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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 l’Hôpital 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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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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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 120–170 mV) corresponds to a 10-fold increase in cation
concentration in mitochondria. Therefore, the concentration of such
cations is 2–3 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 (100–140 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 (60–70 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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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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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
6.0–8.2) (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 15–30 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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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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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
2–3 logs higher in the mitochondrial matrix than in the CS (11). Given
that the resting p of lymphocytes is usually 60–70
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. 1A), 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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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. 2B). 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. 2C) or CMXRos (Fig. 2D), 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. 2F), yet
manifests mitochondrial matrix swelling compared with
DiOC6(3)high cells, which have a normal
phenotype (Fig. 2E). 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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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 3A). This has been shown by
comparative DiOC6(3) staining in the presence or absence of
140 mM NaCl or KCl (Figs. 1A 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. 3C). 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. 3A), TPA does not cause thymocyte apoptosis, as assessed by
measuring nuclear hypoploidy (numbers in cycles in Fig. 3A).
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. 3B), 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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, 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. 4A), the exposure of phosphatidylserine on the
plasma membrane surface (measured by FITC-annexin V conjugates, Fig. 4B), cytosolic K+ efflux (measured with PBFI,
Fig. 4C), and nuclear DNA loss (quantitated by PI staining
of ethanol-fixed cells, Fig. 4D). TPA also prevents the
activation of caspases capable of cleaving the substrate DEVD (Fig. 5A), as well as
oligonucleosomal DNA fragmentation (Fig. 5B). 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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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. 7a). 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. 7a); 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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b), or
anti-Fas (Figs. 4, 5, and 7b). 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. 4B),
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. 3B and 4A), 2)
has no effect when added late (Fig. 7a) 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. 7b), an agent that acts directly on the PT pore. Moreover,
TPA fails to prevent the apoptosis induced by valinomycin, a
K+-specific ionophore (Fig. 7a). 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. 4C), 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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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.
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Acknowledgments |
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Footnotes |
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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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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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 R. Vicente, A. Escalada, M. Coma, G. Fuster, E. Sanchez-Tillo, C. Lopez-Iglesias, C. Soler, C. Solsona, A. Celada, and A. Felipe Differential Voltage-dependent K+ Channel Responses during Proliferation and Activation in Macrophages J. Biol. Chem., November 21, 2003; 278(47): 46307 - 46320. [Abstract] [Full Text] [PDF]
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D. Ekhterae, O. Platoshyn, S. Zhang, C. V. Remillard, and J. X.-J. Yuan Apoptosis repressor with caspase domain inhibits cardiomyocyte apoptosis by reducing K+ currents Am J Physiol Cell Physiol, June 1, 2003; 284(6): C1405 - C1410. [Abstract] [Full Text] [PDF]
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 X. Q. Wang, A. Y. Xiao, A. Yang, L. LaRose, L. Wei, and S. P. Yu Block of Na+,K+-ATPase and Induction of Hybrid Death by 4-Aminopyridine in Cultured Cortical Neurons J. Pharmacol. Exp. Ther., May 1, 2003; 305(2): 502 - 506. [Abstract] [Full Text] [PDF]
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 N. Hail Jr. and R. Lotan Examining the Role of Mitochondrial Respiration in Vanilloid-Induced Apoptosis J Natl Cancer Inst, September 4, 2002; 94(17): 1281 - 1292. [Abstract] [Full Text] [PDF]
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 A. Y. Xiao, L. Wei, S. Xia, S. Rothman, and S. P. Yu Ionic Mechanism of Ouabain-Induced Concurrent Apoptosis and Necrosis in Individual Cultured Cortical Neurons J. Neurosci., February 15, 2002; 22(4): 1350 - 1362. [Abstract] [Full Text] [PDF]
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 S. Krick, O. Platoshyn, M. Sweeney, S. S. McDaniel, S. Zhang, L. J. Rubin, and J. X.-J. Yuan Nitric oxide induces apoptosis by activating K+ channels in pulmonary vascular smooth muscle cells Am J Physiol Heart Circ Physiol, January 1, 2002; 282(1): H184 - H193. [Abstract] [Full Text] [PDF]
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N. Hail Jr. and R. Lotan Mitochondrial Respiration Is Uniquely Associated with the Prooxidant and Apoptotic Effects of N-(4-Hydroxyphenyl)retinamide J. Biol. Chem., November 30, 2001; 276(49): 45614 - 45621. [Abstract] [Full Text] [PDF]
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 J. Pardo, P. Perez-Galan, S. Gamen, I. Marzo, I. Monleon, A. A. Kaspar, S. A. Susin, G. Kroemer, A. M. Krensky, J. Naval, et al. A Role of the Mitochondrial Apoptosis-Inducing Factor in Granulysin-Induced Apoptosis J. Immunol., August 1, 2001; 167(3): 1222 - 1229. [Abstract] [Full Text] [PDF]
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M. Poppe, C. Reimertz, H. Du{beta}mann, A. J. Krohn, C. M. Luetjens, D. Bockelmann, A.-L. Nieminen, D. Kogel, and J. H. M. Prehn Dissipation of Potassium and Proton Gradients Inhibits Mitochondrial Hyperpolarization and Cytochrome c Release during Neural Apoptosis J. Neurosci., July 1, 2001; 21(13): 4551 - 4563. [Abstract] [Full Text] [PDF]
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D. Ekhterae, O. Platoshyn, S. Krick, Y. Yu, S. S. McDaniel, and J. X.-J. Yuan Bcl-2 decreases voltage-gated K+ channel activity and enhances survival in vascular smooth muscle cells Am J Physiol Cell Physiol, July 1, 2001; 281(1): C157 - C165. [Abstract] [Full Text] [PDF]
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 Y. Okada, E. Maeno, T. Shimizu, K. Dezaki, J. Wang, and S. Morishima Receptor-mediated control of regulatory volume decrease (RVD) and apoptotic volume decrease (AVD) J. Physiol., April 1, 2001; 532(1): 3 - 16. [Abstract] [Full Text] [PDF]
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F. Di Virgilio, P. Chiozzi, D. Ferrari, S. Falzoni, J. M. Sanz, A. Morelli, M. Torboli, G. Bolognesi, and O. R. Baricordi Nucleotide receptors: an emerging family of regulatory molecules in blood cells Blood, February 1, 2001; 97(3): 587 - 600. [Abstract] [Full Text] [PDF]
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 C.-C. Shieh, M. Coghlan, J. P. Sullivan, and M. Gopalakrishnan Potassium Channels: Molecular Defects, Diseases, and Therapeutic Opportunities Pharmacol. Rev., December 1, 2000; 52(4): 557 - 594. [Abstract] [Full Text] [PDF]
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X. Wang, A. Y. Xiao, T. Ichinose, and S. P. Yu Effects of Tetraethylammonium Analogs on Apoptosis and Membrane Currents in Cultured Cortical Neurons J. Pharmacol. Exp. Ther., November 1, 2000; 295(2): 524 - 530. [Abstract] [Full Text]
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 K. F. Ferri, E. Jacotot, J. Blanco, J. A. Este, N. Zamzami, S. A. Susin, Z. Xie, G. Brothers, J. C. Reed, J. M. Penninger, et al. Apoptosis Control in Syncytia Induced by the HIV Type 1-Envelope Glycoprotein Complex: Role of Mitochondria and Caspases J. Exp. Med., October 16, 2000; 192(8): 1081 - 1092. [Abstract] [Full Text] [PDF]
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 S. P. Yu and D. W. Choi Ions, cell volume, and apoptosis PNAS, August 15, 2000; 97(17): 9360 - 9362. [Full Text] [PDF]
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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.