Regulation of epithelial transport and barrier function by distinct protein kinase C isoforms

Abstract

The phorbol ester phorbol 12-myristate 13-acetate (PMA) inhibits Cl− secretion (short-circuit current, I sc) and decreases barrier function (transepithelial resistance, TER) in T84 epithelia. To elucidate the role of specific protein kinase C (PKC) isoenzymes in this response, we compared PMA with two non-phorbol activators of PKC (bryostatin-1 and carbachol) and utilized three PKC inhibitors (Gö-6850, Gö-6976, and rottlerin) with different isozyme selectivity profiles. PMA sequentially inhibited cAMP-stimulated I sc and decreased TER, as measured by voltage-current clamp. By subcellular fractionation and Western blot, PMA (100 nM) induced sequential membrane translocation of the novel PKCε followed by the conventional PKCα and activated both isozymes by in vitro kinase assay. PKCδ was activated by PMA but did not translocate. By immunofluorescence, PKCε redistributed to the basolateral domain in response to PMA, whereas PKCα moved apically. Inhibition of I sc by PMA was prevented by the conventional and novel PKC inhibitor Gö-6850 (5 μM) but not the conventional isoform inhibitor Gö-6976 (5 μM) or the PKCδ inhibitor rottlerin (10 μM), implicating PKCε in inhibition of Cl− secretion. In contrast, both Gö-6976 and Gö-6850 prevented the decline of TER, suggesting involvement of PKCα. Bryostatin-1 (100 nM) translocated PKCε and PKCα and inhibited cAMP-elicited I sc. However, unlike PMA, bryostatin-1 downregulated PKCα protein, and the decrease in TER was only transient. Carbachol (100 μM) translocated only PKCε and inhibited I sc with no effect on TER. Gö-6850 but not Gö-6976 or rottlerin blocked bryostatin-1 and carbachol inhibition of I sc. We conclude that basolateral translocation of PKCε inhibits Cl−secretion, while apical translocation of PKCα decreases TER. These data suggest that epithelial transport and barrier function can be modulated by distinct PKC isoforms.