On the mechanism of genistein-induced activation of protein kinase A-dependent Cl- conductance in cardiac myocytes.
Genistein, an inhibitor of protein tyrosine kinase (PTK), enhanced the activation of the cardiac isoform of the protein kinase A (PKA)-regulated cystic fibrosis transmembrane conductance regulator (CFTR) Cl- conductance in guinea-pig ventricular cells. We examined the mechanism(s) underlying this excitatory action of genistein by using patch-clamp techniques. The CFTR Cl- conductance, activated by isoproterenol (ISO, 10 nM; [Cl-] 153 mM extracellular, 21 mM intracellular; 36 degrees C), was enhanced by 20 microM genistein. Daidzein, a structural analogue of genistein with little inhibitory action on PTK, also enhanced CFTR Cl- currents. After maximal activation of the Cl- conductance by a cocktail of adenosine 3', 5'-cyclic monophosphate, 3-isobutyl-1-methylxanthine and okadaic acid or vanadate plus forskolin in the pipette, genistein was no longer stimulatory but was rather slightly inhibitory at 100 microM. Direct exposure of myocytes to higher concentrations of genistein (50-100 microM) elicited outwardly rectifying currents with a reversal potential of -47 mV in the absence of ISO. In the presence of 50 microM H-89, a PKA inhibitor, genistein had no effect. Vanadate in the pipette at a concentration (100 microM) inhibiting phosphotyrosine phosphatases alone did not prevent the action of genistein. In contrast, no conductance was activated by tyrphostins B42 or 51 or lavendustin A, other PTK inhibitors. Genistein's stimulation of cardiac CFTR Cl- conductance appears to be independent of the PTK pathway and to be due to its direct interaction with CFTR Cl- channels.
Division of Cardiac Electrophysiology, Department of Cardiovascular Medicine, Kyoto University School of Medicine, Shogoin, Sakyo-ku, Kyoto 606-01, Japan.
K
K
Obayashi
M
M
Horie
T
T
Washizuka
T
T
Nishimoto
S
S
Sasayama
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D002712:Chlorides / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000502:physiology*; D004553:Electric Conductivity; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D019833:Genistein / Q000494:pharmacology*; D006168:Guinea Pigs; D006321:Heart / Q000502:physiology*; D007529:Isoflavones / Q000494:pharmacology; D007545:Isoproterenol / Q000494:pharmacology; D019319:Okadaic Acid / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D016276:Ventricular Function
D000318:Adrenergic beta-Agonists; D002712:Chlorides; D004791:Enzyme Inhibitors; D007529:Isoflavones; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D019319:Okadaic Acid; C004742:daidzein; D019833:Genistein; D000242:Cyclic AMP; D011505:Protein-Tyrosine Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D007545:Isoproterenol; D015056:1-Methyl-3-isobutylxanthine