Induction of xCT gene expression and L-cystine transport activity by diethyl maleate at the inner blood-retinal barrier

Masatoshi Tomi, Ken Ichi Hosoya, Hitomi Takanaga, Sumio Ohtsuki, Tetsuya Terasaki

Research output: Contribution to journalArticle

46 Citations (Scopus)

Abstract

Purpose. In this study, the expression and regulation of the L-cystine transporter, system xc -, 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 33°C in the presence or absence of diethyl maleate (DEM), and the uptake rate of [14C]L-cystine was measured at 37°C. The mRNA levels of system xc -, 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 [14C]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 μM. At 100 μM 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 [14C]L-cystine was strongly inhibited by L-glutamic acid, L-α-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 xc -. Conclusions. System xc --mediated L-cystine uptake appears to be present at the inner BRB. DEM induces L-cystine transport through system xc - at the inner BRB by enhanced transcription of the xCT gene.

Original languageEnglish
Pages (from-to)774-779
Number of pages6
JournalInvestigative Ophthalmology and Visual Science
Volume43
Issue number3
Publication statusPublished - 2002
Externally publishedYes

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diethyl maleate
Blood-Retinal Barrier
Cystine
Gene Expression
Messenger RNA
Quisqualic Acid
Aspartic Acid
Glutathione
Arginine
Real-Time Polymerase Chain Reaction
Glutamic Acid

ASJC Scopus subject areas

  • Ophthalmology

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Induction of xCT gene expression and L-cystine transport activity by diethyl maleate at the inner blood-retinal barrier. / Tomi, Masatoshi; Hosoya, Ken Ichi; Takanaga, Hitomi; Ohtsuki, Sumio; Terasaki, Tetsuya.

In: Investigative Ophthalmology and Visual Science, Vol. 43, No. 3, 2002, p. 774-779.

Research output: Contribution to journalArticle

Tomi, Masatoshi ; Hosoya, Ken Ichi ; Takanaga, Hitomi ; Ohtsuki, Sumio ; Terasaki, Tetsuya. / Induction of xCT gene expression and L-cystine transport activity by diethyl maleate at the inner blood-retinal barrier. In: Investigative Ophthalmology and Visual Science. 2002 ; Vol. 43, No. 3. pp. 774-779.
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abstract = "Purpose. In this study, the expression and regulation of the L-cystine transporter, system xc -, 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 33°C in the presence or absence of diethyl maleate (DEM), and the uptake rate of [14C]L-cystine was measured at 37°C. The mRNA levels of system xc -, 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 [14C]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 μM. At 100 μM 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 [14C]L-cystine was strongly inhibited by L-glutamic acid, L-α-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 xc -. Conclusions. System xc --mediated L-cystine uptake appears to be present at the inner BRB. DEM induces L-cystine transport through system xc - at the inner BRB by enhanced transcription of the xCT gene.",
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T1 - Induction of xCT gene expression and L-cystine transport activity by diethyl maleate at the inner blood-retinal barrier

AU - Tomi, Masatoshi

AU - Hosoya, Ken Ichi

AU - Takanaga, Hitomi

AU - Ohtsuki, Sumio

AU - Terasaki, Tetsuya

PY - 2002

Y1 - 2002

N2 - Purpose. In this study, the expression and regulation of the L-cystine transporter, system xc -, 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 33°C in the presence or absence of diethyl maleate (DEM), and the uptake rate of [14C]L-cystine was measured at 37°C. The mRNA levels of system xc -, 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 [14C]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 μM. At 100 μM 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 [14C]L-cystine was strongly inhibited by L-glutamic acid, L-α-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 xc -. Conclusions. System xc --mediated L-cystine uptake appears to be present at the inner BRB. DEM induces L-cystine transport through system xc - at the inner BRB by enhanced transcription of the xCT gene.

AB - Purpose. In this study, the expression and regulation of the L-cystine transporter, system xc -, 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 33°C in the presence or absence of diethyl maleate (DEM), and the uptake rate of [14C]L-cystine was measured at 37°C. The mRNA levels of system xc -, 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 [14C]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 μM. At 100 μM 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 [14C]L-cystine was strongly inhibited by L-glutamic acid, L-α-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 xc -. Conclusions. System xc --mediated L-cystine uptake appears to be present at the inner BRB. DEM induces L-cystine transport through system xc - at the inner BRB by enhanced transcription of the xCT gene.

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