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J. Biol. Chem., Vol. 276, Issue 49, 46436-46444, December 7, 2001
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From the Department of Internal Medicine, University of Michigan
Medical Center, Ann Arbor, Michigan 48109
Epidermal growth factor (EGF)
stimulates gastric acid secretion and
H+/K+-ATPase
Functional Role of Protein Kinase B/Akt in Gastric
Acid Secretion*
,
-subunit gene
expression. Because EGF activates the serine-threonine protein kinase
Akt, we explored the role of Akt in gastric acid secretion. Akt
phosphorylation and activation were measured by kinase assays and by
Western blots with an anti-phospho-Akt antibody, using lysates of
purified (>95%) canine gastric parietal cells in primary culture. EGF
induced Akt phosphorylation and activation, whereas carbachol had no
effect. LY294002, an inhibitor of phosphoinositide 3-kinase, completely
blocked EGF induction of Akt phosphorylation, whereas the MEK1
inhibitor PD98059 and the protein kinase C inhibitor GF109203X
had no effect. We examined the role of Akt in
H+/K+-ATPase gene expression by Northern
blotting using a canine H+/K+-ATPase
-subunit cDNA probe. The parietal cells were transduced with a
multiplicity of infection of 100 of the adenoviral vector Ad.Myr-Akt,
which overexpresses a constitutively active Akt gene, or with the
control vector Ad.CMV-
-gal, which expresses
-galactosidase. Ad.Myr-Akt induced
H+/K+-ATPase -subunit gene expression
3-fold, whereas it failed to stimulate the gene cyclooxygenase-2, which
was potently induced by carbachol in the same parietal cells.
Ad.Myr-Akt induced aminopyrine uptake 4-fold, and it potentiated the
stimulatory action of carbachol 3-fold. In contrast, Ad.Myr-Akt failed
to induce changes in either parietal cell actin content, measured by
Western blots with an anti-actin antibody or in the organization of the
actin cellular cytoskeleton, visualized by fluorescein phalloidin
staining and confocal microscopy. Transduction of the parietal cells
with a multiplicity of infection of 100 of the adenoviral vector
Ad.dom.neg.Akt, which overexpresses an inhibitor of Akt, blocked the
stimulatory effect of EGF on both aminopyrine uptake and
H+/K+-ATPase production, measured by Western
blots with an anti-H+/K+-ATPase -subunit
antibody. Thus, EGF induces a cascade of events in the parietal cells
that results in the activation of Akt. The functional role of Akt
appears to be stimulation of gastric acid secretion through induction
of H+/K+-ATPase expression.
*
This work was supported by NIDDK, National Institutes of
Health Grant RO1-DK-058312 (to A. T.), as well as funds from
National Institutes of Health Grant P30DK34933 to the University of
Michigan Gastrointestinal Peptide Research Center.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 an American Gastroenterological Association Industry
Research Scholar Award, a Clinical Investigator Award from the National
Institutes of Health (National Institutes of Health Grant K08DK02336),
and a grant from the Charles E. Culpeper Foundation Health Program. To
whom correspondence should be addressed: 6520 MSRB I, Ann Arbor, MI
48109-0682. Tel.: 734-647-2942; Fax: 734-763-2535; E-mail:
atodisco@umich.edu.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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