cAMP-independent activation of CFTR Cl channels by the tyrosine kinase inhibitor genistein

Abstract

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 125I efflux and by patch clamping in the cell-attached mode. Both forskolin and genistein stimulated 125I efflux and activated a 9-10 pS anion channel in 3T3-CFTR cells but failed to activate 125I 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 125I efflux to approximately 50% of the maximal rate with forskolin. Genistein did not increase 125I 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 microM. These effects suggest that, in addition to activation by protein kinase A, the CFTR is regulated by a tyrosine kinase-dependent pathway.

Development of Substituted Benzo[c]quinolizinium Compounds as Novel Activators of the Cystic Fibrosis Chloride Channel

“…These chemicals include those which can affect CFTR indirectly by interacting with parts of the cAMP signaling mechanism such as phosphodiesterase inhibitors (5,11) and phosphatase inhibitors (12)(13)(14)(15). Direct activation of CFTR has been postulated when using the tyrosine kinase inhibitor flavonoid drug genistein (16,17), xanthine derivatives (15,18) including the adenosine receptor antagonist CPX (19), and the K ϩ channel activators benzimidazolone compounds NS004 (20,21) and 1-EBIO (21). However, the mode of action and the specificity of these latter activators is still debated.…”

The Therapeutic Potential of Small-molecule Modulators of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl− Channel

“…64,77 In the presence of a maximally effective concentration of the cAMP agonist forskolin (10 μM), genistein (50 μM) robustly enhanced F508del-CFTR activity following its delivery to the cell surface by low-temperature incubation (e.g. Hwang et al 78 ).…”

“…genistein) 64 were identified using conventional assays of CFTR function. However, by far the most successful strategy is high-throughput screening (HTS).…”

cAMP- and Ca2+-independent Activation of Cystic Fibrosis Transmembrane Conductance Regulator Channels by Phenylimidazothiazole Drugs

“…The mechanisms of stimulation by 8-cyclopentyl-1,3-dipropylxanthine and ketoconazole are not understood; however, 3-isobutyl-1-methylxanthine appears to act through several mechanisms, one involving inhibition of the membrane associated phosphatase activity (18). There is evidence that the tyrosine kinase inhibitor genistein also activates CFTR by inhibiting a phosphatase (43).…”