Acute ethanol intake induces superoxide anion generation and mitogen-activated

protein kinase phosphorylation in rat aorta: A role for angiotensin type 1 receptor
Alvaro Yogi
a
, Glaucia E. Callera
a
, Andr S. Mecawi
b
, Marcelo E. Batalho
c
, Evelin C. Carnio
c
,
Jos Antunes-Rodrigues
b
, Regina H. Queiroz
d
, Rhian M. Touyz
a
, Carlos R. Tirapelli
e,

a
Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario, Canada
b
Department of Physiology, Faculty of Medicine of Ribeiro Preto, University of So Paulo (USP), Ribeiro Preto, SP, Brazil
c
Department of General and Specialized Nursing, College of Nursing of Ribeiro Preto, USP, So Paulo, Brazil
d
Department of Clinical, Toxicological and Food Science Analysis, Faculty of Pharmaceutical Sciences, USP, So Paulo, Brazil
e
Department of Psychiatric Nursing and Human Sciences, Laboratory of Pharmacology, College of Nursing of Ribeiro Preto, USP, Ribeiro Preto, SP, Brazil
a b s t r a c t a r t i c l e i n f o
Article history:
Received 27 June 2012
Revised 20 August 2012
Accepted 21 August 2012
Available online 6 September 2012
Keywords:
Ethanol
Aorta
Superoxide anion
Blood pressure
Angiotensin II
Losartan
Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothe-
sized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through
reninangiotensin system (RAS) activation. Ethanol (1 g/kg; p.o. gavage) effects were assessed within
30 min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected
by the previous administration of losartan (10 mg/kg; p.o. gavage), a selective AT
1
receptor antagonist.
Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity,
plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxi-
dative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P)
H oxidasemediated vascular generation of superoxide anion and p47phox translocation (cytosol to mem-
brane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed in-
creased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the
phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased
phenylephrine-induced contraction in endothelium-intact aortas. Ethanol signicantly decreased plasma
and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide
metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates
RAS activity and induces vascular oxidative stress and redox-signaling activation through AT
1
-dependent mecha-
nisms. These ndings highlight the importance of RAS in acute ethanol-induced oxidative damage.
2012 Elsevier Inc. All rights reserved.
Introduction
Ethanol consumption is a comorbid variable that increases the in-
cidence of cardiovascular diseases (Di Castelnuovo et al., 2009). While
ethanol predisposes to vascular dysfunction through many mecha-
nisms, oxidative stress may be a key factor (Husain et al., 2005,
2007, 2011). Chronic ethanol consumption causes dose-dependent
blood pressure increase and reactive oxygen species (ROS) genera-
tion (Williams et al., 1990; Husain et al., 2005). Chronic ethanol in-
take was also described to induce vascular inammation, increases
vascular and plasma angiotensin II (ANGII) levels and impairs endo-
thelial functions in association with reduced nitric oxide (NO) bio-
availability (Husain et al., 2008, 2011; Toda and Ayajiki, 2010).
Moreover, ethanol intake is also associated with higher vascular
NAD(P)H oxidase activity and membrane lipid peroxidation (Husain
et al., 2007; Snmez et al., 2009). In addition to its chronic effects,
acute administration of ethanol was shown to induce oxidative stress
in several tissues such as the liver and the cerebellum (Nordmann,
1987; Demori et al., 2006).
ANG II, a key factor in progressive cardiovascular diseases, mediates
its effects through AT
1
receptors and complex downstream signaling
pathways involving redox-sensitive processes, such as mitogen-
activated protein kinase (MAPK) activation. ANG II is an important
mediator of NAD(P)H oxidase-driven generation of ROS in vascular
smooth muscle and endothelial cells (Touyz and Schiffrin, 1999, 2000).
NAD(P)Hoxidase is the major source of ROS in the cardiovascular system
(Lambeth, 2004). The prototypical oxidase is a multisubunit complex
composed of two membrane-associated components, gp91phox (Nox
2) and p22phox, three cytosolic components, p40phox, p47phox and
p67phox, and the small molecular weight protein rac-2 (Lambeth,
2004). All vascular cell types, including endothelial and vascular smooth
Toxicology and Applied Pharmacology 264 (2012) 470478
Correspondingauthor at: Universidade de So Paulo, Escola de Enfermagemde Ribeiro
Preto, Laboratrio de Farmacologia, Avenida Bandeirantes 3900, CEP 14040902, Ribeiro
Preto, SP, Brazil. Fax: +55 16 3633 3271.
E-mail address: crtirapelli@eerp.usp.br (C.R. Tirapelli).
0041-008X/$ see front matter 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.taap.2012.08.029
Contents lists available at SciVerse ScienceDirect
Toxicology and Applied Pharmacology
j our nal homepage: www. el sevi er . com/ l ocat e/ yt aap
muscle cells express components of NAD(P)Hoxidase (Touyz et al., 2002;
Lambeth, 2004).
ANG II has been demonstrated to cause vasoconstriction by increas-
ing the production of superoxide anions via the activation of NAD(P)H
oxidase in the vascular wall (Touyz and Schiffrin, 2000). Interestingly,
several studies demonstrated that chronic ethanol consumption induces
activationof the reninangiotensinsystem(RAS), whichmay play anim-
portant role in tissue oxidative stress and blood pressure increase
(Thevananther and Brecher, 1994; Bechara et al., 2003; Polikandriotis
et al., 2006). However, whether acute administration of ethanol induces
increase in the production of ROS due to RAS activation in vascular tis-
sues remains elusive. In addition, increase in ROS generation could be a
possible event implicated with acute ethanol-induced vasoconstriction
and potential elevation of blood pressure. We hypothesized that acute
administration of ethanol induces ROS generation and activation of
redox signaling in the vasculature through RAS-mediated mechanisms.
Here, we sought toinvestigate the effect of acute administrationof ethanol
on vascular function and ROS levels and the involvement of RAS inthese
responses. Our study demonstratedthat acute ethanol intake stimulates
RAS activity and induces vascular oxidative stress and redox-signaling
activation through AT
1
-dependent mechanisms.
Materials and methods
Acute ethanol administration. Experiments were performed in accor-
dance withthe principles and guidelines of the animal ethics committee
of the University of So Paulo and the University of Ottawa. Male Wistar
rats, initially weighing 350400 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 (Abdel-Rahman et al., 1987; Malinowska et al., 1989;El-Mas and
Abdel-Rahman, 1992, 1999). The oral dose of ethanol (1 g/kg) corre-
sponds to a standard alcoholic drink per day in humans (Beulens et
al., 2007; Thakker, 1998). 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 (Fabiani et al., 2000; Yoshitani et al., 2002).
Cannulation procedure and blood pressure measurements. Thirty six
hours before the experiment, animals were anaesthetized with
ketamine/xylazine (80/10 mg/kg) and a polyethylene catheter was
implanted into the femoral artery for measurement of mean arterial
pressure (MAP), systolic arterial pressure (SAP), diastolic arterial pres-
sure (DAP) and heart rate (HR). The catheters were tunneled subcuta-
neously and exteriorized at the back of the neck between the
scapulae. The blood pressure and HR of unanesthetized freely moving
rats were recorded using a Grass polygraph (Grass P122, USA)
connected to a pressure transducer (Grass, P23XL-1). On the day of
the experiment, the arterial catheter was connected to a pressure
transducer for the measurement of blood pressure and HR, as men-
tioned above. A period of at least 30 min was allowed at the beginning
of the experiment for stabilization of blood pressure and HR. Ethanol
(1 g/kg) was administered by gavage. The administration of similar vol-
ume of water was performed in animals from the control group. This
volume of water appeared to be well tolerated by the rats because it
produced no signicant hemodynamic changes in previous studies
(El-Mas and Abdel-Rahman, 1992, 1999). In another set of experiments
the rats were treated with losartan (10 mg/kg, p.o. gavage) 30 min be-
fore the administration of water or ethanol. The beginning of the exper-
imental protocols, time zero, was dened as the point at which the rat
received ethanol or water. After ethanol or water administration, mea-
surements of blood pressure and HR were made at 5-min intervals for a
period of 120 min.
Blood ethanol measurements. Ethanol (1 g/kg) was administered by
gavage in fasting animals. Rats were anaesthetized with ketamine/
xylazine (80/10 mg/kg) and blood was collected from the aorta 5,
15, 30, 60 and 120 min after ethanol administration using heparin-
ized syringes. Blood samples were transferred to tubes containing
sodium uoride (1 mg/ml). Ethanol analysis was carried out using a
CG-17A gas chromatographer (Shimadzu, Kyoto, Japan) equipped
with a ame-ionization detector and an HSS-4A headspace sampler
(Shimadzu). Injections were made in the split mode onto a
Supelcowax 10 (Supelco, Bellenfonte, PA) column (30 m25 mm
i.d. and 25 m lm thickness). The split ratio was 20:1 and the injec-
tor, column and detector temperatures were 150 C, 50 C and
150 C, respectively. The samples were placed in 10 ml headspace
vials by adding 1 g sodium chloride, 1.0 ml water, 100 l of the inter-
nal standard (acetonitrile, 1 ml/l) solution, and 1 ml of blood. The
samples were sealed using crimp top vial caps with septa and were
placed in the headspace rack at the temperature of 60 C and with
equilibration time of 20 min. Calibrations standards were prepared
in the same headspace vials (0.103.16 mg/ml). 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 the following biochemical assays since peak ethanol levels were
achieved at this time.
Plasma renin activity (PRA). Blood samples were collected in tubes
containing ethylenediamine tetraacetic acid (EDTA). The samples
were centrifuged (20 min, 3000 rpm, 4 C) and PRA was determined
with standard radioimmunoassay (RIA) techniques using commercial
RIA Kit (Immunotech). Results are expressed as ng/ml/h.
Plasma ACE activity. ACE enzymatic activity in plasma was deter-
mined following incubation with intramolecularly quenched syn-
thetic specic substrate (Sentandreu and Toldr, 2006). A working
solution (0.45 mM) of the ACE substrate, Abz-Gly-p-nitro-Phe-Pro-
OH (BACHEM), was prepared in 150 mM Tris buffer (pH 8.3)
containing 1.125 M NaCl. The assay was performed in a black
at-bottom 96-well plate with 5 l of plasma, 95 l of 150 mM Tris
buffer (pH 8.3) and 200 l of Abz-Gly-p-nitro-Phe-Pro-OH, with a
total volume of 300 l. The assay was also performed in the presence
of 10 M captopril. After incubation at 37 C for 4 h, uorescence
was measured using a FLUOstar Galaxy uorescence microplate
reader (BMG Labtechnologies, Durham, NC) at an excitation wave-
length of 320 nm and an emission wavelength of 420 nm. Blank
values were subtracted from all uorescence values. Fluorescence
resulting from ACE-specic activity was determined by subtracting
values obtained in the presence of captopril fromthose in its absence
and results were expressed as relative uorescence unities (RFU).
Plasma and tissue ANG I and ANG II determination. The blood was
collected in chilled tubes containing peptidase inhibitors (1 mM
p-hydroxymercury benzoate, 30 mM 10-phenanthroline, 1 mM PMSF,
1 mM pepstatin A, and 7.5% EDTA 50 l/ml of blood). Plasma was
obtained after centrifugation (20 min, 3000 rpm, 4 C) and stored at
20 C. The aortas were homogenized with 0.045 N HCl in ethanol
(10 ml/g of tissue) containing 0.9 M p-hydroxymercuribenzoate,
131.5 M of 1,10-phenanthroline, 0.9 M phenylmethylsulfonyl uo-
ride, 1.75 M pepstatin A, 0.032% EDTA, and 0.0043% protease-free bo-
vine serum albumin. Protein concentration in the plasma and crude
homogenates were determined by the Bradford method. These homog-
enates were evaporated and after diluted in 2 ml of 0.1% triuoroacetic
acid (TFA). After this, plasma and tissue peptides were extracted onto a
Bond-Elut SEP-COLUMNS (Peninsula Laboratories, San Carlos-CA, USA).
The columns were pre-activated by sequential washes with 4 ml of 60%
acetonitrile/0.1% TFA and 20 ml of 0.1% TFA. After sample application,
the columns were washed with 20 ml of 0.1% TFA. The adsorbed pep-
tides were eluted with 3 ml of 60% acetonitrile/0.1% TFA into polypro-
pylene tubes. After evaporation, angiotensin peptide levels were
measured by RIA. The specic antibodies for ANG I and ANG II were
471 A. Yogi et al. / Toxicology and Applied Pharmacology 264 (2012) 470478
obtained from Peninsula Laboratories (San Carlos-CA, USA; ANG I:
T4166 and ANG II: T4007). The sensitivity of the RIA and coefcient of
variation intra- and 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.
Plasma measurement of thiobarbituric acid-reacting substances. Blood
was collected in tubes containing EDTA, centrifuged and plasma
levels of thiobarbituric acid-reacting substances (TBARS) were mea-
sured by a colorimetric method as previously described (Yogi et al.,
2008). Malondialdehyde (MDA) standards were diluted in the range
of 0 to 4 mol/l. TBARS values were expressed in nmol/ml
malondialdehyde equivalents.
Detection of superoxide anions in aortic endothelial cells and vascular
smooth muscle by lucigenin chemiluminescence. Endothelial cells
were isolated from the aortas mechanically, by gently friction with
plastic stem in plates containing phosphate buffer. The vascular
smooth muscle was frozen in liquid nitrogen. The lucigenin-derived
chemiluminescence assay was used to determine superoxide anion
levels in smooth muscle homogenates 10% (wt/vol) prepared in phos-
phate buffer (20 mM of KH
2
PO
4
, 1 mM of EGTA, and protease inhibi-
tors [pH 7.4]) with a glass-to-glass homogenizer. The reaction was
started by the addition of NAD(P)H (0.1 mM) to the suspension
(250 l of nal volume) containing sample (50 l), lucigenin
(5 M), and assay buffer (50 mM of KH
2
PO
4
, 1 mM of EGTA, and
150 mM of sucrose [pH 7.4]). Luminescence was measured every
1.8 s for 3 min in a luminometer (Orion II Luminometer, Berthold de-
tection systems). Buffer blank was subtracted from each reading. Su-
peroxide production was expressed as relative light unit (RLU)/mg
protein. Protein concentrations were determined with protein assay
reagent (Bio-Rad Laboratories) (Yogi et al., 2008).
Cytosol/membrane fractionation. Endothelium-denuded frozen thoracic
aortas were homogenized in lysis buffer containing 50 mM TrisHCl
(pH 7.4), 2.5 mM EDTA, 5 mM EGTA and protease inhibitor. Homoge-
nates were centrifuged at 100,000 g for 1 h at 4 C. The supernatant
(cytosolic fraction) was collected. The pellet, containing the particulate
fraction, was resuspended inlysis buffer containing 1%TritonX-100 and
centrifuged at 10,000 g for 10 min at 4 C. The resultant supernatant
collected (membrane-enriched fraction) was used as described below.
The protocol for the validation of membrane and cytosolic fractions
was previously published (Callera et al., 2006).
Immunoblotting. Endothelium-denuded thoracic aortas were used
as follows. Frozen tissue was homogenized in lysis buffer [50 mM
Tris/HCl (pH 7.4), NP-40 (1%), sodium deoxycholate (0.5%), SDS
(0.1%)]. Homogenates were centrifuged at 5000 g for 10 min,
the nuclei-rich pellet discarded and the supernatant uid stored at
80 C. Thirty micrograms of protein was separated by electrophore-
sis on a 10% polyacrylamide gel, and transferred onto a nitrocellulose
membrane. Nonspecic binding sites were blocked with 5% skim milk
in Tris-buffered saline solution with Tween 20 for 1 h at 24 C. Mem-
branes were then incubated with specic antibodies overnight at 4 C
as follows: p38MAPK (Thr
180
/Tyr
182
), total p38MAPK, ERK 1/2
(Thr
202
/Tyr
204
), total ERK1/2, SAPK/JNK (Thr
183
/Tyr
185
), total SAPK/
JNK (diluted 1:1000, Cell Signaling) and p47phox (diluted 1:500,
Santa Cruz Biotechnology). After incubation with secondary antibodies
(diluted 1:1000) for 90 minat roomtemperature, signals were revealed
Fig. 1. Effect of acute ethanol intake on mean arterial blood pressure (MAP), systolic arterial pressure (SAP), diastolic arterial pressure (DAP) and heart rate (HR). Results are presented as
meansSEM of 10 to 12 animals. *Compared to control and control+losartan; #Compared to control, ethanol and control +losartan (Pb0.05, repeated measures ANOVA).
472 A. Yogi et al. / Toxicology and Applied Pharmacology 264 (2012) 470478
with chemiluminescence, visualized by autoradiography, and quanti-
ed densitometrically. Results were expressed as a ratio of non
phospho proteins expression that was used as housekeeping proteins.
Measurement of plasma and tissue nitrate. Nitrate (NO
3

, a metabolite of
NO) levels were measured in supernatants fromaorta homogenates. Al-
iquots of 5 l were injected into a Sievers Chemiluminescence Analyzer
(Nitric Oxide Analyzer, NOA280, Sievers Instruments, Colorado, USA)
and pelletedby centrifugationwithVCl
3
and HCl (at 95 C), whichact as
reductants for nitrate. Results were normalized for protein concentra-
tion assessed with the Bradford technique and are expressed as M/mg
protein. To evaluate plasma nitrate levels, blood was collected into chilled
plastic tubes, containing heparin (200 U), centrifuged for 20 min at
2000 g at 4 C for plasma separation and stored at 70 C before dosage.
On the day of the assay, plasma samples were thawed and deproteinized
with 95% ethanol (at 4 C) for 30 min, subsequently centrifuged, and the
supernatant was used for measurement of nitrate as described above.
Results are expressed as M.
Vascular reactivity. The thoracic aorta was isolated and cut into rings
(56 mm in length). The arteries were prepared for organ bath studies
as described previously (Tirapelli et al., 2006). Endothelial integrity was
assessed qualitatively by the degree of relaxation induced by acetylcho-
line (1 M) in the presence of contractile tone induced by phenyl-
ephrine (0.1 M). Cumulative concentrationresponse curves for
phenylephrine (0.000110 M) were determined on endothelium-
intact or -denuded aortic rings. Concentrationresponse curves for
acetylcholine (0.000110 M) and sodium nitroprusside (0.0001
0.3 M) were obtained in endothelium-intact and endothelium-
denuded rings, respectively. Relaxation was expressed as a percentage
change from phenylephrine-contracted levels. Agonist concentration
response curves were tted using a nonlinear interactive tting pro-
gram (Graph Pad Prism 3.0; GraphPad Software Inc., San Diego, CA).
Agonist potencies and maximal responses were expressed as pD
2
(logEC
50
) and E
max
(maximum effect elicited by the agonist),
respectively.
Statistical analysis. Data are presented as meansstandard error of
the mean (SEM). Groups were compared using one-way analysis of
variance (ANOVA) or repeated measures ANOVA. Tukey correction
was used to compensate for multiple testing procedures. Results of
statistical tests with Pb0.05 were considered as signicant.
Fig. 2. Effect of acute ethanol intake in the systemic and local components of RAS. The effect of ethanol was evaluated on: PRA (A), ACE activity (B), plasma ANG I and ANG II levels
(C and D) and aortic ANG I and ANG II levels (E and F). Results are presented as meansSEM of 6 or 7 animals. *Compared to control; #Compared to ethanol; Compared to
control +losartan (Pb0.05, ANOVA followed by Tukey).
473 A. Yogi et al. / Toxicology and Applied Pharmacology 264 (2012) 470478
Results
Blood ethanol measurements
Ethanol levels (g/l) in the blood 5, 15, 30, 60 and 120 min after acute
administrationof ethanol averaged0.280.06, 0.560.07, 1.140.11,
0.600.11 and 0.580.10, respectively (n=6 for each group). The
ethanol level in the blood reached the peak after 30 min. The time
point that presented the higher ethanol content was used for blood
and vascular tissue collection and for the following biochemical and
functional assays. Noethanol was detectedinthe bloodfromcontrol an-
imals as previously described (Schlorff et al., 1999).
Acute ethanol administration decreased blood pressure
Baseline values of MAP and HR were similar in rats from control
(MAP: 944 mm Hg; HR: 34716 bpm; n=11), ethanol (MAP:
964 mm Hg; HR: 35020 bpm; n=12), control plus losartan
(MAP: 956 mm Hg; HR: 36519 bpm; n=10) and ethanol plus
losartan (MAP: 974 mm Hg; HR: 36411 bpm; n=10) groups.
The hemodynamic responses in conscious rats after administration of
water or ethanol are shown in Fig. 1. Compared with the corresponding
control group, ethanol produced a signicant decrease (Pb0.05) in
MAP, SAP and DAP. On the other hand, ethanol administration did not
affect HR. Losartan did not alter blood pressure or HR in control rats
and potentiated the decrease in blood pressure induced by ethanol.
Effect of acute ethanol administration in the components of RAS
Ethanol increased PRA (Fig. 2A) and ACE activities (Fig. 2B). As
expected, losartan signicantly increased PRA and ACE activities
(Pb0.05) because of its stimulatory effect on renal renin release. Plas-
ma ANG I (Fig. 2C) and ANG II (Fig. 2D) levels were signicantly in-
creased (Pb0.05) in ethanol-treated rats compared with control rats.
Losartan signicantly increased (Pb0.05) plasma ANG I and ANG II
levels when compared to control rats. On the other hand, ethanol treat-
ment did not change aortic levels of ANG I (Fig. 2E) or ANG II (Fig. 2F).
Evaluation of oxidative stress
Systemic oxidative stress was evaluated by measuring plasma
TBARS (Fig. 3A). Plasma TBARS levels were signicantly increased
(Pb0.05) in ethanol-treated rats compared with control rats. Treat-
ment with losartan prevented the increase in TBARS levels induced
by ethanol. The effect of ethanol treatment in superoxide anion
generation was evaluated in vascular tissue. Fig. 3B shows that
lucigenin-derived luminescence was signicantly higher (Pb0.05) in
endothelial cells from ethanol-treated rats. Generation of superoxide
anions in endothelial cells from ethanol-treated rats was inhibited by
losartan. Similarly, ethanol induced increase of superoxide anion gen-
eration (Pb0.05) in vascular smooth muscle from ethanol-treated
rats and losartan prevented this effect (Fig. 3C).
Effect of ethanol on p47phox translocation
The translocation of the p47phox subunit from the cytosol to the
membrane is critically involved in the NAD(P)H oxidase activation.
As shown in Fig. 4, aortas from ethanol-treated rats displayed a
Fig. 3. Effect of acute ethanol intake in systemic and tissue oxidative stress. Bar graphs
represent plasma concentration of TBARS, a marker of oxidative stress (A), superoxide
anion levels in aortic endothelial cells evaluated by lucigenin-derived chemiluminescence
assay (B), and superoxide anion levels inaortic smooth muscle homogenates evaluated by
lucigenin-derived chemiluminescence assay (C) in control, ethanol, control plus losartan
and ethanol plus losartan groups. Results are presented as meansSEM of 5 or 6 experi-
ments. *Compared to control, control +losartan and ethanol +losartan (Pb0.05, ANOVA
followed by Tukey).
p47phox
47 kDa
p47phox
47 kDa
Ethanol Control Ethanol Control
Losartan
Membrane
Cytosol
0.0
0.2
0.4
0.6
0.8
Control Ethanol Control Ethanol
Losartan
p
4
7

p
h
o
x

t
r
a
n
s
l
o
c
a
t
i
o
n
(
M
e
m
b
r
a
n
e
/
C
y
t
o
s
o
l
)
*
Fig. 4. Effect of acute ethanol intake on p47phox translocation in the rat aorta. Bar
graphs represent aortic p47phox translocation as a ratio of membrane:cytosol expres-
sion in control, ethanol, control plus losartan and ethanol plus losartan groups. Top
panels: representative immunoblots are shown. Results are presented as means
SEM of 6 experiments. *Compared to control, control +losartan and ethanol +losartan
(Pb0.05, ANOVA followed by Tukey).
474 A. Yogi et al. / Toxicology and Applied Pharmacology 264 (2012) 470478
signicant (Pb0.05) increase in the membrane:cytosol fraction ratio
of p47phox expression, indicating the translocation of the subunit.
Treatment with losartan prevented the increase in p47phox translo-
cation induced by ethanol.
Effect of ethanol on MAPK phosphorylation
Aortas from ethanol-treated rats displayed a signicant (Pb0.05) in-
creased p38MAPK (Fig. 5A) and SAPK/JNK (Fig. 5B) phosphorylation as
compared with controls. Losartan prevent ethanol-induced increase in
the phosphorylation of these kinases. Ethanol did not affect ERK 1/2
phosphorylation (Fig. 5C).
Effect of ethanol on nitrate generation
Ethanol signicantly decreased (Pb0.05) plasma nitrate levels
(Fig. 6A). Nitrate content in endothelium-intact aortas from
ethanol-treated rats was also signicantly lower (Pb0.05) as
compared with control rats (Fig. 6B). Treatment with losartan
did not prevent the decrease in nitrate levels induced by ethanol
in plasma and in vascular tissue.
Effect of ethanol on vascular reactivity
Acute ethanol intake signicantly increased (Pb0.05) phenylephrine-
induced contraction in endothelium-intact rings. Pre-treatment with
losartan did not prevent the enhancement on phenylephrine-induced
contraction induced by ethanol (Fig. 7A; Table 1). Ethanol treatment
did not alter phenylephrine-induced contraction in endothelium-
denuded rings (Fig. 7B; Table 1). Fig. 7C shows that the endothelium-
dependent relaxation induced by acetylcholine signicantly reduced
(Pb0.05) in aortas from ethanol-treated rats as compared with those
from control rats. Pre-treatment with losartan did not prevent the im-
pairment on acetylcholine-induced relaxation induced by ethanol treat-
ment. The relaxation induced by SNP, a NO donor, did not signicantly
vary among the groups (Fig. 7D; Table 1).
Discussion
The present ndings indicate that administration of acute oral
dose of ethanol, which corresponds to a standard alcoholic drink
per day in humans (Thakker, 1998; Beulens et al., 2007), activated
p38
38 kDa
p38 [Thr
180
/Tyr
182
]
38 kDa
SAPK/JNK [Thr
183
/Tyr
185
]
46/52 kDa
SAPK/JNK
46/52 kDa
ERK 1/2 [Thr
202
/Tyr
204
]
42/44 kDa
ERK 1/2
42/44 kDa
0.0
0.5
1.0
1.5
Control Ethanol Control Ethanol
Losartan
p
3
8

M
A
P
K

p
h
o
s
p
h
o
r
y
l
a
t
i
o
n
(
P
h
o
s
p
h
o
/
T
o
t
a
l
)
*
A
0.0
0.5
1.0
1.5
Control Ethanol Control Ethanol
Losartan
S
A
P
K
/
J
N
K

p
h
o
s
p
h
o
r
y
l
a
t
i
o
n
(
P
h
o
s
p
h
o
/
T
o
t
a
l
)
*
B
0.0
0.5
1.0
1.5
Control Ethanol Control Ethanol
Losartan
C
E
R
K
1
/
2

p
h
o
s
p
h
o
r
y
l
a
t
i
o
n
(
P
h
o
s
p
h
o
/
T
o
t
a
l
)
Fig. 5. Effect of acute ethanol intake on MAPK phosphorylation in the rat aorta. Top
panels: representative immunoblots for MAPK protein phosphorylation and expression.
Bottom panels: corresponding bar graphs show densitometric data for phosphorylation
of p38MAPK (A), SAPK/JNK (B) and ERK1/2 (C). Results are presented as meansSEM
of 6 experiments. *Compared to control, control +losartan and ethanol +losartan
(Pb0.05, ANOVA followed by Tukey).
Fig. 6. Effect of acute ethanol intake on plasma and aortic nitrate levels. Bar graphs repre-
sent plasma (A) and aortic levels (B) of nitrate (NO
3

), a metabolite of NO, evaluated by

chemiluminescence assay. Results are presented as meansSEM of 6 to 8 experiments.
*Compared to control and control +losartan (Pb0.05, ANOVA followed by Tukey).
475 A. Yogi et al. / Toxicology and Applied Pharmacology 264 (2012) 470478
the RAS and signicantly increased the generation of superoxide an-
ions in aortic endothelial and vascular smooth muscle cells. However,
the fact that levels of plasma TBARS were increased in ethanol-
treated rats suggest that increased ROS formation is probably global
phenomena. Interestingly, ethanol-induced increase in systemic and
aortic superoxide anion generation was inhibited by losartan, further
suggesting that AT
1
activation plays a key role in this response. Of
note, our data demonstrate that the peak blood concentration of eth-
anol here described (approximately 25 mM) is within those found in
the bloodstream of rats (24 to 50 mM) 30 min or 1 h after the oral
administration of ethanol (Chabielska et al., 1988; Schlorff et al.,
1999; Izbki et al., 2001).
The new nding of our investigation is that acute administration
of ethanol activates RAS. Losartan increased PRA, ACE activity, ANG I
and ANG II levels due to its expected stimulatory effect on renal
renin release as previously described (Pals and Couch, 1993; Peters
et al., 1999). The current study does not address the exact mechanism
whereby ethanol modulates RAS activity. Since renin is produced in
the kidney in response to hypotension, it is possible to suggest that
the hypotensive effect of ethanol, here described, could have been
Fig. 7. Effect of acute ethanol intake on vascular reactivity to phenylephrine (A and B), acetylcholine (C) and SNP (D) in aortic rings. Concentrationresponse curves for phenylephrine
were determined in endothelium-intact (Endo+) or endothelium-denuded (Endo) aortic rings. Concentrationresponse curves for acetylcholine and SNP were determined in
endothelium-intact and endothelium-denuded aortic rings, respectively. Results are presented as meansSEM of 7 to 9 experiments. *Compared to control and control +losartan
(Pb0.05, ANOVA followed by Tukey).
Table 1
Effect of acute ethanol intake on the E
max
and pD
2
values for phenylephrine, acetylcholine and sodium nitroprusside (SNP) in endothelium-intact (Endo+) or
endothelium-denuded (Endo) aortic rings.
Phenylephrine (Endo+) Phenylephrine (Endo) Acetylcholine (Endo+) SNP (Endo)
E
max
(g) pD
2
E
max
(g) pD
2
E
max
(%) pD
2
E
max
(%) pD
2
Control 1.20.1 6.90.2 1.60.1 7.70.2 110.53.1 7.20.2 105.62.9 7.80.1
Ethanol 1.70.1* 7.00.1 1.60.1 7.60.1 74.63.9* 6.40.1* 110.52.4 7.70.1
Control +losartan 1.20.1 7.20.2 1.70.1 7.50.2 107.41.7 7.40.2 104.23.3 8.00.2
Ethanol +losartan 1.60.1* 7.10.2 1.50.3 7.40.2 75.63.9* 6.30.2* 103.34.4 7.60.2
Values are meansSEM of 7 to 9 animals. *Compared to control and control +losartan (Pb0.05; ANOVA followed by Tukey).
476 A. Yogi et al. / Toxicology and Applied Pharmacology 264 (2012) 470478
the trigger for renin release. Moreover, it was suggested that dehy-
dration and increased activity of the sympathetic nervous system
could play a role in the increased PRA observed in humans after
acute ethanol intake (Nieminen et al., 1981). The RAS is a dual system
and all the elements of the RAS are present in the vasculature, indicat-
ing that the vascular system may act independently from the system-
ic RAS to generate ANG II (Cat and Touyz, 2011). Acute ethanol intake
did not change ANG I or ANG II levels in the aorta, suggesting that eth-
anol activates the systemic, but not tissue RAS, which is described to
operate independently of the circulating RAS (Cat and Touyz, 2011).
ANG II stimulates expression of NAD(P)H oxidase subunits and in-
duces superoxide anion production in cells of the vascular wall (Touyz
and Schiffrin, 2000). NAD(P)H oxidase is normally dormant but upon
cellular stimulation, the cytosolic subunits translocate to the membrane
and associate with cytochrome b 558, resulting in the rapid activation of
the oxidase. In endothelial and vascular smooth muscle cells, the
p47phox subunit has been shown to be essential for ROS production
in response to ANGII (Li and Shah, 2003). Inour study, the translocation
of p47phox was increased in aortas from ethanol-treated rats, and this
effect was inhibited by losartan, suggesting that AT
1
receptors were in-
volved in ethanol-induced p47phox translocation and NAD(P)H oxi-
dase activation.
The predominant signaling events in response to ANG II involve
not only the generation of ROS but also the activation of the
non-receptor tyrosine kinase c-Src, which plays an important role in
the activation of other downstream proteins including MAPKs
(Touyz, 2003; Higuchi et al., 2007). MAPKs are a family of serine/
threonine kinases which are classically associated with vascular
smooth muscle cell contraction, migration, adhesion, collagen deposi-
tion, cell growth, differentiation, and survival (Pearson et al., 2001).
Of the major MAPKs, extracellular signal-regulated kinases (ERK1/2),
p38MAPK, and stress-activated protein kinase/c-Jun N-terminal kinases
(SAPK/JNK) are the best characterized (Pearson et al., 2001). In our
study, phosphorylation of p38MAPK and SAPK/JNK was increased in
the aorta of ethanol-treated rats. Losartan prevented p38MAPK and
SAPK/JNK phosphorylation suggesting that ethanol induces MAPKs
phosphorylation via AT
1
dependent pathways. Activation of ERK is an
important process in mitogenesis and hypertrophy in cardiovascular
tissues (Treisman, 1996). In our model, ERK 1/2 phosphorylation was
unaffected in aortas from ethanol-treated rats. The complex signaling
networks that underlie MAPK activation typically require phosphoryla-
tion by a MAPK also known as MEK. The ERK1/2 phosphorylation cas-
cade involves MEK1/2 (MAP/ERK kinase) whereas the signaling
processes leading to SAPK/JNK and p38MAPK activation involve
MEK4/7 and MEK3/6, respectively (Pearson et al., 2001). Such differ-
ences may explain the distinctive effects of ethanol on MAPKs
activation.
Ethanol-induced decrease in plasma and aortic NO levels is not
related to AT
1
receptors activation. Acute ethanol intake impaired
endothelium-dependent, but not endothelium-independent relaxa-
tion as previously described (Hatake et al., 1989). Our data further
show that ethanol administration increased phenylephrine-induced
contraction in an endothelium-dependent manner. The endothelium-
dependent relaxation induced by acetylcholine is mediated by NO and
the contractile response induced by phenylephrine in the aorta is
counteracted by endothelium-derived NO (Tirapelli et al., 2006). Thus,
the impairment in the endothelium-dependent relaxation induced by
ethanol consumption may be related, at least in part, to an increased
generationof superoxide anion, whichmay serve to inactivate endothe-
lial NO. In the present investigation, while acute ethanol ingestion in-
duced endothelial impairment and increased aortic generation of ROS,
a decrease in blood pressure was observed. A possible explanation for
this discrepancy is that the contribution made by conduit vessels, such
as the aorta, to the regulation of systemic blood pressure is not so rele-
vant as compared to vessels of smaller diameter, such as peripheral vas-
cular resistance vessels. Moreover, the effects of ethanol in the
cardiovascular systemare dependent on the pattern of ethanol drinking
(acute or chronic). While chronic ethanol ingestion induces hyperten-
sion (Husain et al., 2007, 2011) acute ethanol intake is described to in-
duce hypotension (Malinowska et al., 1989; Varga and Kunos, 1997;
El-Mas et al., 2006).
Our ndings are in accordance with previous observations describ-
ing that acute administration of ethanol induces hypotension
(Malinowska et al., 1989; Varga and Kunos, 1997; Kawano et al.,
1992; El-Mas et al., 2006). Some mechanisms are proposed in the lit-
erature to explain this response such as direct myocardial depression
(Kelback et al., 1985), vasodilatation (El-Mas et al., 2006), and
-receptor blockade (Abdel-Rahman et al., 1987). Losartan potentiat-
ed ethanol-induced hypotension further discarding a role for RAS in
such response. A possible explanation for this observation is that
under AT
1
blockade, ANG II, which was shown to be increased after
ethanol administration, would act predominantly on AT
2
receptors
to induce vasodilatation and hypotension (Sosa-Canache et al.,
2000). Although we have observed a hypotensive effect of ethanol in
our model, we must be aware of the fact that ethanol induced vascular
oxidative stress and reduced NO levels in the vasculature, actions that
are associated with endothelial dysfunction.
ANG II plays an important role in hypertension and cardiovascular
diseases by promoting changes in vascular reactivity and endothelial
function, tissue remodeling, and oxidative stress (Touyz and Schiffrin,
2000). Accumulating in vivo and in vitro evidence supports the notion
that ANG II may, through c-Srcdependent mechanisms, cause
cardiovascular damage (Touyz, 2003; Higuchi et al., 2007). These
pathologies are associated with increased cellular protein synthesis,
gene expression, and growth, which all depend on MAPKs activation.
Thus, RAS activation with consequent oxidative stress and MAPKs acti-
vation here described could be one mechanism by which ethanol pre-
disposes individuals to vascular dysfunction.
In summary, our study provides novel evidence that acute ethanol
intake stimulates RAS activity and vascular oxidative stress through
an AT
1
-dependent mechanism and implicates translocation of
p47phox and MAPKs phosphorylation as downstream effectors.
These ndings highlight the importance of the RAS in acute
ethanol-induced oxidative damage. Moreover, our ndings raise the
possibility that ethanol consumption increases the risk for acute vas-
cular injury, in part, by altering the RAS and specic NAD(P)H oxidase
component expression and thereby rendering the tissue susceptible
to oxidative stress.
Conict of interest statement
The authors declare that there are no conicts of interest.
Acknowledgments
We thank Juliana T. Rocha and Sonia A. Dreossi for technical sup-
port. This study was supported by grants from the Canadian Institutes
of Health Research, the Conselho Nacional de Desenvolvimento
Cientco e Tecnolgico (process number 470556/2010-2) and the
Fundao de Amparo Pesquisa do Estado de So Paulo (process
number 2010/05815-4).
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