Vol. 302, Issue 1, 225-231, July 2002

Enhancement of L-Cystine Transport Activity and Its Relation to xCT Gene Induction at the Blood-Brain Barrier by Diethyl Maleate Treatment

Ken-ichi Hosoya , Masatoshi Tomi , Sumio Ohtsuki , Hitomi Takanaga , Shigeki Saeki, Yoshikatsu Kanai, Hitoshi Endou, Mikihiko Naito, Takashi Tsuruo and Tetsuya Terasaki

Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama (K.-i.H., M.T.); Core Research for Evolutional Science and Technology of Japan Science and Technology Corporation, Kawaguchi (K.-i.H., M.T., S.O., H.T., T.Te.); Department of Molecular Biopharmacy and Genetics, Graduate School of Pharmaceutical Sciences, and New Industry Creation Hatchery Center (S.O., H.T., T.Te.), Tohoku University, Sendai (S.O., H.T., S.S., T.Te.); Department of Pharmacology and Toxicology, Kyorin University School of Medicine, Tokyo (Y.K., H.E.); and Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, (M.N., T.Ts.) Japan

The purpose of the present study was to elucidate the mechanism of enhancement of L-cystine uptake at the blood-brain barrier (BBB). The uptake of [¹⁴C]L-cystine and [³H]L-glutamic acid (L-Glu) was determined using a mouse brain endothelial cell line (MBEC4) as an in vitro BBB model. The mRNA levels of L-cystine/L-Glu exchanger, system x_c, which consists of xCT and 4F2hc, were determined by quantitative real-time reverse transcription-polymerase chain reaction analysis. The [¹⁴C]L-cystine uptake by MBEC4 cells appeared to be mediated via an Na⁺-independent saturable process. The corresponding Michaelis-Menten constant (K_m) was 63.7 µM. In the presence of L-Glu, there was competitive inhibition with an inhibition constant (K_i) of 83.5 µM. [³H]L-Glu uptake in the absence of Na⁺ was saturable with a K_m of 48.1 µM, and it exhibited competitive inhibition with a K_i of 24.9 µM in the presence of L-cystine. The mutual inhibition between L-cystine and L-Glu and the type of inhibition suggest that system x_c operates in MBEC4 cells. The xCT and 4F2hc mRNAs were expressed in MBEC4 cells and, following diethyl maleate (DEM) treatment, the xCT mRNA level and L-cystine uptake in MBEC4 cells were enhanced in parallel with an increase in DEM concentration (up to 500 µM). Concomitantly, the glutathione concentration in MBEC4 cells was increased. In conclusion, system x_c-mediated L-cystine uptake takes place in MBEC4 cells. L-Cystine transport via system x_c at the BBB is likely to be induced under oxidative stress conditions following DEM treatment due to enhanced transcription of the xCT gene.