Additional studies with superoxide dismutase, ascorbic acid and different specific inhibitors, such as ruthenium red, cyclo-sporin A and Mn2+, allowed us to establish a time sequence of e
Trang 1transition pore and cytochrome c release through
superoxide generation
Marı´a Niklison Chirou, Augusto Bellomio, Fernando Dupuy, Beatriz Arcuri, Carlos Minahk
and Roberto Morero
Departamento de Bioquı´mica de la Nutricio´n, Instituto Superior de Investigaciones Biolo´gicas (Consejo Nacional de Investigaciones
Cientı´ficas y Te´cnicas—Universidad Nacional de Tucuma´n), Instituto de Quı´mica Biolo´gica ‘‘Dr Bernabe Bloj,’’ San Miguel de Tucuma´n, Argentina
Microcin J25 (MccJ25), a 21-amino acid antimicrobial
peptide that is active against certain human pathogens
such as Salmonella and Shigella [1], has an unusual
lasso distinctive structure [2–4] and a dual mechanism
of action Microcin J25 inhibits transcription by
obstructing the RNA polymerase secondary channel
[5] and affects, independently, the cytoplasmic
membrane of Escherichia coli and Salmonella enterica
serovars [6] In this regard, it was shown that MccJ25
disrupts the membrane integrity of S enterica and
therefore causes dissipation of its membrane electrical
potential In addition, MccJ25 inhibits respiratory
enzymes such as NADH, succinate dehydrogenase and
lactate dehydrogenase and alters the oxygen
consumption rate in vivo and in vitro [7] The fact that
MccJ25 is a membrane-active peptide is also supported
by studies carried out on liposomes [8]
Recently, the effect of MccJ25 on intact rat heart mitochondria was explored [9] The peptide modifies the membrane permeability, displays a potent effect as inhibitor of thecomplex III and diminishes drastically the internal ATP level Mitochondria play a vital role
in the regulation of energy metabolism and cell death
by apoptosis and necrosis Mitochondrial function requires a continuous transmembrane potential, which depends on the generation of an electrochemical pro-ton gradient across the inner membrane The loss of the mitochondrial membrane integrity induces the release of pro-apoptotic proteins [10–13] On the one hand, the apoptotic cascade can be triggered by a
Keywords
antibiotics; Ca 2+ ; cytochrome c; microcin;
mitochondria
Correspondence
R Morero, Chacabuco 461,
S.M de Tucuma´n 4000, Argentina
Fax: +54 0381 4248025
Tel: +54 0381 4248921
E-mail: rdmorero@fbqf.unt.edu.ar
(Received 23 April 2008, revised 21 May
2008, accepted 12 June 2008)
doi:10.1111/j.1742-4658.2008.06550.x
Microcin J25, an antimicrobial lasso-structure peptide, induces the opening
of mitochondrial permeability transition pores and the subsequent loss of cytochrome c The microcin J25 effect is mediated by the stimulation of superoxide anion overproduction An increased uptake of calcium is also involved in this process Additional studies with superoxide dismutase, ascorbic acid and different specific inhibitors, such as ruthenium red, cyclo-sporin A and Mn2+, allowed us to establish a time sequence of events starting with the binding of microcin J25, followed by superoxide anion overproduction, opening of mitochondrial permeability transition pores, mitochondrial swelling and the concomitant leakage of cytochrome c
Abbreviations
carboxy-DCF, 5-(-6)-carboxy-2¢,7¢-dichlorofluorescein; carboxy-H 2 DCFDA, 5-(-6)-2¢,7¢-dichloro-dihydrofluorescein diacetate;
carboxy-H2DCFH, 5-(-6)-carboxy-2¢,7¢-dichloro-dihydrofluorescein; CsA, cyclosporin A; DNP, 2,4-dinitrophenol; FITC, fluorescein isothiocyanate; MccJ25, microcin J25; MccJ25F*, fluorescent derivative of microcin J25; MTP, mitochondrial transition pore; ROS, reactive oxygen species;
RR, ruthenium red; SOD, manganese-superoxide dismutase.
Trang 2Ca2+-independent mechanism that involves the Bcl-2
protein family, but this is not necessarily associated
with mitochondrial volume changes [14] On the other
hand, the opening of MTP could be activated by
Ca+2, resulting in mitochondrial swelling with the
con-sequent loss of the electrochemical gradient and the
uncoupling of oxidative phosphorylation [15] As a
result, cytochrome c and other proteins are released
into the cytosol The leakage of cytochrome c from
mitochondria is considered to be an early critical event
in apoptotic cascade induction, which ultimately leads
to programmed cell death [16–19] However, additional
evidence from studies with intact cells and isolated
mitochondria suggests that mitochondrial membrane
permeability changes may also occur by some other
mechanism [20]
In the present study we analysed the effect of
MccJ25 on isolated heart mitochondria Our results
indicated that MccJ25 induces the overproduction of
superoxide anions, thus increasing the mitochondrial
inner membrane permeability and activation of the
mitochondrial transition pore (MTP), resulting in
swelling and cytochrome c release
Results
Mitochondrial uptake of MccJ25
Mitochondrial uptake of MccJ25 was examined using
the peptide fluorescent derivative MccJ25F*, which
showed antibiotic activity and membrane gradient
dis-sipater capability similar to the native peptide (data
not shown) Addition of MccJ25F* to energized
mito-chondria (in the presence of 10 mm succinate) resulted
in the immediate uptake of the microcin analogue
(Fig 1) The uptake was rapid, with a maximal level
reached within 20 min To ensure that the uptake of
MccJ25F* was not an artifact, we confirmed the
results by determining binding displacement with
native MccJ25 (see Fig 1) and fluorescence in the
mitochondrial pellet (results not shown) Studies
car-ried out with nonenergized mitochondria (i.e in the
absence of succinate) showed a marked decrease in the
uptake capability of MccJ25F* Pretreatment of
ener-gized mitochondria with either 200 lm vanadate (an
ATPase inhibitor) or 100 lm 2,4-dinitrophenol
(2,-DNP; anelectrochemical gradient dissipater)
reduced the uptake of MccJ25F* by approximately
85%, suggesting that the uptake of MccJ25F* depends
on both the energy of mitochondria and the
mitochondrial proton membrane gradient, mainly
determined by ATP level and provided by succinate
oxidation, respectively
Effect of MccJ25 on the mitochondrial transition pore of energized mitochondria
The addition of 20 lm MccJ25 induced the swelling of energized mitochondria (Fig 2) The kinetics of swell-ing durswell-ing a period of 5 h had a linear pattern charac-teristic of MTP induction, showing an initial rate of 7.02 4 light scattering ⁄ min · 104 The initial rate of swelling increased as a function of MccJ25 concentra-tion (inset in Fig 2) and became saturated at about
60 lm (results not shown) Similar results were obtained, although with reduced swelling, when the buffer was devoid of succinate Furthermore, the pres-ence of 10 lm antimycin A inhibited completely the swelling, suggesting that the electronic flow through the respiratory chain stimulates the MccJ25 effect (results not shown) We next determined whether MccJ25 was able to induce the permeation of solutes with a relatively low molecular mass To study this we loaded mitochondria with calcein-AM, which is con-verted into calcein by endogenous esterases Loaded mitochondria were treated with increasing concentra-tions of MccJ25 and the release of calcein was studied
A marked calcein release, parallel to the increased swelling, was observed following the addition of 20 lm MccJ25 (Fig 2)
0 5 10 15 20 25 30 35
Time (min)
0 5 10 15 20 25 30 35
Fig 1 Uptake of MccJ25 by isolated mitochondria Energized mitochondria without pre-incubation ( ) or pre-incubated with
200 l M vanadate ( ), 100 l M 2,4-DNP (d) or 5 l M native MccJ25 (h), were suspended in 10 m M Tris–sodium phosphate buffer (pH 7.4), 230 m M mannitol, 70 m M sucrose, 3 m M HEPES, supple-mented with 10 m M succinate and 1 l M rotenone Nonenergized mitochondria (.) were suspended in the same buffer but in the absence of succinate Then, 1.4 l M MccJ25F* (final concentration) was added to the suspensions, which were incubated for different periods of time at 25 C The fluorescence incorporated by mito-chondria was plotted as a function of the incubation time Results are expressed as mean ± SD of five independent experiments.
Trang 3In an attempt to identify the role played by the
MTP in the swelling and permeability changes of
mito-chondria induced by MccJ25, we tested the influence
of the specific pore inhibitor cyclosporin A (CsA), a
calcium chelator (EDTA), a calcium competitor
(Mn2+) and the calcium uniporter inhibitor ruthenium
red (RR) As shown in Table 1, swelling and calcein
release induced by MccJ25 were almost completely
inhibited by 1 lm CsA Additionally, 50 lm EDTA,
125 lm Mn2+ and 2.5 lm RR also almost completely
inhibited the swelling induced by MccJ25, strongly
suggesting that the peptide was able to induce
mito-chondrial swelling through the opening of the MTP
mediated by the uptake of calcium through the uniport
of calcium On the other hand, KCN and antimycin A,
both inhibitors of electron transport, also inhibited the
swelling induced by MccJ25 The inhibition of swelling
induced by ascorbic acid will be discussed later As a
positive control, the swelling and calcein release
induced by calcium, and the inhibitory effect of CsA
and RR, are also shown in Table 1, confirming
previ-ous results [21,22]
Cytochrome c release
An initial aim of this work was to study the influence
of MccJ25 on the release of cytochrome c Therefore,
we investigated whether the indirect opening of MTP
by MccJ25 was followed by the release of cyto-chrome c The experimental results (inset of Fig 3) showed that increasing amounts of cytochrome c were released, showing a positive linear correlation with the
–0.4
–0.3
–0.2
–0.1
0.0
0.1
0 10 20 30 40
0
2
4
6
8
10
12
14
2 4
Time (min)
MccJ25 (µ M )
Fig 2 Effect of MccJ25 on mitochondrial swelling The time
course of swelling (h) and calcein release (D) of energized
mito-chondria induced by 20 l M MccJ25 was assessed by measuring
the change of light scattering at 540 nm or of calcein fluorescence,
respectively, of mitochondria suspended in buffer, as described in
the Materials and methods Controls of swelling ( ) and calcein
release ( ) were performed by incubation in the absence of
MccJ25 The data shown are representative of at least five
sepa-rate studies The inset shows mitochondrial swelling as a function
of MccJ25 concentration.
Table 1 Effect of different drugs on the swelling and calcein release from mitochondria induced by MccJ25 Swelling and calcein release of energized mitochondria were induced by 20 l M MccJ25 and 50 l M Ca +2 in the absence or presence of different inhibitors.
ND, not determined.
Mitochondrial swelling DLight scattering ⁄ min · 10 4
Calcein release (%) MccJ25 (20 l M ) 7.02 ± 0.01 a 52.00 ± 1.00
+Mn2+(125 l M ) 0.01 ± 0.02 0.10 ± 0.10
+Ascorbic acid (0.5 m M ) 0.09 ± 0.02 ND +Antimycin A (10 l M ) 0.50 ± 0.02 ND
Calcium (50 l M ) 15.20 ± 0.02 54.50 ± 2.00
a Results are expressed as means ± SD of three separate experiments.
0 4 8 12 16
0 10 20 30 40 0
2 4 6 8 10 12
Time (min) MccJ25 (µ M )
Fig 3 Effect of MccJ25 on cytochrome c release from mitochon-dria Isolated mitochondria were suspended in 100 m M potassium phosphate buffer (pH 7.4), 10 m M succinate, 1 l M rotenone, and incubated for different periods of time at 37 C in the absence (h) and in the presence of 20 l M MccJ25 ( ) Cytochrome c was determined chromatographically, as described in the Materials and methods Data are the mean ± SD of three independent experi-ments The inset shows cytochrome c release as a function of the MccJ25 concentration after 2 h of incubation at 37 C.
Trang 4concentration of MccJ25 This result agrees with the
swelling effect induced by MccJ25 It should be noted
that at 20 lm MccJ25 the release effect was time
dependent After a lag phase of approximately 50 min,
the release of cytochrome c increased abruptly,
reach-ing a plateau at about 3 h (Fig 3)
Effect of MccJ25 on the production rate of
superoxide anions
It was recently found in our laboratory that the effect
of MccJ25 on the E coli respiratory chain enzymes is
mediated by increased superoxide production [6] As it
is widely accepted that the mitochondrial electron
transport chain is the main source of superoxide
anions, we decided to determine the effect of MccJ25
on the mitochondrial electron transport chain and the
possible implications on the swelling and permeability
changes As shown in Fig 4, the rate of O2 generation
by the submitochondrial particles increased as a
func-tion of time, reaching a plateau at just about 45 min
(data not shown in Fig 4) The effect was dependent
on the MccJ25 concentration (inset of Fig 4) and was
almost completely inhibited by superoxide dismutase
(SOD), ensuring the superoxide anion determination
To demonstrate and better understand the mechanism
of the reactive oxygen species (ROS) overproduction
induced by the antimicrobial peptide, MccJ25, we
examined the effect on isolated mitochondria The
ROS production was monitored using a ROS-sensitive
fluorescent probe [5-(-6)-carboxy-2¢,7¢-dichloro-dihy-drofluorescein diacetate (carboxy-H2DCFDA)] The results shown in Fig 5 indicate that 20 lm MccJ25 induced a clear increment of ROS production com-pared with the control experiment performed in the absence of the peptide Indeed, the rate of ROS pro-duction in the presence of MccJ25 was three times higher than in the absence of this peptide The maxi-mum ROS production rate was obtained after 30 min
of exposure to antibiotic Pretreatment of isolated mitochondria with antioxidants such as 0.5 mm ascor-bic acid or 0.5 mm a-tocoferol acetate almost com-pletely suppressed the ROS production induced by MccJ25 By contrast, pretreatment with 2.5 lm RR was unable to prevent the ROS overproduction induced by MccJ25
Effect of MccJ25 on NADPH oxidation Reactive oxygen species produced in mitochondria are inactivated by a set of protective enzymes, including SOD, glutathione peroxidase and glutathione reductase [23] Glutathione reductase recycles oxidized glutathi-one to its reduced form, using electrons from the NADPH As a consequence, the overproduction of superoxide anions induced by MccJ25 would implicate
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0 10 20 30 40 50 60
0.0
0.4
0.8
1.2
O2
O2
Time (min) MccJ25 (µ M )
Fig 4 Rate of O
2 generation induced by MccJ25 in rat heart
sub-mitochondrial particles The rate of superoxide generation in the
presence of 20 l M MccJ25 (d) or in the presence of 20 l M
MccJ25 and manganese-SOD (300 unitsÆmL)1) ( ) was determined
as described in the Materials and methods Data are mean ± SD of
three independent experiments The rate of superoxide generation
as a function of MccJ25 concentration is shown in the inset.
0 500 1000 1500 2000 2500
Time (min)
Fig 5 Effects of MccJ25 on the generation of ROS in isolated mitochondria A preparation of mitochondria, preloaded with the flu-orescence probe carboxy-H2DCFDA, was suspended in 10 m M Tris–potassium phosphate buffer (pH 7.4), 150 m M sucrose, 50 m M KCl, 1 l M rotenone (h), or in buffer containing 20 l M MccJ25 ( ) Pretreatment with 2.5 l M RR (s), 0.5 m M a-tocopherol acetate (d)
or 0.5 m M ascorbic acid () was also performed The time course
of 5-(-6)-carboxy-2¢,7¢-dichlorofluorescein (carboxy-DCF) fluores-cence (kex, 485 nm; kem, 525 nm) was monitored after the addition
of 10 m M succinate The data shown are representative of at least five separate studies.
Trang 5oxidation of NADPH As shown in Fig 6, 20 lm
MccJ25 markedly increased the oxidation of NADPH
compared with a control in the absence of the peptide
EDTA, RR and ascorbic acid completely inhibited the
effect of MccJ25 Surprisingly, CsA, a specific inhibitor
of the pore, was unable to inhibit the effect of MccJ25,
which strengthens the hypothesis that MccJ25 gener-ates ROS, regardless of the presence of calcium
Discussion Understanding the mechanisms of action of some types
of proteins and peptides on the structural and func-tional state of mitochondria seems to be important in the development of apoptosis-regulating technologies, particularly for anticancer therapy, and for the design
of new classes of antibiotics, taking into account a cer-tain similarity between mitochondria and bacteria In this work we observed that mitochondrial uptake of MccJ25 proceeds very rapidly and in a concentration-dependent manner The peptide inserts into the mem-brane, modifying its permeability and provoking conse-quently an electrical potential dissipation, as described previously [9] Such an effect promotes the uncoupling
of electron transport with a concomitant loss of the efficiency to reduce oxygen and an increase in superox-ide species and subsequently ROS generation Interest-ingly, this MccJ25 effect is similar to that described recently for E coli [6] The membrane perturbations would induce a small increase in the internal calcium concentration, which in turn activates the uniporter of calcium, increasing even more the calcium influx and ROS production Both the increase of intramitochond-rial calcium concentration and the opening of MTP trigger mitochondrial swelling, with the concomitant release of the apoptotic inducer cytochrome c [24] The sequence of these events is schematically represented in Fig 7 and ordered on a timescale according to the
–40
–35
–30
–25
–20
–15
–10
–5
0
5
Time (min)
Fig 6 Effect of MccJ25 on the mitochondrial NADPH level
Mito-chondria suspended (0.5 mgÆmL)1) in Tris–potassium phosphate
buffer (pH 7.4), 10 m M succinate, 1 l M rotenone, 150 m M sucrose,
50 m M KCl were incubated in the absence (d) or in the presence
of 20 l M MccJ25 without any pretreatment (D), or pretreated for
1 min with 1 l M CsA ( ), 2.5 l M RR (s), 0.1 m M EDTA (.) or
0.5 m M ascorbic acid (h) At different time-points NADPH intrinsic
fluorescence was monitored at excitation and emission
wave-lengths of 366 and 450 nm respectively.
60 min
Cyt
r
c
elease
MccJ25
Fluidity increase
Gradient dissipation
SOD
Vit C Vit E
NADPH
uniporter
MTP
RR EDTA
CsA
Timescale events
Uptake
Fig 7 Schematic representation of the time sequence steps triggered by MccJ25 acting on isolated heart mitochondria.
Trang 6kinetics of each process The interaction between
MccJ25 and the membrane is followed by membrane
perturbation, superoxide and ROS overproduction,
stimulation of the calcium uniporter, opening of the
MTP and, finally, mitochondrial swelling with
cyto-chrome c leakage This sequence is supported by the
effect of several specific inhibitors The overproduction
of superoxide and ROS is the first event induced by
MccJ25 and is responsible for triggering the subsequent
effects because the presence of ascorbic acid was
suffi-cient to inhibit mitochondrial swelling (see Table 1)
The kinetic of superoxide and ROS overproduction
clearly demonstrates that the generation of this reactive
species precedes MTP opening and mitochondrial
swelling The inability of RR and CsA, specific
inhibi-tors of the calcium uniport and MTP respectively, to
inhibit the increase of ROS strongly supports the
above-mentioned hypothesis The NADPH oxidation
occurs prior to the opening of the MTP pore and is
coupled to the transport of calcium from the
intermem-brane space to the matrix because it is inhibited by RR
but not by CsA The mitochondrial swelling induced by
MccJ25 was also inhibited by EDTA, Mn2+ and RR,
indicating that the influx of Ca2+to the mitochondrial
matrix was necessary for activation of the MTP These
results indicate that the effect of MccJ25 on the
respira-tory chain forces the opening of the MTP pore, which
is mediated by an increase of the matrix calcium
con-centration, with the concomitant release of
cyto-chrome c
The fact that the mitochondrial swelling induced by
MccJ25 was inhibited by antimycin A allowed us to
presume that the peptide effect takes place only when
the electron flow through the respiratory chain is
oper-ating In addition, and supporting this result, the
swell-ing effect is highly elevated in activated mitochondria
and completely inhibited by rotenone (complex I
inhib-itor) in ‘not activated’ mitochondria Alternatively, we
could consider that in the presence of antimycin A the
mitochondrial membrane potential cannot be built
up and therefore mitochondria would not accumulate
calcium, which is required for MTP induction
In conclusion, we have shown that MccJ25 has a
mitochondrial deleterious effect that is associated with
the induction of the MTP Microcin J25 would target
the site of anion superoxide generation, increasing the
production of ROS However, the results presented
here do not clarify the mechanism by which MccJ25
induces ROS production Superoxide can be produced
at complex I and⁄ or at complex III [25] Our results
clearly indicate that complex III is essentially
impli-cated in the mechanism of superoxide production
because the peptide effect was obtained in the presence
of rotenone, a specific inhibitor of complex I We dem-onstrated that ROS play a major role in mediating mitochondrial dysfunction induced by MccJ25 Taking into account the induced swelling and the cytochrome
c release, we could hypothesize that MccJ25 behaves
as an apoptotic agent However, any disruption in the electrochemical gradient and⁄ or oxidative phosphory-lation, resulting in a decrease of ATP production, could compromise the progression of this form of cell death because an energy requirement is clearly needed for apoptosome formation Moreover, there is contro-versy about whether or not the mitochondria indeed swell during apoptosis Some studies have reported observing mitochondrial swelling [26], whereas others have reported that swelling never occurred [27,28] Recent investigations suggest that cytochrome c release
in apoptosis was not caused by mitochondrial swelling [29] Additional studies will be helpful to understand,
in greater detail, the molecular basis of this effect, its biological significance and the possible peptide bio-applicability mainly in whole human cells These studies are currently underway in our laboratory
Materials and methods Chemicals and reagents
Calcein-AM, CsA, fluorescein isothiocyanate (FITC) and ATP were purchased from Sigma Chemical Co (St Louis,
MO, USA) Carboxy-H2DCFDA-SE was obtained from Molecular Probes Inc (Eugene, OR, USA) All other reagents were of analytical grade or the purest available commercial form
Peptides synthesis and purification
Microcin J25 was obtained from the supernatant of E coli AB259 harboring pTUC200 and was purified according to the procedure previously reported [7] This procedure yielded a preparation that appeared homogeneous in two different systems of analytical RP-HPLC [30,31] A fluores-cent analog containing FITC (MccJ25F*) was prepared for mitochondrial uptake studies A mutated peptide in which isoleucine of position thirteen was replaced with lysine was obtained and purified from the supernatant of E coli DH5a harboring pI13K The strain was generously provided by P Vincent (INSIBIO, CONICET⁄ UNT,
S M Tucuma´n) The fluorescent peptide was obtained by incubation of the mutated peptide with FITC (1 : 3, w⁄ w) for 2 h in alkaline medium and in darkness at room tem-perature (25C) The labeled peptide was purified by chro-matography on a hydrophobic C8 cartridge The fraction eluted with 100% methanol was reduced under vacuum, resuspended in water and chromatographed on a C18
Trang 7column using a Gilson HPLC system, being finally eluted
with a 50 mm sodium phosphate buffer (pH 6.5)⁄ methanol
gradient Peptide concentrations were determined by
mea-surement of the absorbance at 278 nm [32]
Isolation of heart mitochondrial
Winstar rats (250–300 g) were killed by CO2inhalation, in
accordance with the European directive for protection of
vertebrate animals for scientific research Hearts were
rap-idly removed and placed in 10 mL of ice-cold 5 mm
HEPES buffer (pH 7.4), 200 mm mannitol, 10 mm sucrose,
1 mm EDTA, 0.1% BSA The tissue was finely minced with
scissors and then homogenized using an Omni-Mixer
(Sor-vall, Norwalk, CT, USA) The homogenate was centrifuged
at 900· g for 10 min, the pellet was discarded and the
supernatant centrifuged again at 17 000· g for 10 min
The pellet containing the mitochondria was resuspended in
the isolation buffer without EDTA To prepare
submito-chondrial particles, the mitosubmito-chondrial pellet was
resus-pended (20 mgÆmL)1) in 50 mm Tris–HCl (pH 7.6), 230 mm
mannitol, 70 mm sucrose, and sonicated three times (each
consisting of a 30-s pulse burst) at 1-min intervals, at 4C
The sonicated mitochondria were centrifuged at 8500· g
for 10 min to remove the unbroken organelles The
super-natant was centrifuged again at 100 000· g for 60 min,
and the resulting pellet was washed and resuspended in the
same buffer [33] The protein concentration was determined
by the method of Lowry et al., with bovine albumin as the
standard [34]
Mitochondrial uptake studies
For mitochondrial uptake of MccJ25F*, mitochondria
(1 mgÆmL)1) were suspended in 10 mm Tris–sodium
phos-phate buffer (pH 7.4), 230 mm mannitol, 70 mm sucrose,
3 mm HEPES, supplemented with 10 mm succinate and
1 lm rotenone, at 25C, then 1.4 lm MccJ25F* was added
and the solution was incubated at 25C Uptake was
stopped at different time-points by centrifugation
(12 000· g, 5 min, 4 C) and fluorescence of tyrosine (kex:
277 nm, kem: 305 nm) and FITC (kex: 490 nm, kem:
520 nm) in the supernatant was measured using an ISS
(Champaign, IL, USA) PC1 spectrofluorometer at 25C
Uptake experiments were also determined in mitochondrial
samples incubated previously with 200 mm vanadate, or
100 mm DNP, for 5 min at 37C
Mitochondrial swelling assay
Isolated mitochondria (1 mgÆmL)1) were incubated in
10 mm Tris–sodium phosphate buffer (pH 7.4), 230 mm
mannitol, 70 mm sucrose, 3 mm HEPES, supplemented
with 10 mm succinate and 1 lm rotenone at 25C [27]
Different quantities of MccJ25 were added to the incuba-tion buffer In addiincuba-tional experiments, inhibitors such as EDTA, Mn2+, CsA, KCN, antimycin A, ascorbic acid and
RR were added before the addition of MccJ25 Calcium (50 lm) was used as a positive control A control experiment in the absence of MccJ25 and Ca2+ was also performed Swelling was estimated from the changes of light scattering at 540 nm in a DU7500 spectrophotometer (Beckman, Fullerton, CA, USA) equipped with a peltier constant temperature chamber The rate of swelling (Dlight scattering⁄ min) was calculated from the slope of the initial linear portion of the curve
Calcein release from mitochondria
Mitochondria isolated from rat heart were incubated at
25C for 30 min in the suspension buffer containing 2 lm calcein-AM and then washed and resuspended in the same buffer To assess calcein release, loaded mitochondria (0.1 mgÆmL)1) were added to the assay buffer [5 mm Tris– HEPES buffer (pH 7.4), 250 mm sucrose, 0.1% BSA,
10 lm CoCl2] and variations in the fluorescence were mea-sured using an ISS PC1 spectrofluorometer [22] The excita-tion and emission wavelengths were 488 and 530 nm respectively The buffer contained 10 mm CoCl2to quench the fluorescence of calcein released from mitochondria The fluorescence value obtained in the presence of 0.2% Triton X-100 was considered as 100% leakage
Cytochrome c release
Quantification of cytochrome c release from rat heart mito-chondria was performed as described by Crouser et al [24], with minor modifications Essentially, intact mitochondria (1 mgÆmL)1) suspended in 100 mm potassium phosphate buffer (pH 7.4), 10 mm succinate and 1 lm rotenone were incubated at 37C with MccJ25 At different time-points, aliquots were centrifuged (30 000 g, 10 min, 4C) and the supernatant was analyzed using HPLC (Gilson HPLC equipped with a UV-VIS detector) through a C8 reverse-phase analytical column (Waters XTerra MS C8 5 lm, 4.6· 250 mm) preceded by a guard column A linear gradi-ent, increasing from 20 to 60% acetonitrile in water, was employed Both the 20% and the 60% acetonitrile solutions also contained 100 mm KCl and 0.1% trifluoroacetic acid (v⁄ v) The eluted cytochrome c was detected at 393 nm Cytochrome c concentrations were calculated from a standard measurement
Superoxide anion radical generation
The rate of O2 generation by submitochondrial particles was measured as reduction of acetylated ferricytochrome c, which is an excellent quantitative trap for O2[35] The
Trang 8reduction was followed spectrophotometrically at 550 nm
in a Beckman DU 7500 at 25C The reaction mixture
contained 100 mm potassium phosphate buffer (pH 7.4),
10 lm acetylated ferricytochrome c, 1 lm rotenone, small
mitochondrial particles (0.5 mg of proteinÆmL)1) and
20 lm MccJ25 The reaction was started by the addition of
10 mm succinate We also performed experiments in which
the effect of MccJ25 was studied after pre-incubation for
5 min with SOD
Reactive oxygen species production
Reactive oxygen species production was monitored using
the ROS-sensitive fluorescent probe, 5-(-6)-carboxy-2¢,
7¢-dichloro-dihydrofluorescein diacetate (carboxy-H2DCFDA)
Once in the mitochondria, the acetate groups are cleaved
by nonspecific esterases, hence the nonfluorescent
5-(-6)-carboxy-2¢,7¢-dichloro-dihydrofluorescein (carboxy-H2DCFH)
is trapped inside This probe was selected because is well
retained not only in cells but also in mitochondria [36]
Oxidation of carboxy-H2DCFH by ROS yields
carboxy-DCF This fluorescent product indirectly measures the O2
produced that has dismutated to H2O2through the action
of endogenous Mn2+-dependent SOD The suspension of
mitochondria (10 mgÆmL)1) was incubated with
carboxy-H2DCFDA for 30 min at 30C and then washed twice
with 10 mm Tris–sodium phosphate buffer (pH 7.4),
150 mm sucrose and 50 mm KCl to eliminate
nonincorpo-rated probe To monitor ROS production,
(0.1 mgÆmL)1) in 10 mm Tris–sodium phosphate buffer
(pH 7.4), 150 mm sucrose, 50 mm KCl, 10 mm succinate,
1 lm rotenone, and the variation in fluorescence was
followed using an ISS PC1 spectrofluorometer at 25C
after the addition of MccJ25 The excitation and emission
wavelengths were 490 and 520 nm respectively
Determination of NADPH oxidation
Mitochondrial pyridine nucleotides were monitored, in an
ISS PC1 spectrofluorometer at 25C, by measuring their
intrinsic fluorescence at 450 nm after exciting at 340 nm
[37] Mitochondria were suspended (0.1 mgÆmL)1) in 10 mm
Tris–potassium phosphate buffer (pH 7.4), 10 mm
succi-nate, 1 lm rotenone, 150 mm sucrose and 50 mm KCl The
suspension was pre-incubated for 1 min, and the oxidation
of NADPH was started by adding 20 lm MccJ25 or 50 lm
Ca2+
Acknowledgements
Financial support was provided by CONICET (Grant
PIP 4996) and CIUNT (Grant 26⁄ D228) and the
Agencia Nacional de Promocio´n Cientı´fica y Te´cnica
(PICTO 843, PAE 22642) M V N and F D are recipient of a CONICET fellowship R D M., C M and A B are CONICET career investigator We thank Monica Delgado for his generous assistance
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