xCT Expression and Regulation in Retinal Ganglion Cells (RGC-5) Treated with Glutamate

Abstract

Abstract: : Purpose: The purpose of this study was to establish the presence of xCT, a light chain component of cystine/glutamate antiporter (x_c^–) 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 x_c^– 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 x_c^– 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 x_c^– transporter. Future studies will examine the levels of xCT protein in RGC-5 cells exposed to high levels of glutamate.

Keywords: ganglion cells • excitatory neurotransmitters • oxidation/oxidative or free radical damage