title: Lack of involvement of G proteins in the activation of cardiac CFTR Cl- current by genistein.
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 (ICl) 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) (GDPbetaS) and guanosine-5'-0-(3-thiotriphosphate) (GTPgammaS) were used to modify G protein activity, and the efficacy of the treatments determined by examining the activation of ICl by isoproterenol (ISO) and forskolin (FSK), and its inhibition by 1 microM acetylcholine (ACh). GDPbetaS inhibited ISO-activated ICl by 80-90%, but had little effect on ICl activated by different GST regimens (50 microM; 100 microM; 50 microM plus 0.1 microM FSK). GTPgammaS had little effect on the amplitude of ICl activated by 1 microM ISO, whereas it increased the amplitude of the current activated by 50 and 100 microM GST and rendered it insensitive to 1 microM ACh (inhibition of 2+/-2% versus (PTX-sensitive) inhibition of 94+/-3% in control myocytes). Unlike ICl activated by ISO in GTPgammaS-dialysed myocytes, ICl activated by GST deactivated on removal of the drug. GST (50 microM) reversibly increased ICl by nearly 50% in myocytes with Gs selectively activated by 1 microM ISO, and also reversibly increased the ICl that was persistently activated after withdrawal of ISO from GTPgammaS-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 GTPgammaS-dialysed myocytes mediates the potentiated responses to GST.
authors.affiliation: Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada.
authors.forename: L M
authors.initials: LM
authors.lastname: Shuba
authors.forename: T F
authors.initials: TF
authors.lastname: McDonald
mesh_terms: D037361:Adenylate Cyclase Toxin; D000818:Animals; D015220:Calcium Channels / Q000187:drug effects* / Q000502:physiology; D005576:Colforsin / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000502:physiology*; D004553:Electric Conductivity; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000502:physiology*; D019833:Genistein / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006168:Guinea Pigs; D006321:Heart / Q000502:physiology*; D007545:Isoproterenol / Q000494:pharmacology; D037342:Pertussis Toxin; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D013566:Sympathomimetics / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D037361:Adenylate Cyclase Toxin; D015220:Calcium Channels; D004791:Enzyme Inhibitors; D013566:Sympathomimetics; D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006153:Guanosine Diphosphate; D005576:Colforsin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); D019833:Genistein; D037342:Pertussis Toxin; D011505:Protein-Tyrosine Kinases; D019204:GTP-Binding Proteins; D007545:Isoproterenol