A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) due to Ca2+ release from intracellular Ca2+ stores and Ca2+ influx through plasmalemmal Ca2+ channels plays a critical role in mitogen-mediated cell growth. Depletion of intracellular Ca2+ stores triggers capacitative Ca2+ entry (CCE), a mechanism involved in maintaining Ca2+ influx and refilling intracellular Ca2+ stores. Transient receptor potential (TRP) genes have been demonstrated to encode the store-operated Ca2+ channels that are activated by Ca2+ store depletion. In this study, we examined whether CCE, activity of store-operated Ca2+ channels, and humanTRP1 (hTRP1) expression are essential in human pulmonary arterial smooth muscle cell (PASMC) proliferation. Chelation of extracellular Ca2+ and depletion of intracellularly stored Ca2+ inhibited PASMC growth in media containing serum and growth factors. Resting [Ca2+]cyt as well as the increases in [Ca2+]cyt due to Ca2+ release and CCE were all significantly greater in proliferating PASMC than in growth-arrested cells. Consistently, whole cell inward currents activated by depletion of intracellular Ca2+ stores and the mRNA level of hTRP1 were much greater in proliferating PASMC than in growth-arrested cells. These results suggest that elevated [Ca2+]cyt and intracellularly stored [Ca2+] play an important role in pulmonary vascular smooth muscle cell growth. CCE, potentially viahTRP1-encoded Ca2+-permeable channels, may be an important mechanism required to maintain the elevated [Ca2+]cyt and stored [Ca2+] in human PASMC during proliferation.
- transient receptor potential gene
- pulmonary hypertension
- pulmonary artery smooth muscle cells
pulmonary arterial pressure is the function of cardiac output and pulmonary vascular resistance. Pulmonary vasoconstriction and vascular medial hypertrophy (due to smooth muscle cell proliferation) greatly contribute to the increased pulmonary vascular resistance in patients with primary pulmonary hypertension (PPH), a fatal disease with an unknown cause (31, 38). Studies from patients with essential hypertension demonstrate that, at the cellular level, blood pressure (BP) is directly proportional to cytosolic free Ca2+ concentration ([Ca2+]cyt), which is determined primarily by extracellular Ca2+concentration ([Ca2+]o) and intracellularly stored Ca2+ ([Ca2+]SR) in the sarcoplasmic reticulum (SR): BP ∝k[Ca2+]cyt =k(f [Ca2+]o +g[Ca2+]SR), where k,f, and g are proportionality factors (30). Hypertension is then a progressive disease manifested clinically by elevated blood pressure and cellularly by, at least in part, an excessive [Ca2+]cyt in vascular smooth muscle cells and an improper balance of extracellular [Ca2+] and [Ca2+]SR (30). A common hypothesis is that vasoconstriction and cell proliferation, two linked phenomena in the etiology of PPH (31), use overlapping signaling processes that result in parallel intracellular events (35, 38).
Intracellular Ca2+ is a critical signal transduction element in regulating muscle contraction (34), gene expression (8, 19), and cell proliferation (6, 16,28). Because Ca2+ diffuses rapidly between the cytosol and nucleus (1), an increase in [Ca2+]cyt activates Ca2+-dependent events occurring in both the cytosol and nucleus. A variety of mitogenic agents stimulate cell growth by increasing [Ca2+]cyt (6, 16, 23, 28,32). The mitogen-mediated changes in [Ca2+]cyt usually consist of initial Ca2+ release from the SR followed by sustained Ca2+ entry through sarcolemmal Ca2+ channels. Depletion of Ca2+ from the SR due to the agonist-induced Ca2+ release triggers Ca2+ influx through store-operated Ca2+ channels (SOCs) in the plasma membrane, namely capacitative Ca2+ entry (CCE) (29). Therefore, CCE is a mechanism that links [Ca2+]SR to membrane Ca2+permeability (4, 27) and serves as an important pathway to refill intracellular Ca2+ stores and maintain sustained Ca2+ influx (27). A novel gene family, transient receptor potential (TRP) genes, which are essential for agonist-activated CCE, have recently been cloned in mammals and humans (hTRP) (4, 39, 44, 45). Expression of TRP genes has been demonstrated to form the Ca2+-permeable cation channels that are activated by Ca2+ store depletion. This suggests that theTRP-encoded proteins may be the putative channels responsible for CCE (4, 5, 37, 40, 44, 45).
In vascular smooth muscle cells, sustained Ca2+ influx and maintaining sufficient Ca2+ in the SR are both required for cell growth (15, 33). Indeed, removal of extracellular Ca2+ and depletion of the SR Ca2+ significantly inhibited cell growth (33). This study was designed to test the hypothesis that CCE, potentially throughTRP-encoded Ca2+-permeable channels, is an important mechanism to maintain the elevated [Ca2+]cyt and [Ca2+]SR that are necessary for human pulmonary arterial smooth muscle cell (PASMC) proliferation.
MATERIALS AND METHODS
Cell preparation and culture.
Patients undergoing lobectomy for bronchogenic carcinoma and lung/heart transplantation for cardiopulmonary diseases, who had no evidence of pulmonary hypertension, were the source of control lung tissues for preparing primary cultured PASMC. In some cases, patients undergoing lung transplantation with PPH or secondary pulmonary hypertension (SPH; e.g., patients with congenital heart disease and pulmonary thromboembolic disease) were the source of diseased lung tissues. Lung tissues were removed from patients in the operating room and immediately placed in cold (4°C) saline and taken to the laboratory for dissection (41).
Small muscular intrapulmonary arteries were isolated and incubated for 20 min in Hank's balanced salt solution containing 2 mg/ml collagenase (Worthington). After the incubation, the adventitia was carefully stripped off, and the endothelium was removed. Remaining smooth muscle was digested with 2.0 mg/ml collagenase (Worthington), 0.5 mg/ml elastase, and 1 mg/ml bovine albumin (Sigma) at 37°C. Single PASMC were resuspended, plated onto 25-mm cover slips (for fluorescence microscopy and electrophysiological experiments) or 10-cm Petri dishes (for molecular biological experiments), and incubated in a humidified atmosphere of 5% CO2 in air at 37°C (41).
The cells were cultured in smooth muscle growth medium (Clonetics), which consisted of smooth muscle basal medium, 5% fetal bovine serum, 5 μg/ml insulin, 2 ng/ml human fibroblast growth factor, and 0.5 ng/ml human endothelial growth factor (Clonetics), for 5–7 days before each experiment. In some experiments, normal human PASMC (Clonetics) were used at passages 4–6. The cells were stained with the membrane-permeable nucleic acid stain 4′,6′-diamidino-2-phenylindole (5 μM, Molecular Probes). All the 4′,6′-diamidino-2-phenylindole-stained cells in the cultures cross-reacted with the smooth muscle cell α-actin antibody, indicating that the cultures consisted only of smooth muscle cells. There were no significant morphological differences between the cells cultured in media with or without serum and growth factors. Cell counts were determined using a hemocytometer. The viable cell number normalized by size of the Petri dishes (in cells/cm2) was used to compare cell growth rate.
Measurement of [Ca2+]cyt.
The cells were loaded in culture medium containing the acetoxymethyl ester form of fura 2 (fura 2-AM; 3 μM, Molecular Probes) for 30 min at room temperature under an atmosphere of 5% CO2 in air (10). The fura 2-loaded cells were then superfused with standard bath solution for 30 min at 32–34°C to wash away extracellular dye and permit intracellular esterases to cleave cytosolic fura 2-AM into active fura 2. Fura 2 fluorescence (510-nm emission; 380- and 360-nm excitation) from the cells and background fluorescence were imaged using a GEN III charge-coupled device camera (Stanford Photonics) coupled to a Carl Zeiss microscope. Fluorescent images were obtained using a microchannel plate image intensifier (Amperex XX1381) coupled by fiber optics to the charged-coupled device camera. Image acquisition and analysis were performed with a MetaMorph imaging system (Universal Imaging). Video frames containing images of fura 2 fluorescence from cells, as well as the corresponding background images (fluorescence from fields devoid of cells), were digitized at a resolution of 512 horizontal × 480 vertical pixels and an 8-bit grey scale using a Matrix LC imaging board operating in an IBM-compatible PC. To improve the signal-to-noise ratio, four to eight consecutive video frames were usually averaged at a video frame rate of 30 frames/s. Images were acquired at a rate of one averaged image every 3 s when [Ca2+]cyt was changing and every 60 s when [Ca2+]cyt was stable. [Ca2+]cyt was calculated from fura 2 fluorescent emission excited at 380 and 360 nm using the ratio method (11). In most experiments, multiple (6-10) cells were imaged in single field, and one arbitrarily chosen peripheral cytosolic area (4–6 × 4–6 pixels) from each cell was spatially averaged.
Measurement of Ca2+ release-activated Ca2+ currents.
Whole cell and single-channel Ca2+ release-activated Ca2+ currents (I CRAC) were recorded with an Axopatch-1D amplifier and a DigiData 1200 interface (Axonpatch) using patch-clamp techniques (12). Patch pipettes (2–4 MΩ) were fabricated on a Sutter electrode puller using borosilicate glass tubes and fire polished on a microforge (Narishige). For whole cell current recordings, voltage stimuli lasting 300 ms were delivered from a holding potential of 0 mV (to inactivate voltage-gated Ca2+ and Na+ channels) using voltage steps from +80 mV to −80 mV. Traces recorded before the activation of SOCs were used as a template to subtract leak currents. All experiments were performed at room temperature (22–24°C). In both whole cell and single-channel configurations, SOCs were activated by passive depletion of the SR using 10 μM cyclopiazonic acid (CPA; Sigma) dissolved in the Ca2+-free solution.
Solution and reagents.
For [Ca2+]cyt measurement experiments, the cells were superfused (2–3 ml/min) with physiological salt solution (PSS). The PSS contained (in mM) 141 NaCl, 4.7 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose (pH 7.4 with 5 M NaOH). The bath solution for recording whole cellI CRAC contained (in mM) 120 sodium methane sulfonate, 20 calcium aspartate, 0.5 3,4-diaminopyridine, 10 glucose, and 10 HEPES (pH 7.4 with methane sulfonic acid). The pipette (intracellular) solution contained (in mM) 138 cesium aspartate, 1.15 EGTA, 1 Ca(OH)2, 2 Na2ATP, and 10 HEPES (pH 7.2). These ionic conditions eliminated the currents through K+ or Cl− channels. In Ca2+-free PSS, CaCl2 was replaced by equimolar MgCl2, and 1 mM EGTA was added to chelate residual Ca2+. In the high-K+ (40 mM) solution, NaCl was replaced, mole-for-mole, by KCl to maintain the osmolarity of the solution. For cell-attached recording of single-channel I CRAC, the pipette (extracellular) solution contained (in mM) 120 sodium methane sulfonate, 20 calcium aspartate, 0.5 3,4-diaminopyridine, and 10 HEPES (pH 7.4 with methane sulfonic acid). The bath (extracellular) solution contained (in mM) 141 NaCl, 4.7 KCl, 1.2 MgCl2, 1.8 CaCl2, 10 glucose, and 10 HEPES (pH 7.4).
CPA and thapsigargin (Sigma) were dissolved into DMSO to make stock solutions of 30 and 10 mM, respectively. Aliquots of the stock solutions were then diluted 1:3,000–10,000 into the bath solution or culture medium to make final concentrations of 10 μM CPA (pH 7.4) and 1 μM thapsigargin. Nifedipine (Sigma) and Ni2+(Sigma) were directly dissolved in the bath solutions on the day of use. The pH values of all solutions were checked after addition of the drugs and readjusted to 7.4. In [Ca2+]cyt andI CRAC measurement experiments, the same amount of DMSO used for dissolving CPA was added to control solutions (0.03%). Vehicle (DMSO) alone had negligible effects on [Ca2+]cyt and I CRAC in PASMC.
Total RNA (2–3 μg) prepared from the cells by the acid guanidinium thiocyanate-phenol-chloroform extraction method, was reverse-transcribed using the First-Strand cDNA synthesis kit (Pharmacia Biotech). The sense/antisense primers were designed from coding regions ofhTRP1 [5′-CAAGATTTTGGAAAATTTCTTG-3′ (nt. 2,338–2,359)/5′-TTTGTCTTCATGATTTGCTAT-3′ (nt. 2,689–2,709);U31110]; hTRP3 [5′-TGACTTCCGTTGTGCTCAAATATG-3′ (nt. 2,316–2339)/5′-TCTGAAGCCTTCTCCTTCTGC-3′ (nt. 2,610–2,633);Y13758], and the human voltage-dependent Ca2+ channel (VDCC) β2-subunit [5′-TGGTGGATAGGGCGATTGGT-3′ (nt. 808–827)/5′-TGCGTGCTTACTGGGATTGTT-3′ (nt. 1336–1356);U95019], respectively. The first-strand cDNA reaction mixture (2 μl) was used in a 50-μl PCR reaction consisting of 0.1 μM of each primer, 10 mM Tris · HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 200 μN of each dNTP, and 2 units of Taq DNA polymerase (Perkin-Elmer). The cDNA samples were amplified in a DNA thermal cycler (Perkin-Elmer) under the following conditions: the mixture was annealed at 50–61°C (for 1 min), extended at 72°C (for 2 min), and denatured at 94°C (for 1 min) for 25 cycles. This was followed by a final extension at 72°C (for 10 min) to ensure complete product extension. The PCR products were electrophoresed through a 1% agarose gel, and amplified cDNA bands were visualized by ethidium bromide staining. The PCR product was sequenced, and the result indicated that the sequence of our PCR product using the sense and antisense primers designed for hTRP1 matched exactly with the hTRP1 sequence (U31110) in GenBank. To quantify the PCR products, an invariant mRNA of human β-actin was used as an internal control. The OD value for each band on the gel was measured by a Gel Documentation System (UVP). The OD values in the channel signals were normalized to the OD values in the β-actin signals; the ratios are expressed as arbitrary units for quantitative comparison.
The composite data are expressed as means ± SE. Statistical analyses were performed using unpaired Student's t-test or ANOVA and post hoc tests (Student Newman-Keuls) where appropriate. Differences were considered to be significant when P < 0.05.
Requirement of extracellular Ca2+ and intracellularly stored Ca2+ for PASMC growth.
A rise in [Ca2+]cyt can activate cytoplasmic mitogen-activated protein kinase (6) (which is part of the phosphorylation cascade that leads to activation of DNA synthesis-promoting factor), rapidly increase nuclear [Ca2+] (1), and promote cell proliferation (22). The addition of 2 mM EDTA, a Ca2+chelator, to the culture medium (which contains 1.6 mM Ca2+) decreases the free [Ca2+] to 59 μM (9) and significantly inhibited human PASMC growth in media containing 5% fetal bovine serum and growth factors (Fig.1). Depletion of intracellular Ca2+ stores (mainly the SR) with 1 μM thapsigargin further inhibited proliferation of human PASMC cultured in media containing 2 mM EDTA (Fig. 1). These results demonstrate that a sustained Ca2+ influx through sarcolemmal Ca2+channels and sufficient [Ca2+] maintained within the SR (33) are both required for PASMC growth.
Increased resting [Ca2+]cyt and CCE in proliferating PASMC.
Withdrawal of serum and growth factors from culture media abolished cell growth (Fig. 1). This indicates that the cells cultured in smooth muscle basal medium (without serum and growth factors) are growth-arrested cells, whereas the cells cultured in smooth muscle growth medium (smooth muscle basal medium supplemented with 5% fetal bovine serum and growth factors) are proliferating cells.
In the absence of extracellular Ca2+, CPA, by blocking Ca2+ sequestration into the SR, induced a transient [Ca2+]cyt rise due to leakage of Ca2+ from the SR to the cytosol. The CPA-induced [Ca2+]cyt transient declined back to the original baseline level after 5–7 min as the SR Ca2+was depleted. Under these conditions, restoration of extracellular Ca2+ induced a rise in [Ca2+]cyt, which was obviously due to CCE (Fig.2 A)
The peak increase in [Ca2+]cyt due to CCE was significantly enhanced in proliferating human PASMC (1,712 ± 63 nM, n = 98) compared with growth-arrested cells (369 ± 22 nM, n = 92, P < 0.001) (Fig. 2). Furthermore, the resting [Ca2+]cyt(171 ± 6 vs. 88 ± 4 nM, P < 0.01) and the CPA-mediated Ca2+ transients (due to Ca2+release from the SR) (614 ± 31 vs. 301 ± 29 nM,P < 0.01) were also significantly higher in proliferating PASMC (n = 98) than in growth-arrested cells (n = 92).
Inhibitory effects of Ni2+ on CCE and PASMC growth.
Blockade of L-type VDCC with nifedipine (1 μM) abolished the 40 mM K+-induced increase in [Ca2+]cytbut had little effect on the CCE-mediated increase in [Ca2+]cyt (Fig.3 A, top). CCE is largely determined by the conductance of SOCs when the transmembrane Ca2+ electrochemical gradient is constant. Ni2+has been demonstrated to block SOCs (46). Indeed, Ni2+ (0.5 mM) or SKF-96365 (10 μM) (7) significantly attenuated CCE in proliferating PASMC in the presence of nifedipine (Fig. 3 A). Accordingly, treatment of the cells with 0.5 mM Ni2+ for 2 days significantly diminished human PASMC growth in media containing serum and growth factors (Fig.3 B).
Increased SOC activity in proliferating PASMC.
The flux of Ca2+ ions through SOCs has been defined electrophysiologically as I CRAC (14, 17,18, 20, 27). Whole cell I CRAC were recorded in cells superfused with solutions containing 120 mM Na+ and 20 mM Ca2+ and dialyzed with solutions containing 138 mM Cs+ and ∼100 nM free Ca2+. Holding potential was set at 0 mV to inactivate voltage-gated Na+ and Ca2+ channels. Whole cellI CRAC were elicited by 300-ms voltage steps from −80 to +80 mV before and after application of 10 μM CPA (Fig.4 A). Subtracting the currents recorded under control conditions from the currents recorded after 10–15 min application of CPA revealed I CRACthat were activated by CPA-induced depletion of the SR Ca2+(Fig. 4 A, right). The inward current was apparently carried by Ca2+ (and Na+), and the outward current was carried by Cs+ (Cs permeability = Na permeability for SOCs) (2, 17, 18).
Consistent with the results on CCE, whole cellI CRAC were much greater in proliferating cells than in growth-arrested cells (Fig. 4). The averaged amplitudes ofI CRAC, elicited by −80 mV, were −252 ± 69 pA in proliferating cells and −33 ± 12 pA in growth-arrested cells (Fig. 4 B, right). Extracellular application of Mg2+ (2 mM), which has been demonstrated to inhibit CCE in vascular smooth muscle cells (2, 10), significantly reduced whole cell I CRAC in both growth-arrested (by 16 ± 2% at +80 mV, P < 0.01) and proliferating (by 22 ± 2%, P < 0.01) PASMC.
Single-channel I CRAC were recorded in cell-attached patches from proliferating PASMC using the ionic conditions under which inward Ca2+ (or Na+) currents could be displayed at negative holding potentials for the patches. While the cell was held at −45 mV, application of 10 μM CPA (for 4–8 min) gradually induced a small amplitude inward current that was apparently carried by Ca2+ (or Na+) through SOCs (Fig. 5 A). The slope conductance of this current, calculated from the current-voltage relationship curve, is 5.34 pS (Fig. 5 B).
Enhanced hTRP1 expression in proliferating PASMC.
It has been demonstrated that TRP-encoded proteins may be responsible for CCE (4, 5, 37, 44, 45). Human PASMC from nonpulmonary hypertension patients expressed hTRP1 but nothTRP3 (Fig. 6 A). The mRNA level of hTRP1 was significantly higher in proliferating cells than in growth-arrested cells (Fig. 6). The enhanced mRNA expression was selective to hTRP1 because the mRNA level of the hVDCC β2-subunit was unaltered in proliferating PASMC (Fig. 6 B). These results are consistent with the electrophysiological and fluorescence microscopy experiments showing that I CRAC and CCE were enhanced in human PASMC during proliferation.
Temporal relationship between hTRP1 expression and PASMC proliferation.
Whether the mRNA expression of hTRP1 was temporally related to the cell proliferation was examined by comparing the time courses of the change of hTRP1 mRNA levels and the cell growth rate. The cells were first growth arrested by incubation in smooth muscle basal medium (the basal medium without serum and growth factors) for 24–36 h and then cultured in smooth muscle growth medium (smooth muscle basal muscle supplemented with 5% fetal bovine serum and growth factors) for up to 4 days. As shown in Fig.7, the cell number in culture dishes (A) and the mRNA level of hTRP1 (B) were both increased after incubating the cells in media containing serum (5% fetal bovine serum) and growth factors (smooth muscle growth medium). However, the time courses are quite different (Fig.7 C): the mRNA expression of hTRP1 was significantly increased after 6 h of incubation (by 10.1 ± 3.4%, P < 0.05) in the smooth muscle growth medium (5% fetal bovine serum and growth factors), whereas the cell growth was not significantly increased until 24 h (by 15.4 ± 2.4%,P < 0.001) (Fig. 7 C). The calculated times for the 50% increase in the hTRP1 mRNA expression and the cell growth are 18.36 and 46.56 h, respectively (Fig. 7 C). These results suggest that enhancement of hTRP1 mRNA expression precedes the onset of cell proliferation in the presence of fetal bovine serum and growth factors. The precise temporal relationship between hTRP1 expression and actual proliferating process (e.g., different cell cycle phases) needs further study.
Elevated resting [Ca2+]cyt and [Ca2+]SR in growth-arrested PASMC from patients with PPH.
Patients with SPH (n = 18) and PPH (n = 7), from whom PASMC were isolated, had similar mean pulmonary arterial pressure (48 ± 4 vs. 53 ± 4 mmHg, P = 0.30) and total vascular resistance (13.6 ± 3 vs. 11.2 ± 1.7 mmHg · l−1 · min−1,P = 0.48) (41). However, the resting [Ca2+]cyt in quiescent (growth arrested) PASMC (n = 16) from PPH patients was significantly higher than in cells (n = 19) from SPH patients (Fig.8, A andC).
In the presence of extracellular Ca2+, the CPA-induced peak increase in [Ca2+]cyt was significantly greater in PPH PASMC (600 ± 70 nM) than in SPH PASMC (350 ± 25 nM, P < 0.01) (Fig. 8, B,top, and C, right). The CPA-induced transient [Ca2+]cyt rise in the absence of extracellular Ca2+ (by Ca2+ mobilization from the SR) and the CCE-mediated [Ca2+]cytincrease while extracellular Ca2+ was restored (199 ± 23 nM, n = 16, vs. 96 ± 8 nM, n = 19, P < 0.01) were both greater in PPH PASMC than in SPH PASMC. These results indicate that resting [Ca2+]cyt, [Ca2+]SR, and CCE are all higher in PASMC from PPH patients than in cells from SPH patients.
In human PASMC, chelation of extracellular Ca2+ and depletion of intracellularly stored Ca2+ markedly inhibited cell growth in media containing serum and growth factors, whereas resting [Ca2+]cyt was much higher in proliferating cells than in growth-arrested cells. These results imply that elevated [Ca2+]cyt and [Ca2+]SR, due to a constant Ca2+influx, are necessary for human PASMC proliferation. CCE is a critical mechanism involved in maintaining sustained Ca2+ influx and refilling Ca2+ into the SR (4, 27, 36). Indeed, the CCE-mediated increase in [Ca2+]cyt, whole cellI CRAC, and hTRP1 mRNA expression were all significantly enhanced in PASMC during proliferation. Inhibition of SOCs with Ni2+ reduced CCE and significantly attenuated human PASMC growth. These results suggest that CCE, potentially through the upregulated hTRP1 expression and the resultant increase in I CRAC, is a critical mechanism required to maintain the elevated [Ca2+]cyt and [Ca2+]SR in PASMC during proliferation.
Regulation of cytosolic free [Ca2+].
In vascular smooth muscle cells, including PASMC, [Ca2+]cyt is regulated by Ca2+influx through Ca2+-permeable channels in the plasma membrane and Ca2+ release from intracellular Ca2+ stores, such as the sarco(endo)plasmic reticulum (34, 36). Many vasoactive agonists stimulate smooth muscle proliferation by increasing [Ca2+]cyt(6, 16, 23, 28, 32). The mitogen-induced changes in [Ca2+]cyt usually consist of an initial release of Ca2+ from the SR followed by a sustained Ca2+ influx through sarcolemmal Ca2+ channels. In human PASMC, resting membrane potential is approximately −45 mV, and resting [Ca2+]cyt is 50–150 nM. Thus the observations that extracellular Ca2+ chelation inhibited human PASMC growth imply that continuous Ca2+influx is essential for human PASMC.
There are at least three classes of Ca2+-permeable channels in the plasma membrane: 1) VDCC (24),2) receptor-operated channels (36); and3) SOCs (4, 27, 36). Membrane potential regulates [Ca2+]cyt by governing Ca2+ influx via VDCC (24, 42). The agonist-induced Ca2+ influx is mainly caused by the receptor-mediated activation of receptor-operated channels and store depletion-mediated opening of SOCs (4, 27, 36). It is likely that depolarization-activated VDCC, agonist-activated receptor-operated channels, and store depletion-activated CCE all contribute to the sustained Ca2+ influx in PASMC during proliferation. Inhibition of CCE by the SOC blocker Ni2+significantly attenuated human PASMC growth, suggesting that CCE through SOCs is a critical mechanism involved in maintaining sustained Ca2+ influx and refilling Ca2+ into the SR, which are both necessary for PASMC growth.
Molecular identification of CCE.
Electrophysiological studies on the store depletion-activatedI CRAC suggest that SOCs are complex and heterogeneous in molecular composition and in their cellular regulation (4). There are at least seven members (TRP1–TRP7) in the mouse TRP gene family, of which at least four (TRP1, TRP3, TRP4, and TRP6) have been identified in human tissues (4,26, 44, 45). Expression of TRP genes results in the formation of the Ca2+-permeable cation channels that are activated by Ca2+ store depletion, suggesting that SOCs may be composed of subunits encoded in TRP genes (3-5, 25, 37, 44, 45). SOCs are hypothesized to be heterotetramers and/or homotetramers made of different TRPs (3,4, 27). In mammalian tissues, it has been recently demonstrated that TRP1, TRP2, TRP4, and TRP5may encode the endogenous SOCs that are activated by store depletion, whereas TRP3, TRP6, andTRP7 may encode the channels that are activated by inositol 1,4,5-trisphosphate (3, 4, 21, 26) and store depletion.
Patch-clamp studies on the store depletion-activatedI CRAC indicate that there are multiple SOCs based on the single-channel conductance (0.2–110 pS) (14,17, 18, 20, 27). In human PASMC, only the hTRP1transcript (but not hTRP3 transcript) was identified using RT-PCR, suggesting that hTRP1 is a critical TRPgene that encodes SOCs in human PASMC. In proliferating PASMC, we observed a 5.34-pS channel that was activated by CPA-induced depletion of the SR Ca2+. These observations suggest that the 5.34-pS channel may be an important SOC that participates in regulating human PASMC growth.
Role of Ca2+ in PASMC Proliferation.
Cytosolic Ca2+ stimulates cell proliferation (22, 23,28, 32), whereas [Ca2+]SR may regulate cell proliferation by modulating the amplitude and frequency of Ca2+ signals in the cytosol and nucleus (6, 8, 19,33). Maintaining a sufficient level of Ca2+ in the SR is also critical for cell growth; indeed, depletion of the SR Ca2+ stores induces growth arrest (15, 33). In the cell cycle, there are four Ca2+-sensitive steps: transitions 1) from the G0 (resting state) to the G1 phase (the beginning of DNA synthesis);2) from the G1 to the S phase (an interphase during which replication of the nuclear DNA occurs);3) from the G2 to the M phase (mitosis); and4) through the M phase (22). Thus a rise in [Ca2+]cyt plays a critical role in stimulating PASMC proliferation.
A critical signal transduction pathway upon activation of membrane receptors by mitogenic agonists is the increase in [Ca2+]cyt due to 1) Ca2+ release from the SR and 2) Ca2+influx through sarcolemmal Ca2+ channels (4, 13, 32,36). In human PASMC, CCE, potentially throughhTRP1-encoded SOCs, is an important mechanism involved in1) maintaining the sustained increase in [Ca2+]cyt and 2) refilling Ca2+ into the SR. Thus the expression of TRPs and the function of SOCs may play an important role in regulating PASMC growth via modulation of [Ca2+]cyt and [Ca2+]SR (10).
Mechanisms involved in elevated [Ca2+]cytin PASMC from PPH patients.
Pulmonary vasoconstriction and vascular smooth muscle proliferation greatly contribute to the increased pulmonary vascular resistance in patients with PPH (31, 35, 38). Elevated pulmonary arterial pressure in patients with SPH is often caused by primary diseases, such as left to right shunt in congenital heart diseases and vascular embolism in pulmonary thromboembolic disease. The results from this study demonstrate that resting [Ca2+]cytand [Ca2+]SR are higher, and CCE is greater, in PASMC from PPH patients than in the cells from SPH patients. These data suggest that increases in [Ca2+]cyt and [Ca2+]SR due to enhanced CCE may also play a role in the development of pulmonary vascular smooth muscle proliferation in patients with PPH (Fig.9).
We previously reported that the activity of voltage-gated K+ channels is significantly decreased in PASMC from PPH patients compared with PASMC from SPH patients (41, 43). The resultant membrane depolarization increases [Ca2+]cyt by opening VDCC and may play an important role in initiating pulmonary vasoconstriction in PPH patients (41, 43). In PASMC isolated from SPH and PPH patients, 60 mM K+-induced rises in [Ca2+]cytare similar between these two cell types (41). High-K+-induced rises in [Ca2+]cyt mainly result from opening of VDCC via membrane depolarization (24, 42). These results suggest that VDCC function normally in PPH PASMC or at least similarly in SPH and PPH PASMC. The observations from the current study demonstrate that the increase in [Ca2+]cytdue to CCE is much greater in PPH PASMC than SPH PASMC. Therefore, the elevated [Ca2+]cyt and [Ca2+]SR in PPH PASMC could be provoked by1) indirect activation of VDCC via membrane depolarization and 2) direct augmentation of CCE through thehTRP1-encoded SOCs.
In summary, resting [Ca2+]cyt and [Ca2+]SR as well as store depletion-mediatedI CRAC and CCE are significantly enhanced in normal human PASMC during proliferation (compared with growth-arrested cells). Blockade of SOCs diminishes CCE and significantly inhibits human PASMC growth in media containing serum and growth factors. These results suggest that CCE, a novel mechanism essential for agonist-induced increase in [Ca2+]cyt and for refilling Ca2+ into the SR, may play an important role in mediating PASMC growth and stimulating pulmonary vascular medial hypertrophy in patients with PPH (Fig. 9).
We thank S. S. McDaniel, Dr. Ying Yu, and S. Krick for excellent technical assistance.
↵* Authors contributed equally to this work.
This work was supported by National Heart, Lung, and Blood Institute Grants HL-54043 and HL-64945 (to J. X.-J. Yuan) and by the American Heart Association, Mid-Atlantic Affiliate (to V. Golovina). J. X.-J. Yuan is an Established Investigator of the American Heart Association.
Present address of J. Wang: Div. of Gastroenterology, St. Joseph's Health Center, London, Ontario N6A 4V2, Canada.
Address for reprint requests and other correspondence: J. X.-J. Yuan, Dept. of Medicine, Univ. of California at San Diego, 200 West Arbor Dr., San Diego, California 92103-8382 (E-mail:).
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.
- Copyright © 2001 the American Physiological Society