Mechanism of inhibition of cAMP-dependent epithelial chloride secretion by phorbol esters.
In T84 cells, we investigated how stimulation of protein kinase C leads to an inhibition of cAMP-dependent chloride secretion. Specifically, we tested the hypothesis that the inhibition was caused by loss of the cystic fibrosis transmembrane regulator (CFTR), an apical membrane chloride channel. As described by others (Trapnell, B. C., Zeitlin, P. L., Chu, C.-S., Yoshimura, K., Nakamura, H., Guggino, W. B., Bargon, J., Banks, T. C., Dalemans, W., Pavirani, A., Lecocq, J.-P., and Crystal, R. G. (1991) J. Biol. Chem. 266, 10319-10323), we found that treatment with the phorbol ester, phorbol myristate acetate (PMA), reduced CFTR mRNA levels by approximately 80% with a t 1/2 of approximately 2 h. Chloride secretion, measured as forskolin-induced short circuit current, was also abolished by PMA with a t 1/2 of approximately 2 h. Levels of mature glycosylated CFTR measured by Western blotting also declined to 50 +/- 8% (n = 7) of control after a 12-h PMA treatment. However, a 12-h exposure to PMA did not affect the forskolin-stimulated efflux of 125I into high potassium medium, a measure of apical membrane CFTR activity. We conclude that increased turnover of apical membrane CFTR in PMA-treated cells compensates for the decline in anion channel numbers. By contrast to its lack of effect on 125I effluxes, PMA reduced the cAMP-induced increase in 86Rb efflux, suggesting that it inhibits chloride secretion mainly by an action on basolateral potassium channels.
Shen
B Q
BQ
Cystic Fibrosis Research Center, University of California, San Francisco 94143.
Barthelson
R A
RA
Skach
W
W
Gruenert
D C
DC
Sigal
E
E
Mrsny
R J
RJ
Widdicombe
J H
JH
D015153:Blotting, Western; D002463:Cell Membrane Permeability / Q000187:drug effects; D002712:Chlorides / Q000378:metabolism*; D003110:Colonic Neoplasms; D000242:Cyclic AMP / Q000494:pharmacology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004553:Electric Conductivity; D004848:Epithelium / Q000187:drug effects / Q000502:physiology; D006031:Glycosylation; D006801:Humans; D007163:Immunosorbent Techniques; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012414:Rubidium Radioisotopes / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
C505032:CFTR protein, human; D002712:Chlorides; D008565:Membrane Proteins; D015221:Potassium Channels; D012333:RNA, Messenger; D012414:Rubidium Radioisotopes; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000242:Cyclic AMP; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate