Influence of NADPH oxidase inhibition on oxidative stress

Influence of NADPH oxidase inhibition on oxidative stress

Pharmacological Reports
Copyright © 2013
2013, 65, 898–905
by Institute of Pharmacology
ISSN 1734-1140
Polish Academy of Sciences
Influence of NADPH oxidase inhibition
on oxidative stress parameters in rat hearts
Paulina Kleniewska1, Marta Michalska2, Anna Gor¹ca1
1
Experimental and Clinical Physiology, Department of Cardiovascular Physiology, Medical University of Lodz,
Mazowiecka 6/8, PL 92-215 £ódŸ, Poland
2
Department of Biochemical Pharmacy, Department of Pharmacy, Medical University of Lodz, Muszyñskiego 1,
PL 90-151 £ódŸ, Poland
Correspondence: Paulina Kleniewska, e-mail: [email protected]
Abstract:
Background: The aim of this study was to assess whether apocynin, an nicotinamide adenine dinucleotide phosphate (NADPH)
oxidase blocker, influences lipid peroxidation TBARS, hydrogen peroxide (H2O2) content, protein level, heart edema, tumor necrosis factor a (TNF-a) concentration or the glutathione redox system in heart homogenates obtained from endothelin 1 (ET-1)-induced
oxidative stress rats.
Methods: Experiments were carried out on adult male Wistar-Kyoto rats. The animals were divided into 4 groups: Group I: salinetreated control; Group II: saline followed by ET-1 (3 µg/kg b.w., iv); Group III: apocynin (5 mg/kg b.w., iv) administered half an hour
before saline; Group IV: apocynin (5 mg/kg b.w., iv) administered half an hour before ET-1 (3 µg/kg b.w., iv).
Results: Injection of ET-1 alone showed a significant (p < 0.001) increase in thiobarbituric acid reactive substances (TBARS) and
the hydrogen peroxide level (p < 0.01) vs. control, as well as a decrease (p < 0.001) in the GSH level. Apocynin significantly decreased TBARS (p < 0.001) and H2O2 (p < 0.05) level (vs. control) as well as improved protein level (p < 0.001) in the heart. Apocynin also prevented ET-1-induced heart edema (p < 0.05). The presence of ET-1 increased the concentration of TNF-a (p < 0.05)
while apocynin decreased it (p < 0.05). Our results indicate that ET-1 may induce oxidative stress in heart tissue by reducing the
GSH/GSSG ratio, stimulating lipid peroxidation and increasing TNF-a concentration. Apocynin diminished these measures of oxidative stress and TNF-a.
Conclusion: ET-1-induced formation of ROS in the heart is at least partially regulated via NADPH oxidase.
Key words:
apocynin, endothelin 1, reactive oxygen species, heart
Abbreviations: CAT – catalase, ET-1 – endothelin 1, GPx –
glutathione peroxidase, GR – glutathione reductase, GSH – reduced glutathione, GSSG – oxidized glutathione, H2O2 – hydrogen peroxide, ROS – reactive oxygen species, SOD – superoxide dismutase, TBARS – thiobarbituric acid reactive substances,
tGSH – total glutathione, TNF-a – tumor necrosis factor a,
VEGF – vascular endothelial growth factor.
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Pharmacological Reports, 2013, 65, 898–905
Introduction
Endothelins are a family of 21 amino acid peptides
with 3 distinctive isoforms ET-1, ET-2 and ET-3. ET-1
is the most abundant isoform in the cardiovascular
NADPH oxidase inhibition in oxidative stress
Paulina Kleniewska et al.
system and is released from endothelial cells [5], cardiac fibroblasts [6], cardiomyocytes [1] and vascular
smooth muscle cells [21].
The biological effects of ET-1 are mediated
through two G-protein coupled receptors, endothelin
A (ETA) or B (ETB) [29]. ETA receptors appear for the
most part in cardiomyocytes (90%) and are considered as more important for the cardiac effects. Endothelin receptors ETA and ETB1 are expressed on vascular smooth muscle cells and induce vasoconstriction, whereas ETB2 receptors are expressed only on
endothelial cells [20] and mediate vasodilatation [5].
Reports indicate that ET-1 increases reactive oxygen species (ROS) levels in endothelial cells [11],
vascular smooth muscle cells [34] and rat aortic rings
[27] by stimulating nicotinamide adenine dinucleotide
phosphate (NADPH) oxidases (a family of superoxide
generating transmembrane proteins) [13] leading to
endothelial dysfunction, hypertension and atherosclerosis [27]. In the myocardium, NADPH oxidases are
a major source of ROS. It has been shown that isoforms of NADPH oxidases Nox1, Nox2 and Nox4 are
expressed in the heart [3].
Apocynin (4-hydroxy-3-methoxyacetophenone) is
a methoxy-substituted catechol derived from the root
which was discovered during activity-guided isolation
of immunomodulatory constituents from the medical
herb Picrorhiza kurroa [38]. Apocynin is known to be
a potent intracellular inhibitor of superoxide anion
production by activated neutrophils and eosinophils
[38]. It also protects a secretory leukocyte protease inhibitor from oxidative inactivation [38]. Apocynin enhances intracellular GSH by increasing g-glutamylcysteine synthetase activity in human A549 cells by
regulating transcription factors such as activator
protein-1 (AP-1) [25]. Apocynin possesses powerful
antioxidant and anti-inflammatory properties, which
are attributable to its ability to block the activity of
NADPH oxidase; this is achieved by interfering with
the assembly of cytosolic NADPH oxidase components with its membrane components [38]. Apocynin
is capable of reacting with the thiol groups required
for enzyme assembly [38]. It has been effective in
ameliorating neuropathological damage in both in
vivo and in vitro models of Parkinson’s disease [2]
and in preventing ischemic damage and blood-brain
barrier disruption in different animal models of experimental stroke [23]. It has also been seen to retard
disease progression and extend survival in a mouse
amyotrophic lateral sclerosis model [4] and bestow
a protective effect in ischemia-reperfusion lung injury
[9].
The aim of this study was to investigate whether
ET-1-induced ROS generation in rat hearts is regulated by NADPH oxidase inhibition.
Materials and Methods
Chemicals
Apocynin (4-hydroxy-3-methoxy-acetophenone – powder), endothelin-1 (powder), thiobarbituric acid (TBA),
butylated hydroxytoluene (BHT), sodium acetate trihydrate, triethanoloamine hydrochloride (TEA), 5-sulfosalicylic acid hydrate (5-SSA), 5,5’-dithio-bis (2nitrobenzoic acid) (DTNB), b-NADPH (b-nicotinamide
adenine dinucleotide phosphate), glutathione reductase
(GR), 2-vinylpyridine were purchased from Sigma
Chemical Co. (St. Louis, MO, USA). All other reagents
were obtained from POCH (Gliwice, Poland) and were
of analytical grade.
Animals
Experiments were performed on male Wistar rats
weighing 190–210 g, aged 2–3 months. The animals
were housed 6 per cage under standard laboratory
conditions in a 12/12 h light-dark cycle (lights on at
7.00 a.m.) at 20 ± 2°C ambient temperature and air
humidity of 55 ± 5%. All animals received standard
laboratory diet and water ad libitum. All animals were
given a one-week acclimation period before the onset
of the experiment. The experimental procedures followed the guidelines for the care and use of laboratory
animals, and were approved by the Medical University of Lodz Ethics Committee (28/£B 520/2010).
Experimental protocol
Animals were randomly divided into four groups as
follows:
I group (control group, n = 6) received two doses of
0.2 ml of saline, half an hour apart;
II group (ET-1 group, n = 6) received 0.2 ml of saline, and half an hour later, the rats were injected with
a single dose of ET-1 (3 µg/kg);
Pharmacological Reports, 2013, 65, 898–905
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III group (apocynin group, n = 6) were given
0.2 ml of saline, and half an hour later, the rats were
injected with a single dose of apocynin (5 mg/kg);
IV group (apocynin + ET-1, n = 6) received a single
dose of apocynin (3 µg/kg) and after half an hour,
a single dose of ET-1 (3 µg/kg).
All chemicals were injected intravenously into the
femoral vein between 8.00 a.m. and 9.00 a.m.
Animal preparations
Animals were anesthetized by an intraperitoneal injection of 10% urethane (2 ml/100 g b.w.). When
a sufficient level of anesthesia was achieved, a 2-cm
long polyethylene tube (2.00 mm O.D.) was inserted
into the trachea. A polyethylene catheter PE-10 was
inserted into the femoral vein for administration of
experimental drugs.
Tissue preparation and collection of samples
At the end of the experimental period, the animals
were euthanized. The hearts were surgically removed
and cleaned of extraneous tissue. They were rinsed
with cold isotonic saline, dried by blotting between
two pieces of filter paper and weighed on an electronic balance to estimate heart edema. The ratios of
heart weight to body weight (HW/BW) were calculated and used as an index of heart edema. The hearts
were stored at –80°C for further measurement of their
oxidative parameters and tumor necrosis factor a
(TNF-a) concentration.
Preparation of homogenates
An accurately weighed portion of heart (50 mg) was
homogenized in either 0.15 M KCl for estimation of
lipid peroxidation and concentration of H2O2, or 5%
SSA for estimation of glutathione. The resulting supernatant was used for biochemical analyses immediately.
Determination of lipid peroxidation
To determine the degree of oxidative damage in the
heart, lipid peroxidation was measured in heart homogenates. The lipid peroxidation product content in
heart homogenates was assayed as thiobarbituric acid
reactive substances (TBARS), previously described
by Yagi [42]. Briefly, 50 mg of heart tissue was homogenized with 2 ml of 1.15% potassium chloride.
900
Pharmacological Reports, 2013, 65, 898–905
Then, 4 ml of 0.25% hydrochloric acid containing
0.375% TBA, 15% trichloroacetic acid (TCA) and
0.015% BHT were added. The samples were boiled
for 30 min at 100°C in tightly closed tubes. After
cooling to 10°C, 2.5 ml of butanol was added to each
tube and the samples were centrifuged for 10 min
(3,800 rpm, 20°C). TBA-reactive substances in the butanol layer were measured spectrofluorometrically using an LS 50 Perkin Elmer Luminescence Spectrometer (Norwalk, CT, USA). Excitation was set at 515 nm
and emission was measured at 546 nm. Sample
TBARS concentrations were calculated by the use of
the regression equation as follows: Y = 0.43 (X – Xo) –
2.43, where Y = TBARS concentration (µM); X, Xo =
fluorescence intensity of the samples and control, respectively (arbitrary units; AU). The regression equation was prepared from triplicate assays of six increasing concentrations of tetramethoxypropane (range 0.01
– 50 µM) as a standard for TBARS. A mixture of 2 ml
of 1.1% potassium chloride and 4 ml of 0.25 M hydrochloric acid was used as a control. Finally, the results
were calculated for 50 mg of heart tissue.
Determination of H2O2
The heart tissue fragments were washed in ice-cold saline and stored at –80°C for no longer than 2 weeks.
Generation of H2O2 in heart homogenates was determined according to Ruch et al. [33]. Briefly, 50 mg of
the heart tissue fragments were homogenized with 2 ml
of 1.15% potassium chloride. Then, 10 µl aliquot of tissue homogenate was mixed with 90 µl of PBS – physiological buffered saline (pH 7.0) and 100 µl of horseradish peroxidase (1 U/ml) containing 400 µmol homovanillic acid (HRP + HVA assay) or with 90 µl of PBS and
100 µl of 1 U/ml horseradish peroxidase only (HRP) assay. Both homogenates were incubated for 60 min at
37°C. Subsequently, 300 µl of PBS and 125 µl of 0.1 M
glicyne-NaOH buffer (pH 12.0) with 25 mM EDTA
(ethylenediaminetetraacetic acid) were added to each
homogenate sample. Excitation was set at 312 nm and
emission was measured at 420 nm (Perkin Elmer Luminescence Spectrometer, Beaconsfield UK).
Readings were converted into H2O2 concentration
using the regression equation: Y = 0.0361X – 0.081,
where Y = H2O2 concentration in homogenate (µM);
X = intensity of light emission at 420 nm for HRP
+ HVA assay reduced by HRP assay emission (arbitrary units, AU). The regression equation was prepared from three series of calibration experiments
NADPH oxidase inhibition in oxidative stress
Paulina Kleniewska et al.
with 10 increasing H2O2 concentrations (range 10 –
1000 µM). The lowest H2O2 detection was 0.1 nM,
with intraassay variability not exceeding 2%.
Determination of GSH levels
Total glutathione (tGSH), reduced glutathione (GSH)
and oxidized glutathione (GSSG) were measured in
the heart homogenates. Briefly, the hearts were
homogenized in cold 5% 5-SSA and centrifuged
(10,000 × g, 10 min, 4°C). The total GSH content of
the supernatant was measured in a 1 ml cuvette containing 0.7 ml of 0.2 mM NADPH, 0.1 ml of 0.6 mM
DTNB – Ellman’s reagent, 0.150 ml of H2O and 50 µl
of sample. The cuvette with the mixture was incubated for 5 min at 37°C and then supplemented with
0.6 U/l of GR. The reaction kinetics was followed
spectrophotometrically at 412 nm for 5 min by monitoring the increase in absorbance.
GSSG concentration was determined in supernatant
aliquots by the same method after optimalization of pH
to 6–7 with 1 M TEA and derivatization of endogenous
GSH with 2-vinylpyridine (v/v). The reduced GSH
level in the supernatant was calculated as the difference
between total GSH and GSSG. The increments in absorbance at 412 nm were converted to GSH and GSSG
concentrations using a standard curve (3.2 – 500 µM
GSH for total GSH and 0.975 – 62 GSSG µM for
GSSG). The results were expressed in µM.
Determination of total protein
Protein was measured by the method of Lowry et al.
[28], using bovine serum albumin as standard.
TNF-a assay
TNF-a in the heart tissue was assayed by specific enzyme linked immunosorbent assay using a commercially-available ELISA test kit (R&D Systems) containing a monoclonal antibody specific for rat TNF-a.
The results were read using a TEK Instruments
EL340 BIO-spectrophotometer (Winooski VT, USA)
(l = 45 nm). The sensitivity of the kits was 0.037 pg/
ml. The TNF-a concentration was read from standard
curves and expressed in pg/ml. Experiments were repeated twice.
Statistical analysis
The results are presented as the mean ± SEM, if not
stated otherwise, from 6 animals in each group. The
statistical analysis was done by ANOVA followed
by the Duncans multiple range test as post-hoc.
A p value less than 0.05 was considered significant.
Results
Evaluation of heart edema
In ET-1 treated rats, the HW/BW ratio was markedly
higher when compared to control rats (p < 0.001).
Apocynin administration before ET-1 ameliorates
ET-1-induced heart edema (p < 0.05). Despite the increased HW/BW ratio after ET-1 administration,
a slightly decreased protein concentration was observed in this group of animals compared to control
(p < 0.05). However, the increased protein concentration was seen in the apo + ET-1 group, compared to
the ET-1 group (p < 0.001)
Evaluation of lipid peroxidation
Table 1 displays the influence of endothelin-1 (ET-1)
on TBARS level. The concentration of TBARS,
a marker of lipid peroxidation induced by ROS, increased significantly during ET-1 infusion when compared to the control group (20.88 ± 0.75 µmol/l vs.
39.51 ± 1.37 µmol/l). Rats treated with apocynin
alone exhibited a significant decrease in lipid peroxidation when compared with the control group (p <
0.001). Apocynin also significantly attenuated ET-1induced increase in TBARS level in heart homogenates when compared to the ET-1 group (p < 0.001).
Evaluation of H2O2
In rats treated with ET-1, the H2O2 concentration was
increased in comparison to the control group (1.12
± 0.14 µmol/l vs. 0.41 ± 0.08 µmol/l, p < 0.01). In rats
treated with apocynin + ET-1, the H2O2 concentration
was reduced to values similar to those found in control rats (p < 0.01).
Pharmacological Reports, 2013, 65, 898–905
901
Tab. 1. The influence of endothelin-1 (ET-1) and apocynin on thiobarbituric acid reactive substances (TBARS) level, hydrogen peroxide (H2O2)
concentration, protein level and HW/BW ratio in hearts. The results are the mean ± SEM. The data were statistically evaluated by one-way
ANOVA
0.9% NaCl,
n=6
TBARS (µM)
Apocynin
(5 mg/kg), n = 6
Apocynin
(5 mg/kg) + ET-1
(3 µg/kg), n = 6
20.88 ± 0.75
39.51 ± 1.37***
11.04 ± 0.4***###
27.39 ± 1.72*##
0.41 ± 0.08
1.11 ± 0.14**
0.17 ± 0.04###
0.37 ± 0.09##
H2O2 (µM)
Protein (µg/ml)
0.9% NaCl + ET-1
(3 µg/kg), n = 6
223.81 ± 13.31
HW/BW ratio (´ 100)
0.28 ± 0.01
188.46 ± 15.16
0.35 ± 0.01***
365.99 ± 8.7***###
0.30 ± 0.02
307.5 ± 11.06**###
0.31 ± 0.01***#
* p < 0.02; ** p < 0.01; *** p < 0.001 vs. control; # p < 0.05; ## p < 0.01; ### p < 0.001 vs. ET-1
Tab. 2. Total, oxidized and reduced glutathione concentration and glutathione redox ratio in heart homogenates in control, after administration
of endothelin-1 (ET-1) (3 µg/kg b.w.), apocynin (5 mg/kg b.w.) and apocynin + ET-1 (5 mg/kg b.w. and 3 µg/kg b.w., respectively) (n = 6, per
group). Data are shown as the mean ± SEM
tGSH (µM)
0.9% NaCl,
n=6
0.9% NaCl + ET-1
(3 µg/kg), n = 6
Apocynin
(5 mg/kg), n = 6
Apocynin
(5 mg/kg) + ET-1
(3 µg/kg), n = 6
47.34 ± 1.34
30.74 ± 2.58***
48.20 ± 1.96###
43.35 ± 2.57#
5.64 ± 0.24#
GSSG (µM)
6.92 ± 0.24
5.68 ± 0.22**
7.10 ± 0.19###
GSH (µM)
40.42 ± 1.41
25.85 ± 2.22***
41.09 ± 1.77###
37.48 ± 2.46#
5.78 ± 0.13##
6.23 ± 0.41#
GSH/GSSG (µM)
5.89 ± 0.36
4.93 ± 0.20
tGSH – total glutathione; GSH – reduced glutathione; GSSG - oxidized glutathione. ** p < 0.02; *** p < 0.001 vs. control; # p < 0.05; ## p < 0.02;
p < 0.01 vs. ET-1
###
Evaluation of glutathione
Myocardial levels of total glutathione and reduced
glutathione were lower by 35 and 36%, respectively,
in the ET-1 group compared with control (p < 0.001)
(Tab. 2). In rats treated with apocynin plus ET-1, the
concentration of total glutathione and reduced glutathione were enhanced in comparison with ET-1
group (43.35 ± 2.57 µmol/l vs. 30.74 ± 2.58 µmol/l
and 37.481 ± 2.46 µmol/l vs. 25.85 ± 2.22 µmol/l, p <
0.05, respectively).
The GSH/GSSG ratio was slightly decreased in the
ET-1 group when compared to the control (p < 0.05).
In the apo + ET-1 group, the GSH/GSSG ratio was
significantly increased when compared to the ET-1
group (p < 0.05).
Evaluation of TNF-a
Treatment with ET-1 resulted in a significant increase in
the TNF-a concentration in heart homogenates (p <
902
Pharmacological Reports, 2013, 65, 898–905
0.05) (Fig. 1). In apocynin and ET-1-treated rats, the
concentration of TNF-a was significantly reduced when
compared with ET-1 and saline-treated group (p < 0.01).
Fig. 1. TNF-a concentration in heart homogenates in control, after
administration of endothelin-1 (ET-1, 3 µg/kg b.w.), apocynin (apo)
(5 mg/kg b.w.) and apocynin + endothelin-1 (5 mg/kg b.w. and
3 µg/kg b.w., respectively) (n = 6, per group). Data are shown as the
mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. ET-1; # p < 0.01
vs. apo + ET-1
NADPH oxidase inhibition in oxidative stress
Paulina Kleniewska et al.
Discussion
In the present investigation, ET-1 infusion was sufficient to induce oxidative stress in the hearts, manifested by increased formation of TBARS, H2O2 and
decreased glutathione. The measurement of TBARS
is a sensitive index of lipid peroxidation; the TBARS
assay measures secondary products of lipid peroxidation such as 2,4-decadienols and, to a lesser extent,
saturated aldehydes [14]. Lipid peroxidation is a complex process produced by radicals or excited species
that results in toxic oxidized biocompounds and exerts damaging effects on membranes. As a consequence of oxidative alteration, biomembranes lose
their fluidity, their permeability to different ions increases and their membrane potential is reduced. Finally, the organelle membranes are destroyed, which
leads to cell destruction and organ damage [18]. Our
results are consistent with previous reports, which
demonstrate that ET-1 may lead to oxidative stress by
increasing ROS production and stimulating lipid peroxidation [32]. In the present study, ET-1-induced
ROS generation was also manifested by enhanced
H2O2 formation. Wu et al. [41] have indicated that
H2O2 significantly increases the level of malondialdehyde in the vascular smooth muscles (VSCM) and decreases superoxide dismutase (SOD) activity.
H2O2 is formed during superoxide anion dismutation and exerts harmful effects in cells when in excess
amounts. H2O2 is more toxic than oxygen-derived free
radicals and is capable of producing the most toxic
hydroxyl radical (OH•). This radical must be removed
very efficiently. Catalase (CAT) and glutathione peroxidase (GPx) are basic enzymes regulating the intracellular concentration of H2O2. CAT is a highlyreactive enzyme in most tissues which converts toxic
H2O2 to water. GPx, which is almost as efficient as
catalase, removes H2O2 at the expense of glutathione
oxidation. Decline in the activity of these enzymes
during excess ROS production results in intensive
conversion of H2O2 to toxic hydroxyl radicals which
may contribute to ET-1-induced oxidative stress.
Evidence suggests that various enzymatic and
non-enzymatic systems have been developed by the
cell to attenuate ROS, and these defenses against ROS
become insufficient under conditions of oxidative
stress. In this event, ROS affect the antioxidant defense mechanisms, reduce the intracellular concentration of GSH and enhance lipid peroxidation [26]. The
decreased protein concentrations observed in this
study may be due to oxidative damage of protein
molecules [8].
Glutathione is an intracellular reducing agent that
plays a central role in antioxidant defense by detoxifying ROS directly or by a mechanism catalyzed by
GPx. In this study, the decreased glutathione content
and the GSH/GSSG ratio seen after ET-1 administration reflect a decrease in the antioxidant status of the
tissue. These results are in line with previous reports,
which demonstrate that ET-1 may lead to oxidative
stress by reducing glutathione, diminishing the antioxidant GSH/GSSG ratio and stimulating lipid peroxidation in a time-dependent manner [39].
Apocynin, an NADPH oxidase specific inhibitor,
was administered to determine whether NADPH oxidase regulates ET-1-stimulated ROS production; lowered TBARS, H2O2 content and glutathione were
found in heart homogenates. The decrease in lipid
peroxidation may be related to the inhibition of
NADPH oxidase activation by apocynin. Some authors
suggest that apocynin, after metabolic conversion by
peroxidases, inhibits the assembly of NADPH oxidase [19, 37, 38] and attenuates oxidative stress. The
results from this study indicate that decreases in
TBARS concentration are associated with decreases
in H2O2 concentration in heart homogenates. This
situation may result from inhibiting superoxide production by apocynin, as O2– is a key element of ROS.
In our study, the increased concentration of total
protein after the administration of apocynin indicates
that this antioxidant has the ability to reduce heart
damage by inhibiting oxidation of -SH groups in proteins [12], as well as enhancing the synthesis of glutathione and proteins containing sulfhydryl groups.
Konopka et al. [24] have observed increases in -SH
group content after apocynin administration. Moreover, an increased protein concentration level can result in increased synthesis of antioxidant enzymes
such as SOD [17].
Our results show that apocynin administration increases tGSH levels in rats in the control and ET-1
groups. The high glutathione content found in the
heart after apocynin supplementation may reflect an
increase in the antioxidant status of this tissue [31].
Consequently, the increase in heart glutathione levels
may have significantly contributed to the reduction of
TBARS levels in rats receiving ET-1.
The redox status within the cell is reflected by the
GSH/GSSG ratio. In our study, the ibcrease in the
GSH/GSSG ratio after the apocynin administration
Pharmacological Reports, 2013, 65, 898–905
903
suggests that this antioxidant might modulate cardiac
antioxidative defense by elevation of the glutathione
redox status of the tissue.
It has been also shown that ET-1 stimulates the peripheral blood mononuclear cells and tissue cells to release TNF-a [10, 22], which in turn increases the generation of ROS in various cell types via signaling with
nuclear factor k B (NFkB) and NADPH oxidase [36].
In our study, a decrease in the TNF-a concentration in
heart homogenates after the apocynin administration
may result in diminished NADPH oxidase activation,
which is a major source of superoxide generation [7,
38]. Recently, Deng et al. [7] noticed that TNF-a can
also induce ROS generation in endothelial cells via
PKCß2-dependent activation of NADPH oxidase, and
this effect was negated when combined with apocynin.
The present study also revealed an increase in heart
edema after the ET-1 administration. This increase
may be attributed to albumin extravasculation in the
vascular bed [15, 16], an increase in vascular permeability and myocardial water content [30] or to increased VEGF release in the heart after the ET-1 injection [35]. The decrease in heart edema displayed
by animals in the apo + ET-1 group may be explained
by the antioxidant action of apocynin and lowered
vascular permeability or decreased VEGF content in
the heart. Indeed, it has previously been observed that
apocynin reduces pulmonary permeability and edema
[43] and decreases the permeability of the blood-brain
barrier, resulting in decreased brain edema [40].
In conclusion, this report demonstrates that ET-1
may lead to oxidative stress in the heart tissue by reducing the GSH/GSSG ratio, stimulating lipid peroxidation and increasing TNF-a concentration. Apocynin,
an inhibitor of NADPH oxidase, diminished these
measures of oxidative stress, as well as the TNF-a levels. ET-1-induced formation of ROS in heart might be
partially regulated via NADPH oxidase.
Acknowledgment:
This study was supported by grants 503/0-079-03/503-01 and
503/3-015-02/503-01 from the Medical University of Lodz.
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Received: May 18, 2012; in the revised form: February 2, 2013;
accepted: February 16, 2013.
Pharmacological Reports, 2013, 65, 898–905
905