Department of Medicine, University of California, San Diego, California 92103-8382
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ABSTRACT |
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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
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INTRODUCTION |
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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-, 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.
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METHODS AND MATERIALS |
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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 -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
-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
M) 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).
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.
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RESULTS |
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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).
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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
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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).
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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).
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DISCUSSION |
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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 -subunits
and/or the regulatory
-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 -
or
-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
- and
-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
-subunits and the regulatory
-subunits in a
1:1 molar ratio. In other words, each
-subunit associates with a
-subunit to form
4
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).
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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.
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ACKNOWLEDGEMENTS |
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We thank B. R. Lapp, I. Barash, and Y. Zhao for technical assistance.
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FOOTNOTES |
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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.
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