Role of Na+/Ca2+ exchange in regulating cytosolic Ca2+ in cultured human pulmonary artery smooth muscle cells

Shen Zhang,1 Jason X.-J. Yuan,1 Kim E. Barrett,2 and Hui Dong2

Divisions of 1Pulmonary and Critical Care Medicine and 2Gastroenterology, Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California

Submitted 20 August 2004 ; accepted in final form 23 September 2004


    ABSTRACT
 TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) in pulmonary artery smooth muscle cells (PASMC) is an important stimulus for cell contraction, migration, and proliferation. Depletion of intracellular Ca2+ stores opens store-operated Ca2+ channels (SOC) and causes Ca2+ entry. Transient receptor potential (TRP) cation channels that are permeable to Na+ and Ca2+ are believed to form functional SOC. Because sarcolemmal Na+/Ca2+ exchanger has also been implicated in regulating [Ca2+]cyt, this study was designed to test the hypothesis that the Na+/Ca2+ exchanger (NCX) in cultured human PASMC is functionally involved in regulating [Ca2+]cyt by contributing to store depletion-mediated Ca2+ entry. RT-PCR and Western blot analyses revealed mRNA and protein expression for NCX1 and NCKX3 in cultured human PASMC. Removal of extracellular Na+, which switches the Na+/Ca2+ exchanger from the forward (Ca2+ exit) to reverse (Ca2+ entry) mode, significantly increased [Ca2+]cyt, whereas inhibition of the Na+/Ca2+ exchanger with KB-R7943 (10 µM) markedly attenuated the increase in [Ca2+]cyt via the reverse mode of Na+/Ca2+ exchange. Store depletion also induced a rise in [Ca2+]cyt via the reverse mode of Na+/Ca2+ exchange. Removal of extracellular Na+ or inhibition of the Na+/Ca2+ exchanger with KB-R7943 attenuated the store depletion-mediated Ca2+ entry. Furthermore, treatment of human PASMC with KB-R7943 also inhibited cell proliferation in the presence of serum and growth factors. These results suggest that NCX is functionally expressed in cultured human PASMC, that Ca2+ entry via the reverse mode of Na+/Ca2+ exchange contributes to store depletion-mediated increase in [Ca2+]cyt, and that blockade of the Na+/Ca2+ exchanger in its reverse mode may serve as a potential therapeutic approach for treatment of pulmonary hypertension.

sodium-calcium exchange; calcium homeostasis; vascular smooth muscle


INTRACELLULAR CA2+ is a critical second messenger that links external stimuli to cell contraction, proliferation, migration, and gene expression (3, 4). An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) in pulmonary artery smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC proliferation and migration (24, 31). [Ca2+]cyt in PASMC can be increased by Ca2+ release from intracellular stores, such as the sarcoplasmic (or endoplasmic) reticulum (SR), and by Ca2+ influx through Ca2+-permeable cation channels in the plasma membrane (24).

Binding of vasoconstrictive and mitogenic agonists with G protein-coupled receptors (GPCR) and receptor tyrosine kinases in the plasma membrane activates phospholipase C, which causes hydrolysis of phosphatidylinositol and generation of cytosolic inositol 1,4,5-trisphosphate (IP3) and membrane-bound diacylglycerol. IP3 induces a rapid Ca2+ release through IP3 receptors at the SR membrane (14, 51), and the subsequent depletion of Ca2+ from the stores (i.e., the SR) opens a special family of Ca2+-permeable channels, namely, store-operated Ca2+ channels (SOC), and elicits capacitative Ca2+ entry (CCE) (5, 34, 37, 38). The store depletion-activated SOC are believed to be formed heterogeneously by different isoforms of transient receptor potential (TRP) channels (5, 11, 26, 32, 59). The homo- or heterotetrameric TRP channels are generally nonselective cation channels that allow both Ca2+ and Na+ to go through (29, 50, 64). Therefore, opening of TRP channels would increase both [Ca2+]cyt and cytosolic [Na+] as a result of Ca2+ and Na+ influx via the channels.

Mammalian cells maintain a low cytoplasmic concentration of Na+ ([Na+]cyt, ~10 mM) compared with the extracellular concentration of Na+ ([Na+]out, ~140 mM) because of the activity of the Na+-K+-ATPase (25, 58). The transmembrane Na+ gradient can be utilized to energize the Na+/Ca2+ exchanger, which moves Na+ and Ca2+ across the membrane in the opposite direction. Two families of plasma membrane Na+/Ca2+ exchanger proteins have been described in mammalian tissues (8, 30), one in which Ca2+ movement depends only on Na+ (NCX family) and the other in which Ca2+ movement depends on Na+ and K+ (NCKX family). The stoichiometry of NCX is 3 Na+ per 1 Ca2+, whereas that for NCKX is 4 Na+ per 1 Ca2+ and 1 K+. Both NCX and NCKX can operate in either a forward (Ca2+ exit) or reverse (Ca2+ entry) mode, depending on the Na+ and Ca2+ (and K+) gradients and membrane potential (Em) (8, 14, 24). For a constant extracellular [Ca2+] (1.8–2 mM) and [Na+] (~140 mM), [Ca2+]cyt is a cubic function of [Na+]cyt and an exponential function of Em. Therefore, a small change in [Na+]cyt or Em can cause a large change in [Ca2+]cyt (6).

Many functional studies have demonstrated that the plasma membrane Na+/Ca2+ exchanger is involved in regulating Ca2+ homeostasis of blood vessels (1, 8, 44, 45). The functional evidence for the sarcolemmal Na+/Ca2+ exchanger in vessels is also supported by direct demonstration that the Na+/Ca2+ exchanger is expressed in vascular smooth muscle and endothelial cells (23). However, the expression and function of the Na+/Ca2+ exchanger in human PASMC are unknown, although studies show that Na+/Ca2+ exchange activity is involved in the development of hypoxic pulmonary vasoconstriction in animals (8, 12, 27, 42, 57). Recently, it was also suggested that there is a functional and physical interaction of TRP cation channels with NCX proteins (40). Therefore, the purpose of this study was to confirm the functional expression of NCX and NCKX proteins and to characterize the role of the Na+/Ca2+ exchanger in store depletion-mediated Ca2+ entry in human PASMC, as well as the potential role of the Na+/Ca2+ exchange-driven Ca2+ entry in PASMC proliferation.


    METHODS AND MATERIALS
 TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell preparation and culture. Human PASMC from normal subjects were purchased from Cambrex and used at the 4th-6th passages. The cells were plated onto coverslips or petri dishes and incubated in a humidified atmosphere of 5% CO2 in air at 37°C in smooth muscle growth medium (SmGM; Cambrex, Walkersville, MD). The SmGM was composed of smooth muscle basal medium (SmBM) supplemented with 5% fetal bovine serum (FBS), 0.5 ng/ml human epidermal growth factor, 2 ng/ml human fibroblast growth factor, and 5 µg/ml insulin. Cells were subcultured or plated onto 25-mm coverslips in trypsin-EDTA buffer when 70–90% confluence was achieved. The morphology of the cells was examined using an inverted phase-contrast microscope attached to a digital camera. Rat brain and pulmonary artery tissues were used in some experiments. Left and right branches of the main pulmonary artery along with intrapulmonary arterial branches were isolated from male Sprague-Dawley rats (125–250 g) (62). Brain tissues were usually removed from the same rats from which lungs were removed to prepare isolated extra- and intrapulmonary arteries.

Western blot analysis. Human PASMC were gently washed twice in cold PBS, scraped into 0.3 ml of radioimmunoprecipitation assay buffer [1x PBS, 1% Nonidet P-40 (Amaresco, Solon, OH), 0.5% sodium deoxycholate, and 0.1% SDS], and incubated on ice for a 45-min period, during which the cell mixture was shaken for 30 s by vortex three times. Rat brain and pulmonary artery were stored at –80°C and then homogenized in a glass tube with a Teflon Dounce pestle in 5 ml of ice-cold assay buffer as described above. The resulting cell and tissue lysates were sonicated and centrifuged at 14,000 rpm for 15 min at 4°C. The supernatants were collected, and protein concentrations were determined using Coomassie Plus protein assay reagent (Pierce Biotechnology, Rockford, IL) with BSA as a standard. Protein (30 µg) was mixed and boiled in 2x sample buffer (0.25 M Tris·HCl, pH 6.8, 20% glycerol, 8% SDS, and 0.02% bromphenol blue). Protein suspensions were electrophoretically separated on an 8% acrylamide gel, and protein bands were transferred to nitrocellulose membranes by electroblotting in a Mini Trans-Blot cell transfer apparatus (Bio-Rad, Hercules, CA) under conditions recommended by the manufacturer. After 1 h of incubation in a blocking buffer (0.1% Tween 20 and 5% nonfat dry milk powder), the membranes were incubated with R3F1 monoclonal antibody against NCX1 (Swant, Bellinzona, Switzerland) diluted in blocking buffer (1:5,000) overnight at 4°C. Finally, the membranes were washed and exposed to anti-mouse horseradish peroxidase-conjugated IgG for 60 min at room temperature. The bound antibody was detected with an enhanced chemiluminescence detection system (Amersham, Arlington Heights, IL).

RT-PCR. Total RNAs were extracted from human PASMC, rat brain, and rat pulmonary artery using the RNeasy mini kit (Qiagen, Valencia, CA). Genomic DNA was removed with RNase-free DNase according to the manufacturer's instructions. SuperScript reverse transcriptase (Invitrogen, Carlsbad, CA) was used to synthesize cDNA. RNA (2 µg) was first incubated with oligo(dT) (1 µl at 0.5 µg/µl) at 70°C for 10 min, and then 8 µl of a solution that contained 10x buffer, 10 mM dNTP, 20 mM MgCl2, 0.1 M DTT, 40 U/µl RNaseOUT, and 50 U/µl SuperScript II reverse transcriptase were added to the samples and incubated for 10 min at 30°C, 60 min at 42°C, and 5 min at 95°C. RNase-H (1 µl at 2 U/µl; GIBCO, Grand Island, NY) was added to each reaction. The sense and antisense primers were specifically designed from the coding regions of each gene (Table 1). The fidelity and specificity of the sense and antisense oligonucleotides were examined using the BLAST program.


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Table 1. Oligonucleotide sequences of primers used for RT-PCR

 
Immunofluorescence labeling. Human PASMC on slides were fixed in 4% paraformaldehyde for 20 min. After blocking with 4% BSA for 20 min, a specific monoclonal antibody against NCX1 (R3F1; Swant) was applied to the cells, followed by a secondary antibody conjugated with green fluorescence (Alexa Fluor 488; Molecular Probes, Eugene, OR). The cells were then stained with the membrane-permeable nucleic acid stain 4',6'-diamidino-2-phenylindole (DAPI, 5 µM; Sigma, St. Louis, MO), and the blue fluorescence (emitted at 461 nm) was used to detect cell nuclei. The cell images were processed by three-dimensional deconvention fluorescence microscopy with softWoRx (Applied Precision, Issaquah, WA) and analyzed using Matlab (Mathworks, Natick, MA).

Measurement of [Ca2+]cyt. [Ca2+]cyt in single human PASMC was measured using the Ca2+-sensitive fluorescent indicator fura 2-AM. Cells on 25-mm coverslips were loaded with fura 2-AM (3 µM) for 30 min in the dark at room temperature (22–24°C) under an atmosphere of 5% CO2-95% air. The fura 2-AM-loaded cells were then transferred to a perfusion chamber on the microscope stage and superfused with physiological salt solution (PSS) for 30 min to remove extracellular dye and allow intracellular esterases to cleave cytosolic fura 2-AM into active fura 2. The PSS contained (in mM) 141 NaCl, 4.7 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose, pH 7.4. In Ca2+-free PSS, CaCl2 was replaced by equimolar MgCl2, and 0.1 mM EGTA was added to chelate residual Ca2+. Fura 2 fluorescence (510-nm light emission excited by 340- and 380-nm illuminations) from the cells, as well as background fluorescence, was collected at room temperature (22°C) with the use of a x40 Nikon UV-Fluor objective and a charge-coupled device camera. The fluorescence signals emitted from the cells were monitored continuously using an Intracellular Imaging fluorescence microscopy system and were recorded in an IBM-compatible computer for later analysis. [Ca2+]cyt was calculated from fura 2 fluorescent emission excited at 340 and 380 nm (F340/F380) using the ratio method based on the equation [Ca2+]cyt = Kd x (Sf2/Sb2) x (R – Rmin)/(Rmax – R), where Kd (225 nM) is the dissociation constant for Ca2+, R is the measured fluorescence ratio, and Rmin and Rmax are minimal and maximal ratios, respectively (20).

Cell cycle analysis. Human PASMC cell cycle distribution was analyzed using flow cytometry. Cells were first growth arrested in SmBM for 24 h and then cultured in 5% FBS-SmGM with or without KB-R7943 for 24 h. The cells were trypsinized, washed once with PBS, and fixed with 70% ethanol for at least 30 min at room temperature. The fixed cells were washed with PBS and incubated with a solution containing 50 µg/ml propidium iodine (Sigma) and 50 µg/ml RNase A (Sigma) for 30 min at room temperature in the dark. The stained cells were analyzed by FACS-Calibur with excitation at 488 nm and emission at 560–640 nm (FL2 mode) using CellQuest software (Becton Dickinson, Mountain View, CA).

Measurement of cell number. Human PASMC were cultured in growth-arresting medium (SmBM) for 24 h and then switched to culture in 5% FBS-SmGM with or without KB-R7943 for 48 h. Cells were pelleted, washed twice with cold PBS, and resuspended in PBS for counting. Cell numbers were determined using a Z2 Coulter counter.

Chemicals. All chemicals were of analytical grade or better and were obtained from Fisher (Nepean, ON, Canada), BDH (Toronto, ON, Canada), or Sigma unless indicated otherwise. Pharmacological reagents were purchased from LC Laboratories (Woburn, MA), Seikagaku America (Falmouth, MA), or Calbiochem (San Diego, CA). Cell culture reagents were from Life Technologies (Rockville, MD).

Statistics and data analysis. Data are expressed as means ± SE; n represents the number of cells. Statistical analysis was performed using unpaired or paired Student's t-test or ANOVA as indicated. Differences were considered to be significant at P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
mRNA and protein expression of NCX and NCKX. With the use of rat brain tissues as positive controls, our RT-PCR experiments show that human PASMC expressed NCX1 and NCKX3 (Fig. 1A). The PCR products for NCX1 and NCKX3 obtained from human PASMC were sequenced by our core facility; the sequences matched with the GenBank sequences of NCX1 and NCKX3. Furthermore, our Western blot experiments revealed protein expression of NCX1 in human PASMC, although the protein expression level was much less than in rat brain tissues and rat pulmonary arteries (Fig. 1B). R3F1, an anti-NCX1 monoclonal antibody, recognized a pattern of protein expression typical of NCX1 in human PASMC comprising two bands with molecular masses of 120 and 70 kDa (17, 49). The weak band of 70 kDa corresponds to the short form of NCX1, which is either a proteolytically cleaved product or a functional, truncated form of NCX1 (53). In rat brain and pulmonary artery tissues, R3F1 recognized an additional band located between 120 and 70 kDa (that was not present in human PASMC), which is probably another proteolytically cleaved or truncated form of NCX1. In contrast, we could not detect NCKX3 protein expression in human PASMC, although mRNA expression was shown by RT-PCR analysis.



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Fig. 1. Expression of Na+/Ca2+ exchanger (NCX) isoforms in human pulmonary arterial smooth muscle cells (PASMC). A: RT-PCR products of NCX1 (231 bp for rats and 230 bp for humans) in rat (r) brain tissues and human (h) PASMC. M, 100-bp DNA ladder. Result shown is a representative of 3 separate experiments. B: Western blot analysis of NCX1 in rat brain and pulmonary artery (PA) tissues as well as human PASMC. The molecular masses of the bands are shown as standards. The experiment was reproduced 3 times with similar results. C: immunofluorescence analysis of NCX1 in human PASMC. Cells in the top images (+NCX1-Ab) were labeled with the NCX1-specific antibody (R3F1) and the second antibody (2nd-Ab) conjugated with FITC. Cells in the bottom images (–NCX1-Ab) were labeled only with the secondary antibody. 4',6'-Diamidino-2-phenylindole (DAPI) stain was used to identify cell nuclei.

 
The protein expression of NCX1 in human PASMC also was demonstrated using immunofluorescence analysis. As shown in Fig. 1C, human PASMC incubated with both primary (R3F1) and secondary antibodies showed strong fluorescent staining at the cell surface membrane (Fig. 1C, top); cells incubated with only secondary antibody showed no fluorescence (Fig. 1C, bottom). These results further suggest that NCX1 is expressed in human PASMC.

Functional role of Na+/Ca2+ exchanger in regulating [Ca2+]cyt. To test whether the Na+/Ca2+ exchanger is functionally involved in regulating [Ca2+]cyt, we first measured [Ca2+]cyt in human PASMC superfused with solutions with or without extracellular Na+. As shown in Fig. 2, removal of extracellular Na+ [by replacing Na+ in the bath solution with equimolar N-methyl-D-glucamine (NMDG+) or Li+] caused a rapid increase in [Ca2+]cyt as a result of switching the Na+/Ca2+ exchanger from the forward (Ca2+ exit) mode to the reverse (Ca2+ entry) mode (8, 28). Extracellular application of nifedipine (10 µM), a specific blocker of voltage-dependent Ca2+ channels, had no effect on the increase in [Ca2+]cyt driven by the reverse mode of the Na+/Ca2+ exchanger. However, extracellular application of KB-R7943, a potent and selective inhibitor of the Na+/Ca2+ exchanger (especially in its reverse mode), significantly attenuated the increase in [Ca2+]cyt induced by removal of extracellular Na+ (Fig. 2B). These results suggest that the Na+/Ca2+ exchanger can operate actively in the reverse mode in human PASMC.



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Fig. 2. Activation of the reverse mode of Na+/Ca2+ exchange increases the cytosolic free Ca2+ concentration ([Ca2+]cyt) in human PASMC. A: a representative record (a) showing the time course of [Ca2+]cyt changes in cells before, during, and after extracellular Na+ was replaced with equimolar N-methyl-D-glucamine (NMDG+) in the presence (0Na+5K) or absence (0Na+0K) of 5 mM K+. Nifedipine (10 µM) was included in all solutions to block voltage-gated Ca2+ channels. Summarized data (means ± SE, b) show the amplitude of increases in [Ca2+]cyt in cells superfused with 0Na+5K (+K, n = 33) or 0Na+0K (–K, n = 33) solution. B: a representative record (a) showing the time course of [Ca2+]cyt changes induced by 0Na+5K solution in the absence or presence of 10 µM KB-R7943 (KB-R). Summarized data (means ± SE, b) show the amplitude of increases in [Ca2+]cyt in cells superfused with 0Na+5K solution in the absence (Cont, n = 30) or presence (KB-R, n = 50) of 10 µM KB-R7943. ***P < 0.001 vs. Cont.

 
The activity of the Na+/Ca2+ exchanges in human PASMC appeared to be independent of extracellular K+, because removal of extracellular K+ had little effect on the rise in [Ca2+]cyt due to the reverse mode of Na+/Ca2+ exchange (Fig. 2). These results suggest that the Ca2+ entry via the reversed mode of Na+/Ca2+ exchange is likely due to the function of NCX (rather than NCKX) in human PASMC.

Involvement of Na+/Ca2+ exchanger in store depletion-induced Ca2+ entry. Upon activation of GPCR or receptor tyrosine kinases, IP3-mediated Ca2+ release (which induces an early transient increase in [Ca2+]cyt) depletes Ca2+ from intracellular stores (e.g., the SR). The store deletion-mediated opening of TRP channels would promote not only Ca2+ influx but also Na+ influx, because TRP channels are permeable to both Ca2+ and Na+ (35, 54). Because Ca2+ entry via the Na+/Ca2+ exchanger depends greatly on [Na+]cyt, the store depletion-mediated Na+ influx through TRP channels would increase [Na+]cyt, activate the reverse mode of Na+/Ca2+ exchange, and enhance Ca2+ entry. The next set of experiments was designed to test the hypothesis that in addition to triggering capacitative Ca2+ entry, the passive store depletion mediated by cyclopiazonic acid (CPA; 10 µM) induces Ca2+ entry as well via the Na+/Ca2+ exchanger.

In the absence of extracellular Ca2+ and presence of nifedipine (10 µM), extracellular application of CPA, a blocker of the Ca2+-Mg2+-ATPase in the SR (SERCA), induced a transient increase in [Ca2+]cyt due to Ca2+ leakage from the SR to the cytosol. Approximately 5–10 min later when the store was depleted (i.e., when the [Ca2+]cyt transients declined back to the basal level), restoration of extracellular [Ca2+] (to 1.8 mM) induced a large increase in [Ca2+]cyt due to Ca2+ entry (Fig. 3A). Extracellular application of KB-R7943 (10 µM; an inhibitor of the reverse mode of the Na+/Ca2+ exchanger) significantly attenuated the sustained phase of the store depletion-mediated Ca2+ entry (Fig. 3). Furthermore, removal of extracellular Na+ (by replacing external Na+ with equimolar NMDG+ or Li+) also markedly inhibited the store depletion-mediated Ca2+ entry (Fig. 4, A and C). In the absence of extracellular Na+ (i.e., when Na+/Ca2+ exchange was in the reverse mode or Ca2+ entry mode), the CPA-induced transient [Ca2+]cyt rise and baseline [Ca2+]cyt were both enhanced as a result of inhibited Ca2+ extrusion via the forward mode (or Ca2+ exit mode) of Na+/Ca2+ exchange (Fig. 4, A and B). These results indicate that the Ca2+ entry via the reverse mode of Na+/Ca2+ exchange is involved in store depletion-mediated Ca2+ entry in human PASMC.



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Fig. 3. Inhibition of Na+/Ca2+ exchanger with KB-R7943 attenuates store depletion-mediated Ca2+ entry. A: representative records showing the time course of [Ca2+]cyt changes in human PASMC in response to cyclopiazonic acid (CPA; 10 µM) in the presence or absence (0Ca) of extracellular Ca2+. KB-R-7943 (10 µM) or vehicle (DMSO) was applied to the cells when the CPA-mediated [Ca2+]cyt increase reached the maximal level in the presence of extracellular Ca2+. Nifedipine (10 µM) was included in all solutions to eliminate the contribution of voltage-gated Ca2+ channels to the [Ca2+]cyt changes. B: summarized data (means ± SE) showing the amplitude of CPA-mediated increases in [Ca2+]cyt after treatment with vehicle (n = 36) or KB-R7943 (n = 48). ***P < 0.001 vs. vehicle control.

 


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Fig. 4. Switching from the forward mode to the reverse mode of Na+/Ca2+ exchange by removing extracellular Na+ inhibits store depletion-mediated Ca2+ entry. A: representative records showing the time course of [Ca2+]cyt changes in response to CPA (10 µM) in the presence or absence (0Ca) of extracellular Ca2+. Cells were superfused with solutions containing 140 (140Na) or 0 mM Na+ (0Na). In these experiments, extracellular Na+ was replaced with equimolar Li+, and nifedipine (10 µM) was included in all solutions to eliminate the contribution of voltage-gated Ca2+ channels to the [Ca2+]cyt changes. B: summarized data (means ± SE) showing the amplitudes of CPA-induced increases in [Ca2+]cyt due to Ca2+ leakage or release in the absence of extracellular Ca2+ (shaded bars) and the basal levels of [Ca2+]cyt after CPA-induced Ca2+ release (solid bars) in cells bathed in Na+-containing (140Na, n = 38) or Na+-free (0Na–) solution. C: summarized data (means ± SE) showing the amplitudes of CPA-induced increases in [Ca2+]cyt due to Ca2+ entry or influx in the presence of extracellular Ca2+ in cells bathed in Na+-containing (140Na, n = 38) or Na+-free (0Na–) solution. Extracellular Na+ was replaced with NMDG (n = 63) or LiCl (n = 53). ***P <0.001 vs. 140Na.

 
Involvement of Na+/Ca2+ exchangers in human PASMC proliferation. Store depletion-mediated elevation of [Ca2+]cyt is associated with human PASMC proliferation in response to serum and growth factors (19, 48, 60). As mentioned earlier, the store depletion-mediated increase in [Ca2+]cyt is partially due to the KB-R7943-sensitive Ca2+ entry via the reverse mode of Na+/Ca2+ exchange. To examine the possible involvement of the Na+/Ca2+ exchanger in human PASMC proliferation, we tested the effect of KB-R7943 on cell growth rate using flow cytometry. As shown in Fig. 5A, addition of serum and growth factors to culture medium (i.e., switching culture medium from SmBM to SmGM) significantly increased the number of cells that are in S/G2/M phases, whereas treatment of human PASMC with KB-R7943 (10–30 µM for 24 h) significantly reduced the counts of cells in S/G2/M phases. Furthermore, inhibition of the Na+/Ca2+ exchanger with KB-R7943 (3–30 µM for 48 h) also markedly inhibited the increase in cell numbers when human PASMC were cultured in growth medium (SmGM) (Fig. 5B). Compared with cells treated with vehicle, KB-R7943 reduced cell proliferation in a dose-dependent manner. At low doses (<1 µM), KB-R7943 inhibits only the forward mode of Na+/Ca2+ exchange, whereas at high doses (such as those used in the experiments shown in Fig. 5), KB-R7943 is more potent in inhibiting the reverse mode of Na+/Ca2+ exchange (22). Therefore, these results suggest that KB-R7943-mediated antiproliferative effect on human PASMC is likely due to its inhibition of the reverse mode of the Na+/Ca2+ exchanger and to the subsequent decrease in Ca2+ entry.



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Fig. 5. Inhibition of the reverse mode of Na+/Ca2+ exchange attenuates human PASMC proliferation. A: flow cytometry histograms (a) of cell cycle analysis for growth-arrested (smooth muscle basal medium, SmBM) and proliferating (smooth muscle growth medium, SmGM) cells in the absence or presence of 10 or 30 µM KB-R7943 (for 24 h). Numbers of human PASMC in S, G2, and M phases (S/G2/M) when cultured in SmBM and SmGM with or without KB-R7943 (10 and 30 µM) are shown in b. B: summarized data (means ± SE) showing cell numbers (n = 12 experiments) of human PASMC cultured in SmBM and SmGM with or without 3, 10, or 30 µM KB-R7943 (for 48 h). ***P <0.001 vs. SmGM without KB-R7943 (solid bar).

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Pulmonary vasoconstriction and vascular medial hypertrophy (due to inappropriate PASMC hyperplasia and hypertrophy) greatly contribute to the elevated pulmonary vascular resistance and pulmonary arterial pressure in patients with pulmonary arterial hypertension (41, 47). Intracellular Ca2+ is a critical signal transduction element in regulating PASMC contraction (46) and proliferation (3, 18), as well as gene expression (4, 9). A rise in [Ca2+]cyt not only activates Ca2+-dependent function occurring in the cytosol (e.g., contraction) but also activates Ca2+-sensitive events in the nucleus (e.g., expression of the nuclear proteins that are related to the cell cycle) (4, 9). Indeed, the resting [Ca2+]cyt are higher and the agonist-mediated increases in [Ca2+]cyt are greater in PASMC isolated from patients with pulmonary arterial hypertension than in PASMC from normal subjects and patients without pulmonary hypertension (19, 61). Therefore, understanding the cellular mechanisms involved in regulating [Ca2+]cyt in human PASMC would help develop new therapeutic approaches for patients with pulmonary arterial hypertension.

The results from this study demonstrate that 1) human PASMC express NCX1 and NCKX3; 2) removal of extracellular Na+ activates the operation of the Na+/Ca2+ exchanger in the reverse mode to promote Ca2+ entry and increase [Ca2+]cyt; and 3) removal of extracellular K+ has no effect on the Ca2+ entry via the reverse mode of Na+/Ca2+ exchange. These observations provide strong evidence that Ca2+ entry via the reverse mode of the Na+/Ca2+ exchanger is a critical mechanism that regulates intracellular Ca2+ homeostasis in human PASMC. The NCX1 is probably the major Na+/Ca2+ exchanger isoform in human PASMC that functions in the reverse mode when [Na+]cyt is increased but works in the forward mode when [Ca2+]cyt is increased and [Na+]cyt remains unchanged. The protein expression level of NCX1 in cultured human PASMC was noticeably much smaller than that in freshly isolated rat pulmonary arteries (Fig. 1B), although the immunocytochemistry data clearly showed the surface expression of NCX1 in cultured PASMC (Fig. 1C). There are several possibilities that explain the discrepancies: 1) expression level of NCX1 may be significantly different between human and rat PASMC, and 2) expression level of NCX1 in human pulmonary arteries may be changed (e.g., downregulated) in cultured PASMC because of phenotypic changes (e.g., from contractile phenotype to synthetic or proliferative phenotype).

As mentioned earlier, the stoichiometry of the NCX-encoded Na+/Ca2+ exchanger proteins is 3 Na+ per 1 Ca2+. Thus [Ca2+]cyt determined by NCX is mainly related to [Na+]cyt by the following equation:

where F is the Faraday constant, R is the gas constant, and T is absolute temperature. The equation indicates that for a constant [Ca2+]out and [Na+]out, [Ca2+]cyt is positively proportional to the third power of [Na+]cyt; that is, a small increase in [Na+]cyt can cause a large increase in [Ca2+]cyt due to Na+/Ca2+ exchange.

In PASMC, activation of GPCR (e.g., endothelin receptor) or receptor tyrosine kinases (e.g., platelet-derived growth factor receptor) often leads to an increased synthesis of IP3 that mediates Ca2+ mobilization from the SR to the cytosol by activating IP3 receptors (9, 15, 18, 36). The subsequent depletion of Ca2+ from the SR opens SOC in the plasma membrane and causes further Ca2+ influx to maintain a high level of [Ca2+]cyt during contraction or proliferation. The store depletion-mediated Ca2+ entry (SDCaE) has been demonstrated to result mainly from capacitative Ca2+ entry through Ca2+-permeable SOC (5, 56, 63). However, the canonical TRP (TRPC) channels that form functional SOC (5, 10, 33, 52, 55) are also permeable to other cations, including Na+; some of the TRPC channels are actually more permeable to Na+ than to Ca2+ (5, 13, 19). In other words, store depletion-mediated opening of TRPC channels would allow both Ca2+ and Na+ to enter the cell. The store depletion-mediated Na+ entry (SDNaE) would thus induce a local rise in [Na+]cyt, which activates the operation of Na+/Ca2+ exchangers in the reverse mode and subsequently increases [Ca2+]cyt (1, 2).

The results from this study demonstrate that 1) the CPA-induced passive depletion of Ca2+ from the intracellular stores (i.e., the SR) increases [Ca2+]cyt as a result of store depletion-mediated Ca2+ entry, and 2) removal of extracellular Na+ or extracellular application of KB-R7943 (an inhibitor of the Na+/Ca2+ exchanger) (8, 16, 21, 43) significantly attenuates the store depletion-mediated Ca2+ entry. These data suggest that the reverse mode of Na+/Ca2+ exchange participates in store depletion-mediated Ca2+ entry. In other words, the store depletion-mediated Ca2+ entry in human PASMC is composed of two components: 1) capacitative Ca2+ entry through Ca2+-permeable SOC (or TRPC) channels and 2) Ca2+ entry via the reverse model of the Na+/Ca2+ exchanger.

Store depletion-mediated Ca2+ entry is an important stimulus for mitogen-mediated PASMC proliferation (5, 9, 34, 39). Our previous studies demonstrated that functional blockade of SOC with La3+, Ni2+, or SKF-96365 as well as downregulation of TRPC channels with siRNA or antisense oligonucleotides all inhibited PASMC growth in the presence of serum and growth factors (19, 26, 48, 60). Consistent with these observations, we show in this study that inhibition of the reverse mode of Na+/Ca2+ exchange with KB-R7943, in addition to reducing the amplitude of store depletion-mediated Ca2+ entry, significantly inhibits PASMC proliferation. These results suggest that Ca2+ entry via the reverse mode of Na+/Ca2+ exchange is, at least in part, involved in inducing the rise in [Ca2+]cyt required for PASMC proliferation.

Increasing evidence suggests that there is a functional interaction between the Na+/Ca2+ exchanger and SOC (40) or TRPC channels that are activated by store depletion (34). Under physiological conditions, opening of these nonselective cation channels not only results in Na+ influx to the restricted plasma membrane-SR junctional space but also causes membrane depolarization as a result of inward cationic currents. Both the increase in [Na+]cyt and membrane depolarization drive the plasma membrane Na+/Ca2+ exchanger to its reversed mode of operation, thereby transporting more Ca2+ into cell (1, 7, 28). In human PASMC, our data support the contention that store depletion-mediated Ca2+ entry is caused by both capacitative Ca2+ entry and NCX-mediated Ca2+ entry, and the latter is also involved in serum- and growth factor-mediated PASMC proliferation. Whether TRPC isoforms, as well as which ones, are functionally or physically interacted with NCX proteins in human PASMC remains unclear.

Together, our results indicate that function of the Na+/Ca2+ exchanger plays an important role in regulating [Ca2+]cyt in human PASMC. Ca2+ entry via the reverse mode of Na+/Ca2+ exchange is a critical pathway for increasing [Ca2+]cyt, inducing pulmonary vasoconstriction, and stimulating PASMC proliferation. In patients with pulmonary arterial hypertension, sustained pulmonary vasoconstriction and pulmonary vascular medial hypertrophy (due to excessive PASMC proliferation) are the major causes of increased pulmonary arterial pressure (41, 47). Therefore, better understanding the functional role of the Na+/Ca2+ exchanger in the pulmonary vasculature may lead to development of novel therapeutic approaches for treatment of pulmonary vascular diseases such as idiopathic pulmonary arterial hypertension.


    GRANTS
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 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
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 REFERENCES
 
This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-064945, HL-54043, and HL-66012 (to J. X.-J. Yuan) and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-33491 (to K. E. Barrett). Dr. H. Dong serves as a co-investigator on Grant DK-33491.


    ACKNOWLEDGMENTS
 
We thank Dr. J. Lytton (Dept. of Biochemistry and Molecular Biology, Univ. of Calgary, Canada) for kindly providing the R3F1 antibody and A. Nicholson for technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: H. Dong, Division of Gastroenterology (8414), Dept. of Medicine, CTF A-103, Univ. of California, San Diego, 210 Dickinson St., San Diego, CA 92103-8414 (E-mail: h2dong{at}ucsd.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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