Diverse Effects of Hydrogen Peroxide on Cytosolic Ca2+ Homeostasis in Rat Pancreatic .BETA.-cells.

Abstract

ABSTRACT. Oxygen-free radicals are thought to be a major cause of b-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H2O2 on cytosolic Ca 2+ concentration ([Ca 2+ ]i) and the activity of ATP-sensitive potassium (K + ATP) channels in isolated rat pancreatic b-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H2O2 in the presence of 2.8 mM glucose increased [Ca 2+ ]i from 114.3±15.4 nM to 531.1±71.9 nM (n=6) and also increased frequency of K + ATP channel openings. The intensity of NAD(P)H autofluorescence was conversely reduced, suggesting that H2O2 inhibited the cellular metabolism. These three types of cellular parameters were reversed to the control level on washout of H2O2, followed by a transient increase in [Ca 2+ ]i, the transient inhibition of K + ATP channels associated with action currents and increase of the NAD(P)H intensity with an overshoot. In the absence of external Ca 2+ , 0.1 mM H2O2 increased [Ca 2+ ]i from 88.8±7.2 nM to 134.6±8.3 nM. Magnitude of [Ca 2+ ]i increase induced by 0.1 mM H2O2 was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca 2+ pump (45.8±4.9 nM vs 15.0±4.8 nM). Small increase in [Ca 2+ ]i in response to an increase of external Ca 2+ from zero to 2 mM was further facilitated by 0.1 mM H2O2 (330.5±122.7 nM). We concluded that H2O2 not only activates K + ATP channels in association with metabolic inhibition, but also increases partly the Ca 2+ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic b-cells.

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

“…We demonstrated that the modest increase in [Ca2+]i observed after H202 treatment (the first phase) is due to an increased permeability of the thapsigargin-sensitive intracellular Ca2+ stores [24]. The second phase of [Ca2+]i elevation has been suggested to be due to the in- creased Ca2+ permeability of the plasma membrane [24], or alternatively it has been speculated that it is induced by an increased influx of Ca2+ through the non-selective cation channel activated by H2O2 treatment [16] . Our results revealed a remarkable increase in [Ca2+]; after 20 min of exposure to 0.3 mM H2O2 (Fig.…”

“…The first-phase increase in [Ca2+]; is induced by the reduction of cytosolic ATP levels caused by mitochondrial dysfunction after the H202 treatment and the failure of Ca2+-pumping into the endoplasmic reticulum [17]. We demonstrated that the modest increase in [Ca2+]i observed after H202 treatment (the first phase) is due to an increased permeability of the thapsigargin-sensitive intracellular Ca2+ stores [24]. The second phase of [Ca2+]i elevation has been suggested to be due to the in- creased Ca2+ permeability of the plasma membrane [24], or alternatively it has been speculated that it is induced by an increased influx of Ca2+ through the non-selective cation channel activated by H2O2 treatment [16] .…”

Protection of rat pancreatic islets by potassium channel openers against alloxan, sodium nitroprusside and interleukin-1β mediated suppression—possible involvement of the mitochondrial membrane potential

“…In studies on rat beta cells using hydrogen peroxide it was shown that hydrogen peroxide induced rapid increases in Ca 2+ depending on mobilzation of Ca 2+ both from intracellular and extracellular compartments. This was accompanied by an increased frequency of K ATP channel openings, an effect interpreted to depend on a metabolic inhibition induced by the free radical and not a direct effect on the K ATP channels of the beta cells [13,14]. In another study aqueous NO was applied to rat islet cells which led to a rapid and transient increase in intracellular Ca 2+ concentrations probably via a mobilization of Ca 2+ from the endoplasmic reticulum [15].…”

Obligatory Role of Early Ca²⁺ Responses in H₂O₂-Induced β-Cell Apoptosis

“…16) In our previous study in INS-1 β-cells, we showed that H 2 O 2 -induced apoptosis was abolished by 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester (BAPTA/AM), a chelator of intracellular Ca 2+ ; by 2-aminoethoxydiphenylborate (2-APB), a blocker of inositol 1,4,5-trisphosphate (IP 3 ) receptors and cation channels, including SOCs; and by xestospongin D, a blocker of IP 3 receptors and was partially blocked by SKF-96365, a blocker of nonselective cation channels, including SOCs. 17 ), BAPTA/AM, fura-PE3-acetoxymethyl ester (fura-PE3/AM), cremophor EL, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), and the goat anti-mouse immunoglobulin G (IgG) horseradish peroxidase (HRP) antibody were from Sigma-Aldrich (St. Louis, MO, U.S.A.).…”