Structure, function, and regulation of human cystine/glutamate transporterin retinal pigment epithelial cells

PURPOSE. The purpose of this investigation was to provide evidence for the expression of the cystine/glutamate trans porter (x(c)(-)) in the human ret inal pigment epithelial cell line ARPE-19, clone the light chain of the tra nsporter from an ARPE-19 cell cDNA library and study its function, and inve stigate the regulation of this transporter by nitric oxide (NO) in ARPE-19 cells. METHODS. Uptake of radiolabeled cystine and glutamate was measured in ARPE- 19 cells. The functional identity of x(c)(-) in these cells was established by substrate specificity and Na+ - independence of the uptake process. The human x(c)(-) light chain (human xCT) was cloned from an ARPE-19 cell cDNA library. The functional identity of the cloned human xCT was investigated by heterologous coexpression of the light chain with the heavy chain (human 4F2hc) in HeLa cells. ARPE-19 cells were treated with or without the NO do nor 3-nitroso-N-acetylpenicillamine (SNAP) and the expression of x(c)(-) wa s studied at the functional and molecular levels. RESULTS. ARPE-19 cells take up cystine as well as glutamate in the absence of Na+. Substrate specificity studies indicate that although the uptake of cystine in the absence of Na+ is mediated by multiple amino acid transport systems including x(c)(-), the uptake of glutamate in the absence of Na+ oc curs exclusively via x(c)(-). The human xCT cloned from ARPE-19 cells is a protein of 501 amino acids. These cells express the heavy chain 4F2hc as ev idenced from RT-PCR analysis. Coexpression of human xCT with 4F2hc in HeLa cells leads to the induction of cystine and glutamate uptake with character istics similar to that of x(c)(-). The activity of x(c)(-) in ARPE-19 cells is upregulated by SNAP, and the process is associated with an increase in the expression of xCT with no detectable change in the expression of 4F2hc. CONCLUSIONS. ARPE-19 cells express the cystine/glutamate transporter x(c)(- ) (the light chain xCT and the heavy chain 4F2hc) as is evident from functi onal and molecular studies. NO upregulates this transport system and the pr ocess is associated with an increase in xCT mRNA but with no change in 4F2h c mRNA.