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

The phorbol ester phorbol 12-myristate 13-acetate (PMA) inhibits Cl- secret ion (short-circuit current, I-sc) and decreases barrier function (transepit helial resistance, TER) in T84 epithelia. To elucidate the role of specific protein kinase C (PKC) isoenzymes in this response, we compared PMA with t wo non-phorbol activators of PKC (bryo-statin-1 and carbachol) and utilized three PKC inhibitors (Go-6850, Go-6976, and rottlerin) with different isoz yme selectivity profiles. PMA sequentially inhibited cAMP-stimulated I-sc a nd decreased TER, as measured by voltage-current clamp. By subcellular frac tionation and Western blot, PMA (100 nM) induced sequential membrane transl ocation of the novel PKC epsilon followed by the conventional PKC alpha and activated both isozymes by in vitro kinase assay. PKC delta was activated by PMA but did not translocate. By immunofluorescence, PKC epsilon redistri buted to the basolateral domain in response to PMA, whereas PKC alpha moved apically. Inhibition of I-sc by PMA was prevented by the conventional and novel PKC inhibitor Go-6850 (5 muM) but not the conventional isoform inhibi tor Go-6976 (5 muM) or the PKC delta inhibitor rottlerin (10 muM), implicat ing PKC epsilon in inhibition of Cl- secretion. In contrast, both Go-6976 a nd Go-6850 prevented the decline of TER, suggesting involvement of PKC alph a. Bryostatin-1 (100 nM) translocated PKC epsilon and PKC alpha and inhibit ed cAMP-elicited I-sc. However, unlike PMA, bryostatin-1 downregulated PKC alpha protein, and the decrease in TER was only transient. Carbachol (100 m uM) translocated only PKC epsilon and inhibited I-sc with no effect on TER. Go-6850 but not Go-6976 or rottlerin blocked bryostatin-1 and carbachol in hibition of I-sc. We conclude that basolateral translocation of PKC epsilon inhibits Cl- secretion, while apical translocation of PKC alpha decreases TER. These data suggest that epithelial transport and barrier function can be modulated by distinct PKC isoforms.