Effect of chronic apocynin treatment on nitric oxide and reactive oxygen species production in borderline and spontaneous hypertension

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Effect of chronic apocynin treatment on nitric oxide and reactive oxygen species production in borderline and spontaneous hypertension

Olga Pechánová^1,2, Lýdia Jendeková¹, Stanislava Vranková¹

1Institute of Normal and Pathological Physiology, Center of Excellence for Cardiovascular Research, Slovak Academy of Sciences, Sienkiewiczova 1, Bratislava 81371, Slovak Republic

2CRC and Institute of Physiology, AS CR, Videnska 1083, Prague 14220, Czech Republic

Correspondence: Olga Pechánová, e-mail: olga.pechanova@savba.sk

Abstract:

The purpose of this study was to investigate the effect of NAD(P)H oxidase inhibitor – apocynin (4-hydroxy-3-methoxyacetophenone) on the increase of systolic blood pressure (SBP) in borderline (BHR) and spontaneously hypertensive rats (SHR). Young 6-week-old male BHR (offspring of SHR dams and Wistar Kyoto sires) and SHR were treated with apocynin (30 mg/kg/day) for six weeks. SBP was measured by tail-cuff plethysmography. Nitric oxide synthase (NOS) activity was determined in the left ventricle and aorta. Protein expression of nuclear factor kappa B (NF-kB) and NAD(P)H oxidase subunits p67phox and p22phox as well as concentration of cGMP were determined for the left ventricle. Apocynin significantly decreased SBP in all groups investigated. Ad- ministration of apocynin had no effect on NOS activity in either tissue studied. However, apocynin decreased protein expression of NF-kB (p65) and NAD(P)H oxidase subunit p22phox in both hypertensive groups and p67phox subunit in the SHR group. Moreo- ver, apocynin was able to prevent a decrease in cGMP concentration in the left ventricle of both hypertensive groups. In conclusion, our study demonstrated that apocynin treatment partially prevented SBP rise in borderline and spontaneously hypertensive rats, yet without increasing activity of NOS in the left ventricle and aorta. However, apocynin was able to decrease production of reactive oxygen species in hypertensive rats; thus preventing the decrease in cGMP formation.

Key words: experimental hypertension, NO synthase, reactive oxygen species, NAD(P)H oxidase, NF-kappa B, apocynin

Introduction

Recent studies have demonstrated that altered oxygen utilization and/or increased formation of reactive oxygen species (ROS) contribute to the progression of cardiovascular diseases [9]. Oxidative stress promotes endothelial dysfunction, reduced vasorelaxation, vascular media thickening, and vascular lumen narrow- ing. In particular, superoxide anion (^•O₂^–), hydroxyl radical (OH^•) and hydrogen peroxide (H₂O₂) contrib-

ute substantially to this process [3, 9]. On the other hand, endothelium-derived nitric oxide (NO) repre- sents a crucial vasodilator and plays an important role in the control of blood pressure [3]. Acting as a free radical, NO reacts extremely rapidly with superoxide to form further ROS, which are mainly the highly reactive molecule peroxynitrite (ONOO^–) [4]. Increased formation of peroxynitrite results in NO synthase uncoupling and elevated production of superoxide. The classified enzymatic sources of ROS in the vascular wall which are considered to contribute to the hyper-

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tensive process include: NAD(P)H oxidase, mono- meric (uncoupled) NO synthase, xanthine oxidase, and cyclooxygenase. The vascular NADPH oxidase has been identified in all vascular cell types and is the most important source of ^•O₂^– within the vessel [9, 17, 19, 26].

Zalba et al. [32] reported abnormally increased NAD(P)H oxidase activity in the aortic wall of adult SHR. This abnormal activity was associated with upregulated p22phox mRNA expression. Excessive production of superoxide anions occurs in the endothelium of stroke-prone SHR [12]. Generation of superoxide anions causes contractions that are more ex- tensive in the aortas from spontaneously hypertensive rats than normotensive rats [1]. These findings sug- gest that long-term exposure to hypertension, possibly combined with other factors (i.e., local overproduc- tion of angiotensin II), may play a role in overactivity of the NAD(P)H oxidase and vice versa. Enhanced NAD(P)H oxidase mediated ^•O₂^– production might contribute to functional and structural alterations present in the aortas of adult SHR [14].

Apocynin (4’-hydroxy-3’methoxyacetophenone), a methoxy-substituted catechol, is the well-characterized inhibitor of NAD(P)H oxidase [26]. Apocynin has been shown to impede the assembly of the p47phox and p67phox subunits with the membrane complex and therefore effectively eliminates the increase in superoxide production. Through this mechanism, apocynin also increases the bioavailability of NO. More- over, it was found that apocynin reduces COX 2 protein synthesis and activity leading to the decrease of ROS generation [2]. Inhibition of NAD(P)H oxidase with apocynin or diphenyleneiodonium induced re- laxation of endothelium-intact aortic rings of SHR [14].

The purpose of the present study was to investigate the effect of apocynin on blood pressure development, oxidative status, and NO synthase activity in borderline and spontaneously hypertensive rats.

Materials and Methods

Animals

All procedures and experimental protocols were ap- proved by the Ethical Committee of the Institute of

Normal and Pathological Physiology SAS, and con- form to the European Convention on Animal Protec- tion and Guidelines on Research Animal Use.

Young, 6-week-old male rats were divided into 6 groups (n = 6 each): control WKY; WKY receiving a 30 mg/kg/day apocynin dose (WKY+A); control BHR; BHR receiving a 30 mg/kg/day (BHR+A) apocynin dose; control SHR, and SHR receiving a 30 mg/

kg/day (SHR+A) apocynin dose. Rats were housed under standard conditions (temperature 22 ± 2°C, 12-h light-dark cycle), drank tap water, and were fed a standard chowad libitum. Control WKY, BHR, and SHR drank tap water during the experiment, whereas WKY+A, BHR+A, and SHR+A drank apocynin (30 mg/kg/day) dissolved in the tap water.

Systolic blood pressure (SBP) was measured by tail-cuff plethysmography, a non-invasive method, every week. After 6 weeks of treatment, the animals were sacrificed and the body weight (BW) and heart weight (HW) were determined. The heart weight : body weight (HW/BW) ratio was calculated.

Total NO synthase activity assay

Total NOS activity was determined in crude ho- mogenates of the left ventricle and aorta by measuring the formation of [³H]-L-citrulline from [³H]-L-argini- ne as previously described by Bredt and Snyder [6], with modifications according to Pechanova [21]. Total NO synthase activity was expressed as pmol/min/mg protein.

Western blot analysis

Samples of the left ventricle were homogenized in 25 mmol/L Tris-HCl, pH 7.4, containing 5 mmol/l EDTA, 50 mmol/l NaCl, 1mmol/l leupeptin, 0.3 mmol/l aprotinin, 0.1 mmol/l PMSF, 1 mmol/l pepstatin, and 1% sodium dodecyl sulfate. After centrifugation (15000 × g, 20 min, twice) supernatants were sub- jected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 10% gels. Follow- ing the electrophoresis, proteins were transferred to nitrocellulose membranes and were probed with a polyclonal rabbit anti-NFkB antibody (p65 KDa), anti-NAD(P)H oxidase-p67phox antibody, and anti- NAD(P)H oxidase-p22phox antibody (Santa Cruz Biotechnology, CA). Bound antibodies were detected using a secondary peroxidase-conjugated anti-rabbit antibody (Calbiochem, Germany). The bands were

Apocynin and spontaneous hypertension

Olga Pechánová et al.

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Cyclic GMP concentration

Cyclic GMP (cGMP) concentration was determined in the homogenate of the left ventricle using a radio- immunoassay kit (Immunotech, S.A., France) based on the competition between the succinylated cGMP of the sample and¹²⁵I-labeled tracer for binding to polyclonal antibody-coated tubes [22]. The concentration of the cGMP in the sample was calculated from the standard curve and expressed as pmol/g tissue.

Statistical analysis

Results were expressed as the means ± SEM. One- way analysis of variance and the Duncan test were used for statistical analysis. Values were considered to differ significantly if the probability value was less than 0.05 (p < 0.05).

Results

Cardiovascular parameters

Chronic administration of apocynin to SHR and BHR decreased their SBP after the third week of treatment.

in comparison with normotensive WKY. Apocynin treatment decreased BW in all three rat strains (WKY+A, BHR+A, and SHR+A) and lowered heart weight in BHR+A. Similarly, apocynin treatment decreased the heart weight : body weight ratio in the BHR+A and SHR+A groups (Tab. 1).

Tab. 1. Effect of apocynin on body weight (BW), heart weight (HW), HW/BW ratio

Body weight (BW) [g]

Heart weight (HW) [mg]

HW/BW [mg/g]

WKY 339.2 ± 6.8 881.3 ± 18 2.60 ± 0.03 WKY+A 320.8 ± 5.7* 824.3 ± 18 2.57 ± 0.03 BHR 319.0 ± 4.4* 872.8 ± 21 2.74 ± 0.04*

BHR+A 289.2 ± 5.8*⁺ 745.7 ± 11*⁺ 2.58 ± 0.04⁺ SHR 262.3 ± 5.9* 903.6 ± 22 3.45 ± 0.06*

SHR+A 277.3 ± 2.0* 914.9 ± 16 3.30 ± 0.04*⁺

WKY – Wistar Kyoto rats, BHR – borderline hypertensive rats, SHR – spontaneously hypertensive rats, A – apocynin treatment (30 mg/kg/

day), * p < 0.05vs. WKY,⁺p < 0.05vs. untreated rats of the same genotype

Total NO synthase activity

Total NOS activity was significantly increased in the left ventricle and aorta of both hypertensive groups in comparison with WKY. Apocynin did not affect activity of total NO synthase in either strain or tissue investigated (Fig. 2).

Western blot analysis

There were no significant changes in the protein expression of the NAD(P)H oxidase subunit, p67phox, in the left ventricle of BHR in comparison with WKY.

However, in SHR, the protein expression of p67phox was significantly increased by 22%. Apocynin attenuated the expression of p67phox in SHR by 26% in comparison with untreated SHR (Fig. 3A). Protein expression of the NAD(P)H oxidase subunit p22phox in the left ventricle was significantly higher in the BHR and SHR than in the WKY (46% and 78%, respec- tively). Apocynin attenuated protein expression of p22phox in the left ventricle of both hypertensive

Fig. 1. Effect of apocynin treatment on systolic blood pressure (SBP) development in WKY, BHR, and SHR. WKY – Wistar Kyoto rats, BHR – borderline hypertensive rats, SHR – spontaneously hypertensive rats, A – apocynin 30 mg/kg/day. Significant differences: * p < 0.05 vs. WKY,⁺p < 0.05vs. untreated rats of the same genotype

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groups by 6% in BHR+A and by 23% in SHR+A (Fig. 3B).

The protein expression of NF-kB (p65) was signifi- cantly higher in the left ventricle of SHR by 18%. On the other hand, significant reduction of NF-kB (p65) in the left ventricle of BHR in comparison with normotensive WKY was observed. Apocynin attenuated expression of NF-kB in all three rat strains (21% in WKY+A, 12% in BHR+A, and 27% in SHR+A in comparison with untreated animals) (Fig. 3C).

Cyclic GMP concentration

Cyclic GMP concentration in the left ventricle of BHR and SHR was significantly decreased in comparison with WKY. Apocynin was able to significantly increase the concentration of cGMP in SHR+A.

Moreover, the concentration of cGMP in BHR+A and

SHR+A groups reached the same level as in normotensive WKY (Fig. 4).

Correlation between SBP and NF-kB protein ex- pression

A positive correlation between SBP and NF-kB pro- tein expression in the left ventricle of hypertensive

Fig. 2. Effect of apocynin treatment on NO syntase activity in the left ventricle (upper panel) and aorta (lower panel) of WKY, BHR, and SHR. WKY – Wistar Kyoto rats, BHR – borderline hypertensive rats, SHR – spontaneously hypertensive rats, A – apocynin 30 mg/kg/

day. Significant differences: * p < 0.05vs. WKY,⁺p < 0.05vs. untreated rats of the same genotype

Fig. 3. Effect of apocynin treatment on protein expression of NAD(P)H oxidase subunits – p67phox (A) and p22phox (B) and nuclear factor NFkB (C) in the left ventricle of WKY, BHR, and SHR. WKY – Wistar Kyoto rats, BHR – borderline hypertensive rats, SHR – spontaneously hypertensive rats, A – apocynin 30 mg/kg/day. Significant differences: * p < 0.05vs. WKY,⁺p < 0.05vs. untreated rats of the same genotype

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This is the first report to demonstrate that chronic apocynin treatment (for 6 weeks) leads to the decrease of systolic blood pressure in both normotensive and hypertensive rats (BHR and SHR). Moreover, it is evident that the decrease of blood pressure was associated with decreased expression of NF-kB protein as demonstrated by the positive correlation between systolic blood pressure and NF-kB protein expression in the hypertensive groups. Apocynin treatment also decreased the expression of NAD(P)H oxidase subunit p67phox in spontaneously hypertensive rats and p22phox in both borderline and spontaneously hypertensive rats. However, apocynin failed to affect NO synthase activity in the left ventricle and aorta of normotensive Wistar Kyoto rats as well as in BHR and SHR.

Increased levels of reactive oxygen species is one of the major causes of endothelial dysfunction in the cardiovascular diseases [3, 9]. Enhanced production of ROS was documented in different models of experimental hypertension such as stroke-prone hypertension, Angiotensin II-induced hypertension, DOCA- salt, and spontaneous hypertension [8, 27, 28, 32].

NAD(P)H oxidase is considered to be the major pro- ducer of superoxide in the hypertensive processes. In the cardiovascular system, the main catalytic subunits are nox1, nox2 (gp91 phox), and nox4 with the regu- latory subunits including p22phox, p47phox, p67phox, and rac-1 or 2 [13]. Generation of endogenous super- oxides activates ligand and cell-specific mitogen- activated protein kinases, tyrosine kinases, phosphata- ses, transcriptions factors including NF-kB and acti- vator protein 1, early growth response, and hypoxia- inducible factors [10, 16]. In addition, ROS modulate intracellular Ca²⁺ concentration by redox-dependent inositol triphosphate-induced Ca²⁺ mobilization, increased Ca²⁺influx and decreased Ca²⁺-ATPase activation which could contribute to altered vascular con- tractility in hypertension [15]. Moreover, such an increase in ^•O₂^–production accelerates the breakdown of nitric oxide, uncoupling of nitric oxide synthase, decrease of guanylate cyclase activity, and downregu- lation of its subunit beta(1) [18]. Zalba et al. [32]

showed that the overactivity of NAD(P)H oxidase is associated mainly with up-regulation of p22phox mRNA, which results in impaired NO-dependent re- laxation and media hypertrophy in the aorta of SHR.

Accordingly, we observed increased protein expres-

Fig. 4. Effect of apocynin treatment on cGMP concentration in the left ventricle of WKY, BHR, and SHR. WKY – Wistar Kyoto rats, BHR – borderline hypertensive rats, SHR – spontaneously hypertensive rats, A – apocynin 30 mg/kg/day. Significant differences: * p < 0.05 vs. WKY,⁺p < 0.05vs. untreated rats of the same genotype

Fig. 5. Relationship between systolic blood pressure (SBP) and NFkB protein expression in the left ventricle of BHR (A) and SHR (B).

Control rats (¢), apocynin treated rats (¯)

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sions of NAD(P)H oxidase subunits p22phox and p67phox associated with decreased cGMP concentration in the left ventricle of spontaneously hypertensive rats. Increased expression of the p22phox subunit was also determined in the left ventricle of borderline hypertensive rats. Since the increased expression of NAD(P)H oxidase precedes development of hypertension [7], the antioxidant treatment should be more efficient in young SHR before the development of secondary hypertensive changes. Our results support this hypothesis quite well as the antioxidant treatment with apocynin attenuated hypertension development in young BHR and SHR.

We showed similar results using N-acetylcysteine and melatonin treatment in young SHR. Both N-acetylcysteine and melatonin revealed better preventive than therapeutic effects [24, 25]. In contrast to the above substances, apocynin was also able to decrease systolic blood pressure in young normotensive WKY.

Interestingly, this decrease in blood pressure was not associated with increased concentration of cGMP, as it was the case for spontaneously hypertensive rats.

Nevertheless, decreased expression of NF-kB was also demonstrated in WKY treated with apocynin.

Moreover, we have found a positive correlation between systolic blood pressure and NF-kB protein ex- pression in hypertensive rats. Thus, it seems that other factors possibly affected by NF-kB might be partially responsible for blood pressure regulation.

Higher blood pressure and endothelial dysfunction in SHR are associated with enhanced degradation of NO by superoxide rather than with decreased NO generation [31]. This is in agreement with previous studies showing increased urinary excretion and plasma concentration of NO metabolites as well as elevated kidney and aorta eNOS and iNOS protein expression in prehypertensive (3 weeks) and early hypertensive (8–12 weeks) SHR [29]. Similarly, our results showed significantly higher activity of NO synthase in the left ventricle and aorta of SHR and BHR in comparison with normotensive rats. Apocynin did not further increase NO synthase activity in hypertensive rats; rather, it improved NO bioavailabilityvia its inhibitory effect on NAD(P)H oxidase. The improve- ment of NO bioavailability might further lead to increased cGMP concentration. Indeed, we have observed that the cGMP concentration in the left ventricle of hypertensive rats treated with apocynin increased to the level seen in normotensive WKY.

Hayashi et al. [11] investigated the usefulness of apocynin administration in a model of diabetic angio- pathy. In their experiments, apocynin reversed the endothelial dysfunction and prevented the atheroscle- rotic and morphological changes in diabetic rats re- gardless of NO synthase inhibition. The authors suggested that a decrease in the plasma TNF-a con- centration was involved in these beneficial effects [11]. These results are in good agreement with our study, since NF-kB can be involved in the increased production of TNF-a. It was also reported that apo- cynin treatment decreased blood pressure in mineralocorticoid-induced hypertension [5] and in angiotensin II-induced hypertension [30]. Park et al. [20] documented that inhibition of NAD(P)H oxidase with apocynin prevented hypertension and associated cardiac hypertrophy and fibrosis in the aldosterone-infused rat model. Similarly in our experiment, chronic apocynin treatment was able to decrease heart weight : body weight ratio in borderline and spontaneously hypertensive rats.

In conclusion, our study demonstrated that chronic treatment with apocynin partially prevented systolic blood pressure increases in hypertensive rats and decreased systolic blood pressure in normotensive rats.

This effect was associated with decreased expression of NF-kB protein in both normotensive and hyperten- sive rats and increased cGMP concentration in hypertensive rats. Thus, even without affecting NO synthase activity, decreased production of ROS may improve nitric oxide bioavailability and contribute to the partial prevention of hypertension development.

Acknowledgment:

This work was supported in part by research grants VEGA 2/0178/09, 1/0142/09 and APVT-51-017902, APVV-0586-06, and APVV-0538-07.

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Received:

December 1, 2008; in revised form: January 19, 2009.