Korean J Physiol Pharmacol.  2017 Mar;21(2):233-239. 10.4196/kjpp.2017.21.2.233.

Hydrogen peroxide attenuates refilling of intracellular calcium store in mouse pancreatic acinar cells

Affiliations
  • 1Department of Physiology, College of Medicine, Konyang University, Daejeon 35365, Korea. hspark@konyang.ac.kr
  • 2Myunggok Medical Research Institute, Konyang University, Daejeon 35365, Korea.

Abstract

Intracellular calcium (Ca²âº) oscillation is an initial event in digestive enzyme secretion of pancreatic acinar cells. Reactive oxygen species are known to be associated with a variety of oxidative stress-induced cellular disorders including pancreatitis. In this study, we investigated the effect of hydrogen peroxide (Hâ‚‚Oâ‚‚) on intracellular Ca²âº accumulation in mouse pancreatic acinar cells. Perfusion of Hâ‚‚Oâ‚‚ at 300 µM resulted in additional elevation of intracellular Ca²âº levels and termination of oscillatory Ca²âº signals induced by carbamylcholine (CCh) in the presence of normal extracellular Ca²âº. Antioxidants, catalase or DTT, completely prevented Hâ‚‚Oâ‚‚-induced additional Ca²âº increase and termination of Ca²âº oscillation. In Ca²âº-free medium, Hâ‚‚Oâ‚‚ still enhanced CCh-induced intracellular Ca²âº levels and thapsigargin (TG) mimicked Hâ‚‚Oâ‚‚-induced cytosolic Ca²âº increase. Furthermore, Hâ‚‚Oâ‚‚-induced elevation of intracellular Ca²âº levels was abolished under sarco/endoplasmic reticulum Ca²âº ATPase-inactivated condition by TG pretreatment with CCh. Hâ‚‚Oâ‚‚ at 300 µM failed to affect store-operated Ca²âº entry or Ca²âº extrusion through plasma membrane. Additionally, ruthenium red, a mitochondrial Ca²âº uniporter blocker, failed to attenuate Hâ‚‚Oâ‚‚-induced intracellular Ca²âº elevation. These results provide evidence that excessive generation of Hâ‚‚Oâ‚‚ in pathological conditions could accumulate intracellular Ca²âº by attenuating refilling of internal Ca²âº stores rather than by inhibiting Ca²âº extrusion to extracellular fluid or enhancing Ca²âº mobilization from extracellular medium in mouse pancreatic acinar cells.

Keyword

Hydrogen peroxide; Intracellular Ca²⁺ stores; Pancreatic acinar cells; Reactive oxygen species; Sarcoplasmic reticulum Ca²⁺ ATPase

MeSH Terms

Acinar Cells*
Animals
Antioxidants
Calcium*
Carbachol
Catalase
Cell Membrane
Cytosol
Extracellular Fluid
Hydrogen Peroxide*
Hydrogen*
Ion Transport
Mice*
Pancreatitis
Perfusion
Reactive Oxygen Species
Reticulum
Ruthenium Red
Thapsigargin
Antioxidants
Calcium
Carbachol
Catalase
Hydrogen
Hydrogen Peroxide
Reactive Oxygen Species
Ruthenium Red
Thapsigargin

Figure

  • Fig. 1 Effects of hydrogen peroxide (H2O2) and antioxidants on CCh-induced intracellular Ca2+ oscillation in intact pancreatic acinar cells.(A) Representative trace showing the effect of H2O2 on CCh-induced Ca2+ oscillation. (B, C) Representative traces showing the effects of antioxidants (30 µg/ml of catalase and 2 mM of DTT) on H2O2-induced intracellular Ca2+ changes. Oscillatory Ca2+ signals were induced by perfusion with 500 nM of CCh in HEPES buffer containing normal extracellular Ca2+. H2O2 at 300 µM was perfused for 5 minutes. All data were obtained from at least five separate experiments (71~98 cells) and expressed as changes of 340/380 ratio. The perfusion of H2O2 resulted in an elevation of intracellular Ca2+ concentration and a termination of Ca2+ oscillation. Antioxidants completely prevent H2O2-induced Ca2+ accumulation.

  • Fig. 2 H2O2 does not affect Ca2+ entry or Ca2+ extrusion in TG-treated pancreatic acinar cells.(A) Representative trace showing the effect of SERCA inactivation using TG on Ca2+ entry from extracellular medium and Ca2+ extrusion to extracellular medium. To deplete intracellular Ca2+ stores, TG at 1 µM was treated in Ca2+-free medium. After depletion of intracellular Ca2+ stores, 1.28 mM of Ca2+ was added and removed to activate Ca2+ entry and Ca2+ extrusion, respectively. (B, C) Effects of H2O2 on normalized Ca2+ entry and Ca2+ entry rate in TGtreated pancreatic acinar cells. Values are expressed as means±SEM obtained from six separate experiments (76 cells). (D, E) Effects of H2O2 on normalized Ca2+ extrusion and Ca2+ extrusion rate in TG-treated cells. H2O2 at 300 µM did not modify Ca2+ entry or Ca2+ extrusion through plasma membrane in TG-treated pancreatic acinar cells.

  • Fig. 3 Effects of H2O2 and TG on CCh-induced intracellular Ca2+ response in Ca2+-free medium.(A) Representative trace showing CCh-induced intracellular Ca2+ response in Ca2+-free medium. (B) H2O2 -induced additional elevation of intracellular Ca2+ levels. (C) TG mimicked the additional elevation of intracellular Ca2+ levels. (D) Pretreatment of TG with CCh completely abolished H2O2-induced additional elevation of intracellular Ca2+ levels. All data were obtained from at least five separate experiments (74~103 cells). Perfusion of H2O2 at 300 µM resulted in additional elevation of intracellular Ca2+ levels, which was mimicked by 1 µM of TG perfusion in Ca2+-free medium. H2O2-induced additional increase of Ca2+ was completely abolished by inactivation of SERCA with TG pretreatment.

  • Fig. 4 Effect of ruthenium red on H2O2-induced intracellular Ca2+ response in Ca2+-free medium.(A) Representative trace showing the effect of ruthenium red on CCh-induced intracellular Ca2+ response in Ca2+-free medium. (B) Pretreatment of ruthenium red with CCh failed to attenuate H2O2-induced additional elevation of intracellular Ca2+ levels. All data were obtained from six and seven separate experiments (70 and 81 cells). Perfusion of ruthenium red at 50 µM, a mitochondrial Ca2+ uniporter inhibitor, did not mimic H2O2-induced additional elevation of intracellular Ca2+ levels. After pretreatment of ruthenium red with CCh, H2O2 still elevated intracellular Ca2+ concentration.


Cited by  1 articles

Hydrogen peroxide inhibits Ca2+ efflux through plasma membrane Ca2+-ATPase in mouse parotid acinar cells
Min Jae Kim, Kyung Jin Choi, Mi Na Yoon, Sang Hwan Oh, Dong Kwan Kim, Se Hoon Kim, Hyung Seo Park
Korean J Physiol Pharmacol. 2018;22(2):215-223.    doi: 10.4196/kjpp.2018.22.2.215.


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