INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

MEMBRANE POTENTIAL (E_m) controls cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) mainly by modulating activity of sarcolemmal voltage-dependent Ca²⁺ channels (39, 56). Membrane depolarization opens voltage-gated Ca²⁺ channels, increases Ca²⁺ influx, and raises [Ca²⁺]_cyt in smooth muscle cells (18, 39, 63). Membrane depolarization may also promote Ca²⁺ entry via the reverse mode of Na⁺/Ca²⁺ exchange (Ca²⁺-entry/Na⁺-exit mode), which triggers Ca²⁺-induced Ca²⁺ release from ryanodine-sensitive Ca²⁺ stores and further increases [Ca²⁺]_cyt (8, 32, 49). Cytoplasmic ionized Ca²⁺ serves as a critical signal transduction element in a variety of cell functions, such as contraction (55), migration (43), proliferation (5, 37), and gene expression (15, 29, 34). A rise in [Ca²⁺]_cyt triggers vasoconstriction (55), stimulates smooth muscle cell proliferation (5, 37) and migration (43), and, thus, may serve as a shared signal transduction element for pulmonary vasoconstriction and medial hypertrophy observed in patients with pulmonary hypertension. Persistent membrane depolarization causes sustained increase in [Ca²⁺]_cyt (18) and, thus, should have a constant stimulatory effect on pulmonary vasoconstriction and pulmonary artery smooth muscle cell (PASMC) proliferation. Furthermore, maintenance of sufficient Ca²⁺ within sarcoplasmic (or endoplasmic) reticulum (SR), a major intracellular Ca²⁺ store, is necessary for cell growth. Indeed, depletion of the SR Ca²⁺ stores induces growth arrest in vascular smooth muscle cells (53).

Pulmonary vasoconstriction and vascular medial thickening (due to smooth muscle cell proliferation and migration) greatly contribute to the elevated pulmonary vascular resistance in patients with pulmonary hypertension (42, 58). PASMC from patients with primary pulmonary hypertension (PPH) have more depolarized E_m and higher resting [Ca²⁺]_cyt than cells from normal subjects and patients with normotensive cardiopulmonary diseases and secondary pulmonary hypertension (e.g., congenital heart disease, pulmonary thromboembolic disease). The membrane depolarization and elevated resting [Ca²⁺]_cyt in PASMC from PPH patients might be, at least partly, due to inhibited expression and function of voltage-gated K⁺ (Kv) channels (62, 66). Furthermore, increases in [Ca²⁺]_cyt and membrane depolarization due to decreased Kv channel activity in PASMC have also been implicated in hypoxic pulmonary vasoconstriction (19, 24, 36, 45, 46, 59, 64).

An imbalanced ratio of endothelium-derived relaxing factors (EDRF) and constricting factors (EDCF) may contribute to the development of pulmonary hypertension (11, 20, 21). EDRF (e.g., NO and prostacyclin) and EDCF (e.g., endothelin-1 and thromboxane A₂) both affect E_m by modulating K⁺ channel activity in PASMC (2, 38, 51, 65). Therefore, increases in [Ca²⁺]_cyt induced by membrane depolarization, in addition to triggering muscle contraction, may also play an important role in stimulating cell proliferation (48, 50) and migration (43).

The pulmonary medial (and myointimal) hypertrophy, mainly induced by PASMC proliferation and migration, is a critical contributor to the elevated pulmonary vascular resistance in patients with severe pulmonary hypertension. The rationale of this study was to test the hypotheses that 1) an increase in [Ca²⁺]_cyt due to Ca²⁺ influx is essential for PASMC growth, and 2) the elevated [Ca²⁺]_cyt results partly from membrane depolarization induced by Kv channel inhibition.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell preparation and culture. Primary cultures of PASMC were prepared from Sprague-Dawley rats (63). The intrapulmonary artery branches as well as the right branch of the main pulmonary artery were incubated for 20 min in Hanks' balanced salt solution with 1.5 mg/ml collagenase (Worthington). After the incubation, a thin layer of adventitia was carefully stripped off, and endothelium was removed by gently scratching the intimal surface. The remaining smooth muscle was digested with 1.75 mg/ml collagenase, 0.5 mg/ml elastase, and 1 mg/ml albumin (Sigma) for 45 min at 37°C. The cells were plated onto 25-mm coverslips in petri dishes and cultured in 10% fetal bovine serum (FBS)-DMEM in a 37°C, 5% CO₂, humidified incubator.

Immunofluorescence labeling. The primary cultured rat PASMC were fixed in 95% ethanol and stained with the membrane-permeable nucleic acid stain 4',6'-diamidino-2-phenylindole (DAPI; 5 µM; Molecular Probes). The fluorescence emitted at 461 nm was used to visualize the cell nuclei and estimate total cell numbers in the cultures. The specific monoclonal antibody raised against smooth muscle -actin (Boehringer Mannheim) was used to evaluate the cellular purity of cultures (54), and a secondary antibody conjugated with indocarbocyanine (Cy3) (Jackson ImmunoResearch) was used to display the fluorescence image (emitted at 570 nm). The cells were mounted in 10% 1 M Tris · HCl-90% glycerol (pH 8.5) containing 1 mg/ml p-phenylenediamine. The cell images were processed by a MetaMorph Imaging System (Universal Imaging); the Cy3 fluorescence was colored red and the DAPI fluorescence was colored green to display images with red-green overlay. The DAPI-stained cells that also cross-reacted with the smooth muscle cell -actin antibody were defined as smooth muscle cells.

Electrophysiological measurements. Whole cell currents were recorded with an Axopatch-1D amplifier and a DigiData 1200 interface (Axon Instruments) by using patch-clamp techniques (23). Patch pipettes (2-4 M) were fabricated on a Sutter electrode puller with the use of borosilicate glass tubes and were fire polished on a Narishige microforge. Step-pulse protocols and data acquisition were performed with pCLAMP software. Currents were filtered at 1-2 kHz (3 dB) and digitized at 2-4 kHz by using the amplifier. All experiments were performed at room temperature (22-24°C). E_m in single PASMC was measured in current-clamp mode (I = 0) using the patch-clamp technique. In some experiments, E_m was recorded using an intracellular electrode (30-100 M) filled with 3 M KCl. The data were acquired by an electrometer (Electro 705; World Precision) coupled to an IBM-compatible computer and were analyzed using the DATAQ data acquisition software (Dataq Instruments).

Measurement of [Ca²+]_cyt. Cells were loaded with the acetoxymethyl ester form of fura 2 (fura 2-AM; 3 µM) for 30 min at room temperature (24°C) under an atmosphere of 5% CO₂ in air. The fura 2-loaded cells were then superfused with standard bath solution for 20 min at 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) images from the cells and background were obtained with the use of a Gen III charge-coupled device camera (Stanford Photonics) coupled to a Carl Zeiss microscope. 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 gray scale. 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 one every 60 s when [Ca²⁺]_cyt was stable. [Ca²⁺]_cyt was calculated from fura 2 fluorescence emission excited at 380 and 360 nm by using the ratio method (22). In most experiments, multiple cells (usually 6-10) were imaged in single field, and one arbitrarily chosen peripheral cytosolic area (10-12 × 10-12 pixels) from each cell was spatially averaged.

Solution and reagents. A coverslip containing the cells was positioned in the recording chamber (~0.75 ml) and superfused (2-3 ml/min) with the standard extracellular (bath) physiological salt solution (PSS) for recording K⁺ currents or measuring [Ca²⁺]_cyt. The PSS contained (in 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 or 2 M Tris. In Ca²⁺-free PSS, CaCl₂ was replaced by equimolar MgCl₂, and 1 mM EGTA was added to chelate residual Ca²⁺. The internal (pipette) solution for recording whole cell K⁺ currents contained (in mM) 125 KCl, 4 MgCl₂, 10 HEPES, 10 EGTA, and 5 Na₂ATP, buffered to pH 7.2 with 2 M Tris.

Statistical analysis. Data are expressed as means ± SE. Statistical analysis was performed using the unpaired Student's t-test, or analysis of variance, as indicated. Differences were considered to be significant when P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Purity of PASMC in primary cultures. Pulmonary arteries, isolated from rat lungs, have a trilamellar structure that is composed of fibroblasts (adventitia), smooth muscle cells (media), and endothelial cells (intima). Thus smooth muscle cell cultures are often contaminated with other cells, especially fibroblasts because of their rapid growth rate. Morphologically, the primary cultures of fibroblasts and smooth muscle cells are hardly distinguishable when using phase-contrast microscopy (Fig. 1A, top). In this study, adventitia was enzymatically removed before the smooth muscle cell suspension was prepared. In the primary cultures prepared from pulmonary arteries in which adventitia was intentionally removed, virtually all of the cells labeled with DAPI cross-reacted with the smooth muscle -actin antibody (Fig. 1A, left). This indicates that >99.5% of the cells in cultures were smooth muscle cells (Fig. 1B). However, in the primary cultures prepared directly from isolated pulmonary arteries, only 43% of the cells labeled with DAPI cross-reacted with the smooth muscle -actin antibody (Fig. 1A, right), indicating substantial contamination of fibroblasts (Fig. 1B). Thus primary cultures prepared from the adventitia-free rat pulmonary arteries were used in this study.

View larger version (63K): [in this window] [in a new window]   Fig. 1.   Purity of rat pulmonary artery (PA) smooth muscle cells (SMC) in primary cultures. A: phase-contrast photomicrographs (top) showing primary cultured cells prepared from the adventitia-free [PA(Adv)] or adventitia-intact [PA(+Adv)] PA. Primary cultures (bottom) that were stained with the smooth muscle -actin antibody (red) and the nucleic acid dye 4',6'-diamidino-2-phenylindole (DAPI; green) show typical actin filament in PASMC (SMC) but not in fibroblasts (FB). Bar = 20 µm. B: percentage of the cells stained by both -actin antibody and nucleic acid dye (SMC) and the cells stained by only nucleic acid dye (FB) per each detected field in the primary cultures. Data are means ± SE (n = 28 coverslips of cells).

Serum deprivation inhibits PASMC growth. Primary cultured rat PASMC were divided into three groups and incubated in DMEM containing 10%, 0.3%, and 0.1% FBS for 72 h, respectively. As shown in Fig. 2A, reducing serum concentration from 10% to 0.1% in culture media significantly inhibited cell growth, suggesting that the cells cultured in 0.1% FBS or serum-free (data not shown) DMEM are growth arrested after 24-48 h.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Inhibition of PASMC growth by serum deprivation. A: rat PASMC cultured in DMEM containing 10%, 0.3%, and 0.1% fetal bovine serum (FBS). Cell numbers were determined with a hemocytometer after 3 days. Data are means ± SE (n = 18 dishes of cells). ** P < 0.01, *** P < 0.001 vs. 10% FBS. B: human PASMC cultured in smooth muscle basal medium (SMBM) or SMBM supplemented with 5% FBS and growth factors [smooth muscle growth medium (SMGM)]. Cell numbers were determined at 1, 2, and 4 days after they were plated. Data are means ± SE (n = 16 dishes of cells). ** P < 0.01, *** P < 0.001 vs. SMGM.

Resting [Ca²+]_cyt in growth-arrested and proliferating PASMC. There were no obvious morphological changes between growth-arrested and proliferating PASMC from rats (Fig. 3A) and humans. However, resting [Ca²⁺]_cyt, measured in peripheral cytosolic areas, was significantly higher in proliferating cells than in growth-arrested rat (Fig. 3B) and human (Fig. 3D) cells. In rat PASMC, the histogram of resting [Ca²⁺]_cyt indicated a right shift (by ~85 nM) in proliferating cells compared with growth-arrested cells (Fig. 3C). The ratio of [Ca²⁺]_cyt to intracellularly stored [Ca²⁺] in the SR ([Ca²⁺]_SR) is ~1:10,000 (6, 7). Therefore, the sustained elevation of [Ca²⁺]_cyt in proliferating PASMC may cause a large increase in [Ca²⁺]_SR (6, 7), both of which are essential for mitogen-mediated cell growth (5, 37, 53).

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Resting cytosolic free Ca2+ concentration ([Ca2+]cyt) in growth-arrested and proliferating rat and human PASMC. A: fura 2 fluorescence (excitation at 360 nm) images (top) showing the rat cells in which [Ca2+]cyt was measured (bar = 15 µm) and pseudocolor images (bottom) showing resting [Ca2+]cyt in rat PASMC cultured in DMEM containing 0% (left) or 10% (right) FBS. B: resting [Ca2+]cyt in rat PASMC cultured in DMEM contained 0% or 10% FBS. Data are means ± SE. *** P < 0.001 vs. 0% FBS. C: histogram of resting [Ca2+]cyt levels, constructed from the data in B, in growth-arrested (0% FBS) and proliferating (10% FBS) rat PASMC. D: resting [Ca2+]cyt in growth-arrested (SMBM) and proliferating (SMGM) human PASMC. Data are means ± SE, with numbers of cells tested shown in parentheses. *** P < 0.001 vs. SMBM.

Resting E_m in growth-arrested and proliferating PASMC. E_m is an important determinant of resting [Ca²⁺]_cyt in smooth muscle cells because of the voltage dependence of Ca²⁺ influx through L-type voltage-gated Ca²⁺ channels (18, 39). Consistent with the results of [Ca²⁺]_cyt, resting E_m in proliferating PASMC was much more depolarized than that in growth-arrested PASMC from rats and humans (Fig. 4). In smooth muscle cells, the voltage window of sarcolemmal L-type voltage-gated Ca²⁺ channels for sustained elevation of [Ca²⁺]_cyt ranges from 40 to 20 mV and peaks at 30 mV (18). Thus the sustained membrane depolarization in proliferating PASMC may produce a constant Ca²⁺ influx through L-type voltage-gated Ca²⁺ channels and may contribute to maintain the elevated [Ca²⁺]_cyt that is crucial for cell proliferation (29, 48, 50).

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Resting membrane potential (Em) in growth-arrested and proliferating PASMC from rats (A) and humans (B). A: Em was measured in rat PASMC cultured in media containing 0.1% (n = 100 cells) or 10% (n = 115 cells) FBS with the use of intracellular electrodes filled with 3 M KCl. Data are means ± SE. *** P < 0.001 vs. 0.1% FBS. B: Em was measured in human PASMC cultured in SMBM (n = 25 cells) or SMGM (n = 21 cells) with the use of whole cell current clamp techniques. Data are means ± SE. ** P < 0.01 vs. SMBM.

Whole cell Kv currents in growth-arrested and proliferating human PASMC. Resting E_m is regulated by activities of multiple K⁺ channels, including Kv channels in PASMC (17, 44, 45, 63). Whether the membrane depolarization in proliferating cells resulted from inhibition of Kv channels was examined in human PASMC. Whole cell Kv currents [I_K(V)] were elicited by depolarizing the cells from a holding potential of 70 mV to a series of test potentials ranging from 80 to +80 mV in increments of 20 mV (Fig. 5A). The currents appeared to be activated at potentials close to the resting E_m (approximately 40 mV). In these experiments, Ca²⁺-activated (K_Ca) and ATP-sensitive (K_ATP) K⁺ currents were minimized by the removal of extracellular (bath) and intracellular (pipette) Ca²⁺ (plus 1-10 mM EGTA) and the inclusion of 5 mM ATP in the pipette solution (12, 13, 44, 57). In proliferating PASMC, the amplitude of whole cell I_K(V) was significantly reduced and the inactivation of I_K(V) (elicited by a +80 mV test potential) was accelerated compared with growth-arrested cells (Fig. 5). These results suggest that inhibition of Kv channel function may account for the membrane depolarization during PASMC proliferation.

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Whole cell voltage-gated K+ (Kv) currents [IK(V)] in growth-arrested and proliferating PASMC. A: representative families of the currents, elicited by depolarizing the cells from a holding potential of 70 mV to a series of test potentials ranging from 80 to +80 mV in 20-mV increments, in human PASMC cultured in SMBM (without serum and growth factors) and SMGM (with 5% FBS and growth factors) for 2-3 days. B: current-voltage (I-V) relationship of steady-state currents (measured at 290-300 ms) in human PASMC cultured in SMBM (n = 16 cells) or SMGM (n = 17 cells). C: kinetics of the current inactivation. The currents, elicited by a test potential of +80 mV (holding potential, 70 mV), were averaged from all of the cells tested and normalized to the maximal amplitude of each of the current records.

Effects of membrane depolarization on resting [Ca²+]_cyt and ATP-induced rises in [Ca²⁺]_cyt. Cell membrane can be depolarized by either inhibition of K⁺ permeability through membrane K⁺ channels or alteration of the transmembrane K⁺ concentration gradient. Indeed, extracellular application of 4-AP, a blocker of Kv channels, reduced whole cell I_K(V) by 58 ± 7% at +60 mV and 57 ± 8% at +80 mV (n = 9, P < 0.001) in rat PASMC (Fig. 6A). The resultant membrane depolarization induced Ca²⁺-dependent action potentials (Fig. 6B) and increased [Ca²⁺]_cyt by 155 ± 11 nM (n = 17, P < 0.001) (Fig. 6C). These results suggest that activity of Kv channels plays an important role in the regulation of E_m and [Ca²⁺]_cyt in PASMC.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Effects of 4-aminopyridine (4-AP) on whole cell IK(V), Em, and [Ca2+]cyt in rat PASMC. A: representative families of the currents, elicited by depolarizing the cell from a holding potential of 70 mV to a series of test potentials ranging from 60 mV to +80 mV in 20-mV increments, before (control) and during (4-AP) application of 5 mM 4-AP. B: Em was measured using whole cell current-clamp (I = 0) techniques before, during, and after application of 4-AP. C: [Ca2+]cyt in peripheral area of PASMC was measured using fluorescence microscopy before, during, and after application of 4-AP.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Augmenting effects of membrane depolarization on resting [Ca2+]cyt and ATP-induced rises in [Ca2+]cyt in rat PASMC. Cells were cultured in media containing 5 (control) or 40 mM K+ (NaCl was replaced by equimolar KCl) for 2 days. Resting Em (A), [Ca2+]cyt (B), and the ATP (5 µM)-induced [Ca2+]cyt increases in the absence of extracellular Ca2+ (C) were then measured. Cont, control; 40 K, 40 mM K+. Data are means ± SE, with numbers of cells tested shown in parentheses. * P < 0.05, *** P < 0.001 vs. control.

Effects of extracellular Ca²+ chelation and K⁺ ionophore on PASMC growth in media containing serum. In addition to activating contractile proteins in the cytosol, a rise in [Ca²⁺]_cyt due to Ca²⁺ influx through the plasmalemmal Ca²⁺ channels can activate mitogen-activated protein kinase (MAPK) (10) (which is part of the phosphorylation cascade that leads to activation of DNA synthesis-promoting factor) and can rapidly increase nuclear [Ca²⁺] (1). These effects would promote cell proliferation by moving quiescent cells into the cell cycle and propelling the proliferating cells through mitosis (5, 37). The addition of 2 mM EGTA, a Ca²⁺ chelator, to culture media decreased the free [Ca²⁺] from 1.6 mM to ~525 nM and almost abolished rat PASMC growth in the presence of serum and growth factors (Fig. 8). These data are consistent with our previous observations (4) in human PASMC that chelation of extracellular Ca²⁺ with 2 mM EGTA significantly inhibited cell growth in media containing 5% FBS and growth factors.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Inhibition of rat PASMC growth by chelating extracellular Ca2+ and valinomycin. A: cells were cultured in 10% FBS-DMEM in the absence (Cont) and presence of 2 mM EGTA or 100 µM valinomycin (Val). Viable cell numbers were determined 1, 4, and 6 days after cells were plated. Data are means ± SE (n = 12 dishes of cells/group). ** P < 0.01, *** P < 0.001 vs. control.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Excitable and nonexcitable cells both possess a negative resting E_m that is close to the E_K. E_m has been demonstrated to control electrical excitability (e.g., generation and propagation of action potentials) (39), muscle contraction (55), secretion, apoptosis (61), and gene expression (25, 29, 48, 50). The results from this study demonstrate that whole cell I_K(V) was reduced, the cell membrane was depolarized, and resting [Ca²⁺]_cyt was elevated in proliferating PASMC compared with growth-arrested cells. The membrane depolarization apparently resulted from the reduced Kv channel activity and led to the elevated [Ca²⁺]_cyt by activating L-type voltage-gated Ca²⁺ channels (18, 29, 63). Transition of Na⁺/Ca²⁺ exchangers from the Ca²⁺ exit (forward) mode to Ca²⁺ entry (reverse) mode may also partially contribute to the increase in [Ca²⁺]_cyt during membrane depolarization (8, 32, 49). An increase in [Ca²⁺]_cyt is believed to play an important role in stimulating cell growth by activating signal transduction proteins in the cytosol and transcription factors in the nucleus that are essential for the progression of the cell cycle (10, 25, 27, 29, 48, 50). The observations from the present study suggest that activity of Kv channels in PASMC may play an important role in modulating cell growth by regulating E_m and [Ca²⁺]_cyt.

Role of intracellular Ca²+ in cell growth. Reduction of extracellular Ca²⁺ from 1.6 mM to ~525 nM with the Ca²⁺ chelator EGTA significantly inhibits human and rat (Fig. 8) PASMC growth in media containing 5% FBS and growth factors (4, 53). This suggests that a constant Ca²⁺ influx through sarcolemmal Ca²⁺ channels (e.g., voltage-dependent, receptor-operated, and store-operated Ca²⁺ channels) is involved in PASMC proliferation. Ca²⁺ diffuses rapidly between the cytosol and the nucleus (1). Therefore, a rise in [Ca²⁺]_cyt can increase nuclear [Ca²⁺] within 200 ms (1). Ca²⁺ in the nucleoplasm promotes cell proliferation by moving quiescent cells into the cell cycle and propelling the proliferating cells through mitosis (5, 25, 37). Ca²⁺ in the cytoplasm activates the Ca²⁺-sensitive signal transduction proteins that are in the cascade to stimulate cell division. For example, membrane depolarization-induced Ca²⁺ influx activates MAPK, which phosphorylates the downstream protein kinases and stimulates the cell cycle progression (10, 25, 48, 50). Expression of many transcription factors (e.g., c-fos/c-jun, Ras, nuclear factor-B, c-myb) that promote the cell cycle and stimulate the cell division is also Ca²⁺ dependent (25-27, 48).

View larger version (37K): [in this window] [in a new window]   Fig. 9.   Schematic diagram depicting the proposed mechanisms responsible for membrane depolarization-mediated increase in [Ca2+]cyt and PASMC proliferation. Decreased () Kv channel expression and activity lead to the reduction of whole cell IK(V) and membrane depolarization. The resultant opening of voltage-gated Ca2+ channels increases () [Ca2+]cyt. Because of the high ratio of intracellularly stored [Ca2+] in the SR ([Ca2+]SR) to [Ca2+]cyt and the minimal resistance of nuclear membrane to Ca2+, a rise in [Ca2+]cyt subsequently increases [Ca2+]SR and nuclear [Ca2+] ([Ca2+]n). c-fos is a Ca2+-responsive gene that contains 2 Ca2+-sensitive elements in its promotor: the serum response element that binds with serum response factor (SRF) and ternary complex factor (TCF), and the cAMP response element that binds to cAMP response element binding protein (CREB). Activation of the early-responsive gene expression by cytosolic and nuclear Ca2+ as well as increase of the sarcoplasmic/endoplasmic reticular (SR/ER) function (e.g., protein sorting and processing, and lipid synthesis) by [Ca2+]SR would combine to stimulate cell proliferation.

Regulation of [Ca²+]_cyt and E_m in PASMC. In vascular smooth muscle cells, [Ca²⁺]_cyt can be increased by either Ca²⁺ influx through Ca²⁺ channels in the plasma membrane or Ca²⁺ release (mobilization) from intracellular stores (mainly SR). Because of the voltage dependence of the sarcolemmal voltage-gated Ca²⁺ channels (18, 39), E_m serves as a major regulator of [Ca²⁺]_cyt in PASMC. The voltage window for sustained elevation of [Ca²⁺]_cyt of smooth muscle voltage-gated Ca²⁺ channels ranges from 40 to 25 mV (18), suggesting that the sustained membrane depolarization (Fig. 5) may be the cause for the increased resting [Ca²⁺]_cyt in PASMC during proliferation (Fig. 3).

Divergent effects of membrane depolarization on cell growth in excitable and nonexcitable cells. It has been demonstrated that membrane depolarization induced by attenuating Kv (9, 14, 16) and K_Ca (28, 30) channel activity inhibits cell proliferation in nonexcitable cells, such as human T lymphocytes (35), melanoma cells (33, 41), and intestinal epithelial cells (60). However, membrane depolarization induced by attenuating K⁺ channel activity stimulates cell proliferation in excitable cells, such as neurons (29, 50) and smooth muscle cells (e.g., this study). The reason for the opposite effects of membrane depolarization on cell proliferation in nonexcitable (e.g., lymphocytes) and excitable (e.g., neurons and PASMC) cells is that the nonexcitable cells do not express voltage-gated Ca²⁺ channels.