Free Radical-Mediated Tolbutamide Desensitization of K+ATP Channels in Rat Pancreatic β-cells

To study the effects of hydroxyl radicals on the sensitivity of the ATP-sensitive K+ (K+ATP) channel to tolbutamide, we used patch clamp and microfluorometric techniques in pancreatic β-cells isolated from rats. In cell-attached membrane patches, exposure of the cells to 0.3mM H2O2 increased the probability of opening of K+ATP channels in the presence of 2.8mM glucose. Tolbutamide dose-dependently inhibited the K+ATP channel with half-maximal inhibition (IC50) at 0.8μM before and immediately after exposure to H2O2. After prolonged exposure (>20min) to H2O2, the IC50 was increased to 15μM. The presence of both ATP and ADP at concentrations ranging from 0.01 to 0.1mM in the inside-out bath solution significantly enhanced the inhibition of the channels by 10μM tolbutamide. Addition of 0.3mM H2O2 induced a transient minute increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) within 10min, followed by a sustained pronounced increase in [Ca2+]i. After more than 20min of exposure of cells to 0.3mM H2O2, [Ca2+]i was increased to above 2μM. Treatment of the cytoplasmic face of inside-out membrane patches with 1μM Ca2+ attenuated the tolbutamide-sensitivity of the K+ATP channel, but not the ATP-sensitivity of the channel. These findings indicate that H2O2 reduces tolbutamide sensitivity by inducing a sustained increase in [Ca2+]i.