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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The balance between apoptosis and proliferation in pulmonary artery smooth muscle cells (PASMCs) is important in maintaining normal pulmonary vascular structure. Activity of voltage-gated K+ (KV) channels has been demonstrated to regulate cell apoptosis and proliferation. Treatment of PASMCs with staurosporine (ST) induced apoptosis in PASMCs, augmented KV current [IK(V)], and induced mitochondrial membrane depolarization. High K+ (40 mM) negligibly affected the ST-induced mitochondrial membrane depolarization but inhibited the ST-induced IK(V) increase and apoptosis. Blockade of KV channels with 4-aminopyridine diminished IK(V) and markedly decreased the ST-mediated apoptosis. Furthermore, the ST-induced apoptosis was preceded by the increase in IK(V). These results indicate that ST induces PASMC apoptosis by activation of plasmalemmal KV channels and mitochondrial membrane depolarization. The increased IK(V) would result in an apoptotic volume decrease due to a loss of cytosolic K+ and induce apoptosis. The mitochondrial membrane depolarization would cause cytochrome c release, activate the cytosolic caspases, and induce apoptosis. Inhibition of KV channels would thus attenuate PASMC apoptosis.
apoptosis; voltage-gated potassium channels; potassium loss; caspases; mitochondrial membrane potential depolarization
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INTRODUCTION |
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APOPTOSIS IS A PHYSIOLOGICAL MODE of cell death that is triggered by diverse external or internal signals. Apoptosis in pulmonary artery smooth muscle cells (PASMCs) allows the pulmonary vasculature to tightly control the vascular wall tissue mass (or thickness). Dysfunction of this process has been linked to pathogenesis of cancer, atherosclerosis, and pulmonary vascular diseases (15, 19, 25, 40). Apoptosis in PASMCs and endothelial cells has been implicated in the regression of pulmonary arterial hypertrophy (9, 40).
Cell shrinkage is an early hallmark in apoptosis. The
apoptotic volume decrease has been demonstrated to result, at least in part, from a loss of intracellular K+ (6, 10, 35,
46). Therefore, maintenance of a high concentration of
intracellular K+ ([K+]i) is
required to maintain the cytosolic ion homeostasis necessary to
preserve normal cell volume (21, 33, 46). A sufficient [K+]i is also necessary for suppressing the
activity of the caspases and nucleases (6, 22, 23) that
are believed to be the final mediators of apoptosis
(44). An increase in K+ channel activity would
therefore cause a loss of cytoplasmic K+ based on the
outwardly directed electrochemical gradient of K+ across
the plasma membrane. The resultant decrease in
[K+]i would result in cell and nuclear
shrinkage and facilitate the caspase (or nuclease)-mediated
apoptosis (5, 6, 8, 11, 22, 23, 38, 46, 47, 50).
Furthermore, mitochondrial membrane depolarization, i.e., a decrease
(less negative) in the mitochondrial membrane potential
(m), is thought to be one of the contributory
mechanisms in initiating apoptosis (20, 29, 51).
This study was designed to test whether activation of voltage-gated K+ (KV) channels and
m
depolarization are involved in human PASMC apoptosis induced by
staurosporine (ST), a potent inducer of apoptosis in a variety
of cell types (17, 30).
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METHODS AND MATERIALS |
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Cell preparation. Human PASMCs (Clonetics) were seeded in flasks at a density of 2,500-3,500 cells/cm2 and incubated in smooth muscle growth medium (Clonetics). The culture medium was changed after 24 h and every 48 h thereafter. Smooth muscle growth medium is composed of smooth muscle basal medium, 5% fetal bovine serum, 0.5 ng/ml of human epidermal growth factor, 2 ng/ml of human fibroblast growth factor, and 5 µg/ml of insulin. The cells were subcultured and plated onto 10- or 25-mm coverslips with trypsin-EDTA buffer (Clonetics) when 70-90% confluence was achieved. The cells at passages 4-6 were used for experimentation.
Electrophysiological measurement.
K+ currents (IK) were recorded with
an Axopatch-1D amplifier and a DigiData 1200 interface (Axon
Instruments) with patch-clamp techniques (18, 49). Patch
pipettes (2-4 M) were made with a Sutter electrode puller with
borosilicate glass tubes and fire polished on a Narishige microforge.
Command voltage 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 the amplifier. All experiments were
performed at room temperature (22-24°C).
Nuclear morphology determination. The cells grown on 10-mm coverslips were first washed with PBS (Sigma), 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 with a high-resolution Solamere fluorescence imaging system. For each coverslip, 5-10 fields (with ~20-25 cells/field) were randomly selected to determine the percentage of apoptotic cells in the total cells based on the morphological characteristics of apoptosis. The cells with clearly defined nuclear breakage, remarkably condensed nuclear fluorescence, and a significantly shrunken cell body and nucleus were defined as apoptotic cells. To quantify apoptosis, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assays were also performed with an in situ cell death detection kit (TMR red; Boehringer Mannheim).
Measurement of rhodamine fluorescence.
The cells grown on 25-mm coverslips were loaded with rhodamine 123 (R-123; Molecular Probes) by incubation with 10 µg/ml for 30 min at
37°C. R-123 is taken up selectively by mitochondria (13,
14), and its uptake is dependent of m. R-123
fluorescence was excited at 488 nm and measured at 530 nm with a GEN IV
charge-coupled device camera connected to a Nikon TE 300 microscope. In
isolated mitochondria, the relationship between R-123 fluorescence and
m is linear. The R-123 fluorescence, which is
quenched at resting
m, increases with mitochondrial
membrane depolarization (14). The cells were perfused with
PSS to establish a baseline of fluorescence, and then images were
acquired every third second for 3 min during which the perfusate was
changed at selective time points. The R-123 fluorescence signals were
stored in a Macintosh computer and analyzed with QED software
(Solamere) and a National Institutes of Health imaging system.
Colorimetric assay for ST-induced caspase-3 activation. The cells were plated on 10-cm petri dishes or in T-25 flasks. To measure the activity or concentration of caspase-3 in the lysates of PASMCs, we used the colorimetric caspase-3 assay kit (Sigma), and its detailed protocol is shown in the 96-well plate microassay method (Sigma). The cells were lysed, and the cytosolic contents devoid of the membrane and organelles were isolated before and after treatment with ST. A protease inhibitor cocktail for mammalian cells (Sigma), which was prepared as a 1:100 solution ready for use, was added to the lysis buffer to prevent enzymatic caspase-3 degradation. The caspase-3 activity was measured by incubating the cell lysates (for 90 min) with acetyl-Asp-Glu-Val-Asp p-nitroanilide, a caspase-3 substrate; its absorbance was measured at 405 nm after cleavage by caspase-3. The optical density value at 405 nm is thus used as indicative for the amount of caspase-3.
Solutions and chemicals. ST (Sigma) was prepared as a 1 mM stock solution in DMSO and diluted 10,000-50,000 times in PSS or the culture medium to make final concentrations of 0.02-0.1 µM. 4-Aminopyridine (4-AP; Sigma) and TEA (Sigma) were directly dissolved in PSS or the culture medium at concentrations of 1-5 mM on the day of use; the pH of the 4-AP-containing solution was adjusted to 7.4 at room temperature. In high-K+ solution or culture medium, NaCl in PSS or the customized DMEM (Media Tech) was replaced, mole by mole, with KCl to maintain the osmolarity of the solution.
Statistics. The composite data are expressed as means ± SE. Statistical analysis was performed with paired or unpaired Student's t-test or ANOVA and post hoc test (Student-Newman-Keuls) where appropriate. Differences were considered to be significant when P < 0.05.
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RESULTS |
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Inhibitory effects of 40 mM
K+ and 4-AP
on ST-induced apoptosis in human
PASMCs.
Under control conditions, there were 4-7% apoptotic cells in
the culture dishes. Incubation of the cells in the medium containing 0.02 µM ST for 20 h significantly increased the percentage of apoptotic cells to 45-55% based on the morphological changes
in the nuclei (Fig. 1). Similar results
were obtained with the TUNEL assay. Raising extracellular
K+ from 5 to 40 mM (which inhibits K+ efflux by
reducing the outwardly directed K+ driving force) or the
addition of 5 mM 4-AP (which blocks KV channels) markedly
inhibited the ST-induced apoptosis (Fig. 1B). Caspase-3 is a major effector molecule of many apoptotic inducers. An increase in caspase-3 concentration or activity causes
apoptosis in a variety of cell types (26, 44).
Indeed, the ST-induced apoptosis was also accompanied by a
significant increase in caspase-3 concentration; treatment of human
PASMCs with 0.02 µM ST for 20 h increased the concentration of
caspase-3 in PASMC lysates from 21.0 ± 0.6 to 31.0 ± 1.2 µM/105 cells (P < 0.01).
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Augmenting effect of ST on
KV channel activity.
Whole cell IK(V) was isolated in human PASMCs
when the contribution of KCa and KATP channels
to the current was minimized by removal of extracellular (bath) and
intracellular (pipette) Ca2+ and by the intracellular
addition of 5 mM ATP, which completely blocks KATP channels
(7). Extracellular application of 5 mM 4-AP (Fig.
2) or raising the extracellular
K+ concentration to 40 mM (data not shown) significantly
decreased the currents, suggesting that the currents were mainly
generated by K+ efflux through 4-AP-sensitive
KV channels (1, 37, 49). Under these
conditions, extracellular application of 0.1 µM ST significantly
increased whole cell IK(V) in human
PASMCs in the presence of TEA (Fig.
3), which blocks
KCa channels at doses 1 mM (1). As
shown in Fig. 3A, extracellular application of 0.1 µM ST
induced a 1.4-fold increase in the amplitude of
IK(V) at
40 mV (from 16.1 ± 1.1 to
39.1 ± 4.2 pA) and a 2.9-fold increase at +80 mV (from 327.8 ± 38.6 to 1,293.7 ± 146.6 pA; n = 13 experiments; Fig. 3, Aa-Ac). The ST-induced
augmentation of IK(V) appeared to be voltage
dependent (Fig. 3Ad); the effect was significantly enhanced
at positive potentials ranging from +20 to +80 mV. Furthermore, extracellular application of ST (0.1 µM) also increased the time constant of current inactivation at +80 mV (Fig. 3Ae). The
voltage-dependent augmenting effect of ST on
IK(V) was probably related to the decelerating effect on current inactivation at positive potentials.
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The ST-induced increase in
IK(V) precedes the ST-induced
apoptosis.
Incubation of human PASMCs in medium containing the low dose (0.02 µM) of ST that was used for the apoptosis experiments (Fig. 1) for 90 min also significantly increased the amplitude of
IK(V) from 13.7 ± 0.8 to 36.7 ± 1.7 pA at 40 mV and from 377.4 ± 22.9 to 1,094.7 ± 42.4 pA at
+80 mV (P < 0.001; n = 8-14
experiments; Fig. 4A).
Furthermore, ST treatment (0.02 µM for 90 min) markedly decelerated
the rate of IK(V) inactivation; the time
constants of current inactivation at +80 mV were 46.3 ± 3.9 ms
(n = 14 experiments) and 69.6 ± 8.6 ms
(P < 0.001; n = 9 experiments) in
cells treated with vehicle (DMSO) and 0.02 µM ST, respectively (Fig.
4Ac).
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Effect of ST on R-123 fluorescence.
The m is generated primarily by a proton gradient
across the mitochondrial inner membrane (3). Mitochondrial
membrane depolarization (less negative in
m) has been
demonstrated to induce cytochrome c release from the
mitochondrial intermembrane space to the cytosol (2, 9, 29,
51). Cytochrome c activates the cytosolic caspases,
which are believed to be the final mediator of apoptosis in a
variety of cell types (29, 44). Whether ST-mediated
apoptosis is due, in part, to
m
depolarization was examined by measuring the changes in R-123
fluorescence intensity in human PASMCs.
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DISCUSSION |
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The precise control of the balance between PASMC proliferation and apoptosis plays an important role in maintaining normal pulmonary vascular structure and function. An increase in proliferation and a decrease in apoptosis of pulmonary vascular smooth muscle and endothelial cells would lead to pulmonary vascular remodeling and elevation of pulmonary vascular resistance and arterial pressure as observed in patients with pulmonary hypertension (9, 15, 16, 40, 43).
The observations from this study demonstrated that apoptosis
induced by ST is linked to mitochondrial membrane depolarization and
activation of plasmalemmal KV channels in human
PASMCs. The m depolarization would trigger the
release of cytochrome c that activates caspases in the
cytosol and induces apoptosis (20, 29, 51). The
increase in IK(V) would result in a loss of
cytosolic K+, which relieves the tonic suppression of
K+ on the caspases and nucleases and therefore induces DNA
degradation and apoptosis (6, 22, 23).
Furthermore, the K+ loss would also lead to cell shrinkage
(the apoptotic volume decrease), an early characteristic commonly
used to identify apoptotic cells (5, 6, 10, 35, 46).
Inhibition of K+ efflux through KV channels
with 40 mM K+ or 4-AP, a potent blocker of KV
channels, negligibly affected the ST-induced
m
depolarization but significantly attenuated the ST-induced
apoptosis. These results suggest that ST mediates apoptosis in human PASMCs, at least in part, by two independent mechanisms: 1)
m depolarization and
2) activation of KV channels in the plasma membrane.
KV channels are ubiquitously expressed in both excitable
and nonexcitable cells (27, 37) and play important roles
in the regulation of excitability, neurotransmitter release, muscle
contraction, and cell volume, proliferation, and differentiation
(12, 27, 39, 49). At the molecular level, KV
channels are homomeric and heteromeric tetramers composed of the
pore-forming -subunits and the regulatory
-subunits (27,
37). The
-subunit is composed of six membrane-spanning
segments termed S1 through S6, with a protein kinase C (PKC) binding
site located intracellularly between the S4 and S5 segments
(27). The PKC-mediated phosphorylation of the
-subunits
inhibits the K+ currents generated by K+ efflux
through KV channels encoded by Kv1.1, Kv2.1, Kv4.1, or Kv4.2 (4), whereas PKC-induced phosphorylation of the
Kv
1.2 and Kv
1.3 subunits is necessary for the inactivation of
KV channels (31, 32, 34). Therefore,
inhibition of PKC would increase KV channel activity
(4, 31, 32, 34), promote K+ efflux, and induce
a loss of cytosolic K+. ST is a nonspecific inhibitor of
PKC (17, 30), and the ST-induced increase in
IK(V) and apoptosis in human PASMCs may
be, at least in part, due to the inhibition of PKC.
As mentioned earlier, cell shrinkage (the apoptotic volume decrease) due to the alterations in cell volume and the loss of intracellular K+ is an early requisite feature for the activation of the caspases and other cytosolic, proapoptotic enzymes (5, 6, 10, 22, 23, 35, 46). Indeed, our observations indicated that ST-induced apoptosis was accompanied by a significant increase in caspase-3 concentration, and the decrease in K+ concentration in the assay buffer significantly reduced caspase-3 activity (data not shown). These results suggest that blockade of KV channels may attenuate PASMC apoptosis by diminishing the apoptotic volume decrease and caspase activity via maintaining a high concentration of cytosolic K+.
Due to 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. There are at least four types of K+ channels in vascular smooth muscle cells: 1) KV channels, 2) KCa channels, 3) KATP channels, and 4) inward rectifier K+ channels (27, 37). In neurons, serum deprivation- and ST-mediated apoptosis is associated with an early enhancement of the delayed rectifier IK(V), which leads to a net loss in cytosolic K+ and induces the apoptotic volume decrease. Pharmacological blockade of KV channels or attenuation of IK(V) with high extracellular K+ both inhibit the apoptotic volume decrease and apoptosis (8, 46, 47). In Jurkat T cells, Fas/Fas ligand-mediated apoptosis is accompanied by a cytosolic K+ loss due to an increased K+ efflux (17). More recently, activation of KCa and KV channels has been shown to participate in pathways leading to cell apoptosis mediated by tumor necrosis factor (38), ultraviolet radiation (45), and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (28). These observations suggest that all K+ channels expressed in the plasma membrane may be involved in the apoptotic volume decrease and apoptosis.
Our study also showed that ST depolarizes m, which
would trigger the release of cytochrome c and subsequently
activate cytosolic caspases, such as caspase-3, and induce
apoptosis (2, 3, 6, 17, 29, 30, 44). This
m-mediated apoptosis appears to be
independent of cytosolic K+ loss because decreasing the
K+ driving force by perfusing the cells with a 40 mM
K+-containing solution did not affect the ST-induced
m depolarization. It remains to be investigated
1) how ST mediates mitochondrial membrane
depolarization in human PASMCs, 2) whether the ST-induced activation of KV channels interacts with the ST-mediated
m depolarization in initiating apoptosis, and
3) whether ST induces
m depolarization by
affecting K+ channels in the mitochondrial inner membrane.
Nevertheless, our observations imply that ST-induced apoptosis
may involve multiple cellular pathways in human PASMCs. Recently, it
has been found that the inner mitochondrial membrane contains multiple
K+ channels such as KATP and KCa
(11, 24, 36, 41, 42); therefore, it is possible that the
ST-induced mitochondrial membrane depolarization is due to activation
of the K+ channels in the mitochondrial membrane
(11).
The function and expression of KV channels have been demonstrated to be inhibited in PASMCs from patients with primary pulmonary hypertension (48, 50). The decreased activity of the KV channels is reported to be responsible, at least in part, for pulmonary vasoconstriction and PASMC proliferation (16, 39, 48, 50). As shown in this study, inhibition of KV channel activity by pharmacological blockade of KV channels attenuated ST-induced apoptosis in PASMCs. These results direct us to speculate that the inhibited KV channel function and expression in PASMCs from patients with primary pulmonary hypertension may also contribute to enhance pulmonary vascular medial thickening via inhibition of PASMC apoptosis.
Pulmonary vasoconstriction and vascular smooth muscle proliferation
both contribute to the elevated pulmonary vascular resistance and
arterial pressure in patients with pulmonary hypertension (16,
40, 43). Durmowicz and Stenmark (15) recently
provided evidence that a lack of apoptosis plays a role in the
vascular remodeling associated with chronic pulmonary hypertension. Our study demonstrated that ST-mediated activation of KV
channels exerts an antiproliferative effect on human PASMCs by inducing apoptosis. The cellular mechanisms involved in the ST-induced apoptosis include 1) mitochondrial membrane
depolarization, 2) activation of cytosolic caspase-3, and
3) activation of KV channels in the plasma
membrane (Fig. 6). By developing drugs
specifically targeted to KV channels located in the
pulmonary vasculature, we may find an effective alternative treatment
for patients suffering from pulmonary hypertension.
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ACKNOWLEDGEMENTS |
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We thank Ying Zhao for technical assistance.
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FOOTNOTES |
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This work was supported 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, Division 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 6 December 2000; accepted in final form 7 May 2001.
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