Mitochondrialpermeabilitytransition

Mitochondrial permeability transition:a common pathway

to necrosis and apoptosis

Jae-Sung Kim,Lihua He,and John J.Lemasters *

Department of Cell and Developmental Biology,University of North Carolina at Chapel Hill,CB#7090,236Taylor Hall,

Chapel Hill,NC 27599-7090,USA

Received 10February 2003

Abstract

Opening of high conductance permeability transition pores in mitochondria initiates onset of the mitochondrial permeability transition (MPT).The MPT is a causative event,leading to necrosis and apoptosis in hepatocytes after oxidative stress,Ca 2ttoxicity,and ischemia/reperfusion.Cyclosporin A blocks opening of permeability transition pores and protects cell death after these stresses.In contrast to necrotic cell death which is a consequence of ATP depletion,ATP is required for the development of ap-optosis.Reperfusion and the return of normal pH after ischemia initiate the MPT,but the balance between ATP depletion after the MPT and ATP generation by glycolysis determines whether the fate of cells will be apoptotic or necrotic death.Thus,the MPT is a common pathway leading to both necrotic and apoptotic cell death after ischemia/reperfusion.ó2003Elsevier Science (USA).All rights reserved.

Keywords:Mitochondrial permeability transition;Hepatocytes;Ischemia/Reperfusion;Necrosis;Apoptosis

Since Hunter et al.[1]?rst characterized the mito-chondrial permeability transition (MPT),onset of the MPT has been implicated as a key mechanism under-lying both necrotic and apoptotic cell death.Opening of high conductance permeability transition (PT)pores in the mitochondrial inner membrane initiates the MPT.Ca 2t,inorganic phosphate,alkaline pH,and reactive oxygen species (ROS)are a few of the many agents that promote the MPT,whereas the immunosuppressive drug,cyclosporin A (CsA),Mg 2t,acidic pH,and phospholipase inhibitors,including tri?uoperazine,dibucaine,and quinacrine,block opening of the PT pores [2].As a consequence of PT pore opening,solutes with molecular mass of up to 1500Da nonselectively di?use across the mitochondrial inner membrane,lead-ing to mitochondrial depolarization,uncoupling of ox-idative phosphorylation,and large amplitude swelling,which in turn can lead to ATP depletion and cell death.Patch clamp experiments demonstrate that opening of

just one PT pore may be su?cient to cause mitochon-drial depolarization and swelling [3].

Regulated and unregulated opening of permeability tran-sition pores

Accumulating evidence suggests that PT pores have two open conductance modes —regulated and unregu-lated [4].Regulated PT pore opening requires one or another of several chemical inducers and the loading of Ca 2tinto the matrix space.The regulated MPT is the classical MPT that is inhibited by Ca 2tchelators,Mg 2t,and CsA.Although CsA is an immunosuppressive agent that inhibits calcineurin to produce immune suppres-sion,nonimmunosuppressive cyclosporin analogs,such as N -methylvaline cyclosporin and NIM811,block the MPT but do not inhibit calcineurin,whereas other im-munosuppressive agents like tacrolimus inhibit calci-neurin but do not block the MPT [5,6].Thus,the ability of CsA and its analogs to block the MPT is unrelated to their immunosuppressive activity.Unregulated PT pore opening occurs with high concentrations of

various

Biochemical and Biophysical Research Communications 304(2003)463–470

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BBRC

*

Corresponding author.Fax:1-919-966-7197.

E-mail address:lemaster@https://www.sodocs.net/doc/575590842.html, (J.J.Lemasters).

0006-291X/03/$-see front matter ó2003Elsevier Science (USA).All rights reserved.doi:10.1016/S0006-291X(03)00618-1

chemical inducers.Although the size of the regulated and unregulated PT pores is similar,the unregulated pore does not require Ca 2tfor conductance and is not blocked by Ca 2tchelators,Mg 2t,or cyclosporin A [4].

Models of permeability transition pore structure The molecular composition of PT pores remains un-certain.The dominant hypothesis is that the PT pore spans the mitochondrial inner and outer membranes and is composed,in part,of proteins from both membranes and the matrix (Fig.1A).Inhibitor and reconstitution studies implicate that the adenine nucleotide transloca-tor (ANT),located in the mitochondrial inner mem-brane,is one essential component of the PT pore [7,8].Atractyloside,which binds ANT from the cytosolic side,induces the MPT,whereas bongkrekic acid,which binds from the matrix side,blocks the MPT [7].Other proteins proposed to be part of the PT pore complex include the voltage-dependent anion channel (VDAC)of the outer

membrane and cyclophilin D from the matrix [9,10].VDAC has the important role of making the outer membrane permeable to metabolites that must move in and out of mitochondria,such as ATP,ADP,and re-spiratory substrates.Cyclophilin D is a peptidyl prolyl cis –trans isomerase in the matrix space that appears to associate with the PT pore complex to confer CsA sen-sitivity.Other proteins forming the PT pore may include hexokinase,creatine kinase,the peripheral benzodiaze-pine receptor,and proapoptotic proteins like Bax [11,12].Most agree that the PT pore complex must re-side,at least in part,at contact sites between the inner and outer membranes described by Hackenbrock [13].This prevailing model of pore structure (Fig.1A)does not readily explain the existence of the regulated and unregulated modes of PT pore conductance.Moreover,although PT pore-like conductance can be reconstituted with ANT alone or in combination with other proteins [8,9,12],PT pore conductance and onset of the MPT are reported in triple ANT knockout yeast [14].In addition,unspeci?c high conductance pores can be induced in the absence of ANT by other puri?ed mitochondrial carrier proteins such as the aspartate/glutamate and phosphate carriers [15,16].Small exoge-nous pore-forming amphipathic peptides like mito-chondrial targeting peptides,alamethicin and mastoparan,also induce onset of a Ca 2t-dependent and CsA-sensitive regulated MPT at low concentrations and an unregulated MPT at higher concentrations [4,17,18].To explain these discrepancies,we recently proposed a new model of PT pore formation and regulation (Fig.1B)[4].A common feature of many of the agents that induce the MPT,such an ROS,oxidants,and thiol re-active agents,is the ability to attack and modify mem-brane proteins.We proposed therefore that misfolding of native mitochondrial membrane proteins resulting from this chemical attack leads to exposure of hydro-philic residues in lipid layer phase of the membrane.As a consequence,the misfolded proteins aggregate at their hydrophilic surfaces to form aqueous channels that conduct low molecular mass solutes.Initially,chaper-one-like proteins regulate solute conductance by these misfolded protein aggregates to confer the properties of regulated PT pores.One of these chaperones is cyclo-philin D,which is consistent with the activity of cyclo-philin D as a peptidyl prolyl cis –trans isomerase,namely a protein foldase.Cyclophilin D binding gives sensitivity to CsA and likely Ca 2tas well.Under physiological conditions,the complex of chaperones and misfolded membrane proteins remains closed until matrix Ca 2trises substantially.In this way,the mitochondrion pro-tects itself against the devastating consequences of aqueous channels created by misfolded protein clusters that would otherwise cause mitochondrial uncoupling and large amplitude swelling.Thus,proteins like cyclophilin D are not promoting

mitochondrial

Fig.1.Proposed models of the structure of permeability transition pores.(A)The consensus model of the PT pore comprises a voltage-dependent anion channel (VDAC)from the outer membrane,adenine nucleotide translocator (ANT)from the inner membrane,cyclophilin D (CypD)from the matrix,and other proteins such as peripheral benzodiazepine receptor (PBR),hexokinase (HK),and creatine kinase (CK).(B)A new model proposes PT pore formation by misfolding and clustering of mitochondrial membrane proteins and regulation by chaperones.See text for details.Adapted from [4].

464J.-S.Kim et al./Biochemical and Biophysical Research Communications 304(2003)463–470

permeabilization but are defending against it,which is consistent with recent observations that cyclophilin D overexpressing cells are much less sensitive to oxidative stress and mitochondrial dysfunction[19].The identity of the other chaperones involved in the formation of regulated PT pores is unknown,but one candidate is Hsp25(murine analog of human Hsp27)whose expression after heat shock is associated with resistance to onset of both the regulated and unregulated MPT[20].

Chaperone and cyclophilin D binding to misfolded proteins create regulated PT pores,but as the number of misfolded protein aggregates in the membrane exceeds the number of chaperones to regulate them,unregulated pore opening occurs(Fig.1B).Conductance through such unregulated pores occurs independently of Ca2tactivation and is not inhibited by CsA.Although this new model is speculative,it explains a number of puz-zling features of PT pore opening that older models do not account for,including the ability of completely ex-ogenous amphipathic peptides to create regulated and unregulated PT pores.

Cellular changes in necrotic cell death

Hepatic metabolism and tissue viability are highly oxygen-dependent.Oxygen consumption of the liver is 100–150l mol O2/h/g of wet weight.As a consequence, hypoxia,the relative but not necessarily absolute lack of oxygen,develops rapidly when hepatic blood?ow is impaired.Hypoxia rapidly progresses to an absolute anoxia as residual oxygen is consumed by tissue mito-chondria,especially in the pericentral regions of the liver lobule,which are furthest removed from the in?ow of oxygenated blood.

During anoxia,hepatocytes respond quickly by forming blebs of plasma membrane on the cell surface that can project into the sinusoidal lumen.These blebs are a consequence of ATP depletion and the consequent cytoskeletal alterations and loss of volume control.Bleb formation can rapidly reverse after reoxygenation,but after prolonged anoxia irreversible injury develops.Just prior to cell death,hepatocytes and other cells develop a metastable state,characterized by mitochondrial per-meabilization,lysosomal disruption,bleb coalescence, and growth,cell swelling,and leakage of small molec-ular mass anionic solutes[21].Opening of glycine-sen-sitive anion channels that conduct chloride and other anions,including a variety of anionic?uorophores, initiates the metastable state,whereas bursting of the plasma membrane abruptly ends the metastable period and causes onset of necrotic cell death[22].When the permeability barrier of the plasma membranes is broken,cells release a variety of cytosolic enzymes, including lactate dehydrogenase,alanine aminotrans-ferase,and aspartate aminotransferase,into the extra-cellular medium.Simultaneously,the cells take up supravital dyes like trypan blue or propidium iodide that are excluded by viable cells.

pH dependency of ischemia/reperfusion injury Ischemia results from interruption of blood supply to a tissue.Key events during no-?ow ischemia include anoxia,exhaustion of glycolytic substrates,depletion of ATP,and tissue acidosis.Acidosis develops as a con-sequence of the hydrolysis of high energy phosphates, the accumulation of lactic acid from anaerobic metab-olism,and the release of protons sequestered in acidic organelles like lysosomes and endosomes[23,24].The naturally occurring acidosis of ischemia is highly pro-tective against hypoxic and toxic cell killing in liver, myocardium,and other tissues[25–27],but the mecha-nisms conferring protection by acidosis remain incom-pletely understood.Acidotic pH may suppress the activity of degradative enzymes during ischemia,in-cluding proteases,phospholipases,and endonucleases with a neutral pH optimum[28].Another mechanism of acidosis-dependent protection is inhibition of PT pore opening by pH<7[25].

Reperfusion of tissues leads to reoxygenation and recovery of normal physiological pH.After prolonged exposure to ischemia,reperfusion paradoxically wors-ens tissue injury and precipitates cell death.The mechanisms underlying ischemia/reperfusion injury are multifactorial and generation of ROS undoubtedly plays a role.However,the restoration of pH after is-chemia is a major and independent causative factor precipitating cell death[25,26,29–31].Indeed,restora-tion of normal extracellular pH without reoxygenation can produce virtually the same amount of cell killing as restoration of normal pH with reoxygenation,whereas reoxygenation at acidic pH eliminates virtually all cell death,enzyme release,and uptake of supravital dyes like trypan blue and propidium iodide.In the para-doxical cell killing caused by restoration of normal pH after ischemia(‘‘pH paradox’’),the MPT is a major causative event.

The mitochondrial permeability transition in necrotic cell death after ischemia/reperfusion

Acidotic pH and CsA block PT pore opening.Simi-larly,reperfusion with either acidotic pH or CsA pre-vents reperfusion-induced necrotic cell death in models of ischemia/reperfusion to cultured hepatocytes[25]. However,protection against cell death by CsA does not necessarily implicate involvement of the MPT since CsA also inhibits the Ca2t-dependent phosphatase,

J.-S.Kim et al./Biochemical and Biophysical Research Communications304(2003)463–470465

calcineurin [5].That reperfusion actually induces the MPT is con?rmed by confocal microscopic studies [25].Calcein is a green-?uorescing ?uorophore that can be ester-loaded into the cytosol of hepatocytes.Because calcein is a polyanionic solute of 623Da,it does not cross the mitochondrial inner membrane to enter the matrix space.As a result,confocal images of calcein-loaded hepatocytes have a honeycomb appearance with numerous dark round voids corresponding to individual mitochondria.Co-loading experiments with tetrameth-ylrhodamine methylester (TMRM),a red-?uorescing cationic ?uorophore that accumulates into mitochon-dria in response to the negative-inside mitochondrial membrane potential,con?rm that virtually every dark void is a polarized mitochondrion.After prolonged ex-posure of hepatocytes to simulated ischemia (anoxia at acidotic pH),mitochondria depolarize and release TMRM but do not become permeable to calcein,as shown by the persistence of voids in calcein images (Fig.2).However,after conditions simulating reperfusion (reoxygenation at normal pH),mitochondria transiently repolarize but then depolarize within the ?rst several minutes of reperfusion.Concomitant with depolariza-tion is redistribution of calcein into the mitochondria matrix,causing the voids and honeycomb appearance of calcein images to disappear.Subsequently,cell viability is lost as shown by release of cytosolic calcein (Fig.2)and nuclear staining with propidium iodide (not shown).Moreover,when cells are reoxygenated at pH 6.2or at pH 7.4in the presence of CsA,mitochondrial repolari-zation persists after reperfusion,mitochondrial voids in the calcein ?uorescence do not disappear and loss of cell viability is prevented [25].These observations show di-rectly that reperfusion is causing mitochondrial inner membrane permeabilization and depolarization fol-lowed by cell death that is blocked by low pH and CsA.

Thus,onset of the MPT is a key mechanism underlying pH-dependent necrosis after ischemia/reperfusion to

hepatocytes.

Fig.2.Onset of the MPT after ischemia/reperfusion in hepatocytes.See text for details.Adapted from

[25].

Fig.3.Scheme of intracellular changes leading to onset of the MPT after exposure of hepatocytes to t -BuOOH.Hepatocytes were incu-bated with t -BuOOH.Intracellular changes were then monitored by confocal microscopy.Oxidation of NAD(P)H,Ca 2tconcentration,and ROS formation were monitored with auto?uorescence,Rhod-2,and dichloro?uorescein,respectively.Onset of the MPT was evaluated with calcein/TMRM and inhibited by cyclosporin (CsA)plus tri?uo-perazine (TFZ).t -BuOOH causes mitochondrial NAD(P)H oxidation,which is inhibited by b -hydroxybutylate (BHB).Increased mitochon-drial Ca 2tstimulates intramitochondrial ROS generation that in turn initiates the MPT.Onset of the MPT then depolarizes the mitochon-dria,leading to necrotic or apoptotic cell death.Mitochondrial Ca 2tchelator (BAPTA)and antioxidants such as diphenylphenylene di-amine (DPPD)and desferal prevent events upstream to opening of PT pores.Adapted from [32,33].

466J.-S.Kim et al./Biochemical and Biophysical Research Communications 304(2003)463–470

The cellular alterations leading to the MPT after is-chemia/reperfusion remain incompletely understood. The MPT plays a major role in tert-butyl hydroperoxide (t-BuOOH)-induced necrosis in hepatocytes[32,33]. t-BuOOH initiates a series of mitochondrial alterations that culminate in necrotic cell death:oxidation of pyri-dine nucleotides(NADH and NADPH),elevation of mitochondrial Ca2t,and stimulation of mitochondrial formation of ROS to promote onset of the MPT,mi-tochondrial depolarization,ATP depletion,and cell death(Fig.3).A similar sequence of events may con-tribute to pH-dependent necrotic cell death after reper-fusion,since intramitochondrial Ca2tincreases shortly after reperfusion,an event which is followed by in-creased ROS formation,onset of the MPT,and cell death[34].

Mitochondrial permeability transition in apoptotic cell death after ischemia/reperfusion

Necrotic cell death,also called oncosis or oncotic ne-crosis[35],is widely considered to be a distinct entity from apoptosis or programmed cell death.In contrast to ne-crosis,apoptosis is essential for immune surveillance and regulation of tissue mass.As such,apoptotic cell death is tightly regulated by a variety of signals.Characteristics of apoptosis include cell shrinkage,activation of caspases, DNA cleavage,chromatin condensation,and nuclear fragmentation.Despite the di?erences between apoptotic and necrotic cell death,mitochondria can also play a critical role in the development of apoptotic cell death by releasing proapoptotic proteins,such as cytochrome c, apoptosis-inducing factor(AIF),and Diablo-SMAC, that are normally sequestered in the space between the inner and outer membranes[36,37].In particular,cyto-chrome c triggers caspase activation and a cascade of changes that execute apoptosis[38].Two distinct mech-anisms are proposed to explain the release of these proapoptotic factors.The?rst is the formation of speci?c release channels in the outer membrane promoted by proapoptotic bcl-2family members like Bax,Bad,Bak, Bid,and others[39].The other mechanism is outer membrane rupture following large amplitude mitochon-drial swelling due to onset of the MPT,as originally proposed by Kroemer and co-workers[40].Here,we will con?ne our discussion to the role of the latter mechanism.

Although ischemia/reperfusion is usually associated with necrotic cell death,more recent studies suggest that apoptotic death can also occur after ischemia/reperfu-sion in cells from liver,heart,and other tissues[41–44]. The question thus arises as to how ischemia/reperfusion can give rise to both forms of cell death.New data suggest that the MPT is the common factor in ischemia/ reperfusion that initiates both apoptotic and necrotic cell killing[45].ATP supply and the MPT in the switch from necrotic to apoptotic cell death

Cytochrome c released from mitochondria forms a so-called apoptosome complex with apoptosis protease activating factor-1(APAF-1),procaspase9,and ATP (or the less abundant dATP)[38].In an ATP or dATP-dependent fashion,procaspase9is activated to caspase 9,which then activates caspase3to initiate the?nal executioner phase of apoptosis.Consistent with the ATP dependence of caspase9activation,cellular apoptosis in many systems requires ATP[46–48].When ATP is de-pleted,apoptosis is blocked.Instead the same upstream proapoptotic signals induce a form of necrotic cell death.

Similarly,ATP supply can mediate a switch from necrosis to apoptosis.Reperfusion of ischemic hepato-cytes in the presence of fructose,a substrate for glyco-lytic ATP formation,and glycine,a membrane stabilizing amino acid,prevents MPT-dependent ne-crotic cell killing.Instead,apoptosis develops within8h of reperfusion,as shown by nuclear changes and TU-NEL staining(Fig.4).This apoptosis is caspase-de-pendent since pancaspase and caspase3(but not by caspase8)inhibition blocks reperfusion-induced apoptosis(Fig.4and data not shown).In addition,CsA, the MPT inhibitor,also blocks reperfusion-induced apoptosis,whereas tacrolimus,an immunosuppressing agent that does not block the MPT,has no

e?ect Fig.4.Cyclosporin A and caspase inhibitors prevent apoptosis after ischemia/reperfusion in hepatocytes.Hepatocytes were reoxygenated in the presence of fructose and glycine after4h of simulated ischemia. Cells were then treated with no addition,CsA(MPT blocker),ta-crolimus(non-MPT-blocking immunosuppressant),or DEVD(cas-pase3inhibitor)beginning at20min before and then continuously after reperfusion.TUNEL staining was performed to evaluate devel-opment of apoptosis.Note prominent TUNEL staining after reper-fusion,which was blocked by CsA and DEVD,but not by tacrolimus. Thus,reperfusion of ischemic hepatocytes in the presence of glycolytic energy substrate causes MPT-and caspase-dependent development of apoptosis.Adapted from[45].

J.-S.Kim et al./Biochemical and Biophysical Research Communications304(2003)463–470467

(Fig.4).The data support the conclusion that onset of the MPT is a causative factor inducing apoptosis after ischemia/reperfusion through a caspase3-depen-dent pathway.

Impaired mitochondrial ATP formation is the key stress of anoxic and ischemic injury,and necrotic cell death after ischemia/reperfusion is directly linked to ATP depletion[25,49].In anoxia,anaerobic glycolysis is the only major pathway of ATP generation available to cells.However,glycolysis can only restore cellular ATP levels to at most about50%of aerobic levels.None-theless,recovery of15–20%of normal ATP content is su?cient to prevent anoxic cell killing[45].By contrast, glycine does not support signi?cant ATP regeneration and acts downstream of ATP depletion by stabilizing the plasma membranes in a variety of cell types [22,25,50].Glycine thus prevents hepatocellular necrosis of hepatocytes after reperfusion,but the amino acid does not prevent ATP depletion.How glycine stabilizes plasma membranes and suppresses anoxic cell death in hepatocytes remains under study.One proposal is that glycine inhibits an unselective organic anion channel that opens as an penultimate event leading to necrotic cell death[22,51](see above).Together,fructose and glycine prevent reperfusion-induced necrotic killing and instead promote apoptosis.Individually,fructose and glycine also prevent necrotic cell killing,but only ATP-generating protection with fructose leads to the sub-sequent development of apoptosis.These?ndings are consistent with the conclusion that ATP regeneration is essential for MPT-dependent apoptosis to develop after reperfusion,since ATP recovery of ischemic hepatocytes after reperfusion with fructose is much greater than after reperfusion with glycine.

Fig.5illustrates the role of the MPT in initiating both necrosis and apoptosis after reperfusion.If onset of the MPT is widespread and involves most mitochondria in a cell and if glycolytic sources of ATP are unavailable,the cell becomes profoundly ATP depleted,which leads to glycine-sensitive plasma membrane failure and necrotic cell death.By contrast,if the MPT does not involve all mitochondria within a cell or progresses slowly as may occur after less severe ischemia,then ATP levels may recover,at least in part,after reperfusion,especially if glycolytic substrate is abundant.Partial ATP recovery prevents necrotic cell killing.Instead,apoptosis develops triggered by ATP-dependent caspase activation from cytochrome c released by mitochondria undergoing the MPT.In this way,ATP-regenerating glycolytic sub-strates convert cell killing from MPT-dependent necro-sis to MPT-dependent apoptosis.Even after initiation of apoptosis,necrosis may supervene if ATP levels subse-quently fall.This event then produces a pattern of secondary necrosis that is often observed in patho-physiological settings and in cultured cells(Fig.5).In-terconversion between apoptotic and necrotic cell death induced by Ca2tionophore in hepatocytes is similarly controlled by the availability of ATP[52]. Conclusion

Controversies are still ongoing as to whether ische-mia/reperfusion injury to liver causes predominantly apoptotic or necrotic cell death[41,44].However,the controversy becomes moot upon the realization that apoptosis and necrosis are not distinct and independent entities.Shared pathways give rise to both patterns of cell death,a phenomenon we term‘‘necrapoptosis’’[53]. In necrapoptosis,common pathways,such as the MPT, initiate a series of events that culminate in either apop-tosis or necrosis,depending on other variables,such as ATP supply(Fig.5).Necrapoptosis accounts for the apparent switching between modes of cell death and for the frequent co-existence of both apoptotic and necrotic patterns of cellular damage,especially in pathophysio-logic settings such as ischemia/reperfusion.In necra-poptosis,apoptosis and necrosis represent extremes on a continuum.

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