Diverse effects of hydrogen peroxide on cytosolic Ca2+ homeostasis in rat pancreatic beta-cells.
Oxygen-free radicals are thought to be a major cause of beta-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H2O2 on cytosolic Ca2+ concentration ([Ca2+]i) and the activity of ATP-sensitive potassium (K+ATP) channels in isolated rat pancreatic beta-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H2O2 in the presence of 2.8 mM glucose increased [Ca2+]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 [Ca2+]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 Ca2+, 0.1 mM H2O2 increased [Ca2+]i from 88.8+/-7.2 nM to 134.6+/-8.3 nM. Magnitude of [Ca2+]i increase induced by 0.1 mM H2O2 was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ pump (45.8+/-4.9 nM vs 15.0+/-4.8 nM). Small increase in [Ca2+]i in response to an increase of external Ca2+ 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 Ca2+ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic beta-cells.
Nakazaki
M
M
First Department of Internal Medicine, Faculty of Medicine, Kagoshima University, Japan.
Kakei
M
M
Yaekura
K
K
Koriyama
N
N
Morimitsu
S
S
Ichinari
K
K
Yada
T
T
Tei
C
C
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability; D002614:Chelating Agents / Q000378:metabolism; D003592:Cytophotometry; D003600:Cytosol / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016257:Fura-2 / Q000378:metabolism; D006706:Homeostasis; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007515:Islets of Langerhans / Q000187:drug effects* / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D019284:Thapsigargin / Q000494:pharmacology
D002614:Chelating Agents; D004791:Enzyme Inhibitors; D015221:Potassium Channels; D009249:NADP; D019284:Thapsigargin; D006861:Hydrogen Peroxide; D002118:Calcium; D016257:Fura-2