Inhibitory effects of genistein on ATP‐sensitive K + channels in rabbit portal vein smooth muscle

1 Effects on the pinacidil‐induced outward current of inhibitors of tyrosine kinases and phosphatases were investigated by use of a patch‐clamp method in smooth muscle cells of the rabbit portal vein. 2 A specific tyrosine kinase inhibitor, genistein, inhibited the pinacidil‐induced current in a concentration‐dependent manner with an IC 50 of 5.5 μ M . Superfusion of Ca 2+ ‐free solution did not affect this inhibitory effect of genistein. At higher concentrations, genistein inhibited the voltage‐dependent Ba 2+ and K + currents with IC 50 values of >100 μ M and 75 μ M respectively. Tyrphostin B46 (30 μ M ), a tyrosine kinase inhibitor, also inhibited the pinacidil‐induced current by 70 % of the control. 3 Sodium orthovanadate (100 μ M ), an inhibitor of tyrosine phosphatase, slightly but significantly enhanced both the pinacidil‐induced and delayed rectifier K + currents. Daidzein (100 μ M ), an inactive analogue of genistein, did not inhibit these currents. 4 Neither herbimycin A (1 μ M ), lavendustin A (30 μ M ), tyrphostin 23 (10 μ M ), which are also tyrosine kinase inhibitors, nor wortmannin (10 μ M ), a phosphatidylinositol 3‐kinase inhibitor, had an effect on either the pinacidil‐induced or delayed rectifier K + currents. Epidermal growth factor (EGF; 1 μg ml −1 ) did not induce an outward current or enhance the pinacidil‐induced current. 5 Pinacidil alone, in the cell‐attached configuration, or pinacidil with GDP, in the inside‐out configuration, activated a 42 pS channel in the smooth muscle cells of the rabbit portal vein. Genistein (30 μ M ) reduced the channel's open probability without inducing a change in unitary conductance at any holding potential (−30 to +20 mV). 6 In the inside‐out configuration, genistein at 30 μ M did not change the mean channel open time, but reduced the burst duration. At 100 μ M genistein abolished channel opening. The inhibitory potencies with which 30 and 100 μ M genistein acted on the unitary current of the ATP‐sensitive K + channel were similar to those seen in the whole‐cell voltage‐clamp configuration. 7 Although direct inhibitory actions of genistein on the ATP‐sensitive K + channels are not ruled out, our results suggest that a protein tyrosine kinase may play a role in the regulation of ATP‐sensitive K + channel activity in the rabbit portal vein.