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THE EFFECT OF PERFLUORINATED ARYLALKYLSULFONAMIDES ON BIOENERGETICS OF RAT LI[VER MITOCHONDRIA KendallB. Wallaceand AnatoliStarkov DepartmentofbiochemistpayndMolecularBiology UniversitoyfminnesotaSchoolofmedicine Duluth,AM 55812,US.A. Key words: Mitochon&ia,Membrane potentialR,espirationU,ncoupling,Detergents. Supportedby a grantfrom The 3M Company. MATERIALS AND METHODS KBW 2/4/98 The isolatioonf mitoebondria. NfttOchOndnawere isolatedfrom liverof adultmaic Spraguc-Dawleyrats(-200 g body weight)by a conventionaldifferentwcentri%gatiopnrocedure.Animals were killedby decapitatioLni.verwas excisedand weighed and cooledin 40 znlof isolatiomnedium (210 mM mannitol,10 mM sucrose,5mM BEPES-KOH (pH 7.4),1 niM EGTA). Cooled liverwas minced withscissorasnd washed twicewith20 ml ofisolatiomnedium, then dilutedwiththesame medium and homogenized for I mm witha motor-drivenPotterhomogenizer (Teflonpestle, glassbeaker).The tissueto medium ratiowas 1:8 (g:ml).The homogenate was fdteredthroughgauze and centriffigfeodr10 min x 700 g, t'= 40 C, and theniitochondripaelllewtas recoveredfrom thesupernatanbty centrifugatiaotn10,000g x 10 niixlThe pelletwas resuspendedin 10 ml ofwashing medium (210 niM mannitol, 10 mM sucrose,5mM BEPES-KOEL pH 7.4)supplementedwith bovineserum albumin (BSA, I mg x n-d-1T)h.e suspensionof mitochondriawas dilutetdo 35 nilwiththesame medium withoutBSA, and centrifugeadt 10,000g x 10 min.The finalmitochondriaplelletwas resuspendedin washingmedium to a proteinconcentratioonf 70-80 mg x rn]"and storedon ice. Measurements. Mitochondnal membrane potenual (AT) was estimatedfrom TPFI"'ion distributiomneasured with a TW- selectiveelectrodeconstructeadccordingto Kamo et al.,1979. Mtochondrialmembrane potentiawlas calculateads describedelsewhere(Rottenberg,1984 ). The rateof oxygen consumpfionby mitochondriawas measured with a hand-made Clark-t3W oxygen electrode. Both themitochondnalmembrane potentiaalnd therespiratiornatewere recordedsimultaneousluysinga multichanneilncubationchamber equippedwith a magneticstirreTrh.e volume of the chamber was 1.8nil.All experimentswere performedat room temperature(25' C).The TPP' -sensitiveelectrodewas calibratebdy sequentiaaldditionsofknown amounts of TWCI- beforetheadditionof mitochondria(seeFig.1).The respiration rateswere calculateadssuming theinitiaolxygen concentratiotnobe equalto240 IAM. The mitochondrialmembrane potentialswere slightly(-15 O/o)underestimatedand the oxygen consumptionrateswere overestimatebdecauseno correctiownas made forlower TW bindingconstantsand for lower equilibriumconcentratioonf dissolvedoxygen in thehigh ionicstrengthmedium used in our experiments. Thisisfullysatisfactobreycausethevaluesareused tocompare theeffectosf differenctompounds under thesame conditionsratherthanforenergeticalculations. Protein concentrahonwas determinedby the Bradford assay.Bovine serum albumin was used as a standarcl Additions.PF compounds were dissolvedin absoluteethanol(forexceptforPF143 which was dissolved in deionisedwater).Preliminarystudy revealedthat allPF compounds are very hydrophobic and tend to precipitatienour incubationmediurtlDue tothisa, setof dilutionwsas made foreach PF compound to obtainan arrayof concentrationsstartinfgrom 100 pM down to 6.25 W Dilutionwsere made by addinga volume of 2 KBW 2/4/98 as 1.8 absoluteethanolto a volume ofPF compound stocksolutionP.F compounds were added toniitochondria volume of a solutionof desirableinitiaPlF concentratiOlTLbiS approachallowedus to obtainsatisfactory reproduciblreesults. Reagents.Manxutol was from Aldrich,sucroseUltraPure from ICN, allotherreagentswere from Sigrna. Bovine senan albumin was essentialflayttyacidfree. 3 RESULTS AND DISCUSSION KBW 2/4/98 1. The effectosfPF compounds on oxidativephosphorylationin ratlivermitochondria Becausethisin vitrsotudywas aimedtoreveatlhepotentiianlvivotoxicpropertioefsa setof compounds,a bufferehdighionicstrengtphotassiumchlondemedimn resemblincgellulacrytoplasmmilieuwas chosen.By the same reason,glutamateplusmalatewere used as nutochondnalrespiratosruybstrateTsh.e oxidatioonf glum=te plusmalateuwolvesthe most vulnerabloebligatorcyomplex forhydrophobic compounds,Complex I ofmitochondriarlespiratocrhyain,as wellas allotherrespiratoernyzymes.We also developeda speciaalssayprocedureI.tallowsobtainindgetailedataon theeffectosf a compound on n-iitochondreinaelrgeticTsh.e procedurealsoallowsto compareina universawlay thedliciencyof various compounds affectintgheenergyproductioinnmitochondria. Fig. I explains thedesigonfexperiments. !TPP The pica= shows typical i data obtained by ADP N4ito PFI ZM simultaneousrecordingof niitochondrwrespiration ADP and AT. The ffisatddition -MP+ ofADP induceda transient DNP increasienrespiratiroante a,l, increase F@ig.A1typicaelxampleof simultdneausrecordinqof the mrochandfiraelsi3iraton rateand changes,n @@41. incubatiomnadiurncontained 125mM KCL 10rrtMmOPS, 4mM "P04,5 mM glutemedsa,nd 5 mM malam, pH 7.4. Red curve.m*x:hondnaj respiraliobmluecurve, thechanges inAly. AdditonsM:ko,raltiver mkochondrkItmgtrrAdD.P, 100 itK-PF1 2K 6.25tintONP, 40 iiMZ+din&aphonoL-TPPI, 0.2,0.1Ok 0.8,and 0.4@iM(from toptobotiormtoW conc@fweoon 2 ttK. ofmitochondriwahichwas parallelebdy a dropinAT. it indicates that mitochondriaenteredsocalledmetabolicState3, [Ozl-O thatis theyphosphorylate ADP forthe expenseof E 2 min respiratosruybstrates. The values of respirationrate during (Vg3) and after phosphorylation (V.4) were used to calculate respiratory controlindex(RCI)equw totheratioofV,3 to Vt4. The amount ofoxygen consumed by mitochondriaduring phosphorylatiwoans usedtocalculatAeDP:O ratio. Afterthe phosphorylatioonf ADP was completed,the rateof respiratio&nxmased and AT in niitochondrsipaontaneouslryestoreidndicatintgheonsetof State4. The additionofa compound (PF12M in Fig.1)to mitochondriwaas followedby the secondadditionof ADP, and secondRCI (RCI-compound)and ADP:O (ADP:O-compound) ratiowsere calculatecAlftertheonsetofsecond State4,an uncoupicrwas added at 4 KBW 2/4/98 detemiinedby theactivitoyf a concentratiotnhatstimulatetdherespirationf mitochondriatothenwdmal level respiratorcyhain. Thisrelativelsyimpleapproachallowstoestimateseveralessentiaplropertieosfa compound in relatiotno theenergeticosfmitochondria.By theanalysiosf dataobtainedinsuch assay,itispossibletoshow ifa compound (a)inhibitosr stimulatetsherespiratorcyhain; (b)inhibittsheentryofsubstrateisntomitochondria; (c)specificalilnyhibittsheenzymes oftheoxidativephosphorylatiosnystem; (d)uncouplestheoxidationand phosphorylatioinnmitochondda. The approachdescribedabove was appliedtoallPF compounds. Fig,2shows thetypicalrecordforPF 10. Mito AE)P pFl 0 AC)P DNP A4' increese 1021-0 DNP\I Mito E CD w I min Fig.2.The effectofPF] 0 on therespirationrateand A Tof ratlivermitochondfia The compositionof mcubauon medium and otherconditionsa,s in Fig.l.Additions: PF10,62.5pK ADP, 100pK DNP, 40 pM PF10 in relativelhyighconcentratiosntimulatetsherespiratioannd decreasesAT in State4 whereas has no influenceundermetabolicState3.ItaffectendeitherState3 respiratiornatenor ADP:O ratio(Table1.) 5 KBW 2/4/98 Table 1.The effectofPF] 0 on therespiratiornatesofrat liverndtochondriaunder various metabolicstates, The experunentcaolnditionasnd additionwsere as in Fig.2Abbreviatiounsed:V. therateof oxygen consumptionby untochondriabeforeany additionwsere made; Vg3,therespiratiroanteinthe presenceof 100 jiM ADP; V,,4t,herespiratiroanteafterphosphorylatioonfADP has been completed, RCL therespiratocroyntrolindex,calculateadsVa3 to VA4 ratioA,DP:O, theeiticienciyndex,calculated astheratiofADP (mnol)addedtooxygen(mnol0) consuineduringphosphozylatiofnthatamount ofADP; RC4..pl.),therespiratocroyntroilndexcalculateads theratioofttimc-spmtionrateinthe presenceoftheuncoupler(V(..,w))toViE.Index(PF)means thattheparameterwas measuredinthe presenceofthePF compound. The dataof 5 experimentsarepresentedas mean values S.E@ PF10 Parameter Vi.i vso V14 RCI ADP:O V(PF) Vd3(PF) V.t4(PF) RCI(PIF) ADP:O(pF) RC@.coupler) RC@pF): RCI, % ADP:O(pF): ADP:0, % mean F- 18.8 1.9 84.3 3.9 18.6 2.6 4.778 0.4 1.844 0.1 27.3 2.5 83.7 9.6 25.8 1.9 3.256 0.3 1.84 0.1 111.0 10.4 5.992 0.6 71.0 10.4 99.8 3.5 The effecotfPFIOH ispresentedby Fig.3.and Table2.Being added atlow concentrationst,his compound sumulatestheState4respiratioannd decreasestheRCI and ADP:O indexes,whereas itdoes notaffect theState3 respiratio(nseeTable2). 6 KBW 2/4/98 Mito ADP PF1 OH ADP ONP PF1 OH ADF@ increase [021IDNP Mito 1 min Fig.3. The effectof PFIOH on the respirationrate and AW of rat liver mitochondila. 'Me compositioonfincubatiomnedium andotherconditionass,inFig1..AdditionsP:FIOI-L 6.25pK ADP, 100W; DNP, 40 plA 7 KBW Table 2.The effectofPF] OH on therespirafiornatesofratliverntitochon&iaunder various metabolicstates. The experimentalconditionsand additionsa,s inFi&3. Abbreviationss,eeTable 1. The dataof 5 experimentsarepresentedas mean values S.E. 2/4/98 PF10H Parameter Vi.i Va3 V.t4 RCI ADP:O V(PF) V93(PF) V.t4(PF) RCI(PF) ADP:O(pF) V(uncoupjcr) RC4unooupl-) RCI(pn :RCI, % ADP:O(pF): ADP:O, % mean S.F17.7 0.6 81.1 3.8 16.2 0.9 5.036 0.2 1.814 0.1 30.9 1.9 81.7 7.0 23.5 3.4 3.77 0.6 1.602 0.1 101.1 13.0 5.694 0.7 77.6 17.0 88.4 2.5 The compounds PF143 and PF95 exertedsmall stimulatoryeffecton mitochondrialrespiratiobnoth in State3 and 4.PF95 alsoslightliyncreasedRCI, asitisshown inTable 4.However, PF143 induced small decrease inthe AT ofmitochondria(Fig.4)T.he "incrcasd"inAT inducedby PF95 (Fig.5)was not relatedtothe membrane potentialof znitochondnabecause itwas observed also in the absence of mitochondiia.Thus, PF95 apparently interferewsith the TTF" - selectivelectrodew,luch rendersinqx)ssibltehe measurement of AT by tlusmethod. The othereffectsof thesecompounds on mitochondrw nitegntyare describedlaterinthisreporl Mito p PF1 43 PF143 ADP I ONP 1 I- ADP increase DNP [021-C) KBW 2/4/98 1 min Fig.4.The effectofPF]43 on therespirationrate and A Vlofrat livernatochona'@ia The compositionofincubationmedium and otherconditionsa,s inFig.l.AdditionsP:F143, 100 pK ADP, 100pK DNP, 40 pM 9 KBW 2/4/98 ri:>P+Milo TPP+ AE)P PF95 ADP ONP t TPP+ I PF95 t, v ADP [021-0 + @TPF ONP 6 1 min Fig. 5. The effect ofPF95 on the respiration rate and A Vlofrat liver mitochondria. The composition of incubation medium and other conditions, as in Fig. 1. Additions: PF95, 10 pM, ADP, 100 @LM;DNP, 40 pM to KBW 2/4/98 Table 3.The effectofPF]43 on therespiratiornatesofratlivermitochond?iaunder various metabolicstates. The experirnentaclonditionsand additionsa,s inFi&4. Abbreviationss,eeTable 1. The dataof 5 experimentsarepresentedasmean values S.E. PF143 Parameter mean @S.F- Vini vsa V.t4 RCI ADP:O 15.2 2.7 80.4 5.3 16.3 3.1 5.618 1.5 1.898 0.2 V(PF) Va3(PF) V.t4(PF) RCI(PF) 20.7 3.4 94.6 6.1 17.4 2.0 5.98 t Ou4 ADP:O(pF) V(u ...pler) RC@...pi..) 1.858 0.1 103.0 10.9 7.676 1.3 RCI(pF):RCI, % 105.1 lla6 ADP:O(pF):ADP:O, % 98.1 103 11 KBW 2/4/98 Table 4. Yhe effectof PF95 on therespirationratesof ratliverndtochondriaunder vapious metabolicstates. The expenmental conditionsand additionsa,s inFi&5. Abbreviationss,eeTable 1. The dataof7 experimentsarepresentedasmean values S.E. PF95 Parameter mean SE Vi.i 18.3 0.6 V.0 79.0 2.3 V44 18.0 0.8 RCI 4.434 0.2 ADP:O 1.799 0.1 V(I?F) V93(PF) V.t4(PF) RC@PF) 21.0 1.5 98.3 5.1 18.5 1.9 5.549 0.4 ADP:O(pF) 1.819 0.1 V(,Mwupl..) RC@...pi..) 134.9 9.1 7.38 0.4 RCI(pF):RCI, % 126.23 9.9 ADP:O(pF):ADP:0, % 101.31 2,3 Tables 5 and 6 shows the effectsof PF12L and PF12M on the respiratiornateof mitochondria(seealso Fig.I forPF12L effects)T.hese compounds atlow concentrationsstimulatedboth State3 and State4 respiration, decreasedthe AT (seeFig,I),and decreasedboth RCI and ADP:O ratiosT.hus, PF12L and PF12M affectthe mitochondna ina way, which istypicalfora protonophonc uncoupterlike2,4-dinitrophenoolr,FCCP. As itclearlyseen from Tables 1-6,differenPtF compounds exertdifferenetff" on the energeticsof mitochondna. Low concentrationosf PFIOK PF12L, and PF12M sigmficantlystimulatedthe rateof resting respiratioonf nutochondna. These featuresmchcate that PFIOK PF12L, and PF12M are able to increasethe conductivityof innermembrane of mitochondriatoprotonsor otherions.The suggestionfurthersupportedby the measurements of znitochondru AT. All thesecompounds decreasedthe AT of mitochondriain parallelto stimulatioonf respiration. 12 KBW 2/4/98 Table 5. The effectofPF12L on therespirafiornatesofratlivernutochond?launder va?ious metabolicstates. Theexperimentcaolnditioannsdadditionasi,nFig1,.AbbreviatiosneseT,able1. The dataof 3 experimentsarepr=nted asnmn values SE. PF12L Parameter Vi.i Vd3 V94 RCI ADP:O V(PF) Vst3(PF) V,t4(PF) RC@PF) ADP:O(pF) V(uncoupiff) RC@uncoupler) RCI(pF): RCI, % ADP:O(PF):ADP:O, % mean &E 16.1 1.4 80.3 3.0 15.5 1.5 5.23 0.3 1.797 0.1 38.9 1.3 87.0 3.7 31.8 3.1 2.78 0.3 1.403 0.1 95.8 10.7 6.03 0.8 54.1 7.9 79.1 10.9 PF95 stimulatetdherespiratioinallmetabolicstatest,heeffectresemblingthatofethanolT.he effect couldbe suggestedtoreflecatfluidizatioofninnermitochondriamlembrane, which increasebsoth theprotonleak and theactivitoyfmembrane enzymes.Unfoftmtely,itwas notpossiblteomeasureAT changesinducedby PF95 m nutochondriad,ue tothefactthatthecompound exerteda strongeffecotn TPF" -electrodbeo,thinthe presenceand intheabsenceof nutochondria. Low concentradonosf PF143 were withouteffecton any parameterStudid However,thisistheonly water-solublceompound and itwas possibltehatitspartitiocnoefriciemnatde unfavorabltehedistributioofn PF143 intomitochondriamlembranes.Due tothiswe studiedtheeffectosfhigherconcentratioonfsPF143. Table I shows that100 pM ofPF143 slightlsytimulatebdoththerestinagnd State3 respiratiofnmitochondriaA.t this concentratioPnF.143 was withouteffecotn AT ofmitochondriaT.hisalsocouldbe explainebdy a fluidizatioofn innermitochondriamlembrane, asinthecaseof PF95. 13 KBW 2/4/98 Table 6.The effectofPF12M on therespiratiornatesofrat livermitochondha under various metabolicstates. The experimentalconditionsand additionsa,s inFig.1.Abbreviationss,eeTable 1. The dataof 3 experimentsarepresentedasmean values S.E. PF12M Parameter mean L-& E Vini 15.6 143 vso 77.1 2.0 Va4 15.3 0.4 RCI 5.03 7 042 ADP:O 1.82 7 Owl V(PF) V93(PF) 32.6 1.4 81.9 5.1 Vst4(PF) 27.2 1.5 RC@PF) ADP:O(pF) 3.03 0.2 1.56 0.1 V(-cowler) RC@..pl,.) 95.6 6.4 6.25 0*8 RCI(pF):RCI, % 60.1 3.6 ADP:O(pF): ADP:O, % 85.6 4.6 Low concentrationosf PF10 affectedneithernutochondnal respirationnor AT. ffigherconcentrations slightlystimulatedrestingrespirationa,nd induced a small decreasein AT The substances,which are ableto ma-em the conductivityof the innernutochondnal membrane, could be expectedto uncouple oxidativephosphorylationT.he uncouplingcm be observed as a decreaseinRCI and/or in ADP:O ratioinducedby a compouncl However, the &vm in inmmess of nutochondria=umug due to aging of isolatedorganellesh,eterogeneityof mitochondria,and other factorsdecrease the reproducibilitoyf experiments.Our assayprocedureexplainedabove allowsus to eliminatemost ofthesefactors.Two additionsof ADP tonutochondna, firsitn the absence and the second inthe presenceof a compound under studyprovide an internalstandartthus allowingto compare the effectsof differentcompounds almost independentlyon the variationsinmitochondrialpreparationsF.ig.6compares the effectsof PF compounds on niitochondriaolxidative 14 phosphorylationT.he effectsexpressedas percent changes compounds. KBW 2/4/98 differenPtF RC' and ADP:O inducedby 130 120 110 0 80 70 ou 50 40 30 20 10 0 T RCI [3athalnol IIPFIO NPFIOH [3PF12L IPF12M PF143 OPF95 ADP'O(wmpoundt -- ADP:O Fig.6T.heeffecotfPF compoundsontheefficieonfcoyxidatiPvheOsPhOPylatoifon ratlivemritochon&ia. Thecompositioofnincubatimoendiumandothecronditioanssi,nFig1..For concentrationosfpF compounds, seeTables 1-6. The datashown inFig,6arein a good agreementwiththeeffectosf pF compounds on restingrespiration Of mitochondnaand on the AT. These dataRu-therclariftyhe modes of actionof PF compounds on the mitochon&W energeticsF.ig.6shows thatlow concentrationosf PF12L, PF12K and PF10H decreaseboth the RCI and ADp:o ratiosin mitochondria'.Me actionof thesecompounds on Oxidativpehosphorylatiornesembles thatof classicaulncouplerslikeFCCP or dinitrophenoPlF.143 and PF95, as wellas efl=ol increasedRCI whereas theywere practicallwyithouteffecton ADp:o ratioin mitochondriaT-his does not contradicOtur suggestiondig thesecompounds Increasethe fluiditOyf nut0chOndrialmembrane thusacuvaung theenzymes of of theeffecton ADP:O ratioSuggeststhat r,,e,piratorcyhain.The decreaseinRCI inducedbY PF 10 intheabsence pflo can in== theprotonleak(whichisbelievedtobe absentorinsignificaintState3). 15 KBW 2/4/98 2.The mechanisms oftheeffectosfPF compounds on mitochondrialenergetic. In theprevioussectionwe have shown thatPF compounds affecthemitochondrw energeticisnatleast3 differenwtays. Some compounds, such as PF12L, PF12K and PF10H were shown to decreasethe degree of couplingof ATP productionto oxidationof respiratorsyubstratesO.ther compounds (PF143,PF95) were suggestedto affecthefliuchtoyf nutochondnalmembrane, and PFIO apparentlyexertedan effecton theproton (ion)leakinherento theinnermitochondriamlembrane. We ne3dmade an attempttoinvestigattehe mechanisms involvedintheeffectosf PF compound on mitochondria. To studytheactionofPF compounds on theconductivitoyf mitochondrialmembrane, we tookadvantage of simultaneousmeasurements of respu-auonand membrane potentiaclhanges.Variousconcentrauonsof PF compounds were added tomitochondriaand respiratiornatesand AT changeswere recorded.For a comparison, the same experimentswere performed with a classicaulncoupler-protonophore2,4-dinitrophenoAl.typical recordofchangesinrespiratioand AT inducedby theadditionof2,4-DNP isshown on Fig,3. DNP Mito I DNP DNP DNP increelse [021-0 mito E C3 1 min Fig.3 The effectofva?lous 2,4-DNP concentrafionson the rate of o:@vgenconsumption and A Vlin rat liver mitochondria. hicubationmedium (seeFig.1)was supplementedvhth2 4n-il oligomycineA.ll other conditions were as in Fig.1. The concentrations of 2,4-dinitrophenol (DNP) were as following: 5 gK 5 pK 10 @LK 20 liM (40pM total). 16 KBW 2/4/98 Fig.4 shows thechangeisnrespiratriaotnoefmitochondrpilaotteadgainscthangeisnAT. PFIZL E 40 40 0 ate 0 Aty, v Fig7.h4reelatiboentswhmeiieptnsochomne&mibarlpaonteenatnitdahrleatoef oxygceonnsumipntihpoerneseonfcvearcioonucsentorfaPtcFioomnpsounds. incubamteidoinu(mseFeig.w1a)ssupplemewnitte2hdWrnlohgoinyAclilnoet.her conditionswere as in Fig.1. The respiratioannd membrane potentialof mitochondriawere sequentialltyitratebdy additionsof increasingcmcentrafionsof the PF compoimds. The concentrationwsere as followingP:FIO, 0,62.5,125, 197.5,2.50liM;PFI OR 0, 6.25,12.5,25,50 pK PF12L, 0, 2.08,4.16,8.32,16.64pK PF12L, 0,2.08,416, 8.32,16.64plvt;PF 143,0, 100, 200,300,400 W; DNP, 0,2.22,4.44,8.88,17.76,35.52pM. These experunentrsevealedthatforlow concentrationosfPF12K PF12L, and PF10H theconductivity ofmitochondnalmembrane (representebdy therateofrespiratiorne)lateIsmculy tothemembrane potentiailna rangeof concentratioonfsthecompounds.The same relationshiwpesre shown fora classicaplrotonophore2,4DNP. Tbs indicatetshatthemechanism ofincreasientheinnermembrane conductivitiynvolvesprotonshuttling asitisfirn-deystablishefdora representatiuvnecoupler2,4-dinitrophenHoolw.ever,anotherIdndofexperiments, inparuculatrhedirecmteasurementsof conductivitwyithartificbiialayermembranes,arenecessartyo provethis hypothesis. 17 KBW 2/4/98 PF10 (Fi&5) slightlsytimulatetsherespiratiownith a correspondendtecreasein AT at relativelhyigh concentrat-iohno-wever furtherincreaseisn concentratioonf thiscompound produceno furthercffectI.tcorrelates well with our proposalthatPFIO increasetshe protonleakin mitochondrw membranes. The leakis membrane potentiadlependentm such a way thatslightdecreasesm AT can significantsluyppressthe leak(NichollsDG, 1974). Mito PF1 0 PF1 0 pf:l PFJ 0 PF1 0 DNP PF1 0 increase PF1 0 PF1 0 [021-0 DNP Mito E l min., Fig5..TheeffeocftPF]0 ontherespiratainodnA V/. AllconditiwoenrseasinFig3..FachadditioofPnFIOwas62.5pM 18 KBW 2/4/98 PF143 (Fi&6)in theconcentratiornange 100-400 @M was practicallwyithouteffecton therespiration rateof mitochondriaH.owever, as itclearlyseen in Fi&6, the firstadditionof 100 IAM PF143 iduceda slow decreasein AY of mitochondriawhich continuedto declineb,eingapparentlynotaffectedby furtheradditionsof PF143. In theprevioussectionwe proposethatthiscompound affecttshe fluiditoyf mitochondriamlembrane. Due to thisitcan be expectedthatPF143 might act as a detergentat lugherconcentrationWsh.en higher concentration(sup to I mNI) of the compound were added to niitochondriwae, indeedobserveda transient stimulatioonf respiratiofnollowedby inhibitioonf oxygen consumption,which was insensitivteo 2,4-DNP (data notshown). PF1 43 Mito PF1 43 PF143 PF1 43 1021 A'4' increase PF1 43 mito 0 D E I min Fig.6.The effecotfPF]43 on therespiratioannd A VI. All conditionwsere asinFig.3.Each additioonf PF143 was 100 pK theadditioonfDNP was 40 liM. 19 KBW 2/4/98 PFIOH (Fig.7)at concentrationhsigherthen25 pM stronglyinhibitetdhe respiratioonf mitochondria. These experimentswere repeatedwith a low ionicstrengthmechuin and with the use of anotherrespu-atory substrates,uccinateU.nder theseconditions2,5-50 IAM PF10H alsoinhibitetdhe respiratioTmhis indicatesthat thesiteof inhibitioinslocatedin theregionofubiqtiinone:cytochrocmereductm, which istypicalforartificial uncouplers,or itinhibitcsytochromec oxidase.However, itwas notedthatthe additionof 5 pM cytochrome c to rnitochondrinahibitebdy PFIOH partiallryestoretdherespiratio(nFig.8)I.tiswellknown thatin high ionic strengthmedium, swellingofmitochondriainducesthereleaseofcytochromec and theinhibitioonfrespiration. mito PF1 OH PFI OH PF1 OH PFIOH All, increase -6 1 min Mito Fig.7, The effectofPF]OH on the respirationand A T. Allconditionwsereas infig.3.lle additionosfPFIOH were 6.25,6.25,11.5gM(25 pmtotal). 20 KBW 2/4/98 This pointsto a possibilittyhattheapparentinhibitioonf respiratiobny PF10H isdue to thiscompound inducingtheCa?' -dependentpermeabilitytransitioonf mitochondrialinnermembrane, or so-callepdore openin& This would resultsinhigh amplitudeswellingof mitochondriaand inlossof cytochrome c.To clarifythe effect, we investigatetdhe actionofPF IOH on mitochondriausing differenrtespiratorysubstrateasnd the low ionic Mito PF1 OH DNP 0 -6 E c I min cyt.r. Fig.8T.heeffecotfPFIOH on therespiratioofnratliver?Wtochondria. AllconditioansisnFig.3.AdditionPsF:10H,50pK DNP,40ILM-C,ytc,5 t" strenghtincubationmedium which isknown to decreasetheprobabilityofpore opening In theseexperiments,we used the incubationmedium containing210 mM mannitol 10 mM sucrose, 5mM BEPES-Tris, pH 7.4,and 2 mM MgClz with or without2.4 mM EGTA (indicateidn Figurelegends) to chelatethe residualCe. Glutaniateand malate were used as respiratorsyubstratesin the experiment shown on Fig.8,which impliesthatthe inhibitioonfrespiratiobny PF10H isnot due toitinhibitintghe succinatedehydrogenase.Indeed, therewas no inhibitioonf respiratioenven when the concentrationof PFIOH was100@LM (Fig.9)H.owever, 50 pM PFIOH caused a strongprogressiveinhibitionof the succinate-supporterdespiratioonf mitochondriaand completelydischargedtheAT ifrotenonewas excludedfrom theincubationmediuzn (Fig1.0).This istypicalfora protonophorousuncoupler,the mlulntlonexplainedby the accumulationofoxaloacelicacid (a stronginhibitoorf succinatedehydrogenase)inthe mitochondialmatrix. 21 Mito '5 E 1 min PF1 OH KBW 2/4/98 increase PF10H IC, Mito Fig.9. The effectofPF1 OH on therespirationandd 'Pof ratlivermitochondria respiringon succinate. The incubationmediuln contained210 mM mamlitoll lo, mm sucrose,5mM BEPES-Tris, pH 7.4,2 mM MgCI2, 2.4 mM EGTA, 5 mM succinate2 @Lglmlohgomycine, and 2 @LM rotenone.The concent-atioonfmitochondna was Img/ml- AdchtionsP:FlOli,50 pm 22 KBW 214/98 Mfto PFI OH l min increase PF1 OH DNP M40 Fig.10. The inhibitionof respirafionand the decrease in A W induced by PFI OH in the absence ofrotenone. The incubafion medium and other ronditions were as in Fig.9 for except that rotenone was omift& AdditionsP:F IOIL 50 pK DNP, 40 @M. ' These experimentsallow us toruleout the possibilittyhatPFIOH inhibitsComplex IIIof mitochondrial respiratorychain.However, the cytochrome c - reversiblemlubitionof respiratiomnay also occure due to the displacementof cytochromefrom the mitochondriamlembrane (acetyl-ammortiu-xlnikeeffec@the negative chargescreeningby amphiphilicpositivelcyhargedcompounds) or due tocompetitivienhibitioonfcytochrome bindingtoterminaloxidase(apolylysin-elikeeffect)T.o Ru-thecrL%* thisw,e tookadvantageof a clmical redacmg non-cnzymaticsystemascozbate+ TADD toreduceniitochondricayltochromec.This systemisvery sensitivteoany changesincytochromec bindingand/orinteractiwointhcytochromec oxidaw.Fig.II shows that 50 niM PFIOH decreasetsheAT and stimulatetsherespirationfmitochondrioaxidisinagscorbateT.here isno spontaneousinhibitiofn respirati(oansitwould be observedifPF10H interferweisth cytochromec binding). ThesedatahalherprovethatPFIOH doesnotnimbittherespiratocrhyainperse. 23 KBW 2/4/98 ri:>P* mito TPP+ 1 min TPP+ TPP* T TPP + i Mito PF1 GH PFI OH A41 incre&se I [021-0 Fig.11.77ieeffecotfPF] OH on therespiratiaond A V ofratlivenrdtochondriraespiring on ascorbat+e 7MPD. The incubatimoendiuxncontaine2d10 mM niannito1l0, rrimsucrose5,mM BEPES-Tris, pH 7.4,2 mM MgCI2,2.4 mM EGTA, 5 mM ascorbate1,00 @LM TUPD, 2 @Lg/ml oligomycin,and 2 jiM rotenone.The concentratioofnmitochondriwaas I mg/ml.Additions: PF101-L50 @M The effectosf PF compounds were furtherstudiedin experimentsaimed to revealthe actionofthese compounds on mitochondriailntegritFyo.r this,we investigattehde effectosf high concentratioonfsPF12L, PF12M, PF10,PF95,PF143,andPF10H on theswellinogfmitochondriuandervariousconditionsT.he following datashow thatthecompounds mduce theswellmg and disruptioonfnutochondnaby differenmtecharusms. The swewng of mitochondriwaas measuredinhigh and low ionicstrengtmhedium with or without respiratorsyubstrateasnd bothin thepresenceor in theabsenceof a Ca2' chelatoErGTA- Fig,12 shows,thatthe patternosfPFIOH -inducedswehingwere differenutnderdifferenctonditionsI.nthepresenceofEGTA (Fig12, uppercurve)orsuccinat(edatanotshown)PF10H was lessefficientthanintheabsenceofthesesubstancesT.he efficiencoyfPFIOH was independenotn theionicstrengtohftheincubatiomnedium (datanotshown).Itisclearly seen also,thatthe presenceof EGTA in the incubatiomnedium significantsluyppressedthe high-mnplitude 24 swellinginduced by 25 W KBW 214/98 PFIOII, and changed itskinetics.This indicatesthathigh concerarafionsof PFIOK which were shown toinhibithe respiratioonf mitochondriacan induce Ca2+ -dependentpore opening inthe inner mitochondrialmembrane. Itiswell known thatthe opening of such a pore resultsin uncoupling,swellingand eventualdisruptionof mitochondna. 2 ........................ ............................................................................ ... ................................................................................. PF1 OH ............................................................. PF1 OH PF1 OH ............. ................... ........ ...........P..F.L.M................................................................... 2 4 6 8 10 Time, min Fig.12. The effectof PF] OH on the swellingof mitochon&la Incubation medium was as in Fig.1 for except that glummte and malate were excluded. Upper curve,the incubationmedium was supplementedwith I mM EGTA- h&tochondnal proteinwas I mg x ml4. Each additionofPFIOH was 25 IAM. In contrasto the actionofPFlOI4, the swellingof mitochondriainduced by high concentrationsofPF143 was not affectedby the presenceof EGTA or respiratorsyubstratesH.owever, the dfect of PF143 was strongly dependent on the ionicstrengthof the incubationmediunl Fig.13 shows, thatin a low potassium medium, concentrationsofPF143 as high asabout 4 mM were withouteffecton the swellingof mitochondria. For a comparison,the effectof the strongnon-ionicdetergentTritonX-100 on ratlivermitochondriais shown (Fi& 13).In a potassiumchloridemedium, PFI 43 appearedtobe a strongdetergentwith the efficiencoyf about 1/3relativteothatof Tritom All otherPF compounds (PFIO, PF12L, PF12K and PF95) were withouteffecton the swellingof niitochondria. 25 (D 0 2.0 KBW 2/4/98 c PF1 43, KCI o PF1 43. Mannitol m Triton X-1 00, KCI. a TritonX-1 00. Mannitol 0.a 0 200 ... ... 400 600 Boo 1000 1200 1400 1600 1800 2000 [C@ompound], A g/mg protein Fig.13. The effectof PF]43 on the swelling of mitochondria I-Iighionic strength medium was as in Fi&4, lower curve. nw mannitol -conwning medium was 210 rnM mannitol, 10 mN4 sucrose,4 mM KH2PO4, 10 mM MOPS, pH 7.4.Mtochondrial were added at I mg x mi 26 3. Conclusions KBW 2/4/98 nus study revealsthatPF compounds affectvariousaspectsof mitochondrialenergeticsS.ome of compounds, likePF12L, PF12K PFIOK at relativelyow concentrationdsecreasethedegreeof couplingof respiratorsyubstratoexidationtoATP production.The molecularmechanism of theactionof PF12L and PF12M compounds apparentlyinvolvesthe shuttlinogf protonsacrossinnernutochondriamlembrane, althoughfurther experimentsareneeded.The mechanism of actionof PF10H apparentlyinvolvesthe non-specificchanges in the perxneabihtoyf the innerniitochondriamlembrane. Most probably,thiscompound can change the microdomain structuroef mitochondriahlpid membranes (byinducinglipidclusterinogr by changinglipidphase statefrom a bilayertohexagonal)T.he effectosfPF143 and PF95 aresmallbut significan'tM.ese compounds slightliyncrease the activitoyf enzymes of oxidativephosphotylatioannd respiratorcyhain,the most probablemechanism being the flmchsatioonf the innermitochondriamlembrane. Itmight be expectedthatthe accumulationof such compounds in a tissuceouldresultisntissuedamage. The dataobtainedwithPFIO arethemost interestinfgrom a bioenergetipcointof view. Ifour interpretaiiosn correctand thiscompound increasesthe activitoyf the pathway(s)of protonleakin mitochondriamlembrane, itmay be a very usefultootto studyflow/force relationshiposfvariousenergy-dependentprocessesinniitochondria. 4.Literature cited. Kamo N, Mumtsugu M, Hongoh R, Kobatake Y "Membrane potentiaolfmitochondriameasured with an electrode sensitivteotetmphenylphosphomum and relationshibpetween protonelectrochemicaplotentiaalnd phosphorylatiopnotentiailnsteadystate.J"Membr Biol1979 Aug;49(2):105-121 NichollsDG "The non-Ohmic protonleak--25yearson."BiosciRep 1997 Jun;17(3):251-257 RottenbergH "Membrane potentiaalnd surfacepotentiailn mitochondriau:ptakeand bindingof hpophihc cations.J"Membr Biol1984;81(2):127-138 27 KBW 4/24/98 Supplement 1. Effects of "PF95M" and "Sal" on mitochondrial energetics. All the conditions,procedures, and the logics of experiments were as described in "Materialsand Methode'. Being added at relativellyow concentrations",P-F95W' stimulatesthe rate of respiratioannd decreasesmembrane potentialin ratlivermitochondria (Fig.S-1).Other compound, "Sal",exertsthe same effectsbut at much higher concentration(Fig.S-2). Both thesecompounds decreaseRCI and ADP: 0 ratioof mitochondria(Fig.S-3). For these experiments,the unequal concentrationsof "PF95w' and "Sal" were chosen which approximatelydouble (thatmeans the increaseby 100 %) the State 4 respirationrateof mitochondria (see Fig.S-1 and Fig.S-2).An average RCI in these experiments was about 4.16 (416 % increasein respiratiornateunder additionof ADP) so 100 % increaseinduced by "PF95?X' or "Sar' could not mask the State4 - State3 - State 4 transitioannd RCI and ADP:O estimation.At these concentrationsb,oth "PF95Nf' and "Sal" decreasedRCI (by - 40% and - 30%, respectivelyc,omparing to abs. ethanol controlincubation)and ADP:0 ratio(by - 12 % and - 10 %, respectivelyc,omparing to ethanol controlincubation).Thus, both these compounds are uncouplers of oxidative phosphorylation,although of differentefficiency',TF95Nf' being about four hundred . times more strong,then "Sal' These experiments were repeatedwith the use of low ionicstrenghtmedium (225 mM mannitol, 5 mM Hepes (pH 7.4),4 mM KH2P04, 5 mM glutamate, and 5 mM malate).With the use of thismedium, qualitativeltyhe same resultswere obtained, however the concentrationdsoubling the respiratiownere I W for "PF95m', and 400 for"Sal"(datanot shown). In order to revealthe putativemechanism of uncoupling,we compared the effects of differentconcentrationsof "PF95NT' and "Sal" on mitochondrialrespirationand membrane potentialwith these of a "classical"uncoupler 2,4-dinitrophenolT.he changes in respirationrate and in membrane potentialwere recorded simultaneously.A typical record example isshown by Fig.S-4. + ADP TPP II mito PF95M ADP DNP KBW 4/24/98 [021 0 -5 E CI>Rr 1 min i0 Fi&S-1.Effectof ''PF95M"on therateof respiratioannd membrane potentiaolf mt hvernutochondna. Medium composition and other conditionswere as in Fig.1. Red curve, the respirationof mitochondria;blue curve, the changes in membrane potential.Additions: TPP' or "*", 0.2,0.2,0.4,0.8,and 0.2 gM T?P'Cl- (2 @tM, total);Mito, I mg/ml rat liver mitochondria;ADP, 200 gM ADP; PF95K 0.5 gM "PF95MOI; DNP, 40 liM 2,4dinitrophenol. 2 + TPP ADP I I Mito KBW 4/24/98 Sal ADP DNP C:) -a E 1 min [021":0 Fig.S-2.Effectof "Sal" on the rateof respirationand membrane potentialof ratliver mitochondria. Medium composition and otherconditionswere as in Fig.1.Red curve,the respiratioonf mitochondria;blue curve,the changes in membrane potentialA.dditions:TPP+ or "*", 0,2,0.2,0.4,0.8,and0.2liMTWCI- (2@LK total)h;fitoI,mg/ml ratlivemritochondriAaD;P, 200 IAM ADP; SaL 200 pM "Sal";DNP, 40 pM 2,4-dinitrophenol. 3 KBW 4/24/98 13$ 116 lip 00 TO 160 lm 0 4D 4n r,n 20 RCI ratios ADP:O ratios Fig.S-3.Effectsof low concentrationsof "PF95M" and "Sal"on the degree of coupling and efficiencyof oxidativephosphorylationin ratlivermitochondria. Respiratorycontrolindexes(RCI) and ADP:O ratiosbeforeand afteradditionof "PF95M" or "Sal"to mitochondrialsuspensionwere measured as describedin "Materials and Methods" (see Fig.S-1,Fig.S-2).Each column representsaveraged data from 4 experiments and errorbars show S.E. See textfor furtherexplanations. 4 KBW 4/24/98 TPP + I Mito PF95M I PF95M PF95M PF95M PF95M PF95M [021 =0 C,4 0 E lcq 1 min Fig.S-4. Typical record of changes in respirationrate and membrane potentialof mitochondria induced by sequentialadditionsof "PF95M". Medium composition and other conditionswere as in Fig.1. Red curve, the respirationof mitochondria;blue curve,the changes in membrane potentialA.dditions: T?P+ or "*",0.2, 0.2,0.4,0.8,and 0.2 pM TPPCL- (2 AM, total)M;ito, I mg/ml ratliver mitochondria;PF95K 0.5,1, 1, 1,2, and 2 @LM (7.5@tM total)"PF95M". 5 KBW 4/24/98 The valuesof membrane potentiaalnd the ratesof respiratiownere calculateads describedin"Materialand Methods"'.Fig.S-5 shows theeffectof differenctoncentrations ofallthreecompounds ("PF95W', "Sal"'a,nd dinitrophenolo)n mitochondriarlespiration and Fig.S-6show thechangesinmembrane potentiapllottedagainstchangesin loo- 60- C4 C) 40 - 20- o DNP. gM 6 PF95M. gM 0 Sal,gm x 100 0 0 io 20 30 [uncoupler] Fig.S-5.The increaseinrespirationrateof ratlivermitochondria induced by "PF95Nf', "Sal",and 2,4-dinitrophen(oDlNP). Allthe conditionwsere as in Fig.S-4.Note thatthe concentratioonf "Sal" is expressedasVM x 100(10-4M). respirationF.ig.S-5showsthe differeunntcouplinegfficienocfy thecompounds, "PF95W' and "Sal"beingthe most and thelesspotentthen"classicald"initrophenol, respectivelyF.ig.S-6shows thatallthesecompounds atappliedconcentrationdso not inhibitherespiratorcyhainof mitochondria. 6 KBW 4/24/98 170 > 14.0 s ISO 140 130 o DNP 0 PF95M 0 Sal 0 izo 110 too 90 to 70 0 191 29 391 40 99 p 79 to 90 100 110 129 wapiraficm mte, nn3d ()2 x mm x mg pvftin Fig.S-6.Changes in membrane potentialof mitochondriaplottedagainstchangesin respiratiroanteinducedby differenctoncentratioonfs 'TF95Nf',"Sal",and 2-4- dinitrophen(oDlNP). Conclusion. Our experimentrsevealetdhatthecompound named "PF95m' isa verypotent uncouplerof oxidativpehosporylatioin livermitochondriaT.he uncouplingefficiency ofthiscompound iscomparablewiththatofCCCP, oneofpotent"classicauln"couplers. Anothercompound,"Sal",alsoappearedto be an uncoupler,althoughof relativeloyw efficiencTyh.e relationshbieptween changesinmembrane potentiaalnd increaseinthe rateof respiratiofn mitochondriallowsus to proposethe increasein proton permeabilitoyfinnermitochondriamlembrane asthemechanism ofuncouplingactionof thesecompounds.Furtherexperimentsareneeded to elucidattehe mechanism of the uncouplingatthemolecularlevel. 7