Mechanisms and physiological significance of the transport of the glutathione conjugate of 4-hydroxynonenal in human lens epithelial cells

Rajendra Sharma, Yusong Yang, Abha Sharma, Seema Dwivedi, Vsevolod Popov, Paul J. Boor, Sharad S. Singhal, Sanjay Awasthi, Yogesh C. Awasthi

Research output: Contribution to journalArticle

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Abstract

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 (Km = 8.4 μM), DNP-SG (100 μM) as well as to ATP (Km 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.

Original languageEnglish
Pages (from-to)3438-3449
Number of pages12
JournalInvestigative Ophthalmology and Visual Science
Volume44
Issue number8
DOIs
StatePublished - Aug 1 2003

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Lenses
Glutathione
Epithelial Cells
Adenosine Triphosphate
Caspases
Adenosine Triphosphatases
Cell Membrane
Egtazic Acid
Ouabain
Osmolar Concentration
4-hydroxy-2-nonenal
Mass Spectrometry
Electron Microscopy
Immunoglobulin G
High Pressure Liquid Chromatography
Apoptosis
Temperature
anti-IgG

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Mechanisms and physiological significance of the transport of the glutathione conjugate of 4-hydroxynonenal in human lens epithelial cells. / Sharma, Rajendra; Yang, Yusong; Sharma, Abha; Dwivedi, Seema; Popov, Vsevolod; Boor, Paul J.; Singhal, Sharad S.; Awasthi, Sanjay; Awasthi, Yogesh C.

In: Investigative Ophthalmology and Visual Science, Vol. 44, No. 8, 01.08.2003, p. 3438-3449.

Research output: Contribution to journalArticle

Sharma, Rajendra ; Yang, Yusong ; Sharma, Abha ; Dwivedi, Seema ; Popov, Vsevolod ; Boor, Paul J. ; Singhal, Sharad S. ; Awasthi, Sanjay ; Awasthi, Yogesh C. / Mechanisms and physiological significance of the transport of the glutathione conjugate of 4-hydroxynonenal in human lens epithelial cells. In: Investigative Ophthalmology and Visual Science. 2003 ; Vol. 44, No. 8. pp. 3438-3449.
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abstract = "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 (Km = 8.4 μM), DNP-SG (100 μM) as well as to ATP (Km 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.",
author = "Rajendra Sharma and Yusong Yang and Abha Sharma and Seema Dwivedi and Vsevolod Popov and Boor, {Paul J.} and Singhal, {Sharad S.} and Sanjay Awasthi and Awasthi, {Yogesh C.}",
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T1 - Mechanisms and physiological significance of the transport of the glutathione conjugate of 4-hydroxynonenal in human lens epithelial cells

AU - Sharma, Rajendra

AU - Yang, Yusong

AU - Sharma, Abha

AU - Dwivedi, Seema

AU - Popov, Vsevolod

AU - Boor, Paul J.

AU - Singhal, Sharad S.

AU - Awasthi, Sanjay

AU - Awasthi, Yogesh C.

PY - 2003/8/1

Y1 - 2003/8/1

N2 - 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 (Km = 8.4 μM), DNP-SG (100 μM) as well as to ATP (Km 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.

AB - 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 (Km = 8.4 μM), DNP-SG (100 μM) as well as to ATP (Km 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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DO - 10.1167/iovs.03-0051

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JO - Investigative Ophthalmology and Visual Science

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SN - 0146-0404

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