Inhibition of apical Cl-/OH- exchange activity in Caco-2 cells by phorbol esters is mediated by PKCepsilon

The present studies were undertaken to examine the possible regulation of apical membrane Cl-/OH- exchanger in Caco-2 cells by protein kinase C (PKC). The effect of the phorbol ester phorbol 12-myristate 13-acetate (PMA), an in vitro PKC agonist, on OH- gradient-driven 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-sensitive 36Cl uptake in Caco-2 cells was assessed. The results demonstrated that PMA decreased apical Cl-/OH- exchanger activity via phosphatidylinositol 3-kinase (PI3-kinase)-mediated activation of PKCepsilon. The data consistent with these conclusions are as follows: 1) short-term treatment of cells for 1-2 h with PMA (100 nM) significantly decreased Cl-/OH- exchange activity compared with control (4alpha-PMA); 2) pretreatment of cells with specific PKC inhibitors chelerythrine chloride, calphostin C, and GF-109203X completely blocked the inhibition of Cl-/OH- exchange activity by PMA; 3) specific inhibitors for PKCepsilon (Ro-318220) but not PKCalpha (Go-6976) significantly blocked the PMA-mediated inhibition; 4) specific PI3-kinase inhibitors wortmannin and LY-294002 significantly attenuated the inhibitory effect of PMA; and 5) PI3-kinase activators IRS-1 peptide and phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] mimicked the effects of PMA. These findings provide the first evidence for PKCepsilon-mediated inhibition of Cl-/OH- exchange activity in Caco-2 cells and indicate the involvement of the PI3-kinase-mediated pathways in the regulation of Cl- absorption in intestinal epithelial cells.