Experimental Gerontology 97 (2017) 38–48

Contents lists available at ScienceDirect

Experimental Gerontology
journal homepage: www.elsevier.com/locate/expgero

Cardiovascular risk and the effect of nitric oxide synthase inhibition in T

female rats: The role of estrogen
Jaqueline C. Castardo-de-Paulaa, Blenda H. de Camposa, Eric D.T. Amorima, Rosiane V. da Silvab,
Carine C. de Fariasc, Luciana Higachic, Phileno Pinge-Filhob, Décio S. Barbosac,
Marli C. Martins-Pingea,⁎
a
Department of Physiological Sciences, Center of Biological Sciences, State University of Londrina, Londrina, PR, Brazil
b
Department of Pathological Sciences, Center of Biological Sciences, State University of Londrina, Londrina, PR, Brazil
c
Department of Pathology, Clinical and Toxicological Analysis, Center of Health Sciences, University Hospital, State University of Londrina, Londrina, Parana, Brazil

A R T I C L E I N F O A B S T R A C T

Section Editor: Dr Richard Aspinall It is known that autonomic modulation is responsive to ovarian hormone levels and that estrogen increases nitric
Keywords: oxide (NO) bioavailability. However, little is known about the interaction of nitric oxide synthase (NOS) iso-
Estradiol forms with autonomic modulation, oxidative stress and cardiovascular risk in females. This study aimed to
Heart hate variability investigate cardiovascular, autonomic and oxidative parameters after selective NOS inhibition. A spectral ana-
Paraoxonase 1 lysis of systolic arterial pressure (SAP) and heart rate variability (HRV) was performed. NO levels, total anti-
S-methylisothiourea oxidant capacity (TRAP), lipid hydroperoxides (LOOH) and paraoxonase 1 (PON1) activity were measured in the
L-NG-nitroarginine methyl ester plasma of rats treated with L-NG-nitroarginine methyl ester (L-NAME), S-methylisothiourea (SMT) or saline.
Wistar rats, ovariectomized (OVX) with or without estradiol treatment (1 mg/kg/day) or with a false ovar-
iectomy (SHAM), were submitted to artery and vein catheterization. Cardiovascular parameters were evaluated
before and after the administration of saline or NOS inhibitors. After 2 h, plasma samples were collected for
biochemical measurement. At baseline, cardiovascular and autonomic parameters were not different among the
groups. L-NAME, the constitutive NOS isoform (cNOS) inhibitor, promoted an increase in mean arterial pressure
(MAP) and a reduction in the low frequency band (LF) of SAP of SHAM rats, but this increase was smaller in OVX
animals, which also showed a reduction in PON1 activity. The decreased activity of PON1 caused by L-NAME
was prevented in the OVX + E group. SMT, an inducible NOS isoform (iNOS) inhibitor, promoted an increase in
MAP and in the LF of SAP, in interbeat interval (IBI) parameters at LFnu and in LF/HF ratio of HRV in all groups,
but the OVX + E had lower levels of NO when compared with the OVX group. Our data suggest that while cNOS
contributes to maintaining the activity of PON1 in OVX rats, iNOS activity maintains the levels of NO in OVX
+ E rats.

1. Introduction stronger cardiac sympathetic modulation compared with non-age-

It is recognized that gender differences exist in cardiovascular dis-
ease and in cardiovascular function (Hayward et al., 2000). Men are
generally at a greater risk for cardiovascular and renal disease than age-
matched premenopausal women, and physiologically, men's blood
pressure tends to be higher (Reckelhoff, 2001). Premenopausal women
also have greater endothelium-dependent vasodilation compared to
similarly aged men, a difference that disappears after menopause
(Sarabi et al., 1999).
Cardiac autonomic modulation also changes with ovarian hormone
levels: post-menopausal women have weaker vagal modulation and
matched premenopausal women (Liu et al., 2003). Autonomic functions
are altered after an oophorectomy in premenopausal women (Mercuro
et al., 2000), and estrogen replacement therapy restores these altera-
tions (Liu et al., 2003).
A noninvasive method to assess sympathovagal balance is the power
spectral analysis of heart rate variability (HRV) (Petrofsky et al., 2009).
Alterations in HRV, which primarily reflect the tonic autonomic mod-
ulation, may have substantial clinical implications (Campos et al.,
2014). In the spectral analysis, female rats had the most prominent high
frequency (HF) component, which represents the parasympathetic
drive, during estrus compared with the OVX and diestrus groups; the

⁎
Corresponding author at: Departamento de Ciências Fisiológicas, Centro de Ciências Biológicas, Universidade Estadual de Londrina, Rodovia Celso Garcia Cid, Km 380, Campus
Universitário, CEP 86055-900 Londrina, PR, Brazil.
E-mail address: martinspinge@uel.br (M.C. Martins-Pinge).

http://dx.doi.org/10.1016/j.exger.2017.07.016
Received 22 April 2017; Received in revised form 23 July 2017; Accepted 24 July 2017
Available online 28 July 2017
0531-5565/ © 2017 Elsevier Inc. All rights reserved.
J.C. Castardo-de-Paula et al.
estrous cyclicity, as well as the cycle-related HF changes, disappeared
with ovariectomy (Kuo et al., 2010), corroborating estradiol's partici-
pation in autonomic modulation. It was also demonstrated that HRV are
significantly higher in both high-estradiol-dose and low-estradiol-dose
groups compared to the OVX group (Campos et al., 2014).
Estrogen is an important hormone for the cardiovascular system.
This importance comes to light with aging and consequential meno-
pause when estrogen is lost and there are some inflammatory altera-
tions (Knowlton and Lee, 2012). Alterations such as increased TNF, IL-6
(Chung et al., 2009; Donato et al., 2008) and oxidative stress (Jackson
and McArdle, 2011; Stice et al., 2011) are factors that contribute to
cardiovascular dysfunction. Estrogen also promotes the indirect upre-
gulation of antioxidant gene expression and increases endothelial nitric
oxide synthase (eNOS) activity while decreasing superoxide production
(reviewed by Knowlton and Lee, 2012).
Paraoxonase-1 enzyme (PON1) may be involved in the relation
between the estrogen and the antioxidant systems. Synthesized in the
liver, this hydrolase is associated with HDL lipoproteins, functions as a
“bioscavenger” (Bojic et al., 2014; Li et al., 2013) and is considered a
cardiovascular risk marker. It has been observed in humans that sur-
gical menopause reduces the enzyme activity of PON1 (Kumru et al.,
2005), while estradiol stimulates PON1 activity (Ahmad and Scott,
2010). These estrogenic effects are promising for the treatment of dis-
orders related to oxidative stress.
Estrogen is among the many hormones that increase NO production
(Duckles and Miller, 2010). Estrogen receptors, which are alpha, beta
and G-protein coupled (ERalpha, ERbeta and GPER, respectively), have
identified as having NO-linked mechanisms in adult animals. 17Beta-
estradiol stimulates the expression of eNOS and iNOS in cardiac myo-
cytes (Nuedling et al., 1999). ERalpha is co-localized to endothelial
cells where it activates eNOS (Knowlton and Lee, 2012). Additionally,
estradiol increases eNOS expression and NO in cultured human cor-
onary artery endothelial cells (Duckles and Miller, 2010). Production of
NO in the endothelium is also the mechanism of GPER vasodilation.
Aging results in the downregulation of GPER in rats and is asso-
ciated with reduced vascular relaxation responsiveness to estradiol or
ER agonists (Prossnitz and Barton, 2014). Additionally, it was estab-
lished that the majority of estrogen-stimulated NO production in cor-
onary artery smooth muscle is from neural nitric oxide synthase (nNOS)
(White et al., 2010). Additionally, estrogen attenuates vasoconstriction
by an ERbeta-mediated increase in iNOS expression in wild-type mouse
blood vessels (Zhu et al., 2002).
However, the systemic effects of NOS isoforms and their interaction
with autonomic modulation, oxidative stress and cardiovascular risk in
females have not yet been addressed. This study aimed to investigate
cardiovascular, autonomic and oxidative parameters after selective
NOS inhibition.
2. Methods
2.1. Animals
Adult female Wistar rats were maintained in ventilated and con-
trolled temperature chambers (22 ± 1 °C) on a 12-hour light-dark
cycle with food (Nuvilab CR-1; Nuvital®, Colombo, Paraná, Brazil) and
tap water available ad libitum. The experiments were conducted during
the light phase. All experimental protocols were approved by the
Institutional Animal Ethics Committee of the State University of
Londrina, Paraná, Brazil (CEUA-UEL - Comissão de Ética no Uso de
Animais, process number 276.2013.81) and performed in accordance
with the standards established by the National Council for Animal
Testing Control (CONCEA), Brazil.
2.2. Surgical proceedings and experimental groups
Rats were subjected to bilateral ovariectomy or false surgery under
39
Experimental Gerontology 97 (2017) 38–48
ketamine and xylazine anesthesia (100 and 6.7 mg/kg, i.p.; Ceva Santé
Animale, São Paulo, Brazil) and 24 h after were divided into experi-
mental groups (n = 4–8): an ovariectomy control group (OVX) that
received 0.5 mL/kg estradiol vehicle per day, p.o. (almond oil,
Generophlora drugs, Londrina, Paraná, Brazil); an OVX plus estradiol
treatment (OVX + E) group that received estradiol valerate 1 mg/kg
per day, p.o. (Hangzhou Hetd Industry Co., Zhejiang, China) for
8 weeks (Ceylan-Isik et al., 2009); and a SHAM group.
The estrous cycle in the SHAM-operated females was monitored by
microscopic investigation of vaginal smears (Kauser et al., 1997), and
only females in the estrus phase were used in the experiments.
Eight weeks after the surgery and 24 h before the experiments,
under ketamine–xylazine anesthesia, a polyethylene catheter was in-
serted into the femoral artery and vein and externalized dorsally to
record MAP and heart rate (HR) during a conscious state, according to
previous studies (Ariza et al., 2015; da Cunha et al., 2014).
2.3. Measurement of cardiovascular parameters
Twenty-four hours after catheterization, the animals were kept in
their cages, and basal recordings were obtained for at least 20 min
before starting the protocol. MAP and HR were recorded by an
MLT0380 blood pressure transducer connected to a Powerlab system 4/
20 T (ADInstruments®) while the animals were awake and freely
moving (da Cunha et al., 2014).
After the basal recording, NOS isoforms were inhibited by a bolus
injection (i.v.) of either L-NAME (10 mg/kg), a selective inhibitor of
calcium-dependent isoforms (i.e., cNOS) (Vitecek et al., 2012), or of
SMT (3 mg/kg), a potent inhibitor of iNOS (Su et al., 2007; Szabo et al.,
1994), both from Santa Cruz Biotechnology®, Texas, USA. Control
groups received physiological saline 0.9% (1 mL/kg).
Cardiovascular parameters were recorded for 2 h (Mehanna et al.,
2007). At the end of recording, plasma samples were collected for
measurement of the nitrite and lipoperoxidation levels, the plasma
activity of paraoxonase 1 (PON1) and the total antioxidant capacity of
plasma. A standard laboratory scale was used to measure body weight,
tibia length and uterus weights (Gore et al., 2002; Voltera et al., 2008).
2.4. Heart rate and systolic arterial pressure variability
The recordings of arterial pressure, 10 min from basal and the last
3 min of the 2 h post-treatment, were processed using a specific com-
puter program (LabChart 7 Pro®, ADInstruments, Bella Vista, Australia)
that was able to detect inflection points in pressure pulses to generate a
beat-by-beat time series of pulse interval (PI) and systolic pressure
(SAP). The frequency domain (PI and SAP variability) spectral analysis
was performed using custom software (CardioSeries® v2.4). For power
spectral analysis of the PI and SAP variability, the beat-by-beat series of
these parameters were resampled at data points every 100 ms by cubic
spline interpolation (10 Hz). Next, the interpolated series was divided
into half-overlapping segments. All segments were visually inspected,
and those with artifacts or nonstationary data were excluded from
analysis. Then, a Hanning window was used to attenuate the side ef-
fects, and spectra were calculated for all segments using a fast Fourier
transform (FFT) algorithm for a discrete time series. Finally, the spectra
were integrated in low (LF: 0.2–0.75 Hz) and high frequency (HF:
0.75–3.0 Hz) bands. The relative power of the LF and HF bands of the PI
spectra were calculated taking into account the total power of the
spectra minus the power of the very low oscillations (< 0.2 Hz). To
assess cardiac sympathovagal balance, we also calculated the ratio
between the power of the LF and the HF bands (LF/HF ratio) of the PI
spectrum (Dutra et al., 2013).
2.5. Biochemical analysis
Sample nitrite was measured as an estimate of NO levels and
J.C. Castardo-de-Paula et al. Experimental Gerontology 97 (2017) 38–48

determined as previously described (Panis et al., 2012). NO con-

centration in plasma was estimated by measuring nitrite in a Griess
reaction assay with some modifications. To determine sample nitrite
concentration, a calibration curve was prepared by diluting NaNO2 in
distilled sterile water. Griess reagent (100 μL) was added in triplicate to
the wells of the first three columns of each microtiter plate. The ab-
sorbance was read at 505 nm using a standard microplate reader
(Multiskan EX, LabSystems®, Minnesota USA). The final results were
expressed in μM nitrite.
Chemiluminescence reactions were used for analyzing the level of
lipoperoxide formed during exposure to NOS inhibitors. An increase in
the chemiluminescence level is related to previous in vivo oxidative
stress, which leads to antioxidant defense consumption and the for-
mation of lipoperoxides with consequential photon emissions (Gonzalez
Flecha et al., 1991; Panis et al., 2012). The chemiluminescent reaction
was initiated by the addition of tert-butyl (10 μL) at a final con-
centration of 3 mM, and the reaction was read in a GloMax lumin-
ometer (TD 20/20 Turner Designers®). The results are expressed in
relative light units (RLU), and the obtained curve was used as a qua-
litative indicator of lipoperoxidation. Quantitative results were ob-
tained after area under the curve integration using OriginLab 7.5 soft-
ware.
Total plasma activity of PON1 was determined as previously de-
scribed (Richter et al., 2008). The rate of hydrolysis of phenyl acetate
was determined in a microplate reader (EnSpire, Perkin Elmer®, USA)
at 270 nm, and the temperature was maintained at 25 °C. The activity
was expressed in U/mL based on the phenyl acetate molar extinction
coefficient of 1.31 mmol/L/cm.
The TRAP parameter was evaluated according to the method de-
scribed by Panis et al. (2012) in a microplate reader (Victor X-3, Perkin
Elmer®, USA). Experimental conditions were a running time of 25 min,
a response range from 300 to 620 nm and a temperature of 30 °C. This
method detects hydro and/or liposoluble antioxidants present in the
plasma. The results are expressed in μM Trolox.

2.6. Statistical analysis

Initially, an exploratory analysis was conducted to evaluate the

normal distribution (Shapiro-Wilk test) and the homogeneity of var-
iance (Levene test) of each variable. For variables that presented
normal distribution and homogeneity of variance, parametric analysis
was conducted, and values are reported in mean ± standard error
mean. Two-hour response graphs were analyzed with two-way ANOVA
with repeated measures (two-way RMANOVA). The analyzed factors
were hormone treatment (OVX, OVX + E, SHAM), time after NOS in-
hibition, and time x hormone treatment interaction. If significant in-
teractions were detected, a one-way ANOVA was conducted to de-
termine significant changes in dependent variables. Other parameters
were analyzed by one-way ANOVA followed by a Bonferroni test. For
nonparametric data, multiple comparisons were performed using the
Kruskal–Wallis test followed by the Dunn test, and values are reported Fig. 1. Morphometric parameters of sham-operated, ovariectomized (OVX), or estrogen-
as the median (interquartile range values). In all cases, P ≤ 0.05 was treated OVX (OVX + E) rats. Daily estrogen treatment (1 mg/kg, p.o.) began on the day
after the ovariectomy and lasted 8 weeks. OVX rats received almond oil at the same time.
considered statistically significant.
(a) Weight gain, (b) tibia length, (c) relation between uterus weight and tibia length. (a)
and (b) are bar graphs showing the means ± S.E.M. analyzed by one-way ANOVA fol-
lowed by Bonferroni's multiple comparison test. (c) shows medians ± interquartile range
3. Results analyzed by the Kruskal–Wallis test followed by Dunn's test. Weight gain: body weight at
time of euthanasia - body weight at time of surgery. The number of rats in each group is
OVX rats presented significantly greater body weight gain when shown in parentheses. *P < 0.01 versus SHAM; #P < 0.01 versus OVX (one-way
compared to the other two groups (Fig. 1a) with no difference among ANOVA followed by Bonferroni's test).

their sizes, as evidenced by the length of the tibia (Fig. 1b). Like Paigel
et al. (2011), the effectiveness of the ovariectomy (OVX group) was 3.1. Hemodynamic and autonomic parameters
determined by the absence of ovarian tissue and marked atrophy of the
uterus compared to the SHAM group (Fig. 1c), and the uterine hyper- MAP and HR values during the 120 min of recording after injections
trophy was evident in the ovariectomized group treated with estradiol of saline, L-NAME or SMT are shown in Fig. 2. A statistical analysis of
(OVX + E).

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J.C. Castardo-de-Paula et al.
Fig. 2. Changes in mean arterial pressure (MAP, panels a, b and c) and heart rate (HR, panels d, e and f) in catheterized sham-operated, OVX, or estrogen-treated OVX (OVX + E) rats
after intravenous NOS inhibitor injection. Estrogen daily treatment (1 mg/kg, p.o.) began on the day after the ovariectomy and lasted 8 weeks. OVX rats received almond oil at the same
time. (a) and (d) saline (0.9%, 1 mL/kg) injection, (b) and (e) L-NAME (10 mg/kg) injection, (c) and (f) SMT (3 mg/kg) injection. Values are means ± S.E.M. The number of rats in each
group is shown in parentheses. P > 0.05 for area under the curves.
the areas under the curves was carried out and no differences were
observed.
The baseline values of MAP and HR and the spectral measures of
cardiovascular autonomic control can be observed at time zero (T0) in
Figs. 3, 4 and 5. They were similar in SHAM, OVX and OVX + E rats.
Considering that the blood samples for biochemical measurements were
collected only at the end of the protocol, the analysis and comparison at
T0 was done only at 2 h post-injection (T2).
Fig. 3 shows the analysis of the cardiovascular and autonomic
parameters before and after injection of 0.9% saline. The analysis of
variance (two-way RMANOVA) of MAP indicated an interaction be-
tween the time after injection and treatment (P < 0.05, Fig. 3a).
However, Bonferroni's test revealed no significant differences among
groups within each time or between times within each group. As ex-
pected, saline injection did not alter HR parameters (Fig. 3b). Spectral
analysis of the systolic blood pressure (SBP) and the PI were conducted
in order to investigate the autonomic influence on cardiovascular
parameters. Parameters in the frequency domain were not altered in
SBP (low frequency, SBP-LF; 0.2–0.75 Hz, Fig. 3d), in pulse or in in-
terbeat interval (IBI) parameters at high frequency (IBI-HFnu,
0.75–3 Hz, Fig. 3c) or low frequency (IBI-LFnu, Fig. 3e), as well as in
the LF/HF ratio (IBI-LF/HF, Fig. 3f).
L-NAME injection promoted an increase in MAP in all groups at 2 h
when compared to T0 (two-way RMANOVA: effect of time and
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Experimental Gerontology 97 (2017) 38–48
treatment, P < 0.05, Fig. 4a). The analysis among groups (treatment
factor) indicated that after 2 h, MAP increases by L-NAME were smaller
in the OVX group when compared to the SHAM group. Since the MAP
measurements in the OVX + E group were not different from those in
the SHAM group, treatment with estradiol may have prevented this
difference. Spectral analysis showed that L-NAME equivalently reduced
vascular sympathetic modulation in all groups (two-way RMANOVA:
time effect on SBP-LF, P < 0.05, Fig. 4d). However, cardiac para-
meters (HR, IBI-LFnu, IBI-HFnu, IBI-LF/HF) were not altered by L-
NAME injection (Fig. 4b, c, e and f, respectively). This suggests that
females, generally and independently of estrogen, do not present car-
diac alterations after 2 h of exposure to cNOS inhibition.
Cardiovascular and autonomic effects of iNOS inhibition were
analyzed by SMT injection. MAP (Fig. 5a) and vascular sympathetic
modulation (SBP-LF, Fig. 5d) increased after 2 h, indicated by the time
effect of two-way RMANOVA. HR did not change 2 h after the SMT
injection (Fig. 5b); meanwhile, cardiac frequency domain parameters of
the spectral analysis indicated a predominance of sympathetic mod-
ulation without differences among groups, because IBI-HFnu decreased
and IBI-LFnu and IBI-LF/HF increased (two-way RMANOVA, time ef-
fect, P < 0.05, Fig. 5c, e and f, respectively).
J.C. Castardo-de-Paula et al. Experimental Gerontology 97 (2017) 38–48

Fig. 3. Cardiovascular parameters and spectral hemodynamic data for catheterized sham-operated, OVX, or estrogen-treated OVX (OVX + E) rats before and after intravenous saline
injection (0.9%, 1 mL/kg). (a) Mean arterial pressure (MAP), (b) heart rate (HR), (c) spectral densities of interbeat intervals (IBI) in the high-frequency (IBI-HFnu), (d) spectral density of
systolic blood pressure in the low-frequency (SBP-LF), (e) spectral densities of IBI in the low-frequency (IBI-LFnu) and (f) in the LF/HF ratio (IBI-LF/HF). Daily estrogen treatment (1 mg/
kg, p.o.) began one day after the ovariectomy and lasted 8 weeks. OVX rats received almond oil at the same time. Values are means ± S.E.M. The number of rats in each group is shown
in parentheses. P > 0.05 (two-way RMANOVA followed by Bonferroni's test).

3.2. Plasmatic biochemical parameters
Plasma biochemical parameters 2 h after saline injection are shown
in Fig. 6. There were no differences among groups in the nitric oxide
level (Fig. 6a), PON1 activity (Fig. 6c), or plasma lipoperoxidation
(Fig. 6d). The analysis of variance indicated differences among groups
in TRAP (Fig. 6b), but Bonferroni's post-test did not identify them. We
believe that there was a decrease in TRAP in the OVX group in relation
to the SHAM.
Concerning the effects of cNOS inhibition by L-NAME injection on
the plasma biochemical parameters analyzed, OVX rats presented a
decrease in PON1 activity 2 h after L-NAME injection (Fig. 7c), and
estradiol treatment prevented this alteration.
Fig. 8 illustrates the effects of SMT injection on the plasma bio-
chemical parameters of female rats. SMT changed NO levels differently
among groups (Fig. 8a) according to hormonal status because iNOS
inhibition decreased plasma NO in OVX + E rats when compared to
OVX.
4. Discussion
The efficacy of hormonal deprivation, generated by OVX, and
treatment with estradiol were confirmed by the results observed on
uterine and body weights. Data are in accordance with the literature
(Ceylan-Isik et al., 2009; Paigel et al., 2011), where the same time of
evaluation of the OVX and, in the case of Ceylan-Isik et al. (2009), the
same model of hormonal treatment prevented the differences among
groups in the body and uterine weights.
As observed by El-Mas and Abdel-Rahman (2009), the study of
hemodynamic variability is an important measure of cardiovascular
autonomic function. Our study demonstrated that the hemodynamic
response of Wistar rats after 2 h of NOS inhibition changes according to
the inhibited pathway and is independent of estrogen, for the most part.
The L-NAME inhibitor reduced the LF spectral density of the SBP, while
the SMT increased it. The SMT also reduced the HFnu of the PI and
increased the LFnu and the LF/HF ratio. On the other hand, estradiol
influences the formation of plasma NO and the activity of an important
cardioprotective enzyme, PON1. NO from the constitutive pathway is
more important in maintaining PON1 activity in ovariectomized rats,
whereas NO from the inducible pathway is more important for the total
plasma levels of this gas in rats treated with estradiol.
The physiological profile of the cardiovascular response during
pressure recording can be observed in the groups that received 0.9%
saline. Two-way RMANOVA revealed no differences in cardiovascular

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J.C. Castardo-de-Paula et al. Experimental Gerontology 97 (2017) 38–48

Fig. 4. Cardiovascular parameters and spectral hemodynamic data of catheterized sham-operated, OVX, or estrogen-treated OVX (OVX + E) rats before and after intravenous L-NAME
injection (10 mg/kg). (a) Mean arterial pressure (MAP), (b) heart rate (HR), (c) spectral densities of interbeat intervals (IBI) in the high-frequency (IBI-HFnu), (d) spectral density of
systolic blood pressure in the low-frequency (SBP-LF), (e) spectral densities of IBI in the low-frequency (IBI-LFnu) and (f) in the LF/HF ratio (IBI-LF/HF). Daily estrogen treatment (1 mg/
kg, p.o.) began one day after the ovariectomy and lasted 8 weeks. OVX rats received almond oil at the same time. Values are means ± S.E.M. The number of rats in each group is shown
in parentheses. *P ≤ 0.05 versus corresponding SHAM. Differences at time zero inside each group: (S) SHAM, (O) OVX and (E) OVX + E, P ≤ 0.05 (two-way RMANOVA followed by
Bonferroni's test).

or autonomic parameters among groups or between 2 h post-saline and
T0. Similar results have been reported for Sprague-Dawley OVX rats
(16 weeks after surgery) (El-Mas and Abdel-Rahman, 2009) where MAP
and HR remained unchanged. However, estradiol treatment started
during the third postoperative week decreased MAP (El-Mas and Abdel-
Rahman, 2009, 2014). Subramanian et al. (2011) and Subramanian
et al. (2015) observed increased MAP and HR after estradiol treatment
in Sprague-Dawley rats. Other results describe reduction of HR after a
similar hormonal protocol to ours (Ceylan-Isik et al., 2009), which
shows the lack of consensus regarding the effects of estradiol treatment
on hemodynamic variables in rats.
The literature regarding cardiovascular spectral analysis in female
rats is varied. El-Mas and Abdel-Rahman (2009, 2014) reported that
estradiol restored the PI spectrum densities of Sprague-Dawley OVX
rats to the level of SHAM controls, with cardiac parasympathetic pre-
dominance (decreased LF/HF). In addition, estradiol reduced LF oscil-
lations in SBP, which suggests a reduction in sympathetic vasomotor
tone. In contrast to these observations, Dias et al. (2010) reported an
increase in LF and a reduction in HF HRV of estradiol-treated Wistar
rats, concluding that estradiol disturbs the autonomic balance of the HR
towards a sympathetic predominance.
The results presented above used different doses and periods of
estradiol treatment than those in the present study, and even the rat
strains varied, which helps to explain the discrepancies among the
available information. It is important to note that even in studies of the
same duration, different results can be found (Campos et al., 2014; Dias
et al., 2010), suggesting caution in stating that estrogen is good or bad
for the cardiovascular system.
At 2 h, the ovariectomy appears to have promoted a decrease in the
TRAP of the rats (not confirmed by the Bonferroni post-test). The re-
duction in plasma antioxidant capacity by ovarian withdrawal and its
recovery, or prevention, by estradiol treatment has been previously
observed (Hernandez et al., 2000). Estradiol treatment was associated
with an increase in lipoperoxides, and the authors concluded that their
results might reflect an imbalance in the redox state, especially in
oxidative processes, that could reduce NO activity. The oxidative
parameters of the present results do not allow the same inference,
probably because these proposed changes, in the case of Wistar rats, are
located in tissues of importance to the cardiovascular system, such as
resistance vessels, and plasma measurements, due to the influence of
other tissue parameters, would be unable to detect small but not less
important changes.

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J.C. Castardo-de-Paula et al. Experimental Gerontology 97 (2017) 38–48

Fig. 5. Cardiovascular parameters and spectral hemodynamic data for catheterized sham-operated, ovariectomized (OVX), or estrogen-treated OVX (OVX + E) rats before and after
intravenous SMT injection (3 mg/kg). (a) Mean arterial pressure (MAP), (b) heart rate (HR), (c) spectral densities of interbeat intervals (IBI) in the high-frequency (IBI-HFnu, 0.75–3 Hz),
(d) spectral density of systolic blood pressure (SBP) in the low-frequency (SBP-LF, 0.2–0.75 Hz), (e) spectral densities of IBI in the low-frequency (IBI-LFnu) and (f) in the LF/HF ratio (IBI-
LF/HF). Daily estrogen treatment (1 mg/kg, p.o.) began on the day after the ovariectomy and lasted 8 weeks. OVX rats received almond oil at the same time. Values are means ± S.E.M.
The number of rats in each group is shown in parentheses. Differences at time zero inside each group: (S) SHAM, (O) OVX and (E) OVX + E, P ≤ 0.05 (two-way RMANOVA).

Inhibition of NOS, both with L-NAME and with SMT, promoted an
increase in MAP of the rats in the 3 experimental groups, confirming
that the lower availability of NO is an important risk factor for hy-
pertension (Torok, 2008) and demonstrating that NO participation in
cardiovascular control is important for all females.
The pharmacodynamics of L-NAME begin, once within a biological
system, with its prompt hydrolysis to L-NG-nitroarginine (L-NNA). L-
NNA non-covalently interacts with all NOS isoforms, but its coupling to
iNOS is rapidly reversed, whereas binding to cNOS is a relatively
slower, time-dependent process [reviewed by Vitecek et al., 2012].
Chen and Hu (1997) observed in a dose-response curve study of L-
NAME that the maximal response of increased blood pressure and re-
duced HR was to a dose of 10 mg/kg, the dose selected for the present
study.
The MAP and HR results observed after exposure to L-NAME are in
accord with Gardiner et al. (1990), where hypertension without sig-
nificant change to HR was observed at the same dose and period. In
addition, L-NAME promoted vasoconstriction of renal, mesenteric and
lower limb vascular beds in Long Evans rats. Hu et al. (1997) studied
the effects of L-NAME on arterial hemodynamics and concluded that
acute NO blocking predominantly affects peripheral vascular resistance
with relatively weak actions on arterial impedance and ventricular
function.
In this study, L-NAME decreased LF-SBP with no differences among
groups. These LF fluctuations may occur due to a reduction in vaso-
motor sympathetic activity, a reduction in peripheral vascular response
to the neurotransmitter, or saturation of peripheral sympathetic nerve
discharges with a loss of modulatory action (Silva and Januário, 2005).
However, studies have implicated endothelin-1, a physiological an-
tagonist of the vasodilatory actions of NO, in the mechanisms of acute
hypertensive action of L-NAME (Rapoport, 2014). An increase in the
activation of endothelin-1 receptor (ETA), rather than increased acti-
vation of the alpha-1 adrenergic, angiotensin AT1 or vasopressinergic
receptors, has been attributed to L-NAME hypertension (Banting et al.,
1996). In fact, the use of an endothelin receptor antagonist promoted an
increase in LF-SBP (Souza et al., 2008), which suggests that its activa-
tion may lead to a reduction of LF in L-NAME hypertension. Thus, al-
ternative mechanisms of the sympathetic autonomic nervous system
may be more important in the pressor response to L-NAME.
The hypertension generated by L-NAME was less intense in the OVX
group than in the SHAM. OVX rats have reduced cNOS activity in the
aorta, and daily treatment with estradiol or raloxifene prevents this
change (Pavo et al., 2000). Reduced vascular conductance, an index of
vasodilation, has also been reported in OVX rats (Hernandez et al.,

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J.C. Castardo-de-Paula et al. Experimental Gerontology 97 (2017) 38–48

Fig. 6. Plasma biochemical parameters for catheterized sham-operated, ovariectomized (OVX), or estrogen-treated OVX (OVX + E) rats before and after intravenous saline injection
(0.9%, 1 mL/kg). (a) Nitrite levels, (b) total radical-trapping antioxidant parameter (TRAP), (c) paraoxonase 1 (PON1) activity and (d) lipoperoxidation. Daily estrogen treatment (1 mg/
kg, p.o.) began on the day after the ovariectomy and lasted 8 weeks. OVX rats received almond oil at the same time. Values are means ± S.E.M. The number of rats in each group is
shown in parentheses (one-way ANOVA followed by Bonferroni's multiple comparison test).

2000). Similarly, Lamas et al. (2015) observed decreased action of L-
NAME in the mesenteric vessels of OVX rats compared to SHAM or
estradiol-treated rats, and OVX animals showed a reduction in eNOS
expression and an increase in iNOS.
Plasma PON1 enzyme activity was lower in the OVX group under
cNOS inhibition when compared to SHAM or OVX + E. Menopause
reduces the activity of PON1, an important bioscavenger (Kumru et al.,
2005), while estradiol has been shown to stimulate this enzyme
(Ahmad and Scott, 2010), restoring its levels in treated animals.
However, until these data were obtained, there was no reference for
PON1 activity and the acute use of L-NAME in females since this is the
first work to present this measurement.
Reduced activity of PON1 and L-NAME were observed in L-NAME-
induced chronic hypertension in males (Gocmen et al., 2014) where it
was stated that increased lipid peroxidation products could inactivate
PON1, or the increase in MAP could lead to a decrease in the hydrolysis
capacity of the enzyme. This relation between increased oxidative stress
and reduced oxidative activity, due to the progressive increase in MAP,
may have occurred in OVX rats since these animals have a pro-oxidant
profile, including in tissues important for cardiovascular function
(Ceravolo et al., 2013; Claudio et al., 2014).
The literature shows a relation between the presence of physiolo-
gical iNOS in females and estradiol levels. Upmacis et al. (2011)
detected iNOS in the hearts of male and female mice. Nuedling et al.
(1999) observed that estradiol stimulates the expression of eNOS and
iNOS in the cardiomyocytes of Wistar-Kyoto rats of both sexes and that
ovariectomy reduces the levels of both isoforms. It was observed that
estrogen induces the expression of iNOS in aortic rings without en-
dothelium, guaranteeing direct vasodilator action on vascular smooth
muscle (Zhu et al., 2002). However, constitutive activity of iNOS is
present in males as well, suggesting an independent action of ovarian
hormones that is important for the cardiovascular system, where iNOS
tonic activity exists in the central nervous system to inhibit the sym-
pathetic efflux of the rostral ventrolateral medulla (Chan et al., 2001).
The MAP response to administration of the selective inhibitor of
iNOS, SMT, is in agreement with data for anesthetized males where
doses ranging from 1 to 10 mg/kg promoted maximal responses within
5 min (Southan et al., 1995). At a dose of 3 mg/kg, the inhibitory action
on iNOS is maintained for 6 h (Su et al., 2007). Measurements of au-
tonomic modulation indicated a sympathetic prevalence after 2 h, with
increased LF-SBP, LFnu and LF/HF of HRV and decreased HFnu. Data
available for female rats indicates no influence of iNOS inhibition on
hemodynamic or autonomic parameters under physiological conditions
(El-Mas and Abdel-Rahman, 2014; El-Mas et al., 2006). However, these
studies were performed with female Sprague-Dawley rats and used
different inhibitors. The relation between iNOS inhibition and increased

45
J.C. Castardo-de-Paula et al. Experimental Gerontology 97 (2017) 38–48

Fig. 7. Plasma biochemical parameters for catheterized sham-operated, ovariectomized (OVX), or estrogen-treated OVX (OVX + E) rats before and after intravenous L-NAME injection
(10 mg/kg). (a) Nitrite levels, (b) total radical-trapping antioxidant parameter (TRAP), (c) paraoxonase 1 (PON1) activity and (d) lipoperoxidation. Daily estrogen treatment (1 mg/kg,
p.o.) began on the day after the ovariectomy and lasted 8 weeks. OVX rats received almond oil at the same time. Values are means ± S.E.M. The number of rats in each group is shown in
parentheses. *P < 0.01 versus SHAM; #P < 0.01 versus OVX (one-way ANOVA followed by Bonferroni's test).

MAP with activation of the sympathetic autonomic nervous system
observed in the present study has been demonstrated in males in a study
with rats (Chan et al., 2001) and in the analysis of HRV in iNOS
knockout mice (Mani et al., 2006). To date, this is the first observation
of these changes in spectral analysis after acute inhibition of iNOS in
female Wistar rats.
Estrogen stimulation of NO and its importance in the cardiovascular
system is well established (Knowlton and Lee, 2012). Upmacis et al.
(2011) observed higher levels of nitrite and nitrate in female urine
when compared to male urine, and these levels were lower in iNOS
knockout animals. Studies have demonstrated the ability of estradiol to
physiologically increase the expression and activity of iNOS (Nuedling
et al., 1999; Zhu et al., 2002). Therefore, the significant reduction in the
plasma nitrite levels in OVX + E rats when compared to OVX after
inhibition of iNOS with SMT confirms the action of estradiol as a sti-
mulator of NOS activity.
5. Conclusion
This study was carried out to verify cardiovascular, autonomic and
plasma parameter variations after blocking the constitutive and in-
ducible pathways of the NOS enzymes in OVX and estradiol treated rats.
Considering the observed aspects, both the constitutive and the
inducible pathways are important for blood pressure control in females,
and the increase in MAP by cNOS inhibition seems to depend on per-
ipheral vasoconstrictor agents, such as endothelin, while inhibition of
iNOS can be a mediator of a central sympathetic disinhibition. The
smaller increase in MAP by L-NAME observed in OVX rats leads to the
assumption that ovariectomy promotes a reduction in the amount, or
activity, of cNOS and a pro-oxidant state that, after the inhibition of the
few remaining enzymes, contributed to the reduction of PON1 activity.
This state of increased cardiovascular risk, revealed by inhibition of
cNOS in OVX rats, is prevented by treatment with estradiol. It is pos-
sible that the organism, in the absence of ovarian stimulation, main-
tains NO plasma levels through increased expression or activity of
iNOS, and this mechanism could be enhanced by treatment with es-
tradiol, since iNOS inhibition decreased the levels of nitrite in OVX + E
rats.
Conflict of interest
The authors declare no conflicts of interest.
Acknowledgments
Daniel Penteado Martins Dias, for assistance during the execution of

46
J.C. Castardo-de-Paula et al. Experimental Gerontology 97 (2017) 38–48

Fig. 8. Plasma biochemical parameters for catheterized sham-operated, ovariectomized (OVX), or estrogen-treated OVX (OVX + E) rats before and after intravenous SMT injection
(3 mg/kg). (a) Nitrite levels, (b) total radical-trapping antioxidant parameter (TRAP), (c) paraoxonase 1 (PON1) activity and (d) lipoperoxidation. Daily estrogen treatment (1 mg/kg,
p.o.) began on the day after the ovariectomy and lasted 8 weeks. OVX rats received almond oil at the same time. The number of rats in each group is shown in parentheses. Values are
means ± S.E.M., except for (d) medians ± interquartile range analyzed by the Kruskal–Wallis test. #P < 0.01 versus OVX (one-way ANOVA followed by Bonferroni's test).

the spectral analysis.
Funding
This work was supported by the Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq), which provided the
fellowship to JCCP (870009/2009-5) and research fellowships for PPF
(307787/2015-0) and MCMP (307544/2016-8).
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