Activation of K+ channels: an essential pathway in programmed cell death

Carmelle V. Remillard and Jason X.-J. Yuan

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


    ABSTRACT
 TOP
 ABSTRACT
 APOPTOSIS: AN OVERVIEW
 REGULATION OF CELL VOLUME
 MODULATION OF APOPTOTIC STAGES...
 ACTIVATION OF K+ CHANNELS...
 ACTIVATION OF K+ CHANNELS...
 MODULATION OF CYT-C RELEASE...
 MODULATION OF CASPASE ACTIVITY...
 CL- EFFLUX ALSO AFFECTS...
 MODULATION OF K+ CHANNEL...
 ROLE OF MITOCHONDRIAL ION...
 K+ CHANNELS IN THE...
 INHIBITION OF APOPTOSIS...
 INDUCTION OF APOPTOSIS AS...
 CONCLUSIONS
 REFERENCES
 
Cell apoptosis and proliferation are two counterparts in sharing the responsibility for maintaining normal tissue homeostasis. In recent years, the process of the programmed cell death has gained much interest because of its influence on malignant cell growth and other pathological states. Apoptosis is characterized by a distinct series of morphological and biochemical changes that result in cell shrinkage, DNA breakdown, and, ultimately, phagocytic death. Diverse external and internal stimuli trigger apoptosis, and enhanced K+ efflux has been shown to be an essential mediator of not only early apoptotic cell shrinkage, but also of downstream caspase activation and DNA fragmentation. The goal of this review is to discuss the role(s) played by K+ transport or flux across the plasma membrane in the regulation of the apoptotic volume decrease and apoptosis. Attention has also been paid to the role of inner mitochondrial membrane ion transport in the regulation of mitochondrial permeability and apoptosis. We provide specific examples of how deregulation of the apoptotic process contributes to pulmonary arterial medial hypertrophy, a major pathological feature in patients with pulmonary arterial hypertension. Finally, we discuss the targeting of K+ channels as a potential therapeutic tool in modulating apoptosis to maintain the balance between cell proliferation and cell death that is essential to the normal development and function of an organism.

apoptosis; ion channels; cell volume regulation; pulmonary artery smooth muscle cells; pulmonary hypertension


CELL DEATH IS CRITICAL for the normal development and function of multicellular organisms. For a tissue to function properly, removal of excess cells or of cells with genetic damage or improper developmental mutations is crucial. Cancer (59), hypertension (151), cardiac disease (36), viral infections (11, 125), and autoimmune (48) and neurodegenerative disorders (189) are all characterized by abnormal cell death regulation. The cellular turnover that results from the balance between cell death and proliferation is important in maintaining tissue homeostasis. Ion channels in both the sarcolemmal and mitochondrial membranes have been implicated in the signal transduction cascades that regulate apoptosis (64). This review focuses on the role played by K+ and K+ transport in the onset and development of cellular changes typical of the apoptotic process, especially in pulmonary vascular smooth muscle cells, and how modulation of K+ efflux and K+ channel function by both pro- and antiapoptotic proteins is a potential therapeutic target for cardiopulmonary diseases.


    APOPTOSIS: AN OVERVIEW
 TOP
 ABSTRACT
 APOPTOSIS: AN OVERVIEW
 REGULATION OF CELL VOLUME
 MODULATION OF APOPTOTIC STAGES...
 ACTIVATION OF K+ CHANNELS...
 ACTIVATION OF K+ CHANNELS...
 MODULATION OF CYT-C RELEASE...
 MODULATION OF CASPASE ACTIVITY...
 CL- EFFLUX ALSO AFFECTS...
 MODULATION OF K+ CHANNEL...
 ROLE OF MITOCHONDRIAL ION...
 K+ CHANNELS IN THE...
 INHIBITION OF APOPTOSIS...
 INDUCTION OF APOPTOSIS AS...
 CONCLUSIONS
 REFERENCES
 
Apoptosis, or programmed cell death, allows individual cells to die according to a highly controlled series of morphological and biochemical changes (Fig. 1). In the earliest stage of apoptosis, cells undergo shrinkage, the apoptotic cell shrinkage, with little or no change in the structure of intracellular organelles. As will be discussed later, the enhanced activation of ion-selective channels and water-permeable channels (aquaporins) modulates the apoptotic volume decrease (AVD). Nuclear condensation and DNA fragmentation within the nucleus typically occur after apoptotic stimulation and the onset of AVD, as early as 1–4 h after the apoptotic stimulation in the case of human pulmonary artery vascular smooth muscle cells (129), human leukemia HL-60 cells (31), neurons (20), human lymphoid cells (103), and thymocytes (178). The formation of apoptotic bodies (membrane-bound vesicles that pinch off from the dying cell) containing organelles and nuclear fragments constitutes the final step before phagocytosis by resident macrophages and neighboring cells. Apoptotic bodies are then degraded after phagocytosis. Unlike cellular necrosis, apoptosis does not result in an inflammatory response since the intracellular contents are not exposed to the environment prior to phagocytosis, thereby minimizing damage to adjoining healthy cells.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 1. Diagram showing the chronological order of morphological and biochemical changes during apoptotic stimulation. The apoptotic volume decrease (AVD) due to K+, Cl-, and H2O efflux occurs before nuclear condensation and DNA fragmentation. Unlike in necrosis, the cellular contents are never released from the dying cells but are ingested during phagocytosis, thereby preventing any inflammatory response by the host tissue.

 

Increased proteolytic activity following AVD is a key element in the steps leading to nucleotide fragmentation. The biochemical changes inherent to apoptosis are due to the activation of cytoplasmic proteolytic enzymes, the caspases, by proapoptotic stimuli (41, 68, 161) via one of two pathways (Fig. 2). The extrinsic pathway, or the death receptor pathway, is initiated by the activation of transmembrane death receptors by the binding of proteins such as CD95, tumor necrosis factor-{alpha} (TNF-{alpha}), and Fas ligand. Activation of the death receptors activates the membrane proximal initiator caspase-8 (and/or caspase-10), which then cleaves procaspase-3 to generate the active effector caspase-3. The intrinsic pathway, or the mitochondrial death pathway, requires disruption of the mitochondrial membrane [e.g., by staurosporine (ST), actinomycin D, peroxide, ultraviolet (UV) radiation] and/or the release or translocation of cytochrome c (cyt-c) (81, 183) and other apoptosis-inducing factors from the mitochondrial intermembrane space to the cytoplasm. The precise triggering mechanism for cyt-c release is under investigation. There are suggestions that its release results from 1) physical disruption of the mitochondrial membrane, 2) mitochondrial membrane depolarization, and 3) increased mitochondrial permeability transition (10, 60, 67, 81). Whatever its extrusion mechanism, the released cyt-c binds to apoptotic protease-activating factor 1 (APAF-1) and forms a heptameric APAF-1-cyt-c complex with deoxyadenosine triphosphate/adenosine triphosphate (dATP/ATP), the apoptosome. The apoptosome activates procaspase-9, which in turn activates the downstream effector caspases (caspase-3, -6, -7) in the cytoplasm. Activation of the effector caspases-3/-6/-7 by either death receptor stimulation or mitochondrial disruption leads to chromatin degradation and ultimately to apoptosis.



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 2. The two major apoptotic pathways involve either membrane death receptor stimulation (extrinsic pathway) and/or mitochondrial disruption (intrinsic pathway). The major proteins involved are shown, as well as the modulatory sites for selected regulatory proteins. AIF, apoptosis-inducing factor; AKT, protein kinase B; APAF-1, apoptotic protease-activating factor 1; ARC, apoptosis repressor with caspase recruitment domain; tBid, truncated Bid; cyt-c, cytochrome c; DIABLO, direct IAP-binding protein with low pI; FADD, Fas-associated death domain protein; c-FLIP, FADD-like ICE (caspase-8) inhibitory protein; IAPs, inhibitors of apoptosis; ROS, reactive oxygen species; Smac, second-mitochondrial-derived activator of caspase; {Delta}{Psi}m, mitochondrial membrane potential.

 

Two other mitochondrial proteins, Smac/Diablo (39, 165) and apoptosis-inducing factor (AIF) (77), can be released into the cytoplasm and cause apoptosis via caspase-independent pathways. The initiator caspase-8, initially activated by the extrinsic pathway, can also truncate the cytosolic Bid protein, leading to cyt-c release and then to the activation of the effector caspases-3/-6/-7 via the intrinsic pathway. Mitochondrial proteins released into the cytoplasm, such as Smac/Diablo and Omi/HtrA2 (39, 66, 156, 165, 166), can antagonize the actions of the inhibitors of apoptosis, which directly inhibit caspase activity (35, 175). The death receptor and mitochondrial pathways cross talk with each other in multiple steps to achieve the final goal, activation of the effector caspases.

In summary, apoptosis is a process that plays a critical role in embryonic development and tissue homeostasis. The programmed cell death cascade due to activated death receptors can be divided into at least three functionally distinct stages (60, 104): 1) the initiation or signaling phase in which death-promoting molecules (e.g., TNF-{alpha} and Fas ligand) bind to death receptors on the cell surface with subsequent recruitment of death domain proteins for activation of caspase-8; 2) the effector phase during which depolarization of mitochondrial membrane potential ({Delta}{Psi}m), release of cyt-c from the mitochondrial intermembrane space to the cytoplasm, and/or activation of cytoplasmic caspases take place; and c) the structural alteration and DNA degradation phase in which activated effector caspases lead to the cleavage of the lamin proteins that make up the nuclear lamina (the rigid structure that underlies the nuclear membrane and is involved in chromatin organization) and ICAD [an inhibitor of the caspase-activated deoxyribonuclease (CAD or DFF) responsible for DNA fragmentation], and to the fragmentation and degradation of genomic DNA (161). Indeed, it has been well documented that cells undergoing apoptosis show cell shrinkage, chromatin (nuclear) condensation with subsequent internucleosomal fragmentation of DNA, and membrane redistribution of phospholipids.


    REGULATION OF CELL VOLUME
 TOP
 ABSTRACT
 APOPTOSIS: AN OVERVIEW
 REGULATION OF CELL VOLUME
 MODULATION OF APOPTOTIC STAGES...
 ACTIVATION OF K+ CHANNELS...
 ACTIVATION OF K+ CHANNELS...
 MODULATION OF CYT-C RELEASE...
 MODULATION OF CASPASE ACTIVITY...
 CL- EFFLUX ALSO AFFECTS...
 MODULATION OF K+ CHANNEL...
 ROLE OF MITOCHONDRIAL ION...
 K+ CHANNELS IN THE...
 INHIBITION OF APOPTOSIS...
 INDUCTION OF APOPTOSIS AS...
 CONCLUSIONS
 REFERENCES
 
Mammalian cellular membranes are highly permeable to water. The movement of water across the membrane occurs via water channels, or aquaporins, which are highly expressed in virtually all cell types (80, 137). Of the eleven known aquaporins, for example, eight have been identified in human pulmonary artery smooth muscle cells (PASMC) to varying degrees (I. Fantozzi and J. X.-J. Yuan, unpublished observations). Animal cell membranes cannot tolerate the hydrostatic pressure gradients produced by the passive transport of water according to its concentration gradient. Therefore, water movement is largely regulated by osmotic gradients across the cell membrane and is rarely a limiting factor in cellular volume changes. In fact, alterations of intra- or extracellular osmolarity typically precede the movement of water and cellular volume changes (93, 94).

Ion transport contributes greatly to the regulation of the transmembrane osmotic gradient (Fig. 3). Most cells achieve and maintain a physiological osmotic balance through the continuous activity of an electrogenic Na+-K+-ATPase pump (3 Na+ out: 2 K+ in), which creates an intracellular environment high in K+ (~140 mM) and low in Na+ (~10 mM) (79, 181), as well as various anion and cation cotransporters (108, 121). In most excitable and nonexcitable cells, K+ is the dominant cytoplasmic cation (being ~30-fold more concentrated within the cytoplasm than the intercellular space), whereas Na+ and free Ca2+ are more concentrated in the extracellular space (Table 1). Cl- is the major anion in these cells; the cytoplasmic Cl- concentration ([Cl-]cyt) usually ranges from 5 to 15 mM in many excitable cells, although larger variations can be found in smooth muscle cells, neurons, and cardiac muscle depending on species and tissue type (76, 131, 167). [Cl-]cyt in smooth muscle cells, especially vascular smooth muscle cells, however, can be as high as 50 mM (76), suggesting that, in addition to organic anions (such as and nitrates), Cl- is a dominant cytoplasmic anion in smooth muscle cells.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 3. Transmembrane ion transport proteins involved in the regulation of cell volume. Cell swelling (top) due to increased water influx (via aquaporins) is modulated by the enhanced activity of Na+/K+/2Cl- cotransporters, Na+-K+-ATPase pumps, and coupled exchangers. Cell shrinkage (bottom) is characterized by increased cytoplasmic H2O removal (via aquaporins) due to K+ and Cl- efflux via K+ and Cl- channels, K+/Cl- cotransporters, and coupled exchangers.

 

View this table:
[in this window]
[in a new window]
 
Table 1. Ionic distribution in excitable tissues

 

Because the cell membrane is permeable to K+ under resting conditions, i.e., the permeability to K+ is much greater than the permeability to other ions, the activity of membrane K+ channels plays a critical role in the regulation of cellular volume. Whole cell K+ current (IK) at any given time is determined by the following equation

where N denotes the total number of functional K+ channels expressed in the plasma membrane; i is the current through a single K+ channel; and Popen is the steady-state open probability of a K+ channel. Therefore, when K+ channels open (i.e., i or Popen rises) and/or the number of functional K+ channels in the plasma membrane increases (i.e., N increases due to upregulation of K+ channel gene expression), the whole cell IK or transmembrane K+ efflux is increased, which would induce or enhance cell volume decrease. In contrast, when K+ channels close (i.e., i, N, or Popen decline), the whole cell IK or transmembrane K+ efflux is decreased, which would inhibit cell volume decrease.

The efflux of K+ thereby creates a positive potential outside the cell, which would drag Cl- out of the cell according to its electrochemical gradient, and the membrane hyperpolarization induced by K+ efflux would also activate membrane Cl- channels and further enhance Cl- efflux (192). The resultant accumulation of KCl outside the cell thus shifts the osmotic balance such that water is also extruded from the cell in an attempt to reestablish a normal osmotic gradient. The subsequent cell shrinkage may be functionally important since a doubling of extracellular osmolarity has been shown to trigger apoptosis in lymphocytes (15). Accordingly, the modulation of K+ and Cl- movement as well as K+ and Cl- channel activity is thus crucial in initiating and regulating the apoptotic volume decrease in cells undergoing apoptosis.

Although Na+, K+, and Cl- ions have been implicated in cell shrinkage, Ca2+ ions may also play a role in the regulation of cell volume. After cell swelling, intracellular Ca2+ concentration ([Ca2+]i) increases in some cell types, either due to enhanced sarcolemmal Ca2+ influx or due to Ca2+ release from intracellular stores (93). Although increased [Ca2+]i itself may not have a direct role on the regulation of cell volume, it may affect cytoskeletal elements such as the actin filaments or serve as a signal transduction element to activate other membrane ion channels (e.g., Ca2+-activated K+ and Cl- channels) and transporters. Obviously, both cell swelling and shrinkage result in significant changes in the cytoskeletal architecture. Actin filaments have been found to be depolymerized in swollen cells, possibly due to Ca2+ binding to gelsolin (93). An intact actin filament network is required for the activation of some volume regulatory mechanisms. Disruption due to Ca2+-mediated depolymerization will affect many processes, including 1) Na+ channel activity, 2) insertion of volume regulatory channels into the membrane, 3) regulation of channels by kinases and phospholipids, 4) activation of mechanosensitive anion channels by membrane stretch, and 5) activation of the Na+/H+ exchanger and the Na+/K+/2Cl- cotransporter, resulting in volume deregulation (93).


    MODULATION OF APOPTOTIC STAGES BY K+ FLUX ACROSS THE PLASMA MEMBRANE
 TOP
 ABSTRACT
 APOPTOSIS: AN OVERVIEW
 REGULATION OF CELL VOLUME
 MODULATION OF APOPTOTIC STAGES...
 ACTIVATION OF K+ CHANNELS...
 ACTIVATION OF K+ CHANNELS...
 MODULATION OF CYT-C RELEASE...
 MODULATION OF CASPASE ACTIVITY...
 CL- EFFLUX ALSO AFFECTS...
 MODULATION OF K+ CHANNEL...
 ROLE OF MITOCHONDRIAL ION...
 K+ CHANNELS IN THE...
 INHIBITION OF APOPTOSIS...
 INDUCTION OF APOPTOSIS AS...
 CONCLUSIONS
 REFERENCES
 
Cell shrinkage is an early hallmark of apoptosis. Apoptotic cell shrinkage occurs in two distinct stages: the initial phase starting before formation of the cyt-c/APAF-1/caspase-9 apoptosome and cell fragmentation and the late phase that is associated with cell fragmentation (123). The early phase of the apoptotic cell shrinkage when cells undergo apoptosis is mainly regulated by the activity of membrane ion channels and transporters (123, 185, 186). The time courses of the effect of apoptosis inducers on morphological changes, cyt-c translocation, caspase activation, and DNA/cell fragmentation have demonstrated that the initial phase of AVD occurs before the release of cyt-c, activation of cytoplasmic caspases, and breakage of cell nuclei. However, a rise in cytoplasmic cyt-c and an increase in active caspases in the cytoplasm have also been demonstrated to contribute to the apoptotic cell shrinkage, mainly the late phase of the volume decrease (73, 129, 143, 169, 180). These results suggest that the early and late phases of cell shrinkage in apoptotic cells may result from different mechanisms. In both stages, membrane ion channels and transporters appear to be involved.

The apoptotic cell shrinkage has been demonstrated to correlate with increased K+ and Cl- efflux and activation of K+ channels (13, 45, 87, 88, 172, 188). Because high cytoplasmic K+ ([K+]cyt) is required to maintain cytoplasmic ion homeostasis and cell volume, any changes of K+ efflux or influx will influence plasma membrane permeability and cell volume. The link between K+ efflux and apoptosis has been further established by experiments using ionophores. Valinomycin, a K+ ionophore that allows K+ efflux based on the K+ electrochemical gradient, can induce apoptosis in many cell types, including neurons (188), thymocytes (4, 29, 30), ascites hepatoma cells (74), and PASMC (87).

K+ uptake (from extracellular fluid to the cytoplasm) is modulated primarily by the ouabain-sensitive Na+-K+-ATPase, or Na+ pump (181). Recent studies have shown that anti-Fas and dexamethasone treatments inactivated an ouabain-sensitive Na+-K+-ATPase pump in lymphocytes (17) and thymocytes (106), significantly decreasing K+ uptake, irreversibly depolarizing the cell membrane, activating voltage-dependent K+ and Cl- channels, and causing apoptosis. What follows is an overall review of the modalities of cytoplasmic K+ efflux and how they regulate apoptosis.


    ACTIVATION OF K+ CHANNELS INDUCES APOPTOTIC CELL SHRINKAGE
 TOP
 ABSTRACT
 APOPTOSIS: AN OVERVIEW
 REGULATION OF CELL VOLUME
 MODULATION OF APOPTOTIC STAGES...
 ACTIVATION OF K+ CHANNELS...
 ACTIVATION OF K+ CHANNELS...
 MODULATION OF CYT-C RELEASE...
 MODULATION OF CASPASE ACTIVITY...
 CL- EFFLUX ALSO AFFECTS...
 MODULATION OF K+ CHANNEL...
 ROLE OF MITOCHONDRIAL ION...
 K+ CHANNELS IN THE...
 INHIBITION OF APOPTOSIS...
 INDUCTION OF APOPTOSIS AS...
 CONCLUSIONS
 REFERENCES
 
Enhancement of K+ efflux-mediated cell shrinkage is considered to be one of the earliest signs of apoptosis in many cells. The role of K+ channels in apoptosis was proposed by the original work of Yu et al. in 1997 (188) and subsequently supported by other investigators (18, 30, 56, 87, 103, 169). Bortner et al. (18, 169) showed a correlation between the significant decrease in [K+]cyt and the number of shrunken cells when lymphocyte apoptosis was induced by Fas ligand, dexamethasone, ST, and anisomycin (a protein synthesis inhibitor), thereby establishing a link between K+ efflux and AVD. These studies were further reinforced by observations that raising extracellular K+ ([K+]o), which reduces transmembrane K+ concentration gradient and reduces K+ efflux, can inhibit AVD and apoptosis induced by valinomycin, Fas ligand, carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone (FCCP), and ST in neurons (188), PASMC (86, 87), and lymphocytes (18, 56). Inhibition of K+ channel activity with quinine or Ba2+ also prevents cell shrinkage induced by ST or TNF-{alpha}/cycloheximide (103). In addition, treatment of lymphocytes and cortical neurons with tetrapentylammonium (TPA) inhibits the early stages of apoptosis before caspase activation has occurred (30, 176). These results indicate that an increased K+ efflux via sarcolemmal K+ channels is thus a central mediator of AVD and apoptosis.

Okada and Maeno (123) classified the apoptotic cell shrinkage into two stages: the early volume decrease that occurs before cyt-c release and caspase activation and the late volume decrease that occurs concurrently with DNA fragmentation and nuclear breakage. In PASMC, our recent results suggest that, when cells are treated with ST, voltage-gated K+ current (IK(V)) increases within 30 min immediately followed by a reduction of cell size/volume (Fig. 4) (129). The ST-mediated nuclear breakage and condensation, determined by 4',6'-diamidino-2-phenylindole (DAPI) staining (and terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay, occurs after the ST-mediated increase in IK(V) and cell volume decrease (Fig. 4) (129). Furthermore, cytoplasmic application of recombinant cyt-c enhances 4-aminopyridine (4-AP)-sensitive voltage-gated K+ (KV) currents in these cells (Fig. 5) independently of caspase-9 activation (129), suggesting that cyt-c-mediated opening of K+ channels precedes caspase-9 activation. These observations indicate that activation of K+ channels is involved in both the early and late volume decrease. In cells challenged by apoptosis inducers (e.g., ST, TNF-{alpha}, UV light, dexamethasone) or death triggers, K+ channels may be activated by an unknown mechanism to initiate the early volume decrease and further activated by cyt-c to maintain the early stage of cell shrinkage. The cyt-c-mediated KV channel activation may also play an important role in initiating the late volume decrease associated with cell fragmentation.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 4. Whole cell voltage-gated K+ current [IK(V)] increase precedes staurosporine (ST)-induced AVD in rat pulmonary artery smooth muscle cells (rPASMC). A: averaged IK(V), elicited by 300-ms test potentials ranging from -60 to +80 mV (-70 mV holding potential) in control (untreated, Cont) cells and following ST (0.02 µM) treatment for 0.5 (middle) and 2.5 (right) h. B: phase-contrast images of cell before ST (0.02 µM) and after 2.5- and 3.5-h ST treatment (middle and right). C: time courses of ST-induced enhancement of IK(V) (red circles), cell volume (AVD, blue triangles), and nuclear breakage (green bars) show that AVD and IK(V) enhancement occur within a similar short time frame, whereas nuclear breakage starts 4–6 h after ST treatment. D: inverse correlation of the amplitude of IK(V) and cell size in PASMC treated with ST (r2 = 0.91), indicating that increased currents lead to cell shrinkage. [Modified from Platoshyn et al. (129).]

 


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 5. Cytoplasmic application of cyt-c enhances IK(V) in PASMC. A: representative currents were elicited by 300-ms test potentials from -60 to +80 mV from a holding potential of -70 mV. Recordings were obtained immediately (0 min) or 20 min following whole cell access during which recombinant cyt-c (5 µM, dissolved in the pipette solution) was allowed to dialyze into the cell. B: time course of cyt-c-induced IK(V) increase after membrane rupture. [Modified from Platoshyn et al. (129).]

 


    ACTIVATION OF K+ CHANNELS INDUCES APOPTOSIS
 TOP
 ABSTRACT
 APOPTOSIS: AN OVERVIEW
 REGULATION OF CELL VOLUME
 MODULATION OF APOPTOTIC STAGES...
 ACTIVATION OF K+ CHANNELS...
 ACTIVATION OF K+ CHANNELS...
 MODULATION OF CYT-C RELEASE...
 MODULATION OF CASPASE ACTIVITY...
 CL- EFFLUX ALSO AFFECTS...
 MODULATION OF K+ CHANNEL...
 ROLE OF MITOCHONDRIAL ION...
 K+ CHANNELS IN THE...
 INHIBITION OF APOPTOSIS...
 INDUCTION OF APOPTOSIS AS...
 CONCLUSIONS
 REFERENCES
 
In addition to K+-permeable channels, K+ efflux is also controlled by many different mechanisms in excitable cells, such as an electroneutral K+-Cl- symporter and a K+-H+ and exchanger system (121). Enhanced K+ efflux through K+ channels, however, is a major pathway for K+ loss. Five classes of K+ channels have been identified in excitable cells: KV channels, Ca2+-activated K+ (KCa) channels, ATP-sensitive K+ (KATP) channels, inwardly rectifying K+ (KIR) channels, and tandem pore K+ (KT, two-pore six-domain) channels (97, 105, 115). The enhanced activity of four of these channels has been implicated in apoptosis induced by the stimulation of either the mitochondrial or death receptor apoptotic pathways, as is discussed below.

ST, a potent apoptosis inducer in almost all cell types, enhances the activity of a 4-AP-sensitive KV channel in human and rat PASMC (Fig. 6A) (37, 85) and of a tetraethylammonium (TEA)-sensitive K+ channel in mouse neocortical neurons (188). Activation of the 4-AP-sensitive KV channels by the nitric oxide (NO) donor S-nitroso-N-acetyl-penicillamine (SNAP) also causes apoptosis in PASMC (Fig. 6B). In rat and human PASMC treated with ST, the maximum enhancement of KV currents occurs within 6 h, whereas apoptosis is maximal after ~24 h of treatment (Fig. 4C), suggesting that KV channel activation occurs rapidly following the challenge of apoptotic inducers or death triggers and likely precedes caspase activation and DNA degradation (43, 86, 129). A similar 4-AP-sensitive K+ current is activated by UV radiation in myeloblastic leukemia cells (173). In rat fetal neurons, the sulfhydryl-oxidizing agent 2,2'-dithiodipyridine activates a TEA-sensitive K+ current with similar kinetics to that produced by the 4-AP-sensitive KV channels (109), whereas neuronal apoptosis is associated with a significant increase in KV currents (188). K+ currents sensitive to TPA, a TEA analog, were also detected in thymocytes and cortical neurons treated with dexamethasone and ST, respectively (30, 176). In thymocytes, TPA prevented all characteristics of dexamethasone-induced apoptosis, including {Delta}{Psi}m dissipation, cytosolic K+ efflux, chromatin condensation, and caspase and endonuclease activation (30). These results using TPA as a K+ channel inhibitor should be interpreted with caution, as the compound has been shown to have multiple nonspecific effects on voltage-dependent Ca2+ and Na+ channels' activity, as well as on K+ channel activity, in cortical neurons (176).



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 6. Apoptotic inducers increase K+ efflux via sarcolemmal K+ channels in PASMC. ST (0.1 µM, A) and S-nitroso-N-acetyl penicillamine (SNAP, 0.1 mM; B) treatments activate 4-aminopyridine (4-AP)-sensitive KV currents [whole cell IK(V) recordings in A and B] in human PASMC (hPASMC) and rPASMC, respectively. The development of apoptotic nuclei induced by ST and SNAP, characterized by nuclear shrinkage and condensation (quantified using DAPI staining and TUNEL assay), is inhibited by the application of 4-AP, a relatively selective KV channel antagonist (bar graphs). TEA, tetraethylammonium. [Modified from Krick et al. (86, 88).] ***P < 0.001 vs. ST (A) or SNAP (B).

 

In addition to KV channels, activation of KCa channels has also been implicated in AVD and apoptosis. In vascular smooth muscle cells, for example, FCCP, which dissipates the proton gradient across the inner mitochondrial membrane (IM) and disrupts the {Delta}{Psi}m, causes an increase in cytoplasmic free Ca2+ concentration and enhances K+ efflux via iberiotoxinand TEA-sensitive KCa channels (Fig. 7A) (38, 87). Activation of KCa channels by the NO donor SNAP (Fig. 7B) and by diydroepiandrosterone (DHEA) also induces apoptosis in human PASMC (86, 88). TNF-{alpha}, a death receptor agonist, activates Ca2+-dependent and protein kinase C (PKC)-activated KCa channels, increases K+ currents and efflux, and induces apoptosis in rat liver HTC cells (120). Furthermore, hydrogen peroxide (H2O2)-mediated apoptosis (134) is associated with activation of TREK KT channels (162), whereas cromakalim induces neuronal apoptosis by activating KATP channels (188). Conductance of the human ether-a-go-go channels markedly promotes H2O2-induced apoptosis in various tumor cell lines (170). Nevertheless, inhibition of K+ channels by Ba2+ and quinine attenuates apoptosis and increases viability of ST- or TNF-{alpha}/cycloheximide-treated cells in human lymphoid (U-937) and epithelial (HeLa) cells, hybrid neuroblastoma/glioma (NG108-5) cells, rat pheochromoytoma (PC12) cells (103), and liver HTC cells (120).



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 7. Proapoptotic agents stimulate K+ efflux via iberiotoxin (IBTX)-sensitive Ca2+-activated (KCa) channels in PASMC. Unitary KCa currents (IK(CA)) were recorded in hPASMC and rPASMC stimulated with carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone (FCCP, 5 µM; A) or SNAP (0.1 mM, B) and following drug washout. Single channel conductance of the evoked currents was ~250 pS, typical for KCa channels in vascular smooth muscle. The application of 100 nM IBTX, a selective inhibitor of these large-conductance KCa channels, attenuated the apoptosis induced by FCCP and SNAP (histograms) in rPASMC and hPASMC. [Modified from Krick et al. (87, 88).] ***P < 0.001 vs. FCCP (A) or SNAP (B).

 

There is evidence that the proapoptotic stimulation of K+ channels may be mediated by auxiliary modulatory proteins and kinases. KChAP (K+ channel-associated protein/protein inhibitor of activated STAT) is a K+ channel modulatory protein belonging to the protein inhibitor of the STAT family, members of which are known to interact with transcription factors such as the proapoptotic p53 protein (90, 179). KChAP induces apoptosis in prostate cancer cell lines by increasing K+ efflux and causing cell shrinkage; KChAP is also increased by ST treatment (179). On the basis of the latter study, it is believed that KChAP increases p53 levels and stimulates phosphorylation of p53 residue serine 15, leading to elevation of p21 levels and apoptosis (38, 179). Although PKC is involved in numerous cell functions, its role in modulating apoptosis is unclear. There is evidence that the enhanced K+ efflux produced by Fas and TNF-{alpha} can be blocked by PKC stimulation (56, 120), i.e., PKC inhibition promotes cell shrinkage. Tyrosine kinase-mediated phosphorylation appears to play a more important role in modulating cell survival than PKC. In cortical neurons (187) and lymphocytes (157), tyrosine kinase inhibition (by herbimycin A, lavendustin A, or genistein) attenuates Fas- and ceramide-induced apoptosis and upregulation of N-type and delayed-rectifier K+ channels. A more recent study showed that inhibition of tyrosine phosphorylation also suppresses the activity of the Na+-K+-ATPase pump in cortical neurons, leading to apoptosis (177). Although kinases may modulate K+ channel activity, kinase stimulation also may be dependent on K+ channel activation. For example, in myeloblastic leukemia cells, UV-stimulated K+ currents subsequently activate the JNK/SAPK signaling pathway to cause apoptosis (177). These observations indicate that phosphorylation of apoptotic proteins and of membrane channels plays an important role in regulating cell survival and, in particular, in enhancing the proapoptotic role of K+ channels.


    MODULATION OF CYT-C RELEASE AND CASPASE ACTIVATION BY K+ EFFLUX
 TOP
 ABSTRACT
 APOPTOSIS: AN OVERVIEW
 REGULATION OF CELL VOLUME
 MODULATION OF APOPTOTIC STAGES...
 ACTIVATION OF K+ CHANNELS...
 ACTIVATION OF K+ CHANNELS...
 MODULATION OF CYT-C RELEASE...
 MODULATION OF CASPASE ACTIVITY...
 CL- EFFLUX ALSO AFFECTS...
 MODULATION OF K+ CHANNEL...
 ROLE OF MITOCHONDRIAL ION...
 K+ CHANNELS IN THE...
 INHIBITION OF APOPTOSIS...
 INDUCTION OF APOPTOSIS AS...
 CONCLUSIONS
 REFERENCES
 
The release of cyt-c from the mitochondrial intermembrane space is pivotal to apoptosis, since formation of the cyt-c/APAF-1/caspase-9 apoptosome triggers the activation of the effector caspases-3/-6/-7. ST (86, 103), TNF-{alpha}/cycloheximide (103), NO (88), Fas ligand (56), etoposide (183), and UV irradiation (52, 90) all cause cyt-c release into the cytosol. Mitochondrial membrane depolarization induced by FCCP and NO also causes cyt-c release (10, 67). In many of these cases (except Fas ligand), caspase inhibitors do not prevent the release of cyt-c, indicating that cyt-c release occurs before activation of caspase-3 (19, 60, 78, 81, 129, 164, 183).

Both cyt-c release and caspase-3 activation are readily attenuated by inhibition of sarcolemmal K+ channels by quinine and Ba2+ (103), suggesting that activation of K+ channels occurs before cyt-c release and caspase activation in apoptotic cells. In addition, a decrease in [K+]cyt enhances caspase activation and limits cyt-c release in lymphocytes (16). Physiological [K+]cyt also inhibits formation of the APAF-1/cyt-c/caspase-9 apoptosome (21), while increasing [K+]o (which reduces the driving force for K+ efflux) inhibits death receptor-mediated apoptosis before cyt-c release and caspase-8 activation can occur (21, 160). Therefore, maintenance of physiological and high [K+]cyt not only inhibits AVD but also suppresses cyt-c release from the mitochondria and inhibits cytoplasmic caspase activation, the deciding factors in cell death.


    MODULATION OF CASPASE ACTIVITY AND DNA FRAGMENTATION BY CYTOPLASMIC K+
 TOP
 ABSTRACT
 APOPTOSIS: AN OVERVIEW
 REGULATION OF CELL VOLUME
 MODULATION OF APOPTOTIC STAGES...
 ACTIVATION OF K+ CHANNELS...
 ACTIVATION OF K+ CHANNELS...
 MODULATION OF CYT-C RELEASE...
 MODULATION OF CASPASE ACTIVITY...
 CL- EFFLUX ALSO AFFECTS...
 MODULATION OF K+ CHANNEL...
 ROLE OF MITOCHONDRIAL ION...
 K+ CHANNELS IN THE...
 INHIBITION OF APOPTOSIS...
 INDUCTION OF APOPTOSIS AS...
 CONCLUSIONS
 REFERENCES
 
The final phase of apoptosis involves degradation of the nucleus and its contents. Internucleosomal DNA fragmentation is typically visualized as DNA laddering, i.e., DNA fragments that migrate as multiples of ~200 bp during agarose gel electrophoresis correspond to strands of DNA cleaved in internucleosomal sites (112). Cytoplasmic K+ in physiological concentration (~140 mM) inhibits chromatin condensation and DNA fragmentation, likely through suppression of caspase and endonuclease activities (29). The suppression of endonucleases and caspases is mimicked by sarcolemmal K+ channel inhibition (30) during apoptotic stimulation by dexamethasone (a glucocorticoid receptor agonist) and etoposide (a topoisomerase inhibitor and genetoxic agent). Similar effects are also observed with quinine and Ba2+ in ST- or TNF-{alpha}/cycloheximide-treated U-937, HeLa, PC12, and NG108-15 cells (103).

Although K+ efflux may also occur via K+-Cl- cotransporters and the combined K+-H+ exchange/ exchange system, most efflux occurs via K+ channels. Decreased [K+]cyt, due to enhanced K+ efflux through opened sarcolemmal K+ channels, also enhances endonuclease activity. This suggests that the [K+]cyt, transmembrane K+ gradient, and function and expression of sarcolemmal K+ channels all contribute to regulating the early (e.g., by modulating AVD) and late (e.g., by modulating caspase activity) stages of apoptosis (16, 112). Indeed, in a cell-free system (isolated nuclei), a decrease in [K+] from 140 to 80 mM caused a 1.6-fold increase in apoptosis induced by the apoptosis-inducing factor (29). In lymphocytes, an 8-h treatment with ST decreased [K+]cyt from 140 to 50 mM, whereas a decrease in [K+] in assay buffer from 150 to 80 mM caused a 2.4-fold increase in DNA degradation in isolated nuclei (72). Similar experiments performed in rat PASMC show that increasing [K+] in the assay buffer enhanced caspase-3 activity (Fig. 8A) (105). The NO-induced apoptosis (Fig. 6B) and increase in caspase-3 (Fig. 8B) were both attenuated by K+ channel inhibition by 4-AP, TEA, and high [K+]o. These results provide evidence that a high [K+]cyt is required to suppress the activation of apoptotic processes (e.g., activation of caspases and endonucleases), whereas K+ efflux relieves the inhibition on cytoplasmic caspases and nucleases, thereby enhancing apoptosis.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 8. Inverse correlation between [K+] and caspase-3 (Casp-3) activity. A: increasing [K+] in the assay buffer from 0 to 25, 75, 100, and 150 mM causes a marked decrease in caspase-3 activity [means ± SE, determined as the optical density (OD) at 405 nm of the caspase-3-cleaved product]. [Reproduced with permission from Elsevier from Mandegar et al. (105).] B: SNAP (0.1 mM) treatment significantly increases caspase-3 concentration (means ± SE) in PASMC. The SNAP-induced enhancement is attenuated by inhibiting K+ channel activity with increased extracellular K+ concentration ([K+]o) or TEA (1 mM). **P < 0.01 vs. solid bar. NOR, SNAP in physicological solution; 40 K, 40 mM [K+].

 


    CL- EFFLUX ALSO AFFECTS APOPTOSIS
 TOP
 ABSTRACT
 APOPTOSIS: AN OVERVIEW
 REGULATION OF CELL VOLUME
 MODULATION OF APOPTOTIC STAGES...
 ACTIVATION OF K+ CHANNELS...
 ACTIVATION OF K+ CHANNELS...
 MODULATION OF CYT-C RELEASE...
 MODULATION OF CASPASE ACTIVITY...
 CL- EFFLUX ALSO AFFECTS...
 MODULATION OF K+ CHANNEL...
 ROLE OF MITOCHONDRIAL ION...
 K+ CHANNELS IN THE...
 INHIBITION OF APOPTOSIS...
 INDUCTION OF APOPTOSIS AS...
 CONCLUSIONS
 REFERENCES
 
As was discussed earlier, Cl- efflux is tightly coupled to K+ efflux, especially in cells undergoing apoptosis. It is therefore not surprising that numerous apoptosis inducers can trigger Cl- channel activity. ST-induced AVD and apoptosis are significantly reduced by Cl- channel inhibitors such as 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), phloretin, and 4-acetamido-4'-isothiocyanostilbene in HeLa, U-937, PC12, and NG108-15 cells (103). Apoptosis induced by TNF-{alpha} treatment of rat liver HTC cells is reversed by NPPB and N-phenylanthranilic acid (DPC) (120). However, at least in HeLa and U-937 cells, application of anthracene-9-carboxylate and furosemide does not prevent ST-induced apoptosis, thereby eliminating cAMP-activated cystic fibrosis transmembrane regulator (CFTR) channels and Na+-K+-2Cl- and Na+-Cl- symporters as possible Cl- extrusion pathways (103), although the role of CFTR in apoptosis is under debate (58, 110). Fas ligand/CD95 binding-mediated apoptosis is partially inhibited by indanyloxyacetic acid, DPC, and DIDS in lymphocytes (158). Many of these Cl- channel antagonists also attenuate cyt-c release, caspase-3 activation, and DNA fragmentation in the same cells (103, 136). The Cl- channels involved in apoptosis may possibly be members of the Ca2+-activated or volume-sensitive channel families identified in mammalian cells based on their pharmacological properties (95, 152).


    MODULATION OF K+ CHANNEL ACTIVITY BY ANTIAPOPTOTIC PROTEINS
 TOP
 ABSTRACT
 APOPTOSIS: AN OVERVIEW
 REGULATION OF CELL VOLUME
 MODULATION OF APOPTOTIC STAGES...
 ACTIVATION OF K+ CHANNELS...
 ACTIVATION OF K+ CHANNELS...
 MODULATION OF CYT-C RELEASE...
 MODULATION OF CASPASE ACTIVITY...
 CL- EFFLUX ALSO AFFECTS...
 MODULATION OF K+ CHANNEL...
 ROLE OF MITOCHONDRIAL ION...
 K+ CHANNELS IN THE...
 INHIBITION OF APOPTOSIS...
 INDUCTION OF APOPTOSIS AS...
 CONCLUSIONS
 REFERENCES
 
The important role played by [K+]cyt and K+ channel activity in AVD and apoptosis is further enhanced by the fact that the antiapoptotic proteins Bcl-2, an antiapoptotic member of the Bcl-2 family, and ARC (apoptosis repressor with caspase recruitment domain), an antiapoptotic protein in cardiac and skeletal myocytes, modulate sarcolemmal K+ channel function (43, 44).

Bcl-2 is a large family of proteins with contrasting effects on apoptosis. Although structurally similar, some members of the family (Bax, Bak, Bad, Bid, Bim, PUMA, Noxa, Blk, Bik/Nbk, Hrk/DP5, Bok/Mbl, Bcl-xS) promote apoptosis, whereas others (Bcl-2, Bcl-xL, Bcl-2, A1, Mcl-1, Boo) inhibit apoptosis (1, 154). Proapoptotic Bcl-2 proteins are cytoplasmic and activate only with apoptotic stimulation. Some (like Bak, Bax, and truncated Bid) can translocate and insert themselves into the mitochondrial membrane upon apoptotic stimulation, thereby enhancing cyt-c release and causing apoptosis (62, 98, 101). The antiapoptotic protein Bcl-2 is mainly located in the endoplasmic reticulum (ER) membrane, the nuclear envelope, and the outer mitochondrial membrane (OM). The designation of Bcl-2 and Bcl-xL as antiapoptotic proteins has been blurred by recent evidence that cleavage by caspase-3 converts them into proapoptotic proteins similar to Bax (22, 23). Furthermore, mitochondrial Bcl-2 can cause apoptosis, whereas ER Bcl-2 protects against apoptosis induced by Bax overexpression (174). Therefore, the physical origin or location of Bcl-2 may be an important regulator in apoptosis.

Bcl-2 genes are regulated by cytokines and other death-survival signals at different levels. For example, antiapoptotic genes are induced transcriptionally by certain cytokines, whereas antiapoptotic Bax genes are induced as part of the p53-mediated damage response (3, 14). In addition, Bcl-2 can protect against apoptosis induced by {gamma}- and UV-irradiation, cytokine withdrawal, glucocorticoid treatment, and ST (25), but not against apoptosis induced by ligand binding to CD95 death receptors in lymphocytes (153).

Bcl-2 inhibits apoptosis primarily by blocking cyt-c release into the cytoplasm (81, 183), although it can also protect against apoptosis via 1) inhibition of some proapoptotic proteins (see Fig. 2) (62, 98, 101, 155), 2) restoration of the high ATP-to-ADP ratio in the cytosol by facilitating mitochondrial ATP/ADP exchange (163), 3) direct antioxidant effects (24, 69), 4) regulation of Ca2+ content in the mitochondria and sarcoplasmic reticulum (65, 91, 197), and 5) maintenance of a negative {Delta}{Psi}m via enhanced proton efflux or formation of mitochondrial cation channels (5, 142, 145). In addition to these more well-characterized effects, the antiapoptotic Bcl-2 protein has been shown to prevent apoptosis by, at least in part, acting on sarcolemmal K+ channels in vascular smooth muscle cells. In rat PASMC, overexpression of the human bcl-2 gene using an adenoviral vector 1) markedly increases the protein expression of Bcl-2 (Fig. 9Aa), 2) decreases current density of the 4-AP-sensitive KV channels (Fig. 9, Ab–d), 3) downregulates mRNA expression of KV1.1, KV1.5, and KV2.1 channels as well as their representative whole cell KV currents (Fig. 9B), and 4) inhibits ST-mediated apoptosis (Fig. 9C) (43). These results suggest that inhibition of KV channel activity may serve as an additional mechanism involved in the Bcl-2-mediated antiapoptotic effect in vascular smooth muscle cells. The precise mechanisms by which Bcl-2 downregulates mRNA expression of KV channels and inhibits KV channel activity are unknown.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 9. Overexpression of antiapoptotic Bcl-2 inhibits KV channel expression and function. A: Western blot analysis of human Bcl-2 protein levels in rPASMC infected with an empty adenoviral vector (-bcl-2) and an adenovirus carrying the human bcl-2 gene (+bcl-2, a). Families of currents were elicited by test pulses ranging between -40 and +80 mV (-70 mV holding potential) in control (left) and Bcl-2-infected (right) cells (b). Summarized results showing the current (I)-voltage (V) relationship curves in control ({circ}) and bcl-2-transfected ({bullet}) cells (c). Current density of IK(V) at +80 mV is signifi-cantly decreased in cells infected with bcl-2 (d). B: single cell RT-PCR amplified products (a) for human Bcl-2 (267 bp) and rat Kv1.1 (298 bp), Kv1.5 (196 bp), Kv2.1 (269 bp), and {beta}-actin (267 bp) as well as the corresponding whole cell IK (b) in a control cell (-bcl-2) and a bcl-2-infected cell (+bcl-2). -RT, RT performed in the absence of reverse transcriptase. M, 1 kb plus DNA ladder marker. C: ST (0.02 µM)-induced apoptosis is inhibited in cells infected with bcl-2. ***P < 0.001 vs. -bcl-2. [From Ekhterae et al. (43).]

 

The recruitment and activation of caspases is central to the regulation of apoptosis. One of the protein-protein interaction motifs involved in death receptor-mediated apoptosis involves a caspase recruitment domain (CARD). ARC is a cardiac and skeletal muscle CARD-containing protein that binds to the initiator caspases-2/-8 and significantly attenuates death receptor-induced apoptosis (83, 161). Multiple mechanisms are involved in the antiapoptotic effect of ARC on cardiomyocytes: 1) inhibition of caspase activation (83); 2) blockade of hypoxia/ischemia-induced cyt-c release (42); and 3) prevention of H2O2-mediated loss of membrane integrity and disruption of the {Delta}{Psi}m (116).

In addition to the inhibitory effects on cyt-c release (42) and mitochondrial disruption (116), overexpression of ARC in cardiomyocytes 1) blocks sarcolemmal KV channels (Fig. 10A), which possibly contributes to inhibition of the apoptotic cell shrinkage, and 2) inhibits ST-mediated activation of K+ channels (Fig. 10B) and apoptosis (Fig. 10C) (44). The precise mechanism(s) by which ARC blocks KV channels remains unclear. The ARC-mediated inhibition of ST-induced increase in IK(V) may be partially due to its inhibiting cyt-c release (42), because cytoplasmic dialysis of cyt-c increases IK(V) in vascular smooth muscle cells (Fig. 5) (129).



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 10. Overexpression of ARC decreases KV channel activity in embryonic rat heart H9c2 cells. A: Western blot analysis (top) showing the ARC protein levels in Neo cells (empty vector) and cells stably transfected with the human ARC-5 gene. Representative currents (bottom), elicited by test potentials between -60 and +80 mV (holding potential -70 mV), in a Neo cell and an ARC-5-transfected cell. B: averaged currents at +80 mV (holding potential, -70 mV) in Neo cells and ARC-5-transfected cells before (Cont) and after (ST) treatment with ST (0.02 µM). C: summarized data showing the percentage of cells undergoing apoptosis in Neo and ARC-5-transfected cells before (Cont) and after (ST) exposure to ST. ***P < 0.001 vs. Neo. [From Ekhterae et al. (44).]

 

The inhibitory effects of the antiapoptotic proteins Bcl-2 and ARC on plasmalemmal K+ channels in smooth muscle cells and cardiomyocytes further support the theory that activation of K+ channels is a critical step for cells to undergo apoptosis, whereas inhibition of K+ channels attenuates apoptosis, which would facilitate cell proliferation and cause tissue remodeling.


    ROLE OF MITOCHONDRIAL ION FLUX IN APOPTOSIS
 TOP
 ABSTRACT
 APOPTOSIS: AN OVERVIEW
 REGULATION OF CELL VOLUME
 MODULATION OF APOPTOTIC STAGES...
 ACTIVATION OF K+ CHANNELS...
 ACTIVATION OF K+ CHANNELS...
 MODULATION OF CYT-C RELEASE...
 MODULATION OF CASPASE ACTIVITY...
 CL- EFFLUX ALSO AFFECTS...
 MODULATION OF K+ CHANNEL...
 ROLE OF MITOCHONDRIAL ION...
 K+ CHANNELS IN THE...
 INHIBITION OF APOPTOSIS...
 INDUCTION OF APOPTOSIS AS...
 CONCLUSIONS
 REFERENCES
 
Changes in mitochondrial membrane permeability (MMP) determine the ultimate fate of cells irrespective of the nature of the proapoptotic stimuli. Therefore, the mitochondria play a central role in modulating apoptosis by 1) integrating different signal transduction cascades to a common pathway (47) initiated by MMP alterations and 2) releasing soluble proteins (i.e., cyt-c, Smac/Diablo, AIF, procaspases) from the mitochondrial intermembrane space into the cytosol.

The two well-defined compartments (i.e., intermembrane space and matrix) within the mitochondria regulate its activity. Under physiological conditions, the folded IM (Fig. 11) is almost impermeable, allowing the respiratory chain within the matrix (the region surrounded by the IM) to generate an electrochemical gradient that regulates the highly negative (-150 to -200 mV) {Delta}{Psi}m via the production and translocation of H+. Disruption of MMP may result from defective ATP/ADP exchange between the matrix and cytosol mediated by the adenine nucleotide translocase (ANT) on the IM and voltage-dependent anion channels (VDAC) on the mitochondrial OM. Persistent membrane impermeability to ATP/ADP exchange ultimately results in loss of OM integrity, rendering it permeable to soluble proteins. IM permeabilization (visualized by cytofluorometry) results from disruption of {Delta}{Psi}m.



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 11. Cross-sectional view of the mitochondria and the inner and outer membrane channels contribute to the regulation of apoptosis. IM, inner mitochondrial membrane, OM, outer mitochondrial membrane, VDAC, voltage-dependent anion channel; ANT, adenine nucleotide translocase; mtKCa, mitochondrial KCa channel, mtKATP, mitochondrial KATP channel, mtCLIC, mitochondrial intracellular Cl- channel.

 

The numerous channels and exchangers that populate the mitochondrial IM and OM play significant roles in the modulation of {Delta}{Psi}m and apoptosis. What follows is a brief discussion of selected mitochondrial ion channels and how ion permeability within the mitochondria also contributes to cyt-c release and apoptosis. A focused review of mitochondrial K+ channels will follow in a separate section. For more in-depth information, readers should refer to recent publications dealing with mitochondrial cation transport, {Delta}{Psi}m regulation, and control of cellular function (12, 47, 89, 118, 124, 175).

The VDAC, or mitochondrial porin, is a large-diameter OM channel serving as a voltage-dependent permeability pathway for large uncharged molecules (<~5 kDa) such as NADH and metabolites. Together with the ANT on the IM, the VDAC forms the so-called mitochondrial permeability transition pore, a nonselective channel that, when opened, allows for the equilibration of ions within the matrix and intermembrane space, thereby dissipating the H+ gradient and disrupting or depolarizing {Delta}{Psi}m (60, 89, 147). VDAC activity is required for apoptotic {Delta}{Psi}m loss or depolarization to occur (146). Alone, the VDAC is weakly anion selective (i.e., permeable to Cl-, DIDS sensitive) (146).

Bax channels are formed on the OM when soluble Bax proteins translocate from the cytosol upon apoptotic stimulation. Bax channels exhibit cation (K+, Na+) selectivity and, like VDAC, do not allow cyt-c release from the mitochondrial intermembrane space to the cytosol (146). However, both Bax and Bak proteins can interact with OM VDAC to form a large pore. In consequence, disruption of VDAC integrity allows cyt-c to pass into the cytosol at a rate of ~10 molecules·s-1·channel-1 (68, 146).

A mitochondrial intracellular Cl- channel (mtCLIC) has been identified on the mitochondrial IM (46); no other mitochondrial IM Cl- channels have been identified to date. Expression of mtCLIC is regulated by p53 and TNF-{alpha}, two potent proapoptotic agents, suggesting that it may be a common downstream effector for these two apoptotic stimulants. It has been suggested that changes in mitochondrial IM permeability to Cl- via mtCLIC and changes in {Delta}{Psi}m lead to activation of the mitochondrial permeability transition pore and apoptosis.

Bcl-2 channels on the mitochondrial OM are mostly closed at neutral pH. At more acidic pH (pH 5.4), Bcl-2 forms cation channels (142). The antiapoptotic Bcl-2 and Bcl-xL channels increase cell survival by causing {Delta}{Psi}m hyperpolarization, leading to 1) decreased cyt-c release, 2) increased mitochondrial uptake of cationic fluorescent dyes (e.g., rhodamine-123), 3) increased Ca2+ uptake, 4) increased resistance to disruption of {Delta}{Psi}m, 5) enhanced H+ efflux in the presence of {Delta}{Psi}m-depolarizing stimuli (without influencing K+ efflux from the mitochondria), 6) maintained mitochondrial osmotic homeostasis, 7) VDAC closure, and 8) prevention of Bax/Bak dimerization and translocation to the OM (2, 62, 142, 145, 147, 164, 174).

The idea that {Delta}{Psi}m depolarization is required for translocation of cyt-c from the mitochondria is not, however, universally accepted (19). The aforementioned effects are specific to local mitochondrial regulation. Bcl-2 can also modulate MMP and {Delta}{Psi}m indirectly via control of sarcolemmal K+ permeability. When plasma membrane potential (Em) is more depolarized, {Delta}{Psi}m should be more hyperpolarized to maintain mitochondrial membrane integrity and to contain cyt-c within the mitochondria. Because antiapoptotic Bcl-2 can decrease KV currents and cause Em depolarization, it is not surprising that release of cyt-c is also inhibited by Bcl-2 (43, 146). One must not, however, assume that modulation of sarcolemmal K+ channels by apoptotic agents is itself linked to changes in {Delta}{Psi}m. For example, in PASMC, ST-induced {Delta}{Psi}m depolarization (visualized as increased rhodamine-123 fluorescence) is not blocked by decreased K+ efflux due to increased [K+]o (from 5 to 40 mM) (86). Rapid FCCP-induced depolarization of {Delta}{Psi}m is not mimicked by enhanced [K+]o (from 5 to 25 mM), or by inhibition of KCa channels by TEA or iberiotoxin (Fig. 12) (87). In the latter example, FCCP depolarization of {Delta}{Psi}m likely causes Ca2+ release from the mitochondria to the cytosol, thereby activating KCa channels and contributing to K+ efflux, AVD, and apoptosis. Finally, SNAP induces apoptosis and gradual {Delta}{Psi}m depolarization, the latter being unaffected by 40 mM [K+]o or iberiotoxin (88).



View larger version (45K):
[in this window]
[in a new window]
 
Fig. 12. FCCP-induced depolarization of {Delta}{Psi}m occurs independently of sarcolemmal K+ channel blockade. A: pseudocolor images showing rhodamine-123 (Rho-123) fluorescence, used to estimate the relative change of {Delta}{Psi}m, in PASMC before (Control) and during [carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone (FCCP)] treatment with 5 µM FCCP. B: summarized data showing FCCP-enhanced Rho-123 fluorescence, i.e., {Delta}{Psi}m depolarization, is unaffected by increasing [K+]o (from 5 to 25 mM) or by K+ channel inhibition (1 mM TEA) in PASMC. [From Krick et al. (87).]

 


    K+ CHANNELS IN THE MITOCHONDRIA
 TOP
 ABSTRACT
 APOPTOSIS: AN OVERVIEW
 REGULATION OF CELL VOLUME
 MODULATION OF APOPTOTIC STAGES...
 ACTIVATION OF K+ CHANNELS...
 ACTIVATION OF K+ CHANNELS...
 MODULATION OF CYT-C RELEASE...
 MODULATION OF CASPASE ACTIVITY...
 CL- EFFLUX ALSO AFFECTS...
 MODULATION OF K+ CHANNEL...
 ROLE OF MITOCHONDRIAL ION...
 K+ CHANNELS IN THE...
 INHIBITION OF APOPTOSIS...
 INDUCTION OF APOPTOSIS AS...
 CONCLUSIONS
 REFERENCES
 
As previously mentioned, sarcolemmal K+ channel activity does not appear to contribute directly to {Delta}{Psi}m regulation. Therefore, it is possible that K+ flux within intracellular organelles may modulate {Delta}{Psi}m. Mitochondrial KATP (mtKATP) channels have been identified in mitochondrial IM from neurons (33, 99, 118), cardiac cells (71, 100), and liver (75, 126). The primary functions of mtKATP channels are to control mitochondrial volume by maintaining K+ homeostasis and to enable the formation of the pH gradient and the transmembrane electric potential on the IM (33, 53). Based on pharmacological similarities, it has been hypothesized that the molecular structures (sulfonylurea receptor + KIR 6.x channel) of sarcolemmal and mtKATP channels are similar (53, 169). Nonetheless, mtKATP channels differ in that they are selectively activated by diazoxide and inhibited by 5-hydroxydecanoate (33, 100), a distinction that has proven essential in determining the physiological role of mtKATP channels.

In one scenario, activation of neuronal mtKATP channels by diazoxide causes K+ influx into the matrix, thereby causing {Delta}{Psi}m depolarization (12), mitochondrial matrix swelling, cyt-c release, caspase activation, and apoptosis (33, 118, 149). Presumably, {Delta}{Psi}m depolarization leads to more than just the release of cyt-c, since, in the same cells, diazoxide-induced mtKATP activation can also protect against apoptosis during ischemia, hypoxia, or ST treatments (63, 100). The protective mechanism mediated by mtKATP channel opening (which would induce {Delta}{Psi}m depolarization) may involve 1) relief of the mitochondrial Ca2+ overloading typical in cardiac ischemiareperfusion injury (71), 2) gradual oxidation of cardiac cells and neurons (57, 100, 141, 195), 3) elevated Bcl-2 suppressing both Bax translocation to the mitochondrial membrane and cyt-c release in neurons (99), 4) enhanced ATP production (85), or 5) altered reactive oxygen species production (50, 184).

Recently identified neural (148) and cardiac (182) IM mitochondrial large-conductance KCa (mtKCa) channels may also have dual effects, mediating both apoptosis and cardioprotection. The biophysical (unitary conductance and Ca2+ and voltage dependence) and pharmacological (charybdotoxin sensitivity) properties of mtKCa channels are similar to those of sarcolemmal maxi-KCa channels identified in many smooth muscles (115). Cardiac mtKCa channel activity is detectable at physiological (~200 nM) and high (40 µM) cytosolic free Ca2+ concentrations (182), suggesting that it may play an important role in modulating mitochondrial function under physiological situations or during conditions of Ca2+ overload, such as during ischemia. During neuronal apoptosis, activation of mtKCa might be expected to cause complete and irreversible uncoupling of the mitochondria, thereby promoting the effect of mitochondrial apoptosis. However, evidence using isolated perfused hearts suggests that, as for activated mtKATP channels, preischemic exposure to NS-1619, an mtKCa channel opener, results in ~50% protection against myocardial infarction (182).

It appears, therefore, that mitochondrial K+ channels may play a more protective role against ischemia-induced apoptosis, at least in cardiac myocytes. Despite this evidence, it is still unclear as to which trigger, if any, determines the ultimate outcome of mitochondrial membrane depolarization. Further study of the mtKATP and mtKCa channels is required to address the following issues: 1) Do different cell populations within a tissue contribute to the antiapoptotic response? and 2) Does the activation of mtKATP and/or mtKCa channels by environmental (e.g., ischemia/hypoxia) or chemical (e.g., ST) stresses result in protective preconditioning or apoptosis? Although mitochondrial K+ channels have not yet been discovered in vascular smooth muscle cells, it is possible that the relative contributions of sarcolemmal and mitochondrial K+ channel activity to [K+]cyt regulation may determine the fate of cells under pathological situations. Figure 13 provides a schematic overview of the roles of sarcolemmal and mitochondrial channels in cell survival and death. Despite the stark contrast between the proposed roles of mitochondrial and sarcolemmal K+ channels in the regulation of cell death, mitochondrial K+ channels should be regarded as potential therapeutic targets for stroke and neurodegenerative conditions (159).



View larger version (27K):
[in this window]
[in a new window]
 
Fig. 13. Schematic view of how proapoptotic agents ("death inducers") affect sarcolemmal and mitochondrial K+ and Cl- channels' activity, resulting in apoptosis. Also shown are 2 mitochondria-mediated mechanisms that enhance cell survival in cardiac and neuronal ischemia. [Ca2+]cyt, cytosolic Ca2+ concentration; [K+]i, intracellular K+ concentration; Em, membrane potential.

 


    INHIBITION OF APOPTOSIS CONTRIBUTES TO THE DEVELOPMENT OF PULMONARY VASCULAR REMODELING IN PATIENTS WITH PRIMARY PULMONARY HYPERTENSION
 TOP
 ABSTRACT
 APOPTOSIS: AN OVERVIEW
 REGULATION OF CELL VOLUME
 MODULATION OF APOPTOTIC STAGES...
 ACTIVATION OF K+ CHANNELS...
 ACTIVATION OF K+ CHANNELS...
 MODULATION OF CYT-C RELEASE...
 MODULATION OF CASPASE ACTIVITY...
 CL- EFFLUX ALSO AFFECTS...
 MODULATION OF K+ CHANNEL...
 ROLE OF MITOCHONDRIAL ION...
 K+ CHANNELS IN THE...
 INHIBITION OF APOPTOSIS...
 INDUCTION OF APOPTOSIS AS...
 CONCLUSIONS
 REFERENCES
 
Primary pulmonary hypertension (PPH) is a fatal disease of unidentified etiological cause in which increased pulmonary arterial pressure and vascular resistance lead to right heart failure and death. Pulmonary vasoconstriction, pulmonary vascular wall remodeling, and in situ thrombosis are the main causes for the elevated pulmonary vascular resistance in patients with PPH (6, 49, 139). Pulmonary vascular remodeling is characterized by arterial wall thickening as a result of increased fibroblast, PASMC, and endothelial cell proliferation in the tunica adventitia, media, and intima, respectively (151). The pulmonary vascular medial hypertrophy in pulmonary hypertension is mainly due to increased PASMC growth and/or decreased PASMC apoptosis (40, 168). Therefore, the precise control of the balance between PASMC proliferation and apoptosis 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 normal human PASMC, our preliminary data showed that apoptosis inducers, such as ST and cyt-c, increased K+ channel activity (Figs. 4, 5, 6), whereas antiapoptotic proteins (e.g., Bcl-2) decreased K+ channel activity (Fig. 9). Inhibition of K+ currents by pharmacological blockade of KV channels or by reducing the transmembrane K+ gradient (e.g., raising extracellular K+ concentration) attenuated ST-induced apoptosis. In PASMC from PPH patients, expression of functional KV channels and the resulting macroscopic currents were markedly reduced compared with PASMC from normal subjects and patients with secondary pulmonary hypertension (SPH) (190192). Accordingly, ST-induced apoptosis was also signifi-cantly inhibited in PPH-PASMC compared with SPH-PASMC (196). Therefore, inhibition of apoptosis in PASMC as a result of gene downregulation and/or dysfunction of KV channels (8, 190, 192, 193) plays a critical role in the development of pulmonary vascular medial hypertrophy and the increased pulmonary vascular resistance and arterial pressure in PPH patients.

Vasoactive agonists, growth factors, and cytokines regulate PASMC proliferation and apoptosis and pulmonary vascular remodeling (Fig. 14) (96, 111, 114, 127). Bone morphogenetic proteins (BMP) are part of the greater transforming growth factor-{beta} family of polypeptides that regulate a wide spectrum of cellular functions, such as proliferation, differentiation, migration, and apoptosis (107). Mutations of the BMP receptor type II gene (BMP-RII) have been identified in patients with familial and sporadic PPH (34, 92, 102, 117). The protein expression level of BMP-RII is significantly decreased in lung tissues from PPH patients with or without mutations in the BMP-RII gene compared with normal subjects and patients with SPH (9). These observations suggest that dysfunction and/or downregulation of BMP-RII and/or its downstream signaling may play an important role in the development of pulmonary vascular medial hypertrophy.



View larger version (54K):
[in this window]
[in a new window]
 
Fig. 14. Controlling the balance between proliferation and apoptosis in the development of pulmonary hypertension. A: the modulation of K+ efflux (+, increase; -, decrease) by vasoactive agonists, transcription factors (e.g., c-Jun), mitochondrial factors, and {Delta}{Psi}m, is a central control mechanism in maintaining this balance. Also shown are other regulatory therapies being tested for which no involvement of K+ channels has been shown, but that are effective at producing or preventing apoptosis [e.g., matrix inhibitors, bone morphogenetic protein receptor type II (BMP-RII), TNF-related apoptosis-inducing ligand (TRAIL), caspase inhibitors, c-FLIP, c-Abl]. TRAIL and caspase inhibitors are being investigated for use in treatment of certain cancers and autoimmune and neurodegenerative disorders such as rheumatoid arthritis, Parkinson's disease, and amyotrophic lateral sclerosis (117). AP-1, activating protein-1. B: representative histological (hematoxylin and eosin stain, original magnification x200) slides showing cross sections of pulmonary arteries in lung tissues from a normotensive patient and a patient with primary pulmonary hypertension [mean pulmonary arterial pressure (PAP), 51 mmHg].

 

In PASMC from normotensive patients, low doses (10–100 nM) of BMPs (e.g., BMP-2, -4, and -7) inhibit cell proliferation (determined by [3H]thymidine incorporation), whereas high doses (100–200 nM) of BMPs (e.g., BMP-2 and -7) induce cell apoptosis (113, 196). The BMP-mediated antiproliferative and proapoptotic effects on PASMC are significantly inhibited in PASMC from PPH patients compared with PASMC from normal subjects and patients with SPH (196). These results provide compelling evidence that BMPs and their receptors and downstream signal transduction are involved or required for preventing normal PASMC from overgrowth (i.e., hypertrophy and hyperplasia), which is important in maintaining the thin pulmonary vascular wall and low pulmonary vascular resistance under normal conditions. Mutation and/or downregulation of BMP ligands and receptors as well as defects in the downstream signaling pathway would therefore enhance pulmonary vascular remodeling and increase pulmonary vascular resistance and arterial pressure in patients with PPH (9, 32, 113, 140, 196).

The precise mechanisms by which BMPs induce apoptosis in normal human PASMC are still unknown. Our preliminary study indicates that treatment of normal PASMC with BMP-2 decreases the mRNA and protein expression of Bcl-2, an antiapoptotic protein that attenuates apoptosis by inhibiting cyt-c release (81, 183), blocking K+ channels (43), downregulating K+ channel gene expression (43), maintaining Ca2+ in the sarcoplasmic/endoplasmic reticulum (65, 91), and regulating proton flux in mitochondria (145). Geraci et al. (54) show that the mRNA expression of Bcl-2 was upregulated in lung tissues from sporadic and familial PPH patients. The upregulated Bcl-2 gene transcription may be related to the mutations in BMP-RII gene and/or dysfunction of BMP signaling. These results imply that modulation of Bcl-2 gene expression is a critical mechanism in directing human PASMC to undergo proliferation or apoptosis.


    INDUCTION OF APOPTOSIS AS A THERAPEUTIC APPROACH FOR PATIENTS WITH PULMONARY HYPERTENSION
 TOP
 ABSTRACT
 APOPTOSIS: AN OVERVIEW
 REGULATION OF CELL VOLUME
 MODULATION OF APOPTOTIC STAGES...
 ACTIVATION OF K+ CHANNELS...
 ACTIVATION OF K+ CHANNELS...
 MODULATION OF CYT-C RELEASE...
 MODULATION OF CASPASE ACTIVITY...
 CL- EFFLUX ALSO AFFECTS...
 MODULATION OF K+ CHANNEL...
 ROLE OF MITOCHONDRIAL ION...
 K+ CHANNELS IN THE...
 INHIBITION OF APOPTOSIS...
 INDUCTION OF APOPTOSIS AS...
 CONCLUSIONS
 REFERENCES
 
Apoptosis is a highly regulated process in which cells that are no longer needed during development and vascular cells that undergo "misguided" hypertrophy and hyperplasia are removed. Activation of apoptosis is implicated in the regression of pulmonary vascular medial hypertrophy, whereas inhibition of apoptosis may lead to the progression of pulmonary vascular wall thickening (Fig. 14B).

In addition to the protein-protein interactions that regulate apoptosis, protein-matrix interactions are also involved in the cell proliferation and migration associated with vascular wall remodeling. Therefore, another avenue of treatment for pulmonary hypertension is the modulation of extracellular matrix glycoproteins, whose expression and deposition is 1) increased in clinical and experimental progressive pulmonary hypertension and 2) linked to increased cell migration, proliferation, and apoptosis (132, 133). Tenascin-C, one such matrix glycoproteins, is induced by matrix metalloproteinases and amplifies the response of smooth muscle to growth factors. Treatment of pulmonary hypertensive (due to hypoxia or monocrotaline) rats with serine elastase and matrix metalloproteinase inhibitors suppresses tenascin-C induction and collagen and elastin deposition, resulting in complete reversal of pulmonary hypertension (2628, 138). Antisense tenascin-C treatment (with osteopontin blockade) also induces apoptosis in hypertrophied pulmonary arteries. Therefore, disrupted matrix glycoprotein attachment appears to be a powerful trigger for apoptosis, making it a logical target in the treatment of pulmonary intimal hypertrophy.

Similar to pulmonary vascular thickening (or medial hypertrophy) in pulmonary hypertension, cancers arise not only due to unrestrained cell proliferation, but also due to insufficient apoptotic turnover (60). In recent years, the modulation of apoptosis has gained much interest as a potential therapeutic target for diseases, such as cancer, where increased cell proliferation or decreased cell apoptosis is a primary diagnostic tool. As discussed earlier, many enzymes, ligands, receptors, signal transduction proteins, and transcription factors are involved in the apoptotic cascade; very few conventional drugs are available that specifically target these factors. Combined with the fact that drug therapies effective in the brain may not work in the heart and lung, attention has been focused on gene antisense therapies aimed at intermediary apoptotic proteins, particularly Bcl-2. G-3139 is an antisense oligonucleotide that targets the first six codons of human bcl-2; its binding to mRNA precludes translation into Bcl-2 protein (119). As of late 2000, G-3139 was involved in phase III clinical trials for the treatment of malignant melanoma, non-Hodgkin's lymphomas, and leukemia, in which Bcl-2 levels are elevated. Attempts are also underway to combine Bcl-2 antisense therapy with common chemotherapeutic therapies to combat lung, prostate, breast, and colorectal cancers (119).

As with cancers, antisense Bcl-2 strategies may also prove effective in the treatment of pulmonary hypertension. In normal PASMC, overexpression of Bcl-2 inhibits apoptosis by downregulating K+ channel gene expression and by decreasing K+ efflux via sarcolemmal KV channels (43). In familial and sporadic PPH patients, Bcl-2 levels in lung tissues are significantly higher than in normal subjects and patients with SPH (54). In PPH patients, the mRNA expression and activity of KV channels in PASMC are also decreased compared with normal subjects and patients with SPH (190, 191, 194). Therefore, inhibition of bcl-2 gene expression using antisense and overexpression of K+ channels using adenoviral vectors can both be potential therapeutic approaches for patients with PPH. Indeed, Pozeg and colleagues (130) recently reported that in vivo gene transfer of KV1.5, a delayed-rectifier KV channel that is downregulated in PPH patients (190, 194) and in animals with hypoxia-mediated pulmonary hypertension (7, 128, 150, 171), significantly reduced pulmonary hypertension and right heart hypertrophy in rats.

Another avenue that bears more scrutiny is the use of NO as a proapoptotic stimulus. We have shown in the past that NO (SNAP) and DHEA induce apoptosis of human PASMC by promoting K+ efflux through activated KCa and KV channels (88) and that DHEA also has an additive effect on SNAP-induced apoptosis. Recently, one group has shown that DHEA ingestion by hypoxic rats dose dependently inhibits the hypoxia-induced increase in pulmonary artery pressure, total pulmonary resistance, and right ventricular hypertrophy, while having minimal effects on systemic pressure or vascular resistance (122). Therefore, it is possible that treatment with K+ channel openers may reverse the physiological effects of pulmonary hypertension via parallel (but not necessarily related) processes that target both pulmonary vasoconstriction and the increased medial hypertrophy. Figure 15 presents a schematic summary of how modulation of K+ channel expression and function by genetic and environmental factors influences both pulmonary vascular remodeling and hypertrophy in the development of pulmonary hypertension.



View larger version (21K):
[in this window]
[in a new window]
 
Fig. 15. Schematic diagram showing the potential roles played by ion channel activity and intracellular Ca2+ in modulating cell proliferation and apoptosis and in regulating pulmonary vascular tone and remodeling in patients with pulmonary arterial hypertension. CREB, cAMP response element-binding protein; Ca2+/CaM, calcium-calmodulin complex; PVR, pulmonary vascular resistance; VDCC, voltage-dependent Ca2+ channels.

 


    CONCLUSIONS
 TOP
 ABSTRACT
 APOPTOSIS: AN OVERVIEW
 REGULATION OF CELL VOLUME
 MODULATION OF APOPTOTIC STAGES...
 ACTIVATION OF K+ CHANNELS...
 ACTIVATION OF K+ CHANNELS...
 MODULATION OF CYT-C RELEASE...
 MODULATION OF CASPASE ACTIVITY...
 CL- EFFLUX ALSO AFFECTS...
 MODULATION OF K+ CHANNEL...
 ROLE OF MITOCHONDRIAL ION...
 K+ CHANNELS IN THE...
 INHIBITION OF APOPTOSIS...
 INDUCTION OF APOPTOSIS AS...
 CONCLUSIONS
 REFERENCES
 
Ion permeation through transmembrane channels regulates a variety of processes, including, but not limited to, excitation-contraction coupling, cell volume regulation, protein trafficking, DNA replication and fragmentation, nerve transmission, cell metabolism, cell proliferation, and cell death. Apoptosis is a process that plays a critical role in embryonic development and tissue homeostasis; a balance between proliferation and apoptosis controls cell number or density. In humans, dysfunctional apoptosis has been linked to the pathogenesis of cancer, atherosclerosis, and pulmonary vascular disease.

K+ ions, in particular, play an especially important role in the maintenance of so-called "normal" physiology. Regulated K+ transmembrane transport is a major determinant of cell volume regulation, Em control, and the balance of cell proliferation and apoptosis. In this review, we have focused on the modulatory role of transmembrane K+ permeation via sarcolemmal and intraorganelle K+ channels in apoptosis. Evidence has been provided that K+ efflux via sarcolemmal channels may be a key target in the treatment of human diseases where the balance between cell proliferation and cell death has been disturbed, such as cancer and pulmonary hypertension.


    ACKNOWLEDGMENTS
 
We are very grateful to S. Krick for her role in the initiation and continued development of this research project and to J. E. S. Yi for the histological experiments on lung tissues from patients. We are also indebted to O. Platoshyn, S. Zhang, I. Fantozzi, D. Ekhterae, Y. Yu, S. S. McDaniel, B. R. Lapp, P. A. Thistlethwaite, and L. J. Rubin for support in this work.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-64945, HL-54043, HL-66012, HL-69758, and HL-66941.


    FOOTNOTES
 

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).


    REFERENCES
 TOP
 ABSTRACT
 APOPTOSIS: AN OVERVIEW
 REGULATION OF CELL VOLUME
 MODULATION OF APOPTOTIC STAGES...
 ACTIVATION OF K+ CHANNELS...
 ACTIVATION OF K+ CHANNELS...
 MODULATION OF CYT-C RELEASE...
 MODULATION OF CASPASE ACTIVITY...
 CL- EFFLUX ALSO AFFECTS...
 MODULATION OF K+ CHANNEL...
 ROLE OF MITOCHONDRIAL ION...
 K+ CHANNELS IN THE...
 INHIBITION OF APOPTOSIS...
 INDUCTION OF APOPTOSIS AS...
 CONCLUSIONS
 REFERENCES
 

  1. Adams JM and Cory S. The Bcl-2 protein family: arbiters of cell survival. Science 281: 1322-1326, 1998.[Abstract/Free Full Text]
  2. Adrain C, Creagh EM, and Martin SJ. Apoptosis-associated release of Smac/DIABLO from mitochondria requires active caspases and is blocked by Bcl-2. EMBO J 20: 6627-6636, 2001.[Abstract/Free Full Text]
  3. Akashi K, Kondo M, von Freeden-Jeffry U, Murray R, and Weissman IL. Bcl-2 rescues T lymphopoiesis in interleukin-7 receptor-defi-cient mice. Cell 89: 1033-1041, 1997.[ISI][Medline]
  4. Allbritton NL, Verret CR, Wolley RC, and Eisen HN. Calcium ion concentrations and DNA fragmentation in target cell destruction by murine cloned cytotoxic T lymphocytes. J Exp Med 167: 514-527, 1988.[Abstract]
  5. 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]
  6. Archer S and Rich S. Primary pulmonary hypertension: a vascular biology and translational research "work in progress". Circulation 102: 2781-2791, 2000.[Abstract/Free Full Text]
  7. Archer SL, London B, Hampl V, We X, Nsair A, Puttagunta L, Hashimoto K, Waite RE, and Michelakis E. Impairment of hypoxic pulmonary vasoconstriction in mice lacking the voltage-gated potassium channel Kv1.5. FASEB J 15: 1801-1803, 2001.[Abstract/Free Full Text]
  8. 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 Kv1.2, in hypoxic pulmonary vasoconstriction and control of resting membrane potential in rat pulmonary artery myocytes. J Clin Invest 101: 2319-2330, 1998.[Abstract/Free Full Text]
  9. Atkinson C, Stewart S, Upton PD, Machado R, Thomson JR, Trembath RC, and Morrell NW. Primary pulmonary hypertension is associated with reduced pulmonary vascular expression of type II bone morphogenetic protein receptor. Circulation 105: 1672-1678, 2002.[Abstract/Free Full Text]
  10. Bal-Price A, Borutaite V, and Brown GC. Mitochondria mediate nitric oxide-induced cell death. Ann NY Acad Sci 893: 376-378, 1999.[Free Full Text]
  11. Berke G. The binding and lysis of target cells by cytotoxic lymphocytes: molecular and cellular aspects. Annu Rev Immunol 12: 735-773, 1994.[CrossRef][ISI][Medline]
  12. Bernardi P. Mitochondrial transport of cations: channels, exchangers, and permeability transition. Physiol Rev 79: 1127-1155, 1999.[Abstract/Free Full Text]
  13. Bock J, Szabó I, Jekle A, and Gulbins E. Actinomycin D-induced apoptosis involves the potassium channel Kv1.3. Biochem Biophys Res Commun 295: 526-531, 2002.[CrossRef][ISI][Medline]
  14. Boise LH, Minn AJ, Noel PJ, June CH, Accavitti MA, Lindsten T, and Thompson CB. CD28 costimulation can promote T cell survival by enhancing the expression of Bcl-XL. Immunity 3: 87-98, 1995.[ISI][Medline]
  15. Bortner CD and Cidlowski JA. Absence of volume regulatory mechanisms contributes to the rapid activation of apoptosis in thymocytes. Am J Physiol Cell Physiol 271: C950-C961, 1996.[Abstract/Free Full Text]
  16. Bortner CD and Cidlowski JA. A necessary role for cell shrinkage in apoptosis. Biochem Pharmacol 56: 1549-1559, 1998.[CrossRef][ISI][Medline]
  17. Bortner CD, Gómez-Angelats M, and Cidlowski JA. Plasma membrane depolarization without repolarization is an early molecular event in anti-Fas-induced apoptosis. J Biol Chem 276: 4304-4314, 2001.[Abstract/Free Full Text]
  18. Bortner CD, Hughes FMJ, 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]
  19. Bossy-Wetzel E, Newmeyer DD, and Green DR. Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J 17: 37-49, 1998.[Abstract/Free Full Text]
  20. Boutillier A-L, Trinh E, and Loeffler J-P. Caspase-dependent cleavage of the retinoblastoma protein is an early step in neuronal apoptosis. Oncogene 19: 2171-2178, 2000.[CrossRef][ISI][Medline]
  21. Cain K, Langlais C, Sung X-M, Brown DG, and Cohen GM. Physiological concentrations of K+ inhibit cytochrome c-dependent formation of the apoptosome. J Biol Chem 276: 41985-41990, 2001.[Abstract/Free Full Text]
  22. Cheng EH-Y, Kirsch DG, Clem RJ, Ravi R, Kastan MB, Bedi A, Ueno K, and Hardwick JM. Conversion of Bcl-2 to Bax-like death effector by caspases. Science 278: 1966-1968, 1997.[Abstract/Free Full Text]
  23. Clem RJ, Cheng EH-Y, Karp CL, Kirsch DG, Ueno K, Takahashi A, Kastan MB, Griffin DE, Earnshaw WC, Veliuona MA, and Hardwick JM. Modulation of cell death by Bcl-xL through caspase interaction. Proc Natl Acad Sci USA 95: 554-559, 1998.[Abstract/Free Full Text]
  24. Cook SA, Sugden PH, and Clerk A. Regulation of bcl-2 family proteins during development and in response to oxidative stress in cardiac myocytes: association with changes in mitochondrial membrane potential. Circ Res 85: 940-949, 1999.[Abstract/Free Full Text]
  25. Cory S. Regulation of lymphocyte survival by the bcl-2 gene family. Annu Rev Immunol 13: 513-543, 1995.[CrossRef][ISI][Medline]
  26. 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: 698-702, 2000.[CrossRef][ISI][Medline]
  27. Cowan KN, Jones PL, and Rabinovitch M. Elastase and matrix metalloproteinase inhibitors induce regression, and tenascin-C antisense prevents progression, of vascular disease. J Clin Invest 105: 21-34, 2000.[Abstract/Free Full Text]
  28. 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 apoptosis. Circ Res 84: 1223-1233, 1999.[Abstract/Free Full Text]
  29. 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: 5605-5615, 1998.[Abstract/Free Full Text]
  30. Dallaporta B, Marchetti P, de Pablo MA, Maisse C, Duc H-T, Métivier D, Zamzami N, Geuskens M, and Kroemer G. Plasma membrane potential in thymocyte apoptosis. J Immunol 162: 6534-6542, 1999.[Abstract/Free Full Text]
  31. Darzynkiewicz Z, Bruno S, Del Bino G, Gorczyca W, Hotz MA, Lassota P, and Traganos F. Features of apoptotic cells measured by flow cytometry. Cytometry 13: 795-808, 1992.[ISI][Medline]
  32. De Caestecker M and Meyrick B. Bone morphogenetic proteins, genetics and the pathophysiology of primary pulmonary hypertension. Respir Res 2: 193-197, 2001.[CrossRef][ISI][Medline]
  33. Debska G, May R, Kicinska A, Szewczyk A, Elger CE, and Kunz WS. Potassium channel openers depolarize hippocampal mitochondria. Brain Res 892: 42-50, 2001.[CrossRef][ISI][Medline]
  34. Deng Z, Morse JH, Slager SL, Cuervo N, Moore KJ, Venetos G, Kalachikov S, Cayanis E, Fischer SG, Barst RJ, Hodge SE, and Knowles JA. Familial primary pulmonary hypertension (gene PPH1) is caused by mutations in the bone morphogenetic protein receptor II-gene. Am J Hum Genet 67: 737-744, 2000.[CrossRef][ISI][Medline]
  35. Deveraux QL, Stennicke HR, Salvesen GS, and Reed JC. Endogenous inhibitors of caspases. J Clin Immunol 19: 388-398, 1999.[CrossRef][ISI][Medline]
  36. Dispersyn G and Borgers M. Apoptosis in the heart: about programmed cell death and survival. News Physiol Sci 16: 41-47, 2001.[Abstract/Free Full Text]
  37. 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]
  38. D'Souza FM, Sparks RL, Chen H, Kadowitz PJ, and Jeter JRJ. Mechanisms of eNOS gene transfer inhibition of vascular smooth muscle cell proliferation. Am J Physiol Cell Physiol 284: C191-C199, 2003.[Abstract/Free Full Text]
  39. Du C, Fang M, Li Y, Li L, and Wang X. Smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. Cell 102: 33-42, 2000.[ISI][Medline]
  40. Durmowicz AG and Stenmark KR. Mechanisms of structure remodeling in chronic pulmonary hypertension. Pediatr Rev 20: e91-e102, 1999.[Medline]
  41. Earnshaw WC, Martins LM, and Kaufmann SH. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem 68: 383-424, 1999.[CrossRef][ISI][Medline]
  42. Ekhterae D, Lin Z, Lundberg MS, Crow MT, Brosius FCI, and Núñez G. ARC inhibits cytochrome c release from mitochondria and protects against hypoxia-induced apoptosis in heart-derived H9c2 cells. Circ Res 85: e70-e77, 1999.[ISI][Medline]
  43. Ekhterae D, Platoshyn O, Krick S, Yu Y, McDaniel SS, and Yuan JX-J. Bcl-2 decreases voltage-gated K+ channel activity and enhances survival in vascular smooth muscle cells. Am J Physiol Cell Physiol 281: C157-C165, 2001.[Abstract/Free Full Text]
  44. Ekhterae D, Platoshyn O, Zhang S, Remillard CV, and Yuan JX-J. Apoptosis repressor with caspase domain (ARC) inhibits cardiomyocyte apoptosis by reducing voltage-gated K+ currents and increasing capacitative Ca2+ entry. Am J Physiol Cell Physiol 284: C1405-C1410, 2003.[Abstract/Free Full Text]
  45. Fernández-Fernández JM, Nobles M, Currid A, Vázquez E, and Valverde MA. Maxi K+ channel mediates regulatory volume decrease response in a human bronchial epithelial cell line. Am J Physiol Lung Cell Mol Physiol 283: L1705-L1714, 2002.
  46. Fernández-Salas E, Sagar M, Cheng C, Yuspa SH, and Weinberg WC. p53 and tumor necrosis factor {alpha} regulate the expression of a mitochondrial chloride channel protein. J Biol Chem 274: 36488-36497, 1999.[Abstract/Free Full Text]
  47. Ferri KF and Kroemer G. Mitochondria - the suicide organelles. Bioessays 23: 111-115, 2001.[CrossRef][ISI][Medline]
  48. Fisher GH, Rosenberg FJ, Straus SE, Dale JK, Middleton LA, Lin AY, Strober W, Lenardo MJ, and Puck JM. Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome. Cell 81: 935-946, 1995.[ISI][Medline]
  49. Fishman AP. Etiology and pathogenesis of primary pulmonary hypertension. Chest 114: 242S-247S, 1998.[Abstract/Free Full Text]
  50. Forbes RA, Steenbergen C, and Murphy E. Diazoxide-induced cardioprotection requires signaling through a redox-sensitive mechanism. Circ Res 88: 802-809, 2001.[Abstract/Free Full Text]
  51. Gahwiler BH and Brown DA. GABA{beta}-receptor-activated K+ current in voltage-clamped CA3 pyramidal cells in hippocampal cultures. Proc Natl Acad Sci USA 82: 1558-1562, 1985.[Abstract]
  52. Gao W, Pu Y, Luo KQ, and Chang DC. Temporal relationship between cytochrome c release and mitochondrial swelling during UV-induced apoptosis in living HeLa cells. J Cell Sci 114: 2855-2862, 2001.[Abstract/Free Full Text]
  53. Garlid KD. Cation transport in mitochondria - the potassium cycle. Biochim Biophys Acta 1275: 123-126, 1996.[ISI][Medline]
  54. Geraci M, Moore M, Gesell T, Yeager M, Alger L, Golpon H, Gao B, Loyd JE, Tuder RM, and Voelkel NF. Gene expression patterns in the lungs of patients with primary pulmonary hypertension: a gene microarray analysis. Circ Res 88: 555-562, 2001.[Abstract/Free Full Text]
  55. Gerevich Z, Tretter L, Adam-Vizi V, Baranyi M, Kiss JP, Zelles T, and Vizi ES. Analysis of high intracellular [Na+]-induced release of [3H] noradrenaline in rat hippocampal slices. Neuroscience 104: 761-768, 2001.[CrossRef][ISI][Medline]
  56. Gómez-Angelats M, Bortner CD, and Cidlowski JA. Protein kinase C (PKC) inhibits Fas receptor-induced apoptosis through modulation of the loss of K+ and cell shrinkage. J Biol Chem 275: 19609-19619, 2000.[Abstract/Free Full Text]
  57. Goodman Y and Mattson MP. K+ channel openers protect hippocampal neurons against oxidative injury and amyloid {beta}-peptide toxicity. Brain Res 706: 328-332, 1996.[CrossRef][ISI][Medline]
  58. Gottlieb RA and Dosanjh A. Mutant cystic fibrosis transmembrane conductance regulator inhibits acidification and apoptosis in C27 cells: possible relevance to cystic fibrosis. Proc Natl Acad Sci USA 93: 3587-3591, 1996.[Abstract/Free Full Text]
  59. Green DR and Evan GI. A matter of life and death. Cancer Cell 1: 19-30, 2002.[CrossRef][ISI][Medline]
  60. Green DR and Reed JC. Mitochondria and apoptosis. Science 281: 1309-1312, 1998.[Abstract/Free Full Text]
  61. Greger R. Membrane voltage and preservation of the ionic distribution across the cell membrane. In: Comprehensive Human Physiology. From Cellular Mechanisms to Integration, edited by Greger R and Windhorst U. Berlin, Heielberg, Germany: Springer-Verlag, 1996, p. 245-266.
  62. Gross A, Jockel J, Wei MC, and Korsmeyer SJ. Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J 17: 3878-3885, 1998.[Abstract/Free Full Text]
  63. Gross GJ and Fryer RM. Sarcolemmal versus mitochondrial ATP-sensitive K+ channels and myocardial preconditioning. Circ Res 84: 973-979, 1999.[Abstract/Free Full Text]
  64. Gulbins E, Jekle A, Ferlinz K, Grassmé H, and Lang F. Physiology of apoptosis. Am J Physiol Renal Physiol 279: F605-F615, 2000.[Abstract/Free Full Text]
  65. 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]
  66. Hegde R, Srinivasula SM, Zhang Z, Wassell R, Mukattash R, Cilenti L, DuBois G, Lazebnik Y, Zervos AS, Fernandes-Alnemri T, and Alnemri ES. Identification of Omi/HtrA2 as a mitochondrial apoptotic serine protease that disrupts inhibitor of apoptosis protein-caspase interaction. J Biol Chem 277: 432-438, 2002.[Abstract/Free Full Text]
  67. 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]
  68. Hengartner MO. The biochemistry of apoptosis. Nature 407: 770-776, 2000.[CrossRef][ISI][Medline]
  69. Hockenberry DM, Oltvai ZN, Yin XM, Milliman CL, and Korsmeyer SJ. Bcl-2 functions in an antioxidant pathway to prevent apoptosis. Cell 75: 241-251, 1993.[ISI][Medline]
  70. Hodgkin AL. The Conduction of the Nervous Impulse. Liverpool, UK: Liverpool University Press, 1964.
  71. Holmuhamedov EL, Jovanovic S, Dzeja PP, Jovanovic A, and Terzic A. Mitochondrial ATP-sensitive K+ channels modulate cardiac mitochondrial function. Am J Physiol Heart Circ Physiol 275: H1567-H1576, 1998.[Abstract/Free Full Text]
  72. Hughes FMJ, 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]
  73. Hughes FM Jr and Cidlowski JA. Glucocorticoid-induced thymocyte apoptosis: protease-dependent activation of cell shrinkage and DNA fragmentation. J Steroid Biochem Mol Biol 65: 207-217, 1998.[CrossRef][ISI][Medline]
  74. 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]
  75. Inoue I, Hagase H, Kishi K, and Higuti T. ATP-sensitive K+ channel in the mitochondrial inner membrane. Nature 352: 244-247, 1991.[CrossRef][ISI][Medline]
  76. Jones AW. Content and fluxes of electrolytes. In: Handbook of Physiology. The Cardiovascular System. Vascular Smooth Muscle. Bethesda, MD: Am. Physiol. Soc., 1980, sect. 2, vol. II, chapt. 11, p. 253-299.
  77. Joza N, Susin SA, Daugas E, Stanford WL, Cho SK, Li CYJ, Sasaki T, Elia AJ, Cheng H-YM, Ravagnan L, Ferri KF, Zamzami N, Wakeham A, Hakem R, Yoshida H, Kong Y-Y, Mak TW, Zúñiga-Pflücker JC, Kroemer G, and Penninger JM. Essential role of the mitochondrial apoptosis-inducing factor in programmed cell death. Nature 410: 549-554, 2001.[CrossRef][ISI][Medline]
  78. Jurgensmeier JM, Xie Z, Deveraux QL, Ellerby L, Bredesen D, and Reed JC. Bax directly induces release of cytochrome c from isolated mitochondria. Proc Natl Acad Sci USA 95: 4997-5002, 1998.[Abstract/Free Full Text]
  79. Kim KJ, Cheek JM, and Crandall ED. Contribution of active Na+ and Cl- fluxes to net ion transport by alveolar epithelium. Respir Physiol 85: 245-256, 1991.[CrossRef][ISI][Medline]
  80. King LS and Yasui M. Aquaporins and disease: lessons from mice to humans. Trends Endocrinol Metab 13: 355-360, 2002.[CrossRef][ISI][Medline]
  81. 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]
  82. Koch RA and Barish ME. Perturbation of intracellular calcium and hydrogen ion regulation in cultured mouse hippocampal neurons by reduction of the sodium ion concentration gradient. J Neurosci 14: 2585-2593, 1994.[Abstract]
  83. Koseki T, Inohara N, Chen S, and Nunez G. ARC, an inhibitor of apoptosis expressed in skeletal muscle and hart that interacts selectively with caspase. Proc Natl Acad Sci USA 95: 5156-5160, 1998.[Abstract/Free Full Text]
  84. Kostyuk PG. Calcium Ions in Nerve Cell Function. Oxford, UK: Oxford University Press, 1992.
  85. Kowaltowski AJ, Seetharaman S, Paucek P, and Garlid KD. Bioenergetic consequences of opening the ATP-sensitive K+ channel of heart mitochondria. Am J Physiol Heart Circ Physiol 280: H649-H657, 2001.[Abstract/Free Full Text]
  86. Krick S, Platoshyn O, McDaniel SS, Rubin LJ, and Yuan JX-J. Augmented K+ currents and mitochondrial membrane depolarization in pulmonary artery myocyte apoptosis. Am J Physiol Lung Cell Mol Physiol 281: L887-L894, 2001.[Abstract/Free Full Text]
  87. 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: C970-C979, 2001.[Abstract/Free Full Text]
  88. 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: H184-H193, 2002.[Abstract/Free Full Text]
  89. Kroemer G and Reed JC. Mitochondrial control of cell death. Nat Med 6: 513-519, 2000.[CrossRef][ISI][Medline]
  90. Kulms D and Schwarz T. Molecular mechanisms of UV-induced apoptosis. Photodermatol Photoimmunol Photomed 16: 195-201, 2000.[CrossRef][ISI][Medline]
  91. Lam M, Dubyak GR, Ghen L, Nuñez 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: 6569-6273, 1994.[Abstract]
  92. Lane KB, Machado RD, Pauciulo MW, Thomson JR, Phillips JAI, Loyd JE, Nichols WC, and Trembath RC. Heterozygous germline mutations in BMPR2, encoding a TGF-{beta} receptor, cause familial primary pulmonary hypertension. Nat Genet 26: 81-84, 2000.[CrossRef][ISI][Medline]
  93. Lang F, Busch GL, Ritter M, Völkl H, Waldegger S, Gulbins E, and Häussinger D. Functional significance of cell volume regulatory mechanisms. Physiol Rev 78: 247-306, 1998.[Abstract/Free Full Text]
  94. Lang F, Lepple-Wienhues A, Paulmichl M, Szabó I, Siemen D, and Gulbins E. Ion channels, cell volume, and apoptotic cell death. Cell Physiol Biochem 8: 285-292, 1998.[CrossRef][ISI][Medline]
  95. Large WA and Wang Q. Characteristics and physiological role of the Ca2+-activated Cl- conductance in smooth muscle. Am J Physiol Cell Physiol 271: C435-C454, 1996.[Abstract/Free Full Text]
  96. Lawson CA, Yan SD, Yan SF, Liao H, Zhou YS, Sobel J, Kisiel W, Stern DM, and Pinsky DJ. Monocytes and tissue factor promote thrombosis in a murine model of oxygen deprivation. J Clin Invest 99: 1729-1738, 1997.[Abstract/Free Full Text]
  97. Lesage F and Lazdunski M. Molecular and functional properties of two-pore-domain potassium channels. Am J Physiol Renal Physiol 279: F793-F801, 2000.[Abstract/Free Full Text]
  98. Li H, Zhu H, Xu C-J, and Yuan Y. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94: 491-501, 1998.[ISI][Medline]
  99. Liu D, Lu C, Wan R, Auyeung WW, and Mattson MP. Activation of mitochondrial ATP-dependent potassium channels protects neurons against ischemia-induced death by a mechanism involving suppression of Bax translocation and cytochrome c release. J Cereb Blood Flow Metab 22: 431-443, 2002.[CrossRef][ISI][Medline]
  100. Liu Y, Sato T, Seharaseyon J, Szewczyk A, O'Rourke B, and Marbán E. Mitochondrial ATP-dependent potassium channels. Viable candidate effectors of ischemic preconditioning. Ann NY Acad Sci 874: 27-37, 1999.[Abstract/Free Full Text]
  101. Luo X, Budihardjo I, Zou H, Slaughter C, and Wang X. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94: 481-490, 1998.[ISI][Medline]
  102. Machado RD, Pauciulo MW, Thomson JR, Lane KB, Morgan NV, Wheeler L, Phillips JAI, Newman J, Williams D, Galiè N, Manes A, McNeil K, Yacoub M, Mikhail G, Rogers P, Corris P, Humbert M, Dionnai D, Martensson G, Tranebjaerg L, Loyd JE, Trembath RC, and Nichols WC. BMPR2 haploinsufficiency as the inherited mechanism for primary pulmonary hypertension. Am J Hum Genet 68: 92-102, 2001.[CrossRef][ISI][Medline]
  103. Maeno E, Ishizaki Y, Kanaseki T, Akihiro H, and Okada Y. Normotonic cell shrinkage because of disordered volume regulation is an early prerequisite to apoptosis. Proc Natl Acad Sci USA 97: 9487-9492, 2000.[Abstract/Free Full Text]
  104. Mallat Z and Tedgui A. Apoptosis in the vasculature: mechanisms and functional importance. Br J Pharmacol 130: 947-962, 2000.[Abstract/Free Full Text]
  105. Mandegar M, Remillard CV, and Yuan JX-J. Ion channels in pulmonary arterial hypertension. Prog Cardiovasc Dis 45: 81-114, 2002.[CrossRef][ISI][Medline]
  106. Mann CL, Bortner CD, Jewell CM, and Cidlowski JA. Glucocorticoid-induced plasma membrane depolarization during thymocyte apoptosis: association with cell shrinkage and degradation of the Na+/K+-adenosine triphosphatase. Endocrinology 142: 5059-5068, 2001.[Abstract/Free Full Text]
  107. Massagué J and Chen Y-G. Controlling TGF-{beta} signaling. Genes Dev 14: 627-644, 2000.[Free Full Text]
  108. McDonough AA, Velotta JB, Schwinger RH, Philipson KD, and Farley RA. The cardiac sodium pump: structure and function. Basic Res Cardiol 97: I19-I24, 2002.[Medline]
  109. McLaughlin B, Pal S, Tran MP, Parsons AA, Barone FC, Erhardt JA, and Aizenman E. p38 activation is required upstream of potassium current enhancement and caspase cleavage in thiol oxidant-induced neuronal apoptosis. J Neurosci 21: 3303-3311, 2001.[Abstract/Free Full Text]
  110. Meisenholder GW, Martin SJ, Green DR, Nordberg J, Babior GM, and Gottlieb RA. Events in apoptosis. Acidification is downstream of protease activation and BCL-2 protection. J Biol Chem 271: 16260-16262, 1996.[Abstract/Free Full Text]
  111. Michiels C, Arnould T, and Remacle J. Endothelial cell responses to hypoxia: initiation of a cascade of cellular interactions. Biochim Biophys Acta 1497: 1-10, 2000.[ISI][Medline]
  112. Montague JW, Bortner CD, Hughes FMJ, and Cidlowski JA. A necessary role for reduced intracellular potassium during the DNA degradation phase of apoptosis. Steroids 64: 563-569, 1999.[CrossRef][ISI][Medline]
  113. Morrell NW, Yang X, Upton PD, Jourdan KB, Morgan N, Sheares KK, and Trembath RC. Altered growth responses of pulmonary artery smooth muscle cells from patients with primary pulmonary hypertension to transforming growth factor-{beta}1 and bone morphogenetic proteins. Circulation 104: 790-795, 2001.[Abstract/Free Full Text]
  114. Nakaki T, Nakayama M, Yamamoto S, and Kato R. {alpha}1-Adrenergic stimulation and {beta}2-adrenergic inhibition of DNA synthesis in vascular smooth muscle cells. Mol Pharmacol 37: 30-36, 1990.[Abstract]
  115. 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]
  116. Neuss M, Monticone R, Lundberg MS, Chesley AT, Fleck E, and Crow MT. The apoptotic regulatory protein ARC (apoptosis repressor with caspase recruitment domain) prevents oxidant stress-mediated cell death by preserving mitochondrial function. J Biol Chem 276: 33915-33922, 2001.[Abstract/Free Full Text]
  117. Newman JH, Wheeler L, Lane KB, Loyd E, Gaddipati R, Phillips JAI, and Loyd JE. Mutation in the gene for bone morphogenetic protein receptor II as a cause of primary pulmonary hypertension in large kindred. N Engl J Med 345: 319-324, 2001.[Abstract/Free Full Text]
  118. Nicholls DG and Budd SL. Mitochondria and neuronal survival. Physiol Rev 80: 315-360, 2000.[Abstract/Free Full Text]
  119. Nicholson DW. From bench to clinic with apoptosis-based therapeutic agents. Nature 407: 810-816, 2000.[CrossRef][ISI][Medline]
  120. Nietsch HH, Roe MW, Fiekers JF, Moore AL, and Lidofsky SD. Activation of potassium and chloride channels by tumor necrosis factor {alpha}: role in liver death. J Biol Chem 275: 20556-20561, 2000.[Abstract/Free Full Text]
  121. O'Donnell ME and Owen NE. Regulation of ion pumps and carriers in vascular smooth muscle. Physiol Rev 74: 683-721, 1994.[Free Full Text]
  122. Oka M, Nagaoka T, Morris KG, and McMurtry IF. Dehydroepiandrosterone markedly reduces the development of hypoxic pulmonary hypertension in rats (Abstract). Am J Respir Crit Care Med 167: A824, 2003.[CrossRef]
  123. Okada Y and Maeno E. Apoptosis, cell volume regulation and volume-regulatory chloride channels. Comp Biochem Physiol A 130: 377-383, 2001.[ISI]
  124. O'Rourke B. Pathophysiological and protective roles of mitochondrial ion channels. J Physiol 529: 23-36, 2000.[Abstract/Free Full Text]
  125. Oyaizu N, McCloskey TW, Coronesi M, Chirmule N, Kalyanaraman VS, and Pahwa S. Accelerated apoptosis in peripheral blood mononuclear cells (PBMCs) from human immunodeficiency virus type-1 infected patients and in CD4 cross-linked PBMCs from normal individuals. Blood Vessels 84: 3392-3400, 1993.
  126. Paucek P, Mironova G, Mahdi F, Beavis AD, Woldegiorgis G, and Garlid KD. Reconstitution and partial purification of the glibenclamide-sensitive, ATP-dependent K+ channel from rat liver and beef heart mitochondria. J Biol Chem 267: 26062-26069, 1992.[Abstract/Free Full Text]
  127. Pitt BR, Weng W, Steve AR, Blakely RD, Reynolds I, and Davies P. Serotonin increases DNA synthesis in rat proximal and distal pulmonary vascular smooth muscle cells in culture. Am J Physiol Lung Cell Mol Physiol 266: L178-L186, 1994.[Abstract/Free Full Text]
  128. Platoshyn O, Yu Y, Golovina VA, McDaniel SS, Krick S, Li L, Wang JY, Rubin LJ, and Yuan JX-J. Chronic hypoxia decreases KV channel expression and function in pulmonary artery myocytes. Am J Physiol Lung Cell Mol Physiol 280: L801-L812, 2001.[Abstract/Free Full Text]
  129. Platoshyn O, Zhang S, McDaniel SS, and Yuan JX-J. Cytochrome c activates K+ channels before inducing apoptosis. Am J Physiol Cell Physiol 283: C1298-C1305, 2002.[Abstract/Free Full Text]
  130. Pozeg ZI, Michelakis ED, Thebaud B, Wu X-C, Dyck JRB, 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 chronic hypoxic rats. Circulation 106: II-494, 2001.
  131. Purves D, Augustine GJ, Fitzpatrick D, Katz LC, LaMantia A-S, and McNamara JO. Electrical signals of nerve cells. In: Neuroscience, edited by Purves D, Augustine GJ, Fitzpatrick D, Katz LC, LaMantia A-S, McNamara JO, and Williams SM. Sunderland, MA: Sinauer Associates, 2001, p. 37-50.
  132. Rabinovitch M. Elastase and the pathobiology of unexplained pulmonary hypertension. Chest 114: 213S-224S, 1998.[Abstract/Free Full Text]
  133. Rabinovitch M. Pathobiology of pulmonary hypertension. Extracellular matrix. Clin Chest Med 22: 433-439, 2001.[ISI][Medline]
  134. Rabkin SW and Kong JY. Nitroprusside induces cardiomyocyte death: interaction with hydrogen peroxide. Am J Physiol Heart Circ Physiol 279: H3089-H3100, 2000.[Abstract/Free Full Text]
  135. Raley-Susman KM, Kass IS, Cottrell JE, Newman RB, Chambers G, and Wang J. Sodium influx blockade and hypoxic damage to CA1 pyramidal neurons in rat hippocampal slices. J Neurophysiol 86: 2715-2726, 2001.[Abstract/Free Full Text]
  136. Rasola A, Farahi Far D, Hofman P, and Rossi B. Lack of internucleosomal DNA fragmentation is related to Cl- efflux impairment in hematopoietic cell apoptosis. FASEB J 13: 1711-1723, 1999.[Abstract/Free Full Text]
  137. Reuss L and Hirst BH. Water transport controversies - an overview. J Physiol 542: 1-2, 2002.[Abstract/Free Full Text]
  138. Riley DJ, Thakker-Varia S, Wilson FJ, Polani GJ, and Tozzi CA. Role of proteolysis and apoptosis in regression of pulmonary vascular remodeling. Physiol Res 49: 577-585, 2000.[ISI][Medline]
  139. Rubin LJ and Rich S. Primary Pulmonary Hypertension. New York, NY: Dekker, 1997.
  140. Rudarakanchana N, Flanagan JA, Chen H, Upton PD, Machado R, Patel D, Trembath RC, and Morrell NW. Functional analysis of bone morphogenetic protein type II receptor mutations underlying primary pulmonary hypertension. Hum Mol Genet 11: 1517-1525, 2002.[Abstract/Free Full Text]
  141. Sasaki N, Sato T, Ohler A, O'Rourke B, and Marbán E. Activation of mitochondrial ATP-dependent potassium channels by nitric oxide. Circulation 101: 439-445, 2000.[Abstract/Free Full Text]
  142. Schendel SL, Xie Z, Oblatt Montal M, Matsuyama S, Montal M, and Reed JC. Channel formation by antiapoptotic protein Bcl-2. Proc Natl Acad Sci USA 94: 5113-5118, 1997.[Abstract/Free Full Text]
  143. Schrantz N, Blanchard DA, Auffredou MT, Sharma S, Leca G, and Vazquez A. Role of caspases and possible involvement of retinoblastoma protein during TGF{beta}-mediated apoptosis of human B lymphocytes. Oncogene 18: 3511-3519, 1999.[CrossRef][ISI][Medline]
  144. Segal M and Barker JL. Rat hippocampal neurons in culture: properties of GABA-activated Cl- ion conductance. J Neurophysiol 51: 500-515, 1984.[Abstract/Free Full Text]
  145. Shimizu S, Eguchi Y, Kamiike W, Funahashi Y, Mignon A, Lacronique V, Matsuda H, and Tsujimoto Y. Bcl-2 prevents apoptotic mitochondrial dysfunction by regulating proton flux. Proc Natl Acad Sci USA 95: 1455-1459, 1998.[Abstract/Free Full Text]
  146. Shimizu S, Ide T, Yanagida T, and Tsujimoto Y. Electrophysiological study of a novel large pore formed by Bax and the voltage-dependent anion channel that is permeable to cytochrome c. J Biol Chem 275: 12321-12325, 2000.[Abstract/Free Full Text]
  147. Shimizu S, Konishi A, Kodama T, and Tsujimoto Y. BH4 domain of antiapoptotic Bcl-2 family members closes voltage-dependent anion channel and inhibits apoptotic mitochondrial changes and cell death. Proc Natl Acad Sci USA 97: 3100-3105, 2000.[Abstract/Free Full Text]
  148. Siemen D, Loupatatzis C, Borecky J, Gulbins E, and Lang F. Ca2+-activated K channel of the BK-type in the inner mitochondrial membrane of a human glioma cell line. Biochem Biophys Res Commun 257: 549-554, 1999.[CrossRef][ISI][Medline]
  149. Skulachev VP. Cytochrome c in the apoptotic and antioxidant cascades. FEBS Lett 423: 275-280, 1998.[CrossRef][ISI][Medline]
  150. 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: H365-H370, 1994.[Abstract/Free Full Text]
  151. Stenmark KR and Mecham RP. Cellular and molecular mechanisms of pulmonary vascular remodeling. Annu Rev Physiol 59: 89-144, 1997.[CrossRef][ISI][Medline]
  152. Strange K, Emma F, and Jackson PS. Cellular and molecular physiology of volume-sensitive anion channels. Am J Physiol Cell Physiol 270: C711-C730, 1996.[Abstract/Free Full Text]
  153. Strasser A, Harris AW, Huang DC, Krammer PH, and Cory S. Bcl-2 and Fas/APO-1 regulate distinct pathways to lymphocyte apoptosis. EMBO J 14: 136-147, 1995.
  154. Strasser A, O'Connor L, and Dixit VM. Apoptosis signaling. Annu Rev Biochem 69: 217-245, 2000.[CrossRef][ISI][Medline]
  155. Susin SA, Zamzami N, Castedo M, Hirsch T, Marchetti P, Macho A, Daugas E, Geuskens M, and Kroemer G. Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J Exp Med 184: 1331-1341, 1996.[Abstract]
  156. Suzuki Y, Imai Y, Nakayama H, Takahashi K, Takio K, and Takahashi R. A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death. Mol Cell 8: 613-621, 2001.[ISI][Medline]
  157. Szabò I, Gulbins E, Apfel H, Zhang X, Barth P, Busch AE, Schlottmann K, Pongs O, and Lang F. Tyrosine phosphorylation-dependent suppression of a voltage-gated K+ channel in T lymphocytes upon Fas stimulation. J Biol Chem 271: 20465-20469, 1996.[Abstract/Free Full Text]
  158. Szabó I, Lepple-Wienhues A, Kaba KN, Zoratti M, Gulbins E, and Lang F. Tyrosine kinase-dependent activation of a chloride channel in CD95-induced apoptosis in T lymphocytes. Proc Natl Acad Sci USA 95: 6169-6174, 1998.[Abstract/Free Full Text]
  159. Szewczyk A and Marban E. Mitochondria: a new target for potassium channel openers? Trends Pharmacol Sci 20: 157-161, 1999.[CrossRef][ISI][Medline]
  160. Thompson GJ, Langlais C, Cain K, Conley EC, and Cohen GM. Elevated extracellular [K+] inhibits death-receptor- and chemical-mediated apoptosis prior to caspase activation and cytochrome c release. Biochem J 357: 137-145, 2001.[CrossRef][ISI][Medline]
  161. Thornberry NA and Lazebnik Y. Caspases: enemies within. Science 281: 1312-1316, 1998.[Abstract/Free Full Text]
  162. 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: C588-C594, 2002.[Abstract/Free Full Text]
  163. Vander Heiden MG, Chandel NS, Williamson EK, Schumacker PT, and Thompson CB. Bcl-XL prevents cell death following growth factor withdrawal by facilitating mitochondrial ATP/ADP exchange. Cell 91: 627-637, 1999.
  164. 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.[CrossRef][ISI][Medline]
  165. Verhagen AM, Ekert PG, Pakusch M, Silke J, Connolly LM, Reid GE, Moritz RL, Simpson RJ, and Vaux DL. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to and antagonizing IAP proteins. Cell 102: 43-53, 2000.[ISI][Medline]
  166. Verhagen AM, Silke J, Ekert PG, Pakusch M, Kaufmann H, Connolly LM, Day CL, Tikoo A, Burke R, Wrobel C, Moritz RL, Simpson RJ, and Vaux DL. HtrA2 promotes cell death through its serine protease activity and its ability to antagonize inhibitor of apoptosis proteins. J Biol Chem 277: 445-454, 2002.[Abstract/Free Full Text]
  167. Vick RL, Chang DC, Nichols BL, Hazlewood CF, and Harvey MC. Sodium potassium, and water in cardiac tissues. Ann NY Acad Sci 204: 575-592, 1973.[ISI][Medline]
  168. Voelkel NF and Tuder RM. Cellular and molecular biology of vascular smooth muscle cells in pulmonary hypertension. Pulm Pharmacol Ther 10: 231-241, 1997.[CrossRef][ISI][Medline]
  169. Vu CCQ, Bortner CD, and Cidlowski JA. Differential involvement of initiator caspases in apoptotic volume decrease and potassium efflux during Fas- and UV-induced cell death. J Biol Chem 276: 37602-37611, 2001.[Abstract/Free Full Text]
  170. Wang H-W, Zhang Y, Cao L, Han H, Wang J, Yang B, Nattel S, and Wang Z. HERG K+ channel, a regulator of tumor cell apoptosis and proliferation. Cancer Res 62: 4843-4848, 2002.[Abstract/Free Full Text]
  171. Wang J, Juhaszova M, Rubin LJ, and Yuan X-J. Hypoxia inhibits gene expression of voltage-gated K+ channel {alpha} subunits in pulmonary artery smooth muscle cells. J Clin Invest 100: 2347-2353, 1997.[Abstract/Free Full Text]
  172. Wang J, Morishima S, and Okada Y. IK channels are involved in the regulatory volume decrease in human epithelial cells. Am J Physiol Cell Physiol 284: C77-C84, 2003.[Abstract/Free Full Text]
  173. Wang L, Xu D, Dai W, and Lu L. An ultraviolet-activated K+ channel mediates apoptosis of myeloblastic leukemia cells. J Biol Chem 274: 3678-3685, 1999.[Abstract/Free Full Text]
  174. Wang NS, Unkila MT, Reineks EZ, and Distelhorst CW. Transient expression of wild-type of mitochondrially targeted Bcl-2 induces apoptosis, whereas transient expression of endoplasmic reticulum-targeted Bcl-2 is protective against Bax-induced cell death. J Biol Chem 276: 44117-44128, 2001.[Abstract/Free Full Text]
  175. Wang X. The expanding role of mitochondria in apoptosis. Genes Dev 15: 2922-2933, 2001.[Free Full Text]
  176. Wang X, Xiao Y, Ichinose T, and Yu SP. Effects of tetraethylammonium analogs on apoptosis and membrane currents in cultured cortical neurons. J Pharmacol Exp Ther 295: 524-530, 2000.[Abstract/Free Full Text]
  177. Wang XQ and Yu SP. Tyrosine phosphorylation regulates activity of Na+, K+-ATPase in cortical neurons. Soc Neurosci Abstr 446.8, 2002.
  178. Wesselborg S and Kabelitz D. Activation-driven death of human T cell clones: time course kinetics of the induction of cell shrinkage, DNA fragmentation, and cell death. Cell Immunol 148: 234-241, 1993.[CrossRef][ISI][Medline]
  179. Wible BA, Wang L, Kuryshev YA, Basu A, Haldar S, and Brown AM. Increased K+ efflux and apoptosis induced by the potassium channel modulatory protein KCAP/PIAS3{beta} in prostate cancer cells. J Biol Chem 27: 17852-17862, 2003.[CrossRef]
  180. Wolf CM, Reynolds JE, Morana SJ, and Eastman A. The temporal relationship between protein phosphatase, ICE/CED-3 proteases, intracellular acidification, and DNA fragmentation in apoptosis. Exp Cell Res 230: 22-27, 1997.[CrossRef][ISI][Medline]
  181. Xiao AY, Wei L, Xia S, Rothman S, and Yu SP. Ionic mechanism of ouabain-induced concurrent apoptosis and necrosis in individual cultured cortical neurons. J Neurosci 22: 1350-1362, 2002.[Abstract/Free Full Text]
  182. Xu W, Liu Y, Wang S, McDonald T, Van Eyk JE, Sidor A, and O'Rourke B. Cytoprotective role of Ca2+-activated K+ channels in the cardiac inner mitochondrial membrane. Science 298: 1029-1033, 2002.[Abstract/Free Full Text]
  183. 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]
  184. Yao Z, Tong J, Tan X, Li C, Shao Z, Kim WC, Venden Hoek TL, Becker LB, Head CA, and Schumacker PT. Role of reactive oxygen species in acetylcholine-induced preconditioning in cardiomyocytes. Am J Physiol Heart Circ Physiol 277: H2504-H2509, 1999.[Abstract/Free Full Text]
  185. Yu SP, Canzoniero LMT, and Choi DW. Ion homeostasis and apoptosis. Curr Opin Cell Biol 13: 405-411, 2001.[CrossRef][ISI][Medline]
  186. Yu SP and Choi DW. Ions, cell volume, and apoptosis. Proc Natl Acad Sci USA 97: 9360-9362, 2000.[Free Full Text]
  187. Yu SP, Yeh C-H, Gottron F, Wang X, Grabb MC, and Choi DW. Role of the outwardly delayed rectifier K+ current in ceramide-induced caspase activation and apoptosis in cultured cortical neurons. J Neurochem 73: 933-941, 1999.[CrossRef][ISI][Medline]
  188. Yu SP, Yeh 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]
  189. Yuan J and Yankner BA. Apoptosis in the nervous system. Nature 407: 802-809, 2000.[CrossRef][ISI][Medline]
  190. 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: 1400-1406, 1998.[Abstract/Free Full Text]
  191. Yuan JX-J and Rubin LJ. Altered expression and function of Kv channels in primary pulmonary hypertension. In: Potassium Channels in Cardiovascular Biology, edited by Archer SL and Rusch NJ. New York: Kluwer Academic/Plenum, 2001, p. 821-836.
  192. Yuan XJ. Role of calcium-activated chloride current in regulating pulmonary vascular tone. Am J Physiol Lung Cell Mol Physiol 272: L959-L968, 1997.[Abstract/Free Full Text]
  193. Yuan XJ, 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: L116-L123, 1993.[Abstract/Free Full Text]
  194. Yuan XJ, Wang J, Juhaszova M, Gaine SP, and Rubin LJ. Attenuated K+ channel gene transcription in primary pulmonary hypertension. Lancet 351: 726-727, 1998.[CrossRef][ISI][Medline]
  195. Zhang HY, McPherson BC, Liu H, Baman TS, Rock P, and Yao Z. H2O2 opens mitochondrial KATP channels and inhibits GABA receptors via protein kinase C-{epsilon} in cardiomyocytes. Am J Physiol Heart Circ Physiol 282: H1395-H1403, 2002.[Abstract/Free Full Text]
  196. Zhang S, Fantozzi I, Krick S, Rubin LJ, and Yuan JX-J. BMP-mediated apoptosis is inhibited in pulmonary artery smooth muscle cells from the patients with primary pulmonary hypertension. Circulation 106: II-365, 2002.
  197. Zhu L, Ling S, Yu X-D, Venkatesh LK, Subramaniam 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]