Activation of cardiac chloride conductance by the tyrosine kinase inhibitor, genistein

Abstract

1 Genistein (GST), an inhibitor of protein tyrosine kinase (PTK), Na3VO4 (VO4), an inhibitor of phosphotyrosine phosphatase (PTPase), and forskolin (FSK), an activator of the cyclic AMP-dependent, cystic fibrosis transmembrane conductance regulator (CFTR) Cl-channel, were applied to guinea-pig ventricular myocytes to probe for a possible role of tyrosine phosphorylation in the regulation of cardiac Cl-channels. 2 Myocytes in the standard whole-cell configuration were pulsed to various potentials and Cl-current (Icl) measured as the difference from control background current. GST (1-500 gM) activated a current that had similar biophysical properties (time-and voltage-independent; Cl--dependent reversal potential and outward rectification) as Ick activated by 5 gM FSK. The EC_0 for activation of Cl-conductance (ga) by GST was approximately 100 JM, and g0 activated by GST (500 gM) was as large as gcj activated by maximally-effective FSK (5 gM). Daidzein, a GST analogue with little effect on PTK, was at least one order less effective than GST. 3 GST responses were rapidly and reversibly inhibited by 0.1-1 mM V04 treatments that had little effect on FSK-activated Icl.4 Niflumic acid (100-200 gM) reversibly depressed GST (100 uM)-activated gci by 55%. 5 GST (50 gM) strongly incremented current in myocytes with cyclic AMP-dependent CFTR IC, already activated by maximally-effective FSK 5 gM. 6 Based on these results, and on evidence of a synergistic interaction between GST and FSK, we conclude that inhibition of tyrosine phosphorylation by GST causes an activation of cardiac CFTR that is not mediated by an elevation of cyclic AMP.

Cardiac Na+–Ca2+ exchanger current induced by tyrphostin tyrosine kinase inhibitors

“…The genistein-induced shifts were almost certainly due to activation of outwardly rectifying I Cl(CFTR) as previously reported (Shuba et al, 1996;Chiang et al, 1997;Shuba & McDonald, 1997), whereas the A23-induced shifts were unrelated to Cl À pathways because they were unaffected by changes in dialysate Cl À concentration. A further distinction between genistein-and A23-induced current was that A23-induced current was strongly inhibited by Cd 2 þ .…”

“…Verapamil (SigmaAldrich) was dissolved in water and diluted in the bathing solution. Aqueous stock solutions (100 mM) of Na 3 VO 4 (orthovanadate) (Fisher Scientific, Nepeon, ON, Canada) were freshly prepared, and the pH was adjusted to B10 (Gordon, 1991;Shuba et al, 1996). Appropriate amounts of stock solution were added to the superfusate just before use, and the pH was readjusted to 7.4 with NaOH.…”

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

“…Genistein (GST) is a selective inhibitor of tyrosine kinase (TK) [2,20] that has been reported to activate cyclic adenosine monophosphate (cAMP)-dependent cystic fibrosis transmembrane conductance regulator (CFTR) Cl -channels in a variety of cells [26,32,40,52], including guinea-pig ventricular myocytes [7,8,41,43]. Some investigators have suggested that GST "directly" activates CFTR [8,12,52], whereas others have concluded that GST opens CFTR by directly or indirectly inhibiting a phosphatase that dephosphorylates and inactivates the channel [27,38,56].…”

Genistein and daidzein prevent low potassium-dependent apoptosis of cerebellar granule cells

“…Indeed, GEN was shown to be either pro-apoptotic or anti-apoptotic depending both on its concentration and the cell system [14,[22][23][24]. Similarly, the GEN analogue daidzein (DZN), which does not inhibit TK [25], can inhibit D-gal-induced apoptosis via the Bcl-2/Bax apoptotic pathway [26] and may be a potential medical candidate for neurodegeneration therapy. DZN exhibits an antioxidant activity [27] and shows other biological properties, including estrogen-like and estrogenin-dependent effects [for refs see 28].…”