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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
potassium ion channel; pulmonary hypertension
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 -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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 4080% 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 57 h of exposure to the transfection medium, cells were refed with construct-free serum-containing medium and incubated 1224 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 20100 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 2224°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 (23 M) 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 (2224°C). For recording optimal whole cell IK(V), a coverslip containing cells was positioned in a recording chamber and superfused (23 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, 1525 fields (with 2040 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 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 1,0002,000 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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 30150 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).
|
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).
|
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).
|
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.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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-
, H2O2, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and ultraviolet radiation (4, 5 , 11 , 1821 , 23 , 24, 36, 3841). 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 -subunit (e.g., KCNA5) expression and inhibition of Kv channel function in PASMC have been implicated in IPAH and hypoxia-mediated pulmonary arterial hypertension (13, 27, 42, 46). In animal experiments, Pozeg et al. (27) showed that in vivo gene transfer of KCNA5, an important pore-forming
-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 (13, 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.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Archer SL, Huang JM, Hampl V, Nelson DP, Shultz PJ, and Weir EK. Nitric oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K channel by cGMP-dependent protein kinase. Proc Natl Acad Sci USA 91: 75837587, 1994.[Abstract]
3. Archer SL, Souil E, Dinh-Xuan AT, Schremmer B, Mercier JC, El Yaagoubi A, Nguyen-Huu L, Reeve HL, and Hampl V. Molecular identification of the role of voltage-gated K+ channels, Kv1.5 and Kv21, in hypoxic pulmonary vasoconstriction and control of resting membrane potential in rat pulmonary artery myocytes. J Clin Invest 101: 23192330, 1998.
4. Bock J, Szabo I, Jekle A, and Gulbins E. Actinomycin D-induced apoptosis involves the potassium channel Kv1.3. Biochem Biophys Res Commun 295: 526531, 2002.[CrossRef][ISI][Medline]
5. Bortner CD, Hughes FM Jr, and Cidlowski JA. A primary role for K+ and Na+ efflux in the activation of apoptosis. J Biol Chem 272: 3243632442, 1997.
6. Chiche JD, Schlutsmeyer SM, Bloch DB, de la Monte SM, Roberts JD Jr, Filippov G, Janssens SP, Rosenzweig A, and Bloch KD. Adenovirus-mediated gene transfer of cGMP-dependent protein kinase increases the sensitivity of cultured vascular smooth muscle cells to the antiproliferative and pro-apoptotic effects of nitric oxide/cGMP. J Biol Chem 273: 3426334271, 1998.
7. Clapp LH, Finney P, Turcato S, Tran S, Rubin LJ, and Tinker A. Differential effects of stable prostacyclin analogs on smooth muscle proliferation and cyclic AMP-generation in human pulmonary artery. Am J Respir Cell Mol Biol 26: 194201, 2002.
8. Coppock EA, Martens JR, and Tamkun MM. Molecular basis of hypoxia-induced pulmonary vasoconstriction: role of voltage-gated K+ channels. Am J Physiol Lung Cell Mol Physiol 281: L1L12, 2001.
9. Cowan KN, Heilbut A, Humpl T, Lam C, Ito S, and Rabinovitch M. Complete reversal of fatal pulmonary hypertension in rats by a serine elastase inhibitor. Nat Med 6: 698702, 2000.[CrossRef][ISI][Medline]
10. 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: 12231233, 1999.
11. Dallaporta B, Hirsch T, Susin SA, Zamzami N, Larochette N, Brenner C, Marzo I, and Kroemer G. Potassium leakage during the apoptotic degradation phase. J Immunol 160: 56055615, 1998.
12. Haunstetter A and Izumo S. Apoptosis: basic mechanisms and implications for cardiovascular disease. Circ Res 82: 11111129, 1998.
13. Hengartner MO. The biochemistry of apoptosis. Nature 407: 770776, 2000.[CrossRef][ISI][Medline]
14. Hughes FMJ, Bortner CD, Purdy GD, and Cidlowski JA. Intracellular K+ suppresses the activation of apoptosis in lymphocytes. J Biol Chem 272: 3056730576, 1997.
15. Hulme JT, Coppock EA, Felipe A, Martens JR, and Tamkun MM. Oxygen sensitivity of cloned voltage-gated K+ channels expressed in the pulmonary vasculature. Circ Res 85: 489497, 1999.
16. Ishida A, Sasaguri T, Kosaka C, Nojima H, and Ogta J. Induction of the cyclin-dependent kinase inhibitor p21(Sdi1/Cip1/Waf1) by nitric oxide-generating vasodilator in vascular smooth muscle cells. J Biol Chem 272: 1005010057, 1997.
17. Kolpakov V, Gordon D, and Kulik TJ. Nitric oxide-generating compounds inhibit total protein and collagen synthesis in cultured vascular smooth muscle cells. Circ Res 76: 305309, 1995.
18. Krick S, Platoshyn O, Sweeney M, Kim H, and Yuan JX-J. Activation of K+ channels induces apoptosis in vascular smooth muscle cells. Am J Physiol Cell Physiol 280: C970C979, 2001.
19. Krick S, Platoshyn O, Sweeney M, McDaniel SS, Zhang S, Rubin LJ, and Yuan JX-J. Nitric oxide induces apoptosis by activating K+ channels in pulmonary vascular smooth muscle cells. Am J Physiol Heart Circ Physiol 282: H184H193, 2002.
20. Lang F, Busch GL, Ritter M, Volkl H, Waldegger S, Gulbings E, and Haussinger D. Functional significance of cell volume regulatory mechanisms. Physiol Rev 78: 247306, 1996.[ISI]
21. Maeno E, Ishizaki Y, Kanaseki T, Hazama A, and Okada Y. Normotonic cell shrinkage because of disordered volume regulation is an early prerequisite to apoptosis. Proc Natl Acad Sci USA 97: 94879492, 2000.
22. Means AR. Calcium, calmodulin and cell cycle regulation. FEBS Lett 347: 14, 1994.[CrossRef][ISI][Medline]
23. Nietsch HH, Roe MW, Fiekers JF, Moore AL, and Lidofsky SD. Activation of potassium and chloride channels by tumor necrosis factor . Role in liver cell death. J Biol Chem 275: 2055620561, 2000.
24. Okada Y, Maeno E, Shimizu T, Dezaki K, Wang J, and Morishima S. Receptor-mediated control of regulatory volume decrease (RVD) and apoptotic volume decrease (AVD). J Physiol 532: 316, 2001.
25. Platoshyn O, Golovina VA, Bailey CL, Limsuwan A, Krick S, Juhaszova M, Seiden JE, Rubin LJ, and Yuan JX-J. Sustained membrane depolarization and pulmonary artery smooth muscle cell proliferation. Am J Physiol Cell Physiol 279: C1540C1549, 2000.
26. Post JM, Hume JR, Archer SL, and Weir EK. Direct role for potassium channel inhibition in hypoxic pulmonary vasoconstriction. Am J Physiol Cell Physiol 262: C882C890, 1992.
27. Pozeg ZI, Michelakis ED, McMurtry MS, Thebaud B, Wu XC, Dyck JR, Hashimoto K, Wang S, Moudgil R, Harry G, Sultanian R, Koshal A, and Archer SL. In vivo gene transfer of the O2-sensitive potassium channel Kv1.5 reduces pulmonary hypertension and restores hypoxic pulmonary vasoconstriction in chronically hypoxic rats. Circulation 107: 20372044, 2003.
28. Rabinovitch M. Elastase and the pathobiology of unexplained pulmonary hypertension. Chest 114: 213S224S, 1998.
29. Riley DJ, Thakker-Varia S, Wilson FJ, Poiani GJ, and Tozzi CA. Role of proteolysis and apoptosis in regression of pulmonary vascular remodeling. Physiol Res 49: 577585, 2000.[ISI][Medline]
30. Roberts JD Jr, Chiche JD, Weimann J, Steudel W, Zapol WM, and Bloch KD. Nitric oxide inhalation decreases pulmonary artery remodeling in the injured lungs of rat pups. Circ Res 87: 140145, 2000.
31. Runo JR and Loyd JE. Primary pulmonary hypertension. Lancet 361: 15331544, 2003.[CrossRef][ISI][Medline]
32. Santella L. The role of calcium in the cell cycle: facts and hypotheses. Biochem Biophys Res Commun 244: 317324, 1998.[CrossRef][ISI][Medline]
33. Smirnov SV, Robertson TP, Ward JPT, and Aaronson PI. Chronic hypoxia is associated with reduced delayed rectifier K+ current in rat pulmonary artery muscle cells. Am J Physiol Heart Circ Physiol 266: H365H370, 1994.
34. Smith JD, McLean SD, and Nakayama DK. Nitric oxide causes apoptosis in pulmonary vascular smooth muscle cells. J Surg Res 79: 121127, 1998.[CrossRef][ISI][Medline]
35. Stenmark KR and Mecham RP. Cellular and molecular mechanisms of pulmonary vascular remodeling. Annu Rev Physiol 59: 89144, 1997.[CrossRef][ISI][Medline]
36. Trimarchi JR, Liu L, Smith PJS, and Keefe DL. Apoptosis recruits two-pore domain potassium channels used for homeostatic volume regulation. Am J Physiol Cell Physiol 282: C588C594, 2002.
37. Wang BY, Ho HK, Lin PS, Schwarzacher SP, Pollman MJ, Gibbons GH, Tsao PS, and Cooke JP. Regression of atherosclerosis: role of nitric oxide and apoptosis. Circulation 99: 12361241, 1999.
38. Wang J, Juhaszova M, Rubin LJ, and Yuan X-J. Hypoxia inhibits gene expression of voltage-gated K+ channel subunits in pulmonary artery smooth muscle cells. J Clin Invest 100: 23472353, 1997.
39. Wang L, Xu D, Dai W, and Lu L. An ultraviolet-activated K+ channel mediates apoptosis of myeloblastic leukemia cells. J Biol Chem 274: 36783685, 1999.
40. Wang X, Xiao AY, Ichinose T, and Yu SP. Effects of tetraethylammonium analogs on apoptosis and membrane currents in cultured cortical neurons. J Pharmacol Exp Ther 295: 524530, 2000.
41. Yu SP, Yeh CH, 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: 114117, 1997.
42. Yuan JX-J, Aldinger AM, Juhaszova M, Wang J, Conte JVJ, Gaine SP, Orens JB, and Rubin LJ. Dysfunctional voltage-gated K+ channels in pulmonary artery smooth muscle cells of patients with primary pulmonary hypertension. Circulation 98: 14001406, 1998.
43. Yuan X-J. Voltage-gated K+ currents regulate resting membrane potential and [Ca2+]i in pulmonary arterial myocytes. Circ Res 77: 370378, 1995.
44. Yuan X-J, Goldman WF, Tod ML, Rubin LJ, and Blaustein MP. Hypoxia reduces potassium currents in cultured rat pulmonary but not mesenteric arterial myocytes. Am J Physiol Lung Cell Mol Physiol 264: L116L123, 1993.
45. Yuan X-J, Tod ML, Rubin LJ, and Blaustein MP. NO hyperpolarizes pulmonary artery smooth muscle cells and decreases the intracellular Ca2+ concentration by activating voltage-gated K+ channels. Proc Natl Acad Sci USA 93: 1048910494, 1996.
46. Yuan X-J, Wang J, Juhaszova M, Gaine SP, and Rubin LJ. Attenuated K+ channel gene transcription in primary pulmonary hypertension. Lancet 351: 726727, 1998.[CrossRef][ISI][Medline]
47. Yuan X-J, Wang J, Juhaszova M, Golovina VA, and Rubin LJ. Molecular basis and function of voltage-gated K+ channels in pulmonary arterial smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 274: L621L635, 1998.
48. Zhang S, Fantozzi I, Tigno DD, Yi ES, Platoshyn O, Thistlethwaite PA, Kriett JM, Yung G, Rubin LJ, and Yuan JX-J. Bone morphogenetic proteins induce apoptosis in human pulmonary vascular smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 285: L740L754, 2003.
49. Zimmermann KC, Bonzon C, and Green DR. The machinery of programmed cell death. Pharmacol Ther 92: 5770, 2001.[CrossRef][ISI][Medline]