From the Division of Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Box 210, SE-171 77 Stockholm, Sweden
Received for publication, January 23, 2001, and in revised form, February 27, 2001
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ABSTRACT |
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Release of cytochrome c from
mitochondria is a key initiative step in the apoptotic process,
although the mechanisms regulating this event remain elusive. In the
present study, using isolated liver mitochondria, we demonstrate that
cytochrome c release occurs via distinct mechanisms that
are either Ca2+-dependent or
Ca2+-independent. An increase in mitochondrial matrix
Ca2+ promotes the opening of the permeability transition
(PT) pore and the release of cytochrome c, an effect
that is significantly enhanced when these organelles are incubated in a
reaction buffer that is based on a physiologically relevant
concentration of K+ (150 mM KCl)
versus a buffer composed of mannitol/sucrose/Hepes. Moreover, low concentrations of Ca2+ are sufficient to
induce mitochondrial cytochrome c release without measurable manifestations of PT, though inhibitors of PT effectively prevent this release, indicating that the critical threshold for PT
varies among mitochondria within a single population of these organelles. In contrast, Ca2+-independent cytochrome
c release is induced by oligomeric Bax protein and occurs
without mitochondrial swelling or the release of matrix proteins,
although our data also indicate that Bax enhances permeability
transition-induced cytochrome c release. Taken together, our results suggest that the intramitochondrial Ca2+
concentration, as well as the reaction buffer composition, are key
factors in determining the mode and amount of cytochrome c release. Finally, oligomeric Bax appears to be capable of stimulating cytochrome c release via both
Ca2+-dependent and Ca2+-independent mechanisms.
Apoptosis, a gene-regulated form of cell death, is involved in
cell deletion during organogenesis and in the control of cell proliferation and differentiation in adult tissues, as well as in
various diseases (1, 2). The biochemical machinery required for
apoptotic cell death is constitutively present in virtually all
mammalian cells and can be activated by a variety of extra- and
intracellular signals. Attempts to identify a common, unifying step in
the apoptotic program, in response to various cytotoxic stimuli, have
focused on the role of mitochondrial participation in this form of cell
death (3-7).
Specifically, the release of several proteins, normally located in the
intermembrane space of mitochondria, has been observed during the early
stages of apoptotic cell death (8-10). Among these proteins is
cytochrome c, a component of the electron transport chain
that shuttles electrons between complexes III (bc1) and IV (cytochrome
oxidase). Only holocytochrome c, and not apocytochrome c, is able to stimulate procaspase-9 activation, which
occurs when cytochrome c is released into the cytosol and
interacts with Apaf-1 and procaspase-9 in the presence of dATP to form
the apoptosome complex (11). Moreover, some observations indicate that
both reduced and oxidized cytochrome c are able to activate
procaspase-9 with similar efficiencies, an effect that may be related
to the speed with which oxidized cytochrome c is reduced
in vitro (12).
The mechanisms regulating cytochrome c release remain
obscure. However, with the exception of disturbances in osmotic balance accounting for a non-selective release of intramitochondrial proteins (13, 14), two distinct models for cytochrome c release have emerged that can be distinguished on the basis of whether
Ca2+ is required for the event. In one instance,
mitochondrial Ca2+ overload results in swelling and rupture
of the outer membrane followed by the release of cytochrome
c and other intermembrane space proteins (15, 16). The
Ca2+-independent model asserts that a more selective
protein release occurs without changes in mitochondrial volume. This
mechanism involves specific channels/pores in the outer mitochondrial
membrane that may be opened and regulated by certain proapoptotic
members of the Bcl-2 family of proteins, including Bax (17, 18). The precise way Bax modulates cytochrome c release is
controversial. In particular, there are reports asserting that this
protein functions directly on mitochondria to stimulate the release of
cytochrome c by forming a selective pore in the outer
membrane, whereas others argue that Bax interacts with the
voltage-dependent anion channel and/or the adenine
nucleotide translocator
(ANT)1 to facilitate opening
of the permeability transition (PT) pore (19-21). More recent evidence
indicates that truncated Bid induces a conformational change in Bax
that allows this protein to insert in the outer membrane and stimulate
cytochrome c release (22). A different study demonstrated
the ability of Bax to induce a complete loss of cytochrome c
from mitochondria isolated from Xenopus eggs without
otherwise affecting the functional integrity of these organelles
(23).
The present study was undertaken to provide a more definitive
understanding of the mechanisms regulating cytochrome c
release in response to Ca2+-dependent and
Ca2+-independent effects on isolated liver mitochondria.
The results indicate that cytochrome c release triggered by
Ca2+ is significantly enhanced when mitochondria are
incubated in KCl- versus MSH-based reaction buffers.
Moreover, this release of cytochrome c can be observed prior
to any measurable manifestations of MPT, such as swelling or the
release of accumulated Ca2+. In comparison, oligomeric, but
not monomeric, Bax triggers a Ca2+-independent release of
cytochrome c that is not mitigated by inhibitors of MPT.
Importantly, oligomeric Bax was also able to enhance
Ca2+-induced cytochrome c release, pointing to a
possible dual function for this protein. These data clearly demonstrate
that cytochrome c release occurs by distinct
Ca2+-dependent and Ca2+-independent
mechanisms that are each modulated by oligomeric Bax.
Isolation of Rat Liver Mitochondria--
Male Harlan
Sprague-Dawley rats (6 to 8 weeks old) were killed by CO2
inhalation in accordance with the European directive of protection of
vertebrate animals for scientific research. The liver was minced on
ice, resuspended in 50 ml of MSH buffer (210 mM mannitol,
70 mM sucrose, 5 mM Hepes, pH 7.5) supplemented
with 1 mM EDTA and homogenized with a glass Dounce
homogenizer and Teflon pestle. Homogenates were centrifuged at 600 × g for 8 min at 4 °C. The supernatant was decanted and
recentrifuged at 5,500 × g for 15 min to form a
mitochondrial pellet that was resuspended in MSH buffer without EDTA
and centrifuged again at 5,500 × g for 15 min. The
final mitochondrial pellet was resuspended in MSH buffer at a protein
concentration of 80-100 mg/ml. The purity of the mitochondrial
fraction was verified by the absence of both cytosolic
(glyceraldehyde-3-phosphate dehydrogenase) and nuclear (poly(ADP-ribose) polymerase) proteins as determined by Western blot analysis.
Measurement of Functional Activity of Isolated
Mitochondria--
Mitochondria (1 mg/ml) were incubated in MSH buffer
or a buffer containing 150 mM KCl, 1 mM
KH2PO4, 5 mM succinate, and 5 mM Tris-HCl, pH 7.4, at 25 °C. Rotenone (2 µM) was added to maintain pyridine nucleotides in a
reduced form. Estimation of Western Blot Analysis--
Samples were mixed with Laemmli's
loading buffer, boiled for 5 min, and subjected to 15% SDS
polyacrylamide gel electrophoresis at 130 V followed by electroblotting
to nitrocellulose membranes for 2 h at 100 V. Membranes were
blocked for 1 h with 5% nonfat milk in phosphate-buffered saline
at room temperature and subsequently probed overnight with an
anti-cytochrome c (1:2,500), anti-adenylate kinase-2
(1:2,000), anti-adenylate kinase-3 (1:2,000),
anti-glyceraldehyde-3-phosphate dehydrogenase (1:5,000), or
anti-poly(ADP-ribose) polymerase (1:1,000) antibody. The membranes were
rinsed and incubated with a horseradish peroxidase-conjugated secondary
antibody (1:10,000). Following the secondary antibody incubation, the
membranes were rinsed, and bound antibodies were detected using
enhanced chemiluminescence according to the manufacturer's instructions.
Expression and Purification of Bax--
Expression and
purification of full-length Bax protein was performed as described
previously (24). Briefly, the full-length human Bax cDNA sequence
was amplified by standard polymerase chain reaction techniques. The
polymerase chain reaction DNA fragment was isolated using the QIAquick
kit (Qiagen) and subcloned into the NcoI and
HindIII sites of the plasmid pBAD. The plasmid was transformed into Escherichia coli, and transformants
were isolated by selection for ampicillin resistance. Cultures of the
resistant colony were grown to an A650 of ~7.
After induction, the culture was further incubated for several hours,
and cells were harvested by centrifugation. The cells were resuspended
in lysis buffer (100 mM Hepes-NaOH, pH 8.0, 100 mM NaCl, 1 mM MgCl2, 0.1%
2-mercaptoethanol, 1% Triton X-100, a mixture of protease inhibitors,
30 µg/ml DNase I, and 50 µg/ml lysozyme) and broken by
sonication. After centrifugation, Bax was recovered in the
supernatant. Because the protein was expressed with a His tag at the N
terminus, the purification by affinity chromatography on
nickel-nitrilotriacetic acid-agarose (Qiagen) followed by ion-exchange
chromatography on Q-Sepharose (Amersham Pharmacia Biotech) was
performed according to the manufacturer's instructions. The protein
was at least 95.8% pure as determined by SDS polyacrylamide gel
electrophoresis. Multimerization of recombinant Bax to a stable
complex, which did not dissociate into smaller species, was performed
in the presence of 1% octyl glucoside (24).
Statistical Analysis--
Data are presented as means ± S.D., and significance was determined using a Student's t
test. A value of p < 0.05 was considered to be significant.
Mitochondrial Permeability Transition and Cytochrome c
Release--
MPT is a consequence of Ca2+ overload (15,
16), and the sensitivity of mitochondria to permeability transition
(PT) can be enhanced by different factors. Among these factors are an
elevated level of Pi, oxidative stress, the
depletion of adenine nucleotides, and the oxidation of pyridine
nucleotides. Fig. 1 is representative of
a typical response of mitochondria to PT-inducing agents. In particular, when Pi or organic hydroperoxide is added to
Ca2+-loaded isolated mitochondria (Fig. 1), these
organelles swell (panel A),
Although the rate of Ca2+ release during MPT is similar in
KCl versus MSH incubation buffers (Fig.
2A), the amount of cytochrome c released is significantly more pronounced when
mitochondria are incubated in the more physiologically relevant KCl
buffer as compared with MSH buffer (Fig. 2B). Further
evidence of this is reflected in the assorted effects that cytochrome
c release has on mitochondrial respiration in the two
buffers (Fig. 2C). Specifically, the induction of MPT in MSH
buffer resulted in a time-dependent acceleration of oxygen
consumption that persisted until all oxygen was consumed. In contrast,
incubation of mitochondria in KCl buffer resulted in an initial
acceleration of respiration that was followed by its suppression, and
no additional stimulation of oxygen consumption was observed upon the
addition of the uncoupler CCCP. Moreover, Western blot analysis
revealed that most cytochrome c was present in the
supernatant fraction following MPT induction in KCl buffer, whereas
only partial release of cytochrome c was observed in MSH
buffer (Fig. 2D). Taken together, these data indicate that
although PT-induced uncoupling of mitochondria occurs in both buffers,
respiration is suppressed only in KCl buffer, an effect most likely
because of a greater loss of cytochrome c.
Low Doses of Ca2+ Stimulate MPT and Cytochrome c
Release in a Subpopulation of Mitochondria--
In the aforementioned
experiments, cytochrome c release was a consequence of PT
induction, accompanied by swelling of mitochondria and rupture of the
outer membrane. The next step was to determine whether cytochrome
c release could occur when Ca2+ loading was
insufficient to induce observable manifestations of MPT. As seen in
Fig. 3A, Ca2+
loading of mitochondria alone did not induce changes characteristic of
MPT (cf. Fig. 1C), and this cation was retained
by mitochondria unless CCCP, an uncoupler of oxidative phosphorylation,
was added. In addition, Ca2+-loaded mitochondria exhibited
controlled respiration (Fig. 3B), although increasing the
Ca2+ retention time from 1 to 5 min prominently diminished
the rate of CCCP-directed uncoupled respiration (Panel B,
trace a versus trace b). Taking into
consideration that the observed decrease in the rate of uncoupled
respiration might be because of damage of the respiratory chain and/or
the release of cytochrome c, the presence of this protein in
the incubation buffer was analyzed. As seen in Fig. 3C, the
accumulation of Ca2+ by mitochondria in the absence of
observable PT was sufficient to stimulate a release of cytochrome
c, an effect that was considerably more pronounced the
longer mitochondria retained accumulated Ca2+. Inhibitors
of MPT, such as ADP and Mg2+, suppressed this release (Fig.
3C) and restored the rate of uncoupled respiration (Fig.
3D). Additional evidence of cytochrome c release in the absence of observable MPT is presented in Fig.
4 where the sequential addition of
Ca2+ pulses to isolated mitochondria led to a stepwise
decrease in the overall optical density (Fig. 4A,
traces 1-5) without inducing large amplitude swelling
characteristic of PT. Meanwhile, the amount of cytochrome c
released from mitochondria was enhanced as Ca2+ loading
increased (Fig. 4B). This release reflected PT induction in
a subpopulation of mitochondria, which was further supported by the
fact that co-treatment of isolated mitochondria with 1 µM
CsA eliminated cytochrome c release induced by
Ca2+ additions ranging between 20 and 80 nmol/mg protein
(data not shown). Cytochrome c release ultimately reached a
pinnacle at 80 nmol Ca2+/mg protein (Fig. 4B,
lanes 6 and 7) as all mitochondria underwent PT
(Fig. 4A).
Bax-induced Cytochrome c Release Can Occur via Both MPT-independent
and MPT-dependent Mechanisms--
Next, we tested the
effect of recombinant Bax protein on cytochrome c release.
Bax has been reported to facilitate cytochrome c release in
response to different stimuli, although the precise mechanism
responsible for this event remains unclear. Both monomeric and
oligomeric forms of Bax (Fig.
5A) were generated for our
study because of reports indicating that oligomerization of Bax is a critical event for integration of this protein into membranes (22). It
should be noted that our monomeric form of Bax also contained the
oligomeric form (Fig. 5A, lane 2), although it is clear from the data that these different pools of recombinant Bax had
strikingly dissimilar effects on cytochrome c release.
The addition of oligomeric Bax to isolated mitochondria prominently
induced the release of cytochrome c (Fig. 5B).
This release did not depend on MPT, because the incubation buffer used
for these experiments contained 1 mM EGTA, a concentration
sufficient to chelate available Ca2+ and hence prevent MPT.
In contrast to the results obtained with oligomeric Bax, cytochrome
c release was marginal in the presence of the monomeric form
of Bax (Fig. 5B), which is in agreement with other data
indicating that only oligomeric Bax is able to induce cytochrome
c release (25). The slight release observed in the presence
of monomeric Bax was most likely because of the presence of some of the
oligomeric form in this preparation (Fig. 5A, lane
2).
In addition to stimulating an MPT-independent release of cytochrome
c, recombinant oligomeric Bax also facilitated
Ca2+-induced MPT as assessed by both the release of
accumulated Ca2+ (Fig. 5C) and a drop in
mitochondrial membrane potential (Fig. 5D). In contrast, the
monomeric form of this protein had no effect on either parameter as
compared with control mitochondria (Fig. 5, C and
D). Western blot analysis of cytochrome c release
in samples taken 6 min after the addition of Ca2+ revealed
that oligomeric Bax enhanced MPT-dependent cytochrome c release (Fig. 5E). Finally, an attempt to
compare the ability of oligomeric Bax to enhance MPT-independent and
MPT-dependent release of cytochrome c revealed,
as we expected, that cytochrome c release was significantly
more pronounced in samples taken from Bax-treated mitochondria that
were stimulated to undergo PT (Fig. 5F).
To determine whether the combined or individual effects of Bax and
Ca2+ on cytochrome c release were unique for
this apoptogenic protein, we also evaluated the release of other
mitochondrial proteins. Results indicated that the intermembrane space
protein adenylate kinase-2 was released into the cytosol during
MPT-independent and MPT-dependent modes of protein release,
an effect that was consistent with our cytochrome c release
data (Fig. 6, A and
B). In contrast, the mitochondrial matrix protein adenylate
kinase-3 was released only via PT, although oligomeric Bax
significantly enhanced this result by potentiating the onset of PT
(Fig. 6C). Taken together, our data indicate that the effect
of Bax alone is not specific for cytochrome c release
insofar as adenylate kinase-2, a different intermembrane space protein,
is also released. However, treatment of isolated mitochondria with Bax
alone does not appear to compromise matrix integrity, because this
protein did not initiate or enhance the release of adenylate kinase-3 unless PT was induced.
Although it was originally believed that MPT induction was the
root mechanism responsible for cytochrome c release in
response to different cytotoxic stimuli, more recently this notion has been challenged, and the precise mechanisms regulating the release of
this protein are unclear. In fact, many of the early results on
mechanisms of cytochrome c release were generated using
cell-free systems wherein isolated mitochondria and nuclei were treated with different PT pore activators, which, in turn, led to mitochondrial swelling, the release of cytochrome c (and other proteins),
and subsequent changes in nuclear morphology that were characteristic of apoptosis. However, ample evidence from more recent studies suggests
that although MPT is likely to be a mechanism responsible for
cytochrome c release, it is no longer regarded as the
mechanism (26). In particular, Martinou et al. (27)
demonstrated that mitochondria of neurons undergoing apoptosis, induced
by neuronal growth factor depletion, actually reduced in size and
resumed their normal function when reincubated with neuronal growth
factor. A more recent study from our laboratory demonstrated that
etoposide stimulated cytochrome c release from isolated
mitochondria, despite the presence of 1 mM EGTA (a known
inhibitor of MPT) in the reaction buffer (28). Thus, the current study
was undertaken to examine more comprehensively the mechanisms
regulating the release of this important apoptogenic protein and the
potential effect recombinant Bax protein has on this event. We clearly
demonstrate here, using isolated liver mitochondria, that cytochrome
c release occurs via distinct mechanisms that can be divided
into two groups, Ca2+-dependent and
Ca2+-independent.
As mentioned previously, it is well known that Ca2+ is the
basic PT pore activator (16). Coincidentally,
Ca2+-dependent cytochrome c release
occurs when mitochondria experience Ca2+ overload,
resulting in the induction of PT. Yet a number of reports in the
literature make claims of MPT-dependent cytochrome
c release in the absence of Ca2+; in fact, many
of these studies base this claim on the fact that CsA, a
pharmacological inhibitor of MPT, effectively blocks the release of
this apoptogenic protein (29, 30). For example, Kroemer and co-workers
(29) recently reported the ability of Bid (also a proapoptotic Bcl-2
family protein) to induce cytochrome c release by opening
the permeability transition pore, an effect they base solely on the
fact that it was preventable by CsA and other inhibitors of PT.
Although this may be the case, a far more accurate and convincing
determination of MPT would have included the presence of
Ca2+-mediated large amplitude swelling. In short, claims of
cytochrome c release via MPT in the absence of
Ca2+ are at best improbable. This was our rationale for
employing a multiparameter mitochondrial functional analysis, including measurements of swelling, decreases in Consistent with data reported by other investigators, we demonstrate
the ability of mitochondria undergoing PT to release cytochrome
c (31, 32). The fact that this effect was more pronounced in
KCl versus MSH buffers is likely to be related to the
understanding that cytochrome c binding to the inner
mitochondrial membrane is weaker in buffers with higher ionic strength
(33). In addition to cytochrome c release occurring during
full-blown MPT, our results indicate that this protein is released even
when Ca2+ loading is below the threshold needed to elicit
observable manifestations of PT, though different inhibitors of MPT,
including ADP and Mg2+, were able to suppress cytochrome
c release. Thus, it appears that low Ca2+
loading is sufficient to induce PT in a subpopulation of mitochondria, and as recently described by Rizzuto et al. (34) the
released Ca2+ is subsequently taken up by neighboring
polarized mitochondria. This effect can likely be explained by the
inherent heterogeneity of mitochondria (35) that make up any particular
population of these organelles in terms of their sensitivity to PT. In
other words, all mitochondria are not created equally.
The mechanisms responsible for Ca2+-independent cytochrome
c release are less clear, although increasing evidence
indicates that certain proapoptotic members of the Bcl-2 family of
proteins, notably Bax, are able to participate in this process. One
possible, and increasingly popular, mechanism is that in the absence of MPT, or any changes in mitochondrial volume, oligomeric Bax inserts and
forms a channel in the outer membrane large enough to allow the release
of cytochrome c (20, 36). As our data suggest, monomeric Bax
is incapable of stimulating cytochrome c release, which is
consistent with reports indicating that oligomerization of Bax is
required prior to its insertion in the membrane. It should be noted
that in addition to the ability of Bax to stimulate cytochrome
c release in the absence of any changes in mitochondrial volume, this protein also possesses the ability to enhance MPT-mediated release of cytochrome c. In this case, Bax may interact with
the permeability transition pore proteins ANT or
voltage-dependent anion channel and hasten the opening of
the pore. In fact, it was previously reported that Bax interacts
directly with ANT and facilitates ANT opening induced by atractyloside
(an ANT inhibitor) and that ANT-deficient yeast are resistant to
Bax-induced cell death, suggesting that ANT may also be a functional
target of Bax (26, 37). At the same time, it was demonstrated that Bax and Bak directly target and open voltage-dependent anion
channel in liposomes and induce changes in Recently, evidence was provided that cardiolipin, which is present
exclusively in mitochondrial membranes, mediates the targeting of
truncated Bid to mitochondria through a previously unknown three-helix
domain in truncated Bid and that this interaction is critical for
cytochrome c release (38). The involvement of cardiolipin in
the release of cytochrome c was also documented by data
showing that elevated levels of mitochondrial phospholipid hydroperoxide glutathione peroxidase in cells triggered to undergo apoptosis completely suppressed the release of cytochrome c
(39). Taken together, these findings suggest that cytochrome
c release may occur by a two-step process, wherein this
protein is first liberated from cardiolipin and then released via
specific or nonspecific pores/channels in the outer mitochondrial membrane.
In summary, we have demonstrated that mitochondria release cytochrome
c via distinct Ca2+-dependent or
Ca2+-independent mechanisms. In the first case,
mitochondrial Ca2+ overload causes swelling, rupture of the
outer mitochondrial membrane, and the nonspecific release of cytochrome
c. In contrast, Ca2+-independent protein release
is regulated by the oligomeric form of the proapoptotic protein Bax, is
specific for intermembrane space proteins, and occurs without MPT.
Importantly, oligomeric Bax also enhances MPT-induced protein release.
Which, if any, of these mechanisms predominates under physiological
conditions remains unclear. However, it is tempting to speculate that
under pathological conditions when the level of intracellular
Ca2+ increases, such as during ischemia-reperfusion injury,
swelling, rupture, and the release of mitochondrial proteins occurs by
PT, whereas during "authorized" cell death or receptor-mediated
killing this release occurs without swelling and proceeds via a Bax-
and/or truncated Bid-regulated pathway.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was performed using an electrode
sensitive to the lipophilic cation tetraphenylphosphonium (TPP+). Energized mitochondria rapidly accumulate
TPP+ from the incubation buffer and release this cation as
decays. Ca2+ fluxes across the inner mitochondrial
membrane were monitored using a Ca2+-sensitive electrode
(model 97-20; Orion Research, Inc., Beverly, MA). Mitochondrial
swelling was monitored continuously as changes in
A540. Oxygen consumption by isolated rat
liver mitochondria was measured using a Clark-type oxygen electrode
(Yellow Spring Instrument Co., Yellow Springs, OH) at 25 °C.
Mitochondria with a respiratory control ratio (defined as the rate of
respiration in the presence of ADP divided by the rate obtained
following the expenditure of ADP) above 4 were used for all
experiments. In some instances, mitochondrial respiration was uncoupled
by the addition of 1 µM carbonyl cyanide
m-chlorophenylhydrazone (CCCP). Fresh mitochondria were
prepared for each experiment and used within 4 h. At the end of
the incubation period, mitochondrial suspensions were centrifuged at
10,000 × g for 5 min, and the resulting supernatants
and/or pellets were used for Western blot analysis.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
decays (panel
B), and accumulated Ca2+ is released (panel
C). MPT-induced drops in
result in an acceleration of
respiration (panel D), and all of these manifestations of
MPT can be prevented by cyclosporin A (CsA) (panels
A-D).
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Fig. 1.
Induction of MPT in isolated rat liver
mitochondria assessed by estimation of mitochondrial swelling
(A), a drop in mitochondrial membrane potential
(B), a release of accumulated Ca2+
(C), and changes in mitochondrial oxygen consumption
(D). Mitochondria (1 mg/ml) were incubated in MSH
buffer as described under "Experimental Procedures." After a 2-min
stabilization period, mitochondria were loaded with Ca2+
(50 nmol/mg protein), and MPT was induced by adding either 100 µM tert-butyl-hydroperoxide (tBH)
or 5 mM Pi. The concentration of CsA used was 1 µM. Results are typical of five or more independent
experiments.
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Fig. 2.
MPT-induced release of cytochrome
c (Cyt c) from mitochondria incubated
in either MSH or KCl buffer. A, mitochondria (1 mg/ml)
were incubated as described under "Experimental Procedures." After
a 2-min stabilization period, mitochondria were loaded with
Ca2+ (50 nmol/mg protein), and MPT was induced by adding 5 mM Pi. B, mitochondrial suspensions
from A were centrifuged, and the resulting supernatants were
separated by SDS polyacrylamide gel electrophoresis and Western blotted
as described under "Experimental Procedures." C,
mitochondrial respiration following MPT induction in either MSH or KCl
buffer was analyzed as described under "Experimental Procedures."
D, mitochondrial suspensions from C were
centrifuged, and the resulting supernatant (S) and pellet
(P) fractions were separated by SDS polyacrylamide gel
electrophoresis and used for Western blot analysis. Results are typical
of five or more independent experiments.
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Fig. 3.
Ca2+-induced release of
cytochrome c (Cyt c) from
mitochondria in the absence of observable PT. A,
mitochondria (1 mg/ml) were incubated in KCl buffer as described under
"Experimental Procedures." After a 2-min stabilization period,
mitochondria were loaded with Ca2+ (25 nmol/mg protein)
prior to the addition of 1 µM CCCP at 5 min.
B, samples incubated under the same conditions as in
A were used to evaluate the rate of uncoupled respiration
after 1 and 5 min. C, the amount of cytochrome c
released from mitochondria after 1 and 5 min of Ca2+
retention ± 0.5 mM ADP or 1 mM
Mg2+. D, samples incubated under the same
conditions as in C were used to determine the effect of
inhibitors of MPT on the rate of uncoupled respiration of
Ca2+-loaded mitochondria (*, p < 0.05).
Results are typical of five or more independent experiments.
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Fig. 4.
The effect of Ca2+ loading on
mitochondrial swelling and the release of cytochrome
c. Mitochondria (0.5 mg/ml) were incubated as
described under "Experimental Procedures" in 2 ml of KCl buffer.
A, mitochondria were loaded sequentially with varied amounts
of Ca2+ until MPT was induced. B, samples were
taken after 5 (lanes 1-6) or 8 min (lane 7) of
incubation, and the supernatants were evaluated for cytochrome
c (Cyt c) content. Results are typical of five or
more independent experiments.
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Fig. 5.
Bax-induced release of cytochrome
c from mitochondria. A, the different
preparations of Bax protein used in the experiments. Lane 1,
fraction of Bax containing both oligomeric and monomeric forms;
Lane 2, fraction of Bax, containing predominantly the
monomeric form. B, MPT-independent release of cytochrome
c (Cyt c) induced by the different preparations
of Bax. Mitochondria were incubated in KCl buffer containing 1 mM EGTA, and samples were taken for Western blot analysis
after 10 min of incubation. Both forms of Bax (Bax-oligo and
Bax-mono) were added at a final concentration of 20 µg/ml.
C, effect of the different preparations of Bax on
mitochondrial permeability transition assessed by estimation of
accumulated Ca2+ release. After a 2-min stabilization
period, 20 µg/ml Bax protein (oligomeric or monomeric form) was added
to the mitochondrial suspension followed by the addition of
Ca2+ (40 nmol/mg protein), and the organelles were
incubated until MPT occurred. D, same conditions as in
C, except mitochondrial permeability transition was assessed
by estimation of decreases in mitochondrial membrane potential.
E, effect of oligomeric Bax on Ca2+-induced
cytochrome c release after 6 min of incubation.
F, comparison of Bax-induced cytochrome c release
with or without MPT. Results are typical of five or more independent
experiments.
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Fig. 6.
Individual and combined abilities of Bax and
Ca2+ to induce the release of intermembrane space
(A and B) and matrix
(C) proteins. After a 2-min stabilization period,
mitochondria were treated with 20 µg/ml oligomeric Bax and/or
Ca2+ (40 nmol/mg protein), and samples were taken for
Western blot analysis after either 10 (control or Bax alone) or 6 min
(Ca2+ plus Bax or Ca2+ alone) of incubation.
Results are typical of three or more independent experiments. Cyt
c, cytochrome c; AK, adenylate kinase.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, changes in respiration, and the release of accumulated Ca2+, to more accurately
link changes in mitochondrial integrity and the release of cytochrome
c.
(21). Interestingly,
it should be noted that more recent results from our laboratory
indicate that Ca2+-independent cytochrome c
release can also occur via mitochondrial swelling stimulated by
alterations in osmotic
balance.2
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ACKNOWLEDGEMENTS |
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We thank Dr. Bruno Antonsson (Serono Pharmaceutical Research Institute, Geneva, Switzerland) for kindly providing pBadHisBax plasmid and Marina Protopopova (Microbiology and Tumor Biology Center, Karolinska Institutet) and Margareta Sandström (Institute of Environmental Medicine, Karolinska Institutet) for preparation of the recombinant Bax proteins. We also thank Dr. Takafumi Noma (Yamaguchi University School of Medicine, Yamaguchi, Japan) for providing the anti-adenylate kinase-2 and anti-adenylate kinase-3 antibodies.
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FOOTNOTES |
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* This work was supported in part by grants from the Swedish Medical Research Council (03X-2471) and the Swedish Cancer Society (Cancerfonden; 3829-B98-03XAC). V. G. was supported by grants from the Royal Swedish Academy of Sciences and the Russian Foundation of Basic Research (01-04-48191), and J. D. R. was supported by a Visiting Scientist grant from The Swedish Foundation for International Cooperation in Research and Higher Education (STINT).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.
Permanent address: Inst. of Theoretical and Experimental
Biophysics, Pushchino 142290, Russia.
§ To whom correspondence should be addressed. Tel.: 46 8 33 58 74; Fax: 46 8 32 90 41; E-mail: Sten.Orrenius@imm.ki.se.
Published, JBC Papers in Press, March 22, 2001, DOI 10.1074/jbc.M100614200
2 V. Gogvadze, J. D. Robertson, B. Zhivotovsky, and S. Orrenius, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
ANT, adenine
nucleotide translocator;
MPT, mitochondrial permeability transition;
MSH, mannitol/sucrose/Hepes;
TPP+, tetraphenylphosphonium;
CCCP, carbonyl cyanide m-chlorophenylhydrazone;
CsA, cyclosporin A;
, mitochondrial membrane potential;
PT, permeability transition.
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