Purchase this article with an account.
M.S. Ola, D.M. Maddox, H. Ahsan, V. Ganapathy, S.B. Smith; xCT Expression and Regulation in Retinal Ganglion Cells (RGC-5) Treated with Glutamate . Invest. Ophthalmol. Vis. Sci. 2003;44(13):5208.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: The purpose of this study was to establish the presence of xCT, a light chain component of cystine/glutamate antiporter (xc–) system in RGC-5 cells and to investigate its regulation at the functional and molecular levels under oxidative stress caused by L-glutamate. The cystine taken up by system xc– from the extracellular space is reduced to cysteine for use in the synthesis of glutathione (GSH). GSH protects cells from various oxidative stress conditions. Methods: xCT protein was studied immunohistochemically in intact mouse eyes and in cultured RGC-5 cells. Total RNA from RGC-5 cells was isolated and RT-PCR performed using mouse xCT-specific primers. Intracellular GSH levels were quantified in RGC-5 cells treated with 0.5 and 1 mM glutamate for 24 h. Uptake of radiolabeled cystine and glutamate was measured in RGC-5 cells treated 24 h with 0.25, 0.5, 1.0 mM glutamate. The mRNA level of xCT in glutamate treated cells was determined by semiquantitative RT-PCR. Results: xCT protein was detected in several retinal cell types, including RGCs. The presence of xCT mRNA in cultured RGC-5 cells was established by RT-PCR and further confirmed by cloning and sequencing. Intracellular levels of GSH were reduced in RGC-5 cells treated with high concentrations of glutamate. Exposure of RGC-5 cells to increasing concentrations of L-glutamate led to a marked increase of glutamate and cystine uptake. Semiquantitative RT-PCR demonstrated a marked induction of mRNA encoding xCT in cells exposed to increasing concentrations of glutamate. Conclusions: Immunohistochemical, functional and molecular analyses show that RGCs express xCT and utilize the xc– system for glutamate and cystine transport. Exposure of RGC-5 cells to high levels of glutamate leads to a lowering of intracellular GSH levels, which in turn upregulates transcription of the xCT gene and increases the activity of the xc– transporter. Future studies will examine the levels of xCT protein in RGC-5 cells exposed to high levels of glutamate.
This PDF is available to Subscribers Only