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VASCULAR BIOLOGY

Upregulation of Na⁺/Ca²⁺ exchanger contributes to the enhanced Ca²⁺ entry in pulmonary artery smooth muscle cells from patients with idiopathic pulmonary arterial hypertension

Department of Medicine, University of California, San Diego, La Jolla, California

Submitted 12 July 2006 ; accepted in final form 23 December 2006

	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 a trigger for pulmonary vasoconstriction and a stimulus for PASMC proliferation and migration. Multiple mechanisms are involved in regulating [Ca²⁺]_cyt in human PASMC. The resting [Ca²⁺]_cyt and Ca²⁺ entry are both increased in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH), which is believed to be a critical mechanism for sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in these patients. Here we report that protein expression of NCX1, an NCX family member of Na⁺/Ca²⁺ exchanger proteins is upregulated in PASMC from IPAH patients compared with PASMC from normal subjects and patients with other cardiopulmonary diseases. The Na⁺/Ca²⁺ exchanger operates in a forward (Ca²⁺ exit) and reverse (Ca²⁺ entry) mode. By activating the reverse mode of Na⁺/Ca²⁺ exchange, removal of extracellular Na⁺ caused a rapid increase in [Ca²⁺]_cyt, which was significantly enhanced in IPAH PASMC compared with normal PASMC. Furthermore, passive depletion of intracellular Ca²⁺ stores using cyclopiazonic acid (10 µM) not only caused a rise in [Ca²⁺]_cyt due to Ca²⁺ influx through store-operated Ca²⁺ channels but also mediated a rise in [Ca²⁺]_cyt via the reverse mode of Na⁺/Ca²⁺ exchange. The upregulated NCX1 in IPAH PASMC led to an enhanced Ca²⁺ entry via the reverse mode of Na⁺/Ca²⁺ exchange, but did not accelerate Ca²⁺ extrusion via the forward mode of Na⁺/Ca²⁺ exchange. These observations indicate that the upregulated NCX1 and enhanced Ca²⁺ entry via the reverse mode of Na⁺/Ca²⁺ exchange are an additional mechanism responsible for the elevated [Ca²⁺]_cyt in PASMC from IPAH patients.

transient receptor potential channel; reverse and forward mode; proliferation

In addition to the Ca²⁺-permeable channels in the plasma membrane, human PASMC also functionally express multiple Ca²⁺ transporters that allow Ca²⁺ to enter cell against the Ca²⁺ electrochemical gradient. The Na⁺/Ca²⁺ exchanger (NCX) is a ubiquitously expressed protein that transports Ca²⁺ across the plasma membrane based on the transmembrane electrochemical gradient of Na⁺ and Ca²⁺ (9). Na⁺/Ca²⁺ exchangers operate in either a forward (3 Na⁺ entry and 1 Ca²⁺ exit for NCX; or 4 Na⁺ entry and 1 Ca²⁺ and 1 K⁺ exit for NCKX) or reverse (3 Na⁺ exit and 1 Ca²⁺ entry or 4 Na⁺ exit and 1 Ca²⁺ and 1 K⁺ entry) mode based on the transmembrane Na⁺ and Ca²⁺ (and K⁺) concentration gradients and membrane potential (9). Since the stoichiometry of the NCX family of Na⁺/Ca²⁺ exchanger proteins is 3 Na⁺ per 1 Ca²⁺, the [Ca²⁺]_cyt determined by the NCX family members of Na⁺/Ca²⁺ exchangers is thus mainly related to changes in cytosolic [Na⁺] ([Na⁺]_cyt) according to the following equation: [Ca²⁺]_cyt = [Ca²⁺]_out x ([Na⁺]_cyt ÷ [Na⁺]_out)³ x e^(E_mF/RT), where E_m is the membrane potential, F is the Faraday constant, R is the gas constant, T is the absolute temperature, and "out" and "cyt" indicate extracellular or cytosolic concentrations of Ca²⁺ or Na⁺, respectively. The equation indicates that, when extracellular [Ca²⁺] and [Na⁺] are maintained constant, [Ca²⁺]_cyt is positively proportional to the third power of [Na⁺]_cyt. That is, a small increase in intracellular [Na⁺] can significantly increase [Ca²⁺]_cyt due to the reverse mode of Na⁺/Ca²⁺ exchange (9, 29, 63).

In addition to voltage-gated Na⁺ channels (28, 45), human PASMC (30, 60, 62) also express transient receptor potential (TRP) channels that form functional cation channels allowing both Na⁺ and Ca²⁺ to enter cell (8, 13, 24). Therefore, TRP channels may serve as a critical pathway for increasing [Na⁺]_cyt, when cells are stimulated by mitogenic agonists and vasoactive substances (4, 10, 11, 47). The resultant activation of the reverse mode of Na⁺/Ca²⁺ exchange (as a result of increased [Na⁺]_cyt) would thus function as an additional pathway for elevating [Ca²⁺]_cyt in PASMC. Since the Na⁺/Ca²⁺ exchanger is functionally coupled to the sarcoplasmic reticulum (SR) or endoplasmic reticulum (ER) Ca²⁺-Mg²⁺ ATPase (SERCA) (31), the accumulated Ca²⁺ due to Ca²⁺ entry via Na⁺/Ca²⁺ exchanger would be easily and efficiently sequestered to the SR/ER by SERCA and thereby increasing [Ca²⁺] in the SR/ER ([Ca²⁺]_SR/ER).

Activation of G protein-coupled receptors (GPCRs) or tyrosine kinase receptors leads to activation of phospholipase C (PLC- and -) and to synthesis of inositol-1,4,5-trisphosphate (IP₃) and diacylglycerol. IP₃ induces Ca²⁺ mobilization from the SR/ER by activating IP₃ receptors (17, 22, 44), while diacylglycerol mediates Ca²⁺ influx by activating receptor-operated Ca²⁺ channels (ROC) in the plasma membrane (2, 16). Furthermore, the IP₃-mediated store depletion opens store-operated Ca²⁺ channels (SOC) and further increases [Ca²⁺]_cyt by promoting Ca²⁺ influx (8, 46, 55, 64). Therefore, a higher concentration of stored Ca²⁺ in the SR/ER may play an important role in triggering (and maintaining) the signaling cascade involved in agonist-induced vasoconstriction and mitogen-mediated PASMC proliferation (24, 63).

In this study, we examined and compared the protein expression of Na⁺/Ca²⁺ exchanger and the increase in [Ca²⁺]_cyt due to the reverse mode of Na⁺/Ca²⁺ exchanger in PASMC from normal subjects and patients with idiopathic pulmonary arterial hypertension (IPAH). Our results indicate that 1) the reverse mode of Na⁺/Ca²⁺ exchange functions as an important pathway for regulating [Ca²⁺]_cyt in normal human PASMC, 2) the store depletion-mediated Ca²⁺ entry is partially mediated by activating the reverse mode of Na⁺/Ca²⁺ exchange in normal PASMC, 3) the NCX1 isoform of Na⁺/Ca²⁺ exchanger proteins is upregulated and the increase in [Ca²⁺]_cyt due to the reverse mode of Na⁺/Ca²⁺ exchange is enhanced in PASMC from IPAH patients compared with PASMC from normal subjects and control patients. The upregulated Na⁺/Ca²⁺ exchangers, along with the upregulated TRP channels in caveolae (42, 59), in PASMC may play an important pathogenic role in the development of sustained pulmonary vasoconstriction and severe pulmonary vascular remodeling in patients with IPAH.

	METHODS AND MATERIALS

TOP ABSTRACT METHODS AND MATERIALS RESULTS DISCUSSION GRANTS REFERENCES

 
Cell preparation and culture. Primary cultured PASMC from transplant patients and normal human PASMC purchased from Cambrex were used in this study. Lung tissues were obtained from patients during lung/heart transplantation. The diagnosis of IPAH was established on the basis of the criteria used in the National Registry on Primary Pulmonary Hypertension and was confirmed histopathologically. The mean pulmonary arterial pressure of three IPAH patients from whom PASMC were prepared, was 52±1 mmHg. All the patients had been treated with beraprost, warfarin, digoxin, and furosemide before lung transplantation.

Peripheral muscular pulmonary arteries isolated from the explanted lung tissues were first incubated in Hanks' balanced salt solution that contained 2 mg/ml collagenase (Worthington Biochemical) for 20 min to remove adventitia by a fine forceps and to remove endothelium by a surgical scalpel (61). The remaining smooth muscle was then digested for 40–50 min with (in mg/ml) 2.25 collagenase, 0.5 elastase, and 1 albumin (Sigma) at 37°C to make a cell suspension of PASMC. The cells were resuspended in the smooth muscle growth medium (SmGM, Cambrex) and cultured in an incubator under a humidified atmosphere of 5% CO₂-95% air at 37°C. The cells from normotensive patients and IPAH patients were split into new petri dishes when 70–90% confluence was achieved to amplify cell numbers. The subcultured cells were then stored at –80°C and used at passages 4–7 for molecular biological and fluorescence microscopy experiments.

Human PASMC from normal subjects were also cultured in SmGM and used at passages 4–6 for experimentation. The medium was changed after 24 h and every 48 h thereafter. The SmGM was composed of smooth muscle basal medium supplemented with 5% fetal bovine serum, 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 using trypsin-EDTA buffer (Cambrex) when 70–90% confluence was achieved.

Western blot analysis. Cells were gently washed twice in cold PBS, scraped into 0.3 ml of radioimmunoprecipitation assay buffer [1x PBS, 1% Nonidet P-40 (Amaresco), 0.5% sodium deoxycholate, and 0.1% SDS], and incubated on ice for 45 min. The cell lysates were sonicated and centrifuged at 14,000 rpm for 15 min at 4°C. The supernatants were collected, and protein concentration was determined by Coomassie Plus protein assay reagent (Pierce Biotechnology) using 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% bromophenol blue). Protein suspensions were electrophoretically separated on an 8% acrylamide gel, and protein bands were transferred to nitrocellulose membranes by electroblot in a Mini Trans-Blot cell transfer apparatus (Bio-Rad) 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). Negative control experiments for Western blot analysis were performed using only the secondary antibody; there was no detectable band on the membrane if the primary antibody (for NCX1) was not added (data not shown).

Immunofluorescence labeling. Human PASMC on slides were fixed in 4% paraformaldehyde for 20 min. After being blocked with 4% bovine serum albumin 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). The cells were then stained with the membrane-permeable nucleic acid stain 4,6-diamidino-2-phenylindole (5 µM; Sigma), and the blue fluorescence (emitted at 461 nm) was used to detect cell nuclei. The cell images were processed by 3D deconvolution fluorescence microscopy with the SoftWorx (Applied Precision), and analyzed by Matlab (The 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 modified Krebs solution for 30 min to remove extracellular dye and allow intracellular esterases to cleave cytosolic fura 2-AM into active fura 2. The modified Krebs solution contained (in mM) 140 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⁺. In Na⁺-free PSS, NaCl was replaced by equimolar N-methyl-D-glucamine (NMDG⁺) or LiCl.

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) using 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 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 = K_d x (Sf₂ ÷ Sb₂) x (R – R_min) ÷ (R_max – R), where K_d (225 nM) is the dissociation constant for Ca²⁺, R was the measured fluorescence ratio, R_min and R_max were minimal and maximal ratios, respectively.

Chemicals. Cyclopiazonic acid (CPA; Sigma) and KB-R7943 (Tocris, Ellisville, MO) were dissolved in DMSO to make a stock solution of 50–100 mM. Aliquots of the stock solution were then diluted 1:1,000–10,000 in PSS, Ca²⁺-free PSS, or Na⁺-free PSS, respectively, on the day of use to their final concentrations. All chemicals were of analytical grade or better.

Statistics. The composite data are expressed as means ± SE. Statistical analysis was performed using paired or unpaired Student's t-test or ANOVA and post hoc tests (Student-Newman-Keuls) where appropriate. Differences were considered to be significant at P < 0.05.

	RESULTS

TOP ABSTRACT METHODS AND MATERIALS RESULTS DISCUSSION GRANTS REFERENCES

 
Protein expression of Na⁺/Ca²⁺ exchanges is upregulated in PASMC from IPAH patients. To examine whether protein expression of Na⁺/Ca²⁺ exchanges in PASMC is enhanced in IPAH, we compared the protein level of NCX1 in IPAH PASMC using Western blot and immunocytochemistry analyses with PASMC isolated from normal subjects (Nor) and patents with pulmonary hypertension secondary to other disease (SPH), e.g., patients with idiopathic pulmonary fibrosis. As shown in Fig. 1, protein expression levels of NCX1 in PASMC from three IPAH patients were all much higher than in PASMC from three Nor subjects (Nor1, -2, and -3) and a SPH patient (IPF), whereas protein levels of -actin were comparable in PASMC from IPAH patients and normal subjects and SPH patients (Fig. 1A, a and c). The protein expression level of NCX1 in PASMC from normal subjects and SPH patients were comparable to the expression level in lung tissues from normotensive patients with COPD (Fig. 1Ab). In addition, the immunocytochemical experiments also indicate that NCX1 protein level was higher in IPAH PASMC than in normal PASMC (Fig. 1B).

View larger version (45K): [in this window] [in a new window]   Fig. 1. The Na+/Ca2+ exchanger (NCX1) isoform of Na+/Ca2+ exchanger proteins is upregulated in pulmonary artery smooth muscle cells (PASMC) from idiopathic pulmonary arterial hypertension (IPAH) patients. A: Western blot analysis of NCX1 (a, top) and -actin or -actin (bottom) in PASMC from normal subjects (Nor), patients with IPAH, and patients with idiopathic pulmonary fibrosis (IPF). Western blot analysis of NCX1 (top) and -actin (bottom) in lung tissues from two normotensive patients with chronic obstructive pulmonary disease (COPD) is shown in b. The molecular masses (in kDa) of the bands are shown as standards. The experiments were reproduced 3 times with similar results (b; means ± SE). **P < 0.01 vs. control cells. B: immunofluorescent analysis of NCX1 in normal and IPAH PASMC. Cells were labeled with the NCX1-specific antibody (R3F1) and the second antibody (2nd-Ab) conjugated with FITC. 4,6-diamidino-2-phenylindole stain was used to identify cell nuclei.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Activation of the reverse mode of Na+/Ca2+ exchange increases cytosolic Ca2+ concentration ([Ca2+]cyt) in normal PASMC and the rise in [Ca2+]cyt due to Ca2+ entry via the reverse mode of Na+/Ca2+ exchange is enhanced in PASMC from IPAH patients. A: representative records showing the time course of [Ca2+]cyt changes in normal PASMC (left) and IPAH PASMC (middle) superfused with modified Krebs solution (MKS; with 140 mM Na+) or Na+-free (0 Na) solutions. A representative record showing the [Ca2+]cyt changes in IPAH PASMC by removal of extracellular Na+ in the absence or presence of 10 µM KB-R7943 (KB-R; right). B: summarized data (means ± SE) showing amplitude of [Ca2+]cyt increases in normal (open bar; n = 39) and IPAH (solid bar; n = 34) PASMC superfused with 0 Na solution. ***P < 0.001 vs. normal cells.

View larger version (43K): [in this window] [in a new window]   Fig. 3. Ionomycin (IM)-mediated increase in [Ca2+]cyt is comparable in PASMC from normal subjects and IPAH patients. A: representative records showing the time course of [Ca2+]cyt changes in normal PASMC treated with 10 µM IM in MKS (140 Na; a) or Na+-free (0 Na, b) MKS, as well as in IPAH PASMC treated with IM in MKS (140 Na, c). B: summarized data (means ± SE) showing the amplitude of IM-mediated increase in [Ca2+]cyt in normal PASMC bathed in MKS (140 Na) or Na+-free (0 Na) solutions. ***P < 0.001 vs. IM-140 Na. C: histogram showing the amplitude of IM-mediated increases in [Ca2+]cyt in normal (top) and IPAH (bottom) PASMC superfused with MKS (140 Na). The average values of IM-mediated rise [Ca2+]cyt are 402 ± 27 nM in normal PASMC (n = 61 cells) and 395 ± 25 nM in IPAH PASMC (n = 59 cells; P = 0.835). D: kinetics of the decline phase of IM-mediated [Ca2+]cyt transients in normal and IPAH patients. The time constant for the decay was 65.2 ± 2.7 and 53.6 ± 1.9 ms in normal and IPAH PASMC, respectively.

Store-operated Na⁺ influx contributes to the [Ca²⁺]_cyt increase due to activation of the reverse mode of Na⁺/Ca²⁺ exchange in normal and IPAH PASMC. The transmembrane Na⁺ gradient can be changed by a local increase in [Na⁺]_cyt due to Na⁺ influx through Na⁺-permeable channels, such as voltage-gated Na⁺ channels (6, 14, 39, 45, 48) and TRP channels (19, 20, 41). In human PASMC superfused with Ca²⁺-free solution (0 Ca), inhibition of the SR/ER Ca²⁺ pump (SERCA) with CPA (10 µM), first caused a rapid increase in [Ca²⁺]_cyt due to Ca²⁺ leakage from the SR/ER to the cytosol (Fig. 4A, left). The mobilized Ca²⁺ was then extruded by Ca²⁺ pumps and Na⁺/Ca²⁺ exchangers (in the forward mode) in the plasma membrane. After CPA-mediated Ca²⁺ mobilization from the SR/ER was complete, restoration of extracellular Ca²⁺ caused an additional increase in [Ca²⁺]_cyt due apparently to capacitative Ca²⁺ entry (CCE) (Fig. 4A, left, shadowed box) or Ca²⁺ influx through SOCs.

View larger version (29K): [in this window] [in a new window]   Fig. 4. Downregulation of NCX1 inhibits store depletion-mediated increase in [Ca2+]cyt in normal PASMC. A: representative records showing the time course of [Ca2+]cyt changes in control PASMC (left) and in PASMC treated with small interfering (si)RNA for NCX1 (siNCX1, right) in response to cyclopiazonic acid (CPA; 10 µM) in the presence or absence (0 Ca) of extracellular Ca2+. The shadowed area depicts the rise in [Ca2+]cyt or the Ca2+ influx due to CPA-mediated store depletion. B: summarized data (means ± SE) showing the resting [Ca2+]cyt (left) and the amplitude of CPA-mediated Ca2+ leakage or release (Release) and store depletion-mediated Ca2+ entry in normal (open bars) and IPAH (solid bars) PASMC. ***P < 0.001 vs. control.

Passive depletion of intracellular Ca²⁺ stores with CPA caused an increase in [Ca²⁺]_cyt due to Ca²⁺ influx through SOC, usually referred to as capacitative Ca²⁺ entry, in normal PASMC (Fig. 4A, left). Treatment of the cells with small interfering (si)RNA specifically targeting NCX1, however, caused a 56% reduction of the amplitude of store depletion-mediated Ca²⁺ entry, while the resting [Ca²⁺]_cyt level and the amplitude of CPA-mediated Ca²⁺ leakage or release were not significantly affected by NCX1-siRNA treatment (Fig. 4, A and B). These results indicate that the store depletion-mediated [Ca²⁺]_cyt increase is 1) partially caused by the store-operated Ca²⁺ influx through SOC or CCE, and 2) partially caused by Ca²⁺ entry through the reverse mode of Na⁺/Ca²⁺ exchange, which is activated by store depletion-mediated Na⁺ influx via TRP channels and subsequent increase in local [Na⁺]_cyt. Removal of extracellular Na⁺ or inhibition of the reverse mode of Na⁺/Ca²⁺ exchange significantly inhibited store depletion-mediated increase in [Ca²⁺]_cyt (63). In other words, the store depletion-mediated rise in [Ca²⁺]_cyt (e.g., induced by CPA) is significantly inhibited in PASMC superfused with Na⁺-free solution compared with PASMC superfused with 140 mM Na⁺-containing solution (63). These observations further suggest that store depletion-mediated [Ca²⁺]_cyt increase results from both Ca²⁺ influx through SOC (or CCE) and Ca²⁺ entry via the reverse mode of Na⁺/Ca²⁺ exchange.

When PASMC are superfused with Na⁺-free solution, store depletion-mediated opening of TRP channels or SOC would be unable to raise [Na⁺]_cyt and activate the reverse mode of Na⁺/Ca²⁺ exchange. Therefore, store depletion-mediated increase in [Ca²⁺]_cyt in PASMC superfused with Na⁺-free solution would be due solely to Ca²⁺ influx through SOC (or TRP channels) or CCE. The next set of experiments was designed to examine whether store depletion-mediated Ca²⁺ influx through SOC (i.e., CCE) independent of the Ca²⁺ entry via Na⁺/Ca²⁺ exchange was also augmented in PASMC from IPAH patients.

As shown in Fig. 5, the CPA-mediated Ca²⁺ mobilization (or release) from the intracellular stores in PASMC bathed in Ca²⁺-free and Na⁺-free solution was increased in patients with IPAH (Fig. 5, A and B), indicating that [Ca²⁺] in the SR/ER is higher in IPAH PASMC than normal PASMC. Furthermore, the rise in [Ca²⁺]_cyt due to store depletion-mediated Ca²⁺ influx through SOC (or CCE) in IPAH PASMC (523.6 ± 27.5 nM; n = 33 cells) was 81% higher than that in normal PASMC (288.8 ± 13.0 nM; n = 36 cells; P < 0.001) (Fig. 5, A and B). These results are consistent with our previously published data showing that 1) TRPC channels (e.g., TRPC3 and TRPC6) are upregulated and 2) CPA-mediated CCE (i.e., Ca²⁺ influx through SOC) is enhanced in PASMC from IPAH patients (59).

View larger version (23K): [in this window] [in a new window]   Fig. 5. Store depletion-mediated rise in [Ca2+]cyt via Ca2+ entry, independent and dependent of the reverse model of Na+/Ca2+ exchange, is enhanced in PASMC from IPAH patients. A: representative records showing the time course of [Ca2+]cyt changes in normal (left) and IPAH (right) PASMC in response to CPA (10 µM) in the presence or absence (0 Ca) of extracellular Ca2+. Extracellular Na+ was replaced by NMDG+ (0 Na) to eliminate the contribution of the reverse model of Na+/Ca2+ exchange to the [Ca2+]cyt change. B: summarized data (means ± SE) showing the resting [Ca2+]cyt (left) and the amplitudes of CPA-mediated Ca2+ leakage or release (Release) and capacitative Ca2+ entry (CCE) in normal (open bars) and IPAH (solid bars) PASMC superfused with Na+-free (0 Na) solution. C: summarized data (means ± SE) showing the resting [Ca2+]cyt (left) and amplitude of CPA-mediated increases in [Ca2+]cyt (due to release or CCE) in IPAH PASMC in response to CPA in the presence or absence (0 Ca) of extracellular Ca2+. The CPA-mediated response was compared in MKS (140 Na) and Na+-free (0 Na) solutions. ***P < 0.01 vs. IPAH-140Na.

Nevertheless, the amplitude of [Ca²⁺]_cyt rise due to store depletion-mediated Ca²⁺ entry in IPAH-PASMC bathed in Na⁺-free solution (523.6 ± 27.5 nM, n = 66 cells) was 30% less than the amplitude in IPAH-PASMC bathed in 140 mM Na⁺-containing solution (728.7 ± 10.3 nM, n = 37 cells; P < 0.001) (Fig. 5C). These data also imply that the inward transportation of Ca²⁺ via the reverse mode of Na⁺/Ca²⁺ exchange accounts for about one-third to one-fourth of the total Ca²⁺ entry induced by CPA-mediated passive depletion of intracellular stores.

	DISCUSSION

TOP ABSTRACT METHODS AND MATERIALS RESULTS DISCUSSION GRANTS REFERENCES

 
Sustained pulmonary vasoconstriction and thickening of the pulmonary vascular wall, resulting from excessive PASMC proliferation and migration, greatly contribute to the elevated pulmonary vascular resistance in patients with IPAH. When a vasoconstrictive or mitogenic ligand binds with its receptor in the plasma membrane in PASMC, a rise in [Ca²⁺]_cyt due to Ca²⁺ release and/or entry is an important signaling mechanism for agonist-mediated PASMC contraction and mitogen-mediated PASMC proliferation. Compared with PASMC from normal subjects, many studies have demonstrated that 1) the proliferation rate (determined by cell number, DNA content, and ³H-thymidine incorporation) is significantly accelerated (37, 38, 59) and 2) the store depletion-mediated Ca²⁺ entry is enhanced in PASMC from IPAH patients (59), as well as in PASMC from chronically hypoxic animals (32, 57). Therefore, PASMC from IPAH patients may undergo phenotypic changes that make the cells more prone (inclined) to proliferation and contraction or to activation by contractile agonists and mitogenic factors.

The results from this study demonstrate that human PASMC functionally express Na⁺/Ca²⁺ exchangers, e.g., NCX1 (63); removal of extracellular Na⁺ increases [Ca²⁺]_cyt in PASMC by activating the reverse mode of Na⁺/Ca²⁺ exchange. Furthermore, the store depletion-mediated Ca²⁺ entry is caused by at least two mechanisms: 1) CCE, through Ca²⁺ release-activated Ca²⁺ channels, SOC, and/or TRP channels (2, 40); and 2) inward transportation of Ca²⁺ via the reverse mode of Na⁺/Ca²⁺ exchange, which is triggered by a local rise in [Na⁺]_cyt due to store depletion-mediated Na⁺ influx through TRP-encoded SOC. These observations indicate that, in normal PASMC, agonist- or mitogen-mediated Ca²⁺ release from the SR (due to IP₃-mediated activation of IP₃ receptors) causes store depletion, which induces not only Ca²⁺ influx but also Na⁺ influx through TRP-encoded SOC. The subsequent rise in [Na⁺]_cyt reverses the driving force for Na⁺/Ca²⁺ exchange (from the forward mode to the reverse mode), and ultimately enhances inward transportation of Ca²⁺ through the reverse mode of Na⁺/Ca²⁺ exchange as shown in Fig. 6) .

View larger version (32K): [in this window] [in a new window]   Fig. 6. Schematic diagram depicting the proposed mechanisms of store depletion-mediated Ca2+ entry via the reverse mode of Na+/Ca2+ exchange. Binding of ligand with the membrane receptors (e.g., G protein-coupled receptor, GPCR) activates phospholipace C (PLC), which results in hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG). IP3 activates IP3 receptors on the SR/ER membrane and induces Ca2+ mobilization. Depletion of Ca2+ from the SR/ER (store depletion) then activates store-operated Ca2+ channels (SOC), putatively formed by TRPC channel subunits, and causes Na+ and Ca2+ entry. The accumulated intracellular Na+ activates the reverse mode of NCX and induces the inward transportation of Ca2+ via NCX. Furthermore, DG and protein kinase C (PKC) can also open receptor-operated Ca2+ channels (ROC), putatively formed by TRPC channel subunits, cause Na+ (and Ca2+) influx, and subsequently enhance the inward transportation of Ca2+ via the reverse mode of Na+/Ca2+ exchange. An increase in [Ca2+]cyt as a result of Ca2+ release, Ca2+ influx through ROC and SOC, and the inward transportation of Ca2+ through NCX serves as a major trigger for PASMC contraction and migration and an important stimulus for PASMC proliferation. SERCA, SR/ER Ca2+-Mg2+ ATPase.

In PASMC from IPAH patients, we previously reported that mRNA and protein expression of TRPC3 and TRPC6 were upregulated compared with PASMC from normal subjects and patients with normotensive cardiopulmonary diseases and secondary pulmonary hypertension (e.g., due to chronic obstructive pulmonary disease and emphysema, interstitial pulmonary fibrosis) (59). The data from the current study indicate that protein expression of NCX1 is also upregulated in PASMC from IPAH patients. Interestingly, the upregulated NCX1 appear to function mainly in the reverse mode because the Ca²⁺ extrusion due to the forward mode of Na⁺/Ca²⁺ exchange in the presence of high extracellular Na⁺ is not enhanced in IPAH PASMC. However, the inward transportation of Ca²⁺ through the reverse mode of Na⁺/Ca²⁺ exchange, induced by either removal of extracellular Na⁺ or by CPA-induced passive store depletion, was augmented in PASMC from IPAH patients. These results suggest that upregulated TRPC channels and Na⁺/Ca²⁺ exchangers interact functionally with each other in submembrane vicinity close to caveolae in PASMC from IPAH patients to magnify agonist-mediated smooth muscle contraction and mitogen-mediated PASMC proliferation and migration. Indeed, our recent data indicate that mRNA and protein expression of caveolin and number of caveolae in IPAH-PASMC are much greater than those in normal PASMC and PASMC from patients with secondary pulmonary hypertension (42). Treatment of IPAH-PASMC with methyl--cyclodextrin (MCD), a compound that depletes membrane cholesterol and disrupts caveolae, and with siRNA for caveolin-1 resulted in a concentration-dependent decrease in store depletion-mediated Ca²⁺ entry (42). The functional and physical colocalization of receptors and TRPC channels in caveolae by caveolin have also been implicated in systemic arterial smooth muscle cells (5).

As mentioned earlier, the TRP channels that participate in forming SOC or ROC are nonselective cation channels. On the basis of the permeation studies, the permeability for Na⁺ is actually greater than Ca²⁺ for many TRP isoforms (8, 24). Therefore, upon activation of receptors in the plasma membrane in PASMC, when TRP channels are opened, either by store depletion (for SOC) or diacylglycerol and PKC (for ROC), both Ca²⁺ and Na⁺ can, competitively, go through the channels and enter cell. In the case of that TRP channels and Na⁺/Ca²⁺ exchangers are both upregulated, such as in PASMC from IPAH patients, if opened TRP channels (formed in either SOC or ROC) predominantly allow Ca²⁺ to enter cell, it would directly contribute to increasing [Ca²⁺]_cyt. However, if opened, TRP channels predominantly allow Na⁺ to go through and enter cell, the increased Na⁺ influx or increased accumulation of Na⁺ in the area close to Na⁺/Ca²⁺ exchange proteins would indirectly raise [Ca²⁺]_cyt by activating inward transportation of Ca²⁺ through the reverse mode of Na⁺/Ca²⁺ exchange. The remaining question is whether and how extracellular Na⁺ competes with Ca²⁺ for opened TRP channels, and whether TRP channels have a "special" mode to recognize Na⁺ vs. Ca²⁺.

In cells transfected with TRPC channels, the Na⁺ currents through these channels seem to have higher amplitude (which is obviously due to the channels' higher permeability) than the Ca²⁺ currents. Removal of extracellular Na⁺, however, significantly enhances the amplitude of Ca²⁺ currents, indicating that Na⁺ and Ca²⁺ do compete with each other to get into the pore of TRPC channels (19). The ion permeability among different TRP channels seem to be dramatically different (19). These may also indicate that the functional heterotetrameric TRP channels existing in native cells may have very different permeability to Na⁺ and Ca²⁺. In IPAH, upregulation of certain TRPC isoforms may alter the composition of heterotetrameric TRP channels in PASMC, change the relative permeability of the functional channels to Na⁺ and Ca²⁺, and enhance Ca²⁺ entry via direct Ca²⁺ influx through the channels and via indirect Ca²⁺ entry through the reverse mode of Na⁺/Ca²⁺ exchange. Furthermore, it is unclear whether and how NCX1 and TRPC3/6 interact functionally in PASMC from IPAH patients, although they may both colocalize in caveolae by caveolins (3, 5, 19, 33, 47, 53).

In summary, circulating mitogenic factors in blood can penetrate into the smooth muscle layer in the pulmonary vasculature when endothelium is injured and/or endothelial barrier dysfunction takes place (34, 51). In addition, mitogenic factors synthesized and released from endothelial cell, smooth muscle cells and fibroblasts (as well as activated macrophages and platelets entrapped in extracellular matrix) can also activate receptors on the plasma membrane in PASMC via an autocrine or paracrine mechanism (15, 18, 25, 26, 58, 60). In PASMC from IPAH patients, contractile and mitogenic factors may accumulate in microdomains of the plasma membrane, such as caveolae, and consistently stimulate membrane receptors and their downstream signal transduction cascade. One of the downstream signaling pathways of GPCRs and receptor tyrosine kinase, when activated by mitogenic ligands, is to increase [Ca²⁺]_cyt. With upregulated NCX1 (this study) and TRPC channels (43, 59), the mitogen- or agonist-mediated Ca²⁺ entry can be markedly enhanced in PASMC from IPAH patients. Selective blockade of the reverse mode of Na⁺/Ca²⁺ exchange and specific downregulation of NCX1, in combination with selective inhibition of TRPC channels (e.g., TRPC3 and TRPC6), would be a potential therapeutic strategy for patients with severe pulmonary hypertension, such as patients with familial and idiopathic pulmonary arterial hypertension.

	GRANTS

TOP ABSTRACT METHODS AND MATERIALS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-064945, HL-054043, and HL-66012 and the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-33491, and by a Beginning Grant-in-Aid award from the American Heart Association (to H. Dong).

	ACKNOWLEDGMENTS

 
We thank Mr. O. Platoshyn for data analysis, Dr. C. V. Remillard for critical review of the manuscript, and Dr. J. Lytton (University of Calgary, Canada) for kindly providing the R3F1 antibody.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. X.-J. Yuan, Div. of Pulmonary and Critical Care Medicine, Dept. of Medicine, MC 0725, Univ. of California, San Diego, 9100 Gilman Dr., La Jolla, CA 92093-0725 (e-mail: xiyuan{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.

^* S. Zhang and H. Dong contributed equally to this work.

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