CAMP AND GENISTEIN STIMULATE HCO3- CONDUCTANCE THROUGH CFTR IN HUMAN AIRWAY EPITHELIA

We studied the role of the cystic fibrosis transmembrane conductance r egulator (CFTR) Cl- channel as an HCO3- conductor during adenosine 3', 5'-cyclic monophosphate (cAMP)-dependent regulation in human airway ep ithelial cell lines. HCO3- or Cl- currents across the apical membrane were measured in the presence of an HCO3- or Cl- gradient under short- circuit conditions in intact and alpha-toxin-permeabilized monolayers, which allowed manipulation of the intracellular regulators cAMP and A TP. CFTR as the current carrier for HCO3- was identified by 1) stimula tion by cAMP, 2)ATP dependence, 3) blocker sensitivity, 4) stimulation by genistein, and 5) lack of stimulation in CF epithelia bearing muta ted Delta F508 CFTR. In pulmonary alpha-toxin-permeabilized Calu-3 mon olayers, cytosolic addition of 100 mu M cAMP stimulated apical HCO3- c urrents from -9.4 +/- 1.6 to -31.1 +/- 3.9 mu A/cm(2) (n = 18), and ap ical Cl- currents increased from -54.1 +/- 7.1 to -203.2 +/- 15.4 mu A /cm(2) (n = 27). Average relative permselectivity for HCO3- vs. Cl- wa s similar to 15%. Absence of cytosolic ATP resulted in loss of cAMP st imulation of HCO3- and Cl- currents. Genistein (50 mu M), which has be en proposed to inhibit phosphatases controlling apical CFTR, as well a s the alkaline phosphatase inhibitor (-)-p-bromotetramisole (1 mM) fur ther activated cAMP-stimulated HCO3- and Cl- currents. Activated curre nts remained stimulated on removal of cAMP, suggesting inhibition of a protein phosphatase by genistein and bromotetramisole. The Cl- channe l blockers glibenclamide (300 mu M) and N-phenylanthranilic acid (5 mM ), but not 4,4'-dinitro-2,2'-stilbenedisulfonic acid (100 mu M), inhib ited cAMP- and genistein-stimulated HCO3- and Cl- currents. Blocker ef fects were absent in human CF tracheal cells homozygous for the Delta F508 mutation of CFTR (CFT1); Cl- and HCO3- currents were rescued in C FT1 cells recombinantly expressing wild-type CFTR. Thus CFTR functions as a HCO3- and Cl- conductor, and genistein and bromotetramisole maxi mize CFTR activity in airway epithelial cells.