Role of Fyn and PI3K in H2O2-induced inhibition of apical Cl-/OH- exchange activity in human intestinal epithelial cells.
H(2)O(2) 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 H(2)O(2) 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(-) (HCO(3)(-)). The present studies were, therefore, undertaken to examine the direct effects of H(2)O(2) on OH(-) gradient-driven DIDS (4,4'-di-isothiocyanostilbene-2,2'-disulfonate)-sensitive (36)Cl(-) uptake utilizing a post-confluent transformed human intestinal epithelial cell line, Caco-2. Our results demonstrate that H(2)O(2) (1 mM for 60 min) significantly inhibited (approx. 60%; P<0.05) Cl(-)/OH(-) exchange activity in Caco-2 cells. H(2)O(2)-mediated inhibition of Cl(-)/OH(-) exchange activity involved the Src kinase Fyn and PI3K (phosphoinositide 3-kinase)-dependent pathways. H(2)O(2) 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 H(2)O(2), resulting in the activation of the downstream target PLCgamma1 (phospholipase Cgamma1). Elevated intracellular Ca(2+) levels and PKCalpha (protein kinase Calpha) functioned as downstream effectors of H(2)O(2)-induced PLCgamma1 activation. Our results, for the first time, provide evidence for H(2)O(2)-induced Src kinase Fyn/PI3K complex association. This complex association resulted in the subsequent activation of PLCgamma1 and Ca(2+)-dependent PKCalpha, resulting in the inhibition of Cl(-)/OH(-) exchange activity. These findings suggest that H(2)O(2)-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.
Department of Medicine, University of Illinois at Chicago, Jesse Brown VA Medical Center, Chicago, IL 60612, USA.
Seema
S
Saksena
Ravinder K
RK
Gill
Sangeeta
S
Tyagi
Waddah A
WA
Alrefai
Krishnamurthy
K
Ramaswamy
Pradeep K
PK
Dudeja
D026905:Amino Acid Transport Systems / Q000502:physiology; D017920:Antiporters / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D018938:Caco-2 Cells; D027963:Chloride-Bicarbonate Antiporters / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007413:Intestinal Mucosa / Q000166:cytology / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D051966:Phospholipase C gamma / Q000502:physiology; D051571:Protein Kinase C-alpha / Q000502:physiology; D051076:Proto-Oncogene Proteins c-fyn / Q000502:physiology*; D000076988:Sulfate Transporters; D027981:Symporters / Q000502:physiology
D026905:Amino Acid Transport Systems; D017920:Antiporters; D027963:Chloride-Bicarbonate Antiporters; C081011:SLC26A3 protein, human; C475905:SLC36A1 protein, human; D000076988:Sulfate Transporters; D027981:Symporters; C093920:chloride-base exchanger; D006861:Hydrogen Peroxide; D019869:Phosphatidylinositol 3-Kinases; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn; D051571:Protein Kinase C-alpha; D051966:Phospholipase C gamma