Activation of K+ channels induces apoptosis in vascular smooth muscle cells

Stefanie Krick, Oleksandr Platoshyn, Michele Sweeney, Hyong Kim, and Jason X.-J. Yuan

Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California School of Medicine, San Diego, California 92103-8382


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Intracellular K+ plays an important role in controlling the cytoplasmic ion homeostasis for maintaining cell volume and inhibiting apoptotic enzymes in the cytosol and nucleus. Cytoplasmic K+ concentration is mainly regulated by K+ uptake via Na+-K+-ATPase and K+ efflux through K+ channels in the plasma membrane. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), a protonophore that dissipates the H+ gradient across the inner membrane of mitochondria, induces apoptosis in many cell types. In rat and human pulmonary artery smooth muscle cells (PASMC), FCCP opened the large-conductance, voltage- and Ca2+-sensitive K+ (maxi-K) channels, increased K+ currents through maxi-K channels [IK(Ca)], and induced apoptosis. Tetraethylammonia (1 mM) and iberiotoxin (100 nM) decreased IK(Ca) by blocking the sarcolemmal maxi-K channels and inhibited the FCCP-induced apoptosis in PASMC cultured in media containing serum and growth factors. Furthermore, inhibition of K+ efflux by raising extracellular K+ concentration from 5 to 40 mM also attenuated PASMC apoptosis induced by FCCP and the K+ ionophore valinomycin. These results suggest that FCCP-mediated apoptosis in PASMC is partially due to an increase of maxi-K channel activity. The resultant K+ loss through opened maxi-K channels may serve as a trigger for cell shrinkage and caspase activation, which are major characteristics of apoptosis in pulmonary vascular smooth muscle cells.

mitochondrial membrane potential; cytoplasmic calcium; pulmonary artery smooth muscle cells


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ABSTRACT
INTRODUCTION
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PULMONARY ARTERIES have a trilamellar structure that is composed of fibroblasts (adventitia), smooth muscle cells (media), and endothelial cells (intima). In pulmonary artery smooth muscle cells (PASMC), there is a natural balance between proliferation and apoptosis under normal conditions (8, 46). Augmentation of proliferation and inhibition of apoptosis in PASMC would lead to pulmonary medial thickening, which is an early vascular lesion in patients with primary pulmonary hypertension (8, 43, 46, 55). Therefore, an imbalance between PASMC proliferation and apoptosis may play a critical role in the development of pulmonary vascular remodeling. Inhibition of PASMC growth and augmentation of cell apoptosis could also serve as therapeutic approaches for patients with pulmonary hypertension (8, 37, 46, 57).

Cytoplasmic K+ in excitable and nonexcitable cells plays an important role in maintaining intracellular ion homeostasis to control cell volume (4), regulating cell cycle (7, 9), and inhibiting apoptotic enzymes in the cytosol and nucleus (24). Cytoplasmic K+ concentration ([K+]c) is mainly regulated by the activity of Na+-K+-ATPase and various K+ channels in the plasma membrane. The loss of cytoplasmic K+ due to increased K+ efflux through plasmalemmal K+ channels results in cell shrinkage, a major characteristic of apoptosis (4), and caspase activation, a triggering process in apoptosis (24, 45, 52, 56).

The large-conductance, voltage- and Ca2+-sensitive K+ (maxi-K) channels are highly expressed in vascular smooth muscle cells and synergistically regulated by cytoplasmic Ca2+ concentration ([Ca2+]c) and plasma membrane potential (Em) (5, 38, 51, 53, 63). A localized increase in [Ca2+]c (e.g., Ca2+ sparks) in vascular smooth muscle cells, due to Ca2+ release from intracellular Ca2+ stores, opens maxi-K channels, increases K+ currents [IK(Ca)] through maxi-K channels, hyperpolarizes the cell membrane, and causes vasodilation (27, 41). Opening of maxi-K channels would also promote K+ efflux and decrease [K+]c, as a result of their large conductances (200-250 pS), and induce apoptosis (60, 61).

Many metabolic inhibitors increase maxi-K channel activity by releasing Ca2+ from intracellular organelles [e.g., mitochondria and sarcoplasmic/endoplasmic reticulum (S/ER)] (11, 12, 39) and thus induce cell death. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), which uncouples mitochondrial oxidative phosphorylation and inhibits ATP synthesis, is a protonophore that dissipates the proton gradient across the inner membrane of mitochondria (19, 23). The H+ gradient is required for maintaining a transmembrane potential in mitochondria (Delta Psi m), stimulating Ca2+ accumulation in mitochondria (19), and causing oxidative ATP synthesis (23). Therefore, FCCP causes an abolition (i.e., depolarization) of Delta Psi m, which subsequently mobilizes Ca2+ from mitochondria into the cytosol (11, 12). FCCP induces apoptosis in many cell types (10). In this study, we used patch-clamp techniques and digital imaging fluorescence microscopy to test the hypothesis that FCCP-mediated activation of maxi-K channels contributes to induction of apoptosis in human and animal PASMC.


    METHODS
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INTRODUCTION
METHODS
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Cell preparation. Rat PASMC were prepared from pulmonary arteries of Sprague-Dawley rats (150-200 g) (62, 63). The isolated pulmonary arteries were incubated for 20 min in Hanks' balanced salt solution containing 1.5 mg/ml collagenase (Worthington). Adventitia and endothelium were carefully removed after the incubation. The remaining smooth muscles were then digested with 1.5 mg/ml collagenase and 0.5 mg/ml elastase (Sigma Chemical) at 37°C. The cells were plated onto 25-mm coverslips and incubated in DMEM containing 10% fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2 in air at 37°C. Human PASMC (Clonetics) were seeded in flasks at a density of 2,500-3,500 cells/cm2 and incubated in smooth muscle growth medium (Clonetics). The medium was changed after 24 h and every 48 h thereafter. Smooth muscle growth medium is composed of smooth muscle basal medium, 5% FBS, 0.5 ng/ml human epidermal growth factor, 2 ng/ml human fibroblast growth factor, and 5 µg/ml insulin. Cells were subcultured or plated onto 25-mm coverslips using trypsin-EDTA buffer (Clonetics) when 70-90% confluence was achieved. The cells at passages 4-6 were used for experimentation.

Electrophysiological measurement. Whole cell and single-channel K+ currents (IK) were recorded with an Axopatch-1D amplifier and a DigiData 1200 interface (Axon Instruments) using patch-clamp techniques (20, 62). Patch pipettes (2-4 MOmega ) were fabricated 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 software (Axon Instruments). Currents were filtered at 1-2 kHz (-3 dB) and digitized at 2-4 kHz using the amplifier. In experiments with cell-attached patches, a gigaohm seal was achieved using fire-polished glass electrodes filled with a high-K+ (135 mM) solution. The bath solution was the standard physiological salt solution (PSS) with 4.7 mM KCl. Under these conditions, the actual patch membrane potential was unknown; however, it was assumed that the patch membrane potential is equal to the difference between the pipette command potential and the actual resting membrane potential (which is about -40 mV in the cell preparation used in this study) (62, 63). Thus voltages are expressed as pipette (or applied command) potentials. All experiments were performed at room temperature (22-24°C).

Immunocytochemistry. The cells, grown on 10-mm coverslips, were first washed with PBS (Sigma Chemical) and then fixed in 95% ethanol and stained with the membrane-permeable nucleic acid stain 4',6'-diamidino-2-phenylindole (DAPI; Sigma Chemical). 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 fluorescence microscope (model TE 300, Nikon), and the cell (nuclear) images were acquired using a high-resolution fluorescence imaging system (Solamere).

For each coverslip, 5-10 fields (~20-25 cells/field) were randomly selected to determine the percentage of apoptotic cells in total cells on the basis of the morphological characteristics of apoptosis: cell (nuclear) shrinkage, nuclear condensation, and nuclear breakage. The cells with clearly defined nuclear breakage, remarkably condensed nuclear fluorescence, and significantly shrunken cell body and nucleus were defined as apoptotic cells. The relative cross-sectional nuclear area of the DAPI-stained cells (on the basis of the area of pixels) was measured using the NIH Imaging software. 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); the nuclear morphology was examined by labeling with DAPI.

Measurement of rhodamine fluorescence. The cells, grown on 25-mm coverslips, were loaded with rhodamine 123 (R123, Molecular Probes) by incubation with 10 µg/ml for 30 min at 37°C (11, 12). R123 is taken up selectively by mitochondria (29, 30), and its uptake is dependent on Delta Psi m. R123 fluorescence was excited at 488 nm and measured at 530 nm using a GEN IV charge-coupled device camera connected to a microscope (model TE 300, Nikon). In isolated mitochondria, the relationship between R123 fluorescence and Delta Psi m is linear (13). The R123 fluorescence, which is quenched at resting Delta Psi m, increases with mitochondrial membrane depolarization (11, 12). The R123 fluorescence signals were stored in a Macintosh computer and analyzed using QVD software (Solamere). The percent change of the R123 fluorescence from the baseline level is used for comparison between responses.

Measurement of [Ca2+]c. The cells were loaded with fura 2-AM (3 µM) for 30 min at 24°C under an atmosphere of 5% CO2-95% air. The fura 2-loaded cells were then superfused with PSS for 20 min at 32°C to wash away extracellular fura 2-AM and to allow sufficient time for intracellular esterases to cleave cytosolic fura 2-AM into the active fura 2. Fura 2 fluorescence (510-nm emission, 360- and 380-nm excitation) from the cells and background was measured using a charge- coupled device camera connected to a Nikon microscope. The fluorescence signals were collected continuously and stored in an IBM-compatible computer for later analysis. The 360- to 380-nm excitation ratios of the fluorescence images were then calculated and calibrated to express [Ca2+]c (18, 62).

Reagents and solutions. For measuring whole cell IK and [Ca2+]c, 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) 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 IK contained (in mM) 135 KCl, 4 MgCl2, 10 HEPES, 10 EGTA, and 5 Na2ATP (pH 7.2). For single-channel IK recording in cell-attached patches, the pipette (external) solution contained (in mM) 135 KCl, 4 MgCl2, 10 HEPES, and 10 EGTA (pH 7.4).

FCCP (Sigma Chemical) and valinomycin (Sigma Chemical) were prepared as 20 mM stock solutions in DMSO. Aliquots of the stock solutions were diluted 1:1,000-4,000 into PSS (for electrophysiological and fluorescent experiments) or 10% FBS-DMEM (for immunocytochemical experiments). Similar dilutions of DMSO (0.017-0.05%), alone, were used as vehicle control in PSS or the culture media. Tetraethylammonium (TEA; Sigma Chemical) and iberiotoxin (IBTX; Sigma Chemical) were directly dissolved into PSS or culture media on the day of use. The pH values of all solutions were checked after addition of the drugs and readjusted to 7.4. In high-K+ (25 or 40 mM) solution or culture medium, NaCl in PSS and in the customized DMEM (MediaTech) was replaced, mole-for-mole, by KCl to maintain the solution's osmolarity.

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 when P < 0.05.


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Effects of FCCP on R123 fluorescence in PASMC. Delta Psi m is primarily generated by a proton gradient across the mitochondrial inner membrane (3, 19, 23). Changes in Delta Psi m were determined in human PASMC loaded with R123 (11, 12); mitochondrial depolarization increases R123 fluorescence. In human PASMC, FCCP significantly increased R123 fluorescence (i.e., depolarized Delta Psi m; Fig. 1A). Increasing extracellular K+ concentration from 5 to 25 mM, which decreases the driving force for K+ efflux, and extracellular application of 1 mM TEA or 100 nM IBTX, which blocks maxi-K channels, negligibly affected the R123 fluorescence (Fig. 1). Furthermore, pretreatment of the cells with 25 mM K+, 1 mM TEA, or 100 nM IBTX had little effect on the FCCP-induced increases in R123 fluorescence (Fig. 1B). These results indicate that inhibition of K+ efflux across the plasma membrane, as a result of reduced K+ driving force or blocked maxi-K channels, does not interfere with the depolarizing effect of FCCP on Delta Psi m in PASMC.


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Fig. 1.   Effects of carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 25 mM K+, tetraethylammonia (TEA), and iberiotoxin (IBTX) on mitochondrial membrane potential (Delta Psi m) in pulmonary artery smooth muscle cells (PASMC). A, top: pseudocolor images showing rhodamine 123 (R123) fluorescence, used to estimate the relative change of Delta Psi m in PASMC before (control) and during application of 5 µM FCCP. Magnified images show the peripheral area of the cell before (a) and after (b) treatment with FCCP. A, bottom: representative R123 fluorescence recorded in PASMC before and during application of FCCP, 25 mM K+ (25K), 1 mM TEA, and 100 nM IBTX. B: summarized data showing FCCP-induced relative changes of R123 fluorescence in the absence (control) and presence of 25 mM K+, TEA, or IBTX in rat and human PASMC. Values are means ± SE, with number of cells in parentheses.

Maintaining a negative Delta Psi m induces Ca2+ accumulation in mitochondria (11, 12, 19). The FCCP-induced Delta Psi m depolarization in PASMC (Fig. 1) would therefore mobilize Ca2+ from mitochondria to the cytosol. Indeed, extracellular application of FCCP reversibly increased [Ca2+]c in rat PASMC in the presence (by 181 ± 14 nM, n = 28) or absence (by 134 ± 12 nM, n = 25) of extracellular Ca2+. Pretreatment of the cells with 10 µM cyclopiazonic acid (CPA) did not abolish the FCCP-induced rise in [Ca2+]c in human PASMC bathed in Ca2+-free solution, suggesting that FCCP releases Ca2+ from mitochondria (data not shown).

FCCP increases the large-conductance IK(Ca) in rat PASMC. A large-amplitude single-channel IK was observed in cell-attached membrane patches (with symmetrical K+ gradient) of rat PASMC during sustained depolarization to positive potentials (Fig. 2A). Slope conductance of the channels responsible for the current, determined by current-voltage relationships obtained from 12 cells, ranged from 200 to 225 pS (218 ± 8 pS). This is consistent with the slope conductance (200-250 pS) of the large-conductance maxi-K channels that have been identified and characterized in vascular smooth muscle cells (5, 41, 42, 44, 53). Thus this large-amplitude IK in rat PASMC was actually IK(Ca) resulting from K+ efflux through the large-conductance maxi-K channels.


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Fig. 2.   Single-channel Ca2+-sensitive K+ current [IK(Ca)] in cell-attached membrane patches of rat PASMC in symmetrical K+ gradient (A) and effect of FCCP on IK(Ca) (B). Representative current traces were recorded from a PASMC membrane patch at different membrane potentials of +50 to +120 mV (left). Composite current-voltage relationship (I-V curve) indicates that the slope conductance of this channel is 218 ± 8 pS (n = 8; right). B: unitary current recordings (top) and all-points amplitude histograms (bottom) from a cell-attached membrane patch of PASMC before (control), during (FCCP), and after (washout) 3-min application of 5 µM FCCP. The patch membrane potential was held at +70 mV. Horizontal lines denote the current level when the channel is closed. NPo, open channel probability.

Extracellular application of 5 µM FCCP for 1-2 min significantly increased single-channel IK(Ca); the steady-state open probability (NPo) was increased ninefold (from 0.06798 to 0.71057; Fig. 2B). This augmentation was negligibly influenced by pretreatment of the cells with the ATP-sensitive K+ channel blocker glibenclamide (6, 42); NPo was increased 17-fold (from 0.00685 to 0.12665) in the presence of 5 µM glibenclamide (Fig. 3A). In PASMC, extracellular application of FCCP reversibly induced membrane depolarization (from -46 ± 1.6 to -29.4 ± 2.2 mV). Therefore, the FCCP-induced activation of maxi-K channels was likely due to the synergistic effects of 1) a rise in [Ca2+]c due to Ca2+ release from mitochondria and 2) a plasma membrane depolarization. This is why the current amplitudes shown in Fig. 2 were increased during FCCP application.


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Fig. 3.   Effects of FCCP on single-channel IK(Ca) in a cell-attached patch of PASMC pretreated with glibenclamide (A) and in an outside-out patch of PASMC (B). A: single-channel recording (top) and amplitude histograms (bottom) from a cell-attached membrane patch before (control), during (FCCP), and after (washout) 3-min application of 5 µM FCCP in the presence of 4 µM glibenclamide. Patch membrane potential was held at +70 mV. B: single-channel recording (top) and amplitude histograms (bottom) from an outside-out membrane patch before (control) and during (FCCP) application (for 2 min) of 5 µM FCCP. Patch membrane potential was held at +60 mV.

In the excised (outside-out) membrane patches, however, extracellular application of 5 µM FCCP had a negligible effect on single-channel IK(Ca); NPo was slightly increased 0.1-fold (from 0.28367 to 0.30083; Fig. 3B). These results suggest that the FCCP-induced increase in IK resulted primarily from activation of maxi-K channels as a result of Ca2+ release from mitochondria, rather than from activation of ATP-sensitive K+ channels as a result of inhibited ATP synthesis (6, 42).

FCCP increases whole cell IK(Ca) in human PASMC. Extracellular application of IBTX (100 nM) and TEA (1 mM), blockers of maxi-K channels (2, 42), significantly decreased whole cell IK in human PASMC (Fig. 4A, a and b). Consistent with the single-channel results in rat PASMC (Figs. 2 and 3A), application of 5 µM FCCP reversibly increased whole cell IK in human PASMC (Fig. 4Ac). The IBTX-sensitive, TEA-sensitive, and FCCP-activated components of whole cell IK were activated at approximately -40 mV (Fig. 4B) and show marked outward rectification at potentials more positive than +40 mV (Fig. 4C). The kinetics of the IBTX-sensitive, TEA-sensitive, and FCCP-activated components of whole cell IK are very similar to those of the noisy IK(Ca) observed in vascular smooth muscle cells (2, 17). Furthermore, FCCP rapidly decreased membrane input resistance at a holding potential of 0 mV; only K+ channels were active under these conditions (the calculated equilibrium potentials for K+, Cl-, Na+, and Ca2+ were -84, -1.5, +66, and +122 mV, respectively).


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Fig. 4.   Effects of IBTX, TEA, and FCCP on whole cell IK(Ca) in human PASMC. A: representative families of currents, elicited by test potentials ranging from -40 to +80 mV in 20-mV increments (holding potential -70 mV), were recorded before (control), during, and after (recovery) application of 100 nM IBTX (a), 1 mM TEA (b), and 5 µM FCCP (c). The difference currents (subtraction), representing IBTX-, TEA-, and FCCP-sensitive currents, were obtained by subtracting the currents recorded during application of the drugs from the currents recorded under control conditions. Leakage and capacitance currents were subtracted. B: composite current-voltage relationships (I-V curves) obtained from PASMC before (Con) and during application of IBTX, TEA, and FCCP. Values are means ± SE; control I-V curves are significantly different from the I-V curves during application of IBTX, TEA, and FCCP (P < 0.01, Student-Newman-Keuls test). C: I-V curves (means ± SE) representing IBTX-, TEA-, and FCCP-sensitive currents obtained from 8, 6, and 11 PASMC, respectively. Inset: families of averaged currents, showing IBTX-, TEA-, and FCCP-sensitive components of IK. Vertical and horizontal bars denote 100 pA and 50 ms, respectively.

Inhibitory effect of IBTX on FCCP-induced increase in IK(Ca) in human PASMC. In the absence of IBTX in the pipette solution, extracellular application of 5 µM FCCP significantly increased the activity of maxi-K channels in cell-attached membrane patches; NPo was increased 67-fold (from 0.00234 to 0.15999; Fig. 5A). Inclusion of 100 nM IBTX in the pipette solution significantly decreased the activity of maxi-K channels; averaged NPo values were 0.0257 ± 0.0212 and 0.00044 ± 0.00006 in the absence and presence of IBTX, respectively. Furthermore, the FCCP-induced increase in single-channel IK(Ca) was almost abolished when 100 nM IBTX was included in the pipette solution (Fig. 5B). These results suggest that the FCCP-induced increase in IK was mainly due to activation of the IBTX-sensitive maxi-K channels.


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Fig. 5.   Inhibitory effect of IBTX on FCCP-induced increase in single-channel IK(Ca) in human PASMC. A and B: unitary current recordings (top) and amplitude histograms (bottom) from cell-attached membrane patches before (control), during (FCCP), and after (washout) application of 5 µM FCCP in the absence (-IBTX, A) and presence (+IBTX, B) of 100 nM IBTX in the pipette (extracellular) solution. Patch membrane potential was held at +70 mV. Horizontal lines denote the current level when the channel is closed.

Inhibitory effect of 40 mM K+ or TEA on FCCP-induced apoptosis in PASMC. Treatment of rat or human PASMC with FCCP (5-15 µM for 20 h) induced cell (nuclear) shrinkage, nuclear condensation, nuclear breakage, and apoptotic bodies in 15-40% of the cells, while <3% of the untreated control cells showed these apoptotic characteristics (Fig. 6, A and B). Increasing extracellular [K+] from 5 to 40 mM, which attenuates IK by reducing the K+ driving force, decreased the FCCP-induced apoptosis by ~30% in rat PASMC (from 32 ± 5 to 22 ± 5%, P < 0.001) and ~47% in human PASMC (from 38 ± 6 to 18 ± 5%, P < 0.001; Fig. 6C). Furthermore, treatment of the cells with 1 mM TEA or 100 nM IBTX, which blocks maxi-K channels, also significantly inhibited the FCCP-induced apoptosis in rat and human PASMC (Fig. 6C).


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Fig. 6.   Inhibitory effects of 40 mM K+, IBTX, and TEA on FCCP-induced apoptosis in PASMC. A: 4',6'-diamidino-2-phenylindole (DAPI)-stained nuclei of rat (a) and human (b) PASMC cultured in media with and without 15 µM FCCP for 20 h. B: dose-response curves (means ± SE) showing the percentage of apoptotic cells before and after treatment with 5, 10, and 15 µM of FCCP (for 20 h). C: summarized data showing FCCP-induced apoptosis in rat (left) and human (right) PASMC treated with 40 mM K+ (40K), 100 nM IBTX, and 1 mM TEA, respectively. Values are means ± SE, with number of experiments in parentheses. ***P < 0.001 (Student-Newman-Keuls test) vs. FCCP.

In the absence of TEA, treatment of the cells with FCCP decreased the cross-sectional area of nuclei by ~57% in rat PASMC (n = 61, P < 0.001) and by ~50% in human PASMC (n = 42, P < 0.001). In the presence of 1 mM TEA, however, FCCP only decreased the nuclear areas by ~28% in rat PASMC and ~25% in human PASMC, indicating that TEA significantly inhibited the FCCP-mediated nuclear shrinkage. The inhibitory effects of 40 mM K+, TEA, and IBTX on the FCCP-induced apoptosis in PASMC were also observed using TdT-mediated dUTP nick end label staining (data not shown).

Inhibitory effect of 40 mM K+ on valinomycin-induced apoptosis. The transmembrane K+ efflux is determined by the K+ electrochemical gradient (driving force) and the K+ permeability. Valinomycin is a K+ ionophore that increases K+ efflux and induces apoptosis in variety of cell types (16, 25), including rat and human PASMC (Fig. 7A). Increasing extracellular K+ from 5 to 40 mM, which decreases the K+ electrochemical gradient, significantly inhibited the valinomycin-induced apoptosis in rat (from 88 to 63%, P < 0.001) and human (from 81 to 63%, P < 0.001) PASMC (Fig. 7B). These results suggest that FCCP- and valinomycin-induced apoptosis in PASMC is related to increased K+ efflux, which is caused by FCCP-activated K+ channels and valinomycin-formed K+ pores in the plasma membrane.


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Fig. 7.   Inhibitory effect of 40 mM K+ on valinomycin-induced apoptosis in PASMC. A: DAPI-stained nuclei of rat (a) and human (b) PASMC cultured in control medium and medium containing 100 µM valinomycin for 25 h. B: summarized data showing valinomycin-induced apoptosis in rat and human PASMC in media containing 5 (control and valinomycin) or 40 mM K+ (valinomycin-40K). Values are means ± SE, with number of experiments in parentheses. ***P < 0.001 (Student-Newman-Keuls test) vs. valinomycin.


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FCCP induced apoptosis with characteristic cell shrinkage, nuclear condensation, and breakage in PASMC. In rat and human PASMC, FCCP depolarized Delta Psi m, mobilized Ca2+ from the mitochondria to the cytosol, activated maxi-K channels, increased IK(Ca), and induced apoptosis. Blockade of the maxi-K channels by IBTX and TEA or decrease of K+ efflux by reducing the K+ driving force significantly inhibited the FCCP-induced PASMC apoptosis. These results suggest that FCCP-mediated apoptosis in PASMC is partially due to activation of maxi-K channels in the plasma membrane. The resultant K+ loss through opened K+ channels may be a trigger for apoptosis in pulmonary vascular smooth muscle cells (Fig. 8).


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Fig. 8.   Possible involvement of maxi-K channel activation in FCCP-induced apoptosis in PASMC. [K+]c, cytoplasmic K+ concentration; -, inhibition.

Involvement of K+ efflux through sarcolemmal K+ channels in apoptosis. K+ is the predominant cation in the cytosol. Maintenance of a high [K+] in the cytoplasm (140-150 mM) is essential for 1) governing cell excitability (42), 2) setting resting Em (42), 3) regulating apoptotic enzyme activity (24), and 4) controlling cell volume (4). Cytoplasmic K+ at normal concentration (~140 mM) decreases apoptotic DNA fragmentation and caspase-3-like protease activation (24). Decrease in [K+]c, due to elevated K+ efflux through opened K+ channels, results in cell shrinkage (4, 16, 25) and reduces the inhibitory effect of cytoplasmic K+ on caspase-3-like protease and the internucleosomal DNA cleavage nuclease (24). Caspases and nucleases are major inducers of apoptosis (52). Therefore, an increase in K+ efflux, partially due to activated sarcolemmal K+ channels, is necessary for the initiation of apoptosis (24, 60, 61). The observations in PASMC from the present study are consistent with the results observed in neurons and lymphocytes (24, 60, 61): blockade of K+ channels by TEA or decreasing the K+ driving force by raising extracellular K+ significantly attenuated the apoptosis.

It is unknown whether the apoptosis induced by increasing K+ channel activity depends on the time course of K+ efflux. A transient (or short-term) increase in K+ efflux or cytosolic K+ loss would relieve its tonic suppression on caspase activity and thus trigger the caspase-mediated apoptosis (e.g., in the presence of apoptosis inducers). Because apoptosis is an irreversible process, apoptosis may occur any time when K+ efflux is increased.

In in vivo experiments, PASMC apoptosis has been observed in hypertrophied pulmonary arteries (8). Apoptosis can take place in different cell cycle phases; therefore, increasing K+ efflux should be able to work on an already modified pulmonary vascular wall. However, whether apoptosis induced by increased K+ efflux only occurs in the cells that contribute to hypertrophy, but not in the normally controlled cells, is unknown. Further study is needed to define whether apoptosis induced by increasing K+ efflux depends on cell phenotype.

Activation of maxi-K channels by FCCP-induced Ca2+ release. In vascular smooth muscle cells including PASMC, the large-conductance maxi-K channels are regulated by cytoplasmic Ca2+ and Em (5, 42, 53). A localized rise in [Ca2+]c, due to metabolic inhibition-mediated Ca2+ mobilization from mitochondria and the S/ER, activates maxi-K channels and increases IK(Ca) (39, 41, 63). FCCP is a proton ionophore that 1) depolarizes Delta Psi m by dissipating the H+ gradient across the inner membrane of mitochondria (11, 12, 19), 2) releases Ca2+ from the mitochondria into the cytosol (11, 12, 19, 36, 64), and 3) inhibits ATP production by uncoupling oxidative phosphorylation (23). There are numerous close contacts between the mitochondria and S/ER (47), suggesting that these two organelles may coordinate with each other in releasing Ca2+ to or sequestering Ca2+ from the cytosol.

In the presence of other uncouplers (e.g., antimycin, rotenone, and cyanide) and mitochondrial ATPase inhibitors (e.g., oligomycin), FCCP is still able to increase [Ca2+]c in the absence of extracellular Ca2+ (11, 12, 28). In PASMC, pretreatment of the cells with thapsigargin or CPA attenuated, but did not abolish, the FCCP-induced increase in [Ca2+]c in the absence of extracellular Ca2+ (11, 12, 15, 63). These results suggest that FCCP releases Ca2+ from multiple intracellular stores (e.g., mitochondria and S/ER) (28, 34, 63). Ca2+ sparks, caused by the coordinated opening of Ca2+ release channels in the S/ER, activate maxi-K channels and increase whole cell IK(Ca) (27, 41). Taken together, these results suggest that maxi-K channels can be efficiently opened by Ca2+ release from intracellular organelles in vascular smooth muscle cells.

We previously reported that preincubation of PASMC with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM (20 µM for 60 min) completely abolishes the FCCP-induced increases in [Ca2+]c and whole cell IK(Ca) in PASMC (63). BAPTA-AM is a membrane-permeable Ca2+ chelator that enters not only the cytosol but also intracellular stores. Thus, after hydrolysis of the acetoxymethyl ester by intracellular esterases, BAPTA is able to buffer Ca2+ in cytosol and stores, including mitochondria and sarcoplasmic reticulum. As shown in Fig. 3B, FCCP negligibly affected single-channel IK(Ca) in outside-out patches. These observations suggest that FCCP is not a direct activator of the maxi-K channel protein.

TEA is a nonselective blocker of K+ channels in vascular smooth muscle cells. Therefore, the TEA-mediated inhibitory effect on the FCCP-induced apoptosis is potentially due to its blockade of maxi-K and Kv channels in rat and human PASMC. Indeed, our preliminary observations (data not shown) and those from other investigators (60, 61) demonstrated that activation of the TEA-sensitive Kv channels also contributes to induce apoptosis in PASMC and neurons.

Other possible mechanisms involved in apoptosis mediated by FCCP or K+ channel activation. Mitochondrial intermembrane space contains several proteins that are liberated through the outer membrane to participate in initiation of apoptosis (35, 50). Release of cytochrome c to the cytosol (31, 59) and translocation of the apoptosis-inducing factor to the nucleus (50) initiate the apoptotic cascade. A direct relationship between Delta Psi m depolarization and the release of cytochrome c (and apoptosis-inducing factors) has been demonstrated to play an important role in apoptosis (10, 22, 54). However, whether Delta Psi m depolarization is required for apoptosis is still unclear (14, 32).

Mitochondrial K+ channels, which are regulated by ATP, have been identified in the inner membrane of mitochondria (26, 48). The K+ electrochemical gradient across the mitochondrial inner membrane favors K+ flux from the mitochondrial intermembrane space into the mitochondrial matrix. Activation of mitochondrial K+ channels would thus lead to matrix swelling, outer membrane breakdown, release of cytochrome c, and loss of mitochondrial function (3). Many K+-permeable channels have been described in the inner membrane of mitochondria (1, 40), but whether maxi-K (and Kv) channels are also distributed in the mitochondrial inner membrane and participate in the regulation of cytochrome c release and mitochondrial function is unknown.

Maintaining sufficient Ca2+ in the S/ER (21, 33) and mitochondria (64) has been demonstrated to be essential for cell survival. Indeed, depletion of Ca2+ from the S/ER and mitochondria results in growth arrest (49) and induces apoptosis (21, 64). In addition, Bcl-2 represses apoptosis by regulating the S/ER Ca2+ (33). Therefore, in addition to activation of maxi-K channels, the FCCP-induced apoptosis may also be due to 1) direct release of Ca2+ from the S/ER and mitochondria, 2) inhibition of oxidative ATP production (23), and 3) dephosphorylation of BAD induced by Ca2+-activated calcineurin (58).

Summary and conclusion. The results from this study suggest that FCCP-induced apoptosis in rat and human PASMC is partially due to activation of maxi-K channels in the plasma membrane. FCCP depolarizes Delta Psi m and releases Ca2+ from mitochondria to the cytosol. The local rise in [Ca2+]c activates maxi-K channels and increases IK(Ca). The resultant K+ loss due to elevated K+ efflux may play an important role in the onset of apoptosis in pulmonary vascular smooth muscle cells (Fig. 8). Activation of maxi-K channels by Ca2+ sparks due to Ca2+ release from intracellular organelles also triggers vasodilation. Thus development of drugs directed at activation of K+ channels in PASMC would be potentially a useful therapeutic approach for treatment of pulmonary hypertension that is characterized by sustained vasoconstriction and excessive vascular medial hypertrophy.


    ACKNOWLEDGEMENTS

We thank S. S. McDaniel, Y. Yu, and Y. 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 the Rotary International. J. X.-J. Yuan is an Established Investigator of the American Heart Association (Grant 974009N).

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 23 June 2000; accepted in final form 26 October 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Antonsson, B, Conti F, Ciavatta A, Montessuit S, Lewis S, Martinou I, Bernasconi L, Bernard A, Mermod J-J, Mazzei G, Maundrell K, Gambale F, Sadoul R, and Martinou J-C. Inhibition of Bax channel-forming activity by Bcl-2. Science 277: 370-372, 1997[Abstract/Free Full Text].

2.   Beech, DJ, and Bolton TB. Two components of potassium current activated by depolarization of single smooth muscle cells from the rabbit portal vein. J Physiol (Lond) 418: 293-309, 1989[Abstract].

3.   Bernardi, P. Mitochondrial transport of cations: channels, exchangers, and permeability transition. Physiol Rev 79: 1127-1155, 1999[Abstract/Free Full Text].

4.   Bortner, CD, Hughes FM, and Cidlowski JA. A primary role for K+ and Na+ efflux in the activation of apoptosis. J Biol Chem 272: 32436-32442, 1997[Abstract/Free Full Text].

5.   Carl, A, Lee HK, and Sanders KM. Regulation of ion channels in smooth muscles by calcium. Am J Physiol Cell Physiol 271: C9-C34, 1996[Abstract/Free Full Text].

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

7.   Cone, CD, Jr, and Cone CM. Induction of mitosis in mature neurons in central nervous system by sustained depolarization. Science 192: 155-158, 1976[ISI][Medline].

8.   Cowan, KN, Jones PL, and Rabinovitch M. Regression of hypertrophied rat pulmonary arteries in organ culture is associated with suppression of proteolytic activity, inhibition of tenascin-C, and smooth muscle cell apoptosis. Circ Res 84: 1223-1233, 1999[Abstract/Free Full Text].

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

10.   Dispersyn, G, Nuydens R, Connors R, Borgers M, and Geerts H. Bcl-2 protects against FCCP-induced apoptosis and mitochondrial membrane potential depolarization in PC 12 cells. Biochim Biophys Acta 1428: 357-371, 1999[ISI][Medline].

11.   Duchen, MR. Contributions of mitochondria to animal physiology: from homeostasis sensor to calcium signalling and cell death. J Physiol (Lond) 516: 1-17, 1999[Abstract/Free Full Text].

12.   Duchen, MR, and Biscoe TJ. Relative mitochondrial membrane potential and [Ca2+]i in type I cells isolated from the rabbit carotid body. J Physiol (Lond) 450: 33-61, 1992[Abstract].

13.   Emaus, RK, Grunwald R, and Lemasters JJ. Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties. Biochim Biophys Acta 850: 436-448, 1986[ISI][Medline].

14.   Finucane, DM, Waterhouse NJ, Amarante-Mendes GP, Cotter TG, and Green DR. Collapse of the inner mitochondrial transmembrane potential is not required for apoptosis of HL 60 cells. Exp Cell Res 251: 166-174, 1999[ISI][Medline].

15.   Friel, DD, and Tsien RW. An FCCP-sensitive Ca2+ store in bullfrog sympathetic neurons and its participation in stimulus-evoked changes in [Ca2+]i. J Neurosci 14: 4007-4024, 1994[Abstract].

16.   Furlong, IJ, Lopez-Mediavilla C, Ascaso R, Lopez-Rivas A, and Collins MK. Induction of apoptosis by valinomycin: mitochondrial permeability transition causes intracellular acidification. Cell Death Differ 5: 214-221, 1998[ISI][Medline].

17.   Gelband, CH, and Hume JR. Ionic currents in single smooth muscle cells of the canine renal artery. Circ Res 71: 745-758, 1992[Abstract].

18.   Grynkiewicz, G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440-3450, 1985[Abstract].

19.   Gunter, TE, Gunter KK, Sheu SS, and Gavin CE. Mitochondrial calcium transport: physiological and pathological relevance. Am J Physiol Cell Physiol 267: C313-C339, 1994[Abstract/Free Full Text].

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

21.   He, H, Lam M, McCormick TS, and Distelhorst CW. Maintenance of calcium homeostasis in the endoplasmic reticulum by Bcl-2. J Cell Biol 138: 1219-1228, 1997[Abstract/Free Full Text].

22.   Heiskanen, KM, Bhat MB, Wang H-W, Ma J, and Nieminen A-L. Mitochondrial depolarization accompanies cytochrome c release during apoptosis in PC6 cells. J Biol Chem 274: 5654-5658, 1999[Abstract/Free Full Text].

23.   Heytler, PG. Uncouplers of oxidative phosphorylation. Methods Enzymol 55: 462-442, 1979[Medline].

24.   Hughes, FM, Jr, Bortner CD, Purdy GD, and Cidlowski JA. Intracellular K+ suppresses the activation of apoptosis in lymphocytes. J Biol Chem 272: 30567-30576, 1997[Abstract/Free Full Text].

25.   Inai, Y, Yabuki M, Kanno T, Akiyama J, Yasuda T, and Utsumi K. Valinomycin induces apoptosis of ascites hepatoma cells (AH-130) in relation to mitochondrial membrane potential. Cell Struct Funct 22: 555-563, 1997[ISI][Medline].

26.   Inoue, I, Nagase H, Kishi K, and Higuiti T. ATP-sensitive K+ channel in the mitochondrial inner membrane. Nature 352: 244-247, 1991[ISI][Medline].

27.   Jaggar, JH, Porter VA, Lederer WJ, and Nelson MT. Calcium sparks in smooth muscle. Am J Physiol Cell Physiol 278: C235-C256, 2000[Abstract/Free Full Text].

28.   Jensen, JR, and Rehder V. FCCP releases Ca2+ from a non-mitochondrial store in an identified Helisoma neuron. Brain Res 551: 311-314, 1991[ISI][Medline].

29.   Johnson, LV, Walsh ML, Bockus BJ, and Chen LB. Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy. J Cell Biol 88: 526-535, 1981[Abstract].

30.   Johnson, LV, Walsh ML, and Chen LB. Localization of mitochondria in living cells with rhodamine 123. Proc Natl Acad Sci USA 77: 990-994, 1980[Abstract].

31.   Kluck, RM, Bossy-Wetzel E, Green DR, and Newmeyer DD. The release of cytochrome c from mitochondria: a primary site for Bcl-2 regulation of apoptosis. Science 275: 1132-1136, 1997[Abstract/Free Full Text].

32.   Krohn, AJ, Wahlbrink T, and Prehn JHM Mitochondrial depolarization is not required for neuronal apoptosis. J Neurosci 19: 7394-7404, 1999[Abstract/Free Full Text].

33.   Lam, M, Dubyak G, Chen L, Nunez G, Miesfeld RL, and Distelhorst CW. Evidence that BCL-2 represses apoptosis by regulating endoplasmic reticulum-associated Ca2+ fluxes. Proc Natl Acad Sci USA 91: 6569-6573, 1994[Abstract].

34.   Li, CX, and Poznansky MJ. Effect of FCCP on tight junction permeability and cellular distribution of ZO-1 protein in epithelial (MDCK) cells. Biochim Biophys Acta 1030: 297-300, 1990[ISI][Medline].

35.   Liu, X, Kim CN, Yang J, Jemmerson R, and Wang X. Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86: 147-157, 1996[ISI][Medline].

36.   Luo, Y, Bond JD, and Ingram VM. Compromised mitochondrial function leads to increased cytosolic calcium and to activation of MAP kinases. Proc Natl Acad Sci USA 94: 9705-9710, 1997[Abstract/Free Full Text].

37.   Luo, Z, Sata M, Nguyen T, Kaplan JM, Akita GY, and Walsh K. Adenovirus-mediated delivery of Fas ligand inhibits intimal hyperplasia after balloon injury in immunologically primed animals. Circulation 99: 1776-1779, 1999[Abstract/Free Full Text].

38.   McCobb, DP, Fowler NL, Featherstone T, Lingle CJ, Saito M, Krause JE, and Salkoff L. A human calcium-activated potassium channel gene expressed in vascular smooth muscle. Am J Physiol Heart Circ Physiol 269: H767-H777, 1995[Abstract/Free Full Text].

39.   Miller, AL, Morales E, Leblanc NR, and Cole WC. Metabolic inhibition enhances Ca2+-activated K+ current in smooth muscle cells of rabbit portal vein. Am J Physiol Heart Circ Physiol 265: H2184-H2195, 1993[Abstract/Free Full Text].

40.   Murphy, RC, Diwan JJ, King M, and Kinnally KW. Two high conductance channels of the mitochondrial inner membrane are independent of the human mitochondrial genome. FEBS Lett 425: 259-262, 1998[ISI][Medline].

41.   Nelson, MT, Cheng H, Rubart M, Santana LF, Bonev AD, Knot HJ, and Lederer WJ. Relaxation of arterial smooth muscle by calcium sparks. Science 270: 633-637, 1995[Abstract].

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

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

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

45.   Perregaux, D, and Gabel CA. Interleukin-1beta maturation and release in response to ATP and nigericin. Evidence that potassium depletion mediated by these agents is a necessary and common feature of their activity. J Biol Chem 269: 15195-15203, 1994[Abstract/Free Full Text].

46.   Rabinovitch, M. Elastase and the pathobiology of unexplained pulmonary hypertension. Chest 114: 213S-224S, 1998[Abstract/Free Full Text].

47.   Rizzuto, R, Pinton P, Carrington W, Fay FS, Fogarty KE, Lifshitz LM, Ruft RA, and Pozzan T. Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses. Science 280: 1763-1766, 1998[Abstract/Free Full Text].

48.   Sasaki, N, Sato T, Ohler A, Orourke B, and Marban E. Activation of mitochondrial ATP-dependent potassium channels by nitric oxide. Circulation 101: 439-445, 2000[Abstract/Free Full Text].

49.   Short, AD, Bian J, Ghosh TK, Waldron RT, Rybak SL, and Gill DL. Intracellular Ca2+ pool content is linked to control of cell growth. Proc Natl Acad Sci USA 90: 4986-4990, 1993[Abstract].

50.   Susin, SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM, Mangion J, Jacotot E, Costantini P, Loeffler M, Larochette N, Goodlett DR, Aebersold R, Siderovski DP, Penninger JM, and Kroemer G. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 387: 441-446, 1999.

51.   Tanaka, Y, Meera P, Song M, Knaus H-G, and Toro L. Molecular constituents of maxi KCa channels in human coronary smooth muscle: predominant alpha  + beta  subunit complexes. J Physiol (Lond) 502: 545-557, 1997[Abstract].

52.   Thornberry, NA, and Lazebnik Y. Caspases: enemies within. Science 281: 1312-1316, 1998[Abstract/Free Full Text].

53.   Toro, L, Wallner M, Meera P, and Tanaka Y. Maxi-KCa, a unique member of the voltage-gated K channel superfamily. News Physiol Sci 13: 112-118, 1998[Abstract/Free Full Text].

54.   Vander Heiden, MG, Chandel NS, Williamson EK, Schumacker PT, and Thompson CB. Bcl-XL regulates the membrane potential and volume homeostasis of mitochondria. Cell 91: 627-637, 1997[ISI][Medline].

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

56.   Walev, I, Reske K, Palmer M, Valeva A, and Bhakdi S. Potassium-inhibited processing of IL-1beta in human monocytes. EMBO J 14: 1607-1614, 1995[Abstract].

57.   Wang, B-Y, Ho H-KV, Lin PS, Schwarzacher SP, Pollman MJ, Gibbons GH, Tsao PS, and Cooke JP. Regression of atherosclerosis: role of nitric oxide and apoptosis. Circulation 99: 1236-1241, 1999[Abstract/Free Full Text].

58.   Wang, H-G, Pathan N, Ethell IM, Krajewski S, Yamaguchik Y, Shibasaki F, McKeon F, Bobo T, Franke TF, and Reed JC. Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD. Science 284: 339-343, 1999[Abstract/Free Full Text].

59.   Yang, J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, Peng T-I, Jones DP, and Wang X. Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked. Science 275: 1129-1132, 1997[Abstract/Free Full Text].

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

61.   Yu, SP, Yeh C-H, Strasser U, Tian M, and Choi DW. NMDA receptor-mediated K+ efflux and neuronal apoptosis. Science 284: 336-339, 1999[Abstract/Free Full Text].

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

63.   Yuan, X-J, Sugiyama T, Goldman WF, Rubin LJ, and Blaustein MP. A mitochondrial uncoupler increases KCa currents but decreases Kv currents in pulmonary artery myocytes. Am J Physiol Cell Physiol 270: C321-C331, 1996[Abstract/Free Full Text].

64.   Zhu, L, Ling S, Yu X-D, Venkatesh LK, Subramanian T, Chinnadurai G, and Kuo TH. Modulation of mitochondrial Ca2+ homeostasis by Bcl-2. J Biol Chem 274: 33267-33273, 1999[Abstract/Free Full Text].


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