Modulation of Ca²⁺ Signaling by Na⁺/Ca²⁺ Exchangers in Mast Cells

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 Ca_i 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⁺ (K_o), Na⁺ (Na_o), and Ca²⁺ (Ca_o). 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 Ca_i 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 Ca_i 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 (FcεRI) by antigenes leads to activation of store-operated Ca²⁺ channels, elevation of Ca_i, and release of histamine and other mediators of inflammation (see Ref. 13). Low Na_o 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.

RBL cells expressing the human muscarinic receptor (RBL-2H3-hm1) were cultured in α-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 FcεRI, cell aliquots were first exposed to 2.4G2 rat anti-mouse FcγRII/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 FcεRI 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.

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 [α-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.

A single cell imaging system (Till Photonics) was used to measure Ca_i 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 Ca_i. 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 Ca_o 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.

To estimate the contribution of Na⁺/Ca²⁺ exchange mechanisms to the Ca²⁺ homeostasis, we analyzed the Ca²⁺ clearance (i.e., the return to resting Ca_i) as previously described ( 18). Elevations of Ca_i 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 Ca_i decay; 2) The derivative −dCa_i/dt (i.e., clearance rate) was calculated from Ca_i time plots; 3) −dCa_i/dt was plotted as a function of Ca_i and fitted with sigmoid Hill functions.

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 MΩ 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 MΩ 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) (<www.stanford.edu/∼cpatton/>). The osmolarity of internal and external solutions was 300–340 mOsm.

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. 1,A). 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. 1,B). 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. 1 B).

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. 2,A 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. 2,A). 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. 2,B). As expected from the continuous current recordings (Fig. 2,A), 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. 2,C). According to the ionic settings of the external and internal solutions, the difference between the current-voltage relations shown in Fig. 2,C 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 (ΔI) elicited by the switch from Na⁺-containing, Ca²⁺-free solutions to Na⁺-free, Ca²⁺-containing solutions. As illustrated in Fig. 2 D, K⁺-independent Na⁺/Ca²⁺ exchangers appeared to function in RBL cells because we never observed zero ΔI 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 ΔI values in the presence than in the absence of external K⁺.

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. 3,A). 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. 3,A). Apparent differences in Ca_i 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. 3,B, 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. 3,A), 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. 3,B, the dependence of clearance rates (−dCa_i/dt) on Ca_i 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 Ca_i range. The inspection of the −dCa_i/dt curves (Fig. 3 B) indicated 28–51% reduction of clearance rates within the Ca_i 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 Ca_i 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. 4,A). On average, Ca_i 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 Ca_i produced by the removal of external Ca²⁺ (Fig. 4,A). To quantify these effects, normalized average Ca_i decays were fitted with single exponential functions, which accounted for 99% of the decay (Fig. 4,B, inset). A comparison of the exponential time constants indicated that the Ca_i decayed ∼1.2-fold slower in the absence of external Na⁺. The corresponding Ca²⁺ clearance plots indicated that the removal of external Na⁺ reduced −dCa_i/dt by 39–58% preferentially at Ca_i levels above 200 nM (Fig. 4,B). 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 Ca_i was close Ca²⁺ plateau levels.

The basal Ca_i 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. 5,A). Because modification in the composition of the bath solution usually increased or decreased Ca_i only within this range, changes in basal Ca_i (ΔCa_i) were measured with respect to Ca_i 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 Ca_i (Fig. 5,B). The removal of external Ca²⁺ alone induced a rapid drop in Ca_i (Fig. 5,B, 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. 5 B, right panel), however, we were not able to detect changes in the time course of ΔCa_i. Thus, the contribution of Na⁺/Ca²⁺ exchange to Ca²⁺ clearance at basal Ca_i levels appeared to be marginal.

To test further for the operation of Na⁺/Ca²⁺ exchangers at basal Ca_i 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 Ca_i after removal of external Na⁺ (Fig. 5,C), 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 Ca_i induced by the simultaneous removal of external K⁺ and Na⁺ (Fig. 5,C, 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 Ca_i increase that was higher than in 5.4 mM external K⁺ (Fig. 5,C), 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 Ca_i increase after the removal of Na⁺ in 5.4 mM external K⁺ (Fig. 6,A cf. Fig. 5,C). In contrast to nontreated cells (Fig. 5,C), however, the Ca_i increase induced in ouabain-treated cells by Na⁺ removal was similar in the presence of 5.4 and 70 mM external K⁺ (Fig. 6,A). Thus, the ouabain treatment enhanced the Ca²⁺ influx at least at 5.4 mM external K⁺ (nontreated: 14.5 ± 7.1 nM, Fig. 5,C; ouabain-treated: 65.8 ± 6.7 nM, Fig. 6 A), 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 Ca_i increase induced by Na⁺ removal (Figs. 5,C and 6,A). 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. 5,B, 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. 6,B). 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. 6,B). 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 Ca_i shown in Figs. 5,C and 6 A.

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 Ca_i in RBL cells. Each ΔCa_i value was obtained in an independent experiment because we observed that Ca_i hardly returned to basal levels after solution exchange, for instance, after application of Na⁺-free, Ca²⁺-containing solutions (see Figs. 5,C and 6 A). In nontreated cells (without (−) ouabain), ΔCa_i 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 ΔCa_i 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 ΔCa_i values obtained after Na⁺ removal in the presence of 5.4 and 70 mM K⁺ but the removal of Na⁺ and K⁺ induced ΔCa_i 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 ΔCa_i induced by Na⁺ removal at 70 mM external K⁺. By contrast, ouabain enhanced the ΔCa_i 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 ΔCa_i 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 Ca_i.

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. 4,A). 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. 4,A). 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. 4 A, the instantaneous Ca_i 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. 8,A), in line with previous experiments indicating that Na⁺-free solutions modify the time course but not the peak of the Ca²⁺ release component (Fig. 3,A). With thapsigargin, the peaks of Ca²⁺ signals were ∼41% smaller in the Na⁺-free solution (Fig. 8,C; 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 Ca_i 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. 6,B, 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. 8,B, right panel). Thus, the Mn²⁺ quenching experiments demonstrated that Na⁺-free solutions, basically, diminish the activity of store-operated Ca²⁺ channels. Consequently, the lower Ca_i 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⁺.

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 FcεRI 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. 1,B and 9,A indicated that the NCKX3 signals of RBL and BMMC correspond to 88 and 76% of the respective brain NCKX3 signals. The basal Ca_i of BMMC was 197 ± 23 nM (n = 97). In experiments conceptually similar to those shown for RBL cells in Fig. 5,C, we tested the function of K⁺-dependent and K⁺-independent Na⁺/Ca²⁺ exchangers in BMMC. As illustrated in Fig. 9,B, the removal of external Na⁺ was sufficient to induces an increase of Ca_i to 279 ± 11 nM (n = 33). When external K⁺ was raised to 70 mM simultaneously with the Na⁺ removal, Ca_i increased to 319 ± 7 nM (n = 6). The corresponding ΔCa_i 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, Ca_i 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 FcεRI 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. 9,B, the removal of external Na⁺ alone shifted the basal Ca_i to higher values also in the experiments shown in Fig. 10,A. Under these conditions, application of TNP-OVA was still able to induce Ca_i 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 Ca_i lasted no longer than 5 min. In contrast to control cells, no sustained phase of Ca²⁺ signal was detectable and Ca_i returned rapidly to initial values in the absence of external Na⁺. To compare the time courses of Ca²⁺ signals, we constructed ΔCa_i time plots as illustrated in Fig. 10,B. The ΔCa_i 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 Ca_i decay in the absence of external Na⁺ resulted in ΔCa_i 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. 8,C), 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. 10 C 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⁺.

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⁺.

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, 5,C, 6,A, 7, and 9,B). Furthermore, Mn²⁺ quenching experiments ruled out the possible activation of Ca²⁺ entry through ionic channels in Na⁺-free solutions (Fig. 6,B). 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 Ca_i enhancement (Figs. 5,B and 7), indicating that internal Ca²⁺ stores including mitochondria did not contribute to the Ca_i 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 Ca_i in ouabain-treated cells. However, the Ca_i enhancements we report were transient and lasted as long as Na⁺ was absent from the external solution (Fig. 6 A), indicating that the Ca²⁺ influx underlying the Ca_i increase in ouabain-treated cells was dependent on the transmembrane Na⁺ gradient. Thus, the Ca_i 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.

The RT-PCR data indicated that K⁺-dependent (NCKX1, NCKX3) and K⁺-independent (NCX3) Na⁺/Ca²⁺ exchanger isoforms are expressed in RBL cells (Fig. 1,A). However, only the expression of NCKX3 was detected in Northern blots (Fig. 1,B). 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. 9,A). 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. 5,C, 7, and 9,B), 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. 5,C and 7) indicated the functional expression of NCX exchangers in mast cells. According to our RT-PCR data (Fig. 1,A), 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 Ca_i recordings (Figs. 6,A 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 Ca_i responses to Na⁺ removal both at zero and 5.4 mM external K⁺ in ouabain-treated RBL cells (Fig. 7; Fig. 5,C cf. Fig. 6,A), 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. 5 C.

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 FcεRI 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. 6,A). In nontreated cells (Fig. 4,A), 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. 5,B) 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. 3,B and 4 B, we suggest that ∼50% of the Ca²⁺ extrusion is mediated by Na⁺/Ca²⁺ exchangers when Ca_i 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 FcεRI 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.

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 Löhr and S. Buchhlolz for excellent assistance with cell culture.