Synergistic activation of guinea-pig cardiac cystic fibrosis transmembrane conductance regulator by the tyrosine kinase inhibitor genistein and cAMP

1. The regulation of cardiac C1^- current (I(C1)) by tyrosine and serine/threonine phosphorylation was examined in guinea-pig and rat ventricular myocytes. The protein tyrosine kinase (PTK) inhibitor genistein (GST) and phosphotyrosine phosphatase (PTP) inhibitor sodium orthovanadate (VO₄) were used to modify tyrosine phosphorylation, whereas forskolin (FSK), cAMP, and other agents were used to modify cytoplasmic cAMP concentration and protein kinase A (PKA) phosphorylation. 2. Low concentrations (0.1 μM) of FSK did not activate the PKA-regulated cystic fibrosis transmembrane regulator (CFTR) I(C1) in guinea-pig ventricular myocytes, but strongly potentiated activation of an I(C1) by 20-100 μM GST. The potentiation did not occur when GST was replaced by PTK-inactive daidzein, and it was strongly inhibited by 1 mM VO₄. 3. Potentiation by 0.1 μM FSK was linked to a small stimulation of the adenylate cyclase-cAMP-PKA pathway. The potentiation was not mimicked by inactive 1,9-dideoxyforskolin, and was inhibited by muscarinic stimulation (ACh) and by a PKA inhibitor. Internal application of a cAMP solution that alone was too weak to activate CFTR I(C1) strongly potentiated the activation of I(C1) by 50 μM GST and occluded potentiation by 0.1 μM FSK. 4. The foregoing suggests that potentiated I(C1) flows through cAMP-dependent CFTR channels. In agreement with this interpretation, GST did not increase I(C1) when CFTR was maximally activated by a high concentration (5 μM) of FSK and okadaic acid, and neither GST nor GST plus FSK activated an I(C1) in CFTR-deficient rat myocytes. The lack of effect in rat myocytes was not due to the absence of functional, channel-relevant PKA and PTK-PTP systems, because (as in guinea-pig myocytes) L-type Ca²⁺ current (I(Ca,L)) was stimulated by FSK and inhibited in a VO₄-reversible manner by GST. 5. The synergistic activation of CFTR by low concentrations of FSK and GST cannot be explained by either a GST-induced elevation of cAMP concentration or inhibition of serine/threonine phosphatase. Rather, it appears to be due to tyrosine dephosphorylation that facilities PKS-mediated phosphorylation of the channels.