Báo cáo Y học: Changes in ultrastructure and the occurrence of permeability transition in mitochondria during rat liver regeneration

Eur. J. Biochem. 269, 3304–3312 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03010.x Changes 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 Marra3 and Margherita Greco3 1Department 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 theorganellesisolatedfromratliverduringtheprereplicative 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,withspecialinteresttotheroleplayedbyCa2+ inthisprocess.Theresultsshowthatduringthefirstdayafter partialhepatectomy,significantchangesintheultrastructure of mitochondria in situ occur. Mitochondrial swelling and releasefrommitochondriaofbothglutamatedehydrogenase andaspartateaminotransferaseisoenzymeswithanincrease in the mitochondrial Ca2+ content were also observed. Cyclosporin-A proved to be able to prevent the changes in 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 activation of cell proliferation [5,6], mitochondria were investigated as they are directly involved in the process of liver regeneration [4,7–16].Manymitochondrialfunctions,includingoxidative 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 ATPsynthasecomplex content[14],andglutathionecontent 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,aspartateaminotransferase;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) 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, themembranepermeabilitypropertiesandtheCa2+ 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. [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,noreleaseofcytochrome cwasfoundduringthe 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. MATERIALS AND METHODS 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 Ó FEBS 2002 Mitochondria and liver regeneration (Eur. J. Biochem. 269) 3305 sacrificed. 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 0.1 M sodium cacodylate buffer pH 7.4 for 4 h at 4 °C. After fixation and an overnight wash in sodium cacodylate buffer at 4 °C, the specimens were postfixed with 1% osmium tetroxide in sodium cacodylate buffer for 1 h at 4 °C, 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 firstinspectedonsemithinsectionsby lightmicroscopy.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. 3306 F. Guerrieri et al. (Eur. J. Biochem. 269) animal the morphology of about 600 mitochondria in a hepatic lobule was examined. Preparations of cytosolic fraction and mitochondria MitochondriawerepreparedaccordingtoBustamanteet al. [18] using a medium containing 0.25 M sucrose and 5 mM Tris/HCl (pH 7.4) as isolation buffer. After precipitation of mitochondria, the supernatant was used for preparation of cytosolbyultracentrifugationat105 000 gfor1 h.Thefinal 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 Tomonitor themitochondrialswelling properties insucrose solution, mitochondria (0.5 mg proteinÆmL)1) were suspen-ded in a swelling medium [5 mM succinate/Tris, 10 mM Mops/Tris, 0.2 M sucrose, 1 mM phosphate/Tris, 2 lM rotenone and 1 lgÆmL)1 oligomycin (pH 7.4)]. The absorbance was followed at 540 nm and at 25 °C, as described previously [19], using a spectrophotometer equipped with magnetic stirring and thermostatic control. Whereindicated,1 lM CsA(SandozProdottiFarmaceutici, 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 25 °C 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]activitiesweredeterminedinthefinal 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 37 °C and 70 °C for 15 min, then AAT activity in both samples was determined. The AAT activity ofthe sample incubated at 37 °C was taken to be that of both isoenzymes (mitochondrial and cytosolic Ó FEBS 2002 AAT), whereas that of the sample incubated at 70 °C 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 70 °C [22]. The activity of mitochondrial AAT was taken as the difference between the two values. Determination of cytochrome c 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 lgofprotein)preparationswereloadedontoan SDS/polyacrylamide gel. Gels were then incubated in a mediumcontainingtetrametylbenzidinein10%isopropanol and7%aceticacid.After10 min,H2O2 30%wasaddedand, after 1–2 min, the greenish-blue bands of heme-containing peptides,amongwhichwascytochromec,weredeveloped,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 0.25 M 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 lM SDS 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-formedeither inthe presenceor intheabsenceofruthenium 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. RESULTS 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·)wereexaminedfromonehepaticlobuleof five rats for each experimental group (control, 24 and 96 h Ó FEBS 2002 Mitochondria and liver regeneration (Eur. J. Biochem. 269) 3307 after 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 mitochondriafromliverat24 hafterPHshowedalterations 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; P ¼ 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, 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: mitochondriaisolated before PH.Tracea¢:asain thepresenceof1 lM CsA.Traceb:mitochondriaisolated24hafterPH.Traceb¢:asbinthe presence of 1 lM CsA. Trace c: mitochondria isolated 96 h after PH. Trace c¢: as c in the presence of 1 lM CsA. werefoundtoswellatalowrateandextentinabout20 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, traceb). CsAwasfoundtopreventswellingin every case (Fig. 2, traces a¢, b¢, c¢). Liver mitochondria isolated from sham-operated rats at 0, 24 and 96 h after surgery werefoundtoswellpoorlyinamannersimilartothatfound 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 25 °C 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,laneb)andmitochondriaisolatedat96hafterPH (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 3308 F. Guerrieri et al. (Eur. J. Biochem. 269) Ó FEBS 2002 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 for40 s.Thesupernatantswere,then,centrifugedfor10 minat10 000 g.(A)FivemicrolitersofthefinalsupernatantwasanalyzedbySDS/PAGE; lane a, standard Mr proteins; lane b, supernatant from control mitochondria; lane c, supernatant from mitochondria isolated 24 h after PH; lane d, supernatantfrommitochondriaisolated96 hafterPH.(B)GDHandAATactivitiesreleasedinthesupernatantsofcontrolmitochondria(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 presenceof1.7 nmolÆmg)1 protein CsA added to the incubation mediumare reported as filled columns (band 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). of 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 respecttocytosolisolatedfromcontrolrats(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). Asthemitochondrialpermeabilitytransitionisdependent 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)1 protein; this amount remained constant up to 6 h after PH. No differenceinlivermitochondrialCa2+ contentwasobserved 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)1 protein) 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]. DISCUSSION Following PH, the remaining mature hepatocytes enter a complex process, known as liver regeneration, which after aninitialprereplicativephasereconstitutestheoriginalmass 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 ... - tailieumienphi.vn