The Journal of Immunology, 1999, 162: 6534-6542.
Copyright © 1999 by The American Association of Immunologists
Plasma Membrane Potential in Thymocyte Apoptosis1
Bruno Dallaporta*,
Philippe Marchetti*,
,
Manuel A. de Pablo*,
Carine Maisse*,
Huynh-Thien Duc
,
Didier Métivier*,
Naoufal Zamzami*,
Maurice Geuskens§ and
Guido Kroemer2,*
*
Centre National de Recherche Scientifique, Unité Propre de Recherche 420, Villejuif, France;
Institut National de la Santé et de la Recherche Médicale, Unit 459, Lille, France;
Centre Hépatobiliaire de lHôpital Paul Brousse, Villejuif, France; and
§
Department of Molecular Biology, Université Libre de Bruxelles, Rhode-Saint-Genèse, Belgium
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Abstract
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Apoptosis is accompanied by major changes in ion
compartmentalization and transmembrane potentials. Thymocyte apoptosis
is characterized by an early dissipation of the mitochondrial
transmembrane potential, with transient mitochondrial swelling and a
subsequent loss of plasma membrane potential (
p)
related to the loss of cytosolic K+, cellular shrinkage,
and DNA fragmentation. Thus, a gross perturbation of

p occurs at the postmitochondrial stage of apoptosis.
Unexpectedly, we found that blockade of plasma membrane K+
channels by tetrapentylammonium (TPA), which leads to a

p collapse, can prevent the thymocyte apoptosis
induced by exposure to the glucocorticoid receptor agonist
dexamethasone, the topoisomerase inhibitor etoposide,
-irradiation,
or ceramide. The TPA-mediated protective effect extends to all features
of apoptosis, including dissipation of the mitochondrial transmembrane
potential, loss of cytosolic K+, phosphatidylserine
exposure on the cell surface, chromatin condensation, as well as
caspase and endonuclease activation. In strict contrast, TPA is an
ineffective inhibitor when cell death is induced by the potassium
ionophore valinomycin, the specific mitochondrial benzodiazepine ligand
PK11195, or by primary caspase activation by Fas/CD95 cross-linking.
These results underline the importance of K+ channels for
the regulation of some but not all pathways leading to thymocyte
apoptosis.
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Introduction
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Apoptosis
may be defined as a cell death process in which the activation of
catabolic processes and enzymes occurs before cytolysis, thereby
facilitating the recognition, uptake, and digestion of the apoptotic
cell by neighboring cells. For a long time, it has been assumed that
the activation of endonucleases and specific proteases (caspases) would
not only constitute a distinctive feature of apoptosis but also
determine the decisive step (the "decision to die" or "commitment
point") that distinguishes living from dying cells. It is now clear
that, in most models of apoptosis, inhibition of nucleases and caspases
does not prevent cytolysis (1, 2, 3, 4, 5), indicating that the decision to die
is made before catabolic enzymes are activated and that the activation
of such enzymes can be a byproduct of the cell death process rather
than a regulatory event (reviewed in Refs. 6 and 7). Accordingly,
current attempts to understand the decisive step of the apoptotic
process are focusing on the mechanisms leading to caspase and
endonuclease activation rather than on the action of these hydrolases.
We and others have shown that defective ion compartmentalization and
membrane potentials may have a major impact on apoptotic regulation
(7, 8, 9, 10). Lipophilic cations accumulate in the mitochondria matrix,
driven by the electrochemical gradient following the Nernst equation,
according to which every 61.5-mV increase in membrane potential
(usually 120170 mV) corresponds to a 10-fold increase in cation
concentration in mitochondria. Therefore, the concentration of such
cations is 23 logs higher in the mitochondrial matrix than in the
cytosol (CS) (11). During the process of apoptosis, cells reduce the
mitochondrial retention of such lipophilic cations, including the
fluorescent dye 3,3'-dihexyloxacarbocyanine iodide
(DiOC6(3))3 (7, 8).
Thus, the inner mitochondrial transmembrane potential
(
m), which is mainly a proton gradient, dissipates
relatively early during apoptosis (12, 13) via a process that involves
the Bcl-2-inhibited and Bax-facilitated opening of the permeability
transition (PT) pore (14, 15). Opening of the PT pore (which allows for
the free diffusion of solutes of
1500 Da on the inner mitochondrial
membrane) causes an increase in mitochondrial matrix volume (16).
Because the surface of the outer mitochondrial membrane is smaller than
that of the inner membrane, the PT pore opening may cause local
mechanic disruption of the outer mitochondrial membrane (17, 18), with
consequent release of soluble intermembrane proteins (19, 20). Several
of the mitochondrial intermembrane proteins released upon PT pore
opening (e.g., cytochrome c, apoptosis-inducing factor, and
procaspases-2, -3, and -9) are endowed with the capacity to activate
caspases or nucleases (21, 22, 23, 24, 25). As a result, mitochondrial membrane
permeabilization has been viewed as a phenomenon that marks the
"point-of-no-return" of the apoptotic process (6, 7, 12, 26, 27).
A further apoptosis-relevant alteration in ion compartmentalization
affects plasma membrane K+ homeostasis. Under normal
circumstances, the K+ concentration is much higher in the
CS (100140 mM) than in the extracellular (EC) fluid (
5 mM).
A continuous, low K+ efflux, via so-called K+
leakage channels, accounts for the maintenance of the charge
differences on the plasma membrane, the plasma membrane potential
(
p), and thus is vital for ion and volume
homeostasis. A major decrease of the intracellular K+
levels has been observed in several cell types (S49, Jurkat, HL60, and
thymocytes) undergoing apoptosis in response to a variety of different
inducers (glucocorticoids, A23187, anisomycin, thapsigargin,
staurosporine, anti-Fas/CD95, UV irradiation, and etoposide) (10, 28, 29). Complete loss of the K+ gradient may be expected
to have severe metabolic consequences and to be accompanied by a loss
of 
p, because K+ efflux (which normally
follows the concentration gradient) would be interrupted. Moreover, a
loss of intracellular K+ may be linked to cellular
shrinkage (10, 28), one of the hallmarks of apoptosis. Importantly
enough, K+ efflux is mandatory for endonuclease activation,
given that physiological intracellular K+ concentrations
inhibit nuclear endonucleases in both cells and cell-free systems of
apoptosis (10, 29, 30). This has lead to the proposal that the
derepression of endonucleases due to low intracellular K+
concentrations may be a decisive prerequisite for end-stage DNA
fragmentation (29).
The mechanisms accounting for K+ efflux during the
apoptotic process have been elusive thus far. On theoretical grounds,
this K+ efflux could be due to the activation of
K+ channels and/or due to the inhibition of active
K+ transport systems such as K+/Na+
ATPase. Inhibition of K+/Na+ ATPase may be
expected to result in an increase in cellular volume, which is not
found in apoptosis and rather is a feature of necrosis (7). Therefore,
we reasoned that an increased activity of K+ channels
rather than an inhibition of active K+ transport might
account for the loss of cellular K+, a hypothesis that we
have tested in this work using K+ channel inhibitors.
Because K+ efflux causes a local charge asymmetry on the
plasma membrane and is the major determining factor for

p (6070 mV in most cell types, negative inside), we
wondered whether the apoptosis-associated K+ loss would
culminate in a loss of 
p. Therefore, we determined
the retention of DiOC6(3) in the CS of cells undergoing
apoptosis (which, based on the Nernst equation, should reach a
cytosolic concentration
1 log higher than that of the EC medium at a

p of 60 mV) and comparatively monitored the

p and the 
m in cells undergoing
apoptosis.
Like other cells, thymocytes possess voltage-dependent K+
channels that participate in sustaining the resting

p, in regulating cell volume, and in enabling
cellular activation processes (31). Here, we addressed the functional
impact of K+ channels, K+ depletion, and

p breakdown on thymocyte apoptosis. Our results
suggest that gross perturbations of K+ currents leading to

p collapse are only observed at a relatively late,
postmitochondrial stage of apoptosis. However, inhibition of
K+ channels may prevent the apoptosis induced by some but
not all inducers at a premitochondrial stage. These data underscore the
physiological importance of plasma membrane ion gradients in the
control of apoptosis.
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Materials and Methods
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Induction and inhibition of apoptosis
Thymocytes from female 4- to 8-wk-old BALB/c mice were cultured
at 37°C with 5% CO2 in RPMI 1640 supplemented with 10%
FCS, L-glutamine, antibiotics, and 2-ME (50 µM; Sigma,
St. Louis, MO). The following cell death inducers were employed: the
glucocorticoid receptor agonist dexamethasone (DEX) (final
concentration 1 µM; Sigma), etoposide (10 µM; Sigma), irradiation
(10 Gy; RX30/55 irradiator, Gravaton Industries, Gosport, U.K.), C2
ceramide (50 µM; Sigma), valinomycin (100 µM; Sigma), PK11195 (200
µM; Sigma), or an Ab specific for CD95/Apo-1/Fas (clone 154000D, 500
ng/ml; PharMingen, San Diego, CA). The following apoptosis inhibitors
were tested: the K+ channel blockers tetraethylammonium
(TEA) and tetrapentylammonium (TPA) (200-µM standard dose; Sigma),
the glucocorticoid receptor antagonist RU38486 (10 µM; kindly
provided by Roussel-Hoechst-Marion), the broad-spectrum caspase
inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone
(Z-VAD.fmk) (100-µM final concentration; stock 10 mM in ethanol;
Bachem, Basel, Switzerland), or the ligand of the adenine nucleotide
translocator bongkrekic acid (50-µM final concentration; kindly
provided by Dr. Duine, Delft University, Delft, The Netherlands).
Cytofluorometric determination of 
p, viability,
phosphatidylserine exposure, reactive oxygen species (ROS), cytosolic
K+, and DNA loss
For the determination of the 
p,
DiOC6(3) (at a final concentration of 40 nM, stock 40 µM
in ethanol, and an excitation wavelength of 488 nm (emission 529 nm);
Molecular Probes, Eugene, OR) was employed (32). Alternatively, we used
20 nM of chloromethyl-X-rosamine (CMXRos) (fluorescence at 600 nm), as
described previously (33). The fluorescence intensity or emission
spectrum of these dyes is not influenced by variations in the pH (data
not shown), within a pH range relevant for apoptosis regulation (pH
6.08.2) (9). To determine the contribution of the

p, DiOC6 staining was performed in either
complete medium, 140 mM NaCl supplemented with 2 mM glucose and 10 mM
HEPES (pH 7.4), or 140 mM KCl plus 2 mM glucose plus 10 mM HEPES (pH
7.4), as indicated. This latter procedure leads to the collapse of the

p (34). Nonviable cells were excluded by simultaneous
staining with propidium iodine (PI) (final concentration of 5 µM;
stock 10 mM in DMSO; excitation at 488 nM, emission 620 nM; Molecular
Probes). Loss of membrane integrity was determined by means of the
vital dye ethidium (Eth) bromide (200 ng/ml; 5 min at room temperature,
excitation at 480 nm, emission 600 nm). An FITC-annexin V conjugate
(1/400 dilution; 1 µg/ml, 15 min at 4°C; Brand Applications,
Maastricht, The Netherlands; 525 nm) with a high affinity for PS (35, 36) was used for the assessment of aberrant PS exposure. Labeling with
FITC-annexin V was performed after the removal of FCS by washing cells
twice in HEPES buffer (10 mM HEPES-NaOH (pH 7.4), 150 mM NaCl, 5 mM
KCl, 1 mM MgCl2, and 1.8 mM CaCl2). The
generation of ROS was monitored with hydroethidine (HE) (final
concentration of 2 µM; stock 10 mM in DMSO, excitation at 488 nM,
emission 620 nM; Molecular Probes). For the determination of
intracellular K+ levels, cells were loaded for 1530 min
with cell-permeant benzofuran isophtalate (PBFI)-acetoxymethyl ester
(final concentration of 2.5 µM; stock 500 µM in DMSO). The
resulting PBFI fluorescence was elicited at 360 nM and measured at
485 ± 20 nm as described previously (29, 37). The frequency of
hypoploid cells was determined by ethanol fixation followed by staining
with PI as described previously (38) using an Epics Profile II Analyzer
(Coulter, Hialeah, FL). All other stainings were analyzed using a
FACSvantage cytofluorometer (Becton Dickinson, Mountain View, CA),
which was also used for cell sorting.
Confocal microscopy
Cells were stained with DiOC6(3) as described above
and were examined with a Leica TCS NT confocal microscope (Leica
Microsystemes, Rueil Malmaison, France) equipped with a 15-mW
argon-krypton laser configured with an inverted Leica DM IRBE. The
488-nm line was used to excite DiOC6(3). Each image
consisted of the projection of 50 optical sections performed at
intervals of 200 nm in the z-axis. xy fields of 1024 x
1024 pixels were scanned using an oil Pl Apo 100x (not applicable =
1.4) objective. Chats (0.2 x 0.2-µm size) were quantified, and
images were processed using the Leica TCS NT software (PowerScan
module) and printed on a Sony HD-8800 color printer.
Determination of Asp-Glu-Val-Aspase (DEVDase) activity of CS
A total of 100 µl of CS (1 x 107 cells/100
µl in CFS buffer (220 mM mannitol, 68 mM sucrose, 2 mM NaCl, 2.5 mM
PO4H2K, 0.5 mM EGTA, 2 mM MgCl2, 5
mM pyruvate, 0.1 mM PMSF, 1 mM DTT, and 10 mM HEPES-NaOH, pH 7.4)
supplemented with additional protease inhibitors: 1 µg/ml leupeptin,
1 µg/ml pepstatin A, 50 µg/ml antipain, and 10 µg/ml chymopapain)
were prepared by five freeze/thaw cycles in liquid nitrogen, followed
by centrifugation (1.5 x 105 g, 4°C,
1 h) as described previously (39). The capacity of CS or purified
caspase-3 to cleave the caspase-3 recognition site DEVD was determined
using Ac-DEVD-amino-4-methylcoumarin (Bachem) as fluorogenic substrate
(40).
DNA fragmentation analysis and electron microscopy
DNA fragmentation (5 x 105 cells/lane) was
determined by agarose gel electrophoresis (32). Electron microscopy was
performed on ultrathin sections of
glutaraldehyde/osmiumtetroxide-fixed, Epon-embedded cells after uranyl
acetate and lead citrate staining as described previously (41).
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Results and Discussion
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Sequential loss of 
m and 
p in
thymocyte apoptosis
Lipophilic cations such as DiOC6(3) accumulate in the
mitochondrial matrix, driven by the electrochemical gradient following
the Nernst equation. Therefore, the concentration of such cations is
23 logs higher in the mitochondrial matrix than in the CS (11). Given
that the resting 
p of lymphocytes is usually 6070
mV, the cytosolic concentration is
10-fold higher than in the
incubation medium. This implies that two potentials
(
m plus 
p) influence the cellular
uptake of DiOC6 (3). To distinguish the relative
contribution of 
mand 
p to
DiOC6(3) uptake, thymocytes were stained with
DiOC6(3) in the presence of either 140 mM NaCl (which is
compatible with the maintenance of 
p) or 140 mM KCl
(which abolishes 
p). As expected, staining
thymocytes in the presence of high EC K+, which causes a

p depolarization, induces a reduction in
DiOC6(3) fluorescence (Fig. 1
A). This
K+-dependent reduction is not seen when cells are treated
with the K+ channel blocker TPA, indicating that
TPA-inhibitible, voltage-gated K+ channels maintain the

p in this cell type, as reported previously (42).
Moreover, no K+-mediated reduction is found for
DiOC6(3)low thymocytes stimulated with DEX,
indicating that such cells do not have 
p or

m (Fig. 1
A), a finding which is in line
with our previous observation that such cells have undergone massive
K+ efflux (29) and thus must be unable to generate

p. As a result, it appears that both TPA and
DEX cause a 
p collapse, although both reagents affect

m in a differential fashion.

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FIGURE 1. Alterations of  m and  p induced by
DEX or TPA. A, Determination of DiOC6(3)
incorporation into untreated thymocytes (control) or cells cultured in
the presence of DEX (1 µM, 6 h) or TPA (200 µM, 2 h).
Cells were stained with DiOC6(3) in the presence of a
buffer containing either 140 mM NaCl or 140 mM KCl. Results are
representative of five independent experiments. B,
Confocal image of DiOC6(3)-stained control thymocytes,
DEX-treated cells (DiOC6(3)low population), or
TPA-treated cells. Black, background intensity; red, 20100 arbitrary
units of fluorescence intensity; green, 100200 arbitrary units; and
blue, >200 arbitrary units. C, Quantitation of signals
measured by confocal microscopy. The fluorescence intensity of selected
areas of cells (mitochondrion-rich cytoplasm, CS) treated as described
in A as well as DiOC6(3) background
fluorescence (in the EC area) were measured by confocal microscopy as
described in Materials and Methods. Each graph contains
curves for three different representative cells obtained by moving the
cursor measuring the fluorescence intensity on different subcellular
regions. Data are shown on a linear scale of arbitrary units of
fluorescence intensity for the EC medium, the CS, and
mitochondrion-rich areas (Mito). Each point represents a 0.2 x
0.2-µm area within the cell. This experiment has been repeated once,
yielding similar data.
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This interpretation was confirmed using confocal analysis of
DiOC6(3)-stained control, DEX-, or TPA-treated cells. TPA
selectively affects the cytosolic (but not the mitochondrial)
accumulation of DiOC6(3). The gradient between the EC and
the cellular DiOC6(3)-dependent fluorescence disappears
after preincubation of cells with TPA (Fig. 1
C). In
contrast, DEX reduces both the plasma membrane and the mitochondrial
DiOC6(3) concentration gradients (Fig. 1
, B and
C).
Previously, we have reported the existence of a minor (<5%)
DiOC6(3)intermediate population among
preapoptotic thymocytes (4, 43). These cells represent an intermediate
stage of the apoptotic process because, once purified, they quickly
become DiOC6(3)low (44). To clarify the

m/
p status of these cells,
DEX-treated thymocytes were stained with DiOC6(3), followed
by cytofluorometric separation of cells into
DiOC6(3)high (I),
DiOC6(3)intermediate (II), and
DiOC6(3)low (III) cells (Fig. 2
B). These cells were then
restained for 15 min in NaCl (which would maintain the

p) or KCl (which would depolarize the

p) with either DiOC6(3) (Fig. 2
C) or CMXRos (Fig. 2
D), a 
-sensitive dye
emitting a wavelength other than DiOC6(3) (33). As
expected, DiOC6(3)high cells possess

p, whereas DiOC6(3)low cells
have a greatly reduced, if any, 
p.
DiOC6(3)intermediate cells treated with 140 mM
KCl lower their fluorescence upon restaining with DiOC6(3)
or de novo staining with CMXRos (Fig. 2
, C and
D), indicating that the cells have undergone an at least
partial 
m collapse; however, these cells retain their

p. Electron microscopy revealed that this
DiOC6(3)intermediate population lacks advanced
chromatin condensation and cell shrinkage (Fig. 2
F), yet
manifests mitochondrial matrix swelling compared with
DiOC6(3)high cells, which have a normal
phenotype (Fig. 2
E). The mitochondrial swelling of
DiOC6(3)intermediate cells must be transient,
because DiOC6(3)low cells, which represent a
later stage of the process with full nuclear apoptosis, have undergone
shrinkage both of the cytoplasm and of the mitochondria (Refs. 13, 33,
and 44 and data not shown). In conclusion, thymocytes undergoing
apoptosis dissipate their 
m and manifest a
transient mitochondrial swelling before they lose

p.

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FIGURE 2. Temporal sequence of  m and  p
collapse induced by DEX. Untreated cells (A) or
DEX-treated (1 µM, 4 h) thymocytes (B) were
stained with DiOC6(3), followed by cell sorting into
DiOC6(3)high (I, 41% of total),
DiOC6(3)intermediate (II, 3% of total), or
DiOC6(3)low (III, 23% of total) populations,
as indicated by the windows in B. After sorting, cells
were washed in 140 mM NaCl or 140 mM KCl, followed by restaining with
DiOC6(3) (C) or de novo staining with CMXRos
(D) and reanalysis. This experiment was repeated three
times, yielding similar results. In addition,
DiOC6(3)high (E) or
DiOC6(3)intermediate (F) cells
were fixed and subjected to electron microscopy. Mitochondria-rich
areas of representative cells are shown.
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K+ channel blockade inhibits thymocyte apoptosis
induced by DEX and etoposide
The specific K+ channel blocker TPA causes a
dose-dependent loss of 
p without affecting

m, which can be measured independently from

p by assessing the ratio between cytosolic and
mitochondrial DiOC6(3) retention (Figs. 1
and 3
A). This has been shown by
comparative DiOC6(3) staining in the presence or absence of
140 mM NaCl or KCl (Figs. 1
A and 3A) or by
confocal quantification of the DiOC6(3) concentration
gradients on the plasma and mitochondrial membranes (Fig. 1
, B and C and Fig. 3
C). The

p decrease induced by TPA appears to be almost total,
because the DiOC6 (3) retention of TPA-treated cells is not
reduced by treatment with 2 mM ouabain (data not shown), an inhibitor
of the Na/K ATPase that is specifically involved in the maintenance of

p yet dispensable for that of the inner mitochondrial
membrane (45). Even upon prolonged incubation (4 h in Fig. 3
A), TPA does not cause thymocyte apoptosis, as assessed by
measuring nuclear hypoploidy (numbers in cycles in Fig. 3
A).
In contrast, incubating cells with DEX during the same period readily
induced a total collapse in DiOC6 (3) retention and nuclear
apoptosis in
40% of the cells. Unexpectedly, when thymocytes were
kept in culture in the continuous presence of both DEX and TPA (Fig. 3
B), we found that TPA treatment prevented the cells from
responding to DEX by advancing to the
DiOC6(3)low phenotype and caused them to lose
their 
m (Fig. 3
, B and C).
Dose-response analyses revealed a strong correlation between the
TPA-mediated collapse of 
p and the prevention of
DEX-induced 
m dissipation (Fig. 3
, A and
B). Whereas TPA was an efficient inhibitor of the
DEX-triggered 
m collapse at doses as low as 20 µM,
a dose also reported to prevent UV-induced cell shrinkage of HL60 cells
(28), another quaternary ammonium, TEA, which has a lower affinity for
K+ channels than TPA (46), had to be employed at millimolar
concentrations to obtain a similar effect (Fig. 3
, A and
B). Again, this effect correlated with the induction of a

p collapse (Fig. 3
, A and B).

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FIGURE 3. Effect of TPA on DiOC6(3) uptake. Thymocytes were cultured
in the absence (A) or presence (B) of
DEX. In addition, the indicated amount of TPA or TEA was added to the
culture medium. After 4 h of incubation, cells were stained with
DiOC6(3) in complete culture medium (A and
B). The DiOC6(3) incorporation profiles
obtained in the absence of TPA are shown as an internal control in each
individual graph. Alternatively, the concentration of
DiOC6(3) within the mitochondrion-rich and
mitochondria-free cytoplasm was measured by confocal microscopy as
described in the legend to Fig. 1 C. The fluorescence
intensity was measured for each subcellular region of 10 different
cells. Data are expressed as mean values ± SEM
(n = 10).
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In a further series of experiments, we addressed the question of
whether TPA would selectively affect some features of DEX-induced
apoptosis or rather block the entire apoptotic program. As shown in
Fig. 4
, we found that TPA prevented all
characteristics of apoptosis, including the mitochondrial
hypergeneration of ROS (measured by determining the oxidation of HE to
Eth, Fig. 4
A), the exposure of phosphatidylserine on the
plasma membrane surface (measured by FITC-annexin V conjugates, Fig. 4
B), cytosolic K+ efflux (measured with PBFI,
Fig. 4
C), and nuclear DNA loss (quantitated by PI staining
of ethanol-fixed cells, Fig. 4
D). TPA also prevents the
activation of caspases capable of cleaving the substrate DEVD (Fig. 5
A), as well as
oligonucleosomal DNA fragmentation (Fig. 5
B). Electron
microscopy confirmed that thymocytes treated with both TPA and DEX
exhibited a normal morphology (Fig. 6
).
Similar data were obtained when the topoisomerase type II inhibitor
etoposide was used instead of DEX (Fig. 4
). The dose of TPA necessary
for the blockade of nuclear apoptosis was found to correlate with that
causing a 
p collapse (numbers in circles in Fig. 3
, A and B). Thus, TPA can block the thymocyte
apoptosis induced by the glucocorticoid agonist DEX or by the genotoxic
agent etoposide.

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FIGURE 4. Cytofluorometric assessment of various apoptosis-associated parameters
and their modulation by TPA. Thymocytes were cultured for 4 h with
DEX, etoposide, or a mAb specific for Fas/CD95 and/or TPA, followed by
staining with DiOC6(3) plus HE (A), Eth
bromide plus FITC-annexin V (B), or PBFI
(C). Alternatively, cells were permeabilized with
ethanol, and the nuclear DNA content was determined with PI
(D). Results are representative of four independent
experiments.
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FIGURE 5. Effect of TPA on caspase and endonuclease activation. Thymocytes
treated as described in the legend to Fig. 4 were subjected to lysis
and determination of DEVD-aminotrifluoromethylcoumarin cleavage
(A). Alternatively, DNA was extracted (5 x
105 cells/lane) and subjected to agarose gel
electrophoresis for the detection of oligonucleosomal DNA fragmentation
(B). Cells were cultured for 4 h with the indicated
combination of DEX, etoposide, anti-CD95, and/or TPA, followed by
analysis of the indicated parameter. Shaded boxes indicate addition of
the corresponding substance.
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K+ channel blockade blocks a specific rather than
general apoptosis pathway
To determine whether TPA would block DEX-induced apoptosis at an
early or a late stage, we performed time-course inhibition experiments.
RU38486, a glucocorticoid receptor antagonist, can block DEX-induced
thymocyte apoptosis (12, 41) when added early after DEX (Fig. 7
a). In contrast, the caspase
inhibitor Z-VAD.fmk prevents the advent of nuclear apoptosis at a later
stage, in accordance with the idea that caspase-dependent endonuclease
activation occurs after the commitment to apoptosis triggered via
glucocorticoid receptor occupation (4). Compared with Z-VAD.fmk, TPA
has to be added at a relatively early stage to obtain a suppression of
DEX-induced apoptosis (Fig. 7
a); this finding is in line
with the fact that TPA prevents all signs of DEX-induced
apoptosis, including the early mitochondrial changes (Figs. 3
and 4
), whereas Z-VAD.fmk blocks the process leading to DNA fragmentation
after 
m dissipation and mitochondrial swelling (4, 43).

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FIGURE 7. Time course and spectrum of apoptosis-inhibitory action of TPA.
A, Time course of apoptosis inhibition mediated by
RU38480, TPA, or Z-VAD.fmk. Thymocytes were cultured for 5 h in
the presence of DEX. The indicated agents were added 30, 60, 90, 120,
180, or 240 min after initiation of the culture. Hypoploidy was
assessed after 5 h, and the percentage of inhibition of this
parameter was determined. Control values of untreated thymocytes were
15 ± 2%; values of thymocytes treated with DEX only were 52
± 3%. B, TPA-mediated inhibition of apoptosis induced
by a panel of different inducers. Thymocytes were cultured for 4 h
with the indicated apoptosis inducer in the presence or absence of TPA
(200 µM), and the frequency of subdiploid cells was measured. Results
are mean values of three independent determinations ± SEM.
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The above findings indicate that TPA acts at a relatively early stage
of the DEX-induced apoptotic process, suggesting that it could
interfere with the activation of the apoptotic machinery rather than
with the machinery itself. Therefore, we determined whether TPA would
have a vast spectrum of antiapoptotic action or instead whether it
would fail to prevent apoptosis induction by some apoptosis triggers.
We explored the effects of TPA on apoptosis induction by 1) ceramide, a
proapoptotic second messenger elicited by glucocorticoids and genotoxic
stress (47, 48), 2) valinomycin, a K+ ionophore that
dissipates cellular and mitochondrial ion gradients (49), 3) PK11195, a
ligand of the peripheral benzodiazepine receptor that facilitates the
opening of the PT pore (50), and 4) Fas/CD95 cross-linking, a
manipulation leading to primary caspase activation (51). TPA prevents
the apoptosis induced by ceramide but has no inhibitory effect on the
apoptosis caused by valinomycin, PK11195 (Fig. 7
b), or
anti-Fas (Figs. 4
, 5
, and 7
b). Thus, TPA can prevent the
cell death induced by DEX and DNA damage after the stage of ceramide
generation, yet remains ineffective when apoptosis is enforced by
direct K+ depletion, PT pore opening, or caspase
activation.
Concluding remarks
The present work demonstrates that, during thymocyte apoptosis,
gross alterations of plasma membrane function and structure occur after
signs of mitochondrial dysfunction have manifested (Fig. 8
). Thus, dissipation of the

m and transient mitochondrial swelling are observed
before several changes affect the plasma membrane: loss of the

p (Figs. 1
and 2
), reduction of intracellular
K+ (29), cell shrinkage, and loss of membrane asymmetry
with aberrant exposure of phosphatidylserine residues on the plasma
membrane surface (33). TPA collapses the 
p of
thymocytes, indicating that TPA-inhibitible, voltage-gated
K+ channels generate this 
p. However, TPA
has no major toxic effects on thymocytes (Fig. 6
), does not cause a
nonspecific permeabilization of membranes with Ca2+ influx
(data not shown), and does not cause PS exposure (Fig. 4
B),
indicating that 
p collapse by itself does not perturb
the distribution of lipids in the plasma membrane. Unexpectedly, we
found that the prevention of K+ efflux by TPA fully
inhibits the thymocyte apoptosis induced by a variety of stimuli that
may be p53-dependent (etoposide,
-irradiation) or p53-independent
(DEX, ceramide). These pathways have in common that they require de
novo mRNA and protein synthesis (52) and that they are inhibited by PT
pore blockade (26, 41, 53) or the knock-out of Apaf-1 or caspase-9 (54, 55), two molecules which link mitochondrial damage to downstream events
of apoptosis. TPA intercepts the apoptotic pathway at the early,
premitochondrial level, because 1) it prevents the early

m dissipation and later mitochondrial ROS generation
induced by DEX or etoposide (Figs. 3
B and 4A), 2)
has no effect when added late (Fig. 7
a) or when added to
DiOC6(3)intermediate cells that have already
disrupted of their 
m (data not shown), and 3) has no
inhibitory effect when apoptosis is enforced by PK11105 (Fig. 7
b), an agent that acts directly on the PT pore. Moreover,
TPA fails to prevent the apoptosis induced by valinomycin, a
K+-specific ionophore (Fig. 7
a). This latter
negative result suggests that TPA exerts its antiapoptotic effect by
virtue of is modulatory effect on K+ fluxes rather than via
a yet-to-be discovered nonspecific effect. TPA does not suppress the
apoptosis induced by anti-Fas/CD95 (Figs. 4
and 5
), a pathway that
causes direct caspase activation without any need of protein synthesis,
Apaf-1, or caspase-9 (51, 54, 55, 56). Hence, TPA acts on a specific rather
than on a general pathway of apoptosis (Fig. 8
). Of note, TPA does not
prevent the K+ efflux triggered by anti-Fas/CD95 (Fig. 4
C), indicating that other TPA-resistant mechanisms of
K+ efflux can intervene in a late stage of apoptosis, at
least in the Fas/CD95 pathway. The exact mechanisms by which TPA
prevents the early mitochondrial changes triggered by DEX, DNA damage,
or ceramide remain elusive. Potassium channel blockers have been shown
previously to inhibit lymphocyte activation processes (31, 57), and it
may be possible that an analogous effect underlies the
apoptosis-inhibitory effect of TPA.

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|
FIGURE 8. Stages of the common phase of thymocyte apoptosis and effects of TPA on
apoptosis induction. The diagram resumes the data contained in this
paper, as well as information from References 29 and 33. The difference
between stages 2 and 3 is the capacity of enhanced HE Eth conversion
(low in stage 2, high in stage 3; Ref. 29). Note that TPA has the
capacity to prevent apoptosis induction by some but not all inducers.
|
|
Several published reports suggest that the regulation of K+
fluxes has a major impact on apoptosis regulation. Valinomycin, a
K+-specific ionophore, is a potent inducer of apoptosis in
many cell types, including neurons (58), hepatoma cells (59), pre-B
cells (49), and thymocytes (Refs. 60 and 61 and Fig. 7
b).
K+ depletion suffices to cause apoptosis in neurons (3, 62, 63), and K+ efflux underlies a murine model of cerebellar
cell death in which the weaver mutation causes the constitutive
activation of the G protein-regulated inward rectifier 2 K+
channel (64). In neocortical cells, attenuating outward K+
current with TEA or elevated EC K+ rescues the apoptosis
induced by staurosporine, serum withdrawal (58), or ß-amyloid (65, 66). Plasma membrane channels mediating a K+ efflux can
cause apoptosis in a variety of different cell types. Thus,
ATP-activated P2Z/P2X purinergic receptors in macrophages
simultaneously mediate pore formation, 
p collapse,
and apoptosis (67). Similarly, ß-amyloid (68) and the HIV-encoded Vpr
protein (69) can incorporate into plasma membranes, thereby perturbing
ion homeostasis and causing cell death. In strict contrast to these
data, several apoptosis-inducing agents have been reported to inhibit
K+ channels. This applies to ceramide, which inhibits the
voltage-gated potassium channels of T lymphocytes via tyrosine kinases
(70), 4-aminopuridine, which blocks an outward rectifier K+
channel (71), or the apoptogenic peptides Reaper and Grim (72). It
remains to be confirmed that these effects are truly responsible for
apoptosis induction by these agents.
At first glance, the relationship between the mitochondrial PT pore
opening and K+ efflux appears paradoxical. On one hand,

m breaks down before a major CS K+ loss
and concomitant 
p loss occur (Figs. 1
and 2
and 29). On the other hand, a blockade of K+ channels can
prevent the mitochondrial dysfunction required for DEX- or
etoposide-mediated apoptosis (Figs. 3
and 4
). As a possibility, subtle
changes in K+ homeostasis occurring together with the PT
pore opening may participate in a positive feed-forward loop,
facilitating the apoptosis-related mitochondrial dysfunction. This
possibility is suggested by the recent finding that the PT pore
possesses a low conductance mode of opening that is
K+-selective and thus may cause selective alterations of
local K+ concentrations (73). Alternatively, K+
fluxes might play a pleiotropic role at several levels of the apoptotic
cascade, in analogy to cytosolic free Ca2+, which can
function, in the low micromolar range, as a facultative
premitochondrial second messenger-signaling PT pore opening (74) and
increase as an obligate consequence of the PT pore opening to levels of
>200 µM at a late, postmitochondrial stage of the apoptotic process
(75, 76). Thus, subtle changes in K+ fluxes could be
involved in the early apoptotic induction phase, whereas a major
K+ loss would only occur during the late degradation phase
of apoptosis. Irrespective of these theoretical possibilities, the
present data underline the importance of K+ as an
endogenous apoptosis modulator.
 |
Acknowledgments
|
|---|
We thank Martial Flactif (Institut Federatif de Recherche 22,
Lille, France) for confocal microscopy.
 |
Footnotes
|
|---|
1 This work was supported by Agence Nationale pour la Recherche sur le SIDA, Association pour la Recherche contre le Cancer, Centre National de la Recherche Scientifique, Fondation pour la Recherche Médicale, Ligue Française contre le Cancer, Institut National de la Santé et de la Recherche Médicale, and Programme de Recherche Fondamentale en Microbiologie et Maladies Infectieuses et Parasitaires du Ministère de la Recherche, Sidaction (to G.K.). M.G. is a senior research associate of the Belgian National Fund for Scientific Research. 
2 Address correspondence and reprint requests to Dr. Guido Kroemer, 19 rue Guy Môquet, B.P. 8, F-94801 Villejuif, France. E-mail address: 
3 Abbreviations used in this paper: DiOC6(3), 3,3'-dihexyloxacarbocyanine iodide; DEX, dexamethasone; TEA, tetraethylammonium; TPA, tetrapentylammonium; CMXRos, chloromethyl-X-rosamine; 
m, mitochondrial transmembrane potential; 
p, EC, extracellular; CS, cytosol; plasma membrane potential; PI, propidium iodide; Eth, ethidium; HE, hydroethidine; PBFI, benzofuran isophtalate; DEVD, Asp-Glu-Val-Asp; PT, permeability transition; ROS, reactive oxygen species; Z-VAD.fmk, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. 
Received for publication December 21, 1998.
Accepted for publication March 15, 1999.
 |
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