Time-dependent modulation of capacitative Ca2+ entry signals by plasma membrane Ca2+ pump in endothelium

Andrey Klishin, Marina Sedova, and Lothar A. Blatter

Department of Physiology and Cardiovascular Institute, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois 60153

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

In vascular endothelial cells, depletion of intracellular Ca2+ stores elicited capacitative Ca2+ entry (CCE) that resulted in biphasic changes of intracellular Ca2+ concentration ([Ca2+]i) with a rapid initial peak of [Ca2+]i followed by a gradual decrease to a sustained plateau level. We investigated the rates of Ca2+ entry, removal, and sequestration during activation of CCE and their respective contributions to the biphasic changes of [Ca2+]i. Ca2+ buffering by mitochondria, removal by Na+/Ca2+ exchange, and a fixed electrical driving force for Ca2+ (voltage-clamp experiments) had little effect on the CCE signal. The rates of entry of Mn2+ and Ba2+, used as unidirectional substitutes for Ca2+ entry through the CCE pathway, were constant and did not follow the concomitant changes of [Ca2+]i. Pharmacological inhibition of the plasma membrane Ca2+ pump, however, abolished the secondary decay phase of the CCE transient. The disparity between the biphasic changes of [Ca2+]i and the constant rate of Ca2+ entry during CCE was the result of a delayed, Ca2+-dependent activation of the pump. These results suggest an important modulatory role of the plasma membrane Ca2+ pump in the net cellular gain of Ca2+ during CCE.

cytoplasmic calcium concentration; endoplasmic reticulum; thapsigargin; indo 1

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

A WIDE ARRAY OF CELLULAR processes such as contraction, cell development, and secretion is controlled by intracellular Ca2+. One of the most exciting areas in cellular Ca2+ signaling has been the discovery of capacitative Ca2+ entry (CCE) (39), i.e., a Ca2+ entry pathway across the plasma membrane that is activated by depletion of intracellular Ca2+ stores. CCE has been described in many nonexcitable cell types (for recent reviews see Refs. 2, 7, 17, 40), including vascular endothelial cells (16, 31, 47). There is increasing evidence that CCE not only serves to refill intracellular Ca2+ stores (the endoplasmic reticulum) but also is involved in variety of other important cellular functions. For example, CCE has been suggested to play a role in volume regulation, phototransduction, mitogenesis, regulation of adenylate cyclase, and sustained Ca2+ oscillations and Ca2+ waves (2).

Major unanswered questions in the field of CCE deal with the nature of the signal that reports the filling status of the intracellular stores to the surface membrane, the biophysical properties of the entry pathway, and the cellular mechanisms that regulate Ca2+ fluxes through this pathway. Despite the lack of detailed knowledge of many aspects of this important Ca2+ entry pathway, some biophysical properties of this membrane conductance have been established. For example, Hoth and Penner (19) were the first to describe a Ca2+ current, and its gating mechanisms, that was activated by store depletion in mast cells. They referred to this current as Ca2+ release-activated Ca2+ (CRAC) current (ICRAC). To date, Ca2+ release-activated channels have been characterized with different conductances and selectivities in other tissues, and there is increasing evidence that the original CRAC channel represents only one member of an entire family of Ca2+ channels that are controlled by store depletion (7).

Many functions of vascular endothelial cells are mediated and regulated by changes of intracellular Ca2+ concentration ([Ca2+]i). Vascular endothelial cells typically lack voltage-gated Ca2+ channels (5, 15), and it has been suggested that CCE might be the sole Ca2+ entry pathway in certain vascular endothelial cells (31), emphasizing the relevance of this pathway to Ca2+ homeostasis. Changes in [Ca2+]i resulting from CCE typically exhibit kinetics with an initial rapid increase of [Ca2+]i, followed by a slower decay to an elevated plateau level of [Ca2+]i. It remains a matter of debate, however, whether these biphasic kinetics, especially the plateau phase, are the result of inactivation of CCE or a time-dependent activation of Ca2+ removal or sequestration. Indeed, ICRAC has been reported to rapidly inactivate (20). This rapid inhibition of CCE was only partial, and it has been suggested that Ca2+ could enter through this route indefinitely at a lower rate (58). Contributions from Ca2+ influx, removal, and sequestration during CCE, however, are difficult to separate experimentally due to the lack of specific pharmacological tools to inhibit these pathways. To date, there is no specific blocker for the CCE pathway, and many of the compounds that have been shown to exert various degrees of inhibition of CCE also affect other Ca2+-handling systems such as the plasma membrane Ca2+-ATPase (PMCA). The experimental evaluation of these systems is further complicated by the fact that Ca2+ itself often plays an autoregulatory role in the transport of Ca2+. ICRAC, for example, has been reported to be inactivated by Ca2+, and positive and negative feedback loops for the regulation of CCE involving Ca2+ have been documented (2). Furthermore, it has been suggested that the feedback effects of Ca2+ on CCE might be highly localized (22, 34), thereby emphasizing the importance of microdomains for Ca2+ signaling (21).

Despite these experimental difficulties, the goal of the present study was to characterize the cellular mechanisms that maintain a dynamic balance between Ca2+ influx and Ca2+ removal during CCE, resulting in the typical biphasic kinetics of [Ca2+]i transients. With the combined use of electrophysiological methods and fluorescence microscopy, we demonstrate that CCE in vascular endothelial cells displays a rate of Ca2+ entry that is nearly constant over time and that the typical biphasic changes of [Ca2+]i result from a delayed and [Ca2+]i-dependent activation of the PMCA.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cultured Vascular Endothelial Cells

Experiments were performed on cultured calf pulmonary artery endothelial (CPAE) cells. The CPAE cell line was purchased from the American Type Culture Collection (ATCC CCL-209, Rockville, MD). The cells were cultured in Eagle's minimum essential medium, supplemented with 20% fetal bovine serum (GIBCO, Grand Island, NY) and L-glutamine (2 mM) and kept at 37°C in an atmosphere of 5% CO2 and 95% air. Once a week the cells were dispersed using a Ca2+-free (0.1% EDTA) 0.25% trypsin solution and subcultured onto glass coverslips for later experimentation. Cells from passages 2-5 were used. Experiments were carried out within 1 wk after plating the cells onto coverslips. All experiments were performed at room temperature (20-22°C) on single cells in nonconfluent cultures.

Fluorescence Measurements

Spatially averaged [Ca2+]i measurements from single endothelial cells were performed as described previously (16). Briefly, cultured endothelial cells were loaded with the ratiometric Ca2+ indicator indo 1 in standard Tyrode solution containing 5 µM indo 1-AM (Molecular Probes, Eugene, OR) and 5 µl of 20% wt/wt Pluronic F-127 (Molecular Probes; solubilized in DMSO) for 20 min. [Ca2+]i was calculated according to the formula (12) [Ca2+] = KD × beta  × (R - Rmin)/(Rmax - R). R denotes the ratio of the cellular fluorescence signals recorded at two emission wavelengths, Flambda em1 and Flambda em2 (R = Flambda em1/Flambda em2). KD was assumed to be 250 nM (12), and beta  was defined as Flambda em2 at zero Ca2+ concentration divided by Flambda em2 at saturating Ca2+ concentration. The experiments presented in this study were performed on two different experimental setups with slightly different optical components. Indo 1 fluorescence was excited at either 357 or 361 nm. Flambda em1 was 405 or 410 nm, respectively, whereas Flambda em2 was 485 nm on both setups. Values for Rmin, Rmax, and beta  were determined for each individual setup. Rmin and Rmax were obtained by intracellular calibrations using the Ca2+ ionophore ionomycin. Individual endothelial cells were exposed to 10 µM ionomycin and superfused with Ca2+-free (10 mM EGTA) and 10 mM Ca2+-containing Tyrode solution to obtain Rmin and Rmax, respectively.

Simultaneous measurements of [Ca2+]i and Mn2+ entry in indo 1-loaded cells were achieved by recording cellular fluorescence simultaneously at 424 nm (Ca2+-isosbestic, Mn2+-sensitive emission wavelength) and at 485 nm (Ca2+-sensitive wavelength). In control experiments, it was determined that the cytoplasmic fluorescence signal was nearly Ca2+ insensitive (isosbestic) at the emission wavelength of 424 nm. Small cell-to-cell differences for the indo 1 isosbestic emission wavelength for Ca2+, however, were observed. These differences (<= 1 nm) were corrected for by adjusting the angle at which the 424 ± 10-nm interference filter was positioned in the emission pathway to change the peak transmission wavelength of the filter. This adjustment was made in each Mn2+ experiment during the recording of the first control [Ca2+]i transient in the absence of Mn2+. Fluorescence signals were low-pass filtered at 1 kHz and sampled at 0.5 Hz.

Electrophysiological Measurements

Membrane currents were measured using a perforated patch-clamp technique (25). Amphotericin B (0.2 mg/ml; Sigma, St. Louis, MO) was added to the pipette solution containing (in mM) 138 KCl, 1 MgCl2, 10 HEPES, and 10 dextrose, adjusted to pH 7.2 with KOH. Na+ and K+ in the extracellular superfusion solution were substituted with N-methyl-D-glucamine. Membrane currents were measured with an Axopatch 200B patch-clamp amplifier (Axon Instruments, Foster City, CA). Patch pipettes were pulled from glass capillaries (TW150F; World Precision Instruments, Sarasota, FL) and fire polished to give a resistance of 2.5-5.0 MOmega when filled with pipette solution.

Solutions and Chemicals

The cells were superfused continuously with a physiological salt solution (standard Tyrode solution) composed of (in mM) 135 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 10 dextrose, and 10 HEPES, titrated to pH 7.3 with NaOH. In some experiments, extracellular Ca2+ concentration ([Ca2+]o) varied between 0.1 and 10 mM. In these experiments, [Na+]o was increased or decreased accordingly to hold ionic strength constant. In Na+-free Tyrode solution, Na+ was replaced with equimolar Li+ or K+. Carbonyl cyanide p-(trifluoromethoxy)phenyl-hydrazone (FCCP), ionomycin, and oligomycin were obtained from Sigma. Bisindolylmaleimide I [protein kinase C (PKC) inhibitor], H-89 [protein kinase A (PKA) inhibitor], and W-7 (calmodulin antagonist) were from Calbiochem (San Diego, CA), thapsigargin was from Alexis Biochemicals (San Diego, CA), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; Cl- channel inhibitor) was obtained from Research Biochemicals International (Natick, MA).

Statistical Analysis

When appropriate, population results are reported as means ± SE for the indicated number (n) of cells. Statistical significance was determined with the nonparametric one-tailed Wilcoxon rank test for paired observations.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Intracellular Store Depletion and Kinetics of CCE Transients

The standard protocol to deplete intracellular Ca2+ stores and to evoke CCE in CPAE cells is illustrated in Fig. 1. During superfusion with Ca2+-free Tyrode solution, cells were exposed to 1 µM thapsigargin, a selective inhibitor of the endoplasmic reticulum Ca2+ pump (16, 53). After recovery of the thapsigargin-induced [Ca2+]i transient, the cell was exposed to different [Ca2+]o. Reexposure to extracellular Ca2+ caused a typical biphasic [Ca2+]i transient as a result of CCE. This protocol was used for CCE activation in all experiments presented. The CCE transient1 was characterized by an initial rise of [Ca2+]i that rapidly reached a peak, followed by a slower decline of [Ca2+]i to a sustained plateau of elevated [Ca2+]i while extracellular Ca2+ was present. The peak amplitude, the plateau level, and the kinetics of the CCE transient revealed a complex dependence on [Ca2+]o as well as on [Ca2+]i. The steady-state [Ca2+]i level eventually reached during the plateau phase appeared to be directly proportional to [Ca2+]o. The peak amplitude, however, was influenced not only by [Ca2+]o but also by the preceding level of [Ca2+]i. The peak amplitude retained the same tendency of direct proportionality with [Ca2+]o. For a given [Ca2+]o, however, the peak amplitude was inversely proportional to the preceding [Ca2+]i, whereas the plateau level remained constant (illustrated by the shaded areas in Fig. 1 for the case of [Ca2+]o = 10 mM).


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Fig. 1.   Capacitative Ca2+ entry (CCE) in cultured calf pulmonary artery endothelial (CPAE) cells. In all experiments, intracellular Ca2+ stores were first depleted by thapsigargin treatment (Tg; 1 µM; solid bar). Despite removal of thapsigargin from superfusion solution after depletion of stores, endoplasmic reticulum Ca2+ pumps remain blocked due to irreversible binding of inhibitor (16). Protocol for extracellular Ca2+ concentration ([Ca2+]o) changes is shown underneath intracellular Ca2+ concentration ([Ca2+]i) trace. Shaded areas highlight 10 mM [Ca2+]o periods.

These initial experiments suggested that changes of [Ca2+]i during CCE were more complex than expected from mere Ca2+ influx driven by the transmembrane electrochemical gradient for Ca2+. The data implicated the involvement of [Ca2+]i-dependent processes. Potential candidates were a [Ca2+]i-dependent regulation of CCE itself or activation of other Ca2+-transporting systems. To explore these possibilities, we next set out to study the rate of Ca2+ entry during activation of CCE, as well as the involvement and kinetics of Ca2+ removal, sequestration, and buffering that occurred simultaneously during CCE.

Driving Force for Ca2+ Influx During CCE

Our first approach was to determine whether the observed kinetics of the CCE transient could simply be related to concomitant changes of the membrane potential (Em). Specifically, we investigated whether the observed decay phase of the CCE transient might be the result of membrane depolarization caused by a net influx of Ca2+ or by activation of Ca2+-dependent anion or cation conductances that would lead to a reduction of the electrochemical driving force for Ca2+ influx. To test this possibility, CCE recordings were obtained under voltage-clamp conditions. Em was held constant at -30 mV using the perforated patch method. Reexposure to Ca2+ (2 mM) after thapsigargin treatment caused the characteristic biphasic CCE transients (Fig. 2, bottom) that were paralleled by transient inward currents (Fig. 2, top). This current was carried primarily by Cl- because it was sensitive to the Cl- channel inhibitor NPPB, and the reversal potential of this current shifted according to the calculated Cl- equilibrium potential when extracellular Cl- concentration was altered (data not shown). As illustrated by Fig. 2, the kinetics of the CCE transients were qualitatively similar under voltage-clamp conditions. Furthermore, current-clamp experiments (data not shown) revealed that the maximum changes in Em during CCE transients were <10 mV and that Em remained significantly more negative than the equilibrium potential for Ca2+.


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Fig. 2.   Simultaneous recordings of [Ca2+]i and membrane current (Im) in a CPAE cell using perforated patch-clamp technique. Amphotericin B was used as pore-forming compound. Membrane potential was held at -30 mV. Maximum error in voltage-clamp in this experiment was 3.4 mV [series resistance (Rs) = 17 MOmega ; 60% compensated]. Rise of [Ca2+]i due to exposure to thapsigargin (open bar) and during CCE (solid bars) was paralleled by transient inward currents with similar time courses as changes in [Ca2+]i. Additional experiments (data not shown; see text for details) revealed that net inward current was sum of leak current and a Ca2+-activated Cl- current.

With these experiments, we could eliminate the possibility that the biphasic kinetics of the CCE transient, particularly the secondary decay phase, could simply be explained by a reduced electrical driving force for Ca2+ entry. Nevertheless, these experiments did not provide any information on the rate of Ca2+ entry during the different phases of the CCE transient or, specifically, on whether a slowing of the rate of Ca2+ entry could account for the decay phase. Therefore, in the next step, we attempted to directly measure ion transport rates through the CCE pathway.

Divalent Cation Entry Through the CCE Pathway

The measurement of the unidirectional flux of Ca2+ through the CCE channels in intact cells is difficult due to removal and sequestration of cellular Ca2+ occurring simultaneously with CCE. We therefore took advantage of the fact that Ba2+ and Mn2+ are capable of passing through the CCE pathway (40). Both ions are poor substrates for Ca2+ removal systems (28, 48) and can serve as surrogates for Ca2+ to study unidirectional ion permeation through Ca2+ entry pathways, including CCE channels.

Figure 3A shows indo 1 ratio signals obtained from a thapsigargin-treated endothelial cell during exposure to extracellular Ca2+ (2 mM) and subsequently to 5 mM Ba2+. Restoring extracellular Ca2+ led to the typical biphasic CCE transient (Fig. 3A, left). When Ca2+ was replaced with 5 mM Ba2+, a completely different time course of rise of the indo 1 ratio signal was observed. In contrast to Ca2+, Ba2+ entry after store depletion caused a monophasic increase of the ratio signal, consistent with steady entry of Ba2+. Because the Ba2+-indo 1 complex is less fluorescent than the Ca2+-indo 1 complex (37), the indo 1 ratio signal reports changes of [Ca2+]i and [Ba2+]i on different scales. We have estimated the [Ba2+]i by calibrating Rmax for the Ba2+-indo 1 complex intracellularly (for details, see Fig. 3). As shown in Fig. 3B, [Ba2+]i reached substantially higher levels than [Ca2+]i.


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Fig. 3.   Decay phase to a steady-state plateau level of CCE transient is mediated by intracellular Ca2+. A: in contrast to Ca2+, Ba2+ entry after store depletion led to a monophasic increase of [Ba2+]i. Note that indo 1 has a lower affinity for Ba2+ than for Ca2+ and that fluorescence intensity (F410) of Ba2+-indo 1 complex is lower than that of Ca2+-indo 1 complex. Therefore, same changes in indo 1 fluorescence ratio (R = F410/F485) reflect much higher changes of [Ba2+]i than of [Ca2+]i. B: [Ba2+]i was estimated from ratio signal with assumption that [Ca2+]i was constant during Ba2+ application. Resting fluorescence ratio value just before Ba2+ application was used as Rmin for [Ba2+]i calibration. Rmax and beta  were obtained at end of experiment by addition of 10 mM Ba2+ and 1 mM ionomycin; KD was assumed to be 2 µM (37). [Ba2+]i was calculated according to formula used for calculation of [Ca2+]i (see METHODS).

These experiments clearly showed that, at the time when the typical CCE transient had reached its peak and the decay phase of [Ca2+]i was initiated, [Ba2+]i continued to rise at a steady rate. After ~4 min, however, the rate of increase of [Ba2+]i appeared to slow down. This apparent decrease in the rate of Ba2+ entry was most likely due to increasing saturation of indo 1 fluorescence, which had reached Rmax after >= 5 min. The rate of ion entry through the CCE channels remained unchanged over this time frame.

We demonstrated the constant entry rate directly by using Mn2+ to monitor the rate of divalent cation entry through the CCE pathway. Mn2+ entry is commonly measured as the decrease in indo 1 fluorescence due to quenching by Mn2+. Indo 1 fluorescence was measured at the isosbestic (i.e., Ca2+-insensitive) emission wavelength for Ca2+. In our experiments, Mn2+ entry through the CCE pathway was monitored at 424 nm. Changes in [Ca2+]i were derived from the indo 1 ratio F424/F485. Figure 4 shows the time courses of the F424 and F485 (A) and the R424/485 signal (B) during exposure to Mn2+ (50 µM) and Ca2+ (2 mM). Addition of Mn2+ to the extracellular environment caused a steady decrease of the F424 signal in store-depleted cells, consistent with Mn2+ entry at a constant rate. Increasing [Ca2+]o to 2 mM decreased the slope of the F424 signal (i.e., Mn2+ entry). Nevertheless, the rate of Mn2+ entry remained constant, whereas the Ca2+-sensitive signal (F485) and the ratio signal revealed the typical biphasic characteristics of a CCE transient. Figure 4C shows the normalized first derivative of the F424 signal, equivalent to the rate of Mn2+ entry. Although the rate of Mn2+ entry was different in the presence and absence of extracellular Ca2+, the rate remained constant in both cases.


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Fig. 4.   Rate of ion entry during CCE estimated from time course of indo 1 quenching by Mn2+. Rate of indo 1 quenching by Mn2+ was used as a measure of plasma membrane Ca2+ permeability, assuming that Mn2+ penetrates cell through same pathway as Ca2+. A: time course of fluorescence intensities recorded at 424 nm (indo 1 isosbestic wavelength for Ca2+, Mn2+ sensitive) and 485 nm (Ca2+-sensitive emission wavelength); a.u., arbitrary units. B: F424/F485 ratio signal reporting changes of [Ca2+]i. C: normalized first derivative of F424 (F424'/F424) representing rate of Mn2+ entry.

The Ba2+ and the Mn2+ experiments indicated that the entry rate of divalent cations through the CCE pathways was constant over time and was not influenced by changes of [Ca2+]i that had the typical characteristics of a CCE transient. Therefore, we concluded that a time-dependent inactivation of CCE was unlikely to be responsible for the typical biphasic kinetics of the CCE transient, and our focus turned toward the involvement of Ca2+ removal and/or Ca2+ sequestration during CCE and the dependence of these processes on [Ca2+]i.

That the decay phase of the CCE transient might be modulated by [Ca2+]i was suggested by the experiments in Fig. 1, as well as by the observation that the decay phase only occurred when Ca2+ was the charge carrier for CCE. Experiments illustrated in Fig. 5 lent further support for this hypothesis. In this experiment, raising [Ca2+]o to only 0.1 mM caused a [Ca2+]i transient revealing a peak followed by a plateau phase that was only slightly lower than the preceding peak [Ca2+]i. A second exposure to 0.1 mM [Ca2+]o, now in the presence of the Ca2+ ionophore ionomycin (1 µM), caused a biphasic change of [Ca2+]i that was virtually identical to a typical CCE transient. Compared with exposure to Ca2+ alone, the transient in the presence of ionomycin (trace b in Fig. 5) reached a peak about fourfold higher than and a plateau [Ca2+]i level about twice as high as the control transient (trace a in Fig. 5). Addition of ionomycin alone to thapsigargin-treated cells in Ca2+-free solution did not produce any changes in [Ca2+]i. Figure 5, right, shows the difference of the [Ca2+]i transients (trace b - trace a) in the presence and absence of ionomycin. To a first approximation, this difference transient could be interpreted as a change of [Ca2+]i that resulted from Ca2+ entry other than CCE. Because Ca2+ entry occurred through an ionophore and was unlikely to be gated by cellular mechanisms such as intracellular Ca2+, these results strongly suggested that the declining phase was the result of activation of Ca2+ sequestration or Ca2+ removal. Therefore, in the next series of experiments, we sought to investigate the role of Ca2+ sequestration by mitochondria and Ca2+ removal by Na+/Ca2+ exchange and the PMCA.


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Fig. 5.   CCE and Ca2+ entry in ionomycin-treated cells. Ca2+ stores were depleted previously with thapsigargin. Application of low [Ca2+]o (0.1 mM) in absence (left; trace a) and presence (middle; trace b) of Ca2+ ionophore ionomycin (1 µM). Ionomycin greatly increased overall influx of Ca2+ and enhanced peak and plateau phase of transient. Right: arithmetical difference (trace b - trace a) of the 2 transients (shifted vertically to match resting [Ca2+]i).

Mitochondrial Ca2+ Buffering During CCE

In various cell preparations and cell types, mitochondria have been shown to have substantial Ca2+-buffering capacities under conditions in which [Ca2+]i was significantly elevated above basal levels (14). We used FCCP (1 µM) to block mitochondrial Ca2+ uptake in combination with oligomycin (5 µM) to prevent ATP consumption by FCCP-treated mitochondria. Fig. 6A shows that FCCP-oligomycin did not change the typical biphasic shape of the CCE transient. Application of FCCP-oligomycin during the plateau phase of the CCE transient failed to significantly increase [Ca2+]i (Fig. 6B), as would be expected if mitochondria were accumulating substantial amounts of Ca2+. These results excluded the possibility that mitochondria were acting as a Ca2+ sink during CCE transients.


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Fig. 6.   Mitochondrial contribution to CCE transients. FCCP was used to block Ca2+ uptake by mitochondria. ATP consumption by FCCP-treated mitochondria was prevented by oligomycin. A: inhibition of Ca2+ uptake by mitochondria did not eliminate biphasic time course of CCE transient. B: application of FCCP (open bar) during plateau phase of CCE transient failed to significantly elevate [Ca2+]i. Solid bars, application of 2 mM Ca2+.

Role of Na+/Ca2+ Exchange During CCE

We explored the possible contribution of the plasma membrane Na+/Ca2+ exchange to the kinetics of the CCE transient (Fig. 7). After store depletion with thapsigargin (1 µM), repetitive exposures to extracellular Ca2+ (5 mM) caused typical biphasic CCE transients. When extracellular Na+ was replaced with either Li+ (Fig. 7A) or K+ (Fig. 7B) to block Na+/Ca2+ exchange, the peak of the CCE transient became larger; however, the plateau phase remained unaffected. These results indicated that extrusion of Ca2+ via Na+/Ca2+ exchange occurred during the rapid increase of [Ca2+]i and that Na+/Ca2+ exchange activity reduced the peak of the CCE transient under control conditions. Nevertheless, the biphasic nature of the CCE transient was conserved, and the decay phase to the plateau was not affected, suggesting only a minor role of Na+/Ca2+ exchange in Ca2+ efflux during CCE.


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Fig. 7.   Role of Na+/Ca2+ exchange during CCE transients. A and B: recordings from 2 different thapsigargin-treated (shaded bar; 1 µM; 8 min) CPAE cells. Solid bars, application of 5 mM Ca2+; open bars, substitution of extracellular Na+ by equimolar concentrations of Li+ (A) or K+ (B).

Role of PMCA During CCE

The remaining mechanism involved in Ca2+ regulation to be tested for its role in shaping the CCE transient was the PMCA. As mentioned earlier, the study of PMCA activity in intact cells is difficult because of the lack of a selective pharmacological blocker. Unfortunately, among the compounds that exert some inhibitory effect on the PMCA, many block the pump only partially and at the same time block CCE. To circumvent these experimental obstacles, we used three different and unrelated blocking compounds.

The best known and most potent blocker of the PMCA is La3+. To quantify the rate of Ca2+ extrusion by the PMCA, we used the monoexponential time course (tau ) of [Ca2+]i recovery after removal of extracellular Ca2+. Application of La3+ (1 mM) immediately after removal of extracellular Ca2+ (Fig. 8A) slowed the rate of [Ca2+]i recovery approximately sixfold (tau La/tau control = 5.9 ± 1.2; n = 5). Unfortunately, the effect of La3+ on the pump during CCE could not be tested because La3+ abolished CCE completely (Fig. 8A, 3rd exposure to 2 mM extracellular Ca2+; see also Refs. 1, 31, 40). For that reason, we used two other blockers that were likely to affect CCE to a lesser extent.


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Fig. 8.   Inhibition of plasma membrane Ca2+ pump during CCE. Pump inhibition by La3+ (A), VO3-4 (B), and Hg2+ (C) is shown. Solid bars, presence of 2 mM Ca2+; open bars, presence of inhibitors. D: time constant tau  of rate of [Ca2+]i restoration after removal of extracellular Ca2+ under control conditions and in presence of 3 different blockers of plasma membrane Ca2+-ATPase (PMCA). Difference is statistically significant at P < 0.05 (1-tailed nonparametric Wilcoxon rank test for paired observations) for all blockers used.

Figure 8B shows the effect of orthovanadate (VO3-4), which has been shown to block PMCA (8). In the presence of 2 mM VO3-4, the CCE transient failed to decay to a lower plateau level (3rd CCE transient in Fig. 8B). The initial rapid increase in [Ca2+]i was followed by a slower but continuous rise of [Ca2+]i until extracellular Ca2+ was removed. After removal of extracellular Ca2+, [Ca2+]i recovered approximately five times more slowly than under control conditions (tau orthovanadate/tau control = 4.6 ± 0.4; n = 6). Short pretreatment with VO3-4 (Fig. 8B, 2nd CCE transient), however, was not sufficient to abolish the biphasic time course of the CCE transient. The most important finding of these experiments was that during activation of CCE [Ca2+]i continued to rise instead of reaching a peak and then declining to a lower plateau level. Therefore, we concluded that the secondary component of a typical CCE transient, i.e., the decline of [Ca2+]i from the peak to the lower plateau level, was due to Ca2+ removal by the PMCA.

Qualitatively similar results were obtained with Hg2+, a substance that has been shown to block the PMCA (10, 56). As shown in Fig. 8C, a second exposure to Ca2+ in the presence of 0.4 µM Hg2+ caused a rapid (although slower than under control conditions) increase of [Ca2+]i. This rapid phase was followed by a slower but continuous further increase in [Ca2+]i. After removal of extracellular Ca2+, [Ca2+]i decreased at a rate approximately twofold slower than under control conditions (tau Hg/tau control = 1.6 ± 0.1; n = 6).

The effects of Hg2+ and VO3-4 on the time constant tau  of [Ca2+]i recovery after removal of extracellular Ca2+ were similar to La3+ and are consistent with an inhibitory effect on the activity of the PMCA. The observation that both Hg2+ and VO3-4 prevented a decay of [Ca2+]i to the lower plateau level after the initial peak lent further support to the hypothesis that the plasma membrane Ca2+ pump is mainly responsible for removing Ca2+ during and after activation of CCE.

Figure 8D summarizes the effects of Hg2+, VO3-4, and La3+ on the activity of the PMCA. As a measure of pump activity, the time constants tau  of [Ca2+]i restoration in the presence of PMCA blockers were compared with the rate of decay under control condition. All three blockers slowed the rate of decay significantly (P < 0.05), with La3+ having the most profound effect.

Time Dependence of PMCA Activation During the CCE Transient

So far, we have established evidence that the PMCA was the primary mechanism that counteracted the steady entry of Ca2+ during CCE under conditions in which reuptake of Ca2+ into the intracellular stores was permanently blocked with thapsigargin. In the next series of experiments, we tested the hypothesis that time-dependent or delayed changes in PMCA activity during CCE were responsible for the biphasic time course of the CCE transient. We approached the problem by evaluating PMCA activity as a function of [Ca2+]i and the duration for which CCE had been active. Figure 9A shows two consecutive exposures of variable duration (400 s vs. 45 s) of a store-depleted cell to 5 mM extracellular Ca2+. In both cases, at the time extracellular Ca2+ was removed, [Ca2+]i had reached the same level (~400 nM) despite an ~10-fold difference in the duration for which CCE had been active. In the case of the short activation of CCE, the rate of [Ca2+]i recovery was significantly slower than after a long-lasting activation of CCE. The rate of decline of [Ca2+]i after removal of extracellular Ca2+ could be best described by a double-exponential fit. In the experiment shown in Fig. 9A, both the fast (which produced a good fit of >75% of the initial decay phase) and the slow component were significantly faster than when the cell was exposed to extracellular Ca2+ for a longer period of time. The relationship between the time constant tau  of the decline of [Ca2+]i and [Ca2+]i itself is illustrated in Fig. 9B. In this experiment, a store-depleted cell was exposed repetitively to 5 mM extracellular Ca2+ for periods ranging from 10 to 450 s. Figure 9C shows that tau (fast component) of [Ca2+]i recovery decreased as exposure time increased, i.e., there was a time-dependent increase in the PMCA activity. The experiment further revealed that initially the degree of activation (tau ) paralleled the peak of the CCE transient, indicative of a [Ca2+]i dependence of PMCA activation. Most interesting, however, was the observation that, once the PMCA was fully activated (50-70 s), [Ca2+]i could decline from its maximum value to levels as low as 300 nM without affecting the rate of Ca2+ removal by the PMCA (compare 450-s and 70-s points in Fig. 9C). The most straightforward interpretation of these results was that during CCE the increase of [Ca2+]i activated the PMCA. Full activation of the pump, however, occurred with a delay in the range of ~1 min. Because full activation of the PMCA is delayed, removal of Ca2+ by this mechanism becomes prominent only during the secondary phase of the CCE transient. As a result, the net Ca2+ entry during activation of CCE is increasingly diminished by Ca2+ removal by the PMCA, leading to the typical biphasic kinetics of the CCE transient.


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Fig. 9.   Time- and [Ca2+]i-dependent enhancement of PMCA activity during CCE. A: changes in [Ca2+]i in response to exposures to extracellular Ca2+ for 45 s (open circle ) and 400 s (bullet ). Although [Ca2+]i had reached same level at time of removal of extracellular Ca2+, [Ca2+]i decreased more rapidly when cell was exposed to extracellular Ca2+ for a longer time. Rate of this decay was best fit by double-exponential procedure (inset). The first, faster component (tau 1) resulted in a reasonable fit of 75 and 89% of decay phase, for 45 and 400 s, respectively. B: multiple exposures of a single, store-depleted endothelial cell to 5 mM extracellular Ca2+ for variable durations between 10 and 450 s. Dashed line indicates [Ca2+]i value after 20 s and 450 s for comparison. C: final level of [Ca2+]i immediately before removal of extracellular Ca2+ (open circle ) and time constant tau  (bullet ) of decay of [Ca2+]i (tau  = fast component of double-exponential fit of [Ca2+]i decay; see A) plotted as a function of time of exposure to 5 mM extracellular Ca2+ (data taken from experiment shown in B). Data in A and B represent different CPAE cells.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Kinetics of Changes of [Ca2+]i Related to CCE in Vascular Endothelial Cells

Vascular endothelial cells have been the focus of several studies on CCE. As with many other cell types, in endothelial preparations CCE was typically investigated by the characterization of specific changes of [Ca2+]i or Mn2+ entry associated with store depletion (16, 31, 47) or, more directly, by measuring membrane currents related to capacitative Ca2+ influx (51, 55). In intact cells, CCE occurs in the presence of a functional machinery of Ca2+ regulatory mechanisms through which Ca2+ entry, removal, and sequestration contribute at any given point in time to the level of [Ca2+]i. Therefore, the goal of the present study was to investigate the interplay between CCE and other cellular Ca2+ transport and buffering systems that eventually result in the characteristic changes of [Ca2+]i observed on depletion and refilling of intracellular Ca2+ stores.

The most intriguing finding of our study was the apparent discrepancy between the biphasic time course of changes of [Ca2+]i and the constant rate of ion transport into the cell during activation of CCE. The changes of [Ca2+]i during activation of CCE revealed a characteristic biphasic time course with a rapid rise to a peak that slowly decayed to a plateau of elevated [Ca2+]i. The use of Ba2+ and Mn2+, however, which served as Ca2+ substitutes to characterize unidirectional ion flux through the CCE pathway, revealed an entry rate that was constant over time and distinctly different from the time course of the CCE signal (Figs. 2 and 3). From these experiments, it became apparent that other Ca2+ regulatory mechanisms with different activation kinetics from CCE per se had to contribute to the typical biphasic CCE transient. Several mechanisms needed to be considered to explain this biphasic change of [Ca2+]i, including a time-dependent change in electrical driving force for Ca2+ or inhibition of Ca2+ entry, activation of Ca2+ sequestration into organelles, and/or Ca2+ extrusion from the cell.

Factors Responsible for the Biphasic Kinetics of the CCE Transient

Electrical driving force for Ca2+ entry. It has been shown that in CPAE cells an increase in [Ca2+]i activates a Ca2+-dependent Cl- current (Ref. 36; see also Fig. 2, top) that may affect the Em and therefore the driving force for Ca2+ influx. We therefore recorded CCE transients at constant Em using a perforated patch-clamp technique. This method has the advantage of avoiding disruption of the cytoplasmic environment and second messenger systems. Under these conditions, reexposure to extracellular Ca2+ after thapsigargin treatment caused CCE transients that revealed typical biphasic kinetics (Fig. 2, bottom) qualitatively identical to the ones observed in unclamped cells. We also were able to show that Em was only minimally affected by [Ca2+]i during CCE (current-clamp experiments). These findings indicated that the decline of [Ca2+]i after the peak and the development of the plateau phase of the CCE transient were not due to membrane depolarization and reduction of the driving force for Ca2+ entry.

Feedback regulation of CCE by [Ca2+]i. The membrane Ca2+ current activated by store depletion (ICRAC) originally described in mast cells revealed strong [Ca2+]i-induced inactivation (20). Similar feedback control of CCE by [Ca2+]i has been suggested in other studies (29, 32). To investigate the possible role of feedback regulation of CCE by increased [Ca2+]i, we simultaneously measured Mn2+ influx as a measure of unidirectional ion transport through the CCE pathway (28, 48) and changes of [Ca2+]i. If a decrease of [Ca2+]i to a plateau phase were the result of inhibition of CCE by higher [Ca2+]i, one might expect a change in the rate of Mn2+ entry during CCE that would follow the kinetics of the [Ca2+]i signal. Mn2+ influx, however, did not reveal the secondary decay phase to the lower plateau level typical of the [Ca2+]i signal (Fig. 4). Our data provided clear evidence that the rate of Mn2+ entry was constant, both in the presence and in the absence of extracellular Ca2+, although it was reduced in the presence of Ca2+. Possible explanations of this latter result are competition between Ca2+ and Mn2+ for entry or a fast (within milliseconds) Ca2+-dependent inactivation similar to the one that has been reported for ICRAC in Jurkat and mast cells in response to hyperpolarizing pulses (20, 58). We cannot exclude that a fast inhibition of CCE might be present, since events occurring on the time scale of a few hundred milliseconds were not resolved in our experiments. Recent evidence, however, suggests that store-operated Ca2+ channels may exist that have biophysical properties (conductance, open probability, Ca2+ dependence) that are different from the original ICRAC (30, 49). There is indeed increasing experimental evidence that the original ICRAC represents just one member of an entire family of store-operated channels (7). For example, a slow (over tens of seconds) inactivation of ICRAC has been reported in T lymphocytes (59). Nevertheless, direct inhibition of CCE by increased [Ca2+]i appeared unlikely in our experiments because the rate of Mn2+ entry remained constant over the time course of minutes despite significant changes in [Ca2+]i.

However, there are two experimental observations that would lend support to the idea that the biphasic nature of the CCE transient was indeed related to [Ca2+]i, although not through direct inhibition of CCE. First, the use of the Ca2+ ionophore ionomycin resulted in a biphasic [Ca2+]i transient, qualitatively identical to CCE transients (Fig. 5). In these experiments, most of the Ca2+ entered the cell via a pathway different from CCE. These results, therefore, suggested that a rise in [Ca2+]i initiated cellular processes that led to an overall reduction in net Ca2+ influx, which appeared to be directly related to the increased level of [Ca2+]i. Second, our result showed that the pathway activated by store depletion in CPAE cells was permeable to Ba2+. In contrast to Ca2+, the increase of [Ba2+]i in store-depleted cells was monophasic and failed to decline to a lower plateau level. [Ba2+]i continued to rise until extracellular Ba2+ was removed. The rate of Ba2+ entry was constant at the point in time when a typical CCE transient reached its peak and started to decline to the lower plateau level, supporting the notion that CCE occurred at a constant rate. Given that Ca2+ entry from the external medium was nearly constant during the CCE transient, it appeared reasonable to propose that the decrease of [Ca2+]i after the peak resulted either from thapsigargin-insensitive sequestration of Ca2+ by mitochondria or from Ca2+ extrusion via Na+/Ca2+ exchange and/or the PMCA.

Mitochondrial Ca2+ buffering. Mitochondria are known to accumulate large amounts of Ca2+ under certain specific conditions (i.e., cytoplasmic Ca2+ overload; see Refs. 14, 41), and Ca2+ sequestration by mitochondria can influence cellular Ca2+ signaling by changing [Ca2+]i in restricted spaces (4, 23). In this study, inhibition of mitochondrial Ca2+ uptake by treatment with FCCP and oligomycin did not prevent the biphasic kinetics of the CCE transient (Fig. 6A). Dissipating the mitochondrial Em during the plateau had little effect on [Ca2+]i and did not reveal significant mitochondrial Ca2+ uptake (Fig. 6B). Our results are in contrast to the finding that mitochondria were able to dynamically regulate CCE in T lymphocytes (18). This discrepancy might be due to different thresholds for Ca2+ uptake by mitochondria in different cells, the level of [Ca2+]i, and the structural relationships between mitochondria and the source of the [Ca2+]i increase (27, 41). Indeed, during large changes of [Ca2+]i, mitochondrial Ca2+ uptake occurs in endothelial cells, as revealed by confocal microscopy (J. Hüser and L. A. Blatter, unpublished results; see also Ref. 9). However, this was not the case during the plateau phase of the CCE transient (Fig. 6B).

Na+/Ca2+ exchange. Despite evidence for the existence of Na+/Ca2+ exchange in endothelial cells (13, 42), its quantitative relevance for Ca2+ efflux during store-operated Ca2+ entry remains to be determined. Inhibition of this transport system by omission of extracellular Na+ enhanced the amplitude of the initial peak but did not affect the plateau phase and rate of [Ca2+]i decline on removal of extracellular Ca2+ (Fig. 7). These results demonstrate that Na+/Ca2+ exchange contributes to Ca2+ extrusion, particularly under circumstances in which [Ca2+]i is substantially elevated. This is in agreement with findings that Na+/Ca2+ exchange is the major pathway for Ca2+ efflux in neuronal cells and cardiac muscle (3), whereas in nonexcitable cells Ca2+ efflux is almost entirely mediated by the PMCA (52, 57).

Activation of the PMCA Modulates the Net Cellular Gain of Ca2+ During CCE

The participation of the PMCA in Ca2+ extrusion from the cytosol is generally accepted, particularly in nonexcitable cells (33, 35). Studying the contributions of the pump experimentally, however, is hampered by the complication that there are no known specific pharmacological blockers of this pathway and many of the compounds that have been shown to exert some inhibitory effect on pump activity also inhibit CCE, as shown for La3+ in our study (Fig. 8A). To circumvent these experimental difficulties, we employed three unrelated compounds to challenge PMCA activity, namely La3+, Hg2+ (56), and VO3-4 (8). To evaluate the inhibitory effect of these substances, the rate of decline of [Ca2+]i to the plateau phase during CCE and the rate of decline of [Ca2+]i after removal of extracellular Ca2+ were measured.

Both Hg2+ and VO3-4 had qualitatively similar effects on the kinetics of CCE transients. Instead of reaching a peak and decreasing to a lower plateau level, [Ca2+]i continued to rise until extracellular Ca2+ was removed. The second parameter used as a measure of PMCA activity was the rate of [Ca2+]i recovery after removal of extracellular Ca2+. All three compounds, La3+, Hg2+, and VO3-4, significantly slowed the rate of decrease of [Ca2+]i; however, their effectiveness differed (Fig. 8D). The relative effectiveness with which the three compounds blocked the PMCA may be related to their mode of action (46). La3+ inhibits the pump from the inside as well as from the outside, whereas Hg2+ and VO3-4 act on the pump only from the inside. This may explain why La3+ was more effective than either Hg2+ or VO3-4 under our experimental conditions (i.e., extracellular application). In many cases, recovery of [Ca2+]i was incomplete, indicating that the pump remained partially blocked. Despite the complication that La3+, Hg2+, and VO3-4 may interfere with CCE, these experiments, taken together, provided strong evidence that the plasma membrane Ca2+ pump is mainly responsible for the decline of [Ca2+]i to a lower plateau level during CCE.

After we had established a constant rate of Ca2+ entry during CCE and only minimal contributions by mitochondrial Ca2+ sequestration and removal by Na+/Ca2+ exchange, the biphasic nature of the CCE transient suggested to us a time- and [Ca2+]i-dependent activation of the PMCA. The results presented in Fig. 9 are in agreement with this hypothesis. They clearly demonstrate an acceleration of Ca2+ extrusion (evaluated as time constant tau  of [Ca2+]i restoration to the basal level) as a function of the duration of CCE activation as well as the level of [Ca2+]i. Several observations point toward the involvement of calmodulin in the mechanism responsible for this [Ca2+]i-dependent delay. Calmodulin is an important endogenous regulator of PMCA activity and has been shown to rather slowly associate with the pump at low levels of [Ca2+]i (43). Calmodulin has been demonstrated to contribute to the generation of biphasic changes of [Ca2+]i (11, 44), and there is experimental evidence that the decrease of [Ca2+]i after a peak to a sustained plateau level can be eliminated by the calmodulin antagonist W-7 or by intracellular application of the synthetic peptide that antagonized endogenous calmodulin (an analog of the calmodulin-binding domain of protein kinase II). In our experiments, W-7 (50-200 µM) caused a dose-dependent inhibition of Ca2+ entry (unpublished results). This observation is consistent with various reports indicating involvement of calmodulin in activation and regulation of CCE (6, 26, 54). For example, the Drosophila trpl Ca2+ channel, which is considered to be regulated by store depletion, has been shown to have a calmodulin-binding domain (38).

Another feature of calmodulin action is consistent with our results. The Ca2+ dependence of the rate constants for calmodulin-PMCA association/dissociation (43) and the hysteresis-like interaction of the pump with calmodulin (45) may explain how full activation of the pump could be maintained despite a continuous fall in [Ca2+]i (Fig. 9C). Thus low levels of elevated [Ca2+]i were sufficient to keep PMCA fully activated once activation had occurred, although the same levels of [Ca2+] per se could not initiate activation of the pump. By this model, at resting [Ca2+]i the pump is not associated with calmodulin and stimulation of the PMCA is probably negligible. However, if the pump is partially activated by elevated [Ca2+]i, a subsequent rise of [Ca2+]i would be blunted by the prior activation of the pump. This is consistent with the results shown in Fig. 1. The difference in the peak amplitude of the CCE transients during application of the same [Ca2+]o (10 mM; Fig. 1) was inversely related to the preceding [Ca2+]i level, consistent with variable degrees of PMCA activation. Nevertheless, full activation of PMCA at the end of all these CCE transients led to the same [Ca2+]i plateau level.

In addition to calmodulin action, regulation of PMCA activity by PKC and PKA has been reported (for review see Ref. 35). Phosphorylation of the pump by these kinases would be expected to enhance Ca2+ removal during agonist stimulation. In our experiments, however, pharmacological inhibition of PKC (with 250 nM bisindolylmalemide I) or PKA (with 250 nM H-89) had no effect on the kinetics of CCE transients (unpublished observations).

In conclusion, our results document, for the first time, the important role of the PMCA in modulating net Ca2+ influx through CCE. The results identify the PMCA as a major modulator of refilling of Ca2+ stores in endothelial cells. This novel mechanism suggests close cooperation between CCE and removal of Ca2+ by the plasma membrane Ca2+ pump for Ca2+ homeostasis in vascular endothelial cells.

    ACKNOWLEDGEMENTS

We thank the late Christine E. Rechenmacher for expert technical help and Dr. S. L. Lipsius and Jaclyn R. Holda for critical comments on the manuscript. We also thank John J. Payne and Vezetter Whitaker for invaluable contributions made by building customized equipment.

    FOOTNOTES

Financial support was provided by National Heart, Lung, and Blood Institute Grant HL-51941 and by the American Heart Association National Center and the Schweppe Foundation Chicago.

L. A. Blatter is an Established Investigator of the American Heart Association. A. Klishin is a postdoctoral fellow of the Falk Foundation, Cardiovascular Institute, Loyola University Chicago.

Portions of this work have been presented previously in abstract form (24, 50).

1 In the following paragraphs, the characteristic biphasic change of [Ca2+]i observed on reexposure to extracellular Ca2+ after store depletion is referred to as the "CCE transient." The phase of slow decline of [Ca2+]i to a sustained plateau level is referred to as the "decay phase" of the CCE transient. As will be shown, this terminology does not imply that CCE itself is the sole mechanism contributing to the [Ca2+]i transient.

Address for reprint requests: L. A. Blatter, Dept. of Physiology, Loyola University Chicago, 2160 S. First Ave., Maywood, IL 60153.

Received 19 November 1997; accepted in final form 12 January 1998.

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Discussion
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AJP Cell Physiol 274(4):C1117-C1128
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