Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California School of Medicine, San Diego, California 92103
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ABSTRACT |
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Cell
shrinkage is an incipient hallmark of apoptosis in a variety of
cell types. The apoptotic volume decrease has been demonstrated to
attribute, in part, to K+ efflux; blockade of plasmalemmal
K+ channels inhibits the apoptotic volume decrease and
attenuates apoptosis. Using combined approaches of gene
transfection, single-cell PCR, patch clamp, and fluorescence
microscopy, we examined whether overexpression of Bcl-2, an
anti-apoptotic oncoprotein, inhibits apoptosis in pulmonary
artery smooth muscle cells (PASMC) by diminishing the activity of
voltage-gated K+ (Kv) channels. A human bcl-2
gene was infected into primary cultured rat PASMC using an adenoviral
vector. Overexpression of Bcl-2 significantly decreased the amplitude
and current density of Kv currents (IKv). In
contrast, the apoptosis inducer staurosporine (ST) enhanced
IKv. In bcl-2-infected cells,
however, the ST-induced increase in IKv was
completely abolished, and the ST-induced apoptosis was
significantly inhibited compared with cells infected with an empty
adenovirus (bcl-2). Blockade of Kv channels in control cells (
bcl-2) by 4-aminopyridine also inhibited the
ST-induced increase in IKv and
apoptosis. Furthermore, overexpression of Bcl-2 accelerated the
inactivation of IKv and downregulated the mRNA
expression of the pore-forming Kv channel
-subunits (Kv1.1, Kv1.5,
and Kv2.1). These results suggest that inhibition of Kv channel
activity may serve as an additional mechanism involved in the
Bcl-2-mediated anti-apoptotic effect on vascular smooth muscle cells.
apoptotic volume decrease; pulmonary artery smooth muscle cells; current density of voltage-gated potassium channels
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INTRODUCTION |
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APOPTOSIS IS AN EVOLUTIONARILY conserved process that plays a critical role in embryonic development and tissue homeostasis. In humans, dysfunction of this process has been linked to pathogenesis of cancer, atherosclerosis, and pulmonary vascular disease (13, 16, 20, 27, 43, 53). Apoptosis is implemented by death machinery, the executionary arms of which are cysteine proteases known as caspases and nucleases (40). An incipient hallmark of apoptosis is cell shrinkage. The apoptotic volume decrease usually takes place before cytochrome c release, caspase activation, and DNA fragmentation (4, 6, 7, 19, 37, 59).
Cell volume is primarily controlled by intracellular ion homeostasis. Thus ion transport across the plasma membrane plays a critical role in regulating cell volume. K+ is the dominant cation in the cytosol, with a concentration of ~150 mM. Maintenance of a high concentration of intracellular K+ concentration ([K+]i) is required to maintain the cytosolic ion homeostasis necessary to preserve normal cell volume (23, 36, 59). A sufficient [K+]i is also compulsory for suppressing the activity of caspases and nucleases (6, 7, 19, 24, 25). [K+]i is basically regulated by the Na+-K+-ATPase and K+ channels in the plasma membrane. Because of the outwardly directed electrochemical gradient for K+, opening of the plasmalemmal K+ channels would promote efflux or loss of cytosolic K+ and induce the apoptotic volume decrease and apoptosis. In a number of cell types, enhancement of the plasma membrane permeability to K+ has been associated with the early responses to apoptotic stimuli (6, 7, 14, 15, 19, 37, 59).
There are at least four types of K+ channels in vascular smooth muscle cells as follows: 1) voltage-gated K+ (Kv) channels, 2) Ca2+-activated K+ (KCa) channels, 3) ATP-sensitive K+ channels, and 4) inward-rectifier K+ channels (28, 38). In neurons, the serum depravation- and staurosporine (ST)-mediated apoptosis is associated with an early enhancement of delayed-rectifier Kv currents (IKv), which leads to a net loss of cytosolic K+ and induces the apoptotic volume decrease. Pharmacological blockade of Kv channels or attenuation of IKv with high extracellular K+ inhibits the apoptotic volume decrease and apoptosis (9, 60). In Jurkat T cells, the Fas/FasL-mediated apoptosis is accompanied with a cytosolic K+ loss as a result of an increased K+ efflux (19). More recently, activation of KCa and Kv channels has been shown to participate in pathways leading to the programmed cell death mediated by tumor necrosis factor (39), FCCP (32), and ultraviolet radiation (56). K+ channels have also been shown to play an important role in apoptosis of neutrophils (3). These observations suggest that activation of Kv channels in the plasma membrane is an important mechanism involved in the apoptotic volume decrease. Blockade of Kv channels may thus play a critical role in inhibiting the apoptotic volume decrease and attenuating apoptosis (6, 7, 9, 37, 39, 59-61).
Bcl-2 is an oncoprotein with its best known function as a suppressor of cell death (1, 11). The anti-apoptotic effect of Bcl-2 has been attributed to 1) inhibition of the release of proapoptotic mitochondrial proteins (e.g., cytochrome c- and apoptosis-inducing factor; see Refs. 31, 50, 55 and 58), 2) inhibition of the proapoptotic Bcl-2 family proteins (e.g., Bax and Bak; see Ref. 52), 3) restoration of the ability to exchange mitochondrial ATP for cytosolic ADP to maintain a high ATP-to-ADP ratio in the cytosol (54), and 4) the antioxidant activity (11, 22). It has been reported that Bcl-2 could enhance cell survival by forming nonselective ion channels in the organelle membranes and by regulating ion channels in the plasma membrane (2, 46, 57). This study was designed to test the hypothesis that Bcl-2 exerts its antiapoptotic effect on pulmonary artery smooth muscle cells (PASMC) by, at least in part, inhibiting K+ channel activity. The resultant reduction of K+ currents diminishes K+ efflux, inhibits the apoptotic volume decrease, and prevents cells from undergoing apoptosis.
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MATERIALS AND METHODS |
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Cell preparation and culture. Primary cultured rat PASMC were prepared as previously described (41, 64). Briefly, the pulmonary arterial branches (3rd division) were isolated from lung tissues and incubated for 20 min in Hanks' balanced salt solution containing 1.5 mg/ml of collagenase (Worthington). After the incubation, a thin layer of adventitia was carefully stripped off with fine forceps, and the endothelium was removed by gently scratching the intimal surface with a surgical blade. The remaining pulmonary artery smooth muscle was then digested with 1.5 mg/ml of collagenase and 0.5 mg/ml of elastase (Sigma) at 37°C. The cells were plated on 25-mm coverslips (for electrophysiological and fluorescent experiments) and in 10-cm petri dishes (for molecular biological experiments). The cells were incubated in a humidified atmosphere of 5% CO2 in air at 37°C and cultured in 10% FBS-DMEM for 5-6 days before experiment.
Bcl-2 infection protocol. Recombinant adenovirus type 5 expressing human bcl-2 was used to infect bcl-2 into primary cultured PASMC. The detailed methods for bcl-2 infection have been described elsewhere (30, 48). Briefly, the cells were infected with appropriate virus at 500 plaque-forming units/cell in DMEM without serum. After 7 h of infection, the medium was replaced with the growth medium (10% FBS-DMEM) or the basal medium (0% FBS-DMEM). Cells were used 24-48 h after virus infection. The expression of Bcl-2 was verified by Western blot analysis.
Electrophysiological measurement.
Whole cell K+ currents were recorded with an Axopatch-1D
amplifier and a DigiData 1200 interface (Axon Instruments) using
patch-clamp techniques (41, 64). Patch pipettes (1-3
M) were made on a Sutter electrode puller using borosilicate glass
tubes and fire polished on a Narishige microforge. Command voltage
protocols and data acquisition were performed using pCLAMP-8 software
(Axon Instruments). Currents were filtered at 1-2 kHz (
3 dB) and
digitized at 2-4 kHz using the amplifier. All experiments
were performed at room temperature (22-24°C).
Nuclear morphology determination. The cells, grown on 25-mm coverslips, were first washed with PBS, fixed in 95% ethanol, and stained with the membrane-permeable nucleic acid stain 4'-6'-diamidino-2-phenylindole (DAPI; Sigma). DAPI (5 µM) was dissolved in an antibody buffer containing 500 mM NaCl, 20 µM NaN3, 10 µM MgCl2, and 20 µM Tris · HCl (pH 7.4). The blue fluorescence emitted at 461 nm was used to visualize the cell nuclei. The DAPI-stained cells were examined with a Nikon TE 300 fluorescence microscope, and the cell (nuclear) images were acquired using a high-resolution Solamere fluorescence imaging system. For each coverslip, 5-10 fields (with 20-25 cells in each of the fields) were randomly selected to determine the percentage of apoptotic cells in total cells based on the morphological characteristics of apoptosis. The cells with clearly defined nuclear breakage, remarkably condensed nuclear fluorescence, and significantly shrunken cell nucleus were defined as apoptotic cells. To quantify apoptosis, TdT-mediated dUTP nick end labeling assays were also performed with the In Situ Cell Death Detection Kit (TMR Red; Boehringer-Mannheim).
Chemicals. ST (Sigma) was prepared as a 1 mM stock solution in DMSO; aliquots of the stock solution were then diluted by 1:10,000-500,000 times to the culture media or PSS for experimentation. 4-Aminopyridine (Sigma) was directly dissolved in the bath solution or culture media on the day of use; the pH of the solution was readjusted to 7.4 at room temperature. In high-K+ solution or culture medium, NaCl in PSS or the customized DMEM (Media Tech) was replaced by equimolar KCl to maintain the solution's osmolarity.
Western blot analysis. Primary cultured PASMC were gently washed two times in cold PBS, scraped in 0.3 ml lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml phenylmethylsulfonyl fluoride, and 30 µl/ml aprotinin), and incubated for 30 min on ice. The cell lysates were then sonicated and centrifuged at 12,000 rpm for 10 min, and the insoluble fraction was discarded. The protein concentrations in the supernatant were determined by the bicinchoninic acid protein assay (Pierce) using BSA as a standard. Proteins (10 µg) were mixed and boiled in SDS-PAGE sample buffer for 5 min. The protein samples separated on 12% SDS-PAGE were then transferred to nitrocellulose membranes by electroblotting in a Mini Trans-Blot Cell transfer apparatus (Bio-Rad) according to the manufacturer's instructions. After incubation overnight at 4°C in a blocking buffer (0.1% Tween 20 in PBS) containing 5% nonfat dry milk powder, the membranes were incubated with the murine anti-human Bcl-2 monoclonal antibody (Transduction Laboratories). The membranes were then washed and incubated with anti-mouse horseradish peroxidase-conjugated IgG for 90 min at room temperature. The bound antibody was detected using an enhanced chemiluminescence detection system (Amersham).
Single-cell RT-PCR.
To determine the mRNA expression of Kv channel -subunits (Kv1.1,
Kv1.5, and Kv2.1) in the control and bcl-2-infected rat PASMC at the single-cell level, multiplex single-cell RT-PCR was performed according to a modified protocol previously described by
Comer et al. (10). Briefly, after recording
IKv, the whole cell was carefully aspirated into
a collection pipette that contained 12 µl of the pipette solution
supplemented with 10 µM dNTP and 0.5 U/µl RNase inhibitor. The
content in the pipette was then expelled immediately into a 0.2-ml PCR
tube that contained 20 µl of the solution composed of 10 mM
Tris · HCl, 50 mM KCl, 2.5 mM MgCl2, 10 mM
dithothreitol, 1.25 mM oligo(dT), 0.5 mmol/l dNTPs, and 5 units avian
myeloblastosis virus RT XL. The RT was performed for 60 min at 42°C.
Next, first-round PCR with 45 cycles was performed in the same tube by
the addition of 80 µl of the premix PCR buffer containing 10 mM
Tris · HCl, 50 mM KCl, 2.5 mM MgCl2, 20 nM of each
sense and antisense primer (first primers) for all the genes of
interest, and 5 units of Taq polymerase (RNA PCR kit,
version 2.1; Takara, Otsu, Japan). Aliquots (2 µl) of the first-round PCR products were reamplified by the second-round PCR with 25-30 cycles, which was separately carried out using fully nested
gene-specific primers (nested primers) for each target gene.
Second-round PCR-amplified products were separated on 1.5% agarose gel
and visualized with GelStar gel staining. The cell-free samples were
also used in PCR as a negative control. To semiquantify the PCR
products, an invariant mRNA of
-actin was used as an internal
control. The sense and antisense primers (the first and nested primers)
were specifically designed from the coding regions of human Bcl-2
(X06487) and rat Kv1.1 (X12589), Kv1.5 (M27158), and Kv2.1 (X16476; Table 1). The fidelity and specificity of
the sense and antisense oligonucleotides were examined using the BLAST
program.
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Statistics. The composite data are expressed as means ± SE. Statistical analysis was performed using paired or unpaired Student's t-test or ANOVA and post hoc tests (Student-Newman-Keuls) where appropriate. Differences were considered to be significant at P < 0.05.
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RESULTS |
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Overexpression of Bcl-2 decreases whole cell
IKv.
The recombinant adenovirus is highly efficient (>95%) in transfecting
bcl-2 genes in primary cultured cells (30, 48). As shown in Fig. 1A, the human
Bcl-2 was hardly detectable using Western blot analysis in control rat
PASMC infected with an empty adenoviral vector. However, there was a
significant level of Bcl-2 protein expression in rat PASMC infected
with the adenovirus expressing the human bcl-2 (Fig.
1A).
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Overexpression of Bcl-2 downregulates
mRNA expression of Kv channel
-subunits.
The reduction of current density of IKv in rat
PASMC infected with bcl-2 suggests that Bcl-2 may also
affect gene expression of Kv channels, which would subsequently
decrease total numbers of the functional Kv channels and decrease whole
cell IKv. It has been reported that Bcl-2 could
cooperate with transcription factors to regulate gene expression
(44). Using single-cell RT-PCR, we examined whether
overexpression of Bcl-2 was associated with a reduction of mRNA
expression of the pore-forming Kv channel
-subunits.
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Overexpression of Bcl-2 abolishes
ST-mediated activation of Kv channels in
PASMC.
ST is a potent apoptosis inducer (17, 34, 45, 60).
Consistent with the observations in neurons (60),
treatment with ST for 3 h significantly increased whole cell
IKv in control PASMC (proliferating cells that
were cultured in 10% FBS medium; Fig. 4A). Overexpression of Bcl-2
attenuated IKv and completely abolished the
ST-induced increase in IKv (Fig. 4B).
These results suggest that ST and Bcl-2 have opposite effects on the
activity of Kv channels in rat PASMC; the apoptosis inducer ST
increases IKv, whereas the apoptosis
inhibitor Bcl-2 decreases IKv and eliminates the
ST-mediated augmenting effect on IKv. The next
set of experiments was designed to examine whether Bcl-2-mediated
blockade of Kv channels inhibited ST-induced apoptosis.
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Overexpression of Bcl-2 inhibits
ST-induced apoptosis in PASMC.
In control cells and cells infected with bcl-2, there were
3.0 ± 0.6% (n = 31) and 3.2 ± 0.5%
(n = 30) apoptotic cells, respectively. Treatment
of the control cells with ST (0.02 µM, 20 h) significantly increased the percentage of the cells undergoing apoptosis
(Fig. 5, A and B).
Application of 4-aminopyridine (2 mM), a blocker of Kv channels,
significantly attenuated the ST-induced increase in
IKv and inhibited the ST-induced
apoptosis (Fig. 5B). Similar to 4-aminopyridine,
overexpression of Bcl-2 also diminished the ST-induced increase in
IKv and inhibited the ST-induced
apoptosis (Fig. 5C). These results indicate that
overexpression of Bcl-2 in PASMC is functionally capable of suppressing
ST-mediated apoptosis; one of the involving mechanisms may be
the decrease in Kv channel activity.
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The ST-induced apoptosis is preceded by the increase in
IKv.
To determine whether the ST-induced increase in
IKv precedes its apoptotic effect, we
compared the time courses of ST-induced increase in
IKv and apoptosis in proliferating PASMC
(cultured in 10% FBS medium) treated with 0.02 µM ST. The ST-induced
increase in IKv (at +80 mV) took place at 30 min
after application of ST and was maximized at 3-4.5 h (Fig.
6). The time course of the ST-induced
apoptosis indicated that the percentage of apoptotic nuclei
was not significantly increased until 6 h of treatment with 0.02 µM ST; the maximal effect occurred after 15 h of treatment with
ST (Fig. 6). These observations indicate that the ST-induced enhancement of IKv occurs before the onset of
apoptosis in human PASMC.
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DISCUSSION |
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Recent studies have enhanced our understanding of the cellular and
molecular mechanisms of apoptosis in normal and disease conditions. Although much is known about the involvement of
intracellular organelles (e.g., mitochondria) in apoptotic pathways
(33), little is known about the role of the plasma
membrane and the regulation of ion fluxes across the plasma membrane in
this process in pulmonary vascular smooth muscle cells. In the current
study using rat PASMC, we observed that overexpression of the
antiapoptotic protein Bcl-2 1) decreased current density
and accelerated current inactivation of IKv,
2) downregulated mRNA expression of Kv channel -subunits
(Kv1.1, Kv1.5, and Kv2.1), and 3) abolished the ST-induced increase in IKv and inhibited ST-induced
apoptosis. These results suggest that inhibition of the Kv
channel activity is an important mechanism involved in the
antiapoptotic effect of Bcl-2 in vascular smooth muscle cells.
Apoptotic volume decrease resulting from the loss of intracellular ions is an early prerequisite for apoptosis. When cells are targeted by death stimuli, cell shrinkage or the apoptotic volume decrease generally occurs before cytochrome c release, caspase-3 activation, and DNA fragmentation (19, 37, 59). The apoptotic volume decrease is coupled to K+ efflux through the plasmalemmal K+ channels and thus is inhibited by pharmacological blockers of K+ channels (e.g., tetraethylammonium, quinine, Ba2+, glibenclamide, and 4-aminopyridine) or by reducing the K+ transmembrane driving force using high extracellular K+ solution (6, 7, 9, 14, 15, 17, 37, 39, 56, 59-61). However, the apoptotic volume decrease is not inhibited by a caspase inhibitor (37). Pharmacological blockade of K+ efflux and the apoptotic volume decrease prevents the ST-mediated release of cytochrome c, activation of caspase-3, and laddering of DNA (37, 59). These observations suggest that the K+ channel activation-mediated apoptotic volume decrease is an early prerequisite for apoptosis (59).
In rat PASMC, ST increased IKv and induced apoptosis; the ST-induced apoptosis was preceded by the increase in IKv. Blockade of Kv channels with 4-aminopyridine or overexpression of Bcl-2 eliminated the ST-induced increase in IKv and inhibited the ST-induced apoptosis. Consistent with the observations in neurons (9, 59-61), liver cells (39), thymocytes (14, 15), epithelial cells (37), neutrophils (3), and lymphoma cells (6, 7, 24, 25), our results indicate that activation of Kv channels contributes to induce the apoptotic volume decrease and apoptosis, whereas blockade of Kv channels inhibits the apoptotic volume decrease and apoptosis, in pulmonary vascular smooth muscle cells.Activity of K+ channels regulates cell volume and apoptosis. Whole cell IKv in PASMC is determined by the activity of single Kv channels and the total number of functional Kv channels. Opening of a Kv channel and upregulation of Kv channel expression would both contribute to increase whole cell IKv and promote K+ efflux. An increase in whole cell IKv would result in a net K+ loss, leading to cell and nuclear shrinkage, and eventually cause apoptosis. A decrease in IKv would limit K+ loss, inhibit the apoptotic volume decrease, and prevent apoptosis (6, 7, 14, 15, 59-61). The inhibitory effects of Bcl-2 on the ST-induced increase in IKv and apoptosis, observed from this study, further support the contention that K+ efflux through plasmalemmal K+ channels plays an important role in the programmed cell death (59).
The efflux of K+ is obviously insufficient to cause a volume change in cells because it has to be accompanied by an efflux of an anion to achieve a net reduction in the amount of solute within the cell (34, 59). In smooth muscle cells, ClCytoplasmic K+ loss is
accompanied with activation of caspases.
In addition to controlling cell volume, intracellular K+ is
also demonstrated to suppress the activity of caspases and nucleases (6, 7, 24, 25). Activation of caspases and nucleases is a
major signal transduction pathway (40) for
apoptosis induced by a variety of apoptosis inducers,
e.g., ST (34), valinomycin (15, 26), tumor
necrosis factor- (37, 39), and ultraviolet radiation
(31, 56). A high [K+]i is
apparently required for maintaining a low activity of the apoptotic
enzymes (e.g., caspases and endonucleases) under normal conditions. The
net loss of cytoplasmic K+ resulting from increased
K+ efflux through plasmalemmal K+ channels
would relieve the tonic suppression of cytosolic caspases and
endonucleases and thus cause apoptosis (6, 7, 19, 24,
25).
Mechanisms involved in antiapoptotic effect of Bcl-2. The mechanisms by which Bcl-2 suppresses apoptosis have been investigated (1). There are at least six major mechanisms involved in the Bcl-2-mediated antiapoptotic effect as follows: 1) blockade of the release of cytochrome c- and apoptosis-inducing factor from the mitochondria to the cytosol (31, 50, 55, 58), 2) inhibition of the proapoptotic effects of the Bcl-2 family proteins (e.g., Bax and Bak; see Ref. 52), 3) facilitation of mitochondrial ATP-cytosolic ADP exchange via the adenine nucleotide translocator (ANT) and voltage-dependent anion channel (VDAC) in the mitochondrial inner (ANT) and outer (VDAC) membranes (54), 4) maintenance of sufficient Ca2+ in the sarcoplasmic/endoplasmic reticulum (21, 35) and mitochondria (65) by enhancing capacitative Ca2+ entry (57), 5) maintenance of the negative mitochondrial membrane potential by enhancing proton efflux (47) and forming cation-selective ion channels (2, 46) in the inner mitochondrial membrane, and 6) the direct antioxidant activity (11, 22). In rat PASMC, overexpression of Bcl-2 reduced IKv, eliminated the ST-induced increase in IKv, and, accordingly, inhibited the ST-induced apoptosis. These results suggest that Bcl-2-mediated inhibition of Kv channel activity is an additional pathway for the antiapoptotic effect of Bcl-2 in vascular smooth muscle cells.
Overexpression of Bcl-2 reduced the current density and decreased the time constant of current inactivation of IKv in rat PASMC. Furthermore, the mRNA expression of the delayed-rectifier Kv channelPossible role of dysfunctional Kv channels in inhibited PASMC apoptosis. Pulmonary vascular remodeling (e.g., medial and myointimal thickening) is a major cause for the elevated pulmonary vascular resistance in patients with pulmonary hypertension. The mechanism for the medial thickening has been attributed to an increase in proliferation and a decrease in apoptosis of PASMC. Therefore, the precise control of the balance of cell apoptosis and proliferation in PASMC may play an important role in maintaining normal pulmonary vascular structure and function (12, 13, 16, 43, 49).
It has been demonstrated that the function and expression of Kv channels are inhibited in PASMC from patients with primary pulmonary hypertension (62, 63). The decreased activity of Kv channels is reported to be responsible, at least in part, for pulmonary vasoconstriction and PASMC proliferation (18, 41, 62, 63). As shown in this study, inhibition of Kv channel activity by pharmacological blockade of Kv channels or overexpression of Bcl-2 attenuated apoptosis in PASMC. These results direct us to speculate that the inhibited Kv channel function and expression in PASMC from patients with primary pulmonary hypertension may also contribute to enhance vascular medial thickening via inhibition of PASMC apoptosis. In summary, the observations from our study and those reported by others suggest that movement of K+ across the plasma membrane is involved in the initiation and regulation of the apoptotic volume decrease. Overexpression of the antiapoptotic protein Bcl-2 decreased IKv by accelerating the current inactivation and downregulating the mRNA expression of the pore-forming Kv channel ![]() |
ACKNOWLEDGEMENTS |
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We thank Y. Zhao for technical assistance.
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FOOTNOTES |
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* D. Ekhterae, O. Platoshyn, S. Krick, and Y. Yu contributed equally to this work.
This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-54043 and HL-64945 (to J. X.-J. Yuan). S. Krick is an Ambassadorial Scholar of Rotary International. J. X.-J. Yuan is an Established Investigator of the American Heart Association.
Address for reprint requests and other correspondence: J. X.-J. Yuan, Div. of Pulmonary and Critical Care Medicine, UCSD Medical Center, 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 27 November 2000; accepted in final form 8 February 2001.
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