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

ABSTRACT

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.

• Cystine
• EAAT
• epithelial cells
• glutamate
• glutathione
• lens
• Xc⁻ exchanger

ACKNOWLEDGMENTS

The authors are grateful for the technical assistance provided by Christopher Duggan. The data were presented in part at the 2006 meeting of the Association for Research in Vision and Ophthalmology.

DECLARATION OF INTEREST

The authors report no conflict of interest. The authors alone are responsible for the content and writing of the article. This work was supported in part by an unrestricted Continuing Medical Education grants from Allergan and Alcon in support of the education and research activities of the residents and faculty of the Department of Ophthalmology, Louisiana State University Health Sciences Center, Shreveport, LA.