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J. Biol. Chem., Vol. 275, Issue 28, 21169-21176, July 14, 2000
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From the Department of Medicine, University of California, San
Diego, School of Medicine, San Diego, California 92103
Epidermal growth factor (EGF) inhibits
carbachol-induced chloride secretion in T84 colonic
epithelial cells and has been shown to activate phosphatidylinositol
(PI) 3-kinase, leading to inhibition of a basolateral potassium
conductance. We asked whether the inhibitory effect of EGF on secretion
is due to activation of specific isoforms of protein kinase C (PKC) by
PI 3-kinase. Western analysis revealed that PKC
A Role for Protein Kinase C
in the Inhibitory Effect of
Epidermal Growth Factor on Calcium-stimulated Chloride Secretion in
Human Colonic Epithelial Cells*
, and
, 
, 
, 
, µ,
/
, and 
were expressed in T84 cells. Ro318220 (an
inhibitor active against PKC, 10 µM) but not
Gö6983 (an inhibitor active against PKC, 10 µM)
reversed the inhibitory effect of EGF (100 ng/ml) on
carbachol-stimulated chloride secretion. EGF induced the rapid
translocation of PKC from the cytoplasm to the membrane. Wortmannin
(50 µM) and LY294002 (20 nM), which are PI
3-kinase inhibitors that by themselves had no effect on PKC
activity, significantly suppressed PKC translocation activated by
EGF. LY294002 also reversed the inhibitory action of EGF on chloride
secretion. PI (3,4)P2 increased membrane-associated PKC and reduced carbachol-induced 86Rb+ efflux.
Antisense oligonucleotides against PKC decreased PKC mass and
prevented the inhibitory effect of EGF on carbachol-induced 86Rb+ efflux. Thus, the inhibitory effect of
EGF on carbachol-induced chloride secretion involves the activation of
PKC mediated by PI 3-kinase. Our findings contribute to the
understanding of the cellular mechanisms that control chloride secretion.
*
This work was supported by NIDDK, National Institutes of
Health Grant DK28305 (to K. E. B.).The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Recipient of a Predoctoral Fellowship, supported by the
Institutional Training Grant in Digestive Diseases DK07202 while a student in the Biomedical Sciences Ph.D. program of UCSD School of Medicine.
§
Chair of the Biomedical Sciences Ph.D. Program of UCSD School of
Medicine. To whom correspondence should be addressed: Univ. of
California, San Diego Medical Center, Division of Gastroenterology, 8414, 200 West Arbor Dr., San Diego, CA 92103-8414. Tel.: 619-543-3726; Fax: 619-543-6969; E-mail: kbarrett@ucsd.edu.
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