Bcl-2 decreases voltage-gated K+ channel activity and enhances survival in vascular smooth muscle cells

Daryoush Ekhterae*, Oleksandr Platoshyn*, Stefanie Krick*, Ying Yu*, Sharon S. McDaniel, 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


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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 alpha -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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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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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 MOmega ) 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).

For recording optimal whole cell IKv, a coverslip containing the cells was positioned in a recording chamber (~0.75 ml) and superfused (2-3 ml/min) with the standard 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). In Ca2+-free PSS, CaCl2 was replaced by equimolar MgCl2, and 1 mM EGTA was added to chelate residual Ca2+. The pipette (internal) solution for recording whole cell IKv contained (in mM) 135 KCl, 4 MgCl2, 10 HEPES, 10 EGTA, and 5 Na2ATP (pH 7.2).

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 alpha -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 beta -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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Table 1.   Oligonucleotide sequences of the primers used for single-cell RT-PCR

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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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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Fig. 1.   Overexpression of Bcl-2 decreases whole cell voltage-gated K+ (Kv) currents (IKv) in pulmonary artery smooth muscle cells (PASMC). A: Western blot analysis of human Bcl-2 protein levels in primary cultured rat PASMC infected with the empty adenoviral vector (-bcl-2) and the adenovirus carrying the human bcl-2 gene (+bcl-2). The molecular mass of the human Bcl-2 is ~26 kDa. B: representative families of superimposed currents, elicited by test potentials ranging from -40 to +80 mV in 20-mV increments (holding potential -70 mV), in a control cell and a bcl-2-infected cell. Leakage and capacitance currents were subtracted. C: composite current (I)-voltage (V) relationships obtained from control cells (n = 54) and cells infected with bcl-2 (n = 63). Data are means ± SE [P < 0.001, by Student-Newman-Keuls (SNK) test, between the 2 I-V curves].

Overexpression of Bcl-2 significantly decreased the amplitude of whole cell IKv in PASMC (Fig. 1B). The averaged current amplitudes were 25 ± 4 pA at -40 mV and 1,400 ± 23 pA at +80 mV in control cells and 12 ± 2 pA (P < 0.001) and 400 ± 32 pA (P < 0.001) in cells infected with bcl-2, respectively (Fig. 1, B and C). Infection of bcl-2 negligibly affected membrane capacitance of the cells (Fig. 2A) but markedly reduced the amplitude (Fig. 2B) and current density (Fig. 2C) of IKv in rat PASMC. The relationships of current density and voltage (averaged from 54 control cells and 64 bcl-2-infected cells) show that overexpression of Bcl-2 decreased the current density of IKv by ~80% in growth-arrested PASMC (cultured in 0% FBS medium; Fig. 2C, inset). Furthermore, by analyzing the kinetics of IKv elicited by +80 mV, we observed that overexpression of Bcl-2 significantly accelerated the current inactivation; the time constants of inactivation were 225 ± 24 ms in control cells and 150 ± 18 ms (P < 0.05) in bcl-2-infected cells (Fig. 2D). These results suggest that Bcl-2 protein may, directly and/or indirectly, interfere with Kv channel proteins to decrease the function of the channels.


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Fig. 2.   Effects of overexpression of Bcl-2 on membrane capacitance (Cm), current amplitude, and current density of IKv in PASMC. A and B: values of Cm (A) and amplitude of IKv at +80 mV (B) measured from each of the control cells (open circle ) and the cells infected with bcl-2 (). Bars represent the averaged Cm (A) and IKv amplitudes (B; means ± SD). C: averaged families of current density, elicited by test potentials ranging from -40 to +80 mV (holding potential -70 mV) in control cells (n = 54) and bcl-2-infected cells (n = 63). Inset: composite relationships of current density and voltage in control cells and bcl-2-infected cells. Data are means ± SE. P < 0.001 between control and bcl-2-infected curves. D: averaged currents (a) and the normalized currents (b) showing the inactivation of IKv (at +80 mV) in control cells and cells infected with bcl-2; c: time constants (means ± SE) for current inactivation (tau inact) in control cells (open bar) and cells infected with bcl-2 (filled bar). *P < 0.001 vs. bcl-2-infected cells.

Overexpression of Bcl-2 downregulates mRNA expression of Kv channel alpha -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 alpha -subunits.

Subsequent to each patch-clamp experiment, a single PASMC used for measuring IKv was collected, and RT-PCR was performed to determine the mRNA expression of Kv1.1, Kv1.5, and Kv2.1 in the cell. An invariant mRNA of beta -actin was used as an internal control. As shown in Fig. 3A, the mRNA levels of Kv1.1, Kv1.5, and Kv2.1 in a bcl-2-infected cell were much lower than in a control cell, whereas the mRNA levels of beta -actin were similar in the control and bcl-2-infected cells. In contrast, the mRNA level of Bcl-2 in the bcl-2-infected cell was much greater than in the control cell (Fig. 3A).


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Fig. 3.   mRNA level of Bcl-2 inversely correlates to the mRNA levels of Kv channel alpha -subunits and the amplitude of whole cell IKv in single PASMC. A: single-cell RT-PCR amplified products for human Bcl-2 (267 bp) and rat Kv1.1 (298 bp), Kv1.5 (196 bp), Kv2.1 (269 bp), and beta -actin (244 bp) transcripts in a control cell (infected with the empty adenoviral vector) and a bcl-2-infected cell. "M," molecular weight marker. -RT, RT performed in the absence of reverse transcriptase (cDNA). B, top: corresponding whole cell IKv, elicited by test potentials ranging from -40 to +80 mV, in the control cell (left) and the bcl-2-infected cell (right). Bottom: I-V relationships (I-V curves) constructed from the current records shown in top in the control cell (open circle ) and the bcl-2-infected cell (). The experiments were repeated in 5 paired cells.

The reduced mRNA expression of Kv1.1, Kv1.5, and Kv2.1 should result in a decrease in total numbers of the functional Kv channels and thus reduce whole cell IKv. Indeed, in the bcl-2-infected cell, the decreased mRNA expression of Kv channel alpha -subunits (Fig. 3A) was associated with the diminished amplitude of whole cell IKv (Fig. 3B). The same results were reproduced in five pairs of control and bcl-2-infected cells. These results suggest that Bcl-2 decreases IKv by affecting both the function and expression of Kv channels in rat PASMC.

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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Fig. 4.   Inhibitory effect of overexpression of Bcl-2 on staurosporine (ST)-induced increase in IKv in PASMC. A and B: representative families of superimposed current density (a), elicited by test potentials ranging from -40 to +80 mV in 20-mV increments (holding potential -70 mV), and composite I-V relationships (I-V curves, means ± SE, b) in control cells (A) and cells infected with bcl-2 (B). The cells were treated with vehicle (-ST) or 0.02 µM ST (+ST) for 3 h before the currents were measured.

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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Fig. 5.   Inhibitory effects of 4-aminopyridine (4-AP) and overexpression of Bcl-2 on ST-induced apoptosis in PASMC. A: 4',6'-diamidino-2-phenylindole-stained nuclei of PASMC cultured in media with and without 0.02 µM ST for 20 h. B: summarized data (means ± SE) showing ST-induced apoptosis in rat PASMC cultured in the absence (-4-AP) or presence (+4-AP) of 2 mM 4-aminopyridine. **P < 0.001 (SNK) vs. -4-AP. Inset: averaged currents (at +80 mV) in rat PASMC treated with ST in the absence or presence of 4-AP. C: summarized data (means ± SE) showing ST-induced apoptosis in rat PASMC infected with the empty adenoviral vector and the adenovirus carrying bcl-2. **P < 0.001 (SNK) vs. control (-bcl-2). Inset: averaged currents (at +80 mV) in control cells (-bcl-2) and bcl-2-infected cells (+bcl-2) treated with 0.02 µM ST. Vertical and horizontal bars denote 100 pA and 50 ms, respectively.

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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Fig. 6.   Time courses of ST-mediated increase in whole cell IKv and apoptosis in PASMC. Summarized data (means ± SE) showing the current density of IKv at +80 mV () in cells before (0 h) and after treatment with 0.02 µM ST for 0.5, 1.5, 2.5, 3.5, 4.5, and 6.0 h and the percentage of apoptotic nuclei in total cells (open circle ) before (0 h) and after treatment with ST for 3, 6, 9, 15, 20, and 24 h. **P < 0.001 vs. 0 min (SNK). The time course curve of the ST-induced apoptosis is significantly different from the time course curve of the ST-induced increase in IKv (P < 0.001, SNK). IK, K+ currents.


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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 alpha -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, Cl- is a dominant anion in the cytosol, with a concentration of 50-100 mM (8, 29). Indeed, the apoptotic volume decrease induced by ST is significantly attenuated by blocking either K+ or Cl- channels, suggesting that the volume decrease is achieved by a parallel efflux of Cl- and K+ and regulated concurrently by activities of K+ and Cl- channels (34, 51, 59).

Cytoplasmic 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-alpha (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).

In a cell-free system (isolated nuclei from HeLa cells), Dallaporta et al. (14, 15) demonstrated that a decrease in K+ concentration ([K+]) from 140 to 80 mM caused a 1.6-fold increase of apoptosis induced by apoptosis-inducing factor. In lymphocytes, an 8-h treatment with ST decreased intracellular [K+] from 140 to 55 mM, whereas a decrease in [K+] in assay buffer from 150 to 80 mM caused a 2.4-fold increase in DNA degradation in isolated thymocyte nuclei (24, 25). Furthermore, increasing [K+] from 0 to 140 mM in the assay buffer almost completely abolished the cytochrome c-mediated increase in caspase-3 activity. Decreasing [K+] from 140 to 80 mM caused a 2.5-fold enhancement of the cytochrome c-mediated increase in caspase-3 activity (24, 25). These observations suggest that a decrease in [K+] is associated with an increase in caspase activity in the presence of apoptosis inducers (e.g., cytochrome c).

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 channel alpha -subunits Kv1.1, Kv1.5, and Kv2.1 was markedly attenuated in bcl-2-infected PASMC compared with cells infected with the empty adenoviral vector. These results suggest that Bcl-2 may decrease IKv by multiple mechanisms, including 1) direct or indirect blockade of the pore-forming alpha -subunit of Kv channels, 2) acceleration of inactivation kinetics of the channels, and 3) cooperation with certain transcription factors (44) to downregulate gene expression of the Kv channel alpha -subunits.

Possible 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 alpha -subunits in PASMC. The apoptosis inducer ST increased IKv and induced apoptosis. Blockade of Kv channels with 4-aminopyridine and overexpression of Bcl-2 both abolished the ST-mediated increase in IKv and attenuated the ST-induced apoptosis. These observations reveal an important mechanism by which Bcl-2 suppresses apoptosis in vascular smooth muscle cells.

Although apoptosis has long been recognized as a principal mechanism for the elimination of redundant, autoreactive, or neoplastic cells, it may also be a mechanism for the elimination of the "misguided" proliferative PASMC in remodeled pulmonary vasculature (43). Indeed, apoptosis in hypertrophied PASMC has been attributed to the regression in medial hypertrophy, whereas inhibition of apoptosis is related to progression of pulmonary vascular medial thickening (12, 13, 16, 42).


    ACKNOWLEDGEMENTS

We thank Y. Zhao for technical assistance.


    FOOTNOTES

* 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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