Free radical-mediated tolbutamide desensitization of K+ATP channels in rat pancreatic beta-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 beta-cells isolated from rats. cell-attached membrane patches, exposure of the cells to 0.3 mM H2O2 increased the probability of opening of K+ATP channels in the presence of 2.8 mM glucose. Tolbutamide dose-dependently inhibited the K+ATP channel with half-maximal inhibition (IC50) at 0.8 microM before and immediately after exposure to H2O2. After prolonged exposure (>20 min) to H2O2, the IC50 was increased to 15 microM. The presence of both ATP and ADP at concentrations ranging from 0.01 to 0.1 mM in the inside-out bath solution significantly enhanced the inhibition of the channels by 10 microM tolbutamide. Addition of 0.3 mM H2O2 induced a transient minute increase in the cytoplasmic Ca2+ concentration ([Ca2+]i) within 10 min, followed by a sustained pronounced increase in [Ca2]i. After more than 20 min of exposure of cells to 0.3mM H2O2, [Ca2]i was increased to above 2 microM. Treatment of the cytoplasmic face of inside-out membrane patches with 1 microM 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.
Koriyama
N
N
First Department of Internal Medicine, Faculty of Medicine, Kagoshima University, Japan.
Kakei
M
M
Nakazaki
M
M
Yaekura
K
K
Hashiguchi
H
H
Aso
K
K
Fukudome
M
M
Yada
T
T
Ito
S
S
Tei
C
C
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D004361:Drug Tolerance; D004553:Electric Conductivity; D005609:Free Radicals; D006861:Hydrogen Peroxide / Q000008:administration & dosage / Q000494:pharmacology; D007004:Hypoglycemic Agents / Q000008:administration & dosage / Q000494:pharmacology*; D007515:Islets of Langerhans / Q000502:physiology*; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000187:drug effects* / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D014044:Tolbutamide / Q000008:administration & dosage / Q000494:pharmacology*
D005609:Free Radicals; D007004:Hypoglycemic Agents; D015221:Potassium Channels; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D014044:Tolbutamide; D006861:Hydrogen Peroxide