PURPOSE: The purpose of this investigation was to provide evidence for the expression of the cystine/glutamate transporter (x(c)(-)) in the human retinal pigment epithelial cell line ARPE-19, clone the light chain of the transporter from an ARPE-19 cell cDNA library and study its function, and investigate 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 donor 3-nitroso-N:-acetylpenicillamine (SNAP) and the expression of x(c)(-) was 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(+) occurs 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 evidenced from RT-PCR analysis. Coexpression of human xCT with 4F2hc in HeLa cells leads to the induction of cystine and glutamate uptake with characteristics 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 functional and molecular studies. NO upregulates this transport system and the process is associated with an increase in xCT mRNA but with no change in 4F2hc mRNA.
PURPOSE: The purpose of this investigation was to provide evidence for the expression of the cystine/glutamate transporter (x(c)(-)) in the human retinal pigment epithelial cell line ARPE-19, clone the light chain of the transporter from an ARPE-19 cell cDNA library and study its function, and investigate 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 (humanxCT) was cloned from an ARPE-19 cell cDNA library. The functional identity of the cloned humanxCT was investigated by heterologous coexpression of the light chain with the heavy chain (human4F2hc) in HeLa cells. ARPE-19 cells were treated with or without the NO donor3-nitroso-N:-acetylpenicillamine (SNAP) and the expression of x(c)(-) was 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(+) occurs exclusively via x(c)(-). The humanxCT cloned from ARPE-19 cells is a protein of 501 amino acids. These cells express the heavy chain 4F2hc as evidenced from RT-PCR analysis. Coexpression of humanxCT with 4F2hc in HeLa cells leads to the induction of cystine and glutamate uptake with characteristics 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 functional and molecular studies. NO upregulates this transport system and the process is associated with an increase in xCT mRNA but with no change in 4F2hc mRNA.
Authors: Jan Lewerenz; Sandra J Hewett; Ying Huang; Maria Lambros; Peter W Gout; Peter W Kalivas; Ann Massie; Ilse Smolders; Axel Methner; Mathias Pergande; Sylvia B Smith; Vadivel Ganapathy; Pamela Maher Journal: Antioxid Redox Signal Date: 2012-08-03 Impact factor: 8.401
Authors: Alejandra M Pacchioni; Joseph Vallone; Roberto I Melendez; Andy Shih; Timothy H Murphy; Peter W Kalivas Journal: Brain Res Date: 2006-11-17 Impact factor: 3.252
Authors: François Verrey; Ellen I Closs; Carsten A Wagner; Manuel Palacin; Hitoshi Endou; Yoshikatsu Kanai Journal: Pflugers Arch Date: 2003-06-11 Impact factor: 3.657
Authors: Christy C Bridges; Huankai Hu; Seiji Miyauchi; Umapathy N Siddaramappa; Malliga E Ganapathy; Leszek Ignatowicz; Dennis M Maddox; Sylvia B Smith; Vadivel Ganapathy Journal: Invest Ophthalmol Vis Sci Date: 2004-09 Impact factor: 4.799