浙江大学 文本 Mitochondrial Nitric Oxide Synthase Drives Redox Signals for Proliferation and Quiescence in Rat Liver Development_生理学

Mitochondrial Nitric Oxide Synthase Drives Redox Signals for Proliferation and Quiescence in Rat Liver Development Maria C.Carreras,Daniela P.Converso,Alicia S.Lorenti,Mariana Barbich,Damian M.Levisman.Aricl Jaitoich, Valeria G.Antico Ardiuch Soledad Galli,and Juan J.Poderoso Mitochondrial nitric oxide synthase (mtNOS)is a fine regulator of orygen uptake and reactive oxygen species that eventually modulates the activity of regulatory proteins and cell cyce progression.From this perspective,we examined liver mtNOS modulation and mito- chondrial redox changes in developing rats from embryonic days 17-19 and postnatal day 2 (proliferaring heparocyte phenotype)through postnatal days 15-90(quiescent phenorype). mtNOS expression and activity were almost undetectable in feral liver,and progressively increased after birth by tenfold up to adult stage.NO-dependent mitochondrial hydrogen peroxide (H2O2)production and Mn-superoxide dismutase followed the developmental modulation of mtNOS and contributed to parallel variations of cytosolie H2O:concentra- tion ([H2O2])and cell fluorescence.miNOS-dependent [H2Ozl was a good predictor of extracellular signal-regulated kinase (ERK)/p38 activity ratio,cyelin D1,and tissue prolif eration.At low 10-11-10-12 M [HO2 proliferating phenorypes had high cyclin D1 and phospho-ERK1/2 and low phospho-p38 mitogen-activated protein kinase,while at 10-M [HOl quiescent phenotypes had the opposite pattern.Accordingly,leading postnatal day 2-isolated hepatocytes to embryo or adult redox conditions with H2O,or NO-H,O2 scav- engers,or with ERK inhibitor U0126,p38 inhibitor SB202190 or p38 activator anisomycin resulted in correlative changes of ERK/p38 activity ratio,cyclin D1 expression,and PH] thymidine incorporation in the cells.Accordingly,p38 inhibitor SB202190 or N-aceryl. cysteine prevented H2O:inhibitory effects on proliferation.In conclusion,the results sug gest that a synchronized increase of mtNOS and derived H2O,operate on hepatocyte signaling pathways to support the liver developmental transition from proliferation to quiescence.(HEPATOLOGY 2001pi0:157-166.) n normal adult liver,heparocytes are highly differen- riated and rarely undergo cell division,but they retain Afiredenae wuho wurenlowinl cimir aid gateE emerdler a remarkable ability to proliferate in response to acute 红ird hiwoe:Me S0D).goe aneren☆6ts八ig axide rpukuc::migru-ecisered grotre icar:. or chronic injury.1 While liver regenerarion depends on nA,wipl-e-giso线aii aitnr ande the transcriptional effects of cytokines.the mechanisms hte:m eranm'niwvic sie gnute n八easkufniy mint动 muhav:FrS.frrdl cugfaene:LO,siigrinone NAC Naeremrive. that govern developmental hepatocyte proliferation and Fross ak Lakovay hgm Mrakodu,Lainmry Hwpitdf aud dv the transition to the quicscent oondition are not fully mmn afClinna dliudemin.Srbonlof Pivranngr a flivrdemin Linivm characterized. Ar relarively low matrix steady state concentration dlrMu FarnlemL Fowpical点ons Nursat A%Avne Pecrivrd Nmenfrr 17.2003:acve led Marce 2d.2004. NO exerts marked inhibitory effects on the activity of p球d可Bwum&G了M位n.fr Neiud rodox components of the dectron transfer chain,partiou- Apewy far Prowisu ofcun6dd了aka山dral SMarkeyeow (ic.To4 larly on cyrochrome oxise,thus regulating osygen ile Pr Camune,Barer.Ara Arguin uptake.Consequently,the reduction level of mitochoa- Addrrx wgrhe mgann u Proghoo htaris Crib Carone,Iaiaouy drial components increases on the substrate side,leading h Mrteblio.Unvminy Fxpin Carba 2351.1120 Buno Aire rgrn Emu Gmu5kLrc年/小-9a8以. to high superoxide anion()producrion;masr ofO is dismurared by matri manganese superoride dismurase PakCdivd'ouler tu Wiiry fueveiciraer fira.tummerereasrouul (Mn-SOD)to hydrogen peroxide (HO2)that freely dif Dw70a2%2m25 fuses outside the mitochondria. 157

Mitochondrial Nitric Oxide Synthase Drives Redox Signals for Proliferation and Quiescence in Rat Liver Development Marı´a C. Carreras,1,2 Daniela P. Converso,1 Alicia S. Lorenti,3 Mariana Barbich,3 Damian M. Levisman, ´ 1 Ariel Jaitovich,1 Valeria G. Antico Arciuch,1 Soledad Galli,1 and Juan J. Poderoso1 Mitochondrial nitric oxide synthase (mtNOS) is a fine regulator of oxygen uptake and reactive oxygen species that eventually modulates the activity of regulatory proteins and cell cycle progression. From this perspective, we examined liver mtNOS modulation and mito￾chondrial redox changes in developing rats from embryonic days 17–19 and postnatal day 2 (proliferating hepatocyte phenotype) through postnatal days 15–90 (quiescent phenotype). mtNOS expression and activity were almost undetectable in fetal liver, and progressively increased after birth by tenfold up to adult stage. NO-dependent mitochondrial hydrogen peroxide (H2O2) production and Mn-superoxide dismutase followed the developmental modulation of mtNOS and contributed to parallel variations of cytosolic H2O2 concentra￾tion ([H2O2]ss) and cell fluorescence. mtNOS-dependent [H2O2]ss was a good predictor of extracellular signal–regulated kinase (ERK)/p38 activity ratio, cyclin D1, and tissue prolif￾eration. At low 10
11–10
12 M [H2O2]ss, proliferating phenotypes had high cyclin D1 and phospho-ERK1/2 and low phospho-p38 mitogen-activated protein kinase, while at 10
9 M [H2O2]ss, quiescent phenotypes had the opposite pattern. Accordingly, leading postnatal day 2–isolated hepatocytes to embryo or adult redox conditions with H2O2 or NO-H2O2 scav￾engers, or with ERK inhibitor U0126, p38 inhibitor SB202190 or p38 activator anisomycin resulted in correlative changes of ERK/p38 activity ratio, cyclin D1 expression, and [3H] thymidine incorporation in the cells. Accordingly, p38 inhibitor SB202190 or N-acetyl￾cysteine prevented H2O2 inhibitory effects on proliferation. In conclusion, the results sug￾gest that a synchronized increase of mtNOS and derived H2O2 operate on hepatocyte signaling pathways to support the liver developmental transition from proliferation to quiescence. (HEPATOLOGY 2004;40:157–166.) I n normal adult liver, hepatocytes are highly differen￾tiated and rarely undergo cell division, but they retain a remarkable ability to proliferate in response to acute or chronic injury.1 While liver regeneration depends on the transcriptional effects of cytokines, the mechanisms that govern developmental hepatocyte proliferation and the transition to the quiescent condition are not fully characterized. At relatively low matrix steady-state concentration, NO exerts marked inhibitory effects on the activity of redox components of the electron transfer chain, particu￾larly on cytochrome oxidase, thus regulating oxygen uptake.2,3Consequently, the reduction level of mitochon￾drial components increases on the substrate side, leading to high superoxide anion (O2
) production4; most of O2
is dismutated by matrix manganese superoxide dismutase (Mn-SOD) to hydrogen peroxide (H2O2) that freely dif￾fuses outside the mitochondria. Abbreviations: mtNOS, mitochondrial nitric oxide synthase; ERK, extracellular signal–regulated kinase; Mn-SOD, manganese superoxide dismutase; NOS, nitric oxide synthase; MAPK, mitogen-activated protein kinase; PMSF, phenylmethylsul￾fonyl fluoride; L-NMMA, NG-methyl-L-arginine; iNOS, inducible nitric oxide synthase; nNOS, neuronal nitric oxide synthase; eNOS, endothelial nitric oxide synthase; FCS, fetal calf serum; UQ, ubiquinone; NAC, N-acetyl-cysteine. From the 1Laboratory of Oxygen Metabolism, University Hospital and the 2De￾partment of Clinical Biochemistry, School of Pharmacy and Biochemistry, Univer￾sity of Buenos Aires, Buenos Aires, Argentina; and 3Instituto de Ciencias Ba´sicas y Medicina Experimental, Hospital Italiano, Buenos Aires, Argentina. Received November 17, 2003; accepted March 24, 2004. Supported by the University of Buenos Aires (UBACyT M627), the National Agency for Promotion of Scientific and Technological Development (PICT 08468), Consico Nacional de Investigaciones Cientificas y Te´cnicas (PIP 58), and Funda￾cio´n Pe´rez Companc, Buenos Aires, Argentina. Address reprint requests to: Professor Marı´a Cecilia Carreras, Ph.D., Laboratory of Oxygen Metabolism,University Hospital, Co´rdoba 2351, 1120 Buenos Aires, Argentina. E-mail: carreras@ffyb.uba.ar; fax: 54-11-59508811. Copyright © 2004 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hep.20255 157

155 CARRERAS ET AL HEPATOLOGY.Juy 2004 Nitric oxide is vecrorially relased into matrix by mi.of aprotinin and leupeptin.50 mM Nal and 0.1 mM tochondrial nitric oxide synthises (mtNOSs).Different sodium orthovanadate)per 100 mg of tissue.Tween 20 mtNOS isoforms have heen descrihed in rat tissues-7; was then added at a final concentration of0.1%.Homog- liver mtNOS is a Ca+/calmodulin dependent,constiru enates were clarifed by centrifugarion ar 10,000g for 10 tively expressed variant that is localiced in the inner mi- minutes at 4'C and stored at-70"C. tocbondrial membranc.Elfering et al.teported 100% Isolation and Parification of Liver Mitochondria. homology berween liver mrNOS and neuronal nirric Liver was homogenized in MSHE buffer (225 mM man. oxide synthase (NOS)-a by mas spectromet女：differen- nitol,70 mM sucrose,1 mM EGTA,25 mM HEPES) ially,liver mNOS has two posttranslational modifica- pH 7.with 5 ug/mL each of aprotinin and kupeptin. tions:acylation with myrisric acid and phosphorylarion at 100 ug/mL PMSF,10 ug/mL pepstatin,and 0.1%bo- C terminus,and a lower molecular weight (130 vs.157 vine serum albumin.The homogenate was centrifuged at kDa).Additionally,mtNOS is subjected to selective 70g ar 4C for 10 minutes:the supernarant was cenrri- modulation by thyroid stanus,cold acclimation,hypox. fuged at 7000g for 10 minutes3 A mitochondrial pellet and brain plasticity. was further purified using Perroll gradient to completely Hydrogen peroxide and the consequent oxidative remove contaminating organelles and broken mirochon. sre levd play an important role in the activation of dria.Purified mitochondria were tested for contamina signaling molecules thar conrrol the complex machinery tion by comparing lictate dehydrogense activity involved in cell proliferation,differentiation,apoptosis, (cytosolic marker)to succinate-cytochrome e reductase and senescence.12 The major components of the cellcycle activiry (mitochoodrial mrker)minimal comtamination machinery are the cyclins and cyclin-dependent kinases. was found (29-5%). Cydlin DI is implicated in the control of Gl phase pro Mitocbondrial Enzyme Activities.Nicotinamide gression in bepatocytes and other proliferating cell adenine dinucleoride-and succinate-cytochrome c reduc- types.and its expression is positively regulated by the tase activities (complexes 1-lll and I-lll.respectively) extracdlular signal-regulated kinase (ERK)pathway and were assayed by cytochrome c reduction at 550 nm with a antagoniced by stres-activated p38 mitogen-activated Hitachi U3000 spectrophotometer (Hitachi,Tokyo,Ja- procein kinse (MAPK)ccade.During liver develop- pan)ar 30C.Cytochrome oridlase activiry (cample:TV) ment.cyclin DI content is inversely relared ro p38 MAPK was derermined by moniroring cytochrome coxidation ar autivily,which in tum my be tgulaod by reactive 550 nm (esso-21 mM-1.cm);the reaction rare was oygen speciesta.17 and NO.14.1 measured as the pseudo-frst order reaction constant ( Considering the mitochondrial urilization of NO and and cxpressed as min-mg protcin. the NO-derived production of oxygen-active species,the NOSActinity.NOS activity was determined through regulation of mtNOS provides new insight into the phys- the conversion of [H]-L-arginine to [H]-L-citrulline in ological significance of mitochondria in cell biology.We S0 nM of polassium phe可phate bufTer(pH7,5分ime report the modulation of mtNOS activity and the puta- presence of 100 M L-arginine,0.1 M H]-L-arginine tive regulation ofoell cyce rodlox sigraling in the squence (NEN,Bosron,MAl.0.1 mM NADPH.0.3 mM CaCi. of proliferaring to quiescenr cell stages during rar liver 0.1 uM calmodulin,10 AM tetrahydrobiopterin,1 uM development. Havin adenine dinudleotide.I uM Bavin mononucle- otide,50 mM Ivaline and 1 mgfml protcin.s Spacific Materials and Methods activiry was calculated by subtracring the remaining acriv- ity in the presence of the NOS inhbitor Ni-methyl-L- Amfurals.Wistar rats were used in the experiments. arginine (5 mM I-NMMA)or 2 mM EGTA. National Research Council criteria for the care and use of Immoblotting.Cells were disrupeed in lysis huffer Lbaratory animals in reseanh were fallowal. (50 mM 'T'ris buffer pH 7.:0.5%Nonidet P-40.150 Preparation of Whole Liper Homogenates.Pooled mM NaCl:1 mM EDTA:1 mM EGTA:10%glycerol;I liver ofone litter from fetal (cmbryonic days 17 [E17]and mM MgCls 1 mM PMSF;5 ug/mL cach of sprotinin, 19 [E19])and newboen rats ar posmnatal day 2 (P2),or leupeprin,and pepsratin;1 mM sodium orthovanadare; whole liver of young and adult rars (P15-P90)were ho. 25 mM NaF:and 0.1 mM ammonium molybdate).Ly. mopenized in 1 mL of cold lysis buffer (50 mM HEPES sates were cemtrifuged at 12,000g for 30 minutes ar 4C, IpH 7.5],150 mM NaCl,1 mM ethylenediaminetet- and the supernarants were stored ar -70C.Western blot raaceric acid (DT'A),2.5 mM ethyleneglycol terraaceric anzysis was performed is described:membranes were acid [EGTAl,1 mM dithiothreitol,10%glycerol,1 mM stained with Ponceau red to ensure equivalent amounts of phenylmethylsulfonyl fluoride [PMSF],10 ug/mL each protein loading and clecttophorctic transfer among sam-

Nitric oxide is vectorially released into matrix by mi￾tochondrial nitric oxide synthases (mtNOSs). Different mtNOS isoforms have been described in rat tissues5–7; liver mtNOS is a Ca2/calmodulin-dependent, constitu￾tively expressed variant that is localized in the inner mi￾tochondrial membrane. Elfering et al. reported 100% homology between liver mtNOS and neuronal nitric oxide synthase (NOS)-
by mass spectrometry8; differen￾tially, liver mtNOS has two posttranslational modifica￾tions: acylation with myristic acid and phosphorylation at C terminus, and a lower molecular weight (130 vs. 157 kDa). Additionally, mtNOS is subjected to selective modulation by thyroid status,3 cold acclimation,9 hypox￾ia,10 and brain plasticity.11 Hydrogen peroxide and the consequent oxidative stress level play an important role in the activation of signaling molecules that control the complex machinery involved in cell proliferation, differentiation, apoptosis, and senescence.12 The major components of the cell cycle machinery are the cyclins and cyclin-dependent kinases. Cyclin D1 is implicated in the control of G1 phase pro￾gression in hepatocytes and other proliferating cell types,13 and its expression is positively regulated by the extracellular signal–regulated kinase (ERK) pathway and antagonized by stress-activated p38 mitogen-activated protein kinase (MAPK) cascade.14 During liver develop￾ment, cyclin D1 content is inversely related to p38 MAPK activity,15 which in turn may be regulated by reactive oxygen species16,17 and NO.18,19 Considering the mitochondrial utilization of NO and the NO-derived production of oxygen-active species, the regulation of mtNOS provides new insight into the phys￾iological significance of mitochondria in cell biology. We report the modulation of mtNOS activity and the puta￾tive regulation of cell cycle redox signaling in the sequence of proliferating to quiescent cell stages during rat liver development. Materials and Methods Animals. Wistar rats were used in the experiments. National Research Council criteria for the care and use of laboratory animals in research were followed. Preparation of Whole Liver Homogenates. Pooled liver of one litter from fetal (embryonic days 17 [E17] and 19 [E19]) and newborn rats at postnatal day 2 (P2), or whole liver of young and adult rats (P15–P90) were ho￾mogenized in 1 mL of cold lysis buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 1 mM ethylenediaminetet￾raacetic acid (EDTA), 2.5 mM ethyleneglycol tetraacetic acid [EGTA], 1 mM dithiothreitol, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride [PMSF], 10 g/mL each of aprotinin and leupeptin, 50 mM NaF and 0.1 mM sodium orthovanadate) per 100 mg of tissue.14 Tween 20 was then added at a final concentration of 0.1%. Homog￾enates were clarified by centrifugation at 10,000g for 10 minutes at 4°C and stored at
70°C. Isolation and Purification of Liver Mitochondria. Liver was homogenized in MSHE buffer (225 mM man￾nitol, 70 mM sucrose, 1 mM EGTA, 25 mM HEPES), pH 7.4 with 5 g/mL each of aprotinin and leupeptin, 100 g/mL PMSF, 10 g/mL pepstatin, and 0.1% bo￾vine serum albumin. The homogenate was centrifuged at 700g at 4°C for 10 minutes; the supernatant was centri￾fuged at 7000g for 10 minutes.3 A mitochondrial pellet was further purified using Percoll gradient to completely remove contaminating organelles and broken mitochon￾dria. Purified mitochondria were tested for contamina￾tion by comparing lactate dehydrogenase activity (cytosolic marker) to succinate-cytochrome c reductase activity (mitochondrial marker); minimal contamination was found (2%–5%). Mitochondrial Enzyme Activities. Nicotinamide adenine dinucleotide– and succinate-cytochromec reduc￾tase activities (complexes I-III and II-III, respectively) were assayed by cytochrome c reduction at 550 nm with a Hitachi U3000 spectrophotometer (Hitachi, Tokyo, Ja￾pan) at 30°C. Cytochrome oxidase activity (complex IV) was determined by monitoring cytochrome c oxidation at 550 nm (550 21 mM
1
cm
1); the reaction rate was measured as the pseudo-first order reaction constant (k’) and expressed as k’/min
mg protein. 20 NOS Activity. NOS activity was determined through the conversion of [3H]-L-arginine to [3H]-L-citrulline in 50 mM of potassium phosphate buffer (pH 7.5) in the presence of 100 M L-arginine, 0.1 M [3H]-L-arginine (NEN, Boston, MA), 0.1 mM NADPH, 0.3 mM CaCl2, 0.1 M calmodulin, 10 M tetrahydrobiopterin, 1 M flavin adenine dinucleotide, 1 M flavin mononucle￾otide, 50 mM L-valine and 1 mg/ml protein.3 Specific activity was calculated by subtracting the remaining activ￾ity in the presence of the NOS inhibitor NG-methyl-L￾arginine (5 mM L-NMMA) or 2 mM EGTA. Immunoblotting. Cells were disrupted in lysis buffer (50 mM Tris buffer [pH 7.4]; 0.5% Nonidet P-40; 150 mM NaCl; 1 mM EDTA; 1 mM EGTA; 10% glycerol; 1 mM MgCl2; 1 mM PMSF; 5 g/mL each of aprotinin, leupeptin, and pepstatin; 1 mM sodium orthovanadate; 25 mM NaF; and 0.1 mM ammonium molybdate). Ly￾sates were centrifuged at 12,000g for 30 minutes at 4°C, and the supernatants were stored at
70°C. Western blot analysis was performed as described11; membranes were stained with Ponceau red to ensure equivalent amounts of protein loading and electrophoretic transfer among sam- 158 CARRERAS ET AL. HEPATOLOGY, July 2004

HEPATOLOGY,VoL 40,No.1.2004 CARRERAS ET AL 159 ples.Western blotting enhanced chemiluminescence de ference of HO production rate in the presence of 100 tection system and Hybond-P membranes were from uM L-arginine and L-arginine plus 2 mM L-NMMA. Amersham Biosciences (Little Chalfont,United King- Mitochondrial preprations were supplemented with 1 dom).Quintification of bands was performed using dig uM Mn(lll)tetrakis(4-benzoic acid)porphyrin (Cayman ital image anzlysis (Total Lab,Nonlinear Dymamics, Chemical,Ann Arbor,MI)to uniform the masimal Newestle,UK).Liver hamogenates of lipopolysncha- H.O:prudution rate. ride-treared rats cerebellum cytosol,and endothelial cell Cell Isolation d Cultre.Hepatocytes were iso- lysites were used as inducible nitric oxide synthase hted by collagenase digestion as described previously (iNOS)-,neuronal nitric oxide synthase (nNOS)-,and For culture,P2-and P15-isolated hepatocytes were endothelial nitric otide synthise (eNOS)-positive con- seeded in 96-well plates (50,000 cells per well)in medium rol.Praliferating liver from rats irealal with a single 199(Giboo-BRL.Invitrugen Life Tochnologies,Brab, dose of T3(60 ug/100 g body weighr,intraperironeally) the Nerherlands)supplemented with 10%i fetal calfserum wl8名ontrol for对gnaling3y,21 (FCS)and 50 ug/mL genramicin and were allowed to Co-iaopreeipitarion.Mitachondrial or cytoso- artach for 4 hours.Cells were synchronixal by 20-hour lic proteins (500 ug)were incubared with 4 ug of poly. incubarion with 2%FCS and 2 hours withour FCS.He- clonal nNOS intibady and 30 uL protein A/G PLUS patocyte trearments were performed in medium 199 with Agarose (Santa Cruz.CA)at 4'C.The beads were then 10%FCS for 72 hours Proliferation was assayed through washed three times suspended in sample bufTer,boiled, H]thymidine incorporation.The last 24 hours.cells and cenrrifuged,and the supernarants were subjecred to were incubated with 0.8 uCiH]thymidine/well and immunoblorting,against monodonal nNOS antibodies. harvested:cpm were measured in a liquid scinrillation Amtibodies.Polyclonal anti-iNOS and anti-nNOS, counter (Wallac 1414,Turku,Finlind). anri-cyclin D2 and D3.and monoclonal anri-mouse cy- clin DI were obeained from Santa Cruz Biotechnology. Detection of Intreceliular ROS.Intracellular ROS Inc.(Santa Cruz,CAl;polydonal anti-eNOS and mono- were analyzed by fow cytometry.using a 2'.7'dichlo. conal nNOS antibodics were obtained from Transduc- rofuorescin diacetate probe.The cellular fuores- tion Laboratories (Lexingion,KY).Polyconal anribodies cence intensity was measured after 30 minutes of against total and phospho-p38 MAPK,ERK and cJun incubation with 5 M 2',7'-dichlorofuorescin diac- N-terminal kinase,and phospho MAPK kinises were etate by using an Ortho Cytoron Absolute Flow-Cy- from Cell Signaling Technology (Beverly,MA). tometer Johnson Johnson,Raritan,NY). Fmemnoelrctron Microreopy.Purified mitochon- Propidium iodide (0.005%)was used to derect dead dria were suspended in 4%formaldehyde (pH 7.4)for 2 cells.For each analysis,10,000 events were recorded. hours at 4"C,then delydrated in 70%,96%,and 1009 DNA Ayris For mesuring apoptosis,the ploidy ethanol (30 minutes for each step)and embodded in IR dererminarion of hepatocytes was assessed with pro- WWhire.Immunocytochemistry was performed using a pri- pidium iodide staining and Hlow cytometry as docribed mary mouse anti C terminal nNOS (1095-1289)at a p线ly.24 dilution of 1:100 in phosphate-buffered saline (pH 7.4). Antioxidant Ensyme Activities.Mitochondrial Grids were washod in phoophate-buffered saline and Mn-SOD activity was daterminod by inhibition of cyto- counterstained with 1%uranyl acerare.Nonspecific hack. chrome e reduction at 550 nm in 50 mM pocassium phos- ground was blocked by incubation with 5%normal goar phate buffer/0.1 mM EDTA (pH7.8)at 25C.Caralase serm in phosplote-bufTered saline at the beginning of and glutathione peroxidase activities in 7.000g superna- the procedure.Specimens were observed in a Zeiss EM. tanrs were derermined by the decrease in H2O:ahsorp- 109.'I'transmission electron microscope at 80 kv. tion at 240 nm (e2ss-4l uM-1.cm),or by the Mitocbondrial H202 Production.HO:produc- oxidation of NADPH at 340 nm (Mo 6.22 mM-1. tion was continuously monitored usinga Hitachi F-2000 cm). ctrofluoromncter (Hir米hi）with eitation and emis Reagemts.SDS,glycerol,2-(B-mercapeoethinol). sion wavelengths ar 315 and 425 nm,respecrively.The and bromophenol blue were obtaincd from Bio-Rad reaction medium,which consisred of potassium phos (Richinond,CA)SB 202190 was ablained fiom Calbio- phare buffer (50 mM)and 50 mM I-valine,was supple- chem (San Diego,CA):U0126 was obtained from Cell mented with 10 mM succinate,12.5 units/mL Signaling Tochnolugys and other chemicals were from horseradish peroxidise,250 uM-hydroxypbenyl-acetic Sigma Chemical Co.(St Louis,MO) acid,and 0.15 mgof mitochonxrial protein per mLNO- Data Amelyris.Data are expressed as mean SE and dependent H:O:production was determined as the dif were arlyred by ANOVA and SciefTe tot,Simple linear

ples. Western blotting enhanced chemiluminescence de￾tection system and Hybond-P membranes were from Amersham Biosciences (Little Chalfont, United King￾dom). Quantification of bands was performed using dig￾ital image analysis (Total Lab, Nonlinear Dynamics, Newcastle, UK). Liver homogenates of lipopolysaccha￾ride-treated rats, cerebellum cytosol, and endothelial cell lysates were used as inducible nitric oxide synthase (iNOS)-, neuronal nitric oxide synthase (nNOS)-, and endothelial nitric oxide synthase (eNOS)-positive con￾trols. Proliferating liver from rats treated with a single dose of T3 (60 g/100 g body weight, intraperitoneally) was used as a control for signaling assays.21 Co-immunoprecipitation. Mitochondrial or cytoso￾lic proteins (500 g) were incubated with 4 g of poly￾clonal nNOS antibody and 30 L protein A/G PLUS￾Agarose (Santa Cruz, CA) at 4°C. The beads were then washed three times, suspended in sample buffer, boiled, and centrifuged, and the supernatants were subjected to immunoblotting against monoclonal nNOS antibodies. Antibodies. Polyclonal anti-iNOS and anti-nNOS, anti-cyclin D2 and D3, and monoclonal anti–mouse cy￾clin D1 were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); polyclonal anti-eNOS and mono￾clonal nNOS antibodies were obtained from Transduc￾tion Laboratories (Lexington, KY). Polyclonal antibodies against total and phospho-p38 MAPK, ERK 1⁄2 and c-Jun N-terminal kinase, and phospho–MAPK kinases 1⁄2 were from Cell Signaling Technology (Beverly, MA). Immunoelectron Microscopy. Purified mitochon￾dria were suspended in 4% formaldehyde (pH 7.4) for 2 hours at 4°C, then dehydrated in 70%, 96%, and 100% ethanol (30 minutes for each step) and embedded in LR White. Immunocytochemistry was performed using a pri￾mary mouse anti–C-terminal nNOS (1095-1289) at a dilution of 1:100 in phosphate-buffered saline (pH 7.4). Grids were washed in phosphate-buffered saline and counterstained with 1% uranyl acetate. Nonspecific back￾ground was blocked by incubation with 5% normal goat serum in phosphate-buffered saline at the beginning of the procedure. Specimens were observed in a Zeiss EM- 109-T transmission electron microscope at 80 kv. Mitochondrial H2O2 Production. H2O2 produc￾tion was continuously monitored using a Hitachi F-2000 spectrofluorometer (Hitachi) with excitation and emis￾sion wavelengths at 315 and 425 nm, respectively.22 The reaction medium, which consisted of potassium phos￾phate buffer (50 mM) and 50 mM L-valine, was supple￾mented with 10 mM succinate, 12.5 units/mL horseradish peroxidase, 250 M p-hydroxyphenyl-acetic acid, and 0.15 mg of mitochondrial protein per mL. NO￾dependent H2O2 production was determined as the dif￾ference of H2O2 production rate in the presence of 100 M L-arginine and L-arginine plus 2 mM L-NMMA. Mitochondrial preparations were supplemented with 1 M Mn(III)tetrakis(4-benzoic acid) porphyrin (Cayman Chemical, Ann Arbor, MI) to uniform the maximal H2O2 production rate. Cell Isolation and Culture. Hepatocytes were iso￾lated by collagenase digestion as described previously.23 For culture, P2- and P15-isolated hepatocytes were seeded in 96-well plates (50,000 cells per well) in medium 199 (Gibco-BRL, Invitrogen Life Technologies, Breda, the Netherlands) supplemented with 10% fetal calf serum (FCS) and 50 g/mL gentamicin and were allowed to attach for 4 hours. Cells were synchronized by 20-hour incubation with 2% FCS and 2 hours without FCS. He￾patocyte treatments were performed in medium 199 with 10% FCS for 72 hours. Proliferation was assayed through [3H] thymidine incorporation.11 The last 24 hours, cells were incubated with 0.8 Ci[3H] thymidine/well and harvested; cpm were measured in a liquid scintillation counter (Wallac 1414, Turku, Finland). Detection of Intracellular ROS. Intracellular ROS were analyzed by flow cytometry, using a 2,7-dichlo￾rofluorescin diacetate probe. 23 The cellular fluores￾cence intensity was measured after 30 minutes of incubation with 5 M 2,7-dichlorofluorescin diac￾etate by using an Ortho Cytoron Absolute Flow-Cy￾tometer (Johnson & Johnson, Raritan, NY). Propidium iodide (0.005%) was used to detect dead cells. For each analysis, 10,000 events were recorded. DNA Analysis. For measuring apoptosis, the ploidy determination of hepatocytes was assessed with pro￾pidium iodide staining and flow cytometry as described previously.24 Antioxidant Enzyme Activities. Mitochondrial Mn-SOD activity was determined by inhibition of cyto￾chromec reduction at 550 nm in 50 mM potassium phos￾phate buffer/0.1 mM EDTA (pH 7.8) at 25°C.25Catalase and glutathione peroxidase activities in 7,000g superna￾tants were determined by the decrease in H2O2 absorp￾tion at 240 nm (240 41 M
1
cm
1), 26 or by the oxidation of NADPH at 340 nm (340 6.22 mM
1
cm
1).27 Reagents. SDS, glycerol, 2-(-mercaptoethanol), and bromophenol blue were obtained from Bio-Rad (Richmond, CA); SB 202190 was obtained from Calbio￾chem (San Diego, CA); U0126 was obtained from Cell Signaling Technology; and other chemicals were from Sigma Chemical Co. (St Louis, MO). Data Analysis. Data are expressed as mean  SE and were analyzed by ANOVA and Scheffe´ test. Simple linear HEPATOLOGY, Vol. 40, No. 1, 2004 CARRERAS ET AL. 159

160 CARRERAS ET AL HEPATOLOGY.Juy 2004 B the sequential proliferating-differentiating process.It is surmised that promotion of proliferation requires a con- trollod inlibition of mitochondrl ropiration spccifi- cally.low complex acrivities and reduced mitochondrial mass have a negative impact on redor mitochondrial-de. 4 pendent cell signaling. Mitochondrial NOS Is Modulated During Devel- opent The increase or deerease of mNOS content A程ey can be an adaprive mechanism to control mirochondrial f尼1.Lier weight a"d mitoctondrisl moss verig物修dL中ewe functions.3 In this study.liver mrNOS content was develapmert《Al Proge550 n cf tatal1emt他g.Dge clearly modulated during rar development:it was almosr opmental ages ai plotted wih te rabe af liver weigtt gain (L),and the underectable at highly proliferative E19.while it progres. npectiv michardial mass ()mit pot,miechondrial protein. sively increased after birth,up to quiescent P30-P90 (Fig. 2Al;modulation was confrmed by immunoelectron mi- regression was utilized as appropriare.Staristical signif. croeopy (Fig,2E).mINOS ativity prallded protein cance wis accepted atP05. expression-both increased renfold from feral to adult stages (Fig.2A and B).Considering the variarions of mi Results and Discussion tochondrial miss.mtNOS activity per gram ofliver tissue enhanoed in the adult organ by 500-fold.It is then in- Mitochomdrial Maturatione in Rat Liver Develop- ered that,.d小ring liver devclopment,《1)mtri NO memt.Liver mass continuously increases with age due to steady-stare concentrarion raises by sequenrial increase of cell duplication and hypertropby (Fig 1A).From E19 mrNOS content and (2)toral NO liver production is to sulthood.relarive liver weight went from 6.1%to greatly amplified by the expansion of the mitochondrial 3.4%of body weight and liver growth decreased renfold pool. (ig 1B).Likewise,transition from fetal to adult liver is Although it B a INOS vartnt.a 130-kDa liwt inINOS accompanied by a hurst ofheparocyre proliferation in late reacrs wirh both anri-nNOS and anri-iNOS anribodies (see gestation and in the immediare neonatal period (E19- Fig,2A):eNOS was nor detected in liver mitochondria.In P2-3).followed by a decrease of proliferation after the teresingly,cytosol immunoblotting with antibodies aginst first pootnatal weck Instead,mitochondrial biogeneis nNOS revealed two specific bands:dasic nNOS-a with M persistently active throughout devdopment;mitochon- of 157 kDaand a scoond land with 130 kDa,the eaptession drial protein content increased from 0.13(E19)to 34 mg of which was inversely modulated.The specificiry of the per g of liver tissue (P90)(Fig.IB).To evaluate mito- nNOS 130 kDa band variant was validated by immunopre chondril marurarion,enzyme activities were resred in the cipitation of mitochondria and cytosolic proteins from P2 same condirions.Mitochondria isolated from maximally liver (soe Fig.2B).Moroover,130-kDa protein was solcly proliferating liver at E19-P2 retined 10%50%of com evidenced at carly stages while,concomitantly with a higher plex 1.11-111.and IV acrivities of quiescent organelles expression of mNOS.ir disappeared from cytosol after P15 mitochondrial ubiquinone (U)had a similar partern This parern sugpests that cytosal 130-kDa protein is the (Table 1).Previous reports showed an increase of the spe- enzyme rranslocated to mitochondri during devdopment. cific activity of a number of oxidative enxymes during the Campararively,cyosolic NOS activiry.as contribured by early posmnatal development.2 Boch increasing mito- the Ca-dependent isocorms,increased early at birth and re chondrial pool and phenotypic changes may take part in mained stable up to adult stage:no significant modulation of Table 1.Mitochondrial Enzyme Activities and Ubiquinol Content in Rat Liver Derelopment 19 a P15 P30 P90 Corploes HI [rmaymit pret) 170±26 247±65 404±5到 417±27 441±52 7+ 5+8 的一9 m+10 0oT版WKmh门gp 5±1” 11+1" 14±1 1T=2 16+2 U090 ent irnovng p国 ND 11±01° 2±01 1.8=01 19±01 2E0 Ypla HIt NADH-Eytseome e reductrsc.C.weu5且x3ua3ee6cs经.Cag.令102a。.Data are bp2c0s ■P2意5三.1BH2

regression was utilized as appropriate. Statistical signifi- cance was accepted at P  .05. Results and Discussion Mitochondrial Maturation in Rat Liver Develop￾ment. Liver mass continuously increases with age due to cell duplication and hypertrophy (Fig. 1A).28 From E19 to adulthood, relative liver weight went from 6.1% to 3.4% of body weight and liver growth decreased tenfold (Fig. 1B). Likewise, transition from fetal to adult liver is accompanied by a burst of hepatocyte proliferation in late gestation and in the immediate neonatal period (E19 – P2–3), followed by a decrease of proliferation after the first postnatal week.13 Instead, mitochondrial biogenesis is persistently active throughout development; mitochon￾drial protein content increased from 0.13 (E19) to 34 mg per g of liver tissue (P90) (Fig. 1B). To evaluate mito￾chondrial maturation, enzyme activities were tested in the same conditions. Mitochondria isolated from maximally proliferating liver at E19 –P2 retained 40%–50% of com￾plex I, II-III, and IV activities of quiescent organelles; mitochondrial ubiquinone (UQ) had a similar pattern (Table 1). Previous reports showed an increase of the spe￾cific activity of a number of oxidative enzymes during the early postnatal development.29 Both increasing mito￾chondrial pool and phenotypic changes may take part in the sequential proliferating– differentiating process.30 It is surmised that promotion of proliferation requires a con￾trolled inhibition of mitochondrial respiration31; specifi- cally, low complex activities and reduced mitochondrial mass have a negative impact on redox mitochondrial-de￾pendent cell signaling. Mitochondrial NOS Is Modulated During Devel￾opment. The increase or decrease of mtNOS content can be an adaptive mechanism to control mitochondrial functions.3,9 In this study, liver mtNOS content was clearly modulated during rat development: it was almost undetectable at highly proliferative E19, while it progres￾sively increased after birth, up to quiescent P30 –P90 (Fig. 2A); modulation was confirmed by immunoelectron mi￾croscopy (Fig. 2E). mtNOS activity paralleled protein expression— both increased tenfold from fetal to adult stages (Fig. 2A and B). Considering the variations of mi￾tochondrial mass, mtNOS activity per gram of liver tissue enhanced in the adult organ by 500-fold. It is then in￾ferred that, during liver development, (1) matrix NO steady-state concentration raises by sequential increase of mtNOS content and (2) total NO liver production is greatly amplified by the expansion of the mitochondrial pool. Although it is a nNOS variant,3,8 130-kDa liver mtNOS reacts with both anti-nNOS and anti-iNOS antibodies (see Fig. 2A); eNOS was not detected in liver mitochondria. In￾terestingly, cytosol immunoblotting with antibodies against nNOS revealed two specific bands: classic nNOS-
with Mr of 157 kDa and a second band with 130 kDa, the expression of which was inversely modulated. The specificity of the nNOS 130-kDa band variant was validated by immunopre￾cipitation of mitochondria and cytosolic proteins from P2 liver (see Fig. 2B). Moreover, 130-kDa protein was solely evidenced at early stages while, concomitantly with a higher expression of mtNOS, it disappeared from cytosol after P15. This pattern suggests that cytosol 130-kDa protein is the enzyme translocated to mitochondria during development. Comparatively, cytosolic NOS activity, as contributed by the Ca-dependent isoforms, increased early at birth and re￾mained stable up to adult stage; no significant modulation of Fig. 1. Liver weight and mitochondrial mass variations during rat liver development. (A) Progression of total liver weight with age. (B) Devel￾opmental ages are plotted with the rate of liver weight gain (
), and the respective mitochondrial mass (●). mit prot, mitochondrial protein. Table 1. Mitochondrial Enzyme Activities and Ubiquinol Content in Rat Liver Development E19 P2 P15 P30 P90 Complexes I–III (nmol/min.mg prot) 170  28* 247  68* 404  54 417  27 441  52 Complexes II–III (nmol/min.mg prot) 37  7* 48  6* 55  6* 89  9 96  10 Complex IV (k/min.mg prot) 5  1* 11  1* 14  1 17  2 16  2 UQ-9 content (nmol/mg prot) ND 1.1  0.1* 2  0.1 1.8  0.1 1.9  0.1 Abbreviations: E, embryonic; P, postnatal; prot, protein; UQ, ubiquinone; ND, not determined. NOTE: Complexes I–III: NADH-cytochrome c reductase. Complexes II–III: succinate-cytochrome c reductase. Complex IV: cytochrome oxidase. Data are expressed as mean  SE from 4 – 8 different experiments. *P  .05 respect to P90. 160 CARRERAS ET AL. HEPATOLOGY, July 2004

HEPATOLOGY.VaL 40 No.1.2004 CARRERAS ET AL 161 A Mitachondria 1刘kD 157 kDa 1约kD ✉【 EEPPC N满 C P 157 kDa 上 04e C EE.PP.P.P.C KnFaP:P.PaPa D E 3D5 14 。s EE PP.P.P.C E:E.FP..F.r. mtNos copression in embryonic atd pocmnatal mtocnondra wia 2m-INOS and and-nNDS andbocies;ghe dershametry of mtNCS (amt-nNCS:P90 wro aat 11.[B)as by the of PH]-l-nrginina to P-L-cilina.Irset Ca2-dapesdent() 8diep4e线0N08ingg.(GLt9g同uing wil前mt-OS:lg:#ve eopession of0 S verianes:riht co-immunaprecipitstion cf mnathondtal and cytasalc prcteins of P2 lwers wih palydlonal nko aticodies.D)Cytreol immundblotang with astl eNUS anbodes.Data are mean SE at three stparate expermerts.Whbe calunrs rpesent palertg penctypes wty colamns resresemt qaiestent phenaty低“P<.C5s6P0.(用Inuneelectron mic6 opy af isolated p山ed Iiver nbochondda fam P2 and ad山kk.Te 心nhw的ateledl wth ati-N0阁atd14 steal with a paet ani-nu值cmnjgstal with mileidd51-m tamneler paticlo 0 ptl megficstion×85000.}n3et0 S in submitoctondial portices from the对ne的res.iN06,inducible ritic caide世a:n03 neuronal nitric adde syrthase:E.embryoni F.pasttatat mtNOS.mitochondral tit oodde sytase:[Hjtr,I'H]L citulline;prat,protein:NOS. ntric caide symmase:Mt,mboctondia:Dyt,cytreat SMF,submbactondial partides eNOS expresion was detected (Fg 2B [inset]tein increses with developmental passibly account- and D) ing for the decreased HO production rate in less mature The selecrive mtNOS modulation could be relared mitochondra. to developmental activities of membrane mitochon- Inaphysiological tting,supplementation of mitcho drial transporters and chaperone proreins,or to enzyme dria with NO promocesasignificant O burst NO inhibits activation or degradation.It has been reported that cyochrome oxidase and l regica at compkx ll and in arginase and calpain proteases are higher in fetal creases the ubisemiquinone radical level thar prowvides the liver thn in adult liver,contributing to low matrix NO electrons to O.Mimochondrial production of freely diffus levels in fetal hepatocytes by reducing NOS suhsrrare ble H depends on superoide dismutas-cyed dis- and/or by increasing mtNOS degradation by mito- mutarion of NO-dependent O (reacrion 3): chondrial calpains. tNOS Modulation Correlates With Liper Mito- NO+UQH-→NO-+UQ-+H+ (1) cbondrial Hydrogen Peroxide Yield.Immiture mito chordri supplmented with complex Ill inhibitor UQ-+O2→UQ+02 (2) antimycin had a noriceahly slower HO production rate than adult organelles (Fig.3A).According to Table 1. respiratory chain activitics per unit of mitochondrial pro- 20,+2H*00 H02十0， (3)

eNOS expression was detected (Fig. 2B [inset] and D). The selective mtNOS modulation could be related to developmental activities of membrane mitochon￾drial transporters and chaperone proteins, or to enzyme activation or degradation. It has been reported that arginase32 and calpain proteases33 are higher in fetal liver than in adult liver, contributing to low matrix NO levels in fetal hepatocytes by reducing NOS substrate and/or by increasing mtNOS degradation by mito￾chondrial calpains. mtNOS Modulation Correlates With Liver Mito￾chondrial Hydrogen Peroxide Yield. Immature mito￾chondria supplemented with complex III inhibitor antimycin had a noticeably slower H2O2 production rate than adult organelles (Fig. 3A). According to Table 1, respiratory chain activities per unit of mitochondrial pro￾tein increases with developmental age, possibly account￾ing for the decreased H2O2 production rate in less mature mitochondria. In a physiological setting, supplementation of mitochon￾dria with NO promotes a significant O2
burst4; NO inhibits cytochrome oxidase and b-c1 region at complex III and in￾creases the ubisemiquinone radical level that provides the electrons to O2. 22Mitochondrial production of freely diffus￾ible H2O2 depends on superoxide dismutase– catalyzed dis￾mutation of NO-dependent O2
(reaction [3]): NO  UQH  3 NO   UQ   H  (1) UQ   O2 3 UQ  O2  (2) 2O2   2H  O¡ Mn-SOD H2O2  O2 (3) Fig. 2. Modulation of mitochondrial and cytosolic NOS during liver development. Proteins (50 g/lane) were separated in 7.5% gels. (A) Left, mtNOS expression in embryonic and postnatal mitochondria with anti-iNOS and anti-nNOS antibodies; right, densitometry of mtNOS (anti-nNOS; P90 value was set as 1). (B) mtNOS activity as measured by the conversion of [3H]-L-arginine to [3H]-L-citrulline. Inset: cytosolic Ca2-dependent (E) and independent (
) NOS activities. (C) Left, cytosol immunoblotting with anti-nNOS; center, relative expression of nNOS variants; right, co-immunoprecipitation of mitochondrial and cytosolic proteins of P2 livers with polyclonal nNOS antibodies. (D) Cytosol immunoblotting with anti-eNOS antibodies. Data are mean  SE of three separate experiments. White columns represent proliferating phenotypes; grey columns represent quiescent phenotypes. *P  .05 versus P90. (E) Immunoelectron microscopy of isolated purified liver mitochondria from P2 and adult rats. The organelles were labeled with anti-nNOS antibody and treated with a goat anti–mouse serum conjugated with colloidal gold (10-nm diameter particles). (Original magnification 85,000.) Inset: mtNOS in submitochondrial particles from the same organelles. iNOS, inducible nitric oxide synthase; nNOS, neuronal nitric oxide synthase; E, embryonic; P, postnatal; mtNOS, mitochondrial nitric oxide synthase; [3H]citr, [3H]-L-citrulline; prot, protein; NOS, nitric oxide synthase; Mt, mitochondria; Cyt, cytosol; SMP, submitochondrial particles. HEPATOLOGY, Vol. 40, No. 1, 2004 CARRERAS ET AL. 161

162 CARRERAS ET AL HEPATOLOGY.Juy 2004 = C 1a' 09n0 1O,indn 14% 1% 灯% P。preliferation mINOS activiry a节口n天 D E Ew P:Pe PePe Developmental age mINOS activiry pmnel I'kit'nining po 月g3.ct of mN0 modultion on mitschandr内H-0,brocucdon. C (A)NO-dependert H0 pedaction rate is conpard wth madmal H0 li0mp4 th antingen.幻-petdlatt Irpiigan penpi网 g期oc3tlM0,la小琴n the equ)5n阳d in Results 间sTpe1rerB水n bewveen myasol IH0,l.andT时scuw iy.(D)Modulation af Mn-SOD acivty darng rat lwer develapnen (E) come arion with mN05.white ounrs ar cisc sapncart prolfering 内们3pg:urs伊rw时ogt为t同tm8+p .06 versus P90.Dats represent 4-8 samples fiam each goup.prot. pratrit;LAng L artine;INOS,Induc ble ritrc cad:symttace:E.cm ryoric:P.postnatal:mtNOS,mitschardial ntrie coide symhase:500. spenoid8 rfsm;PHok BHl-ciina. Flg 4.Deveiopnemtal varatons of IH/l and hepatrcyte prolmer 1o.,oa■p它0ma1 mturrlula'peraddes in1 estim-is0dP2 prol fereing hepstrecytes and ciferaelialed P15-P90 cel Cels were To invetigate mtNOS contribution,H:O:production incucoed with 5 uM 2'.7-dichloroluosescin docatate,ard Ivve popu- rate was measured in the presence of L arginine alone or lation was tsamined by fow crtemeary.NO dependent cjtosal [H02l in addition to L-NMMA.The NO-dependent mitochon- ard [H,Oa!index the maddo betwcen NO-deperdent (+L-arhnine)and madna(十at的)Hg0 podudtion mate are itd.h4Aes drial H:O:production was undetectable in E17 mito- modulton of P2 and P15 heperocyl prolieration e Msterisls and chondri.hur thereafier ir progressively increased up to Metnods).Syctroneed tepstoa/tes were sueplemerted wih the cifer P30-P90,indicating a linkage with mrNOS modulation ent teztmem也5eAb的tors)and1C%Ft5for3d3:H hymidine incorporarion6■amdd山向the last24 hours (C (see Fig.3A).Funthermore,in embryos low HO:yicld is fegiresemalive frra cytnmnetry DNA snalis nf P2 hepaenas abat cooperatively contribured by low mitochondrial complex oodatwe chalenge.2C dplo d nudei:4C.tetrsploid nuclei.Whie activiries and UQ content (see reacrions 1]and 2 above couunss represent proirerating phenatipes prey colunrs regresent quiestemt phenot pes.Dota are mcan±SE af n=st也ee也ard and Table 1).A parallel increase of mtNOS,UQ,and n 16 (cnmteta).*P <.05 versn tte mspactie basal ronditin.P. HO2 was oberved in rat brain in the transit from neu- p动atsl:L-NANE10H-ig=1:AC.体hS roblast proliferation to neuronal differentiation. teine:GSH patath one;AlZ 3-smno 12,4-ttaml:;L-Ag.L-atz nne; Mitochondrial Contributions to Cytorol Liver GR凡n fusrsce1r. H202 Strudy-State Coneentration.Mitochondria is

To investigate mtNOS contribution, H2O2 production rate was measured in the presence of L-arginine alone or in addition to L-NMMA. The NO-dependent mitochon￾drial H2O2 production was undetectable in E17 mito￾chondria, but thereafter it progressively increased up to P30 –P90, indicating a linkage with mtNOS modulation (see Fig. 3A). Furthermore, in embryos low H2O2 yield is cooperatively contributed by low mitochondrial complex activities and UQ content (see reactions [1] and [2] above and Table 1). A parallel increase of mtNOS, UQ, and H2O2 was observed in rat brain in the transit from neu￾roblast proliferation to neuronal differentiation.11 Mitochondrial Contributions to Cytosol Liver H2O2 Steady-State Concentration. Mitochondria is Fig. 4. Developmental variations of [H2O2]ss and hepatocyte prolifer￾ation. (A) comparison of intracellular peroxides in freshly-isolated P2 proliferating hepatocytes and differentiated P15–P90 cells. Cells were incubated with 5 M 2,7-dichlorofluorescin diacetate, and live popu￾lation was examined by flow cytometry. NO-dependent cytosol [H2O2]ss and [H2O2] index, the ratio between NO-dependent (L-arginine) and maximal (antimycin) H2O2 production rates, are included. (B) Redox modulation of P2 and P15 hepatocyte proliferation (see Materials and Methods). Synchronized hepatocytes were supplemented with the differ￾ent treatments (see Abbreviations) and 10% FCS for 3 days; [3H] thymidine incorporation was measured during the last 24 hours. (C) Representative flow cytometry DNA analysis of P2 hepatocytes after oxidative challenge. 2C, diploid nuclei; 4C, tetraploid nuclei. White columns represent proliferating phenotypes; grey columns represent quiescent phenotypes. Data are mean  SE of n 8 (treatments) and n 16 (controls). *P  .05 versus the respective basal condition. P, postnatal; L-NAME, NG -nitro-L-arginine methyl ester; NAC, N-acetyl-cys￾teine; GSH, glutathione; ATZ, 3-amino 1,2,4-triazole; L-Arg, L-arginine; GR-FL, green fluorescence. Fig. 3. Effect of mtNOS modulation on mitochondrial H2O2 production. (A) NO-dependent H2O2 production rate is compared with maximal H2O2 production rate with antimycin. (B) NO-dependent hydrogen peroxide steady-state concentration ([H2O2]ss) as in the equation noted in Results. (C) Simple linear regression between cytosol [H2O2]ss and mtNOS activ￾ity. (D) Modulation of Mn-SOD activity during rat liver development; (E) correlation with mtNOS. White columns or circles represent proliferating phenotypes; grey columns or circles represent quiescent phenotypes. *P  .05 versus P90. Data represent 4 – 8 samples from each group. prot, protein; L-Arg, L-arginine; iNOS, inducible nitric oxide synthase; E, em￾bryonic; P, postnatal; mtNOS, mitochondrial nitric oxide synthase; SOD, superoxide dismutase; [3H]citr, [3H]-L-citrulline. 162 CARRERAS ET AL. HEPATOLOGY, July 2004

1H7A1O10×Y.l40.1,20 CARRERAS EI AL 165 B Fg互.Nodulatian of cell slzraling n 风K,E,PP,PaP the developing rat图.adB, PL EE F,Fu P.P. -星RK Fver tomeeises (25 ug/lene)wer Cyclis DI seperoned i 12%pels:T3-injemed,pro ===日 Iferadng tat heer was used as cortdl Cyuta B F4.青estom blat deto时on cf cydins D adP&a线gfnI4 d sith spcii正 Cyetis 115. 8atd0dws.(C的Sie物8sn6 A MAPK between cpei D1.actve MAPKs.ard NO-ccpencent cytooal IH/0.lu (cydin C PNKI4 DiC2-aa1:phaspho-Ei减1口j ad2[L户-Q95ml096:al IKI phosgho-p38 MAPK [r0.78:all P .05).Each palt tepreserts tfree Er E.P:Pa P.P.PL indeperdert experinenes nomma laod to F-MEK I F17.F.antrymic:P.p:FK otrecellulat si小-ee时材rds c WMF%,mt也en artiated p加n材 nase;INM,tJun N-teminal knase; XO-depeadent HO,lss the main contributor to liver H,steady state concen. Therefore,we propose a developmental grading of cyto. tration (HOl;only a small fraction of perooisome- solic (HO as bised upon a coordinited increase of derived HO appears to escape peroxisomal ctalas. mitochondrial complex activities mtNOS and Mn-SOD. Depending on matrix NO.in the steady state condition. Redux Modlation of Hepatoeyie Prolfferation.As mitochondrial HO,production equils cytosolic H.O reported,is in neonatal hepatocyres there is srill a synchro- utilication by the two main scavenger enxymes catalase nized high proliferation rate.We therefore atrempted to and glutathione perocidase NO-dependent [HOcan mimic ex wno the redox modulation of proliferation in P2 be calculated according to the following equarion (where and P15 hepatocytes with low and high mtNOS content, +H2O2 a is the rate ofL-arginine-dependent HO2 repectivdy.Supplementation of synchronixad P2-cul- productionis the secdorder rate c for the rured bepwith Hcarse inhibiror 3amino caralase-catalyzed metabolism of HO.and is that for 1.2,d-triaxole,or Larginine invariably determined a the glutathione peroxidase-driven reaction): dose-dependent negative modulation of cell proliferation [H O,1 +d[H,O:ldt/k[catalase] (Fig 4B).In contrast,decreasing cell HO2 levels by con- trolled treatment with savengers or NOS inhibitors such +kaglutathione peroxidase](4) as N-aceryl-cysteine (NAC),glutathione,or N-nirro-L NO-dependent (HO:l was undetectable at E17 and arginine methyl ester increased proliferarion rates by up to increased by two orders of magninxle from E19 to P90 30%.At a higher NAC concentration,a similar response 10-M ro 10-M).paralleling the developmenral was observed in more differentated and less proliferative modulation of mtNOS (Fig.3B and C).Mn SOD activ P15 hepatocytes with higher [HO (e Fig.4B).Inall ity was similarly comodulated and well correlated with conditions.cell viabiliry with Trypan blue was 98%and mtNOS,the net flux of H2O2 into cytosol and the re- LDH in the culrure supernatant was less than 1%of cy sulted [HOl (Fig,3 D and E). tosol values,which indicates that at the utilized concen- Differentil oxidant pruduction was confirmed by How trations the tested compounds were not toxic for cytometry (Fig 4A).The dichlorofluorexin mean fluo- hepatocytes.No hypoploid peak representative of apo- rescence ratio berween P15 and P2 and adult P90 hepa- ptosis was ohserved in the permeahilized P2 heparocyres tocytes was abour 5:15,which is in agreement with that in the different condirions (Fig,4C). obeained from estimted NO-dependent [H2O:(10: These results confirm that(1)hepatocyte proliferation 20).Thereby.the contriburion of urilized NO to maximal at different devclopmental stages depends on a precise mitochondrial H,O,production (namely H,O,index) tisue H.O:concentrarion and (2)in this conrest,prolif. markedly increased from proliferating to quiesoent stages erarion correlares with liver in sino developmental modu- (see Fig,4A). lation of mirochondrial activities and mtNOS. These data suggest that most HO,comes from mito- NO may modulate perse the expression of cell cycle chondrl mctabolism;at P90,NO-dependent HO is regulatory proteins,and it induces cytootasis by inhibi- similar to that obeained by perfising adule rat liver.ss tion of cyrlin DI or hy inhibirion of cle2 (cyclin Fand A

the main contributor to liver H2O2 steady-state concen￾tration ([H2O2]ss); only a small fraction of peroxisome￾derived H2O2 appears to escape peroxisomal catalase.34 Depending on matrix NO, in the steady-state condition, mitochondrial H2O2 production equals cytosolic H2O2 utilization by the two main scavenger enzymes, catalase and glutathione peroxidase. NO-dependent [H2O2]ss can be calculated according to the following equation (where d[H2O2 ]/dt is the rate of L-arginine– dependent H2O2 production, k3 is the second-order rate constant for the catalase-catalyzed metabolism of H2O2, and k4 is that for the glutathione peroxidase– driven reaction): 35 H2O2 ss   dH2O2 /dt/k3catalase  k4glutathione peroxidase (4) NO-dependent [H2O2]ss was undetectable at E17 and increased by two orders of magnitude from E19 to P90 ( 10
11 M to 10
9 M), paralleling the developmental modulation of mtNOS (Fig. 3B and C). Mn-SOD activ￾ity was similarly comodulated and well correlated with mtNOS, the net flux of H2O2 into cytosol and the re￾sulted [H2O2]ss (Fig. 3 D and E). Differential oxidant production was confirmed by flow cytometry (Fig. 4A). The dichlorofluorescin mean fluo￾rescence ratio between P15 and P2 and adult P90 hepa￾tocytes was about 5:15, which is in agreement with that obtained from estimated NO-dependent [H2O2]ss ( 10: 20). Thereby, the contribution of utilized NO to maximal mitochondrial H2O2 production (namely H2O2 index) markedly increased from proliferating to quiescent stages (see Fig. 4A). These data suggest that most H2O2 comes from mito￾chondrial metabolism; at P90, NO-dependent H2O2 is similar to that obtained by perfusing adult rat liver.35 Therefore, we propose a developmental grading of cyto￾solic [H2O2]ss as based upon a coordinated increase of mitochondrial complex activities mtNOS and Mn-SOD. Redox Modulation of Hepatocyte Proliferation. As reported,13 in neonatal hepatocytes there is still a synchro￾nized high proliferation rate. We therefore attempted to mimicex vivo the redox modulation of proliferation in P2 and P15 hepatocytes with low and high mtNOS content, respectively. Supplementation of synchronized P2-cul￾tured hepatocytes with H2O2, catalase inhibitor 3-amino 1,2,4-triazole, or L-arginine invariably determined a dose-dependent negative modulation of cell proliferation (Fig. 4B). In contrast, decreasing cell H2O2 levels by con￾trolled treatment with scavengers or NOS inhibitors such as N-acetyl-cysteine (NAC), glutathione, or NG-nitro-L￾arginine methyl ester increased proliferation rates by up to 30%. At a higher NAC concentration, a similar response was observed in more differentiated and less proliferative P15 hepatocytes with higher [H2O2]ss (see Fig. 4B). In all conditions, cell viability with Trypan blue was 98% and LDH in the culture supernatant was less than 1% of cy￾tosol values, which indicates that at the utilized concen￾trations the tested compounds were not toxic for hepatocytes. No hypoploid peak representative of apo￾ptosis was observed in the permeabilized P2 hepatocytes in the different conditions (Fig. 4C). These results confirm that (1) hepatocyte proliferation at different developmental stages depends on a precise tissue H2O2 concentration and (2) in this context, prolif￾eration correlates with liver in vivo developmental modu￾lation of mitochondrial activities and mtNOS. NO may modulate per se the expression of cell cycle regulatory proteins,19 and it induces cytostasis by inhibi￾tion of cyclin D1 or by inhibition of cdc2 (cyclin E and A Fig. 5. Modulation of cell signaling in the developing rat liver. (A) and (B), liver homogenates (25 g/ lane) were separated in 12% gels; T3-injected, pro￾liferating rat liver was used as control (PL). Western blot detection of cyclins D and MAPKs was performed with specific antibodies. (C) Simple linear regression between cyclin D1, active MAPKs, and NO-dependent cytosol [H2O2]ss (cyclin D1 [E], r2
0.91; phospho-ERK 1 (
) and 2 [‚], r2
0.95 and
0.96; and phospho-p38 MAPK [], r2 0.76; all P  .05). Each point represents three independent experiments normalized to E17. E, embryonic; P, postnatal; ERK, extracellular signal–regulated kinase; MAPK, mitogen-activated protein ki￾nase; JNK, c-Jun N-terminal kinase; MEK, MAPK kinases. HEPATOLOGY, Vol. 40, No. 1, 2004 CARRERAS ET AL. 163

164 CARRERAS ET AL HEPATOLOGY.Juy 2004 pathways).In accordance.NO has antimitogenic effects A on cultured hepatocytes. L Redox Modulation of Cell Signaling.In partial P.Hepatecytes t P. heparectomy or injury,mechanisms regularing bepato cyte proliferation rely on MAPK activation by growth OaDI☐ factor but fetal hepatocytes my praliferate in aboen of Daual I A AA exogenous factors with a consrinrive level of MAPK ac- 属领 tivation.Indeed,cyclin DI is sufficient to promore pro- EK⅓= 口一国4=■ gression of hepatocytes through Gl restriction point.As a14s45c0项tAA4L has been previously reported,cydlin DI is up-regu- A ated during Tiver proliferation (E17-P2)and almost dis- P-p3RAPK appears in the quiescent orgn:expression of cydins D2 and D3 followed that of cydin D1 (Fig.SA).However, cydins D2 and D3 would have a lower contribution to 国 adulr liver proliferarion.as shown during regeneration B and here after 'I'3 treatment. ERK acivation paralleled cycin DI expression while P-KAS p38 MAPK activation followed an inverse pattern (Fig 5And段，Similarly,p38米iviry was repored inverse to cyclin DI conrent in liver regenerarion.s and transfection with p38 activating kinise MKK6 arrests hepatocyte growth.is In addition,P2 heparocytes markedly expressed Bad I +58+C 5 +AC A AA P ERK1/2 upstream kinase phospho-MAPK kinises 1/2 and P90 hepatocytes did pot,while phospho-c-Jun N- P.lleputecytr terminal kinase (a stress-activated kinase related to liver proliferation and spoptosis)was only detectable in the quiescent phenorypes As referred,Phaspho-c-Jun N. terminal kinase increase may express both the late increase of axidant levels and ERK decrease (Fig.5B).Total MAPKs were not modified during development. Liver cydin DI and phospho-ERKs were inverse to wne NO-dependent [HOzl and HO2 index;instead. this correlation was positive for p38 MAPK (Fig,5C). Cansequently,a high ERK/p38 acriviry ratio was repre- 国1w5d05+%C5线1A sentarive of proliferating phenocypes:a low ERK/p38 ra HO,GMI tio was representative of quiescent ones and was related to Fi 6 Padro modalalo uf ir cell sipuing P2 hepalcas were [H2Ol and liver growth.ERK/p38 ratio was considered exposed te H0>a one or with 10 uM SB202190 ar 0.1 mM NAC Iboth a datermirnt of growth and darmancy in human cancer supp emerted 2 houts betor:Hi0l.200 n/mL ancom/dn 2 uM cells:Aguirre-Ghiso er alL showed thar modulrion of artimoin atd 10 uM UO126.(A)Cdin D1 and MAPHs wee detected ERK/p38 activity by pharmcological and genctic inter- m2eusd15ni版s44i.ny immuschloting50吧 eneh中ic.的Epp8P%时io(dersitor时tic messure- ventions predicis the in siwo behavioe in 90%of the mer也t元mh层tead是②petmem也0 he d fferemt5sB eximined cell lines.43 expessed C)P2 hepatocytes were inrubabod in simlar cendions as The interplay berween redox status.signaling and pro- those deser had i超Fg4.“Diflasant frome hasal condtion..DIferent from repediv H.O outcenluation.P05 P.postseat ER taltscelllat liferation was analyzed by exposing P2 bepatocytes to ega-6J与edh8 A anisomycir:AA,antimycir;鬼，202190： HO2,antimycin,and NAC,and/or to the ERK inhibitor NAC,N atrtyl cystrine:WAFK,mbogen acttvaed protein Hnase. U0126.p38 inhibitor 5B202190,and p38 acrivator ani- somycin.Exposure to H:or to anisomycin markedly reduced cyclin D1 expression,kading P2 cells torheadult anisomycin or U0126 was dlearly associated with the low liver level.In these candirions,endogenous or exogenous est hepatocyte proliferation rate (Fig 6B and C).Like- HO,decreased ERK/p38 activiry ratio,an effect equally wise,SB202190 significintly increased cell proliferation prevented by NAC or SB202190 (Fig.6A and B).Simi- by approximately 25%,and SB202190 oe NAC also pre- larly,a marked reduction of ERK/p38 activity ratio by vented the lowering of the proliferation rate induced by

pathways).18 In accordance, NO has antimitogenic effects on cultured hepatocytes.36 Redox Modulation of Cell Signaling. In partial hepatectomy or injury,37 mechanisms regulating hepato￾cyte proliferation rely on MAPK activation by growth factors, but fetal hepatocytes may proliferate in absence of exogenous factors with a constitutive level of MAPK ac￾tivation.38 Indeed, cyclin D1 is sufficient to promote pro￾gression of hepatocytes through G1 restriction point.39 As has been previously reported,13,40 cyclin D1 is up-regu￾lated during liver proliferation (E17–P2) and almost dis￾appears in the quiescent organ; expression of cyclins D2 and D3 followed that of cyclin D1 (Fig. 5A). However, cyclins D2 and D3 would have a lower contribution to adult liver proliferation, as shown during regeneration41 and here after T3 treatment. ERK activation paralleled cyclin D1 expression while p38 MAPK activation followed an inverse pattern (Fig. 5A and B). Similarly, p38 activity was reported inverse to cyclin D1 content in liver regeneration,13 and transfection with p38-activating kinase MKK6 arrests hepatocyte growth.15 In addition, P2 hepatocytes markedly expressed ERK1/2 upstream kinase phospho–MAPK kinases 1/2 and P90 hepatocytes did not, while phospho– c-Jun N￾terminal kinase (a stress-activated kinase related to liver proliferation and apoptosis) was only detectable in the quiescent phenotypes. As referred,42 Phospho– c-Jun N￾terminal kinase increase may express both the late increase of oxidant levels and ERK decrease (Fig. 5B). Total MAPKs were not modified during development. Liver cyclin D1 and phospho-ERKs were inverse to in vivo NO-dependent [H2O2]ss and H2O2 index; instead, this correlation was positive for p38 MAPK (Fig. 5C). Consequently, a high ERK/p38 activity ratio was repre￾sentative of proliferating phenotypes; a low ERK/p38 ra￾tio was representative of quiescent ones and was related to [H2O2]ss and liver growth. ERK/p38 ratio was considered a determinant of growth and dormancy in human cancer cells; Aguirre-Ghiso et al. showed that modulation of ERK/p38 activity by pharmacological and genetic inter￾ventions predicts the in vivo behavior in 90% of the examined cell lines. 43 The interplay between redox status, signaling, and pro￾liferation was analyzed by exposing P2 hepatocytes to H2O2, antimycin, and NAC, and/or to the ERK inhibitor U0126, p38 inhibitor SB202190, and p38 activator ani￾somycin. Exposure to H2O2 or to anisomycin markedly reduced cyclin D1 expression, leading P2 cells to the adult liver level. In these conditions, endogenous or exogenous H2O2 decreased ERK/p38 activity ratio, an effect equally prevented by NAC or SB202190 (Fig. 6A and B). Simi￾larly, a marked reduction of ERK/p38 activity ratio by anisomycin or U0126 was clearly associated with the low￾est hepatocyte proliferation rate (Fig. 6B and C). Like￾wise, SB202190 significantly increased cell proliferation by approximately 25%, and SB202190 or NAC also pre￾vented the lowering of the proliferation rate induced by Fig. 6. Redox modulation of liver cell signaling. P2 hepatocytes were exposed to H2O2 alone or with 10 M SB202190 or 0.1 mM NAC (both supplemented 2 hours before H2O2), 200 ng/mL anisomycin, 2 M antimycin, and 10 M U0126. (A) Cyclin D1 and MAPKs were detected at 2 hours and 15 minutes, respectively, by immunoblotting (50 g/ lane) in triplicate. (B) P-ERK/P-p38 MAPK ratio (densitometric measure￾ments from three separate experiments) of the different assays is expressed. (C) P2 hepatocytes were incubated in similar conditions as those described in Fig. 4. *Different from basal condition. #Different from respective H2O2 concentration. P  .05. P, postnatal; ERK, extracellular signal–regulated kinase; A, anisomycin; AA, antimycin; SB, SB202190; NAC, N-acetyl-cysteine; MAPK, mitogen-activated protein kinase. 164 CARRERAS ET AL. HEPATOLOGY, July 2004

HEPATOLOGY,VoL 40,No.1.2004 CARRERAS ET AL 165 H (p.05:Fig.6B ind C).These dara agree with 12.Hung P,Feng L.Cliham EA.Keating MI.Pharkert W.Supereaid: previous reports that showed that mitochondrial reactive dimue a target St the kctive kilintg of cacer cdh.Nature 2000: 40-50-别5. oxygen spccies initiate phosphorylation of p38 in cardio- 13.Awad MM,Gnppua 2A.Cal cpde coneal daring.linee dewdoprmt in myocytess and thar low-level oxidtive stress acrivates the m rridener indisting a sak for rrdlis Dl pouttraucristioral sepu p38 and leads to growth arrest in U926 cells,an effect that hien-ClGm山Df2止ll:1-4 s inhtibited by NAC.s 14.Lavpi:IN.L'Alkrais G.Brne:A.Malkr B.Peuobgur J.Crdin Dl peoua u retled potich bythe pitipiuard repeivel by the The present resulrs suggest that synchronized increase P/HOGAu.Bid Chem 1936:271-20808-20616 of mitochondrial activities,mrNOS,and (Hl oper 1i.Awad MM,Eraken H,Barian JM.Dnie Rf,Crappato PA.Grwth rg- ates on the halance ofliver signaling pathways todrive the lon ip miga-activprecis kincindrrdepingve.]Biel ham2t士i:/16-%21. transirion from proliferation to quiescence.In the same way,we recently repored that a critical reduction of mt cuned by lov kvd ac dirive stre.Rdl Cher 200-279:1914416. NOS activity and [HO contribute to tumoral persis- tent ("cmbryonic")bchaviot.Further studics could C山hl4dl28e2L.1326- confirm the contribution of mitochondrial redox signal- L1以 ing to the modulation of cyclins D2 and D3,which par- 18.Pervin S,Singa R,Chaudhuri G.Nitie coide-indced and col allds cydin D1 espresion a developmental rodox cr ocll ln:(MDMMB-231): ml:of cvln DL.Piec Narl Acad 5ei U5 A 201:. condirions (Fig 5A and C)and may play a central role in 19.Tarner FC Maier P.Gooutert H.Chanpien C.Nabel EG.Ltocher TF liver development. f Aokmoaledgment The authors thank R Gireco for fron.Crauatae !UO:I0I:1991-199. her secresarial assistance.Dr.M.Barbosa for flow cyrom- etry analysis.Dr.L.Schreier and Dr.P.Argibay for tech E tret.Facc Bad Biol Med: 4-1 nical assisrance,and Dr.F.Cadenas for hdpful 21.Pibiri M,Ledda-Colunbaso GM.Cauu C.Sirbub G.Manegani M. comments. :T0.EsEB】20:I0K6-l01生. References 22.Podes II,Lideno C.Schipfer F,Dcho N,Canea MC.Cadeaa E,e: hendrid asra uptak:by redro reactiom 1.Fao N.Canpbel J5.The rule d hepytos and oval odlb is live rncration ad rpopalurion.Mech Devdopnent 2003:120:117-50. J7716 2.Breris A.Podeuse J.Realaion ol otygen metabelim by sitti toide 21.Herera B,Alaxz AM 5ancher A,Farzinder M,Kancee C,Berit M. al.acygen specins (RO)the rhachondrial-depn hademi:Preu,J0o0955-65. 3.Carreru MC.Peral K Correno DP.Fitocchigto PV.Rebariati 1. 5,FA5EB1201:15741-1 Cire Phrviol 200121:H2252-H2285. Jide saiaing enl How cytometry J lnnnadl Metlock 1991 139:271 4.Poderoe J.Carern MC.Lixdese C.Riobo N.Schopfer F.Beveri A. ductian n rat heat mitochendria ad abrritchoniral partickes Arch ing fres radical reactient.I.Radicale geaerated by the irperacrion af Bochamn Biephra 199628:5-2. dria.J Bil Chen 199273:11038-11043. 6.u6nf,Richeer C.Nitic随id:eymth山ei可in mim四da chenical aabdr.Now Yerk Irrcrockncr,114:405-414. e5L174131-29张 Katai Al.Pearte 1 Cemest ?Bisde:LVa Biblar M.Chai 5,al. Iderrification of a aesmal nitric odde syuthase in lslaned candlac miro temedlabin-fee riad or Iwr.Blal Chera 1974225X45-46 handri uring dertmcheraicul detocrion.Pooe NaAcadSe USA2UUL: 16126-14151. aride cynchane.I Rid Chom 2812:277-5500-1015 cymx in mr liver mbochandru.Fhyed Baheraode 1977:25325-554. mrgy espendine in rar cold acclmatin Ane J Phyddl Hor Ciu d encgrtescJ山rar学Hm1395773355-367 hrl21国：2421-H255 31.Simoeet H.Alacrd N.Pleiffer K.Gallou C.Bevroud C Dertont J. 10.Lao 2.Padd,M J,Zhang )Rap-pboe N,.Mi- lochendrid nitri oide syuthae i corsituvdy active and i lundiondly e山ndd in hypasin.Free Had Hial Mnd20 11:t6o-16l人， 759-715. 11.Riobo NA Mdlaei M.Sarjuin N.Fwemtm Ml.Gitwicle MC.Ceten 52.Dyenegi K.Naker K.Sogree H,'Tseluaki H.Miseni R.Nikao'T. MCet al The modlatioa of rhechrndrial aitric-odde symthaoe acriviry A cmdy of uea-onhedaing enaymes ia pimaal ard human i w lin deedepme.J llil Chen 2002 277-1291742235 Eve.Pafalr Ke 1580 14 236-2411

H2O2 (p  .05; Fig. 6B and C). These data agree with previous reports that showed that mitochondrial reactive oxygen species initiate phosphorylation of p38 in cardio￾myocytes16 and that low-level oxidative stress activates p38 and leads to growth arrest in U926 cells, an effect that is inhibited by NAC.15 The present results suggest that synchronized increase of mitochondrial activities, mtNOS, and [H2O2]ss oper￾ates on the balance of liver signaling pathways to drive the transition from proliferation to quiescence. In the same way, we recently reported that a critical reduction of mt￾NOS activity and [H2O2]ss contribute to tumoral persis￾tent (“embryonic”) behavior.44 Further studies could confirm the contribution of mitochondrial redox signal￾ing to the modulation of cyclins D2 and D3, which par￾allels cyclin D1 expression at developmental redox conditions (Fig. 5A and C) and may play a central role in liver development. Acknowledgment: The authors thank R. 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166 CARRERAS ET AL HEPATOLOGY.Juy 2009 33.Taraka K.Hridk T.Marashi T.Chatpo is comn o clan and ard liver growth m.Catcer Ra 2001:61:5564- a中n in ra:lier duing prowth句ol Cha坠n3dN度 50 1w517357-3. 51.Thunsikal V1.Fmburg BL Hestineoty nie inodlsi和ding AmJ toprltes theesprewienol cplis Dduing Ever desdepnet.J Bidl Chon Mrpiol Lurg Cell Mol Phoidl 2000:279:L1005-L1028. 2002:27736167-36173 4L.Ridkh:m DG.Ncben CL Faoor IT,Tin:hako NA.Harues LK.Al pede in Babue ClrcaLMam Dl,a Corgon Gme Fapradon and brecht IH.[xffreneal negalurion of cyclns DI aad Ds n hepurcyn 36.DluM.pis M.Nimn T,B时sm民日l.Nitric动e 42.Cat MI.Lie H.WatgY.Oaidant-induopl hepitoxyie injury foort men ihibin DNAxathair and induce artiatiae of pelADP-rboiel poh- adiane i regabtd by ERK ard API igralng HDATOLOGY 200:17: a culneed rar Exp Cdl:21K-14-1K 45-1415 37.Br N.Laid AD,'bhr SM.Liwe inamak.2 Rode af powth 43.Agaime-Gihion 1A.Farad Y,lia [(lomki 1.ERKC activry ac a 银inim3ni年natE用i959152-1536 determismt of tu powth and dotanr:inglion by pgsursn 3g.Barln [M Grsppuo队Unpupling of bpatic平der wth fao R20363:l684-l65. 松c1npa-2日ot:n ki1ahh:丘 .GhS女oM,Bdae玉Carerar MC.Peder.De 16dhm1月长17337刷-0. ceaud mitechendrialaitric cde oynthas acthry ard hydrogon peraide 39.Ndvee J.Ridlein DG.Tindealo NA.Suaky MO.Allerh J1 Tramiest capieiien of crcin DI i safficknt to promnete hepmocyie 200366370-6377

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