Expression and regulation of L-cystine transporter, system xc-, in the newly developed rat retinal Muller cell line (TR-MUL)

The purpose of the present study was to elucidate the expression and regulation of the L-cystine transporter, system x(c)(-), in Miffler cells. In this study, newly developed conditionally immortalized rat Miffler cell lines (TR-MUL) from transgenic rats harboring the temperature-sensitive SV 40 large T-antigen gene were used as an in vitro model. TR-MUL cells express large T-antigen and grow well at 33degreesC with a doubling time of 30 h, but do not grow at 39degreesC. TR-MUL cells express typical Mtiller cell markers such as S-100, glutamine synthetase, and EAAT1/GLAST, whereas EAAT2/ GLT-1 and EAAT5 are not detected. TR-MUL cells also exhibit little or no expression of glial fibrillary acidic protein. We found that TR-MUL5 cells exhibited [C-14]L-cystine uptake activity and expressed xCT and 4F2hc, which involve system xc-. The uptake of [C-14]L-eystine was significantly inhibited by L-glutamic acid and L-aspartic acid, whereas L-leucine had no effect. Following diethyl maleate (DEM) treatment, the glutathione concentration in TR-MUL5 cells was reduced in the first 24 h, then gradually recovered for more than 24 h. The L-cystine uptake rate and the xCT expression level in TR-MUL5 cells were enhanced by DEM treatment. In contrast, the 4F2hc expression level was unchanged. In conclusion, TR-MUL cells have the properties of Miffler cells and exhibit system xc(-)-mediated L-cystine uptake activity. The oxidative stress conditions following DEM treatment activate L-cystine transport in TR-MUL cells due to the enhanced transcription of the xCT gene. (C) 2003 Wiley-Liss, Inc.