H2O2 is a highly reactive oxygen metabolite that has been implicated as an important mediator of inflammation-induced intestinal injury associated with ischaemia/reperfusion, radiation and inflammatory bowel disease. Previous studies have shown that H2O2 inhibits NaCl absorption and activates Cl− secretion in the rat and rabbit colon. To date, however, almost no information is available with respect to its effect on the human intestinal apical anion exchanger Cl−/OH− (HCO3−). The present studies were, therefore, undertaken to examine the direct effects of H2O2 on OH− gradient-driven DIDS (4,4′-di-isothiocyanostilbene-2,2′-disulfonate)-sensitive 36Cl− uptake utilizing a post-confluent transformed human intestinal epithelial cell line, Caco-2. Our results demonstrate that H2O2 (1 mM for 60 min) significantly inhibited (approx. 60%; P<0.05) Cl−/OH− exchange activity in Caco-2 cells. H2O2-mediated inhibition of Cl−/OH− exchange activity involved the Src kinase Fyn and PI3K (phosphoinositide 3-kinase)-dependent pathways. H2O2 also induced phosphorylation of Fyn and p85 (the regulatory subunit of PI3K) in Caco-2 cells. Moreover, an increased association of Fyn and p85 was observed in response to H2O2, resulting in the activation of the downstream target PLCγ1 (phospholipase Cγ1). Elevated intracellular Ca2+ levels and PKCα (protein kinase Cα) functioned as downstream effectors of H2O2-induced PLCγ1 activation. Our results, for the first time, provide evidence for H2O2-induced Src kinase Fyn/PI3K complex association. This complex association resulted in the subsequent activation of PLCγ1 and Ca2+-dependent PKCα, resulting in the inhibition of Cl−/OH− exchange activity. These findings suggest that H2O2-induced inhibition of the Cl−/OH− exchange process may play an important role in the pathophysiology of diarrhoea associated with inflammatory disorders, where the amount of reactive oxygen species is markedly elevated.
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Research Article|
October 28 2008
Role of Fyn and PI3K in H2O2-induced inhibition of apical Cl−/OH− exchange activity in human intestinal epithelial cells
Seema Saksena;
Seema Saksena
1Section of Digestive Diseases and Nutrition, Department of Medicine, University of Illinois at Chicago, Jesse Brown VA Medical Center, Chicago, IL 60612, U.S.A.
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Ravinder K. Gill;
Ravinder K. Gill
1Section of Digestive Diseases and Nutrition, Department of Medicine, University of Illinois at Chicago, Jesse Brown VA Medical Center, Chicago, IL 60612, U.S.A.
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Sangeeta Tyagi;
Sangeeta Tyagi
1Section of Digestive Diseases and Nutrition, Department of Medicine, University of Illinois at Chicago, Jesse Brown VA Medical Center, Chicago, IL 60612, U.S.A.
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Waddah A. Alrefai;
Waddah A. Alrefai
1Section of Digestive Diseases and Nutrition, Department of Medicine, University of Illinois at Chicago, Jesse Brown VA Medical Center, Chicago, IL 60612, U.S.A.
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Krishnamurthy Ramaswamy;
Krishnamurthy Ramaswamy
1Section of Digestive Diseases and Nutrition, Department of Medicine, University of Illinois at Chicago, Jesse Brown VA Medical Center, Chicago, IL 60612, U.S.A.
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Pradeep K. Dudeja
Pradeep K. Dudeja
1
1Section of Digestive Diseases and Nutrition, Department of Medicine, University of Illinois at Chicago, Jesse Brown VA Medical Center, Chicago, IL 60612, U.S.A.
1To whom correspondence should be addressed (email pkdudeja@uic.edu).
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Publisher: Portland Press Ltd
Received:
July 18 2007
Revision Received:
June 02 2008
Accepted:
June 18 2008
Accepted Manuscript online:
June 18 2008
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2008 Biochemical Society
2008
Biochem J (2008) 416 (1): 99–108.
Article history
Received:
July 18 2007
Revision Received:
June 02 2008
Accepted:
June 18 2008
Accepted Manuscript online:
June 18 2008
Citation
Seema Saksena, Ravinder K. Gill, Sangeeta Tyagi, Waddah A. Alrefai, Krishnamurthy Ramaswamy, Pradeep K. Dudeja; Role of Fyn and PI3K in H2O2-induced inhibition of apical Cl−/OH− exchange activity in human intestinal epithelial cells. Biochem J 15 November 2008; 416 (1): 99–108. doi: https://doi.org/10.1042/BJ20070960
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