Genistein and tyrphostin 47 stimulate cystic fibrosis transmembrane
conductance regulator-mediated chloride secretion in human intestinal
epithelial cell (t84) monolayers.
Sears, Cynthia L., Farifteh Firoozmand, Annika Mellander, Frances G.
Chambers, Irene G. Eromar, Alice G. M. Bot, Bob Scholte, Hugo R. De
Jonge, Mark Donowitz.
Departments of Medicine and Physiology, The Johns Hopkins
University School of Medicine Baltimore, MD 21205-2196; and the
Departments of Biochemistry and Cell Biology, Medical Faculty,
Erasmus University, 3000 DR. Rotterdam, The Netherlands
APStracts 2:0106G, 1995.
The involvement of tyrosine phosphorylation in the regulation of
epithelial cell chloride secretion is unknown. Therefore, the purpose
of these studies was to determine if tyrosine kinase activation was
involved in the regulation of chloride secretion using the tyrosine
kinase inhibitors, genistein and tyrphostin 47, and T84 cells as an
intestinal chloride secretory model. Genistein rapidly, but
reversibly, stimulated sustained apical chloride secretion in
monolayers of T84 cells without increasing intracellular cyclic
nucleotides or calcium levels. Tyrphostin 47 also stimulated chloride
secretion in T84 monolayers, although it was short-lived.
Transfection experiments in 3T3 fibroblasts and IEC-6 intestinal
cells utilizing wild type cystic fibrosis transmembrane regulator
(CFTR) showed that genistein and tyrphostin 47 stimulated 125I efflux
only in CFTR-transfected cells and not in CFTR negative cells. Thus,
genistein and tyrphostin 47-stimulated chloride secretion involved
CFTR. Genistein also acted synergistically with the calcium/protein
kinase C-dependent acetylcholine analogue, carbachol, to stimulate
chloride secretion in T84 monolayers. However, the chloride secretory
response to saturating concentrations of the cyclic AMP agonist,
forskolin, or the cyclic GMP agonist, Escherichia coli heat-stable
toxin (STa), was not further enhanced by genistein. Although the
mechanism of activation of chloride secretion is unclear, these data
suggest that tyrosine kinase activity limits basal chloride secretion
in T84 cells and that inhibition of T84 cell tyrosine kinase(s)
stimulates apical membrane chloride secretion, most likely through
activation of the CFTR-Cl- channel. Moreover, genistein does not act
itself through cyclic AMP or cyclic GMP elevation but appears to
share a common chloride secretory pathway with cyclic nucleotide
-dependent agonists whereas it augments the secretory responses to a
calcium/protein kinase C-dependent agonist.
Received 18 January 1995; accepted in final form 22 May 1995.
APS Manuscript Number G21-5.
Article publication pending Am. J. Physiol. (Gastrointest. Liver
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 30 May 1995.