Cytochrome c activates K+ channels before inducing apoptosis

Oleksandr Platoshyn, Shen Zhang, Sharon S. McDaniel, and Jason X.-J. Yuan

Department of Medicine, University of California, San Diego, California 92103-8382


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

Cell shrinkage is an early prerequisite for apoptosis. The apoptotic volume decrease is due primarily to loss of cytoplasmic ions. Increased outward K+ currents have indeed been implicated in the early stage of apoptosis in many cell types. We found that cytoplasmic dialysis of cytochrome c (cyt-c), a mitochondria-dependent apoptotic inducer, increases K+ currents before inducing nuclear condensation and breakage in pulmonary vascular smooth muscle cells. The cyt-c-mediated increase in K+ currents took place rapidly and was not affected by treatment with a specific inhibitor of caspase-9. Cytoplasmic dialysis of recombinant (active) caspase-9 negligibly affected the K+ currents. Furthermore, treatment of the cells with staurosporine (ST), an apoptosis inducer that mediates translocation of cyt-c from mitochondria to the cytosol, also increased K+ currents, caused cell shrinkage, and induced apoptosis (determined by apoptotic nuclear morphology and TdT-UTP nick end labeling assay). The staurosporine-induced increase in K+ currents concurred to the volume decrease but preceded the activation of apoptosis (nuclear condensation and breakage). These results suggest that the cyt-c-induced activation of K+ channels and the resultant K+ loss play an important role in initiating the apoptotic volume decrease when cells undergo apoptosis.

apoptotic volume decrease; voltage-gated potassium channels; pulmonary artery smooth muscle cells


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

APOPTOSIS IS A process that plays a critical role in embryonic development and tissue homeostasis. In humans, dysfunction of this process has been linked to the pathogenesis of cancer, atherosclerosis, and pulmonary vascular disease (9, 12, 23, 25). Cell shrinkage is an incipient hallmark of apoptosis and as such is seen in almost all cell types that undergo programmed cell death. The apoptotic volume decrease has been recently demonstrated to be an early prerequisite for apoptosis (1, 3, 19, 22, 30). Cell volume is primarily controlled by the intracellular ion homeostasis; thus, ion transport across the plasma membrane is of primary importance for the regulation of cell volume (17). K+ is the dominant cation in the cytosol and a main contributor to maintaining cell volume. Because of the outwardly directed K+ electrochemical gradient, opening of plasmalemmal K+ channels would increase efflux or loss of cytosolic K+ and induce the apoptotic volume decrease. Indeed, it has been demonstrated that staurosporine-induced apoptosis is associated with an early increase in outward K+ currents through voltage-gated K+ (Kv) channels, which subsequently leads to a net loss of cytoplasmic K+ and thus to the apoptotic volume decrease (6, 19, 31, 32). Attenuation of K+ efflux, by pharmacological blockade of K+ channels or by reducing the K+ electrochemical gradient using high extracellular K+, inhibits apoptosis induced by a variety of apoptosis inducers, such as staurosporine, valinomycin, anti-Fas, the protein kinase C inhibitor Gö-6976, tumor necrosis factor-alpha , H2O2, carbonyl cyanide 4-trifluoromethoxyphenylhydrazone, and ultraviolet radiation (1, 3, 8, 15, 19, 30-32).

Release of cytochrome c (cyt-c) from the mitochondrial intermembrane space to the cytosol is a critical step in initiating apoptosis (14, 16, 29). It has been well documented that an increase in cytosolic cyt-c, in association with Apaf-1, is a trigger for activation of caspase-9, which then activates a set of cysteine proteases (e.g., caspases-3, -5, and -7) that are central executioners of the apoptotic pathway (10, 26). Furthermore, abundant evidence demonstrates that the apoptosis volume decrease and the loss of cytosolic K+ are also required for activation of the effector caspases and apoptotic nucleases in the mitochondria-independent apoptosis mediated by the Fas receptor (8, 10). In spite of extensive studies on the caspase-dependent mechanisms by which cyt-c induces apoptosis, it is unknown whether cyt-c affects plasmalemmal K+ channels. This study was designed to test the hypothesis that cyt-c, in addition to activating caspase-9, activates Kv channels and increases K+ currents through these channels, and ultimately causes net loss of cytosolic K+ and apoptotic cell shrinkage.

Precise control of the balance of cell apoptosis and proliferation in pulmonary artery smooth muscle cells (PASMC) also plays a critical role in maintaining normal function and structure of the pulmonary vasculature. Inhibition of apoptosis and augmentation of proliferation in PASMC would lead to pulmonary vascular-wall remodeling, an important pathological feature in patients with pulmonary hypertension. Thus understanding of the cellular and molecular mechanisms involved in the regulation and control of PASMC apoptosis will provide important information for the development of useful therapeutic approaches for treatment of pulmonary hypertension and other pulmonary vascular diseases.


    METHODS AND MATERIALS
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

Cell preparation and culture. Primary cultured PASMC were prepared from pulmonary arteries of male Sprague-Dawley rats (150-200 g). Animals were killed by decapitation, which has been justified and approved by the Institutional Animal Care and Use Committee at the University of California, San Diego. After decapitation, the heart and lungs were removed and placed in Hanks' balanced salt solution (HBSS). The right and left branches (2nd division) of the main pulmonary artery and intracellular pulmonary arteries (3rd-4th division) were then isolated from the lungs and incubated for 20 min in HBSS containing 1.5 mg/ml collagenase (Worthington). After the incubation, a thin layer of adventitia was carefully stripped off with a fine forceps, and endothelium was removed by gently scratching the intimal surface with a surgical blade. The remaining smooth muscle was then digested with 1.5 mg/ml collagenase and 0.5 mg/ml elastase (Sigma) for 45 min at 37°C. The cells were dispersed and plated on coverslips and incubated in DMEM containing 10% FBS in a humidified atmosphere of 5% CO2 in air at 37°C (33). The purity of PASMC in primary cultures was confirmed by the specific monoclonal antibody raised against smooth muscle alpha -actin (Boehringer Mannheim). Primary cultured cells were first stained with the membrane-permeable nucleic acid stain 4',6'-diamidino-2-phenylindole (DAPI, 5 µM; Molecular Probes) to estimate total cell numbers in the cultures. All the DAPI-stained cells also cross-reacted with the smooth muscle cell alpha -actin antibody, indicating that the cultures were all smooth muscle cells.

Electrophysiological measurements. Whole cell K+ currents were recorded with an Axopatch-1D amplifier and a DigiData 1200 interface (Axon Instruments) using patch-clamp techniques (33). Patch pipettes (2-3 MOmega ) were made on a Sutter electrode puller using borosilicate glass tubes and fire polished on a Narishige microforge. Step-pulse protocols and data acquisition were performed, and leakage and capacitive currents were subtracted using pCLAMP software (Axon Instruments). Currents were filtered at 1-2 kHz (-3 dB) and digitized at 2-4 kHz using the Axopatch-1D amplifier. All experiments were performed at room temperature (22-24°C).

A coverslip containing the cells in the recording chamber (approx 0.75 ml) was superfused (2 ml/min) with the extracellular (bath) solution containing (in mM): 141 NaCl, 4.7 KCl, 3.0 MgCl2, 0.1 EGTA, 10 HEPES, and 10 glucose (pH 7.4 with 1 M NaOH). The internal (pipette) solution contained (in mM): 125 KCl, 4 MgCl2, 10 HEPES, 10 EGTA, and 5 ATP (pH 7.2). Recombinant cyt-c (Sigma) was first dissolved in distilled water to make a stock solution of 5 mM cyt-c. Aliquots of the stock solution were then added to the pipette solution to make a final concentration of 5 µM cyt-c. In some experiments, cyt-c was heat-inactivated by boiling the cyt-c stock solution at 100°C for >3 h before it was diluted in the pipette solution. Recombinant (human) caspase-9 (Calbiochem) was dissolved in PBS to make a stock solution of 5 mM; aliquots of the stock solution were diluted 1:1,000 in the pipette solution to make a final concentration of 5 µM caspase-9. After formation (break-in) of the whole cell recording configuration, cyt-c, heat-inactivated cyt-c, or recombinant caspase-9 was dialyzed gradually in the cell cytosol. The currents recorded immediately (0 min) after break-in were used as control.

A potent, cell-permeable inhibitor of caspase-9 (Calbiochem) was dissolved in dimethyl sulfoxide to make a stock solution of 20 mM; aliquots of the stock solution were then diluted to the culture medium and bath solution to make a final concentration of 10 µM. Cells were pretreated with 10 µM caspase-9 inhibitor dissolved in the culture medium for 12 h before currents were recorded using an electrode in which cyt-c-containing solution was included. In these experiments, the cells were superfused with the bath solution containing the same amount of caspase-9 inhibitor as is in the culture medium.

Nuclear morphology determination. The cells on 25-mm cover slips were first washed with PBS (Sigma), fixed in 95% ethanol, and stained with the membrane-permeable nucleic acid stain DAPI (Sigma). DAPI (5 µM) was dissolved in an antibody buffer containing 500 mM NaCl, 20 µM NaN3, 10 µM MgCl2, and 20 µM Tris · HCl (pH 7.4). The blue fluorescence emitted at 461 nm was used to visualize the cell nuclei. The DAPI-stained cells were examined with a Nikon TE 300 fluorescence microscope, and the cell (nuclear) images were acquired using a high-resolution Solamere fluorescence imaging system and analyzed using the NIH Imaging software. For each coverslip, 5-10 fields (15-20 cells/field) were randomly selected to determine the percentage of apoptotic cells in total cells on the basis of the morphological characteristics of apoptosis. The cells with clearly defined nuclear breakage, remarkably condensed nuclear fluorescence, and a significantly shrunken cell body and nucleus were defined as apoptotic cells. To quantify apoptosis, TdT-UTP nick end labeling (TUNEL) assays were also performed with the In Situ Cell Death Detection Kit (TMR Red; Boehringer-Mannheim) according to the manufacturer's instructions.

The relative cross-sectional nuclear area of the DAPI-stained cells (visualized by a fluorescence objective) and the size of the cells (visualized by a Phase-contrast objective) were measured using the NIH Image software and are expressed as number of pixels (arbitrary unit) or the percentage change of the number of pixels compared with control cells.

Statistical analysis. Data are expressed as means ± SE. Statistical analysis was performed using the unpaired Student's t-test or ANOVA and post hoc tests (Student-Newman-Keuls) as indicated. Differences were considered to be significant at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

Cytoplasmic application of recombinant cyt-c increases Kv currents. Whole cell K+ currents through Kv channels [IK(V)] were isolated in PASMC superfused with Ca2+-free bath solution (plus 0.1 mM EGTA) and dialyzed with Ca2+-free and ATP-containing pipette solution (with 10 mM EGTA). Under these conditions, the contribution of Ca2+-activated and ATP-sensitive K+ currents to the whole cell outward currents was minimized (33). Cytoplasmic dialysis of cyt-c (5 µM) for up to 20 min through an electrode significantly increased the amplitude and current density of whole cell IK(V) elicited by test potentials ranging from -60 to +80 mV (Fig. 1A). The threshold potential for activating the cyt-c-sensitive IK(V) was between -60 and -55 mV (Fig. 1, B and C), which is within the range of resting membrane potential in PASMC. This suggests that the cyt-c-sensitive Kv channels are active under resting conditions. The cyt-c-mediated increase in IK(V) took place ~3-5 min after rupture (break in) of the membrane patch and maximized at 15-20 min (Fig. 1D). Extracellular application of 4-aminopyridine (4-AP), a potent blocker of Kv channels (33), markedly attenuated the cyt-c-induced increase in IK(V) (Fig. 1E).


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 1.   Activation of voltage-gated K+ (Kv) channels by cytochrome c (cyt-c) in pulmonary artery smooth muscle cells (PASMC). A: dialysis of recombinant cyt-c (5 µM) for 20 min after rupture (0 min) of the membrane patch increased the amplitude of whole cell K+ current through Kv channels [IK(V)] elicited by a series of test potentials ranging from -60 to +80 mV. The holding potential was -70 mV. Leakage and capacitive currents were subtracted. Inset: Current-voltage (I-V) relationship curves of IK(V) recorded immediately (0 min) or 20 min after rupture (break in) of the membrane patch. B: summarized data (means ± SE, n = 40) showing the current amplitudes at -60 and -40 mV recorded immediately (0 min) and 20 min after break in. **P < 0.01 vs. 0 min. C: cyt-c-activated currents (inset) were obtained by subtracting the currents recorded immediately after break in from the currents recorded 20 min after break in. Composite I-V curves (means ± SE) of the cyt-c-sensitive currents are based on the experiments obtained from 40 cells. D: time course of cyt-c-induced increase in IK(V) in a cell after rupture (break in) of the membrane patch. E: effect of 4-aminopyridine (4-AP; 5 mM) on cyt-c-mediated increase in IK(V). Current density at +80 mV was measured 20 min after cytoplasmic dialysis of cyt-c (5 µM) in control (Cont) cells (n = 40) and cells treated with 5 mM 4-AP (n = 20). ***P < 0.001 vs. Cont.

Amplitudes of ion currents are somehow affected by washout of the cytoplasmic molecules and organelles with intracellular (pipette) solutions. To confirm that the cyt-c-mediated increase in IK(V) was not due artificially to the washout, we conducted the same experiments as shown in Fig. 1 using the pipette solution that contains either vehicle (distilled water) or heat-inactivated cyt-c (the stock solution of cyt-c was boiled at 100°C for >3 h before being dissolved in the pipette solution). Intracellular dialysis of the vehicle (distilled water) used for dissolving cyt-c negligibly affected IK(V); the current amplitudes at +80 mV were 706 ± 89 pA immediately after the rupture of the membrane patch and 640 ± 83 pA 20 min later (P = 0.59). Furthermore, intracellular dialysis of the heat-inactivated cyt-c for 20 min failed to increase IK(V) (Fig. 2).


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of heat-inactivated cyt-c on IK(V) in PASMC. A: representative currents, elicited by test potentials ranging from -60 to +80 mV (with a holding potential of -70 mV) in PASMC dialyzed with pipette solution including 5 µM heat-inactivated cyt-c, recorded immediately (0 min) or 20 min after break in. The difference currents (Subtraction), representing the currents induced by the heat-inactivated cyt-c, were obtained by subtracting the currents recorded immediately after break in from the currents recorded 20 min after break in. In this experiment, cyt-c was heat inactivated by boiling the cyt-c stock solution at 100°C for >3 h before being dissolved in the pipette solution. B: summarized data (means ± SE, n = 13) showing I-V relationship curves of IK(V) recorded immediately (0 min, open circle ) or 20 min () after break in.

These results demonstrate that cyt-c rapidly increases IK(V) by opening the 4-AP-sensitive Kv channels in PASMC. The activation of K+ channels and increase in IK(V), in addition to causing intracellular K+ loss and the apoptotic volume decrease, would also cause membrane hyperpolarization. In vascular smooth muscle cells, the intracellular Cl- concentration ([Cl-]) is up to 50-100 mM, and the calculated equilibrium potential for Cl- is approximately -25 to -8 mV (13). Thus the membrane hyperpolarization resulting from increased IK(V) should also facilitate the apoptotic volume decrease by increasing Cl- efflux (19, 22).

The cyt-c-mediated increase in IK(V) is independent of caspase-9. Procaspase-9 is an immediate downstream target of endogenous cyt-c released from the mitochondria. The cyt-c-mediated activation of caspase-9 in the presence of Apaf-1 is an initial step for activation of the effector caspases (e.g., caspase-3, -5, and -7; see Refs. 10 and 26) and for cleavage of various proteins and enzymes that are required for cell survival. The next set of experiments was designed to determine whether caspase-dependent proteolysis was involved in the cyt-c-mediated increase in IK(V).

The cells were pretreated with a membrane-permeant, specific caspase-9 inhibitor for 12 h before IK(V) was recorded, with the electrode including recombinant cyt-c (5 µM) in the presence of the caspase-9 inhibitor dissolved in the bath solution. As shown in Fig. 3A, the cyt-c-mediated increase in IK(V) was unaffected by the treatment with the caspase-9 inhibitor. The amplitude of IK(V) at +80 mV was increased by 113 ± 8% (n = 15) by cyt-c in cells treated with the caspase-9 inhibitor (Fig. 3A) and by 131 ± 15% (n = 40) in control cells (P = 0.22; Fig. 1A). The cyt-c-activated currents recorded from the cells treated with the caspase-9 inhibitor (Fig. 3A, bottom) are indeed comparable to the cyt-c-activated currents recorded from control cells (Fig. 1C, inset). The time course of the cyt-c-mediated increase in IK(V) in cells treated with the caspase-9 inhibitor (Fig. 3C) is also similar to that in control cells (Fig. 1D). Moreover, cytoplasmic dialysis for up to 27 min with recombinant active caspase-9 itself failed to increase IK(V) (Fig. 3, B and C). These results indicate that the cyt-c-induced increase in IK(V) is independent of mature caspase-9 and appears not to result from caspase-dependent proteolysis of the Kv channel protein. Consistent with these findings, the preferred cleavage sequence (LEHD-X) of active caspase-9 (5) was not found in the coding regions of most Kv channel alpha - and beta -subunits.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of the caspase-9 (Casp-9) inhibitor on cyt-c-induced increase in IK(V) in PASMC. A: representative superimposed currents (left), elicited by test potentials ranging from -60 to +80 mV (with a holding potential of -70 mV), recorded immediately (0 min) and 20 min after rupture of the membrane patch in PASMC treated with a membrane-permeant inhibitor of caspase-9 (10 µM for 12 h). The cyt-c-activated currents (Subtraction) in the presence of caspase-9 inhibitor were obtained by subtracting the currents recorded immediately after break in from the currents recorded 20 min after break in. A, right: summarized data (means ± SE, n = 15) showing that 20-min dialysis of cyt-c significantly increased current density of IK(V) in the presence of caspase-9 inhibitor. ***P < 0.001 vs. 0 min. B: dialysis of recombinant, active caspase-9 had negligible effect on IK(V) in PASMC. Representative superimposed currents (left), elicited by test potentials ranging from -60 to +80 mV (holding potential was -70 mV), recorded immediately (0 min) and 20 min after break in using an electrode in which 5 µM recombinant, active caspase-9 was included in the pipette solution. B, right: summarized data (means ± SE, n = 14) showing that caspase-9 had no effect on the current density (P = 0.42). C: time courses of the cyt-c-induced increase in IK(V) in a cell treated with the caspase-9 inhibitor (open circle ) and of the caspase-9-induced effect on IK(V) in a control cell (). Arrow, time when the membrane patch was ruptured (break in).

To test the efficiency of the caspase-9 inhibitor used for the electrophysiological experiments, we examined the effect of this inhibitor on staurosporine-induced apoptosis in PASMC, which has been demonstrated to depend on activation of caspase-9 (10, 26). As shown in Fig. 4, treatment of the cells with the caspase-9 inhibitor significantly attenuated the apoptotic effect of staurosporine (0.1 µM for 6 h). These results indicate that the caspase-9 inhibitor used for the electrophysiological experiments is functionally efficient to decrease endogenous caspase activity and attenuate caspase-9-dependent apoptosis in PASMC.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of the caspase-9 inhibitor on staurosporine (ST)-induced apoptosis in PASMC. Summarized data (means ± SE, n = 7 experiments) showing staurosporine (0.1 µM)-induced apoptosis in PASMC treated with (last two bars) or without (second bar) the caspase-9 inhibitor. **P < 0.01 vs. without inhibitor.

Cytoplasmic application of recombinant cyt-c induces apoptosis. To confirm that the cytoplasmic dialysis of cyt-c was sufficient and able to cause apoptosis, we examined nuclear morphological changes in the cells patched for recording IK(V) or, in other words, in the cells to which cyt-c was applied intracellularly by the electrode. Immediately after the patch-clamp experiments, the cells maintained a normal nuclear morphology (Fig. 5Aa, patched cells); no nuclear breakage or DNA fragmentation was observed in any cells tested. However, 12-15 h after the patch-clamp experiments (with the cells maintained in an incubator at 37°C), cells dialyzed with cyt-c showed typical apoptotic nuclear morphology, including nuclear condensation and breakage (Fig. 5A, b-d, patched cells), whereas cells on the same coverslip that were not patched for recording IK(V) showed a normal nuclear morphology (Fig. 5A, b-d, unpatched cells). As shown in Fig. 5B, the cross-sectional nuclear area in the patched cells that were stained with DAPI 12 h after the patch-clamp experiments was significantly smaller than the unpatched cells and the patched cells that were stained with DAPI immediately after the patch-clamp experiments (Fig. 5B). These results suggest that cytoplasmic dialysis of cyt-c via the electrode is sufficient to increase IK(V) and cause apoptosis. However, the time courses of the cyt-c-mediated increases in IK(V) and apoptosis were quite different; the increase in IK(V) (<15 min) preceded the cyt-c-induced apoptosis (~12 h).


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 5.   Apoptotic effect of cytoplasmic dialysis of cyt-c via electrodes in PASMC. A: superimposed currents, elicited by test potentials ranging from -60 to +80 mV (with a holding potential of -70 mV), were first recorded immediately (0 min) and 20 min after rupture of the membrane patch (break in, left). Next, the patched cells were stained with 4',6'-diamidino-2-phenylinodole (DAPI) either immediately after (0 h, a) or 12-15 h after (b-d) the patch-clamp experiments. In b-d, the cells patched for recording IK(V) were placed in an incubator for 12-15 h before being fixed and stained with DAPI. A, right: unpatched cells on the same cover slips as the patched cells were stained with DAPI as control at corresponding time when the patched cells were stained with DAPI. e: Currents were first recorded immediately (0 min) and 20 min after break in in a cell dialyzed with the pipette solution including only vehicle (first two columns), and the cell was stained with DAPI 15 h after the patch-clamp experiment (third column). Last column in e: unpatched cell on the same coverslip shown as control. B: summarized data (means ± SE) showing nuclear areas of the cells that were not patched (Control) and the cells patched for recording IK(V) (cyt-c) immediately after (0 h) or 12 h after the patch-clamp experiments. *P < 0.05 vs. control.

To examine whether dialysis of normal pipette solutions to the cells and/or excision of the electrode from the cell induce apoptosis, we conducted the same experiments as shown in Fig. 5A, a-d using the pipette solution containing vehicle (distilled water). Amplitudes of IK(V) in cells dialyzed with the cyt-c-free pipette solution did not change along time (up to 20 min; Fig. 5Ae, left). Similar to the unpatched cells, the patched cells dialyzed with the cyt-c-free solution did not show apoptotic nuclear morphology 15 h after the electrophysiological experiments (Fig. 5Ae, right). These results rule out the possibility that the cell death processes took place in the patched cells during dialysis with pipette solution in the absence of cyt-c or because of mechanical damage induced by the electrodes (e.g., break in, wash out, and/or excision).

Correlation of staurosporine-induced cell shrinkage and increase in IK(V) . Staurosporine is a potent inducer of apoptosis, which causes translocation of cyt-c from the mitochondrial intermembrane space to the cytosol in many cell types (14, 16, 29). The staurosporine-induced increase in cytosolic cyt-c occurs rapidly (<= 1 h) and is often maximal 3-4 h after treatment (14, 29). Exposure of PASMC to staurosporine also significantly increased IK(V) (Fig. 6A). The time course of the staurosporine-induced increase in IK(V) correlated with that of cell shrinkage induced by the drug but preceded staurosporine-induced apoptosis, defined by positive TUNEL stain (Fig. 6, B-D). In normal cells, the amplitude of IK(V) is, usually, positively proportional to the cell size. However, in cells treated with staurosporine, the amplitude of IK(V) was inversely correlated with the cell size, which further suggests that the staurosporine-induced increase in IK(V) is a trigger for the cell shrinkage. These results also suggest that endogenous cyt-c-mediated apoptosis is preceded by an increase in IK(V).


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 6.   Staurosporine-induced apoptosis is preceded by an increase in IK(V) in PASMC. A: averaged currents, elicited by test potentials ranging from -60 to +80 mV (with a holding potential of -70 mV), from control cells (left, treated with vehicle, n = 25) and cells treated with staurosporine (0.02 µM) for 0.5 h (middle, n = 25) and 2.5 h (right, n = 25). Leakage and capacitive currents were subtracted. B: phase-contrast micrograph of cells before and after treatment with staurosporine for 2.5 and 3.5 h. Magnified images (bottom) show the size change of a cell (arrow) before and after treatment with staurosporine. C: DAPI (a)- and TdT-UTP nick end labeling (TUNEL; b)-stained nuclei of control cells (treated with vehicle) and cells treated with staurosporine for 2.5 and 3.5 h. D: time courses of the ST-induced increase in IK(V) (), decrease in cell volume or size (), and apoptosis (black-triangle). Data are means ± SE. IK, K+ current.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

Apoptosis is a physiological process that is tightly controlled by diverse extracellular and intracellular pathways (10, 12, 25). Cell shrinkage is a major hallmark of programmed cell death or apoptosis (22, 28, 30). The apoptotic cell shrinkage occurs in the following two distinct stages: the initial phase starting before cell fragmentation or formation of the apoptotic body and the late phase that is associated with cell fragmentation. The early phase of cell shrinkage when cells undergo apoptosis is defined as an apoptotic volume decrease that is mainly regulated by the activity of membrane ion channels and transporters (22).

In vascular smooth muscle cells, K+ is the dominant cation and Cl- is the dominant anion in the cytoplasm. Maintenance of a high concentration of intracellular K+ ([K+], ~140 mM) and Cl- (50-100 mM) is required to maintain normal cell volume, whereas loss of intracellular K+ is in part responsible for the apoptotic volume decrease (17, 30). Furthermore, maintenance of a high concentration of intracellular K+ is also necessary for suppression of caspases and nucleases that are believed to be the final mediators of apoptosis (3, 11, 25). In a cell-free system (isolated nuclei), a decrease in [K+] from 140 to 80 mM caused a 1.6-fold increase of apoptosis induced by the apoptosis-inducing factor (3). In lymphocytes, an 8-h treatment with staurosporine decreased intracellular [K+] from 140 to 55 mM, while a decrease in [K+] in assay buffer from 150 to 80 mM caused a 2.4-fold increase in DNA degradation in isolated nuclei (11). These results provide compelling evidence that a high concentration of cytosolic [K+] is required to suppress the activation of apoptosis processes under normal conditions.

Intracellular [K+] is mainly controlled by the activity of Na+-K+-ATPase and K+ channels in the plasma membrane. Efflux of K+ through K+ channels, therefore, plays an important role in initiating the apoptotic volume decrease and apoptosis. Blockade of K+ channels in the plasma membrane significantly attenuated the staurosporine-induced apoptotic volume decrease and apoptosis (19, 30-32). It has been demonstrated in neurons and lymphocytes that activation of K+ channels precedes cleavage of caspases in H2O2-induced apoptosis (20) and that staurosporine-induced apoptosis is associated with an increase in IK(V) (6, 15, 21, 31, 32). Our results provide further evidence that cyt-c, either translocated from the mitochondrial intermembrane space to the cytosol or intracellularly applied through an electrode, may have a direct effect on the pore-forming alpha -subunits and/or the regulatory beta -subunits of Kv channels, which subsequently participate in initiating the apoptotic volume decrease.

The cyt-c is a hemoprotein and an efficient biological electron-transporter that plays a vital role in cellular oxidation because of its ready fluctuation between the ferrous (Fe2+) and ferric (Fe3+) states. Oxidation of the inactivation gate in Kv channels formed by the NH2-terminus of the alpha - or beta -subunits has been demonstrated to increase IK(V) by inhibiting the occlusion of the inactivation gate in the channel pore (24, 34). These direct us to speculate that cyt-c may increase IK(V) by directly interacting with or indirectly oxidizing the cytosolic inactivation gate. PASMC from animals and humans express multiple alpha - and beta -subunits of Kv channels (2). Characterization of cloned Kv channels indicates that the native Kv channels are most likely heteromeric tetramers formed by both the pore-forming alpha -subunits and the regulatory beta -subunits in a 1:1 molar ratio. In other words, each alpha -subunit associates with a beta -subunit to form alpha 4beta 4-heteromultimers in native Kv channels (2). Among the cloned K+ channels invoked to serve cell volume regulation so far are Kv1.3 in T lymphocytes (4), Kv1.5 in fibroblasts (7), and minK channels in epithelial cells (18). Which types of the Kv channels are responsible for volume regulation and which one(s) is responsible for the cyt-c induced increase in IK(V) in PASMC remain to be investigated.

Whether and how the volume decrease or cell shrinkage triggers the apoptosis processes remain unclear. Autocatalysis and chemical amplification are important properties of living cells, including vascular smooth muscle cells. Under normal conditions, the enzymes that have different functions tend to be compartmentarized in the cytoplasm (and nucleus) so that reagents or activators of effector caspases (e.g., cyt-c, Apaf-1, apoptosis-inducing factor, caspase-9, etc.) have no direct contact with products or effector caspases and nucleases. When cells shrink because of loss of intracellular ions and water, the agents that cleave or activate inactive procaspases get close to their targets. This would facilitate and accelerate the activation or cleavage of effector caspases and nucleases, and ultimately induce apoptosis.

In this study, we have demonstrated a novel mechanism by which cyt-c induces cell shrinkage [activation of Kv channels and an increase in IK(V)]. Cytoplasmic application of exogenous cyt-c or translocation of cyt-c from the mitochondrial intermembrane space to the cytosol (14, 16, 29) may directly or indirectly open Kv channels by oxidizing the channel protein. The increased IK(V) or K+ efflux reduces cytoplasmic [K+]. Concurrently, the membrane hyperpolarization induced by the increase in IK(V) would further Cl- efflux and reduce cytoplasmic [Cl-]. The resultant decrease in the cytosolic concentration of KCl mediates water efflux through the plasmalemmal water channels (aquaporins) and eventually leads to the apoptotic volume decrease (Fig. 7). In addition, cyt-c in the cytosol binds to the adaptor protein, Apaf-1, which in turn promotes activation of procaspase-9 to caspase-9. By cleaving procaspases-3, -5, or -7, the active caspase-9 activates the effector caspases, which induce DNA fragmentation and nuclear breakage by cleaving proteins and enzymes that are required for cell survival, and ultimately cause apoptosis (Fig. 7).


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 7.   Schematic diagram showing the proposed mechanisms involved in cyt-c-mediated apoptotic volume decrease and apoptosis in PASMC. [Cyt-c]cyt, cytoplasmic concentration of cyt-c; Em, membrane potential; [Cl-]cyt, cytosolic Cl- concentration; [K+]cyt, cytosolic K+ concentration.

Cytoplasmic K+ at a normal concentration (~140 mM) suppresses apoptosis by inhibiting caspase activation (11). The cyt-c-mediated decrease in cytoplasmic [K+] resulting from opened Kv channels, in addition to inducing the apoptotic volume decrease, may also contribute to induction of DNA fragmentation and apoptosis by reducing the inhibitory effect of cytoplasmic K+ on the cytoplasmic caspases and the internucleosomal DNA cleavage nuclease (Fig. 7 and Ref. 11). Furthermore, apoptosis inducers, such as staurosporine, may also open K+ and Cl- channels through mechanisms independent of cyt-c.

In summary, the results from this study imply that cyt-c-mediated activation of K+ channels, which might be independent of caspase-9, may serve as a critical effector mechanism in the apoptotic volume decrease when cells undergo apoptosis.


    ACKNOWLEDGEMENTS

We thank B. R. Lapp, I. Barash, and Y. Zhao for technical assistance.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-54043, HL-64945, HL-69758, and HL-66012. J. X.-J. Yuan is an Established Investigator of the American Heart Association.

Address for reprint requests and other correspondence: J. X.-J. Yuan, UCSD Medical Center, 200 W. Arbor Dr., San Diego, CA 92103-8382 (E-mail: xiyuan{at}ucsd.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpcell.00592.2001

Received 14 December 2001; accepted in final form 11 June 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

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

2.   Coppock, EA, Martens JR, and Tamkun MM. Molecular basis of hypoxia-induced pulmonary vasoconstriction: role of voltage-gated K+ channels. Am J Physiol Lung Cell Mol Physiol 281: L1-L12, 2001[Abstract/Free Full Text].

3.   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].

4.   Deutsch, D, and Chen LQ. Heterologous expression of specific K+ channels in T lymphocytes: functional consequences for volume regulation. Proc Natl Acad Sci USA 90: 10036-10040, 1993[Abstract].

5.   Earnshaw, WC, Martinas LM, and Kaufmann SH. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu Rev Biochem 68: 383-424, 1999[ISI][Medline].

6.   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].

7.   Felipe, A, Snyders DJ, Deal KK, and Tamkun MM. Influence of cloned voltage-gated K+ channel expression on alanine transport, Rb+ uptake, and cell volume. Am J Physiol Cell Physiol 265: C1230-C1238, 1993[Abstract/Free Full Text].

8.   Gomez-Angelats, M, Bortner CD, and Cidlowski JD. Protein kinase C (PKC) inhibits Fas receptor-induced apoptosis through modulation of the loss of K+ and cell shrinkage: A role for PKC upstream of caspases. J Biol Chem 275: 19609-19619, 2000[Abstract/Free Full Text].

9.   Haunstetter, A, and Izumo S. Apoptosis: basic mechanisms and implications for cardiovascular disease. Circ Res 82: 1111-1129, 1998[Free Full Text].

10.   Hengartner, MO. The biochemistry of apoptosis. Nature 407: 770-776, 2000[ISI][Medline].

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

12.   Isner, JM, Kearney M, Bortman S, and Passeri J. Apoptosis in human atherosclerosis and restenosis. Circulation 91: 2703-2711, 1995[Abstract/Free Full Text].

13.   Jones, AW. Content and fluxes of electrolytes. In: Handbook of Physiology. The Cardiovascular System. Vascular Smooth Muscle. Bethesda, MD: American Physiol Soc, 1980, sect. 2, vol. II, chapt. 11, p. 253-299.

14.   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].

15.   Krick, S, Platoshyn O, McDaniel SS, Rubin LJ, and Yuan JXJ 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].

16.   Kroemer, G, and Reed JC. Mitochondrial control of cell death. Nat Med 6: 513-519, 2000[ISI][Medline].

17.   Lang, F, Busch GL, Ritter M, Volkl H, Waldegger S, Gulbins E, and Haussinger D. Functional significance of cell volume regulatory mechanisms. Physiol Rev 78: 247-306, 1998[Abstract/Free Full Text].

18.   Lock, H, and Valverde MA. Contribution of the IsK (MinK) potassium channel subunit to regulatory volume decrease in murine tracheal epithelial cells. J Biol Chem 275: 34849-34852, 2000[Abstract/Free Full Text].

19.   Maeno, E, Ishizaki Y, Kanaseki T, Hazama A, and Okada Y. Normotonic cell shrinkage because of disordered volume regulation is an early prerequisite to apoptosis. Proc Natl Acad Sci USA 97: 9487-9492, 2000[Abstract/Free Full Text].

20.   McLaughlin, B, Pal S, Tran MP, Parsons AA, Barone FC, Erhardt JA, and Aizenman AE. p38 activation is required upstream of potassium current enhancement and caspase cleavage in thiol oxidant-induced neuronal apoptosis. J Neurosci 21: 3303-3311, 2000[Abstract/Free Full Text].

21.   Niestsch, HH, Roe MW, Fiekers JF, Moore AL, and Lidofsky SD. Activation of potassium and choloride channels by tumor necrosis factor alpha : role in liver cell death. J Biol Chem 275: 20556-20561, 2000[Abstract/Free Full Text].

22.   Okada, Y, Maeno E, Shimizu T, Dezaki K, Wang J, and Morishima S. Receptor-mediated control of regulatory volume decrease (RVD) and apoptotic volume decrease (AVD). J Physiol (Lond) 532: 3-16, 2001[Abstract/Free Full Text].

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

24.   Rettig, J, Heinemann SH, Wunder F, Lorra C, Parcej DN, Dolly JO, and Pongs O. Inactivation properties of voltage-gated K+ channels altered by presence of beta -subunit. Nature 369: 289-294, 1994[ISI][Medline].

25.   Thompson, CB. Apoptosis in the pathogenesis and treatment of disease. Science 267: 1456-1462, 1995[ISI][Medline].

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

27.   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].

28.   Wyllie, AH, Kerr JR, and Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol 68: 251-306, 1980[Medline].

29.   Yang, J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J, Peng TI, 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].

30.   Yu, SP, and Choi DW. Ions, cell volume, and apoptosis. Proc Natl Acad Sci USA 97: 9360-9362, 2000[Free Full Text].

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

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

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

34.   Zhou, M, Morais-Cabral JH, Mann S, and MacKinnon R. Potassium channel receptor site for the inactivation gate and quaternary amine inhibitors. Nature 411: 657-661, 2001[ISI][Medline].


Am J Physiol Cell Physiol 283(4):C1298-C1305
0363-6143/02 $5.00 Copyright © 2002 the American Physiological Society