ion, intracellular thiol levels and enhanced cell survival in oxidative stress. Our data revealed a significant 2-fold increase in cellular GSH levels in G5555 manufacturer a-crystallin overexpressing clones when compared to controls. One of the main mechanisms for elevation of cellular GSH is increased biosynthesis catalyzed by the rate-limiting enzyme glutamate-cysteine ligase . The increase in total GSH levels was associated with significant upregulation of the gene and protein expression of the catalytic unit of GCL but not GCLM, the modifier unit of GCL. Mitochondrial fractions from a-crystallin overexpressing cells had significantly higher GSH levels after treatment with 150 mM H2O2 for 24 h. The magnitude of increase in GSH level in cytosol, MRP-related GSH transporters in RPE cells We then proceeded to characterize the transporter mediating GSH efflux from RPE cells. Several MRPs are known to mediate GSH efflux in mammalian cells. To determine the presence of MRPs in RPE, MRP mRNA levels were analyzed by RT-PCR. RNA isolated from RPE cells was amplified using specific MRP primer sequences. mRNAs encoding for MRP1, MRP2, MRP3, MRP4, MRP5, MRP6, and MRP7 were detected in RPE cells. MRP1 was the most abundant of the MRP family members in RPE. All further experiments were performed with MRP1 because it is the most well characterized MRP 
with respect to efflux of GSH and GSSG. MRP1-Mediated GSH Efflux in RPE Cells Localization of MRP1 in a-Crystallin overexpressing RPE cells In subconfluent ARPE cells, MRP1 is predominantly localized in the plasma membrane and the staining pattern is punctate. In human polarized RPE monolayers, we observed lateral membrane localization of MRP1. Biotinylation of intact 22314911 cells with subsequent immunoblot analysis revealed surface localization of MRP1 in the membrane fraction. These studies further established that membrane expression of MRP1 was almost three fold higher in aB crystallin overexpressing cells than vector control cells which correlated well with the increased GSH efflux in a-crystallin overexpressing cells. In addition, cellular MRP1 expression showed a.2.5 fold increase in aB crystallin overexpressing cells as compared to vector control cells. Furthermore, consistent with GSH efflux under oxidative stress, we observed a.2 fold increase in MRP1 expression only in vector control cells subjected to oxidative stress. Having established that increased a-crystallin levels increased MRP1 expression, we then investigated whether knocking down of aB crystallin could affect the expression of MRP1. As seen in Fig. 5E, a significant suppression of aB crystallin was achieved by siRNA silencing. MRP1 expression tended to increase in aB crystallin knock-down RPE cells and treatment with H2O2 further increased MRP1 expression by 1.5 fold. These studies suggest that MRP1 regulates GSH efflux under conditions of oxidative stress in RPE cells. Effect of MRP1 inhibition on GSH release and cell death To 8199874 further confirm that MRP1 is involved in GSH efflux in RPE cells, we inhibited MRP1 by pharmacological agents and siRNAmediated gene silencing. When serum-starved RPE cells were treated with MRP inhibitors for 5 h, a significant 50% decrease in GSH efflux was observed. MK571 and sulfinpyrazone are non-specific MRP inhibitors and can therefore be expected to inhibit some or all the MRP isoforms present in RPE cells. MRP1-Mediated GSH Efflux in RPE Cells To delineate the specific role of MRP1 in GSH transport, we selectively suppre
