From the Consiglio Nazionale delle Ricerche Unit for the Study of
Biomembranes at the Departments of Biomedical Sciences
and
Biological Chemistry, University of Padova, Viale Giuseppe
Colombo 3, I-35100 Padova, Italy
Received for publication, November 26, 2000, and in revised form, December 26, 2000
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ABSTRACT |
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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-Co2+ 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 tetramethylrhodamine 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 distribution require longer PTP
openings, which cause release of cytochrome c and may
result in cell death. Modulation 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) underlying its release are the
matter of intense investigation and of considerable controversy.
Mitochondria from a variety of tissues can be induced to 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). Since the PT is accompanied by swelling as well as by
cytochrome 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 truncated BID followed by oligomerization of BAX and/or
BAK (15-18 and see Ref. 19 for review). However, it has been reported 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 mutually 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 interpretation of the results obtained with fluorescent probes
(13) and from the fact that detection methods based on cell disruption
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
depolarization, 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 depolarization, these findings suggest
that relatively short PTP openings are detected only by trapped calcein
and have little impact on cell viability, while detectable changes of
TMRM distribution require longer PTP openings, which eventually cause
cytochrome 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.
Cell Cultures--
MH1C1 rat hepatoma cells were seeded onto
uncoated 22-mm (for calcein and annexin-V staining) or 13-mm (for
immunofluorescence) 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 potentiometric 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 µM CsH, which inhibits the MDR pump (13), but not the PTP
(26). MH1C1 cells were loaded with 1 µM
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 acquisition
was started 30 min later, a protocol that made addition of CsH
unnecessary in the experiments with calcein (results not shown).
Cellular fluorescence images were acquired with an Olympus IMT-2
inverted microscope, equipped with a xenon light source (75 watts) for
epifluorescence illumination and with a 12-bit digital cooled CCD
camera (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 immersion objective (Nikon). Data were acquired and
analyzed using Metamorph 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 Nonidet 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 antibody (PharMingen, clone 6H2.B4)
and with an affinity-purified rabbit antibody against the rat
bc1 complex (a generous gift of Prof. Roberto
Bisson, Padova, Italy). Cells were then sequentially incubated for 15 min at 37 °C with tetramethylrhodamine
isothiocyanate-conjugated goat anti-mouse IgG and with
fluorescein isothiocyanate-conjugated goat anti-rabbit IgG.
For cytochrome c and bc1 complex
detection, red and green channel images were acquired simultaneously
using two separate color channels 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 subsequent 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./
Annexin-V and propidium iodide staining and imaging were carried out
exactly as described previously (27).
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 decrease of calcein
fluorescence that was due to PTP opening (panel A,
squares), as indicated here by inhibition of the
fluorescence 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 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).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
)red/(S.D./
)green. A punctate
distribution results in a higher S.D., while normalization allows
correction for different fluorescence 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.
View larger version (21K):
[in a new window]
Fig. 1.
Immunofluorescence detection of the
bc1 complex and cytochrome
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 after immunofluorescence labeling
as described under "Materials and Methods." Secondary antibodies
against the bc1 complex were fluoresceinated
(green color in the images), while antibodies against
cytochrome c were conjugated with tetramethylrhodamine
isothyocyanate (red color in the images). Bar, 10 µm. The fluorescence 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
cytochrome c profiles, respectively.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (15K):
[in a new window]
Fig. 2.
Changes of mitochondrial calcein and TMRM
fluorescence intensities induced by arachidonic acid. MH1C1 cells
were coloaded with calcein-AM and CoCl2 (panel
A) or TMRM (panel B) as described under "Materials
and Methods," and images were collected at 60-s intervals. In the
experiments denoted by circles, cells had been treated with
2 µM CsA. Where indicated (arrows) 200 µM AA and 2 µM 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 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).
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 panel A in Fig. 2), and essentially complete
within about 3 min of the addition of A23817. The fluorescence changes
were still 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).
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We next tested whether depolarization with the protonophore FCCP was
able to cause PTP opening in MH1C1 cells in the absence of inducing
agents like AA or A23187. The experiments of Fig. 4, panel A, indicate that no
changes of calcein fluorescence could be elicited by FCCP either in the
absence or presence of CsA (squares and circles,
respectively) and that, consistently, the FCCP-dependent decrease of TMRM fluorescence was not affected by CsA (panel
B, symbols are the same as in panel A). These
experiments thus allowed to define three clear-cut conditions: (i) PTP
openings matched by a measurable mitochondrial depolarization (addition
of AA, Fig. 2); (ii) PTP openings not matched by a measurable
mitochondrial depolarization (addition of A23187, Fig. 3); and (iii)
mitochondrial depolarization without PTP opening (addition of FCCP,
Fig. 4). The next question we addressed was whether any of these
conditions was associated to release of mitochondrial cytochrome
c and cell death.
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In the experiments of Fig. 5 the
distribution of cytochrome c and of the mitochondrial
bc1 complex were studied in individual cells
in situ with the quantitative technique described under "Materials and Methods." It can be clearly appreciated that the addition of FCCP had negligible effects on the cytochrome 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 cytochrome 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 cytochrome c
redistribution was dependent on PTP opening, as shown by its almost
complete inhibition by CsA (closed symbols in Fig. 5).
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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 experiments 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).
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DISCUSSION |
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Preliminary Considerations-- The PTP has been extensively characterized in mitochondrial suspensions (29-31), in individual organelles (32), and at the single channel level (33 and 34 and see Ref. 8 for a comprehensive review). Although its structural 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 advances, 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 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 depolarization 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 openings 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, 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 membrane, PTP opening can be studied as a CsA-sensitive quenching 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 results 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 correlated better with the decrease of mitochondrial TMRM fluorescence 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 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 shrinkage 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 peripheral (intermembrane) space, and sometimes between themselves, through very narrow tubular regions (42). These findings are in good agreement with earlier work demonstrating 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 instrumental to make cytochrome c available for release.
In summary, the present results demonstrate that PTP opening 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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FOOTNOTES |
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* 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 Membrana" (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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence 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 University Medical School, Dana-Farber Cancer Institute, SM758, One Jimmy Fund Way, Boston, MA 01225.
Published, JBC Papers in Press, December 27, 2000, DOI 10.1074/jbc.M010604200
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ABBREVIATIONS |
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The abbreviations used are: PT, permeability transition; PTP, permeability transition pore; AA, arachidonic acid; CsA and CsH, cyclosporin A and cyclosporin H, respectively; TMRM, tetramethylrhodamine methyl ester; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; MDR, multidrug resistance.
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