Involvement of mitochondrial swelling in cytochrome c release from mitochondria treated with calcium and Alloxan

doi:10.4236/jbpc.2011.21002

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Vol.2, No.1, 10-18 (2011)

doi:10.4236/jbpc.2011.21002

Journal of Biophysical Chemistry

Involvement of mitochondrial swelling in cytochrome c

release from mitochondria treated with calcium and

Alloxan

Takuya Ichimura, Mika Ito, Kiyoshi Takahashi, Kyohei Oyama and Koichi Sakurai*

Division of Biochemistry, Department of Life science, Hokkaido Pharmaceutical University School of Pharmacy, Otaru, Japan;

ks51@hokuyakudai.ac.jp

Received 29 October 2010; revised 18 November 2010; accepted 25 November 2010.

ABSTRACT

An early event in the induction of apoptosis is

cytochrome c (Cyt c) release from mitochondria.

We investigated the involvement of mitochon-

drial permeability transition (MPT) and mito-

chondrial swelling in Cyt c release from mito-

chondria treated with alloxan and/or calcium

(Ca2+). When mitochondria were treated with a

high concentration of Ca2+ alone or Ca2+ with

alloxan (alloxan-Ca2+), the MPT was accompa-

nied by mitochondrial swelling and the release

of Cyt c. Cyclosporin A prevented the induction

of MPT but only slightly decreased the release of

Cyt c. High molecular weight polyethylene glycol

almost completely inhibited MPT-dependent

osmotic mitochondrial swelling and Cyt c re-

lease. However, MPT-independent mitochondrial

swelling and Cyt c release induced by exoge-

nous K+ were inhibited by the high molecular

weight polyethylene glycol. Ruthenium red

strongly decreased the amount of Cyt c released.

These results suggest that mitochondrial swell-

ing but not MPT is necessary for Cyt c release

induced by Ca2+ alone or alloxan and Ca2+.

Keywords: Mitochondrial Swelling; Cytochrome C

Release; Calcium; Alloxan; Mitochondri al

Permeability Transition

1. INTRODUCTION

Mitochondria have a central role in energy metabo-

lism and Ca2+ homeostasis in cells [1-3]. The release of

mitochondrial proteins, such as cytochrome c (Cyt c),

Smac/DIABLO, and apoptosis inducing factor (AIF),

into the cytosol is likely involved in the early events in

cell death [4,5]. Several triggers of mitochondrial per-

meability transition (MPT) result in apoptotic cell death

[6-8]. From these findings, it is proposed that the MPT is

involved in Cyt c release from mitochondria and apop-

tosis [9,10]. MPT is associated with an increase in the

permeability of the mitochondrial inner membrane, al-

lowing the transmission of a solute with a molecular

mass of up to approximately 1.5 k Da. MPT is also asso-

ciated with mitochondrial swelling, followed by the re-

lease of mitochondrial proteins including Cyt c [10].

Cytochrome c with a molecular weight of approximately

12 k Da is loosely bound to phospholipids of the outer

surface of the inner mitochondrial membrane, primarily

cardiolipin, and functions to transmit electrons from

complex III to complex IV of the electron transport

chain in mitochondria [11,12]. There are two distinct

processes in the Cyt c release from the intermembrane

space of mitochondria into the cytosol. One process is

the dissociation of Cyt c from the inner membrane, and

the other process is the transfer of the dissociated Cyt c

through the outer membrane.

Several mechanisms for the release of Cyt c from mi-

tochondria have been proposed. Petrosillo et al. demon-

strated that Cyt c is dissociated from the inner mito-

chondrial membrane response to cardiolipin peroxida-

tion by reactive oxygen species (ROS) prior to the re-

lease into the cytosol [12]. Several researchers have

proposed that the release of Cyt c occurs due to the rup-

ture of the mitochondrial outer membrane [10,13-17].

Apoptotic Bcl-2 family proteins, such as Bax and tBid,

cause Cyt c release from mitochondria by an increase in

outer membrane permeability [18-21]. The induction of

MPT is due to the opening of the permeability transition

pore (PTP), which consists of the voltage-dependent

anion channel (VDAC), adenine nucleotide translocator

(ANT), cyclophilin D (CyD), and other proteins, such as

hexokinase [10]. Because the physiological function of

the PTP is to allow high molecular weight substances to

pass through the mitochondrial outer membrane,

whereas low molecular weight materials such as water,

T. Ichimura et al. / Journal of Biophysical Chemistry 2 (2011) 10-18

oxygen, and carbon dioxide [9] can pass freely through

the mitochondrial inner membrane, it has been suggested

that PTP has a primary role in the matter transport into

mitochondria.

Mitochondria are one of the Ca2+ store sites in cells

and contribute to the maintenance of Ca2+ density in the

cytoplasm [3]. Excessive Ca2+ accumulation in cells in-

duces MPT [1,22,23]. We have previously demonstrated

that the diabetogenic compound, alloxan, causes MPT in

the presence of Ca2+. Quinone compounds that have a

similar structure to alloxan induce MPT in the presence

of Ca2+ [8,24-26]. Mangiferin quinoid compounds trig-

gers MPT via interaction with mitochondrial protein

thiol groups [27]. The MPT was inhibited by several

chemicals, such as inhibitors of the cyclophilin family,

mitochondrial Ca2+ uniporter and mitochondrial respira-

tory chain [28-30], suggesting that the release of Cyt c is

induced through several different pathways. However,

the mechanism by which Cyt c is released from mito-

chondria treated with Ca2+ remains largely unknown.

The present study demonstrates the involvements of

MPT and mitochondrial swelling in Cyt c release from

mitochondria treated with various concentrations of Ca2+

alone or Ca2+ with alloxan (alloxan-Ca2+). The MPT in-

hibitors significantly inhibited the MPT induced by Ca2+

alone or alloxan-Ca2+ but did not inhibit Cyt c release.

High-molecular weight polyethylene glycol (PEG) al-

most completely inhibited MPT-dependent osmotic mi-

tochondrial swelling and Cyt c release caused by the

large amplitude MPT induced by K+ or other inducers.

These results suggest that mitochondrial swelling but not

MPT relates to Cyt c release from mitochondria treated

with alloxan-Ca2+ or high concentrations of Ca2+ alone.

2. MATERIALS AND METHODS

2.1. Materials

Cyt c (from horse heart), rotenone, ethyleneglycol-

bis-(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA),

stigmatellin (STG), and carbonyl cyanide m-chlorophenyl-

hydrazone (CCCP) were purchased from Sigma Chemi-

cal Co. (St. Louis, MO). Calcium chloride (CaCl2), so-

dium succinate, potassium chloride (KCl), potassium

cyanide (KCN), alloxan, cyclosporin A (CsA), and PEG

4000 (molecular weight of 3 kDa) were from Wako Pure

Chemical Industries Ltd. (Osaka, Japan). Ruthenium red

(RuR) was from Kanto Chemical Co. (Tokyo, Japan).

The polyclonal anti-Cyt c antibody and the goat anti-

rabbit IgG HRP were from Santa Cruz Biotechnology

Inc. (Santa Cruz, CA). The ECL plus Western blotting

detection system was from GE Healthcare UK Ltd.

(Buckinghamshire, England). The BCA protein assay kit

was from Pierce Biotechnology Inc. (Rockford, IL). The

other chemicals used in this study were of analytical

grade from commercial suppliers.

2.2. Preparation of Rat Liver Mitochondria

Male Wistar rats that were approximately 200 g were

used for the experiments after an overnight fast. Mito-

chondria were isolated in Chelex-100 treated medium

containing 0.25 M sucrose, 10 mM Tris-HCl (pH 7.4)

and 1 mM EGTA by differential centrifugation of liver

homogenates as previously described [31]. Mitochon-

drial protein concentrations were determined by a BCA

protein assay kit (Pierce, USA) according to the manu-

facturer’s instruction with bovine serum albumin used as

a standard. All rats were treated in accordance with the

guiding principles for care and use of experimental ani-

mals in Hokkaido Pharmaceutical University School of

Pharmacy.

2.3. Measurement of Mitochondrial Swelling

Mitochondria (1 mg of protein/ml) were equilibrated

in Chelex-100 treated medium consisting of 10 mM

Tris-HCl (pH 7.4), 0.25 M sucrose and 0.1 M rotenone

for 5 min at 37˚C. After mitochondria were energized by

the addition of 5 mM succinate to the mitochondrial

suspension for 5 min, mitochondrial swelling was then

initiated by adding various MPT inducers (Ca2+ and al-

loxan-Ca2+) for 10 min. Mitochondrial swelling induced

by K+ was initiated by the addition of 80 mM KCl to

mitochondria suspended in Chelex-100 treated medium

containing 0.25 M sucrose, 10 mM Tris-HCl (pH 7.4)

and 1 mM EGTA. Mitochondrial swelling was measured

by recording the decrease in absorbance at 540 nm with

a spectrophotometer (Hitachi U-2,000, Japan).

2.4. Release of Cyt C

Released Cyt c was detected by Western blot analysis.

After the mitochondrial suspension was treated with

various compounds, the suspension was centrifuged at

10,000 × g for 30 min at 4˚C. The collected supernatant

was mixed with loading buffer, boiled for 5 min and

subjected to 15% SDS-polyacrylamide gel (Atto, Japan)

for 40 min at 200 V followed by electroblotting to nitro-

cellulose membranes for 1.5 h at 2 mA/cm2. Membranes

were blocked for 1 h with 5% skim milk in TBS-T at

room temperature, rinsed and subsequently probed with

a polyclonal anti-Cyt c antibody (1:1,000 dilution) for 1

h at room temperature. The membranes were rinsed and

incubated with a horseradish peroxidase-labeled anti-

rabbit antibody (1:20,000 dilution) for 1 h at room tem-

perature. After incubation with the secondary antibody,

the membranes were rinsed, and the Cyt c blots were

visualized by enhanced chemiluminescence using the

T. Ichimura et al. / Journal of Biophysical Chemistry 2 (2011) 10-18

ECL plus western blotting detection system (GE Health-

care, UK). Individual band densities were integrated using

the Luminous Imager Software (Aisin cosmos, Japan).

2.5. Statistical Analysis

Data were expressed as means ± standard deviations

(S.D.) and were statistically analyzed by either Student’s

t test (in the case of equal variances between two groups)

or Welch’s t test (in the case of unequal variances be-

tween two groups) after the F test. Data with P < 0.05

were considered statistically significant.

3. RESULTS

3.1. MPT and Cyt C Release Induced by

Ca2+ or Alloxan-Ca2+

Initially, we studied the relationship between the in-

duction of MPT and the release of Cyt c from rat liver

mitochondria treated with several concentrations of Ca2+

(25 M-200 M). As shown in Figure 1(a), after the addi-

tion of 50 M Ca2+ to the mitochondrial suspension, the

absorbance at 540 nm decreased by approximately 0.3

for 10 min, indicating the induction of small amplitude

MPT. After the addition of 200 M Ca2+, the absorbance

rapidly decreased during the first 2 min and then de-

creased moderately (A540 nm/10 min was approxi-

mately 0.6) indicating the induction of large amplitude

MPT. These results suggest that the extent of MPT de-

pends on Ca2+ concentration. We then measured the

concentrations of Cyt c released from mitochondria by

Western blotting. Addition of Ca2+ (25 M-200 M) to the

mitochondrial suspension clearly induced the release of

Cyt c from mitochondria. The concentrations of released

Cyt c increased with increasing Ca2+ concentrations but

reached a plateau at Ca2+ concentrations of 100 M or

more. Moreover, we studied the relationship between

MPT and Cyt c release induced by alloxan together with

20 M Ca2+ (alloxan-Ca2+). As shown in Figure 1(b),

addition of alloxan-Ca2+ to the mitochondrial suspension

caused a rapid decrease in absorbance at 540 nm for ap-

proximately 4 min and, subsequently, a moderate de-

crease in absorbance ( A540 nm/10 min was approxi-

mately 1) Furthermore, the addition of alloxan-Ca2+ ini-

tiated the Cyt c release from mitochondria (Figure 1(b)).

Fluorescence intensity of the bands was correlated with

the increase in concentrations of authentic Cyt c (1-100

pmol) in image of Western blotting (Figure 1(c)), and

the coefficient of determination (R) between the concen-

trations of Cyt c and the band intensities was 0.99. These

results indicate that both Ca2+ alone and alloxan-Ca2+

cause the induction of MPT and the release of Cyt c

from mitochondria. To elucidate the relationship between

MPT and Cyt c release, we studied the effect of CsA, an

100

200

2min

(μM)

02550 100 200

Cyt

ΔA

540 nm

=0.2

concentration (μM)

(a)

Cyt

Alloxan + Ca

ΔA

540 nm

=0.2

blank

Alloxan-Ca

2 min

(b)

Cyt

(pmol)

0.5 15 102550 100

(c)

Mitochondria were incubated in a medium at a concentration of 1

mg protein/ml. The incubation medium consisted of 0.25 M sucrose,

0.1 M rotenone, and 10 mM Tris-HCl (pH 7.4). MPT was induced

by (a) 25 M-200 M Ca2+ or (b) 1 mM alloxan and 20 M Ca2+ (al-

loxan-Ca2+) in the presence of 5 mM succinate. In blank neither

Ca2+ nor alloxan-Ca2+ was included in the medium. Typical traces

from three independent experiments are shown. Cyt c was detected

by Western blotting. Similar results were obtained in two additional

independent experiments; (c) Several concentrations (0-100 pmol)

of Cyt c were measured by Western blotting. Typical results of one

experiment are shown, and similar results were obtained three dif-

ferent preparations.

Figure 1. Induction of MPT and release of Cyt c.

inhibitor of classical MPT, on MPT and Cyt c release. As

shown in Figure 2(a), CsA prevented approximately

68% of total MPT and approximately 26% of total Cyt c

release from mitochondria in the presence of 50 M Ca2+

(Figure 2(a)). However, in high concentrations of Ca2+

(200 M), CsA led to a decrease in total MPT by ap-

proximately 82% but did not significantly affect Cyt c

release (Figure 2(b)). Together, these data suggest that

Cyt c release from mitochondria is not necessarily re-

lated to MPT.

T. Ichimura et al. / Journal of Biophysical Chemistry 2 (2011) 10-18

3.2. Effects of Inhibitors of Mitochondrial

Function on MPT and Cyt C Release

As shown in Figure 3, we studied the effects of sev-

eral inhibitors of mitochondrial function including CCCP,

STG, and KCN on MPT and Cyt c release from mito-

chondria treated with Ca2+. Under the condition of 50 M

Ca2+, an uncoupler, CCCP, and an inhibitor of b-c1 com-

plex, STG, significantly prevented both MPT and Cyt c

release, but KCN did not affect either one (Figu re 3(a) ).

With 200 M Ca2+, CCCP, KCN, and STG signifi-

Blank-CsA +CsA

+CsA

Blank

2min

Cytc

(50 μM)

ΔA

540nm

=0.2

(a)

2min

+CsA

Blank

Cyt c

Blank-CsA +CsA

(200 μM)

ΔA

540 nm

=0.2

Ca2+

(b)

MPT was induced by (a) 50 M or (b) 200 M Ca2+ in the pres-

ence of 5 mM succinate. The incubation medium contained 1

M CsA from the beginning of the experiments. In blank Ca2+

was not included in the medium. Typical traces from three

independent experiments are shown. Released Cyt c from

mitochondria was detected by Western blotting. Similar re-

sults were obtained in two additional independent experi-

ments. The other conditions were the same as previously de-

scribed in Figure 1.

Figure 2. Effects of CsA on Ca2+-induced MPT and

Cyt c release.

cantly inhibited MPT, whereas STG, but not CCCP or

KCN, strongly decreased Cyt c release (Figure 3(b)).These

results suggest that the inhibitors of mitochondrial func-

tion have different effects on MPT and Cyt c release

caused by low (50 M) or high (200 M) concentrations of

Ca2+. We then examined the effects of CsA and the in-

hibitors of mitochondrial function on MPT and Cyt c

release from mitochondria treated with alloxan-Ca2+. In

alloxan-Ca2+ conditions, CsA and the inhibitors of mito-

chondrial function significantly prevented MPT but not

Cyt c release (Table 1). These data suggest that proc-

esses other than MPT may be involved in Cyt c release.

3.3. Correlation Between Mitochondrial

Swelling and Cyt C Release

Mitochondrial swelling is caused by the increase in

mitochondrial volume, which is determined by a balance

Blank +CCCP

Cyt c

0.5

ΔA

540 nm

/10 min

(50 μM)

Blank +KCN

Blank +STG

Cyt c

(a)

ΔA

540nm

/10 min

0.5

Cyt c

(200μM)

Blank +CCCP

Blank+KCN

Blank+STG

(b)

Inhibitors of mitochondrial function including 0.05 M CCCP, 300 M

KCN, and 5 M STG were added to the incubation medium from the

beginning of the experiments. MPT was induced by (a) 50 M or (b)

200 M Ca2+ in the presence of 5 mM succinate. Value of ordinate refers

to the A540 nm for 10 min. Cyt c released was detected by Western blot-

ting. In blank Ca2+ was not included in the medium. Similar results

were obtained in two additional independent experiments. Data values

are represented as means ± S.D. of 3 to 11 experiments. * represents P

< 0.05 compared with mitochondria treated with Ca2+ alone. The other

conditions were the same as previously described in Figure 1.

Figure 3. Effects of the inhibitors of mitochondrial function

on MPT and Cyt c release induced by Ca2+.

T. Ichimura et al. / Journal of Biophysical Chemistry 2 (2011) 10-18

Table 1 .Effect of CsA and mitochondrial function inhibi-

tors on swelling and Cyt c release induced by alloxan-Ca2+.

Conditions ΔA540 nm Cyt c release (a.u.)

Alloxan-Ca2+ 1.008 ± 0.079 2.8 ± 0.6

+ CsA 0.482 ± 0.155* 2.9 ± 0.5

+ CCCP 0.647 ± 0.068* 2.2 ± 0.8

+ KCN 0.428 ± 0.193* 2.9 ± 0.6

+ STG 0.073 ± 0.031* 2.2 ± 0.6

Mitochondrial swelling was induced by 1 mM alloxan and 20 mM Ca2+

(alloxan-Ca2+) in the presence of 5 mM succinate. Swelling data refer to the

DA540 nm for 10 min. The amount of released Cyt c was analyzed by West-

ern blotting and quantified by densitometry, and is relatively represented as

compared to that of mitochondria treated with neither alloxan-Ca2+ nor

the inhibitors. Data values are represented as a mean ± S.D. of three to eight

experiments. * represents P < 0.05 when compared to alloxan-Ca2+. The

other conditions were the same as previously described in Figure 1 and

Figure 3.

Blank

2 min

+PEG

Blank

+PEGCa

Cytc

ΔA

540 nm

=0.2

(a)

2 min

+PEG

Blank

ΔA=0.2

Cytc

Blank +PEGAlloxan-C a

Alloxan-C a

540 nm

(b)

Mitochondrial swelling was induced by (a) 200 M Ca2+ (b)

or 1 mM alloxan and 20 M Ca2+ in the presence of 5 mM

succinate. Forty M of PEG 4000 was included in the in-

cubation medium from the beginning of the experiments.

Cyt c was detected by Western blotting. In blank neither

Ca2+ nor alloxan-Ca2+ was included in the medium. Simi-

lar results were obtained in two additional independent

experiments. The other conditions were the same as pre-

viously described in Figure 1.

Figure 4. Effects of PEG on MPT and Cyt c re-

lease induced by Ca2+ and alloxan-Ca2+.

of osmotic pressure across the inner mitochondrial

membrane. To examine the correlation between mito-

chondrial swelling and Cyt c release from mitochondrial

intermembrane space, we examined the effect of PEG on

mitochondrial swelling and Cyt c release from mito-

chondria induced by high concentrations of Ca2+ alone

or alloxan-Ca2+. PEG strongly inhibited mitochondrial

swelling induced by Ca2+ and completely inhibited Cyt c

release from mitochondria (Figure 4(a)). In addition,

PEG significantly prevented mitochondrial swelling and

Cyt c release from mitochondria treated with al-

loxan-Ca2+ (Figure 4(b)). These results indicate that

mitochondrial swelling associated with MPT is con-

nected with the release of Cyt c from mitochondria.

We next investigated the regulation of Cyt c release

from mitochondria with mitochondrial swelling induced

by another mechanism that is not mediated by MPT:

hypotonic shock triggered by low K+ concentrations [32].

As shown in Figure 5, 80 mM K+ significantly induced

mitochondrial swelling and Cyt c release from mito-

chondria when compared to a blank which neither K+

nor CsA is included. CsA did not inhibit K+-induced

mitochondrial swelling and Cyt c release, indicating that

Cyt c release from mitochondria may be due to mito-

chondrial swelling regardless of functional MPT. CsA

Blank

Blank +CsA

0.2

ΔA

540nm

/10 min

0.1

+CsA

Cyt c

Mitochondria were incubated in a medium at a concentration

of 1 mg protein/ml. The incubation medium consisted of 0.25

M sucrose, 0.1 M rotenone, 5 mM succinate, 0.1 mM EGTA,

and 10 mM Tris-HCl (pH 7.4). Mitochondrial swelling was

induced by 80 mM K+. The incubation medium contained 1 M

CsA from the beginning of the experiments. Value of ordinate

refers to the A540 nm for 10 min. Data values are represented as

means ± S.D. of three separate experiments. Cyt c was de-

tected by Western blotting. In blank neither Ca2+ nor K+ was

included in the medium. Similar results were obtained in two

additional independent experiments. * represents P < 0.05

when compared to the blank.

Figure 5. K+-induced mitochondrial swelling and

Cyt c release.

T. Ichimura et al. / Journal of Biophysical Chemistry 2 (2011) 10-18

alone did not cause MPT or Cyt c release from mito-

chondria (data not shown). These results suggest that

mitochondrial swelling may act as a major regulator of

Cyt c release from mitochondria treated with K+.

3.4. VDAC Opening May Be Involved in Cyt

C Release

To investigate the involvement of the passage process

through the mitochondrial outer membrane in Cyt c re-

lease, we studied the effect of RuR that inhibits both the

mitochondrial Ca2+ uniporter and the VDAC opening. As

shown in Figure 6(a), RuR significantly inhibited MPT

caused by high concentrations of Ca2+ or alloxan-Ca2+.

The degree of MPT inhibition by RuR was almost equal

to the CsA inhibition (Figure 2). In addition, RuR

strongly inhibited the Cyt c release caused by the induc-

0.5

Blank +RuR

+RuR

ΔA

540 nm

/10 min

A llo x a n - C a

(a)

Cyt crelease (a.u.)

Blank +RuR+RuRCa2+ Alloxan-Ca2+

(b)

The incubation medium contained 25 M RuR from the beginning of

the experiments. (a) MPT and (b) release of Cyt c were induced by 200

M Ca2+ or 1 mM alloxan and 20 M Ca2+ in the presence of 5 mM suc-

cinate. In blank neither Ca2+, alloxan-Ca2+ nor RuR was included in the

medium. Data are represented as means ± S.D. of 3 to 11 experiments.

* represents P < 0.05 compared with mitochondria treated with Ca2+

alone. ** represents P < 0.05 compared with mitochondria treated with

alloxan-Ca2+. The other conditions were the same as previously de-

scribed in Figure 1.

Figure 6. Effects of RuR on MPT and Cyt c release.

ers (Figure 6(b)), but CsA did not (Figure 2). These

results indicate that RuR significantly inhibits mito-

chondrial swelling and Cyt c release in mitochondria

treated with high concentrations of Ca2+ or alloxan-Ca2+,

and these data suggest that VDAC opening may be in-

volved in Cyt c release from mitochondria.

4. DISCUSSION

The present study shows that the addition of MPT in-

ducers, such as high concentrations of Ca2+ alone or al-

loxan-Ca2+, to mitochondrial suspension causes mito-

chondrial swelling and then Cyt c release from mito-

chondria isolated from rat livers. The release of Cyt c,

which is bound to the outer surface of the inner mito-

chondrial membrane, into the cytosol has an important

role in an early event in apoptosis [4,5,7]. CsA, a repre-

sentative MPT inhibitor, significantly attenuated the ex-

tent of MPT induced by Ca2+ alone or alloxan-Ca2+,

whereas Cyt c release from mitochondria was still ob-

served (Figure 2 and Table 1).

When the osmotic pressure in the matrix and inter-

membranous space of mitochondria was equilibrated

with PEG that cannot pass the mitochondrial inner

membrane, mitochondrial swelling and Cyt c release

induced by Ca2+ alone or alloxan-Ca2+ were almost

completely prevented (Figure 4). Brustovetsky and

Dubinsky demonstrated that high molecular compounds

of PEG inhibit mitochondrial swelling induced by high

concentrations of Ca2+ but do not inhibit MPT or the

collapse of the mitochondrial membrane potential [28].

From these results, we propose that MPT is not required

to release Cyt c from mitochondria. K+ causes mito-

chondrial swelling without the induction of MPT in mi-

tochondria isolated from adult rabbit hearts [33]. In our

study, the addition of K+ to the mitochondrial suspension

resulted in the release of Cyt c, which was not inhibited

by CsA (Figure 5). These results suggest that mitochon-

drial swelling that is induced through a process other

than MPT is involved in the release of Cyt c from mito-

chondria treated with alloxan- Ca2+ or high concentra-

tions of Ca2+ alone. Halestrap et al. showed that swelling

was slightly induced by the addition of 20 mM KCl [32].

Crouser et al. demonstrated that the swelling and the

release of Cyt c were induced at ionic strengths that var-

ied from 5 to 150 mM of KCl with 0.1 mol CaCl2, but

the activity of an intermembrane space marker adenylate

kinase was not released during the swelling at interme-

diate values of ionic strength [34], suggesting that the

rupture of mitochondrial outer membrane might not de-

velop in mitochondria treated with intermediate strength

of KCl. Further studies are required to elucidate the

mechanism by which Cyt c is released from mitochon-

dria incubated with various ionic strengths of KCl.

T. Ichimura et al. / Journal of Biophysical Chemistry 2 (2011) 10-18

Accumulating evidence indicates that there are

mechanisms for passage through the outer mitochondrial

membrane including the rupture of the outer membrane

or VDAC opening for the release of Cyt c [10,13-17].

Normally, VDAC monomers form an aqueous pore (2.5

to 3 nm in diameter) that allows uncharged polymers,

such as dextran and PEG with a molecular mass of ap-

proximately 5 kDa, to pass the membranes [35,36]. Cyt c

has a molecular mass of 12 kDa and should be unable to

pass through the mitochondrial outer membrane by

VDAC opening alone. Alternatively, oligomeric forms of

VDAC create a large pore, such as the Bax-VDAC pore,

with conductance levels 4-fold greater than the levels of

VDAC monomers allowing Cyt c translocation across

the outer membrane [19,37]. Ca2+ binding site(s) of

VDAC is involved in the regulation of VDAC opening

[38]. RuR, an inhibitor of MPT and of VDAC opening,

strongly inhibited the Cyt c release (Figure 6). RuR

binds to Ca2+ binding site(s) of VDAC and completely

closes VDAC [38,39]. These data suggested that RuR

may inhibit the opening of VDAC and oligomerization

with MPT induction in mitochondria treated with high

concentrations of Ca2+ alone or alloxan- Ca2+. However,

another VDAC inhibitor, DIDS [12], did not decrease

Cyt c release with high concentrations of Ca2+ (date not

shown). G3139 caused a closure of VDAC and increased

the sensitivity of MPT induction to high concentrations

of Ca2+ (200 M) [40]. Additional studies are needed to

elucidate the mechanism by which Cyt c travels through

the outer mitochondrial membrane to the cytosol.

Alloxan has cytotoxicity against pancreatic-cells and,

thus, causes type 1 (insulin-dependent) diabetes mellitus.

Although detailed mechanisms of the cytotoxicity are

not yet clearly understood, alloxan causes apoptotic cell

death with the release of Cyt c [7]. We previously dem-

onstrated that in mitochondria, which were previously

incubated with alloxan, enhanced MPT was clearly ob-

served after addition of succinate [8]. The characteristics

of MPT in the present study agreed with our previous

report [8]. The inhibitors of mitochondrial function, such

as CCCP, STG, and KCN, significantly inhibited MPT

but did not inhibit the release of Cyt c (Figure 3(b) and

Table 1). These findings suggest that the release of Cyt c

from mitochondria is not parallel with MPT induced by

alloxan-Ca2+ and may be initiated independently of en-

ergy status. The present study demonstrates that mito-

chondrial swelling directly correlates with Cyt c release

from mitochondria treated with high concentrations of

Ca2+ alone or alloxan-Ca2+. There are several lines of

evidence to support that overexpression of calmodulin in

mice results in insulin secretion defects, loss of pancre-

atic-cells and diabetes [41,42]. The elevation of cytoso-

lic Ca2+ concentrations by free fatty acid induces-cells

apoptosis [43]. Several quinones, which have similar

structures to alloxan, reduce the threshold of Ca2+ con-

centrations to induce mitochondrial swelling [26]. These

findings lead us to speculate that alloxan may reduce the

threshold of Ca2+ concentration to induce MPT accom-

panied by swelling and that the subsequent mitochon-

drial dysfunction resulting from Cyt c release may be a

cause of pancreatic-cell death.

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