Alternate stimulation of apical CFTR by genistein in epithelia

The cystic fibrosis transmembrane regulator (CFTR) is a Cl- channel regulated by adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase A. A cAMP-independent activation has been recently shown for the protein tyrosine kinase inhibitor genistein in CFTR-transfected NIH/3T3 fibroblasts. We further studied the role of genistein on Cl- secretion in HT-29/B6 and T84 colonic epithelial cells, which express native CFTR in their apical membranes. Transepithelial Cl- secretion was more effectively stimulated in T84 cells when compared with 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 and 9.2 +/- 0.3 pS, respectively. Monolayers in Ussing chambers were basolaterally permeabilized with the pore former cr-toxin, and gradient-driven Cl- current across the apical membrane (I-Cl) was measured. I-Cl was stimulated by serosal (i.e., cytosolic) cAMP (half-maximal stimulatory concentration = 9.8 +/- 1.9 mu M). In the presence of cAMP (>5 mu M), subsequent mucosal, but not serosal, addition of genistein further increased I-Cl by similar to 16%; in the absence of cytosolic cAMP, genistein had no effect on I-Cl The inactive analogue daidzein had no effect. When cAMP agonists were removed in the continued presence of genistein, I-Cl 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 (half maximal inhibitory concentration = 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.