European Journal of Pharmacology 588 (2008) 267–276

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European Journal of Pharmacology

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Role of superoxide and hydrogen peroxide in hypertension induced by an antagonistof adenosine receptors

Teresa Sousa a,b, Dora Pinho a, Manuela Morato a,b, José Marques-Lopes a, Eduarda Fernandes c, Joana Afonso a,Sofia Oliveira a, Félix Carvalho d, António Albino-Teixeira a,⁎a Institute of Pharmacology and Therapeutics, Faculty of Medicine of Porto and IBMC, University of Porto, Porto, Portugalb Pharmacology Department, Faculty of Pharmacy, University of Porto, Porto, Portugalc REQUIMTE, Physical Chemistry Department, Faculty of Pharmacy, University of Porto, Porto, Portugald REQUIMTE, Toxicology Department, Faculty of Pharmacy, University of Porto, Porto, Portugal

⁎ Corresponding author. Institute of PharmacologyMedicine of Porto, Alameda Prof. Hernâni Monteiro, 420022 551 36 42; fax: +351 22 551 36 43.

E-mail address: albinote@med.up.pt (A. Albino-Teixe

0014-2999/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.ejphar.2008.04.044

A B S T R A C T

A R T I C L E I N F O

Article history:

Treatment of Wistar rats f Received 5 November 2007Received in revised form 2 April 2008Accepted 9 April 2008Available online 24 April 2008

Keywords:Adenosine receptorsHypertensionRenin–angiotensin systemOxidative stressH2O2

or 7 days with 1,3-dipropyl-8-sulfophenylxanthine (DPSPX), an antagonist ofadenosine receptors, induces long-lasting hypertension associated with marked changes in vascular structureand reactivity and renin–angiotensin system activation. This study aimed at evaluating the role of oxidativestress in the development of DPSPX-induced hypertension and also at identifying the relative contribution ofsuperoxide radical (O2

•−) vs hydrogen peroxide (H2O2). Vascular and systemic prooxidant/antioxidant statuswas evaluated in sham (saline, i.p., 7 days) and DPSPX (90 μg/kg/h, i.p., 7 days)-treated rats. Systolic bloodpressure was determined by invasive and non-invasive methods. The activity of vascular NADPH oxidase,superoxide dismutase (SOD), catalase and glutathione peroxidase was assayed by fluorometric/spectro-photometric methods. H2O2 levels were measured using an Amplex Red Hydrogen Peroxide kit. Plasmathiobarbituric acid reactive substances and plasma antioxidant capacity were also measured. In addition wetested the effects of antioxidants or inhibitors of reactive oxygen species generation on blood pressure,vascular hyperplasia and oxidative stress parameters. DPSPX-hypertensive rats showed increased activity ofvascular NADPH oxidase, SOD, catalase and glutathione peroxidase, as well as increased H2O2 generation.DPSPX-hypertensive rats also had increased plasma lipid peroxidation and decreased plasma antioxidantcapacity. Treatment with apocynin (1.5 mmol/l, per os, 14 days), or with polyethylene glycol (PEG)-catalase(10,000 U/kg/day, i.p., 8 days), prevented the DPSPX-induced effects on blood pressure, vascular structure andH2O2 levels. Tempol (3 mmol/l, per os, 14 days) failed to inhibit these changes, unless PEG-catalase was co-administered. It is concluded that O2

•− generation with subsequent formation of H2O2 plays a major role in thedevelopment of DPSPX-induced hypertension.

© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Continuous blockade of adenosine receptors for 7 days with 1,3-dipropyl-8-sulfophenylxanthine (DPSPX), a non-selective A1/A2

antagonist of adenosine receptors, induces hypertension (Albino-Teixeira et al., 1991; Matias et al., 1991), endothelial dysfunction (Paivaet al., 1997), altered vascular reactivity (Morato et al., 2002) andmarked cardiovascular hypertrophic and hyperplastic changes(Albino-Teixeira et al., 1991; Matias et al., 1991; Sousa et al., 2002;Morato et al., 2003) in Wistar rats. This hypertensive state lasts for atleast 7 weeks after the end of the infusion of the drug. The renin–angiotensin system is activated in this model of hypertension, aspreviously shown by the increased plasma renin activity (Sousa et al.,

and Therapeutics, Faculty of-319 Porto, Portugal. Tel.: +351

ira).

l rights reserved.

2002) and plasma angiotensin II levels (Morato et al., 2002) andcontributes to the rise of blood pressure and to the alterations invascular morphology (Morato et al., 2003; Sousa et al., 2002). The factthat the hypertensive state as well as the renin–angiotensin systemactivation are maintained after the end of DPSPX infusion, stronglysuggests the existence of amplificating mechanisms prolonging theeffects of DPSPX infusion on vascular structure and reactivity.

Reactive oxygen species have been identified as major contributorsto the development and progression of cardiovascular diseases.Among biological reactive oxygen species, superoxide radical (O2

•−)and hydrogen peroxide (H2O2) are especially relevant since they canpromote changes in the vascular tone and structure (Rao and Berk,1992; Rajagopalan et al., 1996; Lounsbury et al., 2000; Touyz, 2003).Several studies have attributed a pivotal role for O2

•− in mediatinghypertension, since it reduces nitric oxide bioavailability leading toendothelial dysfunction. However, emerging evidence suggests thatH2O2 also plays a major role in the development of cardiovasculardiseases such as atherosclerosis and arterial hypertension. Of note,

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H2O2 seems to be more harmful than O2•−, since it is uncharged,

relatively longer-lived and freely diffusible within and between cells(Griendling and Harrison, 1999; Cai, 2005).

Angiotensin II and adenosine produce opposite effects in proox-idant/antioxidant balance. Angiotensin II stimulates the activity ofNADPH oxidase, which is the major source of reactive oxygen speciesgeneration in the vasculature (Griendling et al., 1994; Griendling et al.,2000; Cai et al., 2003). The blockade of angiotensin II effects byinhibitors of angiotensin converting enzyme or by antagonists ofangiotensin II AT1 receptors has been shown to reduce oxidative stressand improve antioxidant status (Donmez et al., 2002; Khattab et al.,2004; Bayorh et al., 2005; Bolterman et al., 2005; Flammer et al.,2007). In contrast to angiotensin II, adenosine provides protectionagainst oxidative stress by increasing the expression and the activityof antioxidant enzymes in the cardiovascular system (Husain andSomani, 2005; Zhang et al., 2005).

Alterations in the production or metabolism of reactive oxygenspecies may underlie the changes in the vascular reactivity andstructure observed in DPSPX-hypertensive rats, namely decreasedcontractile response to angiotensin II (Morato et al., 2002), increasedcontraction to noradrenaline (Morato et al., 2002), endothelialdysfunction (Paiva et al., 1997), increasedmedia thickness and smoothmuscle cell width and reduced lumen by proliferation of subintimalcells (Albino-Teixeira et al., 1991; Matias et al., 1991; Sousa et al., 2002;Morato et al., 2003). Furthermore, the prevention of DPSPX-inducedhypertension and vascular hypertrophy/hyperplasia by captopril orlosartan (Sousa et al., 2002; Morato et al., 2003) might be related tothe antioxidant effects of angiotensin II inhibition.

Keeping in mind these facts, we aimed at evaluating the role ofoxidative stress in the development of DPSPX-induced hypertensionand also at identifying the relative contribution of O2

•− vs H2O2 in thesepathophysiological processes.

2. Materials and methods

2.1. Animals and experimental design

Experiments were conducted in male Wistar rats (250–300 g). Theanimals were maintained under constant photoperiod conditions(12 h dark, 12 h light) at 21 °C temperature and 60% relative humidity.Standard laboratory rat chow and tap water were available ad libitum.Housing and experimental treatment of the animals were conductedunder the European Community guidelines for the use of experi-mental animals (European convention for the protection of vertebrateanimals used for experimental and other scientific purposes,1986, andProtocol of amendment to the European convention for the protectionof vertebrate animals used for experimental and other scientificpurposes, 1998).

The present study was divided into two parts:

Part I: Characterization of prooxidant and antioxidant status inDPSPX-hypertensive rats.Part II: Evaluation of the effects of antioxidants or inhibitors ofreactive oxygen species generation on blood pressure, vascularhyperplasia and oxidative stress parameters in DPSPX-treated rats.

2.1.1. Part I: Characterization of prooxidant and antioxidant status inDPSPX-hypertensive rats

Animals were randomly divided in two groups: DPSPX (90 μg/kg/h)-treated (DPSPX) and sham-operated (sham). DPSPX (90 μg/kg/h) orsaline (vehicle) were infused for 7 days through Alzet osmoticminipumps (model 2ML1; Alza, Palo Alto, CA, U.S.A.), intraperitoneallyimplanted under sodium pentobarbitone anaesthesia (60 mg/kg, i.p.).In addition, another rats were treated simultaneously with DPSPX(90 μg/kg/h, i.p.) and captopril (100 mg/kg/day, per os). Systolic bloodpressure was measured in conscious animals with a tail cuff using a

photoelectric pulse detector (LE 5000, LETICA, Barcelona, Spain). Fivedeterminations were made each time and the means used for furthercalculations. Normotensive rats were selected after a 7 day period ofadaptation to the blood pressure measurement methodology. Directdeterminations of systolic blood pressure were made in some ratsfrom each group by implantation of an indwelling catheter. The ratswere anaesthetized with a mixture of ketamine (60 mg/kg) andmedetomidine (0.25 mg/kg) (i.p.) and a polyethylene catheter (PE-10)was placed in the abdominal aorta via the left femoral artery. Thedistal end was joined to PE-50 tubbing and tunneled subcutaneouslyto the dorsum of the neck where it was exteriorised. After a recoveryperiod, the arterial catheter was connected to a pressure transducer(Letica, TRA-021) and blood pressure signals were recorded on apolygraph (Letica, Unigraph 2000-5.6) in conscious, unrestrainedanimals. One week after the end of DPSPX infusion (day 14), rats wereanaesthetized with sodium pentobarbitone (60 mg/kg, i.p.), the bloodwas withdrawn from the left ventricle into heparinized tubes and themain branch of the mesenteric artery was collected and dissected freefrom fat and connective tissue. Plasmawas obtained by centrifugationof blood at 1000 ×g, 4 °C, for 20 min, and subsequent separation of thesupernatant. To avoid further oxidation in plasmas for the evaluationof lipid peroxidation, the antioxidant butylated hydroxy toluene(0.005%, w/v) was added before storage. Samples used for morpho-logical study and H2O2 measurement were promptly processed. Theother samples were stored at −80 °C until further analysis.

2.1.2. Part II: Evaluation of the effects of antioxidants or inhibitors ofreactive oxygen species generation on blood pressure, vascularhyperplasia and oxidative stress parameters in DPSPX-treated rats

Animals were randomly distributed among the evaluated groups.Hypertension was induced in Wistar rats by the infusion of DPSPX(90 µg/kg/h, from day 0 to day 7) through Alzet osmotic minipumps(model 2ML1; Alza, Palo Alto, CA, U.S.A.), intraperitoneally implantedunder pentobarbitone sodium anaesthesia (60 mg/kg, i.p.). Sham-operated rats received an infusion of saline. To determine the role ofO2•−, rats were treated for 14 days with apocynin (1.5 mmol/l), which

inhibits O2•− generation by NADPH oxidase, or with tempol (3 mmol/l),

a superoxide dismutase (SOD) mimetic that increases O2•− degradation.

To study the contribution of H2O2 to the DPSPX-induced changes inblood pressure and vascular morphology, rats were treated for 8 dayswith polyethylene glycol catalase (PEG-catalase, 10,000 U/kg/day) orwith tempol (3 mmol/l) plus PEG-catalase (PEG-catalase, 10,000 U/kg/day). Tempol and apocynin were administered in drinking water.Apocynin was first solubilized in DMSO and then added to tap water(final concentration of DMSO, 0.06%, v/v). The chosen doses ofapocynin and tempol have been shown to reduce blood pressure inother experimental models of hypertension (Beswick et al., 2001a;Kobori and Nishiyama, 2004). PEG-catalase was intraperitoneallyadministered after solubilization in saline. PEG-catalase was preferredto catalase, since the conjugation of the enzyme with PEG increasesthe circulatory half-life of catalase (Beckman et al., 1988). The dose ofPEG-catalase was chosen for its ability to reduce oxidative stress invarious pathologic conditions (Birtwistle et al., 1989; Liu et al., 1989)though never tested before in hypertensive states. All the treatmentsstarted on the day of implantation of the Alzet pumps (day 0). Thetested drugs were given to both sham- and DPSPX-treated animals.Systolic bloodpressurewasmeasured throughout the study by the tail-cuff method, as previously described in Part I. At the end of thetreatments, rats were anaesthetized with sodium pentobarbitone(60mg/kg, i.p.) and the blood and arteries (mesenteric and tail arteries)were collected and processed as previously described in Part I.

2.2. Enzyme assays

Mesenteric arteries were homogenized in a glass-to-glass homo-genizer. Those for SOD and catalase assays were homogenized in a

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cold phosphate buffer (50 mM, 7.4) containing Triton 0.1% (v/v) andcentrifuged for 10 min, at 15700 ×g, 4 °C, while vessels for NADPHoxidase evaluation were homogenized in a cold HEPES buffer(25 mmol/l) containing EDTA (1 mmol/l) and phenylmethyl-sulfonylfluoride (PMSF) (0.1 mmol/l). Protein content was assayed by theBradford method (Bradford, 1976). A dihydroethidium-based fluores-cence assay was used to evaluate O2

•− production from NADPH oxidase(Yi et al., 2006; Zou et al., 2001). Mesenteric artery homogenates(≈20 µg) were incubated with dihydroethidium (20 µM) and salmontestes DNA (0.5 mg/ml), with or without NADPH (1 mM) in amicroplate, at 37 °C for 30 min. Ethidium fluorescence was measuredat 475 nm-excitation and 610 nm-emission using a fluorescencemicroplate reader (Spectromax Gemini, Molecular Devices). Tiron(20 mM), a O2

•− scavenger, was used to confirm the specificity of themethod. To determine whether NADPH oxidase was the source of O2

•−,a specific inhibitor of NADPH oxidase (diphenylene iodonium, DPI,500 µM) was used. The effects of a xanthine oxidase inhibitor(allopurinol, 500 µM) and a nitric oxide synthase inhibitor (nitro-L-arginine methylester, L-NAME, 500 µM) were also tested to excludeother possible contributors for the O2

•−mediated ethidium fluores-cence. Background activity, which corresponds to the fluorescencegenerated by the sample homogenate in the absence of NADPH, wassubtracted to the activity generated by the sample in the presence ofNADPH. Results were expressed as fluorescence arbitrary units permin per mg of protein.

Total SOD activity was assayed by the method of Flohe and Otting(1984) with slight modifications. The rate of inhibition of nitro bluetetrazolium (NBT, 50 μM) reduction by SOD was monitored during2 min, at 540 nm. O2

•− required for NBT reduction was generated by axanthine (44 μM)/xanthine oxidase (0.29 U/ml) system. SOD activitywas calculated against a standard curve of SOD (0.0078–2 μg/ml) frombovine erythrocytes. Results are expressed as Units of SOD per mg ofprotein. One unit of SOD was defined as the concentration required toinhibit by 50% the rate of the xanthine/xanthine oxidase mediated

Fig. 1. Systolic blood pressure (SBP) (mmHg). (A) Tail-cuff measurements of SBP in Sham- andcaptopril treated rats (n=4); (C) Correlation between tail-cuff and intra-arterial pressures; (Dare mean±S.E.M. ⁎Pb0.05 vs sham; #Pb0.05 vs DPSPX.

reduction of NBT. Catalase activity was evaluated by monitoring H2O2

decomposition at 240 nm, at 25 °C (Aebi, 1984). Measurements wererestricted to the optical density values over the initial 40 sec of theassay which corresponded to the linear portion of the absorbancecurve. One unit of catalase was defined as the amount of enzyme thatdecomposes 1 μmol of H2O2 per min. Results were calculated using anextinction coefficient of 0.0394mM−1 cm−1 and are expressed as Unitsper mg of protein. Glutathione peroxidase activity was assayedspectrophotometrically by following NADPH oxidation at 340 nmwhen glutathione peroxidase/H2O2-produced oxidized glutathione isreverted back by glutathione reductase (Flohe and Gunzler, 1984).Results were calculated using an extinction coefficient of 6.22 mM−1.cm−1 and are expressed as nmol of oxidized NADPH per min per mg ofprotein.

2.3. Measurement of urinary excretion of H2O2 and vascular H2O2

production

To collect urine samples, rats were placed in metabolic cages.Urinary excretion of H2O2 was evaluated in 50 μl aliquots of 24-h urinesamples diluted 50 fold with 50 mM sodium phosphate buffer pH 7.4.H2O2 production was also evaluated in the mesenteric artery and tailartery. After removal of the connective tissue and blood, the arterieswere placed in Krebs-HEPES medium (mM: NaCl 118, KCl 4.5, CaCl22.5, MgCl2 1.2, KH2PO41.2, Na-HEPES 25, NaHCO3 25 and glucose 5; pH7.4) at 37 °C. Ninety minutes later duplicate 50 μl aliquots were takenfrom each of the tubes to determine the concentration of H2O2. Someexperiments were also performed in the presence of the NADPHoxidase inhibitor, DPI (500 μM).

Urinary and vascular concentrations of H2O2 were measured usingan Amplex Red Hydrogen Peroxide Assay kit (Molecular Probes),according to the protocol provided by the manufacturer. Results areexpressed as μmol of H2O2 per kg per day (urinary H2O2 excretion) oras nmol of H2O2 per mg of protein (vascular production of H2O2).

DPSPX-treated rats (n=10); (B) Tail-cuff measurements of SBP in DPSPX and DPSPX plus) Tail-cuff and intra-arterial measurements of SBP in DPSPX-treated rats (n=4). Results

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2.4. Evaluation of plasma lipid peroxidation—measurement ofthiobarbituric acid reactive substances

Plasma levels of thiobarbituric acid reactive substances weremeasured according to the method of Ohkawa et al. (1979) withslight modifications. Briefly, 100 μl of plasma were added to 50 μl of8.1% sodium dodecyl sulfate, vortexed and incubated for 10 min atroom temperature. 375 μl of 28% (w/v) trichloroacetic acid and 375 μlof 0.6% thiobarbituric acid (w/v) were added and placed in a waterbath at 95 °C for 60 min. The samples were allowed to cool at roomtemperature. 1.25 ml of a butanol:pyridine mixture (15:1, v/v) wasadded, vortexed and centrifuged at 100 g for 5 min. The absorbancewas measured at 532 nm in the organic layer. The breakdownproduct of malondialdehyde bis(diethyl acetal) was used as astandard. The results are expressed in nmol of malondialdehydeequivalents per ml.

2.5. Evaluation of plasma antioxidant status

The plasma antioxidant status was evaluated according to adescribed procedure called the oxygen radical absorbance capacity(Dogra et al., 2001; Fernandes et al., 2004). This assay measures thedecrease of fluorescence of fluorescein added to plasma in thepresence of a free radical generator and corresponds to the ability ofplasma components to trap free radicals. It is measured against aTrolox standard (1 μM) and a phosphate buffer blank. Reactionmixtures contained, in a final volume of 200 μl, the following reagentsdissolved in 75 mM potassium phosphate buffer, pH 7.4: fluorescein(61 nM), plasma (diluted 900 fold with phosphate buffer) and α,α'-azodiisobutyramidine dihydrochloride (AAPH) (19 mM). Fluorescence

Fig. 2. (A) Generation of O2•− after addition of NADPH, under control conditions or in the prese

(allopurinol) or NO synthase (L-NAME); (B) NADPH oxidase activity in DPSPX and DPSPX pluand DPSPX-treated rats (n=6); (D) Effect of DPI on H2O2 production in mesenteric arteriesDPSPX.

measurements were performed on a microplate reader (HT Synergy,BIO-TEK) with excitation and emission wavelenghts of 485 nm and528 nm, respectively. The area under the fluorescence decay curve(AUC) was calculated and compared to the AUC for Trolox standard.The oxygen radical absorbance capacity values were calculated as theratio of the plasma AUC to the Trolox AUC and are expressed as μM ofTrolox equivalents.

2.6. Morphological study

Tail arteries were cut into small fragments and fixed for 48 h inBouin's solution (mixture of picric acid saturated aqueous solution,formaldehyde 37–40%, glacial acetic acid, 75:25:5 v/v). After fixation,the tissues were dehydrated using graded ethanol washes for 3 h,followed by benzol washes for 5 min, and embedded in paraffin wax.Semi-thin sections of 6 µmwere obtained from five blocks represent-ing each experimental condition, taken at random, with an LKBUltrotome. Sections were deparaffinised, hydrated, mounted on slidesand stained with hematoxylin and eosin. The slides of the arterieswere analysed using the Leica Image Analysis System QWin Q550software (Leica, Germany).

2.7. Statistical analysis

Results are expressed as mean±S.E.M. and n corresponds to thenumber of rats per group. Statistical analysis of the data wasperformed using unpaired Student's t test (when comparing twogroups) or using one-way analysis of variance (ANOVA) followed byNewman-Keuls test (when comparing three or more groups). P valuesof less than 0.05 were considered significant.

nce of the O2•− scavenger (Tiron) and inhibitors of NADPH oxidase (DPI), xanthine oxidase

s captopril treated rats (n=4–5). (C) H2O2 production in mesenteric arteries from Sham-from DPSPX-treated rats (n=4). Results are mean±S.E.M. ⁎Pb0.05 vs sham; #Pb0.05 vs

Fig. 3. Antioxidant enzyme activity in mesenteric arteries from Sham- and DPSPX-treated rats (n=6–9). (A) SOD; (B) Catalase; (C) Glutathione peroxidase. Results are mean±S.E.M.⁎Pb0.05 vs sham.

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2.8. Drugs

Ethanol, benzol, glacial acetic acid, butanol and pyridine werepurchased from Merck (Darmstadt, Germany). Other drugs werepurchased from Sigma (St. Louis, MO, USA).

3. Results

3.1. Part I

3.1.1. Systolic blood pressureContinuous treatment with DPSPX caused a significant increase in

systolic blood pressure (146.3±1.7 mmHg), when compared to sham-operated group (121.7±0.9 mmHg) (Pb0.05) (Fig. 1). There was ahighly significant correlation (r2=0.9886) between tail–cuff andintra–arterial measurements of systolic blood pressure. Tail-cuffsystolic blood pressure values of DPSPX-treated rats were similarto the values obtained by intra-arterial measurement (147.3±2.9 vs151.8±5.2 mmHg, n=4, P=0.4795) (Fig. 1). Simultaneous treatmentwith DPSPX and captopril prevented the DPSPX-induced rise inblood pressure (109.7±3.1 mmHg) (Fig. 1).

3.1.2. NADPH oxidase activityNADPH oxidase activity was significantly increased in the

mesenteric artery of DPSPX-hypertensive rats (550.97±40.43 vs385.15±40.16 fluorescence units/min/mg protein, n=5, Pb0.05)(Fig. 2). DPI (a NADPH oxidase inhibitor) and Tiron (a O2

•− scavenger)inhibited O2

•− -mediated ethidium fluorescence by 50% respectively(Fig. 2), while allopurinol and L-NAME had no effect (Fig. 2). In ratstreated with DPSPX+captopril, NADPH oxidase activity was reducedby 35%, compared to DPSPX group (Fig. 2).

Fig. 4. Plasma lipid peroxidation (A); plasma antioxidant status (B), in Sham- andDPSPX-treated rats (n=7–9). Results are mean±S.E.M. ⁎Pb0.05 vs sham.

3.1.3. H2O2 productionDPSPX-hypertensive rats showed approximately a two-fold

increase in mesenteric artery production of H2O2, when comparedto sham-operated rats (0.092±0.021 vs 0.040±0.008 nmol/mg ofprotein, n=6, Pb0.05) (Fig. 2). In the presence of DPI (an inhibitor ofNADPH oxidase) there was a significant reduction of H2O2 productionin mesenteric arteries from DPSPX-hypertensive rats (Fig. 2).

3.1.4. Antioxidant enzyme activityDPSPX-hypertensive rats had increased activities of SOD when

compared to sham-operated rats (3.05±0.66 vs 1.08±0.32 U SOD/mgprotein, n=9, Pb0.05) (Fig. 3), catalase (5.01±0.22 vs 3.07±0.45 Ucatalase/mg protein, n=6, Pb0.01) (Fig. 3) and glutathione peroxidase(79.73±2.40 vs 60.39±2.73 nmol/min/mg protein, n=6, Pb0.001)(Fig. 3) in the mesenteric artery.

3.1.5. Plasma prooxidant and antioxidant statusDPSPX treatment caused an elevation of plasma lipid peroxidation

(27.17±3.39 vs 18.46±0.92 nmol/ml, n=9, Pb0.05) (Fig. 4) andsignificantly reduced the plasma antioxidant potential assessed by

Fig. 5. (A) Effect of apocynin and tempol on systolic blood pressure (mmHg) of Sham-and DPSPX-treated rats. (n=7–10). (B) Effect of PEG-catalase and PEG-catalase plustempol on systolic blood pressure (mmHg) of DPSPX-treated rats (n=4). Results aremean±S.E.M. ⁎Pb0.05 vs respective sham. #Pb0.05 vs DPSPX.

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oxygen radical absorbance capacity assay (1.27±0.12 vs 1.69±0.04 µMTrolox equivalents, n=7–9, Pb0.05) (Fig. 4).

3.2. Part II

3.2.1. Effect of apocynin, tempol and PEG-catalase on systolic bloodpressure

DPSPX induced a significant increase in systolic blood pressure fromday 3 (135.44±0.98 vs 124.06±0.8 mmHg, Pb0.001) after the start of

Fig. 6. Representative photomicrographs of vascular morphology in Sham (A), DPSPX (C, D)catalase+tempol (H). Shown are tail artery sections of 6 μm stained with hematoxylin and eosprotrude into the lumen, are denoted by arrows. D and F correspond to vascular smooth mu

the infusion until the end of the experiments (147.30±1.69 vs 122.58±1.12 mmHg, Pb0.001) (Fig. 5). DPSPX-induced hypertension wasprevented by the treatment with apocynin (128.90±1.20 mmHg,Pb0.001) (Fig. 6) or with PEG-catalase (122.33±1.08 mmHg, Pb0.001)(Fig. 5). Tempol failed to abolish the blood pressure rise (144.65±2.05mmHg) (Fig. 5) unless PEG-catalasewas co-administered (123.46±0.74mmHg, Pb0.001) (Fig. 5). Systolic blood pressure of sham-operatedanimals was not affected by apocynin (124.47±1.01 mmHg), tempol(125.10±0.67 mmHg) or PEG-catalase (125.17±0.17 mmHg).

, DPSPX+tempol (E, F), DPSPX+apocynin (B); DPSPX+PEG−catalase (G); DPSPX+PEG−in. Proliferation of subintimal cells, with the formation of smoothmuscle cell buttons thatscle cell buttons details from DPSPX and DPSPX+tempol, respectively.

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3.2.2. Vascular morphologyDPSPX induced the proliferation of subintimal cells, leading to the

development of smooth muscle cells buttons that protruded into thelumen of the arteries (Fig. 6). Treatment with apocynin or PEG-catalase prevented the vascular hyperplasia induced by DPSPX-treatment (Fig. 6). Tempol failed to avoid the proliferation of vascularsmooth muscle cells unless PEG-catalase was co-administered (Fig. 6).Neither apocynin, tempol or PEG-catalase had effect on vascularmorphology of sham-operated animals (data not shown).

3.2.3. Effects of apocynin and tempol on plasma lipid peroxidationproducts, vascular glutathione peroxidase activity, urinary excretion ofH2O2 and vascular production of H2O2

Apocynin and tempol prevented the rise of plasma lipid peroxida-tion products in DPSPX-treated rats (19.15±0.80 and 21.37±1.17 vs30.16±3.61 nmol malondialdehyde equivalents/ml plasma, respec-tively, Pb0.01) (Fig. 7). DPSPX-induced increase of vascular glu-tathione peroxidase activity (86.01±3.90 vs 62.64±4.58 nmol NADPH/min/mg protein, Pb0.01) was prevented by apocynin (61.87±4.87 nmol NADPH/min/mg protein, Pb0.01), but not by tempol(76.89±2.54 nmol NADPH/min/mg protein) (Fig. 7). Urinary excretionof H2O2 was significantly higher in DPSPX-infused rats (5.46±0.61 vs2.32±0.39 µmol H2O2/kg/day, Pb0.001) and in rats treated simulta-neously with DPSPX and tempol (4.02±0.51 µmol H2O2/kg/day,Pb0.05) (Fig. 7). Apocynin prevented the increase in urinary excretionof H2O2 in DPSPX-treated rats (1.35±0.24 µmol H2O2/kg/day, Pb0.001)(Fig. 7). DPSPX increased the vascular production of H2O2 (0.088±0.009 vs 0.053±0.007, nmol H2O2/mg protein, Pb0.01) (Fig. 7). TheDPSPX-induced rise in vascular production of H2O2 was prevented byapocynin (0.044±0.014 nmol H2O2/mg protein, Pb0.05), but not by

Fig. 7. Effect of apocynin and tempol on: (A) plasma lipid peroxidation (expressed as nmol(nmol/min/mg of protein) in the mesenteric artery (n=6–7). (C) urinary excretion of H2O2 (μmoResults are mean±S.E.M. ⁎Pb0.05 vs sham. #Pb0.05 vs DPSPX.

tempol (0.093±0.013 nmol H2O2/mg protein) (Fig. 7). Neitherapocynin, nor tempol had effect on plasma lipid peroxidationproducts, glutathione peroxidase activity, urinary and vascular H2O2

levels in sham-operated rats (data not shown).

4. Discussion

The present work aimed at evaluating the role of oxidative stress inthe development of DPSPX-induced hypertension and also atcomparing the relative contribution of O2

•− and H2O2 in thesepathophysiological processes. In the first part, we evaluated theeffects of DPSPX infusion on vascular and systemic prooxidant andantioxidant status. These parameters were evaluated on day 14 afterthe beginning of DPSPX infusion, which corresponds to a periodwhere the hypertension and the alterations in vascular structure andreactivity are maintained, even though DPSPX is no longer beinginfused. Mesenteric arteries from DPSPX-hypertensive rats hadenhanced activity of NADPH oxidase, SOD, catalase and glutathioneperoxidase, as well as increased H2O2 generation. DPSPX-hypertensiverats also showed increased plasma lipid peroxidation and decreasedplasma antioxidant capacity. These results reflect vascular andsystemic oxidative stress.

The rise of NADPH oxidase activity can be explained by the increaseof angiotensin II plasma levels previously observed in DPSPX-hypertensive rats (Morato et al., 2002). Indeed, activation ofangiotensin II receptors results in the activation of NADPH oxidase(Cai et al., 2003; Griendling et al., 1994; Griendling et al., 2000). Theseresults are in accordance with previous reports showing thathypertension is associated with increased NADPH oxidase activityand oxidative stress (Rajagopalan et al., 1996; Zalba et al., 2000; Virdis

malondialdehyde equivalents/ml plasma) (n=6–8); (B) glutathione peroxidase activityl/kg/day) (n=5–12); (D) H2O2 production in the tail artery (nmol/mg of protein) (n=4–11).

274 T. Sousa et al. / European Journal of Pharmacology 588 (2008) 267–276

et al., 2004). NADPH oxidase plays a critical role in mediating thevascular oxidative stress and the blood pressure response toangiotensin II (Landmesser et al., 2002). In fact, we also observedthat DPSPX-infused rats treated with the angiotensin-convertingenzyme inhibitor, captopril, had lower NADPH oxidase activity incomparison to DPSPX-treated rats. However, since captopril alsoprevented the blood pressure rise and has intrinsic antioxidantproperties (Bagchi et al., 1989; Chopra et al., 1989), we cannot attributethe effect on NADPH oxidase activity solely to the angiotensinconverting enzyme inhibition. Besides the increase in prooxidantactivity, several works have also described an impairment ofantioxidant enzyme defenses in hypertension (Ito et al., 1995;Lassegue and Griendling, 2004). However, we found that DPSPX-hypertensive rats have increased antioxidant enzyme activity in themesenteric artery. It has been described that angiotensin II stimulatesNADPH oxidase-dependent accumulation of H2O2, which is formedthrough dismutation of O2

•−, either spontaneous or catalyzed by SOD(Zafari et al., 1998). Our results are consistent with this observation.Vascular SOD activity and H2O2 generation were significantlyincreased in DPSPX-hypertensive rats and incubation with theNADPH oxidase inhibitor, DPI, markedly reduced H2O2 generation inthe mesenteric artery. Although SOD is usually seen as a defensemechanism against oxidative stress, large increments in the activity ofthis enzymemay lead to tissue injury by accelerating H2O2 production(Harris, 1992). The increases in vascular catalase and glutathioneperoxidase activities are not surprising in conditions of increasedH2O2 production, since both enzymes are responsible for itsdetoxification. Furthermore, catalase is known to be of specialimportance when the clearance of high concentrations of H2O2 isrequired (Scandalios, 2005; Wassmann et al., 2004). An adaptiveincrease of catalase and glutathione peroxidase activities has alreadybeen shown after H2O2 infusion in isolated perfused hearts fromspontaneously hypertensive rats (Csonka et al., 2000). Moreover, thereare some studies describing increases in antioxidant enzyme activitiesin other experimental models of hypertension. Animals with lead-induced hypertension show increased SOD and catalase activities(Farmand et al., 2005). L-NAME-induced hypertension is also asso-ciated with increased SOD and glutathione peroxidase activities (Sainzet al., 2005). These changes in antioxidant enzymes are likely to becompensatory responses to oxidative stress.

The blockade of adenosine physiological effects should also beconsidered when interpreting these results. The antagonism of A1

adenosine receptors is responsible for the renin–angiotensin systemactivation in DPSPX-induced hypertension (Morato et al., 2002; Sousaet al., 2002), since adenosine inhibits renin release (Jackson, 1991).There might be other adenosine actions involved in the redox changesobserved in this model of hypertension. Adenosine is known to becytoprotective and confers increased tissue resistance to oxidativeinjury (Ramkumar et al., 2001). The blockade of adenosine protectiveeffects may adversely affect outcome in conditions of enhancedreacyive oxygen species generation. In animals subjected to con-cussive head injury the administration of caffeine, a non-selectiveantagonist of A1/A2 adenosine receptors, exacerbates brain injury byenhancing oxidative stress (Al Moutaery et al., 2003). A similarmechanism could account for the redox dysfunction in the early phaseof DPSPX-induced hypertension. However, once DPSPX infusion stopssuch a direct effect of the drug is no longer expected. In this latterphase, adenosine is more likely to be involved in the adaptiveresponse to enhanced reactive oxygen species generation. Oxidativestress increases the expression of A1 adenosine receptors, which areknown to contribute to the cytoprotective role of adenosine (Nie et al.,1998). In addition, adenosine has been shown to promote the increasein the expression and activity of cardiovascular antioxidant defenses,such as SOD, catalase and glutathione peroxidase (Husain and Somani,2005; Zhang et al., 2005). Therefore, it is possible that the enhance-ment of vascular antioxidant enzyme activity observed after the end of

DPSPX infusion may be induced by adenosine to counteract theincreased generation of reactive oxygen species by NADPH oxidase.

It is known that changes in the structure or in the reactivity ofsmall arteries can contribute to increased peripheral vascularresistance, thus affecting blood pressure. Some authors have proposedthat H2O2 mediates the vascular hypertrophic and contractileresponses to angiotensin II (Zafari et al., 1998; Torrecillas et al.,2001). As DPSPX-induced hypertension is accompanied by activationof the renin–angiotensin system (Morato et al., 2002; Sousa et al.,2002) it seems conceivable that H2O2 also functions as a mediator ofangiotensin II-induced effects in the vascular structure and reactivityof these rats. The increase of H2O2 levels in hypertension mayaccelerate vascular smooth muscle cell proliferation and hypertrophy(Touyz, 2003), since H2O2 stimulates DNA synthesis and proliferationand induces the expression of growth-related genes (Rao and Berk,1992; Touyz, 2003). Previous studies in the DPSPX model ofhypertension have shown marked functional and structural changesin the mesenteric and tail arteries, namely increased media thicknessand smooth muscle cell width (Sousa et al., 2002; Morato et al., 2003)and reduced lumen by proliferation of subintimal cells (Albino-Teixeira et al., 1991; Morato et al., 2003).

In order to evaluate the role of oxidative stress and compare theimportance of H2O2 vs O2

•− in the pathogenesis of DPSPX-inducedhypertension, we treated DPSPX-infused rats with drugs (apocynin,tempol, PEG-catalase) that interfere with the bioavailability of suchreactive oxygen species. We found that apocynin and tempol, despitehaving a similar effect in reducing plasma oxidative stress, do notshare the ability to counteract the hypertension and the vascularhyperplasia induced by DPSPX. Tempol failed to prevent both theblood pressure rise and the vascular hyperplasia, while apocynineffectively blocked these effects. Treatment with these agents hasbeen reported to be effective in reducing blood pressure in othermodels of hypertension (Schnackenberg andWilcox, 1999; Beswick etal., 2001a; Beswick et al., 2001b; Dobrian et al., 2001). Thisantihypertensive effect is attributed to a reduction of O2

•− levels(Schnackenberg and Wilcox, 1999; Beswick et al., 2001a; Beswicket al., 2001b; Dobrian et al., 2001). However, while apocynin inhibitsO2•− generation by NADPH oxidase (Stolk et al., 1994; Hamilton et

al., 2004), tempol, as a SOD mimetic, converts the generated O2•−

into H2O2 (Chen et al., 2003), which is a much more stablemolecule and seems to be more harmful than O2

•− (Cai, 2005). Thelack of antihypertensive effect by tempol has also been observed inother models of hypertension with marked oxidative stress. In ratstreated with the inhibitor of nitric oxide synthase, nitro-L-argininemethylester, or with the inhibitor of SOD, diethyldithiocarbamate,treatment with tempol did not prevent the hypertension, despite areduction in renal O2

•− and systemic oxidative stress biomarkers(Makino et al., 2003; Yanes et al., 2005).

The rapid development of marked structural changes observed inthe arteries of DPSPX-infused rats (Albino-Teixeira et al., 1991; Matiaset al., 1991; Morato et al., 2003) may be due to increased generation ofH2O2. In the present studywe observed that DPSPX-treated rats have ahuge increase in vascular SOD activity and H2O2 productionaccompanied by an adaptive rise in vascular catalase and glutathioneperoxidase. Tempol, besides lacking a protective effect on bloodpressure and vascular morphology, also failed to prevent the DPSPXinduced-rise on vascular H2O2 production and glutathione peroxidaseactivity. Treatment with apocynin effectively prevented the increasein vascular H2O2 production and glutathione peroxidase activity.

In addition, we also found that DPSPX-treated rats excrete moreurinary H2O2 and that apocynin, but not tempol, prevented the rise inH2O2 levels. These results favour the interpretation that H2O2

contributes to the pathophysiology of DPSPX-induced hypertension.To confirm this hypothesis we treated DPSPX- and DPSPX+tempol ratswith PEG-catalase. The prevention of both hypertension and vascularhyperplasia by PEG-catalase or by tempol plus PEG-catalase further

275T. Sousa et al. / European Journal of Pharmacology 588 (2008) 267–276

demonstrates that H2O2 plays a major role in the development ofDPSPX-induced hypertension. Recent reports have highlighted theimportance of H2O2 in the pathophysiology of cardiovascular diseasessuch as hypertension and atherosclerosis (Lacy et al., 2000; Makinoet al., 2003; Yang et al., 2004; Cai, 2005; Suvorava et al., 2005). H2O2

activates various signalling cascades that mediate changes in vascularstructure including vascular smooth muscle cell hypertrophy andproliferation (Griendling and Harrison, 1999; Touyz, 2003; Cai, 2005).It also acts as a vasoconstrictor (Suvorava et al., 2005; Torrecillaset al., 2001), probably by modulating an increase in intracellularcalcium concentration. Furthermore, the long-term infusion of H2O2

directly into the renal medulla results in sustained hypertension(Makino et al., 2003). These facts stress the idea that H2O2 is animportant hypertensive factor. Modulation of catalase activity seemsto be a promising strategy in the prevention or treatment ofcardiovascular diseases. Overexpression of catalase has been shownto inhibit vascular smooth muscle cell hypertrophy and proliferation,to retard atherogenesis and to reduce vasoconstriction and systolicblood pressure (Brown et al., 1999; Suvorava et al., 2005; Yang et al.,2004). In vitro studies also demonstrated that treatment with catalaseprevents the vascular constriction and hypertrophy induced byangiotensin II (Zafari et al., 1998; Torrecillas et al., 2001). In ratstreated with diethyldithiocarbamate, tempol failed to counteract thehypertension unless catalase was co-infused (Makino et al., 2003). Inthe present study, we treated rats with a PEG-catalase combinedform, since the conjugation of the enzyme with PEG increases thestability in aqueous solution, reduces immunogenicity and decreasessensitivity to proteolysis, thus increasing the circulatory half-life ofcatalase from less than 10 min to 40 h (Beckman et al., 1988). Asreferred previously, PEG-catalase completely prevented the hyper-tension and the vascular hyperplasia induced by DPSPX. Takentogether, these data indicate that the reduction of H2O2 levels isimportant to counteract the development or the progression ofhypertension and pathological cardiovascular structural changes.NADPH oxidase seems to be the primary source of reactive oxygenspecies in DPSPX-induced hypertension. Accumulating evidencesupports our interpretation. First, DPSPX-hypertensive rats havesignificantly increased levels of plasma angiotensin II (Morato et al.,2002), a peptide known to be a major stimulus for the activation ofthis enzyme (Griendling et al., 1994; Griendling et al., 2000; Cai et al.,2003). Second, these animals have increased activity of vascularNADPH oxidase, and DPI, an inhibitor of this enzyme, markedlyreduces this effect and also the vascular generation of H2O2. In thepresent study we also showed that apocynin, which blocks theassembly of NADPH oxidase subunits thus preventing its activation(Stolk et al., 1994; Hamilton et al., 2004), inhibits the DPSPX-inducedeffects on systolic blood pressure, vascular structure and H2O2

production. Furthermore, since we have previously demonstratedthat DPSPX inhibits xanthine oxidase in vitro and in vivo (Sousa et al.,2004), it is not expectable that this enzyme primarily contributes tothe increase in reactive oxygen species generation in DPSPX-inducedhypertension, at least in the early phase of this hypertensive state.

It has been recently described that NADPH oxidase derived-H2O2

propagates its own production through the feed-forward activation ofreactive oxygen species sources, such as mitochondrial enzymes,xanthine oxidase, uncoupled endothelial nitric oxide synthase andalso NADPH oxidase itself (Cai, 2005). This self-amplificating effectseems to prolong H2O2 induced changes in the vascular structure andreactivity, and may explain why the hypertensive state induced byDPSPX lasts for at least 7 weeks after the end of the infusion of thedrug.

In summary, DPSPX-induced hypertension is associated withmarked vascular and systemic redox changes. Treatment withapocynin, a NADPH oxidase inhibitor, or with PEG-catalase, a H2O2

detoxifying enzyme, prevented the DPSPX-induced effects on bloodpressure, vascular structure and H2O2 levels. Tempol, a SOD mimetic,

failed to inhibit these changes, unless PEG-catalase was co-adminis-tered. It is concluded that NADPH oxidase mediated generation of O2

•−

with subsequent formation of H2O2 contributes to the pathophysiol-ogy of DPSPX-induced hypertension.

Acknowledgements

This work was supported by the Fundação Calouste Gulbenkianand Fundação para a Ciência e a Tecnologia (SFRH/BD/7055/2001,SFRH/BPD/21863/2005 and POCTI/NSE/45409/2002). The authors aregrateful to the Institute of Histology and Embriology, Faculty ofMedicine of Porto, for the preparation of slides for the morphologicalstudy, and to the Biochemistry Department, Faculty of Pharmacy ofPorto, for the use of equipment (Leica Image Analysis System QWinQ550 software). The excellent technical assistance of Mrs. Elisa Nova,Dr. Luís Belo, Eng. Paula Serrão and Mrs Mabilde Gomes is greatlyappreciated.

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