaelis-Menten constant was 9.18 microM. At 100 microM DEM, the xCT mRNA level and L-cystine uptake activity in TR-iBRB2 cells were enhanced in a time-dependent manner. Concomitantly, the glutathione concentration in TR-iBRB2 cells was increased. In contrast, the 4F2hc mRNA level was unchanged up to 24 hours and was induced for more than 24 hours by DEM treatment. Under both normal and DEM treatment conditions, the uptake of [(14)C]L-cystine was strongly inhibited by L-glutamic acid, L-alpha-aminoadipic acid, L-homocysteic acid, and L-quisqualic acid, whereas L-aspartic acid and L-arginine had no effect, which is evidence of the induction of system x(-)(c).
  
  
  CONCLUSIONS
  System x(-)(c)-mediated L-cystine uptake appears to be present at the inner BRB. DEM induces L-cystine transport through system x(-)(c) at the inner BRB by enhanced transcription of the xCT gene.
authors.affiliation: Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama, Japan.
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Tomi
authors.forename: Ken-ichi
authors.initials: K
authors.lastname: Hosoya
authors.forename: Hitomi
authors.initials: H
authors.lastname: Takanaga
authors.forename: Sumio
authors.initials: S
authors.lastname: Ohtsuki
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Terasaki
mesh_terms: D000818:Animals; D001692:Biological Transport; D001813:Blood-Retinal Barrier / Q000502:physiology*; D002196:Capillaries; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics*; D002478:Cells, Cultured; D003553:Cystine / Q000378:metabolism*; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D005786:Gene Expression Regulation; D005978:Glutathione / Q000378:metabolism; D008297:Male; D008298:Maleates / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D012171:Retinal Vessels / Q000187:drug effects* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
chemical_list: D002352:Carrier Proteins; D008298:Maleates; D012333:RNA, Messenger; D003553:Cystine; C014476:diethyl maleate; D005978:Glutathione