Augmented K+ currents and mitochondrial membrane depolarization in pulmonary artery myocyte apoptosis

Stefanie Krick, Oleksandr Platoshyn, Sharon S. McDaniel, Lewis J. Rubin, and Jason X.-J. Yuan

Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California School of Medicine, San Diego, California 92103


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

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 (Delta Psi 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 Delta Psi 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).


    METHODS AND MATERIALS
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ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
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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 MOmega ) 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).

For measurement of whole cell IK, a coverslip containing the cells was positioned in the recording chamber (approx 0.75 ml) and superfused (2 ml/min) with an extracellular (bath) physiological salt solution (PSS). The PSS contained (in mM) 141 NaCl, 4.7 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose (pH 7.4). For 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 IK contained (in mM) 135 KCl, 4 MgCl2, 10 HEPES, 10 EGTA, and 5 Na2ATP (pH 7.2). To isolate the optimal KV current [IK(V)], the cells were superfused with Ca2+-free PSS containing 1 mM tetraethylammonium chloride [TEA; which predominantly blocks Ca2+-activated K+ (KCa) channels at doses <=  1 mM] (1) and dialyzed with Ca2+-free pipette solutions including 5 mM ATP [which completely blocks ATP-sensitive K+ (KATP) channels] (7).

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 Delta Psi 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 Delta Psi m is linear. The R-123 fluorescence, which is quenched at resting Delta Psi 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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
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REFERENCES

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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Fig. 1.   Inhibitory effects of 40 mM K+ (40K) and 4-aminopyridine (4-AP) on staurosporine (ST)-induced apoptosis in human pulmonary artery smooth muscle cells (PASMCs). A: 4',6'-diamidino-2-phenylindole-stained nuclei of the cells cultured in medium with and without (control) 0.02 µM ST for 20 h. B: summarized data showing ST-induced apoptosis in PASMCs treated with 40 mM K+ or 5 mM 4-AP. Data are means ± SE; nos. in parentheses, no. of experiments. *** P < 0.001 vs. ST (by Student-Newman-Keuls test).

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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Fig. 2.   Inhibitory effect of 4-AP on whole cell voltage-gated K+ current [IK(V); IK] in human PASMCs. Aa: representative families of superimposed currents elicited by depolarizing the cell with a series of test potentials from -40 to +80 mV from a holding potential of -70 mV in 20-mV increments. The currents were recorded before (control), during, and after (recovery) a 3-min application of 5 mM 4-AP. Ab: difference currents, representing the 4-AP-sensitive components of whole cell IK, were obtained by subtracting the currents recorded during 4-AP application from the currents recorded under control conditions. Leakage and capacitive currents were subtracted. B: composite current-voltage relationships (I-V curves) obtained from PASMCs before, during, and after application of 4-AP. Data are means ± SE; n = 10 experiments. Control I-V curve is significantly different from the I-V curve during 4-AP application (P < 0.001 by Student-Newman-Keuls test).



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Fig. 3.   Augmenting effect of ST on whole cell IK(V) in human PASMCs. Aa: families of superimposed currents averaged from 14 cells before (control) and during 15-min application of 0.1 µM ST in the absence and presence of 5 mM 4-AP. The currents were elicited by test potentials ranging from -40 to +80 mV (holding potential -70 mV). Leakage and capacitive currents were subtracted. Ab: composite I-V curves obtained from PASMCs before [control (Cont)] and during ST application in the absence and presence of 4-AP. Data are means ± SE; n = 14 experiments. Control I-V curve is significantly different from the I-V curve during ST application (P < 0.001 by Student-Newman-Keuls test). Summarized data show the amplitude of IK(V) at -40 mV (Ac) before and during application of 0.1 µM ST, the ST-induced percent increase in IK(V) at various test potentials (Ad), and the time constant (tau inact) of current inactivation at +80 mV (Ae). Data are means ± SE; n = 14 experiments. *** P < 0.001 vs. Cont. B: representative time courses of the effect of ST on IK(V) in the presence of tetraethylammonium chloride (TEA). Top: changes in the amplitude of IK(V) at +80 mV before, during, and after application of 0.1 µM ST. 4-AP (5 mM) was applied to the cells when ST-induced increase in IK(V) reached the plateau. Bottom: changes in the amplitude of IK(V) at +80 mV before, during, and after application of ST in solutions containing 5 or 40 mM K+.

Interestingly, the time course of the ST-induced activation of KV channels indicated a 15- to 20-min delay between the application of ST and the onset of the increase in IK(V) (Fig. 3B). The ST-mediated activation of KV channels was almost completely blocked by extracellular application of 5 mM 4-AP (Fig. 3B, top) or by raising extracellular K+ from 4.7 to 40 mM (Fig. 3B, bottom), suggesting that the ST-induced increase in whole cell IK was predominantly due to activation of the 4-AP-sensitive KV channels.

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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Fig. 4.   Time course of ST-mediated increase in IK(V) and apoptosis in human PASMCs. Aa: averaged current records at +80 mV from 14 control cells and 20 cells treated with 0.02 µM ST for 90 min. Summarized data show the amplitude of IK(V) at -40 mV (Ab) and the tau inact of current inactivation at +80 mV (Ac) in control cells (n = 14 experiments) and cells treated with ST (n = 20 experiments). Values are means ± SE. Significantly different from control: *** P < 0.001; * P < 0.05. B: summarized data showing the current density of IK(V) at +80 mV before (0 h) and after treatment with 0.02 µM ST for 0.5, 1.5, 2.5, and 4.5 h and the percentage of apoptotic nuclei in total cells before (0 h) and after treatment with 0.02 µM ST for 3, 6, 9, 15, 20, and 24 h. Data are means ± SE. Significantly different from 0 h: *** P < 0.001; ** P < 0.01.

To determine whether the ST-induced increase in IK(V) preceded its apoptotic effect, we compared the time courses of the ST-induced increase in IK(V) and apoptosis in human PASMCs treated with 0.02 µM ST for up to 24 h. The ST-induced increase in IK(V) took place ~30 min after treatment of the cells with 0.02 µM ST (Fig. 4B). The time course of the ST-induced apoptosis indicated that the percentage of apoptotic nuclei was not significantly increased until 6 h after treatment with 0.02 µM ST; the maximal effect occurred 20 h after treatment with ST (Fig. 4B). These observations indicate that the ST-induced enhancement of IK(V) occurred before the onset of apoptosis in human PASMCs, suggesting that the increased IK(V) may play a causal role in initiating apoptosis (5, 6, 8, 10, 35, 38, 46, 47).

Effect of ST on R-123 fluorescence. The Delta Psi m is generated primarily by a proton gradient across the mitochondrial inner membrane (3). Mitochondrial membrane depolarization (less negative in Delta Psi 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 Delta Psi m depolarization was examined by measuring the changes in R-123 fluorescence intensity in human PASMCs.

As shown in Fig. 5, extracellular application of ST (0.02 µM) rapidly increased R-123 fluorescence in human PASMCs. Delta Psi m is linearly related to the R-123 fluorescence intensity in vitro. The increase in R-123 fluorescence and the decrease in Delta Psi m indicated that the mitochondrial membrane was depolarized (13, 14). However, increasing the extracellular K+ concentration from 5 to 40 mM, which decreases the driving force for K+ efflux across the plasma membrane, had negligible effect on the ST-induced increase in R-123 fluorescence (Fig. 5, B and C). These results indicate that inhibition of K+ efflux across the plasma membrane (e.g., by reducing the transmembrane K+ driving force or blocking sarcolemmal K+ channels) does not interfere with the ST-induced depolarizing effect on Delta Psi m in PASMCs.


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Fig. 5.   Depolarizing effect of ST on the mitochondrial membrane potential (Delta Psi m) in human PASMCs. A: pseudocolor images showing rhodamine 123 (R-123) fluorescence, used to estimate the relative change in Delta Psi m, in PASMCs before (control) and during application of 0.02 µM ST. B: representative time course of the ST-induced changes in R-123 fluorescence recorded in PASMCs bathed in medium containing 5 (blue circles) or 40 (red circles) mM K+. C: summarized data showing the increase in R-123 fluorescence intensity in cells treated with 0.02 µM ST for 1 or 2 min in medium containing 5 (5K) or 40 mM K+. Values are means ± SE; n = 8 experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
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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 Delta Psi 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 Delta Psi 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) Delta Psi 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 alpha -subunits and the regulatory beta -subunits (27, 37). The alpha -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 alpha -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 Kvbeta 1.2 and Kvbeta 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 Delta Psi 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 Delta Psi 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 Delta Psi 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 Delta Psi m depolarization in initiating apoptosis, and 3) whether ST induces Delta Psi 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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Fig. 6.   Schematic diagram illustrating the possible mechanisms involved in ST-induced apoptosis in PASMCs. Cyto-c, cytochrome c; -, inhibition.


    ACKNOWLEDGEMENTS

We thank Ying Zhao for technical assistance.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
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