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

Abstract

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.