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FEBS Volume 795. number 1.1,3, 51-54 0 1991 Fcdcra:ion or European Uncoupling Biochemical Socictics 10502 December effect of fatty acids on heart muscle mitochondria submitochondrial particles V.I. Dedukhova’, E.N. Mokhova’, 1991 00145793/9l/S3.50 V.P. Skulachev’, A.A. Starkov’, and V.A. Bobyleva3 and E. Arrigoni-Martel!i? ’Dcpartrmwr qf Bioettergerics, A.N. Belozersk_v htitm of Pll~sico-Ci~er,ricaI Biology. Moscow State Uttiwrsity, Moscow USSR, ‘Siptu- Tall, Potncsia, Ronru. Italy and ‘ltrstitutc of Gcrrcral Patlrolqy. Modctta, Ilaly. Received IO October 119899. 1991 The effect oiATP/ADP-antiporter inhibitors on palmi1atc-induced uncoupling was studied in heart muscle mitochondria and inside-out submitochondrial particles. In both systems palmitatc is found to decrease the respiration-generated membrane potential. In mitochondria. this effect is spccitically abolished by carboxyatractylatc (CAtr) a non-pcnctrating inhibitor ofantiporter. In submitochondrial particles, CAtr does not abolish the palmitatc-induced potential decrease. At the same time, bongkrckic acid, a penetrating inhibitor ofthc antiporter.supprcsscs the palmitatceffcct on the potential both in mitochondria and particles. Palmitoyl-CoA which is known to inhibit the antiporter in rnitochondria as well as in particles decreases the palmi:ate uncouplingcfficiency in both these systems. Thcsc data arc in agreement with the bypothcsis that the ATP/ADP-antiporter is involved in the action of lice l’atty acids as natural uncouplers of oxidativc phosphorylation. Uncoupling; Fatty acid; ATPIADP-antiportcr; 1. INTRODUCTION Fatty acids are known to differ from classical protonophore uncouplers in that they penetrate through a phospholipid bilayer only in their protonated form. Their anions do not penetrate the bilayer [1,2]. This is why they fail to uncouple, i.e. to increase the I-I’ conductance, in cytochrome oxidase protcoliposomes [2]. To explain the mechanism of fatty acid uncoupling in mitochondria. we suggested that there are protein(s) in the inner mitochondrial membrane that facilitate transport of the fatty acid anionic species [2-51. In brown fat, which is specialized for heat production by means of fatty acid-mediated uncoupling, the role of fatty acid anion porter was postulated to be performed by the ‘uncoupling protein’ thermogenin [5]. Recently this suggestion seems to have been confirmcd by Jczek and Garlid [6,?] who found that thermogenin transports various monovalent anions which can substitute for fatty acids in actuating thermogeninAbbr~sihms: dy/. transmcmbranc electric potential difrcrcncc; Atr, atractylate; USA. bovine serum albumin; CAtr. carboayatractylate; DNP. 2,4-dinitrophenol; EDTA, cthylencdiaminc-N,~V,WN’-tctraacetic acid; EGTA. cthylcnc glycol-his@aminoethyl cthcr)rV,IV,N’,N’-tctraacetic acid; FCCP, p-trifluoromcthoxycarbonylcyanide phcnylhydrazonc; MOPS, morpholinopropanc sulphonate: PCB-, phcnyldicarbaundccaboranc; SDS. sodium dodccyl sulfalc; TPP+. tctraphcnyl phosphonium C’orrqwmkm-e r~r/dress:Y.P. Skttlnchcv, Department oT Biocncrgctics, A.N. Bclozcrsky lnsthu1c oTPhysico-Chemical Biology. Moscow State University, Moscow 119899, USSR Heat L mitochondria; Submitochondrial particle linked H--conducting activity. The anion efficiency was shown to increase with the increase in the length of hydrocarbon chain of the anion. Thermogenin is absent from tissue other than brown fat, but nevertheless fatty acids can uncouple in these other tissues (for reviw, see [S]). We assumed [2,5] that in these cases the role of the fatty acid anion porter is performed by ATP/ADP antiporter, a protein which is very similar to thermogenin in amino acid sequence, domain structure, and some other properties [8.9]. It was suggested that the adenine nucleotide anion-translocnting machinery of the antiporter can transport hy drophobic anions without the involvement ATP’(ADP”-)-specific gate [2,5]. In agreement with this hypothesis, the following observations were made by our group. (1) The ATP/ADP antiporter inhibitors (CAtr. Atr, bongkrekic acid and pyridoxal 5-phosphate) and its substrate (ADP anion) rpecifica!!y inhibit fatty acidinduced uncoupling in liver and skeletal muscle mitochondria, while having no effect on FCCP-induced uncoupling [2-4]. (2) CAtr-sensitive uncoupling is inherent not only to fatty acids but also to other hy&ophobic monovalent anions such as dodecyl sulfate and cholate [IO]. (3) The burst in heat production during arousal of the hibernating ground squirrels is accompanied by (i) appearance of CAtr-sensitive uncoupling in liver, heart and skeletal muscle mitochondria and (ii) an increase of the free fatty acid concentration in tissues and isolated mitochondria [l l-121. The observation (1) was recently confirmed and extended by Schonfeld 1131who showed that the uncou- 51 Volume 295. number 12.3 FEBS LETTERS December I99 I pling activity of e Patty acid wries in different tissues in proportion to the ATP/ADP antiporter content (heart > kidney > iiver). In this paper, we compared the uncoupling effect of fatty acids in heart muscle mitochondria and inside-out submitochondrial particles. 2. MATERIALS AND METHODS hlitochondri;: were isolated from rabbit or heart muwle. Chilled muscle tissur. previously scparalcd ti-om 1;lt and tendons. wcx minced and then passed throu_rh u staicless-steel press with holes oi about I nm in diamtcr. The tissue was then homogcnizcd using a Teflon pcstlc in a gl;~ss (Pvrcs) homogcnircr Ibr about 3 min. The tissuciisolation medium radio WIS I :8. After the first ccntril’ugalion (IO min. 600~s). the supcrnalant was decanted and filtcrcd through 3 layers of g;~uzc. The filtrate was cermifugcd (IO min. I2 000x&. the pcllct resuspcndcd in I ml of ~hc isolation medium (250 mM sucrose, IO mM MOPS. I mM EDTA. pH 7.4) supplemcntcd with BSA (3 m&ml). and then medium wilhoul BSA was added. The final mitochondrial prccipitalc (IO min, I:! OOOxg)was suspended in isolation medium with BSA. The mitochondrial suspension (iO-90 mg/ml) was stored on ice. Inside-out AS-type submitochondrial particles wcrc prcparcd according to Kollar and Vinogradov [l4]. In the majorhy or cxpcrimcnts. the incubation medium contained 250 m&l sucrose. IO mM MOPS. ;lnd 0.5 mM EGTA. .aH 7.4. Glutamate (5 mbl) and malatc (5 mill) or succinatc (5 mM) wre used as substrates. I PM rotcnone was added IO the medium wh:n succinatc oxidation ws studied. Oxygen consuniption was rrcordcd with a Clark-type oxygen clcctrode and LP-7E polarograph. The incubation misturc was stirred with glass-covcrcd magnetic stirring bar. The concentration of mitochondrial protein WIS I mg/ml (glutamate plus ~nelt~tc)or 0.4 m$ml (succinatc); the temperature was 26°C. The sycthaic pcnetraling ions (TPP‘ and PCB-) wcrc used as dy probes [l5]. The penetrating ion concentration in solution was measured with TPP’ and PCB-- sensitive electrodes [IG]. Oligomycin. blOPS. palmitic acid. palmitcgl-CoA. CAtr. Atr and Dug’ acid-l’rcc BSA wcrc from Sigma; EDTA, EGTA, ADP, GDP, rotcnonc. palmitoyl-L-carnitinc and DNP rrom Scrva; malats from Fluka; FCCP rrom Bochringcr. 3. RESULTS AND DISCUSSION As seen in Fig. I, curves a-c, CAtr was strongly inhibitory for respiration of rat heart mitochondria, stimuiated by paimirare in the presence of OiigOt@ii. The inhibition was relcascd by DNP. ADP (curve c), Atr (curve d), palmitoyl CoA and bongkrekic acid (not shown) were also inhibitory but their effects were not as pronounced as that of CAtr. Moreover, CAtr added after Atr failed to inhibit the palmitate-stimulated respiration as strongly as without Atr (curve d). CAtr-sensitive stimulation of respiration could also be induced by SDS but at a concentration higher than that of palmitate (curves e and f). Pnlmitoyl carnitine could not substitute for either palmitatc or CAtr (not shown). Stimulation of the mitochoncirial respiration by palmitate was accompanied by a dty decrcasc which was completely or partially reversed by CAtr or bongkrekic acid, rcspectivcly (Fig. 2). 52 Fig. I. Efrccls of CAtr. Air and SDS on 111~respiration or heart mitochondria. Incubation mixlurc: 0.25 A1sucrose. IO mM MOPS.0.5 mbl EGTA. 5 mXl p.lutamatc, 5 mM :na!atc, oligomycin (I ,@ml). BSA (0.2 mgml). pH 7.4. 27°C. Additions. mite. a-d. rat heart mitochol:dria (I mg protein/ml): c.l: rabbit heart mitochondria (I mg protein/ml): Palm. 30~1kf palmitic acid; 2pXl CAtr; 5p.M Air; 40,uM AD!? 90 phi SDS: 5 PM DNP (a-d) ;III~ 8 PM DNP (c,f). Figures above cuwcs. respiration ralcs (nmol 0: x min-’ x mg prolcin-‘). Inside-out beef heart submitochondrial particles were also found to respond to the p;llmitate addition by a lowering of 4,~. However, in this case CAtr did not SUppXSS ihC action Of pLliXitZte. On tile other hand: bongkrekic acid suppressed the palmitate effect. Again, as in mitochondria, subsequent addition of an artificial protonophore (FCCP) caused a dly decrease (Fig. 3). Palmitoyl CoA proved to decrease the palmitate effect in both rnitochondria and submitochondrial pnrticles (not shown). The data presented above clearly indicate that membrane sidedncss is critical for CAtr inhibition of palmiLate-induced uncoupling in heart muscle mitochondria and their inside-out particles, CAtr effectively abolishes the uncoupling in the milochondria but not in the particles. On the other hand, some inhibiting effects of bongkrckic acid and palmitoyl-CoA were seen in both mitochondria and particlcs. Volume 295, number 1,2.3 FEBS December 1991 LETTERS 6 Palm OS25 0125 s 0.25, zc c & c.5 %c 7 % L!zi 0.25 0.5 1.0 Fig. 2. Efl’ccls oTCArr and bongkrckic acid (BA) on palmitawinduced dl dccrcasc in mitochondrin. Incubation mixture: 0.25 M sucrose. 10 mhl MOPS. 0.5 mM EGTA. IO mM succinatc. I pM rownonc. oligo:llyc;:r (2,@ml). BSA (0.4 m/ml). l.SpM TPP’. pH 7.4. 26°C. Additions. mite. rat hoart mimchondria (0.8 m_cprolcin/ml); I phi C&r: 20 @I DNP: 5 PM B& 30 phi (A) and 20 @l (B) palmitic acid. Just these relationships could be predicted assuming that it is the ATPlADP antiporter that mediates the uncoupling effect of free fatty acids. It is known that the CAtr and bongkrekate binding sites of the antiporter arc localized on outer and inner surfaces of the inner 3.0 -I succinate 2 3.0 1 s 2.0. z I!% l.O0.5 Fig. 3. Efrccts of bongkrekic acid. CAtr and FCCP on palmilatcinduced dy dccrcasc in subnlitochondrial parliclcs. lncubalion mixture: 0.25 M sucrose, IO mM MOPS, 0.5 mM EGTA, and submilocbondrial parliclcs (0.45 mg protcinlml); I PM rolenone. oligomycin (4ggh1l), I5 AM PCB-, p1-I 7.3, 26°C. Additions, 5 mM succinalc. I ,uM CA& BA. 8 PM bongkrckic ilcid; palm. 30 @l palmilic acid; I /rM FCCP. mitochondrial membrane. respectively, whereas sites for palmitoyl-CoA are on both membrane surfaces. It is also found that CAtr cannot penetrate through the membrane whereas bongkrekic acid can (for review see [W. Thus comparison of heart mitochondria and particles gives one more piccc of evidence that the ATP/A.DP antiporter is somehow involved in the fatty acid uncoupling. The results described for heart mitochondria confirmed our previous observations made with liver and skeletal muscle mitochondria [2-4], i.e. among the specific inhibitors of the antiporter, CAtr is the most potent agent for suppressing fatty acid-induced uncoupling, whereas ADP, Atr. palmitoyl CoA and bongkrekic acid are less effective. Atr added before CAtr prevents, at least for some time, complete inhibition of the palmitate-stimulated respiration by CAtr (see also [2]). It should be emphasized that the Atr concentration used was quite sufficient to completely inhibit oxidative phosphorylation. A probable explanation for this fact is t!~at a!! !!re tested inhibitors combine with the interface-localized, substrate-specific antiporter gates rather than with the non-specific anion-translocating mechanism inlaid in the membrane core. Apparently, modification of gate decelerates the translocator mechanism and, as a result, inhibits the fatty acid anion-translocation rate. This deceleration may vary depending on the kind of inhibitor. nckrro~~lc~l~or~orfs: The authors arc very gralcful lo Prof. A.D. Vinogradov for providing us with a preparation oTsubmilochondrial particlcs and to Sigma-Tay Ibr financial support. REFERENCES [I] W;dwr, A. and Gulknccht. J. (1984) J. Mcmbr. Biol. 77.355-261. 53 Volume 295, number 123 FEBS LETTERS Andrcyev. A.Yu.. Bondareva, T.O.. Dedukhova. V.I., Mokhova. E.N.. Skulachev. V.P.. Tsofina, L.M.. Volkov. N.I. and Vygodina. T.V. (19S9) Eur. J. Biochem. 182. 585-592. [31 Andrevcv. A.Yu.. Volkov. N.I.. Mokhova. E.N. and Skulachev, V.P. (1987) Biol. .Membrany 4,474-178 (Russ.). PI Andrcyev. A.Yu.. Bondarcva. T.O.. Dcdukhova. V.I.. Mokhova, E.N.. Skulachev. V.P. and Volkov. N.I. (1987) FEBS Lett. 226. 265-269. PI Skulachcv. V.P. (1988) Mcmbranc Bioenergetics. Springer. Berlin. Garlid, K.D. (1990) Biochim. Biophys. Acta 1018. 151-154. it; Jezck. P. and Garlid. K.D. (1990) J. Biol. Chem. 265. 1930319311. Bl Aquila, H.. Link, T.A. and Klingcnberg. M. (1985) EMBO J. 4. 2369-2376. PI Bouilland. F.. Weissenbach. J. and Ricquier. D. (1986) J. Biol. Chem. 261, 1487-1490. PI 54 December 1991 [IO] Brustovctsky. N.N.. Dedukhova. V.I.. Yegorova. M.N., Mokhow E.N. and Skulachcv. V.P. (1990) FEBS Lctt. 272, 187-189. [I I] Brustovclsky. N.N., Amerkhilnov, Z.G., Ycgorova, M.N., Mokhova. E.N. and Skulachcv. V.P. (1990) FEES Leti. 272, 190-192. [I21 Brustovcrsky. N.N.. Amcrkhanov. Z.G., Yegorova. M.N.. MOkhova. E.N. and Skulaclw. V.P. (1991) Biokhimiya 56, 947-953 (Russ.). II 31 Schonfeld. P. (1990) FEBS Lett. 264, 246-24R. .-. [l4] Kotlar, A.B. and Vinogradov, A.D. (1990) Biochim. Biophys. Acta 101% 151-158. [IS] Grinius. L.L.. Jasaibs. A.A.. Kadziauskas, J.P., Libcrman, E.A., Skulachcv. V.P., Topali, V.P. and Vladimirova. %l.A. (1970) Biochim. Biophys. Acla 2lG, l-12. [I61 Kamo. N.. Muratsuga, Y., Hongoh, R. and Kobalake, Y. (1979) J. Membr. Biol. 49, 105-121.