Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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(Journal of Leukocyte Biology. 2002;71:611-617.)
© 2002 by Society for Leukocyte Biology

Capacitative Ca2+ influx and activation of the neutrophil respiratory burst. Different regulation of plasma membrane- and granule-localized NADPH-oxidase

Daniel Granfeldt*, Marie Samuelsson{dagger} and Anna Karlsson*

The Phagocyte Research Laboratory, Departments of
* Rheumatology, and
{dagger} Medical Microbiology and Immunology, University of Göteborg, Sweden

Correspondence: Anna Karlsson, The Phagocyte Research Laboratory, Department of Rheumatology, Guldhedsgatan 10, S-413 46 Göteborg, Sweden. E-mail: anna.karlsson{at}microbio.gu.se


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The neutrophil NADPH-oxidase may be activated in the plasma membrane, resulting in release of oxygen metabolites extracellularly, or in the granule or phagosomal membranes, giving intracellular production of oxidants. An increase in [Ca2+]i mediated through binding of fMLF to its receptor is part of a signaling cascade that activates the plasma membrane-localized oxidase. In contrast, a rise in [Ca2+]i induced by a Ca2+ ionophore results in activation of the intracellular pool of oxidase. We mimicked fMLF-induced emptying of intracellular Ca2+ stores with thapsigargin. This induced a pronounced intracellular oxidase activity but no extracellular release of oxidants. The thapsigargin-induced effect was dependent on capacitative Ca2+ influx, because the effect was inhibited dose-dependently by EGTA and the Ca2+ channel blocker La3+. At La3+ concentrations between 200 and 400 µM, thapsigargin also induced a massive extracellular production of superoxide anion. No other channel blockers tested induced a similar effect. We conclude that elevation in [Ca2+]i by capacitative Ca2+ influx induces NADPH-oxidase activation at an intracellular site. Further, activation of the plasma membrane-localized NADPH-oxidase is regulated by a more complex Ca2+ signaling, involving capacitative Ca2+ influx and possibly the specific action of La3+-sensitive Ca2+ channels.

Key Words: thapsigargin • signal transduction • fMLF • [Ca++]i


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophils are of major importance in the innate protection of the host against microbial infections. Apart from the antibacterial defense exerted by the wide array of bactericidal proteins and peptides stored in the neutrophil granules and released into the phagosome upon phagocytosis of a prey, the production of oxygen free radicals (superoxide anion and hydrogen peroxide) is of utmost importance. This respiratory burst is performed by the phagocyte NADPH-oxidase, an enzyme system that transfers electrons from cytosolic NADPH to oxygen on the opposite side of the membrane. For the NADPH-oxidase to become activated, a stimulus has to induce a signal transduction cascade that in turn causes the translocation of several cytosolic oxidase components to the membrane-bound part of the oxidase, the b-cytochrome [1 ]. Assembly of the NADPH-oxidase in the plasma membrane leads to release of the superoxide anion to the extracellular compartment or into a preformed phagosome. However, 80–85% of the b-cytochrome is localized in granule membranes [2 ], and increasing evidence shows that the NADPH-oxidase can be activated in these membranes [3 4 5 6 7 ] and that the produced oxygen metabolites are not released extracellularly but are retained in the cell [3 , 7 ] (for a review, see ref. [8 ]). Neutrophils are not only of importance in the defense toward infection but may also be a potential threat to the host by inducing pathophysiological effects. The same mechanisms that are responsible for bacterial killing, including oxygen radical formation, are also involved in the destruction of endogenous, healthy tissue [9 ]. The localization of the formed radicals most probably influences the potential degree of tissue damage and subsequently, the outcome of the inflammatory process [8 ].

Different stimuli induce different patterns of extracellular versus intracellular production of superoxide anion, suggesting a diversity in the regulating pathways leading to activation of the plasma membrane-bound and the granule-localized pools of NADPH-oxidase, respectively [3 , 10 11 12 ]. Direct activation of protein kinase C (PKC) by phorbol myristate acetate (PMA) induces assembly of the oxidase both in the plasma membrane and the specific granule membrane [3 ]. Recently, we have demonstrated that the intracellular oxidase activity triggered by PMA is dependent on phosphatidylinositol 3-kinase (PI 3-kinase) activity in contrast to the superoxide release that is induced without PI 3-kinase activation [13 ]. Thus, the PKC-dependent signal-transduction pathway may be involved in differential regulation of the two NADPH-oxidase pools.

The best-characterized signal-transduction event leading to activation of the neutrophil respiratory burst is that induced by agonists binding to G-protein-coupled receptors activating phospholipase C (PLC), which cleaves phosphatidylinositol 3,4 bisphosphate (PIP2) into diacylglycerol (DAG), a PKC agonist, and inositol 1,4,5-trisphosphate (IP3) [14 ]. IP3 induces [Ca2+]i elevations by releasing Ca2+ from intracellular stores into the cytoplasm. This Ca2+ release is followed by an influx of Ca2+ over the plasma membrane, a process termed store-operated or capacitative Ca2+ influx (reviewed in ref. [15 ]). The involved Ca2+ channels are subsequently called store-operated cation channels (SOCs). This is probably the predominant type of Ca2+ influx in neutrophils, since voltage-activated Ca2+ channels are absent in inflammatory cells [16 , 17 ].

The early work by Pozzan et al. [18 ] stated that a [Ca2+]i elevation is not sufficient for activation of the NADPH-oxidase. However, the technique to determine oxygen radical production used by Pozzan et al., as well as by many other investigators [18 19 20 ], does not allow for radicals to be measured at an intracellular site. In fact, the [Ca2+]i increase induced by a Ca2+ ionophore is sufficient to achieve an intracellular production of oxygen radicals [7 ]. Thus, it would be of great interest to investigate the roles played by intracellular Ca2+ stores and Ca2+ influx in regulating NADPH-oxidase activity.

Ligand-induced emptying of intracellular Ca2+ stores can be mimicked by using thapsigargin [21 22 23 ], an inhibitor of the Ca2+-ATPase that is responsible for refilling these stores [24 ]. In neutrophils, thapsigargin induces a rapid depletion of the stores, subsequently leading to Ca2+ entry through the plasma-membrane channels. In combination with Ca2+ channel inhibitors and extracellular Ca2+ chelators, thapsigargin can thus be used to study the participation of Ca2+ in various cellular processes. Using this approach, we found that the granule-localized NADPH-oxidase is activated by thapsigargin alone, through the induction of capacitative Ca2+ influx. Regulation of the plasma membrane-localized NADPH-oxidase is even more complex, involving release of intracellular stores, capacitative Ca2+ influx, as well as action of La3+-sensitive Ca2+ channels.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of neutrophils
Human neutrophils were isolated from buffy coats obtained from healthy blood donors. After dextran sedimentation at 1 g, hypotonic lysis of the remaining erythrocytes, and centrifugation in a Ficoll-Paque gradient [25 ], the neutrophils were washed twice and resuspended (1x107/ml) in Krebs-Ringer phosphate buffer containing glucose (10 mM), Ca2+ (1 mM), and Mg2+ [1.5 mM; Krebs-Ringer glucose buffer (KRG), pH 7.3]. The cells were kept on melting ice until use.

Inhibitors
Econazole was diluted in dimethyl sulfoxide (DMSO) and stored in stock solution at -20°C. A fresh working solution in KRG was prepared new for every experiment. Solutions of NiCl2 and LaCl3 were prepared in KRG, fresh for every experiment.

[Ca2+]i measurements
Neutrophils (PMN; 2x107/ml) were loaded with fura-2 by incubation with the cell-permeable acetoxymethylated derivative FURA-2/AM (2 µM) in Ca2+-free KRG with 0.1% bovine serum albumin at room temperature for 30 min. After incubation, the cells were washed once with RPMI and once with KRG. The cells were adjusted to a concentration of 2 x 107/ml and kept in KRG at room temperature until use.

The fura-2 fluorescence was measured with a luminescence spectrometer (LS50B, Perkin Elmer Corp., Wellesley, MA), thermostatized to 37°C at 340 nm excitation and 510 nm emission wavelength. After preincubation of the neutrophils (2x106/ml) for 5 min at 37°C in the presence or absence of La3+ (50–500 µM) or ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid (EGTA; 5 mM), respectively, thapsigargin (300 nM) was added, and fluorescence was measured for 700 s. The extracellular Ca2+ concentration was 1 mM throughout the experiments.

Superoxide production
The superoxide anion produced by the neutrophil NADPH-oxidase was determined using a luminol/isoluminol-enhanced chemiluminescence (CL) system [8 ]. The CL activity was measured in a six-channel Biolumat LB 9505 (Berthold Co., Wildbad, Germany), using disposable, 4 ml polypropylene tubes with a 0.90 ml reaction mixture containing 106 neutrophils. The tubes were equilibrated in the Biolumat at 37°C, after which the stimulus (0.1 ml) was added. The light emission was recorded continuously. To quantify intracellularly and extracellularly generated reactive oxygen species, respectively, two different reaction mixtures were used. Tubes used for measurement of extracellular release of superoxide anion contained neutrophils, horseradish peroxidase (HRP; a cell-impermeable peroxidase; 4U), and isoluminol (a cell-impermeable CL substrate; 2x10-5 M) [8 ]. Tubes used for measurement of intracellular generation of reactive oxygen species contained neutrophils, superoxide dismutase (SOD; a cell-impermeable scavenger for O2-; 50 U), catalase (a cell-impermeable scavenger for H2O2; 2000 U), and luminol (a cell-permeable CL substrate; 2x10-5 M).

Hydrogen peroxide production
Generation of hydrogen peroxide was assayed by measuring the HRP-dependent oxidation of p-hydroxyphenyl acetic acid (PHPA) by fluorometry [8 ]. Samples containing neutrophils (106/ml), PHPA (3.3 mM), and HRP (4 U/ml) in KRG were equilibrated for 10 min at 37°C, after which the stimulus was added. The increase in fluorescence intensity (excitation wavelength, 317 nm; emission wavelength, 400 nm) was measured continuously. Some samples contained NaN3 (1 mM), which inhibits the hydrogen peroxide-consuming enzymes catalase and myeloperoxidase, allowing for intracellularly produced hydrogen peroxide to leak out of the cell and be determined extracellularly.

Reagents
The PHPA, LaCl3, NiCl2, econazole, isoluminol, and luminol were obtained from Sigma Chemical Co. (St. Louis, MO). Fura-2/AM was from Molecular Probes (Eugene, OR). Catalase, SOD, and HRP were purchased from Boehringer Mannheim (Mannheim, Germany). Dextran and Ficoll-Paque were from Pharmacia (Uppsala, Sweden). thapsigargin was obtained from Calbiochem (San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
thapsigargin induces increase in neutrophil [Ca2+]i
In Ca2+-containing medium, addition of thapsigargin to neutrophils induced an initial rise in [Ca2+]i, which was followed by a sustained, elevated phase (Fig. 1 ). The amplitude of the initial response and the duration of the Ca2+ signal decreased when the extracellular Ca2+ was chelated with EGTA for 5 min prior to thapsigargin addition (Fig. 1) . The same effect was seen when EGTA was added 30 s prior to thapsigargin (unpublished results). These results are in agreement with previously published data, showing that the initial rise in [Ca2+]i is caused by emptying intracellular Ca2+ stores, while the later part of the [Ca2+]i rise and the sustained phase are a result of influx of Ca2+ from the extracellular medium [16 , 23 ]. Replacing EGTA with La3+, a known blocker of Ca2+ influx [17 , 20 , 26 ], gave a similar effect with respect to amplitude and duration of the [Ca2+]i rise (Fig. 1) .



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  		Figure 1. Time-course of the thapsigargin-induced Ca2+ response in the presence or absence of EGTA and La3+, respectively. Fura-2-loaded neutrophils (106/ml) werepreincubated for 10 min at 37°C in the presence or absence of EGTA (5 mM) or La3+ (500 µM), after which thapsigargin (300 nM) was added (indicated by the arrow). Representative Ca2+ measurements are shown, and the Ca2+ concentration is given in arbitrary units (AU). Calibration levels of maximal and minimal [Ca2+] levels are shown. To investigate the dose dependency for La3+, concentrations between 50 and 500 µM were used. At 300–500 µM, the Ca2+ influx was decreased to approximately equal extents, and little or no effect could be detected at 200 µM and below.

 
Release of intracellular Ca2+ stores induces activation of granule-localized NADPH-oxidase
It has been proposed that emptying intracellular Ca2+ stores by thapsigargin does not induce superoxide anion production in neutrophils [19 , 27 ]. However, these studies suffer from the same limitation as the study by Pozzan et al. [18 ], in that the technique used for determination of oxygen radicals only detects radicals released by the activated plasma membrane-localized NADPH-oxidase. Luminol/isoluminol-amplified CL permits the detection of extracellularly released as well as intracellularly produced oxygen radicals. Here, we used this technique to investigate the effect of Ca2+ release and influx on the two pools of NADPH-oxidase.

The thapsigargin-induced increase in intracellular [Ca2+] was accompanied by an intracellular production of superoxide anion (Fig. 2 ). The response amounted to approximately 40% of an intracellular NADPH-oxidase response to PMA. Contrarily, very little extracellular release of superoxide was detected (approximately 5% of an extracellular PMA response), indicating that the thapsigargin-induced elevation of intracellular [Ca2+] activates the intracellular pool of NADPH-oxidase specifically and not the plasma membrane-bound pool.



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  		Figure 2. Effect of thapsigargin on the neutrophil NADPH-oxidase activity. Neutrophils (106/ml) were preincubated for 5 min at 37°C in Ca2+-containing medium and were treated with thapsigargin (300 nM) at time 0. The intracellularly produced superoxide (solid line) was measured using luminol in the presence of SOD and catalase. The extracellularly released superoxide (dashed line) was measured using isoluminol-amplified CL in the presence of HRP. The amount of CL is given in Mcpm (106 cpm).

 
Capacitative Ca2+ influx is necessary for activation of the intracellular NADPH-oxidase
As discussed above, the thapsigargin-induced emptying of intracellular Ca2+ stores leads to opening plasma membrane-localized Ca2+ channels, SOCs, resulting in influx of Ca2+ over the membrane. To investigate how Ca2+ influx affects the thapsigargin-induced NADPH-oxidase activity, we used two approaches: chelation of the extracellular Ca2+ with EGTA and blocking of Ca2+ influx by a Ca2+ channel blocker. Ca2+ chelation by pretreatment with EGTA for 5 min resulted in a dose-dependent inhibition of the intracellular NADPH-oxidase activity induced by thapsigargin (Fig. 3A and 3C ). The same effect was seen when adding EGTA 30 s prior to stimulation. This effect indicates that the Ca2+ influx over the plasma membrane is necessary for the response. This was supported by the results obtained with La3+, showing that blocking the capacitative Ca2+ changes (Fig. 1) by this ion prior to thapsigargin stimulation resulted in a dose-dependent inhibition of the intracellular NADPH-oxidase activity (Fig. 4A and 4C ). Taken together, we conclude that the intracellular oxidative response is dependent on capacitative Ca2+ influx.



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  		Figure 3. Effect of EGTA on the thapsigargin-induced NADPH-oxidase activation. Neutrophils (106/ml) were preincubated in the absence or presence of EGTA at various concentrations (1, control; 2, 0.03 mM; 3, 0.1 mM; 4, 0.3 mM; 5, 1 mM) for 5 min at 37°C and were treated with thapsigargin (300 nM) at time 0. The NADPH-oxidase activity was measured as CL, and the intracellular production (A) and extracellular release (B) of superoxide anion were recorded as described in Figure 2 . (C) The dose-response curves for the extracellular (open symbols) and intracellular (closed symbols) response are shown, also giving the maximal CL responses, set to 100%.

 


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  		Figure 4. Effect of La3+ on the thapsigargin-induced NADPH-oxidase activation. Neutrophils (106/ml) were preincubated in the absence or presence of La3+ at various concentrations (1, control; 2, 50 µM; 3, 100 µM; 4, 200 µM; 5, 400 µM; 6, 600 µM; 7, 1000 µM) for 5 min at 37°C and were treated with thapsigargin (300 nM) at time 0. The NADPH-oxidase activity was measured as CL, and the intracellular production (A) and extracellular release (B) of superoxide anion were recorded as described in Figure 2 . (C) The dose-response curves for the extracellular (open symbols) and intracellular (closed symbols) response are shown, also giving the maximal CL responses, set to 100%.

 
We confirmed that all decreases in intracellular CL caused by addition of chelators and channel blockers were a result of decreases in intracellular oxidants by using the PHPA oxidation technique (unpublished results; see ref. [8 ]).

The thapsigargin-induced, extracellular NADPH-oxidase activity involves Ca2+ influx through La3+-sensitive Ca2+ channels
Although the extracellular NADPH-oxidase response induced by thapsigargin was minute, we investigated whether chelating extracellular Ca2+ and blocking Ca2+ channels would also have an effect on this low response. Treatment with EGTA resulted in a dose-dependent inhibition of the small thapsigargin-induced extracellular response (Fig. 3B) . When adding La3+ to the cells prior to thapsigargin activation, a striking effect was seen. At La3+ concentrations between 200 and 400 µM, thapsigargin induced a massive, extracellular production of superoxide anion, but lower as well as higher concentrations had no or very small effects (Fig. 4B and 4C) . The effect was Ca2+-dependent, because adding thapsigargin to the La3+-pretreated cells in the absence of extracellular Ca2+ did not induce a response (unpublished results).

There are at least two possible explanations for the phenomenon of thapsigargin-induced, extracellular release of oxidants in the presence of La3+. Either the plasma membrane NADPH-oxidase could be activated in a narrow, extracellular Ca2+ concentration range, or La3+-sensitive Ca2+ channels may be involved as a necessary and regulating part of the signaling to the plasma membrane-bound NADPH-oxidase. To test the first hypothesis, a wider range (1–1000 µM) of EGTA concentrations was used to modulate the extracellular Ca2+ concentration and thereby the influx. However, no extracellular response could be detected, independent of the EGTA concentration used (unpublished results). Using another approach, we exchanged La3+ for two other Ca2+ channel blockers, Ni2+ [16 , 20 ] and the imidazole derivative econazole [21 , 26 ]. However, no increased extracellular response could be detected, regardless of concentration of the blockers. Ni2+ and econazole inhibited the small, extracellular response induced by thapsigargin alone dose-dependently (Fig. 5 ), correlating with the results achieved with EGTA (Fig. 2) . Thus, the La2+-induced effect is most probably caused by (modulated) blocking of a La3+-sensitive Ca2+ channel. Exposing the cells to either of the alternative blockers (Ni2+ or econazole) also gave a dose-dependent decrease of the thapsigargin-induced, intracellular NADPH-oxidase activity (Fig. 5) .



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  		Figure 5. Effect of Ni2+ and econazole on the thapsigargin-induced NADPH-oxidase activation. NADPH-oxidase activity was measured in neutrophils pretreated with econazole (left) and Ni2+ (right), respectively, as described in Figures 3 and 4 . The dose-response curves for the inhibition of the extracellular (open symbols) and intracellular (closed symbols) response are shown, and the maximal CL responses, set to 100%, are given.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Calcium signaling in neutrophils has been studied extensively, but there is no consensus on the role played by Ca2+ in the induction of various effector responses of the cell. Among the effector functions most intensely studied is the superoxide production. Yet, the involvement of Ca2+ in activation of the responsible enzyme system, the NADPH-oxidase, has not been clarified entirely. To do so, it is important to make a distinction between the NADPH-oxidase localized in the plasma membrane and that localized in intracellular membranes, because these two pools are regulated separately and by distinct signal-transduction pathways [13 ]. Phagocytosis-independent, intracellular (granule-dependent) activation of the NADPH-oxidase can be induced through occupation of the galectin-3 receptor as well as of the complement receptor 3 [10 , 28 ]. The function of intracellular oxidants that are formed independent of phagosome formation is largely unknown so far. However, mounting evidence suggests roles for oxygen radicals in intracellular signaling [29 , 30 ] and as inducers of apoptosis [31 ]. The fact that intracellular activation of the oxidase also can be induced by a Ca2+-specific ionophore [7 ] suggests that Ca2+ ions participate in regulation of the system. Here, we make an attempt to define the role played by Ca2+ release from intracellular stores as well as by capacitative Ca2+ influx in activation of the NADPH-oxidase.

The intracellular Ca2+ fluxes resulting from cell activation by chemoattractants or a phagocytizable prey have been shown to result both from Ca2+ release from intracellular stores and from capacitative influx over the plasma membrane [17 , 21 , 32 , 33 ]. With regard to the NADPH-oxidase, it has been suggested that the elevation of the [Ca2+]i is not a sufficient signal to induce activation [18 , 19 , 27 ]. However, in this study, we present data showing that in fact, the [Ca2+]i elevation per se induces superoxide anion production, although not at the plasma membrane but at an intracellular site. As has been discussed above, the reason for the discrepancy between our study and previously published work is the use of different techniques to measure oxidants. Recently, several different research groups have shown that the NADPH-oxidase can assemble and become activated in membranes of intracellular granules [3 4 5 6 7 ]. To be able to measure this intracellular activation, care must be taken to choose a proper measuring technique such as luminol-amplified CL [8 ].

The induction of exclusive, intracellular superoxide production by emptying intracellular Ca2+ stores is in strong contrast to the chemoattractant-induced NADPH-oxidase activity that takes place mainly in the plasma membrane. The fMLF-induced activation of the formyl peptide receptor not only causes a [Ca2+]i elevation but also induces additional signals, e.g., through the PKC activation by DAG. In fact, Foyouzi-Youssefi et al. [22 ] have shown convincingly that an additional signal in combination with the [Ca2+]i elevation is necessary for oxidase activation induced by fMLF. The identity of this signal is unknown so far. However, it is characterized to be dependent on a [Ca2+]i flux, and it is inactivated by high levels of [Ca2+]i. The inactivation by high [Ca2+]i could correlate to our finding that in the absence of La3+, no extracellular NADPH-oxidase activity is seen, but lowering the [Ca2+]i by addition of La3+ allows for the plasma membrane-localized oxidase to be activated. At the higher La3+ concentrations, the inhibitory effect is probably a result of the lesser influx of Ca2+ into the cell and thus, a similar effect to that seen for the intracellular response. Since we could not induce the signal only by altering the [Ca2+]i by addition of EGTA but needed to add La3+ to achieve the effect, we suggest that La3+-sensitive Ca2+ channels are involved in the signal leading to activation of the plasma membrane-localized NADPH-oxidase. It is possible that these channels are part of the second signal induced by fMLF in addition to a [Ca2+]i increase, suggested by Foyouzi-Youssefi et al. [22 ].

Elevation of intracellular Ca2+ by treating cells with Ca2+ ionophores will induce intracellular NADPH-oxidase activation in analogy with the thapsigargin-induced activation [11 ]. Ionomycin releases Ca2+ from intracellular stores as well as allowing for influx over the membrane [34 ], thus making it possible that its effects are similar to those of thapsigargin. In fact, Nusse et al. [35 ] have shown that ionomycin may induce an extracellular NADPH-oxidase activation at specific extracellular Ca2+ concentrations. This is in line with our results showing that modulated inhibition of Ca2+ influx (i.e., by adding La3+ at concentrations between 200 and 400 µM) induces activation of the plasma membrane-bound NADPH-oxidase. However, the study by Nusse et al. is done in HL-60 cells, and since these cells lack specific granules, the NADPH-oxidase activation may be turned to the plasma membrane as a result of deficiency in intracellular b-cytochrome.

We chose the Ca2+ channel inhibitors based on the following: La3+ has been shown to be a very efficient Ca2+ channel blocker, and Ni2+, although a blocker of Ca2+ channels, has low affinity and therefore, is quite unspecific. Econazole is an imidazole derivative shown to be an inhibitor of cytochrome P-450. However, it has been shown convincingly that the effect of econazole in neutrophils is not a result of this effect but because of an unspecific inhibition of Ca2+ influx [26 ]. Our data from using these inhibitors can be interpreted to mean that several Ca2+ channels are involved; all channels are inhibited by Ni2+ and econazole, giving a total block of the Ca2+ influx while some channels are specifically La3+-inhibited, giving only partial inhibition of Ca2+ influx.

The identity of the Ca2+ channels involved in the capacitative Ca2+ influx has not been clarified. Some distinct SOCs are known, e.g., the so-called Icrac channels from mast cells and T lymphocytes [36 , 37 ]. The transient receptor potential (TRP) channels have also been suggested as candidate SOCs. None of the TRP family members possess characteristics identical to those of SOCs. Nonetheless, they are discussed extensively in the literature as possible candidates, based on data showing that heterologous expression of TRP family members leads to the appearance of Ca2+-permeable channels [38 ], and antisense constructs of TRP cDNAs inhibit capacitative Ca2+ influx [39 40 41 42 ]. Further, endogenous TRP4 has been shown to contribute to the capacitative Ca2+ influx in adrenal cells [43 ]. Recently, TRP3, which exhibits store-dependent activation mechanisms [42 ] and appears to be regulated through interaction with the IP3 receptor [44 ], has been proven very sensitive to blocking by La3+. Whether this channel or any other now-identified SOC-like channels are involved in the neutrophil Ca2+ influx remains to be investigated.

In conclusion, this study shows that elevations in [Ca2+]i by capacitative Ca2+ influx induces NADPH-oxidase activation at an intracellular site. Further, activation of the plasma membrane-localized NADPH-oxidase is regulated by a more complex Ca2+ signaling, involving capacitative Ca2+ influx and possibly the specific action of La3+-sensitive Ca2+ channels. Furthermore, it should be noted that the thapsigargin-induced release of oxygen radicals is rapidly transient, suggesting that an elevation of [Ca2+]i or emptying of the Ca2+ stores also regulates a hitherto-unknown termination system.


   ACKNOWLEDGEMENTS
 
This work was supported by the Swedish Medical Research Council, the Swedish Rheumatism Association, the Swedish Society of Medicine, the King Gustaf V 80-Year Foundation, and the Fredrik and Ingrid Thuring Foundation.

Received October 25, 2000; revised December 7, 2001; accepted December 14, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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