Modulation of Ca2+ Signaling by Na+/Ca2+ Exchangers in Mast Cells
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免疫学杂志 2005年第4期
Abstract
Mast cells rely on Ca2+ signaling to initiate activation programs leading to release of proinflammatory mediators. The interplay between Ca2+ release from internal stores and Ca2+ entry through store-operated Ca2+ channels has been extensively studied. Using rat basophilic leukemia (RBL) mast cells and murine bone marrow-derived mast cells, we examine the role of Na+/Ca2+ exchangers. Calcium imaging experiments and patch clamp current recordings revealed both K+-independent and K+-dependent components of Na+/Ca2+ exchange. Northern blot analysis indicated the predominant expression of the K+-dependent sodium-calcium exchanger NCKX3. Transcripts of the exchangers NCX3 and NCKX1 were additionally detected in RBL cells with RT-PCR. The Ca2+ clearance via Na+/Ca2+ exchange represented 50% of the total clearance when Ca2+ signals reached levels 200 nM. Ca2+ signaling and store-operated Ca2+ entry were strongly reduced by inverting the direction of Na+/Ca2+ exchange, indicating that Na+/Ca2+ exchangers normally extrude Ca2+ ions from cytosol and prevent the Ca2+-dependent inactivation of store-operated Ca2+ channels. Working in the Ca2+ efflux mode, Na+/Ca2+ exchangers such as NCKX3 and NCX3 might, therefore, play a role in the Ag-induced mast cell activation by controlling the sustained phase of Ca2+ mobilization.
Introduction
Calcium signals are essential in the maturation, differentiation, and activation of cells of the immune system. Generally, stimulation of membrane receptors activates phopholipase C (PLC)2 and promotes the production of inositol 1,4,5-trisphosphate (IP3) that in turn induces the release of Ca2+ ions from the endoplasmic reticulum ( 1). The Ca2+ release is accompanied by the entry of Ca2+ ions from the extracellular space through so-called store-operated Ca2+ channels, which are activated through an unknown mechanism initiated by the depletion of IP3-sensitive Ca2+ stores and represent the principal Ca2+ entry pathway ( 2, 3). Both, Ca2+ release and Ca2+ entry underlie the changes in the cytosolic-free Ca2+ concentration Cai that can be spatially localized or summated to give global Ca2+ signals with a variety of temporal patterns including repetitive spikes and sustained plateaus ( 4). The physiological role of various Ca2+ homeostatic mechanisms in the PLC-dependent Ca2+ signaling has been also recognized. For instance, mitochondria and the plasma membrane Ca2+-ATPase (PMCA) interact functionally with store-operated Ca2+ channels ( 5, 6). However, possible interactions between Na+/Ca2+ exchange, Ca2+ release, and store-operated Ca2+ entry in immune cells are less understood, although Na+/Ca2+ exchangers appear to be for the most part ubiquitously expressed (see Ref. 7).
Two types of Na+/Ca2+ exchanger have been described in mammalian tissues: K+-independent and K+-dependent Na+/Ca2+ exchangers, exemplified by the cardiac and retinal exchangers, respectively ( 7). The K+-independent Na+/Ca2+ exchangers are encoded by a family of three genes (NCX1, NCX2, and NCX3) and a structurally unrelated family of at least four genes (NCKX1, NCKX2, NCKX3, and NCKX4) encodes for K+-dependent Na+/Ca2+ exchangers ( 8, 9). NCX1 is nearly ubiquitous, NCKX3 and NCKX4 are also widely expressed whereas NCKX2, NCX2, and NCX3 appear to have a more restricted tissue distribution. NCKX1 has been found so far only in retinal rods and, interestingly, in megakaryocytes and platelets. Both members of the NCX and NCKX families are bidirectional transporters driven by the transmembrane gradient of Na+ and Ca2+, and NCXK isoforms are additionally dependent on the transmembrane K+ gradient. Thus, any Na+/Ca2+ exchanger can in principle mediate both Ca2+ influx and Ca2+ efflux under appropriated settings of the external concentrations of K+ (Ko), Na+ (Nao), and Ca2+ (Cao). For instance, the direction of exchange can be inverted from Ca2+ efflux to Ca2+ influx by removing external Na+. However, Ca2+ extrusion is the main function of Na+/Ca2+ exchangers in mammalian tissues especially when Cai is elevated and the membrane potential is more negative than the reversal potential of the exchanger ( 7). The exceptions are cardiac cells and some mammalian erythrocytes, in which the Ca2+ influx via Na+/Ca2+ exchange play an important physiological role. The function of Na+/Ca2+ exchangers becomes more prominent when voltage-dependent Ca2+ channels and Na+/Ca2+ exchangers are closely localized such as in synaptic buttons in which Na+/Ca2+ exchangers play an important role controlling exocytosis ( 10). Similarly, the Na+/Ca2+ exchange activity might potentially interact with the store-operated Ca2+ entry. For instance, over-expressed cardiac Na+/Ca2+ exchangers might counteract store-operated Ca2+ entry or enhance the rise of Cai supported by store-operated Ca2+ entry in subplasma membrane regions ( 11, 12), depending probably on expression levels.
In mast cells, cross-linking of IgE receptors (FcRI) by antigenes leads to activation of store-operated Ca2+ channels, elevation of Cai, and release of histamine and other mediators of inflammation (see Ref. 13). Low Nao reduces the secretory response ( 14, 15), suggesting an important role for the Na+/Ca2+ exchange in antigene-stimulated degranulation and secretion of inflammatory mediators from mast cells. In the present study, we analyzed the possible role of Na+/Ca2+ exchange in the Ca2+ homeostasis of mast cells using a rat basophilic leukemia (RBL) cell model and untransformed murine bone marrow-derived mast cells (BMMC). We present for the first time evidences for the expression of the K+-independent (NCX3) and K+-dependent (NCKX1, NCKX3) Na+/Ca2+ exchangers in mast cells. Accordingly, the corresponding Na+/Ca2+ exchange activity was not only dependent on the transmembrane gradient for Na+ and Ca2+ but also for K+. The Na+/Ca2+ exchange working in the Ca2+ efflux mode was required to ensure sustained store-operated Ca2+ entry and Ca2+ plateaus in mast cells.
Materials and Methods
Cell culture
RBL cells expressing the human muscarinic receptor (RBL-2H3-hm1) were cultured in -MEM medium (Invitrogen Life Technologies) supplemented with 15% FBS and 1% L-glutamine at 37°C/5% CO2, as previously described ( 13). Isolation, differentiation and FACS analysis of BMMC were essentially performed as described ( 16). In brief, BMMC were obtained from the femur of mice C57BL6/129SvJ and cultured at 37°C/5% CO2, in IMDM (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FCS, 50 μM 2-ME (Sigma-Aldrich), 10 U/ml penicillin/streptomycin, and 2 ng/ml recombinant murine IL-3 (R&D Systems). To determine the expression of FcRI, cell aliquots were first exposed to 2.4G2 rat anti-mouse FcRII/RIII Ab (BD Pharmingen) and subsequently incubated with mouse IgE (Sigma-Aldrich). Cell staining was performed with FITC-labeled monoclonal rat anti-mouse IgE Ab (BD Pharmingen) and PE-labeled rat anti-c-Kit mAb (BD Pharmingen). As analyzed with a Galaxy Flow Cytometry System (DAKO), >98% of the cells expressed both FcRI and c-Kit after 4 wk of differentiation. Calcium imaging experiments were conducted with BMMC maintained in culture for 14–20 wk. BMMC were attached to coverslips coated with poly-L-lysine (0.5 mg/ml; Sigma-Aldrich) just before start recording Ca2+ signals. The experiments with RBL cells were performed 1–3 days after plating the cells on coverslips. All experiments were conducted at room temperature and repeated with cells of at least two independent batches.
Detection of NCX/NCKX transcripts by RT-PCR and Northern blot
For RT-PCR 5 μg of total RNA from RBL cells and rat brain was reversed transcribed using random primers. One of seven of the reversed transcribed cDNA was applied to PCR to amplify fragments with a length of 440 bp (NCX1), 436 bp (NCX2), 437 bp (NCX3), and 456 bp (NCKX1) and (NCKX3). The following primer pairs specific for each sequence and each separated by at least one intron in the rat genomic sequence were applied to 15 cycles (94°C, 15 s; 62°C, 45 s; 72°C, 45 s) followed by 30 cycles (94°C, 15 s; 62°C, 30 s; 72°C, 45 s plus 2 s/cycle): 5'-GCT TCA TTG TCT CCA TCC TCA TG-3' and 5'-GGA AGA TGT GAG GAG CTT GGC-3' for NCX1 (GenBank accession no. NM_019268); 5'-CTT TGG TGT CTG CAT CCT GGT C-3' and 5'-GGT GGT GGC TAG CTT GGG TC-3' for NCX2 (GenBank accession no. NM_078619); 5'-CTT CGT GGT CTC CAT CCT CAT C-3' and 5'-ACG TTG TGG CAA GCT TGC AGC-3' for NCX3 (GenBank accession no. AJ006781); 5'-CAC CTT CCT GGG ATC CAT CAT C-3' and 5'-CGA TCT TCT AAC ATC ACA CTG ATC-3' for NCKX1 (GenBank accession no. NM_020090); 5'-GAC GTT TGC TTC CTC TAC ACT G-3' and 5'-AAC TCC GTC ATG ATG GAG AAG C-3' for NCKX3 (GenBank accession no. XM_342533). Reaction products were separated by electrophoresis in 2.0% agarose and stained with ethidium bromide. Northern blots were performed as described ( 17) using 10 μg of poly(A)+ RNA from RBL cells, rat brain, liver, and kidney, as well as using 5 μg of poly(A)+ RNA from BMMC, mice brain and rabbit heart. For detection of NCX3 (SLC8A3) transcripts, we used a BspE1/HindIII cDNA fragment from rat NCX3 (kindly provided by D. A. Nicoll and K. D. Philipson, University of California, Los Angeles, School of Medicine, Los Angeles, CA) corresponding to nt 1268–3530 of U53420. For detection of NCKX1 (SLC24A1) and NCKX3 (SLC24A3) the cDNA fragments obtained by RT-PCR using RBL RNA were subcloned, sequenced on both strands and were then used as probes. The probes were labeled by random priming using [-32P]dCTP and hydridized under stringent conditions. The filters were exposed to x-ray films with intensifying screens at –80°C for 3 days (NCKX3) and 21 days (NCX3, NCKX1) before they were hybridized with a 239-bp cDNA fragment of the human GAPDH.
Calcium imaging
A single cell imaging system (Till Photonics) was used to measure Cai as previously described ( 13). Cells were loaded with fura 2-AM (5 μM; Molecular Probes) for 40 min at room temperature. Sequences of paired images containing 5–20 cells/frame were obtained every 3 s with excitation wavelengths of 340 and 380 nm (510 nm emission filter). The corresponding ratios (F340/F380) were used to obtain Cai. In the Mn2+ quench experiments, the fluorescence images were obtained every 3 s with an excitation wavelength of 360 nm (510 nm emission filter). Following a baseline recording to determine the spontaneous decrease of fluorescence, Mn2+ was introduced into the bath. The mean fluorescence intensities were normalized to initial intensities (F/F0). Statistical data and are given as mean (±SEM).
The standard bath solution in the Ca2+ imaging and Mn2+ quenching experiments contained 130 mM NaCl, 5.4 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM glucose and 10 mM HEPES (pH 7.4/NaOH). The various Na+/Ca2+ exchange mechanisms were studied using bath solutions with modified concentrations of CaCl2, KCl, and/or NaCl whereas the concentrations of MgCl2 and glucose were maintained constant. N-methyl-D-glucamine (NMDG) was used to replace Na+ and K+. The Na+-free solutions contained 0, 5.4, or 70 mM KCl and the NMDG concentrations were 135, 130, or 65 mM, respectively (pH 7.4/HCl). In the Ca2+-free solutions with no added CaCl2, 500 μM EGTA were used to ensure Cao values below 10 nM. The Ca2+-free solutions contained various concentrations of K+ and Na+ (5.4 mM KCl and 130 mM NaCl; 5.4 or 70 mM KCl with Na+ replaced by 130 or 65 mM NMDG, respectively, pH 7.4/HCl). The Na+ entry through store-operated channels was prevented by the presence of Mg2+ (>1.9 mM) in the Ca2+-free solutions ( 3). When required, 2 mM LaCl3 were added to nominally Ca2+-free solutions. In the Mn2+ quench experiments, 500 μM MnCl2 were added to the standard bath solutions. Ca2+ signaling was initiated by application of either 2 μM thapsigargin or 1 mM carbachol. Alternatively, cells were primed overnight with 0.3–3 μg/ml anti-2,4,6-trinitrophenyl (TNP) IgE and Ca2+ mobilization was induced by the addition of 50 ng/ml TNP-conjugated-OVA (TNP-OVA). The osmolarity of the bath solutions was 300–340 mOsm. Only ionic concentrations that differ from those of the standard bath solution are indicated in the figures.
Ca2+ clearance
To estimate the contribution of Na+/Ca2+ exchange mechanisms to the Ca2+ homeostasis, we analyzed the Ca2+ clearance (i.e., the return to resting Cai) as previously described ( 18). Elevations of Cai were induced by Ca2+ entry or by depletion of internal Ca2+ stores. Clearance plots were built as follows using the decay phase of Ca2+ transients in Ca2+-free solutions with and without external Na+: 1) the decay phases of Ca2+ transients were fitted with single or double exponential functions to obtain the time constants of Cai decay; 2) The derivative –dCai/dt (i.e., clearance rate) was calculated from Cai time plots; 3) –dCai/dt was plotted as a function of Cai and fitted with sigmoid Hill functions.
Whole-cell patch clamp
Ionic currents were recorded in the whole-cell mode of the patch-clamp technique ( 19) using an EPC 9-2 amplifier (Heka Electronics). Recording pipettes with resistances of 2.5–3 M were pulled from borosilicate glass capillaries (Kimax-51; Kimble Products). Series resistances and capacitive components were compensated using the internal circuit of the amplifier. Only experiments with series resistances below 10 M were used for further analysis. The average capacitance of the RBL cells was 12.6 ± 0.6 pF (n = 35). Voltage-clamp steps (100 ms) were applied every 2 s to potentials between –100 and +50 mV from a holding potential of 0 mV. Membrane currents were sampled at 4 kHz and 33.3 Hz (filter cutoff: 33 kHz, 6.67 Hz) in the pulse and continuous mode, respectively. To allow the exchange of the external solutions, the glass coverslips containing RBL cells were placed into a recording chamber that had a volume of 600 μl and was connected to a gravity-driven perfusion system. Data were analyzed offline using a combination of the software packages Pulse/Pulsefit (Heka Electronics). Statistical data are given as mean (±SEM).
Magnesium-inhibited currents and store-operated Ca2+ currents were suppressed by high cytosolic Mg2+ (2 mM) and low buffering of cytosolic Ca2+ (1 μM), respectively. Additionally, pipette and bath solutions contained tetraethylammonium (TEA)+ to block K+ channel currents. As in previous studies of recombinant Na+/Ca2+ exchangers (e.g., Ref. 20), we used a Na+-based pipette solution containing 120 mM NaCl, 40 mM KCL, 20 mM TEA-Cl, 2 mM MgCl2, 2 mM Mg-ATP, 8 mM glucose, 10 mM HEPES (pH 7.2/CsOH), and 1 μM free Ca2+. The external solution in the patch clamp experiments contained 130 mM NaCl, 40 mM KCl, 20 mM TEA-Cl, 2 mM MgCl2, 10 mM glucose and 10 mM HEPES (pH 7.2/CsOH), and 500 μM EGTA. With this set of solutions, we recorded nearly linear currents at potential between –100 and +50 mV in nearly every analyzed RBL cell, suggesting a complete block of ionic channel currents. Na+/Ca2+ exchange currents were elicited by switching to a bath solutions that contained 130 mM LiCl, 20 mM TEA-Cl, 2 mM MgCl2, 2 mM CaCl2, 10 mM glucose and 10 mM HEPES (pH 7.2/CsOH). The KCl content of the bath solutions was either 0 or 40 mM. Free Ca2+ concentrations in the pipette solutions were adjusted using CaCl2/EGTA mixtures calculated with the MaxChelator (v2.10) (
Results
Expression of Na+/Ca2+ exchangers in RBL cells
Because it is not known whether mast cells express Na+/Ca2+ exchangers, we first determine the expression of NCX and NCKX isoforms in RBL cells with RT-PCR and Northern blot analysis. Using specific primers for the various NCX and NCKX exchangers, we detected a prominent expression of the K+-dependent Na+/Ca2+ exchanger NCKX3 (Fig. 1). Lower expression levels of the K+-independent Na+/Ca2+ exchanger NCX3 and the K+-dependent Na+/Ca2+ exchanger NCKX1 became also visible after RT-PCR amplification (Fig. 1A). We were not able to detect the expression of NCX1 and NCX2. In Northern blots, the expression of NCKX3 was readily detected after 3 days exposure of the filter (Fig. 1B). By contrast, the expression of NCX3 and NCKX1 was not detected in Northern blots even after 21 days filter exposure. In line with previous reports ( 20), the major NCKX3 transcript was 4.4 kb in length and was abundantly expressed in RBL cells as well as in brain, whereas kidney and liver apparently lacked expression of NCKX3 (Fig. 1B).
Next, we measure Na+/Ca2+ exchange currents to determine whether the detected NCX and NCKX isoforms are functional in RBL cells. The selected experimental settings ensured the blockade of ionic channel currents previously described in RBL cells (see Materials and Methods). Because all Na+/Ca2+ exchangers are bidirectional transporters, the rational in the patch-clamp experiments was basically to switch between the Ca2+ efflux and Ca2+ influx modes and record the resulting inward and outward currents, respectively. We switched into Ca2+ efflux mode by removing external Ca2+ in the presence of external Na+ and, conversely, into the Ca2+ influx mode by removing external Na+ in the presence of external Ca2+. Furthermore, high internal Na+ was used to enhance outward currents in Ca2+ influx mode, also to slow down the Ca2+ efflux and, consequently, to minimize inward currents. Thus, the design of the patch-clamp experiments allowed primarily the detection of outward currents, i.e., Ca2+ influx. Fig. 2A illustrates experiments in which whole-cell membrane currents were recorded continuously at –80 mV during the exchange of solutions. In these experiments, we used high external K+ to ensure the function of K+-dependent Na+/Ca2+ exchangers. Independently of the bath solution used, we recorded negative currents at negative membrane potentials. The main effect of the removal of external Na+ in the presence of Ca2+ was an upward shift of currents levels, which was reversed upon re-addition of Na+ and removal of Ca2+ (Fig. 2A). Likely, this shift to less negative current levels reflected the appearance of outward currents that superimposed on baseline currents. Furthermore, we also determine current-voltage relations using voltage-clamp steps (Fig. 2B). As expected from the continuous current recordings (Fig. 2A), the removal of Na+ in the presence of Ca2+ induced a reduction of the current amplitudes at potentials below –25 mV (Fig. 2, B and C). At 0 mV, no current was detected in the Ca2+-free solution but we consistently observed the appearance of small detectable outward currents after the switch to the Na+-free solution (Fig. 2, B and C). Thus, the exposure to the Na+-free, Ca2+-containing solution produced a shift of the current-voltage relations into the upward direction at potentials 0 mV (Fig. 2C). According to the ionic settings of the external and internal solutions, the difference between the current-voltage relations shown in Fig. 2C likely represented an outward current component supported by any Na+/Ca2+ exchanger because all NCX and NCKX isoforms allow Ca2+ influx in the absence of external Na+. In an effort to distinguish between K+-dependent and K+-independent components of Na+/Ca2+ exchange currents, we omitted K+ ions from the external solutions to suppress Ca2+ influx through K+-dependent Na+/Ca2+ exchangers in further whole-cell current recordings. Other experimental conditions were as in Fig. 2, A–C. Outward current components were estimated as difference currents (I) elicited by the switch from Na+-containing, Ca2+-free solutions to Na+-free, Ca2+-containing solutions. As illustrated in Fig. 2D, K+-independent Na+/Ca2+ exchangers appeared to function in RBL cells because we never observed zero I values in the absence of external K+ and, similarly, K+-dependent Na+/Ca2+ exchangers must also function in RBL cells because we usually observed higher I values in the presence than in the absence of external K+.
Ca2+ clearance
Because generally the main function of Na+/Ca2+ exchangers is the extrusion of Ca2+ ions from cytosol, we analyzed in RBL cells the clearance of Ca2+ ions released from intracellular stores (Fig. 3) and the clearance of Ca2+ ions entering the cell throughstore-operated Ca2+ channels (Fig. 4). For this purpose, we separated experimentally the components of Ca2+ signals, i.e., Ca2+ release from Ca2+ entry, using the so called Ca2+ re-addition protocols ( 2). Thapsigargin or carbachol were applied to RBL cells exposed to external Ca2+-free solutions to isolate the Ca2+ release component (Fig. 3). Subsequently, external Ca2+ was re-added to the bath solution to visualize the Ca2+ entry during Ca2+ plateaus (Fig. 4). The Ca2+ clearance supported by Na+/Ca2+ exchangers was worked out by comparing clearance rates measured in the absence of external Ca2+ with and without external Na+ because the removal of external Ca2+ alone is expected to reinforce Ca2+ efflux and the additional removal of external Na+ to suppress Ca2+ efflux through Na+/Ca2+ exchangers. Because the K+ dependence of Na+/Ca2+ exchange can be hardly analyzed in the Ca2+ efflux mode using calcium imaging, external K+ was maintained constant at 5.4 mM.
In the absence of external Ca2+, the Ca2+ release signals induced by carbachol attained roughly identical peaks independently of whether Na+ was present (0.42 ± 0.02 μM, n = 36) or absent (0.47 ± 0.04 μM, n = 30) in the external solution (Fig. 3A). Similar results were obtained with thapsigargin, indicating that the Ca2+ release was not affected by the removal of external Na+. Basically, the removal of external Na+ slowed down the time course of Ca2+ release signals (Fig. 3A). Apparent differences in Cai baselines were not statistically significant. To compare time courses, Ca2+ release signals were normalized to peak values and the decay phase was fitted with single exponential functions that were sufficient to account for 90% of the decay. As illustrated in Fig. 3B, inset, the decay time constant of average Ca2+ release signals was 1.7-fold slower in the absence of Na+, suggesting inhibition of a Ca2+ clearance component supported by Na+/Ca2+ exchange mechanisms. Clearly, this inhibition was small compared with the inhibition produced by 2 mM La3+ (Fig. 3A), indicating that various Ca2+ clearance mechanisms were still functioning in the absence of external Na+. The Ca2+ clearance component inhibited by the removal of external Na+ was visualized using Ca2+ clearance plots, as previously described ( 18). As shown in Fig. 3B, the dependence of clearance rates (–dCai/dt) on Cai followed a sigmoid Hill function both in the presence and absence of external Na+. In the absence of Na+, however, the clearance rates were lower within a wide Cai range. The inspection of the –dCai/dt curves (Fig. 3B) indicated 28–51% reduction of clearance rates within the Cai range from 200 to 400 nM, when the cells were exposed to Na+-free solutions. Thus, the inhibition of Na+/Ca2+ exchange in Na+- and Ca2+-free solutions slowed down the clearance of Ca2+ ions released from intracellular stores preferentially when Cai was close to the peak of Ca2+ release signals.
The effects of external Na+ on the clearance of Ca2+ ions entering the cell via store-operated Ca2+ channels were analyzed in Ca2+ re-addition experiments (Fig. 4). We observed that the levels and time courses of Ca2+ plateaus were not dependent on external Na+ (Fig. 4A). On average, Cai was 0.56 ± 0.01 μM and 0.50 ± 0.01 μM (n = 31) 2 min after re-addition of Ca2+ in the presence and absence of external Na+, respectively. The effects of Na+ removal became evident during the decay of Cai produced by the removal of external Ca2+ (Fig. 4A). To quantify these effects, normalized average Cai decays were fitted with single exponential functions, which accounted for 99% of the decay (Fig. 4B, inset). A comparison of the exponential time constants indicated that the Cai decayed 1.2-fold slower in the absence of external Na+. The corresponding Ca2+ clearance plots indicated that the removal of external Na+ reduced –dCai/dt by 39–58% preferentially at Cai levels above 200 nM (Fig. 4B). As for the clearance of Ca2+ ions released from intracellular stores (Fig. 3), it appeared therefore that Na+/Ca2+ exchangers removed the Ca2+ ions that entered the cell, primarily when Cai was close Ca2+ plateau levels.
Na+/Ca2+ exchange at basal Cai
The basal Cai of RBL cells exposed to the standard bath solution ranged from 18 to 171 nM with a mean of 71.7 ± 1.6 nM (Fig. 5A). Because modification in the composition of the bath solution usually increased or decreased Cai only within this range, changes in basal Cai (Cai) were measured with respect to Cai values obtained at the beginning of recordings. In analogy to previous experiments (Figs. 3 and 4), external Ca2+ and/or Na+ were removed to test whether Na+/Ca2+ exchangers contribute to the maintenance of basal Cai (Fig. 5B). The removal of external Ca2+ alone induced a rapid drop in Cai (Fig. 5B, left panel), which can be explained by the suppression of basal Ca2+ entry into the cell and/or by the enhancement of Ca2+ extrusion supported by Na+/Ca2+ exchangers. When external Na+ was additionally removed to suppress Ca2+ efflux via Na+/Ca2+ exchangers after the removal of Ca2+ (Fig. 5B, right panel), however, we were not able to detect changes in the time course of Cai. Thus, the contribution of Na+/Ca2+ exchange to Ca2+ clearance at basal Cai levels appeared to be marginal.
To test further for the operation of Na+/Ca2+ exchangers at basal Cai levels, we removed external Na+ in the presence of external Ca2+, a maneuver that was expected to invert the direction of transport from Ca2+ efflux to Ca2+ influx via all Na+/Ca2+ exchangers ( 7). With 5.4 mM external K+, we observed a continuous increase of Cai after removal of external Na+ (Fig. 5C), suggesting the presence of functional Na+/Ca2+ exchangers in RBL cells. The operation of K+-independent Na+/Ca2+ exchangers was indicated by the transient increase of Cai induced by the simultaneous removal of external K+ and Na+ (Fig. 5C, inset), because Ca2+ influx via K+-dependent Na+/Ca2+ exchangers is inhibited in the absence of external K+ ( 9). Furthermore, we tested the effects of Na+ removal in the presence of 70 mM external K+ because the K+-dependent Na+/Ca2+ exchange can be enhanced by increasing K+. Under these conditions, we observed a transient Cai increase that was higher than in 5.4 mM external K+ (Fig. 5C), indicating the presence of functional K+-dependent Na+/Ca2+ exchangers in RBL cells. To test further the activity of Na+/Ca2+ exchangers, we induced Na+ overload by exposing the cells to ouabain, a blocker of the Na+/K+ ATPase. High internal Na+ was expected to increase the driving force for Ca2+ influx via Na+/Ca2+ exchangers. Accordingly, we observed in ouabain-treated cells a prominent Cai increase after the removal of Na+ in 5.4 mM external K+ (Fig. 6A cf. Fig. 5C). In contrast to nontreated cells (Fig. 5C), however, the Cai increase induced in ouabain-treated cells by Na+ removal was similar in the presence of 5.4 and 70 mM external K+ (Fig. 6A). Thus, the ouabain treatment enhanced the Ca2+ influx at least at 5.4 mM external K+ (nontreated: 14.5 ± 7.1 nM, Fig. 5C; ouabain-treated: 65.8 ± 6.7 nM, Fig. 6A), as expected if Na+/Ca2+ exchangers are functionally expressed in RBL cells.
A distinct possibility that we considered was that Ca2+ release from intracellular stores and/or Ca2+ entry through ionic channels contributed to the Cai increase induced by Na+ removal (Figs. 5C and 6A). The possible mobilization of Ca2+ from intracellular stores was unlikely because no Ca2+ release signal was observed when external Na+ was removed from Ca2+-free solutions in the presence of 5.4 mM (data not shown) or 70 mM external K+ (Fig. 5B, right panel). In Mn2+ quenching experiments with 5.4 mM external K+, the fluorescence decreased linearly after application of Mn2+ into the bath, indicating a basal ionic leak in ouabain-treated cells (Fig. 6B). The quenching rates, however, remained unchanged after removal of external Na+ (0.064 ± 0.007 s–1 and 0.077 ± 0.005 s–1; Fig. 6B). Similar results were obtained in Mn2+ quenching experiments with nontreated cells (data not shown), suggesting that Na+ removal did not activate Ca2+/Mn2+ permeable channels. Furthermore, Ca2+ entry was not expected in experiments with 70 mM external K+ because high K+ depolarizes the cells and, consequently, reduces the driving force for Ca2+ entry. Thus, it was unlikely that an enhancement of Ca2+ entry through ionic channels contributed to the changes of Cai shown in Figs. 5C and 6A.
Fig. 7 shows the summary of the experiments in which the transmembrane gradients for Ca2+, Na+, and K+ were systematically changed to test the function of Na+/Ca2+ exchangers at basal Cai in RBL cells. Each Cai value was obtained in an independent experiment because we observed that Cai hardly returned to basal levels after solution exchange, for instance, after application of Na+-free, Ca2+-containing solutions (see Figs. 5C and 6A). In nontreated cells (without (–) ouabain), Cai values recorded after the removal of Na+ and K+ were on average 28% of those obtained after removing only Na+, suggesting an important contribution of K+-independent Na+/Ca2+ exchangers in these experiments. Furthermore, the Cai induced by Na+ removal was potentiated about 7-fold by increasing external K+ from 5.4 to 70 mM (p < 0.001), as expected for the functional expression of K+-dependent Na+/Ca2+ exchangers. In ouabain-treated cells (with (+) ouabain), no difference was observed between Cai values obtained after Na+ removal in the presence of 5.4 and 70 mM K+ but the removal of Na+ and K+ induced Cai values that amounted 50% of corresponding values measured in 5.4 mM external K+. Comparing ouabain-treated and nontreated cells, we found that ouabain had no effect on the Cai induced by Na+ removal at 70 mM external K+. By contrast, ouabain enhanced the Cai responses to Na+ removal by 9-fold with no K+ and by 4.5-fold in 5.4 mM external K+ (p < 0.001). According to the latter results, it appeared that the ouabain treatment was more effective on the K+ independent Na+/Ca2+ exchange. The Cai measured after removal of external Ca2+ was not dependent on external Na+ and K+, supporting our suggestion that Na+/Ca2+ exchangers contribute marginally to the maintenance of basal Cai.
Na+/Ca2+ exchange during Ca2+ signaling
Finally, we explored the function of Na+/Ca2+ exchangers during the generation of Ca2+ signals in RBL cells (Fig. 8), a process that is believed to involve Ca2+ release from intracellular stores and store-operated Ca2+ entry ( 3). An analysis of the K+ dependence of Na+/Ca2+ exchange during Ca2+ signaling was precluded because changes in the transmembrane gradient for K+ modify the driving force for Ca2+ entry. We indeed observed in preliminary experiments (data not shown) that the increase of external K+ from 5.4 to 70 mM abolished Ca2+ plateaus completely in a similar way as when external Ca2+ was removed (Fig. 4A). To keep constant the store-operated Ca2+ entry, external K+ and Ca2+ were therefore maintained at 5.4 and 2 mM, respectively. Because the removal of external Na+ is sufficient to invert the transport mode of K+-dependent and K+-independent Na+/Ca2+ exchangers ( 7), we analyzed Ca2+ signaling in standard and Na+-free solutions, i.e., with Na+/Ca2+ exchangers working in Ca2+ efflux and Ca2+ influx modes, respectively. The advantage was that such Ca2+ influx was expected to be insignificant under these conditions because Ca2+ plateaus were nearly identical in standard and Na+-free solutions (Fig. 4A). When external Na+ was removed in the middle of Ca2+ plateaus i.e., 3 min after Ca2+ re-addition in experiments conceptually similar to those shown in Fig. 4A, the instantaneous Cai increase was 16 ± 6 nM (n = 31). Thus, the absence of external Na+ was expected to prevent Ca2+ efflux rather than to induce Ca2+ influx via Na+/Ca2+ exchangers during Ca2+ signaling.
The Ca2+ signals were initiated in RBL cells either by the blockade of sarco(endo)plasmic reticulum Ca2+-ATPases pumps in the internal Ca2+ stores with thapsigargin or by activation of the stably expressed muscarinic receptors with carbachol ( 13, 19). Fig. 8 illustrates the effects of the Na+-free solution on Ca2+ signals. The initial peaks of Ca2+ signals induced by carbachol were almost identical with and without external Na+ (Fig. 8A), in line with previous experiments indicating that Na+-free solutions modify the time course but not the peak of the Ca2+ release component (Fig. 3A). With thapsigargin, the peaks of Ca2+ signals were 41% smaller in the Na+-free solution (Fig. 8C; 1.20 ± 0.05 μM vs 0.71 ± 0.04 μM). During the sustained phase of the Ca2+ signals, however, we observed 34–65% lower Cai levels in absence of external Na+ both after stimulation with thapsigargin and carbachol (Fig. 8, A and C; carbachol: 1.09 ± 0.14 μM vs 0.38 ± 0.07 μM; thapsigargin: 0.41 ± 0.02 μM vs 0.27 ± 0.02 μM). Because store-operated Ca2+ entry has been usually associated with the sustained phase of Ca2+ signals ( 3), we next performed Mn2+ quenching experiments (Fig. 8, B and D) to estimate the activity of store-operated Ca2+ channels in the Na+-free solution. As predicted from the experiments shown in Fig. 6B, the Na+-free solution had no effect on spontaneous quenching rates, i.e., on the fluorescence decay after addition of Mn2+ and before application of Ca2+ mobilizing agents. Indicating activation of store-operated Ca2+ channels, carbachol and thapsigargin enhanced the Mn2+ quenching in the standard solution (Fig. 8, B and D, left panels). In the absence of external Na+, however, the enhancement of quenching rates was less pronounced both after application of carbachol and thapsigargin (Fig. 8, B and D; carbachol: 1.74 ± 0.02 s–1 vs 0.16 ± 0.001 s–1; thapsigargin: 0.39 ± 0.02 s–1 vs 0.19 ± 0.02 s–1). In fact, the quenching rates were almost identical before and after application of carbachol (Fig. 8B, right panel). Thus, the Mn2+ quenching experiments demonstrated that Na+-free solutions, basically, diminish the activity of store-operated Ca2+ channels. Consequently, the lower Cai levels during the sustained phase of Ca2+ signals (Fig. 8, A and C, right panels) likely reflect the reduced Ca2+ entry in the absence of external Na+.
Na+/Ca2+ exchange in murine BMMC
In the experiments with RBL cells shown in Fig. 8, stably transfected muscarinic receptors were stimulated with carbachol and internal Ca2+ stores were depleted with thapsigargin. To test whether Na+/Ca2+ exchangers are involved in regulating store-operated Ca2+ entry upon FcRI cross-linking in nontransformed mast cells, we next performed experiments with murine BMMC.
Fig. 9 illustrates that the major Na+/Ca2+ exchanger found in RBL cells is also expressed in BMMC. The predominant NCKX3 transcript was 4.4 kb in length in BMMC and brain, whereas heart apparently lacks NCKX3 expression. Taking into account the GAPDH signals, a densitometric analysis of the Northern blots shown in Fig. 1B and 9A indicated that the NCKX3 signals of RBL and BMMC correspond to 88 and 76% of the respective brain NCKX3 signals. The basal Cai of BMMC was 197 ± 23 nM (n = 97). In experiments conceptually similar to those shown for RBL cells in Fig. 5C, we tested the function of K+-dependent and K+-independent Na+/Ca2+ exchangers in BMMC. As illustrated in Fig. 9B, the removal of external Na+ was sufficient to induces an increase of Cai to 279 ± 11 nM (n = 33). When external K+ was raised to 70 mM simultaneously with the Na+ removal, Cai increased to 319 ± 7 nM (n = 6). The corresponding Cai values induced by removal of external Na+ in the presence of 5.4 and 70 mM were 81 ± 9 nM and 108 ± 1 nM, respectively. Furthermore, Cai increased with a much faster time course in the experiments with high external K+. Thus, BMMC appeared to express functional K+-dependent and K+-independent Na+/Ca2+ exchangers as RBL cells. Fig. 10 illustrates the functional consequences of removing external Na+ previously to cell activation via FcRI cross-linking. In BMMC primed with anti-TNP IgE, the Ca2+ signals initiated by the application of TNP-OVA showed the typical sustained phase that lasted >12 min. Generally, the sustained phase of antigene-induced Ca2+ mobilization is much lower in BMMC than in RBL cells ( 13 cf.16). As expected from the experiments shown in Fig. 9B, the removal of external Na+ alone shifted the basal Cai to higher values also in the experiments shown in Fig. 10A. Under these conditions, application of TNP-OVA was still able to induce Cai mobilization in BMMC, but the time courses of the Ca2+ signals were disrupted in the Na+-free solutions. After the application of TNP-OVA, Ca2+ oscillations were observed in a considerable number of cells. The oscillations and rise of Cai lasted no longer than 5 min. In contrast to control cells, no sustained phase of Ca2+ signal was detectable and Cai returned rapidly to initial values in the absence of external Na+. To compare the time courses of Ca2+ signals, we constructed Cai time plots as illustrated in Fig. 10B. The Cai peak values attained immediately after application of TNP-OVA appeared to be not dependent on external Na+. At times longer than 5–7 min, however, the different time courses became evident. The faster Cai decay in the absence of external Na+ resulted in Cai values lower that 50 nM after 10 min of exposure to TNP-OVA, whereby the sustained Ca2+ signals in the presence of external Na+ maintained values of 93 ± 6 nM (n = 15) for >10 min. Similarly, the removal of external Na+ resulted in a 75% reduction of Ca2+ plateau levels in RBL cells primed with anti-TNP IgE and stimulated with TNP-OVA (174 ± 8 nM, n = 40 vs 44 ± 12 nM, n = 20; 10 min TNP-OVA stimulation). As for RBL cells (Fig. 8C), the sustained phase of Ca2+ signals induced by the application of 1 μM thapsigargin to BMMC was reduced 79% by the removal of external Na+ (403 ± 24 nM, n = 35 vs 318 ± 64 nM, n = 25; 10 min thapsigargin exposure). To test whether the reduced Ca2+ plateaus levels of BMMC exposed to Na+-free solutions were due to a reduced activity of store-operated Ca2+ channels, we performed Mn2+ quenching experiments. Fig. 10C illustrates that the quenching rates measured in BMMC were, in fact, less pronounced in the absence of external Na+ (0.23 ± 0.01 s–1, n = 20 vs 0.11 ± 0.01 s–1, n = 15). As in RBL cells, thus, the main effect of removing external Na+ was a reduction store-operated Ca2+ entry in BMMC, which correlated with the inability of BMMC to maintain Ca2+ plateaus for longer times in the absence of external Na+.
Discussion
In the present study, we combined calcium imaging and the patch clamp technique with Northern blot and RT-PCR analysis to establish the presence and function of Na+/Ca2+ exchangers in mast cells using the RBL model and murine BMMC. Our data suggested an important role for K+-dependent (NCKX3, NCKX1) and K+-independent (NCX3) Na+/Ca2+ exchangers in the Ca2+ clearance at Ca2+ plateau levels. Accordingly, Ca2+ signals were strongly dependent on external Na+ and, more interestingly, the activity of store-operated Ca2+ channels appeared to be diminished in the absence of external Na+.
Detection of Na+/Ca2+ exchange activity
As in previous studies of Na+/Ca2+ exchangers ( 20), we detected Na+/Ca2+ exchange activity in RBL cells and BMMC by taking advantage of the fact that the direction of ionic transport through all NCX and NCKX isoforms changes from Ca2+ efflux to Ca2+ influx when external Na+ is removed (Figs. 2, 5C, 6A, 7, and 9B). Furthermore, Mn2+ quenching experiments ruled out the possible activation of Ca2+ entry through ionic channels in Na+-free solutions (Fig. 6B). However, side effects of Na+ removal need to be considered. For instance, it is well known that the Ca2+ efflux from mitochondria is mediated by Na+/Ca2+ exchangers ( 21). Because the removal of external Na+ might reduce the intracellular Na+ concentration, thus, an inhibition of the Ca2+ efflux from mitochondria can be expected in Na+-free solutions. In the absence of external Ca2+, in fact, the removal of external Na+ was not able to induce any detectable Cai enhancement (Figs. 5B and 7), indicating that internal Ca2+ stores including mitochondria did not contribute to the Cai enhancement induced by Na+ removal. Furthermore, we challenged the RBL cells with ouabain to strengthen our evidences favoring the existence of Na+/Ca2+ exchangers in mast cells. As in previous studies of Na+/Ca2+ exchange with a number of cells including those that express exclusively NCKX isoforms ( 22), the rational behind this approach was to induce intracellular accumulation of Na+ and test whether the Ca2+ influx via Na+/Ca2+ exchange increases according to the enhanced driving force. In this context, we note that such increase of internal Na+ might also force the Ca2+ efflux from mitochondria. Because the exposure to ouabain lasted >1 h, such effect might produce a sustained increase of basal Cai in ouabain-treated cells. However, the Cai enhancements we report were transient and lasted as long as Na+ was absent from the external solution (Fig. 6A), indicating that the Ca2+ influx underlying the Cai increase in ouabain-treated cells was dependent on the transmembrane Na+ gradient. Thus, the Cai and membrane currents recordings with inverted Na+ gradients strongly support our suggestion that Na+/Ca2+ exchangers are present and function in RBL cells and BMMC.
K+-dependent and K+-independent Na+/Ca2+ exchangers
The RT-PCR data indicated that K+-dependent (NCKX1, NCKX3) and K+-independent (NCX3) Na+/Ca2+ exchanger isoforms are expressed in RBL cells (Fig. 1A). However, only the expression of NCKX3 was detected in Northern blots (Fig. 1B). Because the expression analysis by Northern blot is less sensitive than by RT-PCR, NCKX3 might be the predominant Na+/Ca2+ exchanger in RBL cells. Similarly, BMMC appear to express the Na+/Ca2+ exchanger NCKX3 (Fig. 9A). As NCX3 expression was only detected by RT-PCR in RBL cells, the question raised on whether this K+-independent Na+/Ca2+ exchanger is functionally expressed in mast cells. At physiological levels of internal Na+, the Ca2+ influx via Na+/Ca2+ exchange was largely dependent on external K+ (Figs. 5C, 7, and 9B), in line with the prominent expression of NCKX3. Additional experiments will be needed to proof the functional expression of NCKX1. Because NCKX exchangers do not mediate Ca2+ influx in the absence of external K+ ( 20), the Ca2+ influx we observed via Na+/Ca2+ exchange in the absence of external K+ (Figs. 5C and 7) indicated the functional expression of NCX exchangers in mast cells. According to our RT-PCR data (Fig. 1A), the most likely candidate to support such Na+/Ca2+ exchange is NCX3. In patch clamp experiments, we were also able to detect membrane currents under conditions specific for the detection of NCX and NCKX isoforms (Fig. 2). All in all, these data indicated that both K+-dependent and K+-independent Na+/Ca2+ exchangers are functionally expressed in RBL cells and BMMC.
In some experiments, we treated RBL cells with ouabain to test whether the activity of the expressed NCX and NCKX exchangers depends on the Na+ gradient. Not surprisingly, the K+ dependence of Na+/Ca2+ exchange was not evident under these conditions, although ouabain was present in the bath during Cai recordings (Figs. 6A and 7). In fact, interactions with external K+ were expected because ouabain and K+ ions compete at the K+ binding site of the Na+/K+ ATPase, in such a way, that the effects of ouabain are diminished by increasing external K+ ( 23). Accordingly, the Na+ overload induced by ouabain was likely lowered during the exposure to 70 mM external K+ and the different levels of internal Na+ explain why the ouabain treatment distorted the K+ dependence of Na+/Ca2+ exchange at high external K+. Conversely, ouabain was expected to be more effective at low K+ levels and, in fact, we observed strong effects on Cai responses to Na+ removal both at zero and 5.4 mM external K+ in ouabain-treated RBL cells (Fig. 7; Fig. 5C cf. Fig. 6A), indicating increased Ca2+ influx both via K+-dependent and K+-independent Na+/Ca2+ exchange. Furthermore, it appeared that the ouabain treatment enhanced more effectively the K+-independent Na+/Ca2+ exchange (Fig. 7). By way of caution we note that the removal of external K+ alone also induces an intracellular accumulation of Na+ by blocking the plasma membrane Na+/K+ ATPase ( 24) and, therefore, mimics to some extent the effects of ouabain. Thus, the activity of K+-independent Na+/Ca2+ exchangers might be overestimated when judged from experiments in which external K+ and Na+ are removed simultaneously such as shown in Fig. 5C.
Role of Na+/Ca2+ exchangers in mast cells
The transport stoichiometry of 4 Na+ (1 Ca2+ plus 1 K+) predicts reversal potentials above 50 mV for K+-dependent Na+/Ca2+ exchangers under most physiological conditions (see Ref. 25). Considering that mast cells have a resting potential of about –70 mV and FcRI receptor stimulation depolarizes the cells by <20 mV ( 26, 27), an inversion of the transport is very unlikely and, therefore, NCKX exchangers might in the main support Ca2+ efflux from mast cells. Conversely, the K+-independent exchanger NCX1 can mediate Ca2+ efflux, electroneutral Ca2+ influx as well as Na+ influx ( 28). Based on the stoichiometry of 3 Na+:1 Ca2+ for the major transport mode, it can be predicted that the reversal potential of K+-independent Na+/Ca2+ exchangers is close to –60 mV under physiological conditions ( 7). In mast cells, NCX exchangers might therefore support not only Ca2+ efflux but eventually also Ca2+ influx. When Na+ permeable channels induce Na+ accumulation close to NCX1 exchangers, for instance, the direction of transport is inverted and the resulting Ca2+ influx contributes to Ca2+ plateaus in fibroblasts ( 29). We also detected such Ca2+ influx in RBL cells treated with ouabain (Fig. 6A). In nontreated cells (Fig. 4A), however, the contribution of Ca2+ influx to Ca2+ plateaus was insignificant (20 nM) even under the extreme condition of Na+ removal. We suggest, therefore, that the function of Na+/Ca2+ exchange in mast cells is to support Ca2+ extrusion rather than Ca2+ influx. Na+/Ca2+ exchangers participated marginally or not at all in the basal Ca2+ homoeostasis (Fig. 5B) but played a central role in Ca2+ clearance after stimulation of the PLC signaling pathway (Figs. 3, 4, 8, and 10). We also intended to distinguish between the clearance of Ca2+ ions released from internal stores (Fig. 3) and the clearance of Ca2+ ions entering the cell via store-operated Ca2+ channels (Fig. 4). Based on the Ca2+ clearance plots shown in Figs. 3B and 4B, we suggest that 50% of the Ca2+ extrusion is mediated by Na+/Ca2+ exchangers when Cai is higher than 200 nM in RBL cells.
In the absence of external Na+, we observed reduced Ca2+ levels during the sustained phase of Ca2+ signaling both in RBL cells and BMMC (Figs. 8 and 10). Then, the question arises on how the omission of Ca2+ extrusion via Na+/Ca2+ exchange reduces rather than increases Ca2+ signals. The most like explanation is given by the Mn2+ quenching experiments in Figs. 8 and 10, which suggested that the activity of store-operated Ca2+ channels is diminished when external Na+ is removed. Due to the low Ca2+ clearance, this inhibition likely reflects accumulation of Ca2+ in the cytosol. Several mechanisms have been identified whereby cytosolic Ca2+ inactivates store-operated Ca2+ channels ( 30). These include a direct Ca2+-dependent inactivation and an indirect mechanism in which the Ca2+-dependent refilling of Ca2+ stores diminishes the activity of store-operated Ca2+ channels. Our experiments distinguish between these two possible mechanisms (Fig. 8). By inhibiting store refilling with thapsigargin, we were able to eliminate the store-dependent component and to unmask the Ca2+-depedent inactivation. Under these conditions, we observed strong effects of Na+ removal on Mn2+ quenching and Ca2+ signals in RBL cells and BMMC, indicating that the Ca2+ clearance supported by Na+/Ca2+ exchangers is needed to prevent the Ca2+-dependent inactivation of store-operated Ca2+ channels. So far, it has been shown that the function of store-operated Ca2+ channels is coupled to mitochondrial Ca2+ uptake and Ca2+ extrusion by the PMCA (see Ref. 30). On the basis of the present study, we propose that Na+/Ca2+ exchangers also belong to the group of plasma membrane proteins that shape PLC-dependent Ca2+ signals.
Fig. 11 integrates our findings to previously described mechanisms that control Ca2+ signaling in mast cells (see Ref. 3). Following PLC activation (e.g., via cross-linking of FcRI receptors), two components underlie the enhancement of internal Ca2+: Ca2+ release from internal stores and Ca2+ entry via store-operated Ca2+ channels. The mechanisms that reduce cytosolic Ca2+ include re-uptake into the endoplasmic reticulum by sarco(endo)plasmic reticulum Ca2+-ATPases pumps, Ca2+ uptake into mitochondria mainly via the Ca2+ uniporter and the Ca2+ clearance supported by the PMCA. Our experiments with ouabain indicated that RBL cells express Na+/K+ ATPases in the plasma membrane, which likely control the gradients for Na+ and K+. The Na+/Ca2+ exchangers NCKX3 and NCX3, basically, prevent the Ca2+-dependent inactivation of store-operated Ca2+ channels by extruding Ca2+ ions from cytosol. Accordingly, the Ca2+ entry during the sustained phase of Ca2+ signals might be reduced or enhanced depending on the Ca2+ clearance capacity of the Na+/Ca2+ exchangers. In this regard, it can be expected that antigene-induced mast cell activation depends not only on the transmembrane gradient for Ca2+ but also on the gradients for Na+ and K+. Similarly, the chemotaxis of mucosal mast cells toward adenine nucleotides appears to be dependent on Na+ and K+ gradients ( 31). Finally, it has to be mentioned that NCX exchangers are activated by phosphatidylinositol 4,5-bisphosphate ( 32). Because PLC activation might decrease the phosphatidylinositol 4,5-bisphosphate pool, future experiments are required to study the modulation of Na+/Ca2+ exchangers during mast cell activation.
Acknowledgments
We thank Veit Flockerzi and Peter Lipp for comments on the manuscript, Deborah A. Nicoll and Kenneth D. Philipson for providing NCX cDNAs, M. Wymann for advice on BMMC culture, and Heidi L?hr and S. Buchhlolz for excellent assistance with cell culture.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Address correspondence and reprint requests to Dr. Adolfo Cavalié, Pharmakologie und Toxikologie, Universit?t des Saarlandes, D-66421 Homburg, Germany. E-mail address: adolfo.cavalie@uniklinik-saarland.de
2 Abbreviations used in this paper: PLC, phopholipase C; IP3, inositol 1,4,5-trisphosphate; PMCA, plasma membrane Ca2+-ATPases; Cai, intracellular Ca2+ concentration; BMMC, bone marrow-derived mast cell; RBL, rat basophilic leukemia; NMDG, N-methyl-D-glucamine; TNP, 2,4,6-trinitrophenyl.
Received for publication July 13, 2004. Accepted for publication October 1, 2004.
References
Mikoshiba, K., M. Hattori. 2000. IP3 receptor-operated calcium entry. Sci. STKE 2000:PE1.
Putney, J. W., Jr, L. M Broad, F. J. Braun, J. P. Lievremont, G. S. Bird. 2001. Mechanisms of capacitative calcium entry. J. Cell Sci. 114:2223.
Parekh, A. B., R. Penner. 1997. Store depletion and calcium influx. Physiol. Rev. 77:901.-930.
Berridge, M. J., P. Lipp, M. D. Bootman. 2000. The versatility and universality of calcium signalling. Nat. Rev. Mol. Cell Biol. 1:11.
Glitsch, M. D., D. Bakowski, A. B. Parekh. 2000. Store-operated Ca2+ entry depends on mitochondrial Ca2+ uptake. EMBO J. 21:6744.
Bautista, D. M., R. S. Lewis. 2004. Modulation of plasma-membrane calcium-ATPase activity by local calcium microdomains near CRAC channels in human T cells. J. Physiol. 556:805.
Blaustein, M. P., W. J. Lederer. 1999. Sodium/calcium exchange: its physiological implications. Physiol. Rev. 79:763.
Philipson, K. D., D. A. Nicoll. 2000. Sodium-calcium exchange: a molecular perspective. Annu. Rev. Physiol. 62:111.
Prinsen, C. F., C. B. Cooper, R. T. Szerencsei, S. K. Murthy, D. J. Demetrick, P. P. Schnetkamp. 2002. The retinal rod and cone Na+/Ca2+-K+ exchangers. Adv. Exp. Med. Biol. 514:237.
Reuter, H., H. Porzig. 1995. Localization and functional significance of the Na+/Ca2+ exchanger in presynaptic boutons of hippocampal cells in culture. Neuron 15:1077.
Chernaya, G., M. Vazquez, J. P. Reeves. 1996. Sodium-calcium exchange and store-dependent calcium influx in transfected Chinese hamster ovary cells expressing the bovine cardiac sodium-calcium exchanger: acceleration of exchange activity in thapsigargin-treated cells. J. Biol. Chem. 271:5378.
Brini, M., S. Manni, E. Carafoli. 2002. Recombinant expression of the plasma membrane Na+/Ca2+ exchanger affects local and global Ca2+ homeostasis in Chinese hamster ovary cells. J. Biol. Chem. 277:38693.
Djouder, N., E. Aneiros, A. Cavalié, K. Aktories. 2003. Effects of large clostridial cytotoxins on activation of RBL 2H3-hm1 mast cells indicate common and different roles of Rac in FcRI and M1-receptor signaling. J. Pharmacol. Exp. Ther. 304:1243.
Rumpel, E., U. Pilatus, A. Mayer, I. Pecht. 2000. Na+-dependent Ca2+ transport modulates the secretory response to the Fc receptor stimulus of mast cells. Biophys. J. 79:2975.
Alfonso, A., J. Lago, M. A. Botana, M. R. Vieytes, L. M. Botana. 1999. Characterization of the Na+/Ca2+ exchanger on rat mast cells: evidence for a functional role on the regulation of the cellular response. Cell. Physiol. Biochem. 92:53.
Laffargue, M., R. Calvez, P. Finan, A. Trifilieff, M. Barbier, F. Altruda, E. Hirsch, M. P. Wymann. 2002. Phosphoinositide 3-kinase is an essential amplifier of mast cell function. Immunity 16:441.
Philipp, S., A. Cavalié, M. Freichel, U. Wissenbach, S. Zimmer, C. Trost, A. Marquart, M. Murakami, V. Flockerzi. 1996. A mammalian capacitative calcium entry channel homologous to Drosophila TRP and TRPL. EMBO J. 15:6166.
Fierro, L., R. DiPolo, I. Llano. 1998. Intracellular calcium clearance in Purkinje cell somata from rat cerebellar slices. J. Physiol. 510:499.
Djouder, N., U. Prepens, K. Aktories, A. Cavalié. 2000. Inhibition of calcium release-activated calcium current by Rac/Cdc42-inactivating clostridial cytotoxins in RBL cells. J. Biol. Chem. 275:18732.
Kraev, A., B. D. Quednau, S. Leach, X. F. Li, H. Dong, R. Winkfein, M. Perizzolo, X. Cai, R. Yang, K. D. Philipson, J. Lytton. 2001. Molecular cloning of a third member of the potassium-dependent sodium-calcium exchanger gene family, NCKX3. J. Biol. Chem. 276:23161.
Bernardi, P.. 1999. Mitochondrial transport of cations: channels, exchangers, and permeability transition. Physiol. Rev. 79:1127.
Lee, S. H., M. H. Kim, K. H. Park, Y. E. Earm, W. K. Ho. 2002. K+-dependent Na+/Ca2+ exchange is a major Ca2+ clearance mechanism in axon terminals of rat neurohypophysis. J. Neurosci. 22:6891.
Clausen, T.. 2003. Na+-K+ pump regulation and skeletal muscle contractility. Physiol. Rev. 83:1269.
Harootunian, A. T., J. P. Kao, B. K. Eckert, R. Y. Tsien. 1989. Fluorescence ratio imaging of cytosolic free Na+ in individual fibroblasts and lymphocytes. J. Biol. Chem. 264:19458.
Dong, H., P. E. Light, J. R. French, J. Lytton. 2001. Electrophysiological characterization and ionic stoichiometry of the rat brain K+-dependent Na+/Ca2+ exchanger, NCKX2. J. Biol. Chem. 276:25919.
Duffy, S. M., W. J. Lawley, E. C. Conley, P. Bradding. 2001. Resting and activation-dependent ion channels in human mast cells. J. Immunol. 167:4261.
Gericke, M., O. Dar, G. Droogmans, I. Pecht(Eduardo Aneiros, Stephan )
Mast cells rely on Ca2+ signaling to initiate activation programs leading to release of proinflammatory mediators. The interplay between Ca2+ release from internal stores and Ca2+ entry through store-operated Ca2+ channels has been extensively studied. Using rat basophilic leukemia (RBL) mast cells and murine bone marrow-derived mast cells, we examine the role of Na+/Ca2+ exchangers. Calcium imaging experiments and patch clamp current recordings revealed both K+-independent and K+-dependent components of Na+/Ca2+ exchange. Northern blot analysis indicated the predominant expression of the K+-dependent sodium-calcium exchanger NCKX3. Transcripts of the exchangers NCX3 and NCKX1 were additionally detected in RBL cells with RT-PCR. The Ca2+ clearance via Na+/Ca2+ exchange represented 50% of the total clearance when Ca2+ signals reached levels 200 nM. Ca2+ signaling and store-operated Ca2+ entry were strongly reduced by inverting the direction of Na+/Ca2+ exchange, indicating that Na+/Ca2+ exchangers normally extrude Ca2+ ions from cytosol and prevent the Ca2+-dependent inactivation of store-operated Ca2+ channels. Working in the Ca2+ efflux mode, Na+/Ca2+ exchangers such as NCKX3 and NCX3 might, therefore, play a role in the Ag-induced mast cell activation by controlling the sustained phase of Ca2+ mobilization.
Introduction
Calcium signals are essential in the maturation, differentiation, and activation of cells of the immune system. Generally, stimulation of membrane receptors activates phopholipase C (PLC)2 and promotes the production of inositol 1,4,5-trisphosphate (IP3) that in turn induces the release of Ca2+ ions from the endoplasmic reticulum ( 1). The Ca2+ release is accompanied by the entry of Ca2+ ions from the extracellular space through so-called store-operated Ca2+ channels, which are activated through an unknown mechanism initiated by the depletion of IP3-sensitive Ca2+ stores and represent the principal Ca2+ entry pathway ( 2, 3). Both, Ca2+ release and Ca2+ entry underlie the changes in the cytosolic-free Ca2+ concentration Cai that can be spatially localized or summated to give global Ca2+ signals with a variety of temporal patterns including repetitive spikes and sustained plateaus ( 4). The physiological role of various Ca2+ homeostatic mechanisms in the PLC-dependent Ca2+ signaling has been also recognized. For instance, mitochondria and the plasma membrane Ca2+-ATPase (PMCA) interact functionally with store-operated Ca2+ channels ( 5, 6). However, possible interactions between Na+/Ca2+ exchange, Ca2+ release, and store-operated Ca2+ entry in immune cells are less understood, although Na+/Ca2+ exchangers appear to be for the most part ubiquitously expressed (see Ref. 7).
Two types of Na+/Ca2+ exchanger have been described in mammalian tissues: K+-independent and K+-dependent Na+/Ca2+ exchangers, exemplified by the cardiac and retinal exchangers, respectively ( 7). The K+-independent Na+/Ca2+ exchangers are encoded by a family of three genes (NCX1, NCX2, and NCX3) and a structurally unrelated family of at least four genes (NCKX1, NCKX2, NCKX3, and NCKX4) encodes for K+-dependent Na+/Ca2+ exchangers ( 8, 9). NCX1 is nearly ubiquitous, NCKX3 and NCKX4 are also widely expressed whereas NCKX2, NCX2, and NCX3 appear to have a more restricted tissue distribution. NCKX1 has been found so far only in retinal rods and, interestingly, in megakaryocytes and platelets. Both members of the NCX and NCKX families are bidirectional transporters driven by the transmembrane gradient of Na+ and Ca2+, and NCXK isoforms are additionally dependent on the transmembrane K+ gradient. Thus, any Na+/Ca2+ exchanger can in principle mediate both Ca2+ influx and Ca2+ efflux under appropriated settings of the external concentrations of K+ (Ko), Na+ (Nao), and Ca2+ (Cao). For instance, the direction of exchange can be inverted from Ca2+ efflux to Ca2+ influx by removing external Na+. However, Ca2+ extrusion is the main function of Na+/Ca2+ exchangers in mammalian tissues especially when Cai is elevated and the membrane potential is more negative than the reversal potential of the exchanger ( 7). The exceptions are cardiac cells and some mammalian erythrocytes, in which the Ca2+ influx via Na+/Ca2+ exchange play an important physiological role. The function of Na+/Ca2+ exchangers becomes more prominent when voltage-dependent Ca2+ channels and Na+/Ca2+ exchangers are closely localized such as in synaptic buttons in which Na+/Ca2+ exchangers play an important role controlling exocytosis ( 10). Similarly, the Na+/Ca2+ exchange activity might potentially interact with the store-operated Ca2+ entry. For instance, over-expressed cardiac Na+/Ca2+ exchangers might counteract store-operated Ca2+ entry or enhance the rise of Cai supported by store-operated Ca2+ entry in subplasma membrane regions ( 11, 12), depending probably on expression levels.
In mast cells, cross-linking of IgE receptors (FcRI) by antigenes leads to activation of store-operated Ca2+ channels, elevation of Cai, and release of histamine and other mediators of inflammation (see Ref. 13). Low Nao reduces the secretory response ( 14, 15), suggesting an important role for the Na+/Ca2+ exchange in antigene-stimulated degranulation and secretion of inflammatory mediators from mast cells. In the present study, we analyzed the possible role of Na+/Ca2+ exchange in the Ca2+ homeostasis of mast cells using a rat basophilic leukemia (RBL) cell model and untransformed murine bone marrow-derived mast cells (BMMC). We present for the first time evidences for the expression of the K+-independent (NCX3) and K+-dependent (NCKX1, NCKX3) Na+/Ca2+ exchangers in mast cells. Accordingly, the corresponding Na+/Ca2+ exchange activity was not only dependent on the transmembrane gradient for Na+ and Ca2+ but also for K+. The Na+/Ca2+ exchange working in the Ca2+ efflux mode was required to ensure sustained store-operated Ca2+ entry and Ca2+ plateaus in mast cells.
Materials and Methods
Cell culture
RBL cells expressing the human muscarinic receptor (RBL-2H3-hm1) were cultured in -MEM medium (Invitrogen Life Technologies) supplemented with 15% FBS and 1% L-glutamine at 37°C/5% CO2, as previously described ( 13). Isolation, differentiation and FACS analysis of BMMC were essentially performed as described ( 16). In brief, BMMC were obtained from the femur of mice C57BL6/129SvJ and cultured at 37°C/5% CO2, in IMDM (Invitrogen Life Technologies) supplemented with 10% heat-inactivated FCS, 50 μM 2-ME (Sigma-Aldrich), 10 U/ml penicillin/streptomycin, and 2 ng/ml recombinant murine IL-3 (R&D Systems). To determine the expression of FcRI, cell aliquots were first exposed to 2.4G2 rat anti-mouse FcRII/RIII Ab (BD Pharmingen) and subsequently incubated with mouse IgE (Sigma-Aldrich). Cell staining was performed with FITC-labeled monoclonal rat anti-mouse IgE Ab (BD Pharmingen) and PE-labeled rat anti-c-Kit mAb (BD Pharmingen). As analyzed with a Galaxy Flow Cytometry System (DAKO), >98% of the cells expressed both FcRI and c-Kit after 4 wk of differentiation. Calcium imaging experiments were conducted with BMMC maintained in culture for 14–20 wk. BMMC were attached to coverslips coated with poly-L-lysine (0.5 mg/ml; Sigma-Aldrich) just before start recording Ca2+ signals. The experiments with RBL cells were performed 1–3 days after plating the cells on coverslips. All experiments were conducted at room temperature and repeated with cells of at least two independent batches.
Detection of NCX/NCKX transcripts by RT-PCR and Northern blot
For RT-PCR 5 μg of total RNA from RBL cells and rat brain was reversed transcribed using random primers. One of seven of the reversed transcribed cDNA was applied to PCR to amplify fragments with a length of 440 bp (NCX1), 436 bp (NCX2), 437 bp (NCX3), and 456 bp (NCKX1) and (NCKX3). The following primer pairs specific for each sequence and each separated by at least one intron in the rat genomic sequence were applied to 15 cycles (94°C, 15 s; 62°C, 45 s; 72°C, 45 s) followed by 30 cycles (94°C, 15 s; 62°C, 30 s; 72°C, 45 s plus 2 s/cycle): 5'-GCT TCA TTG TCT CCA TCC TCA TG-3' and 5'-GGA AGA TGT GAG GAG CTT GGC-3' for NCX1 (GenBank accession no. NM_019268); 5'-CTT TGG TGT CTG CAT CCT GGT C-3' and 5'-GGT GGT GGC TAG CTT GGG TC-3' for NCX2 (GenBank accession no. NM_078619); 5'-CTT CGT GGT CTC CAT CCT CAT C-3' and 5'-ACG TTG TGG CAA GCT TGC AGC-3' for NCX3 (GenBank accession no. AJ006781); 5'-CAC CTT CCT GGG ATC CAT CAT C-3' and 5'-CGA TCT TCT AAC ATC ACA CTG ATC-3' for NCKX1 (GenBank accession no. NM_020090); 5'-GAC GTT TGC TTC CTC TAC ACT G-3' and 5'-AAC TCC GTC ATG ATG GAG AAG C-3' for NCKX3 (GenBank accession no. XM_342533). Reaction products were separated by electrophoresis in 2.0% agarose and stained with ethidium bromide. Northern blots were performed as described ( 17) using 10 μg of poly(A)+ RNA from RBL cells, rat brain, liver, and kidney, as well as using 5 μg of poly(A)+ RNA from BMMC, mice brain and rabbit heart. For detection of NCX3 (SLC8A3) transcripts, we used a BspE1/HindIII cDNA fragment from rat NCX3 (kindly provided by D. A. Nicoll and K. D. Philipson, University of California, Los Angeles, School of Medicine, Los Angeles, CA) corresponding to nt 1268–3530 of U53420. For detection of NCKX1 (SLC24A1) and NCKX3 (SLC24A3) the cDNA fragments obtained by RT-PCR using RBL RNA were subcloned, sequenced on both strands and were then used as probes. The probes were labeled by random priming using [-32P]dCTP and hydridized under stringent conditions. The filters were exposed to x-ray films with intensifying screens at –80°C for 3 days (NCKX3) and 21 days (NCX3, NCKX1) before they were hybridized with a 239-bp cDNA fragment of the human GAPDH.
Calcium imaging
A single cell imaging system (Till Photonics) was used to measure Cai as previously described ( 13). Cells were loaded with fura 2-AM (5 μM; Molecular Probes) for 40 min at room temperature. Sequences of paired images containing 5–20 cells/frame were obtained every 3 s with excitation wavelengths of 340 and 380 nm (510 nm emission filter). The corresponding ratios (F340/F380) were used to obtain Cai. In the Mn2+ quench experiments, the fluorescence images were obtained every 3 s with an excitation wavelength of 360 nm (510 nm emission filter). Following a baseline recording to determine the spontaneous decrease of fluorescence, Mn2+ was introduced into the bath. The mean fluorescence intensities were normalized to initial intensities (F/F0). Statistical data and are given as mean (±SEM).
The standard bath solution in the Ca2+ imaging and Mn2+ quenching experiments contained 130 mM NaCl, 5.4 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM glucose and 10 mM HEPES (pH 7.4/NaOH). The various Na+/Ca2+ exchange mechanisms were studied using bath solutions with modified concentrations of CaCl2, KCl, and/or NaCl whereas the concentrations of MgCl2 and glucose were maintained constant. N-methyl-D-glucamine (NMDG) was used to replace Na+ and K+. The Na+-free solutions contained 0, 5.4, or 70 mM KCl and the NMDG concentrations were 135, 130, or 65 mM, respectively (pH 7.4/HCl). In the Ca2+-free solutions with no added CaCl2, 500 μM EGTA were used to ensure Cao values below 10 nM. The Ca2+-free solutions contained various concentrations of K+ and Na+ (5.4 mM KCl and 130 mM NaCl; 5.4 or 70 mM KCl with Na+ replaced by 130 or 65 mM NMDG, respectively, pH 7.4/HCl). The Na+ entry through store-operated channels was prevented by the presence of Mg2+ (>1.9 mM) in the Ca2+-free solutions ( 3). When required, 2 mM LaCl3 were added to nominally Ca2+-free solutions. In the Mn2+ quench experiments, 500 μM MnCl2 were added to the standard bath solutions. Ca2+ signaling was initiated by application of either 2 μM thapsigargin or 1 mM carbachol. Alternatively, cells were primed overnight with 0.3–3 μg/ml anti-2,4,6-trinitrophenyl (TNP) IgE and Ca2+ mobilization was induced by the addition of 50 ng/ml TNP-conjugated-OVA (TNP-OVA). The osmolarity of the bath solutions was 300–340 mOsm. Only ionic concentrations that differ from those of the standard bath solution are indicated in the figures.
Ca2+ clearance
To estimate the contribution of Na+/Ca2+ exchange mechanisms to the Ca2+ homeostasis, we analyzed the Ca2+ clearance (i.e., the return to resting Cai) as previously described ( 18). Elevations of Cai were induced by Ca2+ entry or by depletion of internal Ca2+ stores. Clearance plots were built as follows using the decay phase of Ca2+ transients in Ca2+-free solutions with and without external Na+: 1) the decay phases of Ca2+ transients were fitted with single or double exponential functions to obtain the time constants of Cai decay; 2) The derivative –dCai/dt (i.e., clearance rate) was calculated from Cai time plots; 3) –dCai/dt was plotted as a function of Cai and fitted with sigmoid Hill functions.
Whole-cell patch clamp
Ionic currents were recorded in the whole-cell mode of the patch-clamp technique ( 19) using an EPC 9-2 amplifier (Heka Electronics). Recording pipettes with resistances of 2.5–3 M were pulled from borosilicate glass capillaries (Kimax-51; Kimble Products). Series resistances and capacitive components were compensated using the internal circuit of the amplifier. Only experiments with series resistances below 10 M were used for further analysis. The average capacitance of the RBL cells was 12.6 ± 0.6 pF (n = 35). Voltage-clamp steps (100 ms) were applied every 2 s to potentials between –100 and +50 mV from a holding potential of 0 mV. Membrane currents were sampled at 4 kHz and 33.3 Hz (filter cutoff: 33 kHz, 6.67 Hz) in the pulse and continuous mode, respectively. To allow the exchange of the external solutions, the glass coverslips containing RBL cells were placed into a recording chamber that had a volume of 600 μl and was connected to a gravity-driven perfusion system. Data were analyzed offline using a combination of the software packages Pulse/Pulsefit (Heka Electronics). Statistical data are given as mean (±SEM).
Magnesium-inhibited currents and store-operated Ca2+ currents were suppressed by high cytosolic Mg2+ (2 mM) and low buffering of cytosolic Ca2+ (1 μM), respectively. Additionally, pipette and bath solutions contained tetraethylammonium (TEA)+ to block K+ channel currents. As in previous studies of recombinant Na+/Ca2+ exchangers (e.g., Ref. 20), we used a Na+-based pipette solution containing 120 mM NaCl, 40 mM KCL, 20 mM TEA-Cl, 2 mM MgCl2, 2 mM Mg-ATP, 8 mM glucose, 10 mM HEPES (pH 7.2/CsOH), and 1 μM free Ca2+. The external solution in the patch clamp experiments contained 130 mM NaCl, 40 mM KCl, 20 mM TEA-Cl, 2 mM MgCl2, 10 mM glucose and 10 mM HEPES (pH 7.2/CsOH), and 500 μM EGTA. With this set of solutions, we recorded nearly linear currents at potential between –100 and +50 mV in nearly every analyzed RBL cell, suggesting a complete block of ionic channel currents. Na+/Ca2+ exchange currents were elicited by switching to a bath solutions that contained 130 mM LiCl, 20 mM TEA-Cl, 2 mM MgCl2, 2 mM CaCl2, 10 mM glucose and 10 mM HEPES (pH 7.2/CsOH). The KCl content of the bath solutions was either 0 or 40 mM. Free Ca2+ concentrations in the pipette solutions were adjusted using CaCl2/EGTA mixtures calculated with the MaxChelator (v2.10) (
Results
Expression of Na+/Ca2+ exchangers in RBL cells
Because it is not known whether mast cells express Na+/Ca2+ exchangers, we first determine the expression of NCX and NCKX isoforms in RBL cells with RT-PCR and Northern blot analysis. Using specific primers for the various NCX and NCKX exchangers, we detected a prominent expression of the K+-dependent Na+/Ca2+ exchanger NCKX3 (Fig. 1). Lower expression levels of the K+-independent Na+/Ca2+ exchanger NCX3 and the K+-dependent Na+/Ca2+ exchanger NCKX1 became also visible after RT-PCR amplification (Fig. 1A). We were not able to detect the expression of NCX1 and NCX2. In Northern blots, the expression of NCKX3 was readily detected after 3 days exposure of the filter (Fig. 1B). By contrast, the expression of NCX3 and NCKX1 was not detected in Northern blots even after 21 days filter exposure. In line with previous reports ( 20), the major NCKX3 transcript was 4.4 kb in length and was abundantly expressed in RBL cells as well as in brain, whereas kidney and liver apparently lacked expression of NCKX3 (Fig. 1B).
Next, we measure Na+/Ca2+ exchange currents to determine whether the detected NCX and NCKX isoforms are functional in RBL cells. The selected experimental settings ensured the blockade of ionic channel currents previously described in RBL cells (see Materials and Methods). Because all Na+/Ca2+ exchangers are bidirectional transporters, the rational in the patch-clamp experiments was basically to switch between the Ca2+ efflux and Ca2+ influx modes and record the resulting inward and outward currents, respectively. We switched into Ca2+ efflux mode by removing external Ca2+ in the presence of external Na+ and, conversely, into the Ca2+ influx mode by removing external Na+ in the presence of external Ca2+. Furthermore, high internal Na+ was used to enhance outward currents in Ca2+ influx mode, also to slow down the Ca2+ efflux and, consequently, to minimize inward currents. Thus, the design of the patch-clamp experiments allowed primarily the detection of outward currents, i.e., Ca2+ influx. Fig. 2A illustrates experiments in which whole-cell membrane currents were recorded continuously at –80 mV during the exchange of solutions. In these experiments, we used high external K+ to ensure the function of K+-dependent Na+/Ca2+ exchangers. Independently of the bath solution used, we recorded negative currents at negative membrane potentials. The main effect of the removal of external Na+ in the presence of Ca2+ was an upward shift of currents levels, which was reversed upon re-addition of Na+ and removal of Ca2+ (Fig. 2A). Likely, this shift to less negative current levels reflected the appearance of outward currents that superimposed on baseline currents. Furthermore, we also determine current-voltage relations using voltage-clamp steps (Fig. 2B). As expected from the continuous current recordings (Fig. 2A), the removal of Na+ in the presence of Ca2+ induced a reduction of the current amplitudes at potentials below –25 mV (Fig. 2, B and C). At 0 mV, no current was detected in the Ca2+-free solution but we consistently observed the appearance of small detectable outward currents after the switch to the Na+-free solution (Fig. 2, B and C). Thus, the exposure to the Na+-free, Ca2+-containing solution produced a shift of the current-voltage relations into the upward direction at potentials 0 mV (Fig. 2C). According to the ionic settings of the external and internal solutions, the difference between the current-voltage relations shown in Fig. 2C likely represented an outward current component supported by any Na+/Ca2+ exchanger because all NCX and NCKX isoforms allow Ca2+ influx in the absence of external Na+. In an effort to distinguish between K+-dependent and K+-independent components of Na+/Ca2+ exchange currents, we omitted K+ ions from the external solutions to suppress Ca2+ influx through K+-dependent Na+/Ca2+ exchangers in further whole-cell current recordings. Other experimental conditions were as in Fig. 2, A–C. Outward current components were estimated as difference currents (I) elicited by the switch from Na+-containing, Ca2+-free solutions to Na+-free, Ca2+-containing solutions. As illustrated in Fig. 2D, K+-independent Na+/Ca2+ exchangers appeared to function in RBL cells because we never observed zero I values in the absence of external K+ and, similarly, K+-dependent Na+/Ca2+ exchangers must also function in RBL cells because we usually observed higher I values in the presence than in the absence of external K+.
Ca2+ clearance
Because generally the main function of Na+/Ca2+ exchangers is the extrusion of Ca2+ ions from cytosol, we analyzed in RBL cells the clearance of Ca2+ ions released from intracellular stores (Fig. 3) and the clearance of Ca2+ ions entering the cell throughstore-operated Ca2+ channels (Fig. 4). For this purpose, we separated experimentally the components of Ca2+ signals, i.e., Ca2+ release from Ca2+ entry, using the so called Ca2+ re-addition protocols ( 2). Thapsigargin or carbachol were applied to RBL cells exposed to external Ca2+-free solutions to isolate the Ca2+ release component (Fig. 3). Subsequently, external Ca2+ was re-added to the bath solution to visualize the Ca2+ entry during Ca2+ plateaus (Fig. 4). The Ca2+ clearance supported by Na+/Ca2+ exchangers was worked out by comparing clearance rates measured in the absence of external Ca2+ with and without external Na+ because the removal of external Ca2+ alone is expected to reinforce Ca2+ efflux and the additional removal of external Na+ to suppress Ca2+ efflux through Na+/Ca2+ exchangers. Because the K+ dependence of Na+/Ca2+ exchange can be hardly analyzed in the Ca2+ efflux mode using calcium imaging, external K+ was maintained constant at 5.4 mM.
In the absence of external Ca2+, the Ca2+ release signals induced by carbachol attained roughly identical peaks independently of whether Na+ was present (0.42 ± 0.02 μM, n = 36) or absent (0.47 ± 0.04 μM, n = 30) in the external solution (Fig. 3A). Similar results were obtained with thapsigargin, indicating that the Ca2+ release was not affected by the removal of external Na+. Basically, the removal of external Na+ slowed down the time course of Ca2+ release signals (Fig. 3A). Apparent differences in Cai baselines were not statistically significant. To compare time courses, Ca2+ release signals were normalized to peak values and the decay phase was fitted with single exponential functions that were sufficient to account for 90% of the decay. As illustrated in Fig. 3B, inset, the decay time constant of average Ca2+ release signals was 1.7-fold slower in the absence of Na+, suggesting inhibition of a Ca2+ clearance component supported by Na+/Ca2+ exchange mechanisms. Clearly, this inhibition was small compared with the inhibition produced by 2 mM La3+ (Fig. 3A), indicating that various Ca2+ clearance mechanisms were still functioning in the absence of external Na+. The Ca2+ clearance component inhibited by the removal of external Na+ was visualized using Ca2+ clearance plots, as previously described ( 18). As shown in Fig. 3B, the dependence of clearance rates (–dCai/dt) on Cai followed a sigmoid Hill function both in the presence and absence of external Na+. In the absence of Na+, however, the clearance rates were lower within a wide Cai range. The inspection of the –dCai/dt curves (Fig. 3B) indicated 28–51% reduction of clearance rates within the Cai range from 200 to 400 nM, when the cells were exposed to Na+-free solutions. Thus, the inhibition of Na+/Ca2+ exchange in Na+- and Ca2+-free solutions slowed down the clearance of Ca2+ ions released from intracellular stores preferentially when Cai was close to the peak of Ca2+ release signals.
The effects of external Na+ on the clearance of Ca2+ ions entering the cell via store-operated Ca2+ channels were analyzed in Ca2+ re-addition experiments (Fig. 4). We observed that the levels and time courses of Ca2+ plateaus were not dependent on external Na+ (Fig. 4A). On average, Cai was 0.56 ± 0.01 μM and 0.50 ± 0.01 μM (n = 31) 2 min after re-addition of Ca2+ in the presence and absence of external Na+, respectively. The effects of Na+ removal became evident during the decay of Cai produced by the removal of external Ca2+ (Fig. 4A). To quantify these effects, normalized average Cai decays were fitted with single exponential functions, which accounted for 99% of the decay (Fig. 4B, inset). A comparison of the exponential time constants indicated that the Cai decayed 1.2-fold slower in the absence of external Na+. The corresponding Ca2+ clearance plots indicated that the removal of external Na+ reduced –dCai/dt by 39–58% preferentially at Cai levels above 200 nM (Fig. 4B). As for the clearance of Ca2+ ions released from intracellular stores (Fig. 3), it appeared therefore that Na+/Ca2+ exchangers removed the Ca2+ ions that entered the cell, primarily when Cai was close Ca2+ plateau levels.
Na+/Ca2+ exchange at basal Cai
The basal Cai of RBL cells exposed to the standard bath solution ranged from 18 to 171 nM with a mean of 71.7 ± 1.6 nM (Fig. 5A). Because modification in the composition of the bath solution usually increased or decreased Cai only within this range, changes in basal Cai (Cai) were measured with respect to Cai values obtained at the beginning of recordings. In analogy to previous experiments (Figs. 3 and 4), external Ca2+ and/or Na+ were removed to test whether Na+/Ca2+ exchangers contribute to the maintenance of basal Cai (Fig. 5B). The removal of external Ca2+ alone induced a rapid drop in Cai (Fig. 5B, left panel), which can be explained by the suppression of basal Ca2+ entry into the cell and/or by the enhancement of Ca2+ extrusion supported by Na+/Ca2+ exchangers. When external Na+ was additionally removed to suppress Ca2+ efflux via Na+/Ca2+ exchangers after the removal of Ca2+ (Fig. 5B, right panel), however, we were not able to detect changes in the time course of Cai. Thus, the contribution of Na+/Ca2+ exchange to Ca2+ clearance at basal Cai levels appeared to be marginal.
To test further for the operation of Na+/Ca2+ exchangers at basal Cai levels, we removed external Na+ in the presence of external Ca2+, a maneuver that was expected to invert the direction of transport from Ca2+ efflux to Ca2+ influx via all Na+/Ca2+ exchangers ( 7). With 5.4 mM external K+, we observed a continuous increase of Cai after removal of external Na+ (Fig. 5C), suggesting the presence of functional Na+/Ca2+ exchangers in RBL cells. The operation of K+-independent Na+/Ca2+ exchangers was indicated by the transient increase of Cai induced by the simultaneous removal of external K+ and Na+ (Fig. 5C, inset), because Ca2+ influx via K+-dependent Na+/Ca2+ exchangers is inhibited in the absence of external K+ ( 9). Furthermore, we tested the effects of Na+ removal in the presence of 70 mM external K+ because the K+-dependent Na+/Ca2+ exchange can be enhanced by increasing K+. Under these conditions, we observed a transient Cai increase that was higher than in 5.4 mM external K+ (Fig. 5C), indicating the presence of functional K+-dependent Na+/Ca2+ exchangers in RBL cells. To test further the activity of Na+/Ca2+ exchangers, we induced Na+ overload by exposing the cells to ouabain, a blocker of the Na+/K+ ATPase. High internal Na+ was expected to increase the driving force for Ca2+ influx via Na+/Ca2+ exchangers. Accordingly, we observed in ouabain-treated cells a prominent Cai increase after the removal of Na+ in 5.4 mM external K+ (Fig. 6A cf. Fig. 5C). In contrast to nontreated cells (Fig. 5C), however, the Cai increase induced in ouabain-treated cells by Na+ removal was similar in the presence of 5.4 and 70 mM external K+ (Fig. 6A). Thus, the ouabain treatment enhanced the Ca2+ influx at least at 5.4 mM external K+ (nontreated: 14.5 ± 7.1 nM, Fig. 5C; ouabain-treated: 65.8 ± 6.7 nM, Fig. 6A), as expected if Na+/Ca2+ exchangers are functionally expressed in RBL cells.
A distinct possibility that we considered was that Ca2+ release from intracellular stores and/or Ca2+ entry through ionic channels contributed to the Cai increase induced by Na+ removal (Figs. 5C and 6A). The possible mobilization of Ca2+ from intracellular stores was unlikely because no Ca2+ release signal was observed when external Na+ was removed from Ca2+-free solutions in the presence of 5.4 mM (data not shown) or 70 mM external K+ (Fig. 5B, right panel). In Mn2+ quenching experiments with 5.4 mM external K+, the fluorescence decreased linearly after application of Mn2+ into the bath, indicating a basal ionic leak in ouabain-treated cells (Fig. 6B). The quenching rates, however, remained unchanged after removal of external Na+ (0.064 ± 0.007 s–1 and 0.077 ± 0.005 s–1; Fig. 6B). Similar results were obtained in Mn2+ quenching experiments with nontreated cells (data not shown), suggesting that Na+ removal did not activate Ca2+/Mn2+ permeable channels. Furthermore, Ca2+ entry was not expected in experiments with 70 mM external K+ because high K+ depolarizes the cells and, consequently, reduces the driving force for Ca2+ entry. Thus, it was unlikely that an enhancement of Ca2+ entry through ionic channels contributed to the changes of Cai shown in Figs. 5C and 6A.
Fig. 7 shows the summary of the experiments in which the transmembrane gradients for Ca2+, Na+, and K+ were systematically changed to test the function of Na+/Ca2+ exchangers at basal Cai in RBL cells. Each Cai value was obtained in an independent experiment because we observed that Cai hardly returned to basal levels after solution exchange, for instance, after application of Na+-free, Ca2+-containing solutions (see Figs. 5C and 6A). In nontreated cells (without (–) ouabain), Cai values recorded after the removal of Na+ and K+ were on average 28% of those obtained after removing only Na+, suggesting an important contribution of K+-independent Na+/Ca2+ exchangers in these experiments. Furthermore, the Cai induced by Na+ removal was potentiated about 7-fold by increasing external K+ from 5.4 to 70 mM (p < 0.001), as expected for the functional expression of K+-dependent Na+/Ca2+ exchangers. In ouabain-treated cells (with (+) ouabain), no difference was observed between Cai values obtained after Na+ removal in the presence of 5.4 and 70 mM K+ but the removal of Na+ and K+ induced Cai values that amounted 50% of corresponding values measured in 5.4 mM external K+. Comparing ouabain-treated and nontreated cells, we found that ouabain had no effect on the Cai induced by Na+ removal at 70 mM external K+. By contrast, ouabain enhanced the Cai responses to Na+ removal by 9-fold with no K+ and by 4.5-fold in 5.4 mM external K+ (p < 0.001). According to the latter results, it appeared that the ouabain treatment was more effective on the K+ independent Na+/Ca2+ exchange. The Cai measured after removal of external Ca2+ was not dependent on external Na+ and K+, supporting our suggestion that Na+/Ca2+ exchangers contribute marginally to the maintenance of basal Cai.
Na+/Ca2+ exchange during Ca2+ signaling
Finally, we explored the function of Na+/Ca2+ exchangers during the generation of Ca2+ signals in RBL cells (Fig. 8), a process that is believed to involve Ca2+ release from intracellular stores and store-operated Ca2+ entry ( 3). An analysis of the K+ dependence of Na+/Ca2+ exchange during Ca2+ signaling was precluded because changes in the transmembrane gradient for K+ modify the driving force for Ca2+ entry. We indeed observed in preliminary experiments (data not shown) that the increase of external K+ from 5.4 to 70 mM abolished Ca2+ plateaus completely in a similar way as when external Ca2+ was removed (Fig. 4A). To keep constant the store-operated Ca2+ entry, external K+ and Ca2+ were therefore maintained at 5.4 and 2 mM, respectively. Because the removal of external Na+ is sufficient to invert the transport mode of K+-dependent and K+-independent Na+/Ca2+ exchangers ( 7), we analyzed Ca2+ signaling in standard and Na+-free solutions, i.e., with Na+/Ca2+ exchangers working in Ca2+ efflux and Ca2+ influx modes, respectively. The advantage was that such Ca2+ influx was expected to be insignificant under these conditions because Ca2+ plateaus were nearly identical in standard and Na+-free solutions (Fig. 4A). When external Na+ was removed in the middle of Ca2+ plateaus i.e., 3 min after Ca2+ re-addition in experiments conceptually similar to those shown in Fig. 4A, the instantaneous Cai increase was 16 ± 6 nM (n = 31). Thus, the absence of external Na+ was expected to prevent Ca2+ efflux rather than to induce Ca2+ influx via Na+/Ca2+ exchangers during Ca2+ signaling.
The Ca2+ signals were initiated in RBL cells either by the blockade of sarco(endo)plasmic reticulum Ca2+-ATPases pumps in the internal Ca2+ stores with thapsigargin or by activation of the stably expressed muscarinic receptors with carbachol ( 13, 19). Fig. 8 illustrates the effects of the Na+-free solution on Ca2+ signals. The initial peaks of Ca2+ signals induced by carbachol were almost identical with and without external Na+ (Fig. 8A), in line with previous experiments indicating that Na+-free solutions modify the time course but not the peak of the Ca2+ release component (Fig. 3A). With thapsigargin, the peaks of Ca2+ signals were 41% smaller in the Na+-free solution (Fig. 8C; 1.20 ± 0.05 μM vs 0.71 ± 0.04 μM). During the sustained phase of the Ca2+ signals, however, we observed 34–65% lower Cai levels in absence of external Na+ both after stimulation with thapsigargin and carbachol (Fig. 8, A and C; carbachol: 1.09 ± 0.14 μM vs 0.38 ± 0.07 μM; thapsigargin: 0.41 ± 0.02 μM vs 0.27 ± 0.02 μM). Because store-operated Ca2+ entry has been usually associated with the sustained phase of Ca2+ signals ( 3), we next performed Mn2+ quenching experiments (Fig. 8, B and D) to estimate the activity of store-operated Ca2+ channels in the Na+-free solution. As predicted from the experiments shown in Fig. 6B, the Na+-free solution had no effect on spontaneous quenching rates, i.e., on the fluorescence decay after addition of Mn2+ and before application of Ca2+ mobilizing agents. Indicating activation of store-operated Ca2+ channels, carbachol and thapsigargin enhanced the Mn2+ quenching in the standard solution (Fig. 8, B and D, left panels). In the absence of external Na+, however, the enhancement of quenching rates was less pronounced both after application of carbachol and thapsigargin (Fig. 8, B and D; carbachol: 1.74 ± 0.02 s–1 vs 0.16 ± 0.001 s–1; thapsigargin: 0.39 ± 0.02 s–1 vs 0.19 ± 0.02 s–1). In fact, the quenching rates were almost identical before and after application of carbachol (Fig. 8B, right panel). Thus, the Mn2+ quenching experiments demonstrated that Na+-free solutions, basically, diminish the activity of store-operated Ca2+ channels. Consequently, the lower Cai levels during the sustained phase of Ca2+ signals (Fig. 8, A and C, right panels) likely reflect the reduced Ca2+ entry in the absence of external Na+.
Na+/Ca2+ exchange in murine BMMC
In the experiments with RBL cells shown in Fig. 8, stably transfected muscarinic receptors were stimulated with carbachol and internal Ca2+ stores were depleted with thapsigargin. To test whether Na+/Ca2+ exchangers are involved in regulating store-operated Ca2+ entry upon FcRI cross-linking in nontransformed mast cells, we next performed experiments with murine BMMC.
Fig. 9 illustrates that the major Na+/Ca2+ exchanger found in RBL cells is also expressed in BMMC. The predominant NCKX3 transcript was 4.4 kb in length in BMMC and brain, whereas heart apparently lacks NCKX3 expression. Taking into account the GAPDH signals, a densitometric analysis of the Northern blots shown in Fig. 1B and 9A indicated that the NCKX3 signals of RBL and BMMC correspond to 88 and 76% of the respective brain NCKX3 signals. The basal Cai of BMMC was 197 ± 23 nM (n = 97). In experiments conceptually similar to those shown for RBL cells in Fig. 5C, we tested the function of K+-dependent and K+-independent Na+/Ca2+ exchangers in BMMC. As illustrated in Fig. 9B, the removal of external Na+ was sufficient to induces an increase of Cai to 279 ± 11 nM (n = 33). When external K+ was raised to 70 mM simultaneously with the Na+ removal, Cai increased to 319 ± 7 nM (n = 6). The corresponding Cai values induced by removal of external Na+ in the presence of 5.4 and 70 mM were 81 ± 9 nM and 108 ± 1 nM, respectively. Furthermore, Cai increased with a much faster time course in the experiments with high external K+. Thus, BMMC appeared to express functional K+-dependent and K+-independent Na+/Ca2+ exchangers as RBL cells. Fig. 10 illustrates the functional consequences of removing external Na+ previously to cell activation via FcRI cross-linking. In BMMC primed with anti-TNP IgE, the Ca2+ signals initiated by the application of TNP-OVA showed the typical sustained phase that lasted >12 min. Generally, the sustained phase of antigene-induced Ca2+ mobilization is much lower in BMMC than in RBL cells ( 13 cf.16). As expected from the experiments shown in Fig. 9B, the removal of external Na+ alone shifted the basal Cai to higher values also in the experiments shown in Fig. 10A. Under these conditions, application of TNP-OVA was still able to induce Cai mobilization in BMMC, but the time courses of the Ca2+ signals were disrupted in the Na+-free solutions. After the application of TNP-OVA, Ca2+ oscillations were observed in a considerable number of cells. The oscillations and rise of Cai lasted no longer than 5 min. In contrast to control cells, no sustained phase of Ca2+ signal was detectable and Cai returned rapidly to initial values in the absence of external Na+. To compare the time courses of Ca2+ signals, we constructed Cai time plots as illustrated in Fig. 10B. The Cai peak values attained immediately after application of TNP-OVA appeared to be not dependent on external Na+. At times longer than 5–7 min, however, the different time courses became evident. The faster Cai decay in the absence of external Na+ resulted in Cai values lower that 50 nM after 10 min of exposure to TNP-OVA, whereby the sustained Ca2+ signals in the presence of external Na+ maintained values of 93 ± 6 nM (n = 15) for >10 min. Similarly, the removal of external Na+ resulted in a 75% reduction of Ca2+ plateau levels in RBL cells primed with anti-TNP IgE and stimulated with TNP-OVA (174 ± 8 nM, n = 40 vs 44 ± 12 nM, n = 20; 10 min TNP-OVA stimulation). As for RBL cells (Fig. 8C), the sustained phase of Ca2+ signals induced by the application of 1 μM thapsigargin to BMMC was reduced 79% by the removal of external Na+ (403 ± 24 nM, n = 35 vs 318 ± 64 nM, n = 25; 10 min thapsigargin exposure). To test whether the reduced Ca2+ plateaus levels of BMMC exposed to Na+-free solutions were due to a reduced activity of store-operated Ca2+ channels, we performed Mn2+ quenching experiments. Fig. 10C illustrates that the quenching rates measured in BMMC were, in fact, less pronounced in the absence of external Na+ (0.23 ± 0.01 s–1, n = 20 vs 0.11 ± 0.01 s–1, n = 15). As in RBL cells, thus, the main effect of removing external Na+ was a reduction store-operated Ca2+ entry in BMMC, which correlated with the inability of BMMC to maintain Ca2+ plateaus for longer times in the absence of external Na+.
Discussion
In the present study, we combined calcium imaging and the patch clamp technique with Northern blot and RT-PCR analysis to establish the presence and function of Na+/Ca2+ exchangers in mast cells using the RBL model and murine BMMC. Our data suggested an important role for K+-dependent (NCKX3, NCKX1) and K+-independent (NCX3) Na+/Ca2+ exchangers in the Ca2+ clearance at Ca2+ plateau levels. Accordingly, Ca2+ signals were strongly dependent on external Na+ and, more interestingly, the activity of store-operated Ca2+ channels appeared to be diminished in the absence of external Na+.
Detection of Na+/Ca2+ exchange activity
As in previous studies of Na+/Ca2+ exchangers ( 20), we detected Na+/Ca2+ exchange activity in RBL cells and BMMC by taking advantage of the fact that the direction of ionic transport through all NCX and NCKX isoforms changes from Ca2+ efflux to Ca2+ influx when external Na+ is removed (Figs. 2, 5C, 6A, 7, and 9B). Furthermore, Mn2+ quenching experiments ruled out the possible activation of Ca2+ entry through ionic channels in Na+-free solutions (Fig. 6B). However, side effects of Na+ removal need to be considered. For instance, it is well known that the Ca2+ efflux from mitochondria is mediated by Na+/Ca2+ exchangers ( 21). Because the removal of external Na+ might reduce the intracellular Na+ concentration, thus, an inhibition of the Ca2+ efflux from mitochondria can be expected in Na+-free solutions. In the absence of external Ca2+, in fact, the removal of external Na+ was not able to induce any detectable Cai enhancement (Figs. 5B and 7), indicating that internal Ca2+ stores including mitochondria did not contribute to the Cai enhancement induced by Na+ removal. Furthermore, we challenged the RBL cells with ouabain to strengthen our evidences favoring the existence of Na+/Ca2+ exchangers in mast cells. As in previous studies of Na+/Ca2+ exchange with a number of cells including those that express exclusively NCKX isoforms ( 22), the rational behind this approach was to induce intracellular accumulation of Na+ and test whether the Ca2+ influx via Na+/Ca2+ exchange increases according to the enhanced driving force. In this context, we note that such increase of internal Na+ might also force the Ca2+ efflux from mitochondria. Because the exposure to ouabain lasted >1 h, such effect might produce a sustained increase of basal Cai in ouabain-treated cells. However, the Cai enhancements we report were transient and lasted as long as Na+ was absent from the external solution (Fig. 6A), indicating that the Ca2+ influx underlying the Cai increase in ouabain-treated cells was dependent on the transmembrane Na+ gradient. Thus, the Cai and membrane currents recordings with inverted Na+ gradients strongly support our suggestion that Na+/Ca2+ exchangers are present and function in RBL cells and BMMC.
K+-dependent and K+-independent Na+/Ca2+ exchangers
The RT-PCR data indicated that K+-dependent (NCKX1, NCKX3) and K+-independent (NCX3) Na+/Ca2+ exchanger isoforms are expressed in RBL cells (Fig. 1A). However, only the expression of NCKX3 was detected in Northern blots (Fig. 1B). Because the expression analysis by Northern blot is less sensitive than by RT-PCR, NCKX3 might be the predominant Na+/Ca2+ exchanger in RBL cells. Similarly, BMMC appear to express the Na+/Ca2+ exchanger NCKX3 (Fig. 9A). As NCX3 expression was only detected by RT-PCR in RBL cells, the question raised on whether this K+-independent Na+/Ca2+ exchanger is functionally expressed in mast cells. At physiological levels of internal Na+, the Ca2+ influx via Na+/Ca2+ exchange was largely dependent on external K+ (Figs. 5C, 7, and 9B), in line with the prominent expression of NCKX3. Additional experiments will be needed to proof the functional expression of NCKX1. Because NCKX exchangers do not mediate Ca2+ influx in the absence of external K+ ( 20), the Ca2+ influx we observed via Na+/Ca2+ exchange in the absence of external K+ (Figs. 5C and 7) indicated the functional expression of NCX exchangers in mast cells. According to our RT-PCR data (Fig. 1A), the most likely candidate to support such Na+/Ca2+ exchange is NCX3. In patch clamp experiments, we were also able to detect membrane currents under conditions specific for the detection of NCX and NCKX isoforms (Fig. 2). All in all, these data indicated that both K+-dependent and K+-independent Na+/Ca2+ exchangers are functionally expressed in RBL cells and BMMC.
In some experiments, we treated RBL cells with ouabain to test whether the activity of the expressed NCX and NCKX exchangers depends on the Na+ gradient. Not surprisingly, the K+ dependence of Na+/Ca2+ exchange was not evident under these conditions, although ouabain was present in the bath during Cai recordings (Figs. 6A and 7). In fact, interactions with external K+ were expected because ouabain and K+ ions compete at the K+ binding site of the Na+/K+ ATPase, in such a way, that the effects of ouabain are diminished by increasing external K+ ( 23). Accordingly, the Na+ overload induced by ouabain was likely lowered during the exposure to 70 mM external K+ and the different levels of internal Na+ explain why the ouabain treatment distorted the K+ dependence of Na+/Ca2+ exchange at high external K+. Conversely, ouabain was expected to be more effective at low K+ levels and, in fact, we observed strong effects on Cai responses to Na+ removal both at zero and 5.4 mM external K+ in ouabain-treated RBL cells (Fig. 7; Fig. 5C cf. Fig. 6A), indicating increased Ca2+ influx both via K+-dependent and K+-independent Na+/Ca2+ exchange. Furthermore, it appeared that the ouabain treatment enhanced more effectively the K+-independent Na+/Ca2+ exchange (Fig. 7). By way of caution we note that the removal of external K+ alone also induces an intracellular accumulation of Na+ by blocking the plasma membrane Na+/K+ ATPase ( 24) and, therefore, mimics to some extent the effects of ouabain. Thus, the activity of K+-independent Na+/Ca2+ exchangers might be overestimated when judged from experiments in which external K+ and Na+ are removed simultaneously such as shown in Fig. 5C.
Role of Na+/Ca2+ exchangers in mast cells
The transport stoichiometry of 4 Na+ (1 Ca2+ plus 1 K+) predicts reversal potentials above 50 mV for K+-dependent Na+/Ca2+ exchangers under most physiological conditions (see Ref. 25). Considering that mast cells have a resting potential of about –70 mV and FcRI receptor stimulation depolarizes the cells by <20 mV ( 26, 27), an inversion of the transport is very unlikely and, therefore, NCKX exchangers might in the main support Ca2+ efflux from mast cells. Conversely, the K+-independent exchanger NCX1 can mediate Ca2+ efflux, electroneutral Ca2+ influx as well as Na+ influx ( 28). Based on the stoichiometry of 3 Na+:1 Ca2+ for the major transport mode, it can be predicted that the reversal potential of K+-independent Na+/Ca2+ exchangers is close to –60 mV under physiological conditions ( 7). In mast cells, NCX exchangers might therefore support not only Ca2+ efflux but eventually also Ca2+ influx. When Na+ permeable channels induce Na+ accumulation close to NCX1 exchangers, for instance, the direction of transport is inverted and the resulting Ca2+ influx contributes to Ca2+ plateaus in fibroblasts ( 29). We also detected such Ca2+ influx in RBL cells treated with ouabain (Fig. 6A). In nontreated cells (Fig. 4A), however, the contribution of Ca2+ influx to Ca2+ plateaus was insignificant (20 nM) even under the extreme condition of Na+ removal. We suggest, therefore, that the function of Na+/Ca2+ exchange in mast cells is to support Ca2+ extrusion rather than Ca2+ influx. Na+/Ca2+ exchangers participated marginally or not at all in the basal Ca2+ homoeostasis (Fig. 5B) but played a central role in Ca2+ clearance after stimulation of the PLC signaling pathway (Figs. 3, 4, 8, and 10). We also intended to distinguish between the clearance of Ca2+ ions released from internal stores (Fig. 3) and the clearance of Ca2+ ions entering the cell via store-operated Ca2+ channels (Fig. 4). Based on the Ca2+ clearance plots shown in Figs. 3B and 4B, we suggest that 50% of the Ca2+ extrusion is mediated by Na+/Ca2+ exchangers when Cai is higher than 200 nM in RBL cells.
In the absence of external Na+, we observed reduced Ca2+ levels during the sustained phase of Ca2+ signaling both in RBL cells and BMMC (Figs. 8 and 10). Then, the question arises on how the omission of Ca2+ extrusion via Na+/Ca2+ exchange reduces rather than increases Ca2+ signals. The most like explanation is given by the Mn2+ quenching experiments in Figs. 8 and 10, which suggested that the activity of store-operated Ca2+ channels is diminished when external Na+ is removed. Due to the low Ca2+ clearance, this inhibition likely reflects accumulation of Ca2+ in the cytosol. Several mechanisms have been identified whereby cytosolic Ca2+ inactivates store-operated Ca2+ channels ( 30). These include a direct Ca2+-dependent inactivation and an indirect mechanism in which the Ca2+-dependent refilling of Ca2+ stores diminishes the activity of store-operated Ca2+ channels. Our experiments distinguish between these two possible mechanisms (Fig. 8). By inhibiting store refilling with thapsigargin, we were able to eliminate the store-dependent component and to unmask the Ca2+-depedent inactivation. Under these conditions, we observed strong effects of Na+ removal on Mn2+ quenching and Ca2+ signals in RBL cells and BMMC, indicating that the Ca2+ clearance supported by Na+/Ca2+ exchangers is needed to prevent the Ca2+-dependent inactivation of store-operated Ca2+ channels. So far, it has been shown that the function of store-operated Ca2+ channels is coupled to mitochondrial Ca2+ uptake and Ca2+ extrusion by the PMCA (see Ref. 30). On the basis of the present study, we propose that Na+/Ca2+ exchangers also belong to the group of plasma membrane proteins that shape PLC-dependent Ca2+ signals.
Fig. 11 integrates our findings to previously described mechanisms that control Ca2+ signaling in mast cells (see Ref. 3). Following PLC activation (e.g., via cross-linking of FcRI receptors), two components underlie the enhancement of internal Ca2+: Ca2+ release from internal stores and Ca2+ entry via store-operated Ca2+ channels. The mechanisms that reduce cytosolic Ca2+ include re-uptake into the endoplasmic reticulum by sarco(endo)plasmic reticulum Ca2+-ATPases pumps, Ca2+ uptake into mitochondria mainly via the Ca2+ uniporter and the Ca2+ clearance supported by the PMCA. Our experiments with ouabain indicated that RBL cells express Na+/K+ ATPases in the plasma membrane, which likely control the gradients for Na+ and K+. The Na+/Ca2+ exchangers NCKX3 and NCX3, basically, prevent the Ca2+-dependent inactivation of store-operated Ca2+ channels by extruding Ca2+ ions from cytosol. Accordingly, the Ca2+ entry during the sustained phase of Ca2+ signals might be reduced or enhanced depending on the Ca2+ clearance capacity of the Na+/Ca2+ exchangers. In this regard, it can be expected that antigene-induced mast cell activation depends not only on the transmembrane gradient for Ca2+ but also on the gradients for Na+ and K+. Similarly, the chemotaxis of mucosal mast cells toward adenine nucleotides appears to be dependent on Na+ and K+ gradients ( 31). Finally, it has to be mentioned that NCX exchangers are activated by phosphatidylinositol 4,5-bisphosphate ( 32). Because PLC activation might decrease the phosphatidylinositol 4,5-bisphosphate pool, future experiments are required to study the modulation of Na+/Ca2+ exchangers during mast cell activation.
Acknowledgments
We thank Veit Flockerzi and Peter Lipp for comments on the manuscript, Deborah A. Nicoll and Kenneth D. Philipson for providing NCX cDNAs, M. Wymann for advice on BMMC culture, and Heidi L?hr and S. Buchhlolz for excellent assistance with cell culture.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Address correspondence and reprint requests to Dr. Adolfo Cavalié, Pharmakologie und Toxikologie, Universit?t des Saarlandes, D-66421 Homburg, Germany. E-mail address: adolfo.cavalie@uniklinik-saarland.de
2 Abbreviations used in this paper: PLC, phopholipase C; IP3, inositol 1,4,5-trisphosphate; PMCA, plasma membrane Ca2+-ATPases; Cai, intracellular Ca2+ concentration; BMMC, bone marrow-derived mast cell; RBL, rat basophilic leukemia; NMDG, N-methyl-D-glucamine; TNP, 2,4,6-trinitrophenyl.
Received for publication July 13, 2004. Accepted for publication October 1, 2004.
References
Mikoshiba, K., M. Hattori. 2000. IP3 receptor-operated calcium entry. Sci. STKE 2000:PE1.
Putney, J. W., Jr, L. M Broad, F. J. Braun, J. P. Lievremont, G. S. Bird. 2001. Mechanisms of capacitative calcium entry. J. Cell Sci. 114:2223.
Parekh, A. B., R. Penner. 1997. Store depletion and calcium influx. Physiol. Rev. 77:901.-930.
Berridge, M. J., P. Lipp, M. D. Bootman. 2000. The versatility and universality of calcium signalling. Nat. Rev. Mol. Cell Biol. 1:11.
Glitsch, M. D., D. Bakowski, A. B. Parekh. 2000. Store-operated Ca2+ entry depends on mitochondrial Ca2+ uptake. EMBO J. 21:6744.
Bautista, D. M., R. S. Lewis. 2004. Modulation of plasma-membrane calcium-ATPase activity by local calcium microdomains near CRAC channels in human T cells. J. Physiol. 556:805.
Blaustein, M. P., W. J. Lederer. 1999. Sodium/calcium exchange: its physiological implications. Physiol. Rev. 79:763.
Philipson, K. D., D. A. Nicoll. 2000. Sodium-calcium exchange: a molecular perspective. Annu. Rev. Physiol. 62:111.
Prinsen, C. F., C. B. Cooper, R. T. Szerencsei, S. K. Murthy, D. J. Demetrick, P. P. Schnetkamp. 2002. The retinal rod and cone Na+/Ca2+-K+ exchangers. Adv. Exp. Med. Biol. 514:237.
Reuter, H., H. Porzig. 1995. Localization and functional significance of the Na+/Ca2+ exchanger in presynaptic boutons of hippocampal cells in culture. Neuron 15:1077.
Chernaya, G., M. Vazquez, J. P. Reeves. 1996. Sodium-calcium exchange and store-dependent calcium influx in transfected Chinese hamster ovary cells expressing the bovine cardiac sodium-calcium exchanger: acceleration of exchange activity in thapsigargin-treated cells. J. Biol. Chem. 271:5378.
Brini, M., S. Manni, E. Carafoli. 2002. Recombinant expression of the plasma membrane Na+/Ca2+ exchanger affects local and global Ca2+ homeostasis in Chinese hamster ovary cells. J. Biol. Chem. 277:38693.
Djouder, N., E. Aneiros, A. Cavalié, K. Aktories. 2003. Effects of large clostridial cytotoxins on activation of RBL 2H3-hm1 mast cells indicate common and different roles of Rac in FcRI and M1-receptor signaling. J. Pharmacol. Exp. Ther. 304:1243.
Rumpel, E., U. Pilatus, A. Mayer, I. Pecht. 2000. Na+-dependent Ca2+ transport modulates the secretory response to the Fc receptor stimulus of mast cells. Biophys. J. 79:2975.
Alfonso, A., J. Lago, M. A. Botana, M. R. Vieytes, L. M. Botana. 1999. Characterization of the Na+/Ca2+ exchanger on rat mast cells: evidence for a functional role on the regulation of the cellular response. Cell. Physiol. Biochem. 92:53.
Laffargue, M., R. Calvez, P. Finan, A. Trifilieff, M. Barbier, F. Altruda, E. Hirsch, M. P. Wymann. 2002. Phosphoinositide 3-kinase is an essential amplifier of mast cell function. Immunity 16:441.
Philipp, S., A. Cavalié, M. Freichel, U. Wissenbach, S. Zimmer, C. Trost, A. Marquart, M. Murakami, V. Flockerzi. 1996. A mammalian capacitative calcium entry channel homologous to Drosophila TRP and TRPL. EMBO J. 15:6166.
Fierro, L., R. DiPolo, I. Llano. 1998. Intracellular calcium clearance in Purkinje cell somata from rat cerebellar slices. J. Physiol. 510:499.
Djouder, N., U. Prepens, K. Aktories, A. Cavalié. 2000. Inhibition of calcium release-activated calcium current by Rac/Cdc42-inactivating clostridial cytotoxins in RBL cells. J. Biol. Chem. 275:18732.
Kraev, A., B. D. Quednau, S. Leach, X. F. Li, H. Dong, R. Winkfein, M. Perizzolo, X. Cai, R. Yang, K. D. Philipson, J. Lytton. 2001. Molecular cloning of a third member of the potassium-dependent sodium-calcium exchanger gene family, NCKX3. J. Biol. Chem. 276:23161.
Bernardi, P.. 1999. Mitochondrial transport of cations: channels, exchangers, and permeability transition. Physiol. Rev. 79:1127.
Lee, S. H., M. H. Kim, K. H. Park, Y. E. Earm, W. K. Ho. 2002. K+-dependent Na+/Ca2+ exchange is a major Ca2+ clearance mechanism in axon terminals of rat neurohypophysis. J. Neurosci. 22:6891.
Clausen, T.. 2003. Na+-K+ pump regulation and skeletal muscle contractility. Physiol. Rev. 83:1269.
Harootunian, A. T., J. P. Kao, B. K. Eckert, R. Y. Tsien. 1989. Fluorescence ratio imaging of cytosolic free Na+ in individual fibroblasts and lymphocytes. J. Biol. Chem. 264:19458.
Dong, H., P. E. Light, J. R. French, J. Lytton. 2001. Electrophysiological characterization and ionic stoichiometry of the rat brain K+-dependent Na+/Ca2+ exchanger, NCKX2. J. Biol. Chem. 276:25919.
Duffy, S. M., W. J. Lawley, E. C. Conley, P. Bradding. 2001. Resting and activation-dependent ion channels in human mast cells. J. Immunol. 167:4261.
Gericke, M., O. Dar, G. Droogmans, I. Pecht(Eduardo Aneiros, Stephan )