Influence of NADPH oxidase inhibition on oxidative stress parameters in rat hearts

Pharmacological Reports 2013, 65, 898–905 ISSN 1734-1140

Copyright © 2013 by Institute of Pharmacology Polish Academy of Sciences

Influence of NADPH oxidase inhibition on oxidative stress parameters in rat hearts Paulina Kleniewska1, Marta Michalska2, Anna Gor¹ca1 

Experimental and Clinical Physiology, Department of Cardiovascular Physiology, Medical University of Lodz, Mazowiecka 6/8, PL 92-215 £ódŸ, Poland 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, H O – 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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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 (ET)) or B (ET*) [29]. ET) receptors appear for the most part in cardiomyocytes (90%) and are considered as more important for the cardiac effects. Endothelin receptors ET) and ET* are expressed on vascular smooth muscle cells and induce vasoconstriction, whereas ET* 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);

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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 H O , 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.

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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 H O 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 H O concentration using the regression equation: Y = 0.0361X – 0.081, where Y = H O 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 H O concentrations (range 10 – 1000 μM). The lowest H O 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 H O 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 H O 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 H O concentration was reduced to values similar to those found in control rats (p < 0.01).

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Tab. 1. The influence of endothelin-1 (ET-1) and apocynin on thiobarbituric acid reactive substances (TBARS) level, hydrogen peroxide (H O ) 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

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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, H O 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 H O formation. Wu et al. [41] have indicated that H O significantly increases the level of malondialdehyde in the vascular smooth muscles (VSCM) and decreases superoxide dismutase (SOD) activity. H O is formed during superoxide anion dismutation and exerts harmful effects in cells when in excess amounts. H O 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 H O . CAT is a highlyreactive enzyme in most tissues which converts toxic H O to water. GPx, which is almost as efficient as catalase, removes H O at the expense of glutathione oxidation. Decline in the activity of these enzymes during excess ROS production results in intensive conversion of H O 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, H O 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 H O concentration in heart homogenates. This situation may result from inhibiting superoxide production by apocynin, as O ` 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

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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 PKCß -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.

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