Lack of involvement of G proteins in the activation of cardiac CFTR Cl^- current by genistein

The involvement of guanine nucleotide-binding proteins (G proteins) in the activation of cardiac adenosine 3',5'-cyclic monophosphate (cAMP)- dependent cystic fibrosis transmembrane conductance regulator (CFTR) Cl^- current (I(Cl)) by the tyrosine kinase inhibitor genistein (GST) was investigated in guinea-pig ventricular myocytes. Pertussis toxin (PTX) and intracellular application of 1 mM non-hydrolysable guanosine-5'-0(2- thiodiphosphate) (GDPβS) and guanosine-5'-0-(3-thiotriphosphate) (GTPγS) were used to modify G protein activity, and the efficacy of the treatments determined by examining the activation of I(Cl) by isoproterenol (ISO) and forskolin (FSK), and its inhibition by 1 μM acetylcholine (ACh). GDPβS inhibited ISO-activated I(Cl) by 80-90%, but had little effect on I(Cl) activated by different GST regimens (50 μM; 100 μM; 50 μM plus 0.1 μM FSK). GTPγS had little effect on the amplitude of I(Cl) activated by 1 μM ISO, whereas it increased the amplitude of the current activated by 50 and 100 μM GST and rendered it insensitive to 1 μM ACh (inhibition of 2±2% versus (PTX-sensitive) inhibition of 94±3% in control myocytes). Unlike I(Cl) activated by ISO in GTPγS-dialysed myocytes, I(Cl) activated by GST deactivated on removal of the drug. GST (50 μM) reversibly increased I(Cl) by nearly 50% in myocytes with G(s) selectively activated by 1 μM ISO, and also reversibly increased the I(Cl) that was persistently activated after withdrawal of ISO from GTPγS-dialysed myocytes. These results indicate that G proteins are not involved in the pathway between GST binding and CFTR opening, and suggest that enhanced adenylate cyclase activity in GTPγS- dialysed myocytes mediates the potentiated responses to GST.