Department of Physiology, University College London, London, WC1E 6BT, UK
* Author for correspondence (e-mail:m.duchen{at}ucl.ac.uk )
Accepted 19 December 2001
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Summary |
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Key words: Mitochondria, Oxidative stress, Permeability transition pore, Photosensitization, Intracellular calcium
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Introduction |
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The mitochondrial permeability transition pore (mPTP) represents a
fundamental pathological process that can initiate pathways to cell death,
either by causing ATP depletion and energetic collapse
(Qian et al., 1999) or by
promoting the release of cytochrome c and/or apoptosis-inducing factor (AIF)
and precipitating apoptotic cell death (for reviews, see
Bernardi et al., 1999
;
Crompton, 1999
;
Kroemer, 1999
). ROS are key
inducers of mPTP opening in isolated mitochondria, as oxidation of critical
mitochondrial thiol groups may trigger pore opening
(Crompton and Costi, 1988
). In
the current study, we present a model that permits the controlled generation
of ROS specifically from mitochondria within cells. This allows examination of
the specific consequences of mitochondrial ROS generation for both
mitochondrial and cellular fate and function. Furthermore, the degree of ROS
production is `tunable' so that the consequences of different loads of radical
species can be readily studied.
Several authors have described `spontaneous', reversible mitochondrial
depolarisations in cells loaded with fluorescent, potentiometric dyes. Such
fluctuations of mitochondrial membrane potential (m) have been
described in cardiomyocytes (Duchen et
al., 1998
; Leyssens et al.,
1995
; Zorov et al.,
2000
), neurons (Buckman and
Reynolds, 2001
), the COS-7 cell line
(De Giorgi et al., 2000
) and
isolated mitochondria (Hüser et al.,
1998
). The depolarisations are sensitive (to varying degrees) to
antioxidants, blockers of the mPTP and Ca2+ chelators. We have seen
similar
m fluctuations in primary cortical astrocytes
(Jacobson and Duchen, 1998
)
and here we have purposely used these ROS-induced depolarisations to further
explore the consequences of varying degrees of mitochondrial ROS formation in
astrocytes.
Our data lead us to propose a model in which mitochondrial ROS generation
promotes Ca2+ release from nearby ER. As this probably occurs in
microdomains close to mitochondria, it results in mitochondrial calcium
accumulation. This will further increase the rate of ROS production
(Dykens, 1994), initiating a
positive feedback cycle. In addition, the combination of michondrial calcium
loading and ROS generation will increase the probability of mPTP opening,
initially as transient and reversible events, but later as an irreversible
state. We have then proceeded to use this approach to examine the consequences
of these two states of the mPTP for cell fate. Remarkably, transient,
reversible flickering of the pore appears quite innocuous and has no impact on
cell fate. By contrast, once mPTP opening was sustained, necrotic cell death
followed inexorably.
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Materials and Methods |
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Dye loading and drug application
All solutions were based on a saline that comprised 156 mM NaCl; 3 mM KCl;
2 mM MgSO4; 1.25 mM KH2PO4; 2 mM
CaCl2; 10 mM D-glucose; 10 mM Hepes; pH adjusted to 7.35. Cells
were loaded with TMRE (1.5 µM) for 15 minutes at room temperature followed
by washing. Cells were loaded with rhod-2 as the AM ester (4.4 µM) for 30
minutes before washing (Boitier et al.,
1999). Short-acting drugs (e.g. FCCP, rotenone) were applied
locally close to cells from pressurised micropipettes. In some cases (e.g. as
with BAPTA-AM and antioxidants) drugs were added to the bathing saline.
Drugs and dyes
Fluorescent dyes were purchased from Molecular Probes Europe BV (Leiden,
The Netherlands). N-methyl 4-valine cyclosporin was the gift of Novartis
Pharma AG (Basel, Switzerland) and all other chemicals were purchased from
Sigma (Poole, UK).
Imaging
Digital imaging of cells loaded with TMRE, Fura-2 or rhod-2 was performed
using either a cooled CCD camera (Hamamatsu 4880) or a Zeiss 510 CLSM confocal
microscope equipped with x40 and x63 oil immersion, quartz
objective lenses (NA 1.3 and 1.4, respectively) as well as a x40 long
working distance lens (NA 0.68). For the cooled CCD system, coverslips were
placed in a homemade chamber mounted on the stage and fluorescence excited
using a 75 W xenon arc lamp, the light from which passed through a 546 nm band
pass filter and then a 580 nm dichroic mirror. Light longer than 590 nm was
collected and focussed on the camera. Fura-2 was sequentially excited at 340
nm and 380 nm and the fluorescence beyond 530 nm was imaged. On the confocal
system, cells were illuminated using the 543 nm emission line of a HeNe laser
and the intensity of the incident light was maintained between 0.5 and 5% of
the total laser output. The fluorescence of TMRE or rhod-2 was collected at
wavelengths longer than 585 nm.
To image NADH autofluorescence, cells were illuminated using the 351 nm laser line of the Zeiss 510 CLSM, and the fluorescence signal was collected through a bandpass filter between 375 and 488 nm. In order to avoid any crosstalk in cells co-loaded with TMRM, `multitracking' was used, in which images at each optical configuration are collected successively, rather than simultaneously, on two channels. The pinhole was opened to a confocal thickness of about 5 µm to optimise light collection for the autofluorescence signal.
Image processing and statistical analysis
The incidence of mitochondrial depolarisations was assessed as follows:
background-subtracted images were differentiated in time using image analysis
software (Lucida 5.0, Kinetic Imaging, Liverpool, UK), emphasising only those
pixels that had changed since the preceding frame
(Fig. 3B). Mitochondrial
depolarisations were revealed as localised, bright areas in a dark background,
whereas mitochondria with a stable m were not
discernible and mitochondrial movement caused only small changes in signal.
The peak digital value over individual cells was plotted for each frame of the
differentiated image series and after normalisation to a baseline, the area
under each plot was calculated (Microcal Origin 4.1 software, Northampton,
MA). Hence a high frequency of mitochondrial depolarisations produced a higher
integrated value than seen in quiescent cells
(Fig. 5B). This value was
consistent with the subjective observation of `active' and `quiescent' cells.
In cells where the fluorescent flickers summated rapidly to produce a steadily
rising global signal, the fluorescence was measured over the whole cell
(omitting the mitochondrion-free nucleus) and the rate of rise was measured by
fitting a line to the steepest part of the plot. Results, either the
integrated values or the slopes of the fitted lines, were statistically
compared using GraphPad Instat 4.00 software (San Diego, CA); data with a
homogenous variance were assessed by non-paired two-tailed Student's
t-test, those with a heterogeneous variance were assessed by
Mann-Whitney U test. All data are presented as mean±standard deviation
(s.d.) and the point of minimum acceptable statistical significance was taken
to be 0.05.
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To represent changing intensities in both space and time, we have presented
data from image sequences as surface plots derived from line images. Each line
of the line image displays the colour-coded fluorescence intensity profile
along a line drawn through the length of the cell for one frame of an image
sequence. Plotting all the lines together in temporal sequence produces a 2D
image where the fluorescence values along the line are represented against
time. The surface plots shown in this paper were constructed as follows:
images were background-subtracted and divided by a digitally constructed
`darkest image' to represent baseline values
(Duchen et al., 1998). A line
was drawn along the axis of a cell and the pixel values along the line for the
full image sequence were then read using Lucida software. The resultant data
were imported into MatLab software (The Mathworks, Natick, MA) the ASCII
matrices were used to create the surface plots shown. The colour-coded look-up
table applied runs through the spectrum, with blue representing the lowest
ratio value and red the highest.
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Results |
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When concentrated, TMRE exhibits autoquenching, such that the fluorescence
signal becomes a nonlinear function of dye concentration
(Bunting et al., 1989;
Duchen, 1992
;
Duchen and Biscoe, 1992
);
mitochondrial depolarisation causes loss of mitochondrial dye, and dilution
into the cytosol where the signal therefore increases. The TMRE fluorescence
changed systematically in response to biochemical agents as predicted from
chemiosmotic principles, increasing with manipulations expected to cause
mitochondrial depolarisation. Thus, application of the uncoupler FCCP (1
µM), which collapses
m, caused a brisk increase of fluorescence
while the definition of mitochondrial structures was lost
(Fig. 1Cii,D). Similar changes
were seen in response to inhibition of mitochondrial respiration with rotenone
(Fig. 1E). The images shown as
Fig. 1Ciii and iv were obtained
by dividing the image sequence by the first image of the sequence
(Fig. 1Ci) so that the signal
is normalised for each pixel. After mitochondrial depolarisation, the signal
over the cytosol increased, whereas that over mitochondria had changed little
so that the mitochondria appear dark against a bright cytosol. This process
emphasises the proportional change in TMRE signal over each intracellular
compartment.
Transient mitochondrial depolarisations in quiescent cells
When collecting imaging series with time, brief flashes of increased
fluorescence were frequently seen over individual mitochondria reflecting
transient mitochondrial depolarisations.
Fig. 2A shows the time course
of these depolarisations of individual mitochondria.
Fig. 2C and D were extracted
from the same image sequence, emphasising that the depolarisations were
independent in time and space. Analysis of the time course of 91 mitochondrial
transient depolarisations in 58 cells showed a mean time from baseline to peak
fluorescence of 257.5±153.5 mseconds (mean±s.d.).
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Single mitochondria could depolarise repeatedly
(Fig. 2B,C). The signal over
individual organelles often returned to (or even below) baseline implying that
the recovery of potential could be complete. The mean signal rise was only
39±17% of that seen following complete dissipation of m by
FCCP (n=100, Fig. 2E).
That the signal change induced by the transient depolarisations was much
smaller than that seen on complete dissipation with FCCP argues that the
transient depolarisations either reflect only small changes in
m
or that they are so brief that the dye does not have time to reequilibrate. In
some cells, the transient depolarisations were overwhelmed by an increase in
signal over the whole cell, although transient depolarisations of individual
mitochondria were evident in the initial images. Because TMRE diffuses out of
depolarised mitochondria but is retained in the short term within the cell by
the plasma membrane potential (Nicholls
and Ward, 2000
), the global signal rise reflected the
depolarisations of many mitochondria and accumulation of dye within the
cytosol. Thus, the rate of rise of whole-cell fluorescence reflected the
number, or magnitude, of multiple mitochondrial depolarisations. Application
of FCCP (1 µM) to cells in which the fluorescence had reached a plateau
failed to evoke any further signal (Fig.
2F), suggesting that the mitochondrial depolarisation was global
and complete.
The sequential subtraction of successive images from an image series was used to highlight only those pixels in which the signal had changed between image frames (Fig. 3B). This process revealed `puffs' of fluorescence around individual mitochondria, consistent with local loss of dye from the organelles, and emphasies that changes in TMRE signal are due to fluorophore redistribution rather than mitochondrial movement.
The frequency of transients and the rate of global depolarisation
were dependent on illumination intensity
Operationally, it rapidly became apparent that the incidence of
mitochondrial depolarisations was related to the intensity of illumination.
Thus, reduction of the excitation light intensity significantly increased the
time taken to establish global mitochondrial depolarisation
(Fig. 4A). Systematically
decreasing the intensity of illumination by using a range of neutral density
filters significantly increased the time taken for the TMRE signal to reach an
FCCP-insensitive plateau (P<0.0001, Kruskal-Wallis non-parametric
ANOVA test, Fig. 4B). Three
neutral density filters were used, giving 1% transmission (46 cells), 3.5%
transmission (27 cells) and 5% transmission (38 cells) of excitation light.
When all but 1% of the excitation light was excluded, individual mitochondria
depolarised occasionally, but the global signal remained stable
(Fig. 4A).
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Thus the frequency of the mitochondrial depolarisations and the time to global mitochondrial depolarisation could be controlled by varying the illumination intensity. Minimising the excitation light allowed mitochondria to be observed repeatedly depolarising and repolarising with a minimal change in overall fluorescence, permitting us to explore the nature of the depolarisations and their consequences for cell function more fully.
Focal mitochondrial depolarisations were independent of external
Ca2+, but dependent on stored Ca2+
In a previous paper (Duchen et al.,
1998), we described similar mitochondrial depolarisations in
single rat cardiomyocytes that were dependent upon ryanodine-sensitive
sarcoplasmic reticulum Ca2+-release and subsequent mitochondrial
Ca2+-uptake. We therefore investigated the
Ca2+-dependence of the depolarisations in astrocytes in which
Ca2+ stores are largely inositol (1,4,5)-trisphosphate
[Ins(1,4,5)P3]-dependent
(Boitier et al., 1999
;
Langley and Pearce, 1994
;
Peuchen et al., 1996
). The
mitochondrial depolarisations were unaffected by removal of extracellular
Ca2+. In the presence of a Ca2+-free saline containing 1
mM EGTA the integrated activity (see were identified in the control group and
only one was seen in the imaged group. Exposure of the astrocytes to 500 µM
staurosporine revealed that the majority of treated cells were apoptotic after
4 hours, as defined by morphology and Hoechst staining (data not shown). Image
processing and statistical analysis) was 13.7±2.4 (n=19),
compared with matched controls of 13.2±3.2 (n=10)
(P=0.703, Mann-Whitney U test). To investigate the role of
intracellular Ca2+, the cells were loaded with the
membrane-permeant Ca2+ chelator, BAPTA-AM (10 µM). Again this
had no impact on the transient depolarisations (the integrated activity of the
BAPTA-treated group was 11.8±2.7 with controls of 12.2±4.2,
(n=21; P=0.7, Mann-Whitney U test). These data could suggest
that the events are independent of [Ca2+]cyt. However,
in some cell types, recent data suggest a privileged access of ER
Ca2+ to mitochondria (Rizzuto
et al., 1998
), and if applicable to cortical astrocytes might
explain mitochondrial Ca2+-accumulation insensitive to BAPTA.
Mitochondrial Ca2+-uptake may be blocked by ruthenium red, but
this is membrane impermeant, not very selective, and interferes with TMRE
fluorescence (e.g. Chamberlain et al.,
1984) (M.R.D., unpublished). We therefore tested a derivative of
ruthenium red, Ru-360, which blocks mitochondrial Ca2+-uptake in
cardiomyocytes (Matlib et al.,
1998
). Unfortunately, in our hands this agent induced depletion of
Ca2+ stores in the astrocytes (data not shown), and so could not be
used.
Intracellular Ca2+ stores can be depleted by inhibition of the
sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) with
thapsigargin. However, thapsigargin may induce mitochondrial
Ca2+-loading (Ricken et al.,
1998; Sheu and Sharma,
1999
), and so sensitise the mitochondria to ROS production. We
therefore loaded cells with BAPTA-AM before exposure to thapsigargin (200 nM),
in order to chelate Ca2+ released from the ER by thapsigargin and
so limit mitochondrial Ca2+-loading. The BAPTA loading prevented
any rise in [Ca2+]cyt in response to thapsigargin
the resting fura-2 ratio was unchanged in treated cells compared with
controls (P=0.540, unpaired t-test, n=6). The
efficient and complete emptying of stores by the manoeuvre was confirmed as
the response to ATP, a Ca2+ mobilising agonist was abolished
(Fig. 5A). This protocol
greatly suppressed the transient events, giving an integrated activity of
10.7±2.4 (n=117; P<0.0001, Mann-Whitney U test),
significantly reduced from the matched controls (14.6±5.4,
n=68; Fig. 5B). Thus,
ER Ca2+ was required for the transient mitochondrial
depolarisations in cells with predominantly
Ins(1,4,5)P3-sensitive Ca2+ stores, illustrated
further by the surface plot in Fig.
5C.
The depolarisations reflect transient openings of the mitochondrial
permeability transition pore
Uptake of Ca2+ by mitochondria is electrogenic and results in a
reversible depolarisation of m (e.g.
Duchen, 1992
).
Ca2+-dependent mitochondrial depolarisations may therefore result
from the electrogenic uptake of Ca2+ by mitochondria in response to
local Ca2+ transients. Alternatively, mitochondrial Ca2+
accumulation could promote opening of the mPTP, particularly if mitochondria
are also sensitised by oxidative stress
(Hüser et al., 1998
). We
therefore asked whether the depolarisations reflect opening of the mPTP.
Cyclosporin A, the classical inhibitor of mPTP opening
(Crompton et al., 1988
)
significantly reduced the rate of progression of the global mitochondrial
depolarisation (slope of onset of TMRE dequench in controls was
0.058±0.031, n=15; in 500 nM CsA-treated, 0.004±0.005,
n=17. P<0.0001, unpaired Mann-Whitney U test).
Cyclosporin A binds all cyclophilins and so is not selective for the mPTP. We
therefore also used N-methyl 4-valine cyclosporin (meth-v-Cs), a
non-immunosuppressive analogue of cyclosporin A. Meth-v-Cs also binds
cyclophilins, but the cyclophilin A/meth-v-Cs complex does not bind
Ca2+-sensitive calcineurin
(Petronilli et al., 1994b
).
400 nM meth-v-Cs significantly reduced the integrated activity
(Fig. 6A) to 9.8±2.5,
n=122, compared with a matched control of 12.0±2.6,
n=82 (P<0.0001, two-tailed, unpaired t-test).
Trifluoperazine (10 µM), another inhibitor of pore formation
(Lemasters et al., 1998
;
Bernardi et al., 1993
) also
reduced the rate of global depolarisation
(Fig. 6B) to a slope of
0.008±0.006 (n=60 cells; P<0.0001), compared with
0.041±0.018 (n=27) cells for the matched controls
(Mann-Whitney U test). Thus, the concordance of data strongly suggests that
the transient, Ca2+-dependent mitochondrial depolarisations
signalled reversible openings of the mPTP.
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mPTP openings were suppressed by antioxidants
Illumination of fluorophores causes production of ROS. Indeed,
photobleaching of fluorophores is largely an oxidative reaction and
commercially available `antifade' compounds are in fact cocktails of
antioxidants. Several mitochondrial probes, including TMRE, produce singlet
oxygen upon illumination (Bunting,
1992). As mPTP opening is induced by oxidative stress we explored
the effects of a range of antioxidants on the events. Exposure of cells to a
solution of ascorbic acid (1 mM), catalase (250 units ml-1), the
-tocopherol analogue Trolox (1 mM) and the spin trap TEMPO (500 µM)
markedly reduced the flickering depolarisations
(Fig. 7A) with a reduction in
integrated activity to 6.4±1.9 (n=57) from a matched control
of 10.0±5.6 (n=53, P<0.001, Mann-Whitney U test).
Similarly, the time taken to global depolarisation in antioxidant-treated
cells was significantly prolonged (Fig.
7B) the slope of the onset of the TMRE signal in controls
was 0.094±0.034, (n=9) and in the antioxidant-treated cells,
0.002±0.003 (n=12), P<0.0001, Mann-Whitney U
test.
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Mitochondrial oxidative stress causes mitochondrial calcium
loading
We have noticed, when using the mitochondrial Ca2+-sensitive
indicator rhod-2 AM, that the mitochondrial signal tends to become brighter
with time, even in unstimulated cells (Fig.
8A,B), unless the illumination intensity is kept to a minimum. As
fluorophores are more likely to bleach with illumination, we wondered whether
ROS could be sensitising Ca2+-release from intracellular stores
causing mitochondrial Ca2+ loading. We therefore loaded astrocytes
with rhod-2 AM and examined the effects of antioxidants (as used above) on the
rate of signal rise with illumination. Over a period of illumination, the
rhod-2 signal rose initially over the whole cell, during which the
mitochondria were visible as bright organelles. The cytosolic and nuclear
signals then slowly decreased, leaving bright, Ca2+-loaded
mitochondria. These observations were reminiscent of the rhod-2 signal changes
seen when astrocytes are stimulated with ATP, an agonist of
Ins(1,4,5)P3-mediated Ca2+ release
(Boitier et al., 1999). The
mean rate of rise of the cytosolic/mitochondrial rhod-2 signal was
0.011±0.006 arbitrary units/millisecond (n=63). However, the
antioxidants significantly slowed the rate of rise to 0.002±0.001
arbitrary units/millisecond (n=62; P<0.0001, Mann-Whitney
U test). Thus, illumination of cells loaded with a Ca2+-sensitive
fluorophore induced a mitochondrial Ca2+-loading that was
ROS-dependent.
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Transient mPTP opening is innocuous; global mitochondrial
depolarisation causes necrotic cell death
We used the ability to induce mPTP opening in single astrocytes to
investigate whether transient openings of the pore induced cell death. Cells
were stained with propidium iodide (PI) and Hoechst 33342 to differentiate
between apoptotic and necrotic endpoints. Cells were plated onto coverslips to
which an etched grid (`CELLocate', Eppendorf) was attached. Cells could then
be relocated by the grid reference hours after illumination and examined for
morphological and staining changes. Conditions were established to generate
only transient depolarisations (using a 1% transmission neutral density
filter), keeping the global depolarisation to a minimum. The cells were then
returned to culture medium and replaced in an incubator (36°C, 5%
CO2) and examined again 4 hours later using PI (10 µM) and
Hoechst 33342 (15 µM). Nuclei that stained brightly with Hoechst 33342
showing chromatin condensation were scored as apoptotic if they excluded PI,
while those nuclei that showed PI staining were deemed necrotic
(Fig. 9A). In a further series
of experiments, cells were illuminated using a 30% neutral density filter to
induce a global mitochondrial depolarisation. Again, non-illuminated areas of
the same dish were compared with the imaged cells 4 hours later.
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In those cells in which frequent transient depolarisations had been documented without global depolarisation, no significant increase in cell death, apoptotic or necrotic, was detectable above the control levels (Fig. 9B). Of 2083 illuminated cells, only 10% displayed necrotic staining or morphology after 4 hours, while only four cells displayed apoptotic staining or morphology, compared with non-illuminated controls in which 12% of 1826 cells counted were necrotic and only three were apoptotic (P=0.962, unpaired t-test). By contrast, after sufficient illumination to induce a global mitochondrial depolarisation the incidence of necrotic cell death increased significantly after 4 hours to 35% (of 718 cells) compared with 1% of controls (of 983 counted, P=0.0002, unpaired t-test). In these cells, no apoptotic cells were identified in the control group and only one was seen in the imaged group. Exposure of the astrocytes to 500 µM staurosporine revealed that the majority of treated cells were apoptotic after 4 hours, as defined by morphology and Hoechst staining (data not shown).
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Discussion |
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Mitochondria and free radicals
Although 95-98% of the molecular oxygen that is consumed by mitochondria is
reduced to water, the remainder is partially reduced by the addition of an
electron, forming the oxyradical superoxide
(Chance et al., 1979).
Although mitochondrial antioxidant defences are extensive, excess
mitochondrial ROS production or depletion of antioxidant defences cause
oxidative stress and, ultimately, cellular dysfunction. Indeed, mitochondrial
ROS production may be a major mechanism of cell injury following anoxia and
reperfusion. Many fluorophores generate ROS upon illumination
(Bunting, 1992
) and the
consequent photosensitization causes apoptotic cell death in some cell lines
(Minamikawa et al., 1999
).
Indeed, this is probably the basis for cell killing by sensitisers used in
photodynamic therapy for some cancers
(Fuchs and Thiele, 1998
). ROS
production by illuminated TMRE is specifically intramitochondrial, reflecting
the mitochondrial localisation of the dye, and so this approach provides a
useful model with which to generate a specific mitochondrial oxidative
stress.
These data do not simply reflect the random toxicity of a fluorescent dye.
The model of oxidative stress presented here deliberately uses two key
properties of the potentiometric probe, TMRE. First, the well-described
production of ROS upon illumination of TMRE provides a source of ROS
(principally singlet oxygen) (Bunting,
1992) that varies with the intensity of illumination. Thus the
oxidative stress may be `tuned' by varying the incident excitation light.
Second, the selective partitioning of TMRE into mitochondria means that the
ROS are generated specifically from within the mitochondria. This therefore
provides a controlled model of specific mitochondrial oxidative stress,
perhaps the most appropriate model to study the metabolic oxidative stress
seen for example in reperfusion injury. Oxidative stress that results from the
exogenous application of pro-oxidants such as tertbutyl hydroperoxide or UV
light may be significantly different because the immediate target of such
treatments is likely to be the plasma membrane. The model presented here may
be most instructive in understanding the pathophysiology of the mitochondrial
oxidative stress, implicated in ischaemia reperfusion
(Halestrap et al., 1998
),
neurodegenerative disorders (Beal et al.,
1997
) and sepsis (Kantrow and
Piantadosi, 1997
).
The mitochondrial permeability transition pore
The opening of a high conductance mitochondrial pathway by a combination of
high [Ca2+]mit, high inorganic phosphate and adenine
nucleotide depletion has been recognised for many years
(Hunter and Haworth, 1979).
The pathway is now thought to reflect formation of a membrane-spanning
proteinaceous pore, the mPTP, comprising (at least) the voltage dependent
anion channel (VDAC), the adenine nucleotide translocase (ANT) and the
mitochondrial cyclophilin D (Cyp D). Opening of the mPTP results in rapid
dissipation of
m, cessation of ATP synthesis, and mitochondrial
swelling (Crompton et al.,
1987
; Halestrap et al.,
1997
; Hunter and Haworth,
1979
; Zoratti and
Szabó, 1994
). Comprehensive investigation of isolated
organelles has established a range of inducers and inhibitors of pore opening
(for a review, see Zoratti and
Szabò, 1995
). Classic inducers of mPTP opening include
mitochondrial Ca2+-overload
(Crompton and Costi, 1988
),
oxidative stress (Byrne et al.,
1999
; Nieminen et al.,
1995
) and depletion of adenine nucleotides
(Crompton and Costi, 1990
;
Novgorodov et al., 1991
),
whereas the decapeptide cyclosporin A and related compounds, and agents that
block the ANT reduce the probability of pore opening
(Crompton et al., 1988
;
Halestrap and Davidson,
1990
).
Extrapolating data acquired from isolated mitochondria to the complexity of
the intact cell is not straightforward, although elegant models have been
devised by using the redistribution of calcein into
(Nieminen et al., 1995) and
out of (Petronilli et al.,
1998
) mitochondria, to reveal openings of the pore. Mitochondrial
Ca2+-loading is essential for mPTP opening in isolated
mitochondria, but intracellular mitochondrial Ca2+ uptake may
depend upon a variety of factors including ER-mitochondrial architecture
(Rizzuto et al., 1993
;
Rizzuto et al., 1998
), local
redox state (Byrne et al.,
1999
; Jornot et al.,
1999
) and
m
(Nicholls and Crompton, 1980
).
Although it is clear that oxidative stress sensitises isolated mitochondria to
Ca2+ and mPTP opening
(Petronilli et al., 1994a
;
Valle et al., 1993
), ROS may
have a range of molecular targets, including mitochondrial membrane lipids
(Nomura et al., 1999
) and the
respiratory chain complexes (Heales et
al., 1995
).
Mitochondrially generated ROS causes mitochondrial
Ca2+-loading from ER stores
It is clear that mPTP opening in isolated mitochondria requires
Ca2+-loading (Crompton et al.,
1987). Similarly, in models of oxidative stress, mitochondrial
Ca2+-loading was necessary for pore formation
(Byrne et al., 1999
). Our
present data strongly suggest that the source of that Ca2+ is the
ER: the transient openings of the mPTP and the global depolarisation were both
independent of external Ca2+, but were dramatically attenuated by
emptying ER Ca2+ stores and chelating Ca2+. Furthermore,
mitochondrial Ca2+ loading by illumination was demonstrable,
dependent on ER Ca2+ stores and inhibited by antioxidants. The fact
that neither ER Ca2+ depletion or Ca2+ chelation was
sufficient alone but that both together stopped mitochondrial
Ca2+-loading and inhibited mPTP opening strongly argues that ER and
mitochondria must be closely apposed in these cells, as described for HeLa
cells (Rizzuto et al., 1998
),
RBL-2H3 cells (Csordas et al.,
1999
) and cardiomyocytes
(Ramesh et al., 1998
). The
impact of mitochondrial Ca2+ uptake on the rate of propagation of
calcium waves in astrocytes would also support this proposition
(Boitier et al., 1999
). These
data add further to a body of literature suggesting the expression of a
privileged pathway of ER Ca2+-release and mitochondrial
Ca2+-uptake at least in some cell types. It is also interesting
that several recent reports have emphasised the organisation of mitochondria
as a contiguous network (e.g. Rutter and
Rizzuto, 2000
). In astrocytes, confocal imaging allows individual
mitochondrial structures to be seen clearly, and the mitochondrial
depolarisations were evidently independent in time and space, suggesting that
here the mitochondrial organisation is more complex.
A substantial literature suggests that ROS may modulate the gating of
ryanodine-sensitive Ca2+ channels in the SR
(Boraso and Williams, 1994;
Holmberg and Williams, 1992
)
probably through modification of critical thiol groups
(Kourie, 1998
). Data that
indicate modulation of Ins(1,4,5)P3-mediated
Ca2+ channels by ROS are less abundant. Oxidised glutathione
(Henschke and Elliott, 1995
)
and ROS derived from xanthine/xanthine oxidase
(Wesson and Elliott, 1995
)
have been shown to induce Ca2+-release from
Ins(1,4,5)P3-mediated stores in endothelial cells and
superoxide may stimulate Ins(1,4,5)P3-mediated
Ca2+ release in vascular smooth muscle cells
(Suzuki and Ford, 1992
). As
the opening of the mPTP is also regulated by thiols
(Kowaltowski et al., 1998
), it
seems that the sensitivity of both the Ins(1,4,5)P3
receptor and the mPTP to redox state renders this whole system susceptible to
oxidative damage through the initiation of a destructive feedback cycle,
whereby increased mitochondrial ROS production increases the local probability
of ER Ca2+ release and mitochondrial Ca2+ loading. This
may both increase ROS generation further
(Dykens, 1994
) and sensitise
the mitochondria to ROS, culminating in mPTP opening, initially in a transient
and reversible mode and later in a global irreversible mode. We have then
asked what the implications of mPTP opening in these different states are for
cell fate.
Flickering openings of the mPTP were innocuous but sustained pore
opening induced necrotic death
Studies of the mPTP in isolated mitochondria showed that the pore may
flicker before opening fully, and the existence of subconductance states has
been suggested (Zoratti and Szabó,
1994). Recently, transient opening of the mPTP has been suggested
to underlie initiation of apoptosis in single hepatocytes
(Szalai et al., 1999
), but it
is unclear whether these were reversible openings of the pore in full or
flickering openings of a sub-conductance state. Opening of the mPTP is
expected to have catastrophic consequences for cell fate. As the driving force
of the ATP-synthase is the mitochondrial potential, complete dissipation of
the
m can cause reversal of the ATP synthase, which now consumes
ATP while pumping protons from the mitochondrial matrix. Rapid consumption of
cellular ATP may in turn lead to energetic collapse and necrotic cell death
(Leyssens et al., 1996
).
Additionally, cytochrome c may be released from the mitochondrial
intermembrane space, possibly after mitochondrial swelling and outer membrane
disruption, activating the caspase cascade and initiating apoptotic cell death
(Liu et al., 1996
). Opening of
the mPTP may also release apoptosis-inducing factor (AIF), a soluble protein
that activates caspase 3 (Susin et al.,
1997
). Therefore, opening of the mPTP may predict either necrotic
or apoptotic cell death.
Using our model of mitochondrial oxidative stress and mPTP opening we found that single mitochondria may undergo repeated, transient openings of the pore that seem to have no deleterious effect on cell function. Among those cells in which pore openings were transient and reversible, there was no evidence of either necrotic or apoptotic cell death at 4 hours. However, sustained mPTP opening clearly increased the incidence of necrotic cell death. It seems likely that, in primary cells, where oxidative phosphorylation is the dominant source of intracellular ATP, mPTP opening will cause rapid ATP consumption and therefore predict a necrotic death.
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