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Iron Regulates L-Cystine Uptake and Glutathione Levels in Lens Epithelial and Retinal Pigment Epithelial Cells by Its Effect on Cytosolic Aconitase

From the Department of Molecular Biomedical Sciences, North Carolina State University, Raleigh, North Carolina.

PURPOSE. The authors previously published the novel finding that iron regulates L-glutamate synthesis and accumulation in the cell-conditioned medium (CCM) by increasing cytosolic aconitase activity in cultured lens epithelial cells (LECs), retinal pigment epithelial (RPE) cells, and neurons. The present study was designed to determine whether iron-induced L-glutamate accumulation in the CCM regulates L-cystine uptake and glutathione (GSH) levels through the aconitase pathway in LECs and RPE cells.

METHODS. The presence of xCT, the light chain of X_c^–, a glutamate/cystine antiporter, was analyzed by RT-PCR, immunoblotting, and immunocytochemistry. Uptake of L-[³⁵S]cystine and L-[³H]glutamate was measured in the presence or absence of transporter inhibitors. L-cystine uptake and intracellular GSH concentration were measured in the presence or absence of iron-saturated transferrin, the iron chelator dipyridyl (DP), or oxalomalic acid (OMA), an aconitase inhibitor.

RESULTS. LECs and RPE cells express xCT, as evidenced by RT-PCR analysis and immunoblotting. xCT was localized by immunocytochemistry. The authors found that the iron-induced increase in L-glutamate availability increased L-cystine uptake, with subsequent increases in GSH levels. In addition, L-glutamate production, L-cystine uptake, and GSH concentration were inhibited by OMA and DP, indicating a central role for iron-regulated aconitase activity in GSH synthesis in LECs and RPE cells.

CONCLUSIONS. These results demonstrate for the first time that iron regulates L-cystine uptake and the downstream production of GSH in two mammalian cell types. It is possible that the increase in intracellular antioxidant concentration induced by iron serves as a protective mechanism against the well-established capacity of iron to induce oxidative damage.