Abstract
Purpose: :
Our laboratory previously showed that depletion of glutathione (GSH) causes apoptosis and induces upregulation of transcription factors and GSH-related genes in RPE. The aim of the present study is to understand the mechanism of GSH efflux in stressed and non-stressed human RPE particularly with respect to specific transporter(s) mediating efflux.
Methods: :
The expression of MRP family members (MRP1- MRP8) was determined with RT-PCR and real time RT-PCR in early passage human RPE. The cellular localization of MRP1 in RPE was studied by confocal and transmission electron microscopy. The effect of oxidative stimuli [tert.butyl hydroperoxide (tBH); buthionine sulfoximine (BSO)) on levels of cellular GSH and release of GSH to extracellular medium was measured using a colorimetric assay kit. The expression of MRP1 was determined by Western blot analysis. Furthermore, the effect of silencing MRP1 and inhibition of MRP (MK571, sulfinpyrazone) on intracellular GSH and efflux from RPE was determined.
Results: :
Among the MRP family members, MRP1 is the most abundantly expressed in RPE followed by MRP5 and low expression of MRP2,3,6 and 7. Confocal microscopy revealed that MRP1 was localized to the plasma membrane. MRP1 siRNA and MRP inhibitors lead to accumulation of RPE cellular GSH and a decrease in GSH efflux. There was a significant 68% decrease in GSH efflux with 50 nM MRP1 siRNA compared to nontargeting siRNA. The MRP inhibitors MK571 (75µM, 5h) and sulfinpyrazone (5mM, 5h) resulted in 25% and 65% reduction in GSH efflux from ARPE-19 cells.
Conclusions: :
Our studies show that MRP1 is expressed abundantly in RPE and is localized to the plasma membrane. Our data also show for the first time that MRP1mediates GSH efflux under unstimulated and oxidant-stimulated conditions.
Keywords: antioxidants • retinal pigment epithelium • apoptosis/cell death