GENISTEIN AND TYRPHOSTIN 47 STIMULATE CFTR-MEDIATED CL- SECRETION IN T84 CELL MONOLAYERS

The involvement of tyrosine phosphorylation in the regulation of epith elial cell Cl- secretion is unknown. Therefore, the purpose of these s tudies 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 (T 84 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 Ca2levels. Tyrphostin 47 also stimulated Cl- secretion in T84 monolayers, although it was short-lived. Transfection experiments in 3T3 fibrobla sts and IEC-B intestinal cells utilizing wild-type cystic fibrosis tra nsmembrane conductance regulator (CFTR) showed that genistein and tyrp hostin 47 stimulated I-125 efflux only in CFTR-transfected cells and n ot in CFTR-negative cells. Thus genistein- and tyrphostin 47-stimulate d Cl- secretion involved CFTR. Genistein also acted synergistically wi th the Ca2+- and protein kinase C-dependent acetylcholine analogue, ca rbachol, 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 coil heat-stable enterotoxin, was not further enhanced by genistein. Although the mecha nism of activation of Cl- secretion is unclear, these data suggest tha t tyrosine kinase activity limits basal Cl- secretion in T84 cells and that inhibition of T84 cell tyrosine kinase(s) stimulates apical memb rane Cl- secretion, most likely through activation of the CFTR-Cl- cha nnel. Moreover, genistein does not itself act through cAMP or cGMP ele vation but appears to share a common Cl- secretory pathway with cyclic nucleotide-dependent agonists, whereas it augments the secretory resp onses to a Ca2+- and protein kinase C-dependent agonist.