Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of California, San Diego, California 92103
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Pulmonary vascular medial hypertrophy due to proliferation of pulmonary artery smooth muscle cells (PASMC) greatly contributes to the increased pulmonary vascular resistance in pulmonary hypertension patients. A rise in cytosolic free Ca2+ concentration ([Ca2+]cyt) is an important stimulus for cell growth in PASMC. Resting [Ca2+]cyt, intracellularly stored [Ca2+], capacitative Ca2+ entry (CCE), and store-operated Ca2+ currents (ISOC) are greater in proliferating human PASMC than in growth-arrested cells. Expression of TRP1, a transient receptor potential gene proposed to encode the channels responsible for CCE and ISOC, was also upregulated in proliferating PASMC. Our aim was to determine if inhibition of endogenous TRP1 gene expression affects ISOC and CCE and regulates cell proliferation in human PASMC. Cells were treated with an antisense oligonucleotide (AS, for 24 h) specifically designed to cleave TRP1 mRNA and then returned to normal growth medium for 40 h before the experiments. Then, mRNA and protein expression of TRP1 was downregulated, and amplitudes of ISOC and CCE elicited by passive depletion of Ca2+ from the sarcoplasmic reticulum using cyclopiazonic acid were significantly reduced in the AS-treated PASMC compared with control. Furthermore, the rate of cell growth was decreased by 50% in AS-treated PASMC. These results indicate that TRP1 may encode a store-operated Ca2+ channel that plays a critical role in PASMC proliferation by regulating CCE and intracellular [Ca2+]cyt.
store-operated calcium channels; transient receptor potential channel
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PULMONARY VASOCONSTRICTION and vascular smooth muscle proliferation greatly contribute to the elevated pulmonary vascular resistance and arterial pressure in patients with pulmonary hypertension (45, 52). Vasoconstriction and cellular proliferation may share a common pathway, involving signaling processes that result in parallel intracellular events in pulmonary vascular remodeling and in the development of pulmonary hypertension (58). Intracellular Ca2+ is a critical signal transduction element in regulating muscle contraction (51), cell proliferation (3, 15, 33), and gene expression (19). Cytoplasmic ionized Ca2+ diffuses rapidly between cytosol and nucleus (1); therefore, a rise in cytosolic free Ca2+ concentration ([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, 6, 19, 51).
In pulmonary artery smooth muscle cells (PASMC), [Ca2+]cyt is increased mainly by Ca2+ influx through Ca2+-permeable channels in the plasma membrane and Ca2+ release from intracellular Ca2+ stores, such as the sarcoplasmic reticulum (SR). Mitogen- or agonist-induced increases in [Ca2+]cyt usually consist of an initial release of Ca2+ from the SR followed by a sustained Ca2+ influx through sarcolemmal Ca2+-permeable channels (6, 12, 15, 31, 41, 48). Removal or chelation of extracellular Ca2+ abolishes pulmonary vasoconstriction and significantly inhibits PASMC growth (16, 35), suggesting that a constant influx of Ca2+ from extracellular fluid to the cytosol is required for vasoconstriction and PASMC proliferation. There are at least three classes of Ca2+-permeable channels functionally expressed in the plasma membrane in vascular smooth muscle cells: 1) voltage-dependent Ca2+ channels (VDCC), which are regulated by membrane potential, 2) receptor-operated Ca2+ channels, which are regulated by binding ligand with respective receptors, and 3) store-operated Ca2+ channels (SOC), which are regulated by capacity of Ca2+ in the SR (5, 37, 40, 43, 54). Depletion of Ca2+ from the SR activates SOC and triggers capacitative Ca2+ entry (CCE), a mechanism involved in maintaining sustained Ca2+ influx and refilling Ca2+ into the SR (5, 6, 40, 43). In other words, CCE is an important mechanism that links intracellularly stored [Ca2+]cyt in the SR to membrane Ca2+ permeability. The amplitude of CCE is mainly dependent on 1) the level of [Ca2+] in the SR, 2) activity of the sarcoplasmic Ca2+ channels that are activated by store depletion (SOC), 3) total number of functional SOC in the plasma membrane, and 4) signal transduction from emptied SR to the membrane SOC.
The molecular basis of SOC responsible for CCE is still not completely understood. It has been demonstrated that SOC may be composed of subunits encoded in the transient receptor potential (TRP) channel genes. Indeed, expression of TRP genes in mammalian cells and Xenopus oocytes results in the formation of Ca2+-permeable cation channels, which are activated by Ca2+ store depletion, whereas the blockade of TRP channel gene expression attenuates the cation currents through the store depletion-activated Ca2+ channels and the increase in [Ca2+]cyt due to CCE (5, 7, 38, 56, 57, 61, 64-66). TRP1, a member of the short TRP subfamily (10, 20), is highly expressed in the plasma membrane of vascular smooth muscle cells (16, 59, 61). Overexpression of TRP1 in mammalian cell lines and Xenopus oocytes enhances CCE induced by store depletion. This suggests that the TRP1 gene encodes subunits involved in forming native SOC, which is believed to be composed of four heterogenous or homologous TRP subunits (10, 23, 32, 53, 60, 64-66). In human PASMC, we previously demonstrated that the mRNA and protein expression of TRP1 and the amplitudes of SOC currents (ISOC) and CCE were much greater in proliferating cells than growth-arrested cells (16). These results suggest that TRP1 gene product participates in forming SOC that are involved in elevating [Ca2+]cyt through CCE when cells proliferate. In this study, using an antisense (AS) oligonucleotide that specifically cleaves mRNA of the human TRP1 gene, we examined whether inhibition of the endogenously expressed TRP1 affected activity of SOC, amplitude of CCE, and PASMC proliferation in the presence of serum and growth factors.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture and AS oligonucleotide. Human PASMC from normal subjects were purchased from Clonetics (Clonetics, BioWhittaker) and used at the 4th-6th passage. The cells were plated onto coverslips (for electrophysiological and fluorescence microscopy experiments) or petri dishes (for molecular biological experiments) and were cultured at 37°C in smooth muscle growth medium (SMGM), which is composed of smooth muscle basal medium (SMBM), 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 (Clonetics) when 70-90% confluence was achieved. The cells isolated for study were all synchronized or growth arrested by incubating them in SMBM for 48 h before biochemical characterization of TRP1 expression and functional assessment of SOC activity.
Second Generation AS oligonucleotides that contain nine phosphorothioate DNA linkages to activate RNase H were purchased from Sequitur (Natick, MA). The AS oligonucleotides were designed to cleave mRNA of the human TRP1 gene (GenBank accession number U31110) by activating endogenous RNase H and have a unique combination of specificity, efficacy, and reduced toxicity. The AS oligonucleotides were screened against the GenBank database, and no matches were found to other nontargeted genes. The negatively charged oligonucleotide S12174 (TRP1 Ultramer) from Sequitur was transfected into cells with Lipid 2012-G according to the manufacturer's protocol (Sequitur). The transfection efficiency, determined by measuring uptake of a fluorescent control oligomer in a separate plate, was ~95% using 40 nM of the oligomer and 2.5 g/ml of Lipid 2012-G (for 12 h) in human PASMC (65-70% confluence). An oligonucleotide with the same base composition as S12174, but with scrambled sequence, was used as a control for nonspecific or toxic effects of the oligomers. The sequence of the AS oligonucleotide for human TRP1 was compared with known sequences in GenBank using the National Center for Biotechnology Information web-blasting program to ensure that no homologies to any other human TRP genes were found. For each treatment, the cells were first rinsed with Opti-MEM (GIBCO-BRL), and then oligos in SMGM were added to the cells. After 24 h of incubation with the oligomers, the medium was aspirated and replaced with SMGM without oligomers for 48-72 h before the experiments were performed. The final concentration of the oligos was 40 nM.Cell cycle analysis. The human PASMC cell cycle distribution was analyzed by flow cytometry. Briefly, cells were first cultured in SMBM or SMGM (Clonetics) for 24-42 h. Then, the cells were trypsinized, washed one time with PBS, and fixed with 70% ethanol for at least 1 h at 4°C. The fixed cells were washed with PBS and incubated with a solution containing 0.05 mg/ml propidium iodine, 0.1% sodium citrate, and 50 µg/ml RNase A for 30 min at 4°C in the dark. The stained cells were analyzed by FACSCalibur using CellQuest software (Becton Dickinson, Mountain View, CA).
Electrophysiological measurements.
Whole cell ISOC was recorded with an Axopatch-1D
amplifier using patch-clamp techniques (16, 35). Patch
pipettes (2-4 M) were made on a Sutter electrode puller using
borosilicate glass tubes and fire-polished on a Narishige microforge.
Voltage stimuli were delivered from a holding potential of 0 mV using voltage steps from
100 to 0 or +100 mV. Current traces recorded before the activation of SOC were used as a template to subtract leak
currents. SOC was activated by passive depletion of the SR Ca2+ using 10 µM cyclopiazonic acid (CPA). The bath
(extracellular) solution for recording optimal
ISOC contained (mM) 120 Na methane sulfonate, 20 Ca aspartate, 0.5 3,4-diaminopyridine, 10 glucose, and 10 HEPES (pH 7.4 with methane sulfonic acid). The (intracellular) pipette solution
contained (mM) 138 Cs aspartate, 1.15 EGTA, 1 Ca(OH)2, 2 Na2-ATP, and 10 HEPES (pH 7.2). These ionic conditions eliminated the currents through K+ and Cl
channels. In Ca2+-free bath solution, Ca aspartate was
replaced by equimolar Na aspartate to maintain osmolarity. In
Na+-free bath solution, Na methane sulfonate was replaced
by equimolar N-methyl-D-glucamine
(NMDG+).
Measurement of cytosolic [Ca2+].
[Ca2+]cyt in single human PASMC was measured
using the Ca2+-sensitive fluorescent indicator fura 2 (16, 17). Cells on 25-mm coverslips were loaded with the
acetoxymethyl ester form of fura 2, 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-loaded cells were then
transferred to a recording cell 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 (mM) 141 NaCl, 4.7 KCl,
1.8 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose,
buffered to pH 7.4 with 5 M NaOH. 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 32°C using the Nikon UV-Fluor objectives and a
charge-coupled device camera. The fluorescence signals emitted from the
cells were monitored and recorded continuously on an Intracellular
Imaging fluorescence microscopy system and recorded in an
IBM-compatible computer for later analysis. The 340- to 380-nm ratios
(R) of the fluorescence images were then calculated and
subsequently calibrated. On the basis of these R,
[Ca2+]cyt was calculated by the
equation
![]() |
RT-PCR.
Total RNA (3 µg) was first reverse-transcribed using random
hexamers [pd(N)6 primer]. The sense
[5'-CAAGATTTTGGAAAATTTCTTG-3'; nucleotide (nt) 2338-2359] and AS
(5'-TTTGTCTTCATGATTTGCTAT-3'; nt 2689-2709) primers were
designed from the coding region of the human TRP1 gene (U31110). The
sense and AS primers for the human glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) gene (M33197) are
5'-CTTTGGTATCGTGGAAGGACTC-3' (nt 561-582) and 5'-TCTTCCTCTTGTGCTCTTGCTG-3' (nt 1,089-1,110). The sense and AS primers for the human -actin gene (M10277) are
5'-GACGGGGTCACCCACACTGTGCCCATCTA-3' (nt 2134-2162) and
5'-CTAGAAGCATTTGCGGTGGACGATGGAGG-3' (nt 2971-3000). PCR was
performed by a GeneAmp PCR System using AmpliTaq DNA polymerase and
accompanying buffers. Three microliters of the first-strand cDNA
reaction mixture were used in a 50-µl PCR reaction consisting of 0.2 nmol/l of each primer, 10 mM Tris · HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2, 200 µM each of 2-deoxynucleotide
5'-triphosphates, and 2 units of Taq DNA polymerase.
The cDNA samples were amplified in a DNA thermal cycler under the
following conditions: the mixture was annealed at 52-61°C,
extended at 72°C, and denatured at 94°C for 20-30 cycles. This
was followed by a final extension at 72°C to ensure complete product
extension. The PCR products were electrophoresed through a 2% agarose
gel, and amplified cDNA bands were visualized by ethidium bromide
staining. To quantify the PCR products of TRP1, we used an invariant
mRNA of smooth muscle
-actin as an internal control. Immediately
after each experiment, the optical density (OD) value for each band on
the gel was measured by a gel documentation system. The OD
values in the TRP1 signals were normalized to the OD values in the
-actin or GAPDH signals. The normalized values are expressed as
arbitrary units for quantitative comparison.
Immunoblot analysis. Cells were washed with PBS, scraped into PBS (2 ml/dish), and centrifuged at 3,500 rpm. The cell pellets were homogenized in 10 mM HEPES-KOH (pH 7.0) containing the protease inhibitor cocktail (Complete tablets) for 10 s at 7,000 rpm. Nuclei and plasma membrane were removed by centrifugation in a microfuge at 4°C for 10 min (10,000 rpm). Protein concentrations were determined by the Coomassie BA protein assay, using bovine serum albumin as a standard. Proteins solubilized in SDS-sample buffer were separated by SDS-PAGE on 10% gels, which were calibrated with prestained protein molecular weight markers. The separated proteins were then transferred to the Hybond-C extra nitrocellulose membrane. The efficiency of the transfer was verified by Ponceau S staining. Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline and 0.1% Tween 20 for 40 min at room temperature. The blots were then incubated with the affinity-purified polyclonal antibodies specific for TRP1 (Alomone Labs, Jerusalem, Israel) for ~1 h at room temperature. The membranes were washed (3× 5 min) and incubated with anti-rabbit horseradish peroxidase-conjugated IgG for 1 h, and an enhanced chemiluminescence detection system was used for detection of the bound antibody. Finally, the filters were placed in a plastic sheet protector and exposed to Kodak X-Omat film for 10-60 s.
Statistical analysis. Data are expressed as means ± SE. Statistical analysis was performed using the unpaired Student's t-test or ANOVA and post hoc tests (Student Newman-Keuls) as indicated. Differences are considered to be significant when P < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Enhancement of TRP1 expression during PASMC proliferation. Expression of TRP genes in mammalian cells enhances CCE, suggesting that TRP-encoded proteins may form the putative SOC responsible for CCE (5, 7, 38, 56, 57, 61, 64-66). TRP1 is a member of the short TRP channel subfamily, which is ubiquitously expressed in a variety of tissue types, including lung, heart, and brain (10). It has been demonstrated that TRP1 encodes subunits involved in forming native SOC, which are activated by store depletion in many cell types, including vascular smooth muscle cells (8, 10, 23, 32, 53, 60, 64-66).
In human PASMC cultured in SMGM, which contains serum and growth factors, 36.3% of the cells were in S and G2/M phases, whereas in PASMC cultured in SMBM without serum or growth factors, 94% of the cells were in G0/G1 phase (Fig. 1A). This suggests that the cells cultured in SMGM are proliferating cells, whereas the cells cultured in SMBM are mostly growth-arrested cells. To examine the possible association of TRP1 expression and SOC activity with PASMC proliferation, we measured and compared 1) mRNA and protein expression of TRP1 and 2) whole cell cation currents through store depletion-activated channels in proliferating and growth-arrested PASMC.
|
Enhancement of SOC activity and CCE amplitude during PASMC proliferation. Activity of SOC, or the whole cell cation current through SOC (ISOC), in PASMC is partially determined by the total number of functional SOC proteins expressed in the plasma membrane. If TRP1 encodes the protein subunits involved in forming native SOC, the upregulated expression of TRP1 would increase whole cell ISOC in PASMC during proliferation. To determine whether the upregulated TRP1 expression associates with an increase in SOC activity, we measured and compared whole cell ISOC in proliferating and growth-arrested PASMC.
Whole cell ISOC was elicited in PASMC held at 0 mV to inactivate voltage-dependent Na+ and Ca2+ channels by a series of test potentials ranging from
|
Inhibition of endogenous TRP1 expression reduced ISOC and CCE. It has been demonstrated that SOC is composed of subunits encoded in TRP genes (5, 7, 38, 56, 57, 61, 64-66). Therefore, gene transcription and expression of TRPs should be involved in long-term electrophysiological change of SOC. Similar to molecular topology of voltage-gated K+ channels, TRP1 has six transmembrane domains (S1-S6) with both carboxy and amino termini located intracellularly and a pore-forming loop between the S5 and S6 domains. The native TRP channels are believed to be formed heteromerically by different TRP subunits (5, 10). To test whether TRP1 is involved in forming functional native SOC, we measured and compared whole cell ISOC and CCE in control PASMC and cells in which TRP1 gene expression was inhibited.
To selectively inhibit the gene expression of TRP1, we used the AS oligonucleotide (S12174, TRP1 Ultramer) specifically targeted on the human TRP1 gene (U31110). Treatment of human PASMC with the AS oligonucleotide (40 nM, for 24 h) caused a 67% decrease in mRNA level (0.896 ± 0.046 vs. 0.299 ± 0.015 arbitrary units, n = 8, P < 0.001) and a 40% decrease in protein level (1.097 ± 0.029 vs. 0.667 ± 0.081 arbitrary units, n = 6, P < 0.01) of TRP1 channels but had little effect on mRNA and protein expression of GAPDH and
|
|
|
|
|
Pharmacological blockade of SOC with
Ni2+ decreased CCE and inhibited PASMC
growth.
Ni2+ is a potent cationic blocker of SOC (16,
67). In proliferating human PASMC cultured in SMGM,
extracellular application of Ni2+ decreased
ISOC in a dose-dependent manner; the
EC50 is ~0.38 mM at 80 mV (Fig.
8A).
Moreover, extracellular application of Ni2+ reversibly
attenuated the increase in [Ca2+]cyt due to
CCE (Fig. 8B), suggesting that Ni2+ is a potent
blocker of native SOC that are responsible for CCE in these cells.
Treatment of PASMC with SMGM containing 0.5 mM Ni2+ for
12-72 h significantly reduced the resting
[Ca2+]cyt (Fig. 8C) and inhibited
cell proliferation in the presence of serum and growth factors (Fig.
8D).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
An important signal transduction pathway upon activation of receptors by mitogenic agonists is the increase in [Ca2+]cyt due to Ca2+ release from the SR and Ca2+ influx via Ca2+ channels in the plasma membrane (5, 15, 31, 41, 48, 51, 54). Ionized or free Ca2+ diffuses quickly between the cytosol and nucleus (1); therefore, a rise in [Ca2+]cyt would rapidly increase nuclear [Ca2+]. Modulation of the amplitude or frequency of Ca2+ signals (or spatially distinguishable Ca2+ signals) and sustained increases in [Ca2+]cyt are all involved in regulating gene expression and cell growth (11-15, 18, 25, 49). Ca2+ is believed to be necessary for transitions from G0 to G1 phase, G1 to S phase, and G2 to M phase, and for going through mitosis in the cell cycle (3, 33). Maintenance of sufficient Ca2+ within the SR, an intracellular organelle important in the synthesis of many membrane lipids and proteins, is also required for cell growth; depletion of the SR Ca2+ store induces growth arrest (16, 25, 49) and triggers apoptosis (21). When ionized Ca2+ is depleted from the SR by inositol 1,4,5-trisphosphate (IP3) during activation of mitogenic receptors, the emptied SR needs to be refilled to further (or ensure) the mitogen-mediated cell proliferation and prevent cell apoptosis.
CCE generated by Ca2+ influx through SOC is a critical mechanism involved in maintaining sustained increase in [Ca2+]cyt and in refilling Ca2+ into the SR (5, 6, 40, 43). In human PASMC, amplitudes of CCE and ISOC were both increased when cells proliferated, suggesting that an increase in expression and/or function of SOC is involved in cell growth. Because of the diversity and variability of the biophysical and pharmacological properties of whole cell and single-channel ISOC (16, 24, 27, 30, 34), SOC are believed to be complex and heterogeneous in molecular composition and in cellular regulation (5, 10).
What the native SOC are molecularly made of has been a question for many investigators. Electrophysiological studies demonstrate that expression of TRP genes in mammalian cells results in the formation of Ca2+-permeable cation channels that are activated by store depletion. This suggests that SOC may be composed of subunits encoded in TRP genes (5, 7, 38, 56, 57, 64-66). The TRP gene family can be divided into three subfamilies based on sequence homology: short TRP, long TRP, and osm-9-like TRP (10, 20). TRP1 is a member in short TRP subfamily (termed as TRPC1), which contains an amino terminal ankyrin domain and a proline-rich cytosolic segment in the proximal carboxy terminus. Some of the six transmembrane domains (S1-S6) of TRP channels are homologous to transmembrane segments of voltage-dependent channels; however, the putative S4 domain of TRP channels lacks the positive amino acids involved in the voltage-sensing function (5, 10). SOC are thought to be heterotetramers or homotetramers made up of different TRP subunits (5, 10, 20, 32, 53).
In vascular smooth muscle cells isolated from human mammary arteries and aortas, Xu and Beech (61) provided compelling evidence that TRP1 gene encodes native subunits of SOC, which are responsible for CCE. In pulmonary vascular endothelial cells, Stevens and his associates (8, 36, 39) demonstrated that endogenously expressed TRP1 channel subunits contribute to form a Ca2+-selective, store-operated Ca2+ entry pathway that plays a central role in regulating endothelial permeability and in response to inflammation. These results also indicate that expression of TRP1 gene and function of the SOC-containing TRP1 subunit play critical roles in regulating [Ca2+]cyt and [Ca2+]SR in many cell types.
TRP1 is highly expressed in PASMC isolated from humans (16) and animals (59). We have demonstrated that gene expression of TRP1 and SOC activity are related to PASMC proliferation (16). The mRNA and protein levels of TRP1 and amplitudes of ISOC were both significantly enhanced in proliferating human PASMC cultured in growth medium containing serum and growth factors, compared with growth-arrested cells cultured in basal medium without serum and growth factors (see Figs. 1 and 2A). Moreover, the rise in [Ca2+]cyt due to CCE, elicited by passive depletion of Ca2+ from the SR with CPA (Fig. 2B) and thapsigargin (data not shown), was greatly augmented in proliferating PASMC, in which more than one-third of the cells undergo DNA synthesis and mitosis. These results suggest that, when PASMC are activated by mitogens, the gene expression of TRP1 is triggered to produce more TRP1 channels for the need of Ca2+ in cytosol, nucleus, and the SR.
The molecular identity of native SOC in human PASMC is poorly understood, although multiple TRP gene transcripts have been described in lung tissues or PASMC isolated from humans and animals (10, 16, 59, 61). On the basis of the AS oligomer specifically targeted on the human TRP1 gene (Figs. 3-7), the observations from this study provide crucial evidence for the important role of endogenously expressed TRP1 in forming native SOC in human PASMC. Inhibition of endogenous TRP1 gene expression not only reduced activity of SOC but also decreased amplitude of CCE mediated by store depletion. The reduced ISOC and CCE also led to a reduced [Ca2+]SR. The CPA-induced initial increase in [Ca2+]cyt in the absence of extracellular Ca2+ is mainly due to leakage of Ca2+ from the SR to the cytosol. Its amplitude is thus positively proportional to the level of [Ca2+]SR. In cells treated with the AS oligomer specifically targeted on the human TRP1 gene, the CPA-induced Ca2+ mobilization from the SR was markedly reduced compared with control cells. Because refilling Ca2+ into the SR is an important function of CCE, a reduced number of functional TRP1 channels may likely be involved in the decreased [Ca2+]SR. These results suggest that, in human PASMC, 1) TRP1-encoded proteins are subunits of native SOC, 2) upregulated TRP1 expression plays an important role in cell growth, and 3) development of drugs specifically targeted on the gene expression of TRP1 may be a potential therapeutic approach for inhibiting the progression of pulmonary vascular remodeling in patients with primary and secondary pulmonary hypertension.
Removal of extracellular Ca2+ almost abolished PASMC growth in the presence of serum and growth factors (16, 49), whereas pharmacological blockade of SOC and CCE using Ni2+ only partially inhibited cell growth (by 50%, Fig. 8D). This suggests that Ca2+ influx through CCE or SOC is not the only pathway for raising [Ca2+]cyt (and/or [Ca2+]SR) during cell proliferation. In addition to SOC, PASMC also express voltage-dependent (VDCC) and receptor-operated Ca2+ channels (37, 44). By governing Ca2+ influx via VDCC, membrane potential plays a critical role in regulating [Ca2+]cyt (37). The additive effect of nifedipine, a dihydropyridine blocker of VDCC, on Ni2+-induced inhibition of human PASMC growth (Fig. 8D) indicates that multiple Ca2+ influx pathways (e.g., via SOC and VDCC) are involved in raising [Ca2+]cyt when cells proliferate (29, 47, 62).
Pulmonary vascular remodeling due to smooth muscle proliferation and hypertrophy greatly contributes to the elevated pulmonary vascular resistance observed in patients with pulmonary hypertension. The high levels of vasoactive substances (e.g., endothelin-1, serotonin) and growth factors in plasma and lung tissues have been implicated in pulmonary hypertension (9, 22, 28, 55). The agonist- and mitogen-mediated cell growth depends on extracellular Ca2+ and on the level of intracellularly stored [Ca2+]. CCE, potentially and partially through TRP1-encoded subunits contained in heterotetrameric SOC, is an important mechanism involved in maintaining the agonist-induced increase in [Ca2+]cyt and in refilling Ca2+ into the SR emptied by IP3 in human PASMC. Thus expression of the TRP1 gene and function of TRP1-encoded SOC may also play critical roles in regulating the progression and regression of pulmonary vascular remodeling in patients with pulmonary hypertension via modulation of [Ca2+]cyt and [Ca2+]SR in hypertrophied PASMC.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank B. R. Lapp for technical assistance.
![]() |
FOOTNOTES |
---|
* Michele Sweeney, Ying Yu, and Oleksandr Platoshyn contributed equally to this work.
This work was supported by National Heart, Lung, and Blood Institute Grants HL-66012, HL-54043, and HL-64945 to J. X.-J. Yuan. J. X.-J. Yuan is an Established Investigator of the American Heart Association.
Address for reprint requests and other correspondence: J. X.-J. Yuan, Div. of Pulmonary & Critical Care Medicine, UCSD Medical Center, MC 8382, 200 W. Arbor Dr., San Diego, CA 92103-8382 (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.
First published February 8, 2002;10.1152/ajplung.00412.2001
Received 23 October 2001; accepted in final form 6 February 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abritton, NL,
Oancea E,
Kuhn MA,
and
Meyer T.
Source of nuclear calcium signals.
Proc Natl Acad Sci USA
91:
12458-12462,
1995
2.
Arnon, A,
Hamlyn JM,
and
Blaustein MP.
Na+ entry via store-operated channels modulates Ca2+ signaling in arterial myocytes.
Am J Physiol Cell Physiol
278:
C163-C173,
2000
3.
Berridge, MJ.
Calcium signalling and cell proliferation.
Bioessays
17:
491-500,
1995[ISI][Medline].
4.
Berridge, MJ.
Inositol trisphosphate and calcium signalling.
Nature
361:
315-325,
1993[ISI][Medline].
5.
Birnbaumer, L,
Zhu X,
Jiang M,
Boulay G,
Peyton M,
Vannier B,
Brown D,
Platano D,
Sadeghi H,
Stefani E,
and
Birnbaumer M.
On the molecular basis and regulation of cellular capacitative calcium entry: roles for Trp proteins.
Proc Natl Acad Sci USA
93:
15195-15202,
1996
6.
Bootman, MD,
Lipp P,
and
Berridge MJ.
The organisation and functions of local Ca2+ signals.
J Cell Sci
114:
2213-2222,
2001
7.
Boulay, G,
Brown DM,
Qing N,
Jiang M,
Dietrich A,
Zhu MX,
Chen Z,
Birnbaumer M,
Mikoshiba K,
and
Birnbaumer L.
Modulation of Ca2+ entry by polypeptides of the inositol 1,4,5-trisphosphate receptor (IP3R) that bind transient receptor potential (TRP): evidence for roles of TRP and IP3R in store depletion-activated Ca2+ entry.
Proc Natl Acad Sci USA
96:
14955-14960,
1999
8.
Brough, GH,
Wu S,
Cioffi D,
Moore TM,
Li M,
Dean N,
and
Stevens T.
Contribution of endogenously expressed Trp1 to a Ca2+-selective, store-operated Ca2+ entry pathway.
FASEB J
15:
1727-1738,
2001
9.
Cacoub, P,
Dorent R,
Maistre G,
Nataf P,
Carayon A,
Piette C,
Godeau P,
Cabrol C,
and
Gandjbakhch I.
Endothelin-1 in primary pulmonary hypertension and the Eisenmenger syndrome.
Am J Cardiol
71:
448-450,
1993[ISI][Medline].
10.
Clapham, DE,
Runnels LW,
and
Strübing C.
The TRP ion channel family.
Nat Rev Neurosci
2:
387-396,
2001[ISI][Medline].
11.
Cruzalegui, FH,
Hardingham GE,
and
Banding H.
c-Jun functions as a calcium-regulated transcriptional activator in the absence of JNK/SAPK1 activation.
EMBO J
18:
1335-1344,
1999
12.
Doi, S,
Damron DS,
Horibe M,
and
Murray PA.
Capacitative Ca2+ entry and tyrosine kinase activation in canine pulmonary arterial smooth muscle cells.
Am J Physiol Lung Cell Mol Physiol
278:
L118-L130,
2000
13.
Dolmetsch, RE,
Lewis RS,
Goodnow CC,
and
Healy MI.
Differential activation of transcription factors induced by Ca2+ response amplitude and duration.
Nature
386:
855-858,
1997[ISI][Medline].
14.
Dolmetsch, RE,
Xu K,
and
Lewis RS.
Calcium oscillations increase the efficiency and specificity of gene expression.
Nature
392:
933-936,
1998[ISI][Medline].
15.
Frey, N,
McKinsey TA,
and
Olson EN.
Decoding calcium signals involved in cardiac growth and function.
Nat Med
6:
1221-1227,
2000[ISI][Medline].
16.
Golovina, VA,
Platoshyn O,
Bailey CL,
Wang J,
Limsuwan A,
Sweeney M,
Rubin LJ,
and
Yuan JX-J.
Upregulated TRP and enhanced capacitative Ca2+ entry in human pulmonary artery myocytes during proliferation.
Am J Physiol Heart Circ Physiol
280:
H746-H755,
2001
17.
Grynkiewicz, G,
Poenie M,
and
Tsien RY.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J Biol Chem
260:
3440-3450,
1985[Abstract].
18.
Hardingham, GE,
Chawla S,
Cruzalegui FH,
and
Bading H.
Control of recruitment and transcription-activating function of CBP determines gene regulation by NMDA receptors and L-type calcium channels.
Neuron
22:
789-798,
1999[ISI][Medline].
19.
Hardingham, GE,
Chawla S,
Johnson CM,
and
Bading H.
Distinct functions of nuclear and cytoplasmic calcium in the control of gene expression.
Nature
385:
260-265,
1997[ISI][Medline].
20.
Harteneck, C,
Plant TD,
and
Schultz G.
From worm to man: three subfamilies of TRP channels.
Trends Neurosci
23:
159-166,
2000[ISI][Medline].
21.
He, H,
Lam M,
McCormick TS,
and
Distelhorst CW.
Maintenance of calcium homeostasis in the endoplasmic reticulum by Bcl-2.
J Cell Biol
138:
1219-1228,
1997
22.
Herve, P,
Launay JM,
Scrobohaci ML,
Brenot F,
Simonneau G,
Petitpretz P,
Poubeau P,
Cerrina J,
Duroux P,
and
Drouet L.
Increased plasma serotonin in primary pulmonary hypertension.
Am J Med
99:
249-254,
1995[ISI][Medline].
23.
Hofmann, T,
Schaefer M,
Schultz G,
and
Gudermann T.
Transient receptor potential channels as molecular substrates of receptor-mediated cation entry.
J Mol Med
78:
14-25,
2000[ISI][Medline].
24.
Hoth, M,
and
Penner R.
Calcium release-activated calcium current in rat mast cells.
J Physiol
465:
359-386,
1993[Abstract].
25.
Husain, M,
Bein K,
Jiang L,
Alper SL,
Simons M,
and
Rosenberg RD.
c-Myb-dependent cell cycle progression and Ca2+ storage in cultured vascular smooth muscle cells.
Circ Res
80:
617-626,
1997
26.
Kerschbaum, HH,
and
Cahalan MD.
Monovalent permeability, rectification, and ionic block of store-operated calcium channels in Jurkat T lymphocytes.
J Gen Physiol
111:
521-537,
1998
27.
Kerschbaum, HH,
and
Cahalan MD.
Single-channel recording of a store-operated Ca2+ channel in Jurkat T lymphocytes.
Science
283:
836-839,
1999
28.
Kourembanas, S,
Marsden PA,
McQuillan LP,
and
Faller DV.
Hypoxia induces endothelin gene expression and secretion in cultured human endothelium.
J Clin Invest
88:
1054-1057,
1991[ISI][Medline].
29.
Kuga, T,
Kobayashi S,
Hirakawa Y,
Kanaide H,
and
Takeshita A.
Cell cycle-dependent expression of L- and T-type Ca2+ currents in rat aortic smooth muscle cells in primary culture.
Circ Res
79:
14-19,
1996
30.
Kunze, DL,
Sinkins WG,
Vaca L,
and
Schilling WP.
Properties of single Drosophila Trpl channels expressed in Sf9 insect cells.
Am J Physiol Cell Physiol
272:
C27-C34,
1997
31.
Lee, S-L,
Wang WW,
Lanzillo JJ,
and
Fangurg BL.
Serotonin produces both hyperplasia and hypertrophy of bovine pulmonary artery smooth muscle cells in culture.
Am J Physiol Lung Cell Mol Physiol
266:
L46-L52,
1994
32.
Lintschinger, B,
Balzer-Geldsetzer M,
Baskaran T,
Graier WF,
Romanin C,
Zhu MX,
and
Groschner K.
Coassembly of Trp1 and Trp3 channels generates diacylglycerol and Ca2+-sensitive cation channels.
J Biol Chem
275:
27799-27805,
2000
33.
Lu, KP,
and
Means AR.
Regulation of the cell cycle by calcium and calmodulin.
Endocr Rev
14:
40-58,
1993[ISI][Medline].
34.
Luckhoff, A,
and
Clapham DE.
Calcium channels activated by depletion of internal calcium stores in A431 cells.
Biophys J
67:
177-182,
1994[Abstract].
35.
McDaniel, SS,
Platoshyn O,
Wang J,
Yu Y,
Sweeney M,
Krick S,
Rubin LJ,
and
Yuan JX-J.
Capacitative Ca2+ entry in agonist-induced pulmonary vasoconstriction.
Am J Physiol Lung Cell Mol Physiol
280:
L870-L880,
2001
36.
Moore, TM,
Norwood NR,
Creighton JR,
Babal P,
Brough GH,
Shasby DM,
and
Stevens T.
Receptor-dependent activation of store-operated calcium entry increases endothelial cell permeability.
Am J Physiol Lung Cell Mol Physiol
279:
L691-L698,
2000
37.
Nelson, MT,
Patlak JB,
Worley JF,
and
Standen NB.
Calcium channels, potassium channels, and voltage dependence of arterial smooth muscle tone.
Am J Physiol Cell Physiol
259:
C3-C18,
1990
38.
Niemeyer, B,
Suzuki E,
Scott K,
Jalinik K,
and
Zuker CS.
The Drosophila light-activated conductance is composed of the two channels TRP and TRPL.
Cell
83:
837-848,
1996.
39.
Norwood, N,
Moore TM,
Dean DA,
Bhattacharjee R,
Li M,
and
Stevens T.
Store-operated calcium entry and increased endothelial cell permeability.
Am J Physiol Lung Cell Mol Physiol
279:
L815-L824,
2000
40.
Parekh, AB,
and
Penner R.
Store depletion and calcium influx.
Physiol Rev
77:
901-930,
1997
41.
Pitt, BR,
Weng W,
Steve AR,
Blakely RD,
Reynolds I,
and
Davies P.
Serotonin increases DNA synthesis in rat proximal and distal pulmonary vascular smooth muscle cells in culture.
Am J Physiol Lung Cell Mol Physiol
266:
L178-L186,
1994
42.
Philipp, S,
Cavalie A,
Freichel M,
Wissenbach U,
Zimmer S,
Trost C,
Marquart A,
Murakami M,
and
Flockerzi V.
A mammalian capacitative calcium entry channel homologous to Drosophila TRP and TRPL.
EMBO J
15:
6166-6171,
1996[Abstract].
43.
Putney, JW, Jr,
Broad LM,
Braun F-J,
Lievremont J-P,
and
Bird GSJ
Mechanisms of capacitative calcium entry.
J Cell Sci
114:
2223-2229,
2001
44.
Rich, S,
Kaufmann E,
and
Levy PS.
The effect of high doses of calcium-channel blockers on survival in primary pulmonary hypertension.
N Engl J Med
327:
76-81,
1992[Abstract].
45.
Rubin, LJ.
Primary pulmonary hypertension.
N Engl J Med
336:
111-117,
1997
46.
Runnels, LW,
Yue L,
and
Clapham DE.
TRP-PLIK, a bifunctional protein with kinase and ion channel activities.
Science
291:
1043-1047,
2001
47.
Sheng, M,
McFadden G,
and
Greenberg ME.
Membrane depolarization and calcium induce c-fos transcription via phosphorylation of transcription factor CREB.
Neuron
4:
571-582,
1990[ISI][Medline].
48.
Shimoda, LA,
Sylvester JT,
and
Sham JSK
Mobilization of intracellular Ca2+ by endothelin-1 in rat intrapulmonary arterial smooth muscle cells.
Am J Physiol Lung Cell Mol Physiol
278:
L148-L156,
2000
49.
Short, AD,
Bian J,
Ghosh TK,
Waldron RT,
Rybak SL,
and
Gill DL.
Intracellular Ca2+ pool content is linked to control of cell growth.
Proc Natl Acad Sci USA
90:
4986-4990,
1993[Abstract].
50.
Sinkins, WG,
Estacion M,
and
Schilling WP.
Functional expression of TrpC1: a human homologue of the Drosophila Trp channel.
Biochem J
331:
331-339,
1998[ISI][Medline].
51.
Somlyo, AP,
and
Somlyo AV.
Signal transduction and regulation in smooth muscle.
Nature
372:
231-236,
1994[ISI][Medline].
52.
Stenmark, KR,
and
Mecham RP.
Cellular and molecular mechanisms of pulmonary vascular remodeling.
Annu Rev Physiol
59:
89-144,
1997[ISI][Medline].
53.
Strubing, C,
Krapivinsky G,
Krapivinsky L,
and
Clapham DE.
TRPC1 and TRPC5 form a novel cation channel in mammalian brain.
Neuron
29:
645-655,
2001[ISI][Medline].
54.
Tsien, RW,
and
Tsien RY.
Calcium channels, stores, and oscillations.
Annu Rev Cell Biol
6:
715-760,
1990[ISI].
55.
Tuder, RM,
Flook BE,
and
Voelkel NF.
Increased gene expression for VEGF and the VEGF receptors KDR/Flk and Flt in lungs exposed to acute or to chronic hypoxia. Modulation of gene expression by nitric oxide.
J Clin Invest
95:
1798-1807,
1995[ISI][Medline].
56.
Vaca, L,
Sinkins WG,
Hu Y,
Kunze D,
and
Schilling WP.
Activation of recombinant trp by thapsigargin in Sf9 insect cells.
Am J Physiol Cell Physiol
267:
C1501-C1505,
1994
57.
Vannier, B,
Peyton M,
Boulay G,
Brown D,
Qin N,
Jiang M,
Zhu X,
and
Birnbaumer L.
Mouse trp2, the homologue of the human trp2 pseudogene, encodes mTrp2, a store depletion-activated capacitative Ca2+ entry channel.
Proc Natl Acad Sci USA
96:
2060-2064,
1999
58.
Voelkel, NF,
Tuder RM,
and
Weir KE.
Pathophysiology of primary pulmonary hypertension: from physiology to molecular mechanisms.
In: Primary Pulmonary Hypertension, edited by Rubin LJ,
and Rich S.. New York: Marcel Dekker, 1997, p. 83-133.
59.
Walker, RL,
Hume JR,
and
Horowitz B.
Differential expression and alternative splicing of TRP channel genes in smooth muscles.
Am J Physiol Cell Physiol
280:
C1184-C1192,
2001
60.
Wes, PD,
Chevesich J,
Jeromin A,
Rosenberg C,
Stetten G,
and
Montell C.
TRPC1, a human homolog of a Drosophila store-operated channel.
Proc Natl Acad Sci USA
92:
9652-9656,
1995[Abstract].
61.
Xu, S-Z,
and
Beech DJ.
TrpC1 is a membrane-spanning subunit of store-operated Ca2+ channels in native vascular smooth muscle cells.
Circ Res
88:
84-87,
2001
62.
Yuan, JX-J,
Aldinger AM,
Juhaszova M,
Wang J,
Conte JV,
Gaine SP,
Orens JB,
and
Rubin LJ.
Dysfunctional voltage-gated K+ channels in pulmonary artery smooth muscle cells of patients with primary pulmonary hypertension.
Circulation
98:
1400-1406,
1998
63.
Yue, L,
Peng JB,
Hediger MA,
and
Clapham DE.
CaT1 manifests the pore properties of the calcium-release-activated calcium channel.
Nature
410:
705-709,
2001[ISI][Medline].
64.
Zhu, X,
and
Birnbaumer L.
Calcium channels formed by mammalian trp homologues.
News Physiol Sci
13:
211-217,
1998
65.
Zhu, X,
Jiang M,
Peyton M,
Boulay G,
Hurst R,
Stefani E,
and
Birnbaumer L.
trp, a novel mammalian gene family essential for agonist-activated capacitative Ca2+ entry.
Cell
85:
661-671,
1996[ISI][Medline].
66.
Zitt, C,
Zobel A,
Obukhov AG,
Harteneck C,
Kalkbrenner F,
Luckhoff A,
and
Schultz G.
Cloning and functional expression of a human Ca2+-permeable cation channel activated by calcium store depletion.
Neuron
16:
1189-1196,
1996[ISI][Medline].
67.
Zweifach, A,
and
Lewis RS.
Calcium-dependent potentiation of store-operated calcium channels in T lymphocytes.
J Gen Physiol
107:
597-610,
1996[Abstract].