Published online before print December 19, 2005
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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
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Key Words: signal transduction • Ca2+ • mitochondria • permeability transition pore
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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 FcRI 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.
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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 FcRI. 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 (
m) with JC-1
Changes in the 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.
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RI.
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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.
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m under these conditions, we measured m using JC-1. The carbocyanine fluorescent dye JC-1 exists as a monomer and emits green light at low concentrations or at low m, whereas at high concentrations or at high 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 m. Treatment with IgE antigen or thapsigargin led to a marginal collapse (<30% decrease) of m within 5 min after stimulation; 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 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.
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Figure 2. Effects of mitochondrial metabolic inhibitors on 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 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.
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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.
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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. ( ) Basal level.
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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. () Basal level.
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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.
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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. () Basal level.
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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 FcRI 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 FcRI 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.
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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. () Basal level.
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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.
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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.
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Figure 8. The effect of mPTPs on mast cell degranulation. (A) In Ca2+-containing medium, FcRI 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+.
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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 FcRI 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 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 FcRI 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 FcRI 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 FcRI 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.
Received July 26, 2005; revised October 30, 2005; accepted October 31, 2005.
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RI Nature 402,B24-B30[CrossRef][Medline]RI): from physiology to pathology Annu. Rev. Immunol. 17,931-972[CrossRef][Medline]
S from a permeabilized rat basophilic leukaemia cell line (RBL-2H3) Cell. Signal. 6,223-231[CrossRef][Medline] RI-activated response J. Immunol. 169,3954-3962RI signaling of mast cells activates intracellular production of hydrogen peroxide: role in the regulation of calcium signals J. Immunol. 171,6119-6127This article has been cited by other articles:
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