Changes in ultrastructure and the occurrence of permeabilitytransition in mitochondria during rat liver regeneration Ferruccio Guerrieri1,*, Giovanna Pellecchia1, Barbara Lopriore1, Serg
Trang 1Changes in ultrastructure and the occurrence of permeability
transition in mitochondria during rat liver regeneration
Ferruccio Guerrieri1,*, Giovanna Pellecchia1, Barbara Lopriore1, Sergio Papa1, Giuseppa Esterina Liquori2, Domenico Ferri2, Loredana Moro3, Ersilia Marra3and Margherita Greco3
1
Department of Medical Biochemistry and Biology, University of Bari, Italy;2Department of Zoology, Laboratory of Histology and Comparative Anatomy, University of Bari, Italy;3Center for the Study of Mitochondria and Energy Metabolism (CNR) Bari, Italy
Mitochondrial bioenergetic impairment has been found in
the organelles isolated from rat liver during the prereplicative
phase of liver regeneration To gain insight into the
mech-anism underlying this impairment, we investigated
mito-chondrial ultrastructure and membrane permeability
properties in the course of liver regeneration after partial
hepatectomy, with special interest to the role played by Ca2+
in this process The results show that during the first day after
partial hepatectomy, significant changes in the ultrastructure
of mitochondria in situ occur Mitochondrial swelling and
release from mitochondria of both glutamate dehydrogenase
and aspartate aminotransferase isoenzymes with an increase
in the mitochondrial Ca2+ content were also observed
Cyclosporin-A proved to be able to prevent the changes in
mitochondrial membrane permeability properties At 24 h after partial hepatectomy, despite alteration in mitochon-drial membrane permeability properties, no release of cyto-chrome c was found The ultrastructure of mitochondria, the membrane permeability properties and the Ca2+content returned to normal values during the replicative phase of liver regeneration These results suggest that, during the prereplicative phase of liver regeneration, the changes in mitochondrial ultrastructure observed in liver specimens were correlated with Ca2+-induced permeability transition
in mitochondria
Keywords: liver regeneration; mitochondria ultrastructure; membrane permeability; calcium; cyclosporin-A
Seventy percent partial hepatectomy (PH) induces cell
proliferation until the original mass of the liver is restored
[1] The tissue regeneration process consists of two phases:
the prereplicative phase, the duration of which depends on
the age of the animal [2,3] as well as on hormones and
dietary manipulation [2,4] and the replicative phase, during
which a sharp increase in DNA synthesis occurs with active
mitosis [2] In the light of early changes in ATP
concentra-tion found in liver after PH, before activaconcentra-tion of cell
proliferation [5,6], mitochondria were investigated as they
are directly involved in the process of liver regeneration
[4,7–16] Many mitochondrial functions, including oxidative
phosphorylation [11–13] and generation of reactive oxygen
species [14,15], were investigated in some detail in the
prereplicative phase of liver regeneration In isolated
mitochondria, a decrease in the respiratory control index
[12], ATP synthesis, probably due to a decrease in the
ATPsynthase complex content [14], and glutathione content
[13] as well as an increase in malondialdehyde production [14] and oxidant production [15] were found This suggests the occurrence in the prereplicative phase of liver regener-ation of a transient mitochondrial oxidative stress in which mitochondria can also release proteins from the matrix [16] Despite this, mitochondria recover their functions in the replicative phase of liver regeneration [12,14–16]
In this paper, we investigated whether and how the mitochondrial structure can change in the prereplicative phase of liver regeneration and whether mitochondrial permeability properties are somehow affected in this phase
of the process In the prereplicative phase of liver regener-ation, we found the occurrence of a number of mitochon-dria with dilated, paled and vacuolized matrix The isolated mitochondria showed impairment in membrane permeab-ility properties, which were prevented by cyclosporin-A (CsA) An increase in Ca2+ content was also observed Despite alteration in mitochondrial membrane permeability properties, no release of cytochrome c was found during the prereplicative phase of liver regeneration The mitochond-rial ultrastructure, the membrane permeability properties and the Ca2+ content showed normal values during the replicative phase of liver regeneration when a progressive recovery of liver mass is observed
M A T E R I A L S A N D M E T H O D S
Partial hepatectomy Three-month-old male Wistar rats were anaesthetized with
an ether/oxygen mix (at variable ratios) and the median and left lateral lobes of the liver were excised [12] After surgery, the rats were kept on a standard diet until they were
Correspondence to M Greco, Center for the Study
of Mitochondria and Energy Metabolism CNR BARI,
Via Amendola 165/A I-70126 Bari, Italy.
Fax: + 39 080 5443317, Tel.: + 39 080 5443316,
E-mail: csmmmg14@area.area.ba.cnr.it
Abbreviations: AAT, aspartate aminotransferase; CsA, cyclosporin-A;
GDH, glutamate dehydrogenase; PH, partial hepatectomy; EU,
enzyme units.
Enzymes: aspartate aminotransferase (EC 2.6.1.1); glutamate
dehydrogenase (EC 1.4.1.2).
*Note: deceased in November 2000.
(Received 8 February 2002, revised 20 May 2002,
accepted 22 May 2002)
Trang 2sacrificed The livers were removed, weighed, and processed
as follow: one-third were cut into sections for electron
microscopy studies and two-thirds were used for the
isolation of mitochondria Sham-operated rats, obtained
after a small midline abdominal incision without excision of
the liver, were used as a control and killed at 0, 24 and 96 h
after the surgical operation In all the assays reported, no
difference between sham-operated and rats that did not
receive any surgical operation was observed
All operations were carried out under sterile conditions
The animals received humane care and the study was
approved by the State Commission on animal
experimen-tation
Electron microscopy
Ultrastructural morphology of mitochondria was
deter-mined by electron microscopy Liver specimens from
control rats and from rats at 24 and 96 h after PH, were
fixed with 4% glutaraldehyde in 0.1Msodium cacodylate
buffer pH 7.4 for 4 h at 4C After fixation and an overnight wash in sodium cacodylate buffer at 4C, the specimens were postfixed with 1% osmium tetroxide in sodium cacodylate buffer for 1 h at 4C, dehydrated in alcohol and embedded in araldite resin (Taab Laboratories Equipment LTD, Aldermaston, Berkshire, England) and semithin sections (1 lm) were removed for optical micros-copy Ultra-thin sections were mounted on copper mesh grids and stained with uranyl acetate and lead citrate, according to Reynolds [17], before examination with a Zeiss EM 109 electron microscope All tissue samples were first inspected on semithin sections by light microscopy The ultrastructural morphology of mitochondria was evaluated
on five rats for each experimental group (control, 24 and
96 h after PH) and 10 randomly selected electron micro-graphs of a hepatic lobule were observed in each animal (7000· magnification)
Five morphological groups of mitochondria were defined and divided into two types according to the observed conformation: normal and altered (*) (Fig 1) For each
Fig 1 Electron micrographs of normal and altered (*) mitochondria during liver regeneration Representative electron micrographs of normal and altered (*) mitochondria (A) Detail of hepatocyte in control rat (B–D) Detail of hepatocytes at 24 h after PH, showing normal and altered (*) mitochondria (E) Detail of hepatocyte at 96 h after PH Bars ¼ 0.5 lm.
Trang 3animal the morphology of about 600 mitochondria in a
hepatic lobule was examined
Preparations of cytosolic fraction and mitochondria
Mitochondria were prepared according to Bustamante et al
[18] using a medium containing 0.25Msucrose and 5 mM
Tris/HCl (pH 7.4) as isolation buffer After precipitation of
mitochondria, the supernatant was used for preparation of
cytosol by ultracentrifugation at 105 000 g for 1 h The final
supernatant was used as cytosolic fraction In the
prepara-tions used for measurements of mitochondrial Ca2+
content, 1.6 lM ruthenium red and 1 mM EDTA were
added in the isolation buffer to restrict Ca2+ movement
during the subfractionation technique As preliminary
analyses showed that there was no statistically significant
difference in the Ca2+content of mitochondria whether the
buffers used for the subfractionation procedure contained
either 1 mM EDTA alone, or 1 mM EDTA and 1.6 lM
ruthenium red or 1 mM EGTA, for all subsequent
prepa-rations, 1 mM EDTA and 1.6 lM ruthenium red were
included in the subfractionation buffers
Protein concentration was determined using the Bio-Rad
kit (Bio-Rad Laboratories Inc., Milan, Italy)
Swelling assay
To monitor the mitochondrial swelling properties in sucrose
solution, mitochondria (0.5 mg proteinÆmL)1) were
suspen-ded in a swelling medium [5 mM succinate/Tris, 10 mM
Mops/Tris, 0.2M sucrose, 1 mM phosphate/Tris, 2 lM
rotenone and 1 lgÆmL)1oligomycin (pH 7.4)]
The absorbance was followed at 540 nm and at 25C, as
described previously [19], using a spectrophotometer
equipped with magnetic stirring and thermostatic control
Where indicated, 1 lMCsA (Sandoz Prodotti Farmaceutici,
Milano, Italy) was added to the reaction medium
Matrix proteins release assay
For the assay of the in vitro release of matrix proteins,
mitochondria (10 mg proteinÆmL)1) were suspended in the
swelling medium, above reported, and incubated at 25C
for 8 min After incubation, the mitochondria were
preci-pitated by centrifugation at 8000 g for 40 s The
superna-tants were then centrifuged for 10 min at 10 000 g Five
microliters of the final supernatants were used for SDS/
PAGE analysis with a linear gradient of polyacrylamide
(10–15%) [20] After the run, the gel was stained with
Coomassie Brilliant Blue Where indicated, mitochondrial
aspartate-aminotransferase [16] (AAT) or
glutamate-dehy-drogenase (GDH) [21] activities were determined in the final
supernatants When indicated, CsA (1.7 nmolÆmg)1
mito-chondrial proteins) was added The activities of the two
enzymes were also determined in the mitochondrial and
cytosolic fractions, and in the whole liver homogenate The
enzyme activity of mitochondrial AAT in the cytosol was
determined as described by Greco et al [16] Briefly, two
aliquots of either cytosolic fraction or whole homogenate
were incubated separately at 37C and 70 C for 15 min,
then AAT activity in both samples was determined The
AAT activity of the sample incubated at 37C was taken to
be that of both isoenzymes (mitochondrial and cytosolic
AAT), whereas that of the sample incubated at 70C was assumed to be solely due to cytosolic isoenzyme In fact, under conditions where the cytosolic AAT was stable, there was a thermal instability of mitochondrial AAT at 70C [22] The activity of mitochondrial AAT was taken as the difference between the two values
Determination of cytochromec content The amount of cytochrome c in cytosol and mitochondria during rat liver regeneration was determined by SDS polyacrylamide gel electrophoresis analysis, as described by Schaegger et al [23] Mitochondrial (20 lg of protein) or cytosolic (90 lg of protein) preparations were loaded onto an SDS/polyacrylamide gel Gels were then incubated in a medium containing tetrametylbenzidine in 10% isopropanol and 7% acetic acid After 10 min, H2O230% was added and, after 1–2 min, the greenish-blue bands of heme-containing peptides, among which was cytochrome c, were developed, as described by Broger et al [24] The bands were analyzed by laser densitometry at 595 nm, using a CAMAG TLC scanner II densitometer (Merck–Hitachi) Commercially purified horse cytochrome c (Sigma–Aldrich) was used as standard
Determination of mitochondrial Ca2+content For determination of the endogenous Ca2+ content, mitochondria (0.1 mg proteinÆmL)1) were suspended in 0.25M sucrose in the presence of 40 lM Arsenazo III (Sigma–Aldrich, Milan, Italy) The absorbance change at 675–685 nm, was monitored by dual wavelength spectro-photometry After reading a baseline for 1 min, Triton X-100 (0.2%) plus 3.3 lMSDS were added to disrupt the mitochondrial membranes [25] The absorbance change was calibrated by addition of standard aliquots of Ca2+to the medium A standard curve was obtained from the pooled results of five independent series of determinations and used for analysis of mitochondrial Ca2+content, which for the control was 8 ± 0.2 nmol per mg mitochondrial protein
No statistically significant differences in Ca2+content were observed when the mitochondrial preparation was per-formed either in the presence or in the absence of ruthenium red and EDTA in isolation buffer
Statistical analysis Data are reported as the mean ± SEM of five experiments performed using liver sections or mitochondria and cytosol obtained from five different animals for each experimental group (control, 24 and 96 h after PH) Statistical analysis was performed using the Student’s t-test
R E S U L T S
Mitochondrial ultrastructure during liver regeneration after PH
In order to find out whether and how mitochondria structure changes occur during liver regeneration, 10 randomly selected electron micrographs of the same mag-nification (7000·) were examined from one hepatic lobule of five rats for each experimental group (control, 24 and 96 h
Trang 4after PH), and the morphology of about 600 mitochondria
in a hepatic lobule of each animal was analyzed The typical
mitochondrial morphology of control liver is shown in
Fig 1A Liver mitochondria of rats at 24 h after PH
were quite variable in morphology and ultrastructure
(Fig 1B–D) Three different mitochondrial morphologies
were observed: (a) normal mitochondria (Fig 1B)
charac-terized by the same basic architecture of the typical liver
mitochondria with a folded internal membrane and a dense
matrix; (b) altered mitochondria (*) with a marked decrease
in the area of the inner membrane, reduction in the number
of cristae, destructurization of the matrix compartment, a
dilated and paled matrix, lack of dense granules (Fig 1C);
and (c) altered mitochondria (*) with clear vacuolization of
the matrix compartment (Fig 1D) No evident rupture of
mitochondrial outer membrane integrity was observed in
altered mitochondria At 96 h after PH (Fig 1E),
mito-chondria were nearly normal in morphology, cristae-rich,
and with an electron-dense matrix Quantitation of normal
and altered mitochondria in control liver and in liver at
24 and 96 h after PH was performed The majority of liver
mitochondria from control rats presented a normal
mor-phology; only a small fraction (3.0 ± 0.6%) belonged to
the altered type A large proportion (41.0 ± 6.6%) of
mitochondria from liver at 24 h after PH showed alterations
in mitochondrial ultrastructure At 96 h after PH, only a
small fraction (3.0 ± 0.05%) belonged to the altered type
The differences between the number of altered
mitochon-dria at 24 h after PH and the number of altered
mito-chondria in control rats were statistically significant
(P < 0.0001) Furthermore, in liver at 24 h after PH the
total number of mitochondria, counted in 10 randomly
selected electron micrographies of a hepatic lobule, was less
than the total number present in either control liver (11%
decrease; P¼ 0.001) or in liver at 96 h after PH (17%
decrease; P < 0.001) The decrease in the mitochondria
number corresponds to a decrease in the mitochondrial
proportion of the cell volume at 24 h after PH This was
correlated with a decrease in the activity of the
mitochon-drial marker enzymes GDH and mAAT in the total liver
homogenate at 24 h after PH (15% and 24% decrease
for GDH and mAAT, respectively) Moreover, in the
hepatocytes of liver at 24 h after PH, a small increase in
the number of lysosomes and the presence of
autophago-somes were also observed (data not shown) No significant
change in the number of apoptotic nuclei was found with
respect to control liver and liver at 96 h after PH (data not
shown)
Mitochondrial membrane permeability during liver
regeneration after PH
As the ultrastructure of 40% of liver mitochondria at 24 h
after PH is suggestive of changes in membrane permeability
of the organelles, we followed the swelling of mitochondria
isolated during liver regeneration (0, 24, 96 h after PH) in
isotonic sucrose medium supplemented with succinate and
phosphate Mitochondria were suspended in the swelling
medium and the absorbance of the mitochondrial
suspen-sion as a function of time was monitored either in the
absence or in the presence of CsA (1 lM), the specific
inhibitor of the mitochondrial transition pore [26]
Mito-chondria isolated from control rats and at 96 h after PH,
were found to swell at a low rate and extent in about 20 min (Fig 2, traces a and c); mitochondria isolated at 24 h after
PH showed, in contrast, a high rate and extent of swelling (Fig 2, trace b) CsA was found to prevent swelling in every case (Fig 2, traces a¢, b¢, c¢) Liver mitochondria isolated from sham-operated rats at 0, 24 and 96 h after surgery were found to swell poorly in a manner similar to that found for control liver mitochondria (data not shown)
The CsA capability to prevent mitochondrial swelling is indicative of the occurrence of permeability transition in mitochondria during the prereplicative phase of liver regeneration Thus we checked whether the isolated mito-chondria could release matrix proteins into the external medium Incubation of rat liver mitochondria, isolated at
24 h after PH, at 25C for 8 min in the swelling medium, resulted in an increased and nonspecific release of mito-chondrial proteins in the suspension medium (Fig 3A, lane c) compared to mitochondria isolated from control rats (Fig 3A, lane b) and mitochondria isolated at 96 h after PH (Fig 3A, lane d), as revealed by SDS/PAGE of the supernatants obtained after precipitation of mitochondria
by centrifugation This release of proteins at 24 h after PH was associated with the appearance, in the supernatant, of typical matrix enzyme activity, such as GDH (3.5 ± 0.26-fold increase vs control mitochondria; 23 ± 2.5% of the total mitochondrial activity) and AAT (3.15 ± 0.23-fold increase vs control mitochondria; 5.1 ± 0.1% of the total mitochondrial activity) (Fig 3B, empty columns b) CsA, added to the mitochondrial suspensions before incubation, inhibited the release of enzyme activities (Fig 3B, filled columns b) At 96 h after PH, the activities of the enzymes released in the supernatant (1.8 ± 0.1 and 0.8 ± 0.04% of the total mitochondrial activity of GDH and AAT, respectively), were as low as those found in the supernatant
Fig 2 Absorbance changes at 540 nm of rat liver mitochondria isolated during liver regeneration Mitochondria (0.5 mg proteinÆmL)1) isolated
at 0, 24, 96 h after PH were suspended in swelling medium and the absorbance change at 540 nm at 25 C was monitored Trace a: mitochondria isolated before PH Trace a¢: as a in the presence of 1 l M
CsA Trace b: mitochondria isolated 24 h after PH Trace b¢: as b in the presence of 1 l M CsA Trace c: mitochondria isolated 96 h after PH Trace c¢: as c in the presence of 1 l M CsA.
Trang 5of mitochondria isolated from control rats (2.2 ± 0.1 and
0.8 ± 0.05% of the total mitochondrial activity of GDH
and AAT, respectively) (Fig 3B, columns a and c)
As shown in Fig 4, the total activities of the matrix
enzymes GDH and AAT were found to decrease in
mitochondria isolated 24 h after PH, with respect to
mitochondria isolated from control rats (Fig 4, columns
b) (3.07 ± 0.85-fold decrease for GDH and 1.67 ±
0.3-fold decrease for AAT) An increase in enzymatic activities
in the corresponding cytosol (Fig 4, columns b¢) with
respect to cytosol isolated from control rats (Fig 4, columns
a¢) was observed (4.75 ± 0.59-fold increase for GDH and
2.28 ± 0.13-fold increase for AAT) Mitochondria and
cytosols obtained 96 h after PH show a pattern similar to
that of mitochondria and cytosols obtained from control
rats (Fig 4, columns c, c¢)
The amount of cytochrome c in mitochondria did not
change during liver regeneration after PH (Fig 4B;
P> 0.1) Accordingly, no release of cytochrome c was
observed in cytosols isolated from liver control and liver at
24 and 96 h after PH (Fig 4B)
Ca2+content in mitochondria during liver regeneration
after PH
The occurrence of mitochondrial permeability transition is
due to an increase in mitochondrial Ca2+ content [27]
Consistently, Ca2+pulse to mitochondria isolated before
PH or from sham-operated rats and suspended in an
isotonic sucrose medium supplemented with succinate and
phosphate, caused mitochondrial swelling (Fig 5A), which
reflects a change in mitochondrial membrane permeability
[19] Such a mitochondrial swelling was inhibited by the
addition to the mitochondrial suspension of CsA (Fig 5A),
the specific inhibitor of the permeability transition pore of mitochondria [26] This change in permeability of the inner mitochondrial membrane due to Ca2+loading was accom-panied by a nonspecific release of mitochondrial proteins in the suspension medium [28] with the appearance, in the supernatants, of typical matrix enzyme activities, such as mitochondrial AAT, the release of which was also inhibited
by the addition of CsA (Fig 5B)
As the mitochondrial permeability transition is dependent
on the Ca2+content of mitochondria, we checked whether the mitochondrial Ca2+content could change during liver regeneration (Fig 6) The mitochondrial Ca2+content in sham-operated rats was about 8 ± 0.2 nmolÆmg)1protein; this amount remained constant up to 6 h after PH No difference in liver mitochondrial Ca2+content was observed between sham-operated rats and animals that did not receive any surgical intervention (data not shown) A large increase in Ca2+content (17.7 ± 0.4 nmolÆmg)1protein) was found at 24 h after PH The Ca2+content at 72–96 h after PH was the same as the control (Fig 6) The increase
in liver weight after PH showed a biphasic pattern A low rate of increase was measured up to 24 h After this interval the liver weight increased linearly with the time (Fig 6) [16]
D I S C U S S I O N
Following PH, the remaining mature hepatocytes enter a complex process, known as liver regeneration, which after
an initial prereplicative phase reconstitutes the original mass
of the liver [1,2] The residual hepatocytes re-enter the cell cycle while the normal homeostatic mechanisms that couple cell cycle re-entry to cell death are suspended [29,30] The present study shows that after surgical removal of two-thirds of the mass of rat liver, mitochondria in the
Fig 3 Release of matrix proteins from rat liver mitochondria isolated during liver regeneration (A,B) Mitochondria (10 mg proteinÆmL)1) were suspended in the swelling medium and incubated at 25 C for 8 min After incubation, mitochondria were precipitated by centrifugation at 8000 g for 40 s The supernatants were, then, centrifuged for 10 min at 10 000 g (A) Five microliters of the final supernatant was analyzed by SDS/PAGE; lane a, standard M r proteins; lane b, supernatant from control mitochondria; lane c, supernatant from mitochondria isolated 24 h after PH; lane d, supernatant from mitochondria isolated 96 h after PH (B) GDH and AAT activities released in the supernatants of control mitochondria (columns a), mitochondria isolated 24 h after PH (empty columns b), mitochondria isolated 96 h after PH (empty columns c) The enzyme activities in the presence of 1.7 nmolÆmg)1protein CsA added to the incubation medium are reported as filled columns (b and c) The data are the means (± SEM)
of five different mitochondrial preparations The differences between both GDH and AAT activity at 24 h after PH and the same activities in the supernatants of control mitochondria are statistically significant (*P< 0.001).
Trang 6Fig 4 Glutamate-dehydrogenase, mitochondrial aspartate
amino-transferase activities and cytochrome c content in mitochondria and
cytosol prepared during liver regeneration (A) Mitochondrial AAT and
GDH activities were measured in mitochondria and cytosol isolated
from liver control (columns a, a¢), at 24 h (columns b, b¢) and 96 h
(columns c, c¢) after PH The data reported are expressed as lmol of
productÆmin)1per mg of mitochondrial or cytosolic proteins and are
the means (± SEM) of five different preparations The differences
between GDH and AAT activity in mitochondria and cytosols isolated
at 24 h after PH and the enzyme activities in mitochondria and
cyto-sols isolated from control rats or at 96 h after PH are statistically
significant (*P< 0.001) (B) Mitochondrial (20 lg protein) and
cyto-solic (90 lg protein) preparations were loaded on an
SDS/polyac-rilamide gel Gels were then incubated in a medium containing
tetramethylbenzidine in 10% isopropanol and 7% acetic acid After
10 min, H 2 O 2 (30% v/v) was added to reveal cytochrome c The bands
were analyzed by laser densitometry at 595 nm m, mitochondria; c,
cytosol (C) control mitochondria or cytosol; 24 h, mitochondria or
cytosol at 24 h after PH; 96 h, mitochondria or cytosol at 96 h after
PH; S, standard cytochrome c (500 ng) In the bottom panel,
mitochondrial cytochrome c (cyt c) content values are reported as
percentage of those detected in control mitochondria, taken as 100.
The values reported are the means (± SEM) of three different
preparations.
Fig 5 Ca 2+ -induced swelling and externally release of aspartate-ami-notransferase in control liver mitochondria suspended in swelling medium (A) Where indicated, isolated rat liver mitochondria (0.5 mg proteinÆmL)1) were added to the isotonic sucrose medium (swelling medium) reported in Materials and methods and the absorbance change at 540 nm at 25 C was monitored After 4 min, 150 l M CaCl 2 was added The dotted line shows the same experiment run in the presence of 1 l M CsA added to the suspension medium before mito-chondria (B) AAT activity in the supernatant of liver mitochondria incubated 8 min in the swelling medium (column a) or in the swelling medium after a Ca 2+ pulse (70 nmolÆmg protein)1) (column b) Column c: as column b in the presence of CsA (1.7 nmolÆmg pro-tein)1) The data reported are means (± SEM) of five different experiments The differences between AAT activity in the presence of
Ca2+and AAT activity in the absence of Ca2+pulse are statistically significant (*, P < 0.001).
Fig 6 Mitochondrial Ca 2+ content and recovery of liver mass during liver regeneration The mass of the liver at different time points after
PH (open symbols) is expressed as a percentage of the weight of the liver of sham-operated rats (11 ± 1.1 g) For determination of Ca2+ content at different time points after PH (closed symbols), mito-chondria (0.1 mgÆprotein mL)1) were suspended in 0.25 M sucrose in the presence of 40 l M Arsenazo III and the absorbance change at 675–685 nm was monitored After reading a baseline for 1 min, Triton-X100 (0.2%) plus 3.3 l M SDS were added In the mito-chondrial preparation, 1.6 l M ruthenium red and 1 m M EDTA were added to the isolation buffer The difference between mitochondrial
Ca 2+ content at 24 h after PH and control rats is statistically signi-ficant (*, P < 0.001).
Trang 7remaining hepatocytes undergo, in the first 24 h after
hepatectomy, i.e in the prereplicative phase, ultrastructural
changes These are associated with enhancement of the
mitochondrial Ca2+content and increase of CsA-sensitive
permeability to sucrose of the mitochondria isolated from
the residual liver mass
Analysis of the structural and functional state of
mito-chondria in the liver mass which is reconstituted in the
successive 96 h, shows, on the other hand, normal
mito-chondrial ultrastructure, return of mitomito-chondrial Ca2+
content and CsA-sensitive sucrose permeability to the
normal values observed in the liver before hepatectomy or
in sham-operated rats
Previous electron microscopy studies [15,31–33] had
revealed changes in the residual hepatocytes after PH but
less attention was paid to elucidating the correlation
between the changes occurring in the ultrastructure of
mitochondria and biochemical parameters during liver
regeneration The present electron microscopy study shows
that the general organization of the mitochondrial inner
membrane cristae into the typical transverse alignment in
control animals was absent in about 40% of the
dria in the hepatocytes at 24 h after PH These
mitochon-dria were characterized by highly fractured and degenerated
cristae and a clear vacuolation This suggests that the
decrease in ATP synthesis rate observed in mitochondria
isolated during the prereplicative phase of liver regeneration
[12] is probably a result of the decrease in the surface area of
the inner membrane
The ultrastructural changes observed in liver
mito-chondria at 24 h after PH are consistent with the changes
found in the membrane permeability properties of the
mitochondria isolated from the residual liver mass The
in vitro experiments show, in fact, that mitochondria
isolated from rat liver at 24 h after PH exhibit high
CsA-sensitive permeability to sucrose It has been suggested that
permeabilization of the inner mitochondrial membrane
could be required for the turnover of matrix proteins [28] A
release of mitochondrial AAT into the extramitochondrial
phase has been observed following oxygen radical injury of
mitochondria during hypoxic liver reoxygenation [34] Our
data show a release of the mitochondrial matrix enzymes
GDH and AAT into the cytosol of liver at 24 h after PH A
CsA-sensitive release of the same matrix enzymes can be
observed in vitro, following swelling of mitochondria,
isolated 24 h after PH This suggests an involvement of
the inner mitochondrial membrane transition pore in the
release of matrix enzymes in vivo
Our study shows that, during the prereplicative phase of
liver regeneration, the mitochondrial Ca2+content
increa-ses, reaching a maximum (17.75 nmolÆmg)1of protein) at
24 h after PH, when oxidative alteration of mitochondria is
also observed [14,15] Following PH, an increase in cell
Ca2+content has been observed during the prereplicative
phase of liver regeneration [35] HGF, the most important
in vitromitogen for primary hepatocytes and whose plasma
level increases within 1 h upon PH [29,36], has been shown
to induce Ca2+ entry across the hepatocyte plasma
membrane [37] Furthermore, some hormones, that are
known to modulate liver regeneration acting as mitogens or
comitogens [29,36], raise the liver cytosolic Ca2+
concen-tration and cause an increase in the mitochondrial matrix
volume as a consequence of Ca2+entry from cytosol into mitochondria [38]
Both mitochondrial Ca2+ accumulation and oxida-tive stress increase the probability that changes in the mitochondrial membrane permeability occur [25,38,39] Oxidative stress, Ca2+uptake and opening of the transition pore in mitochondria are signals for cell death [40–42] However, only a transient small increase in the number of apoptotic cells ( 5%) has been reported at 1 h after PH [15] Three to six hours after PH, the level of apoptotic cells was as low as that observed in control liver and no increase
in apoptosis was observed at 24 h after PH [15] The present ultrastructural analysis does not show any detectable alteration in mitochondrial outer membrane integrity at
24 h after PH The increase in the number of lysosomes, even if at a low extent, the presence of autophagosomes and the reduction in the number of mitochondria that we observe in hepatocytes at 24 h after PH, suggest that autophagic processes could occur in the prereplicative phase
of liver regeneration
It has been proposed that if the permeability transition occurs only for brief periods, its activity would not create survival problems for mitochondria and cells [43] The mitochondria in intact cells may undergo permeability transition and swelling in a fully reversible manner without progressing to cell death [44–46] Furthermore, it has been observed that mitochondrial swelling is not sufficient to affect cytochrome c release, and thus to trigger apoptosis processes [45] We show here that no release of cytochrome c occurs in the prereplicative phase of liver regeneration This finding is in agreement with the electron microscopy observations showing that neither evident breakage of the mitochondrial outer membrane nor increased number of apoptotic nuclei are present at 24 h after PH We suggest that the mitochondrial permeability transition occurring in the prereplicative phase of liver regeneration is a transient event and that, with the exception of irreparably damaged mitochondria that could be eliminated by autophagy, a great proportion of mitochondria undergoing permeability transition recover in a fully reversible manner Future studies will be needed to ascertain the fate of mitochondrial subpopulations during liver regeneration
A C K N O W L E D G E M E N T
This work was partially supported by a grant within the National Research Project PRIN: Bioenergetics and Membrane Transport of Murst, Italy.
R E F E R E N C E S
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