THE JOURNAL OF BIOLOGICAL CHEMISTRY
12030 –12034, 2001
© 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
The Mitochondrial Permeability Transition, Release of
Cytochrome c and Cell Death
CORRELATION WITH THE DURATION OF PORE OPENINGS IN SITU*
Received for publication, November 26, 2000, and in revised form, December 26, 2000
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M010604200
Valeria Petronilli‡§, Daniele Penzo‡, Luca Scorrano‡¶, Paolo Bernardi‡§, and Fabio Di Lisa§储
From the Consiglio Nazionale delle Ricerche Unit for the Study of Biomembranes at the Departments of ‡Biomedical
Sciences and 储Biological Chemistry, University of Padova, Viale Giuseppe Colombo 3, I-35100 Padova, Italy
We investigated the relationship between opening of
the permeability transition pore (PTP), mitochondrial
depolarization, cytochrome c release, and occurrence of
cell death in rat hepatoma MH1C1 cells. Treatment with
arachidonic acid or A23187 induces PTP opening in situ
with similar kinetics, as assessed by the calcein loading-
Co2ⴙ quenching technique (Petronilli, V., Miotto, G.,
Canton, M., Colonna, R., Bernardi, P., and Di Lisa, F.
(1999) Biophys. J. 76, 725–734). Yet depolarization, as
assessed from the changes of mitochondrial tetrameth-
ylrhodamine methyl ester (TMRM) fluorescence, is
rapid and extensive with arachidonic acid and slow and
partial with A23187. Cyclosporin A-inhibitable release of
cytochrome c and cell death correlate with the changes
of TMRM fluorescence but not with those of calcein
fluorescence. Since pore opening must be accompanied
by depolarization, we conclude that short PTP openings
are detected only by trapped calcein and may have little
impact on cell viability, while changes of TMRM distri-
bution require longer PTP openings, which cause re-
lease of cytochrome c and may result in cell death. Mod-
ulation of the open time appears to be the key element in
determining the outcome of stimuli that converge on the
PTP.
One of the key events in the course of apoptosis is the release
of cytochrome c from mitochondria (1), which is able to activate
procaspase 9 (2) and thus downstream caspases that amplify
the death process (see Ref. 3 for review). Other mitochondrial
proteins can be released as well, including apoptosis-inducing
factor (4) and Smac/DIABLO, which promotes apoptosis by
inactivating inhibitors of apoptosis proteins (5–7). Cytochrome
c remains by far the most studied, and the mechanism(s) un-
derlying its release are the matter of intense investigation and
of considerable controversy.
Mitochondria from a variety of tissues can be induced to
* This work was supported by grants from the Consiglio Nazionale
delle Ricerche, the Ministero per l’Università e la Ricerca Scientifica e
Tecnologica “Il Mantenimento della Vitalità Miocardica a Discapito
della Necrosi” (to F. D. L.), and “Bioenergetica e Trasporto di Mem-
brana” (to P. B.), by Telethon-Italy Grant 1141 (to P. B.), and by the
Armenise-Harvard foundation (to P. B.). The costs of publication of this
article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence and reprints requests may be addressed.
E-mail: petro@civ.bio.unipd.it; bernardi@civ.bio.unipd.it; dilisa@civ.
bio.unipd.it.
¶ Present address: Dept. of Pathology and Medicine, Harvard Uni-
versity Medical School, Dana-Farber Cancer Institute, SM758, One
Jimmy Fund Way, Boston, MA 01225.
12030
Vol. 276, No. 15, Issue of April 13, pp.
Printed in U.S.A.
undergo an inner membrane permeability increase, the PT,1
which allows diffusion of solutes with molecular mass up to
about 1,500 Da. It is widely accepted that this transition is
mediated by opening of an inner membrane high conductance
channel, the PTP (see Ref. 8 for a recent review). The PTP has
been shown to be implicated in ischemic cell death through
dysregulation of Ca2⫹ homeostasis and ATP depletion (9 –11).
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Since the PT is accompanied by swelling as well as by cyto-
chrome c release in vitro (12), it also represents an excellent
candidate for the release of intermembrane proteins in the
course of apoptosis as well (13, 14). An alternative mechanism
for cytochrome c release is outer membrane insertion of trun-
cated BID followed by oligomerization of BAX and/or BAK
(15–18 and see Ref. 19 for review). However, it has been re-
ported that insertion of BAX/BAK in the outer mitochondrial
membrane can also cause cytochrome c release and cell death
through a PT (20, 21), and BNIP3 (a BCL-2 family member) can
cause a PT and cell death without release of cytochrome c and
caspase activation (22). The mechanistic relationships between
PT, cytochrome c release, and cell death remain therefore a
matter of intense debate. It is conceivable that an overlap may
exist between different mechanisms (which often are not mu-
tually exclusive); but it should also be recognized that different
pathways may be activated in different paradigms of cell death.
Finally, apparent discrepancies may also arise from the inter-
pretation of the results obtained with fluorescent probes (13)
and from the fact that detection methods based on cell disrup-
tion can cause rather than measure cytochrome c release (23).
In this study we investigated the occurrence of cell death in
rat hepatoma MH1C1 cells treated with AA, a potent PTP
inducer that is characterized in the accompanying article (24),
or with the ionophore A23187. We found that both treatments
cause PTP opening in situ with similar kinetics, as assessed by
the calcein loading-Co2⫹ quenching technique (25), while depo-
larization, as assessed from the fluorescence changes of the
potentiometric probe TMRM, was rapid and extensive with AA
and slow and partial with A23187. A parallel assessment of cell
viability and of CsA-inhibitable cytochrome c release with a
quantitative in situ method showed that cell death correlates
with the TMRM rather than with the calcein fluorescence
changes. Since pore opening must be accompanied by depolar-
ization, these findings suggest that relatively short PTP open-
ings are detected only by trapped calcein and have little impact
on cell viability, while detectable changes of TMRM distribu-
tion require longer PTP openings, which eventually cause cy-
1
The abbreviations used are: PT, permeability transition; PTP, per-
meability transition pore; AA, arachidonic acid; CsA and CsH, cyclos-
porin A and cyclosporin H, respectively; TMRM, tetramethylrhodamine
methyl ester; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydra-
zone; MDR, multidrug resistance.
This paper is available on line at http://www.jbc.org
 PTP Opening and Cell Death
FIG. 1. Immunofluorescence detec-
tion of the bc1 complex and cyto-
chrome c: effects of arachidonic acid.
MH1C1 cells were treated for 30 min with
vehicle (ethanol: 0.02% v/v; panel A) or
with 0.2 mM AA (panel B). Images were
acquired with the confocal microscope af-
ter immunofluorescence labeling as de-
scribed under “Materials and Methods.”
Secondary antibodies against the bc1 com-
plex were fluoresceinated (green color in
the images), while antibodies against cy-
tochrome c were conjugated with tetram-
ethylrhodamine isothyocyanate (red color
in the images). Bar, 10 ␮m. The fluores-
cence intensity profiles reported in panels
A⬘ and B⬘ correspond to the lines drawn in
panels A and B, respectively; and green
and red lines refer to the bc1 and cyto-
chrome c profiles, respectively.
tochrome c release and cell death. Thus, modulation of the open
time appears to be the key element in determining the outcome
of stimuli that impinge on the PTP.
MATERIALS AND METHODS
Cell Cultures—MH1C1 rat hepatoma cells were seeded onto un-
coated 22-mm (for calcein and annexin-V staining) or 13-mm (for im-
munofluorescence) diameter round glass coverslips and grown for 2
days in Ham’s F-10 nutrient mixture supplemented with 10% fetal calf
serum in a humidified atmosphere of 95% air, 5% CO2 at 37 °C in a
Forma tissue culture water-jacketed incubator.
TMRM and Calcein Staining and Imaging—MH1C1 cells were
loaded with 10 nM TMRM and incubated as specified in the legend of
Fig. 2. The extent of cell and hence mitochondrial loading with poten-
tiometric probes is affected by the activity of the plasma membrane
MDR P-glycoprotein, which is inhibited by CsA (13). Treatment with
this drug therefore causes an increased mitochondrial fluorescence that
can be erroneously interpreted as an increase of the mitochondrial
membrane potential (see Ref. 13 for discussion). To prevent this artifact
and to normalize the loading conditions, in all experiments with TMRM
the medium was supplemented with 1.6 ␮M CsH, which inhibits the
MDR pump (13), but not the PTP (26). MH1C1 cells were loaded with 1
␮M calcein-acetomethoxy ester for 30 min at 37 °C in 1 ml of Hanks’
balanced salt solution supplemented with 10 mM Hepes, pH 7.4, and 1
mM CoCl2 (25). Cells were then washed free of calcein and Co2⫹ and
maintained in Hanks’ balanced salt solution-Hepes. When specified,
CsA was added to the cells after probe loading, and fluorescence acqui-
sition was started 30 min later, a protocol that made addition of CsH
unnecessary in the experiments with calcein (results not shown). Cel-
lular fluorescence images were acquired with an Olympus IMT-2 in-
verted microscope, equipped with a xenon light source (75 watts) for
epifluorescence illumination and with a 12-bit digital cooled CCD cam-
era (Micromax, Princeton Instruments). For detection of fluorescence,
488 ⫾ 25 nm excitation and 525 nm longpass emission and 568 ⫾ 25 nm
excitation and 585 nm longpass emission filter settings were used for
calcein and TMRM, respectively. Images were collected with exposure
times ranging between 50 and 100 ms using a 40⫻, 1.3 NA oil immer-
sion objective (Nikon). Data were acquired and analyzed using Meta-
morph software (Universal Imaging). Clusters of several mitochondria
(10 to 30) were identified as regions of interest, and fields not containing
cells were taken as the background. Sequential digital images were
acquired every 60 s or every 3 min for the experiments with a time
course of 20 and 60 min, respectively, and the average fluorescence
intensity of all relevant regions was recorded and stored for subsequent
analysis. Mitochondrial fluorescence intensities minus background are
reported in Figs. 2– 4 after normalization of the initial fluorescence for
comparative purposes, and they represent the mean of 10 regions of
interest.
Immunodetection of bc1 Complex and Cytochrome c—MH1C1 cells
were incubated as detailed in the figure legends and then washed. Cells
were fixed for 30 min at room temperature with 3.7% (v/v) ice-cold
formaldehyde, permeabilized for 20 min with 0.01% (v/v) ice-cold Non-
idet P-40, and incubated for 15 min with a 0.5% solution of BSA and
then for 15 min at 37 °C with a mouse monoclonal anti-cytochrome c
12031
antibody (PharMingen, clone 6H2.B4) and with an affinity-purified
rabbit antibody against the rat bc1 complex (a generous gift of Prof.
Downloaded from http://www.jbc.org/ by guest on November 17, 2019
Roberto Bisson, Padova, Italy). Cells were then sequentially incubated
for 15 min at 37 °C with tetramethylrhodamine isothiocyanate-conju-
gated goat anti-mouse IgG and with fluorescein isothiocyanate-conju-
gated goat anti-rabbit IgG.
For cytochrome c and bc1 complex detection, red and green channel
images were acquired simultaneously using two separate color chan-
nels on the detector assembly of a Nikon Eclipse E600 microscope
equipped with a Bio-Rad MRC-1024 laser scanning confocal imaging
system and with 488/522 ⫾ 25 nm bandpass and 568/605 nm longpass
filter settings, and a 60⫻, 1.4 NA oil immersion objective (Nikon).
Twenty randomly chosen fields in each coverslip were stored for sub-
sequent analysis.
Fig. 1 shows an example of the quantitative analysis carried out on
a control (panel A) and AA-treated MH1C1 cell (panel B). Using the
Bio-Rad LaserSharp analysis program a set of lines was drawn across
the cells (only two such lines are illustrated in panels A and B for the
sake of clarity). Using the appropriate function of the analysis program,
the fluorescence intensity of each pixel along the line in both the green
and the red channel was measured, and panels A⬘ and B⬘ report the
fluorescence intensity profiles along the lines drawn in panels A and B,
respectively. The localization index is defined as the ratio of the S.D. of
the fluorescence intensity divided by the total fluorescence for each
channel: (S.D./⌺)red/(S.D./⌺)green. A punctate distribution results in a
higher S.D., while normalization allows correction for different fluores-
cence intensities in the two channels. A localization index of 1 indicates
that cytochrome c and the bc1 complex have the same distribution,
which is expected in normal cells, while an index lower than 1 means
that the distribution of cytochrome c is more homogeneous than that of
the bc1 complex. In the example of Fig. 1 the localization index is 1 for
the cell of panel A and 0.6 for the cell in panel B.
Annexin-V and propidium iodide staining and imaging were carried
out exactly as described previously (27).
RESULTS
The experiments of Fig. 2, panel A, report the fluorescence
changes of mitochondria loaded with calcein in the presence of
Co2⫹ in intact cells, a method that allows detection of PTP
openings in situ (25). Addition of AA resulted in a large de-
crease of calcein fluorescence that was due to PTP opening
(panel A, squares), as indicated here by inhibition of the fluo-
rescence changes by CsA (panel A, circles; see also Ref. 24). A
similar experiment was carried out in cells loaded with TMRM,
a probe that accumulates within polarized mitochondria
wherefrom it is released upon depolarization (28). It can be
seen (panel B) that the addition of AA was followed by a large,
CsA-sensitive depolarization with the same time course as that
displayed by the changes of calcein fluorescence (compare with
panel A) and comparable in extent to that observed upon the
addition of FCCP to CsA-treated cells (panel B) or to cells that
had not been treated with AA (see Fig. 4, panel B). These
12032 PTP Opening and Cell Death

FIG. 2. Changes of mitochondrial calcein and TMRM fluores- FIG. 3. Changes of mitochondrial calcein and TMRM fluores-
cence intensities induced by arachidonic acid. MH1C1 cells were cence intensities induced by A23187. Experimental conditions and
coloaded with calcein-AM and CoCl2 (panel A) or TMRM (panel B) as symbols were exactly as in Fig. 2. Where indicated (arrows) 2 ␮M
described under “Materials and Methods,” and images were collected at A23187 and 2 ␮M FCCP were added.
60-s intervals. In the experiments denoted by circles, cells had been
treated with 2 ␮M CsA. Where indicated (arrows) 200 ␮M AA and 2 ␮M
FCCP were added. The initial fluorescence intensities were normalized
for comparative purposes, and values on the ordinate report the mean ⫾
S.D. of four independent experiments. Within the time course of this
experiment, no significant changes of mitochondrial calcein or TMRM

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fluorescence intensities were observed when cells were treated with
vehicle (ethanol: 0.02%, v/v) or CsA in the absence of further additions
(omitted for clarity).

results indicate that PTP opening by AA causes mitochondrial

depolarization in situ, a finding that was also obtained in
nominally Ca2⫹-free media (results not shown).
A similar experiment was carried out with the divalent metal
ionophore A23187. Fig. 3, panel A, shows that addition of
A23187 caused a rapid drop of calcein fluorescence (squares),
which was much faster than that caused by AA (compare with FIG. 4. Changes of mitochondrial calcein and TMRM fluores-
cence intensities induced by FCCP. Experimental conditions and
panel A in Fig. 2), and essentially complete within about 3 min symbols were exactly as in Fig. 2. Where indicated (arrows) 2 ␮M FCCP
of the addition of A23817. The fluorescence changes were still was added.
due to PTP opening, as indicated by their sensitivity to CsA
(circles). Quite unexpectedly, however, the changes of TMRM
fluorescence were negligible over the same time frame and
slowly decreased by only 20% in about 20 min of incubation
(panel B, squares). These TMRM fluorescence changes were
also due to the PTP, since they could be blocked by CsA, which
instead did not affect the probe response to FCCP (panel B,
circles). These experiments demonstrate that PTP openings
may occur that are accompanied by negligible changes of
TMRM fluorescence and suggest that the time required for
redistribution of potentiometric probes like TMRM may be too
long (28) to report these PTP openings, which we assume to be
of short duration. At variance from the case of AA, the PTP-
inducing effects of A23187 disappeared in Ca2⫹-free media
(results not shown).
We next tested whether depolarization with the protono-
phore FCCP was able to cause PTP opening in MH1C1 cells in
the absence of inducing agents like AA or A23187. The exper-
iments of Fig. 4, panel A, indicate that no changes of calcein FIG. 5. Effects of arachidonic acid, A23187, or FCCP treatment
fluorescence could be elicited by FCCP either in the absence or on the intracellular cytochrome c localization. MH1C1 cells were
presence of CsA (squares and circles, respectively) and that, treated for the indicated times with 0.2 mM AA (squares), 2 ␮M A23187
consistently, the FCCP-dependent decrease of TMRM fluores- (circles), 2 ␮M FCCP (triangles) or vehicle (0.02% v/v ethanol, dia-
monds). In the experiments denoted by solid symbols, cells were pre-
cence was not affected by CsA (panel B, symbols are the same treated with 2 ␮M CsA for 15 min. Cells were fixed and treated with
as in panel A). These experiments thus allowed to define three antibodies against the bc1 complex and cytochrome c, and the cyto-
clear-cut conditions: (i) PTP openings matched by a measurable chrome c localization index was determined exactly as described under
mitochondrial depolarization (addition of AA, Fig. 2); (ii) PTP “Materials and Methods” and in the legend to Fig. 1. Values are mean ⫾
S.D. of five different experiments. No significant changes of the local-
openings not matched by a measurable mitochondrial depolar- ization index were observed when cells were treated with CsA in the
ization (addition of A23187, Fig. 3); and (iii) mitochondrial absence of further additions (omitted for clarity).
depolarization without PTP opening (addition of FCCP, Fig. 4).
The next question we addressed was whether any of these and of the mitochondrial bc1 complex were studied in individ-
conditions was associated to release of mitochondrial cyto- ual cells in situ with the quantitative technique described
chrome c and cell death. under “Materials and Methods.” It can be clearly appreciated
In the experiments of Fig. 5 the distribution of cytochrome c that the addition of FCCP had negligible effects on the cyto-
 PTP Opening and Cell Death
FIG. 6. Effects of arachidonic acid, A23187, and FCCP on cells
staining with annexin-V and propidium iodide. MH1C1 cells
grown on coverslips were treated for 45 min with 0.2 mM AA, 2 ␮M
A23187, 2 ␮M FCCP, or vehicle only (control, 0.02% v/v ethanol). When
indicated cells were pretreated with 2 ␮M CsA for 15 min. Cells were
stained with annexin-V-FLUOS and propidium iodide, and images of
cells fields on coverslips were acquired with the epifluorescence micro-
scope as described previously (27). The fraction of annexin-V-positive
cells (filled bars) and double positive cells (hatched bars) was calculated
from 20 randomly chosen fields from four independent experiments,
and values are mean ⫾ S.D.
chrome c distribution (open triangles). On the other hand,
within 30 min of the addition of A23187 the localization index
decreased from 1.0 to 0.8 (open circles), a finding that closely
matched the A23817-dependent fluorescence changes of TMRM
but not those of calcein (compare with Fig. 3). Finally, the
addition of AA caused a drop in the localization index of cyto-
chrome c to 0.5 within 10 min (open squares), which matched
most closely the kinetics of PTP-dependent changes of TMRM
fluorescence (compare with Fig. 2). In these protocols cyto-
chrome c redistribution was dependent on PTP opening, as
shown by its almost complete inhibition by CsA (closed symbols
in Fig. 5).
We finally assessed the consequences of treatment with AA,
A23187, and FCCP on cell viability by double staining with
fluoresceinated annexin-V and propidium iodide. The experi-
ments of Fig. 6 document that within 30 min of treatment with
AA about 50% of the cells displayed an apoptotic phenotype (i.e.
they were positive for Annexin-V only), while this figure was
less than 30% after treatment with A23187 and negligible
relative to control cells after treatment with FCCP, and CsA
effectively prevented cell death by both AA and A23187. As the
incubation proceeded, most AA-treated cells became permeable
to propidium iodide (results not shown).
DISCUSSION
Preliminary Considerations—The PTP has been extensively
characterized in mitochondrial suspensions (29 –31), in individ-
ual organelles (32), and at the single channel level (33 and 34
and see Ref. 8 for a comprehensive review). Although its struc-
tural features are still a matter of debate (see e.g. Ref. 35), the
functional properties of the PTP and the consequences of a PT
in vitro are relatively well understood (8). Despite these ad-
vances, it remains extremely difficult to make predictions
about the occurrence and regulation of the PT in situ, in part
because the PTP is detected by indirect means, most often
based on the expected changes of the mitochondrial membrane
potential, and in part because multiple PTP modulatory factors
change at the same time. A striking example is represented by
12033
the effects of FCCP. The pore is voltage-dependent, in the sense
that depolarization favors PTP opening (36), yet the effect of
depolarization may be offset by matrix acidification (37) and by
the increase of matrix ADP and Mg2⫹, which all prevent pore
opening. The experiments reported here indicate that depolar-
ization with FCCP is not followed by PTP opening in MH1C1
cells, suggesting that PTP inhibitory factors prevail despite
depolarization. Two main issues should be considered.
(i) The open-closed transitions of individual channels occur
well below the time range of the msec (33), which imposes
obvious constraints in measurements based on redistribution
of potentiometric fluorescent probes in situ. Indeed, transient
PTP openings may not be detected when the probe response
time is below the PTP open time, implying that only relatively
long lasting PTP openings may be reliably detected by these
probes in situ. Furthermore, even TMRM-detectable PTP open-
ings may occur asynchronously in individual mitochondria (32)
and would be missed unless single mitochondria are resolved.
(ii) The cellular accumulation of potentiometric probes is not
a simple function of the mitochondrial membrane potential. It
also depends on net transport across the plasma membrane,
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which is determined by the plasma membrane potential (which
drives the accumulation) and by the activity of the MDR pump
(which extrudes the probe). Mitochondrial accumulation will be
affected by changes of the plasma membrane potential (38, 39),
and inhibition of the MDR pump will inevitably increase the
mitochondrial probe accumulation (13). It is unfortunate that
both the PTP and the MDR pump are inhibited by CsA (13), a
finding that calls into question the interpretation of a large
number of experiments where the PTP has been considered as
a causative event in cell death (13).
Because of these problems, we have developed a method for
in situ PTP detection that is based on trapping of calcein
followed by quenching of cytosolic fluorescence by Co2⫹. Since
calcein and Co2⫹ cannot cross the mitochondrial inner mem-
brane, PTP opening can be studied as a CsA-sensitive quench-
ing of mitochondrial calcein fluorescence (25). To address the
second problem we have included CsH (which inhibits the MDR
pump but not the PTP) in our measurements. Given that the
MDR pump is already inhibited by CsH, the effects of CsA on
the TMRM signal coming from mitochondria must be due to
effects on the PTP. A contribution from variations of the
plasma membrane potential cannot be excluded easily, yet this
was not the major cause of the TMRM fluorescence changes in
our protocols, because they were also observed in KCl-based
media (results not shown).
PTP Opening and Cytochrome c Release—Based on the re-
sults of the present paper, we conclude that the changes of
calcein fluorescence are able to detect PTP openings of shorter
duration than those detectable by TMRM. Indeed, addition of
A23187 induced large CsA-sensitive fluorescence changes of
calcein but not of TMRM (Fig. 3). Cytochrome c release corre-
lated better with the decrease of mitochondrial TMRM fluores-
cence than with changes of calcein fluorescence, suggesting
that only relatively long lasting pore openings eventually cause
cytochrome c release. The mechanism through which PTP
opening may cause cytochrome c release remains unsolved.
One possibility is that osmotic swelling causes outer membrane
rupture and release of other intermembrane proteins as well,
such as Smac/DIABLO (5, 7) and AIF (40). Outer membrane
rupture may not occur in all mitochondria at the same time,
nor necessarily cause major structural damage. Indeed, Farber
and co-workers (41) have shown that transient PTP openings in
vitro are not followed by detectable swelling in saline media,
yet cause CsA-sensitive cytochrome c release. This could occur
through swelling-shrinkage cycles of individual mitochondria
12034 PTP Opening and Cell Death
in a nonsynchronized fashion (41). It is noteworthy that even
after PTP-dependent large amplitude swelling of the whole
mitochondrial population pore closure was followed by shrink-
age with full functional recovery provided that cytochrome c
was added back (12). This finding indicates that no permanent
damage to the inner membrane is caused by even long lasting
PTP openings (12).
An alternative possibility is that subtle changes of matrix
volume may make more cytochrome c available for selective
release (13). Tomographic reconstruction of thick sections of
mitochondria after high voltage electron microscopy has indeed
revealed that the intercristal spaces, which contain cytochrome
c, are pleiomorphic structures that communicate with the pe-
ripheral (intermembrane) space, and sometimes between
themselves, through very narrow tubular regions (42). These
findings are in good agreement with earlier work demonstrat-
ing that only 10 –15% of cytochrome c is available for reduction
by outer membrane NADH-cytochrome b5 reductase and that
this fraction can be effectively increased by matrix swelling
(43). If this compartmentation also occurs in vivo, the matrix/
intercristal volume changes caused by a PT could be instru-
mental to make cytochrome c available for release.
In summary, the present results demonstrate that PTP open-
ing can be a causative event in cytochrome c release in situ
irrespective of whether the latter occurs through a selective
pathway or because of outer membrane rupture. Further work
will be required to define the relevance of this mechanism to
endogenous signaling pathways of cell death.
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 The Mitochondrial Permeability Transition, Release of Cytochrome c and Cell
Death: CORRELATION WITH THE DURATION OF PORE OPENINGS IN SITU
Valeria Petronilli, Daniele Penzo, Luca Scorrano, Paolo Bernardi and Fabio Di Lisa
J. Biol. Chem. 2001, 276:12030-12034.
doi: 10.1074/jbc.M010604200 originally published online December 27, 2000

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