Journal
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY
Volume 281, Issue 2, Pages C649-C661Publisher
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajpcell.2001.281.2.C649
Keywords
isoenzymes; intercellular junctions; cytoskeleton; colonic neoplasms; intestinal mucosa; chlorides; intestinal secretion; cystic fibrosis; endocytosis; cell membranes
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Funding
- NIDDK NIH HHS [DK-51630, DK-48010] Funding Source: Medline
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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 (bryo-statin-1 and carbachol) and utilized three PKC inhibitors (Go-6850, Go-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 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 redistributed 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 inhibitor Go-6976 (5 muM) or the PKC delta inhibitor rottlerin (10 muM), implicating PKC epsilon in inhibition of Cl- secretion. In contrast, both Go-6976 and Go-6850 prevented the decline of TER, suggesting involvement of PKC alpha. Bryostatin-1 (100 nM) translocated PKC epsilon and PKC alpha and inhibited cAMP-elicited I-sc. However, unlike PMA, bryostatin-1 downregulated PKC alpha protein, and the decrease in TER was only transient. Carbachol (100 muM) 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 inhibition 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.
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