1521-0103/12/3422-568–575$25.00
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
JPET 342:568–575, 2012
Vol. 342, No. 2
191940/3785022
Estrogen Provokes the Depressant Effect of Chronic Nicotine
on Vagally Mediated Reflex Chronotropism in Female Rats
Mahmoud M. El-Mas, Hanan M. El-Gowelli, Sahar M. El-Gowilly, Mohamed A. Fouda, and
Mai M. Helmy
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt
Received January 13, 2012; accepted May 21, 2012
Introduction
Tobacco smoking is a major risk factor for cardiovascular
diseases including hypertension, atherosclerosis, coronary
heart disease, acute myocardial infarction, and sudden cardiac death (Barnoya and Glantz, 2005; Bullen, 2008). The
nicotine content of tobacco smoke is highly blamed for the
deleterious cardiovascular consequences of cigarette smoking
(Balakumar and Kaur, 2009). The increased cardiovascular
risk is also seen in people using nicotine replacement therapy
to facilitate tobacco cessation (Schnoll and Patterson, 2009).
The adverse effects of nicotine have been attributed largely to
increased activity of the sympathetic nervous system (Narkiewicz et al., 1998). Arterial baroreceptor dysfunction is another important mechanism that contributes to the increased
vulnerability of smokers to cardiovascular risk (Mancia et al.,
1997). Nicotine diminishes the baroreflex gain through direct
This work was supported by the Science and Technology Development Fund
of Egypt [Grant STDF 502].
Article, publication date, and citation information can be found at
http://jpet.aspetjournals.org.
http://dx.doi.org/10.1124/jpet.112.191940.
attenuated by nicotine in a dose-related fashion. In nicotinetreated rats, blockade of ␤-adrenergic (propranolol), but not
muscarinic (atropine), receptors caused additional reductions
in reflex chronotropic responses, implying that nicotine selectively impairs reflex vagal activity. OVX selectively decreased
BRSPE but not BRSSNP and abolished the nicotine-induced
impairment of either response. These effects of OVX were
reversed after treatment with estrogen or the estrogen receptor
modulator raloxifene. In atropine-treated rats, comparable BRS
values were demonstrated in all rat preparations regardless of
the estrogen or nicotine milieu. Collectively, the inhibition of
vagal activity accounts for the depressant effect of chronic
nicotine on baroreflex activity. Furthermore, contrary to nicotine’s acute effects, the baroreflex-attenuating effect of chronic
nicotine is exacerbated by estrogen.
interaction with central mechanisms integrating the baroreceptor input into autonomic responses (Ashworth-Preece et
al., 1998) or reducing arterial compliance and stretch receptor responsiveness (Giannattasio et al., 1994). Nicotine also
modifies effector responsiveness to reflex autonomic modulation (Niedermaier et al., 1993).
Recent experimental reports from our laboratory showed
that the interaction of acutely administered nicotine with
reflex chronotropic responses depended on the animal’s sex,
hormonal milieu, and nature of the baroreflex heart rate
(HR) response (El-Mas et al., 2011a, 2012). In male rats,
nicotine impaired the baroreceptor-mediated control of reflex
tachycardia, but not bradycardia, through a mechanism that
involved disruption of adenosine A2A receptor-mediated facilitation of reflex cardiac sympathoexcitation (El-Mas et al.,
2011a). The depressant effect of nicotine on reflex tachycardia was also demonstrated in female rats with depleted estrogen levels (OVX or diestrus rats), but not physiological
estrogen levels (proestrus rats or estrogen-replaced OVX
rats), suggesting a protective effect for estrogen against the
nicotine-baroreflex interaction (El-Mas et al., 2012). Pharma-
ABBREVIATIONS: HR, heart rate; BP, blood pressure; MAP, mean arterial pressure; BRS, baroreflex sensitivity; OVX, ovariectomy (ovariectomized); SO, sham-operated; E2, estradiol; PE, phenylephrine; SNP, sodium nitroprusside; nAChR, nicotinic acetylcholine receptor.
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ABSTRACT
We recently reported that acute nicotine impairs reflex tachycardic activity in estrogen-depleted, but not estrogen-repleted,
female rats, suggesting a restraining influence for estrogen
against the nicotine effect. In this study, we tested whether the
baroreflex-protective effect of estrogen can be replicated when
nicotine was administered chronically. We also report on the
dose dependence and autonomic modulation of the nicotinebaroreflex interaction. The effects of nicotine (0.5, 1, or 2 mg/
kg/day for 14 days) on baroreflex curves relating changes in
heart rate to increases [phenylephrine (PE)] or decreases [sodium nitroprusside (SNP)] in blood pressure were evaluated in
sham-operated (SO), ovariectomized (OVX), and estrogen-replaced OVX (OVXE2) rats. Slopes of the curves were taken as a
measure of baroreflex sensitivity (BRSPE and BRSSNP). In SO
rats, both reflex bradycardic and tachycardic responses were
Estrogen Exacerbates Nicotine-Evoked Baroreflex Dysfunction
Materials and Methods
Female Wistar rats (200 –250 g; Faculty of Pharmacy animal
facility, Alexandria University, Alexandria, Egypt) were used. All
experiments were approved by the institutional animal care and use
committee and carried out in accordance with the Declaration of
Helsinki and the Guide for the Care and Use of Laboratory Animals
(Institute of Laboratory Animal Resources, 1996) as adopted and
promulgated by the National Institutes of Health.
Intravascular Cannulation
The method described in our previous studies (El-Mas and AbdelRahman, 1993; El-Mas et al., 2011a) was adopted. In brief, rats were
anesthetized with thiopental (50 mg/kg i.p.). Catheters (each consisting of 5-cm polyethylene-10 tubing bonded to 15-cm polyethylene-50
tubing) were placed in the abdominal aorta and vena cava via the
femoral artery and vein for measurement of blood pressure (BP) and
intravenous administration of drugs, respectively. The polyethylene-10 portion was used for the intravascular segment of the catheter. The arterial catheter was connected to a blood pressure transducer (model P23XL; Astro-Med, West Warwick, RI) that was
attached through MLAC11 Grass adapter cable to a computerized
data acquisition system with LabChart-7 pro software (Power Lab
4/35, model ML866/P; ADInstruments Pty Ltd., Castle Hill, Australia). HR was computed from BP waveforms and displayed on another
channel of the recording system.
Finally, catheters were tunneled subcutaneously and exteriorized
at the back of the neck between the scapulae. The catheters were
flushed with heparin (0.2 ml; 100 U/ml) and plugged by stainlesssteel pins, and incisions were closed by surgical clips. Each rat
received an intramuscular injection of 60,000 U of penicillin G benzathine and penicillin G procaine (Penicid; Cid Pharmaceutical Co.,
Cairo, Egypt) and housed in a separate cage. Experiments started 2
days later in conscious freely moving rats. This time period has been
shown to be adequate for recovery from surgery and restoration of
normal rat activity (El-Mas and Abdel-Rahman, 1993; El-Mas et al.,
2011a).
Ovariectomy
Bilateral ovariectomy was performed 14 days before experimentation, i.e., 12 days before intravascular cannulation, as described
previously (El-Mas and Abdel-Rahman, 1998, 2001).
Measurement of Plasma Cotinine
A blood sample was drawn from the arterial line 2 to 3 h after the
last dose of nicotine (day 14) and centrifuged at 800g for 10 min. The
plasma was aspirated and stored at ⫺20°C until analyzed for cotinine levels, the main metabolite of nicotine, by chemiluminescent
immunoassays (EURO/DPC Ltd, DPC Scandinavia, Mölmdal, Denmark) as reported elsewhere (El-Mas et al., 2011a).
Protocols and Experimental Groups
Cardiovascular Effects of Chronic Nicotine in Female Rats.
In this experiment, we investigated the effect of chronic nicotine on
cardiovascular and baroreflex-HR responses and the modulation of
these responses by cardiac vagal and sympathetic activities in conscious female rats. Four groups of female rats (n ⫽ 6 – 8 each) were
used to determine the effects of intraperitoneal nicotine (0.5, 1, or 2
mg/kg/day for 14 days) or equal volumes of saline on systolic BP, HR,
and reflex chronotropic responses. Tail cuff measurements of systolic
BP were performed before the start of the nicotine or saline regimen
(baseline values) and 5, 10, and 14 days thereafter. BP was measured
2 to 3 h after nicotine or saline administration. A computerized data
acquisition system with LabChart-7 pro software (Power Lab 4/30,
model ML866/P; ADInstruments Pty Ltd.) was used for data recording as in our previous studies (El-Mas et al., 2011a, 2012). HR was
computed from BP waveforms and displayed on another channel of
the recording system.
For baroreflex testing, rats were instrumented for direct BP and
HR measurements 2 days before experimentation (12 days after
nicotine administration). On the experiment day, the arterial catheter was connected to the pressure transducer and Power Lab data
acquisition system for the measurement of BP and HR as detailed
above. A period of 30 min was allowed at the beginning of the
experiment to permit hemodynamic stabilization. Baroreflex sensitivity (BRS) was assessed by the vasoactive (Oxford) method (El-Mas
and Abdel-Rahman, 1998; Saleh et al., 2000; El-Mas et al., 2011a),
which measures bradycardic or tachycardic responses to reciprocal
peripherally mediated BP changes evoked by bolus intravenous injections of randomized doses of PE or SNP (1–16 ␮g/kg, every 5 min).
PE and SNP were dissolved in saline, and the injection volume was
kept constant at 0.05 ml/100 g body weight with a flush volume of
approximately 0.1 ml of saline. The mean arterial pressure (MAP;
computed via integration of the area under the pulse pressure curve
by using the LabChart software) and HR values before and after PE
or SNP administration were determined, and peak changes in both
variables (⌬MAP and ⌬HR) were used for the construction of the
baroreflex curves (El-Mas and Abdel-Rahman, 1998; El-Mas et al.,
2011a). Slopes of the regression line (BRSPE and BRSSNP) were taken
as an index of baroreflex sensitivity.
To determine the role of cardiac autonomic control in the nicotinebaroreflex interaction, baroreflex curves of PE and SNP were reestablished in rats (the saline and nicotine 2 mg/kg/day groups) 10
min after intravenous administration of 1 mg/kg dose of atropine
(muscarinic blocker) or propranolol (␤-adrenergic blocker) (El-Mas et
al., 2002b, 2011a). Each rat in a particular group was used in two
experiments (2 and 4 days after intravascular cannulation, i.e., 14
and 16 days after nicotine or saline treatment) to test the effect of
atropine or propranolol. In the first experiment, approximately 50%
of rats in a given group (saline or nicotine, 2 mg/kg/day) received
atropine, and the other 50% received propranolol. In the second
experiment, the administration of atropine and propranolol was
crossed over. Comparison of the BRS before and after atropine or
propranolol would allow a proper assessment of the relative contri-
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cological evidence also implicated central neural pools of
estrogen receptors in the protection offered by estrogen
against nicotine-induced baroreceptor dysfunction in estrogen-depleted female rats (El-Mas et al., 2012). Clinically, the
attenuation of compensatory sympathoexcitation by nicotine
may have detrimental consequences in conditions such as
hypothalamic defense response, posture changes, and ventricular rhythms (Hayano et al., 1990; Duan et al., 1999;
Smith et al., 1999).
Because nicotine was given acutely in our previous studies
(El-Mas et al., 2011a, 2012), the issue of whether the preferential depressant action of nicotine on reflex tachycardia
could be replicated when nicotine is administered on a
chronic basis has not been explored. Therefore, this study
reports on the dose dependence of chronic nicotine on cardiovascular and baroreflex functions in conscious female rats.
More importantly, the estrogenic and autonomic modulation
of the nicotine-baroreflex interaction was also investigated.
The results showed that, unlike nicotine’s acute effects, 1) both
reflex tachycardic and bradycardic responses were attenuated
by chronic nicotine, 2) estrogen exacerbated, rather than protected against, the nicotine-evoked baroreflex dysfunction, and
3) the inhibition of cardiac vagal activity accounted for the
baroreflex-depressant effect of nicotine.
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Results
Effect of Chronic Nicotine on Baseline Systolic BP
and HR. The tail-cuff measurements showed that, compared
with saline-treated values, systolic BP was not altered by the
1 or 2 mg/kg/day dose of nicotine over the 2-week duration of
the study (Fig. 1A). Except for a significant increase in HR
caused by the 2 mg/kg/day dose of nicotine after 10 days, the
HR of female rats treated chronically with nicotine was not
statistically different from that of saline-treated rats (Fig.
1B). Plasma cotinine measured 14 days after nicotine administration (0.5, 1, or 2 mg/kg/day) amounted to 75 ⫾ 27, 163 ⫾
63, and 217 ⫾ 78 ng/ml, respectively.
Effect of Chronic Nicotine on Baroreflex Responsiveness. Figures 2 to 5 depict the effects of nicotine on
peripherally mediated pressor (PE) and depressor (SNP) responses and associated baroreflex-mediated chronotropic responses before and after autonomic blockade. The intravenous administration of PE or SNP (1–16 ␮g/kg each) elicited
Fig. 1. Tail-cuff measurements of systolic blood pressure (A) and HR
(B) in female rats treated with nicotine (0.5, 1, or 2 mg/kg/day) or
saline for 14 consecutive days. Values are means ⫾ S.E.M. of six to
eight observations. ⴱ, P ⬍ 0.05 versus saline values.
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butions of the vagal and sympathetic autonomic components to reflex
HR responses (El-Mas et al., 2002a,b, 2011a).
Another group of rats (n ⫽ 7) was used to test the effect of total
autonomic blockade with atropine and propranolol on reflex chronotropic activity. HR responses to PE and SNP were evaluated before
and after simultaneous intravenous administration of atropine and
propranolol (1 mg/kg each).
Estrogenic Modulation of the Autonomic and Baroreflex
Effects of Nicotine. This experiment tested the hypothesis that the
inhibition of the facilitatory baroreflex and autonomic effects of
estrogen mediates the depressant effect of nicotine on reflex chronotropic responses in female rats. Four groups of rats (n ⫽ 6 – 8 each)
were used to determine the effect of intraperitoneal nicotine (2 mg/
kg/day for 14 days) or saline on baroreflex gain in ovariectomized
rats treated with estrogen (OVXE2) or without estrogen (OVX). For
estrogen replacement, OVX rats received a daily subcutaneous dose
of 17␤-estradiol of 50 ␮g/kg for 5 consecutive days starting from the
ninth day after OVX. The last dose of 17␤-estradiol was administered 24 h before the experiment. This dose regimen of 17␤-estradiol
has been shown in our previous studies to produce physiological
estrogen levels (El-Mas et al., 2009, 2011b). Rats in the OVX group
received the vehicle (sesame oil) instead of 17␤-estradiol. All rats
were instrumented for BP and HR measurements 2 days before
experimentation (12 days after OVX). On the experiment day (day 14
after OVX), the arterial catheter was connected to the pressure
transducer and Power Lab data acquisition system for the measurement of BP and HR, and baroreflex curves of PE and SNP were
generated as described above. Afterward, atropine (1 mg/kg) was
administered intravenously, and 10 min later the baroreflex curves
of PE and SNP were re-established. The effect of atropine was tested
because results of preceding experiments implicated vagal cardiomotor dysfunction in the baroreflex-depressant action of nicotine.
The possibility that the selective estrogen receptor modulator
raloxifene mimics the effect of estrogen on the nicotine-baroreflex
interaction in OVX rats was investigated. Two groups of OVX rats
(n ⫽ 7 each) were used and given raloxifene (10 mg/kg/day s.c.) alone
or combined with nicotine (2 mg/kg/day i.p.). The nicotine regimen
continued for 14 days after OVX, whereas raloxifene was given for 5
days starting from day 9 of OVX. On day 14, baroreflex curves of PE
and SNP were constructed.
To further support the importance of estrogen in baroreflex dysfunction caused by nicotine, four groups of female rats (two proestrus
and two diestrus; n ⫽ 6 each) were used to determine the influence
of the phase of the estrus cycle on the nicotine-baroreflex interaction.
Diestrus rats exhibit significantly lower estrogen levels compared
with proestrus rats (El-Mas et al., 2011b). The phase of the cycle was
identified through microscopic examination of vaginal smears. Two
groups of rats (one proestrus and one diestrus) received nicotine (2
mg/kg/day) for 2 weeks. The other two groups received saline and
served as controls. Reflex chronotropic responses to PE and SNP
were assessed as described earlier.
Drugs. Phenylephrine hydrochloride, sodium nitroprusside, 17␤estradiol, raloxifene (Sigma, St. Louis, MO), nicotine (Merck Schuchardt OHG, Hohenbrunn, Germany), thiopental (Thiopental, Biochemie
GmbH, Vienna, Austria), povidone-iodine solution (Betadine; Nile
Pharmaceutical Co., Cairo, Egypt), and penicillin G procaine (Penicid;
Cid Pharmaceutical Co.) were purchased from commercial vendors.
Statistical Analysis. Values are expressed as means ⫾ S.E.M.
The relationship between changes in MAP evoked by PE or SNP and
associated reciprocal changes in HR was assessed by regression
analysis for individual animals as described in our previous studies
(El-Mas et al., 2002b, 2011a). The regression coefficient (slope of the
regression line, BRSPE and BRSSNP) expressed as beats/min/mm Hg
was taken as an index of baroreflex responsiveness. Analysis of
variance followed by a Newman-Keuls post hoc analysis was used for
two-way repeated measures or multiple comparisons with the level
of significance set at P ⬍ 0.05.
Estrogen Exacerbates Nicotine-Evoked Baroreflex Dysfunction
⫺0.5 ⫾ 0.1 mm Hg/beats/min). This represented an approximately 85% reduction in baroreflex gain. In nicotine-treated
rats (2 mg/kg/day for 14 days), reflex HR responses and
slopes of the regression lines (BRS) were significantly reduced in the presence of propranolol (Figs. 4B and 5) but not
atropine (Figs. 4A and 5). The treatment with atropine or
propranolol caused increases and decreases, respectively, in
basal HR that were of similar magnitudes in sham rats
treated with or without nicotine (Table 1). On the other hand,
autonomic blockade by atropine or propranolol elicited minimal changes in basal MAP (Table 1).
Estrogenic Modulation of the Autonomic and Baroreflex Effects of Nicotine. The effects of OVX and treatments with 17␤-estradiol or the estrogen receptor modulator
raloxifene on BRS in the absence and presence of atropine
are illustrated in Fig. 6. OVX significantly reduced BRSPE
(Fig. 6A) but not BRSSNP (Fig. 6B). The OVX-evoked reductions in BRSPE were abolished after 5-day treatment with
17␤-estradiol (50 ␮g/kg/day) or raloxifene (10 mg/kg/day)
(Fig. 6A). Furthermore, although nicotine (2 mg/kg/day for 14
days) failed to alter reflex chronotropic responses in OVX
rats, it significantly reduced both BRSPE (Fig. 6A) and
Fig. 2. Effect of nicotine (0.5, 1, or 2
mg/kg/day for 14 days) on increases or
decreases in MAP (A and B) elicited
by phenylephrine and sodium nitroprusside (1–16 ␮g/kg each), respectively, and associated changes in HR
(C and D) in female rats. Values are
means ⫾ S.E.M. of six to eight observations. ⴱ, P ⬍ 0.05 versus saline values.
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dose-dependent pressor (Fig. 2A) and depressor (Fig. 2B)
responses, respectively, which were associated with reciprocal changes in HR (Fig. 2, C and D). The pressor effects of PE
were not altered by nicotine, except for a significant decrease
in the response to the 16 ␮g/kg dose of PE in rats receiving
the middle dose of nicotine (Fig. 2A). Furthermore, the depressor actions generated by the 8 and 16 ␮g/kg doses of SNP
were increased by highest dose of nicotine (Fig. 2B).
On the other hand, reflex bradycardic (PE; Fig. 2C) and
tachycardic (SNP; Fig. 2D) responses were significantly reduced in nicotine-treated rats. This resulted in upward (PE)
and downward (SNP) shifts in baroreflex curves (Fig. 3), and
dose-related reductions in the slopes of the curves (regression
coefficient), which represented the BRS (Fig. 3B). Similar
upward and downward shifts in baroreflex curves of PE and
SNP, respectively (Fig. 4) and reductions in BRS (Fig. 5) were
seen when control rats were treated with atropine or propranolol, suggesting the importance of cardiac autonomic
reflexes (vagal and sympathetic) in these responses. Simultaneous treatment with atropine and propranolol significantly reduced reflex HR responses (BRSPE from ⫺2.5 ⫾ 0.3
to ⫺0.4 ⫾ 0.1 mm Hg/beats/min; BRSSNP from ⫺2.8 ⫾ 0.1 to
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El-Mas et al.
BRSSNP (Fig. 6B) in 17␤-estradiol- or raloxifene-treated OVX
rats. In the presence of atropine, similar BRSPE and BRSSNP
values were demonstrated in all rat preparations, including
sham-operated and OVX rats treated with or without nicotine and/or 17␤-estradiol (Fig. 6). The increases caused by
atropine in basal HR were similarly seen in OVX and OVXE2
rats (estrogen-independent) and were comparable with those
observed in intact sham-operated rats (Table 1). Chronic
nicotine significantly attenuated reflex bradycardic and
tachycardic responses in proestrus rats in contrast to no
effect in diestrus rats (Fig. 7).
Discussion
The current study reports on the dose, hormonal, and autonomic dependence of the interaction of chronic nicotine
with reflex HR control in female rats. Chronic nicotine dosedependently attenuated reflex HR responses in sham-operated rats. The inhibition of the estrogen-mediated vagal facilitation constitutes the underlying mechanism of the
baroreflex action of nicotine because: 1) blockade of ␤-adrenergic, but not muscarinic, receptors caused more deterioration of baroreflex responsiveness in nicotine-treated rats,
which highlights the impairment of vagal activity by prior
exposure to nicotine, 2) baroreflex impairment by nicotine
disappeared in OVX rats and was restored upon treatment
with estrogen or raloxifene, and 3) similar baroreflex gain
was demonstrated in all atropine-treated rats regardless of
the nicotine or estrogen status.
Fig. 4. Effect of muscarinic (atropine; A) or ␤-adrenergic (propranolol; B)
blockade on baroreflex curves generated by phenylephrine and sodium nitroprusside in female rats treated with nicotine (2 mg/kg/day) or saline for 14
days. Values are means ⫾ S.E.M. of six to eight observations.
The dose and duration of the smoking or nicotine regimen
greatly affect the nature and magnitude of the evoked cardiovascular abnormalities (Czernin and Waldherr, 2003;
Hanna, 2006). In agreement with this, findings of the current
and previous (El-Mas et al., 2012) studies established distinct effects for acute and chronic nicotine on reflex chronotropic responses and autonomic and estrogenic modulation of
these responses. For instance, in contrast to no effect for
acute nicotine on baroreflexes in female rats (El-Mas et al.,
2012), the current study demonstrated that chronic nicotine
dose-dependently impaired reflex bradycardic and tachycardic responses. Another important difference resided in the
way by which the estrogen status modulated the baroreflexdepressant effects of acute and chronic nicotine. Estrogen
protected against the acute nicotine-evoked baroreflex impairment because the latter was observed in OVX rats but
not in SO or OVXE2 rats (El-Mas et al., 2012). This contrasts
with the current observation that chronic nicotine reduced
BRS in estrogen-repleted (OVXE2 and proestrus) but not
estrogen-depleted (OVX and diestrus) rats, thereby inferring
the importance of estrogen in uncovering the nicotine-induced baroreflex dysfunction. Together, these findings suggest
contrasting modulatory effects for estrogen on the baroreflex
depressant of acute (protection) and chronic (exacerbation)
nicotine.
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Fig. 3. A, baroreflex curves generated by phenylephrine and sodium
nitroprusside in female rats treated with nicotine (0.5, 1, or 2 mg/kg/day)
or saline for 14 days. B, the slopes of baroreflex curves, which represent
BRS. Values are means ⫾ S.E.M. of six to eight observations. ⴱ, P ⬍ 0.05
versus saline values.
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Estrogen Exacerbates Nicotine-Evoked Baroreflex Dysfunction
TABLE 1
Changes in mean arterial pressure (mm Hg) and heart rate (beats/min)
caused by muscarinic (atropine) or ␤-adrenergic (propranolol) blockade
Values are means ⫾ S.E.M. of six to eight observations.
Atropine
Mean arterial pressure
Sham
Sham/nicotine
OVX
OVXE2
Heart rate
Sham
Sham/nicotine
OVX
OVXE2
Propranolol
2 min
10 min
2 min
10 min
⫺3 ⫾ 2
⫺3 ⫾ 1
1⫾2
1⫾1
⫺3 ⫾ 3
⫺1 ⫾ 1
⫺2 ⫾ 2
⫺1 ⫾ 1
3⫾1
1⫾2
4⫾2
3⫾1
20 ⫾ 4
21 ⫾ 4
33 ⫾ 6
26 ⫾ 10
16 ⫾ 2
12 ⫾ 2
19 ⫾ 3
14 ⫾ 6
⫺55 ⫾ 7
⫺52 ⫾ 6
⫺68 ⫾ 7
⫺79 ⫾ 8
Reflex HR responses to abrupt perturbations in BP are
believed to involve opposite changes in cardiac vagal and
sympathetic activities (El-Mas et al., 2001, 2002a; Michelini,
2007). This is supported by the current observations that
reflex changes in HR were 1) remarkably reduced after muscarininc (atropine) or ␤-adrenergic (propranolol) blockade,
and 2) virtually abolished after total autonomic blockade. It
is noteworthy that the residual HR activity demonstrated in
rats treated simultaneously with atropine and propranolol
may suggest the incomplete obliteration of autonomic activity or possibly reflects the involvement of direct cardiac effects in chronotropic response to phenylephrine (Posner et
al., 1984) or nitroprusside (Herring et al., 2001).
Comparisons of current and previous studies (El-Mas et
al., 2012) also revealed strikingly different contributions of
the two divisions of the autonomic nervous system to baroreflex dysfunction caused by acute and chronic regimens of
nicotine. In our previous studies (El-Mas et al., 2012), pharmacologic interruption of reflex cardiac sympathetic, and not
vagal, activity mediated the depressant effect of acute nicotine on BRS. Conversely, the current observation that the
baroreflex-depressant effect of nicotine was abolished in at-
Fig. 6. Effect of muscarinic blockade with atropine (1 mg/kg) on the
nicotine-induced (2 mg/kg/day for 14 days) reductions in reflex bradycardic (BRSPE) and tachycardic (BRSSNP) responses in female rats. Values are means ⫾ S.E.M. of six to seven observations. ⴱ, P ⬍ 0.05 versus
sham. $, P ⬍ 0.05 versus OVX. ⫹, P ⬍ 0.05 versus OVXE2. &, P ⬍ 0.05
versus respective OVX ⫹ raloxifene values. #, P ⬍ 0.05 versus respective
before atropine values.
ropine-treated SO rats highlights a pivotal role for the inhibition of the estrogen-dependent cardiac vagal activity in
baroreflex dysfunction caused by chronic nicotine. Indeed,
our data showed that the BRS-depressant effect of atropine
in animals treated with nicotine or saline was similar (⬃65%
reduction). It is reasonable, therefore, that nicotine produced
a maximal inhibition of reflex vagal activity in such a way
that the concomitant use of atropine could not depress it
further. By the same token, the preservation of the baroreflex-attenuating capacity of nicotine in propranolol-treated
rats rules out a potential role for reflex sympathetic activity
in the nicotine-baroreflex interaction. It is conceivable, there-
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Fig. 5. Effect of muscarinic (1 mg/kg atropine; A) or ␤-adrenergic (1 mg/kg
propranolol, Prop; B) blockade on the nicotine-induced (2 mg/kg/day for
14 days) attenuation of BRS in female rats. Values are means ⫾ S.E.M.
of six to eight observations. ⴱ, P ⬍ 0.05 versus saline values. ⫹, P ⬍ 0.05
versus saline ⫹ atropine or saline ⫹ propranolol values.
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El-Mas et al.
fore, to suggest that the presence of functional cardiomotor
vagal activity is mandatory for the elicitation of the baroreflex-depressant action of chronic nicotine. It is noteworthy
that because the changes in basal HR caused by atropine
(increases) or propranolol (decreases) were similarly demonstrated in sham rats treated with or without nicotine, they
are unlikely to contribute to the depressant effect of nicotine
on baroreflexes. Consistent with our previous report (El-Mas
et al., 2011a), these data infer a role for central rather than
peripheral (e.g., abundance of cardiac muscarinic or ␤1-adrenergic receptors) mechanisms in the nicotine-baroreflex
interaction. Alternatively, the data also indicate that, unlike
its depressant effect on rapid adjustments in baroreceptor
tone, nicotine does not seem to modify the tonic autonomic
control of HR.
The current findings that reflex bradycardia was preferentially impaired in OVX rats and restored to SO levels after
estrogen replacement is consistent with a tonic facilitatory effect of estrogen on baroreceptor-mediated HR control. Similar
observations were reported in previous studies in which baroreflex function was assessed by vasoactive methods (Mohamed
et al., 1999; Saleh et al., 2000) or spectral methods (El-Mas and
Abdel-Rahman, 2009). The demonstration by Pamidimukkala
et al. (2005) that estrogen replacement enhances baroreflex
gain in wild-type OVX rats but not in estrogen receptor-␣
knockout OVX rats directly implicates estrogen receptor-␣ in
the baroreflex response to estrogen. In addition, we report here
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 17, 2017
Fig. 7. A, baroreflex curves generated by phenylephrine and sodium
nitroprusside in proestrus and diestrus rats treated with nicotine (2
mg/kg/day) or saline for 14 days. B, the slopes of baroreflex curves, which
represent BRS. Values are means ⫾ S.E.M. of six observations. ⴱ, P ⬍
0.05 versus values in saline-treated proestrus rats.
that the estrogen status of female rats modulates the vagally
dependent baroreflex-depressant effect of nicotine. This is convincingly supported by the observations that the atropine-sensitive baroreflex-depressant effect of nicotine disappeared in
OVX rats and reappeared after replacement of these rats with
estrogen or the estrogen receptor modulator raloxifene. In fact,
the findings that comparable BRS values were seen in all atropine-treated preparations (SO, OVX, and OVXE2 with or without nicotine; Fig. 6) indicate that differences in baroreflex responsiveness among these preparations relate, at least partly,
to discrepancies in vagal activity.
Recent anatomical and functional evidence implicates
homomeric and heteromeric nAChRs in central autonomic
homeostasis. For example, ␣7 and ␤2 nAChRs have been
identified in the nucleus of the solitary tract and dorsal
motor nucleus of the vagus, medullary nuclei that are
fundamentally involved in the processing of visceral sensory information (Browne et al., 2010; Anfinogenova et al.,
2011). Moreover, the area postrema is endowed with heteromeric ␣3␤4 nAChRs, which modulate inhibitory
GABAergic transmission (Kawa, 2007). The following observations demonstrate that the interaction of nicotine
with nAChRs varies depending on receptor, gender, and
hormonal profiles: 1) the medullary expression of ␣7 and
␤2 nAChRs is decreased and increased, respectively, by
nicotine (Browne et al., 2010), 2) nicotine increases ␣4␤2
nAChR binding sites in mouse brain (Marks et al., 2011),
3) compared with males, nicotine-treated females have
higher medullary ␤2 nAChRs (Browne et al., 2010), and 4)
estradiol increases ␣7 nAChRs in dorsal raphe and locus
coeruleus neurons (Centeno et al., 2006). More studies are
obviously needed to determine the relative contributions of
central nicotinic receptors in the gender and hormonally
dependent nicotine-baroreflex interaction.
The arterial baroreflex system is a powerful homeostatic
mechanism that negatively correlates with BP. Whereas persistent falls in BP develop upon long-term baroreceptor activation (Lohmeier and Iliescu, 2011), impairment of baroreceptor
function usually is associated with hypertension in human
studies (Bristow et al., 1969; Goldstein, 1983) and animal studies (Gordon et al., 1981; Gordon and Mark, 1983). In the current
study, despite the noticeable impairment of baroreceptor function in nicotine-treated rats, tail cuff measurements revealed no
changes in systolic BP. Because nicotine was administered in
single daily doses, it could be argued that the rapid elimination
of nicotine might have hampered the development of hypertension. Remarkably, the single daily-dose regimen has been repeatedly used for studying the biological effects of nicotine (Hui
and Ogle, 1991; Ferrari and Fior-Chadi, 2007; El-Gowilly et al.,
2008). Moreover, pharmacokinetic studies showed that, despite
the relatively short half-life of nicotine (⬃1 h), the half-life of
cotinine, the principal metabolite and pharmacologically active
form of nicotine, ranges from 10 to 24 h (Miller et al., 1977;
Buccafusco and Terry, 2003). In the current study, plasma
cotinine levels measured 2 to 3 h after dosing with nicotine
mimic levels achieved in humans after moderate cigarette
smoking (Roethig et al., 2009; Morin et al., 2011), which establishes the clinical relevance of the current study. Our findings,
nevertheless, should not be interpreted to preclude a possible
correlation between BP and baroreflex activity. Indeed, baroreflex dysfunction precedes the development of hypertension
(Gordon et al., 1981; Gordon and Mark, 1983; Shaltout et al.,
Estrogen Exacerbates Nicotine-Evoked Baroreflex Dysfunction
2012). Probably, a longer period of nicotine exposure might be
necessary for revealing nicotine hypertension as reported elsewhere (Ferrari and Fior-Chadi, 2007).
Collectively, the current study showed that the baroreflex
effects elicited by chronic nicotine in female rats were qualitatively and quantitatively different from those caused by acute
nicotine in rats of the same sex and strain. Although reflex
chronotropic responses were not affected by acute nicotine (ElMas et al., 2012), they were remarkably and dose-dependently
attenuated by chronic nicotine (this study). Moreover, the data
revealed different modulatory roles for estrogen on baroreflex
impairment caused by acute nicotine (protection; El-Mas et al.,
2012) and chronic nicotine (exacerbation; current study). Finally, the inhibition of cardiomotor vagal activity mediated the
estrogen-dependent baroreflex suppression caused by chronic
nicotine. The data emphasize the importance of nicotine regimen in defining the autonomic and cardiovascular consequences of the drug.
Participated in research design: El-Mas, El-Gowelli, and El-Gowilly.
Conducted experiments: Fouda and Helmy.
Performed data analysis: El-Mas, El-Gowelli, and El-Gowilly.
Wrote or contributed to the writing of the manuscript: El-Mas,
El-Gowelli, and El-Gowilly.
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Address correspondence to: Dr. Mahmoud M. El-Mas, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Alexandria University, Alexandria, Egypt. E-mail: [email protected]
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Authorship Contributions
575