Sustained membrane depolarization and pulmonary artery smooth muscle cell proliferation

Oleksandr Platoshyn1, Vera A. Golovina2, Colleen L. Bailey1, Alisa Limsuwan1, Stefanie Krick1, Magdalena Juhaszova3, Jan E. Seiden2, Lewis J. Rubin1, and Jason X.-J. Yuan1

1 Department of Medicine, University of California School of Medicine, San Diego, California 92103-8382; 2 Departments of Physiology and Medicine, University of Maryland, Baltimore 21201; and 3 National Institute on Aging, Gerontology Research Center, Baltimore, Maryland 21224


    ABSTRACT
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Pulmonary vasoconstriction and vascular medial hypertrophy greatly contribute to the elevated pulmonary vascular resistance in patients with pulmonary hypertension. A rise in cytosolic free Ca2+ ([Ca2+]cyt) in pulmonary artery smooth muscle cells (PASMC) triggers vasoconstriction and stimulates cell growth. Membrane potential (Em) regulates [Ca2+]cyt by governing Ca2+ influx through voltage-dependent Ca2+ channels. Thus intracellular Ca2+ may serve as a shared signal transduction element that leads to pulmonary vasoconstriction and vascular remodeling. In PASMC, activity of voltage-gated K+ (Kv) channels regulates resting Em. In this study, we investigated whether changes of Kv currents [IK(V)], Em, and [Ca2+]cyt affect cell growth by comparing these parameters in proliferating and growth-arrested PASMC. Serum deprivation induced growth arrest of PASMC, whereas chelation of extracellular Ca2+ abolished PASMC growth. Resting [Ca2+]cyt was significantly higher, and resting Em was more depolarized, in proliferating PASMC than in growth-arrested cells. Consistently, whole cell IK(V) was significantly attenuated in PASMC during proliferation. Furthermore, Em depolarization significantly increased resting [Ca2+]cyt and augmented agonist-mediated rises in [Ca2+]cyt in the absence of extracellular Ca2+. These results demonstrate that reduced IK(V), depolarized Em, and elevated [Ca2+]cyt may play a critical role in stimulating PASMC proliferation. Pulmonary vascular medial hypertrophy in patients with pulmonary hypertension may be partly caused by a membrane depolarization-mediated increase in [Ca2+]cyt in PASMC.

intracellular calcium; voltage-gated potassium channels; membrane potential


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MEMBRANE POTENTIAL (Em) 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 Em 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 Em 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.


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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.

Human PASMC (Clonetics) were seeded in flasks at a density of 2,500-3,500 cells/cm2 and incubated in smooth muscle growth medium (SMGM, Clonetics), and the medium was changed after 24 h, followed by every 48 h thereafter. SMGM is composed of smooth muscle basal medium (SMBM) supplemented with 5% FBS, 0.5 ng/ml human epidermal growth factor (hEGF), 2 ng/ml human fibroblast growth factor (hFGF), and 5 µg/ml insulin. Cells were subcultured or plated onto 25-mm coverslips by using trypsin-EDTA buffer (Clonetics) when 70-90% confluence was achieved. The cells at passages 5-8 were used for experimentation. A hemocytometer was used to determine cell counts. The cell number was normalized to the area of the petri dishes (expressed as cells/cm2).

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 alpha -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 alpha -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 MOmega ) 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). Em in single PASMC was measured in current-clamp mode (I = 0) using the patch-clamp technique. In some experiments, Em was recorded using an intracellular electrode (30-100 MOmega ) 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.

Valinomycin (Sigma) was prepared as a 100 mM stock solution in DMSO. Aliquots of the stock solution were diluted 1:1,000 into 0% FBS-DMEM to make a final concentration of 100 µM valinomycin. Similar dilution of DMSO alone was used as a vehicle control in culture media. 4-Aminopyridine (4-AP; Sigma) was directly dissolved into PSS on the day of use. The pH values of all solutions were checked after the addition of drugs and were readjusted to 7.4.

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.


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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 alpha -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 alpha -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.


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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 alpha -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 alpha -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).

The purity of human PASMC in cultures was confirmed by positive staining with smooth muscle alpha -actin antibody compared with a known positive control of smooth muscle cells. The cells also tested negative for factor VIII (von Willebrand's) antigen expression.

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.


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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.

Human PASMC were first plated in petri dishes and cultured in the growth medium SMGM (containing 5% FBS and growth factors). After 24 h, the cells were divided into two groups and cultured in SMGM and SMBM (basal medium without serum and growth factors) for 72 h, respectively. Similarly to the primary cultured rat PASMC, removal of serum and growth factors from culture medium significantly inhibited human PASMC growth (Fig. 2B). These results suggest that cells cultured in serum-free medium are growth-arrested cells and that cells cultured in serum-containing medium are proliferating cells.

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).


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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 Em in growth-arrested and proliferating PASMC. Em 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 Em 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).


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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 Em 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 [IK(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 Em (approximately -40 mV). In these experiments, Ca2+-activated (KCa) and ATP-sensitive (KATP) 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 IK(V) was significantly reduced and the inactivation of IK(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.


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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 IK(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 Em and [Ca2+]cyt in PASMC.


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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.

Increasing the extracellular K+ concentration from 5 to 40 mM shifts the K+ equilibrium potential (EK) from -83 to -31 mV and, therefore, causes membrane depolarization. When rat PASMC were cultured in medium containing 40 mM K+ (NaCl was replaced with equimolar KCl in the customized DMEM), the membrane depolarization (Fig. 7A) significantly increased resting [Ca2+]cyt (Fig. 7B). Because of the large ratio of [Ca2+]SR to [Ca2+]cyt (~10,000:1), a very small change in [Ca2+]cyt would result in a large change in [Ca2+]SR. Indeed, the membrane depolarization-mediated increase in resting [Ca2+]cyt significantly enhanced the ATP (5 µM)-induced increase in [Ca2+]cyt in the absence of extracellular Ca2+ (Fig. 7C). These results suggest that a membrane depolarization-mediated rise in resting [Ca2+]cyt may also increase [Ca2+]SR.


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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.


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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.

In the presence of extracellular Ca2+, treatment of rat PASMC with valinomycin, a K+ ionophore, also significantly inhibited cell growth in media containing serum (Fig. 8). These results imply that valinomycin-induced increase of K+ efflux attenuates PASMC growth in the presence of extracellular Ca2+ and serum by preventing membrane depolarization.


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Excitable and nonexcitable cells both possess a negative resting Em that is close to the EK. Em 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 IK(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 Em 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-kappa B, c-myb) that promote the cell cycle and stimulate the cell division is also Ca2+ dependent (25-27, 48).

In vascular smooth muscle cells, maintaining sufficient Ca2+ in the SR, a cytoplasmic organelle involved in protein sorting and processing as well as lipid synthesis, by the Ca2+ pump in the SR membrane (SERCA) is required for cell growth. Indeed, depletion of the SR Ca2+ by thapsigargin significantly inhibits vascular smooth muscle cell growth (4, 26, 27, 53). A modest change in [Ca2+]cyt would result in a large change in [Ca2+]SR because of the great [Ca2+]SR-to-[Ca2+]cyt ratio (6, 7, 56). The resting [Ca2+]cyt in proliferating PASMC is ~75-85 nM higher than that in growth-arrested cells (Fig. 3), and this would result in a marked increase in [Ca2+]SR. Indeed, sustained membrane depolarization increased resting [Ca2+]cyt and enhanced the ATP-induced rise in [Ca2+]cyt due to Ca2+ mobilization from the SR (Fig. 7). These results suggest that sustained membrane depolarization not only increases [Ca2+]cyt but also augments [Ca2+]SR. Accordingly, persistent increases in [Ca2+]cyt, nuclear [Ca2+], and [Ca2+]SR during membrane depolarization all may be involved in PASMC proliferation (Fig. 9) (5, 25-27, 34, 53).


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Fig. 9.   Schematic diagram depicting the proposed mechanisms responsible for membrane depolarization-mediated increase in [Ca2+]cyt and PASMC proliferation. Decreased (down-arrow ) 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 (up-arrow ) [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 Em 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), Em 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).

Resting Em in vascular smooth muscle cells is primarily determined by the activity of Na+-K+-ATPase and the K+ permeability through K+ channels in the plasma membrane. When K+ channels close, whole cell K+ currents decrease and the cell membrane depolarizes. There are at least four types of K+ channels described in vascular smooth muscle cells (40): 1) voltage-gated K+ (Kv) channels; 2) Ca2+-activated K+ (KCa) channels; 3) inward rectifier K+ (KIR) channels; and 4) ATP-sensitive K+ (KATP) channels. It has been demonstrated that Em in PASMC is regulated by activities of Kv (17, 19, 44-46, 63), KCa (3), and KATP (12) channels. Under resting conditions in which [Ca2+]cyt is low (~100 nM) and ATP level is high (~3 mM), K+ current through Kv channels [IK(V)] is thought to be one of the important determinants of Em in PASMC (17, 19, 44-46, 63). Indeed, inhibition of Kv channels by 4-AP depolarizes PASMC (Fig. 6) (44, 62, 63), whereas activation of Kv channels by nitric oxide hyperpolarizes the cells (31, 65). These results suggest that expression and function of Kv channels may play an important role in the cell proliferation by regulating Em and [Ca2+]cyt.

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 KCa (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.

The transmembrane flux of Ca2+ is mainly determined by the Ca2+ driving force (i.e., the transmembrane electrical potential, Delta E, and the chemical gradient, Delta [Ca2+]). Extracellular [Ca2+] is ~20,000-fold greater than [Ca2+]cyt, which favors the entry of Ca2+ into cells. Therefore, Ca2+ influx is predominantly regulated by the Ca2+ permeability and the transmembrane electrical potential (Delta E = Em - ECa, where ECa is the Ca2+ equilibrium potential). Whereas the Ca2+ permeability is constant, the more negative Delta E is, the greater the inward driving force is for positively-charged ions (e.g., Ca2+). When cells are hyperpolarized (Em becomes more negative), the driving force for Ca2+ influx rises (because Delta E is more negative); when cells are depolarized (Em becomes less negative), the driving force decreases (because Delta E is less negative). Therefore, in cells that do not express L-type voltage-gated Ca2+ channels (e.g., lymphocytes and epithelial cells), Ca2+ influx is decreased by membrane depolarization but increased by membrane hyperpolarization. In these cells, passive Ca2+ leakage, receptor-operated Ca2+ channels, nonselective cation channels, and store-operated Ca2+ channels are the major pathways for Ca2+ to enter the cells (47). In lymphocytes, Kv channels are also permeable to Ca2+ ions, suggesting that the enhanced Ca2+ influx through Kv channels may serve as an additional mechanism involved in increases in [Ca2+]cyt (9).

Conversely, in cells that express L-type voltage-gated Ca2+ channels (e.g., neurons, cardiomyocytes, and smooth muscle cells), membrane depolarization increases [Ca2+]cyt by opening the voltage-gated Ca2+ channels, a major pathway for Ca2+ entry in excitable cells (39, 55, 56, 63). Thus the presence of voltage-gated Ca2+ channels in excitable cells (e.g., neurons and smooth muscle cells), but not in nonexcitable cells (e.g., lymphocytes and epithelial cells), explains why membrane depolarization increases PASMC proliferation (this study), whereas it inhibits lymphocyte proliferation (35). These observations also imply that membrane depolarization may cause opposites effect on Ca2+-dependent cell functions (e.g., proliferation, motility, migration, and contractility) in different cell types.

In summary, a common hypothesis is that vasoconstriction and cell proliferation use overlapping signaling processes that result in parallel intracellular events in pulmonary hypertension. Cytosolic ionized Ca2+ is involved in triggering cell contraction, proliferation, migration, and gene expression. Therefore, abnormalities in regulating cytosolic, nuclear, and SR Ca2+ all may contribute to the elevated pulmonary vascular resistance in patients with pulmonary hypertension. Inhibited Kv channel function and expression as well as membrane depolarization in PASMC have been implicated in primary pulmonary hypertension (62, 66) and hypoxic pulmonary vasoconstriction (17, 19, 24, 36, 44-46, 52, 59, 64). The results from this study suggest that regulation of K+ channel function also may play an important role in PASMC proliferation by modulating Em and [Ca2+]cyt.


    ACKNOWLEDGEMENTS

We thank Y. Yu, S. S. McDaniel, M. A. Sweeney, and N. Kim for assistance.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-54043 and HL-64945 (to J. X.-J. Yuan) and grants from the American Heart Association Mid-Atlantic Affiliate (to V. A. Golovina). J. X.-J. Yuan is an Established Investigator of the American Heart Association (9740091N).

Address for reprint requests and other correspondence: J. Yuan, Dept. of 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.

Received 4 February 2000; accepted in final form 9 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Allbritton, NL, Oancea E, Kuhn MA, and Meyer T. Source of nuclear calcium signals. Proc Natl Acad Sci USA 91: 12458-12462, 1994[Abstract/Free Full Text].

2.   Archer, SL, Huang JM, Hampl V, Nelson DP, Shultz PJ, and Weir EK. Nitric oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K channel by cGMP-dependent protein kinase. Proc Natl Acad Sci USA 91: 7583-7587, 1994[Abstract].

3.   Bae, YM, Park MK, Lee SH, Ho WK, and Earm YE. Contribution of Ca2+-activated K+ channels and nonselective cation channels to membrane potential of pulmonary arterial smooth muscle cells of the rabbit. J Physiol (Lond) 514: 747-758, 1999[Abstract/Free Full Text].

4.   Bailey, CL, MacDaniel SS, Rubin LJ, and Yuan XJ. Role of calcium in human pulmonary artery smooth muscle cell proliferation. FASEB J 14: A578, 2000[ISI].

5.   Berridge, MJ. Calcium signalling and cell proliferation. Bioessays 17: 491-500, 1995[ISI][Medline].

6.   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[Abstract/Free Full Text].

7.   Blaustein, MP. Physiological effects of endogenous ouabain: control of intracellular Ca2+ stores and cell responsiveness. Am J Physiol Cell Physiol 264: C1367-C1387, 1993[Abstract/Free Full Text].

8.   Blaustein, MP, and Lederer WJ. Sodium/calcium exchange: its physiological implications. Physiol Rev 79: 763-854, 1999[Abstract/Free Full Text].

9.   Cahalan, MD, Chandy KG, De Coursey TE, and Gupta S. A voltage-gated potassium channel in human T lymphocytes. J Physiol (Lond) 358: 197-237, 1985[Abstract].

10.   Chao, TS, Byron KL, Lee KM, Villereal M, and Rosner MR. Activation of MAP kinases by calcium-dependent and calcium-independent pathways. Stimulation by thapsigargin and epidermal growth factor. J Biol Chem 267: 19876-19883, 1992[Abstract/Free Full Text].

11.   Christman, BW, McPherson CD, Newman JH, King GA, Bernard GR, Groves BM, and Loyd JE. An imbalance between the excretion of thromboxane and prostacyclin metabolites in pulmonary hypertension. N Engl J Med 327: 70-75, 1992[Abstract].

12.   Clapp, LH, and Gurney AM. ATP-sensitive K+ channels regulate resting potential of pulmonary arterial smooth muscle cells. Am J Physiol Heart Circ Physiol 262: H916-H920, 1992[Abstract/Free Full Text].

13.   Davis, NW, Standen NB, and Stanfield PR. ATP-dependent potassium channels of muscle cells: their properties, regulation, and possible functions. J Bioenerg Biomembr 23: 509-535, 1991[ISI][Medline].

14.   DeCoursey, TE, Chandy KG, Gupta S, and Cahalan MD. Voltage-gated K+ channels in human T lymphocytes: a role in mitogenesis? Nature 307: 465-468, 1984[ISI][Medline].

15.   Dolmetsch, RE, Lewis RS, Goodnow CC, and Healy JI. Differential activation of transcription factors induced by Ca2+ response amplitude and duration. Nature 386: 855-858, 1997[ISI][Medline].

16.   Ehring, GR, Kerschbaum HH, Eder C, Neben AL, Fanger CM, Khoury RJ, Negulescu PA, and Cahalan MD. A nongenomic mechanism for progesterone-mediated immunosuppression: inhibition of K+ channels, Ca2+ signaling, and gene expression in T lymphocytes. J Exp Med 188: 1593-1602, 1998[Abstract/Free Full Text].

17.   Evans, AM, Osipenko ON, and Gurney AM. Properties of a novel K+ current that is active at resting potential in rabbit pulmonary artery smooth muscle cells. J Physiol (Lond) 496: 407-420, 1996[Abstract].

18.   Fleischmann, BK, Murray RK, and Kotlikoff MI. Voltage window for sustained elevation of cytosolic calcium in smooth muscle cells. Proc Natl Acad Sci USA 91: 11914-11918, 1994[Abstract/Free Full Text].

19.   Gelband, CH, and Gelband H. Ca2+ release from intracellular stores is an initial step in hypoxic pulmonary vasoconstriction of rat pulmonary artery resistance vessels. Circulation 96: 3647-3654, 1997[Abstract/Free Full Text].

20.   Giaid, A, and Saleh D. Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. N Engl J Med 333: 214-221, 1995[Abstract/Free Full Text].

21.   Giaid, A, Yanagisawa M, Langleben D, Michel RP, Levy R, Shennib H, Kimura S, Masaki T, Duguid WP, and Stewart DJ. Expression of endothelin-1 in the lungs of patients with pulmonary hypertension. N Engl J Med 328: 1732-1739, 1993[Abstract/Free Full Text].

22.   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].

23.   Hamill, OP, Marty A, Neher E, Sakmann B, and Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Arch 391: 85-100, 1981[ISI][Medline].

24.   Harder, DR, Madden JA, and Dawson C. Hypoxic induction of Ca2+-dependent action potentials in small pulmonary arteries of the cat. J Appl Physiol 59: 1389-1393, 1985[Abstract/Free Full Text].

25.   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].

26.   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[Abstract/Free Full Text].

27.   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[Abstract/Free Full Text].

28.   Jensen, BS, Odum N, Jorgensen NK, Christophersen P, and Olesen SP. Inhibition of T cell proliferation by selective block of Ca2+-activated K+ channels. Proc Natl Acad Sci USA 96: 10917-10921, 1999[Abstract/Free Full Text].

29.   Johnson, CM, Hill CS, Chawla S, Treisman R, and Bading H. Calcium controls gene expression via three distinct pathways that can function independently of the Ras/mitogen-activated protein kinases (ERKs) signaling cascade. J Neurosci 17: 6189-6202, 1997[Abstract/Free Full Text].

30.   Khanna, R, Chang MC, Joiners WJ, Kaczmareks LK, and Schlichter LC. hSK4/hIK1, a calmodulin-binding KCa channel in human T lymphocytes. J Biol Chem 274: 14838-14849, 1999[Abstract/Free Full Text].

31.   Koh, SD, Campbell JD, Carl A, and Sanders KM. Nitric oxide activates multiple potassium channels in canine colonic smooth muscle. J Physiol (Lond) 489: 735-743, 1995[Abstract].

32.   Kohomoto, O, Levi AJ, and Bridge JH. Relation between reverse sodium-calcium exchange and sarcoplasmic reticulum calcium release in guinea pig ventricular cells. Circ Res 74: 550-554, 1994[Abstract].

33.   Lepple-Wienhues, A, Berweck S, Bohmig M, Leo CP, Meyling B, Garbe C, and Wiederholt M. K+ channels and the intracellular calcium signal in human melanoma cell proliferation. J Membr Biol 151: 149-157, 1996[ISI][Medline].

34.   Li, W, Llopis J, Whitney M, Zlokarnik G, and Tsien RY. Cell-permeant caged InsP3 ester shows that Ca2+ spike frequency can optimize gene expression. Nature 392: 936-941, 1998[ISI][Medline].

35.   Lin, CS, Boltz RC, Blake JT, Nguyen M, Talento A, Fischer PA, Springer MS, Sigal NH, Slaughter RS, and Garcia ML. Voltage-gated potassium channels regulate calcium-dependent pathways involved in human T lymphocyte activation. J Exp Med 177: 637-645, 1993[Abstract].

36.   Madden, JA, Vadula MS, and Kurup VP. Effects of hypoxia and other vasoactive agents on pulmonary and cerebral artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 263: L384-L393, 1992[Abstract/Free Full Text].

37.   Means, AR. Calcium, calmodulin and cell cycle regulation. FEBS Lett 347: 1-4, 1994[ISI][Medline].

38.   Moncada, S, Palmer RM, and Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 43: 109-142, 1991[ISI][Medline].

39.   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[Abstract/Free Full Text].

40.   Nelson, MT, and Quayle JM. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol Cell Physiol 268: C799-C822, 1995[Abstract/Free Full Text].

41.   Nilius, B, and Wohlrab W. Potassium channels and regulation of proliferation of human melanoma cells. J Physiol (Lond) 445: 537-548, 1992[Abstract].

42.   Palevsky, HI, Schloo BL, Pietra GG, Weber KT, Janicki JS, Rubin E, and Fishman AP. Primary pulmonary hypertension. Vascular structure, morphometry, and responsiveness to vasodilator agents. Circulation 80: 1207-1221, 1989[Abstract].

43.   Pauly, RR, Bilato C, Sollott SJ, Monticone R, Kelly PT, Lakatta EG, and Crow MT. Role of calcium/calmodulin-dependent protein kinase II in the regulation of vascular smooth muscle cell migration. Circulation 91: 1107-1115, 1995[Abstract/Free Full Text].

44.   Peng, W, Karwande SV, Hoidal JR, and Farrukh IS. Potassium currents in cultured human pulmonary arterial smooth muscle cells. J Appl Physiol 80: 1187-1196, 1996[Abstract/Free Full Text].

45.   Post, JM, Gelband CH, and Hume JR. [Ca2+]i inhibition of K+ channels in canine pulmonary artery, novel mechanisms for hypoxia-induced membrane depolarization. Circ Res 77: 131-139, 1995[Abstract/Free Full Text].

46.   Post, JM, Hume JR, Archer SL, and Weir EK. Direct role for potassium channel inhibition in hypoxic pulmonary vasoconstriction. Am J Physiol Cell Physiol 262: C882-C890, 1992[Abstract/Free Full Text].

47.   Putney, JW, Jr, and Bird GS. The inositol phosphate-calcium signaling system in nonexcitable cells. Endocr Rev 14: 610-631, 1993[ISI][Medline].

48.   Rosen, LB, Ginty DD, Weber MJ, and Greenberg ME. Membrane depolarization and calcium influx stimulate MEK and MAP kinase via activation of Ras. Neuron 12: 1207-1221, 1994[ISI][Medline].

49.   Sham, JS, Cleemann L, and Morad M. Gating of the cardiac Ca2+ release channel: the role of Na+ current and Na+-Ca2+ exchange. Science 255: 850-853, 1992[ISI][Medline].

50.   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].

51.   Shimoda, LA, Sylvester JT, and Sham JS. Inhibition of voltage-gated K+ current in rat intrapulmonary arterial myocytes by endothelin-1. Am J Physiol Lung Cell Mol Physiol 274: L842-L853, 1998[Abstract/Free Full Text].

52.   Shimoda, LA, Sylvester JT, and Sham JS. Chronic hypoxia alters effects of endothelin and angiotensin on K+ currents in pulmonary arterial myocytes. Am J Physiol Lung Cell Mol Physiol 277: L431-L439, 1999[Abstract/Free Full Text].

53.   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].

54.   Skalli, O, Ropraz P, Trzeciak A, Benzonana G, Gillessen D, and Gabbiani G. A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation. J Cell Biol 103: 2787-2796, 1986[Abstract].

55.   Somlyo, AP, and Somlyo AV. Signal transduction and regulation in smooth muscle. Nature 372: 231-236, 1994[ISI][Medline].

56.   Tsien, RW, and Tsien RY. Calcium channels, stores, and oscillations. Annu Rev Cell Biol 6: 715-760, 1990[ISI].

57.   Volk, KA, Matsuda JJ, and Shibata EF. A voltage-dependent potassium current in rabbit coronary artery smooth muscle cells. J Physiol (Lond) 439: 751-768, 1991[Abstract].

58.   Wagenvoort, CA. Vasoconstriction and medial hypertrophy in pulmonary hypertension. Circulation 22: 535-546, 1960[ISI].

59.   Wang, J, Juhaszova M, Rubin LJ, and Yuan X-J. Hypoxia inhibits gene expression of voltage-gated K+ channel alpha subunits in pulmonary artery smooth muscle cells. J Clin Invest 100: 2347-2353, 1997[Abstract/Free Full Text].

60.   Wang, JY, Wang J, Golovina VA, Li L, Platoshyn O, and Yuan JX-J. Role of K+ channel expression in polyamine-dependent intestinal epithelial cell migration. Am J Physiol Cell Physiol 278: C303-C314, 2000[Abstract/Free Full Text].

61.   Yu, SP, Yeh CH, Sensi SL, Gwag BJ, Canzoniero LM, Farhangrazi ZS, Ying HS, Tian M, Dugan LL, and Choi DW. Mediation of neuronal apoptosis by enhancement of outward potassium current. Science 278: 114-117, 1997[Abstract/Free Full Text].

62.   Yuan, JX-J, Aldinger AM, Juhaszova M, Wang J, Conte JV, Jr, 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[Abstract/Free Full Text].

63.   Yuan, X-J. Voltage-gated K+ currents regulate resting membrane potential and [Ca2+]i in pulmonary arterial myocytes. Circ Res 77: 370-378, 1995[Abstract/Free Full Text].

64.   Yuan, X-J, Goldman WF, Tod ML, Rubin LJ, and Blaustein MP. Hypoxia reduces potassium currents in cultured rat pulmonary but not mesenteric arterial myocytes. Am J Physiol Lung Cell Mol Physiol 264: L116-L123, 1993[Abstract/Free Full Text].

65.   Yuan, X-J, Tod ML, Rubin LJ, and Blaustein MP. NO hyperpolarizes pulmonary artery smooth muscle cells and decreases the intracellular Ca2+ concentration by activating voltage-gated K+ channels. Proc Natl Acad Sci USA 93: 10489-10494, 1996[Abstract/Free Full Text].

66.   Yuan, X-J, Wang J, Juhaszova M, Gaine SP, and Rubin LJ. Attenuated K+ channel gene transcription in primary pulmonary hypertension. Lancet 351: 726-727, 1998[ISI][Medline].


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