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.

Mechanisms of lysophosphatidic acid (LPA) mediated stimulation of intestinal apical Cl−/OH− exchange

“…Our data showed that the effects of LPA on Cl Ϫ /OH Ϫ exchange activity were blocked in the presence of PI3 kinase and Akt inhibitors, indicating the involvement of PI3 kinaseAkt pathway. The involvement of PI3 kinase-dependent pathway is in agreement with other previous studies from our laboratory that have shown the involvement of PI3 kinase in the regulation of Cl Ϫ /OH Ϫ exchange activity by Lactobacillus acidophilus, taurodeoxycholic acid, and PMA (1,8,33). Furthermore, PI3 kinase has been shown to be involved in medi- Fig.…”

“…Cl Ϫ /OH Ϫ exchangers or anion exchangers are known to play a pivotal role in the vectorial transport of Cl Ϫ across the plasma membrane of polarized epithelial cells. Previously, we have shown that apical Cl Ϫ / OH Ϫ exchange activity is inhibited by various inflammatory mediators, such as nitric oxide, serotonin, and phorbol esters, and also by infection with enteropathogenic Escherichia coli in Caco-2 cells and is stimulated by probiotic bacterium, Lactobacillus acidophilus (8,14,(33)(34)(35). In the present studies, our data for the first time demonstrate that LPA significantly stimulates Cl Ϫ /OH Ϫ exchange activity in Caco-2 cells via activation of PI3 kinase/Akt pathways, suggesting that this effect of LPA could contribute to potential antidiarrheal effects of LPA.…”

The Role of Protein Kinase C Isoforms in Modulating Injury and Repair of the Intestinal Barrier

“…Through a series of pharmacological experiments using a monolayer model, Yoo et al (2001) showed that PKC-⑀ may inhibit hypoxia-induced secretory responses in epithelial cells. This finding was later reproduced by Saksena et al (2002), who showed that EGF inhibited basolateral Cl Ϫ secretion through activation of PKC-⑀. Later studies suggested that PKC-⑀ has an inhibitory role in TNF-induced barrier dysfunction (Yoo et al, 2001).…”

Involvement of c-Src and Protein Kinase Cδ in the Inhibition of Cl-/OH- Exchange Activity in Caco-2 Cells by Serotonin

“…Chloride uptake experiments were performed essentially as described by Olsnes et al (37) with some modifications (38,39). Caco-2 cells were incubated with Dulbecco's modified Eagle's base media containing 20 mM Hepes/KOH, pH 8.5, for 30 min at room temperature.…”

“…The cells were then incubated with the uptake buffer for a 5-min period. This period was chosen because it falls within the linear range of Cl Ϫ uptake in this system (38,39). For 36 Cl Ϫ uptake studies, the uptake buffer was the mannitol buffer containing 1.4 Ci of 36 Cl Ϫ (2.9 mM) of hydrochloric acid (specific activity-17.12 mCi/g) Ϯ 0.3 mM DIDS.…”