Genistein and tyrphostin 47 stimulate CFTR-mediated Cl^- secretion in T84 cell monolayers

The involvement of tyrosine phosphorylation in the regulation of epithelial cell Cl^- secretion is unknown. Therefore, the purpose of these studies was to determine if tyrosine kinase activation was involved in the regulation of Cl^- secretion, using the tyrosine kinase inhibitors, genistein and tyrphostin 47, and human intestinal epithelial cells (T84 cells) as an intestinal Cl^- secretory model. Genistein rapidly but reversibly stimulated sustained apical Cl^- secretion in monolayers of T84 cells without increasing intracellular cyclic nucleotides or Ca²⁺ levels. Tyrphostin 47 also stimulated Cl^- 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 conductance 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 Cl^- secretion involved CFTR. Genistein also acted synergistically with the Ca²⁺- and protein kinase C-dependent acetylcholine analogue, carbachol, to stimulate Cl^- secretion in T84 monolayers. However, the Cl^- secretory response to saturating concentrations of the adenosine 3',5'-cyclic monophosphate (cAMP) agonist, forskolin, or the guanosine 3',5'- cyclic monophosphate (cGMP) agonist, Escherichia coli heat-stable enterotoxin, was not further enhanced by genistein. Although the mechanism of activation of Cl^- secretion is unclear, these data suggest that tyrosine kinase activity limits basal Cl^- secretion in T84 cells and that inhibition of T84 cell tyrosine kinase(s) stimulates apical membrane Cl^- secretion, most likely through activation of the CFTR-Cl^- channel. Moreover, genistein does not itself act through cAMP or cGMP elevation but appears to share a common Cl^- secretory pathway with cyclic nucleotide-dependent agonists, whereas it augments the secretory responses to a Ca²⁺- and protein kinase C-dependent agonist.