Effects of a calcium-channel blocker (CV159) on hepatic ischemia/reperfusion injury in rats: Evaluation with selective NO/pO2 electrodes and an electron paramagnetic resonance spin-trapping method

Article abstract of DOI:10.1248/bpb.33.77

Nitric oxide (NO) and the partial pressure of oxygen (pO2) in the liver were simultaneously quantified in rats with partial hepatic ischemia/reperfusion injury (PHIRI). Real-time NO/pO2 monitoring and immunohistochemical analysis for superoxide dismutase and inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS) were performed to evaluate the protective effects of a dihydropyridine-type calcium-channel blocker-CV159-on PHIRI. Serum high-mobility-group box-1 (HMGB-1) was measured to assess cellular necrosis. Moreover, we used in vitro/ex vivo electron paramagnetic resonance spin trapping to assess the hydroxyl radical ( ?OH)-scavenging activity (OHSA) of CV159 and the liver tissue. The NO levels were significantly higher in CV159-treated rats than in control rats throughout the ischemic phase. Immediately after reperfusion, the levels temporarily increased in waves and then gradually decreased in the treated rats but remained constant in the control rats. pO ₂ was continually higher in the treated rats. In these rats, hepatic eNOS expression increased, whereas iNOS expression decreased. The treated rats exhibited significantly higher cytosolic and mitochondrial concentrations NOx (NO₂+NO₃). The serum HMGB-1 levels significantly decreased in the treated rats. Moreover, CV159 directly scavenged ·OH and both mitochondrial and cytosolic OHSA were preserved in the treated rats. Thus, CV159-mediated inhibition of intracellular Ca²⁺ overloading may effectively minimize organ damage and also have ·OH-scavenging activity and the cytoprotective effects of eNOS-derived NO.

Full text of DOI:10.1248/bpb.33.77

January 2010
Biol. Pharm. Bull. 33(1) 77—83 (2010)
77
Effects of a Calcium-Channel Blocker (CV159) on Hepatic
Ischemia/Reperfusion Injury in Rats: Evaluation with Selective NO/pO2
Electrodes and an Electron Paramagnetic Resonance Spin-Trapping
Method
a
Keizo HATAJI, Taiji WATANABE, ,b Shigeru OOWADA,a Masaki NAGAYA,c Masato KAMIBAYASHI,d
*
Eiichi MURAKAMI,e Hiroyoshi KAWAKAMI,f Atsuko ISHIUCHI,a Toshio KUMAI,g Hiroshi NAKANO,a
Shinichi KOBAYASHI,g and Takehito OTSUBO
a
a Department of Gastroenterological and General Surgery, St. Marianna University Hospital; g Department of
Pharmacology, St. Marianna University Hospital; 2–16–1 Sugao, Miyamae-ku, Kawasaki 216–8511, Japan:
b Hamamatsucho 1st Clinic Yokohama; Kamiya building 4F, 2–19–4 Tsuruya-cho, Kanagawa-ku, Yokohama; 221–0835,
Japan: c Department of Surgery, Pittsburgh, University of Pittsburgh; PA 15201, U.S.A.: d Kyoto Pharmaceutical
University; Yamashina, Kyoto 607–8412, Japan: e Eikokagaku; Tokyo 156–0041, Japan: and f Department of Applied
Chemistry, Tokyo Metropolitan University; 1–1 Minami-Osawa, Hachioji, Tokyo 192–0397, Japan.
Received July 13, 2009; accepted October 10, 2009; published online October 16, 2009
Nitric oxide (NO) and the partial pressure of oxygen (pO2) in the liver were simultaneously quantified in rats
with partial hepatic ischemia/reperfusion injury (PHIRI). Real-time NO/pO2 monitoring and immunohistochem-
ical analysis for superoxide dismutase and inducible nitric oxide synthase (iNOS) and endothelial NOS (eNOS)
were performed to evaluate the protective effects of a dihydropyridine-type calcium-channel blocker—CV159—
on PHIRI. Serum high-mobility-group box-1 (HMGB-1) was measured to assess cellular necrosis. Moreover, we
used in vitro/ex vivo electron paramagnetic resonance spin trapping to assess the hydroxyl radical (·OH)-scav-
enging activity (OHSA) of CV159 and the liver tissue. The NO levels were significantly higher in CV159-treated
rats than in control rats throughout the ischemic phase. Immediately after reperfusion, the levels temporarily in-
creased in waves and then gradually decreased in the treated rats but remained constant in the control rats. pO2
was continually higher in the treated rats. In these rats, hepatic eNOS expression increased, whereas iNOS ex-
pression decreased. The treated rats exhibited significantly higher cytosolic and mitochondrial concentrations
NOx (NO2ꢀNO3). The serum HMGB-1 levels significantly decreased in the treated rats. Moreover, CV159 di-
rectly scavenged ·OH and both mitochondrial and cytosolic OHSA were preserved in the treated rats. Thus,
CV159-mediated inhibition of intracellular Ca2ꢀ overloading may effectively minimize organ damage and also
have ·OH-scavenging activity and the cytoprotective effects of eNOS-derived NO.
Key words hepatic ischemia/reperfusion injury; nitric oxide; endothelial nitric oxide; high-mobility-group box-1; hydroxyl
radical-scavenging activity
Nitric oxide (NO) is an important bioregulatory molecule pyridinedicarboxylic acid methyl 6-(5-phenyl-3-pyrazolyl-
that was originally known as the endothelium-derived relax- oxy) hexyl ester (designated as CV159), which is a 1,4-dihy-
ing factor. Its effects in modulating vascular tone have been dropyridine derivative, represents a new class of calcium
well documented.1—3) NO is generally known to inhibit reac- antagonists that function via the selective blockade of
tive oxygen species (ROS)-mediated reactions, and its pro- Ca2ꢁ/Calmodulin (Ca2ꢁ/CaM).10,11) By performing in vivo
tective effects under various conditions are presumably and ex vivo electron paramagnetic resonance (EPR) measure-
attributable to its ability to detoxify ROS such as superoxide ments, we found that CV159 retained its organ-reducing ac-
(O2ꢀ ) and hydroxyl radicals (·OH) in hepatic ischemia/reper- tivity against radicals in hepatic I/R injury. In ischemic liver
·
fusion (I/R) injury.2—4) Immediately after the initiation of injury, CV159 prevents intracellular Ca2ꢁ overloading and
reperfusion, endothelial nitric oxide synthase (eNOS) be- may thus effectively minimize organ damage by inhibiting
comes dysfunctional and stops generating NO, this increases ROSs.12,13) On the other hands, our previous study revealed
ROS production by Kupffer cells and induces neutrophil re- that activation of high-mobility-group box-1 (HMGB-1) is
cruitment.4—7) However, the endogenous levels of eNOS dur- involved in the immediate proinflammatory stress response
ing hepatic I/R injury have not been examined under condi- to hepatic I/R injury and that treatment with an anti-HMGB-
tions of ROS inhibition. In order to better understand the bio- 1 antibody significantly improves survival. We proposed that
logical roles of NO, its levels should be measured under con- serum HMGB-1 levels can be clinically used as a marker of
ditions of hepatic I/R injury with significant ROS suppres- liver injury, especially in hepatocellular necrosis.14)
sion.8)
The present study was designed to investigate the kinetics
We previously reported that NO and partial pressure of of HMGB-1 and the effects of CV159 treatment in hepatic
oxygen (pO2) levels can feasibly be monitored under I/R injury. We examined the organ-reducing ability of CV159
ischemic conditions in rats with I/R injury of the small bow- in rats by using a real-time system for simultaneously moni-
els by using electrodes selective for NO and O2 molecules.9) toring the NO concentrations, the dynamics of the pO2, and
We have been studying the advanced treatment of hepatic the concentration of NOx—the final metabolite of NO—
I/R injury. 1,4-Dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5- under conditions of ROS suppression. Further, we performed
© 2010 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: twhama-1@honey.ocn.ne.jp

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Vol. 33, No. 1
immunohistochemical analysis to identify the mechanisms ryl)-5-methyl-1-pyrroline N-oxide (CYPMPO).16) When
by which CV159 maintains eNOS expression and inhibits in- phosphate buffer (100 mM) solution containing 10 mM H2O2
ducible NOS (iNOS) expression in hepatic cells under condi- and 10 mM CYPMPO (spin trap) was subjected to ultraviolet
tions of I/R injury. In addition, because the ·OH is very illumination for 5 s, the EPR spectrum shown in Fig. 1 was
harmful and impairs cellular functions, we assessed the ·OH- obtained. The spectrum was reproduced with the computer
scavenging activity of CV159 and the live tissue by using an spectrum simulation and hyperfine splitting constants were
in vitro EPR spin-trapping method.
listed in previous article.16) When CV159 was introduced
into this system, the signal intensity of the CYPMPO-OH
adduct decreased. For comparison, we also examined the
·OH-scavenging activity (OHSA) of nicardipine—a known
MATERIALS AND METHODS
Animals Male Sprague-Dawley rats (8—10 weeks old) calcium antagonist. For measurement of OHSA in mitochon-
were used for this study. All procedures were carried out in drial and cytosolic fractions of the liver, tissue samples were
accordance with the guidelines of the St. Marianna Univer- treated by the method described following part. The meas-
sity Institution Animal Care and Use Committee.
urement of the hydroxyl radical scavenging activity was
Synthesis of CV159 CV159 was synthesized according based on the competitive reaction between CPMPO and the
to a previously described method.11) In brief, the reaction of sample for scavenging ·OH. The hydroxyl radical scavenging
6-chlorohexanol with 3-phenyl-5-pyrazolone in the presence activity was expressed as reduced glutathione (GSH; Sigma
of K2CO3 in dimethylformamide (DMF) produces 6-(5- Chemical, St. Louis, MO, U.S.A.) concentration equivalent
phenyl-3-pyrazolyloxy) hexyl alcohol. This compound in (mM/mg proteins) using the standard curve of GSH. Linear
turn reacts with ammonium carbonate in refluxing methanol correlation between OH radical peak height (4th or 5th signal
to produce 3-amino-crotonic acid 6-(5-phenyl-3-pyrazolyl- peak height used) and GSH concentration was obtained (data
oxy) hexyl ester, which is finally cyclized with methyl 3-ni- not shown). EPR conditions were as follows; magnetic fields
trobenzylidene acetoacetate in ethanol.
Surgical Preparation General anesthesia was induced power 6 mW, time constant 0.1 s, and scanning time 2 min.
in the rats by administering an intraperitoneal (i.p.) injection Electrodes and Measurement Instruments The NO
336.2ꢃ7.5 mT, modulation width 1.0ꢂ0.1 mT, microwave
of 40 mg/kg sodium pentobarbiturate (Dainippon Pharma- levels and pO2 were measured using novel NO- and pO2-se-
ceutical Co., Ltd., Osaka, Japan). The abdomen was shaved, lective microelectrodes that were developed in our laboratory
and a midline incision extending from the xiphoid process to for the real-time monitoring of NO levels and pO2. In design-
the pubis was made. The xiphoid process was clamped, and ing a working microelectrode, we combined a commercially
the thoracic and abdominal cavities were further exposed by available NO-selective microelectrode (NOE-47; Eikouka-
using retractors along the sides. The bowel loops were exteri- gaku, Tokyo, Japan) and an amperometric oxygen microelec-
orized and placed on saline-soaked gauze to gain access to trode (POE-20W, Eikoukagaku). The respective counter elec-
the liver and accessory structures.
trodes employed are commercially available (NOR-20 and
Partial Hepatic I/R Injury We induced partial hepatic POR-10DEK, Eikoukagaku). The principle of the method
ischemia in the rats by maintaining ischemia for 60 min, fol- used is as described previously.9,17) NO and oxygen diffuse
lowed by reperfusion, as described previously.15) In brief, the through the gas-permeable membranes of the working elec-
rats were prepared for surgery, and the portal triad (the portal trodes and are oxidized at the electrodes, thus generating
vein, hepatic artery, and bile duct) was subsequently identi- electrical current. The redox currents between the working
fied. We then occluded the blood flow to the median and left and counter electrodes were detected using a current–voltage
hepatic lobes while preserving adequate blood flow to the converter circuit, an NO meter (NO-502, Eikoukagaku), and
right and caudate lobes by placing a microvascular clamp on a pO2 meter (PO2-150S, Eikoukagaku). The currents were
the respective vascular branches.
recorded on a data acquisition system (PowerLab/8sp;
Experimental Protocol The rats were divided into a ADInstruments Pty, Castle Hill, NSW, Australia) connected
CV159-treated group and a control group. During the induc- to a computer (Chart software program, ver. 5.4.1; ADInstru-
tion of partial hepatic I/R injury (PHIRI), the rats in the ments Pty). The electrodes were calibrated daily before each
CV159-treated group were intraperitoneally administered experiment. 1-Hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-
CV159 (60 mg/kg) 1 h before clamping. The control rats were 3-methyl-1-triazene (NOC-7) was diluted to 50, 100, and
treated with an equal volume of the vehicle [phosphate- 200 mM in an aqueous solution of 0.1 N NaOH and immedi-
buffered saline (PBS)].
ately used as an NO donor. A calibration curve was plotted
Blood Sampling Blood samples were collected in sterile by measuring the current generated when NOC-7 was added
heparinized tubes. The serum was separated by centrifuga- to Krebs-4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
tion at 10000ꢂg for 10 min and stored at ꢀ80 °C until the (HEPES) solution. The electrode currents generated with
assay was performed. The HMGB-1 levels were determined various concentrations of NO and NOC-7 were plotted on the
in blood samples collected before and 24 h after reperfusion.
X and Y axes, respectively. The final standard curve for
In Vitro EPR Spin Trapping To measure the direct NOC-7 was expressed in terms of pA and mM. The NO–pO2
·OH-scavenging activity of CV159 and the liver tissue, we microelectrode was calibrated to a pO2 value of 150 mmHg
employed an EPR spin-trapping method. The ·OH radical in Krebs–HEPES solution under atmospheric pressure.
was produced by photolysis of hydrogen peroxide (H2O2),
NOx Measurement Cytosolic and mitochondrial frac-
and the activity of CV159 in scavenging this radical was then tions of rat liver were prepared according to previously de-
measured by setting up a competitive reaction with the spin- scribed methods.12,13) In brief, 10 volumes of homogenization
trapping agent 5-(2,2-dimethyl-1,3-propoxy cyclophospho- buffer A (0.25 M sucrose, 100 (50) mM Tris–HCl (pH 7.4),

January 2010
79
0.1 mM EDTA-2Na, and a protease inhibitor) were added to The standard dilutions and samples were introduced into
frozen rat liver samples, and the tissue was homogenized for solid-phase microtiter plates (100 ml/well) coated with an
30 s in an ultrasonic homogenizer. The homogenate was fil- anti-HMGB-1 monoclonal antibody (clone FBH7). The reac-
tered through a nylon mesh and centrifuged at 50ꢂg for tion was allowed to proceed for 1 h at room temperature. The
10 min at 4 °C. The supernatant was collected, an equal vol- plates were then washed 4 times with PBS, and rabbit anti-
ume of homogenization buffer B (0.34 M sucrose, 100 HMGB-1/2 antibodies adjusted with PBS containing 1% g-
(50) mM Tris–HCl (pH 7.4), 0.1 mM EDTA-2Na, and a pro- globulin (pH 7.4) were added to the plates (100 ml/well); this
tease inhibitor) was gently added to it, and the solution was reaction was allowed to proceed for 1 h at room temperature.
centrifuged at 700ꢂg for 10 min to remove the nuclear com- The plates were then washed 4 times with PBS, and peroxi-
ponents.18) The supernatant was further centrifuged at dase-labeled anti-rabbit IgG (HꢁL) was added to the plates
8000ꢂg for 10 min to separate the mitochondrial fraction, (100 ml/well). The plates were incubated for 1 h at room tem-
and the supernatant thus obtained was centrifuged at perature and then washed 4 times with PBS. Next, tetram-
100000ꢂg for 60 min to obtain the cytosolic fraction. The ethylbenzidine (TMB) was added to the plates (100 ml/well)
NOx concentrations in both the mitochondrial and cytosolic and allowed to react for 30 min at room temperature; the re-
fractions were determined using a NOx Visible Detector/S- action was arrested by adding 2 N sulfuric acid (100 ml/well).
3210 system (Tokyo Kasei Kogyo, Tokyo, Japan).
Immunohistochemical Analysis The liver tissue speci- mined by comparison with the standard curve.14)
mens were fixed in 10% phosphate-buffered formalin, dehy- Statistical Analysis All data were stored and statistically
The HMGB-1 concentrations in the samples were then deter-
drated, and embedded in paraffin. Further 5-mm sections analyzed using the Statistical Analysis System. The results
were cut and stained with hematoxylin and eosin. For im- are expressed as the meansꢃstandard deviation (S.D.).
munohistochemical analysis of iNOS and eNOS, the sections Groups were compared by performing nonparametric
were treated with anti-iNOS and anti-eNOS polyclonal anti- ANOVA, depending on the type of variable. Significance was
bodies (1 : 100; Santa Cruz Biotechnology, Santa Cruz, CA, assumed at pꢄ0.05.
U.S.A.) and stained using an En Vision kit (Dako, Tokyo,
Japan). The iNOS-positive regions were observed under a RESULTS
Leica DM3000 optical microscope. For each rat, 20—30 sec-
tions were randomly selected, and iNOS expression was
quantified using a digital image analysis software program rate was 100% in both the control and CV159-treated groups.
(Leica QWin V3). iNOS expression was calculated as a per- ·OH Radical-Scavenging Activity A typical ·OH radi-
Survival Rate after PHIRI The postoperative survival
centage of the area with positive iNOS staining to the area of cal adduct was obtained by photolysis of H2O2 solution (Fig.
the whole section. Further, eNOS expression was quantified 1; blank). This spectrum consists of two identical groups of
in a similar manner.
lines which are split by a large hyperfine coupling with phos-
HMGB-1 Measurement For measuring HMGB-1 ex- phorus 31. The spectrum was reproduced with the computer
pression, rat serum was diluted 5-fold with PBS containing spectrum simulation using following hyperfine splitting con-
1% casein-Na (pH 7.4). A complex of bovine HMGB-1 and stants (AN; 1.37/1.35, AH; 1.37/1.23, AP; 4.88/4.70, respec-
HMGB-2 (hereafter referred to as HMGB-1/2) was used as a tively). CV159 could scavenge the ·OH radical when used at
standard and was serially diluted from 200 to 0 ng/ml (spe- a concentration of 15 mmol/mg protein. In contrast, nicardi-
cific dilutions: 200, 100, 50, 25, 12.5, 6.3, 3.1, and 0 ng/ml). pine did not exhibit ·OH-scavenging activity even at a con-
Fig. 1. ·OH-Scavenging Activity
A typical ·OH radical adduct was obtained by photolysis of H2O2 solution (blank). This spectrum consists of two identical groups of lines which are split by a large hyperfine
coupling with phosphorus 31. The spectrum was reproduced with the computer spectrum simulation using following hyperfine splitting constants (AN; 1.37/1.35, AH; 1.37/1.23,
AP; 4.88/4.70, respectively). CV159 exhibited ·OH-scavenging activity when used at a concentration of 15 mmol/mg protein, while nicardipine did not exhibit such activity even
when used at a concentration of 150 mmol/mg protein. The ·OH-scavenging activity of CV159 was dose dependent.

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Fig. 2. Mitochondrial and Cytosolic OHSA in the 24 h after Reperfusion
OH radical scavenging activity in the liver mitochondrial and cytosolic fractions after 24 h experiment. OHSA were expressed as GSH concentration equvalent (mmol/mg pro-
teins).
centration of 150 mmol/mg protein. Mitochondrial and cy- staining with the anti-iNOS antibody (Fig. 5).
tosolic OHSA were decreased in control rats while they were
preserved in CV159-treated rats (Fig. 2).
Assessment of Hepatocellular Injury To determine
whether CV159 has protective effects against hepatic I/R in-
Real-Time Monitoring of NO Levels, pO2, and NOx jury, we administered this agent to rats with induced hepatic
Levels in the Liver The NO levels (pA) in the control I/R injury. In the control group, the serum HMGB-1 levels at
group at various time points were as follows: baseline, 6 and 24 h after reperfusion were significantly higher than
1863.8ꢃ697.9; after clamping, 1696.8ꢃ375.5; before de- those at baseline. Further, the levels at 24 h after reperfusion
clamping, 1127.5ꢃ125.3; and after declamping, 1799.5ꢃ were significantly lower in the CV159-treated group than in
486.2. The levels in the CV159-treated group were as fol- the control group. The serum HMGB-1 level in the sham
lows: baseline, 3437.5ꢃ298.3; after clamping, 2064.5ꢃ group was 3.0ꢃ1.42 mg/ml. In the control group, the levels
428.6; before declamping, 2908ꢃ835.4; and after declamp- were 2.48ꢃ1.25 and 9.86ꢃ2.64 mg/ml at 6 and 24 h after
ing, 4363.3ꢃ1420.8.
reperfusion, respectively (pꢄ0.01). In the CV159-treated
The pO2 (mmHg) values in the control group at various group, the levels were 2.64ꢃ1.93 and 2.25ꢃ1.35 mg/ml at 6
time points were as follows: baseline, 8.37ꢃ2.72; after and 24 h after reperfusion, respectively (Fig. 6).
clamping, 4.73ꢃ2.26; before declamping, 4.63ꢃ1.65; and
after declamping, 5.30ꢃ2.45. The values in the CV159- DISCUSSION
treated group were as follows: baseline, 17.40ꢃ8.96; after
clamping, 14.88ꢃ9.22; before declamping, 18.37ꢃ12.48;
and after declamping, 29.93ꢃ8.39.
CV159 represents a new class of calcium antagonists that
function by selective blockade of Ca2ꢁ/CaM. Previous stud-
The NO levels and pO2 in the liver were simultaneously ies have shown that CV159 at a micromolar-order concentra-
monitored. In the PBS-treated controls, the NO levels re- tion inhibits the expression of Ca2ꢁ/CaM-activated myosin
mained constant throughout the measurement, and the pO2 light chain kinase (MLCK); however, it does not inhibit
current decreased after clamping. The NO level at baseline trypsin-treated MLCK, which is active in the absence of
was significantly higher in the CV159-treated group than in Ca2ꢁ/CaM.10,11) Moreover, the precise role of CaM in intra-
the control group. In the CV159-treated group, the NO level cellular signaling has gradually been elucidated. Large in-
decreased soon after clamping, remained constant under is- creases in the free Ca2ꢁ concentration in the cytosol activate
chemic conditions, and was significantly higher than that in several kinases that are important for neural plasticity, in-
the control group just before unclamping. Immediately after cluding Ca2ꢁ/CaM-dependent kinase II (CaM II), protein ki-
reperfusion, the NO level in the CD159-treated group tem- nase A (PKA), and protein kinase C (PKC). Further, Oꢀ2 · is
porarily increased in a wave-like manner and then gradually known to activate these kinases, mainly in non-neuronal sys-
decreased. After a momentary time lag, pO2 similarly began tems, and Oꢀ2 · production in the mitochondria partly pro-
to increase in a wave-like manner (Fig. 3).
ceeds independent of [Ca2ꢁ].10,11) We reported that CV159
NOx Concentrations The cytosolic NOx level in the exhibits considerable organ-reducing activity in hepatic I/R
CV159-treated group (2.33ꢃ0.63 nmol/mg protein) was injury using an in vivo and ex vivo EPR technique with a spin
lower than that in the control group (3.58ꢃ0.27 nmol/mg probe.12,13)
protein) and almost equal to that in the sham group
NO, especially when present in excess, is cytotoxic and
(1.31ꢃ0.21 nmol/mg protein; Fig. 4). The mitochondrial causes lipid peroxidation, peroxynitrite (ONOOꢀ) formation,
NOx level in the CV159-treated group (9.37ꢃ0.17 nmol/mg and the inhibition of mitochondrial respiration.19—22) In the
protein) was considerably lower than that in the control late stages of reperfusion, activation of iNOS in Kupffer
group (18.69ꢃ0.54 nmol/mg protein) and slightly lower than cells, neutrophils, and hepatocytes drastically increases the
that in the sham group (6.94ꢃ0.28 nmol/mg protein; Fig. 4).
production of NO, causing injury to the surrounding cells.4,8)
Pathological and Immunohistochemical Findings In Conversely, eNOS-derived NO may exert protective effects
the CV159-treated group, the liver samples exhibited strong in models of I/R injury by inducing vasodilation and enhanc-
positive staining with the anti-superoxide dismutase (SOD) ing perfusion in postischemic tissue.1—3) However, because
and anti-eNOS antibodies and weak staining with the anti- NO is extremely labile and difficult to measure indepen-
iNOS antibody. In contrast, the control group samples exhib- dently, its role and dynamics in the pathogenesis of hepatic
ited weak staining with the anti-eNOS antibody and strong I/R injury have not been fully elucidated.

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Fig. 4. Hepatic NOx Levels (nmol/mg protein) 24 h after Reperfusion
NOx levels in the sham group (open bars), the group with induced I/R (gray bar),
and the group with induced I/R and CV159 treatment (filled bars). The level in the
CV159-treated group was lower than that in the control group and almost equal to that
in the sham group. Data are presented as the meansꢃS.D. Each group contained 6 ani-
mals. ∗ pꢄ0.01.
In the present study, we gained some novel insights into
the action mechanism of CV159 administered after hepatic
I/R injury. First, we studied the real-time dynamics of NO in
rats with hepatic I/R injury, under conditions of ROS sup-
pression. During the ischemic phase of PHIRI, the NO level
was significantly higher in the CV159-treated rats than in the
control rats at all time points considered. Immediately after
reperfusion, the NO level temporarily increased in a wave-
like manner and then gradually decreased in the CV159-
treated group but did not change noticeably in the control
group. Moreover, immunohistochemical analysis revealed
that at 24 h after reperfusion, eNOS expression in the liver
samples was markedly higher, and the iNOS expression sig-
nificantly lower, in the CV-treated group than in the control
group. These findings suggest that CV159 can increase NO
production through eNOS activation. Studies have revealed
that some dihydropyridine-type calcium-channel antagonists,
such as amlodipine, exert vasodilative effects by activating
eNOS and inhibiting the production of the superoxide anion
radical (O2ꢀ·).21,23,24) Secondly, in our in vitro experiments,
CV159 exhibited ·OH-scavenging activity in a dose-depend-
ent manner. In the early stage of PHIRI, both NO production
and the ·OH-scavenging activity of CV159 may have benefi-
cial effects: The superoxide anion radical reacts with NO to
produce peroxynitrite, thus yielding a highly toxic species
such as the hydroxyl radical (·OH)—the start point of lipid
peroxidation and/or endothelial injury. We also found that
CV159 directly scavenged the ·OH radical in vitro and both
mitochondria and cytosolic OHSA in the liver were pre-
served in CV-159 treated rats. Thirdly, we measured the con-
centrations of NOx—the final metabolite of NO—in cytoso-
lic and mitochondrial fractions of rat liver. Interestingly, we
found that at 24 h after reperfusion, these concentrations in
the CV159-treated group were significantly lower than those
in the control group and almost equal to those in the sham
group. iNOS is known to produce NO in large amounts. Our
results suggest that CV159 exerts protective effects against
the toxicity of iNOS-derived NO. In our previous study, we
found that the superoxide-scavenging activity (SSA) in
CV159-treated rats was higher than that in untreated rats and
almost equal to that in a sham group of rats.12,13) In the pres-
ent study, the observed increase in the NOx levels and SOD
in the liver indicated the protective effects of CV159 on cells.
Fig. 3. (a) Representative Chart of Hepatic NO and pO2 Levels in CV159
and PBS Treated Rat
A typical graph showing the real-time dynamics in the 2 groups. In the PBS-treated
controls, the NO level remained constant. The pO2 current decreased after clamping.
The NO level at baseline was significantly higher in the CV159-treated group than in
the control group. The NO level temporarily increased in a wave-like manner immedi-
ately after clamping and was then restored to the baseline level. Immediately after
reperfusion, the NO level temporarily increased in a wave-like manner and then gradu-
ally decreased. After a momentary time lag, pO2 began to increase in a wave-like man-
ner.
(b) Hepatic NO Levels in CV159 and PBS Treated Rats
The NO levels (pA) in the control group at various time points were as follows: base-
line, 1863.8ꢃ697.9; after clamping, 1696.8ꢃ375.5; before declamping, 1127.5ꢃ125.3;
and after declamping, 1799.5ꢃ486.2. The levels in the CV159-treated group were as
follows: baseline, 3437.5ꢃ298.3; after clamping, 2064.5ꢃ428.6; before declamping,
2908ꢃ835.4; and after declamping, 4363.3ꢃ1420.8. NO levels in the control and
CV159-treated groups at 4 time points (before clamping, after clamping, before un-
clamping, and after unclamping). Data are presented as the meansꢃS.D. Each group
contained 4 animals.
(c) Hepatic pO2 Levels in CV159 and PBS Treated Rats
The pO2 (mmHg) values in the control group at various time points were as follows:
baseline, 8.37ꢃ2.72; after clamping, 4.73ꢃ2.26; before declamping, 4.63ꢃ1.65; and
after declamping, 5.30ꢃ2.45. The values in the CV159-treated group were as follows:
baseline, 17.40ꢃ8.96; after clamping, 14.88ꢃ9.22; before declamping, 18.37ꢃ12.48;
and after declamping, 29.93ꢃ8.39. pO2 in the control and CV159-treated groups at 4
time points (before clamping, after clamping, before unclamping, and after unclamp-
ing). Data are presented as the meansꢃS.D. Each group contained 4 animals.

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Fig. 6. Serum HMGB-1 Levels (mg/ml)
The HMGB-1 level in the sham group was 3.0ꢃ1.42 mg/ml. At 6 and 24 h after
reperfusion, the serum HMGB-1 levels were 2.48ꢃ1.25 and 9.86ꢃ2.64 mg/ml, respec-
tively, in the control group (pꢄ0.01) and 2.64ꢃ1.93 and 2.25ꢃ1.35 mg/ml, respec-
tively, in the CV159-treated group. At 24 h after reperfusion, the serum HMGB-1 level
in the control group was significantly higher than that in the CV159-treated group
(pꢄ0.01). Data are presented as the meansꢃS.D. Each group contained 6 animals.
∗ pꢄ0.01.
Although the NO level was significantly high in the CV159-
treated rats, NOx production was reduced. Thus, a small
amount of NO appears to have reacted with Oꢀ2 · to form per-
oxynitrite (ONOOꢀ), because CV159-induced ROS suppres-
sion inhibited Oꢀ2 · formation. Finally, however HMGB-1 has
recently received increasing attention as a late mediator of
endotoxin lethality, we previously demonstrated that sys-
temic accumulation of HMGB-1 occurs within 24 h after he-
patic I/R injury.14) In the present study, we found that (1)
HMGB-1 expression exhibited time-dependent kinetics in
hepatic I/R injury in rats and (2) at 24 h after reperfusion,
the serum HMGB-1 levels were significantly lower in the
CV159-treated group than in the control group.
Ischemic postconditioning—a novel strategy involving the
Pringle maneuver (clamping of the portal pedicle)—is com-
monly used to occlude the blood flow to the liver during sur-
gery.25) The implicated factors include ROS generation,
leukocyte migration and activation, microcirculatory abnor-
malities, and sinusoidal endothelial cell damage.25) Pharma-
cological postconditioning, which involves the administration
of an agent that mediates the protective effects of postcondi-
tioning, is a more practical option, and CV159 might be used
as an adjuvant in the Pringle maneuver.13) Further clinical
studies would shed light on the potential clinical applications
of CV159 as a pharmacological postconditioning agent in the
Pringle maneuver.
In conclusion, the results of this study reveal that in he-
patic I/R injury, CV159-mediated inhibition of intracellular
Ca2ꢁ overloading may effectively minimize organ damage
and have also both (1) hydroxyl-scavenging activity and (2)
the cytoprotective effects of eNOS-derived NO.
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January 2010
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