Document yk4ZjRbG2zdmzJYR6DLmQjbkD

J AR2G-0 IF THE EFFECT OF PERFLUORINATED ARYLALKYLSULFONAMIDES ON BIOENERGETICS OF RAT LIVER MITOCHONDRIA Kendall B. Wallace and Anatoli Starkov DepatrtwmetntorfBiiomchesmitstary SanodoMtolaefcduloarnBisology Duluth, MN 55812, USA. Key words: Mitochondria, Membranepotential, Respiration, Uncoupling, Detergents. `Supported by a grant from The 3M Company. 04150 MATERIALSAND METHODS Kew ms "The isolofamittocihonodrnia Mitochwoernedisroliataed fiom Iver of cumlalt Spagae-Dawley rts (-200 bodyweighbtya) convifteeennilcteniifgaoionneoacedlur,Animalswerekilledbydecapitation, Liverwasexcisedand weiagndhcoeolded in 40 ml ofisolation medium (210 mM manitl 10 mM sucrose, Sm HEPES-KOH Gi 7.4), 1mMEGTA, Cooledliverwasmincweitdh sciansdwsashoedrvisce with20mlofsolaton medium, then itedwiththesamemediumandhomogenizedfor 1minwithamotor-drivenPoterhomogenizer(Teflonpestle gasbeaker),Thetissue mediumratiowas 1:8 (gm).Thehomwaosfergedtehrounghgtauzeeand cecnetnrtirfiufguagteidofnoart101m0,i0n0x7x0100gm,in#.T=h4epC,laendwtahsermeiutsopcheonnddardiailn p1e0llmeltowfawsarsehcionvgemreeddfiruommt2h1e0sumpeMrnmaatnasnittbly, 10mMsucrose, mMHEPES-KOH,pH7.4)supplemented withbovineserumalbumin(BSA, 1 mg xml"). The suspensionofmitoshondiawasdied10 35mlwiththe sammediumwithout BSA,andcetifgedat 10000 x 10min.Thefinalmitochondrialpelletwasresuspendedinwashingmedium10 a proteinconcentration of 70-80 `mg xmI"andstoredonice. Measurements. Mitochondrial membrane potential (A) was estimated from TPP" ion distribution measured with a TPP.seletivelectrode onsructedaccording toKamo ota, 197. Mitochondiamembranepoteaial was cal Thacesrdu aetsecl roifba edet glesenewchoed nrseu(mRpottieonnbberygm,i1t9o8c4ho)n. driawas messurodwith ahand-madeClark ypeoxygen letrode. Boththemitochondrialmembranepotential an therespiration stewere recordedsimultaneously sing `multichannelincubation chamberequippedwith a magneticstirrer.Thevolumeofthechamberwas 1.8ml. All experiments were performedatroom temperature (25 C).TheTPP -sensitive electrodewas calibratbeyd `sequential additionsofknownamountsof TPP*CIbeforetheadditionofmitochondria(sceFig.1).Therespiration `rateswerecalculatedassumingtheinitialoxygenconcentrationtobeequal 0240 WM. The mitochondrialmembranepotentials wereslightly (~15 %) underesatnidtmheaotxyegedn `consumptionrateswereoverestimatedbecausenocorrectionwasmadeforlowerTPP"bindingconstants andfor lohwiesriesquuillyisbriisuamccotnrceynbtercataiuosneothfedivsaslouelsveadeoxuysgeenicnotmhpeahrieghtihoeneiffcescttrseonfgtihmfedriucmoumspeoduinndosuurnedxeprerhiemseanmtse. conditionsratherthanfo energetic calcltins. Proteinconcenratonwasdeterminedbythe Bradfordasa. Bovineserumalbwuasumseid35n3 standardAdditions.PFcompoundswere dissolvedinabsolute thanol (fo exceptfor PF143 whichwsdisalved indeionisedwater).Preliminarystudy revealedtha allPFcompoundsareveryhydrophobicand tend to prcipiate inou incubationmedium.Du oti, a stof ition wasmadeforeachPFcompound10 bainan amayofconcentrations startingfrom 100 uM downto6.25 iM.Dilutions weremadebyadding avolumeof ? 04151 KBW 2498 absoluteethanol {0.2volumeofPFcompoundstock solution. PFcompoundswereaddedtomitochondraisa 1.8 ul voluomfea solouftdeisiroabnle initalPFconcentration.Thisapproachallowedustoobtainsatisfactory reproducible results. `Reagents. Mannitol was from Aldrich, sucrose UraPure from ICN, allotherreagentswere from Sigma. Bovineserum albuminwasessentially fayacidfre. 3 04152 RES ANU DDIL SCUT SSIS ON KBW 21498 1. The effects of PF compounds on oxidative phosphorylation in rat liver mitochondria compoBuencdsa,usaebtuhfiesreidnhviigthrositcusdryewnagshapiomtaesdstioumrcehvleoarlidtehmeepdotieunmtiraelsiemnbvliivngotceolxliacprrcoypteorptliaessmomfilaiseeutwoasf chosen. Bythesamereason,glutamateplusmalatewereusedasmitochondrialrespiratorysubstrates. The cooxmidpaotuionndso,fCogmlpulteamxatIeofpmliutsocmhaolnadtreiailnrveoslpviersatotrhye cmhoasitn,avsuwlneelrlabalsealolbolitghaetrorreyspcioramtpolreyexnzfoyrmehsy.dWroephaolbsioc developed aspecialassay procedure. 1.allowsobtainingdetailed dataontheeffects of 3compoundon mitochondrialenergetics.Theprocedurealsoallows ocompar in auniversalwayth efficiency of various `compoundsaffectingtheenergyproduction inmitochondria hedesignFiogf 1 explains in or SRTE T "Thedpeicsuroefshoewxspetryipimceanl || | per b N\A r EeoeR smsacTomocaSenss Gan obained by | | Tp [2 rgSnwr simultancous recording of mitochondrial respiration and AY. The first addition ointcnrevaspipinnraescpisrattisonmeatne | ofmitochondria whichwas ! op w{] 7e -- ' f [1o oe Pl \ Pol i IX sna Sain tae 6oE R0s4a2aI R veoeers , S eToaore. `paralleledby adropin AY. no indcmes that mitochondria teed s0- called metabolic State 3, that is they phosphonate ADP for the expense of wTo! et | | /310 i oo ol of Ti \ ba Dol am respiratory substrates. his "Thevaluesofrespirationraeduring(Va) andaferphosphorylation (Vi wereusedtocalculaterespiratory `controlindex (RCI)equal 10theratioof Voto Vr. Theamountofoxygen consumedbymitochondriaduring phospwh aso user d toycal lculaatet ADi P:Ooratnio. Afrthephosphorylationof ADPwascompleted,therte ofrespiration decreased andAY in mitochondriaspontaneouslyrestoredindicatingtheonsetof State4. Theaddion of acompound (PFI2M in Fig.)tomitochwaosfnoldloeweidbaythesecondadditionofADP,andsecond RCI (RClcompounadn)d ADP.O (ADP:0-<ompound) roswerecalculated.AflertheonsetofsecondState 4,an uncouplerwsaddedat * 04153 8concentrationthatsimulate therespirationofmitochondria 0themaximalleveldeterminedbytKheeacwti2vitymof respiratory chain. "Tisrelativelysimp approachallowsto estimateseveral essentialpropertiesof compoundinrelationto `t(he@enienrhgiebtiitcssoorfsmiimtuolcshtoensdrthiea.reBsypitrhaetoarnyalcyhsaiisn;ofdataobtainedin suchassay,itispossible toshowif a compound (b)inhibitsthe entry of substrates intomitochondria; (c) specifically inhibits the enzymesofthe oxidative phosphorylation system; ( d)uncTouhpeleasptrhoesocxhiddaetsiroinbaonddapbhoosvpehwoaryslsaptpiloineid nm itochondria. all PF compo und s Fig 2 showsth typical ordfo PFI. Mio 5 P10 Ie be \ (MA one i A) | | oUNN Toe] N! | \\ aLll NEL fe J \ voi \A PFFiIg0.2,. 6oT2he5eeefafetActDeoFf,rP1F0o10f04on6btDehNetFr,sesp4oi0raMtt.ionsoratoeaond oArofnratleivwermPitochMondrsia. PF10 inrelativelyhigh concentrationstimulatestherespirationanddecreases A inState 4 whereashas noinfluenceundermetabolicState 3. ItaffectedneitherState 3respirationratenorADP:Oratio(Table1.) 5 04154 van m a sounsie i t er pAea st E B Eti n EE a Spe R i S aTE [Janse| mise RClgr: RC%L 71.0104 ADP:O:AgDPe:0r,%,|99.83.5 E FTC -- theState 3respir(seaeTtaibloe2n). ` 04155 owas "1 ul NY + erin - NorTone JPF10H - } oMrf od [E {ea IYNJ dl N\ MJita . Ay EI oe ~ Hys nct of 11 on 0 in rk cma 89 fr om , 04156 "Table 2. Theefct ofPFI0H on the respiration ratesof ra iver mitochondria under variKouBs W 24198 metaboli"csThaeteex.perimenialconditionsandaditons,asin Fig. AbbreviacteiToabnlse1, "Thedtof experimentsaeprsenedsmeanvalues SE. [roPF10H ns| [oJroronsweoesr| RClpn : RCL % "ADP-Ogr): ADP:0%,| 88.4 25 "ThecompoundsPF143andPF9Sexertedsmallstimulatoryeffectonmilochondrial respirationbothin State and. FOSalo lightlyincreasedRCI, ast sshown in Table 4.However, PF143inducedsmaldecrease inthe AYofmitochon(dFrigi)a.The "inicnArYienduacesdbyePF"9S(Fig 5)wasnotrelate tothemembrane potentialofmitochondriabecause itwasobservedalso in the absenceofmitochondria. Thus, PF9S apparently interfereswiththeTPP" -selectiveelectrode,whichrenders impossiblethemeasurementof A' bythismethod. "Theothereffects of thesecompoundsonmilochondialintegrityaredescatreriin hbisreepodrt. 3 04157 KBW 21498 3 TM ADP PF143 NIE ET a = 0|p ow a0F| | i| [ome || o J <I Li J)ONP\ [071-0 ~ Fi4g."TThhecoemfpfocstioifonPoFIf1i4n3countitohnermeesdpiiratiaonndroatheracnonddiAtioonsf, aot3IvFiegr.1m.iAtodcdhoonndsriPaFI, 100 ADE,10004DNP,60 04158 9 IN Tr vo be [Ne \ u IN oe h== UN\ (|a0 bi fe mm. } Ww i. \ = Wi aAnt or 0 04159 misyEe1rmmmi--"f--re bn i --_-- `Theexperconiditmionesanndat ddia tiol ns,asinFi4g. Abbreviations,seeTable 1. m [pm e s ors ren | } 04160 C niFAo RA rATEmSamAar J [e ror we RClgn RC%L 1262399 ADP:Ogr) :ADP:O%,| 1013123 r m seta a A r m e rs d u Arheeres `mitochondriain a way,whichistypicalfor aprotonophoricuncouplerlike2,4-dinitrophenol, orFCCP. . 04161 -- me arae ---- [Joris| 3550 RClgr, 27803 RClpn: RCL % 541179 ADP::OADgP-rO,) % | 79.1 109 r AE i err n ST 'PF95inmitochondria, duetothefactthatthecompoundexerted astrongeffectonTPP-clectrode,bothinthe a C a osa A sh rTN SHA ES concentration,PF143waswithouteffecton A' ofmitochondria.Thisalsocouldbeexplainedby afluidizationof es . 94182 Kew 2458 m"eTtaabbolle.iTcshaeteef:fctof P1204onthrespiration res ofrat vermitochon undervarious T"Thhedeuxpoerfimexntpalrceonsdsitionrsaensdaidditoionos,aveinsFiSg515. AbbreviasteieToanlse1, I EE [Je RClgp) : RCL % 60.1136 ADP:Ogrr): ADP:0, % | 85.6 4.6 Lowconcentrations ofPFI0affctdnethermitochondrialrespirationnor &. Higher concentrations slightly s"tTihmeulsautbesdtraenscteisn,g wrhesipcihraateioanb,alndionindcurceeadseatshmeaclolnddeuccrteiavsietiynofA'th.e innermitochondrial membrane,could beexpected1 uncoupleaida phosphoniation.Theuncoupling canbe observedas 8doses in RCTandlor inADP.Oratoinducedby compound.However,thdees inintactnessofmilochondia occuring ue 0 agin ofisolatedorganelles, heterogeneityofmitochondria,andotherfcorsdecreasethe reproducibility of experiments,Ourssayprocedure explsinedaboveallowsusto eliminatemostofthsefactors,Twoaiionsof ADPtomitochon,frstinth ascncsandthscondinthe eensof acompoundundesyprovidan internalstand thusallowingtocomparetheeffetsof ifleent compoundsalmostindependently on he varitonsin itochondsalpreparations.Fi 6compare thseectsofPF compoundsonmitochondrialaxidaive !. 04163 phosphonyation The effitsexpressed a pcent changes in RCI and ADP indbyKuiefcwfren2t4Pd8F compounds i [" iy x" 5 ow 4 Low ela ge HBtz oe | . HEanElReE w ET .| HjalaLH2gER:Ee13 FlecwSrs Detuncl HBePrFiIoOH OPFiL 3g BPFH th EE] Berio HoEEeEE! [ers 4gJ2adh| RE: 4B2aaeE 4Td 2S050ee Foitg.veTrhmeicofcchoofnPiFcompoundosn th ficiofeonidctyv phosphorylation of co"TnheccomepoosnfitPiFtocnormofpiaonucnutbdasti,isonocmTenadbiesusma1n.6d. therconditions a nF. For hedatashow in Fig arinagodsgrcmeat withthefect ofPFcompounds on resingrespistion ofmitochondriaandonthe AY. Thesedaafuther claythemodesofaction ofPF compoundsonthe mitochondrial ccrgetc, Fig shows tat low concentrationsof P12L, PFIZM, and PFIOH dees bh he RCIandADP:0ratiosinmiochondeiaTheaction ofthesecompoundsonoxidativephosphorylationrescmbes that ofcasical uncapierslikeFCCPordiniophenal. PE143and PFS,aswell asethanol,increasedRCT whereastheywerepracticallywithouteffec on ADP.O ratoin mitochon.Thisdoesnotcontradict our suggestionthtthesecompoundsness the icityofmitochondrialmembranethus acivatingthenzymesof vespimory cin.Thedecreas inRClinducedbyPFI0 ithe ascncs of he effec on ADP.O alo suggestsUt PFI0can creasetheprotonlek (whichisbelievedtobeabsentorinsignificantinSse3). " 04164 Kew24m 2. The mechanisms of the effectsof PF compounds on mitochondrial energetic. ntheprevioussectionwehave shown haPFcompoundsaftthemitochondrialenergetics nat east 3 different ways. Some compounds, such as PFI2L, PFI2M, and PFIOHwereshown to decrease the degree of coupling of ATP production to oxidation of respiratory substats. Other compounds (PF143, PF9S) were sugge0asfftectetdhe fidiyofmitomcemhbraone,nanddPFr10iapaarenltly excarnecffdetontheproton Gonleknbernt theneemitochondeialmembre. Wenestmadeansept investiga themechanisms involvedintheeffectsofPFcompound onmitochondria. "Tostudy theaction of PFcompoundsonthecondctvtyofmitochondrialmembrane,wetookadvantage of simultaneous measurementsofrespiration and membrane potential changes. Various concentatons of PF compoundswereadd tomitochondriaandrespiration atesand AYchangeswer recorded.Foracomparison, thesame experimentswereperformedwith aclassical uncouplr-protonophors2,4-diniropbenl. A typical `recordof changesinrespiraatndiAo'ninducedbytheadditionof 2,4-DNPisshownonFig3. Tbieo ~~~_NP "L NP E 4 DNP \ og1:0 / 4]] , 1 min, and inFritg vTheeramiftccohofnvdrtooius 24-DNPconcentrations onthe rte of oxygen consumption w30e04irT0nnFcteiuga1a.iToyhnecmeoin(cuenFio.f1 2va4sdsispoeephcentl(NwiPt)h v2sernelssofollgloowmiynig.n.5 Allothier, ,co1nd0iAibns. w 94185 Fi.4h th tron Soave, xa am i. -- z Ln E EgE. \y 3c 2" - aor ate 2 ne te te AY. V cnonnhoenmptnohxe)rw ssfesndre eesss2dpormasPt irhn ednaetped oaAend c Em E2 eee ETs eca t erdr bmr aot pr E SFnsTs A S s-- A ---- E s-- E y oecten--a T----H--r--.--Ton--se--I --------r iy---------- nraei er asitisfirmlyestablisfhoread fr representative uncoupler ER 2,4-dinitrophenol. aHowever, avy mere te anko indt ofexh pere imenr ts, " 04186 Kew240s concentratPiFoIn0,h(owFei5vg)ersfluirgththleyrisntcirmeulaasteessintcheorenspcireatrionn wfitihsa ccoormrpeosuponnddepnrtoddueccerenaosfe ien rAYefaftertelaItcioveelylthicgsh Well withourproposalthat FIOincreasesthe proton ea in ioehonsialmembranesThelak ismembrane potentialdependentinsuch awaythatslightdecreasesin AYcansignificantlysuppressthe leak(NichollsDG, 1974). Mito RETTp TM~ oe ~~ | . PF10 pFi0 Pro Frio / /f | | [tune || mio! Pro ~~ NN)\ Peo | NI | Re ope Cu Now!\e | 3 ;E " \ Fig.cTh foic own fePsF10sonthe Fri.Eecshiraonadnd 4e%F.IOvnas62o.504 1 04187 PFI43 (Fig) in th conenirnion range 100-400 uM was prcially without eft on (hKarwes2p4o8n raeofmitochondria. However, a it clery sen in Fig, the first addon of 100 uM PFI&3 duced a ow decreasein AY ofmiachonsa wichconinue BFL. Intheprevious sectionwepropose hat 1 deci,bengapparently not alfeied byfurtheradiosof siscompoundafectsthe iyofmitochondialmembrane cDounceetnoztahtiisonist c(a1nbtoee1xmpeMc)teodftthhaetcPoFm1p4o3umnidgwhetraectaaddsedadetotmeirtgoecnhtoantdhriiag,hewrecionncdeenetdraotbisoensr.v`eWdh8entrhaingshieenrt mnoutlsahtoiwonn.of respiration follbyoinwieodn ofoxygen consumpon, which was seni t2,6 DNP (daa MI io ~ rePFI "HJnp| h PFIi 43 dbh JI \ J NN ~ [20 ese \_NpIris ld2 $\ \\\ Mio i) J|/ sf$ \ Z N |, L1mmn, NX Fi"gAsl.lTohe edfecwtooePrFs1r4s3naFnsi.t3he.rFesapihrsathioinanndof4F%H.I43ws 160M, he dion of NP wa 40 usM. w 04168 PFIOH (Fig. at concentrations higher then 25 4M srongly inhibited the respiration of miBtoWchon2d4r9ia8. These experimentswererepeatedwith a low ionicstrength mediumandwiththeuseofanoher respiratory substrate, succinate. Unde these conditions, 25-50 4M PFIOH also inhibited the respiration. This indicates hat the it of inhibition i locatedintheregionofubiquinane:cytochrome reductase, which typical faortifical uncouplrsortinhibitscytochroxoidmasee.However, iwasnotedthat th adlionof uM cytochrom(e0 mitochondria inhibitedbyPFIOHpartiallyrestoredtherespiration(Fig).Itiswellknownthatinhigh onic strength medium, swelling ofmitochondria indtuhecreleeasseofcytochranodmhee inibiion ofrespiration. A Mio ON \. PFIOH NT UN | PFI0H / N= PFIOH Jz |i ||| 3 #] tm Mio | \\ \,\| \ | 8vo FigA.lThceoecntdoifvePrfeFs1i0iKonoFnngt.sh.eTrhesepaidradtiinonsaonfdPAPI.OH were6.25, 625, 15kM (2 uM otal). * 04169 "Thispoinstoapossibiltytha theapparentnitionofrespirationbyPFIOFisdue totKisecwom2po4un%d i"nTdiusciwnogultdhreesCualTMtsdienpheingdheanmtplpiertmuedaebislwietlyitng rof amitnoocfshmoiintdotrcihiaonadonrdiinanllionnseorfmceymtborcahnre,omoercs.oT-coalcledlpoaretrhoepieefnffientyg,. weinvestigatedtheaction ofPF10Hon mitochondriausingdiffrentrspirstorysubstraisandthelow ionic --P Mito 8of 2 "\_" PF|IOH ~~ \ DNP ~~.) one Te~~. - N\ 1 min \, N FigA.lThoeenfeictoaosPniFnIs Foi.nAtdhdeornessp.irPaItGiFon,of53r3a0t4vDrNmi4to0cAhoEndrCiota.6,5kM. strenghtincubationmediumwhichisknowntodecreasetheprobability ofporeopening. In these experiments, we use the incubation medium containing 210 mM mannitol, 10 mM sucrose, SmMHEPES-Tris,pH 7.4,and 2 mM MgClwit orwithou2.t4mM EGTA (indicatedinFigureLegends)to chte hGeil rtesaia mdutat leCaane TMd.malatewere usedasrepisorysubstratesintheexperimentshownon Fig, which impli thattheinhibitionof esiraton byPFI0Hisnotceto i inhibitingthesucinatedehydrogenase.Indeed, `therewasnoinhibitionofrespirationevenwhentheconcentration ofPFI0H was 100 uM (Fig.9). However, 50 4M PFIOH caused a srong progressive inhibitionofthe succinat-supporte respirtionofmitochondiia and completelydischargedtheAY ifrotenonewasexcludedfrom theincubation medium(Fig10).Thisis typicfaorla protonophorous coupe, the initionexplainedbyth accumulationofoxalosceic acid (a strong ilibitor of `succinate dehydr intoheg miteochnona dias l maetri)x. 21 04170 A Mito NL TM\,"~ i ime, S iia ~IA A \ Prion E NJ \ \ | 4 \ Mio \ J| | KBW 24/98 sFuicgci9.nTathee.effect of PFI0Hon the respiration a4n%ofdat lve mitochondriarespiring on "Theincubationmediumcontained 210 mM mannitol, 10mMsucrose,SM HEPES-Tris, PrHo7.4t, 2eTmhneMcooMngcnCenlea,to.2n.4ofmmMitEoGcThAo,nd5wmrasiMa|smucgcmi.natAedd,i2tionusg:/mPlFIolOiHg,o5m0y,c0i.ne,and 2 uM 2 04171 Kew2m ni Mio ss ~~~PFIOH a e H1e00a / ee \\ \ \ prion . ~L \ WN O\ NP h aFibg0s. Toefhentiencnhiobenit.ionofrespiration and the decrease in AYinduced by PFIOH in the mit"ThAidncduobantiso:nmPoFdIGiH,5a0n04d,otDhAPrc,o4nd0i4t0i0o.nswesasinFig foexepttha rlenenewas "These experimentsallowust uleout the possibilty tha PFIOH inhibits Compl Iofmitchondeil respiratorychain.Hoveres, thecytochrome -reversibleinhibition of respirationmayasooccurdi tothe displacement of cytochrome from the mitochondrial membrane (scetyl-ammonium lke eect the negative charge crecningbyamphiphilicposiivelychargedcompound)or ueto competi inhibitionofcytochrome binding(0 teminaloxidase (apollysine lke lec). To urherclarity is,wetook achariageof aclassical reducing non-enzymatic ysiem ascorbate + TMPD to reduce mitochondrial cytochrome . This system is very sensitiv 0anychangesincytochrome bindingandrinteractionwithcytochrome oxidase.Fig11showsthat 50mMPFIOHdecreasestheAYandsimulatesthe respirationofmitochondriaoxidisingascorbate,Theris10 sponancous inibilonofresp(aiitrwoaudtbeiobsoernveifPFIOH interes with cytochrome binding) ThesedatfutherprovethaPFI0Hdossno iii th respiratorycha ers. 5 04172 In Mio TPP N\ oo LA] od ton eet | N N\ PFIOH I \ \ \PFI0H 3 Kew24 TPR* N\ \ ncraso 7 R Foinga1s1. cThoero+bfcHaPotfDe.PFI0Hon the respirationand 43ofrat Iver mitochondria respiring PH "T7he,i2ncumbatMioMn gmCeli2c.a4nmiMneE2G1T0Am.M5mmanMnitaosl,co1r0bamtMe,s1u0c0r4oseM, STmtPHEP2DESp-gT,/rmsl, POlFgOom.ySci0n0.and 2 M rotenone. The consentofmitochondria vas | myn Addsions: "TheeffectsofPFcompoundswerefurtherstudi inexperiments simed 10reveal th actionof these compounds on mitochondrial inegiy. For tis, we investigated the effectofhigh concentrationsofPFIZL, PFI2M, PF10, PFS, PF1an4dP3F10,H ontheswellingofmitochunodenrvdariiouas condiTtheifoolnloswi.ng atshow thatthecompoundsinduc theswellinganddisrupionofmitochondriabydiferentmechanisms. "Theswellingofmitochondriawas measuredinhighandlowionicsiengih mediumwith orwithout respiratorysubstratesandbothinth presenceofinth absenceof Ca?chelator EGTA.Fig12shohwa ,the patoifePFmI0sH induced swelling were diffrent under differnt condition. In the presence ofEGTA(Fig 12, uppercurve)o succinate(datanotshown) PFIOHwaslessefficientthanintheabsence ofthesesubstances. The efficiencyofPFIOHwas independoentnhet incstrengthofthe incubsion medium(daanot shown) Is clearly sn als, tha the presence of EGTA in the incubation medium significantly suppressed the high-ampliuce 04173 Kew ms W`shwielclhinwgeirnedsucheodwbnyo2i5 uiM PhFeIr0eHs,piaantdiocnhaonf gmeidtoitcshoknidnreitaiccsa.nTihnidsucindCicaa2te+sthpaethnidegnhtcopnrceentpreantiinogns ofhPeFiI1e0Hr, e`vmeinttocuhaolnddriailmsembuorfanmpiet.aicIhtoionsdwenieal.l knownthattheopening of such aporeresultsinuncoupling,swelling and 8 fan oilf, 1 FRO rns : e1} r] ik PFIwOitHSR . ; 0 2 a 8 1 Fig 12. Theefictof FIO onthesveoflmitiochnondgria Time, min curven,cuthbeaiinocnubmateidoinummowdesu 4waisnsFuipgp1emoenriaxdtwpitt(hh1tmgMhiEaGTA.aMnidomcahloantdeewaelrpereotxecilnuwdaeds.1U7pgpexr `mi. Each addoifPtFIi0Howans 25 uM. Incontrasttothe actionofPFLOH, theswellingofmitochondriainducedbyhighconcentrationsofPF143 asnotaffectedbythe presenceof EGTAorrespiatoysubstses.Hoveve,th fectofPFLE3wasstrongly ependentonthedoncstrengthoftheincubationmedium.Fg13 shows,tatin alowpotassiummedium, Concenirationsof F143 a igh as about 4 m wer withouteffecto heswelingof tochondra For a comprison, the effect ofthe strong nonionic detergent Tron X-100 an rat vr mitochon i shown(Fi 13.In potasium chloridemedium,PFL43appeared bea srongdeergendwiththe efficiencyof about 1 relivetothtofTron. Al ther PF compounds (PFI0, PFIZL, PFIZM, and PFS) were without elit on the swelling of mitochondria. 04174 8a 2 Ree . B20 Kew 2458 10 A P 2 TPPFe11n453s,ie MKoaCIrnniNolano! \ : . 0 Tm wa o so em ow im eo ven ev wm [Compound], pg/mg protein Fig 13. The effectofPF143 on the swellingofmitochondria. "High ionicstrengthmediumwasasinFig 4, lower curve.Themannitol ~contmeadiiumnwiasn21g0 `mMmannitol, 10mMsucrose, 4mMKH;PO,, 10mM MOPS,pH 7.4.Mitochondrial wereaddedat 1 mg x mi'. % 04175 PP, 1 A|DP | Mito | PFO5M | T DNP Kew avon So [02]]=0 | 2EE | | A1mminn Fig-1. EctofPROM"an he re fiction and membraneptioft ver ccd Medium composition and other conditions were as in Fig.l. Red curve, the respiration of mitochondria; blue curve, the changes in membrane potential. Additions: TPP or "*", 02, 0.2, 04, 0.8, and 0.2 uM TPP'CI' (2 uM, total); Mito, 1 mg/ml rat liver `mitochondria, ADP, 200 uM ADP; PF95M, 0.5 uM "PF9SM"; DNP, 40 uM 24dinitrophenol. 04178 2 Kew 21495 This study revels hatPFcompound3s.afCofnclvuasriioonssase of itochondril energetics. Someof compounds, lke PFIZL, PFI2M, PFIOH, a reaively low conenraions decrease the degre of coupling of respintorysubstioxidation toATPproduction. Themolecularmechani ofthe action fPEL and PFIZM `compounds apparently involves the shuttlingofprotons across inner mitochondrial membrane, although further experiamreenenedteds. The mechaonfaictsiomnofPF10H apparently involves the non-specific changes in the permeabilityoftheinnermitochondrialmembrane.Mostprobably, thiscompoundcanchangethemicrodomain Sitlraucyteurreoofhemxiatgoocnhaoln),dTrihaelelfifpeicdtsmoefmbFr1a4n3esn(dbyPiFndSucainegslmiaplild cbltsusitgeaornrib,nygThcehasnegcionmglpiopuidnpdhsassleighsltaytienfcrroemasae hestvityofenzymes of oxidativephosphorylationand responchain, hemosrboblemechanismbeing he Nuidision of theincrmitochonmedmrbriaanel. Itmightbeexpectedtht hecumulatofisoucnh compoundsin suecoldelsi suedamage.Thedata andwithPF10arethe most eres from oenergec.pin ofview. Ifouiferpetaon i come andticompoundfrases testy ofthe athway() of proton lea in michondeal membrane, i may be a vey eel fel fo study flowlores relaonshipofvariousenergy-dependentpussin tochondei, 4.Literature cited. Kamo, Mitsu M, Hongo R,Kotak Y "Menian potential of mitochondriameasured wil anlcd `sentsotiettraiphveney] phosphoniumandrelationshipbetween protonelectrochemicalpotentialand phosphorylationpotentialinsteadystate." J Membr Biol 1979 Aug:49(2):105-121 NichoDGl"lThse nonpr-otoOnlekh-2m5yeairs ocn." Bi Rep 1997 Ja 1)251257 caRtioonts eMHne"mbMbeemBbirrolang1e98p4o81t02e:1an2n7dt-s1ui3rf8aacle potentialinmitoc upth akeo andn bind dinr g ofi lipa ophi: lic " 04176  A KBW 424/98 Supplement 1. Effects of"PFISM" and "Sal" on mitochondrial energetics. All the conditions, procedures, and the logicsofexperiments were as described in "Materials and Methods". Being added at relatively low concentrations, "PFOSM" stimulates the rate of respiration and decreases membrane potential in rat liver mitochondria (Fig.S-1). Other compound, "Sal", exerts the same effects but at much higher concentration (Fig. 5-2). Both these compounds decrease RCI and ADP:O ratioofmitochondria (Fig.-3). For these experiments, the unequal concentrations of "PFOSM" and "Sal" were chosen which approximately double (that means the increase by 100 %) the State 4 respiration rate of mitochondria (see Fig.S-1 and FigS-2). An average RCI in these experiments was about 4.16 (416 % increase in respiration rate under addition of ADP) so 100 % increase induced by "PFISM" or "Sal" could not mask the State 4 - State 3 -- State 4 transition and RCI and ADP:O estimation. At these concentrations, both "PF9SM" and "Sal" decreased RCI (by ~ 40% and ~ 30%, respectively, comparing to abs. ethanol control incubation) and ADP:O ratio (by ~ 12 % and ~ 10 %, respectively, comparing to ethanol control incubation). Thus, both these compounds are uncouplers of oxidative phosphorylation, although of different efficiency, "PE9SM" being about four hundred times more strong, then "Sal". These experiments were repeated with the use of low ionic strenght medium (225 mM mannitol, 5 mM Hepes (pH 7.4), 4 mM KH;PO, 5 mM glutamate, and 5 mM malate). With the use of this medium, qualitatively the same results were obtained, however the concentrations doubling the respiration were 1 uM for "PFOSM", and 400 1M for "Sal" (data not shown) Inorderto reveal the putative mechanismof uncoupling, we compared the effects of different concentrations of "PFOSM" and "Sal" on mitochondrial respiration and `membrane potential with these ofa "classical" uncoupler 2,4-dinitrophenol. The changes in respiration rate and in membrane potential were recorded simultaneously. A typical record example is shown by Fig. 5-4. 1 04177 TPP* T 4 Mito | Kew anus Sal | Tr DNP * Ss | 5 E <| 1 min [02]=0 | Fig.S-2. Effect of "Sal" on the rate of respiration and membrane potential of rat liver mitochondria. Medium composition and other conditions were as in Fig. 1. Red curve, the respiration of `mitochondria; blue curve, the changes in membrane potential. Additions: TPP" or "*", ADP, Sa 200 kM Sa; 0.2,0.2, 0.4, 0.8,and 0.2 NuMP,TP4P0CI0 24diepeel. (2uM,total);Mito, | mg/mlratlivermitochondria; ADP, 200 uM , 04179 KBW 4124198 % : 0A 3 RCI ratios ~~ ADP:O ratios Faingd Se-f3f.icEifefneccytosfoofxildoawticvoencpehnotsrpahtoiroynlsatoifon"PinFIraStMl"ivaerndmi"tSoaclh"onodnritahe degree of coupling Respiratory control indexes (RCI) and ADP:O ratios before and after addition of "PF95M" or "Sal" to mitochondrial suspension were measured as described in "Materials and Methods" (see Fig.S-1, Fig.S-2). Each column represents averaged data from 4 experiments and error bars show S.E. See text for further explanations. 04180 4 p7pt | 0 | | -- PRISM | prosu | PF95M PFo5M | prosm | prosu | [02]=0 | S' E8 E | m1mnin, Fig 5-4. Typical record of changes in respiration rate and membrane potential of `mitochondria induced by sequential additionsof "PFOSM" Medium composition and other conditions were as in Fig.l. Red curve, the respiration of mitochondria; blue curve, the changes in membrane potential. Additions: TPP" or "*", 0.2, 0.2, 0.4, 0.8, and 0.2 jM TPP"CT (2 uM, total); Mito, 1 mg/ml rat liver `mitochondria; PF9SM, 0.5, 1, 1, 1, 2, and 2 uM (7.5 uM total) "PFOSM". 5 04181 : KBW 424/98 The values of membrane potential and the rates of respiration were calculated as described in "Material and Methods". Fig.S-5 shows the effectofdifferent concentrations ofall three compounds ("PF9SM", "Sal", and dinitrophenol) on mitochondrial respiration and Fig. $-6 show the changes in membrane potential plotted against changes in JOR 8 100 x -5 0 x Sw g" , i 5 | wl 0 DNP,uM # PFOSM, uM *Sal,iM x100 05 2) % El [uncoupler] Fig.S-5. The increase in respiration rate of rat liver mitochondria induced by "PFISM", "Sal", and 2,4-dinitrophenol (DNP). All the conditions were as in Fig.S-4. Note that the concentration of "Sal" is expreasssuMexd 100 (10M). respiration. Fig-5 shows the different uncoupling efficiency of the compounds, "PE9SM" and "Sal" being the most and the less potent then "classical" dinitrophenol, respectively.Fig5-6 shows that all these compounds at applied concentrations do not inhibit the respiratory chainofmitochondria. 04182 6 Kew 42498 2 ww - 150 gE . .130 E 0 DNP # PFO5M * Sal 5 . 3 s * 1 2 3 oe 50 0 T0990 1 110 10 respirationrate,nmolOp xmin" x mgprotein"! Fig S-6. Changes in membrane potential of mitochondria plotted against changes in respiration rate induced by different concentrations of "PFOSMTM, "Sal", and 2-4dinitrophenol (DNP). Conclusion. Our experiments revealed that the compound named "PFOSM" is a very potent uncoupler of oxidative phosporylation in liver mitochondria. The uncoupling efficiency ofthis compound is comparable with that of CCCP, oneofpotent "classical" uncouplers. Another compound, "Sal", also appeared to be an uncoupler, although of relatively low efficiency. The relationship between changes in membrane potential and increase in the rate of respiration of mitochondria allows us to propose the increase in proton permeability of inner mitochondrial membrane as the mechanism of uncoupling action of these compounds. Further experiments are needed to elucidate the mechanism of the uncoupling at the molecular level 0413