Activation of cardiac chloride conductance by the tyrosine kinase inhibitor, genistein

1. Genistein (GST), an inhibitor of protein tyrosine kinase (PTK), Na₃VO₄ (VO₄), an inhibitor of phosphotyrosine phosphatase (PTPase), and forskolin (FSK), an activator of the cyclic AMP-dependent, cystic fibrosis transmembrane conductance regulator (CFTR) Cl^- channel, were applied to guinea-pig ventricular myocytes to probe for a possible role of tyrosine phosphorylation in the regulation of cardiac Cl^- channels. 2. Myocytes in the standard whole-cell configuration were pulsed to various potentials and Cl^- current (I(Cl)) measured as the difference from control background current. GST (1-500 μM) activated a current that had similar biophysical properties (time- and voltage-independent; Cl^--dependent reversal potential and outward rectification) as I(Cl) activated by 5 μM FSK. The EC₅₀ for activation of Cl^- conductance (g(Cl)) by GST was approximately 100 μM, and g(Cl) activated by GST (500 μM) was as large as g(Cl) activated by maximally-effective FSK (5 μM). Daidzein, a GST analogue with little effect on PTK, was at least one order less effective than GST. 3. GST responses were rapidly and reversibly inhibited by 0.1-1 mM VO₄ treatments that had little effect on FSK-activated I(Cl). 4. Niflumic acid (100-200 μM) reversibly depressed GST (100 μM)-activated g(Cl) by 55%. 5. GST (50 μM) strongly incremented current in myocytes with cyclic AMP-dependent CFTR I(Cl) already activated by maximally-effective FSK 5 μM. 6. Based on these results, and on evidence of a synergistic interaction between GST and FSK, we conclude that inhibition of tyrosine phosphorylation by GST causes an activation of cardiac CFTR that is not mediated by an elevation of cyclic AMP.