Korean J Physiol Pharmacol.  2018 Mar;22(2):215-223. 10.4196/kjpp.2018.22.2.215.

Hydrogen peroxide inhibits Ca²⁺ efflux through plasma membrane Ca²⁺-ATPase in mouse parotid acinar cells

• Kim MJ
• Choi KJ
• Yoon MN
• Oh SH
• Kim DK
• Kim SH
• Park HS

• KMID: 2410111
• DOI: http://doi.org/10.4196/kjpp.2018.22.2.215

Abstract

Intracellular Ca²âº mobilization is closely linked with the initiation of salivary secretion in parotid acinar cells. Reactive oxygen species (ROS) are known to be related to a variety of oxidative stress-induced cellular disorders and believed to be involved in salivary impairments. In this study, we investigated the underlying mechanism of hydrogen peroxide (Hâ‚‚Oâ‚‚) on cytosolic Ca²âº accumulation in mouse parotid acinar cells. Intracellular Ca²âº levels were slowly elevated when 1 mM Hâ‚‚Oâ‚‚ was perfused in the presence of normal extracellular Ca²âº. In a Ca²âº-free medium, 1 mM Hâ‚‚Oâ‚‚ still enhanced the intracellular Ca²âº level. Ca²âº entry tested using manganese quenching technique was not affected by perfusion of 1 mM Hâ‚‚Oâ‚‚. On the other hand, 10 mM Hâ‚‚Oâ‚‚ induced more rapid Ca²âº accumulation and facilitated Ca²âº entry from extracellular fluid. Ca²âº refill into intracellular Ca²âº store and inositol 1,4,5-trisphosphate (1 µM)-induced Ca²âº release from Ca²âº store was not affected by 1 mM Hâ‚‚Oâ‚‚ in permeabilized cells. Ca²âº efflux through plasma membrane Ca²âº-ATPase (PMCA) was markedly blocked by 1 mM Hâ‚‚Oâ‚‚ in thapsigargin-treated intact acinar cells. Antioxidants, either catalase or dithiothreitol, completely protected Hâ‚‚Oâ‚‚-induced Ca²âº accumulation through PMCA inactivation. From the above results, we suggest that excessive production of Hâ‚‚Oâ‚‚ under pathological conditions may lead to cytosolic Ca²âº accumulation and that the primary mechanism of Hâ‚‚Oâ‚‚-induced Ca²âº accumulation is likely to inhibit Ca²âº efflux through PMCA rather than mobilize Ca²âº ions from extracellular medium or intracellular stores in mouse parotid acinar cells.

Keyword

Calcium; Hydrogen peroxide; Parotid acinar cells; Plasma membrane calcium ATPase; Reactive oxygen species

MeSH Terms

Acinar Cells*
Animals
Antioxidants
Calcium
Catalase
Cell Membrane*
Cytosol
Dithiothreitol
Extracellular Fluid
Hand
Hydrogen Peroxide*
Hydrogen*
Inositol 1,4,5-Trisphosphate
Ions
Manganese
Mice*
Perfusion
Plasma Membrane Calcium-Transporting ATPases
Plasma*
Reactive Oxygen Species
Antioxidants
Calcium
Catalase
Dithiothreitol
Hydrogen
Hydrogen Peroxide
Inositol 1,4,5-Trisphosphate
Ions
Manganese
Plasma Membrane Calcium-Transporting ATPases
Reactive Oxygen Species

Figure

• 

  Fig. 1 Hydrogen peroxide (H2O2) produced intracellular Ca2+ accumulation in intact parotid acinar cells. (A) Effects of various concentrations (0.1–10 mM) of H2O2 (filled circles) for 10 min on Ca2+ accumulation in the presence of normal extracellular Ca2+. (B) The peak (open bars) and recovery (filled bars) values of intracellular Ca2+ accumulation after 10 min of the perfusion and the removal of H2O2. The changes of the 340/380 ratio were expressed as means±SE obtained from at least seven separate experiments. The perfusion of H2O2 resulted in slow increases of intracellular Ca2+ concentrations. H2O2 at concentration of 1 mM effectively accumulate intracellular Ca2+ and nearly recovered to baseline after withdraw of H2O2. The sustained Ca2+ increase was still observed at higher concentration over 3 mM of H2O2 even if H2O2 was removed from the perfusate. The relatively rapid Ca2+ accumulation was observed in 10 mM H2O2. Asterisk indicates the recovery value obtained at 10 min after termination of H2O2 treatment is significantly different from the peak value obtained during H2O2 treatment (p<0.05).

• 

  Fig. 2 H2O2 still enhanced Ca2+ accumulation in the absence of extracellular Ca2+ in intact parotid acinar cells. (A) Effects of 1 mM H2O2 on Ca2+ accumulation in the absence (open circles) or the presence (filled circles) of normal extracellular Ca2+. (B) Intracellular Ca2+ accumulation at 300 s and 600 s after perfusion of 1 mM H2O2 in the absence (open bars) or the presence (filled bars) of normal extracellular Ca2+. The values were expressed as means±SE obtained from seven (control) and five (H2O2) experiments. Although a slight difference was detected in initial values of Ca2+ accumulation at 300 s, the entire Ca2+ accumulation at 600 s was no difference. Asterisk indicates the value obtained in Ca2+-free buffer is significantly different from the corresponding value obtained in normal Ca2+ buffer (p<0.05).

• 

  Fig. 3 Effects of H2O2 on Ca2+ entry using Mn2+ quenching test in intact parotid acinar cells. (A) Effects of various concentrations (1–10 mM) of H2O2 for 10 min on normalized Ca2+ entry. (B) Effects of H2O2 on relative Ca2+ entry at the end of experiments. The values were expressed as means±SE obtained from at least six separate experiments. H2O2 at the concentration of 1 mM (open squares) and 3 mM (open triangles) failed to facilitate Ca2+ entry, whereas 10 mM H2O2 (open diamonds) remarkably accelerated Ca2+ entry through plasma membrane. Asterisk indicates the value obtained in H2O2-treated experiments (filled bars) is significantly different from the corresponding value obtained in control experiments (open bar) (p<0.05).

• 

  Fig. 4 H2O2 failed to modulate Ca2+ transport through endoplasmic reticulum (ER) membrane in permeabilized parotid acinar cells. (A)The effect of H2O2 on Ca2+ refill and InsP3-induced Ca2+ release through ER membrane in permeabilized cells. (B) The effect of H2O2 on Ca2+ refill rates into intracellular Ca2+ stores. (C) The effect of H2O2 on InsP3-induced Ca2+ release from Ca2+ stores. The values were expressed as means±SE obtained from five (control) and six (H2O2) experiments. ER Ca2+ stores were loaded with the buffer containing MgCl2, Na2ATP and CaCl2. After Ca2+ loading, MgCl2 was eliminated to SERCA inactivation at 60 s prior to 1 µM InsP3 application (open circles and bars). The same procedure was repeated in H2O2-treated permeabilized cells (filled circles and bars). The perfusion of H2O2 resulted in change neither Ca2+ refill rate nor InsP3-induced Ca2+ release rate through ER membrane in permeabilized cells.

• 

  Fig. 5 H2O2 attenuated Ca2+ efflux in thapsigargin (TG)-treated intact acinar cells. (A) The effect of H2O2 on Ca2+ efflux during the depletion of ER Ca2+ store by TG treatment in Ca2+-free buffer. (B) The effect of H2O2 on the peak time of intracellular Ca2+ elevation. (C) The effect of H2O2 on the relative Ca2+ efflux at the end of experiments. The values were expressed as means±SE obtained from seven (control) and five (H2O2) experiments. When H2O2 was treated with TG (filled circles and bars), the Ca2+ elevation was delayed and Ca2+ efflux was not fully recovered to baseline compared with control values (open circles and bars). Asterisks indicate the value obtained in H2O2-treated experiments is significantly different from the corresponding value obtained in control experiments (p<0.05).

• 

  Fig. 6 H2O2 suppressed Ca2+ efflux through PMCA in intact parotid acinar cells. (A) The effect of H2O2 and antioxidants on Ca2+ efflux through PMCA. (B) The effect of H2O2 and antioxidants on relative Ca2+ efflux at the end of experiments. The values were expressed as means±SE obtained from at least five separate experiments. Ca2+ store were initially depleted with 1 µM TG, and then Ca2+ entry and Ca2+ efflux was fully stimulated by adding and removing extracellular 1.28 mM Ca2+. Application of H2O2 (filled circles and bar) resulted in remarkable inhibition of Ca2+ efflux through PMCA compared with control values (open circles and bar) in TG-treated acinar cells. The antioxidants, 30 µg/ml catalase (filled triangles and gray bar) or 2 mM dithiothreitol (DTT, filled diamonds and gray bar), completely protected these diminished Ca2+ efflux by H2O2 in TG-treated intact acinar cells. Asterisk indicates the value obtained in H2O2-treated experiments is significantly different from the corresponding value obtained in control experiments (p<0.05).

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