Am J Physiol Cell Physiol 292: C2297–C2305, 2007.
First published December 27, 2006; doi:10.1152/ajpcell.00383.2006.
Upregulation of Na⫹/Ca2⫹ exchanger contributes to the enhanced Ca2⫹ entry
in pulmonary artery smooth muscle cells from patients with idiopathic
pulmonary arterial hypertension
Shen Zhang,* Hui Dong,* Lewis J. Rubin, and Jason X.-J. Yuan
Department of Medicine, University of California, San Diego, La Jolla, California
Submitted 12 July 2006; accepted in final form 23 December 2006
transient receptor potential channel; reverse and forward mode; proliferation
and severe pulmonary
vascular remodeling contribute to the elevated pulmonary vascular resistance and pulmonary arterial pressure in patients
with pulmonary arterial hypertension (50). An increase in
cytosolic Ca2⫹ concentration ([Ca2⫹]cyt) in pulmonary artery
smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction (49) and an important stimulus for pulmonary vascular medial hypertrophy by stimulating PASMC
proliferation (7, 22). Removal of extracellular Ca2⫹ or
SUSTAINED PULMONARY VASOCONSTRICTION
* S. Zhang and H. Dong contributed equally to this work.
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:
[email protected]).
http://www.ajpcell.org
blockade of Ca2⫹ influx significantly inhibits agonist-mediated pulmonary vasoconstriction (35) and markedly attenuates mitogen-mediated PASMC proliferation (24, 52, 60).
These observations indicate that Ca2⫹ entry through Ca2⫹
channels and/or Ca2⫹ transporters in the plasma membrane is
one of the necessary prerequisites for the control of pulmonary
vascular contractility and PASMC proliferation and growth.
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 ⫻ ([Na⫹]cyt ⫼ [Na⫹]out)3 ⫻ e(E F/RT),
where Em 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 reticum
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C2297
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Zhang S, Dong H, Rubin LJ, Yuan JX. Upregulation of
Na⫹/Ca2⫹ exchanger contributes to the enhanced Ca2⫹ entry in
pulmonary artery smooth muscle cells from patients with idiopathic pulmonary arterial hypertension. Am J Physiol Cell Physiol
292: C2297–C2305, 2007. First published December 27, 2006;
doi:10.1152/ajpcell.00383.2006.—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 ␮M) 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.
C2298
NCX AND Ca2⫹ ENTRY IN IPAH
METHODS AND MATERIALS
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
AJP-Cell Physiol • VOL
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 ␮g/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 [1⫻
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 ␮g) was mixed and boiled
in 2⫻ sample buffer (0.25 M Tris 䡠 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
␮M; 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 ␮M) 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 ⫻40 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
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lum (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 receptoroperated Ca2⫹ channels (ROC) in the plasma membrane (2,
16). Furthermore, the IP3-mediated store depletion opens storeoperated 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.
NCX AND Ca2⫹ ENTRY IN IPAH
C2299
380 nm (F340/F380) using the ratio method based on the equation
[Ca2⫹]cyt ⫽ Kd ⫻ (Sf2 ⫼ Sb2) ⫻ (R ⫺ Rmin) ⫼ (Rmax ⫺ R), where
Kd (225 nM) is the dissociation constant for Ca2⫹, R was the
measured fluorescence ratio, Rmin and Rmax 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.
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).
Increase in [Ca2⫹]cyt due to Ca2⫹ entry through the reverse
mode of Na⫹/Ca2⫹ exchanger is enhanced in PASMC from
IPAH patients. Activity of Na⫹/Ca2⫹ exchanger depends predominantly on the transmembrane Na⫹ gradient. Removal of
extracellular Na⫹ reverses the transmembrane Na⫹ gradient to
favor Na⫹ extrusion and Ca2⫹ entry via Na⫹/Ca2⫹ exchange
(9, 31). As shown in Fig. 2A, left, removal of extracellular Na⫹
(Na⫹ was replaced by equimolar NMDG⫹) induced a rapid
increase in [Ca2⫹]cyt, indicating that the reverse mode of
Na⫹/Ca2⫹ exchanger is active in normal PASMC. Compared
with normal PASMC, the increase in [Ca2⫹]cyt due to Ca2⫹
entry via the reverse mode of Na⫹/Ca2⫹ exchange was significantly enhanced (by ⬎50%) in PASMC from IPAH patients
(Fig. 2A, middle, and 2B). The amplitude of [Ca2⫹]cyt increase
mediated by removal of extracellular Na⫹ was 578.8 ⫾ 30.2
nM in normal PASMC (n ⫽ 39 cells) vs. 906.7 ⫾ 60.7 nM in
IPAH PASMC (n ⫽ 34 cells; P ⬍ 0.001). Extracellular
application of 10 ␮M KB-R7943, a selective inhibitor of the
reverse mode of Na⫹/Ca2⫹ exchange (27), markedly attenuated the increase in [Ca2⫹]cyt via the reverse model of Na⫹/
Ca2⫹ exchange induced by removal of extracellular Na⫹ (Fig.
2A, right).
Nonetheless, the increase in [Ca2⫹]cyt due to extracellular
application of 10 ␮M ionomycin, a Ca2⫹ ionophore, in 140
mM Na⫹-containing solution (140 Na) was comparable in
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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.
normal and IPAH PASMC, although ionomycin-mediated rise
in [Ca2⫹]cyt was enhanced in normal PASMC superfused with
Na⫹-free (0 Na) solution (Fig. 3, A–C). These results strongly
suggest that the increase in [Ca2⫹]cyt due to Ca2⫹ entry through
the reverse mode of Na⫹/Ca2⫹ exchange is selectively enhanced in PASMC from IPAH patients compared with
PASMC from normal subjects. The enhanced Ca2⫹ entry was
apparently related to the upregulation of NCX1 in IPAH
PASMC.
As mentioned earlier, the Na⫹/Ca2⫹ exchanger has a dual
function in terms of transporting Ca2⫹: it either pumps Ca2⫹
out of cell (the forward mode) or into cell (the reversed mode)
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RESULTS
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NCX AND Ca2⫹ ENTRY IN IPAH
depending mainly on the transmembrane Na⫹ gradient. When
[Na⫹]cyt is low and extracellular [Na⫹] is high, the Na⫹/Ca2⫹
exchanger is predominantly in the forward mode. To examine
whether upregulated NCX1 also enhanced Ca2⫹ extrusion by
the forward mode when [Ca2⫹]cyt was increased, we compared
the time constants (or the decay kinetics) of ionomycin-mediated Ca2⫹ transients (Fig. 3D). Interestingly, there was no
significant difference of the decay kinetics or time constants
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 ␮M 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 IMmediated 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.
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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 ␮M 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.
between normal and IPAH PASMC (Fig. 3D). These results
indicate that the upregulated NCX1 predominantly contributes
to increasing the function of NCX in the reversed mode. It is
unclear, however, why upregulated NCX1 in IPAH PASMC
negligibly affected the function of NCX in the forward mode.
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 ␮M), 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.
TRP channels, which are permeable to both Na⫹ and Ca2⫹
(PNa ⬎ PCa for most TRP isoforms) (8, 13, 24), are believed to
participate in the formation of functional SOC in systemic and
pulmonary vascular smooth muscle cells (30, 32, 35, 52, 56,
60, 62) and endothelial cells (1, 12, 21, 54). Therefore, store
depletion-mediated activation of TRP-encoded SOC would
also mediate Na⫹ influx (in addition to Ca2⫹ influx), increase
[Na⫹]cyt, reduce transmembrane Na⫹ gradient, and ultimately
activates the reverse mode of Na⫹/Ca2⫹ exchange causing
inward transportation of Ca2⫹ through the exchange. Moreover, the store depletion-mediated increase in [Ca2⫹]cyt should
be attenuated in cells in which Na⫹/Ca2⫹ exchange is inhibited
by downregulating NCX expression and/or attenuating NCX
function.
Passive depletion of intracellular Ca2⫹ stores with CPA
caused an increase in [Ca2⫹]cyt due to Ca2⫹ influx through
NCX AND Ca2⫹ ENTRY IN IPAH
C2301
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
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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 ␮M) 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.
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 depletionmediated 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 depletionmediated 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
AJP-Cell Physiol • VOL
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 ␮M) 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 CPAmediated 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.
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C2302
NCX AND Ca2⫹ ENTRY IN IPAH
DISCUSSION
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
AJP-Cell Physiol • VOL
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⫹ releaseactivated 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).
With the use of human embryonic kidney-293 cells transfected with TRP channels (e.g., TRPC3), Groschner and his
associates provide compelling evidence that store depletionmediated Ca2⫹ entry involves both TRP channels and Na⫹/
Ca2⫹ exchangers, and that TRPC3 and Na⫹/Ca2⫹ exchangers
are functionally coupled and physically colocalized with each
other in caveolae via caveolin (19, 47). These results are in
good agreement with our observations that downregulation of
NCX1 with siRNA attenuated store depletion-mediated Ca2⫹
entry. In other words, the store-operated TRP channels and
Na⫹/Ca2⫹ exchangers (e.g., NCX1) appear to be functionally
coupled in human PASMC.
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 IPAHPASMC are much greater than those in normal PASMC and
PASMC from patients with secondary pulmonary hypertension
(42). Treatment of IPAH-PASMC with methyl-␤-cyclodextrin
(M␤CD), a compound that depletes membrane cholesterol and
disrupts caveolae, and with siRNA for caveolin-1 resulted in
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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).
Store-operated Ca2⫹ entry independent of Na⫹/Ca2⫹ exchange is enhanced in PASMC from IPAH patients. When
PASMC are superfused with Na⫹-free solution, the reverse
mode of Na⫹/Ca2⫹ exchange is fully activated (as shown in
Fig. 2) because of the increased “reverse” driving force (or the
increased ratio of cytosolic and extracellular [Na⫹]), where the
forward mode of Na⫹/Ca2⫹ exchange is inactivated because of
the reduced “forward” driving force (or the reduced ratio of
extracellular and cytosolic [Na⫹]). Under these conditions, the
inward transportation of Ca2⫹ via the reverse mode of Na⫹/
Ca2⫹ exchange would be accumulated in the cytosol and
sequestered into the SR/ER by SERCA (4, 31), and subsequently increase [Ca2⫹]SR/ER. Indeed, as shown in Fig. 5C, the
amplitude of [Ca2⫹]cyt rise due to CPA-mediated Ca2⫹ release
in IPAH PASMC bathed in Na⫹-free solution (636.0 ⫾ 7.5
nM, n ⫽ 66 cells) was ⬃3.3⫻ the amplitude in IPAH PASMC
bathed in 140 mM Na⫹-containing solution (192.5 ⫾ 12.6 nM,
n ⫽ 37; P ⬍ 0.001). These results not only present additional
indirect evidence that the activity of the reverse mode of
Na⫹/Ca2⫹ exchange is augmented in IPAH PASMC, but also
suggest that NCX1 may be functionally coupled and physically
close to TRPC channels in the plasma membrane and SERCA
in the SR/ER membrane (5, 9, 23, 31, 36).
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.
NCX AND Ca2⫹ ENTRY IN IPAH
C2303
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).
AJP-Cell Physiol • VOL
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
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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.
C2304
NCX AND Ca2⫹ ENTRY IN IPAH
patients with severe pulmonary hypertension, such as patients
with familial and idiopathic pulmonary arterial hypertension.
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.
GRANTS
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).
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