J Physiol Biochem (2014) 70:509–523 DOI 10.1007/s13105-014-0331-6

ORIGINAL PAPER

Acute ethanol intake induces mitogen-activated protein kinase activation, platelet-derived growth factor receptor phosphorylation, and oxidative stress in resistance arteries Natália A. Gonzaga & Glaucia E. Callera & Alvaro Yogi & André S. Mecawi & José Antunes-Rodrigues & Regina H. Queiroz & Rhian M. Touyz & Carlos R. Tirapelli Received: 2 October 2013 / Accepted: 10 March 2014 / Published online: 15 April 2014 # University of Navarra 2014

Abstract In the present study, we investigated the role of angiotensin type I (AT1) receptor in reactive oxygen species (ROS) generation and mitogen-activated protein kinases (MAPK) activation induced by acute ethanol intake in resistance arteries. We also evaluated the effect of ethanol on platelet-derived growth factor receptors (PDGF-R) phosphorylation and the role of this receptor on ROS generation by ethanol. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. Acute ethanol intake did not alter angiotensin I or angiotensin II levels in the rat mesenteric arterial bed (MAB). Ethanol induced vascular oxidative stress, and this response was not prevented by losartan (10 mg/kg; p.o. gavage), a selective AT1 receptor antagonist. MAB from ethanol-treated rats displayed increased SAPK/JNK and PDGF-R phosphorylation, responses that were not prevented by losartan. The phosphorylation levels of protein kinase B (Akt) and eNOS were not affected by acute ethanol intake. MAB nitrate

levels and the reactivity of this tissue to acetylcholine, phenylephrine, and sodium nitroprusside were not affected by ethanol intake. Ethanol did not alter plasma antioxidant capacity, the levels of reduced glutathione, or the activities of superoxide dismutase and catalase in the rat MAB. Short-term effects of ethanol (50 mmol/l) were evaluated in vascular smooth muscle cells (VSMC) isolated from rat MAB. Ethanol increased ROS generation, and this response was not affected by AG1296, a PDGF-R inhibitor, or losartan. Finally, ethanol did not alter MAPK or PDGF-R phosphorylation in cultured VSMC. Our study provides novel evidence that acute ethanol intake induces ROS generation, PDGF-R phosphorylation, and MAPK activation through AT(1)-independent mechanisms in resistance arteries in vivo. MAPK and PDGF-R play a role in vascular signaling and cardiovascular diseases and may contribute to the vascular pathobiology of ethanol.

N. A. Gonzaga : C. R. Tirapelli Departamento de Enfermagem Psiquiátrica e Ciências Humanas, Laboratório de Farmacologia, Escola de Enfermagem de Ribeirão Preto, USP, Ribeirão Preto, SP, Brazil

A. S. Mecawi : J. Antunes-Rodrigues Departamento de Fisiologia, Faculdade de Medicina de Ribeirão Preto, USP, Ribeirão Preto, SP, Brazil

N. A. Gonzaga Programa de Pós-Graduação em Farmacologia, Faculdade de Medicina de Ribeirão Preto, USP, Ribeirão Preto, SP, Brazil G. E. Callera : A. Yogi : R. M. Touyz Kidney Research Centre, Ottawa Health Research Institute, University of Ottawa, Ottawa, ON, Canada

R. H. Queiroz Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP, São Paulo, Brazil C. R. Tirapelli (*) Laboratório de Farmacologia, Escola de Enfermagem de Ribeirão Preto, Universidade de São Paulo, Avenida Bandeirantes 3900, 14040-902 Ribeirão Preto, SP, Brazil e-mail: [email protected]

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Keywords Ethanol . Resistance arteries . Superoxide anions . Platelet-derived growth factor . Mitogen-activated protein kinases

Introduction Epidemiological studies in humans [23, 42] and experimental studies in animals [19–21, 39] have shown the link between chronic ethanol consumption and the prevalence of cardiovascular diseases, such as hypertension. Vascular inflammation and the increase of vascular and plasma angiotensin II (ANG II) levels are important molecular factors implicated with chronic ethanolinduced hypertension [19, 20, 49]. ANG II, the active component of the reninangiotensin system (RAS), increases reactive oxygen species (ROS) generation in vascular smooth muscle and endothelial cells via angiotensin type I (AT1) receptor [50]. ROS generation mediated by AT1 receptors influence many downstream signaling targets of ANG II, including mitogen-activated protein kinases (MAPK) [50]. MAPK cascade is proven to be involved in the pathogenesis of cardiovascular dysfunctions such as diabetic cardiomyopathy [25], myocardial hypertrophy [52], vascular fibrosis [44], and hypertension [50]. Although the relation between chronic ethanol consumption and RAS activation is well established [19, 20, 45], there are few reports describing the effects of acute ethanol intake in this system. In this line, increased plasma renin activity was described in humans after acute ethanol intake [27, 37]. Recent findings from our laboratory showed that acute ethanol intake induces RAS activation, aortic oxidative stress, and MAPK phosphorylation through an AT1-dependent mechanism [48, 57]. Ethanol-induced MAPK stimulation in the vasculature is not solely mediated by ANG II [40]. Ethanol effects on MAPK signaling may occur through modulation of several upstream components including receptor tyrosine kinases. This family includes receptors for growth factors, such as platelet-derived growth factor (PDGF) [58]. Exposure of astrocytes to ethanol in vitro results in activation of MAPK by PDGF [28]. In the vasculature, ethanol was described to enhance tyrosine kinase activity in cerebral vessels with consequent MAPK activation [55]. Moreover, PDGF-mediated NAD(P)H oxidase-driven generation of ROS is known to stimulate MAPK in vascular smooth muscle cells

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(VSMC) [6]. However, the role of PDGF receptors (PDGF-R) on MAPK phosphorylation in the vasculature after acute ethanol intake remains unclear. The actions of ethanol in the vasculature are vesselspecific. For example, ethanol-induced vascular dysfunction in conduit (aorta) and resistance arteries (mesenteric arteries) is different, suggesting that a different contribution of signaling cascades in the pathobiology of ethanol in arteries with different physiological function exists [47]. While acute ethanol intake increased ROS generation and decreased NO levels in the rat aorta [48, 57], the oxidative effect of acute ethanol intake in resistance arteries and the participation of RAS in this response remain unclear. Thus, the effect of acute ethanol intake in arteries with different physiological function is a plausible hypothesis that deserves investigation. Here, we sought to investigate the effect of acute administration of ethanol on ROS levels and MAPK activation in the rat mesenteric arterial bed (MAB) and the involvement of AT1 receptors in these responses. We also evaluated the effect of ethanol on PDGF-R phosphorylation. Short-term effects of ethanol on ROS generation, MAPK activation, and PDGF-R phosphorylation were evaluated in VSMC isolated from rat MAB.

Materials and methods Acute ethanol administration Experiments were performed in accordance with the principles and guidelines of the animal ethics committee of the University of São Paulo and the University of Ottawa. Male Wistar rats, initially weighing 350–400 g, were randomly divided into two groups: control and ethanol. Ethanol (1 g/kg; 10 ml/kg of 13 % ethanol diluted in water) was administered by gavage in animals fasted for 12 h [12, 13, 30, 57]. The control group received vehicle (water, p.o. gavage). In another set of experiments, the rats were treated with losartan (10 mg/kg, p.o. gavage) 30 min before the administration of water or ethanol [13, 59]. Blood ethanol measurements Rats were anesthetized, and blood was collected from the aorta 30 min after ethanol administration using heparinized syringes. The samples were analyzed using a CG-17A gas chromatographer (Shimadzu, Kyoto,

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Japan) as previously described [57]. The results are expressed as grams of ethanol per liter of blood. The period of 30 min was chosen for the collection of blood and vascular tissue for biochemical assays since peak ethanol levels were achieved at this time as previously described [48, 57]. Tissue determination of ANG I and ANG II The MAB was homogenized, and tissue peptides were extracted onto a Bond-Elut SEP-COLUMNS (Peninsula Laboratories, San Carlos-CA, USA) as previously described [57]. Protein concentration was determined by the Bradford method. The specific anti-bodies for angiotensin I (ANG I) and ANG II were obtained from Peninsula Laboratories (San Carlos-CA, USA; ANG I: T4166 and ANG II: T4007). The sensitivity of the radioimmunoassay and coefficient of variation intraand inter-assay 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. Detection of superoxide anions in the rat MAB by lucigenin enhanced chemiluminescence Thirty minutes after ethanol administration, the rat MAB was isolated, cleaned, and frozen in liquid nitrogen. The lucigenin-derived chemiluminescence assay was used to determine superoxide anion levels in MAB homogenates 10 % (w/v) prepared in phosphate buffer (20 mmol/l of KH2PO4, 1 mmol/l of EGTA, and protease inhibitors [pH 7.4]) with a glass-to-glass homogenizer. The reaction was started by the addition of NADPH (0.1 mmol/l) to the suspension (250 μl of final volume) containing sample (50 μl), lucigenin (5 μmol/l), and assay buffer (50 mmol/l of KH2PO4, 1 mmol/l of EGTA, and 150 mmol/l of sucrose [pH 7.4]). Luminescence was measured in a luminometer (Orion II Luminometer, Berthold detection systems). Buffer blank was subtracted from each reading. Superoxide production was expressed as relative light unit per microgram protein. Protein concentrations were determined with protein assay reagent (Bio-Rad Laboratories) [56]. Western immunoblotting Frozen tissue was homogenized in lysis buffer [50 mmol/l Tris/HCl (pH 7.4), NP-40 (1 %), sodium deoxycholate (0.5 %), SDS (0.1 %)]. Homogenates were centrifuged at 5,000×g for 10 min, and the

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supernatant was stored at −80 °C. Thirty micrograms of protein was separated by electrophoresis on a 10 % polyacrylamide gel and transferred onto a nitrocellulose membrane. Nonspecific binding sites were blocked with 5 % skim milk in Tris-buffered saline solution with Tween for 1 h at 24 °C. Membranes were then incubated with specific antibodies overnight at 4 °C as follows: p38MAPK (Thr180/Tyr182), total p38MAPK, ERK 1/2 (Thr202/Tyr204), total ERK1/2, SAPK/JNK (Thr183/ Tyr185), total SAPK/JNK, total PDGR-R, PDGF-Rα/β (Tyr849/857), eNOS (Ser1177), total eNOS, Akt (Ser473), and total Akt (diluted 1:1,000, Cell Signaling). After incubation with secondary antibodies (diluted 1:2,000) for 90 min at room temperature, signals were revealed with chemiluminescence, visualized by autoradiography, and quantified densitometrically. Results were expressed as ratio of non-phospho proteins expression that was used as load control. Measurement of basal MAB nitrate Basal nitrate (NO3−, a metabolite of NO) levels were measured in supernatants from MAB homogenates. Aliquots of 5 μl were injected into a Sievers Chemiluminescence Analyzer (Nitric Oxide Analyser, NOA™ 280, Sievers Instruments, Colorado, USA) and pelleted by centrifugation with VCl3 and HCl (at 95 °C), which act as reductants for nitrate. Results were normalized for protein concentration assessed with the Bradford method and are expressed as micromoles per liter per milligram protein. Determination of superoxide dismutase (SOD) activity in the rat MAB Superoxide dismutase (SOD) activity was assayed by the inhibition of pyrogallol autoxidation [31]. MAB was homogenized in 0.2 ml of phosphate buffer (68.9 mmol/l of NaCl, 4.08 mmol/l of Na2HPO4, 0.73 mmol/l of KH2PO4, and 1.34 mmol/l of KCl [pH 7.4]) with a glass-to-glass homogenizer. The homogenates were centrifuged at 10,000 rpm for 10 min at 4 °C. Tris EDTA (442.5 μl) was added to 20 μl of the supernatant. Two types of reactions were performed. One reaction was initiated with 25 μl of pyrogallol and stopped immediately with the addition of 12.5 μl of HCl (1 N). The other reaction was initiated with 25 μl of pyrogallol, and after 30 min, the reaction was stopped with the addition of 12.5 μl of HCl (1 N). The samples

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were centrifuged at 14,000 rpm for 4 min at 4º C. The samples were analyzed spectrophotometrically (Cirrus 80ST, Femto, Brazil), and the results are expressed as units per microgram protein (1 unit indicates an inhibition of 50 % pyrogallol autoxidation). Determination of catalase (CAT) activity in the rat MAB Catalase activity was assayed by H2O2 consumption [1]. MAB was homogenized in 0.2 ml of phosphate buffer (68.9 mmol/l of NaCl, 4.08 mmol/l of Na2HPO4, 0.73 mmol/l of KH2PO4, and 1.34 mmol/l of KCl [pH 7.4]) with a glass-to-glass homogenizer. One hundred microliters of phosphate buffer (K2HPO4, 0.1 mol/l, KH2PO4, 0.1 mol/l [pH 6.5]) was added to the homogenates that were centrifuged at 9,000 rpm for 10 min at 4 °C. Reaction buffer (2.5 ml of Tris EDTA buffer [1 mol/l Trizma and 5 mmol/l of EDTA], 47.35 ml of MilliQ water, and 175.5 μl of H2O2 30 %) was used to read the samples. Reaction buffer (980 μl) was added to quartz cuvettes containing 20 μl of the supernatant. The absorbance was read for 1 min at 240 nm. One catalase (CAT) unit (U) was defined as the amount of enzyme required to decompose 1 mmol/l of H2O2/min. Determination of reduced glutathione (GSH) in the rat MAB Glutathione (GSH) was evaluated through its interaction with 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB). MAB was homogenized in 0.2 ml of phosphate buffer (68.9 mmol/l of NaCl, 4.08 mmol/l of Na2HPO4, 0.73 mmol/l of KH2PO4, and 1.34 mmol/l of KCl [pH 7.4]) with a glass-to-glass homogenizer. Trichloroacetic acid 12.5 % (1v/1v) was added to homogenates that remained 30 min on ice. Homogenates were centrifuged at 3,000 rpm for 15 min at 4 °C. The supernatant (80 μl) was analyzed spectrophotometrically (Cirrus 80ST, Femto, Brazil), and GSH was used as standard to calculate the results. The assay for analyzing the GSH involves oxidation of GSH by sulfhydryl reagent DTNB to form the product 5′-thio-2-nitrobenzoic (TNB-yellow product) measured at 415 nm. Results are expressed as micrograms GSH per milligram protein. Plasma total antioxidant capacity The total antioxidant capacity was measured using an antioxidant assay kit (Cayman®, Ann Arbor, USA-Nº

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709001). The assay relies on the ability of antioxidants in the sample to inhibit the oxidation of ABTS® (2,2′azino-di-[3-ethylbenzthiazoline sulphonate]) to ABTS®+by metmyoglobin. The capacity of the antioxidants in the sample to prevent ABTS oxidation is compared with that of Trolox, a water-soluble tocopherol analogue, and is quantified as millimolar Trolox equivalents. MAB reactivity The rat MAB isolated and perfused in vitro was used as a model of resistance vascular territory as previously described [47]. In brief, the superior mesenteric artery was cannulated with a PE-50 polyethylene catheter and coupled to a perfusion pump (LKB 2215 Multiperpex pump, Broma, Sweden). The MAB was perfused with Krebs solution bubbled with 95 % O2 and 5 % CO2, pH 7.4, at a constant flow of 4 ml/min. Dose–response curves for phenylephrine (0.5 to 50 μg or 2.8 nmol to 0.3 μmol), acetylcholine (0.5 to 50 μg or 2.8 nmol to 0.3 μmol), and sodium nitroprusside (SNP, 0.5 to 50 μg or 1.7 nmol to 0.2 μmol) were determined in the rat MAB. In some experiments, the endothelium was removed with a solution of sodium deoxycholate (1 mg/ml–2 ml, in bolus). Endothelial integrity was assessed qualitatively by the degree of relaxation caused by acetylcholine in the presence of contractile tone induced by phenylephrine. For studies of endotheliumdenuded MAB, the tissues were discarded if there was any degree of relaxation with acetylcholine. Relaxation w a s e x pr es s e d a s pe r c en t c h an ge f r o m th e phenylephrine-contracted levels. A dose of phenylephrine capable of increasing the basal perfusion pressure by 60 mmHg was used in the relaxation protocols. Agonist dose–response curves were fitted using a nonlinear interactive fitting program (Graph Pad Prism 3.0; GraphPad Software Inc., San Diego, CA). Agonist potencies and maximal responses were expressed as EC50 (concentration of agonist producing 50 % of the maximal response) and Emax (maximum effect elicited by the agonist), respectively. Cell culture VSMC derived from MAB of male Wistar-Kyoto rats were isolated and characterized as described previously [5]. In brief, arteries were cleaned of adipose and connective tissue, and VSMC were dissociated by digestion

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of vascular arcades with enzymatic solution (at 37 °C) containing collagenase, elastase, soybean trypsin inhibitor, and bovine serum albumin type I; 60 min. The MAB was filtered, and the cell suspension centrifuged and resuspended in Dulbecco’s modified Eagle’s medium containing 10 % fetal bovine serum, 20 mmol/l HEPES (pH 7.4), 2 mmol/l glutamine, and antibiotics. At subconfluence, the culture medium was replaced with serum-free medium for 24 h to render the cells quiescent. Low-passage cells (passages 4 to 6), from at least four different primary cell cultures, were used in the experiments. Cell stimulation VSMC were stimulated with ethanol (25, 50, or 100 mmol/l) for 5 or 10 min. Superoxide anion production was measured with the use of lucigenin-enhanced chemiluminescence as described above. In another set of experiments, superoxide production was measured in response to ethanol 50 mmol/l after pre-incubation for 30 min with losartan (AT1 receptor antagonist, 1 μmol/l), AG1296 (PDGF-R inhibitor, 1 μmol/l), or vehicle. Superoxide anion production was expressed as percentage increase from baseline values. For Western blotting experiments, VSMC were stimulated with ethanol (50 mmol/l) for 5 and 10 min. In some experiments, cells were incubated for 30 min with losartan (1 μmol/l), AG1296 (1 μmol/l), or vehicle. Membranes were incubated with specific antibodies: p38MAPK (Thr180/Tyr182), total p38MAPK, ERK 1/2 (Thr202/Tyr204), total ERK1/2, SAPK/JNK (Thr183/ Tyr185), total SAPK/JNK, total PDGR-R, PDGF-Rα/β (Tyr849/857), Akt (Ser473), and total Akt (diluted 1:1,000, Cell Signaling). Visualization of superoxide anion generation by intact cells detected with fluorescent dye dihydroethidium (DHE) Generation of intracellular superoxide anion in living cells was measured with the fluoroprobe DHE (exCitation at 488 nm and emission at 610 nm, Molecular Probes). VSMC were incubated in Hank’s balanced salt solution (HBSS) supplemented with 2 μmol/l DHE in a light protected chamber at 37 °C, for 10 min. Cells were rinsed in HBSS, and images were obtained before and after cells exposition to 50 mmol/l ethanol before or after incubation for 30 min with

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losartan (1 μmol/l) or AG1296 (1 μmol/l). Cells were imaged on a wide field epifluorescence microscope equipped with ×40 oil immersion lens using the Stallion live-cell Digital Hi-Speed Multi-Channel Imaging System (Zeiss, Germany). Statistical analysis Data are presented as means±standard error of the mean (SEM). Groups were compared using one-way analysis of variance (ANOVA). Tukey’s correction was used to compensate for multiple testing procedures. Results of statistical tests with P < 0.05 were considered as significant.

Results Blood ethanol levels 30 min after acute administration of ethanol averaged 1.10±0.08 g/l (n=8) (approximately 24 mmol/l). No ethanol was detected in the blood from control animals. ANG I levels (picograms per milligram protein) in the rat MAB from ethanol-treated rats (3.9±0.9, n=7) were not different from those found in the control group (4.1±0.8, n=6). Similarly, treatment with losartan did not alter ANG I levels in both control (4.7±0.8, n=6) and ethanol-treated rats (4.0±0.6, n=6). ANG II levels (picograms per milligram protein) in the rat MAB from ethanol-treated rats (0.50±0.13, n=7) were not different from those found in the control group (0.49±0.08, n=5). Similarly, treatment with losartan did not alter ANG II levels in both control (0.41±0.10, n=5) and ethanoltreated rats (0.40±0.10, n=4). Figure 1a shows that lucigenin-derived luminescence was significantly higher in MAB from ethanol-treated rats. Treatment with losartan did not prevent the increase in superoxide anions levels induced by ethanol. No differences were found on nitrate levels in the rat MAB among the groups (Fig. 1b). As shown in Fig. 2b, MAB from ethanol-treated rats displayed increased SAPK/JNK phosphorylation as compared with controls. Losartan did not prevent ethanol-induced increase in SAPK/JNK phosphorylation. Phosphorylation of ERK1/2 and p38MAPK were not affect by acute ethanol intake (Fig. 2a and c). PDGFR phosphorylation was increased in MAB from ethanoltreated rats, and pre-treatment with losartan did not prevent this response (Fig. 3a). On the other hand,

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As shown in Fig. 6, phosphorylation of p38MAPK, SAPK/JNK, or ERK1/2 was not affected by ethanol. Similarly, ethanol did not affect PDGF-R or Akt phosphorylation in cultured VSMC (Fig 7a and b).

Discussion

Fig. 1 Effect of acute ethanol administration on ROS generation and basal nitrate levels in the rat MAB. a Superoxide generation was determined by lucigenin chemiluminescence. b Basal nitrate was measured in supernatants from MAB homogenates by chemiluminescence. Results are presented as means±SEM of six to eight experiments. *Compared with control and control+losartan (P