Role of Fyn and PI3K in H2O2-incluced inhibition of apical Cl-/OH- exchange activity in human intestinal epithelial cells

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 Cl-36(-) 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 (phospho-inositide 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 PLC gamma 1 (phospholipase C gamma 1). Elevated intracellular Ca2+ levels and PKC alpha (protein kinase C alpha) functioned as downstream effectors of H2O2-incluced PLC gamma 1 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 PLC gamma 1 and Ca2+-dependent PKC alpha, 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.