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

Abstract

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 PKCε. 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 (4α-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 PKCε (Ro-318220) but not PKCα (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)P3] mimicked the effects of PMA. These findings provide the first evidence for PKCε-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.