Overexpression of human KCNA5 increases IK(V) and enhances apoptosis

Elena E. Brevnova, Oleksandr Platoshyn, Shen Zhang, and Jason X.-J. Yuan

Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California, San Diego, La Jolla, California 92093-0725

Submitted 26 January 2004 ; accepted in final form 5 May 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Apoptotic cell shrinkage, an early hallmark of apoptosis, is regulated by K+ efflux and K+ channel activity. Inhibited apoptosis and downregulated K+ channels in pulmonary artery smooth muscle cells (PASMC) have been implicated in development of pulmonary vascular medial hypertrophy and pulmonary hypertension. The objective of this study was to test the hypothesis that overexpression of KCNA5, which encodes a delayed-rectifier voltage-gated K+ (Kv) channel, increases K+ currents and enhances apoptosis. Transient transfection of KCNA5 caused 25- to 34-fold increase in KCNA5 channel protein level and 24- to 29-fold increase in Kv channel current (IK(V)) at +60 mV in COS-7 and rat PASMC, respectively. In KCNA5-transfected COS-7 cells, staurosporine (ST)-mediated increases in caspase-3 activity and the percentage of cells undergoing apoptosis were both enhanced, whereas basal apoptosis (without ST stimulation) was unchanged compared with cells transfected with an empty vector. In rat PASMC, however, transfection of KCNA5 alone caused marked increase in basal apoptosis, in addition to enhancing ST-mediated apoptosis. Furthermore, ST-induced apoptotic cell shrinkage was significantly accelerated in COS-7 cells and rat PASMC transfected with KCNA5, and blockade of KCNA5 channels with 4-aminopyridine (4-AP) reduced K+ currents through KCNA5 channels and inhibited ST-induced apoptosis in KCNA5-transfected COS-7 cells. Overexpression of the human KCNA5 gene increases K+ currents (i.e., K+ efflux or loss), accelerates apoptotic volume decrease (AVD), increases caspase-3 activity, and induces apoptosis. Induction of apoptosis in PASMC by KCNA5 gene transfer may serve as an important strategy for preventing the progression of pulmonary vascular wall thickening and for treating patients with idiopathic pulmonary arterial hypertension (IPAH).

potassium ion channel; pulmonary hypertension


APOPTOSIS REGULATES CELL HOMEOSTASIS by removal of excess cells or cells with genetic damage and developmental mutations (49). Dysfunction or abnormal regulation of this process has been implicated in atherosclerosis, cancer, neurodegenerative disorders, and pulmonary vascular disease (12, 28, 29). At the cellular and molecular levels, apoptosis is characterized by a distinct series of morphological and biochemical changes that include cell shrinkage, caspase activation, and DNA fragmentation (13, 49).

Apoptotic cell shrinkage or volume decrease, an early hallmark of apoptosis, is a necessary prerequisite for the programmed cell death to occur (5, 11, 21, 24). Cell volume is primarily controlled by intracellular ion homeostasis; thus ion transport across the plasma membrane is important for the regulation of cell volume (20, 24). K+ is the dominant cation in the cytoplasm (~140 mM) and thus plays a critical role in maintaining cell volume. Opening of sarcolemmal K+ channels increases efflux or loss of cytoplasmic K+ and induces apoptotic volume decrease (AVD), whereas closure or downregulation of K+ channels decelerates apoptotic cell shrinkage and attenuates apoptosis (4, 5, 11, 18, 21, 23, 36, 38, 40, 41). In addition to its role in the control of cell volume, maintenance of a high cytosolic K+ concentration ([K+]c) is required for suppression of caspases and nucleases (14), the final mediators of apoptosis (13, 49). Therefore, enhanced K+ efflux is an essential mediator not only of early apoptotic cell shrinkage but also of downstream caspase activation and DNA fragmentation (24).

Pulmonary vasoconstriction and vascular remodeling are major causes for the elevated pulmonary vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Pulmonary vascular remodeling is characterized by a combined adventitial, medial, and intimal hypertrophy. The pulmonary artery medial hypertrophy is mainly due to increased proliferation and/or decreased apoptosis of pulmonary artery smooth muscle cells (PASMC) (28, 29, 31, 35).

Downregulation and dysfunction of voltage-gated K+ (Kv) channels in PASMC have been implicated in animals with hypoxia-mediated pulmonary hypertension (8, 15, 26, 33, 38, 44) and patients with IPAH (42, 46). The decreased Kv channel activity not only causes pulmonary vasoconstriction by inducing membrane depolarization and increases in cytoplasmic Ca2+ concentration ([Ca2+]cyt) in PASMC (43) but also contributes to pulmonary vascular medial hypertrophy by inhibiting apoptotic cell shrinkage and apoptosis (48).

KCNA5 (Kv1.5) is a pore-forming {alpha}-subunit that forms hetero- or homotetrameric Kv channels in many cell types including vascular smooth muscle cells (3, 8, 47). Normal expression and function of KCNA5 channels in PASMC are necessary for the regulation of resting membrane potential and pulmonary vascular tone (3, 43). It has been reported that KCNA5 channel expression is downregulated and Kv currents are inhibited in PASMC from animals and patients with hypoxia-mediated pulmonary hypertension (3, 27, 38) and IPAH (46). In vivo gene transfer of KCNA5 with an adenoviral vector can inhibit hypoxia-mediated pulmonary arterial medial hypertrophy (27), suggesting that enhancing KCNA5 protein expression is a potential therapeutic approach for pulmonary arterial hypertension. This study was designed to test the hypothesis that overexpression of human KCNA5 gene, in addition to causing pulmonary vasodilation due to increased Kv channel current (IK(V)) and subsequent membrane hyperpolarization, enhances apoptosis in PASMC, which may contribute to the regression of PASMC hypertrophy and hyperplasia in pulmonary hypertension.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell preparation and culture. All animal procedures in this study conform to the "Guiding Principles for Research Involving Animals and Human Beings" of the American Physiological Society. PASMC were prepared from pulmonary arteries of male Sprague-Dawley rats (43). Briefly, the isolated pulmonary arteries were incubated for 20 min in Hanks' balanced salt solution containing 1.5 mg/ml collagenase (Worthington Biochemical). Adventitia and endothelium were carefully removed after the incubation. The remaining smooth muscle was then digested with 1.5 mg/ml collagenase and 0.5 mg/ml elastase (Sigma) at 37°C. Approximately 45–50 min later, PASMC were sedimented by centrifugation, resuspended in fresh media, and placed onto petri dishes or coverslips. The monkey kidney COS-7 cells (American Type Culture Collection, Manassas, VA) and rat PASMC were both cultured in high-glucose (4.5 g/l) DMEM supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (BioFluids) and incubated in 5% CO2 at 37°C in a humidified atmosphere.

Constructs. In the KCNA5-pBK construct (kindly provided by Dr. M. Tamkun from Colorado State University, Fort Collins, CO), the coding sequence of the human KCNA5 gene was subcloned into XbaI and KpnI sites of multiple cloning site (MCS) of the phagemid expression vector pBK-CMV (Stratagene). For electrophysiological experiments, a KCNA5-GFP construct was designed to visualize the transfected cells. In the KCNA5-GFP construct, the coding sequence of the human KCNA5 gene was subcloned into EcoRI and XbaI sites of MCS of the pCMS-EGFP mammalian expression vector (Clontech). In the pCMS-EGFP vector, the EGFP gene [which encodes the enhanced green fluorescent protein (GFP), a red-shifted variant of wild-type GFP from Aquorea victoria] is expressed separately from the gene of interest and is used as a transfection marker.

Transfection of KCNA5. COS-7 cells and rat PASMC were transiently transfected with the expression constructs by using Lipofectamine reagent according to the manufacturer's instruction. Briefly, cells were first split and then cultured for 24 h. Transfection was performed on 40–80% confluent cells at 37°C in serum-free Opti-MEM I medium (Invitrogen) with 1.6 µg/ml DNA and 4 µl/ml of Lipofectamine reagent. After 5–7 h of exposure to the transfection medium, cells were refed with construct-free serum-containing medium and incubated 12–24 h before experiments. The transfection efficiency was consistently >30% with the Lipofectamine reagents.

Western blot analysis. Cells were scraped from 10-cm petri dishes and collected into 15-ml tubes, centrifuged, and washed two times with cold PBS. Cell pellets were resuspended in 20–100 µl of lysis buffer [1% Triton X-100, 150 mM NaCl, 5 mM EDTA, and 50 mM Tris·HCl (pH 7.4)] supplemented with 1x protease inhibitor cocktail (Sigma) and 100 µg/ml PMSF before use. Cells were incubated in the lysis buffer for 30 min on ice. The cell lysates were then centrifuged at 14,000 rpm for 15 min, and the insoluble fraction was discarded. The protein concentrations in the supernatant were determined by the Coomassie Plus protein assay (Pierce) with BSA as a standard. Proteins (20 µg) were mixed and boiled in SDS-PAGE sample buffer for 2 min. The protein samples separated on 8% 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 for 1 h at 22–24°C in a blocking buffer (0.1% Tween 20 in PBS) containing 5% nonfat dry milk powder, the membranes were incubated with a polyclonal rabbit anti-Kv1.5 antibody (Alomone Labs) overnight at 4°C. The membranes were then washed with the blocking buffer and incubated with corresponding horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. After unbound antibodies were washed with the blocking buffer, the bound antibodies were detected with an enhanced chemiluminescence detection system (Amersham).

Electrophysiological measurement. Whole cell K+ currents were recorded with an Axopatch-1D amplifier and a DigiData 1200 interface (Axon Instruments) with patch-clamp techniques (43). Patch pipettes (2–3 M{Omega}) were fabricated on an electrode puller (Sutter) with borosilicate glass tubes and fire polished on a microforge (Narishige). Command voltage protocols and data acquisition were performed with pCLAMP 8 software (Axon Instruments). All experiments were performed at room temperature (22–24°C). For recording optimal whole cell IK(V), a coverslip containing cells was positioned in a recording chamber and superfused (2–3 ml/min) with the standard extracellular (bath) solution, which contained (in mM) 141 NaCl, 4.7 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose (pH 7.4). For the Ca2+-free solution, 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(V) contained (in mM) 135 KCl, 4 MgCl2, 10 HEPES, 10 EGTA, and 5 Na2ATP (pH 7.2). The green fluorescence emitted at 507 nm was used to visualize the cells transfected with KCNA5-GFP or pCMS-EGFP constructs.

Nuclear morphology determination. Cells grown on 25-mm coverslips were washed with PBS, fixed in 95% ethanol for 15 min at –20°C, and stained with 100 µM 4',6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma) for 8 min at 24°C. The blue fluorescence emitted at 461 nm was used to visualize the cell nuclei. The DAPI-stained cells were examined with a Nikon fluorescence microscope, and the cell (nuclear) images were acquired with a high-resolution Solamere fluorescence imaging system. For each coverslip, 15–25 fields (with 20–40 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. Cells with clearly defined nuclear breakage, remarkably condensed nuclear fluorescence, and significantly shrunken cell nuclei were defined as apoptotic cells.

Measurement of caspase-3 activity. The protein samples were prepared and protein concentration was measured as for Western blot analysis. Proteins (40 µg) were diluted by the lysis buffer to a final volume of 50 µl and subjected to caspase-3 measurements with a caspase-3 colorimetric assay kit (Assay Designs, Ann Arbor, MI), following the instructions provided by the manufacturer. Briefly, 75 µl of a caspase-3 substrate was added to 50 µl of the protein sample in a microtiter plate. The caspase-3 substrate, Ac-DEVD, was labeled with the chromophore p-nitroaniline (pNA). A colorimetric substrate (Ac-DEVD-pNA) releases free pNA from the substrate on cleavage by DEVDase. Free pNA produces a yellow color that was monitored by a spectrophotometer at 405 nm after 3 h of incubation of the plate at 37°C. The amount of yellow color produced on cleavage is proportional to the amount of caspase-3 activity present in the sample.

Cell volume evaluation. The cell volume (V) is proportional to the area (S) and radius (r) of the inscribed circle of a cell as estimated by the following equation: V {propto} S x r. Consequently, cell geometry allows us to evaluate cell volume changes by measuring the cell surface area (which is similar to the area of the inscribed circle because the cultured cells attached onto coverslips are very flat) on the cell images acquired with a high-resolution Solamere fluorescence imaging system. Only transfected cells, visualized by green fluorescence, were used for measurement of the cell surface area with Kodak 1D 3.6 software. Furthermore, a decrease in the inscribed circle area in a cell not only reflects cell volume decrease but also indicates a progression of cell "rounding" (less adherence), which is another characteristic of AVD and apoptosis.

To determine and compare the changes of cell volume in control and KCNA5-transfected cells, the cell surface area values measured after treatment with staurosporine (ST) were normalized to the area value before ST treatment and expressed as a percentage of the initial area value. Using percent changes of cell volume to compare AVD in control and KCNA5-transfected cells also minimizes the potential errors stemming from variation of cell sizes.

Chemicals. ST (Sigma) was prepared as a 1 mM stock solution in DMSO; aliquots of the stock solution were then diluted 1,000–2,000 times to the culture media for experiments. 4-Aminopyridine (4-AP; Sigma) was directly dissolved in the culture media or bath solutions on the day of use. The membrane-permeant DAPI was prepared as a 10 mM stock solution in an antibody buffer containing 500 mM NaCl, 20 µM NaN3, 10 µM MgCl2, and 20 µM Tris·HCl (pH 7.4) and diluted 1:100 in PBS before use.

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 tests (Student-Newman-Keuls) where appropriate. Differences were considered to be significant at P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Functional expression of human KCNA5 gene. To define an optimal time for electrophysiological and fluorescent microscopy experiments, we first determined the time course of KCNA5 protein expression in COS-7 cells and rat PASMC transiently transfected with KCNA5. As shown in Fig. 1A, KCNA5 protein was heterologously expressed at a very high level in both cell types transfected with KCNA5 construct compared with cells transfected with an empty vector. The expression level of KCNA5 protein was maximal 24 h after transfection and was maintained for up to 48 h (Fig. 1A, a and b). The heterologous KCNA5 protein level in KCNA5-transfected COS-7 cells and rat PASMC (24 h after transfection) was 34 and 14 times greater, respectively, than the endogenous KCNA5 protein levels in cells transfected with a control or empty vector (a pCMS-EGFP vector without KCNA5).



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Fig. 1. Expression and functional characterization of the human KCNA5 channels in COS-7 cells and rat pulmonary artery smooth muscle cells (PASMC). A: Western blot analysis of KCNA5 protein levels in COS-7 cells (a) and rat PASMC (b) 1–3 days after transfection with the control pCMS-enhanced green fluorescent protein (EGFP) vector (vector) and KCNA5. B: representative image of cells showing a KCNA5-GFP-transfected COS-7 cell that was patched for recording K+ currents. C: representative currents (a and b), elicited by depolarizing the cells from a holding potential of –80 mV to test potentials ranging from –60 to +60 mV, and composite current-voltage (I-V) relationships (c and d) in control (vector) and KCNA5-transfected cells.

 
To characterize the function of heterologous KCNA5, whole cell IK(V) were recorded and compared in cells transiently transfected with the control vector and the KCNA5-GFP construct. Twenty-four to thirty-two hours after transfection, the cells emitting green fluorescence were selected for recording IK(V) (Fig. 1B). As shown in Fig. 1C, the whole cell IK(V), elicited by depolarizing the cells from a holding potential of –70 mV to a series of test potentials ranging from –60 to +60 mV, were significantly increased in KCNA5-transfected COS-7 cells (Fig. 1Ca) and rat PASMC (Fig. 1Cb); the amplitude of IK(V) was increased by 22–29 times compared with the empty vector-transfected COS cells (Fig. 1Cc) and rat PASMC (Fig. 1Cd). These results show that 1) the whole cell IK(V) in the KCNA5-transfected cells are dominantly generated by the heterologous KCNA5 channels, whereas the contribution of endogenous K+ channels to the total IK(V) is minimal, and 2) the optimal time for maximal expression of KCNA5 channels is 24–48 h.

Overexpression of KCNA5 accelerates AVD. Increased KCNA5 channel expression and subsequent augmentation of IK(V) would promote loss of intracellular K+ and enhance AVD induced by apoptosis inducers such as ST. To investigate whether overexpression of KCNA5 influences ST-induced cell volume decrease, we first transfected COS-7 cells and rat PASMC with the control vector (pCMS-EGFP construct) and the KCNA5-GFP construct. Twenty-four hours after transfection the cells were treated with 1 µM ST for 30–150 min, and the cells emitting green fluorescence (representing transfected cells with either control vector or KCNA5-GFP construct) were selected for cell volume measurement (Fig. 2A).



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Fig. 2. Apoptotic volume decrease (AVD) is accelerated in KCNA5-transfected cells. A: rat PASMC transfected with the control pCMS-EGFP vector before (control) and after treatment with 1 µM staurosporine (ST) for 1 and 3 h. Center and right, phase contrast images of the cells. Left, combination of the phase contrast image overlapping with a fluorescent image of GFP. The photographs were taken at a magnification of x20. The cells emitting green fluorescence were selected for the cell volume measurements. B: summarized data showing the decrease in cell volume in COS-7 cells (a) and rat PASMC (b) transfected with the control vector and KCNA5 before (0 min) and during treatment with 1 µM ST for 30–150 min. The time-course curves for ST-mediated cell volume decrease in control and KCNA5-transfected cells are significantly different (P < 0.01) in COS-7 cells and rat PASMC.

 
Treatment with 1 µM ST for 30–120 min caused significant cell shrinkage in COS-7 cells (Fig. 2Ba) and rat PASMC (Fig. 2Bb); the maximal decrease in cell volume was 46.3% and 61.5%, respectively. The ST-induced AVD was significantly accelerated in KCNA5-transfected COS-7 cells (Fig. 2Ba) and rat PASMC (Fig. 2Bb) compared with the control GFP vector-transfected cells. For example, the time to reach EC50 for ST-induced AVD was shortened from 47.2 ± 4.1 (n = 26) to 22.1 ± 2.2 (n = 26) min (P < 0.001) in COS-7 cells and from 46.5 ± 4.5 (n = 19) to 24.2 ± 3.6 (n = 17) min in rat PASMC by overexpression of KCNA5 (Fig. 2B). These results suggest that increased whole cell IK(V) in KCNA5-transfected cells promote apoptotic cell shrinkage.

Overexpression of KCNA5 gene enhances apoptosis. To examine whether KCNA5 overexpressed in mammalian cells influences apoptosis, we first transfected COS-7 cells with the control vector and the KCNA5 expression construct. Twenty-four hours after transfection, the cells were treated with vehicle (DMSO) or ST. The percentage of cells that exhibited apoptotic nuclear morphology (i.e., nuclear condensation, shrinkage, and breakage) was then determined by fluorescence microscopy (Fig. 3Aa).



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Fig. 3. Apoptosis is enhanced in KCNA5-transfected cells. A: 4',6-diamidino-2-phenylindole dihydrochloride (DAPI)-stained nuclei (a) of COS-7 cells treated with (ST) or without (control) 1 µM ST for 3 h. Arrows indicate the apoptotic nuclei (photographs were taken at a magnification of x40). Magnified images (bottom) show a normal nucleus (left) and an apoptotic nucleus (right). b: Summarized data showing % of apoptotic cells before (control) and after (ST) 3-h treatment with 1 µM ST in COS-7 cells transfected with the control pCMS-EGFP vector and KCNA5. **P < 0.01, ***P < 0.001 vs. control. NS, no statistical significance (P > 0.5). c: Normalized increase of % of apoptotic cells after treatment with ST showing that ST-induced apoptosis is significantly greater in KCNA5-transfected cells (n = 25) than in control cells (vector; n = 24). ##P < 0.01 vs. vector. B: summarized data (a) showing % of apoptotic cells before (control) and after (ST) 1.5-h treatment with 0.5 µM ST in rat PASMC transfected with the control vector and KCNA5. **P < 0.01, ***P < 0.001 vs. control. b: Normalized increase of % of apoptotic cells after treatment with ST in control (n = 10) and KCNA5-transfected (n = 10) rat PASMC. ##P < 0.01 vs. vector.

 
In COS-7 cells, overexpression of KCNA5 alone had little effect on basal apoptosis, the percentage of cells undergoing apoptosis in cells that were not treated with ST (10.6 ± 0.9% in control cells vs. 12.6 ± 1.5% in KCNA5-transfected cells; P = 0.26; Fig. 3Ab), whereas ST-induced apoptosis in KCNA5-transfected cells (21.3 ± 2.5%; n = 24) was almost two times greater than in control cells (11.9 ± 1.9%; n = 25, P < 0.01) (Fig. 3A, b and c). The basal apoptosis was determined 24 h after transfection, when the transfection level of KCNA5 was maximal. In rat PASMC, however, overexpression of KCNA5 alone significantly increased the basal apoptotic rate (determined 24 h after transfection) from 8.2 ± 1.1% (control cells) to 21.3 ± 2.0% (P < 0.001) (Fig. 3Ba), in addition to enhancing ST-induced apoptosis (from 31.3 ± 1.5% to 52.3 ± 3.2%; P < 0.05; Fig. 3Bb). These results suggest that overexpression of KCNA5 channels increases whole cell IK(V), accelerates ST-induced AVD, and enhances ST-induced apoptosis in COS-7 cells and rat PASMC, whereas KCNA5 overexpression alone makes rat PASMC prone to undergo apoptosis.

Caspase-3 activation is increased in KCNA5-transfected cells. Cleavage of procaspase-3 to generate the active effector caspase-3 is an important step that leads to chromatin degradation and ultimately to apoptosis (13). To confirm that the apoptotic morphological changes that we observed in the previous experiments are associated with caspase-3 activation, we measured and compared the caspase-3 activity in total protein samples obtained from the control vector- and KCNA5-transfected COS-7 cells and rat PASMC (24 h after transfection). Consistent with the effect on apoptosis (Fig. 3), overexpression of KCNA5 had a negligible effect on basal caspase-3 activity in COS-7 cells (194 ± 30 vs. 212 ± 20 U/mg total protein in vector- and KCNA5-transfected cells) but significantly increased basal caspase-3 activity in rat PASMC (152 ± 19 vs. 234 ± 19 U/mg; P < 0.05) (Fig. 4).



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Fig. 4. Increased caspase-3 activation in KCNA5-transfected cells. Summarized data show the caspase-3 activity in total cell protein samples obtained from COS-7 cells (A) and rat PASMC (B) transfected with the control pCMS-EGFP vector and KCNA5 before (control) and after (ST) 6-h treatment with 1 µM ST. ***P < 0.05 vs. control.

 
Treatment of the cells with 1 µM ST for 6 h markedly increased caspase-3 activity in both COS-7 cells and rat PASMC, and the ST-mediated caspase activation was significantly greater in KCNA5-transfected COS-7 cells (622 U/mg) than control vector-transfected cells (416 U/mg; P < 0.05) (Fig. 4). The reason we could not detect a significant difference in ST-induced caspase-3 activation between control vector- and KCNA5-transfected rat PASMC was probably a high basal level of caspase-3 activity in KCNA5-transfected cells.

Blockade of KCNA5 channels decelerates ST-induced AVD and inhibits ST-induced apoptosis. Overexpression of KCNA5 increased whole cell IK(V), accelerated AVD, enhanced caspase-3 activation, and induced apoptosis. To verify that the proapoptotic effect of KCNA5 overexpression is due to increased K+ efflux or cytoplasmic K+ loss, we examined the effect of 4-AP, a Kv channel blocker, on KCNA5 currents and ST-induced AVD and apoptosis. Extracellular application of 3 mM 4-AP significantly and reversibly decreased whole cell IK(V) in KCNA5-GFP-transfected COS-7 cells (Fig. 5, A and C) and rat PASMC (Fig. 5, B and D), indicating that 4-AP is a potent blocker of KCNA5 channels. In these experiments, whole cell KCNA5 currents were recorded in KCNA5-GFP-transfected cells both superfused and dialyzed with Ca2+-free solutions.



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Fig. 5. Inhibitory effect of 4-aminopyridine (4-AP) on whole cell K+ currents in KCNA5-transfected cells. A and B: representative currents, elicited by depolarizing the cells from a holding potential of –80 mV to a series of test potentials ranging from –60 to +60 mV in 20-mV increments, in KCNA5-transfected COS-7 cells (A) and rat PASMC (B) before (Cont), during (4-AP), and after (washout) extracellular application of 3 mM 4-AP. C and D: composite I-V relationships (means ± SE) from KCNA5-transfected COS-7 cells (C; n = 9) and rat PASMC (D; n = 7) before, during, and after 4-AP treatment.

 
We then investigated whether 4-AP-mediated blockade of KCNA5 channels influences ST-induced AVD and apoptosis. Empty vector-transfected and KCNA5-GFP-transfected (Fig. 6A) rat PASMC (24 h after transfection) were first treated with 3 mM 4-AP for 30 min and then treated with 1 µM ST for 30–150 min in the presence of 4-AP. As shown in Fig. 6, inhibition of KCNA5 channel activity with 4-AP markedly attenuated or decelerated ST-induced AVD in both vector-transfected (Fig. 6Ba) and KCNA5-transfected (Fig. 6Bb) rat PASMC. The differences of ST-induced AVD (calculated by subtracting the time course curves in cells without 4-AP treatment from the curves in cells with 4-AP treatment) in vector- or KCNA5-transfected cells indicate that 4-AP-mediated inhibition of AVD is much greater in KCNA5-transfected cells than in vector-transfected cells at 30–90 min of ST treatment (Fig. 6C). In control vector-transfected cells, for example, 4-AP attenuated AVD from 63.1 ± 1.0% (n = 38) to 76.2 ± 1.1% (n = 50; a 21% inhibition) 60 min after ST treatment, whereas in KCNA5-GFP-transfected cells, 4-AP decreased AVD from 46.2 ± 1.3% (n = 33) to 67.2 ± 1.2% (n = 36; a 45% inhibition) (Fig. 6D). These results further demonstrate that 4-AP-sensitive native Kv channels in rat PASMC contribute to ST-induced AVD and overexpressed KCNA5 channels are responsible for the acceleration or augmentation of ST-induced AVD in KCNA5-transfected rat PASMC.



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Fig. 6. Functional blockade of KCNA5 channels by 4-AP decelerates ST-induced AVD. A: representative images showing rat PASMC (24 h after transfection with KCNA5-GFP) with (4-AP) or without (control) pretreatment with 3 mM 4-AP. These cells were not treated with ST. Only transfected cells, recognized by green fluorescence, were used to measure volume changes before and after treatment with ST. B: summarized data (means ± SE) showing the % decrease in cell volume of vector (a)- and KCNA5 (b)-transfected PASMC in response to 1 µM ST (treated for 30–150 min) in the absence (control) and presence (4-AP) of 3 mM 4-AP. C: differences of 4-AP-mediated inhibition of AVD (subtracting the time-course curve in control cells from the curve in 4-AP-treated cells) in vector- and KCNA5-transfected cells. D: % changes (mean ± SE) of cell volume in PASMC treated with and without 4-AP 1 h after ST treatment for vector- and KCNA5-transfected cells. ***P < 0.001 vs. ST alone. The 4-AP-mediated inhibition of ST-induced AVD is significantly greater in KCNA5-transfected cells than in vector-transfected cells.

 
To examine whether blockade of KCNA5 channels with 4-AP affects ST-induced apoptosis, the KCNA5-GFP-transfected COS-7 cells (24 h after transfection) were first treated with 3 mM 4-AP for 30 min and then treated with 1 µM ST for 3 h in the presence of 4-AP. Inhibition of KCNA5 channel activity with 4-AP markedly inhibited ST-induced apoptosis (Fig. 7A). In control KCNA5-GFP-transfected cells, treatment with 1 µM ST increased the percentage of apoptotic cells from 12.1 ± 0.8% to 41.2 ± 4.0% (a 3.4-fold increase), whereas in KCNA5-GFP-transfected cells treated with 4-AP, ST increased the percentage of apoptotic cells from 13.7 ± 0.9% to 29.4 ± 2.5% (a 2.1-fold increase) (Fig. 7A). The ST-induced apoptosis in KCNA5-GFP-transfected cells was reduced by ~46% after treatment of the cells with 4-AP (from 29.1 ± 4.0% to 15.7 ± 2.5%; n = 7; P < 0.01; Fig. 7B). These results suggest that the increase in IK(V) due to overexpressed KCNA5 channels results in the enhancement of ST-induced apoptosis.



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Fig. 7. Functional blockade of KCNA5 channels by 4-AP inhibits ST-induced apoptosis. A: summarized data showing % of apoptotic cells before (Cont) and after (ST) 3-h treatment with 1 µM ST in KCNA5-transfected COS-7 cells in the absence (–4-AP; n = 7) or presence (+4-AP; n = 7) of 3 mM 4-AP. ***P < 0.001 vs. Cont. B: normalized increase of % of apoptotic cells after treatment with ST showing that ST-induced apoptosis is significantly inhibited by 4-AP in KCNA5-transfected cells. **P < 0.01 vs. –4-AP.

 
It is noted that treatment with 4-AP, a potent blocker of native Kv channels and KCNA5 channels, only blocked 46% of ST-induced apoptosis (Fig. 7B). The remaining 54% of apoptosis induced by ST may result from 1) activation of 4-AP-sensitive K+ channels that were not completely blocked by the dose of 4-AP we used in these experiments, 2) activation of 4-AP-insensitive K+ channels and other cation or anion (e.g., Cl) channels, and 3) a possible apoptotic effect of 4-AP per se.


    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Pulmonary vascular medial hypertrophy, an important pathological feature in patients with pulmonary hypertension, is mainly due to unbalanced PASMC proliferation and apoptosis. Increased PASMC growth and/or decreased PASMC apoptosis can concurrently mediate thickening of the pulmonary vascular wall, subsequently reducing the lumen diameter of pulmonary arteries, increasing pulmonary vascular resistance, and raising pulmonary arterial pressure (1, 9, 10, 28, 29, 35, 48). Precise control of the balance of cell apoptosis and proliferation in PASMC thus plays a critical role in maintaining 1) the normal structural and functional integrity of the pulmonary vasculature and 2) the low pulmonary arterial pressure in normal subjects. In animal experiments, it has been demonstrated that inducing apoptosis of hypertrophied PASMC in intact pulmonary vessels can prevent the progression of the medial hypertrophy (9, 10, 28). Therefore, it is important to define the genes and gene products that participate in regulating PASMC apoptosis and proliferation.

Apoptotic cell shrinkage, an incipient prerequisite for apoptosis that precedes most other morphological alterations and caspase activation during the apoptotic process, results from a loss of cytosolic ions (e.g., K+ and Cl) and water in response to apoptosis inducers (21, 24). Therefore, the transmembrane K+ transport and activity of Kv channels play an important role in the regulation of AVD (4, 5, 11, 1821, 23, 24, 36, 38, 40, 41). In addition to regulating cell volume, K+ in the cytosol also serves as an inhibitor of caspases and nucleases (14), the central executioners of the apoptotic pathway (13). In other words, maintaining a high [K+]c (i.e., ~140 mM) is necessary for both the maintenance of normal cell volume or K+ homeostasis and the suppression of caspases and nucleases (5, 11, 14). Activation of K+ channels in the plasma membrane increases K+ efflux or loss and plays an important role in initiating AVD and apoptosis, whereas blockade of K+ channels inhibits the apoptotic cell shrinkage and attenuates apoptosis induced by a variety of apoptosis inducers, such as ST, valinomycin, anti-Fas, tumor necrosis factor-{alpha}, H2O2, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and ultraviolet radiation (4, 5 , 11 , 1821 , 23 , 24, 36, 38–41). These results suggest that cytosolic K+ homeostasis and sarcolemmal K+ channel activity are both involved in the regulation of apoptosis.

The inability of 4-AP, a potent blocker of Kv channels in vascular smooth muscle cells, to abolish ST-induced apoptosis suggests that AVD or apoptosis is not only regulated by 4-AP-sensitive Kv (e.g., KCNA5) channels but also regulated by 4-AP-insensitive K+ channels as well as Cl channels. In other words, multiple mechanisms are involved in regulating apoptotic cell shrinkage and apoptosis; activity of Kv channels may serve as one of the important mechanisms to regulate programmed cell death.

Downregulated and dysfunctional Kv channels have been implicated in PASMC from patients with IPAH (42, 46). Acute hypoxia decreases Kv channel activity and chronic hypoxia downregulates Kv channel expression in PASMC, suggesting that hypoxia mediates pulmonary vasoconstriction and vascular medial hypertrophy by, in part, inhibiting Kv channel activity (8, 15, 26, 33, 38, 44). The decreased Kv currents due to downregulated expression and/or attenuated Kv channel function depolarize PASMC, open voltage-dependent Ca2+ channels, promote Ca2+ influx, increase [Ca2+]cyt, and ultimately cause pulmonary vasoconstriction and stimulate PASMC proliferation (22, 25, 32, 43). The inhibited Kv channels in PASMC (42, 46) may also be involved in the attenuated PASMC apoptosis in IPAH patients (48) and subsequently contribute to the excessive pulmonary arterial medial hypertrophy observed in these patients.

A common hypothesis is that enhanced PASMC proliferation and inhibited PASMC apoptosis both contribute to pulmonary vascular medial hypertrophy. Therefore, inhibition of PASMC proliferation and induction of apoptosis in hypertrophied PASMC may both be beneficial for treatment of severe pulmonary arterial hypertension (9, 10, 28). For example, NO and prostacyclin (PGI2) are potent endothelium-derived vasodilators and inhibitors of smooth muscle cell growth (7, 16, 17, 30). Short-term infusion of PGI2 and inhalation of NO decrease pulmonary vascular resistance, whereas long-term therapy with PGI2 improves survival in IPAH patients (1). Furthermore, NO induces apoptosis in vascular smooth muscle cells (6, 19, 30, 34, 37). In PASMC, both NO and PGI2 activate K+ channels (e.g., voltage-gated, Ca2+-activated, and ATP-sensitive K+ channels) (2, 45). These results suggest that activation of sarcolemmal K+ channels may serve as an important therapeutic target for pulmonary hypertension because of its 1) vasodilative effect on pulmonary arteries by causing membrane hyperpolarization, closing voltage-dependent Ca2+ channels, attenuating Ca2+ influx, and decreasing [Ca2+]cyt in PASMC, 2) antiproliferative effect on PASMC by reducing cytoplasmic and nuclear [Ca2+], and 3) proapoptotic effect on PASMC by inducing apoptotic volume decrease and facilitating caspase activation. Inhibition of proliferation or induction of apoptosis in "misguided" hypertrophied PASMC leads to the regression of pulmonary medial hypertrophy (9, 10, 28).

As mentioned above, downregulation of Kv channel {alpha}-subunit (e.g., KCNA5) expression and inhibition of Kv channel function in PASMC have been implicated in IPAH and hypoxia-mediated pulmonary arterial hypertension (1–3, 27, 42, 46). In animal experiments, Pozeg et al. (27) showed that in vivo gene transfer of KCNA5, an important pore-forming {alpha}-subunit that forms delayed-rectifier Kv channels (8, 15), increased IK(V) in PASMC, decreased pulmonary vascular thickness, reduced pulmonary vascular resistance, and lowered pulmonary arterial pressure. These results provide compelling evidence that overexpression of Kv channels in PASMC is an efficient approach for treatment of pulmonary arterial hypertension.

In summary, we showed in this study that in vitro overexpression of human KCNA5 in COS-7 cells and rat PASMC increases whole cell IK(V), accelerates ST-induced apoptotic cell shrinkage, and enhances ST-induced caspase-3 activation and apoptosis. Functional blockade of KCNA5 channels with 4-AP reduced IK(V) and inhibited ST-induced apoptosis in COS-7 cells, confirming that the proapoptotic effect of KCNA5 overexpression is due to an increased K+ efflux. Furthermore, overexpression of the human KCNA5 in rat PASMC induced "basal" apoptosis or, in other words, made PASMC inclined to undergo apoptosis in the absence of apoptosis inducers. These results suggest that, compared with COS-7 cells, PASMC may rely more on K+ channel activity and the apoptotic process to remove unnecessary (e.g., misguided or hypertrophied) cells under normal conditions to maintain a thin vascular wall. Genetic abnormalities (e.g., bone morphogenetic protein receptor II mutations) and KCNA5 downregulation and dysfunction may lead to the removal or inhibition of the K+ channel-dependent apoptotic process, thereby contributing to the development of pulmonary vascular medial hypertrophy.

Further studies are necessary to determine whether the apoptotic effect of KCNA5 overexpression occurs in normal human PASMC and whether overexpression of Kv channels in PASMC from IPAH patients is able to restore normal K+ function and facilitate apoptosis. The results from this study also suggest that normal expression and function of KCNA5 channels are not only necessary for maintaining and regulating resting membrane potential and [Ca2+]cyt (1–3, 8, 15, 43, 47) but also essential for promoting cells to undergo apoptosis. The therapeutic effect of KCNA5 gene transfer on pulmonary arterial hypertension (27) may be partially due to enhanced PASMC apoptosis, which leads to the regression of pulmonary vascular remodeling and reduction of pulmonary vascular resistance.


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This work was supported by grants from the National Heart, Lung, and Blood Institute (HL-64945, HL-66012, HL-54043, HL-69758, and HL-66941).


    ACKNOWLEDGMENTS
 
We thank Dr. M. Tamkun for providing the Kv1.5 plasmid and A. Nicholson for technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. X.-J. Yuan, Division of Pulmonary and Critical Care Medicine, Dept. of Medicine, Medical Teaching Facility, University of California, San Diego, #0725, 9500 Gilman Dr., La Jolla, CA 92093-0725 (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.


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