From the Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709
Received for publication, June 14, 2000, and in revised form, October 23, 2000
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ABSTRACT |
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The movement of intracellular monovalent cations
has previously been shown to play a critical role in events leading to
the characteristics associated with apoptosis. A loss of
intracellular potassium and sodium occurs during apoptotic cell
shrinkage establishing an intracellular environment favorable for
nuclease activity and caspase activation. We have now investigated the
potential movement of monovalent ions in Jurkat cells that occur prior
to cell shrinkage following the induction of apoptosis. A rapid
increase in intracellular sodium occurs early after apoptotic stimuli
suggesting that the normal negative plasma membrane potential may
change during cell death. We report here that diverse apoptotic stimuli
caused a rapid cellular depolarization of Jurkat T-cells that occurs
prior to and after cell shrinkage. In addition to the early increase in
intracellular Na+, 86Rb+
studies reveal a rapid inhibition of K+ uptake in response
to anti-Fas. These effects on Na+ and K+ ions
were accounted for by the inactivation of the
Na+/K+-ATPase protein and its activity.
Furthermore, ouabain, a cardiac glycoside inhibitor of the
Na+/K+-ATPase, potentiated anti-Fas-induced
apoptosis. Finally, activation of an anti-apoptotic signal,
i.e. protein kinase C, prevented both cellular
depolarization in response to anti-Fas and all downstream characteristics associated with apoptosis. Thus cellular depolarization is an important early event in anti-Fas-induced apoptosis, and the
inability of cells to repolarize via inhibition of the
Na+/K+-ATPase is a likely regulatory component
of the death process.
Apoptosis is a fundamental physiological process where activation
of specific biochemical and morphological events results in cellular
suicide. Although programmed cell death is a normal physiologic process
observed during development and cellular homeostasis, insufficient or
excessive apoptosis can lead to various pathological conditions, such
as Alzheimer's and Parkinson's disease, cancer, and AIDS. The loss of
cell volume, chromatin condensation, and internucleosomal DNA
fragmentation are all defining characteristics of this mode of cell
death. Recently, a loss of intracellular monovalent ions has been shown
to play a pivotal role in apoptosis (1-11). A major loss of both
intracellular potassium and sodium occurs when apoptotic cells shrink
and prior to the loss of membrane integrity (6, 7). Maintenance of the
normal physiologic intracellular concentration of these monovalent ions
was also shown to inhibit the activation of effector caspases
(caspase-3-like enzymes) and the apoptotic nuclease activity during
cell death, suggesting that the role ions play during apoptosis is more
extensive than simply facilitating the loss of cell volume (7).
In most excitable cells, cellular depolarization occurs as a result of
a movement of sodium ions, which can occur through a variety of
mechanisms including opening of voltage-gated sodium channels (12),
suppression of the Na+/K+-ATPase activity (13,
14), and activation of Na+-dependent amino acid
co-transport systems, which can act like sodium ionophores (15, 16). In
contrast to excitable cells, lymphocytes have a relatively stable
sodium concentration, and very little detail is known about the
movement of sodium ions in these cells, although several studies have
suggested that changes in sodium levels in lymphocytes may occur by
similar mechanisms as in other cell types (17, 18). Nonetheless, the
movement of ions, especially sodium, in lymphocytes would be likely to be reflected in a change in plasma membrane potential
(PMP).1
The loss of the mitochondrial membrane potential has been shown to
occur in a variety of apoptotic model systems (19-21). Mitochondrial depolarization is proposed to occur through the opening of permeability transition pores, located on the inner mitochondrial membrane, thus
disrupting the member potential by permitting the redistribution of
ions across the membrane (22-26). Recent evidence has shown that
changes in the mitochondrial membrane potential, along with several
other characteristics of apoptosis, appear to be restricted to the
shrunken population of cells (27). These observations led us to
investigate whether an early transit of monovalent ions, prior to the
loss of cell volume, might promote the activation of apoptosis and lead
to the downstream movement of ions and apoptotic events associated with
cell death.
Flow cytometry allows multiple cell death characteristics to be
analyzed at the single cell level and thus has been an invaluable tool
in the study of apoptosis. We have used this technology to ascertain if
an early movement of monovalent ions occurs during apoptosis. By using
a fluorescent dye that measures changes in intracellular sodium, we
show that an early increase in intracellular sodium occurs prior to the
loss of cell volume. We hypothesized that this increase in
intracellular sodium would be reflected in changes in the plasma
membrane potential. By using flow cytometry and a dye that responds to
acute changes in the plasma membrane potential (PMP), we show that
cells depolarize very early during apoptosis, prior to a loss in cell
volume, and in response to various apoptotic stimuli. We also observed
that changes in PMP correlated with a population of cells with
increased intracellular sodium. K+ uptake studies using
86Rb+ suggested the rapid inactivation of the
Na+/K+-ATPase, which was confirmed by using a
functional enzyme activity assay and Western blot analysis. We also
show that inhibition of the Na+/K+-ATPase by
using ouabain enhances cellular depolarization and apoptosis, whereas
treatment with an anti-apoptotic PKC activator prevents
anti-Fas-induced cellular depolarization and cell death. These studies
indicate that an early movement of monovalent ions, particularly
sodium, results in plasma membrane depolarization that may orchestrate
subsequent movement of ions during apoptosis.
Cell Culture and Reagents--
Jurkat cells, E6.1 (human
lymphoma), were cultured in RPMI 1640 medium containing 10%
heat-inactivated fetal calf serum, 4 mM glutamine, 31 mg/liter penicillin, and 50 mg/liter streptomycin at 37 °C, 7%
CO2 atmosphere. Induction of apoptosis in Jurkat cells
(5 × 105 cells per ml) was accomplished using either
10 or 50 ng/ml anti-human Fas IgM (Kamiya Biomedical), 2 µM A23187 (Calbiochem), or 10 µM
thapsigargin (Sigma). The cells were incubated at 37 °C, 7% CO2 atmosphere for the specified periods. The caspase-8
inhibitor benzyloxycarbonyl-IETD-fluormethyl ketone was
purchased from Kamiya Biomedical. Ouabain and the protonophore carbonyl
cyanide m-chlorophenylhydrazone (CCCP) were purchased from
Sigma. Phorbol 12-myristate 13-acetate (PMA; synthetic analog of
diacylglycerol) was purchased from Calbiochem.
Determination of Cell Size by Flow Cytometry--
Cell size and
changes in the light scattering properties of the cell were determined
by flow cytometry as described previously using a Becton Dickinson
FACSort (28). Briefly, 7,500 cells were examined by exciting the cells
with a 488 nm argon laser and determining their position on a
forward-scatter versus side-scatter dot plot. Light
scattered in the forward direction is roughly proportional to cell
size, whereas light scattered at a 90° angle (side scatter) is
proportional to cell density or granularity (29). Therefore, as a cell
shrinks or loses cell volume, a decrease in the amount of
forward-scattered light is observed, along with a slight change in
side-scattered light. A gate based on the properties of the control
cells was set on each forward-scatter versus side-scatter dot plot to separate the normal and apoptotic populations of cells and
remained constant throughout the analysis.
Measurement of Acute Changes in Plasma Membrane
Potential--
Acute changes in the plasma membrane potential were
measured by flow cytometry using DiBAC4(3) (Molecular
Probes). DiBAC4(3) was prepared in Me2SO
according to the manufacturer's instructions. Graded potassium media
were made by altering the KCl and NaCl concentrations in RPMI 1640 media containing glutamine and antibiotics. The normal KCl and NaCl
concentrations in RPMI 1640 are 5.4 and 102.7 mM,
respectively, totaling 108.1 mM for these salts. For graded
potassium media, the KCl concentration was set at 5.4 (normal), 25, 50, 75, or 102.7 mM, whereas the NaCl concentration was
adjusted such that the combined monovalent salt concentration equaled
108.1 mM. Heat-inactivated fetal calf serum, dialyzed
against several changes of the KCl/NaCl-free RPMI 1640, was added to a
final concentration of 10%. Jurkat cells were resuspended in 1 ml of
the various graded potassium media or in normal RPMI 1640 containing
150 nM DiBAC4(3) at a density of 5 × 105 cells per ml. All samples were incubated for 10 min at
37 °C, 7% CO2 atmosphere and were immediately examined
by flow cytometry using a Becton Dickinson FACSort, with excitation
performed using a 488 nm argon laser, and fluorescent emission was
detected at 530 nm (FL-1). Ten thousand cells were examined under each
condition, and all flow cytometric analyses were accomplished using
CellQuest software.
Determination of Changes in Plasma Membrane Potential during
Apoptosis--
Jurkat cells treated with either 10 ng/ml of an
anti-Fas antibody, 2 µM A23187, or 10 mM
thapsigargin were incubated at 37 °C, 7% CO2
atmosphere. Stock solutions of 20 µM
DiBAC4(3) and DiOC6(3) (Molecular Probes) was
prepared in Me2SO. Thirty minutes prior to each time of
examination, either DiBAC4(3) or DiOC6(3) was
added to 1 ml of cells at a final concentration of 150 ng/ml, and
incubation was continued at 37 °C, 7% CO2 atmosphere.
Cells were examined as changes in their plasma membrane potential by flow cytometry using either a Becton Dickinson FACSort or FACSVantage SE as described above. Ten thousand cells were examined under each
condition, and all flow cytometric analyses were accomplished using
CellQuest software.
Determination of Intracellular Sodium--
Jurkat cells treated
with either 10 ng/ml of an anti-Fas antibody, 2 µM
A23187, or 10 mM thapsigargin were incubated at 37 °C,
7% CO2 atmosphere. One hour prior to each time of
examination, SBFI-AM (Na+) was added to 1 ml of cells at a
final concentration of 5 µM, and incubation was continued
at 37 °C, 7% CO2 atmosphere. Immediately prior to flow
cytometric examination, propidium iodide (PI, Sigma) was added to a
final concentration of 10 µg/ml. Ten thousand cells were analyzed by
sequential excitation of the cells containing SBFI-AM and PI at
340-350 and 488 nm, respectively, using a FACSVantage SE flow
cytometer (Becton Dickinson) and CellQuest software.
Measurement of K+ Efflux and Uptake Using
86Rb+--
For the
86Rb+ efflux experiments, Jurkat cells (5 × 105 cells per ml) loaded overnight with 12.5 µCi of
86Rb+ were washed twice in normal RPMI 1640 and
then split into 2 samples at a final cell density of 1 × 106 cells per ml. Anti-Fas antibody was added to one sample
at a final concentration of 100 ng/ml, and all samples were incubated at 37 °C, 7% CO2 atmosphere. At 1-h intervals, 3 separate 1-ml aliquots of cells were harvested for each sample, and 800 µl of the supernatant was removed to be counted. The pellet was
washed in RPMI 1640 and finally resuspended in RPMI 1640 containing
0.5% Triton X-100. Both the pellet and supernatant were counted in triplicate, and the average 86Rb+ in the pellet
fraction from two independent experiments is shown ± S.E. For the
86Rb+ uptake experiments, 5 µCi of
86Rb+ was added to Jurkat cells (5 × 105 cells per ml) in the presence or absence of 100 ng/ml
of an anti-Fas antibody. All samples were incubated at 37 °C, 7%
CO2 atmosphere. At 1-h intervals, 3 separate 1-ml aliquots
of cells were harvested for each sample. The pellets were washed twice
in RPMI 1640 and then resuspended in RPMI 1640 containing 0.5% Triton
X-100 and counted in triplicate, and the average
86Rb+ in the pellet fraction from two
independent experiments is shown ± S.E.
DNA Analysis--
The DNA content for each sample was determined
as described previously by flow cytometry (28). Briefly, 5 ml of cells
were pelleted from the culture medium and fixed by the slow addition of
cold 70% ethanol to a volume of ~1.5 ml. The volume of each sample
was adjusted to 5 ml with cold 70% ethanol, and the cells were stored
at 4 °C overnight. For flow analysis, the fixed cells were pelleted,
washed once in 1× phosphate-buffered saline (PBS), and stained in 1 ml
of 20 µg/ml PI, 1 mg/ml RNase in 1× PBS for 20 min. Seven thousand
five hundred cells were examined by flow cytometry using a Becton
Dickinson FACSort by gating on an area versus width dot plot
to exclude cell debris and cell aggregates. The percentage of degraded
DNA was determined by the number of cells with subdiploid DNA divided
by the total number of cells examined under each experimental condition.
Functional Expression Assay for
Na+/K+-ATPase--
The functional expression
of the Na+/K+-ATPase was assessed by the
ouabain-sensitive uptake of Rb+. Cells cultured in RPMI
1640 were treated in the presence or absence of 100 ng/ml of an
anti-Fas antibody for 3 h. Thirty minutes prior to the assay, 100 µM ouabain was added to the medium in the fraction of
cells used for ouabain-insensitive transport. Cells were pelleted,
washed with 1× PBS, and then resuspended in RPMI 1640 without
K+ but containing 2.5 mM RbCl. After 10 min at
37 °C (time of transport), triplicates of 250-µl cell aliquots
were immediately transferred to Eppendorf tubes on ice, centrifuged at
1000 × g for 2 min, and washed 3 times with cold 0.1 M MgCl2. The pellet was resuspended in 250 µl
of 0.1 M trichloroacetic acid. Cell extracts were analyzed for Rb+ by emission flame photometry in an atomic
absorption spectrophotometer AA100 (PerkinElmer Life Sciences).
Transport activity is expressed as µmol of Rb+ uptake per
million cells in 10 min.
Western Blot Analysis--
Jurkat cells were treated in the
presence or absence of 50 ng/ml of an anti-Fas antibody, harvested at
the indicated times, and washed once in cold PBS. Protein extracts for
each sample were prepared by resuspending the cells in a chilled lysis
buffer (20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 150 mM NaCl, and 0.5% Triton X-100) containing a mixture of
protease inhibitors (1 µM pepstatin, 1 µM
leupeptin, 1 µg/ml aprotinin, 1 µM pepstatin, and 1 mM phenylmethylsulfonyl fluoride) and were homogenized with
a Dounce homogenizer. After 15 min of centrifugation at 13,000 rpm in a
microcentrifuge, the supernatant was collected and assayed for
protein concentration by the method of Bradford using the Bio-Rad
system. 20-50 µg of protein per sample equally diluted in Laemmli
loading buffer and denatured for 5 min were examined by gel
electrophoresis at 120 V for 2 h using 12% SDS-polyacrylamide gel
electrophoresis gels (NOVEX, San Diego, CA). The gels were then
electrophoretically transferred to nitrocellulose membranes (Schleicher
& Schuell) at 42 V for 1.5 h and stained with Ponceau S (Sigma) to
verify the equal amount and quality of protein between lanes prior to Western blotting. Membranes were blocked overnight at 4 °C in Tris-buffered saline (TBS) containing 0.05% Tween (Sigma) and 5%
nonfat dried milk. Monoclonal anti-Na+/K+
recognizing human An Increase in Intracellular Sodium Occurs Early during
Apoptosis--
The maintenance of a homeostatic balance of
intracellular and extracellular ions is crucial for cell survival.
Alterations in this ionic balance can signal a cell to divide,
differentiate, or even to undergo cell death. We have previously shown
that a dramatic loss of intracellular ions, particularly sodium and
potassium, is associated with the shrinkage of cells during apoptosis,
thus altering the intracellular environment and permitting nuclease activity and effector caspase activation (6, 7). In this study, we were
interested in determining if a change in monovalent cations could be
detected in response to apoptotic stimulation, prior to cell shrinkage.
Thus, we analyzed Jurkat cells treated with anti-Fas for changes in
intracellular sodium using the sodium-binding fluorescent indicator
SBFI-AM (Na+) (6). Flow cytometric analysis of Jurkat cells
treated with an anti-Fas antibody showed a time-dependent
increase in a population of cells that had an increase in intracellular
sodium, which occurred prior to the loss of membrane integrity (Fig.
1). Interestingly, we failed to detect
any change in intracellular potassium in these cells (data not shown).
Therefore, we hypothesized that this increase in intracellular sodium
may be reflected in a change in the plasma membrane potential.
Plasma Membrane Depolarization Occurs with a Variety of Apoptotic
Stimuli--
We initially used the plasma membrane-specific dye,
DiBAC4(3), to examine apoptotic cells for changes in their
PMP at the single cell level by flow cytometry. DiBAC4(3)
is an anionic oxonal dye that responds with an increase in fluorescent
intensity at 530 nm upon membrane depolarization. We determined the
utility of this membrane potential dye in our model system, Jurkat
T-cells, by analyzing cells for acute changes in their PMP. Jurkat cell were depolarized with increasing concentrations of extracellular KCl.
In the presence of DiBAC4(3), these KCl-treated Jurkat
cells responded with a stepwise increase in DiBAC4(3)
fluorescence, indicating cellular depolarization (Fig.
2). To determine the specificity of this
dye to measure changes specific to the PMP, we examined the response of
DiBAC4(3) to acute changes in the mitochondrial membrane
potential (MMP) by using various concentrations of the protonophore
CCCP to collapse the membrane potential of these organelles. We have
previously shown that the concentrations of CCCP used in this study
were effective in uncoupling the MMP when either JC-1, a mitochondrial
membrane specific dye, or DiOC6(3), a dye which responds to
both changes in the mitochondrial and plasma membrane potential, were
used to access changes in the MMP (27). In contrast to the results
shown for acute plasma membrane depolarization, DiBAC4(3)
did not respond to changes in the mitochondrial membrane potential
indicating a distinct ability of DiBAC4(3) to measure
strictly changes in the PMP (Fig. 2).
To determine whether changes in the PMP occur during apoptosis, Jurkat
cells were treated with an anti-Fas antibody, the calcium ionophore
A23187, or thapsigargin, all known apoptotic agents that
differ in their mode of cell death activation (27). Under each
apoptotic condition, a population of cells with an increase in
DiBAC4(3) fluorescence, indicating plasma membrane
depolarization, was observed in a time-dependent manner
(Fig. 3). Interestingly, the observed
cellular depolarization was not a transient event, as might occur in
electrically excitable cells, but rather was sustained, as the
population of cells with increased DiBAC4(3) fluorescence
increased over time. This sustained cellular depolarization suggests
that upon apoptotic stimulation, the ability of cells to
repolarize is lost, thus maintaining a constant state of depolarization throughout the cell death process. In addition to the
time-dependent nature of this cellular depolarization
observed during cell death, we determined that this event was also
sensitive to the concentration of apoptotic stimulus employed.
Increasing concentrations of anti-Fas antibody added to Jurkat cells
3 h prior to flow cytometric examination in the presence of
DiBAC4(3) resulted in a concentration-dependent increase in the number of cells with increased DiBAC4(3)
fluorescence (Fig. 4A), thus
indicating that cellular depolarization is intrinsically linked to the
degree of apoptotic stimulation. The onset of plasma membrane
depolarization is rapid, occurring between 1 and 2 h after
stimulation with anti-Fas (Fig. 4B).
Plasma Membrane Depolarization Is Not Restricted to the Shrunken
Population of Apoptotic Cells--
Many characteristics of apoptosis
such as changes in the mitochondrial membrane potential, the loss of
intracellular ions, effector caspase activation, and DNA degradation
have been shown to be restricted to the shrunken population of cells
(6, 7, 27, 28). Thus, we determined if cellular depolarization was also
restricted to the shrunken apoptotic cells or if it occurred prior to
the loss of cell volume. Flow cytometry, which permits the simultaneous
examination of multiple cellular characteristics at the single cell
level, was used to determine the relationship between cellular
depolarization and cell size by examining DiBAC4(3) fluorescence and the forward light scattering property of the cell,
respectively. When control Jurkat cells were examined on a
forward-scatter versus side-scatter dot plot in the presence of DiBAC4(3), a single major population of cells was
observed (Fig. 5). Gating on this single
population of cells, we examined these cells on a forward-scatter
versus a DiBAC4(3) fluorescence contour plot.
Analysis of these control Jurkat cells showed only a single level of
DiBAC4(3) fluorescence. In contrast, gating on the minor
population of cells with a decrease in forward-scattered light in the
control sample, denoting cells with a decreased cell size, showed that
the shrunken cells had an increase in DiBAC4(3) fluorescence or a depolarized plasma membrane (Fig. 5). Treatment of
Jurkat cells with an anti-Fas antibody resulted in an increase in the
number of shrunken cells (Fig. 5). Similar to the control sample, which
contained some spontaneously dying apoptotic cells, the shrunken,
anti-Fas-induced apoptotic cells showed an increase in
DiBAC4(3) fluorescence, again suggesting that the cells
fail to repolarize during the cell death process. However, gating on the normal or nonshrunken anti-Fas-treated cells resulted in 2 distinct
similar sized cell populations on a forward-scatter versus DiBAC4(3) fluorescence contour plot, one having a control
level of DiBAC4(3) fluorescence and a second having an
increase in DiBAC4(3) fluorescence (Fig. 5). These data
suggest that cellular depolarization occurs in the normal population of
cells, prior to the loss of cell volume, and is sustained after cell
shrinkage.
Plasma Membrane Depolarization and Increased Intracellular Sodium
Occur in the Same Population of Apoptotic Cells--
In view of
cellular depolarization and increased intracellular sodium being early
occurrences during the apoptotic process, we subsequently wanted to
determine whether these two events were related. Initial experiments
using DiBAC4(3) and SBFI-AM (Na+) showed that
these two fluorescent probes are incompatible when used simultaneously
in flow cytometric experiments. Therefore, we examined the response of
a different membrane potential dye, DiOC6(3), in relation
to changes in the PMP. DiOC6(3) is a positively charged dye
that has been shown to respond to changes in both the plasma and
mitochondrial membrane potential by a loss of fluorescent emission upon
membrane depolarization (27). We have previously shown that changes in
the MMP resulting from the treatment of Jurkat cells with apoptotic
agents used in this study are completely restricted to the shrunken
population of cells (27). Therefore, by using flow cytometry to
separate the normal and shrunken populations of cells, we can use this
dye to examine only changes in the PMP by gating solely on the normal
or nonshrunken cells. Preliminary experiments using
DiOC6(3) in Jurkat cells treated with an anti-Fas antibody,
A23187, or thapsigargin showed a decrease of this positively charged
dye in a time-dependent manner, indicating plasma membrane
depolarization with this different dye (data not shown). Examination of
only the normal or nonshrunken Jurkat cells using a variety of
apoptotic stimuli resulted in an increase in SBFI-AM (Na+)
fluorescence indicating a rise in intracellular sodium prior to the
loss of cell volume (Fig. 6A).
Additionally, examination of these normal or nonshrunken apoptotically
treated cells in the presence of DiOC6(3) showed a decrease
in DiOC6(3) fluorescence suggesting cellular depolarization
prior to cell shrinkage (Fig. 6A). Simultaneous comparison
of DiOC6(3) fluorescence with SBFI-AM (Na+)
fluorescence showed that cells that were depolarized had a rise in
intracellular sodium (Fig. 6B), suggesting that apoptotic
depolarization and increased intracellular sodium are linked. It is
important to note that DiOC6(3) and SBFI-AM are excited at
2 spatially separate wavelengths (488 and 350-360 nm, respectively),
thus no spectral overlap exists when using these dyes in
combination.
Na+/K+-ATPase Inactivation Occurs during
Anti-Fas-induced Apoptosis--
Mammalian cells normally have an
extreme concentration gradient of Na+ and K+
across their plasma membrane which contributes significantly to the
distinctive cellular negative resting membrane potential. Flow
cytometric analysis of the normal or nonshrunken population of cells
treated with various apoptotic agents failed to reveal any significant
change in the concentration of intracellular potassium prior to the
loss of cell volume (data not shown); however, previously we showed
that a dramatic decrease in intracellular potassium occurs coincident
with cell shrinkage during apoptosis (6, 7). We chose to
concentrate on anti-Fas-induced apoptosis due to its importance in the
normal physiological turnover of lymphoid cells and the well defined
receptor-mediated signal transduction pathway. Therefore, to determine
whether changes in intracellular potassium occur early in the cell
death process, we used 86Rb+ as a tracer for
K+ (30) in Jurkat cells treated with an anti-Fas antibody.
In experiments examining 86Rb+ uptake, anti-Fas
treated Jurkat cells showed a striking decrease in
86Rb+ uptake, which could be detected as early
as 2 h after apoptotic stimulation (Fig.
7). This finding suggests that a major
disruption in the normal ionic balance across the plasma membrane
occurs early in the cell death process. Since maintenance of a
sodium/potassium gradient across the plasma membrane is primordial for
cellular homeostasis and is closely regulated by the ubiquitous plasma membrane Na+/K+-ATPase, we examined the role of
the Na+/K+-ATPase during anti-Fas-induced
apoptosis.
To address this issue we examined anti-Fas-treated Jurkat cells in the
presence or absence of the cardiac glycoside ouabain, a known inhibitor
of the Na+/K+-ATPase, for changes in the PMP,
cell size, and DNA content by flow cytometry. Treatment of Jurkat cells
with ouabain resulted in a dramatic loss of PMP in the entire
population of cells, suggesting that inhibition of the
Na+/K+-ATPase induces cellular depolarization
(Fig. 8). Interestingly, this complete
cellular depolarization alone increased apoptosis, although modestly.
However, the presence of ouabain in Jurkat cells treated with 10 ng/ml
anti-Fas also resulted in a similar loss in PMP in the entire
population of cells, but dramatically potentiated the extent of DNA
degradation compared with either ouabain or anti-Fas alone, which was
also reflected in the overall changes in cell size (Fig. 8). The
occurrence of complete cellular depolarization in the presence of
ouabain alone without a major enhancement of degraded DNA suggests that
increased PMP alone is not by itself a sufficient activator of
apoptosis but rather is necessary for cell death. To substantiate these
inhibitor studies, we examined the functional expression of the
Na+/K+-ATPase during apoptosis using a well
established ouabain-sensitive uptake of Rb+ assay (31-33).
Total Rb+ uptake along with the Rb+ fraction
that was ouabain-insensitive were measured in both control and
anti-Fas-treated Jurkat cells (Fig. 9).
The difference between these two fractions for each individual sample
indicates the net ouabain-sensitive
Na+/K+-ATPase activity. As shown in Fig. 9,
anti-Fas treatment of Jurkat cells resulted in an overall net decrease
in ouabain-sensitive Na+/K+-ATPase activity
compared with control cells. Western blot analysis of both the
To determine whether a change in the
Na+/K+-ATPase subunit expression was associated
with the depolarized population of cells, we simultaneously sorted
depolarized and nondepolarized anti-Fas-treated Jurkat cells by flow
cytometry. Western blot analysis of the PMA Prevents Plasma Membrane Depolarization during Anti-Fas-induced
Apoptosis--
We have recently shown that PKC stimulation is
anti-apoptotic in Jurkat cells and affords a protective effect during
anti-Fas-induced cell death at a site upstream of caspase-8 (34).
Anti-Fas-induced cell shrinkage and loss of intracellular potassium
were both blocked in the presence of the phorbol ester PMA, an
activator of PKC. Additionally, all downstream characteristics of
apoptosis were also inhibited, indicating a profound effect of PKC
stimulation on preventing cell death (34). We now examined if PKC
stimulation also prevented changes in the PMP as observed during
anti-Fas-induced apoptosis. Jurkat cells treated with 50 ng/ml anti-Fas
alone showed cellular depolarization, the loss of cell volume, and the
expected increase in degraded DNA (Fig.
11). PMA alone resulted in a slight hyperpolarization of the PMP; however, the presence of this agent had
no effect on cell size or the extent of degraded DNA (Fig. 11). When
anti-Fas-treated Jurkat cells were examined in the presence of PMA for
changes in their PMP, cell size, and DNA content, a striking inhibition
of the apoptotic process was observed (Fig. 11). This inhibition
included both early apoptotic events such as cellular depolarization,
along with late apoptotic characteristics such as DNA degradation and
cell viability (data not shown), suggesting that prevention of cellular
depolarization can control the apoptotic process and perhaps promote
tumor growth by its ability to inhibit apoptosis.
In this study, we show that an early increase in intracellular
sodium induced by a variety of apoptotic stimuli is associated with
plasma membrane depolarization and occurs prior the loss of cell
volume. Although changes in the PMP have been previously suggested
during cell death (35-37), here we report the first demonstration that
cellular depolarization directly correlates with an early increase in
intracellular sodium upon apoptotic stimulation.
Additionally, we show that cellular depolarization is not a transient
event during apoptosis but is sustained throughout the early activation phase of the cell death process, asserting that cells that become depolarized are prevented from repolarizing back to a normal PMP. We
have also observed plasma membrane depolarization and increased intracellular sodium in a variety of other lymphoid cells including primary rat thymocytes and S49 Neo cells induced to undergo apoptosis with dexamethasone, A23187, thapsigargin, or UV
irradiation.3 Additionally,
we observed plasma membrane depolarization in anti-Fas-treated HeLa
cells undergoing apoptosis. These findings suggest that
cellular depolarization is a general component of the apoptotic process in both lymphoid and nonlymphoid cells.
We also show that a primary target associated with this early change in
intracellular ions is the Na+/K+-ATPase, whose
inhibition contributes to sustained apoptotic depolarization and thus
failure of the cells to repolarize. We have made similar observations
in dexamethasone-treated primary rat
thymocytes.4 Furthermore,
activation of PKC, which has previously been shown to inhibit
anti-Fas-induced apoptosis, also prevented cellular depolarization,
indicating a critical role of the PMP in regulating cell death.
The contribution of ion movement, especially potassium, to the
mitogenic activation of lymphoid cells has been previously documented
(38, 39). Potassium channel inhibitors such as 4-aminopyridine and
tetraethylammonium at a one-to-one drug molecule to channel
stoichiometry prevent mitogenesis induced by phytohemagglutinin. In
addition, phytohemagglutinin-induced protein synthesis and interleukin-2 production are also inhibited by potassium channel blockers, emphasizing the physiological importance of ion gradients in
regulating mitogenic activation. Our current study showing an early
increase in intracellular sodium, cellular depolarization, along with
inhibition of the Na+/K+-ATPase during
apoptosis supports the notion of the importance of maintaining a
normal, homeostatic balance of ions. However, the mechanism underlying
PMA blockade of anti-Fas-mediated depolarization and cell death remains
unclear. Recent studies from our laboratory suggest that PMA may effect
both potassium efflux and uptake and that inhibition of PKC activity
enhances the loss of intracellular potassium and apoptosis (34).
Several studies suggest a potential role for an early movement of
monovalent ions in apoptosis. Several members of the anti- and
pro-apoptotic Bcl-2 protein family are capable of modulating the cell
membrane potential by hyperpolarizing the cells thus increasing their
resistance to apoptosis, and these proteins have the potential to
function as selective ion channels (40-43). Additionally, voltage-dependent n-type K+
channels, which are proposed to function to return the plasma membrane
potential to control levels after cellular depolarization, are
inhibited by ligation of the Fas receptor in Jurkat cells, ceramide (a
lipid metabolite synthesized upon Fas receptor ligation), and by the
generation of reactive oxygen species (44-47). Recently, the
Drosophila genes reaper, grim, and
hid, which induce apoptosis and contain N-terminal sequences
similar to the N-terminal inactivation domains of voltage-gated
potassium channels, are capable of fast inactivation of Shaker-type
potassium channels (48). These studies along with our present data
suggest that an early movement of monovalent ions plays a strategic
role in the activation of apoptosis.
Our current data also provides direct evidence that plasma membrane
depolarization plays a critical role during apoptosis and is mediated
by an early increase in intracellular sodium along with inactivation of
the Na+/K+-ATPase. However, the rise in
intracellular sodium and the inhibition of the
Na+/K+-ATPase may contribute to different
aspects of this early change in monovalent ions in relation to plasma
membrane depolarization. Interestingly, upon apoptotic stimulation,
cellular depolarization is not transient but is maintained through the
initial stages of apoptosis. Since the addition of ouabain, which
inhibits the Na+/K+-ATPase, does not by itself
dramatically induce cell death in Jurkat cells, the early loss of this
ion transport mechanism may play a more important role in sustaining
cellular depolarization during apoptosis. Maintaining a sustained state
of depolarization for a period after apoptotic stimulation could also
occur through the inhibition of the voltage-activated potassium
channels (46). Therefore, cellular depolarization may serve as a check
point in the activation of apoptosis. Additionally, the rise in
intracellular sodium which occurs in the presence of ouabain is not
sufficient to fully activate the apoptotic process. Thus, inhibition of
the Na+/K+-ATPase does not trigger apoptosis
but enhances the early activation of the cell death signal (Fig. 8).
This implies that the early rise in intracellular sodium must occur
through a separate or independent sodium transport mechanism, which is
intimately linked to the activation of the cell death process.
Therefore, our demonstration that the
Na+/K+-ATPase is inhibited during
anti-Fas-induced apoptosis in Jurkat cells combined with the an early
rise in intracellular sodium suggests a previously unexplored level of
control during programmed cell death.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and
isoforms (Affinity Bioreagents, Golden, CO) and monoclonal anti-caspase-8 (Calbiochem) were diluted 1:250 in
TBS, 0.05% Tween, 0.5% milk and membranes were blotted with the
correspondent antibody for 1 h at room temperature. Blots were
washed 3 times with TBS, 0.05% Tween and incubated for 1 h with
peroxidase-linked anti-mouse IgG (Amersham Pharmacia Biotech) diluted
1:5000 in TBS, 0.05% Tween, 0.5% milk. Following washes with TBS,
membranes were treated with ECL chemiluminescence detection system,
exposed to hyperfilm (ECL, PerkinElmer Life Sciences), and developed.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Analysis of intracellular sodium occurs
during anti-Fas-induced apoptosis in Jurkat cells. Jurkat cells
were incubated in the presence and absence of 10 ng/ml anti-Fas for the
times shown. For intracellular Na+ analysis, 2 µl of 2.5 mM SBFI-AM stock (5 µM final) was added to 1 ml of cells for each sample 1 h prior to the time of examination.
Incubation was continued at 37 °C, 7% CO2 atmosphere.
Immediately prior to flow cytometric examination, PI was added to a
final concentration of 10 µg/ml. Samples were analyzed on a
FACSVantage SE flow cytometer examining 10,000 cells per sample on an
SBFI-AM (Na+) versus PI fluorescence dot plot.
The data was transformed into contour plots using CellQuest software.
Flow cytometric analysis of intracellular sodium using SBFI-AM
(Na+) showed a time-dependent increase in the
number of cells that had an increase in intracellular sodium, which
occurred prior to the loss of membrane integrity. The contour plots are
representative of at least 3 independent experiments.
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Fig. 2.
The response of DiBAC4(3) to
measure acute changes in the plasma or mitochondrial membrane
potential. Graded potassium media were made by altering the KCl
and NaCl concentrations in RPMI 1640 media containing glutamine and
antibiotics. The normal KCl and NaCl concentrations in RPMI 1640 are
5.4 and 102.7 mM, respectively, totaling 108.1 mM for these salts. For graded potassium media, the KCl
concentration was set at 5.4 (normal), 25, 50, 75, or 102.7 mM, whereas the NaCl concentration was adjusted such that
the combined monovalent salt concentration equaled 108.1 mM. Heat-inactivated fetal calf serum, dialyzed against
several changes of the KCl/NaCl-free RPMI 1640, was added to a final
concentration of 10%. Upon increasing the extracellular potassium
concentration, a stepwise increase in DiBAC4(3)
fluorescence was observed. In contrast, DiBAC4(3) did not
respond to acute changes in the mitochondrial membrane potential upon
addition of the cyanide compound CCCP, under conditions that have
previously been shown to uncouple the mitochondrial membrane potential
using the mitochondrial specific dye JC-1 (27).
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Fig. 3.
Response of DiBAC4(3) to measure
changes in the plasma membrane potential during apoptosis. Jurkat
cells treated with either 10 ng/ml of an anti-Fas antibody, 2 µM A23187, or 10 mM thapsigargin were
incubated at 37 °C, 7% CO2 atmosphere. Thirty minutes
prior to each time of examination, DiBAC4(3) was added to 1 ml of cells at a final concentration of 150 ng/ml, and incubation was
continued at 37 °C, 7% CO2 atmosphere. Flow cytometric
analysis of the plasma membrane potential using DiBAC4(3)
showed a time-dependent increase in the number of cells
that had an increase in DiBAC4(3) fluorescence, indicating
plasma membrane depolarization, under each apoptotic condition. The
histograms show a representative change in DiBAC4(3)
fluorescence for each experimental condition, and the percentage of
cells with an increase in DiBAC4(3) fluorescence reflects
the average ± S.E. of 3 independent experiments.
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Fig. 4.
Response of DiBAC4(3) to measure
changes in the plasma membrane potential upon increasing concentrations
of anti-Fas antibody after 3 h. A, Jurkat cells
treated with 10, 25, 50, or 100 ng/ml anti-Fas were incubated at
37 °C, 7% CO2 atmosphere for 2.5 h. At this time,
DiBAC4(3) was added to 1 ml of cells to a final
concentration of 150 ng/ml, and incubation was continued at 37 °C,
7% CO2 atmosphere for an additional 30 min. Flow
cytometric analysis showed an increase in DiBAC4(3)
fluorescence, indicating cellular depolarization occurred in a
concentration-dependent manner. The bar graph
shows the results of 3 independent experiments ± S.E.
B, a time course of Jurkat cells treated with 50 ng/ml
anti-Fas in the presence of DiBAC4(3) showed a rapid,
time-dependent increase in the number of depolarized cells.
The graph shows the results of 3 independent
experiments ± S.E.
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Fig. 5.
Anti-Fas induces a rapid change in the plasma
membrane potential prior to cell shrinkage. Jurkat cells treated
with and without 10 ng/ml of an anti-Fas antibody for 6 h were
examined by flow cytometry on a forward-scatter versus
side-scatter dot plot. In the control sample, a gate was drawn around
the normal population of cells, which indicated a single
DiBAC4(3) fluorescence on a forward-scatter
versus DiBAC4(3) fluorescent contour plot.
Gating on the shrunken population of control cells, an increase in
DiBAC4(3) fluorescence was observed indicating plasma
membrane depolarization. When cells treated with an anti-Fas antibody
for 6 h were examined under the same flow cytometric conditions as
the control cells, an increase in DiBAC4(3) fluorescence
was again observed in the shrunken population of cells. However, in the
gate that comprised the normal population of cells, 2 distinct
DiBAC4(3) fluorescent populations were observed. One cell
population had a lower DiBAC4(3) fluorescence, similar to
the control cells; however, a second cell population was observed that
had an increase in DiBAC4(3) fluorescence, occurring prior
to the loss of cell volume. The quadrants shown on the
forward-scatter versus DiBAC4(3) fluorescence
contour plots were positioned to mark the center of the control,
nonshrunken population of cells and remained constant throughout the
analysis.
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Fig. 6.
An early rise in intracellular sodium
occurs prior to the loss of cell volume and correlates with the
depolarized population of apoptotic cells. A, the
response of SBFI-AM (Na+) to measure changes in
intracellular sodium prior to the loss of cell volume under various
apoptotic conditions. Jurkat cells were treated with various apoptotic
stimuli and examined for changes in intracellular sodium as described
in Figs. 1 and 3. Cells were initially examined on a forward-scatter
versus a side-scatter dot plot. An analysis gate was drawn
around the normal or nonshrunken control cells. Treatment of Jurkat
cells with various apoptotic agents after 6 h were then examined
in the presence of the sodium indicator dye on an SBFI-AM
(Na+) versus PI dot plot. Cells that had a
normal and an increase in intracellular sodium were independently gated
and subsequently examined on a SBFI-AM (Na+)
versus forward-scatter dot plot. Results of this analysis
showed a population of cells with increased intracellular sodium
(green) occurred prior to the loss of cell volume.
DiOC6(3) was used to examine changes in the PMP in the
normal or nonshrunken cells under various apoptotic conditions. Jurkat
cells treated with various apoptotic agents were examined by flow
cytometry for changes in their plasma membrane potential after 6 h
using the positively charged membrane potential dye,
DiOC6(3). Thirty minutes prior to flow cytometric
examination, DiOC6(3) was added to a final concentration of
150 ng/ml, and incubation was continued at 37 °C, 7%
CO2 atmosphere. Gating on only the normal or nonshrunken
cells resulted in a population of cells (green) with had a
decrease in DiOC6(3) fluorescence, indicating plasma
membrane depolarization prior to the loss of cell volume. B,
relationship between increased intracellular sodium and cellular
depolarization. Jurkat cells were treated with a variety of apoptotic
agents for 6 h. Prior to flow cytometric examination, SBFI-AM
(Na+) and DiOC6(3) were added as described
above. Analysis of the normal or nonshrunken cells on a
DiOC6(3) fluorescence versus a SBFI-AM
(Na+) fluorescence dot plot showed that only the
depolarized population of cells had an increase in intracellular sodium
(green). The dot plots are representative of at least 2 independent experiments.
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Fig. 7.
Anti-Fas stimulation regulates K+
uptake. 86Rb+ uptake experiments were
accomplished by adding 5 µCi of 86Rb+ to
Jurkat cells (5 × 105 cells per ml) in the presence
or absence of 100 ng/ml of an anti-Fas antibody. All samples were
incubated at 37 °C, 7% CO2 atmosphere. At 1-h
intervals, 3 separate 1-ml aliquots of cells were harvested for each
sample. The pellets were washed twice in RPMI 1640 and then resuspended
in RPMI 1640 containing 0.5% Triton X-100 and counted in triplicate,
and the average 86Rb+ in the pellet fraction
from two independent experiments is shown ± S.E. In the presence
of anti-Fas stimulation, a dramatic decrease in
86Rb+ uptake was observed.
and
subunits of the Na+/K+-ATPase during
anti-Fas-induced apoptosis revealed a rapid time-dependent decrease in both the
and
subunit steady state levels of the proteins (Fig. 10A).
Interestingly, we also detected the specific, apparently proteolytic,
cleavage of the
subunit (Fig. 10A). Congruent with our
results on the uptake of 86Rb+ during
anti-Fas-induced cell death (Fig. 7), the decrease in
subunit
protein and the cleavage of
subunit of the
Na+/K+-ATPase could be detected as early as
1 h after apoptotic treatment, suggesting that the
Na+/K+-ATPase is a specific target for
inactivation during anti-Fas-induced apoptosis.
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Fig. 8.
Ouabain potentiates anti-Fas-induced
apoptosis in Jurkat cells. Control and anti-Fas (10 ng/ml)-treated
Jurkat cells were examined in the presence and absence of 100 µM ouabain after 6 h. Thirty minutes prior to flow
cytometric analysis, DiBAC4(3) was added to 1 ml of each
sample, and incubation at 37 °C, 7% CO2 atmosphere was
continued. Analysis of degraded DNA was accomplished as described
previously (28). Representative histograms and three-dimensional plots
are shown under each experimental condition. The percentage of cellular
depolarization and degraded DNA are the average ± S.E. of 3 independent experiments. The presence of ouabain in the anti-Fastreated
Jurkat cells enhanced both early and late apoptotic
characteristics.
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Fig. 9.
The functional expression of the
Na+/K+-ATPase in the presence or absence of
anti-Fas antibody. The functional expression of the
Na+/K+-ATPase was assessed by the
ouabain-sensitive uptake of Rb+. Cells cultured in RPMI
1640 were treated in the presence or absence of 100 ng/ml of an
anti-Fas antibody. Thirty minutes prior to the assay, 100 µM ouabain was added in the medium in the fraction of
cells used for ouabain-insensitive transport. Cells were pelleted,
washed with 1× PBS, and then resuspended in RPMI 1640 without
K+ but containing 2.5 mM RbCl. After 10 min at
37 °C (time of transport), triplicates of 250-µl cell aliquots
were immediately transferred to Eppendorf tubes on ice, centrifuged at
1000 × g for 2 min, and washed 3 times with cold 0.1 M MgCl2. The pellet was resuspended in 250 µl
of 0.1 M trichloroacetic acid. Cell extracts were analyzed
for Rb+ by emission flame photometry in an atomic
absorption spectrophotometer AA100 (PerkinElmer Life Sciences).
Transport activity is expressed as micromoles of Rb+ uptake
per million cells in 10 min. Data are the average of 3 independent
experiments ± S.E.
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Fig. 10.
Western blot analysis shows a decrease in
the subunit and cleavage of the
subunit of the
Na+/K+-ATPase during anti-Fas-induced
apoptosis. A, Jurkat cells were treated in the presence
or absence of 50 ng/ml of an anti-Fas antibody, harvested at the
indicated times, and washed once in cold PBS. Protein extracts for each
sample were prepared and examined on a 12% SDS-polyacrylamide gel
electrophoresis gel. The gels were transferred to nitrocellulose
membranes and examined for the expression of either the
or
subunit of the Na+/K+-ATPase. Ponceau S
staining was employed to verify equal amounts and quality of protein
between lanes prior the Western blotting. An early decrease in the
subunit of the Na+/K+-ATPase was observed along
with the specific cleavage of the
subunit of the
Na+/K+-ATPase. B, examination of
individual depolarized and nondepolarized anti-Fas-treated Jurkat cells
revealed that the decrease in the
subunit of the
Na+/K+-ATPase occurred in the depolarized
population of cells. C, kinetic analysis of caspase-8
activation during anti-Fas-induced apoptosis. Jurkat cells were treated
with anti-Fas and prepared for Western blot analysis as described
above. Activation of caspase-8 occurs by cleavage of the pro-form of
enzyme (56-58 Kd) and was detected by the
appearance of the cleaved active fragments (43-41 kDa). Cleaved
caspase-8 products were detected within 2 h of anti-Fas treatment.
Each Western analysis is representative of 2 independent
experiments.
subunit of the
Na+/K+-ATPase showed a visible decrease in the
steady state level of this protein in the depolarized cells, compared
with the nondepolarized cells (Fig. 10B), suggesting that
the loss of Na+/K+-ATPase expression, and thus
activity, is related to the depolarized apoptotic cells. For comparison
we analyzed the activation of caspase-8 over the same time and under
the same conditions as shown for the expression of the
Na+/K+-ATPase. These data suggest that in
anti-Fas-treated Jurkat cells cleavage of the
subunit and decreased
levels of the
subunit of the Na+/K+-ATPase
occur prior to detectable levels of active caspase 8 (Fig. 10C). In Fas-induced cell death, both plasma membrane
depolarization and increased intracellular sodium are prevented in the
presence of either the pan-caspase inhibitor benzyloxycarbonyl-VAD or
the caspase-8 inhibitor IETD (data not shown). However, UV-induced caspase-8-deficient cells readily undergo a sustained plasma membrane depolarization, suggesting that more than one pathway likely exists to
drive the degradation of the
Na+/K+-ATPase.2
Together, these data suggest that the
Na+/K+-ATPase plays a vital role in the early
movement of monovalent ions during anti-Fas-induced apoptosis by
contributing to an early change in PMP.
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Fig. 11.
PMA prevents anti-Fas-induced plasma
membrane depolarization and apoptosis. Control and anti-Fas (50 ng/ml)-treated Jurkat cells were examined in the presence and absence
of 20 nM PMA after 3 h by flow cytometry for changes
in the their PMP, cell size, and DNA content as described in Fig. 8.
Representative histograms and three-dimensional plots are shown under
each experimental condition. The percentage of cellular depolarization
and degraded DNA are the average ± S.E. of 3 independent
experiments. The presence of PMA in the anti-Fas-treated Jurkat cells
inhibited both early and late apoptotic characteristics.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
---|
* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 919- 541-1564;
Fax: 919-541-1367.
Published, JBC Papers in Press, October 24, 2000, DOI 10.1074/jbc.M005171200
2 C. Vu, C. D. Bortner, and J. A. Cidlowski, unpublished observations.
3 C. D. Bortner, M. Gómez-Angelats, and J. A. Cidlowski, unpublished observations.
4 C. Mann and J. A. Cidlowski, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: PMP, plasma membrane potential; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; CCCP, carbonyl cyanide m-chlorophenylhydrazone; DiBAC4(3), bis-(1,3-dibutylbarbituric acid) trimethine oxonol; DiOC6, 3,3'-dihexyloxacarbocyanine iodide; SBFI-AM, sodium-binding benzofuran isophthalate acetyloxymethylester; PI, propidium iodide; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; MMP, mitochondrial membrane potential.
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REFERENCES |
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---|
1. |
Wang, L.,
Xu, D.,
and Lu, L.
(1999)
J. Biol. Chem.
274,
3678-3685 |
2. |
Yu, S. P.,
Yeh, C.-H.,
Strasser, M.,
Tian, M.,
and Choi, D. W.
(1999)
Science
284,
336-339 |
3. |
Dallaporta, B.,
Hirsch, T.,
Susin, S. A.,
Zamzami, N.,
Larochette, N.,
Brenner, C.,
Marzo, I.,
and Kroemer, G.
(1998)
J. Immunol.
160,
5605-5615 |
4. | Bilney, A. J., and Murray, A. W. (1998) FEBS Lett. 424, 221-224[CrossRef][Medline] [Order article via Infotrieve] |
5. | Yu, S. P., Farhangrazi, Z. S., Ying, H. S., Yeh, C.-H., and Choi, D. W. (1998) Neurobiol. Dis. 5, 81-88[CrossRef][Medline] [Order article via Infotrieve] |
6. |
Bortner, C. D.,
Hughes, F. M., Jr.,
and Cidlowski, J. A.
(1997)
J. Biol. Chem.
272,
32436-32442 |
7. |
Hughes, F. M., Jr.,
Bortner, C. D.,
Purdy, G. D.,
and Cidlowski, J. A.
(1997)
J. Biol. Chem.
272,
30567-30576 |
8. |
Yu, S. P.,
Yeh, C.-H.,
Sensi, S. L.,
Gwag, B. J.,
Canzoniero, L. M. T.,
Farhangrazi, Z. S.,
Ying, H. S.,
Tian, M.,
Dugan, L. L.,
and Choi, D. W.
(1997)
Science
278,
114-117 |
9. | McCarthy, J. V., and Cotter, T. G. (1997) Cell Death Differ. 4, 756-770[CrossRef] |
10. | Barbiero, G., Duranti, F., Bonelli, G., Amenta, J. S., and Baccino, F. M. (1995) Exp. Cell Res. 217, 410-418[CrossRef][Medline] [Order article via Infotrieve] |
11. | Jonas, D., Walev, I., Berger, T., Liebetrau, M., Palmer, M., and Bhakdi, S. (1994) Infect. Immun. 62, 1304-1312[Abstract] |
12. | Kallen, R. G., Cohen, S. A., and Barchi, R. L. (1993) Mol. Neurobiol. 7, 383-428[Medline] [Order article via Infotrieve] |
13. | Jones, G. S., Van Dyke, K., and Castranova, V. (1981) J. Cell. Physiol. 106, 75-83[Medline] [Order article via Infotrieve] |
14. | Geck, P., Pietrzyk, C., Burckhardt, B. C., Pfeiffer, B., and Heinz, E. (1980) Biochim. Biophys. Acta 600, 432-447[Medline] [Order article via Infotrieve] |
15. | Hacking, C., and Eddy, A. (1981) Biochem. J. 194, 415-426[Medline] [Order article via Infotrieve] |
16. | Philo, R. D., and Eddy, A. (1978) Biochem. J. 174, 801-810[Medline] [Order article via Infotrieve] |
17. |
Senn, N.,
and Garay, R. P.
(1989)
Am. J. Physiol.
257,
C12-C18 |
18. | Adebodun, F., and Post, J. F. M (1993) J. Cell. Physiol. 154, 199-206[Medline] [Order article via Infotrieve] |
19. | Inai, Y., Yabuki, M., Kanno, T., Akiyama, J., Yasuda, T., and Utsumi, K. (1997) Cell Struct. Funct. 22, 555-563[Medline] [Order article via Infotrieve] |
20. | Zamzami, N., Marchette, P., Castedo, M., Hirsch, T., Susin, S. A., Masse, B., and Kroemer, G. (1996) FEBS Lett. 384, 53-57[CrossRef][Medline] [Order article via Infotrieve] |
21. | Cossarizza, A., Kalashnikova, G., Grassilli, E., Chiappelli, F., Salviolo, S., Capri, M., Barbieri, D., Troiano, L., Monti, D., and Franceschi, C. (1994) Exp. Cell Res. 214, 323-330[CrossRef][Medline] [Order article via Infotrieve] |
22. | Lemasters, J. J., Nieminen, A.-L., Qian, T., Trost, L. C., Elmore, S. P., Nishimura, Y., Crowe, R. A., Cascio, W. E., Bradham, C. A., Brenner, D. A., and Herman, B. (1998) Biochim. Biophys. Acta 1366, 177-196[Medline] [Order article via Infotrieve] |
23. | Bernardi, P. (1996) Biochim. Biophys. Acta 1275, 5-9[Medline] [Order article via Infotrieve] |
24. | Zamzami, N., Marchetti, P., Castedo, M., Zanin, C., Vayssière, J. L., Petit, P. X., and Kroemer, G. (1995) J. Exp. Med. 181, 1661-1672[Abstract] |
25. |
Kroemer, G.,
Petit, P. X.,
Zamzami, N.,
Vayssière, J.-L.,
and Mignotte, B.
(1995)
FASEB J.
9,
1277-1287 |
26. | Zoratti, M., and Szabo, I. (1995) Biochim. Biophys. Acta 1241, 139-176[Medline] [Order article via Infotrieve] |
27. |
Bortner, C. D.,
and Cidlowski, J. A.
(1999)
J. Biol. Chem.
274,
21953-21952 |
28. |
Bortner, C. D.,
and Cidlowski, J. A.
(1996)
Am. J. Physiol.
271,
C950-C961 |
29. | Willman, C. L., and Stewart, C. C. (1989) Semin. Diagnostic Pathol. 6, 3-12 |
30. |
Jaunin, P.,
Horisberger, J.-D.,
Richter, K.,
Good, P. J.,
Rossier, B. C.,
and Geering, K.
(1992)
J. Biol. Chem.
267,
577-585 |
31. | Marakhova, I. I., Vereninov, A. A., Toropova, F. V., and Vinogradova, T. A. (1998) Biochim. Biophys. Acta 1368, 61-72[Medline] [Order article via Infotrieve] |
32. |
Longo, N.,
Griffin, L. D.,
and Elsas, L. J.
(1991)
Am. J. Physiol.
260,
C1341-C1346 |
33. |
Takeyasu, K.,
Tamkun, M. M.,
Renaud, K. J.,
and Fambrough, D. M.
(1988)
J. Biol. Chem.
263,
4347-4354 |
34. |
Gómez-Angelats, M.,
Bortner, C. D.,
and Cidlowski, J. A.
(2000)
J. Biol. Chem.
275,
19609-19619 |
35. | Ferlini, C., De-Angelis, C., Biselli, R., Distefano, M., Scambia, G., and Fattorossi, A. (1999) Exp. Cell Res. 247, 160-167[CrossRef][Medline] [Order article via Infotrieve] |
36. |
Ferrari, D.,
Wesselborg, S.,
Bauer, M. K. A.,
and Schulze-Ossthoff, K.
(1997)
J. Cell Biol.
139,
1635-1643 |
37. | Deckers, C. L., Lyons, A. B., Samuel, K., Sanderson, A., and Maddy, A. H. (1993) Exp. Cell Res. 208, 362-370[CrossRef][Medline] [Order article via Infotrieve] |
38. | Chandy, K. D., DeCoursey, T. E., Cahalan, M. D., McLaughlin, C., and Gupta, S. (1984) J. Exp. Med. 160, 369-385[Abstract] |
39. | DeCoursey, T. E., Chandy, K. D., Gupta, S., and Cahalan, M. D. (1984) Nature 307, 465-468[Medline] [Order article via Infotrieve] |
40. |
Schlesinger, P. H.,
Gross, A.,
Yin, X.-M.,
Yamamoto, K.,
Saito, M.,
Waksman, G.,
and Korsmeyer, S. J.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
11357-11362 |
41. | Minn, A. J., Velez, P., Schendel, S. L., Liang, H., Muchmore, S. W., Fesik, S. W., Fill, M., and Thompson, C. B. (1997) Nature 385, 353-357[CrossRef][Medline] [Order article via Infotrieve] |
42. | Muchmore, S. W., Sattler, H. L., Meadows, R. P., Harlan, J. E., Yoon, H. S., Nettesheim, D., Chang, B. S., Thompson, C. B., Wong, S.-L., Ng, S.-C., and Fesik, S. W. (1996) Nature 381, 335-341[CrossRef][Medline] [Order article via Infotrieve] |
43. | Gilbert, M. S., Saad, A. H., Rupnow, B. A., and Knox, S. J. (1996) J. Cell. Physiol. 168, 114-122[CrossRef][Medline] [Order article via Infotrieve] |
44. | Szabo, I., Nilius, B., Zhang, X., Busch, A. E., Gulbins, E., Suessbrich, H., and Lang, F. (1997) Pfluegers Arch. Eur. J. Physiol. 433, 626-632[CrossRef][Medline] [Order article via Infotrieve] |
45. |
Gulbins, E.,
Szabo, I.,
Baltzer, K.,
and Lang, F.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
7661-7666 |
46. |
Szabo, I.,
Gulbins, E.,
Apfel, H.,
Zhang, X.,
Barth, P.,
Busch, A. E.,
Schlottmann, K.,
Pongs, O.,
and Lang, F.
(1996)
J. Biol. Chem.
271,
20465-20469 |
47. | Duprat, F., Guillemare, E., Romey, G., Fink, M., Lesage, F., Lazdunski, M., and Honore, E. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11796-11800[Abstract] |
48. |
Avdonin, V.,
Kasuya, J.,
Ciorba, M. A.,
Kaplan, B.,
Hoshi, T.,
and Iverson, L.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
11703-11708 |