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Parameswaran G. Sreekumar, Chris Spee, Stephen J. Ryan, Ram Kannan, David R. Hinton; Role of Multidrug Resistance Protein 1 in Regulation of Glutathione in RPE Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):887.
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© ARVO (1962-2015); The Authors (2016-present)
Glutathione (GSH) is an endogenous tripeptide essential for a variety of cellular functions. Release of GSH and its extracellular functions have been reported in several cell types. We have previously shown that RPE cells expressed MRP family members, among which MRP1 is the most abundant. The purpose of the present study is to test the hypothesis that manipulation of MRP1 could result in altered GSH efflux under unstressed and stressed conditions.
Confluent ARPE-19 cells were used in the present study. MRP1 localization was performed by confocal microscopy and biotin labeling. MRP1 was inhibited by pharmacological inhibitors (MK571 or sulfinpyrazone) and by RNA-mediated gene silencing using specific siRNA sequences. Human MRP1 was cloned to pcDNA and transfected to ARPE-19 cells. MRP1 mRNA and protein levels were quantified by PCR and Western blot analysis. Cells were challenged with or without 150µM H2O2 for 24 h in serum-free medium. GSH efflux and intracellular levels were measured by a colorimetric assay.
MRP1 was predominantly localized to the plasma membrane in RPE cells. Inhibition of MRP1 by MK571 or sulfinpyrazone significantly (p <0.01 vs. controls) inhibited GSH efflux. When MRP1 inhibited cells were challenged with oxidative stress, no further increase in GSH efflux was observed. Further, these cells were resistant to cell death as a result of higher cellular GSH due to higher conversion of oxidized to reduced GSH by increased glutathione reductase (GR). No further increase in MRP1 expression in stressed cells was found. A significant (p <0.01 vs. controls) increase in GSH efflux was noticed in unstressed MRP1 overexpressing RPE cells with no significant change in cellular GSH. However, no significant change in GR expression could be seen. When treated with H2O2, GSH release was increased in both control and MRP1 overexpressing cells with no significant upregulation of cellular GSH levels.
Overall, our study provides evidence that MRP1 is an effective mediator in maintaining cellular redox status by regulating GSH release under both unstimulated and stressed conditions.
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