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Rajendra Sharma, Yusong Yang, Abha Sharma, Seema Dwivedi, Vsevolod L. Popov, Paul J. Boor, Sharad S. Singhal, Sanjay Awasthi, Yogesh C. Awasthi; Mechanisms and Physiological Significance of the Transport of the Glutathione Conjugate of 4-Hydroxynonenal in Human Lens Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2003;44(8):3438-3449. doi: https://doi.org/10.1167/iovs.03-0051.
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purpose. To study the mechanism and physiological significance of the transport of the glutathione (GSH) conjugate of 4-hydroxynonenal (4-HNE) from lens epithelial cells.
methods. HLE B-3 cells were treated with [3H] 4-HNE and efflux of its GSH conjugate, [3H] GS-HNE, into the medium was quantitated and characterized by HPLC and mass spectrometry. Inside-out vesicles (IOVs) were prepared from HLE B-3 cell membranes. The kinetics of adenosine triphosphate (ATP)-dependent uptake of GS-HNE and dinitrophenyl S-glutathione (DNP-SG) by these IOVs and inhibition of GS-HNE uptake by anti-RLIP76 IgG was studied. Localization of RLIP76 was studied by immunogold electron microscopy and kinetics of the adenosine triphosphatase (ATPase) activity of purified RLIP76 was determined. 4-HNE–induced apoptosis was compared in HLE-B3 cells coated with anti-RLIP76 IgG or preimmune IgG, by caspase activation assay.
results. The results showed the presence of RLIP76 in plasma membranes of HLE B-3 cells and that it mediated ATP-dependent transport of GS-HNE as well as of DNP-SG. The transport was saturable with respect to GS-HNE (K m = 8.4 μM), DNP-SG (100 μM) as well as to ATP (K m 0.45 mM) and was sensitive to temperature and osmolarity of the medium. Anti-RLIP76 IgG inhibited approximately 65% of the transport of GS-HNE and DNP-SG, indicating that most of the transport was mediated by RLIP76. Compatible with its transport function, the EGTA- and ouabain-insensitive ATPase activity of purified RLIP76 was stimulated by DNP-SG and GS-HNE. 4-HNE–induced caspase activation in HLE-B3 cells was exacerbated when the transport of GS-HNE from these cells was blocked by anti-RLIP76 IgG.
conclusions. RLIP76 provides a defense against the deleterious effects of 4-HNE by transporting GS-HNE and can modulate apoptotic signaling by regulating the intracellular concentrations of 4-HNE.
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