VPH-06175; No of Pages 11 Vascular Pharmacology xxx (2015) xxx–xxx

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Vascular Pharmacology journal homepage: www.elsevier.com/locate/vph

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Patrícia Passaglia a,b, Carla S. Ceron b, André S. Mecawi c, José Antunes-Rodrigues c, Eduardo B. Coelho c, Carlos R. Tirapelli b,⁎

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Article history: Received 28 November 2014 Received in revised form 9 March 2015 Accepted 4 April 2015 Available online xxxx

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Keywords: Ethanol Oxidative stress Superoxide anion Hypertension Angiotensin II

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Objectives: We hypothesized that chronic ethanol intake enhances vascular oxidative stress and induces hypertension through renin–angiotensin system (RAS) activation. Methods and results: Male Wistar rats were treated with ethanol (20% v/v). The increase in blood pressure induced by ethanol was prevented by losartan (10 mg/kg/day; p.o. gavage), a selective AT1 receptor antagonist. Chronic ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels and serum aldosterone levels. No differences on plasma osmolality and sodium or potassium levels were detected after treatment with ethanol. Ethanol consumption did not alter ACE activity, as well as the levels of ANG I and ANG II in the rat aorta or mesenteric arterial bed (MAB). Ethanol induced systemic and vascular oxidative stress (aorta and MAB) and these effects were prevented by losartan. The decrease on plasma and vascular nitrate/nitrite (NOx) levels induced by ethanol was prevented by losartan. Ethanol intake did not alter protein expression of ACE, AT1 or AT2 receptors in both aorta and MAB. Aortas from ethanol-treated rats displayed decreased ERK1/2 phosphorylation and increased protein expression of SAPK/JNK. These responses were prevented by losartan. MAB from ethanol-treated rats displayed reduced phosphorylation of p38MAPK and ERK1/2 and losartan did not prevent these responses. Conclusions: Our study provides novel evidence that chronic ethanol intake increases blood pressure, induces vascular oxidative stress and decreases nitric oxide (NO) bioavailability through AT1-dependent mechanisms. © 2015 Published by Elsevier Inc.

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Programa de pós-graduação em Toxicologia, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo (USP), Ribeirão Preto, São Paulo, Brazil Escola de Enfermagem de Ribeirão Preto, Laboratório de Farmacologia, USP, Ribeirão Preto, São Paulo, Brazil Faculdade de Medicina de Ribeirão Preto, USP, Ribeirão Preto, São Paulo, Brazil

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Angiotensin type 1 receptor mediates chronic ethanol consumption-induced hypertension and vascular oxidative stress

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1. Introduction

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Chronic ethanol consumption is a comorbid variable that increases the incidence of cardiovascular diseases [1,2]. In this sense, epidemiologic studies have found a positive association between high ethanol consumption and arterial hypertension [3,4]. However, although the link between ethanol consumption and arterial hypertension is well established, the mechanism through which ethanol increases blood pressure remains elusive. Chronic ethanol consumption induces dose-dependent blood pressure increase, which is associated with increased vascular reactive oxygen species (ROS) generation [5,6]. Ethanol intake reduces nitric oxide (NO) bioavailability in the vasculature and this response is associated with increased arterial blood pressure [5,7,8]. Moreover, chronic

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⁎ Corresponding author at: Escola de Enfermagem de Ribeirão Preto, Laboratório de Farmacologia, Universidade de São Paulo, Avenida Bandeirantes 3900, CEP 14040-902, Ribeirão Preto, SP, Brazil. Tel.: +55 16 33150532; fax: +55 16 33150518. E-mail address: [email protected] (C.R. Tirapelli).

ethanol consumption is associated with membrane lipid peroxidation and increased vascular NAD(P)H oxidase activity [7,9]. Chronic ethanol consumption increases plasma renin activity (PRA) and angiotensin II (ANG II) levels in humans [10–12]. Similarly, increased vascular and plasma ANG II levels were described in rats chronically treated with ethanol [6,13]. The renin–angiotensin system (RAS) is critically involved in the control of blood pressure. ANG II, the major bioactive peptide of the RAS, is produced systemically and locally within the vascular wall [14]. The peptide exerts its biological actions via two G-protein-coupled receptors, named AT1 and AT2 [15]. Most of the actions of ANG II including sodium retention, aldosterone secretion and vasoconstriction are mediated by AT1 receptors [14]. The vascular system may act independently from the systemic RAS to generate ANG II [16]. The vascular ANG II generating system may be activated even when the systemic RAS is suppressed or normal. The exact function of the vascular RAS is not fully understood, but it may amplify the effects of the systemic RAS, particularly in pathological conditions, such as in hypertension [17].

http://dx.doi.org/10.1016/j.vph.2015.04.002 1537-1891/© 2015 Published by Elsevier Inc.

Please cite this article as: Passaglia P, et al, Angiotensin type 1 receptor mediates chronic ethanol consumption-induced hypertension and vascular oxidative stress, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.04.002

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2.1. Ethanol administration

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Experiments were performed in accordance with the principles and guidelines of the animal ethics committee of the University of São Paulo — Campus Ribeirão Preto (#11.1.1103.53.1). Male Wistar rats, initially weighing 250–280 g (60–70 days old), were randomly divided into four groups: control, ethanol, control + losartan (10 mg/kg/day, p.o. gavage) [25,26], and ethanol + losartan. Control rats received water ad libitum, whereas rats from the ethanol group received 20% (v/v) ethanol in their drinking water [8,24,27,28]. All animals had free access to Purina Lab ChowR. To avoid a considerable loss of animals, the ethanoltreated group was submitted to a brief and gradual adaptation period. The animals received 5% ethanol in their drinking water during the first week, 10% in the second and 20% in the third week. At the end of the third week, the experimental stage was initiated and lasted for 2 weeks.

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2.2. Systolic blood pressure measurements

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Plasma ACE activity was determined as previously described [26]. Results are expressed as relative fluorescence unities (RFU). Tissue ACE activity was determined in the aorta and MAB. The tissues were homogenized in buffer containing zinc (10 ml of Tris buffer [150 mM– pH 8.3] and 10 μl of zinc 1 mM diluted in water). ACE enzymatic activity in aorta and MAB was determined following incubation with intramolecularly quenched synthetic specific substrate [29]. A working solution (0.45 mM) of the ACE substrate, Abz-Gly-p-nitro-Phe-Pro-OH (BACHEM, Bubendorf, Switzerland), was prepared in 150 mM Tris buffer (pH 8.3) containing 1.125 M NaCl. The assay was performed in a black flatbottom 96-well plate with 10 μl of sample homogenate, 90 μl of Tris buffer and 200 μl of Abz-Gly-p-nitro-Phe-Pro-OH, with a final volume of 300 μl. The assay was also performed in the presence of 10 μM captopril, an ACE inhibitor. After incubation for 24 h at room temperature, the fluorescence was measured using a fluorometer (excitation wavelength: 365 nm; emission wavelength: 415 nm). Blank values were subtracted from all fluorescence values. Fluorescence resulting from ACE-specific activity was determined by subtracting values obtained in the presence of captopril from those in its absence. Results are expressed as relative fluorescence unities (RFU)/mg protein.

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2.5. Plasma and tissue ANG I and ANG II determination

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Blood, aorta and MAB were collected as previously described [26]. Aorta and MAB were homogenized and peptides were extracted onto a bond Elut SPE-Column (Peninsula Laboratories INC., Belmont, CA, USA) as described previously [26]. The specific anti-bodies for ANG I (#T4166) and ANG II (#T4007) were purchased from Peninsula Laboratories (San Carlos, CA, USA). The sensitivity of the RIA and coefficient of variation intra- and inter-assays were 1.2 pg/ml, 12.2 and 15.2% for ANG I and 0.39 pg/ml, 10.9 and 17.1% for ANG II. Results are expressed as pg/ml or pg/mg protein.

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20 min, 4 °C) and aldosterone was determined using a commercial 131 RIA kit (Immunotech SA, Marseille, France). Results are expressed as 132 ng/dl. 133

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In addition to its biological effects, ANG II has been implicated in the progression and/or onset of cardiovascular diseases, including hypertension [14,15]. ANG II has been demonstrated to cause vasoconstriction partially by increasing the production of superoxide anions (O− 2 ) via the activation of the enzyme NAD(P)H oxidase in the vascular wall [14]. Activation of AT1 receptors leads to O− 2 generation, which will in turn influence downstream signaling targets of ANG II, including mitogenactivated protein kinases (MAPK) [18,19]. MAPK are a family of serine/threonine kinases, which are involved in the pathogenesis of cardiovascular dysfunctions such as vascular fibrosis and hypertension [20,21]. While there are reports describing that ethanol increases plasma ANG II levels, the role of RAS activation on ethanol-induced vascular oxidative stress and hypertension remains elusive. We hypothesized that ethanol consumption induces hypertension, ROS generation and activation of redox-sensitive signaling pathways in the vasculature through RAS-mediated mechanisms. Here, we attempted to investigate the role of RAS in chronic ethanol consumption-induced hypertension and vascular oxidative stress. A second purpose of this study was to assess the effects of chronic ethanol consumption on vascular RAS, based upon experimental evidence on the contribution of this system to vascular oxidative stress and hypertension [22,23]. Since the effects of ethanol are vessel-specific [24], in the present investigation we evaluated the effects of chronic ethanol consumption in conduit (aorta) and resistance arteries (mesenteric arteries).

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2.6. Plasma atrial natriuretic peptide (ANP) and vasopressin (AVP) 165 determination 166 Plasma levels of ANP and AVP were measured by RIA. Details are 167 available in the Supplementary material online. 168 169

The blood was collected in tubes containing heparin (10 μl/ml) and centrifuged (1000 ×g, 10 min, 4 °C). Plasma osmolality was measured in an osmometer (Digimatic model 3D2, Advanced Instruments, Norwood, MA, USA) by freezing point depression. Results are expressed as mOsm/kg H2O.

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Systolic blood pressure was measured weekly by tail-cuff plethysmography (Plethysmograph EFF306, Insight, Ribeirão Preto, Brazil). Before starting the blood pressure measurements, the rats were trained in the apparatus for three continuous days. Systolic blood pressure measurements were recorded weekly. The rats were maintained for 5– 10 min in a warm chamber and three consecutive recordings (∼2 min apart) were performed. Systolic blood pressure is expressed in mm Hg.

2.8. Determination of plasma levels of sodium and potassium

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Animals were anesthetized intraperitoneally with urethane (1.25 g/kg, Sigma-Aldrich, St. Louis, MO, USA) and decapitated. Blood samples were collected in tubes containing ethylenediamine tetraacetic acid (EDTA). The samples were centrifuged (1000 ×g, 3 min, 4 °C) and PRA was determined with standard radioimmunoassay (RIA) techniques using a commercial RIA kit (Immunotech, Swanton, Vermont, USA). Results are expressed as ng/ml/h. For aldosterone determination, blood samples were collected in tubes without anticoagulant. The samples were centrifuged (1000 ×g,

Blood samples were collected in tubes without heparin to avoid contamination of samples with sodium present in anticoagulant. The tubes were kept at room temperature for 2 h and then centrifuged (1000 ×g, 20 min, 4 °C). Plasma sodium concentrations were determined using a flame photometer (model b262, Micronal, São Paulo, Brazil). The instrument was calibrated with a standard solution containing 140 mEq/l dilute sodium MilliQ water (1:100). Results are expressed as mEq/l. For potassium determination, blood was collected in tubes containing coagulum activator and centrifuged (1000 ×g, 10 min, 4 °C). Plasma potassium was measured in a flame photometer (model b262, Micronal, São Paulo, SP, Brazil). Results are expressed as mEq/l.

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Please cite this article as: Passaglia P, et al, Angiotensin type 1 receptor mediates chronic ethanol consumption-induced hypertension and vascular oxidative stress, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.04.002

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The lucigenin-derived chemiluminescence assay was used to determine O2− levels in aortic and MAB homogenates as previously described [26,30]. Luminescence was measured in a luminometer (Orion II luminometer, Berthold detection systems, Pforzheim, Germany). Results are expressed as relative light unit (RLU)/mg protein.

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2.11. Measurement of plasma and vascular nitrate/nitrite (NOx)

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Blood was collected in tubes containing EDTA and centrifuged at 10,000 ×g for 20 min at 4 °C. Tissues (aorta and MAB) were homogenized in 200 μl PBS buffer (pH 7.4) and centrifuged at 10,000 × g (10 min, 4 °C). Then, the supernatant or plasma were ultrafiltered (#UFC5010BK Amicon Ultra-0.5 ml 10 kDa, Millipore, Billerica, MA, USA) at 14,000 × g for 15 min at room temperature. Nitrate/nitrite (NOx) was measured colorimetrically following instructions of a commercially available kit (#780001, Cayman Chemical, Ann Arbor, MI, USA). The results are expressed as NOx concentration (μM) or nmol/mg protein.

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2.12. Western immunoblotting

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Frozen aorta or MAB were homogenized in lysis buffer [50 mM Tris/ HCl (pH 7.4), NP-40 (1%), sodium deoxycholate (0.5%), SDS (0.1%)]. Homogenates were centrifuged (5000 ×g, 10 min, 4 °C) and the supernatant stored at −80 °C. Forty micrograms of protein was separated by electrophoresis on a 10% polyacrylamide gel, and transferred onto a nitrocellulose membrane. Nonspecific binding sites were blocked with 7% skim milk in Tris-buffered saline solution with Tween 20 for 1 h at room temperature. Membranes were then incubated with specific antibodies overnight at 4 °C as follows: p38MAPK (Thr180/Tyr182) (diluted 1:1000, 9211S, Cell Signaling, Danvers, Massachusetts, USA), total p38MAPK (diluted 1:1000, 9212S, Cell Signaling), ERK 1/2 (Thr202/Tyr204) (diluted 1:1000, 9101S, Cell Signaling), total ERK1/2 (diluted 1:1000, 9102, Cell Signaling), SAPK/JNK (Thr183/Tyr185) (diluted 1:1000, 4668S, Cell Signaling), total SAPK/JNK (diluted 1:1000, 9252S, Cell Signaling), anti-AT1 (diluted 1:1000 [aorta]/1:500 [MAB], sc-579, Santa Cruz Biotechnology, Dallas, Texas, USA), anti-AT2 (diluted 1:1000 [aorta]/1:500 [MAB], sc-9040, Santa Cruz Biotechnology), and anti-ACE (diluted 1:500 [aorta]/1:250 [MAB], sc-16496, Santa Cruz Biotechnology). After incubation with secondary antibodies for 90 min at room temperature, signals were revealed with chemiluminescence, visualized using a ChemiDoc™ XRS + (Bio-Rad, Hercules, CA, USA), and quantified by densitometry. Beta-actin (diluted 1:5000, sc-47778, Santa Cruz Biotechnology) was used as an internal control.

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2.13. Statistical analysis

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Data are presented as means ± standard error of the mean (SEM). Groups were compared using two-way or one-way analysis of variance (ANOVA). Tukey correction was used to compensate for multiple testing procedures. Results of statistical tests with p b 0.05 were considered as significant.

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3.2. Effect of chronic ethanol consumption on systolic blood pressure

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Baseline values of systolic blood pressure were similar in rats from control (123 ± 2 mm Hg, n = 6), ethanol (126 ± 2 mm Hg, n = 6), control plus losartan (125 ± 2 mm Hg, n = 6) and ethanol plus losartan (125 ± 2 mm Hg, n = 6) groups. Ethanol treatment for 2 weeks produced a significant increase in systolic blood pressure. Losartan prevented the increase in blood pressure induced by chronic ethanol consumption (Fig. 1).

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3.3. Effect of chronic ethanol administration on the systemic and vascular 259 components of the RAS 260 Ethanol consumption increased PRA (Fig. 2A) and plasma ACE activity (Fig. 2B). As expected, losartan treatment increased PRA and ACE activity because of its stimulatory effect on renal renin release. Plasma ANG I (Fig. 2C) and ANG II (Fig. 2D) levels were significantly increased in ethanol-treated rats compared with control rats. Losartan treatment did not alter plasma ANG I and ANG II levels when compared to control rats. Ethanol consumption increased aldosterone levels and treatment with losartan prevented this response (Fig. 2E). Treatment with ethanol did not change ANG I or ANG II levels as well as ACE activity in the aorta or MAB (Fig. 3).

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3.4. Effect of chronic ethanol administration on plasma ANP and AVP levels 271 Ethanol consumption increased ANP levels and losartan treatment did not prevent this response (Supplementary material online, Fig. 1A). On the other hand, treatment with ethanol decreased plasma AVP levels (Supplementary material online, Fig. 1B). Losartan did not prevent the decrease in plasma levels of AVP.

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Before treatment, body mass averaged 257 ± 5 g in rats from the control group, 255 ± 9 g in rats from the ethanol group, 256 ± 4 g in rats from the control plus losartan group and 261 ± 5 g in rats from ethanol plus losartan group. After treatment with ethanol, rats from the ethanol group (432 ± 23 g) as well as from the ethanol plus losartan group (432 ± 30 g) showed a significant reduction in body mass when compared to the control rats (554 ± 24 g) and the losartantreated animals (533 ± 34 g) (p b 0.05; ANOVA followed by Tukey).

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TBARS levels were determined in plasma, aorta and MAB following instructions of a commercially available kit (#10009055, Cayman Chemical, Ann Arbor, MI, USA). TBARS concentration was determined using a standard curve of malondialdehyde (MDA). Results are expressed as nmol/ml of plasma or nmol/mg protein.

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Fig. 1. Effect of chronic ethanol consumption on systolic blood pressure. Systolic blood pressure was evaluated weekly by plethysmography (n = 6 for each group). *Compared to control, control + losartan and ethanol + losartan (p b 0.05, two-way ANOVA followed by Tukey).

Please cite this article as: Passaglia P, et al, Angiotensin type 1 receptor mediates chronic ethanol consumption-induced hypertension and vascular oxidative stress, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.04.002

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Fig. 2. Effect of chronic ethanol consumption on the systemic components of the RAS. The effect of chronic ethanol consumption was evaluated on PRA (A), plasma ACE activity (B), plasma ANG I and ANG II levels (C and D) and serum aldosterone levels (E). Results are presented as means ± SEM of 6 to 10 animals. *Compared to control; **compared to control and control + losartan; #compared to control, control + losartan and ethanol + losartan (p b 0.05, ANOVA followed by Tukey).

3.5. Effect of chronic ethanol consumption on plasma sodium, potassium and osmolality

3.6. Effect of chronic ethanol consumption on oxidative stress and nitrate/ 291 nitrite (NOx) levels 292

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Ethanol consumption did not change sodium levels (146 ± 4 mEq/l, n = 9), as compared to control (150 ± 4 mEq/l, n = 9), control plus losartan (141 ± 4 mEq/l, n = 10) and ethanol plus losartan groups (140 ± 5 mEq/l, n = 10). Similarly, ethanol consumption did not change potassium levels (5.5 ± 0.3 mEq/l, n = 4), as compared to control (4.9 ± 0.2 mEq/l, n = 6), control plus losartan (4.9 ± 0.2 mEq/l, n = 5) and ethanol plus losartan groups (5.6 ± 0.4 mEq/l, n = 4). Finally, treatment with ethanol did not alter plasma osmolality (294 ± 1 mOsm/kg H2O, n = 9), as compared to control (287 ± 1 mOsm/kg H2 O, n = 9), control plus losartan (285 ± 5 mOsm/kg H2O, n = 10) and ethanol plus losartan groups (290 ± 3 mOsm/kg H2O, n = 9).

Systemic oxidative stress was evaluated by measuring plasma TBARS. Plasma (Fig. 4A) and vascular TBARS levels (Fig. 4E and F) were increased in ethanol-treated rats compared with control rats. Treatment with losartan prevented the increase in TBARS levels induced by ethanol. Ethanol significantly decreased plasma NOx levels and losartan prevented this response (Fig. 4B). Lucigenin-derived luminescence was higher in aortas and MAB from ethanol-treated rats. The increased generation of O− 2 in aortas and MAB from ethanol-treated rats was prevented by losartan (Fig. 4C and D). NOx content in aortas and MAB from ethanoltreated rats was lower as compared with control rats. Treatment with losartan prevented the decrease on vascular NOx levels induced by ethanol (Fig. 4G and H).

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Please cite this article as: Passaglia P, et al, Angiotensin type 1 receptor mediates chronic ethanol consumption-induced hypertension and vascular oxidative stress, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.04.002

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Fig. 3. Effect of chronic ethanol consumption on the vascular components of the RAS. The effect of chronic ethanol consumption was evaluated in the rat aorta and MAB. ACE activity (A and B) was determined fluorometrically. ANG I (C and D) and ANG II levels (E and F) were determined by RIA. Results are presented as means ± SEM of 6 to 9 animals.

3.7. Effect of chronic ethanol consumption on protein expression of AT1 and AT2 receptors and ACE

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Ethanol did not affect the protein expression of AT1 and AT2 receptors or ACE in the rat aorta (Fig. 5A, B and C). Similarly, no differences were observed on the expression of ACE, AT1 and AT2 receptors in the rat MAB among the four experimental groups (Fig. 5D, E and F).

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3.8. Effect of chronic ethanol consumption on MAPK phosphorylation and expression

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Aortas from ethanol-treated rats displayed decreased ERK1/2 phosphorylation as compared with control ones. Treatment with losartan prevented this response (Fig. 6A). Ethanol treatment did not alter p38MAPK or SAPK/JNK phosphorylation in the rat aorta (Fig. 6B and C).

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In the rat MAB, ethanol decreased ERK1/2 and p38MAK phosphorylation. These responses were not prevented by losartan (Fig. 6D and F). Treatment with ethanol did not affect SAPK/JNK phosphorylation in the rat MAB (Fig. 6E). Ethanol consumption did not alter the expression of p38MAPK or ERK1/2 in the rat aorta (Fig. 7A and C). On the other hand, ethanol consumption increased protein expression of SAPK/JNK in the rat aorta. Treatment with losartan prevented this response (Fig. 7B). In the rat MAB, no differences on protein expression of p38MAPK, SAPK/JNK or ERK1/2 were detected among the experimental groups (Fig. 7D, E and F).

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4. Discussion

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The present findings show that chronic ethanol consumption 328 activated the systemic RAS, increased the vascular generation of O− 2 329 and decreased NO bioavailability in the vasculature. Ethanol-induced 330

Please cite this article as: Passaglia P, et al, Angiotensin type 1 receptor mediates chronic ethanol consumption-induced hypertension and vascular oxidative stress, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.04.002

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the range of 35–40 mM [8,24,27,28,31], which are within those found in the bloodstream of humans after ethanol consumption [32]. Our results are in accordance with previous observations describing that chronic ethanol consumption increases blood pressure [5,6,8,31, 33]. The new finding of our study is that losartan prevented ethanolinduced hypertension, further implicating AT1 receptors in such response. An important finding of our investigation is that chronic ethanol consumption increased plasma ANG II. In addition, our study provides novel evidence that ethanol consumption also affects other components of the systemic RAS since increased PRA, plasma ACE activity and plasma ANG I levels as well as serum aldosterone levels were detected after treatment with ethanol. Treatment with losartan did not prevent ethanol-induced increase in PRA, plasma ACE activity and plasma ANG I and ANG II levels. The lack of effect of losartan is probably related to its expected stimulatory effect on renal renin release [34,35]. The current study does not address the exact mechanism whereby ethanol consumption modulates RAS activity. Increased activity of the sympathetic nervous system could play a role in RAS activation here described since it has been previously reported that ethanol consumption increases plasma catecholamine levels [33]. Taken together, our findings show that ANG II/AT1 receptors play a role in ethanol-induced hypertension. The anti-diuretic action of AVP is the main physiological effect of this hormone but AVP may also participate in the regulation of blood pressure through its vasoconstrictor effect, which subsequently increases peripheral vascular resistance and blood pressure [36]. AVP seems not to contribute to the hypertensive state induced by ethanol since reduced levels of this hormone were observed in our study. This finding is in accordance with previous studies showing that ethanol reduces plasma AVP levels [37,38]. In addition to its potent vasoconstrictive effects, ANG II also induces aldosterone release. In our study, serum aldosterone levels increased after ethanol treatment and losartan prevented this response as described previously [35]. Aldosterone contributes to blood pressure levels by stimulating renal re-absorption of sodium ions through a Na+/K+-ATPase [36]. Despite the increase in serum aldosterone levels, no alteration on the levels of plasma sodium and potassium was detected. Increased serum aldosterone with no change on sodium and potassium levels was previously described [39,40]. Moreover, the effect of aldosterone on blood pressure and Na+/K+-ATPase activity in the cortical-collecting tubule is time and dose-dependent [41,42]. Finally, it is important to consider that the regulation of electrolyte balance is complex and involves several interacting systems. For example, ANP, which is increased in our model, has the opposite function of aldosterone being able to abolish much of adosterone's anti-natriuretic effect [43]. Thus, although increased serum aldosterone concentrations during ethanol consumption is a response mediated by ANG II, this mechanism does not seem to contribute to the increase in blood pressure since no alterations in electrolyte balance were evidenced. Blood vessels are an important site of ANG II production and the vascular ANG II generating system may assume a functional significance without a parallel change in the systemic RAS [16]. Importantly, it has been described that the vascular RAS contributes to vascular oxidative stress and hypertension [22,23]. Our findings show that ethanol consumption did not change ANG I and ANG II levels as well as ACE activity and expression in the vasculature. Moreover, since no differences on the vascular expression of both AT1 and AT2 receptors were detected in the present study, we conclude that ethanol intake activates the systemic, but not the vascular ANG II generating system. ANG II, the principal effector peptide of the RAS, plays a major role in the initiation and progression of vascular diseases such as hypertension. The pathophysiological effects of ANG II are partially mediated by ROS, which are generated by membrane-bound NAD(P)H oxidases localized in the vascular wall [14,19]. The lucigenin-derived chemiluminescence assay is based on the enzymatic action of the enzyme NAD(P)H oxidase [44]. In this sense, the increased lucigenin-derived chemiluminescence here described suggests that ethanol consumption induces O− 2 generation

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Fig. 4. Effect of chronic ethanol consumption on systemic and vascular oxidative stress and on plasma and vascular nitrate/nitrite (NOx) levels. Bar graphs represent plasma concentration of TBARS, a marker of oxidative stress (A), superoxide anion levels in aortic tissue (C) and MAB (D) evaluated by lucigenin-derived chemiluminescence assay. TBARS concentration in the aorta (E) and MAB (F) was determined colorimetrically. NOx levels in the plasma (B), aorta (G) and MAB (H) were determined colorimetrically. Results are presented as means ± SEM of 5 to 9 experiments. *Compared to control, control + losartan and ethanol + losartan (p b 0.05, ANOVA followed by Tukey).

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hypertension and increased systemic and vascular oxidative stress were prevented by losartan, further suggesting that AT1 activation plays a key role in these responses. Of note, our data is relevant since using this same model of ethanol intake, we have show blood ethanol levels in

Please cite this article as: Passaglia P, et al, Angiotensin type 1 receptor mediates chronic ethanol consumption-induced hypertension and vascular oxidative stress, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.04.002

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Fig. 5. Effect of chronic ethanol consumption on AT1, AT2 and ACE expression in the rat aorta (left) and MAB (right). Top panels: representative immunoblots for AT1, AT2 and ACE protein expression. Bottom panels: corresponding bar graphs show densitometric data for AT1 (A and B), AT2 (C and D) and ACE (E and F) protein expression. Results are presented as means ± SEM of n = 4 for each group.

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in the vasculature through activation of NAD(P)H oxidase. Moreover, evaluation of TBARS levels revealed increased lipid peroxidation in aortas and MAB from ethanol-treated rats. Thus, our results provide evidence that ethanol-induced NAD(P)H oxidase activation, O− 2 generation and vascular lipid peroxidation are processes mediated by AT1 receptors since losartan prevented these responses. The fact that levels of plasma TBARS were increased in ethanol-treated rats suggests that increased

ROS generation is probably a global phenomenon. Moreover, our findings also implicate AT1 receptors in the increased plasma lipoperoxidation induced by ethanol consumption. Chronic ethanol intake was described to reduce plasma and vascular NO levels [5,7]. The present findings corroborate these previous results and additionally show that AT1 receptors play a role in such response. In general, reduced NO bioavailability may result from an increase in NO

Please cite this article as: Passaglia P, et al, Angiotensin type 1 receptor mediates chronic ethanol consumption-induced hypertension and vascular oxidative stress, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.04.002

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Fig. 6. Effect of chronic ethanol consumption on MAPK phosphorylation in the rat aorta (left) and MAB (right). Top panels: representative immunoblots for MAPK protein phosphorylation and expression. Bottom panels: corresponding bar graphs show densitometric data for phosphorylation of ERK1/2 (A and D), SAPK/JNK (B and E) and p38MAPK (C and F). Results are presented as means ± SEM of 6 experiments. *Compared to control, control + losartan and ethanol + losartan; **compared to control (p b 0.05, ANOVA followed by Tukey).

inactivation due to enhanced O–2 production. Thus, the reduced levels of NO detected after treatment with ethanol may be related, at least in part, to an increased generation of O–2, which may serve to inactivate NO. In this sense, the protective effect of losartan on ethanol-induced reduction of NO levels may be related to the ability of this drug to prevent − O− 2 generation. The reaction of NO with O2 leads to an impaired vascular vasodilatation and to the generation of peroxynitrite (ONOO−). The latter is a powerful oxidant molecule that causes a number of potentially deleterious actions in the vasculature, including lipid peroxidation [45]. The vascular endothelium plays a pivotal role in the regulation of vascular function and structure by releasing various biochemical mediators, such as NO, prostanoids and endothelin-1. Endothelial dysfunction is caused by an increase in ROS generation and a reduction of endothelial

NO bioavailability, either by increasing its oxidative inactivation and/or by decreasing its synthesis [46]. Endothelial dysfunction, observed in hypertension and diabetes mellitus, is characterized by decreased vascular NO bioavailability and increased oxidative stress, which is similar to the findings here described. As a result, we conclude that chronic ethanol consumption is an important risk factor for endothelial dysfunction and vascular damage. The signaling events in response to ANG II involve activation of redox-sensitive signaling molecules such as MAPK [18,19]. MAPK comprise a family of serine/threonine kinases which are associated with vascular smooth muscle cell contraction, migration, collagen deposition, cell growth, and differentiation [47]. Extracellular signal-regulated kinases (ERK1/2), p38MAPK, and stress-activated protein kinase/c-Jun

Please cite this article as: Passaglia P, et al, Angiotensin type 1 receptor mediates chronic ethanol consumption-induced hypertension and vascular oxidative stress, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.04.002

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Fig. 7. Effect of chronic ethanol consumption on MAPK expression in the rat aorta (left) and MAB (right). Top panels: representative immunoblots for MAPK protein phosphorylation and expression. Bottom panels: corresponding bar graphs show densitometric data for protein expression of ERK1/2 (A and D), SAPK/JNK (B and E) and p38MAPK (C and F). Results are presented as means ± SEM of 6 experiments. *Compared to control, control + losartan and ethanol + losartan (p b 0.05, ANOVA followed by Tukey).

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N-terminal kinases (SAPK/JNK) are the best characterized MAPK in the vasculature [47]. In our study, the increased expression of SAPK/JNK in the aorta of ethanol-treated rats was prevented by losartan, suggesting that this effect is mediated by AT1-dependent pathways. This result is in line with a recent finding showing that ANG II increases the expression

of SAPK/JNK in endothelial cells [48]. The effects of ANG II on SAPK/JNK phosphorylation in the vasculature are time and concentrationdependent [49]. This could be the possible explanation for the lack of effect of ethanol on SAPK/JNK phosphorylation in spite of the increased expression of this kinase after treatment with ethanol. SAPK/JNK

Please cite this article as: Passaglia P, et al, Angiotensin type 1 receptor mediates chronic ethanol consumption-induced hypertension and vascular oxidative stress, Vascul. Pharmacol. (2015), http://dx.doi.org/10.1016/j.vph.2015.04.002

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This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico [grant number 470556/ 2010-2] and Fundação de Amparo à Pesquisa do Estado de São Paulo [grant numbers 2010/05815-4 and 2012/10096-2].

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ethanol intake. The major new finding of our study is that ethanolinduced hypertension and vascular oxidative stress are mediated by AT1 receptors. Moreover, results presented here evidenced that ethanol consumption activates the systemic, but not the vascular RAS. ANG II is a multifunctional peptide that modulates vasomotor tone, cell growth, apoptosis, cell migration and extracellular matrix deposition. For those reasons, ANG II plays an important role in hypertension and cardiovascular diseases. Furthermore, ANG II elicits many of its pathophysiological actions by stimulating ROS generation through activation of vascular NAD(P)H oxidase. Thus, RAS activation with consequent oxidative stress here described could be one mechanism by which ethanol predisposes to vascular dysfunction and cardiovascular damage. Targeting such pathways/molecules could prevent or induce regression of hypertensive vascular damage induced by ethanol.

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regulates vascular smooth muscle cell growth and promotes apoptosis [50]. The cellular functions of this kinase are potentially important in vascular hyperplasia and/or hypertrophy in hypertension [18,21]. Thus, RAS activation with consequent oxidative stress and up-regulation of SAPK/ JNK here described could be one mechanism by which ethanol predisposes to vascular dysfunction and hypertension. In our study, phosphorylation of ERK1/2 was decreased in the aorta of ethanol-treated rats. Losartan prevented the decrease in ERK1/2 phosphorylation, suggesting that this response is modulated by AT1dependent pathways. This result is in agreement with a previous finding showing that ANG II, through AT1 activation, inactivates ERK1/2 by increasing mitogen-activated protein kinase phosphatase-1 (MKP-1) expression [51]. The latter is a protein phosphatase that negatively modulates MAPK activity through dephosphorylation of both tyrosine and threonine residues of MAPK [52]. Importantly, MKP-1 is the primary enzyme in the vasculature that dephosphorylates MAPK in response to ANG II [53]. ANG II stimulates ROS generation in the vasculature and oxidative stress was described to increase the expression of MKP-1 [54]. Thus, the reduction in MAPK phosphorylation induced by ethanol could be related to the increase in oxidative stress induced by ANG II/AT1 receptors. However, further investigations are necessary to elucidate the role of the AT1/ROS/MKP-1 pathway in our model. Our results also show that p38MAPK and SAPK/JNK phosphorylation were unaffected in aortas from ethanol-treated rats. The signaling networks that underlie MAPK activation require phosphorylation by a MAPK known as MEK. While MEK1/2 (MAP/ERK kinase) is responsible for ERK1/2 phosphorylation, the signaling processes leading to SAPK/JNK and p38MAPK activation involve MEK4/7 and MEK3/6, respectively [47]. Such differences may explain the distinctive effects of chronic ethanol consumption on MAPK in our model. In the rat MAB, ethanol intake did not affect the protein expression of ERK1/2, SAPK/JNK or p38MAPK, but reduced phosphorylation of ERK1/2 and p38MAPK was detected. The lack of effect of losartan on ethanol-induced decrease in MAPK phosphorylation suggests that this response is not mediated by AT1 receptors. However, this result does not rule out the participation of ANG II in such response since it was previously demonstrated that ANG II induces MAPK dephosphorylation via AT2 receptors [51]. ERK1/2 and p38MAPK-dependent signaling pathways have been associated with cellular differentiation, cellular growth and apoptosis, and with vascular contraction. These signaling processes are important in enhanced hypertrophy, hyperplasia, and/or vascular contractility in hypertension. The exact functional meaning of ethanolinduced reduction in the signaling of ERK1/2 and p38MAPK in vascular smooth muscle cells is ill-defined, but reduction of cell growth, apoptosis and vascular contraction could be possible consequences of this response. Decreased MAPK activation was described as a compensatory mechanism to reduce blood pressure in hypertensive rats [55]. Thus, reduced ERK1/2 and p38MAPK phosphorylation here described may be a compensatory mechanism to the increased vascular resistance and blood pressure in ethanol-treated rats. ANP is a cardiovascular hormone that mediates inhibition of p38MAPK and ERK1/2 phosphorylation via MKP-1 [56,57]. In this sense, the increased plasma levels of ANP here described could explain the inhibitory effects of ethanol on MAPK phosphorylation in the rat MAB. Additionally, it is well established that AVP induces MAPK activation in the vasculature [58]. In this line, reduced plasma AVP levels here described could account for the reduction in MAPK phosphorylation in the rat MAB induced by ethanol. However, the role of ANP and AVP on MAPK inactivation needs further investigation. Based on our findings, we can conclude that the effects of ethanol consumption on vascular MAPK are different in conduit and resistance arteries. This result corroborates previous findings showing that a different contribution of signaling cascades in the pathobiology of ethanol in arteries with different physiological function exists [26,30]. The present study offers new insights into the cardiovascular effects of chronic ethanol consumption. Our findings highlight the importance of the systemic RAS in the cardiovascular effects displayed by chronic

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