August 2006 Biol. Pharm. Bull.

29(8) 1613—1617 (2006) 1613

MCI-186 (3-Methyl-1-phenyl-2-pyrazolin-5-one) Attenuated Simulated

Ischemia/Reperfusion Injury in Cultured Rat Hippocampal Cells
Ting WU, Xin-Sheng DING,* Wei WANG, and Jin WU
Department of Neurology, the First Affiliated Hospital of Nanjing Medical University; 300 Guangzhou Road, Nanjing
210029, China. Received December 1, 2005; accepted April 17, 2006; published online May 29, 2006

The reactive oxygen species and Ca2
overload play a critical role in ischemia/reperfusion (I/R) injury. MCI-
186 has potent effects in the brain as a free radical scavenger in ischemia-reperfusion. Acute glucose–oxygen dep-
rivation and subsequent reoxygenation were used to model ischemia/reperfusion injury in cultured hippocampal
cells. MCI-186 reduced malondialdehyde level and raised the SOD activity when applied upon reoxygenation in a
dose-dependent manner compared with the untreated group. The peak neuroprotective effects occurred at 100
and 300 m M. Intracellular free calcium concentration ([Ca2
]i) was significantly reduced in the 100 m M MCI-186-
treated group compared to the untreated group (32.54.0 versus 50.23.6, p0.01). Treatment with 100 m M
MCI-186 significantly inhibited the decrease of mitochondria membrane potential after simulated ischemia/
reperfusion (20411.6% compared with the untreated group, p0.01). Cell apoptotic rate was significantly
decreased following MCI-186 treatment from 33.72.3% (untreated group) to 16.61.4% (100 m M MCI-186
treated group). There was no significantly protective difference between 100 and 300 m M MCI-186. MCI-186
effectively protects neuron injury after simulated ischemia/reperfusion by inhibiting lipid peroxidation, reducing
Ca2
overload, elevating mitochondria membrane potential, and decreasing apoptosis.
Key words reperfusion injury; MCI-186; reactive oxygen species; apoptosis

It has been indicated that reactive oxygen species (ROS)
and Ca2
overload play a critical role in ischemia/reperfusion
(I/R) injury. Oxidative stress can be neurotoxic by inducing
lipid peroxidation and apoptosis of neurons. Lipid peroxida-
tion, followed by interaction of free radicals and unsaturated
fatty acids, has been assumed to be a major mechanism in
the pathogenesis of brain edema and tissue injury in is-
chemia/reperfusion.1) Although endogenous antioxidants
such as superoxide dismutase, glutathione, and catalase are
activated because of oxidative stress following ischemia,
their levels are not high enough to prevent reperfusion injury.2)
Thus, there is always a need for exogenous radical scav-
engers to supplement against I/R injury.
Recently, edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one
(MCI-186)), a potent free radical scavenger, has been suc-
cessfully applied in treatment of patients with acute cerebral
ischemia.3) Previous studies showed that edaravone (MCI-
186) attenuated ischemic brain injury in adult and immature
animal models,4,5) and several clinical trials demonstrated the
neuroprotective effects of edaravone in patients with acute
cerebral infarction.6) However, the precise mechanism of
neuroprotection afforded by MCI-186 remains obscure.
Therefore, in the present study, we attempted to clarify
whether MCI-186 might directly provide neuroprotection
against I/R injury by inhibiting lipid peroxidation in cultured
neural cells.
Intracellular Ca2
overload results in mitochondria dam-
age and activation of a series of proteolytic enzymes which
consequently produce excessive ROS. Mitochondria mem-
brane potential (MMP), the driving force of cellular ATP for-
mation, is generated by the unsymmetrical distribution of
protons and ions between the inner part and the outer part of
the mitochondrial membrane. Mitochondrial depolarization
can potentially be triggered by tissue ischemia and reperfu-
sion7) and by oxidative stress.8) A decrease of MMP impairs
ATP production and is involved in neuron apoptosis. There-
fore, we also investigated the effects of MCI-186 on Ca2

∗ To whom correspondence should be addressed. e-mail: xsding@public1.ptt.js.cn
overload, MMP and apoptosis in primary cultured hippocam-
pal cells.
MATERIALS AND METHODS
All procedures were performed in accordance with the
Nanjing Medical University animal care guidelines, which
conform to the Guide for the Care and Use of Laboratory
Animals (NIH publication No. 85—23, revised, 1985). Every
effort was made to minimize animal suffering and to reduce
the number of animals used.
Materials MCI-186 was provided by BIOMOL Re-
search Laboratories Inc. (U.S.A.); trypsin, Dulbecco’s modi-
fied Eagle’s medium, fetal horse serum, neurobasal medium,
and B27 were purchased from Gibco (U.S.A.); Fluo-3 AM,
Rhodamine-123 and ascorbate were purchased from Sigma
Chemical (U.S.A.); fetal bovine serum was obtained from
Hangzhou Sijiqing Biotechnical Engineering Company
(China); MDA content and SOD activity assay kits were ob-
tained from Nanjing Jiancheng Bioengineering Institute
(China). Annexin V/FITC kit was obtained from Bender
Medsytems Inc. (U.S.A.). All other chemicals were commer-
cial products with analytic purity.
Hippocampal Cell Culture Hippocampal cells were pre-
pared as described previously with some modifications.9—11)
Briefly, the hippocampi of the 1-day-postnatal Sprague-Daw-
ley rats were microscopically collected, digested in 0.025%
trypsin and mechanically dissociated, and the cells were
plated on poly-L-lysine-coated plastic dishes (at a final den-
sity of 2106 cells/ml) or glass coverslips (at a final density
of 2.5105 cells/ml). Cultures were maintained in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10%
fetal bovine serum and 10% fetal horse serum. The plating
medium was replaced after 24 h incubation with serum free
Neurobasal medium containing supplement B27 and 20 m M
cytosine arabinoside. Cell cultures were kept in humidified
95% air–5% CO2 at 37 °C. The medium was changed every
© 2006 Pharmaceutical Society of Japan
1614
third day, and the cells were grown for at least 10 to 12 d be-
fore experiments were performed.
Simulated Ischemia/Reperfusion Model in Cultured
Cells An in vitro model of acute glucose–oxygen depriva-
tion and subsequent reoxygenation was developed in an
attempt to determine the mechanisms underlying I/R injury.
Firstly, the culture medium was switched to serum-free, glu-
cose-free DMEM. The resultant cultures were placed in
specifically designed hypoxic chambers, subjected to succes-
sive rounds of evacuation followed by flushing with 95% N2/
5% CO2 for 15 min, then sealed. These cells were further cul-
tured for 2 h in an incubator at 37 °C.12) Then the cells were
removed from the chamber and maintained in the regular
incubator with the addition of 5.5 mM glucose for 24 h of
reoxygenation. All parameters were detected after 24 h of
reoxygenation. To examine the dose–response relationship
for the neuroprotective effects of MCI-186, cultured cells
were separated into seven groups: normal group, that was in
normal culture condition without any treatment; simulated
I/R control group that underwent simulated I/R injury; four
MCI-186 groups that were exposed to edaravone upon reoxy-
genation at the concentration of 1 m M, 10 m M, 100 m M, and
300 m M, respectively. In the seventh group, an additional
well-known radical scavenger, ascorbate (100 m M),13) was ap-
plied as a reference drug upon reoxygenation.
Assay of MDA Content and SOD Activity To estimate
the antioxidant effect of MCI-186, we measured the concen-
tration of lipid peroxide in hippocampal cells, estimated in
terms of malondialdehyde (MDA). MDA is used as a well-
established indicator of oxidative stress in cells and tissues.
The superoxide dismutase (SOD) is a pivotal enzyme scav-
enging ROS in vivo. MDA level and SOD activity in the cell
culture supernatant were measured by assay kits (Nanjing
Jiancheng Bioengineering Institute, China) according to the
manufacturer’s instructions.14,15) The content of MDA was
determined by the thiobarbituric acid method. Briefly, the
MDA content was assayed in the form of thiobarbituric acid
reacting substances (TBARS). One milliliter of 20%
trichloroacetic acid and 1.0 ml of 1% TBARS reagent were
added to 100 m l cell culture supernatant, then mixed and in-
cubated at 95 °C for 80 min. After cooling on ice, samples
were centrifuged at 1000 g for 20 min, and then the ab-
sorbance of the supernatant was read at 532 nm (Pharmacia
Biotech, Ultrospec 2000, U.S.A.). TBARS results were ex-
pressed as MDA equivalents using tetraethoxy-propane as
standard. MDA content in the cell culture supernatant was
expressed as millimole per milliliter (mmol/ml). SOD activ-
ity was determined by inhibition of nitroblue tetrazolium re-
duction due to superoxide anion generation by a
xanthine–xanthine oxidase system. Briefly, 500 m l of cell cul-
ture supernatant together with 50 m M xanthine and 2.5 m M
xanthine oxidase in 50 m M potassium phosphate buffer were
mixed and incubated at 37 °C for 40 min, then nitro blue
tetrazolium (NBT) was added. The product (nitrite) produced
by the oxidation of oxyamine was measured by monitoring
the absorbance at 550 nm (Pharmacia Biotech, Ultrospec
2000, U.S.A.). One nitrite unit (NU) of SOD activity was de-
termined as the amount of the enzyme to reach an inhibition
of 50% NBT reduction rate. The SOD activity in supernatant
was expressed as nitrite units per milliliter (NU/ml).
Measurement of Intracellular [Ca2
] Hippocampal
Vol. 29, No. 8
cells on coverslips were loaded with 10 m M calcium-sensitive
dye fluo-3 AM at 37 °C for 30 min, then washed three times
with Ca2
/Mg2
-free phosphate-buffered saline to remove
the extracellular fluo-3 AM. The fluorescence was detected
with a laser scanning confocal microscope (ZEISS LSM 510,
German). An argon laser was used to excite fluo-3 AM at
488 nm and emit it at 525 nm. [Ca2
]i changes were repre-
sented by fluorescence intensity.
Detection of Mitochondria Membrane Potential
(MMP) Rhodamine-123, a mitochondria-binding specific
cationic fluorescent dye, was used to measure MMP with a
laser scanning confocal microscope (ZEISS LSM 510, Ger-
man). This dye was released from the mitochondria when
MMP decreased due to mitochondria membrane damage.
The cells were loaded with 20 m M Rhodamine-123 for
30 min. After treatment, the cells were washed twice with
0.1 mM phosphate buffer and fluorescence was then detected
in the dark at an excitation wavelength of 488 nm and an
emission wavelength of 525 nm. The fluorescent intensity of
Rhodamine-123 was used to reflect changes in MMP.
Flow Cytometric Analysis for Cell Apoptosis Cells un-
dergoing apoptosis were detected with the use of double
staining with Annexin V-FITC/PI in the dark according to the
manufacturer’s instructions. Briefly, cells attached to plastic
dishes were harvested by 0.25% trypsin and washed twice
with cold phosphate buffer solution (PBS). The cell pellets
were suspended in 1 binding buffer (10 mM HEPES/NaOH,
pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) at a concentration of
1106 cells/ml. Then the cells were incubated with Annexin
V-FITC and propidium iodide (PI) for 15 min at room tem-
perature (22—25 °C) in the dark. The stained cells were im-
mediately analyzed by flow cytometry (FAC Svantage SE,
U.S.A.). Annexin V-FITC selectively passed through the
plasma membranes of apoptotic cells and stained them with
green fluorescence. Necrotic cells were stained fluorescent
red with propidium iodide. Apoptosis was considered to have
taken place in cells positive for Annexin V-FITC and nega-
tive for PI.
Statistical Analysis All data are expressed as the
meanS.D. Statistical comparisons were evaluated by one-
way ANOVA followed by Student–Newman–Keuls test, with
a value of p0.05 considered significant. All statistical
analyses were performed with software of SPSS 12.0 (SPSS
Inc., U.S.A.).
RESULTS
Effects of MCI-186 on MDA Content and SOD Activity
in Hippocampal Cells MDA content elevated and SOD
activity declined in the simulated I/R control group as com-
pared with the normal group (p0.05, Table 1). Our data ob-
tained after treatment with 100, 300 m M MCI-186 revealed
significant reduction of MDA level and elevation of SOD ac-
tivity versus the simulated I/R control group (p0.05), and
the peak neuroprotective effects were exhibited at 100 m M. A
higher concentration (300 m M) of MCI-186 did not exhibit
stronger protection than that of 100 m M MCI-186 (p0.05).
The effect of the positive reference drug ascorbate (100 m M)
was significantly lower than that of 100 m M MCI-186, and
equal to that of only 10 m M MCI-186. These results indicated
that MCI-186 inhibited lipid peroxidation and restored the
August 2006 1615

activity of SOD. (p0.05). 1 m M, 10 m M MCI-186 and 100 m M ascorbate

Effects of MCI-186 on Intracellular Free Calcium Con- tended to decrease [Ca2
]i, but showed no statistical signifi-
centration ([Ca2
]i) Simulated I/R induced a remarkable cance compared with simulated I/R control.
[Ca2
]i increase in hippocampal cells (simulated I/R: Effects of MCI-186 on MMP in Hippocampal Cells
50.23.6 versus normal: 27.73.8, p0.05). MCI-186 de- After simulated I/R, the green fluorescence intensity of MMP
creased [Ca2
]i in a dose-dependent manner (Fig. 1). [Ca2
]i in hippocampal cells was weaker than that in normal group
was more significantly reduced in the MCI-186-treated group cells (simulated I/R: 54.14.79 vs. normal: 134.38.79, p
(100 m M : 32.54.0 and 300 m M : 32.33.6) than in that of 0.01), indicating the MMP was obviously decreased. The flu-
the simulated I/R group (50.23.6, p0.05). The inhibitory orescence intensity of Rhodamine-123 in the normal group
effect of 300 m M MCI-186 was similar to that of 100 m M was assigned to be 100%. As shown in the summarized data
(Fig. 2), 1 m M, 10 m M MCI-186 and 100 m M ascorbate showed
Table 1. MDA Content and SOD Activity in Cultured Hippocampal Cells no significant effects on elevating MMP (p0.05). However,
the fluorescent intensity was elevated significantly when hip-
Groups MDA (mmol/ml) SOD (NU/ml)
pocampal neurons were treated with 100 and 300 m M MCI-
Normal 0.570.08** 54.365.58** 186, with the maximum level (20411.6%, 2149% com-
Simulated I/R control 1.470.15 26.403.77 pared with simulated I/R control, respectively. p0.01, n
1 m M MCI-186 1.460.15 27.883.57 4). There was no statistical significance between the two con-
10 m M MCI-186 1.370.15 32.293.79* centrations. This result demonstrated that MCI-186 attenu-
100 m M MCI-186 0.870.11** 48.115.54**
300 m M MCI-186 0.900.13** 47.325.82**
ated the reduction of MMP after simulated I/R.
Ascorbate 1.490.16 35.233.45* Effects of MCI-186 on Apoptosis in Hippocampal Cells
After hippocampal cells were exposed to simulated I/R, an-
Data are expressed as meanS.D., and obtained from 10 independent experiments
(n10 ). ∗ p0.05 compared with simulated I/R control group, ∗∗ p0.01 versus simu-
lated I/R control group.

Fig. 1. Effects of MCI-186 on Intracellular Free Calcium of Hippocampal
Cells
MCI-186 induced a [Ca2
]i decrease in simulated I/R in a dose-dependent manner.
Averaged intracellular Ca2
levels are shown as meanS.D. of four separate experi-
ments. ∗ p0.01 versus simulated I/R control.
Fig. 2. Effects of MCI-186 on Mitochondria Membrane Potential
Histograms indicating dose-dependent attenuation of simulated I/R induced MMP
reduction by MCI-186. The fluorescence intensity of Rhodamine-123 in the normal
group was assigned as 100%. Data are meanS.D. of four separate experiments. ∗ p
0.01 versus simulated I/R control.

Fig. 3. Effects of MCI-186 on Apoptosis in Hippocampal Cells

Contour diagram of annexin V/PI flow cytometry of hippocampal cells. Cells under a normal culture condition without any treatment (G); upon reoxygenation cells untreated
with MCI-186 (A), or treated with MCI-186 1 m M (B), 10 m M (C), 100 m M (D), 300 m M (E), or 100 m M ascorbate (F). The lower right quadrant represents the early apoptotic cells
(Annexin V-positive, PI-negative).
1616
nexin-V and PI double-staining flow cytometry analysis
showed that the apoptotic rate was 33.72.3%, which was
significantly higher than that of the normal group (5.60.8%,
p0.01). When treated with 100 and 300 m M MCI-186, the
percentage of cells in the apoptotic region attenuated to
16.61.4% and 14.21.7%, respectively, but MCI-186 ap-
plied at lower concentrations (1, 10 m M) was not neuroprotec-
tive. The well-known radical scavenger ascorbate also failed
to reduce the apoptotic rate (302.6%, p0.05 vs. simulated
I/R control). Data are meanS.D. of four separate experi-
ments.
DISCUSSION
The results of our study demonstrated that the free radical
scavenger MCI-186 exerted direct protection from simulated
I/R injury. Our data revealed that MCI-186 significantly re-
duced MDA level and enhanced SOD activity of hippocam-
pal cells compared with simulated I/R control. This directly
validated that MCI-186 removed free radicals and so inhib-
ited lipid peroxidation. In primary experiments, we detected
the total SOD activity in both cell culture supernatant and
cell lysates of seven groups of cells. Although the actual data
obtained in culture supernatant was different from that of cell
lysates, the SOD activity changing trend in cell culture su-
pernatant and cell lysates was similar (data not shown). We
also found that MCI-186 prevented pathological apoptosis of
hippocampal cells. The neuroprotective effects of MCI-186
were exhibited at least partially in a dose-dependent manner,
and low concentration of MCI-186 showed no protective
effect. In order to optimize incubations of the hippocampal
cells in terms of MCI-186 concentrations, we performed pre-
liminary experiments where the cells were cultured with dif-
ferent concentrations (1, 10, 100, 300, 1000 m M), and found
that the protective effect of 1000 m M MCI-186 was similar to
that of 300 m M MCI-186 (data not shown). We also con-
firmed that 100 and 1000 m mol/l MCI-186 did not change
cell viability when applied alone under normoxic conditions
(data not shown). Ascorbic acid is a well-known free radical
scavenger. The molecular weight of MCI-186 and ascorbate
is similar, the former 174.2 and the latter 176.12. Ascorbate
is a water-soluble free radical scavenger. MCI-186 is lipid-
soluble and has improved cell membrane permeability com-
pared with ascorbate.13) Any delay and decrease in the deliv-
ery of the drug to the site of oxidant generation will likely at-
tenuate the extent of protection. Therefore, it is probable that
the lower membrane permeability results in the protective
discrepancy between MCI-186 and ascorbic acid.
It was reported that the pathophysiology of the reperfusion
injury might be involved in calcium overload, osmotic pres-
sure overload, and particularly in free radicals that produced
excessively in I/R. Intracellular reactive oxygen species
(ROS) sharply increase after reperfusion. SOD catalyzes the
dismutation of the highly reactive superoxide radical anion to
hydrogen peroxide and molecular oxygen. Although endoge-
nous antioxidant SOD is activated because of oxidative stress
following I/R, much of the SOD activity will be consumed
by the burst of ROS; so SOD activity is greatly reduced after
reperfusion. ROS, including hydroxy radical, hydrogen per-
oxide, and hydrogen peroxide, are demonstrated to be one of
the major factors in reperfusion injury.16,17) The main produc-
Vol. 29, No. 8
tive sources of free radicals in I/R are suggested to be in-
volved in the xanthine oxidase–hypoxanthine system,18) the
electron transmission system in mitochondria, the arachi-
donic cascade,19) and the NADPH oxidase system in leuko-
cytes.20) The increased ROS contribute to various conse-
quences responsible for I/R injury, such as modification of
phospholipids and proteins leading to lipid peroxidation and
oxidation of thiol groups.21) Oxidative stress combined with
decreased ATP level results in depression of the sarcolemmal
Ca2
-pump ATPase and Na
-K
-ATPase activities, thus
leading to increased Ca2
-influx and decreased MMP. Exces-
sive ROS, lipid peroxidation and Ca2
overload can interact
with each other, eventually resulting in irreversible neuron
damage.
MCI-186 has been reported to eliminate hydrogen oxide
radicals that have higher reactivity to trigger the lipid peroxi-
dation and so to inhibit the initiation and propagation of lipid
peroxidation by hydrogen peroxide radicals.21) MCI-186 can
interact with both peroxyl and hydroxyl radicals, and then
format a stable oxidation product (OPB: 2-oxo-3-(phenylhy-
drazono)-butanoic acid) through a radical intermediate.22)
Many damaging factors such as insufficient oxygen, oxida-
tive stress and defective blood perfusion can induce the rising
of intracellular calcium. And Ca2
overload results in inhibi-
tion of the oxidative phosphorylation process in mitochon-
dria and in the decrease of MMP. Mitochondrial depolariza-
tion may initiate cellular apoptosis by disturbing cellular
ATP production or by releasing mitochondrial pro-apoptotic
factors.23,24) Our study showed simulated I/R -induced Ca2

overload and MMP decrease were attenuated by MCI-186.
After stroke, nerve cell death now appears more likely to un-
derlie apoptosis, rather than necrosis. Using Annexin-V and
PI double stain, we subsequently studied the inhibitive effects
of edaravone on apoptosis. Our data showed MCI-186 effec-
tively decreased apoptosis of hippocampal cells after simu-
lated I/R. Our results strongly suggested that MCI-186 ex-
erted its brain-protective effects by targeting the mitochon-
dria at the time of reoxygenation. However, further studies
are needed to identify the precise mechanism.
Despite substantial research in the field of drug-induced
neuroprotection in vivo and in vitro studies, few agents with
potentially neuroprotective effects are available for clinical
practice. Thus, it is recognized that MCI-186 can act as a po-
tential neuroprotective agent. Furthermore, recent studies
have shown that MCI-186 has protective effects against I/R
injury of other organs, including heart, kidney, liver, and
ileum.25—27) Despite the prevalence of brain pathophysiology
that encompasses acute and acquired as well as chronic and
congenital processes, the therapeutic options available to
counteract neuronal damage are still limited. From the clini-
cal standpoint, elucidation of these precise mechanisms of
neuroprotective drugs that may lead to therapeutic manipula-
tion is an urgent agenda in neurology. In conclusion, MCI-
186 is a potent neuroprotective agent against simulated I/R
injury. We demonstrated here that MCI-186 protects neuron
cells from simulated I/R injury by scavenging free radicals,
inhibiting lipid peroxidation and Ca2
overload, elevating
MMP reduction, as well as attenuating pathological apopto-
sis due to reperfusion injury following ischemia.
Acknowledgements We thank Ms. Zhang Hong and Dr.
August 2006
Xiao Hang for their secretarial and technical support, and we
also thank Dr. Martin Reivich for helpful discussion.
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