Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0705412 on December 19, 2005

Published online before print December 19, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0705412v1
79/3/508    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Suzuki, Y.
Right arrow Articles by Ra, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Suzuki, Y.
Right arrow Articles by Ra, C.
(Journal of Leukocyte Biology. 2006;79:508-518.)
© 2006 by Society for Leukocyte Biology

Mitochondrial Ca2+ flux is a critical determinant of the Ca2+ dependence of mast cell degranulation

Yoshihiro Suzuki1, Tetsuro Yoshimaru, Toshio Inoue and Chisei Ra

Division of Molecular Cell Immunology and Allergology, Advanced Medical Research Center, Nihon University Graduate School of Medical Sciences, Tokyo, Japan

1 Correspondence: Division of Molecular Cell Immunology and Allergology, Advanced Medical Research Center, Nihon University Graduate School of Medical Sciences, 30-1 Oyaguchikami-cho Itabashi-ku, Tokyo 173-8610, Japan. E-mail: ysuzuki{at}med.nihon-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
An increase in intracellular Ca2+ ([Ca2+]i) is necessary for mast cell exocytosis, but there is controversy over the requirement for Ca2+ in the extracellular medium. Here, we demonstrate that mitochondrial function is a critical determinant of Ca2+ dependence. In the presence of extracellular Ca2+, mitochondrial metabolic inhibitors, including rotenone, antimycin A, and the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), significantly reduced degranulation induced by immunoglobulin E (IgE) antigen or by thapsigargin, as measured by ß-hexosaminidase release. In the absence of extracellular Ca2+; however, antimycin A and FCCP, but not rotenone, enhanced, rather than reduced, degranulation to a maximum of 76% of that observed in the presence of extracellular Ca2+. This enhancement of extracellular, Ca2+-independent degranulation was concomitant with a rapid collapse of the mitochondrial transmembrane potential. Mitochondrial depolarization did not enhance degranulation induced by thapsigargin, irrespective of the presence or absence of extracellular Ca2+. IgE antigen was more effective than thapsigargin as an inducer of [Ca2+]i release, and mitochondrial depolarization augmented IgE-mediated but not thapsigargin-induced Ca2+ store release and mitochondrial Ca2+ ([Ca2+]m) release. Finally, atractyloside and bongkrekic acid [an agonist and an antagonist, respectively, of the mitochondrial permeability transition pore (mPTP)], respectively, augmented and reduced IgE-mediated Ca2+ store release, [Ca2+]m release, and/or degranulation, whereas they had no effects on thapsigargin-induced Ca2+ store release. These data suggest that the mPTP is involved in the regulation of Ca2+ signaling, thereby affecting the mode of mast cell degranulation. This finding may shed light on a new role for mitochondria in the regulation of mast cell activation.

Key Words: signal transduction • Ca2+ • mitochondria • permeability transition pore


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mast cell is a key effector of allergic reactions in humans and other animals. Mast cells express the high-affinity Fc immunoglobulin E (IgE) receptor (Fc{epsilon}R)I on the cell surface. Its cross-linking by IgE antigen initiates a cascade of intracellular signaling events, which leads to degranulation, inflammatory mediator release, and cytokine production [1 , 2 ]. It is generally accepted that IgE antigen induces the generation of inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG); IP3 binds to its receptor in the membrane of the endoplasmic reticulum (ER) and induces Ca2+ release, whereas DAG together with Ca2+ activates certain isoforms of protein kinase C (PKC) [3 4 5 ]. In nonexcitable cells including mast cells, IP3-induced depletion of internal Ca2+ stores activates movement of extracellular Ca2+ across the plasma membrane, through store-operated Ca2+ channels (SOCs) [6 , 7 ]. Ca2+ and PKC appear to be required for optimal mast cell degranulation [8 ].

The release of preformed granular mediators such as histamine, serotonin, and ß-hexosaminidase from mast cells is a consequence of complicated biochemical events. Studies to date indicate that the degranulation process is strictly dependent on the influx of extracellular Ca2+ [8 ]. Although little is known about the molecular identity of the machinery for degranulation [9 ], recent studies have revealed the molecular machinery that regulates the final steps of the fusion between the granular and plasma membrane. These studies also provide a molecular basis for the requirement for increased intracellular Ca2+ ([Ca2+]i). The soluble N-ethylmaleimide attachment protein receptors (SNAREs), such as syntaxin 4, play a key role in membrane fusion, through formation of a stable complex with S-nitroso-N-acetylpenicillamine (SNAP)-23 [10 11 12 ]. It is important that the rise in Ca2+ resulting from Ca2+ influx modulates the phosphorylation of SNAREs, thereby affecting their capacity to bind to SNAP-23 [10 ].

It has been accepted that degranulation induced by aggregation of Fc{epsilon}RI is dependent on the influx of extracellular Ca2+ across the cell membrane. In contrast, nonimmunological secretogogues can induce degranulation independently of extracellular Ca2+. A variety of pharmacological reagents, including compound 48/80, ammonium chloride, nigericin, and adenosine-5'-O-(3-thio-phosphate), has been reported to evoke histamine release from rat peritoneal mast cells in the absence of extracellular Ca2+ [13 14 15 ]. In addition, we recently demonstrated that ions of the heavy metal silver induce substantial histamine release from rat basophilic leukemia (RBL)-2H3 mast cells, independently of thapsigargin-sensitive Ca2+ stores and Ca2+ influx [16 ]. Taken together, these observations suggest that mast cell degranulation may be dependent or independent of Ca2+. However, little is known about the mechanisms that determine which mode of degranulation occurs, except that intracellular alkalinization has been shown to play a facilitating role in the Ca2+-independent response [13 , 14 ].

Earlier work has suggested a role for mitochondria in mast cell degranulation. Studies with mitochondrial metabolic inhibitors show a close link between energy production and histamine release [17 ]. Furthermore, several lines of evidence indicate that Ca2+ influx is associated with mitochondrial Ca2+ ([Ca2+]m) uptake and refilling of IP3-sensitive stores [18 , 19 ]. Some investigators have shown that [Ca2+]m stores play a crucial role in the regulation of influx through SOCs [20 21 22 ], although the requirement is still controversial [23 ]. This led us to elucidate the possible involvement of mitochondrial function in the regulation of mast cell degranulation. Here, we demonstrate that mitochondrial function is a critical determinant of the Ca2+ dependence of mast cell degranulation, with selective enhancement of the Ca2+-independent pathway. Our data show that such enhancement is relevant to [Ca2+]m handling through the mitochondrial permeability transition pore (mPTP), a putative, fast Ca2+-releasing channel.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Antimycin A, oligomycin, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), rotenone, thapsigargin, atractyloside, and ubiquinone 0 (Ub0) were obtained from Sigma Chemical Co. (St. Louis, MO). Bongkrekic acid (BKA), dissolved in water, was purchased from BioMol (Plymouth Meeting, PA). Antimycin A, FCCP, rotenone, and Ub0 were dissolved in dimethyl sulfoxide (DMSO) and diluted with Hanks’ balanced salt solution (HBSS) to a final concentration of <0.1% before use. DMSO, at the concentration of 0.1% (the vehicle) by itself, had no effects throughout the present study. Atractyloside was dissolved in HBSS. Monoclonal anti-2,4,6-trinitrophenyl (TNP) IgE (clone IgE-3) was from BD PharMingen Japan (Tokyo). TNP-bovine serum albumin (BSA) conjugate (25 molecules TNP coupled to one molecule BSA) was purchased from Cosmo Bio (Tokyo, Japan). Recombinant interleukin-3 (rIL-3) was purchased from LSL (Tokyo, Japan). Fluo-3-acetoxyl-methyl ester (fluo-3/AM) was from Dojindo (Kumamoto, Japan). Rhod-2/AM and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenz-imidazolocarbocyanine iodide (JC-1) were obtained from Molecular Probes (Eugene, OR).

RBL-2H3 cells
The RBL-2H3 cells, obtained from National Institute of Health Sciences [Japan Collection of Research Biosources (JCRB); cell number JCRB0023], were grown in Dulbecco’s modified Eagle’s medium (DMEM; Sigma Chemical Co.) supplemented with 10% fetal bovine serum (FBS; JRH Biosciences, Lenexa, KS) in 5% CO2-containing atmosphere. The RBL-2H3 cells were harvested by incubating them in HBSS supplemented with bicarbonate (pH 7.4) containing 1 mM EDTA and 0.25% trypsin for 5 min at 37°C. For IgE sensitization, cells (1x107/5 ml) suspended in complete DMEM were plated on a 100-mm culture dish or in a 24-well plate (2x105 cells/well) and incubated with anti-TNP IgE (1 µg/dish or 0.1 µg/well) at 37°C overnight. IgE-sensitized cells were washed with phosphate-buffered saline and suspended in HBSS and then stimulated with TNP-BSA at the concentrations indicated at 37°C. Oligomycin, an inhibitor of the mitochondrial FoF1 adenosine 5'-triphosphate (ATP) synthase, was added in experiments using mitochondrial inhibitors to prevent the mitochondrial consumption of cellular ATP. Oligomycin by itself had no significant biological effects in any of the experiments.

Bone marrow-derived mast cells (BMMCs)
BMMCs were prepared from femurs of 4- to 8-week-old C57BL/6 mice as described previously [24 ]. Cells were cultured in RPMI 1640 (Sigma Chemical Co.) supplemented with 10% FBS, 100 U/ml penicillin and streptomycin (Gibco, Grand Island, NY), 10–4 M 2-mercaptoethanol (Wako, Tokyo, Japan), 110 µg/ml sodium pyruvate (Gibco), 1% minimal essential medium nonessential amino acid solution (Gibco), and 5 ng/ml rIL-3 in a 5% CO2-containing atmosphere at 37°C. After 4–6 weeks of culture, cells were stained for cell-surface expression of Fc{epsilon}RI. BMMCs were used for experiments after 4–8 weeks of culture (>95% mast cells).

ß-Hexosaminidase release assay
Degranulation was determined by ß-hexosaminidase release as described [24 ]. Briefly, 40 µl supernatant or cell lysates and 100 µl 2 mM p-nitrophenyl-N-acetyl-ß-D-glucosaminide (in 0.4 M citrate and 0.2 M phosphate buffer, pH 4.5, Sigma Chemical Co.) were added to each well of a 96-well plate, and color was developed for 30 min at 37°C. The enzymatic reaction was terminated by adding 200 µl 0.2 M glycine-NaOH, pH 10.7. The absorbance at 405 nm was measured in a microplate reader (Bio-Rad 550, Nippon Bio-Rad Laboratories, Osaka, Japan).

Measurement of mitochondrial transmembrane potential ({Delta}{Psi}m) with JC-1
Changes in the {Delta}{Psi}m were measured using the lipophilic cation JC-1 as described previously [25 ]. Cells (5x105/500 µl) were loaded with 2 µM JC-1 for 15 min in a CO2 incubator, washed, and resuspended in HBSS. After cell stimulation, the green fluorescence (the monomeric JC-1) and red fluorescence (J-aggregates) were measured using the FL-1 and FL-2 channels, respectively, with a FACSCalibur (Becton Dickinson, San Jose, CA).

Measurement of [Ca2+]m with rhod-2/AM
Measurement of [Ca2+]m was performed using the mitochondrially localizing Ca2+-reactive fluorescence probe, rhod-2/AM. To improve the discrimination between cytosolic and mitochondrially localized dye [26 ], 5 µM rhod-2/AM was reduced to the colorless, nonfluorescent dihydorhod-2/AM by sodium borohydride, according to the manufacturer’s protocol. Cells were loaded with the dye for 40 min at 37°C, washed, and resuspended in HBSS in a 24-well plate. After the cells were stimulated, rhod-2 fluorescence was monitored at 5 s intervals up to 3 min by a microplate fluorometer (Fluoroskan Ascent CF, Labsystems, Helsinki, Finland; excitation and emission at 544 and 590 nm, respectively) using 5 µM A23187 and 5 µM A23187 in the presence of 10 mM EGTA, respectively, as a positive and negative control.

Measurement of [Ca2+]i with fluo-3/AM
Measurement of [Ca2+]i was performed using the Ca2+-reactive fluorescence probe, fluo-3/AM, as described [24 ]. Briefly, a cell suspension (1x106 cells/ml in HBSS) was incubated with 4 µM fluo-3/AM for 30 min at 37°C and then washed with HBSS and resuspended in HBSS supplemented with 1 mM CaCl2 (Ca2+-containing buffer). To study Ca2+ store release and Ca2+ entry separately, aliquots of the fluo-3-loaded cells were resuspended in the medium supplemented with 1 mM EGTA in place of 1 mM CaCl2 (Ca2+-free buffer). Fluo-3 fluorescence was monitored at 5 s intervals up to 3 min in a microplate fluorometer (Fluoroskan Ascent CF, Labsystems; excitation and emission at 485 and 527 nm, respectively). [Ca2+]i was calculated using the equation: [Ca2+]i = Kd [(F–Fmin)/(Fmax–F)], where Kd is the dissociation constant of the Ca2+-fluo-3 complex (450 nM). Fmax represents the maximum fluorescence (obtained by treating cells with 5 µM A23187), and Fmin represents the minimum fluorescence (obtained for A23187-treated cells in the presence of 10 mM EGTA). F is the actual sample fluorescence.

Statistical analysis
Student’s t-test was performed to determine the statistical significance of differences among the experimental groups; P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mitochondrial metabolic inhibitors modulate mast cell degranulation
When RBL-2H3 mast cells were placed in a Ca2+-containing solution (HBSS supplemented with 1 mM CaCl2), the cells released a substantial amount of the granular enzyme ß-hexosaminidase in response to IgE antigen with an optimal antigen concentration of 30 ng/ml (45.5±0.2% vs. spontaneous release 6.6±0.6%, n=6). When exposed to rotenone to inhibit the complex I of the electron transport chain or a mixture of antimycin A (5 µg/ml) and oligomycin (0.5 µg/ml; in short, referred to as antimycin A) to inhibit complex III and the mitochondrial FoF1 ATP synthase, respectively, there was a significant reduction in the amplitude of secretion (Fig. 1A ). More pronounced effects were observed with the protonophore FCCP (5 µM), which collapses the proton motive force across the inner mitochondrial membrane. The receptor-independent agonist thapsigargin also induced ß-hexosaminidase release in a dose-dependent manner with an optimal concentration of 1 µM (45.9±4% vs. spontaneous release 8.7±3.4%, n=6). The secretion was also reduced differently by these mitochondrial metabolic inhibitors (Fig. 1B) . Unlike IgE antigen, FCCP and to a lesser extent, rotenone reduced the release, whereas antimycin A had no effect. Accordingly, mitochondrial function may be important in degranulation, whether or not induced through Fc{epsilon}RI.


Figure 1
View larger version (23K):
[in this window]
[in a new window]
  		Figure 1. Mitochondrial metabolic inhibitors exhibit opposing effects on mast cell degranulation in the presence or absence of extracellular Ca2+. IgE-sensitized RBL-2H3 cells (5x105/200 µl/well) were washed and placed in a Ca2+-containing medium (HBSS supplemented with 1 mM CaCl2; A, B) or in Ca2+-free medium (HBSS with 1 mM EGTA; C, D). Cells were incubated with 5 µM rotenone (ROT), 5 µg/ml antimycin A and 0.5 µg/ml oligomycin (AM), or with 5 µM FCCP at 37°C for 30 min and then stimulated with 30 ng/ml TNP-BSA (A, C) or with 1 µM thapsigargin (B, D) at 37°C for 30 min. ß-Hexosaminidase release from the stimulated cells was measured spectrophotometrically as described in Materials and Methods. The data are shown as the percentage of the control stimulated with IgE antigen or thapsigargin alone and represent the mean ± SE of three to five independent experiments with similar results. Statistical significance versus control was determined using an unpaired Student’s t-test. NS, Not significant, *, P < 0.05; ***, P < 0.001.

 
Consistent with a strict dependence on extracellular Ca2+, IgE antigen had little effect on ß-hexosaminidase release when cells were placed in a Ca2+-free solution (HBSS supplemented with 1 mM EGTA instead of CaCl2; 13.9±3.1% vs. spontaneous release of 7.1±1%, n=5). It is unexpected that we found that mitochondrial dysfunction enhanced rather than suppressed the effect of IgE antigen. As shown in Figure 1C , when cells were treated with FCCP, IgE-mediated degranulation was increased up to 3.4-fold (76% of the value observed in the presence of extracellular Ca2+), although the drug by itself had no effect on degranulation. Antimycin A also had a significant stimulatory effect (2.5-fold), whereas rotenone had no effects. None of these mitochondrial metabolic inhibitors by themselves could induce degranulation. Thapsigargin also induced ß-hexosaminidase release from RBL-2H3 cells only in Ca2+-containing solution (52.1±8.4%, n=6). FCCP and antimycin A, but not rotenone, also enhanced thapsigargin-induced degranulation, but their effects were marginal (1.4-fold, at most; Fig. 1D ). These observations indicate that the markedly increased degranulation is not a result of cell killing. To evaluate the collapse of {Delta}{Psi}m under these conditions, we measured {Delta}{Psi}m using JC-1. The carbocyanine fluorescent dye JC-1 exists as a monomer and emits green light at low concentrations or at low {Delta}{Psi}m, whereas at high concentrations or at high {Delta}{Psi}m, JC-1 forms J-aggregates, which emit red light [25 ]. Thus, this dye has been used widely as a sensitive indicator for change in {Delta}{Psi}m. Treatment with IgE antigen or thapsigargin led to a marginal collapse (<30% decrease) of {Delta}{Psi}m within 5 min after stimulation; {Delta}{Psi}m then returned to almost resting levels until the final time-point (35 min). Conversely, FCCP and antimycin A caused almost complete loss (>95% decrease) of {Delta}{Psi}m throughout the time periods monitored, whereas rotenone had no such effect (Fig. 2A ). FCCP and antimycin A induced mitochondrial depolarization in a dose-dependent manner, irrespective of the presence or absence of extracellular Ca2+ (Fig. 2B and 2C) . Collectively, these results indicate that mitochondrial depolarization has opposing effects on extracellular Ca2+-dependent or -independent mast cell degranulation, with selective enhancement of the Ca2+-independent degranulation. Similar results were obtained with BMMCs (data not shown). These observations led us to elucidate mechanisms of this enhancement in the following experiments.


Figure 2
View larger version (17K):
[in this window]
[in a new window]
  		Figure 2. Effects of mitochondrial metabolic inhibitors on {Delta}{Psi}m. (A) IgE-sensitized RBL-2H3 cells (5x105/500 µl) were loaded with JC-1, and the JC-1-loaded cells suspended in Ca2+-containing medium were treated with 30 ng/ml TNP-BSA (IgE antigen), 5 µM rotenone, 5 µg/ml antimycin A and 0.5 µg/ml oligomycin, or with 5 µM FCCP for 5, 20, and 35 min. (B, C) The collapse of {Delta}{Psi}m in the presence or absence of extracellular Ca2+. The JC-1-loaded cells suspended in Ca2+-containing medium [Ca (+)] or Ca2+-free medium [Ca (–)] were incubated with 30 ng/ml TNP-BSA (IgE antigen) and 5 µg/ml antimycin A and 0.5 µg/ml oligomycin (B) or with 5 µM FCCP (C) for 5 min. The green and red dye fluorescence was then measured by flow cytometry. The data are shown as percentages of the basal level and represent the mean ± SE of three to five separate experiments with similar results.

 
IgE antigen induces Ca2+ store release more effectively than thapsigargin
As increased [Ca2+]i is a critical signal for degranulation, we hypothesized that in extracellular, Ca2+-independent degranulation, this requirement might be met by mobilization of Ca2+ from internal stores, which is affected somewhat more readily by IgE antigen than by thapsigargin. To test this possibility, we compared Ca2+ store release between these two stimuli. As shown in Figure 3A , even in the absence of extracellular Ca2+, a substantial elevation in [Ca2+]i was observed following stimulation with IgE antigen, indicating the induction of Ca2+ store release. The IgE-mediated Ca2+ store release was observed immediately, reaching its peak within 30 s after stimulation and declining rapidly thereafter. The response was antigen dose-dependent, and the highest peak height of [Ca2+]i (188±8 nM, n=8) was at 30 ng/ml, the optimal concentration for degranulation. Although thapsigargin caused Ca2+ store release with a similar time course, the effect was always smaller than that of IgE antigen. With stimulation by 1 µM thapsigargin, the peak height of [Ca2+]i was 68 ± 9 nM (n=8), which was lower than that of a suboptimal antigen dose (3 ng/ml; Fig. 3B ). Conversely, in the presence of extracellular Ca2+, thapsigargin was always more potent than IgE antigen in increasing [Ca2+]i (Fig. 3C) . Similar results were obtained with BMMCs (the [Ca2+]i peak heights for IgE antigen and thapsigargin were ~120 nM and <50 nM, respectively), although again, in the presence of extracellular Ca2+, thapsigargin was more potent than IgE antigen in increasing [Ca2+]i (data not shown). These results demonstrate that as expected, IgE antigen is somewhat more effective than thapsigargin in causing Ca2+ store release.


Figure 3
View larger version (19K):
[in this window]
[in a new window]
  		Figure 3. IgE antigen induces Ca2+ store release more effectively than thapsigargin. (A, B) IgE-sensitized RBL-2H3 cells (1x106/ml) loaded with fluo-3/AM were suspended in a Ca2+-free medium and stimulated with 3, 30, and 300 ng/ml TNP-BSA (IgE antigen; A) or with 30 ng/ml TNP-BSA or 1 µM thapsigargin (Tg; B), and then, fluo-3 fluorescence was monitored by a microplate fluorometer. (C) Fluo-3-loaded RBL-2H3 cells (1x106/ml) were suspended in a Ca2+-containing medium and stimulated with 30 ng/ml TNP-BSA (IgE antigen) or 1 µM thapsigargin, and then, fluo-3 fluorescence were monitored. The data shown as calculated [Ca2+]i are representative of three to five experiments with similar results. ({lozenge}) Basal level.

 
IgE-mediated but not thapsigargin-induced Ca2+ store release is enhanced by mitochondrial depolarization in a mPTP-dependent manner
We next compared the effects of mitochondrial depolarization on Ca2+ store release induced by IgE antigen and thapsigargin. FCCP and antimycin A significantly enhanced IgE-mediated Ca2+ store release to a similar extent, whereas rotenone had no effect (Fig. 4A and 4B ). Conversely, these agents had no stimulatory effect on thapsigargin-induced Ca2+ store release (Fig. 4C and 4D) . These results demonstrate that mitochondrial depolarization enhances IgE-mediated Ca2+ store release selectively. Together with the greater effectiveness of IgE antigen in inducing Ca2+ store release compared with thapsigargin, IgE antigen appears to induce Ca2+ release from an intracellular store other than the ER, which is insensitive to thapsigargin but sensitive to mitochondrial depolarization.


Figure 4
View larger version (31K):
[in this window]
[in a new window]
  		Figure 4. Mitochondrial depolarization enhances IgE- but not thapsigargin-mediated Ca2+ store release. IgE-sensitized RBL-2H3 cells (1x106/ml), loaded with fluo-3/AM, were suspended in a Ca2+-free medium and incubated with 5 µM rotenone, 5 µg/ml antimycin A plus 0.5 µg/ml oligomycin, or 5 µM FCCP at 37°C for 30 min and then stimulated with 30 ng/ml TNP-BSA (IgE antigen; A, B) or with 1 µM thapsigargin (C, D); then, fluo-3 fluorescence was monitored by a microplate fluorometer. The data shown as calculated [Ca2+]i are representative of three to five experiments with similar results. ({lozenge}) Basal level.

 
Mitochondrial depolarization has been shown to result in the induction of mPT, an in vitro increase in the permeability of the inner mitochondrial membrane to solutes with molecular masses of ~1500 daltons or less [27 , 28 ]. It is now widely believed that mPT is a result of the opening of the mPTP, a putative, fast Ca2+-releasing channel [27 28 29 30 ]. Thus, there was the possibility that mitochondria are involved in the thapsigargin-insensitive Ca2+ store release. To test this possibility, we examined the effects of BKA and atractyloside, an antagonist and an agonist of the mPTP, respectively [30 , 31 ], on Ca2+ release. When mPTP was blocked by BKA, IgE-mediated Ca2+ store release was reduced significantly, whereas thapsigargin-induced Ca2+ store release was unaffected (Fig. 5A and B). In addition, stimulation of mPTP opening by atractyloside augmented IgE-mediated Ca2+ store release in a dose-dependent manner, and it had no effects on thapsigargin-induced Ca2+ store release (Fig. 5C and 5D) . Neither BKA nor atractyloside had an effect on basal levels of [Ca2+]i (data not shown). These results demonstrate that IgE antigen but not thapsigargin induces Ca2+ store release in a mPTP-dependent manner. This is consistent with the view that mitochondria are one of the thapsigargin-insensitive Ca2+ stores.


Figure 5
View larger version (31K):
[in this window]
[in a new window]
  		Figure 5. IgE- but not thapsigargin-mediated Ca2+ store release is mPTP-dependent. IgE-sensitized RBL-2H3 cells (1x106/ml), loaded with fluo-3/AM, were suspended in a Ca2+-free medium and incubated with 1 µM BKA (A, B) or with 10 or 100 µM atractyloside (ATC) and stimulated with 30 ng/ml TNP-BSA (IgE antigen; A, C) or with 1 µM thapsigargin (B, D), and then, fluo-3 fluorescence was monitored by a microplate fluorometer. The data shown as calculated [Ca2+]i are representative of three to five independent experiments with similar results. ({lozenge}) Basal level.

 
IgE antigen induces [Ca2+]m release in a mPTP-dependent manner
To obtain more direct evidence for the involvement of mitochondria, we monitored [Ca2+]m by using rhod-2/AM, a Ca2+-sensitive fluorescent indicator that accumulates in mitochondria depending on {Delta}{Psi}m [32 ]. As shown in Figure 6A , IgE antigen induced a drop in [Ca2+]m in a dose-dependent manner. When cells were stimulated with a suboptimal dose of antigen (0.3 ng/ml), [Ca2+]m decreased until 50 s and then returned to the resting level. As the concentration of antigen increased, the magnitude of the decrease grew larger, and the time to recovery became prolonged. In some experiments, no significant recovery was observed during 3 min at 30 ng/ml (see Fig. 6C ). The initial [Ca2+]m levels were similar in antigen-stimulated cells and resting cells, indicating that Fc{epsilon}RI engagement has a minimal effect on [Ca2+]m uptake when the resting [Ca2+]m levels are relatively high. Conversely, a substantial [Ca2+]m increase was observed after Fc{epsilon}RI engagement in the cells with lower resting [Ca2+]m levels (data not shown). Simultaneous monitoring of [Ca2+]i revealed that in accordance with the decrease in [Ca2+]m, [Ca2+]i rose with time to its peak at ~30 s (Fig. 6B) . The concerted behavior of these two Ca2+ signals strongly suggests their connection, although the changes in Ca2+ levels at these two intracellular sites were not completely parallel. To determine whether the fall in [Ca2+]m results from mPTP opening, we examined the effect of BKA. As shown in Figure 6C , antimycin A significantly enhanced the IgE-mediated decrease in [Ca2+]m, and treatment with BKA prior to antimycin A treatment abolished this enhancement. BKA treatment also abolished the enhancing effect of antimycin A on IgE-mediated [Ca2+]i elevation in parallel (Fig. 6D) . These results indicate that IgE antigen can induce [Ca2+]m release in a mPTP-dependent manner.


Figure 6
View larger version (33K):
[in this window]
[in a new window]
  		Figure 6. Blockade of mPTP counteracts the enhancement of IgE-mediated [Ca2+]mrelease and Ca2+ store release. IgE-sensitized RBL-2H3 cells (1x106/ml), which had been loaded with rhod-2/AM (A, C) and fluo-3/AM (B, D) were suspended in a Ca2+-free medium. The cells were incubated at 37°C for 30 min with HBSS or with 1 µM BKA at 37°C for 30 min and treated with 5 µg/ml antimycin A and 0.5 µg/ml oligomycin. Cells were then stimulated with 30 ng/ml TNP-BSA (IgE antigen), and rhod-2 fluorescence (A, C) and fluo-3 fluorescence (B, D) were monitored by a microplate fluorometer. The data are expressed as relative fluorescence units (RFU) or calculated [Ca2+]i and are representative of three independent experiments with similar results. ({lozenge}) Basal level.

 
Blockade of the mPTP abolishes the enhancement of Ca2+-independent degranulation
To elucidate the role of the mPTP in extracellular, Ca2+-independent degranulation, we examined the effects of BKA on the response. When cells were treated with BKA prior to the treatment with mitochondrial metabolic inhibitors, the enhancement of the response was abolished, although the mPTP antagonist by itself had a minimal effect on degranulation (Fig. 7 ). Similar effects were observed with another mPTP antagonist, Ub0, which has been shown to impair mPTP opening, probably by affecting the binding affinity between the mPTP and Ca2+ [33 ]. These results demonstrate that the mPTP plays a critical role in the enhancement of Ca2+-independent degranulation, providing more support for the role of mitochondria.


Figure 7
View larger version (21K):
[in this window]
[in a new window]
  		Figure 7. Blockade of mPTP abolishes the enhancement of extracellular, Ca2+-independent degranulation. IgE-sensitized RBL-2H3 cells (1x106/ml) in a Ca2+-free medium were incubated with HBSS or with 1 µM BKA or 10 µM Ub0 at 37°C for 30 min and treated with 5 µM rotenone, 5 µg/ml antimycin A plus 0.5 µg/ml oligomycin, or 5 µM FCCP. Cells were then stimulated with 30 ng/ml TNP-BSA (IgE antigen), and ß-hexosaminidase release was measured. The data represent the mean ± SE of four independent experiments with similar results. Statistical significance versus control not treated with BKA or Ub0 was determined using an unpaired Student’s t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The data presented in this paper clearly demonstrate that mitochondrial dysfunction has opposing effects on mast cell degranulation, depending on the presence or absence of extracellular Ca2+ (illustrated in Fig. 8 ). Most studies to date indicate that IgE-dependent mast cell degranulation is strictly extracellular, Ca2+-dependent, whereas various nonimmunological secretogogues can induce degranulation independently of extracellular Ca2+ [13 14 15 16 17 18 ]. These observations suggest that mast cells can use two pathways of degranulation with different extracellular Ca2+ dependence. Consistent with the widely accepted role of extracellular Ca2+, we found that IgE-mediated and thapsigargin-induced degranulation are strictly dependent on extracellular Ca2+. Drugs causing mitochondrial depolarization by distinct mechanisms, such as FCCP and antimycin A, inhibited extracellular, Ca2+-dependent degranulation, irrespective of the stimulus used. Rotenone, which impairs complex I of the electron transport chain but has no effect on {Delta}{Psi}m, was less effective than these two agents, indicating that mitochondrial metabolic dysfunction, including reduced energy production, may be responsible for the inhibitory action. Early studies with mitochondrial inhibitors suggested a relationship between energy metabolism and the secretory activity of rat peritoneal mast cells. However, our preliminary experiments showed that intracellular ATP was not significantly altered following treatment with mitochondrial metabolic inhibitors under our experimental conditions. This is consistent with the view that granulocytes rely heavily on glycolysis for their energy production as a result of lowered mitochondrial function. Furthermore, given that mitochondrial inhibitors impair degranulation by reducing the production of ATP, they are expected to suppress degranulation constantly. The present study revealed that this is not in the case. Thus, mitochondria appear to play a more fundamental role in the regulation of degranulation by a Ca2+-linked mechanism. In this respect, it should be noted that early studies suggest an important role for [Ca2+]m stores in the regulation of SOCs in RBL-2H3 cells. We are currently attempting to reveal the mechanisms underlying the inhibition of extracellular, Ca2+-dependent degranulation.


Figure 8
View larger version (18K):
[in this window]
[in a new window]
  		Figure 8. The effect of mPTPs on mast cell degranulation. (A) In Ca2+-containing medium, Fc{epsilon}RI aggregation can induce the influx of extracellular Ca2+, which is required for the induction of degranulation. Ca2+ released via the mPTP appears to regulate this induction negatively. (B) In Ca2+-free medium, [Ca2+]m release facilitates the induction of degranulation in place of the influx of extracellular Ca2+.

 
The enhancement of mast cell degranulation by mitochondrial depolarization in the absence of extracellular Ca2+ reveals a novel degranulation pathway, which is probably only evident when mitochondrial metabolism is impaired. As mitochondrial depolarization per se had little effect on spontaneous and thapsigargin-induced Ca2+-independent degranulation, Fc{epsilon}RI-mediated signals to mitochondria may also be required for this alternative pathway. Increased [Ca2+]i is widely considered to be a critical signal for degranulation. It may occur by release of Ca2+ from certain organelles when degranulation is independent of extracellular Ca2+. In addition to being the main energy-producing machinery of eukaryotic cells, mitochondria play an important role in the regulation of cellular Ca2+ homeostasis. Mitochondria can take up Ca2+ rapidly in response to physiological stimuli and subsequently release this Ca2+ slowly back into the cytosol [25 26 27 28 29 ]. Our data indicate that [Ca2+]m release concomitant with [Ca2+]i elevation occurs rapidly following Fc{epsilon}RI stimulation. Furthermore, the fact that mitochondrial depolarization promotes these two Ca2+ signals and Ca2+ influx-independent degranulation in parallel provides further support for the role of [Ca2+]m flux in this unusual response. Hence, although the requirement for microdomains of high Ca2+ for exocytosis [20 21 22 23 24 25 ] is debated, our results suggest that at least in Ca2+ influx-independent degranulation, [Ca2+]m stores play a crucial role.

Another important finding is that the mPTP appears to be a key component in the regulatory system of mast cell degranulation. It is now widely accepted that mPT induction is a result of the opening of the mPTP, which may function as a fast Ca2+-releasing channel [25 26 27 28 29 ]. The adenine nucleotide translocase (ANT) and the voltage-dependent anion channel, an outer membrane protein, can form a complex to which cyclophilin D is recruited from the matrix, forming the basic unit of the mPTP [34 , 35 ]. mPT induction in vitro causes the collapse of the proton-motive force and subsequent dissipation of {Delta}{Psi}m, disruption of ion homeostasis, and mitochondrial swelling. Consequently, mPTP opening results in release of cytochrome C from the inner mitochondrial membrane and the activation of caspases in apoptosis or results in a loss of cellular energy production, causing necrosis [36 ]. However, increasing evidence suggests that mPT induction is more selective and more importantly, reversible [26 , 37 38 39 ], enabling mitochondria to act as a fast Ca2+-releasing store, which may be relevant to [Ca2+]m flux and cellular Ca2+ homeostasis. Consistent with this view, our data indicate that blocking mPT induction counteracts [Ca2+]m release and the concomitant rise in [Ca2+]i.

The mechanism of Fc{epsilon}RI signal transduction for [Ca2+]m release remains unclear. The differential effects of mitochondrial depolarization on [Ca2+]m release by IgE anigen and thapsigargin suggest that these two stimuli elicit their effects in different ways. It should be noted that IgE antigen and thapsigargin elicit increases in cytosolic Ca2+ by different mechanisms: IgE antigen induces release of Ca2+ from intracellular stores through generation of IP3, and thapsigargin depletes the [Ca2+]i stores by blocking uptake of Ca2+ into these stores by inhibiting sarco/ER Ca2+-ATPase activity in the ER. Influx of external Ca2+ occurs following depletion of the intracellular stores regardless of mechanism of depletion. The differential effects of mitochondrial depolarization on [Ca2+]m release by IgE antigen and thapsigargin might be relevant to this difference in the mechanism of depletion. In the absence of extracellular Ca2+, IgE antigen but not thapsigargin causes an elevation of [Ca2+]i, which is highly thiol-oxidation-dependent and reversed by dithiothreitol. It should be noted that ANT has three cysteine residues whose oxidation is critical for the mPTP open-closed transitions and the [Ca2+]m release [40 , 41 ]. These observations also suggest that reactive oxygen species (ROS) play a critical role in mPTP opening. Increasing evidence indicates that endogenous ROS are a critical regulator of mast cell responses, including Ca2+ response [42 43 44 ]. We previously demonstrated that Fc{epsilon}RI stimulation causes a rapid generation of intracellular ROS, which plays a role in the regulation of [Ca2+]i elevation [24 ]. In addition, our recent experiments showed that IgE antigen but not thapsigargin can evoke the generation of ROS independently of Ca2+ and that ebselen, the selective scavenger for peroxides including H2O2, can abolish [Ca2+]m release and [Ca2+]i elevation (unpublished results). Collectively, these observations are consistent with the hypothesis that ROS produced by Fc{epsilon}RI stimulation directly or indirectly cause a reversible oxidation of critical ANT thiols, thereby inducing a rapid and reversible mPTP opening. Further studies to test this hypothesis are under way in our laboratory.

In conclusion, the present study reveals the critical role of the mPTP in selecting the mode of Ca2+ dependence of mast cell degranulation. This finding may shed light on a new role for mitochondria regulating mast cell activation.


   ACKNOWLEDGEMENTS
 
This work was partially supported by a Grant-in-Aid to promote advanced scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and grants from Nihon University School of Medicine and the Uehara Memorial Foundation. We thank National Institute of Health Sciences (JCRB) for providing RBL-2H3 (cell number JCRB0023).

Received July 26, 2005; revised October 30, 2005; accepted October 31, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Turner, H., Kinet, P. (1999) Signaling through the high-affinity IgE receptor Fc{epsilon}RI Nature 402,B24-B30[CrossRef][Medline]
  2. Kinet, J. P. (1999) The high-affinity IgE receptor (Fc{epsilon}RI): from physiology to pathology Annu. Rev. Immunol. 17,931-972[CrossRef][Medline]
  3. Berridge, M. J., Irvine, R. F. (1989) Inositol phosphates and cell signaling Nature 341,197-205[CrossRef][Medline]
  4. Hoth, M., Penner, R. (1992) Depletion of intracellular calcium stores activates a calcium current in mast cells Nature 355,353-356[CrossRef][Medline]
  5. Putney, J. W., Jr, Bird, G. S. (1993) The signal for capacitative calcium entry Cell 75,199-201[CrossRef][Medline]
  6. Zweifach, A., Lewis, R. S. (1996) Calcium-dependent potentiation of store-operated calcium channels in T lymphocytes J. Gen. Physiol. 107,597-610[Abstract/Free Full Text]
  7. Parekh, A. B., Fleig, A., Penner, R. (1997) The store-operated calcium current I (CRAC): nonlinear activation by InsP3 and dissociation from calcium release Cell 89,973-980[CrossRef][Medline]
  8. Ozawa, K., Szallasi, Z., Kazanietz, M. G., Blumberg, P. M., Mischak, H., Mushinski, J. F., Beaven, M. A. (1993) Ca2+-dependent and Ca2+-independent isozymes of protein kinase C mediate exocytosis in antigen-stimulated rat basophilic RBL-2H3 cells. Reconstitution of secretory responses with Ca2+ and purified isozymes in washed permeabilized cells J. Biol. Chem. 268,1749-1756[Abstract/Free Full Text]
  9. Logan, M. R., Odemuyiwa, S. O., Moqbel, R. (2003) Understanding exocytosis in immune and inflammatory cells: the molecular basis of mediator secretion J. Allergy Clin. Immunol. 111,923-932[Medline]
  10. Pombo, I., Martin-Verdeaux, S., Iannascoli, B. J., Mao, L., Deriano, L., Rivera, J., Blank, U. (2001) IgE receptor type I-dependent regulation of a Rab3D-associated kinase: a possible link in the calcium-dependent assembly of SNARE complexes J. Biol. Chem. 276,42893-42900[Abstract/Free Full Text]
  11. Baram, D., Mekori, Y. A., Sagi-Eisenberg, R. (2001) Synaptotagmin regulates mast cell functions Immunol. Rev. 179,25-34[CrossRef][Medline]
  12. Blank, U., Cyprien, B., Martin-Verdeaux, S., Paumet, F., Pombo, I., Rivera, J., Roa, M., Varin-Blank, N. (2002) SNAREs and associated regulators in the control of exocytosis in the RBL-2H3 mast cell line Mol. Immunol. 38,1341-1345[CrossRef][Medline]
  13. Botana, L. M., Alfonso, A., Botana, M. A., Vieytes, M. R., Louzao, M. C., Cabado, A. G. (1992) Influence of protein kinase C, cAMP and phosphatase activity on histamine release produced by compound 48/80 and sodium fluoride on rat mast cells Agents Actions 37,1-7[CrossRef][Medline]
  14. Alfonso, A., Cabado, A. G., Vieytes, M. R., Botana, L. M. (2000) Calcium-pH crosstalks in rat mast cells: cytosolic alkalinization, but not intracellular calcium release, is a sufficient signal for degranulation Br. J. Pharmacol. 130,1809-1816[CrossRef]
  15. Ludowyke, R. I., Scurr, L. L. (1994) Calcium-independent secretion by ATP {gamma} S from a permeabilized rat basophilic leukaemia cell line (RBL-2H3) Cell. Signal. 6,223-231[CrossRef][Medline]
  16. Suzuki, Y., Yoshimaru, T., Matsui, T., Ra, C. (2002) Silver activates calcium signals in rat basophilic leukemia-2H3 mast cells by a mechanism that differs from the Fc {epsilon} RI-activated response J. Immunol. 169,3954-3962[Abstract/Free Full Text]
  17. Johansen, T. (1987) Energy metabolism in rat mast cells in relation to histamine secretion Pharmacol. Toxicol. 61(Suppl. 2),1-20[Medline]
  18. Mohr, F. C., Fewtrell, C. (1990) The effect of mitochondrial inhibitors on calcium homeostasis in tumor mast cells Am. J. Physiol. 258,C217-C226[Medline]
  19. Ali, H., Maeyama, K., Sagi-Eisenberg, R., Beaven, M. A. (1994) Antigen and thapsigargin promote influx of Ca2+ in rat basophilic RBL-2H3 cells by ostensibly similar mechanisms that allow filling of inositol 1,4,5-trisphosphate-sensitive and mitochondrial Ca2+ stores Biochem. J. 304,431-440[Medline]
  20. Hoth, M., Fanger, C. M., Lewis, R. S. (1997) Mitochondrial regulation of store-operated calcium signaling in T lymphocytes J. Cell Biol. 137,633-648[Abstract/Free Full Text]
  21. Makowska, A., Zablocki, K., Duszynski, J. (2000) The role of mitochondria in the regulation of calcium influx into Jurkat cells Eur. J. Biochem. 267,877-884[Medline]
  22. Glitsch, M. D., Bakowski, D., Parekh, A. B. (2002) Store-operated Ca2+ entry depends on mitochondrial Ca2+ uptake EMBO J. 21,6744-6754[CrossRef][Medline]
  23. Mahmoud, S. F., Fewtrell, C. (2001) Microdomains of high calcium are not required for exocytosis in RBL-2H3 mucosal mast cells J. Cell Biol. 153,339-349[Abstract/Free Full Text]
  24. Suzuki, Y., Yoshimaru, T., Matsui, T., Inoue, T., Niide, O., Nunomura, S., Ra, C. (2003) Fc{epsilon}RI signaling of mast cells activates intracellular production of hydrogen peroxide: role in the regulation of calcium signals J. Immunol. 171,6119-6127[Abstract/Free Full Text]
  25. Smiley, S. T., Reers, M., Mottola-Hartshorn, C., Lin, M., Chen, A., Smith, T. W., Steele, G. D., Jr, Chen, L. B. (1991) Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1 Proc. Natl. Acad. Sci. USA 88,3671-3675[Abstract/Free Full Text]
  26. Hajnoczky, G., Robb-Gaspers, L. D., Seitz, M. B., Thomas, A. P. (1995) Decoding of cytosolic calcium oscillations in the mitochondria Cell 82,415-424[CrossRef][Medline]
  27. Bernardi, P., Broekemeier, K. M., Pfeiffer, D. R. (1994) Recent progress on regulation of the mitochondrial permeability transition pore; a cyclosporin-sensitive pore in the inner mitochondrial membrane J. Bioenerg. Biomembr. 26,509-517[CrossRef][Medline]
  28. Zoratti, M., Szabo, I. (1995) The mitochondrial permeability transition Biochim. Biophys. Acta 1241,139-176[Medline]
  29. Hunter, D. R., Haworth, R. A. (1979) The Ca2+-induced membrane transition in mitochondria. III. Transitional Ca2+ release Arch. Biochem. Biophys. 195,468-477[CrossRef][Medline]
  30. Henderson, P. J., Lardy, H. A. (1970) Bongkrekic acid. An inhibitor of the adenine nucleotide translocase of mitochondria J. Biol. Chem. 245,1319-1326[Abstract/Free Full Text]
  31. Brandolin, G., Le Saux, A., Trezeguet, V., Lauquin, G. J., Vignais, P. V. (1993) Chemical, immunological, enzymatic, and genetic approaches to studying the arrangement of the peptide chain of the ADP/ATP carrier in the mitochondrial membrane J. Bioenerg. Biomembr. 25,459-472[CrossRef][Medline]
  32. Duchen, M. R. (2000) Mitochondria and Ca2+ in cell physiology and pathophysiology Cell Calcium 28,339-348[CrossRef][Medline]
  33. Walter, L., Nogueira, V., Leverve, X., Heitz, M. P., Bernardi, P., Fontaine, E. (2000) Three classes of ubiquinone analogs regulate the mitochondrial permeability transition pore through a common site J. Biol. Chem. 275,29521-29527[Abstract/Free Full Text]
  34. Brini, M. (2003) Ca2+ signaling in mitochondria: mechanism and role in physiology and pathology Cell Calcium 34,399-405[CrossRef][Medline]
  35. Minta, A., Kao, J. P., Tsien, R. Y. (1989) Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores J. Biol. Chem. 264,8171-8178[Abstract/Free Full Text]
  36. Halestrap, A. P., McStay, G. P., Clarke, S. J. (2002) The permeability transition pore complex: another view Biochimie 84,153-166[Medline]
  37. Newmeyer, D. D., Ferguson-Miller, S. (2003) Mitochondria: releasing power for life and unleashing the machineries of death Cell 112,481-490[CrossRef][Medline]
  38. Broekemeier, K. M., Pfeiffer, D. R. (1995) Inhibition of the mitochondrial permeability transition by cyclosporin A during long time frame experiments: relationship between pore opening and the activity of mitochondrial phospholipases Biochemistry 34,16440-16449[CrossRef][Medline]
  39. Ichas, F., Jouaville, L. S., Mazat, J. P. (1997) Mitochondria are excitable organelles capable of generating and conveying electrical and calcium signals Cell 89,1145-1153[CrossRef][Medline]
  40. Petronilli, V., Costantini, P., Scorrano, L., Colonna, R., Passamonti, S., Bernardi, P. (1994) The voltage sensor of the mitochondrial permeability transition pore is tuned by the oxidation-reduction state of vicinal thiols. Increase of the gating potential by oxidants and its reversal by reducing agents J. Biol. Chem. 269,16638-16642[Abstract/Free Full Text]
  41. Halestrap, A. P., Woodfield, K. Y., Connern, C. P. (1997) Oxidative stress, thiol reagents, and membrane potential modulate the mitochondrial permeability transition by affecting nucleotide binding to the adenine nucleotide translocase J. Biol. Chem. 272,3346-3354[Abstract/Free Full Text]
  42. Suzuki, Y., Yoshimaru, T., Inoue, T., Niide, O., Ra, C. (2005) Role of oxidants in mast cell activation Chem. Immunol. Allergy 87,32-42[Medline]
  43. Heneberg, P., Draber, P. (2005) Regulation of cys-based protein tyrosine phosphatases via reactive oxygen and nitrogen species in mast cells and basophils Curr. Med. Chem. 12,1859-1871[CrossRef][Medline]
  44. Yoshimaru, T., Suzuki, Y., Matsui, T., Yamashita, K., Ochiai, T., Yamaki, M., Shimizu, K. (2002) Blockade of superoxide generation prevents high-affinity immunoglobulin E receptor-mediated release of allergic mediators by rat mast cell line and human basophils Clin. Exp. Allergy 32,612-618[Medline]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
T. Inoue, Y. Suzuki, T. Yoshimaru, and C. Ra
Nitric oxide protects mast cells from activation-induced cell death: the role of the phosphatidylinositol-3 kinase-Akt-endothelial nitric oxide synthase pathway
J. Leukoc. Biol., May 1, 2008; 83(5): 1218 - 1229.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0705412v1
79/3/508    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Suzuki, Y.
Right arrow Articles by Ra, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Suzuki, Y.
Right arrow Articles by Ra, C.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS