Camp and genistein stimulate hco3 conductance through cftr in human
airway epithelia.
Illek, Beate, James R. Yankaskas, and Terry E. Machen.
University of California at Berkeley, Department of Molecular and
Cell Biology, Division of Cell and Developmental Biology, 231 Life
Science Addition, Berkeley, California 94720-3200, USA, Phone: (412)
648 8963, Fax: (412) 648 8330, E-mail: Beate+@Pitt.EDU or
Machen@garnet.Berkeley.EDU
APStracts 3:0202L, 1996.
We studied the role of the CFTR Cl channel as a HCO3 conductor during
cAMP dependent regulation in human airway epithelial cell lines. HCO3
or Cl currents across the apical membrane were measured in the
presence of a HCO3 or Cl gradient under short-circuit conditions in
intact and [alpha]-toxin-permeabilized monolayers, which allowed
manipulation of the intracellular regulators cAMP and ATP. CFTR as
the current carrier for HCO3 was identified by: 1) stimulation by
cAMP, 2) ATP dependency, 3) blocker sensitivity, 4) stimulation by
genistein and 5) lack of stimulation in CF epithelia bearing mutated
DF508-CFTR. In pulmonary [alpha]-toxin-permeabilized Calu-3
monolayers, cytosolic addition of 100 [mu]M cAMP stimulated apical
HCO3 currents from -9.4+/-1.6 [mu]A/cm2 to -31.1+/-3.9 [mu]A/cm2
(n=18), and apical Cl currents increased from -54.1+/-7.1 [mu]A/cm2
to -203.2+/-15.4 [mu]A/cm2 (n=27). Average relative permselectivity
for HCO3 vs. Cl was 15%. Absence of cytosolic ATP resulted in loss of
cAMP stimulation of HCO3 and Cl currents. Genistein (50 [mu]M), which
has been proposed to inhibit phosphatases controlling apical CFTR, as
well as the alkaline phosphatase inhibitor (-)-p-bromotetramisole (1
mM) further activated cAMP-stimulated HCO3 and Cl currents. Activated
currents remained stimulated upon removal of cAMP suggesting
inhibition of a protein phosphatase by genistein and
bromotetramisole. The Cl channel blockers glibenclamide (300 [mu]M)
and DPC (5 mM), but not DNDS (100 [mu]M) inhibited cAMP- and
genistein-stimulated HCO3 and Cl currents. Blocker effects were
absent in human cystic fibrosis tracheal cells homozygous for the
DF508 mutation of CFTR (CFT1); Cl and HCO3 currents were rescued in
CFT1 cells recombinantly expressing wildtype CFTR. Thus, CFTR
functions as a HCO3 and Cl conductor, and genistein and
bromotetramisole maximize CFTR activity in airway epithelial cells.
Received 15 July 1996; accepted in final form 1 November 1996.
APS Manuscript Number L218-6.
Article publication pending Am. J. Physiol. (Lung Cell. Mol.
Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 31 December 1996