EAAT and Xc− Exchanger Inhibition Depletes Glutathione in the Transformed Human Lens Epithelial Cell Line SRA 01/04

• Published in: Current eye research Vol. 41; no. 3; pp. 357 - 366
• Main Authors: Langford, Marlyn P., Redens, Thomas B., Liang, Chanping, Kavanaugh, A. Scott, Texada, Donald E.
• Format: Journal Article
• Language: English
• Published: England Taylor & Francis 01.01.2016
• Subjects: Amino Acid Transport System y+ - antagonists & inhibitors; Aspartic Acid - analogs & derivatives; Aspartic Acid - pharmacology; Blotting, Western; Cell Line, Transformed; Cystine; Dose-Response Relationship, Drug; EAAT; epithelial cells; Epithelial Cells - drug effects; Epithelial Cells - metabolism; exchanger; Excitatory Amino Acid Antagonists - pharmacology; glutamate; Glutamate Plasma Membrane Transport Proteins - antagonists & inhibitors; glutathione; Glutathione - metabolism; Humans; Kainic Acid - pharmacology; lens; Lens, Crystalline - cytology; Xc− exchanger; glutathione; epithelial cells; lens; glutamate; Cystine; EAAT
• ISSN: 0271-3683; 1460-2202
• DOI: 10.3109/02713683.2015.1017651

Purpose: Maintaining the high glutathione (GSH; tripeptide of glutamate, cysteine and glycine) levels in the lens cortex promotes lens health. The role of glutamate/aspartate (Glu/Asp) transporters and the cystine (Cys)/Glu exchanger (Xc − exchanger) in maintaining GSH in transformed human lens epithelial cells (SRA 01/04) was investigated. Methods: Detection and differentiation of excitatory amino acid transporters (EAAT1-5) and the Xc − exchanger was performed by the uptake of radiolabeled l-Glu, d-Asp and l-Cys in the presence and absence of Na + , substrate-specific inhibition studies and Western-blot analysis. Reductions in GSH levels post-inhibition of Xc − exchanger and EAAT activities by substrate inhibitors demonstrated the roles of EAAT and Xc − exchanger in maintaining GSH. Results: Glu and d-Asp uptake in HLEC was Na + -dependent. Strong inhibition by substrate-specific Glu/Asp uptake inhibitors and weak inhibition by kainic acid (KA) was consistent with Na + -dependent EAAT1/3/4/5 activity and weak EAAT2 activity, respectively. Na + -independency and Glu inhibition of Cys uptake were consistent with Xc − exchanger activity, but inhibition of Na + -dependent Cys uptake by N-acetylcysteine suggests Cys uptake by EAAT3. EAAT1-5 and xCT (Xc − exchanger light chain) immunoreactive peptides were detected by Western-blot analysis of HLEC lysates. EAAT and Xc − exchanger inhibition by substrate antagonists depleted GSH concentrations by 15-28% (p's ≤ 0.02), while GSH synthesis inhibition by buthionine sulfoximine depleted GSH by 33% (p = 0.008). Conclusion: Inhibition of Glu and Cys uptake by EAAT and Xc − exchanger antagonists depletes GSH in human lens epithelial cells. These in vitro results support pivotal roles for EAAT and Xc − exchanger activities in maintaining GSH and protection against oxidative stress in cortical lens epithelium.