Multiple effects of DiS-C 3 (5) on mitochondrial structure and function Takenori Yamamoto 1,2 , Aiko Tachikawa 1,2 , Satsuki Terauchi 1,2 , Kikuji Yamashita 3 , Masatoshi Kataoka 1 , Hiroshi Terada 4 and Yasuo Shinohara 1,2,5 1 Institute for Genome Research, 2 Faculty of Pharmaceutical Sciences and 3 School of Dentistry, University of Tokushima, Japan; 4 Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda, Japan; 5 Single-Molecule Bioanalysis Laboratory, National Institute of Advanced Industrial Science and Technology, Takamatsu, Japan 3,3¢-Dipropyl-2,2¢-thiadicarbocyanine iodide [DiS-C 3 (5)], often u sed as a tracer dye t o assess t he mitochondrial mem- brane potential, w as investigated in detail regarding its effects on the structure and function of isolated mitochon- dria. As reported previously, DiS-C 3 (5) had an inhibitory effect on NADH-driven mitochondrial electron transfer. On the contrary, in the presence of inorganic phosphate, DiS-C 3 (5) s howed dose-dependent biphasic effects on mito- chondria energized by succinate. At higher concentrations, such as 50 l M ,DiS-C 3 (5) accelerated m itochondrial oxygen consumption. Measurements of the permeability of DiS- C 3 (5)-treated m itochondrial membranes to poly(ethylene glycol) and analysis of mitochondrial configuration by transmission electron microscopy revealed that the a cceler- ating effect of DiS-C 3 (5) on mitochondrial oxygen con- sumption reflects t he induction of the mitochondrial permeability transition (PT). When the mitochondrial PT was induced by DiS-C 3 (5), release of mitochondrial cyto- chrome c was observed, as in the c ase of t he PT induced by Ca 2+ . O n t he contrary, at a low concentration such as 5 l M , DiS-C 3 (5) showed an inhibitor y effect on the latent oxygen consumption by mitochondria. This effect was shown to reflect inhibition of the PT induced by a low concentration of Ca 2+ . F urthermore, in t he absence o f inorganic phosphate, DiS-C 3 (5) caused mitochondrial swelling. Under this condition, DiS-C 3 (5) caused changes in the membrane status of the mitochondria, but did not induce a release of mitochondrial cytochrome c. Keywords: cyanine dye; cytochrome c;DiS-C 3 (5); mito- chondria; p ermeability transition. The mitochondrial inner membrane is highly impermeable even to small solutes and ions. However, under certain conditions, such as in the presence of Ca 2+ and inorganic phosphate (P i ), the inner mitochondrial membrane becomes permeable to solutes and ions up to 1500 Da. This phenomenon is referred to as the mitochondrial permeab- ility transition (PT), and PT is believed t o refle ct the opening of a proteinaceous pore [1–3]. In the field of biochemistry, cyanine dyes are often employed as an indicator dye to assess the mitochondrial membrane potential [4,5]. In our previous studies, we characterized the e ffects of cyanine dyes such as 2,2¢-{3- [2-(3-butyl-4-methyl-2-thiazolin-2-ylidene)ethylidene]pro- penylene}-bis(3-butyl-4-methyl thiazolinium iodide) [ TriS- C 4 (5)] and 2,2¢-{3-[2-(3-hepty l-4-methyl-2-thiazolin-2-ylid- ene) ethylidene] propenylene}-bis(3-heptyl-4-methyl thiazo- linium i odide) [TriS-C 7 (5)], both of which have three heterocylic groups, on mitochondrial structure and func- tion. These cyanine dyes accelerated mitochondrial oxygen consumption only in the presence of P i in the incubation medium [6–8]. Furthermore, the accelerating effects of these cyanine dyes on the mitochondrial oxygen consumption were attributable mainly to the induction of the mito- chondrial PT [9]. However, different from the classical P T induced by Ca 2+ , that induced by these cyanine dyes was only partially sensitive to a specific PT inhibitor, cyclosporin A (CsA) [9,10]. On the contrary, a series of cyanine dyes used for measurement of m itochondrial membrane potential such as 3,3¢-diethyloxadicarbocyanine were reported t o show inhib- itory effects on c omplex I of the mitochondrial respiratory chain [11]. Furthermore, more recently, Scorrano et al. reported that chloromethyltetramethylrosamine (Mito- tracker Orang 2 e TM , Molecular Probes, Inc., Eugene, OR, USA), often used to monitor mitochondrial membrane potential in situ, showed both inhibitory effects on respir- atory complex I and PT-inducing e ffects on isolated mitochondria [12]. These results seem to indicate that hydrophobic cations used for measurement of mitochondrial membrane potential have the dual effects of (i) inhibiting complex I and (ii) inducing the mitochondrial PT, even though their chemical struc tures are markedly different from each other. In the present study, to examine the validity of the above Correspondence to Y. Shinohara, Institute for Genome Research, University of Tokushima, Kuramotocho-3, Tokushima 770-8503, Japan. Fax: +81 8 8 633 9146 1 , E-mail: yshinoha@genome.tokushima-u.ac.jp Abbreviations: CsA, cyclosporin A; DiS-C 3 (5), 3,3¢-dipropyl-2, 2¢- thiadicarbocyanine iodide; PT, permeability transition; SF6847, 3,5-di-tert-butyl-4-hydroxy-benzylidene malononitrile; TEM, trans- mission electron microscopy; TriS-C 4 (5), 2,2¢-{3-[2-(3-butyl-4-methyl- 2-thiazolin-2-ylidene)ethylidene]propenylene}-bis(3-butyl-4-methyl thiazolinium iodide); TriS-C 7 (5), 2,2¢-{3-[2-(3-heptyl-4-methyl-2- thiazolin-2-ylidene) ethylidene] propenylene}-bis(3-heptyl-4-methyl thiazolinium iodide). (Received 1 6 June 200 4, accepted 19 July 2004) Eur. J. Biochem. 271, 3573–3579 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04294.x interpretation; we characterized the effects of yet another cyanine dye, 3,3¢-dipropyl-2,2¢-thiadicarbocyanine iodide [DiS-C 3 (5); Fig. 1], on mitochondrial structure and function. Materials and methods Materials DiS-C 3 (5) and cyclosporin A (CsA) were kindly provided by Hayashibara Biochemical Laboratories, Inc. (Okayama, Japan) and Novartis Pharma Inc. (Tokyo), respectively. Preparation of mitochondria Mitochondria were isolated from the liver of normal male Wistar rats, as described previously [13]. 3,43,4 Animals were killed by cerv ical dislocation to a void the effects o f a nesthetics on membrane systems. All animal experiments were performed according to the guidelines for the care and use of laboratory animals of the University of Tokushima. Protein concentra- tions of mitochondrial p reparations were determined b y the Biuret method with bovine serum albumin as a standard. Measurement of mitochondrial oxygen consumption and swelling For measurements of oxygen consumption and turbidity of mitochondria, mitochondria were suspended in +P i medium (250 m M sucrose, 10 m M K/P i 5 buffer, pH 7.4) to make their final protein concentration of 0 .7 mgÆmL )1 . Then, they were energized by the addition of either 10 m M succinate (plus 0.5 lgÆmg )1 protein rotenone) or 10 m M glutamate and 10 m M malate as respiratory substrates. Rates o f m itochondrial oxygen consumption a t 2 5 °Cwere measured by use of a Clark oxygen electrode (YSI 5331;Yellow Springs Instrument Co., Yellow Springs, OH, USA) 6 . When the inhibitory effects of D iS-C 3 (5) on the mitochondrial oxygen consumption were evaluated, the protonophoric uncoupler 3,5-di-tert-butyl-4-hydroxy-ben- zylidene malononitrile (SF6847) was utilized to induce maximum oxygen consumption. Mitochondrial swelling was monitored at 25 °C by measuring the t urbidity of the reaction mixture at 440 nm with a Shimadzu dual-wave- length spectrophotometer, model UV-3000. WhentheeffectofP i was examined, experiments were performed using –P i medium (200 m M sucrose, 10 m M KCl, 10 m M Tris/Cl buffer; pH 7.4) instead of +P i medium. Measurement of permeability of mitochondrial membrane to poly(ethylene glycol) To examine the permeability of the mitochondrial mem- brane, we measured the effects of poly(ethylene glycol)s of various molecular sizes on the turbidity of mitochondrial suspensions, as d escribed by Pfeiffer et al. [14]. Briefly, mitochondria were first treated with a certain r eagent; and then, after complete induction of swelling, 1.1 mL of 300 mOsmol solution of poly(ethylene glycol) of a given molecular size was added. Changes i n the turbidity o f reaction mixture were monitored at 4 40 nm. Analysis of mitochondrial configuration by transmission electron microscopy Transmission electron microscopy (TEM) a nalysis of mito- chondria under various conditions was performed, essen- tially as described p reviously [13], using an Hitachi electron microscope model H-800MT. Release of mitochondrial cytochrome c To assess whether cytochrome c is released from mitochon- dria, we t reated mitochondria with DiS-C 3 (5)inanoxygen chamber at 25 °C as stated above. After certain periods of incubation, a 500 lL aliquot of the reaction mixture was taken i nto an E ppendorf tube, a nd the mitochond rial pellet and supernatant were obtained by prompt centrifugation 7 at 15 000 g for 2 mins at 4 °C. After complete removal of the supernatant, the mitochondria were resuspended in the original volume of incubation medium. Two microliters of mitochondrial suspension and 5 lLofsupernatantwere subjected to SDS/PAGE and subsequent Western analysis using a specific antibody against cytochrome c,preparedas described previously [13]. Results Effects of DiS-C 3 (5) on the rate of mitochondrial oxygen consumption DiS-C 3 (5) was reported to show inhibitory effects on the mitochondrial NAD-linked respiratory system [15]. A s shown in Fig. 2 , we confirmed the inhibitory effect of DiS-C 3 (5) on the glutamate/malate-driven mitochondrial electron transfer. Under the experimental conditions used, its concentration producing 50% inhibition (IC 50 ) 8 was about 8 l M . The observed inhibition of the NAD-linked respiratory system seemed to reflect a direct effect on complex I and was not attributable to inhibition of the transport s ystem o f t he respiratory substrate, because similar e ffects were also obtained when freeze/thawed mitochondria were used (data not shown). When succinate was added to mitochondria as the respiratory substrate, even in the absence of DiS-C 3 (5), slow oxygen consumption was observed, reflecting oxida- tion of the respiratory substrate to compensate for the leakage of H + across the inner membrane. Furthermore, this slow oxygen c onsumption g radually accelerated during the incubation period, possibly due to the i nduction of the PT by endogenous Ca 2+ (Fig. 3 , broken line). Upon addi- tion of DiS-C 3 (5) to the mitochondria energized by succi- nate, two oppo site actions were observed , depending on the concentration. The addition of DiS-C 3 (5) £ 10 l M caused deceleration of mitochondrial oxygen consumption but >20 l M caused acceleration. These actions of DiS-C 3 (5) on Fig. 1. Chemical structur e of DiS-C 3 (5). 3574 T. Yamamoto et al.(Eur. J. Biochem. 271) Ó FEBS 2004 mitochondria energized by succinate were further charac- terized, as described below in the f ollowing sections. Characterization of mitochondrial PT induced by DiS–C 3 (5) In our previous studies, cyanine dyes such as TriS-C 4 (5) were found to accelerate mitochondrial oxygen consump- tion [6–8]. These actions of cyanine dyes were attributable mainly to the results of induction of the mitochondrial PT [9,10]. Thus, acceleration of mitochondrial oxygen con- sumption by DiS-C 3 (5) was expected to be due to the induction of mitochondrial PT. To validate this interpret- ation, we further characterized the actions of DiS-C 3 (5) on the mitochondrial s tructure and function and com- pared them with those of Ca 2+ , known as a typical PT inducer. Like that of Ca 2+ , the addition of 50 l M DiS-C 3 (5) to the mitochondrial suspension caused a massive decrease in its turbidity, reflecting induction of mitochondrial swelling (Fig. 4 A). In general, t he induction of mitochondrial swelling is one of the criteria used to judge whether the mitochondrial PT is induced. However, as reported previ- ously, s welling can oc cur e ven under conditions where the mitochondrial PT doe s not occ ur [13]. Thus, permeability of the inner mitochondrial membrane was directly evaluated by measuring the responses of preswollen mitocho ndria to the addition of poly(ethylene glycol) of various molecular sizes. A s shown in Fig. 4B, when m itochondria were Fig. 3. Effects of DiS-C 3 (5) on succinate-driven mitochondrial oxygen consumption. Effects of DiS-C 3 (5) on the oxygen consumption of mitochondria energized by succinate were measured. Experiments were performed as shown in the legend of Fig. 2 except f or use of succinate ( plus 0.5 lgÆmL )1 rotenone) as a substrate i nstead of glu - tamate and malate. Broken line represents the oxygen consumption of nontreated mitochondria. Fig. 4. Effects o f DiS-C 3 (5) on the turbidity of mitochondrial suspen- sions (A) and on permeability of mitochondrial inner membrane (B). (A)Theeffectof50l M DiS-C 3 (5) on the turbidity of mitochondrial suspensions (right trace) was compared with that of 100 l M Ca 2+ (left trace). Experimental conditions are as those described in the legend for Fig. 3, and changes in turbidity of mitochondrial suspension were monitored a t 440 nm. ( B) Permeability of DiS-C 3 (5)-pretreated i nn er membranes of mitochondria to poly(ethylene glycol) of various molecular sizes was evaluated. For this, m itochondria w ere fi rst pre- swollen by C a 2+ (left traces) or by DiS-C 3 (5) (right traces) as stated above. Then, absorbance changes in the mitochondrial suspensions that accompanied the addition of solution s of poly(ethylen e glycol) of various molecular sizes w ere r ecorded at 440 nm. The vertical arrow indicates the addition of a poly(ethylene glycol) solution. Trace ÔaÕ represents the result obtained by the addition of medium not con- taining poly(ethylene glycol), used as a negative control. Traces b–g represent the results observed with the addition of solutions of PEG600, PEG1000, PEG2000, PEG4000, PEG6 000 and P EG10000, respectively. Fig. 2. Inhibitory effects of DiS-C 3 (5) on NADH-driven electron transfer. For evaluation o f the inhibitory effect o f DiS-C 3 (5) o n NADH- driven electron transfer, mitocho ndria were suspended in +P i medium at 25 °C. Then, t h ey were ene rgized by additio n of 10 m M glutamate and 10 m M malate (glu/mal) as respiratory substrates and measured their rates of oxygen consumption. The maximum rate of oxygen con- sumption was induced by the addition of 50 n M SF6847, and this value was utilized as the noninhibited rate o f o xygen c onsumption. I nhibitory effects of DiS-C 3 (5) on electron t ran sfer were evaluated by measuring the rates of oxygen consumption in the presence of both 50 n M SF6847 and various amounts of DiS-C 3 (5). Typical t races of oxygraphs are shown i n (A). Dose–response curve of the effect of DiS-C 3 (5) o n t h e rate of mitochondrial oxygen consumption is shown in (B), in which the results are shown as mean values ± SD 12 of thre e in depe ndent runs (bars of SD are smaller t han t he symbols). Ó FEBS 2004 Effects of DiS-C on mitochondrial structure and function (Eur. J. Biochem. 271) 3575 preswollen w ith Ca 2+ , t he addition of poly(ethylene glycol) having a molecular size of more than 4000 (PEG4000) caused increased turbidity of the m itochondrial suspension, reflecting induction of shrinkage of preswollen mitochon- dria; whereas those smaller than 1000 did not, as reported previously [16]. These results are thought to indicate that the mitochondrial membrane became permeable to the mole- cules smaller than a molecular size of 1500 by the Ca 2+ treatment. When poly(ethylene glycol) solutions were added to the m itochondrial suspensions pretreated with DiS-C 3 (5), massive shrinkage was not observed, even with PEG6000 or PEG10000, indicatin g that the m embrane o f the mitochon- dria treated with DiS-C 3 (5) became permeable to large r molecules than Ca 2+ -treated mitochondria. Furthermore, the results of TEM observation also supported the changes in the permeability of the inner mitochondrial membrane caused by DiS-C 3 (5). Compared with the appearance of nontreated control mitochondria (Fig. 5 A), when mitochondria were treated with Ca 2+ (Fig. 5 B), the mitochondrial inner membrane structure disappeared s ignificantly, a s reported previously [13,17–19]. Mitochondria treated with DiS-C 3 (5) showed essentially the same TEM features as those treated with Ca 2+ (Fig. 5C). These r esults indicate clearly t hat acceleration of mitoch- ondrial oxygen consumption induced by DiS-C 3 (5)at50l M is due to the induction of the mitochondrial PT. However, as stated above, the membranes of mitochondria treated with DiS-C 3 (5) became p ermeable to larger molec u les than the Ca 2+ -treated ones. Furthermore, the increase in the permeability of the mitochondrial membranes caused by DiS-C 3 (5) was only partially sensitive for CsA, known as an inhibitor of the classical PT induced by Ca 2+ (data not shown). T hus, t he PT induced by DiS-C 3 (5) w as concluded to be different from that indu ced by Ca 2+ . The induction of PT is generally believed to b e associated with the release of apoptogenic mitochondrial p roteins such as cytochrome c [20]. Thus, we n ext examined whether mitochondrial cytochrome c would b e released w hen mito- chondria were treated with DiS-C 3 (5). As shown in Fig. 6, treatment of mitochondria with 50 l M DiS-C 3 (5) caused a massive release of cytochrome c,aswellaswithCa 2+ . Inhibition of PT induction by DiS-C 3 (5) at low concentration As stated above, DiS-C 3 (5) at low concentrations preven- ted progression of intrinsic oxygen consumption by mitochondria. However, this effect of DiS-C 3 (5) did not reflect inhibition of the mitochondrial respiratory chain, as the addition of the protonophoric uncoupler SF6847 to the mitochondrial suspension treated with DiS-C 3 (5) at a low concentration caused maximum acceleration of mito- chondrial oxygen consumption as effectively as that observed with mitochondria not treated with DiS-C 3 (5) (data not shown). Based on these results, we considered that the protective effects of DiS-C 3 (5) at low concentra- tions on the progression of intrinsic oxygen consumption by mitochondria might reflect induction of the PT by endogenous Ca 2+ . So next we examined the validity of this interpretation. First, we tested the effect of DiS-C 3 (5) o n the Ca 2+ - induced acceleration of mitochondrial respiration and mitochondrial swelling. As shown in Fig. 7, when D iS- C 3 (5) was added to mitochondria pretreated with 10 l M Ca 2+ , it prevented not only acceleration of oxygen consumption (Fig. 7A) but also the turbidity decrease of mitochondrial suspensions (Fig. 7B), resulting in recovery to the same level as found for the nontreated control mitochondria. The protective effects of DiS-C 3 (5)atalow concentration o n t he spontaneous induction of mitochond- rial PT was also confirmed by observing mitochondria by TEM (Fig. 8). The disappearance of the inner mitochond- rial membrane structure induced by 10 l M Ca 2+ was strongly suppressed by treatment of the m itochon dria with 5 l M DiS-C 3 (5). Thus, we concluded that the inhibitory effect of DiS- C 3 (5) on the spontaneous acceleration of mitochondrial Fig. 5. TEM app earances of mitochondria treated with D iS-C 3 (5). Mitochondria were treated with Ca 2+ or DiS-C 3 (5) as described in thelegendinFig.4andsubjectedtoTEM analysis. ( A) The appearance of n ontreated control mitochondria. (B) and (C) The appearances o f mitochondria t reated with 100 l M Ca 2+ and 50 l M DiS-C 3 (5), respect- ively.Barunder(C)indicates1lmforall panels. 13 Fig. 6. Effects of DiS-C 3 (5) on the release of mitochondrial cyto- chrome c. Release of mitoch ondr ial c yto chr ome c was examined as describedinMaterialsandmethods. Briefly, mitochondria were first treated w ith 50 l M DiS-C 3 (5), then they were precipitated by centrif- ugation. Samples of p ellet ( P) and supernatant (S) were subj ected to Western blotting using specific antibody against cytochrome c.Sam- ples of nontreated mitochondria or of Ca 2+ -treated mitochondria were also analyzed as controls. Typical results of more than three independent experiments are s hown. 3576 T. Yamamoto et al.(Eur. J. Biochem. 271) Ó FEBS 2004 oxygen consumption could b e a ttributable to the i nhibition of spontaneous induction of the PT by endogenous Ca 2+ . However, it should be noted that this inhibitory effect of DiS-C 3 (5) was only observed when the PT was i nduced by a relatively low concentration C a 2+ such as 10 l M ; i.e. it was not observed at a concentration of Ca 2+ such as 50 l M (data not shown). Furthermore, the protective effect of DiS-C 3 (5) on t he Ca 2+ -induced PT was not attributable to the inhibition of C a 2+ uptake (data not shown). Effects of DiS-C 3 (5) on mitochondria in the absence of P i All of the above experiments were performed in +P i medium con taining 10 m M phosphate buffer. However, the PT-inducing effects of Ca 2+ and cyanine dyes such as Tri-S-C 4 (5) are known to be dependent on the absence/ presence of P i in the incubation medium. Thus, it was of interest to us to examine the effects of DiS-C 3 (5) on mitochondrial structure and function in the absence of P i . Where Ca 2+ had no effect on the m itochondrial oxygen 9 consumption in the absence o f P i ,DiS-C 3 (5) moderately accelerated mitochondrial oxygen consumption e ven in t he absence of P i (Fig.9A).Furthermore,50l M DiS-C 3 (5) caused massive swelling even in the absence of P i (Fig. 9B). To examine whether the observed mitochondrial swelling induced by DiS-C 3 (5) in this case was attributable to the induction of the m itochondrial PT, we examined the permeability o f D iS-C 3 (5)-treated mitochondrial mem- branes to poly(ethylene glycol). 10 Unfortunately, no c lear conclusion could be obtained, as mito chondria preswollen by DiS-C 3 (5) did not show any clear response upon the addition of poly(ethylene glycol) (data not shown). However, mitochondria treated with 50 l M DiS-C 3 (5) showed morphology d ifferent from that of the nontreated control ( Fig. 10), strongly suggesting alteration of mem- brane status. Their appearance was also apparently different from that of mitochondria treated with Ca 2+ or DiS-C 3 (5) i n the presence of P i (Fig. 5 B,C). Finally, we also tested whether the release of mitochondrial cyto- chrome c could be induced by DiS-C 3 (5) in the absence of P i . As shown in Fig. 11, when mitochondria were incubated with 50 l M DiS-C 3 (5)intheabsenceofP i , most of the cytochrome c was retained in these mito- chondria as well as in the nontreated mitochondria. Discussion Cyanine dyes a re often used to evaluate the mitochondrial membrane potential [4,5]. However, as reported p reviously, some of them are also reported to show in hibitory effects on NAD-linked electron transfer [11,12] and Ca 2+ -like uncoupling actions [9,12]. DiS-C 3 (5) is often used for measurements of mitochondrial membrane potential, and its methods of interaction with mitochondria have been Fig. 7. Inhibitory e ffects of a low concentration DiS-C 3 (5) on the Ca 2+ -induced P T. To examine the effects of DiS-C 3 (5) on the Ca 2+ - induced PT, we measured its effects o n the oxygen consumption of mitochondria (A) and turbidity change in mitochondrial suspensions (B) treated with 10 l M Ca 2+ in +P i medium. Results obtained without the a ddition of Ca 2+ and DiS-C 3 (5) are shown by broken lines (controls). Fig. 8. TEM analysis of mitochondria treated with a l ow concentration of DiS-C 3 (5). To examine the prote ctive effect o f DiS-C 3 (5) on the Ca 2+ -induced PT, we also observed the electron microscopic appear- ances of mitochondria. (A) and (B) show the appearance of mito- chondria treated with 10 l M Ca 2+ andwithboth5l M DiS-C 3 (5) and 10 l M Ca 2+ , respectively. B ar under (B) indicates 1 lmforallpanels. Fig. 9. Effects of DiS-C 3 (5) on the rate of mitochondrial oxygen con- sumption (A) and turbidity of m itochondrial suspensions (B) in t he ab- sence of P i . Experiment s were performed as described in the legends for Figs 3and4exceptthat–P i medium was used inste ad of +P i medium. Ó FEBS 2004 Effects of DiS-C on mitochondrial structure and function (Eur. J. Biochem. 271) 3577 studied [15,21]. H owever, characterization of its effects with respect to PT induction had not been achieved earlier. Thus, in the present study, we investigated in great detail the actions of DiS-C 3 (5) on the structure and function of isolated mitochondria. First, we confirmed the previously reported inhibitory effects of DiS-C 3 (5) on NAD-linked electron transfer. Th is inhibitory effect was con sidered to reflect its direct action on complex I. On the contrary, when DiS-C 3 (5) was added to the mitochondria energized by succinate, both acceleration and deceleration of oxygen consumption were observed, depending on the concentration of the dye. At higher concentrations such as 50 l M ,DiS-C 3 (5) c aused acceler- ation of mitochondrial oxygen consumption. This effect of DiS-C 3 (5) was further characterized and concluded to be attributable to the induction of the mitochondrial PT. PT induced by DiS-C 3 (5) was associated with release of cytochrome c, as was that induced by Ca 2+ . However, it was d ifferent from the ordinary P T i nduced by Ca 2+ in the aspects of pore size and sensitivity for CsA, known as a specific inhibitor of the ordinary PT. P ossibly, these differences may reflect the differe nces in the features of the proteinaceous PT pores formed. Cytochrome c is one of the components comprising the respiratory chain. Thus, r elease of cytochrome c from mitochondria would be expected to cause deceleration of mitochondrial o xygen consumption. However, as seen with the e ffects of 50 l M DiS-C 3 (5), this was not the case. Release of cytochrome c without causing deceleration of mito- chondrial oxygen consumption was also observed when mitochondria were treated with Ca 2+ or valinomycin [13]. However, under these conditions, at least half of the total cytochrome c still remained in the mitochondria. Possibly, this cytochrome c remaining in the mitochondria was sufficient to account for t he electron t ransfer. Further- more, for the release of cytochrome c, permeability of the outer mitochondrial membrane to cytochrome c must be increased, as cytochrome c is present in the intermem- brane s pace o f mitochondria. Several mechanisms concern- ing the release process of cytochrome c have been proposed, but this problem is still under debate. Until now, there was n o detailed study on the PT-indu- cing effects of c hemicals actually used as a t racer dye of t he mitochondrial membrane potential except f or that on Mitotracker Orange TM [12]. Possibly, induction of the PT is one of the common a ctions of hydrophobic cations that are u tilized as a tracer of mitochondrial membrane poten- tial, as s imilar activities w ere observed with these c hemicals regardless their s tructural diversity [9,12]. Further studies on the actions of a series of hydrophobic cations will be necessary for validation of this interpretation a nd for b etter understanding of the features of the mitochondrial P T. Furthermore, we observed two additional novel effects of DiS-C 3 (5) on mitochondria: (i) inhibition of the Ca 2+ - induced PT by a low concentration of DiS-C 3 (5) and (ii) induction of swelling in the absence of P i .Withrespectto the former feature, attention must be paid to it when this dye is employed as a tracer for mitochondrial membrane potential, a s it shows a protective e ffect on the induction of PT at the concentration utilized for monitoring membrane potentials. For the latter action, DiS-C 3 (5) caused r emark- able swelling a nd changes in t he status of the m itochondrial inner membrane without accompanying release o f mitoch- ondrial c ytochrome c (Figs 9,10,11). F urther studies on the status of the inner membrane of m itochondria treated with DiS-C 3 (5) in the absence of P i may give u s insight into the mechanisms causing configurational changes in mito- chondria. Until now, both CsA-sensitive and insensitive PT have been shown to be a ssociated with the release of mitochond- rial cytochrome c. Recently, however, we reported that mitochondrial cytochrome c could be released even w ithout the induction of the m itochondrial PT [13]; and this observation was supported by another group [22]. Thus, detailed studies on the relationship between PT induction and release of mitochondrial cytochrome c remain to be conducted. In conclusion, we found DiS-C 3 (5) to show multiple effects on the mitochondrial structure and function, effects dependent on both its concentration and the P i status. 11 Acknowledgements This work was supported by grants-in-aid for scientific research (no. 14370746 to Y.S.) from t he Ministry of Education, Science, and Culture of Japan, and a fellowship from Katayama Chemical Industries, Co., Ltd (O sa ka) to T.Y. Fig. 10. TEM appearance of mitochondria in the absence of P i . The effects of DiS-C 3 (5) on the mitochondrial morphology in –P i medium were also exa mined by TEM analysis. (A) and (B) s how t he appear- ance of mitochondria incub ated in the absence an d presence of 50 l M DiS-C 3 (5), respective ly. Bar under ( B ) indicates 1 lm for all panels. Fig. 11 . Effects of DiS-C 3 (5) on the allocation of mitochondrial cytochrome c in the absence of P i . Release of mitochondrial cyto- chrome c was examined as described in the legend for Fig. 6. In addition to the samples of pellet (P) and supernatant (S) of mito- chondria treated with 50 l M DiS-C 3 (5), those of nontreated mito- chondria wer e also analyzed. 3578 T. Yamamoto et al.(Eur. J. Biochem. 271) Ó FEBS 2004 References 1. Gunter, T.E. & Pfeiffer, D.R. (1990) Mechanisms by which mitochondria transport calcium. Am.J.Physiol.258, C755–C786. 2. Zo ratti, M . & Szabo, I. (1995) The mitochondrial pe rmeability transition. Biochim. Biophys. Acta 12 41, 139–176. 3. Bernardi, P. 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