1 Medical Research Council Secretory Control Research Group, Physiological
Laboratory, University of Liverpool L69 3BX, UK
2 Department of Medicine, University of Liverpool L69 3BA, UK
* Author for correspondence (e-mail: alastair.watson{at}liv.ac.uk)
Accepted 1 November 2001
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Summary |
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Key words: Calcium, Apoptosis, Menadione, Mitochondria, Permeability transition pore
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Introduction |
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Elevations of the cytosolic Ca2+ concentration are used as
general signalling mechanisms, which activate different processes even in the
same cell. Ca2+ can interact with many different molecular targets,
and it has therefore become increasingly clear in recent years that signal
specificity requires subcellular localization and/or special temporal
patterns. A number of different mechanisms allowing such spatiotemporal
specificity have been identified (Berridge
et al., 2000; Cancella et al., 2000;
Parekh, 2000
;
Petersen et al., 2001
).
Pancreatic acinar cells have proved useful models for the study of
Ca2+ signalling. Physiological stimulation, with the
neurotransmitter acetylcholine (ACh) or the circulating peptide hormone
cholecystokinin (CCK), evokes repetitive cytosolic Ca2+ spikes, and
these are mostly confined to the apical granular pole
(Petersen et al., 2001). This
is partly due to the clustering of Ca2+ release channels in the
endoplasmic reticulum (ER) extensions in the apical pole
(Petersen et al., 2001
) and
partly due to the firewall of active mitochondria around the apical granular
pole, which acts as a Ca2+ buffer barrier
(Tinel et al., 1999
). Each
local Ca2+ spike evokes an exocytotic secretory response
(Maruyama and Petersen, 1994
).
Supramaximal agonist stimulation, eliciting a sustained global cytosolic
Ca2+ elevation, has recently been shown to induce intracellular
enzyme activation and vacuole formation in the apical granular pole
(Parekh, 2000
;
Raraty et al., 2000
).
How do Ca2+ signals that cause apoptosis differ from those that
have other effects? Thapsigargin, a specific inhibitor of sarcoendoplasmic
reticulum Ca2+-ATPase, can induce apoptosis in a wide variety of
epithelial and non-epithelial cell lineages
(Baffy et al., 1993;
Qi et al., 1997
). Recent
evidence suggests this may involve activation of caspase 12, which is
localised at the ER. This mechanism is stimulus specific in that other
inducers of apoptosis not causing stress within the ER do not activate caspase
12 (Nakagawa et al., 2000
).
Mitochondria also play a critical role in another pathway and orchestrate a
wide range of stimuli leading to activation of caspase 9. In response to
appropriate intracellular signals, which remain to be clearly defined,
cytochrome c and other proteins are released into the cytosol from the
intermembrane space of the mitochondria
(Liu et al., 1996
;
Susin et al., 1999
). In the
cytosol, a complex is formed containing cytochrome c, APAF-1 and procaspase 9,
which processes and activates caspase 9
(Zou et al., 1997
). Caspase 9
then activates caspase 3 and other downstream caspase family members that
cleave specific protein targets causing apoptosis
(Li et al., 1997
). There is
also evidence that opening of the PTP in mitochondria is important for the
release of cytochrome c. The PTP is a pore formed from a complex of the
voltage-dependent anion channel (VDAC), the adenine nucleotide translocase and
cyclophilin-D at contact sites between the inner and outer mitochondrial
membrane (Crompton, 1999
). PTP
activators, including Ca2+, induce an open-pore state, possibly
causing swelling of the mitochondrial matrix and rupture of the outer membrane
with release of apoptogenic proteins from the intermembrane space
(Martinou et al., 2000
).
How do cytosolic Ca2+ signals that cause, for example, secretion
or intracellular activation of digestive enzymes, act without inducing
apoptosis? To answer this question we have investigated how the cytosolic
Ca2+ response in intact normal pancreatic acinar cells elicited by
ACh and CCK differs from the Ca2+ response caused by the
proapoptotic oxidant menadione. Menadione is a quinone that is metabolised by
flavoprotein reductase to semiquinone, which can be oxidised back to quinone
in the presence of molecular oxygen. In this redox cycle the superoxide anion
radical, hydrogen peroxide and other reactive oxygen species are generated
(Monks et al., 1992).
Menadione can cause elevations in the cytosolic Ca2+ concentration
contributing to cell death (Nicotera et
al., 1992
). The exact in vivo mechanisms remain to be fully
defined, although studies of permeabilised cultured hepatocytes suggest that
IP3-driven Ca2+ signals can trigger the PTP and induce
apoptosis (Chernyak and Bernardi,
1996
; Costantini et al.,
2000
; Szalai et al.,
1999
). A further goal was to determine whether oxidants and
Ca2+ could interact to induce the PTP and initiate apoptosis.
We show that menadione induces cytosolic Ca2+ spikes initiated in the secretory pole. However, in contrast to those generated by ACh, there is no tendency to restrict the Ca2+ elevation to this part of the cell. Menadione induces partial mitochondrial depolarisation through induction of the PTP, preventing Ca2+ buffering by mitochondria and permitting spread of Ca2+ throughout the cell. Blockade of either cytosolic Ca2+ spikes or induction of the PTP prevents menadione-induced apoptosis. Mitochondrial depolarisation without induction of the PTP does not induce apoptosis even in the presence of an elevated cytosolic Ca2+ concentration. Together these data indicate that both a rapid rise in the cytosolic Ca2+ concentration and induction of the PTP are essential and act cooperatively to mediate menadione-elicited apoptosis.
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Materials and Methods |
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Cell preparation
Single pancreatic cells or small clusters (two or three cells) were acutely
isolated from CD1 mouse pancreas as previously described
(Thorn et al., 1993). Briefly,
the pancreas was injected with 200 units/ml collagenase solution, incubated
for 10-15 minutes at 37°C then agitated manually and by pipette to obtain
the isolated cells. Isolation procedure and experiments were performed in a
standard buffer containing 140 mM NaCl, 1.13 mM MgCl2, 1 mM
CaCl2, 4.7 mM KCl, 10 mM glucose, 10 mM HEPES, pH 7.2 adjusted with
NaOH. After the isolation procedure, cells were washed with standard
buffer.
Ca2+ measurements
Cytosolic Ca2+ was measured by loading pancreatic acinar cells
with either Fura Red or Fluo 4 by incubation in standard buffer containing 10
µM Fura Red AM or Fluo 4 AM for 45 minutes at 22°C. After incubation,
cells were washed with standard solution and used for experiments (excitation
488 nm, emission >515 nm). Fluorescence measurements were done using a
Noran confocal microscope with a 60x Nikon objective (1.4 NA). Linescan
(4 ms per line) in slow mode (6400 ns) was used with a slit of 25 µm.
Images were processed using TwoD Analysis software (Noran): shade correction
(dividing by first image) and 3x3(7) low pass filtering were used.
Images of Fura-Red-loaded cells were also inverted (using subtraction from
saturated image) and shown with linear colour scale. The maximal and minimal
values of the Fura Red fluorescence were determined at the end of each
experiment by applying 20 µM ionomycin with 1 mM CaCl2 or 10 mM
EGTA. Calculations of Ca2+ concentration in single cells were
performed in the conventional way for single wavelength indicators
(Takahashi et al., 1999). Kd
for Fura Red was assumed to be 140 nM and 345 nM for Fluo 4 (Molecular
Probes).
Mitochondrial Ca2+
Pancreatic acinar cells were loaded with Rhod-2 AM (5 µM) for 30 minutes
at 37°C in standard buffer (see cell preparation procedure). After washing
with the same buffer, cells were observed by confocal microscopy (excitation
543 nm, emission >560 nm).
Calcein loading procedure
Cells were loaded with 1 µM calcein AM in the presence of 1 mM
CoCl2 for 15 minutes at 37°C
(Petronilli et al., 1999) in
standard buffer solution followed by washing with the same buffer.
Mitochondrial calcein fluorescence was measured using confocal microscopy
(excitation 488 nm, emission > 515 nm).
Detection of apoptosis by ApoAlert Annexin V FITC apoptosis kit
Isolated cells were divided into several samples: (1) control cells; (2)
cells incubated with 20 µM menadione for 15 minutes or (3) for 30 minutes
or (4) 5 pM CCK or (5) 20 nM ACh or (6) 100 nM ACh or (7) 1 µM thapsigargin
for 30 minutes or (8) for 3 hours. All the cells were incubated at 22°C
and subsequently washed using centrifugation at 200 g for 1
minute. After this, ApoAlert Annexin V FITC kit was applied according to
manufacturer's instructions. Briefly, the pellet of the cells from each sample
was resuspended in the provided binding buffer and centrifuged at 200
g for 1 minute, again resuspended in 200 µl of binding
buffer, and 5 µl of Annexin V FITC conjugate was added. The cells plus
conjugate were incubated for 15 minutes at 22°C, washed by centrifugation
and resuspended in binding buffer and observed by confocal microscopy
(excitation 488 nm, emission >515 nm). To distinguish late apoptotic and
necrotic cells, we used staining with 1 µM of propidium iodide. Early
apoptotic cells and nonapoptotic cells do not show positive staining with this
dye (excitation 363 nm, emission >400 nm). Cells whose nuclei stained with
propidium iodide were assumed to be necrotic and excluded from analysis. In
experiments using apoptosis inhibitors BAPTA or ZVADfmk cells were incubated
for 1 hour in a solution containing 25 µM BAPTA AM or 250 µM ZVADfmk
followed by application of 20 µM menadione.
Subcellular fractionation of immunoblotting for cytochrome c
Cytosolic and mitochondrial fractions from control and menadione-treated
cells (treated for 2 minutes) were prepared according to Schuler et al.
(Schuler et al., 2000). Cells
were isolated from the pancreas of two mice in ice-cold buffer containing 150
mM KCl, 10 mM Hepes (pH 7.2), 1 mM EDTA, 1 mM DTT and protease inhibitor
cocktail tablets (Roche, USA). The protein content of the fractions was
determined by Bradford assay (Sigma, UK). All subcellular fractions were
analysed by dot-blot and western blot analysis. Samples were boiled in Laemmli
buffer for 5 minutes and subjected to electrophoresis in 10-20%
SDS-polyacrylamide gels using Bio-Rad Mini-PROTEAN 3 Cell, followed by
transfer on nitrocellulose membranes (0.2 µm, Sigma). After blocking with
tris buffered saline containing 5% nonfat dry milk and 0.05% Tween 20,
nitrocellulose membranes were exposed to the primary antibodies (cytochrome-c
H-104, rabbit polyclonal, Santa Cruz Biotechnology, Inc.) and horseradish
peroxidase-conjugated anti-rabbit secondary antibodies (Sigma, UK). Antibody
binding was detected using SuperSignal West Pico Chemiluminescent Substrate
according the protocol (SuperSignal, Pierce).
Measurement of caspase 3 and caspase-3-like activity using
fluorescent caspase 3 substrate
A pellet of pancreatic acinar cells (treated with menadione alone or
pre-treated with BAPTA AM before menadione application or untreated (control
cells)) was resuspended in small volume of media containing 10 µM
fluorogenic caspase 3 substrate PhiPhiLux (OncoImmunin). Following incubation
for 1 hour at 37°C and 5% CO2, the pellet was washed twice with
standard buffer and observed under confocal microscopy. The excitation of
PhiPhiLux substrate was 488 nm, emission >515 nm.
Measurement of caspase 9 activity
Caspase 9 activity was measured using a carboxyfluorescein derivative of
benzyloxycarbonyl leucylglutamylhistidylaspartic acid fluoromethyl ketone
(zLEHD-FMK), which is a potent inhibitor of caspase 9 (FAM-LEHD-FMK).
FAM-LEHD-FMK enters the cells and covalently binds to caspase 9 to activate it
as well as to caspase 4, 5 and 6. The caspase 9 detection procedure was
performed according the protocol of CaspaTag caspase 9 (LEHD) Activity Kit
(Intergen). Briefly, 300 µl aliquots of induced and control cells were
transferred to fresh tubes and 10 µ1 of 30x Working Dilution
FAM-LEHD-FMK was added directly to the cell suspension followed by mixing and
incubation for 1 hour at 37°C. After incubation, cells were washed with
1x Working Dilution Wash Buffer and resuspend in standard buffer. Live
cells (propidium iodide negative) containing bound caspase 9 inhibitor were
analysed using confocal microscopy (excitation 488 nm, emission > 515
nm).
Measurements of mitochondrial membrane potential changes
Changes of mitochondrial membrane potential were detected with
tetramethylrhodamine ethylester (TMRE) using Leica SP-2 confocal microscope.
TMRE accumulates in mitochondria according to the Nernst equation and has been
used previously to assess relative changes in mitochondrial potential. No
quenching of cellular fluorescence with TMRE was detected in our experiments.
Decrease in fluorescence corresponds to depolarisation of mitochondrial
membrane potential. Cells were loaded with TMRE at a concentration of 0.5
µM in standard solution for 30 minutes at 37°C (excitation 488 nm,
emission >550 nm). To determine the subcellular localisation of
mitochondria, cells were coloaded with 0.1 µM of Mito Tracker Green FM for
15 minutes (excitation 488 nm, emission <550 nm) and then washed in
standard buffer.
Statistical analysis
We considered that a cell was positively stained with Annexin V FITC if the
fluorescence of this cell was higher than the averaged value for the control
group of cells plus 3 standard deviations of fluorescence of this control
group. The same criterion was applied in experiments with FAM-LEHD-FMK and
PhiPhiLux. Significance was determined using an analysis of variance using
SPSS software. A value of P<0.05 was considered to be
significant.
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Results |
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Menadione induces apoptosis
We then investigated whether menadione induces apoptosis in normal
pancreatic acinar cells. Six hours after isolation, 64±8%
(n=128) of the menadione-treated cells were propidium-iodide positive
compared with 15±6% (n=109) of the untreated cells
(Fig. 2C). All subsequent
studies were then undertaken less than 1 hour after isolation, when less than
5% of the cells treated with menadione were propidium-iodide positive. In
order to identify individual cells undergoing apoptosis, we employed two
techniques based on fluorescence confocal microscopy. First, phosphatidyl
serine becomes redistributed to the outer leaflet of the plasma membrane in
cells undergoing apoptosis and can be detected with fluorescently labelled
Annexin V (Martin et al.,
1995). Cells were incubated for 30 minutes with menadione at
22°C with subsequent application of Annexin V FITC to menadione-treated
and control cells to detect apoptosis. All propidium-iodide negative cells in
a microscope field were analysed in a double blind manner. At least five
microscope fields of view were evaluated. The result for each cell is
presented separately as a single bar (Fig.
2A). No cells were Annexin V FITC positive in the control group.
The average fluorescence intensity of the Annexin V FITC stained control
(non-apoptotic) cells was 71.5±12.3s.d. (arbitrary units),
n=69. Cells with fluorescence higher than 108.5 (average value plus
3xs.d. value) were considered apoptotic. After incubation with
menadione, the fluorescence intensity of Annexin V FITC increased markedly in
comparison with control cells (86.3% of Annexin V FITC positive cells in a
population of cells treated with menadione for 30 minutes and 66.7% of Annexin
V FITC positive cells in population of cells treated with menadione for 15
minutes). The intensity of fluorescence of control group cells and cells
incubated with menadione for 30 minutes (n=96) or 15 minutes
(n=13) were significantly different (P<0.001). Cells
incubated for 30 minutes with either CCK at its physiological concentration (5
pM) or 25 nM ACh or 100 nM ACh did not display statistically significant
changes in Annexin V FITC fluorescence compared with control cells
(Fig. 2A) (n>7 for
each group). It has been reported previously that the specific inhibitor of
the sarcoendoplasmic reticulum Ca2+-ATPase, thapsigargin, can
induce apoptosis in pancreatic islet and haemopoietic cells
(Baffy et al., 1993
;
Qi et al., 1997
;
Zhou et al., 1998
). Treatment
of pancreatic acinar cells with thapsigargin (1 µM) for 30 minutes did not
induce apoptosis within the timeframe of the experiment
(Fig. 2A) (n=15). The
thapsigargin concentration used is maximal and effectively releases all
Ca2+ stored in the endoplasmic reticulum in pancreatic acinar cells
within about 15 minutes (Mogami et al.,
1998
; Toescu et al.,
1992
). However, after treatment of cells with thapsigargin (1
µM) for 3 hours, 37.9% of the cells in the population became apoptotic
(n=30) (Fig. 2A).
Hoechst staining of the nuclei of cells treated with 20 µM menadione for 3
hours demonstrated chromatin condensation characteristic of apoptosis
(Fig. 2D,E)
(Gukovskaya et al., 1997
). No
nuclear condensation could be detected at earlier time points.
|
To check that the increase in Annexin binding to menadione-treated cells
was due to apoptosis, the effect of Z-VADfmk, a cell membrane permeable
inhibitor of caspases 1-10, was studied
(Garcia-Calvo et al., 1998).
Treatment of cells with 250 µM Z-VADfmk for 1 hour reduced the percentage
of apoptotic cells in the menadione-treated population from 86.3% to 21.4%
(Fig. 2A). Furthermore,
Z-VADfmk significantly reduced absolute Annexin fluorescence compared with
cells treated with menadione alone (n=15, P<0.001).
We then determined whether a rise in the cytosolic Ca2+
concentration is required for menadione-induced apoptosis. We have
demonstrated previously that cytosolic Ca2+ spikes induced by ACh
can be abolished by buffering cytosolic Ca2+ with BAPTA, in spite
of the fact that the rate of Ca2+ release from the ER is actually
enhanced under this condition (Mogami et
al., 1998). BAPTA prevented any significant change in Annexin V
FITC fluorescence of cells exposed to menadione (no Annexin V FITC positive
cells in population, n=12) (Fig.
2A). Similar results have been noted in the pancreatic cell line
AR4-2J (Sata et al., 1997
).
BAPTA loading abolished the menadione-elicited cytosolic Ca2+ spike
but did not prevent a very slow rise in the cytosolic Ca2+
concentration (Fig. 2B). These
data, together with data showing that neither CCK nor ACh induce apoptosis,
suggest that a rise in the cytosolic Ca2+ concentration is
essential, but insufficient, for the induction of apoptosis by menadione.
Menadione-induced Ca2+ signals are not restricted to the
apical secretory pole
We have previously shown that at the peak of the ACh-induced cytosolic
Ca2+ response, there can be a very significant cytosolic
Ca2+ concentration gradient along a line connecting the secretory
granular pole with the nucleus (up to 400 nM/µm)
(Gerasimenko et al., 1996). We
therefore examined the spatial distribution of the menadione-induced
Ca2+ signals using confocal linescan measurements. The cytosolic
Ca2+ concentrations were determined along a line connecting the
secretory granule region and the nucleus
(Fig. 3e-h). The location of
the granular region can be seen in the transmitted light images
(Fig. 3a-d) although the
nucleus is the bright area in the Fura-red images on the right side of the
cell (Fig. 3e-h). A low ACh
concentration (25 nM) induced localised Ca2+ spikes. These
Ca2+ signals remained confined to the apical granule-containing
area, owing to the perigranular mitochondrial belt
(Cancela et al., 2000
;
Tinel et al., 1999
), and
therefore did not enter the nucleus (Fig.
3i). Higher doses of ACh (100 nM) usually induced large responses,
which also started in the secretory granule area but then spread into the
basal area. Spreading of the Ca2+ signal into the nucleus was
delayed by 1-7 seconds (n=10) compared to entry into the non-nuclear
region (Fig. 3j). The ratio of
Ca2+ wave speed propagation into the nuclear region (angle ß)
compared to speed into the non-nuclear region (angle
) was
0.4±0.1. This delayed entry into the nucleus is most likely due to the
recently discovered mitochondrial ring surrounding the nucleus
(Park et al., 2001
).
Menadione-induced Ca2+ signals were also initiated in the secretory
granular zone, but the response spread into the nuclear area without delay
with nearly the same speed as into the non-nuclear region (ratio:
0.8±0.1, n=7). The ratio was significantly different when
compared to ACh (P<0.01) (Fig.
3k,m-q).
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We have recently shown that active mitochondria, localized in a ring
surrounding the granular region, serve as a buffer barrier for Ca2+
signal progression from the granular region to the basolateral areas
(Tinel et al., 1999). To test
the proposition that the Ca2+ wave elicited by menadione can spread
fast to the nuclear region because of inhibition of mitochondrial function,
antimycin A, a well characterized inhibitor of mitochondrial electron
transport (Singer, 1979
), was
applied together with ACh. In this case the cytosolic Ca2+ response
was very similar to its response to menadione
(Fig. 3l) (speed ratio:
0.9±0.1, n=5, P>0.3), confirming the mitochondrial
involvement in the delayed appearance of the Ca2+ signal in the
nucleus in response to ACh stimulation under normal conditions. Mitochondrial
Ca2+ uptake (Park et al.,
2000
) could result in a slower rate of Ca2+ elevation
in the nucleus (Fig. 3), which
could be important by preventing activation of Ca2+-dependent
nuclear enzymes.
Menadione causes initial Ca2+ loading into mitochondria
followed by unloading
The results above suggest that ACh causes Ca2+ loading into
mitochondria, thereby delaying the spread of Ca2+ away from the
secretory pole, whereas menadione prevents buffering of Ca2+ by
mitochondria. To test this hypothesis, we compared the effects of ACh and
menadione on mitochondrial Ca2+ loading with Rhod-2. This
fluorescent dye has been well characterised as a reporter of changes in the
Ca2+ concentration inside mitochondria in living cells
(Duchen, 1999). We therefore
measured changes in rhod-2 fluorescence from the perigranular mitochondrial
belt (Tinel et al., 1999
). ACh
(100 nM) caused an initial rapid increase in the mitochondrial Ca2+
concentration followed by a slower decrease back towards basal Ca2+
levels (Fig. 4A). In contrast,
menadione only caused a very small and transient increase in the mitochondrial
Ca2+ concentration followed by a prolonged and marked reduction
(Fig. 4A, n=19). These
results indicate that although menadione causes a small initial uptake of
Ca2+ into the mitochondria, this is countered by induction of a
Ca2+ efflux pathway. This is likely to account for the rapid
menadione-induced spread of Ca2+ through the cell
(Fig. 3k).
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Menadione induces depolarisation of mitochondria
To gain further insight into potential Ca2+ efflux pathways from
mitochondria, we measured changes in the potential across the inner
mitochondrial membrane in isolated pancreatic acinar cells using the
membrane-potential-sensitive fluorescent dye TMRE. Cells were loaded with TMRE
and MitoTracker Green to check the localisation of the mitochondria. The TMRE
distribution was identical to the distribution of Mito Tracker Green
(Fig. 4Ba-c). Figs. 4Ba and 4Be show the TMRE
fluorescence intensity from the mitochondria (green box) and the nucleus
(yellow box) of the same cell measured simultaneously. The mitochondrial TMRE
fluorescence intensity decreased after application of menadione, indicating a
reduction in the mitochondrial membrane potential, whereas the nuclear TMRE
fluorescence, which is close to background, remained constant
(Fig. 4Be) (n=20). The
depolarisation was transient and the mitochondria regained their full
electrical potential some 500 seconds after the menadione application.
Furthermore, the depolarisation was only partial as the protonophore mCCCP (10
µM) had a much greater effect (Fig.
4Be). The menadione-induced depolarisation was markedly reduced,
but not abolished, by loading the cells with BAPTA
(Fig. 4C, n=20). It
has been suggested that the mitochondrial depolarisation may be the result of
caspase activity (Marzo et al.,
1998b), but this is not the case in isolated pancreatic acinar
cells, as Z-VADfmk did not block the mitochondrial depolarisation
(Fig. 4C, n=20).
In order to explore the relationship between changes in the cytosolic
Ca2+ concentration and the reduction in mitochondrial membrane
potential further, these two parameters were measured simultaneously in
mitochondrial regions of cells loaded with Fluo 4 and TMRE. Application of 100
nM ACh induced a substantial elevation in the cytosolic Ca2+
concentration but no change in the mitochondrial membrane potential.
Subsequent application of menadione induced both a renewed Ca2+
rise and a clear decrease in the mitochondrial membrane potential
(Fig. 4E-G) (n=7). In
agreement with these data, CCK in concentrations from 5 pM to 10 nM also
failed to elicit mitochondrial depolarization, although the protonophore FCCP,
in the same experiments, induced collapse of the potential across the inner
mitochondrial membrane (Raraty et al.,
2000). Taking all the data presented in
Fig. 4 together, it is apparent
that menadione causes both a rise in the cytosolic Ca2+
concentration and a reduction in the mitochondrial membrane potential. Since
ACh fails to elicit mitochondrial depolarization, it is clear that this
phenomenon cannot solely be a consequence of the cytosolic Ca2+
rise.
Menadione induces opening of the permeability transition pore
Mitochondrial depolarisation during apoptosis has been reported to be the
result of induction of the PTP (Marzo et
al., 1998a; Szalai et al.,
1999
). To test whether this is true in pancreatic cells, we
preincubated cells for 45 minutes with the PTP inhibitor bongkrekic acid at a
concentration of 50 µM before exposing the cells to menadione. Treatment
with bongkrekic acid either completely prevented mitochondrial depolarisation
or reduced it dramatically (Fig.
4C, n=23) suggesting that the mitochondrial
depolarisation is caused by PTP induction. Furthermore, antimycin A causes
mitochondrial depolarization, and this is not blocked by bongkrekic acid,
suggesting that menadione and antimycin A are causing mitochondrial
depolarization through different mechanisms (n=11)
(Fig. 4D). To test the PTP
induction hypothesis further, cells were loaded with calcein-AM in the
presence of cobalt chloride to quench fluorescence from all cellular domains
except from within mitochondria
(Petronilli et al., 1999
).
Using this protocol there was punctate calcein fluorescence around the
secretory pole and nuclei, consistent with mitochondrial staining
(Fig. 5A,B)
(Tinel et al., 1999
).
Menadione caused a loss of mitochondrial calcein fluorescence, which was
completely blocked by 50 µM bongkrekic acid
(Fig. 5C,D). Although this
protocol does not distinguish between calcein efflux and Co2+
influx, it is consistent with induction of the PTP. Loss of mitochondrial
calcein fluorescence was not simply a result of mitochondrial depolarisation,
as neither antimycin A (Fig.
5E) nor FCCP (data not shown) caused significant loss of calcein
fluorescence.
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Menadione causes efflux of cytochrome c into the cytoplasm and
activates caspase 9 and 3
To determine whether mitochondrial cytochrome c translocates from the
mitochondria to the cytoplasm, mitochondrial and cytoplasmic fractions were
collected following exposure to menadione or vehicle control for 2 minutes.
Cytochrome c was then detected and quantified by immunoblotting. Following
exposure to menadione for 2 minutes only, cytochrome c was released into the
cytosolic fraction (Fig. 6A,B).
BAPTA significantly reduced cytochrome c release from the mitochondria
(Fig. 6A,B) (n=5,
P<0.005).
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A family of cysteine proteases called caspases are known to be activated in
apoptosis and are among the principal effector systems of apoptotic cell
death. We therefore determined the role of caspase 9, a caspase at the apex of
the mitochondrial caspase cascade
(Thornberry and Lazebnik,
1998). Caspase 9 activity was determined using the fluorescent
probe FAM-LEHD-FMK (see Materials and Methods). The averaged value of
fluorescence of FAM-LEHD-FMK-stained control cells was 55.4±9.1.s.d.
arbitrary units (n=20). Cells with fluorescence higher than 82.7
arbitrary units (average value plus 3xs.d. values) were considered to be
FAM-LEHD-FMK positive. A significant increase in FAM-LEHD-FMK fluorescence was
detected in all cells after incubation with menadione for 30 minutes
(n=15, P<0.001) (Fig.
6C). Intracellular caspase 3 activity was determined using
PhiPhiLux-G2D2 - a fluorogenic substrate for caspase-3-like proteases
(Zapata et al., 1998
). The
averaged value of fluorescence of PhiPhiLux-stained control cells was
54.7±7.4s.d. arbitrary units (n=32). Cells with fluorescence
higher than 76.8 arbitrary units (average value plus 3xs.d. values) were
considered PhiPhiLux positive. A rise in the fluorescence intensity was
observed owing to an increase in caspase-3-like activity (n=44,
P<0.001) (95.5% of PhiPhiLux positive cells in population)
(Fig. 6D). Buffering of
cytosolic Ca2+ with BAPTA completely prevented activation of both
caspase 9 (n=15) and 3 (n=20)
(Fig. 6C,D), demonstrating that
Ca2+ is acting at a point upstream of caspase activation. Blockade
of caspase activation with Z-VADfmk did not prevent Ca2+ spike
formation (data not shown, n=13). These data exclude the possibility
that the menadione-elicited rise in the cytosolic Ca2+
concentration is the result of caspase 3 activation. Activation of caspase 9
was not simply the result of inhibition of the electron transport associated
with oxidative phosphorylation, as a combination of ACh and antimycin A did
not activate caspase 9 significantly (n=16)
(Fig. 3l,
Fig. 6C). Applications of 25
µM ACh or 5 pM CCK did not activate caspase 3 significantly
(Fig. 6D) (n=10 for
each group).
Bongkrekic acid inhibits menadione-induced apoptosis
Previous studies have suggested that induction of PTP is essential for the
full induction of apoptosis, though controversy surrounds this point
(Martinou et al., 2000). We
therefore determined whether bongkrekic acid prevents apoptosis induced by
menadione. Preincubation with bongkrekic acid before exposure to menadione
prevented apoptosis in the majority of cells as measured by Annexin V FITC
staining (n=29) (Fig.
2A). Only 14.3% of cells were apoptotic. Bongkrekic acid
completely prevented activation of caspase 9 as measured by FAM-LEDH-FMK
fluorescence (no FAM-LEDH-FMK positive cells, n=16)
(Fig. 6C) and markedly reduced
caspase 3 activity (19% of PhiPhiLux positive cells) as measured by
Phiphilux-G2D2 fluorescence (n=31)
(Fig. 6D). Together these data
suggest that activation of the PTP is an essential element in the process of
apoptosis induction elicited by menadione.
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Discussion |
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Menadione induces apoptosis as defined by the release of cytochrome c from
mitochondria, activation of caspase 9 and 3, Annexin V staining and, at later
time points, condensation of nuclear chromatin. Although nuclear condensation
often occurs simultaneously with Annexin staining, this depends on the cell
type and stimulus (Boersma et al.,
1996). Menadione in concentrations above 50 µM can induce
necrosis, but in our experiments all cells that were stained positive with
propidium iodide (and therefore were potentially necrotic) were excluded from
analysis. In some cell types, including ß-cells from pancreatic islets,
depletion of Ca2+ in the endoplasmic reticulum can induce apoptosis
after 6 to 48 hours of exposure (Baffy et
al., 1993
; Qi et al.,
1997
; Zhou et al.,
1998
). However, in the pancreatic acinar cells, loss of
Ca2+ from the ER was, by itself, insufficient to induce apoptosis.
This was seen in the experiments with specific blockade of the ER
Ca2+ ATPase, in which 30 minutes of exposure to the ER
Ca2+ pump inhibitor thapsigargin failed to induce apoptosis
(Fig. 2A). Even after 3 hours
of thapsigargin treatment, only 37.9% of the cells were Annexin V FITC
positive as compared with 86.3% of the cells treated by menadione for 30
minutes. Although a longer exposure to thapsigargin can induce apoptosis in
pancreatic acinar cells, the time course and the mechanism are probably
different from the apoptosis induced by menadione. Buffering cytosolic
Ca2+ with BAPTA does not prevent stimulant-evoked emptying of the
Ca2+ stores in the ER but actually enhances Ca2+ release
owing to a lack of Ca2+-mediated negative feedback on the
Ca2+ release channels (Mogami
et al., 1998
). This indicates that it is the menadione-elicited
rise in the cytosolic Ca2+ concentration, rather than the reduction
in the Ca2+ concentration within the ER, that is needed for the
induction of apoptosis.
Our studies show that ACh induces a significant uptake of Ca2+ into the mitochondria (Fig. 4A). In contrast, menadione only stimulates a very transient uptake of Ca2+ into the mitochondria followed by a sustained efflux (Fig. 4A). The spatially unrestricted cytosolic Ca2+ concentration rise elicited by menadione could play a causal role in the induction of apoptosis, but this is unlikely, as ACh combined with antimycin A, which also elicits a spatially unrestricted Ca2+ rise (Fig. 3l), does not activate caspase 9 or apoptosis (Fig. 4D, Fig. 6B). Furthermore bongkrekic acid does not prevent the antimycin-A-induced mitochondrial depolarization, although it blocks the menadione-elicited reduction of the mitochondrial membrane potential. This demonstrates that antimycin A does not induce the PTP. These data indicate that it is induction of the PTP that is important for apoptosis and not mitochondrial depolarisation. Furthermore, loss of ATP is excluded as a potential trigger for apoptosis within the time frame of these experiments. It is interesting to note that high concentrations of ACh and antimycin A cause an increase in the nuclear Ca2+ concentration, but this rise would appear not to be sufficient on its own to activate nuclear endonucleases, as these agents do not evoke apoptosis.
The menadione-induced mitochondrial depolarisation was partial, transient
and somewhat dependent on the cytosolic Ca2+ concentration rise,
but not influenced by caspase inhibition
(Fig. 2B,
Fig. 5, Fig. 6). As the normal
Ca2+ spikes evoked by ACh did not induce mitochondrial
depolarization (Fig. 6), it is
clear that the ability of menadione to elicit a reduction in the potential
across the inner mitochondrial membrane, even under conditions of cytosolic
BAPTA loading, could not be explained simply as a consequence of the cytosolic
Ca2+ signal generation. These results contrast with those of Szalai
et al. (Szalai et al., 1999),
who found that Ins(1,4,5)P3-driven Ca2+ signals
were sufficient to induce mitochondrial depolarisation in a permeabilised
hepatocyte cell line.
The mitochondrial depolarization caused by menadione is probably due to
induction of the PTP, as bongkrekic acid, a specific inhibitor of the PTP
(Crompton, 1999;
Marzo et al., 1998b
), markedly
reduced or abolished the menadione-elicited mitochondrial depolarization
(Fig. 4C). Further evidence for
induction of the PTP was obtained with the fluorescent probe calcein
(Petronilli et al., 1999
).
Menadione, but neither FCCP nor antimycin A, caused loss of calcein
fluorescence, which indicated that a large enough pore had been induced to
allow either calcein efflux or Co2+ influx. Either way, this pore
was blocked by bongkrekic acid, providing further evidence for PTP induction.
It has been reported that mitochondrial depolarisation could be induced by the
action of caspases (Marzo et al.,
1998b
), but this was not the case in our experiments, as ZVADfmk
did not prevent depolarisation (Fig.
4C). We hypothesise that the transient uptake of Ca2+
into mitochondria is sufficient to induce the PTP in the presence of
oxidants.
The role of the PTP in apoptosis is controversial. Evidence has been
reported that induction of the PTP is tightly coupled to cytochrome c release
(Fulda et al., 1998;
Heiskanen et al., 1999
).
Furthermore, it has been shown that the proapoptotic protein Bax binds to the
adenine nucleotide translocator, a component of the PTP, to induce apoptosis
(Marzo et al., 1998a
).
However, apparently conflicting results have also been reported, where loss of
mitochondrial potential occurs after cytochrome c release, and it is prevented
by inhibition of caspases (Bossy-Wetzel and
Green, 1999
; Bossy-Wetzel et
al., 1998
; Finucane et al.,
1999
; Nomura et al.,
1999
). In our studies we found that bongkrekic acid blocked the
induction of apoptosis by menadione in the majority of cells studied
(Fig. 6). Within the resolution
of our analysis, mitochondrial depolarisation occurred simultaneously with the
initiation of Ca2+ oscillations induced by menadione
(Fig. 4G).
A remarkable feature of menadione-induced apoptosis is that it is very
rapid. Cytochrome c release from mitochondria can be detected within 2 minutes
of drug application (Fig. 6A).
Similar results have been reported in experiments on isolated mitochondria
where substantial cytochrome c release was found 5 minutes after exposure to
Ca2+ (Gogvadze et al.,
2001). Caspase 9 and 3 activations can be detected 30 minutes
after exposure to menadione. This time course of activation is faster than
many other examples of apoptosis but is not without precedence. For example,
in apoptosis induced by Ins(1,4,5)P3-linked mitochondrial
calcium signals, similar short periods of caspase induction have also been
described (Szalai et al.,
1999
). Furthermore activation of caspase 9 and 3 is consistent
with the kinetics for assembly of the apoptosome, which can occur within 5
minutes (Cain et al., 2000
).
However it should be noted that chromatin condensation could not be detected
until 3 hours after exposure to menadione. Thus our data are consistent with
previous studies of the induction of apoptosis in pancreatic cell lines
(Sata et al., 1997
).
Our data are consistent with a model in which the Ca2+ released
from the ER by menadione is transiently taken up by the mitochondria. In the
presence of oxidants, this causes induction of the PTP
(Chernyak and Bernardi, 1996)
and therefore releases Ca2+ from the mitochondria, allowing a wave
of Ca2+ to traverse the rest of the cell
(Fig. 7). Induction of the PTP
allows release of cytochrome c, which leads to the processing and activation
of caspase 9 and caspase 3, externalisation of phosphatidylserine and
apoptosis (Fig. 7). We cannot
exclude the possibility that elevations in the cytosolic Ca2+
concentration have additional actions such as regulation of the
phosphorylation of proteins critical for apoptosis, for instance the
pro-apoptotic protein BAD (Wang et al.,
1999
). Neither mitochondrial depolarization per se or loss of ATP
is responsible for the triggering of apoptosis, as treatment with ACh and
antimycin A do not cause apoptosis, because the PTP is not induced under these
non-oxidising conditions. This contrasts with the action of submaximal ACh
concentrations. In these cases, Ca2+ released from the ER into the
granular area is trapped by perigranular mitochondria, which, by means of
Ca2+ uptake, act as a firewall and thereby prevent spread to the
rest of the cell (Fig. 7).
|
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Acknowledgments |
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