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

摘要

References(31) 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.