Mechanisms and physiological significance of the transport of the glutathione conjugate of 4-hydroxynonenal in human lens epithelial cells.
OBJECTIVE
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 [(3)H] 4-HNE and efflux of its GSH conjugate, [(3)H] 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 micro M), DNP-SG (100 micro M) 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.
Sharma
Rajendra
R
Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas, USA.
Yang
Yusong
Y
Sharma
Abha
A
Dwivedi
Seema
S
Popov
Vsevolod L
VL
Boor
Paul J
PJ
Singhal
Sharad S
SS
Awasthi
Sanjay
S
Awasthi
Yogesh C
YC
D018528:ATP-Binding Cassette Transporters; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000447:Aldehydes / Q000378:metabolism* / Q000633:toxicity; D019138:Antibodies, Blocking / Q000494:pharmacology; D017209:Apoptosis; D001693:Biological Transport, Active / Q000187:drug effects; D002352:Carrier Proteins / Q000276:immunology / Q000502:physiology*; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002851:Chromatography, High Pressure Liquid; D004847:Epithelial Cells / Q000378:metabolism* / Q000648:ultrastructure; D020690:GTPase-Activating Proteins; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism*; D006801:Humans; D007908:Lens, Crystalline / Q000378:metabolism* / Q000648:ultrastructure; D013058:Mass Spectrometry; D016253:Microscopy, Immunoelectron
D018528:ATP-Binding Cassette Transporters; D000447:Aldehydes; D019138:Antibodies, Blocking; D002352:Carrier Proteins; D020690:GTPase-Activating Proteins; C095637:RALBP1 protein, human; C030190:S-(2,4-dinitrophenyl)glutathione; D000255:Adenosine Triphosphate; D020169:Caspases; D000251:Adenosine Triphosphatases; D005978:Glutathione; C027576:4-hydroxy-2-nonenal