Abstract
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 Gs 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.
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Received: 11 September 1998 / Received after revision: 6 January 1999 / Accepted: 12 January 1999
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Shuba, L., McDonald, T. Lack of involvement of G proteins in the activation of cardiac CFTR Cl– current by genistein. Pflügers Arch 437, 796–803 (1999). https://doi.org/10.1007/s004240050848
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DOI: https://doi.org/10.1007/s004240050848