CAMP-INDEPENDENT ACTIVATION OF CFTR CL CHANNELS BY THE TYROSINE KINASE INHIBITOR GENISTEIN

Genistein, a protein tyrosine kinase inhibitor, activates the cystic fibrosis transmembrane conductance regulator (CFTR) in transfected NIH-3T3 fibroblasts that express the CFTR (3T3-CFTR). CFTR activity was assayed by I-125 efflux and by patch clamping in the cell-attached mode. Both forskolin and genistein stimulated I-125 efflux and activated a 9-10 pS anion channel in 3T3-CFTR cells but failed to activate I-125 efflux in mock-transfected NIH-3T3 cells. Genistein, unlike forskolin and 3-isobutyl-1-methylxanthine, did not increase intracellular adenosine 3',5'-cyclic monophosphate (cAMP) above control levels. This demonstrates that genistein-dependent activation does not involve inhibition of phosphodiesterase activity and suggests that stimulation does not involve a direct activation of protein kinase A. Genistein stimulated I-125 efflux to similar to 50% of the maximal rate with forskolin. Genistein did not increase I-125 efflux at saturating forskolin but decreased the concentration of forskolin required for half-maximal stimulation. Orthovanadate (VO4), a phosphotyrosine phosphatase inhibitor, inhibited genistein-induced channel activation with an inhibition constant of approximately 20 mu M. These effects suggest that, in addition to activation by protein kinase A, the CFTR is regulated by a tyrosine kinase-dependent pathway.