Regional Loss of the Mitochondrial Membrane Potential in the
Hepatocyte Is Rapidly Followed by Externalization of
Phosphatidylserines at That Specific Site during Apoptosis*
W. Marty
Blom,
Hans J. G. M.
de Bont, and
J. Fred
Nagelkerke
From the Division of Toxicology, Leiden-Amsterdam Center for Drug
Research, Leiden University, 2300 RA Leiden, The Netherlands
Received for publication, February 7, 2002, and in revised form, December 6, 2002
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ABSTRACT |
The spatio-temporal relationship between a
decrease in the mitochondrial membrane potential (MMP) and
externalization of phosphatidylserines (PS) during induction of
apoptosis was investigated in single freshly isolated hepatocytes.
Apoptosis was induced in the hepatocytes in three different ways:
attack by activated Natural Killer cells, exposure to ATP, or exposure
to the inhibitor of protein synthesis cycloheximide. Fluorescence
microscopy showed staining of externalized PS at those areas where the
staining for MMP was lost whereas in other areas the mitochondria
remained intact for longer periods of time, indicating coupling between
local loss of MMP and local PS exposure. To discriminate whether the
decrease in MMP itself or a decrease in ATP induced PS externalization,
hepatocytes were treated with rotenone, which resulted in a rapid
collapse of cellular ATP but left the MMP intact for a much longer
period. Addition of fructose prevented the decrease of ATP to ~30%
and also delayed the collapse of the MMP. This indicates that ATP was
needed for the maintenance of the MMP probably via reverse action of
the ATP synthase. In a subsequent study hepatocytes were incubated with
Natural Killer cells for induction of apoptosis followed by addition of
rotenone to deplete ATP. Under these conditions the PS staining
co-localized with mitochondrial MMP indicating that PS externalization
does not require a collapse in MMP. Moreover, exposure of PS was evenly
distributed over the whole plasma membrane. In conclusion, we
propose that after an apoptotic stimulus some mitochondria start to
loose their MMP, which results in cessation of ATP production and
perhaps even consumption of ATP. This results in an overall decrease in
cellular ATP. ATP-consuming enzyme reactions most distal from still
intact mitochondria will be most sensitive to such a decrease.
Apparently the translocase that keeps phosphatidylserines inward-oriented is such a sensitive enzyme.
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INTRODUCTION |
It has been demonstrated in many cell types, using a large
variety of inducers, that mitochondria often play a crucial role in
development of apoptosis and necrosis (reviewed in Refs. 1 and 2).
Already in the 1980s a relation was observed between elevated calcium
concentrations and the mitochondrial membrane potential
(MMP)1 on the one hand and
cell death on the other hand upon exposure of hepatocytes to toxic
compounds or after ischemia reperfusion (3). It was shown that opening
of a high conductance permeability transition pore after
oxidative stress in the mitochondrial inner membrane abruptly increases
the permeability of the mitochondrial inner membrane to solutes of
molecular mass up to 1500 Da (4, 5). The opening of the pore is
associated with a collapse of the MMP, perturbation of intracellular
and mitochondrial Ca2+ homeostasis, and subsequently cell
death. The mitochondrial outer membrane is also the site of competition
between the pro- and anti-apoptotic proteins of the Bcl-2 family and is
associated with the opening of the pore (6-9). Moreover, the
mitochondria are a source of a number of the Bcl-2 family members and,
in lymphoid cells, of the apoptosis inducing factor, a protein
associated with opening of the pore and induction of apoptosis (10).
Other mitochondrial factors that are associated with liver apoptosis in vivo are proteases that are released during cholestasis
(11). Finally several studies showed that after induction of apoptosis mitochondria release cytochrome c (12), which is associated with opening of the mitochondrial pore (13). Cytochrome c
forms a complex with apoptotic protease activating factor-1,
procaspase-9, and ATP. Formation of this complex, the apoptosome, leads
to formation of active caspase-9, which that can subsequently activate
other caspase proteins. In this way the apoptotic signal is amplified (reviewed in Ref. 14). Associated with the activation of the caspases
is exposure of phosphatidyl- serines (PS) in the outer leaflet of cells
(15, 16). These molecules function as a signal for macrophages or other
cells from the reticuloendothelial system to engulf and digest
apoptotic bodies (17). In this way release of intracellular components
and a subsequent immunological reaction is prevented. The exposure of
PS is either the result of inhibition of an ATP-dependent
aminophospholipid transporter (18, 19) or activation of a
calcium-dependent scramblase (20-22). In addition, synthesis of PS through a calcium-dependent exchange of the
polar head group of pre-existing phospholipids has been described
(23).
In thymocytes exposure of PS occurs only in those cells that have lost
their MMP (15, 24). In other cell types the opposite was found; in
L929sAh cells transfected with the FAS receptor and treated with
anti-FAS initially PS were exposed and followed later by a drop in MMP
(25). We described the involvement of the mitochondria in the induction
of apoptosis in hepatocytes after attack by interleukin-activated
Natural Killer (A-NK) cells (16). Fluorescence-activated cell sorter
analysis after staining for extracellular-oriented PS with
fluorescent-labeled annexin V (ANV) demonstrated that A-NK cells
induced apoptosis in the hepatocytes (10% apoptotic cells after 30 min
and 38% after 60 min of co-incubation). In these apoptotic cells the
overall MMP was ~60% of the value as compared with non-apoptotic
hepatocytes. The decrease in MMP and exposure of PS in hepatocytes is
apparently tightly coupled, because all cells with a lowered MMP
exposed PS. In comparison with other studies (15, 24, 25) the decrease in MMP was relatively moderate, and the exposure of PS was very rapid.
Microscopical examination showed that ANV staining was unevenly
distributed over the cell membrane.
To gain more insight in the spatio-temporal relationship between
a decrease in the MMP on the one hand and exposure of PS on the other
hand the present study was undertaken. Time-lapse video microscopy and
confocal laser scan microscopy of hepatocytes that were attacked by
A-NK cells was performed. In addition, two totally unrelated
apoptosis-inducing stimuli were applied: addition of extracellular ATP
or cycloheximide (26, 27). We report that in all cases a local loss of
MMP of a limited number of mitochondria in the cell results, within
minutes, in exposure of phosphatidylserines at that particular spot.
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MATERIALS AND METHODS |
Collagenase, recombinant protein ANV, and
HEPES were obtained from Roche Molecular Biochemicals.
Tetramethylrosamine (TMR), rhodamine 123, propidium iodide (PI),
TOTO-3, and the AlexaTM protein labeling kit were from
Molecular Probes. Fluorescent ANV was prepared by labeling with
AlexaTM488 or AlexaTM633. Bovine serum albumin
(type V), the luciferin/luciferase kit, and poly-L-lysine
were from Sigma. Mouse anti-rat monoclonal antibody OX18 (anti-total
rat major histocompatibility complex class I) was prepared as described
before (28).
Isolation and Activation of Natural Killer Cells
Isolation of natural killer cells and activation of these cells
were done as described (16). Briefly, a cell suspension was prepared
from spleen isolated from a 4-5-month-old male Wag rat
(RT1u), a Wistar-derived strain, purchased from Charles
Rivers Wiga (Schulzfeld, Germany). The splenocytes were separated from
other cells (i.e. B cells, macrophages) by nylonwool
adherence. The non-adherent cells contained ~30% A-NK cells, which
were collected and cultured in RPMI 1640, Dutch modification
(Invitrogen), supplemented with 10% (v/v) heat-inactivated fetal calf
serum, 2 mM glutamine, 50 µg/ml streptomycin, and 50 units/ml penicillin (all Invitrogen). The culture medium was
supplemented with 1000 Cetus units/ml human recombinant interleukin-2
(Chiron) and 50 µM 2-mercaptoethanol. After 24 h
non-adhering cells were removed, and the remaining adhering cells, A-NK
cells, were cultured for another 6 days. The population thus obtained
consisted for
95% of CD161A-positive cells and for
5% of T cell
receptor-positive cells.
Isolation and Incubation of Rat Hepatocytes
Liver parenchymal cells were isolated by collagenase perfusion
from male Wistar rats (200-230 g) (29), purchased from Charles Rivers Wiga (Schulzfeld, Germany) and housed at least 1 week at the
animal facilities of the Sylvius Laboratories. The rats were fed
ad libitum and kept at a 12-h day-night cycle. Prior to the experiment the rats were fasted for 24 h. Viability of the freshly isolated cells was >95% as determined by trypan blue exclusion. After
isolation cells were kept on ice until use.
Incubation of Hepatocytes and A-NK Cells
To allow non-self recognition and induce the cytotoxic response
by A-NK cells, the major histocompatibility complex class I protein of
hepatocytes was blocked with the OX18 antibody (27). This masking leads
to recognition of the target cell as foreign and activation of the
killing machinery (29, 30). The liver cells were preincubated with the
OX18 antibody at 4 °C for 45 min in Hanks'/HEPES buffer (pH 7.4, 4 °C) composed of 120 mM NaCl, 5 mM KCl, 4.2 mM NaHCO3, 1.2 mM
NaH2PO4, 1.3 mM CaCl2,
0.4 mM MgSO4, 25 mM HEPES
supplemented with 10 mM glucose and 1% (w/v) bovine serum
albumin and gassed for 30 min with 95% O2/5%
CO2. Then, cells were washed and resuspended in either
William's E supplemented with 10% fetal calf serum or the
Hanks'/HEPES buffer supplemented with 1% (w/v) bovine serum albumin.
For microscopical experiments isolated liver cells were allowed to
attach to circular glass coverslips coated with
poly-D-lysine (1 mg/ml in water) (16). The coverslips were
placed in a microscope chamber, and 500 µl of cell suspension
(3.0 × 105cells/ml) was carefully added to the glass
coverslip; the hepatocytes were allowed to attach for 45 min. The cells
were kept at 37 °C throughout the experiment. The experiments with
the A-NK cells were started by removal of the medium and addition of
A-NK cells in William's E supplemented with 10% fetal calf serum in
the effector:target ratio of 20:1. This ratio was chosen, because
previous experiments showed that it resulted in apoptosis in a large
number of hepatocytes (16). In other experiments ATP or cycloheximide
were added directly to the cells.
Batch incubations were performed in the same Hanks' buffer on a rotary
shaker that was kept at 37 °C. Cells were incubated at a density of
3.0 × 105 cells/ml. At the selected intervals 0.5-ml
samples were taken for flow cytometry and ATP determinations.
Imaging Techniques
The video microscopy system consisted of an IM35 inverted
microscope with a 100-watt mercury arc lamp (Zeiss) and a Nikon ×40/1.4 NA numerical aperture Fluor objective. ANV staining was detected using a 475-nm band pass filter for excitation, a 510-nm dichroic mirror, and a 540-nm band pass emission filter. TMR was visualized using a 535-nm band pass filter for excitation, a 580-nm dichroic mirror, and a 590-nm long pass filter for emission. Images were recorded using a CCD instrumentation camera, controlled by a CC200
camera controller (Photometrics, Tucson, AZ).
For confocal laser fluorescence microscope (CLSM) an upgraded Bio-Rad
MRC-600 system was used (31). The first filter block of the CLSM
contained a triple dichroic mirror (488/543/633) and an emission filter
(488/543/633). ANV was excited with the 488-nm argon laser, TMR
with the 543-nm HeNe laser, and TOTO-3 with the 633-nm HeNe laser.
Staining Techniques
PS Externalization and Plasma Membrane
Permeabilization--
Exposition of PS on the extracellular side of
the plasma membrane of hepatocytes was visualized by staining PS with
ANV labeled with fluorescent AlexaTM488 (1 µg/ml ANV and
AlexaTM488 in a stoichiometric complex of 1:1) (4). 0.2 µl ANV and 2 µl of a 5 mM solution of the
cell-impermeable dye TOTO-3 were added to 500 µl of cell suspension
in the incubation chamber of the microscope.
Determination of the Relation among Externalization of the
Phosphatidylserines, Mitochondrial Membrane Potential, and Cell
Death--
Hepatocytes were pre-incubated for 15 min with 0.2 µM TMR. Subsequently ANV and TOTO-3 were added. After
recording of the baseline level of TMR the apoptotic stimuli were given.
Determination of the Degree of Co-localization of Fluorescent
Signals of Different Dyes--
Co-localization of the signals was
measured by comparing the equivalent pixel positions in each image and
generation of a co-localization scatter plot using Image Pro software
(Media Cybernetics, Silver Spring, MD). These scatter plots were
analyzed using Pearson's correlation. The result of this
analysis is a number between +1 and
1. The former indicates perfect
correlation, and the latter indicates no correlation (32,
33).
Determination of the Relation among Externalization of the
Phosphatidylserines, Intracellular Free Calcium
([Ca2+]i), and Cell Permeabilization
For determination of intracellular free calcium the hepatocytes
were loaded with 40 µM Fura-2/AM for 30 min. Then, the
cells were washed carefully with Hanks'/HEPES buffer at 37 °C.
Next, ANV and A-NK cells were added. From a group of cells the 470-nm emission images after 340- and 380-nm excitation were recorded using a
dichroic mirror of 395 nm and a 470-nm long pass emission filter.
Images were corrected by background subtraction before calculation of
the ratio images. Ratio images of the 340/380-nm excitation were
determined by division of the 340-nm image by the 380-nm image on a
pixel-to-pixel basis. The intracellular free calcium concentration was
calculated using the equation, [Ca2+]i = Kd*
*[(R
Rmin)/(Rmax
R)], with Kd = 224 nM as the
equilibrium dissociation constant for Ca2+ and Fura-2.
Rmin is the ratio
F340/F380 at zero
calcium; Rmax is the ratio
F340/F380 at saturating
calcium;
is the ratio F380 (zero
calcium)/F380 (saturating calcium), and
F is the pixel fluorescence intensity.
Flow Cytometric Analysis of the Percentage of Viable, Apoptotic,
and Dead Hepatocytes
The MMP of viable, apoptotic, and dead cells was determined by
flow cytometric analysis using ANV, PI, and rhodamine 123 as described
earlier (16). Briefly, after addition of ANV, rhodamine 123, and PI the
hepatocytes were incubated for 15 min on ice in the dark. The
fluorescence of individual cells was analyzed using a FACScalibur flow
cytometer (BD Biosciences), using the CellQuest program.
Determination of ATP in the Hepatocytes
Samples of the hepatocytes were snap-frozen in liquid
N2. Determination of ATP was started by addition of
HClO4 to the frozen cells. Subsequently KPO4
was added, and after 10 min pH was neutralized with KOH. The tubes were
centrifuged, and ATP was determined in the supernatant using the Sigma
luciferin/luciferase kit.
Statistics
Values are expressed as mean ± SD. The statistical
evaluation was performed with an unpaired two-tailed Student's
t test.
 |
RESULTS |
Freshly isolated rat hepatocytes were loaded with TMR to visualize
effects on the MMP; in addition, ANV labeled with Alexa 488 was added
to stain external PS. The hepatocytes were attached to a coverslip
mounted at the bottom of the incubation chamber; after recording of
baseline values the A-NK cells were added as a suspension to the
chamber, and therefore, it took some time before a contact between the
hepatocytes and A-NK cells had been established. Fig.
1 shows that 17 min after the addition of
the A-NK cells such a contact was made. At this time point the
morphology of the hepatocyte was still normal (Fig. 1, bottom
row). However, 15 min later (32') the hepatocyte had become
apoptotic showing numerous blebs. At this time point at certain sites
the MMP was dissipated (Fig. 1, third row). This was
followed 6 min (38') later by the first staining of PS (Fig. 1,
second row, hardly visible). These processes,
decrease in MMP and externalization of PS, continued progressively.
Because TOTO-3 was added the absence of a nuclear staining in the
images indicates that until 94 min no secondary necrosis had occurred;
the cells were still intact. The merged images of the green
MMP signal and the red PS signal are shown in the top
row. If these signals co-localize the merged signal becomes
yellow. The absence of yellow staining indicates that
only at the area where mitochondria had lost their MMP externalization of PS occurred.

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Fig. 1.
Time course of disruption of the MMP and the
externalization of PS in isolated hepatocytes exposed to A-NK
cells. Freshly isolated rat hepatocytes were exposed to
A-NK cells. ANV and PI were added to hepatocytes that were preloaded
with 0.2 mM tetramethylrosamine for 15 min; subsequently
images of the same cell were taken using video microscopy. At
t = 0 A-NK cells were added, and at the indicated time
points (in min) images were taken of the PS externalization
(red ANV staining), MMP (green TMR staining), and
morphology. The non-permeable probe PI was added to the cells to verify
that the loss off MMP was not caused by a leakage of fluorescent probe
because of plasma membrane permeabilization. The upper row
shows the merged images of PS externalization and decrease in
MMP.
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To acquire more detailed images we used the CLSM. Hepatocytes were
incubated under the same conditions as described above. Fig.
2A shows the MMP, and Fig.
2B shows the externalized PS. As can be seen in the merged
Fig. 2C the signals do not co-localize confirming the
finding that PS only externalize in the vicinity of defective
mitochondria. The co-localization was analyzed further using Pearson's
correlation, which is discussed at the end of the "Results."

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Fig. 2.
Confocal laser scan photomicrograph of
disruption of the MMP and the externalization of PS in isolated
hepatocytes exposed to A-NK cells. Conditions were the same as for
Fig. 1. TMR was excited with the green laser (A),
and ANV was excited with the blue laser (B).
These images were pseudo-colored and merged (C). The
morphology is shown in D; arrows indicate
apoptotic cells. Hepatocytes were exposed to A-NK cells for 95 min.
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To test the specificity of the response, apoptosis was induced in
hepatocytes with cycloheximide, a protein synthesis inhibitor. Fig.
3 shows a CLSM image taken 55 min after
the addition of cycloheximide. The arrows in Fig.
3D point at the apoptotic cells. These cells have a lowered
MMP and are high in ANV staining; the merged image again indicates no
co-localization of the two signals. Identical results were found when
apoptosis was induced using addition of extracellular ATP (Fig.
4). These results show that three
completely different apoptotic signals had the same effect:
externalization of PS in the near vicinity of defective mitochondria. A
possible explanation for this phenomenon could be a local decrease in
ATP around defective mitochondria. The enzyme that maintains the
asymmetric orientation of PS, aminophospholipid translocase, needs ATP
for its activity. The most direct approach to investigate the role of
ATP would have been intracellular determination of the concentration using a cell-permeable probe like those used for determination of
Ca2+ or MMP. Unfortunately such a probe does not yet exist.
Therefore, we chose another approach: depletion of ATP without collapse
of the MMP by using the mitochondrial inhibitor rotenone. First, in
batch incubations of hepatocytes, the effect of rotenone on intracellular ATP and MMP was determined. Cells were incubated, and at
different time points two samples were taken. One was immediately snap-frozen for ATP determination. The second was incubated with ANV,
rhodamine 123, and PI for flow cytometric analysis as described under
"Materials and Methods." Fig. 5 shows
that even at 10 µM rotenone ATP levels were less than
15% of control within 30 min. In contrast the MMP remained largely
intact for more than 2 h (Fig. 5). Interestingly, addition of
fructose prevented the decrease in ATP to about 50% and moreover,
delayed the decrease in the MMP. A significant increase in the number
of dead cells as a result of rotenone exposure occurred in parallel
with the (late) decrease in MMP. At all time points neither in the
control nor in the cells incubated with rotenone more than 1% of the
cells were apoptotic, indicating that a decrease in cellular ATP is not
sufficient to induce apoptosis. Subsequently hepatocytes were
co-incubated with A-NK cells to induce apoptosis, and after 15 min
rotenone was added to deplete ATP, and the localization of PS staining
and MMP staining was determined in apoptotic cells. Fig.
6A shows the MMP, and Fig.
6B shows the localization of the PS. In Fig. 6C
these images were merged; it is evident that both stainings overlap. To
further substantiate this the Pearson's correlations of this image and
of the image depicted in Fig. 1C were calculated. The
average value in Fig. 1C was 0.15 ± 0.05 whereas the
value for Fig. 6C was 0.75 ± 0.07. This indicates that
indeed there was much more overlap in staining after treatment with
rotenone. Furthermore, comparison of Fig. 1B and Fig.
6B shows that PS staining was patchy in the former whereas
in rotenone-treated cells the whole plasma membrane was stained
uniformly. When hepatocytes were incubated with rotenone and fructose
and A-NK cells similar images were obtained in which MMP and PS
staining were co-localized. The prevention of the decrease in ATP was
not sufficient to prevent the PS externalization. In the presence of
rotenone and fructose or rotenone alone the same percentage of the
cells became apoptotic and necrotic. A three-dimensional image of Fig.
6B is available on our web site,
www.pharm.leidenuniv.nl/lacdrhomepage/divisions/toxicology/nk2.htm.

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Fig. 3.
Confocal laser scan photomicrograph of
disruption of the MMP and the externalization of PS in isolated
hepatocytes exposed to ATP. Conditions were the same as for Fig.
1. Hepatocytes were incubated with 0.4 mM ATP.
A, TMR; B, ANV; C, merge;
D, morphology is shown. Arrows indicate apoptotic
cells. Hepatocytes were exposed to ATP for 27 min.
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Fig. 4.
Confocal laser scan photomicrograph of
disruption of the MMP and the externalization of PS in isolated
hepatocytes exposed to cycloheximide. Conditions were the same as
for Fig. 1. Hepatocytes were incubated with 30 µg/ml cycloheximide.
A, TMR; B, ANV; C, merge;
D, morphology is shown. Arrows indicate apoptotic
cells. Hepatocytes were exposed to cycloheximide for 55 min.
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Fig. 5.
ATP concentrations in hepatocytes and MMP
exposed to fructose and/or rotenone. Hepatocytes were incubated,
and samples were drawn. One-half of the sample was immediately frozen
in liquid nitrogen. In this sample ATP was determined using the
luciferin/luciferase assay. In the other half of the sample the MMP was
determined using the flow cytometer. A, control;
B, 10 µM rotenone; C, 3 mM fructose; D, 10 µM rotenone and
3 mM fructose.
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Fig. 6.
Confocal laser scan photomicrograph of
disruption of the MMP and the externalization of PS in isolated
hepatocytes exposed to A-NK cells after addition of rotenone.
Conditions were the same as for Fig. 1. TMR was excited with the
green laser (A), and ANV was excited with the
blue laser (B). These images were pseudo-colored
and merged (C). The morphology is shown in D;
arrows indicate apoptotic cells. Hepatocytes were exposed to
A-NK cells for 70 min. Rotenone was added 20 min after the start of the
incubation.
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To investigate the role of intracellular free calcium hepatocytes were
loaded with Fura-2/AM and incubated with the A-NK cells in the presence
of ANV. Initially the same magnification was used as in Figs. 2-4.
Using video microscopy, in many cells an increase in the intracellular
calcium concentration was observed, but no shifts in the Fura-2 signal
at specific locations inside the cell occurred; the calcium changes
were homogeneous within the cells. Therefore, a lower magnification was
used to allow us to follow more cells. We found that addition of A-NK
resulted in a significant increase of
[Ca2+]i from the resting
[Ca2+]i of 205 ± 102 nM (n = 56) to 540 ± 102 nM in 60% (34 of 56) of the observed hepatocytes followed
by externalization of the PS. However, in the other 40% no increase in
intracellular free calcium occurred before PS were externalized.
Moreover, in 17% of the hepatocytes PS were not externalized despite a
[Ca2+]i response.
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DISCUSSION |
Apoptosis was induced in the hepatocytes after attack by A-NK
cells (16) and exposure to ATP (26) or cycloheximide (27). All three
conditions resulted in a rapid decrease of the MMP followed by
externalization of the PS. As shown in the time-lapse series of the
attack by A-NK cells, contact between some hepatocytes and A-NK cells
was made after 17 min. Already at 32 min mitochondria in some areas of
the hepatocytes had lost their MMP, and at 38 min exposure of PS at
sites with affected mitochondria began. As is evident from the
photomicrographs externalization of PS took place at those areas or
sites of the cell where mitochondria had lost their MMP. The most
straightforward explanation for PS externalization at these sites is
that ATP levels in the immediate surroundings of defective mitochondria
rapidly decreases resulting in inhibition of the translocase that
normally translocates phosphatidylserines to the inner leaflet.
Unfortunately no cell-permeable dyes are yet available to determine
local changes in ATP within the cell. ATP has been determined in
hepatocytes, but this involved microinjection of luciferase and
immobilization of the cells in agar, which are both conditions that
will probably effect membrane structure (34) and interaction of
hepatocytes with A-NK cells. Similarly, microinjection of a vector for
luciferase (35) will affect membrane structure, and, in addition, it
requires cell culture that certainly affects energy metabolism in
hepatocytes. Therefore, to discriminate between effects of a collapse
of the MMP itself or the resulting decrease in ATP, we chose the
approach to deplete ATP from cells while keeping the MMP intact, by
treatment of the cells with rotenone. Alternatively we depleted ATP
with rotenone in the presence of fructose to replete it again. Under
ATP-depleted conditions the staining for externalized PS co-localized
with mitochondrial MMP indicating that PS externalization does not
require a collapse in MMP. We found, as reported before (36), that
depletion of ATP by itself does not induce apoptosis; an apoptotic
signal is needed. Very recently (37) a similar finding was reported;
Fas-triggered PS exposure was enhanced by depletion of intracellular
ATP. In cells incubated with rotenone in the presence of fructose the ATP level remained partly intact, but, importantly, the MMP was at the
same level as in control cells indicating that locally generated ATP
from fructose is used for maintenance of the MMP. Such a mechanism has
been described in osteosarcoma cells with defective mitochondria and is
based on reverse action of the ATP synthase (38). The enzyme that
ultimately produces ATP from 3-phosphoglycerolphosphate,
phosphoglyceratekinase, is located throughout the cytosol and often in
the direct vicinity of mitochondria (39). Therefore these organelles
have direct access to newly formed ATP. We therefore hypothesize that
locally produced ATP is used for maintenance of the MMP rather than
transported to peripheral regions of the cell.
Only a part of the mitochondrial population in a cell lost its
potential in our experiments, resulting in a population of cells that
have a relative high overall MMP but do expose PS locally. This is in
contrast with the situation in thymocytes and lymphoma cells (6, 40) in
which the MMP needs to be fully disrupted before externalization of PS
occurs. Above we hypothesized that the link between MMP and PS is a
local drop in ATP. The ability of cells to generate ATP by mitochondria
or glycolysis and the rate of ATP consumption determine the ultimate
local ATP level. In particular, local consumption of ATP by
mitochondria and the presence of high ATP consumption by the plasma
membrane Na+-K+- and
Ca2+-ATPase at the periphery may result in a lower
[ATP] in this domain than in the bulk of the cell cytosol (41). This
may differ strongly between different cell types and, therefore,
influence the coupling between MMP and PS. As the phospholipid
translocase is also an ATP-dependent enzyme this may
explain why locally, where the mitochondria do not function anymore, PS
externalization takes place.
Next to ATP, calcium-dependent processes have also been
described to play a role in PS externalization by activation of a calcium-dependent scramblase or synthesis of PS through a
calcium-dependent exchange of the polar head group of
pre-existing phospholipids. We measured the calcium concentration in
hepatocytes loaded with Fura-2 during attack by A-NK cells. ANV and
TOTO-3 were added to the medium, and, therefore, we could monitor when
cells became apoptotic or necrotic. We found no straightforward
relation between elevation of intracellular calcium and PS exposure. In
60% of the hepatocytes calcium was elevated before exposure of PS, but in 40% it was not. Also, in a number of cells, the calcium levels decreased to baseline again before PS exposure occurred. In addition, some hepatocytes had elevated calcium levels but did not expose PS.
Therefore, in the experiments with the A-NK cells the activation of a
calcium-activated scramblase is probably not essential (but cannot be
excluded). Similarly the involvement of calcium-dependent synthesis is doubtful.
In conclusion we show that in hepatocytes during apoptosis mitochondria
in certain areas within the cell lose their MMP resulting in local
exposure of the PS whereas in other areas mitochondria remain intact.
Although the former apparently leads to exposure of PS, which is
essential for removal of the apoptotic bodies, the latter could be
important for proper assembly of cytochrome c, apoptotic
protease activating factor-1, procaspase-9, and ATP into an apoptosome.
We used in this study A-NK cells that secrete the apoptosis-inducing
molecules granzyme B/perforin and produce FAS ligand and tumor necrosis
factor-related apoptosis-inducing ligand. In addition, ATP was
used, which induces an immediate influx of calcium, resulting in a loss
of MMP (42) and cycloheximide that is a protein synthesis inhibitor.
All these stimuli produced the same result, and, therefore, the
spatio-temporal relationship between a decrease in MMP and exposure of
PS could be a general phenomenon in cells that depend on mitochondria
for their ATP supply.
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FOOTNOTES |
*
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 should be addressed: Division Toxicology,
LACDR, Gorlaeus Laboratories, P. O. Box 9502, 2300 RA Leiden, The
Netherlands. Tel.: 31-71-5276226; Fax: 31-71-5274277; E-mail: nagelker@lacdr.Leidenuniv.nl.
Published, JBC Papers in Press, January 21, 2003, DOI 10.1074/jbc.M201264200
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ABBREVIATIONS |
The abbreviations used are:
MMP, mitochondrial membrane potential;
A-NK, interleukin-2-activated natural
killer cells;
ANV, annexin V;
CLSM, confocal laser scan microscopy;
PI, propidium iodide;
PS, phosphatidylserine(s);
TMR, tetramethylrosamine;
TOTO-3, 1,1'-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)bis[4-(3-methyl-2,3-dihydrobenzo-1,3-thiazolyl-2-methylidene)quinolinium] tetraiodide.
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