Under this condition, DiS-C35 caused changes in the membrane status of the mitochondria, but did not induce a release of mitochondrial cytochrome c.. Measurement of mitochondrial oxygen
Trang 1Multiple effects of DiS-C3(5) on mitochondrial structure and function Takenori Yamamoto1,2, Aiko Tachikawa1,2, Satsuki Terauchi1,2, Kikuji Yamashita3, Masatoshi Kataoka1, Hiroshi Terada4and Yasuo Shinohara1,2,5
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-C3(5)],
often used as a tracer dye to assess the mitochondrial
mem-brane potential, was investigated in detail regarding its
effects on the structure and function of isolated
mitochon-dria As reported previously, DiS-C3(5) had an inhibitory
effect on NADH-driven mitochondrial electron transfer
On the contrary, in the presence of inorganic phosphate,
DiS-C3(5) showed dose-dependent biphasic effects on
mito-chondria energized by succinate At higher concentrations,
such as 50 lM, DiS-C3(5) accelerated mitochondrial oxygen
consumption Measurements of the permeability of
DiS-C3(5)-treated mitochondrial membranes to poly(ethylene
glycol) and analysis of mitochondrial configuration by
transmission electron microscopy revealed that the
acceler-ating effect of DiS-C3(5) on mitochondrial oxygen
con-sumption reflects the induction of the mitochondrial
permeability transition (PT) When the mitochondrial PT was induced by DiS-C3(5), release of mitochondrial cyto-chrome c was observed, as in the case of the PT induced by
Ca2+ On the contrary, at a low concentration such as 5 lM, DiS-C3(5) showed an inhibitory effect on the latent oxygen consumption by mitochondria This effect was shown to reflect inhibition of the PT induced by a low concentration of
Ca2+ Furthermore, in the absence of inorganic phosphate, DiS-C3(5) caused mitochondrial swelling Under this condition, DiS-C3(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-C3(5); mito-chondria; permeability transition
The mitochondrial inner membrane is highly impermeable
even to small solutes and ions However, under certain
conditions, such as in the presence of Ca2+and inorganic
phosphate (Pi), 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 to reflect 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 effects 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-C4(5)] and 2,2¢-{3-[2-(3-heptyl-4-methyl-2-thiazolin-2-ylid-ene) ethylidene] propenylene}-bis(3-heptyl-4-methyl thiazo-linium iodide) [TriS-C7(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 Piin 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 PT induced by Ca2+, 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 mitochondrial membrane potential such as 3,3¢-diethyloxadicarbocyanine were reported to show inhib-itory effects on complex I of the mitochondrial respiratory chain [11] Furthermore, more recently, Scorrano et al reported that chloromethyltetramethylrosamine (Mito-tracker Orang
2 eTM, 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 effects 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 structures 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 88 633 9146
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 16 June 2004, accepted 19 July 2004)
Trang 2interpretation; we characterized the effects of yet another
cyanine dye, 3,3¢-dipropyl-2,2¢-thiadicarbocyanine iodide
[DiS-C3(5); Fig 1], on mitochondrial structure and function
Materials and methods
Materials
DiS-C3(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]
by cervical dislocation to avoid the effects of anesthetics 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 preparaconcentra-tions were determined by 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 +Pi
medium (250 mMsucrose, 10 mMK/Pi
make their final protein concentration of 0.7 mgÆmL)1
Then, they were energized by the addition of either 10 mM
succinate (plus 0.5 lgÆmg)1 protein rotenone) or 10 mM
glutamate and 10 mM malate as respiratory substrates
Rates of mitochondrial oxygen consumption at 25C were
measured by use of a Clark oxygen electrode (YSI
5331;Yellow Springs Instrument Co., Yellow Springs, OH,
USA)
6 When the inhibitory effects of DiS-C3(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 25C by measuring the turbidity of the
reaction mixture at 440 nm with a Shimadzu
dual-wave-length spectrophotometer, model UV-3000
When the effect of Piwas examined, experiments were
performed using –Pimedium (200 mMsucrose, 10 mMKCl,
10 mMTris/Cl buffer; pH 7.4) instead of +Pimedium
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
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 in the turbidity of reaction mixture were monitored at 440 nm
Analysis of mitochondrial configuration by transmission electron microscopy
Transmission electron microscopy (TEM) analysis of mito-chondria under various conditions was performed, essen-tially as described previously [13], using an Hitachi electron microscope model H-800MT
Release of mitochondrial cytochromec
To assess whether cytochrome c is released from mitochon-dria, we treated mitochondria with DiS-C3(5) in an oxygen chamber at 25C as stated above After certain periods of incubation, a 500 lL aliquot of the reaction mixture was taken into an Eppendorf tube, and the mitochondrial pellet and supernatant were obtained by prompt centrifugation
15 000 g for 2 mins at 4C After complete removal of the supernatant, the mitochondria were resuspended in the original volume of incubation medium Two microliters of mitochondrial suspension and 5 lL of supernatant were subjected to SDS/PAGE and subsequent Western analysis using a specific antibody against cytochrome c, prepared as described previously [13]
Results Effects of DiS-C3(5) on the rate of mitochondrial oxygen consumption
DiS-C3(5) was reported to show inhibitory effects on the mitochondrial NAD-linked respiratory system [15] As shown in Fig 2, we confirmed the inhibitory effect of DiS-C3(5) on the glutamate/malate-driven mitochondrial electron transfer Under the experimental conditions used, its concentration producing 50% inhibition (IC50)
about 8 lM 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 system of the respiratory substrate, because similar effects 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-C3(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 consumption gradually accelerated during the incubation period, possibly due to the induction of the
PT by endogenous Ca2+(Fig 3, broken line) Upon addi-tion of DiS-C3(5) to the mitochondria energized by succi-nate, two opposite actions were observed, depending on the concentration The addition of DiS-C3(5)£ 10 lMcaused deceleration of mitochondrial oxygen consumption but
> 20 lMcaused acceleration These actions of DiS-C (5) on
Fig 1 Chemical structure of DiS-C 3 (5).
Trang 3mitochondria energized by succinate were further
charac-terized, as described below in the following sections
Characterization of mitochondrial PT induced
by DiS–C3(5)
In our previous studies, cyanine dyes such as TriS-C4(5)
were found to accelerate mitochondrial oxygen
consump-tion [6–8] These acconsump-tions 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-C3(5) was expected to be due to the induction of mitochondrial PT To validate this interpret-ation, we further characterized the actions of DiS-C3(5)
on the mitochondrial structure and function and com-pared them with those of Ca2+, known as a typical PT inducer
Like that of Ca2+, the addition of 50 lMDiS-C3(5) to the mitochondrial suspension caused a massive decrease in its turbidity, reflecting induction of mitochondrial swelling (Fig 4A) In general, the induction of mitochondrial swelling is one of the criteria used to judge whether the mitochondrial PT is induced However, as reported previ-ously, swelling can occur even under conditions where the mitochondrial PT does not occur [13] Thus, permeability of the inner mitochondrial membrane was directly evaluated
by measuring the responses of preswollen mitochondria to the addition of poly(ethylene glycol) of various molecular sizes As shown in Fig 4B, when mitochondria 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 for use of
succinate (plus 0.5 lgÆmL)1rotenone) as a substrate instead of
glu-tamate and malate Broken line represents the oxygen consumption of
nontreated mitochondria.
Fig 4 Effects of DiS-C 3 (5) on the turbidity of mitochondrial suspen-sions (A) and on permeability of mitochondrial inner membrane (B) (A) The effect of 50 l 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 at 440 nm (B) Permeability of DiS-C 3 (5)-pretreated inner membranes of mitochondria to poly(ethylene glycol) of various molecular sizes was evaluated For this, mitochondria were first pre-swollen by Ca 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 solutions of poly(ethylene glycol) of various molecular sizes were recorded at 440 nm The vertical arrow indicates the addition of a poly(ethylene glycol) solution Trace a 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, PEG6000 and PEG10000, respectively.
Fig 2 Inhibitory effects of DiS-C 3 (5) on NADH-driven electron
transfer For evaluation of the inhibitory effect of DiS-C 3 (5) on
NADH-driven electron transfer, mitochondria were suspended in +P i medium
at 25 C Then, they were energized by addition 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 of oxygen consumption Inhibitory
effects of DiS-C 3 (5) on electron transfer 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 traces of oxygraphs are
shown in (A) Dose–response curve of the effect of DiS-C 3 (5) on the rate
of mitochondrial oxygen consumption is shown in (B), in which
the results are shown as mean values ± SD
12 of three independent runs
(bars of SD are smaller than the symbols).
Trang 4preswollen with Ca2+, the addition of poly(ethylene glycol)
having a molecular size of more than 4000 (PEG4000)
caused increased turbidity of the mitochondrial 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 Ca2+
treatment When poly(ethylene glycol) solutions were added
to the mitochondrial suspensions pretreated with DiS-C3(5),
massive shrinkage was not observed, even with PEG6000 or
PEG10000, indicating that the membrane of the
mitochon-dria treated with DiS-C3(5) became permeable to larger
molecules than Ca2+-treated mitochondria
Furthermore, the results of TEM observation also
supported the changes in the permeability of the inner
mitochondrial membrane caused by DiS-C3(5) Compared
with the appearance of nontreated control mitochondria
(Fig 5A), when mitochondria were treated with Ca2+
(Fig 5B), the mitochondrial inner membrane structure
disappeared significantly, as reported previously [13,17–19]
Mitochondria treated with DiS-C3(5) showed essentially the
same TEM features as those treated with Ca2+(Fig 5C)
These results indicate clearly that acceleration of
mitoch-ondrial oxygen consumption induced by DiS-C3(5) at 50 lM
is due to the induction of the mitochondrial PT However,
as stated above, the membranes of mitochondria treated
with DiS-C3(5) became permeable to larger molecules than
the Ca2+-treated ones Furthermore, the increase in the
permeability of the mitochondrial membranes caused by
DiS-C3(5) was only partially sensitive for CsA, known as an
inhibitor of the classical PT induced by Ca2+ (data not
shown) Thus, the PT induced by DiS-C3(5) was concluded
to be different from that induced by Ca2+
The induction of PT is generally believed to be associated
with the release of apoptogenic mitochondrial proteins such
as cytochrome c [20] Thus, we next examined whether
mitochondrial cytochrome c would be released when
mito-chondria were treated with DiS-C3(5) As shown in Fig 6,
treatment of mitochondria with 50 lMDiS-C3(5) caused a
massive release of cytochrome c, as well as with Ca2+
Inhibition of PT induction by DiS-C3(5) at low
concentration
As stated above, DiS-C3(5) at low concentrations
preven-ted progression of intrinsic oxygen consumption by
mitochondria However, this effect of DiS-C(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-C3(5) at a low concentration caused maximum acceleration of mito-chondrial oxygen consumption as effectively as that observed with mitochondria not treated with DiS-C3(5) (data not shown) Based on these results, we considered that the protective effects of DiS-C3(5) at low concentra-tions on the progression of intrinsic oxygen consumption
by mitochondria might reflect induction of the PT by endogenous Ca2+ So next we examined the validity of this interpretation
First, we tested the effect of DiS-C3(5) on the Ca2+ -induced acceleration of mitochondrial respiration and mitochondrial swelling As shown in Fig 7, when
DiS-C3(5) was added to mitochondria pretreated with 10 lM
Ca2+, 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-C3(5) at a low concentration on the 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 lM Ca2+ was strongly suppressed by treatment of the mitochondria with
5 lMDiS-C3(5)
Thus, we concluded that the inhibitory effect of
DiS-C(5) on the spontaneous acceleration of mitochondrial
Fig 5 TEM appearances of mitochondria treated with DiS-C 3 (5) Mitochondria were treated with Ca2+or DiS-C 3 (5) as described in the legend in Fig 4 and subjected to TEM analysis (A) The appearance of nontreated control mitochondria (B) and (C) The appearances of mitochondria treated with
100 l M Ca 2+ and 50 l M DiS-C 3 (5), respect-ively Bar under (C) indicates 1 lm for all panels.
13
Fig 6 Effects of DiS-C 3 (5) on the release of mitochondrial cyto-chrome c Release of mitochondrial cytocyto-chrome c was examined as described in Materials and methods Briefly, mitochondria were first treated with 50 l M DiS-C 3 (5), then they were precipitated by centrif-ugation Samples of pellet (P) and supernatant (S) were subjected 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 shown.
Trang 5oxygen consumption could be attributable to the inhibition
of spontaneous induction of the PT by endogenous Ca2+
However, it should be noted that this inhibitory effect of
DiS-C3(5) was only observed when the PT was induced by a
relatively low concentration Ca2+such as 10 lM; i.e it was
not observed at a concentration of Ca2+such as 50 lM
(data not shown) Furthermore, the protective effect of
DiS-C3(5) on the Ca2+-induced PT was not attributable to
the inhibition of Ca2+uptake (data not shown)
Effects of DiS-C3(5) on mitochondria in the absence of Pi
All of the above experiments were performed in +Pi
medium containing 10 mMphosphate buffer However, the
PT-inducing effects of Ca2+ and cyanine dyes such as
Tri-S-C4(5) are known to be dependent on the absence/
presence of Piin the incubation medium Thus, it was of
interest to us to examine the effects of DiS-C3(5) on
mitochondrial structure and function in the absence of Pi
Where Ca2+had no effect on the mitochondrial oxygen
9consumption in the absence of Pi, DiS-C3(5) moderately
accelerated mitochondrial oxygen consumption even in the
absence of Pi (Fig 9A) Furthermore, 50 lM DiS-C3(5)
caused massive swelling even in the absence of Pi(Fig 9B)
To examine whether the observed mitochondrial swelling
induced by DiS-C3(5) in this case was attributable to the induction of the mitochondrial PT, we examined the permeability of DiS-C3(5)-treated mitochondrial mem-branes to poly(ethylene glycol)
conclusion could be obtained, as mitochondria preswollen
by DiS-C3(5) did not show any clear response upon the addition of poly(ethylene glycol) (data not shown) However, mitochondria treated with 50 lM DiS-C3(5) showed morphology different 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 Ca2+
or DiS-C3(5) in the presence of Pi(Fig 5B,C) Finally, we also tested whether the release of mitochondrial cyto-chrome c could be induced by DiS-C3(5) in the absence
of Pi As shown in Fig 11, when mitochondria were incubated with 50 lM DiS-C3(5) in the absence of Pi, most of the cytochrome c was retained in these mito-chondria as well as in the nontreated mitomito-chondria Discussion
Cyanine dyes are often used to evaluate the mitochondrial membrane potential [4,5] However, as reported previously, some of them are also reported to show inhibitory effects on NAD-linked electron transfer [11,12] and Ca2+-like uncoupling actions [9,12] DiS-C3(5) is often used for measurements of mitochondrial membrane potential, and its methods of interaction with mitochondria have been
Fig 7 Inhibitory effects of a low concentration
DiS-C 3 (5) on the Ca2+-induced PT To
examine the effects of DiS-C 3 (5) on the Ca 2+
-induced PT, we measured its effects on 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 addition of Ca2+
and DiS-C 3 (5) are shown by broken lines
(controls).
Fig 8 TEM analysis of mitochondria treated with a low concentration
of DiS-C 3 (5) To examine the protective effect of DiS-C 3 (5) on the
Ca2+-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 Ca2+and with both 5 l M DiS-C 3 (5) and
10 l M Ca2+, respectively Bar under (B) indicates 1 lm for all panels.
Fig 9 Effects of DiS-C 3 (5) on the rate of mitochondrial oxygen con-sumption (A) and turbidity of mitochondrial suspensions (B) in the ab-sence of P i Experiments were performed as described in the legends for Figs 3 and 4 except that –P i medium was used instead of +P i medium.
Trang 6studied [15,21] However, 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-C3(5) on the structure and function of
isolated mitochondria
First, we confirmed the previously reported inhibitory
effects of DiS-C3(5) on NAD-linked electron transfer This
inhibitory effect was considered to reflect its direct action on
complex I On the contrary, when DiS-C3(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 lM, DiS-C3(5) caused
acceler-ation of mitochondrial oxygen consumption This effect of
DiS-C3(5) was further characterized and concluded to be
attributable to the induction of the mitochondrial PT
PT induced by DiS-C3(5) was associated with release of
cytochrome c, as was that induced by Ca2+ However, it
was different from the ordinary PT induced by Ca2+in the
aspects of pore size and sensitivity for CsA, known as a
specific inhibitor of the ordinary PT Possibly, these
differences may reflect the differences in the features of the
proteinaceous PT pores formed
Cytochrome c is one of the components comprising the
respiratory chain Thus, release of cytochrome c from
of cytochrome c without causing deceleration of mito-chondrial oxygen consumption was also observed when mitochondria were treated with Ca2+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 the electron transfer 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 space of 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 no detailed study on the PT-indu-cing effects of chemicals actually used as a tracer dye of the mitochondrial membrane potential except for that on Mitotracker OrangeTM[12] Possibly, induction of the PT
is one of the common actions of hydrophobic cations that are utilized as a tracer of mitochondrial membrane poten-tial, as similar activities were observed with these chemicals regardless their structural diversity [9,12] Further studies on the actions of a series of hydrophobic cations will be necessary for validation of this interpretation and for better understanding of the features of the mitochondrial PT Furthermore, we observed two additional novel effects of DiS-C3(5) on mitochondria: (i) inhibition of the Ca2+ -induced PT by a low concentration of DiS-C3(5) and (ii) induction of swelling in the absence of Pi With respect to the former feature, attention must be paid to it when this dye is employed as a tracer for mitochondrial membrane potential, as it shows a protective effect on the induction of
PT at the concentration utilized for monitoring membrane potentials For the latter action, DiS-C3(5) caused remark-able swelling and changes in the status of the mitochondrial inner membrane without accompanying release of mitoch-ondrial cytochrome c (Figs 9,10,11) Further studies on the status of the inner membrane of mitochondria treated with DiS-C3(5) in the absence of Pi may give us insight into the mechanisms causing configurational changes in mito-chondria
Until now, both CsA-sensitive and insensitive PT have been shown to be associated with the release of mitochond-rial cytochrome c Recently, however, we reported that mitochondrial cytochrome c could be released even without the induction of the mitochondrial 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-C3(5) to show multiple effects on the mitochondrial structure and function, effects dependent on both its concentration and the Pistatus
11
Acknowledgements This work was supported by grants-in-aid for scientific research (no 14370746 to Y.S.) from the Ministry of Education, Science, and Culture of Japan, and a fellowship from Katayama Chemical Industries, Co., Ltd (Osaka) 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 examined by TEM analysis (A) and (B) show the
appear-ance of mitochondria incubated in the absence and presence of 50 l M
DiS-C 3 (5), respectively 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 were also analyzed.
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