Published online before print August 7, 2007
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Laboratory of Molecular Pharmacology, Institute of Pharmacology, Faculty of Veterinary Science, Universidad Austral de Chile, Valdivia, Chile
1 Correspondence: Laboratory of Molecular Pharmacology, Institute of Pharmacology, Faculty of Veterinary Science, Universidad Austral de Chile, P.O. Box 567, Valdivia, Chile. E-mail: rburgos1{at}uach.cl
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Key Words: calcium channel inhibitor • signal transduction • platelet-activating factor
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In neutrophils, SOCE can be activated by chemotactic factors such as the platelet-activating factor (PAF), a widely known, biologically active phospholipid [1-O-alkyl-2(R) acetyl-glyceryl-3-phosphorylcholine], liberated by platelets, leukocytes, and smooth muscle cells [7 ]. PAF, via a G protein-coupled receptor, mobilizes cytosolic-free [Ca2+]i from inositol 1,4,5 triphosphate (InsP3)-sensitive ER Ca2+ stores through the phospholipase C–InsP3 pathway. After [Ca2+]i release, PAF initiates Ca2+ entry [8 ] via SOCE [9 ]. Simultaneously, chemoattractants are able to induce an intracellular, biphasic pH change, a fast and transient acidification period followed by a sustained, intracellular alkalinization [10 ]. The balance between cytosolic proton loading and extrusion is critical, as many neutrophil functions, including microbiocidal behavior, cell migration, intracellular oxidant generation, tumor cell cytotoxicity, and azurophil granule exocytosis, are pH-dependent [11 12 13 ]. The intracellular pH (pHi) changes, induced by chemoattractants such as fMLP, leukotriene B4 (LTB4), PAF, and C5a, are controlled mostly by an amiloride-sensitive Na+/H+ exchanger (NHE) [10 , 14 , 15 ]. The signal transduction pathways involved in the NHE activation in neutrophils are poorly known [16 ]. Several authors have proposed that Ca2+ could also be involved in the pHi changes in liver cells, platelets, and eosinophils [17 18 19 ]; however, in human neutrophils, other authors have shown that changes in [Ca2+]i do not affect the NHE function [20 ]. Previously, we suggested that PAF induces intracellular alkalinization through NHE via Akt/protein kinase B and ERK1/2 phosphorylation in neutrophils, as this effect upstream is regulated by the PAF receptor, pertussis toxin-sensitive G protein, and tyrosine kinase protein [10 ].
At present, studies examining the relationship among SOCE, PI-3K–MAPK, and pHi changes in neutrophils are not available. In the present study, using several putative SOCE inhibitors, we support the evidence that ERK1/2, Akt phosphorylation, and the control of intracellular alkalinization are dependent on capacitative Ca2+ entry in bovine neutrophils.
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Neutrophil isolation
Adult Holstein cows were obtained from the university herd. The cattle were maintained on an ad lib grass diet with grain supplementation. All experiments were conducted in accordance with institutional review board-approved protocols. Blood was collected by jugular venipuncture, and PMNs were isolated according to the method of Roth and Kaeberle [21
]. Briefly, following collection into acid citrate dextrose tubes (Becton Dickinson, San Jose, CA, USA), the blood was gently rocked for 5 min (Nutator, Becton Dickinson) and then centrifuged at 1000 g at 20°C for 20 min. The plasma and buffy coat were aspirated, and the remaining RBC and PMN pellet were resuspended in HBSS. The RBCs were removed by flash hypotonic lysis using a pH 7.2, cold, phosphate-buffered water solution (5.5 mM NaH2PO4, 8.4 mM HK2PO4). Once returning to isotonicity using a pH 7.2, hypertonic phosphate-buffer solution (5.5 mM NaH2PO4, 8.4 mM HK2PO4, 0.46 M NaCl), the sample was centrifuged at 600 g at 20°C for 10 min. The remaining PMN pellet was then washed three times with HBSS.
Viability was determined by trypan blue exclusion and was never less than 97%. Purity was at least 94%, as assessed by light microscopy following cytospin and differential staining.
Dye loading
Neutrophils were suspended in HBSS at a concentration of 1 x 107 cells/ml, incubated with 1 µM Fura-2-AM (Molecular Probes) for 30 min at 37°C, and washed twice. Specimens were divided into 2 x 106 cells/ml aliquots and placed on ice in the dark until ready for use. Just before each experiment, individual aliquots were incubated at 37°C for 5 min. Cells were then pelleted by centrifugation at 350 g for 2 min and resuspended in 2 ml HEPES buffer [20 mM HEPES, pH 7.2, 140 mM NaCl, 10 mM glucose, 1 mM KCl, 1 mM Ca2+ chloride (CaCl2), 1 mM MgCl2, 20 mM]. In some experiments, the cells were suspended in Ca2+-free HEPES with 0.3 mM Tris-EGTA added 30 s before the experiment.
The sole exceptions were the experiments involving cationic inhibitors (Gd3+) or influx of strontium ions (Sr2+). These were performed in Ca2+-free HEPES medium and without EGTA to avoid the chelating effect on these compounds.
For pH determinations, PMNs (2x107 cells/ml) were suspended in HEPES buffer, pH 7.2, and incubated with 2.5 µM BCECF-AM (Molecular Probes) for 30 min at 37°C. The cells were then washed twice and suspended at 2 x 106 cells/ml.
Quantification of [Ca2+]i by spectrofluorometry
[Ca2+]i was monitored by measuring Fura-2-AM fluorescence at 509 nm, using 340/380 nm dual-wavelength excitation in a LS55 thermoregulated spectrofluorimeter (PerkinElmer Life Science). Cuvette temperatures were kept at 37°C with constant stirring. Calibration was performed at the end of each experiment by the addition of 100 µM digitonin (Molecular Probes) for the maximum fluorescence ratio and then 15 mM EGTA for the minimum fluorescence ratio. The autofluorescence was measured in the cellular suspension during 200 s under the same experimental conditions but without probe, and the value was subtracted from the total fluorescence.
[Ca2+]i was then calculated from the 340/380-nm excitation fluorescence ratio (Kd=220 nM) according to the methods of Grynkiewicz et al. [22 ]. Dye leakage was small and had no influence on [Ca2+]i calculations. The study sequence of the inhibitors was alternated to avoid bias related to duration of dye loading or time of cell study.
Modifications of these methods were used to assess the apparent influx of [Sr2+]i, according to Itagaki et al. [23 ].
Measurement of Ca+2, Sr+2, and barium ion (Ba2+) influx
To assess SOCE, Fura-2-AM-loaded neutrophils (2x106 cells/ml) were suspended in Ca2+-free HEPES buffer, and 0.3 mM EGTA was added to each cuvette 30 s before the experiment. Following 5 min of incubation with 1 µM Tg and after 200 s of recording, three pulses of 1 mM Ca2+ or Sr2+ were administered and recorded during 600 s. To demonstrate that PAF activates SOCE, Fura-2-AM-loaded neutrophils (2x106 cells/ml) were suspended in Ca2+-free HEPES and stimulated with 100 nM PAF after 200 s of recording. Later, three pulses of Ca2+ or one pulse of Sr2+ or Ba2+ were added. Gd3+ was incubated for 10 min and used in some experiments to demonstrate that the Ca2+, Sr2+, or Ba2+ influx is SOCE-dependent. The Sr2+ and Ba2+ influx was recorded as a 340:380 ratio at 509 nm emission.
Measurement of manganese ion (Mn2+) influx
Neutrophils were loaded with Fura-2-AM as described above for Ca2+. Quenching of Fura-2-AM fluorescence in 1 x 106 cells/ml was recorded at 360 nm excitation and 505 nm emission wavelengths at 37°C under constant stirring. Where indicated, cells were incubated with 1, 5, and 10 µM Gd3+ for 10 min and 1 µM Tg for 3 min before adding 1 mM Mn2+ chloride (MnCl2).
Assessment of SOCE putative inhibitors on Ca2+ influx
To assess the effect of SOCE putative inhibitors, different approaches were used. First, Fura-2-AM-loaded neutrophils (2x106 cells/ml) were suspended in HEPES buffer without Ca2+, and 0.3 mM EGTA was added to each cuvette 30 s before the experiment. After 60 s of recording, 1 µM Tg, a pulse of 2 mM CaCl2 at 250 s, and 100 µM each putative inhibitor at 400 s were added.
Second, Fura-2-AM-loaded neutrophils (2x106 cells/ml) were suspended in HEPES with Ca2+ buffer, then the cells were preincubated in the presence of the inhibitors for 10 min, and 60 s later, 100 nM PAF (Fig. 1a ) or 1 µM Tg (Fig. 1b) was added. Third, 2 x 106 cells/ml were suspended in Ca2+-free HEPES buffer, preincubated in the presence of the inhibitors for 10 min, and then 0.3 mM EGTA was added to each cuvette 30 s before the experiment. After 60 s of recording, 100 nM PAF was added, and at 200 s, a pulse of 1 mM CaCl2 was given and registered for 340 s (Fig. 1c) .
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Figure 1. Analysis of area under the curve (AUC) in Fura-2-AM- and BCECF-AM-loaded bovine neutrophils. Illustration of the [Ca2+]i signaling AUC assessed for 340 s in response to 100 nM PAF stimulation (AUC340). [Ca2+]i sprectrofluorometric recordings from a single bovine PMN isolate, with the area to be integrated hatch-marked, are shown. Note that the area above baseline [Ca2+]i is used rather than the area above the x-axis, as the aim was to assess net response to the agonist rather than total Ca2+ activity. Three different protocols are shown: Neutrophils incubated in the presence of Ca2+ and the effect of 100 nM PAF (a) or 1 µM Tg (b) were recorded. One set of experiments was developed in the absence of external Ca2+ and in the presence of 0.3 mM EGTA; 100 nM PAF was added, followed by a 2 mM Ca2+ pulse (c). The AUC520 of pHi was measured as the area above the maximum acidification during 520 s to include the total pHi recovery (d).
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ERK1/2, p38 MAPK, pAkt immunoblot
A fixed number of PMNs (5x106) were incubated with 100 nM PAF for 2 min at 37°C. In some experiments, neutrophils were preincubated in HBSS without Ca2+ and in the presence of 0.3 mM EGTA for 1 min and 50 µM BAPTA-AM for 30 min afterward, were stimulated with 100 nM PAF. In another set of experiments, the cells were preincubated in HBSS with 0.9 mM CaCl2 and in the presence or absence of SOCE inhibitors, incubated for 10 min. They were then treated with PAF for 2 min, and the cells were lysed in buffer [50 mM Tris-HCl, pH 7.4, 50 mM EDTA, 1 mM EGTA, protease inhibitors (leupeptin, aprotinin, and pepstatin), 10 µg/ml, 25 mM NaF, 2 mM NaVO4, 0.1 mM PMSF, 25 mM DTT, and 1.5% Triton X-100]. The proteins (50 µg) were resolved in 12% SDS/PAGE and transferred to a nitrocellulose membrane. Phosphorylation of ERK1/2, p-p38 MAPK, and pAkt was analyzed with antibodies anti-p-ERK1/2, anti-p-p38 MAPK, and anti-pAkt, according to the manufacturer’s instructions. Detection was done with an ECL system. As a control for p-ERK or p-p38 MAPK, the antibody was removed by incubation with a stripping solution (100 mM 2-ME, 2% SDS, 62.5 mM Tris–HCl, pH 6.7) for 2 h at 50°C with agitation, followed by several washes with TBS-Tween 0.1%. The membrane was then incubated with anti-ERK or anti-p38 MAPK antibody at a dilution of 1:5000 using a procedure similar to that described above. In addition, total p38 MAPK was used as control for pAkt.
Data analysis
[Ca2+]i responses were expressed as the maximum 
peak [Ca2+]i (nM) measured between the maximum peak induced by PAF and basal register of [Ca2+]i without stimuli (Fig. 1a)
, and the integrated AUC in nM x s, corresponding to the [Ca2+]i response AUC340 after stimulation and above the mean baseline [Ca2+]i (Fig. 1a
1b
1c)
. The maximum peak [Ca2+]i induced by PAF is a reproducible assessment of initial Ca2+ increase, more associated with intracellular store release [24
]. AUC340 represents the total net [Ca2+]i flux during the experiment and would be more representative of SOCE changes [9
]. AUC measurements diminish the effect of artifacts on assessments and avoid the use of curve-smoothing programs. The pHi changes induced by PAF were registered as maximum difference of intracellular acidification and intracellular alkalinization ( pHi), measured between the maximum response recorded and the basal value, previous PAF stimuli. The pHi recovery AUC was also measured after the maximum intracellular acidification and during 520 s (Fig. 1d)
. All this analysis was done using FL WINLAB 4.00.02. software (PerkinElmer Life Science).
The results are shown as mean ± SEM. A one-way ANOVA was performed, and a Dunnett’s multiple comparison test was applied using a significance level of 5%.
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Figure 2. Ca2+ chelating interferes with Ca2+ movement and pHi changes. The effect of 100 nM PAF in Fura-2-AM (a) or BCECF-AM (b) loaded neutrophils in the presence of Ca2+ is shown. The effect of free Ca2+ solution plus 0.3 mM EGTA (c and d) and Ca2+-free/0.3 mM EGTA plus 50 µM BAPTA-AM (e and f) on Ca2+ movements and pHi changes is shown. (g) The effect of EGTA and BAPTA-AM on the maximum pHi peak of acidification and alkalinization induced by 2 µM ionomycin is shown. Each bar represents the mean ± SEM of at least four independent experiments. **, P < 0.01, compared with control.
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pHi of acidification could be associated with [Ca2+]i release [25
]. To support this suggestion, we used ionomycin, a widely known ionophore, which induces [Ca2+]i release and SOCE, confirming that in the absence of Ca2+ and in the presence of 5 mM EGTA, only the intracellular alkalinization but not the acidification is reduced. As previously suggested for PAF, 50 µM BAPTA-AM also reduced the intracellular acidification induced by ionomycin (Fig. 2g)
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Tg or PAF induces SOCE in neutrophils
Ca2+ ATPase inhibitor Tg is able to induce influx of Ca2+ through SOCE by blocking ER Ca2+ re-uptake, thus elevating [Ca2+]i and depleting ER Ca2+ stores [26
, 27
]. In Fura-2-AM-loaded and Tg-treated neutrophils, an increase of Ca2+ influx is observed (Fig. 3a
). In human neutrophils, SOCE is highly sensitive to inhibition by lanthanides such as Ga3+ [23
]. Ca2+ influx was reduced significantly by 10 µM Gd3+ at three pulses of Ca2+ in Tg-treated bovine neutrophils; however, this inhibition was partial (Fig. 3a)
. SOCE can increase the influx of other divalent cations such as Sr2+; in fact, previous studies have shown that cells transfected with transient receptor potential 3 (TRP3) had enhanced permeability to Sr2+ after store depletion [28
]. Sr2+ has a lesser affinity than Ca2+ for Fura-2-AM, and its binding causes less fluorescence; however, its isobestic point and 340:380 ratio profile are similar [29
]. Clearly in Fura-2-AM-loaded and Tg-treated bovine neutrophils, an increase of Sr2+ influx was observed, and Gd3+ was able to inhibit this response almost completely at micromolar concentration only (Fig. 3c)
. However, this is not sustained, as Sr2+ can be pumped into the store, thus declining the influx of the divalent cation. For this reason, Ca2+ influx was monitored by following the quenching of intracellular Fura-2, which results from the entry of Mn2+ through the Ca2+ channels. As expected, the addition of Tg to a suspension of neutrophils loaded with Fura-2 increased within 
40 s the rate of quenching of the Ca2+ probe (Fig. 3e)
. Moreover, Gd3+ reduced the influx of Mn2+ in a dose-dependent manner.
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Figure 3. Gd3+ inhibits Ca2+, Sr2+, Mn2+, and Ba2+ influx in bovine neutrophils. A representative spectrofluorometry recording of the 10-µM Gd3+ effect on the influx of 1 mM Ca2+ and Sr2+ in neutrophils pretreated with Tg (a and c) or PAF (b and d) is shown. SrCl2, Sr2+ chloride. A representative spectrofluorometry recording of the effect of Gd3+ on one pulse of 1 mM MnCl2 in neutrophils treated with Tg (e) and on one pulse of 10 mM Ba2+ after 100 nM PAF (f) is depicted.
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Putative SOCE inhibitors 2-APB, capsaicin, and flufenamic acid consistently reduce the Ca2+ influx induced by PAF or Tg
We next tested the effect of different putative SOCE inhibitors 2-APB, capsaicin, and flufenamic acid in bovine neutrophils treated with PAF and Tg. 2-APB is considered a potent inhibitor of SOCE [30
]. 2-APB (75 µM) reduces the Sr2+ more strongly than Ca2+ influx induced by ionomycin in human neutrophils [23
], whereas 0.1 µM 2-APB completely abolished the Ca2+ influx induced by 10 nM PAF [9
]. Similarly, capsaicin has been described as a putative SOCE inhibitor in neutrophils by also reducing the superoxide production induced by PAF [24
]. Flufenamic acid is a nonsteroidal, anti-inflammatory drug from the fenamates family with SOCE-blocking properties in neutrophils [31
] and lymphocytes [32
].
We developed three experimental protocols (see Materials and Methods) to assess the drug efficacy. In the absence of external Ca2+, Tg or PAF induces a mild increase in [Ca2+]i; however, a strong Ca2+ influx by the SOCE mechanism is observed after a Ca2+ pulse [23 , 24 ]. We added 1 µM Tg to bovine neutrophils in the absence of external Ca2+, and after a period of 200 s, a pulse of 2 mM Ca2+ was included. Clearly, Tg activated the influx of external Ca2+, and after a period of equilibration, 2-APB and to a lesser extent, capsaicin were able to reduce the influx of Ca2+ (Fig. 4a 4b 4c 4d ).
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Figure 4. SOCE inhibitors reduce Ca2+ influx induced by Tg. A representative spectrofluoremetry recording of neutrophils treated during 10 min with the vehicle (a), 100 µM 2-APB (b), 100 µM capsaicin (CAP; c), and 100 µM of flufenamic acid (FFA; d) is shown. The neutrophils were stimulated with Tg, and after 150 s, one pulse of 1 mM CaCl2 was added.
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Figure 5. SOCE inhibitors reduced AUC340 of Ca2+ influx induced by Tg. A representative spectrofluoremetry recording of [Ca2+]i movements induced by Tg in neutrophils is shown. The cells were treated for 10 min with the vehicle, 100 µM 2-APB, 100 µM capsaicin, or 100 µM flufenamic acid, followed by the stimulation with 1 µM Tg (a). A characteristic dose response, inhibitory effect of 2-APB, capsaicin, and flufenamic acid on Tg was observed as AUC340 (b). Each bar represents the mean ± SEM of at least four independent experiments. *, P < 0.05; **, P < 0.01, compared with "0" (solid bar).
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Figure 6. SOCE inhibitors reduce Ca2+ influx and AUC340 induced by PAF. A representative spectrofluorimeter recording of the 2-APB, capsaicin, and flufenamic acid effect on the Ca2+ influx is shown. The cells were stimulated with 100 nM PAF in Ca2+-free HEPES/0.3 mM EGTA, and then a 2-mM CaCl2 pulse was added (a), or [Ca2+]i movements were induced by 100 nM PAF in HEPES 1 mM CaCl2 (b). Dose response of putative SOCE inhibitors on AUC340 and peak of [Ca2+]i induced by PAF in Ca2+-free HEPES/0.3 mM EGTA (c and e) or in HEPES, 1 mM CaCl2 solution (d and f), respectively, is shown (see Materials and Methods for details). Each bar represents the mean ± SEM of at least four independent experiments. *, P < 0.05; **, P < 0.01, compared with "0" (solid bars).
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SOCE controls the alkalinization and did not affect the intracellular acidification induced by PAF
As a result of the fact that 0.3 mM EGTA reduced the pH recovery in neutrophils treated by PAF, we used the putative SOCE inhibitors to assess the role of Ca2+ influx on intracellular alkalinization. 2-APB and flufenamic acid reduced, in a dose-dependent manner, the AUC520 of the pH recovery in BCECF-AM-loaded cells treated with PAF 100 nM (Fig. 7a
) and did not affect the maximum intracellular acidification (Fig. 7b
and 7c)
. Capsaicin (10 µm) significantly reduced the pH recovery measured as AUC520 (Fig. 7a)
; however, this was not observed as pHi changes (Fig. 7d)
and did not affect the maximum intracellular acidification. This could be attributed to a reduction in the pH recovery velocity observed only at 10 µM capsaicin. SK&F 96365 reduced the pHi of acidification in a dose-dependent manner and partially reduced the pHi of alkalinization (Fig. 7e)
. It is probable that SK&F 96365 can affect pHi by the inhibition of Ca2+ influx or release from an intracellular store. BTP2 did not produce modifications on pHi (Fig. 7f)
.
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Figure 7. Effect of SOCE inhibitors on AUC520 pHi changes and maximum pHi changes induced by PAF. The inhibitory effect of 2-APB, flufenamic acid, and capsaicin on the AUC520 pHi changes induced by 100 nM PAF in neutrophils is shown (a). The neutrophils were incubated in HEPES solution with 1 mM CaCl2 and the inhibitor by 10 min (see Materials and Methods for details). The effect of 2-APB (b), flufenamic acid (c), capsaicin (d), SK&F 96365 (e), and BTP2 (f) on the maximum pHi peak of acidification and alkalinization induced by 100 nM PAF is depicted. Each bar represents the mean ± SEM of at least four independent experiments. *, P < 0.05; **, P < 0.01, compared with "0."
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100 µM induced an increase in ERK1/2 phosphorylation at 2 min, independent of Ca2+ fluxes (data not shown). ERK1/2 and Akt phosphorylation are upstream controlled by PI-3K in bovine neutrophils activated by PAF, suggesting that PI-3K–ERK1/2 are closely related [10
]. In murine bone marrow-derived mast cells activated by IgE, Akt phosphorylation is dependent on the influx of external Ca2+ and reduced by 2-APB [34
]. We demonstrated that 2-APB, capsaicin, and flufenamic acid significantly reduced the Akt phosphorylation induced by 100 nM PAF in neutrophils (Fig. 8c)
, indicating a potential role of SOCE in this pathway.
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Figure 8. Effect of Ca2+ chelating on ERK1/2 phosphorylation and SOCE inhibitors on MAPK and Akt phosphorylation. The effect of free Ca2+/0.3 mM EGTA and 50 µM BAPTA-AM on ERK1/2 phosphorylation induced by 100 nM PAF is shown (a). The effect of 2-APB, capsaicin, and flufenamic acid on ERK1/2 (b), Akt, and p38 MAPK (c) phosphorylation induced by PAF in HEPES 1 mM CaCl2 is shown. Neutrophils were pretreated with 2-APB, capsaicin, and flufenamic acid (10–100 µM) for 10 min and then stimulated with 100 nM PAF for 2 min. ERK1/2, Akt, and p38 MAPK phosphorylation was detected by immunoblotting (see Materials and Methods for details). The mean ± SEM of four independent experiments is shown. *, P < 0.05; **, P < 0.01, compared with "0" (solid bars).
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We demonstrated that EGTA reduced Ca2+ influx and simultaneously inhibited the intracellular alkalinization and ERK1/2 phosphorylation in PAF-treated bovine neutrophils. Recently, we described that ionomycin increased ERK1/2 phosphorylation, and this was reduced successfully by EGTA, suggesting the dependence of a Ca2+ influx in MAPK activation [25 ]. Other authors demonstrated that the absence of external Ca2+ and EGTA reduces the Ca2+ influx induced by fMLP in human neutrophils [40 ]; however, this condition did not affect the ERK1/2 phosphorylation induced by 2 µM PAF or 1 µM fMLP [41 ]. This discrepancy could be associated with the existence of two PAF receptors with different affinity proposed in human neutrophils [42 ]. We stimulated PMN with 100 nM PAF, where maximum ERK1/2 phosphorylation is observed in bovine neutrophils [10 ]. Furthermore, we described that bovine PMN possess only one high-affinity receptor [43 ]. BAPTA-AM also inhibited the ERK1/2 phosphorylation and reduced the intracellular acidification, indicating that the intracellular source of Ca2+ has an important role in the intracellular acidification phase induced by PAF and ionomycin. Similar findings have been described before in bovine neutrophils activated by propionate [25 ].
Clearly Tg activated SOCE by increasing the entry of Ca2+, Sr2+, and Mn2+, and it was successfully reduced by Gd+3, an effective SOCE inhibitor. Gd+3 inhibited the influx of Sr2+ more strongly than Ca2+ in bovine neutrophils. In human neutrophils, 10 µM Gd3+ blocked the influx of Ca2+ almost completely after fMLP or Tg treatment; however, at nanomolar concentration, Gd3+ is a more selective Sr2+ influx inhibitor [23 ]. Therefore, this suggests that SOCE depends on at least two divalent cation influx pathways in human neutrophils: One of these is nonspecific and Sr2+-permeable; the other is Ca2+-specific [23 ]. We observed that Sr+2 influx was not sustained; this can be explained, as Sr2+ is pumped to an intracellular store, reducing the cation influx [44 ]. If Tg induces the SOCE phenomenon, it is also predictable that Mn2+ influx into the neutrophils would be increased. We confirmed the increase of Mn2 influx following Tg treatment and demonstrated that Gd3+ reduced SOCE. The SOCE response was also activated by PAF, increasing the Ca2+ and Sr2+ influxes, and inhibited by Gd3+ in bovine neutrophils. Finally, we tested the Ba2+ entry, as it has been demonstrated that Ba2+ influx is SOCE-dependent and a poor substrate for Ca2+-pumping ATPases. A sustained influx of Ba2+ was induced by PAF and inhibited by Gd3+, thus corroborating the existence of SOCE.
We assessed several putative SOCE inhibitors such as 2-APB, capsaicin, and flufenamic acid. These compounds also reduced the influx of Ca2+ induced by Tg and PAF, thus confirming the existence of SOCE in bovine neutrophils. 2-APB, at doses that inhibited the AUC340, did not reduce the [Ca2+]i peak (
50 µM); therefore, these data suggest that 2-APB is a potent SOCE inhibitor in bovine neutrophils. It has been described that 2-APB reduces the influx of Ca2+ induced by PAF [9
] or fMLP [23
] in human neutrophils; however, these authors proposed that the effect of 2-APB is by inhibiting InsP3 receptors from the ER. Nevertheless, several reports indicate that the principal antagonistic effect of 2-APB is on Ca2+ entry rather than Ca2+ release, and for this reason, this compound is also used as SOCE inhibitors in nonexcitable cells [30
]. Our results showed that the 2-APB response to higher concentrations (100 µM) inhibits the peak of [Ca2+]i only partially, suggesting a more specific effect on bovine neutrophil SOCE than in humans. In connection with this, it has been described in neutrophils treated with fMLP that 100 µM 2-APB can reduce the Ca2+ influx and release from an intracellular store simultaneously [40
]. Moreover, other authors demonstrated that 100 µM 2-APB or 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (a drug, which prevents store-emptying and hence, abolishes SOCE) inhibits the Ca2+ store release from ER completely, interfering with SOCE in human neutrophils stimulated with PAF [9
]. We demonstrated that capsaicin reduced the AUC340 but not the [Ca2+]i peak induced by PAF. Capsaicin is a well-known SOCE inhibitor in neutrophil-like HL-60 cells; moreover, this compound can interfere with the superoxide production induced by PAF [24
], a SOCE-dependent response [6
]. Flufenamic acid also reduced SOCE in bovine neutrophils induced by PAF and Tg. This inhibition of SOCE has been described earlier in human neutrophils treated with fMLP, A23187, and scarcely with Tg [4
, 31
]. However, at higher doses (100 µM), flufenamic acid can reduce the maximum peak of [Ca2+]i in the presence or absence of external Ca2+, thus indicating a possible effect on [Ca2+]i. However, we did not discard that part of the [Ca2+]i peak could be dependent on the external influx, as reported earlier [31
].
Recent work has involved Ca2+ channel proteins from the TRP superfamily in the mediation of SOCE in human neutrophils [45 46 47 ]. Several reports suggest that 2-APB can inhibit a number of TRP channels at micromolar concentrations [48 49 50 ], proteins normally expressed on the PMN cell membrane [45 , 51 ]. Flufenamic acid can also reduce the Ca2+ entry via TRP inhibition [52 53 54 ]; however, some authors have demonstrated an increase in Ca2+ entry by the same channels [52 , 55 ]. 2-APB or flufenamic acid could reduce SOCE by other mechanism, independent of TRP channels. Recently, two new proteins, stromal interacting molecule 1 (the ER Ca2+ sensor) and Orai1 (the Ca2+ channel), have been suggested as the missing links in SOCE [56 ].
BTP-2 has been described as a SOCE inhibitor in neutrophils at doses of 10 µM [6 ]; however, we observed in bovine neutrophils that BTP2 did not reduce the AUC340 or maximum [Ca2+]i peak induced by PAF. On the contrary, it produced an increase of the [Ca2+]i peak in the absence of external Ca2+, thus suggesting an effect on [Ca2+]i release. Another SOCE putative inhibitor described in neutrophils is SK&F 96365 [57 , 58 ]; this compound reduced the [Ca2+]i peak at doses of 25 µM in the presence or absence of external Ca2+ but only partially inhibited the AUC340 induced by PAF (data not shown). Today, SK&F 96365 is considered a nonselective SOCE inhibitor, as it also produces inhibition of voltage-operated Ca2+ entry, receptor-operated Ca2+ entry, and [Ca2+]i release [58 ] and shows a contradictory effect, such as Ca2+ influx or [Ca2+]i increase [59 ].
PAF can modulate the pHi simultaneously with the [Ca2+]i movement; however, the relationship between these signals has been poorly studied. One report has proposed the existence of a Ca2+/H+ exchanger controlling pHi in human neutrophils stimulated with immune complex, suggesting that the inactivation of this exchanger by EGTA reduces the intracellular alkalinization and increases the intracellular acidification [60 ]. However, we found that EGTA reduced the intracellular alkalinization but did not produce intracellular acidification induced by ionomycin in bovine neutrophils, suggesting a specific role of SOCE in the intracellular alkalinization. In support of this, ionomycin produced a depletion of the [Ca2+]i store, followed by SOCE activation in bovine PMN [25 ]. This assumption was confirmed using putative SOCE inhibitors. 2-APB and flufenamic acid reduced, in a dose-dependent manner, the alkalinization induced by PAF in bovine neutrophils, suggesting that SOCE is key in the regulation of alkalinization. Moreover, we demonstrated that 2-APB and flufenamic acid reduced the phosphorylation of ERK1/2 and Akt induced by PAF. These proteins have been involved in pHi recovery via NHE after PAF, ionomycin, or propionic acid treatments in bovine neutrophils [10 , 25 ]. 2-APB partially reduced at higher concentrations (100 µM) the phosphorylation of p38 MAPK and the [Ca2+]i peak, suggesting a close dependence of ER Ca2+ store release in this pathway. Previously, we proposed that p38 MAPK activation is associated with [Ca2+]i release in neutrophils treated with propionate [25 ]. In support of this, BAPTA-AM strongly reduced the p38 MAPK phosphorylation induced by ionomycin, fMLP, and PAF in human neutrophils [41 ]
Capsaicin (10 µM) slightly reduced the alkalinization following PAF stimulation; however, this was more associated with a reduction in the rate of the pHi recovery, as it reduced the AUC520 but did not reduce the alkalinization maximum pHi. This lack of effect is correlated with the lack of ERK1/2 phosphorylation inhibition; however, an inhibition of Akt phosphorylation was shown. We observed that capsaicin induced ERK1/2 phosphorylation (data not shown), and therefore, it could explain these contradictory results. Moreover, other authors suggest that capsaicin at higher doses can activate neutrophils, stimulating non-SOCE [33
]. Our results corroborate that an inhibition of ERK1/2 phosphorylation would be necessary to reduce NHE activity and the intracellular alkalinization induced by PAF [10
]; thus, this mechanism could contribute to the control of the inflammatory process. In addition, several lines of evidence suggest a relevant role of NHE in inflammation, regulating chemotaxis [61
] or controlling the LTB4, prostaglandin, and cytokine production in leukocytes [37
] via NF-
B activation [62
].
The strong inhibition of Akt phosphorylation induced by 2-APB, capsaicin, and flufenamic acid in PAF-stimulated neutrophils could affect other cellular functions. In these cells, PI-3K/Akt plays an important role in reactive oxygen species production [63 , 64 ] and survival [65 ]. This mechanism would explain the superoxide-decreased production and apoptosis observed by the inhibition of Ca2+ influx in neutrophils [6 , 24 , 66 ].
In conclusion 2-APB and flufenamic acid were the most consistent SOCE inhibitors; these compounds inhibited the intracellular alkalinization and Akt and ERK1/2 phosphorylation induced by PAF. The inhibition of SOCE by 2-APB could represent an important tool to characterize Ca2+ signaling in neutrophils.
Received March 31, 2007; revised June 27, 2007; accepted July 4, 2007.
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receptors and tyrosine kinases J. Cell Biol. 132,1037-1052
(1)-adrenoceptor-activated Ca(2+)-permeable cation channel Circ. Res. 88,325-332]B: an unsung crosstalk and missing link between fluid dynamics and oxidative stress-related inflammation—controversy or pseudo-controversy? Biochem. Biophys. Res. Commun. 327,373-381[CrossRef][Medline]This article has been cited by other articles:
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