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

doi:10.4196/kjpp.2018.22.2.215

Journal List > Korean J Physiol Pharmacol > v.22(2) > 1026241

Go to TopGo to Top Go to BottomGo to Bottom

TOOLS

Kim, Choi, Yoon, Oh, Kim, Kim, and Park: Hydrogen peroxide inhibits Ca2+ efflux through plasma membrane Ca2+-ATPase in mouse parotid acinar cells

Original Article

The Korean Journal of Physiology & Pharmacology 2018; 22(2): 215-223.

Published online: 23 February 2018

DOI: https://doi.org/10.4196/kjpp.2018.22.2.215

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

Min Jae Kim¹, Kyung Jin Choi¹, Mi Na Yoon¹, Sang Hwan Oh², Dong Kwan Kim¹, Se Hoon Kim¹, Hyung Seo Park^1,³

¹Department of Physiology, College of Medicine, Konyang University, Daejeon 35365, Korea.

²Department of Dental Hygiene, College of Medical Science, Konyang University, Daejeon 35365, Korea.

³Myunggok Medical Research Institute, Konyang University, Daejeon 35365, Korea.

Correspondence: Hyung Seo Park. hspark@konyang.ac.kr

Received 9 November 2017 Revised 29 December 2017 Accepted 4 January 2018

The Korean Physiological Society and The Korean Society of Pharmacology

(open-access):

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Intracellular Ca²⁺ 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 (H₂O₂) on cytosolic Ca²⁺ accumulation in mouse parotid acinar cells. Intracellular Ca²⁺ levels were slowly elevated when 1 mM H₂O₂ was perfused in the presence of normal extracellular Ca²⁺. In a Ca²⁺-free medium, 1 mM H₂O₂ still enhanced the intracellular Ca²⁺ level. Ca²⁺ entry tested using manganese quenching technique was not affected by perfusion of 1 mM H₂O₂. On the other hand, 10 mM H₂O₂ induced more rapid Ca²⁺ accumulation and facilitated Ca²⁺ entry from extracellular fluid. Ca²⁺ refill into intracellular Ca²⁺ store and inositol 1,4,5-trisphosphate (1 µM)-induced Ca²⁺ release from Ca²⁺ store was not affected by 1 mM H₂O₂ in permeabilized cells. Ca²⁺ efflux through plasma membrane Ca²⁺-ATPase (PMCA) was markedly blocked by 1 mM H₂O₂ in thapsigargin-treated intact acinar cells. Antioxidants, either catalase or dithiothreitol, completely protected H₂O₂-induced Ca²⁺ accumulation through PMCA inactivation. From the above results, we suggest that excessive production of H₂O₂ under pathological conditions may lead to cytosolic Ca²⁺ accumulation and that the primary mechanism of H₂O₂-induced Ca²⁺ accumulation is likely to inhibit Ca²⁺ efflux through PMCA rather than mobilize Ca²⁺ ions from extracellular medium or intracellular stores in mouse parotid acinar cells.

Keywords: Calcium, Hydrogen peroxide, Parotid acinar cells, Plasma membrane calcium ATPase, Reactive oxygen species

INTRODUCTION

In the salivary gland, agonist-induced Ca²⁺ mobilization is the initial cellular event for fluid and amylase secretions [1]. Cytosolic Ca²⁺ can be mobilized from both external fluid and internal stores, and then rapidly eliminated to internal store and external space in parotid acinar cells [2]. It has been reported that oxidative stress is involved in salivary dysfunction caused by drugs and irradiation [3]. Reduction of submandibular saliva secretion has been observed in the rat treated with lead acetate, which induces oxidative stress [4]. Irradiation-induced hypofunction of the salivary glands has been believed to be involved with oxidative stress [5]. Furthermore, antioxidants have a protective effect on oxidative stress-induced salivary dysfunctions [6 7]. The impairment of salivary secretion in Sjogren's syndrome, an autoimmune disease which progressively destroys salivary glands, has been known to be generated by oxidative stress and be related to intracellular Ca²⁺ accumulation in parotid acinar cells [8 9].

Although reactive oxygen species (ROS) are normally generated from partial reduction of oxygen during the aerobic respiration, they cause oxidative damage to various biological molecules, thereby disrupting normal cellular function and integrity [10 11]. The superoxide (O₂^·−), hydrogen peroxide (H₂O₂) and hydroxyl radical (OH^·) are considered as primary ROS which interact with ion transporters in surface and internal membrane [12 13]. ROS are controlled by intracellular antioxidant enzymes and free radical scavengers which protect cells from oxidative stress under physiological conditions [14]. However, the imbalanced states by excessive production of ROS or reduction of antioxidants leading to morphological and functional damage of cells [15]. Although hydrogen peroxide-induced oxidative stress are correlated with overloaded intracellular Ca²⁺ levels, the mechanism of sustained Ca²⁺ overload has still been unclear due to cell-to-cell difference in Ca²⁺ transport molecules as shown by the following evidence: 1) The enhanced Ca²⁺ release from intracellular store [16 17 18], 2) The stimulated Ca²⁺ entry from extracellular medium [19 20 21 22] and 3) The attenuated Ca²⁺ efflux by inactivation of plasma membrane Ca²⁺-ATPase (PMCA) or sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) [23 24 25] in various cell types. In the present study, we have therefore characterized the effect of hydrogen peroxide on intracellular Ca²⁺ signals and the underlying mechanism of Ca²⁺ accumulation in mouse parotid acinar cells. Here we report that hydrogen peroxide could accumulate intracellular Ca²⁺ by reducing Ca²⁺ efflux through PMCA, rather than by enhancing Ca²⁺ mobilization from extracellular fluid or intracellular store in pathological conditions.

METHODS

Materials

Collagenase P was purchased from Roche Diagnostics GmbH (Mannheim, Germany), fura-2/AM and magfura-2/AM were from Thermo Fisher Scientific (Waltham, MA, USA), inositol 1,4,5-trisphosphate (InsP₃) was from Enzo Life Sciences (Farmingdale, NY, USA) and thapsigargin (TG) was from Tocris (Avonmouth, BS, UK). All other materials were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Isolation of parotid acinar cells

Small cluster of parotid acinar cells (8–15 cells per experiment) were freshly isolated by collagenase digestion as described previously [26]. Briefly, the parotid gland was removed from male Balb/c mice (8–10 weeks) after CO₂ asphyxiation and cervical dislocation. The tissues were enzymatically digested with collagenase P in HEPES-buffered physiological saline for 30 min following gentle agitation. After isolation, parotid acinar cells were suspended in HEPES-buffered physiological saline containing 137 mM NaCl, 4.7 mM KCl, 0.56 mM MgCl₂, 1 mM Na₂HPO₄, 1.28 mM CaCl₂, 10 mM HEPES and 5.5 mM glucose (PH 7.4) until ready for use. To ensure examination in a Ca²⁺-free condition, HEPES-buffered physiological saline containing no added Ca²⁺ was replaced with 5 mM ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetra acetic acid (EGTA). All experimental procedures were performed in accordance with the Guideline for the Care and Use of Laboratory Animal provided by NIH. All experiments adhered to Konyang University policies regarding the care and use of animals.

Intracellular Ca²⁺ measurements

The isolated parotid acinar cells were loaded with 5 µM Fura-2/AM for 1 h at room temperature for measurements of intracellular Ca²⁺. Fura-2/AM loaded cells were mounted on cover glass in the perfusion chambers. Acinar cells were continuously perfused with HEPES-buffered physiological saline. The perfusion rate (1 ml/min) was controlled using an electronic perfusion system (Warner Instrument, CT, USA). Intracellular Ca²⁺ imaging was conducted using a TILL Photonics imaging system. Fura-2/AM loaded cells were excited alternately with light at 340 nm and 380 nm using a Polychrome V monochrometer (TILL Photonics, CA, USA). Fluorescence images emitted at 510 nm were captured using a Cool-SNAP HQ₂ camera (Photometrics, AZ, USA) attached to inverted microscope (Olympus Corp., Tokyo, JP).

Mn²⁺ quenching study to measurement of Ca²⁺ entry

Fura-2 is known to have a high affinity with Mn²⁺, and thereby fura-2 fluorescence is easily quenched by binding to Mn²⁺. Therefore, we used this property of Mn²⁺ to quench fura-2 fluorescence as an indicator of Ca²⁺ influx through plasma membrane. Fura-2/AM loaded cells were perfused with HEPES-buffered physiological saline. The perfusion solution was then switched to 1 mM Mn²⁺-containing solution without EGTA or Ca²⁺ for 10 min to observe the extent of Mn²⁺ entry. Fura-2/AM loaded cells were excited with light at 360 nm, a wavelength insensitive to intracellular Ca²⁺ changes. Fluorescence images emitted at 510 nm were captured. Mn²⁺ quenching the fluorescence signal was normalized using values determined by treatment of 20 µM β-escin to permeabilize cell membrane at the end of the experiments.

Luminal Ca²⁺ measurements in endoplasmic reticulum

The isolated acinar cells were loaded with 3 µM magfura-2/AM for 1 h at room temperature and then attached on cover glass in the perfusion chambers. Cells were permeabilized by perfusion with 20 µM β-escin for 2 min in intracellular medium (ICM) containing 19 mM NaCl, 125 mM KCl, 10 mM HEPES and 1 mM EGTA (pH 7.3) as described previously [27]. To remove intracellular dye, the permeabilized cells were washed with ICM no containing β-escin for 15 min. Intracellular Ca²⁺ stores were subsequently refilled with Ca²⁺ by activation of SERCA. To activate SERCA, cells were perfused with ICM containing 0.650 µM CaCl₂ (free Ca²⁺=200 nM), 3 mM Na₂ATP and 1.4 mM MgCl₂. After the stores were refilled with Ca²⁺, SERCA activity was effectively inactivated by removal of Mg²⁺ from ICM. The free Ca²⁺ was constantly maintained at 200 nM throughout all experiments. Fluorescence images emitted 505 nm were captured following alternate excitation at 340 nm and 380 nm using a TILL Photonics imaging system.

Statistical analysis

All results were presented as mean±S.E. Data were analyzed using the Student's t test. Differences were considered significant when the p value was less than 0.05. Ca²⁺ refill rates and Ca²⁺ release rates were estimated by fitting the changing fluorescence to a single exponential function. Relative Ca²⁺ entry and efflux were normalized to maximum value in each experiments using Origin program.

RESULTS

Hydrogen peroxide (H₂O₂)-induced intracellular Ca²⁺ accumulation

Initial experiments were performed to investigate the effect of H₂O₂ on the intracellular Ca²⁺ level in parotid acinar cells. The change of intracellular Ca²⁺ concentration was monitored in various concentrations of H₂O₂ (0.1–10 mM) in the presence of 1.28 mM extracellular Ca²⁺ in intact cells. As shown in Fig. 1A, the perfusion of H₂O₂ for 10 min resulted in slow increases of intracellular Ca²⁺ concentrations. The significant Ca²⁺ elevations were observed from 1 mM of H₂O₂ and more rapid Ca²⁺ accumulation was observed in 10 mM H₂O₂. After the cessation of H₂O₂ perfusion, the augmented Ca²⁺ was nearly returned to baseline at 1 mM of H₂O₂. Contrastively, the sustained Ca²⁺ increase was observed at 3 mM and 10 mM of H₂O₂, even if H₂O₂ was removed from the perfusate (Fig. 1B). These results suggest that H₂O₂ could remarkably accumulate in intracellular Ca²⁺ and that an excess dose of H₂O₂ could irreversibly alter Ca²⁺ homeostasis in parotid acinar cells.

Effects of H₂O₂ on Ca²⁺ entry from extracellular medium

Next, we compared the intracellular Ca²⁺ accumulation in the presence and the absence of extracellular Ca²⁺ to confirm whether H₂O₂ could facilitate Ca²⁺ entry from the extracellular medium. Intracellular Ca²⁺ accumulation steadily increased during treatment of H₂O₂ in both normal Ca²⁺ and Ca²⁺-free mediums (Fig. 2A). Although, a slight difference was detected in initial values of Ca²⁺ accumulation, the final values of Ca²⁺ accumulation showed no difference as shown in Fig. 2B (values at 300 s; 0.064±0.012 Δ ratio vs 0.034±0.007 Δ ratio, values at 600 s; 0.123±0.023 Δ ratio vs 0.122±0.015 Δ ratio, presence and absence of extracellular Ca²⁺, respectively). Thus, the entire Ca²⁺ accumulation was observed regardless of external Ca²⁺ existence.

In another experiment, the Mn²⁺ quenching test was performed to confirm Ca²⁺ entry. As shown in Fig. 3A, the perfusion of 1 mM and 3 mM H₂O₂ for 10 min failed to facilitate quenching of fura-2 fluorescence, whereas 10 mM ₂O₂ markedly accelerated quenching of fura-2 fluorescence. The relative Ca²⁺ entries at the end of the experiments were 57.14±8.73%, 62.99±6.03%, 63.56±6.79% and 86.21±1.77% in control, 1 mM, 3 mM and 10 mM H₂O₂-treated groups, respectively (Fig. 3B). These data indicate that the primary origin of accumulated Ca²⁺ induced by 1 mM H₂O₂ may not come from extracellular fluid because Ca²⁺ accumulation was still observed in Ca²⁺-free solution and Mn²⁺ quenching of fura-2 fluorescence was not facilitated in the presence of 1 mM H₂O₂. Therefore, we used a concentration of 1 mM H₂O₂ in the following experiments to identify the primary mechanism of Ca²⁺ accumulation. The next experiment was planned to evaluate whether H₂O₂ can influence Ca²⁺ transport through intracellular Ca²⁺ stores membrane.

Effects of H₂O₂ on Ca²⁺ transport through intracellular Ca²⁺ stores membrane

To determine the influence of H₂O₂ on Ca²⁺ transport through endoplasmic reticulum (ER) membrane, we employed unidirectional fluorescent ER Ca²⁺ measurements in permeabilized cells. Parotid acinar cells were loaded with a low-affinity Ca²⁺ dye magfura-2/AM and then permeabilized with β-escin to release the cytosolic dye. As shown in Fig. 4A, the perfusion with intracellular medium containing CaCl₂, MgCl₂ and ATP resulted in an increase of the fluorescence ratio that indicates an effective Ca²⁺ refill into the ER stores. In these conditions, the perfusion of 1 mM H₂O₂ failed to change Ca²⁺ refill rate into the ER stores (Fig. 4B). After the stores were refilled with Ca²⁺, MgCl₂ was eliminated from the buffer to provide SERCA inactivation. Application of 1 µM InsP₃ caused significant Ca²⁺ release from the stores, but H₂O₂ had no effect on InsP₃-induced Ca²⁺ release rates (Fig. 4C). These findings indicate that 1 mM H₂O₂ led to neither Ca²⁺ refill nor InsP₃-induced Ca²⁺ release through the ER membrane.

H₂O₂ inhibits Ca²⁺ efflux through PMCA inactivation

To confirm the effect of H₂O₂ on PMCA activity, we evaluated the effect of H₂O₂ in Ca²⁺-free conditions during the depletion of Ca²⁺ from the ER store by the treatment of 1 µM TG, a SERCA blocker. As shown in Fig. 5A, TG induced a transient Ca²⁺ elevation that indicates spontaneous Ca²⁺ release from the ER stores, and these responses were completely returned to baseline, which means there was Ca²⁺ efflux by plasma membrane Ca²⁺-ATPase (PMCA) in control experiments. When H₂O₂ was treated with TG, the peak time of Ca²⁺ elevation was delayed (Fig. 5B, control; 24.83±2.76 s vs. H₂O₂; 37.17±3.17 s) and Ca²⁺ efflux was not fully returned to the control value in 1 mM H₂O₂-treated group (Fig. 5C, control; 103.95±7.62% vs. H₂O₂; 39.58±9.33%).

To further test the effect of H₂O₂ on PMCA activity in another set of 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⁺ in intact cells, respectively. In the control experiment, as shown in Fig. 6A, the addition of extracellular Ca²⁺ remarkably stimulated Ca²⁺ entry, and the elimination of extracellular Ca²⁺ resulted in a clear extrusion of intracellular Ca²⁺ to external space. In 1 mM H₂O₂-treated cells, the elevated intracellular Ca²⁺ level was not returned to baseline by withdrawal of extracellular Ca²⁺. That means the extrusion of intracellular Ca²⁺ was markedly disrupted by treatment of 1 mM H₂O₂. Additionally, the perfusion of antioxidants with H₂O₂, either 30 µg/ml catalase or 2 mM dithiothreitol, completely protected the diminished Ca²⁺ efflux in TG-treated intact acinar cells (Fig. 6B). These evidences suggest that H₂O₂ may induce Ca²⁺ accumulation by inhibition of PMCA activity in mouse parotid acinar cells.

DISCUSSION

The present study provides evidence that the reactive oxygen species H₂O₂ accumulates cytosolic Ca²⁺ by attenuating Ca²⁺ efflux through PMCA rather than by mobilizing the Ca²⁺ from the extracellular space or intracellular Ca²⁺ store in mouse parotid acinar cells. Cytosolic free Ca²⁺ plays a crucial role in the salivary secretion of parotid acinar cells, and Ca²⁺ can be mobilized from both the external fluid and the internal Ca²⁺ stores such as endoplasmic reticulum, acidic store and mitochondria to elicit physiological responses [28 29 30]. Acetylcholine, a major agonist in the parotid gland, is known to initially mobilize Ca²⁺ from internal stores through activation of InsP₃ receptors, and to subsequently activate the store-operated Ca²⁺ entry from the external medium to refill the depleted stores [31 32]. After Ca²⁺ mobilization, cytosolic Ca²⁺ was rapidly eliminated to internal store and external space by SERCA and PMCA, respectively. Since the accumulation of intracellular Ca²⁺ causes cellular toxicity, including apoptosis and necrosis, basal intracellular Ca²⁺ concentrations are finely regulated to under about 10,000 folds compare to the extracellular space [33]. The balance between Ca²⁺ mobilization and Ca²⁺ elimination are regulated by various Ca²⁺ transporters expressed both in plasma membrane and ER membrane [34]. Oxidative stress is well known risk factor which induces cellular dysfunction of several tissues and organs [10 11], and oxidants-induced cellular dysfunction are closely linked with intracellular Ca²⁺ accumulation [12 13]. In this study, when parotid acinar cells were exposed to 1 mM H₂O₂ in normal Ca²⁺ buffer, there was a significant intracellular Ca²⁺ accumulation, and the elevated Ca²⁺ was nearly returned to baseline after the cessation of H₂O₂ perfusion. Generally, H₂O₂ is known to accumulate cytosolic Ca²⁺ without acute cell death at concentrations from 10 µM to 5 mM in various cell types [16 17 18 19 20 21 22 23 24 25]. Thus, mouse parotid acinar cells are thought to be relative resistance to H₂O₂.

To confirm the underlying mechanism of H₂O₂-induced Ca²⁺accumulation, we measured Ca²⁺ transport through plasma membrane and ER membrane. Actually, H₂O₂-induced Ca²⁺ entry through plasma membrane is important for intracellular Ca²⁺ accumulation, and transient-receptor potential channels and store-operated Ca²⁺ channels are known to involved in Ca²⁺ overload in various cell types [19 20 21 22]. However, in our study, the entire Ca²⁺ accumulation induced by 1 mM H₂O₂ was not suppressed by the elimination of extracellular Ca²⁺ in intact acinar cells and Mn²⁺ quenching property was not facilitated by the perfusion of 1 mM H₂O₂. Since 10 mM H₂O₂ produced more rapid cytosolic Ca²⁺ elevation and accelerated Mn²⁺ quenching fura-2 fluorescence, higher concentrations of H₂O₂ may be required to promote Ca²⁺ influx in parotid acinar cells. Moreover, H₂O₂ failed to stimulate InsP₃-induced Ca²⁺ release for measurement Ca²⁺ transport through ER membrane in permeabilized acinar cells. These results suggest that H₂O₂ could accumulate cytosolic Ca²⁺ irrelevant of Ca²⁺ entry from external fluid and Ca²⁺ release from ER stores. Since H₂O₂ could mobilize Ca²⁺ from other TG-insensitive intracellular stores such as mitochondria [35 36], we evaluated the effect of H₂O₂ in Ca²⁺-free conditions after the depletion of Ca²⁺ from the ER store by the pretreatment of 1 µM TG (Supplementary Fig. 1). TG induced a transient Ca²⁺ elevation, and these responses were completely returned to baseline. After return to baseline, the perfusion of 1 mM H₂O₂ failed to increase the Ca²⁺ release. It has been proposed that mitochondrial Ca²⁺ release by H₂O₂ could conceivably contribute to Ca²⁺ accumulation when cytosolic Ca²⁺ is high, since H₂O₂ evoked cytosolic Ca²⁺ elevation when cells were pre-stimulated by cholecystokinin (CCK) but failed to evoke cytosolic Ca²⁺ elevation without prestimulation of CCK in ER Ca²⁺ stores were depleted pancreatic acinar cells [24]. Thus, 1 mM H₂O₂-induced Ca²⁺ accumulation is not due to mobilize Ca²⁺ from other intracellular Ca²⁺ stores or mitochondria in our study. On the other hand, when TG were treated with 1 mM H₂O₂ in the absence of extracellular Ca²⁺, cytosolic Ca²⁺ elevation was delayed and enhanced Ca²⁺ was not fully recovered to baseline. Actually, slow intracellular Ca²⁺ elevation by SERCA inhibition was likely due to spontaneous release or leak of Ca²⁺ from the ER stores, and slow Ca²⁺ decline was due to extrusion of Ca²⁺ to the external space by PMCA or Na⁺/Ca²⁺ exchanger (NCX) activation [37]. Although, NCX are also known to sensitive to oxidants [12 13], there is no evidence of the expression and the participation of NCX for Ca²⁺ extrusion in parotid acinar cells at the present time. Furthermore, Ca²⁺ extrusion was not affected when Na⁺ was replaced with NMDG⁺ to NCX inactivation in our preliminary study (data not shown). Since the removal of elevated Ca²⁺ from cytosol to the extracellular site was carried out mainly by PMCA under inhibition of SERCA by TG treatment in parotid acinar cells, this finding strongly suggests that H₂O₂ effectively suppressed PMCA activity. In another set of experiment, we further investigated the effect of H₂O₂ on Ca²⁺ efflux through PMCA. PMCA activity was assessed by a Ca²⁺ decrease to baseline by removing extracellular Ca²⁺ after store-operated Ca²⁺ entry was fully stimulated by adding extracellular Ca²⁺ in TG-treated cells. Here we show that the extrusion of intracellular Ca²⁺ was markedly disrupted by pretreatment of 1 mM H₂O₂ in TG-treated intact acinar cells. Actually, the initial Ca²⁺ levels were slightly higher in H₂O₂-treated cells than in control cells. These results were considered caused by blocking of Ca²⁺ efflux through PMCA, rather than by enhancing of the Ca²⁺ entry through activation of store-operated Ca²⁺ channels. Furthermore, the inhibitory effects of H₂O₂ on Ca²⁺ efflux were strongly protected by the adding of catalase, an enzyme degrading hydrogen peroxide, and DTT, a sulfhydryl reducing agent.

In fact, it has been reported that functionally important sulfhydryl group are present within PMCA and SERCA molecules that is localized within the catalytic and controlling centers of Ca²⁺-ATPase [38 39 40]. We previously reported that H₂O₂ accumulates intracellular Ca²⁺ by attenuating SERCA activity in pancreatic acinar cells [25]. Interestingly, in this study, H₂O₂ failed to change Ca²⁺ refilling into the ER store through SERCA by the application of Ca²⁺ and Mg-ATP in permeabilized parotid acinar cells. As shown in supplement Fig. 2, three folds higher concentration of H₂O₂ are need to inhibit Ca²⁺efflux through PMCA in pancreatic acinar cells compared to parotid acinar cells. Ca²⁺-ATPase has distinct isoforms with different expression and regulation properties that caused diversity of Ca²⁺ signaling in various cell types [41]. It has been known that distinct Ca²⁺-ATPase isoforms have different sensitivity to ROS due to locational difference of sulfhydryl group [42,43,44]. Thus, the different sensitivities of Ca²⁺-ATPase to H₂O₂ between parotid and pancreatic acinar cells are thought to be due to differences in expression and regulation of Ca²⁺-ATPase. In immunofluorescence study, PMCA1 is distributed throughout the plasma membrane, PMCA2 is localized to the basolateral membrane and PMCA4 is localized to the apical membrane in parotid acinar cell [45]. Even though PMCAs are dominantly expressed in apical membrane in pancreatic acinar cells [46 47], isoform specific distributions are not clear at the present time. Although the further studies are need to elucidate this issues, we predict from the present results that H₂O₂ could lead to accumulate cytosolic Ca²⁺ by attenuation of Ca²⁺ efflux through the oxidation of functional sulfhydryl groups of PMCA in parotid acinar cells.

From the above results, we concluded that H₂O₂ can accumulate intracellular Ca²⁺ by attenuating Ca²⁺ efflux through PMCA, rather than by mobilizing Ca²⁺ from extracellular medium or intracellular stores in parotid acinar cells. Thus the primary target for H₂O₂ excessively generated in pathological conditions is considered PMCA in mouse parotid acinar cells.

Figures and Tables

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).

ACKNOWLEDGEMENTS

This work was supported by the 2015 Konyang University Myunggok Research Fund.

Notes

^{Author contributions} M.J.K., K.J.C. and M.N.Y. acquired and analyzed the data. M.J.K. drafted the manuscript. S.H.O., D.K.K. and S.H.K contributed to conception and design of study. H.S.P. coordinated the study and reviewed the manuscript.

^{CONFLICTS OF INTEREST} The authors declare no conflicts of interest.

References

1. Ambudkar IS. Calcium signalling in salivary gland physiology and dysfunction. J Physiol. 2016; 594:2813–2824.

2. Petersen OH. Stimulus-secretion coupling: cytoplasmic calcium signals and the control of ion channels in exocrine acinar cells. J Physiol. 1992; 448:1–51.

3. Fox PC. Acquired salivary dysfunction. Drugs and radiation. Ann N Y Acad Sci. 1998; 842:132–137.

4. Abdollahi M, Fooladian F, Emami B, Zafari K, Bahreini-Moghadam A. Protection by sildenafil and theophylline of lead acetate-induced oxidative stress in rat submandibular gland and saliva. Hum Exp Toxicol. 2003; 22:587–592.

5. Acauan MD, Figueiredo MA, Cherubini K, Gomes AP, Salum FG. Radiotherapy-induced salivary dysfunction: Structural changes, pathogenetic mechanisms and therapies. Arch Oral Biol. 2015; 60:1802–1810.

6. Yamada T, Ryo K, Tai Y, Tamaki Y, Inoue H, Mishima K, Tsubota K, Saito I. Evaluation of therapeutic effects of astaxanthin on impairments in salivary secretion. J Clin Biochem Nutr. 2010; 47:130–137.

7. Abedi SM, Yarmand F, Motallebnejad M, Seyedmajidi M, Moslemi D, Ashrafpour M, Bijani A, Moghadamnia A, Mardanshahi A, Hosseinimehr SJ. Vitamin E protects salivary glands dysfunction induced by ionizing radiation in rats. Arch Oral Biol. 2015; 60:1403–1409.

8. Ryo K, Yamada H, Nakagawa Y, Tai Y, Obara K, Inoue H, Mishima K, Saito I. Possible involvement of oxidative stress in salivary gland of patients with Sjogren's syndrome. Pathobiology. 2006; 73:252–260.

9. Pagano G, Castello G, Pallardó FV. Sjøgren's syndrome-associated oxidative stress and mitochondrial dysfunction: prospects for chemoprevention trials. Free Radic Res. 2013; 47:71–73.

10. Gardner AM, Xu FH, Fady C, Jacoby FJ, Duffey DC, Tu Y, Lichtenstein A. Apoptotic vs. nonapoptotic cytotoxicity induced by hydrogen peroxide. Free Radic Biol Med. 1997; 22:73–83.

11. Aruoma OI. Free radicals, oxidative stress, and antioxidants in human health and disease. J Am Oil Chem Soc. 1998; 75:199–212.

12. Kourie JI. Interaction of reactive oxygen species with ion transport mechanisms. Am J Physiol. 1998; 275:C1–24.

13. Kiselyov K, Muallem S. ROS and intracellular ion channels. Cell Calcium. 2016; 60:108–114.

14. Yu BP. Cellular defenses against damage from reactive oxygen species. Physiol Rev. 1994; 74:139–162.

15. Dröge W. Free radicals in the physiological control of cell function. Physiol Rev. 2002; 82:47–95.

16. Roveri A, Coassin M, Maiorino M, Zamburlini A, van Amsterdam FT, Ratti E, Ursini F. Effect of hydrogen peroxide on calcium homeostasis in smooth muscle cells. Arch Biochem Biophys. 1992; 297:265–270.

17. Gen W, Tani M, Takeshita J, Ebihara Y, Tamaki K. Mechanisms of Ca²⁺ overload induced by extracellular H₂O₂ in quiescent isolated rat cardiomyocytes. Basic Res Cardiol. 2001; 96:623–629.

18. Sun L, Yau HY, Lau OC, Huang Y, Yao X. Effect of hydrogen peroxide and superoxide anions on cytosolic Ca²⁺: comparison of endothelial cells from large-sized and small-sized arteries. PLoS One. 2011; 6:e25432.

19. Giambelluca MS, Gende OA. Hydrogen peroxide activates calcium influx in human neutrophils. Mol Cell Biochem. 2008; 309:151–156.

20. Liu X, Cotrim A, Teos L, Zheng C, Swaim W, Mitchell J, Mori Y, Ambudkar I. Loss of TRPM2 function protects against irradiation-induced salivary gland dysfunction. Nat Commun. 2013; 4:1515.

21. Kozai D, Ogawa N, Mori Y. Redox regulation of transient receptor potential channels. Antioxid Redox Signal. 2014; 21:971–986.

22. Santiago E, Climent B, Muñoz M, García-Sacristán A, Rivera L, Prieto D. Hydrogen peroxide activates store-operated Ca²⁺ entry in coronary arteries. Br J Pharmacol. 2015; 172:5318–5332.

23. Zaidi A, Barŕon L, Sharov VS, Schöneich C, Michaelis EK, Michaelis ML. Oxidative inactivation of purified plasma membrane Ca²⁺-ATPase by hydrogen peroxide and protection by calmodulin. Biochemistry. 2003; 42:12001–12010.

24. Bruce JI, Elliott AC. Oxidant-impaired intracellular Ca²⁺ signaling in pancreatic acinar cells: role of the plasma membrane Ca²⁺-ATPase. Am J Physiol Cell Physiol. 2007; 293:C938–C950.

25. Yoon MN, Kim DK, Kim SH, Park HS. Hydrogen peroxide attenuates refilling of intracellular calcium store in mouse pancreatic acinar cells. Korean J Physiol Pharmacol. 2017; 21:233–239.

26. Park HS, Betzenhauser MJ, Zhang Y, Yule DI. Regulation of Ca²⁺ release through inositol 1,4,5-trisphosphate receptors by adenine nucleotides in parotid acinar cells. Am J Physiol Gastrointest Liver Physiol. 2012; 302:G97–G104.

27. Choi KJ, Kim KS, Kim SH, Kim DK, Park HS. Caffeine and 2-aminoethoxydiphenyl borate (2-APB) have different ability to inhibit intracellular calcium mobilization in pancreatic acinar cell. Korean J Physiol Pharmacol. 2010; 14:105–111.

28. Gallacher DV, Petersen OH. Stimulus-secretion coupling in mammalian salivary glands. Int Rev Physiol. 1983; 28:1–52.

29. Yule DI, Straub SV, Bruce JI. Modulation of Ca²⁺ oscillations by phosphorylation of Ins(1,4,5)P₃ receptors. Biochem Soc Trans. 2003; 31:954–957.

30. Melvin JE, Yule D, Shuttleworth T, Begenisich T. Regulation of fluid and electrolyte secretion in salivary gland acinar cells. Annu Rev Physiol. 2005; 67:445–469.

31. Berridge MJ, Bootman MD, Roderick HL. Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol. 2003; 4:517–529.

32. Won JH, Cottrell WJ, Foster TH, Yule DI. Ca²⁺ release dynamics in parotid and pancreatic exocrine acinar cells evoked by spatially limited flash photolysis. Am J Physiol Gastrointest Liver Physiol. 2007; 293:G1166–G1177.

33. Clapham DE. Calcium signaling. Cell. 2007; 131:1047–1058.

34. Petersen OH. Calcium signalling and secretory epithelia. Cell Calcium. 2014; 55:282–289.

35. Pariente JA, Camello C, Camello PJ, Salido GM. Release of calcium from mitochondrial and nonmitochondrial intracellular stores in mouse pancreatic acinar cells by hydrogen peroxide. J Membr Biol. 2001; 179:27–35.

36. Baggaley EM, Elliott AC, Bruce JI. Oxidant-induced inhibition of the plasma membrane Ca²⁺-ATPase in pancreatic acinar cells: role of the mitochondria. Am J Physiol Cell Physiol. 2008; 295:C1247–C1260.

37. Strehler EE, Caride AJ, Filoteo AG, Xiong Y, Penniston JT, Enyedi A. Plasma membrane Ca²⁺ ATPases as dynamic regulators of cellular calcium handling. Ann N Y Acad Sci. 2007; 1099:226–236.

38. Bellomo G, Mirabelli F, Richelmi P, Orrenius S. Critical role of sulfhydryl group(s) in ATP-dependent Ca²⁺ sequestration by the plasma membrane fraction from rat liver. FEBS Lett. 1983; 163:136–139.

39. Zaidi A, Barŕon L, Sharov VS, Schöneich C, Michaelis EK, Michaelis ML. Oxidative inactivation of purified plasma membrane Ca²⁺-ATPase by hydrogen peroxide and protection by calmodulin. Biochemistry. 2003; 42:12001–12010.

40. Pengpanichpakdee N, Thadtapong T, Auparakkitanon S, Wilairat P. Plasma membrane Ca²⁺-ATPase sulfhydryl modifications: implication for oxidized red cell. Southeast Asian J Trop Med Public Health. 2012; 43:1252–1257.

41. Strehler EE, Zacharias DA. Role of alternative splicing in generating isoform diversity among plasma membrane calcium pumps. Physiol Rev. 2001; 81:21–50.

42. Grover AK, Samson SE, Misquitta CM. Sarco(endo)plasmic reticulum Ca²⁺ pump isoform SERCA3 is more resistant than SERCA2b to peroxide. Am J Physiol. 1997; 273:C420–C425.

43. Barnes KA, Samson SE, Grover AK. Sarco/endoplasmic reticulum Ca²⁺-pump isoform SERCA3a is more resistant to superoxide damage than SERCA2b. Mol Cell Biochem. 2000; 203:17–21.

44. Yin D, Kuczera K, Squier TC. The sensitivity of carboxyl-terminal methionines in calmodulin isoforms to oxidation by H₂O₂ modulates the ability to activate the plasma membrane Ca-ATPase. Chem Res Toxicol. 2000; 13:103–110.

45. Baggaley E, McLarnon S, Demeter I, Varga G, Bruce JI. Differential regulation of the apical plasma membrane Ca²⁺-ATPase by protein kinase A in parotid acinar cells. J Biol Chem. 2007; 282:37678–37693.

46. Belan PV, Gerasimenko OV, Tepikin AV, Petersen OH. Localization of Ca²⁺ extrusion sites in pancreatic acinar cells. J Biol Chem. 1996; 271:7615–7619.

47. Lee MG, Xu X, Zeng W, Diaz J, Kuo TH, Wuytack F, Racymaekers L, Muallem S. Polarized expression of Ca²⁺ pumps in pancreatic and salivary gland cells. Role in initiation and propagation of [Ca²⁺]_i waves. J Biol Chem. 1997; 272:15771–15776.

SUPPLEMENTARY MATERIALS

Supplementary data including two figures can be found with this article online at http://pdf.medrang.co.kr/paper/pdf/Kjpp/Kjpp022-02-11-s001.pdf.

Supplementary Fig. 1

H₂O₂ failed to accumulate intracellular Ca²⁺ after depletion of ER Ca²⁺ stores by TG treatment in intact acinar cells.

TOOLS

Similar articles