Alternate stimulation of apical cftr by genistein in epithelia.
Illek, Beate, Horst Fischer, and Terry E. Machen.
University of California at Berkeley, Department of Molecular and
Cell Biology, Division of Cell and Developmental Biology, 235 Life
Science Addition, Berkeley, California 94720-3200, USA, Phone: (510)
642 4380/642 2983, Fax: (510) 643 6791, E-mail:
Fisch@UCLink.Berkeley.EDU or Terry_Machen@MailLink.Berkeley.EDU
APStracts 2:0281C, 1995.
The cystic fibrosis transmembrane regulator (CFTR) is a Cl- channel
regulated by cAMP-dependent protein kinase A (PKA). A cAMP
-independent activation has been recently shown for the protein
tyrosine kinase (PTK) inhibitor genistein in CFTR-transfected NIH-3T3
fibroblasts. We further studied the role of genistein on Cl-
secretion in HT-29/B6 and T-84 colonic epithelial cells, which
express native CFTR in their apical membranes. Transepithelial Cl
secretion was more effectively stimulated in T-84 cells when compared
to HT-29/B6 cells by mucosal perfusion with 50 [mu]M genistein.
Genistein, like the cAMP agonist forskolin stimulated CFTR activity
in cell-attached patches of single cells with similar slope
conductances of 8.5+/-0.5 pS and 9.2+/-0.3 pS, respectively.
Monolayers in Ussing chambers were basolaterally permeabilized with
the pore former [alpha]-toxin, and gradient-driven Cl- current across
the apical membrane (ICl) was measured. ICl was stimulated by serosal
(i.e. cytosolic) cAMP (K1/2=9.8+/-1.9 [mu]M). In the presence of cAMP
(>5 [mu]M), subsequent mucosal, but not serosal, addition of
genistein further increased ICl by 16%; in the absence of cytosolic
cAMP, genistein had no effect on ICl. The inactive analog daidzein
had no effect. When cAMP agonists were removed in the continued
presence of genistein, Cl- currents remained elevated in both
permeabilized and intact monolayers as well as in cell-attached
patches of single cells. In addition, genistein blocked K+ currents
across the basolateral membrane in apically amphotericin B
-permeabilized monolayers (KI=44.2+/-8.1 [mu]M). Therefore, in intact
epithelia the overall secretory response to genistein is composed of
stimulatory effects on the apical CFTR and inhibitory effects on the
basolateral K+ conductance. We propose that genistein blocks a
phosphatase, which regulates CFTR during cAMP-dependent stimulation.
Received 1 February 1995; accepted in final form 11 July 1995.
APS Manuscript Number C62-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 30 July 1995.