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

Abstract

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.