Effects of the Menopause, Gender, and Estrogen Replacement Therapy on Vascular Nitric Oxide Activity
The Journal of Clinical Endocrinology & Metabolism
Copyright © 2000 by The Endocrine Society
Effects of the Menopause, Gender, and Estrogen Replacement Therapy on Vascular Nitric Oxide Activity
N. G. MAJMUDAR 0
S. C. ROBSON 0
G. A. FORD 0
0 Departments of Pharmacological Sciences (N. G.M., G.A.F.), Obstetrics (S.C.R.), and Medicine (N.G.M., G.A.F.) , University of Newcastle Upon Tyne , NE2 4HH Newcastle Upon Tyne , United Kingdom
Changes in vascular nitric oxide (NO) activity may contribute to cardiovascular risk. We determined the effect of the menopause, gender, and estrogen replacement therapy on arterial vascular NO activity. Vascular NO activity and sensitivity were determined in 15 healthy premenopausal women (mean age, 48 yr), 12 postmenopausal women (51 yr), and 14 men (51 yr). The effects of 14 days of estrogen replacement therapy (625 mg conjugated estrogens) were studied in 20 healthy postmenopausal women (60 yr). Forearm blood flow responses to brachial arterial infusions of L-NG-monomethyl-arginine (L-NMMA), norepinephrine, glyceryl trinitrate (GTN), and serotonin were determined using venous occlusion plethysmography. Constrictor responses to L-NMMA were reduced in postmenopausal women (82 6 14, summary response, mean 6 SEM) and men (89 6 6) compared to premenopausal women (118 6 10; P , 0.05). Constrictor responses
ARDIOVASCULAR disease is the leading cause of
death in women in the developed world.
Premenopausal women in particular have significantly reduced risk
compared to men, although the incidence increases greatly
after the menopause (
). Women who experience a surgical
menopause without estrogen replacement have twice the
risk of coronary heart disease compared with their
premenopausal peers (
). In women undergoing natural menopause,
each years of delay in age of onset of the menopause reduces
cardiovascular mortality risk by 2% (
). The increased risk is
thought to be due to withdrawal of estrogenic effects.
Epidemiological studies suggest that estrogen replacement in
postmenopausal women is associated with a 50% reduction
in the incidence of cardiac events (
). However, the recent
Heart and Estrogen/Progestin Study found no benefit from
4 yr of treatment with conjugated equine estrogens and
medroxyprogesterone acetate in postmenopausal women with
established coronary disease, and the results of other
ongoing randomized controlled trials are awaited (
Estrogen has several actions that may contribute to a
cardioprotective effect: lowering of total and low density
lipoprotein cholesterol, elevation of high density lipoprotein
), and reductions in fibrinogen and factor VII
). However, lipid changes only appear to account for 25%
of the protective effect (
). Nitric oxide (NO), synthesized by
the vascular endothelium, plays a major physiological role in
to norepinephrine were increased in males (125 6 13) compared to
premenopausal (81 6 8) and postmenopausal (88 6 16) women (P ,
0.05). No differences were observed in GTN or serotonin
responsiveness. Constrictor responses to L-NMMA increased after estrogen
replacement (132 6 7 vs. 89 6 14; P , 0.05), with no change in
norepinephrine, GTN, or serotonin responses. The menopause and male
gender were associated with reduced arterial NO activity. Two weeks
of estrogen replacement therapy restored vascular NO activity to
premenopausal levels. Changes in vascular NO activity may
contribute to changes in cardiovascular risk associated with male gender,
postmenopausal status, and estrogen replacement therapy. Increased
a-adrenoceptor responsiveness may contribute to increased
cardiovascular risk in males. (J Clin Endocrinol Metab 85: 1577–1583,
maintaining basal vascular arterial tone, inhibiting platelet
aggregation and adhesion (
), inhibiting the development of
atherosclerotic plaques (
), and inhibiting vascular smooth
muscle proliferation (
). Some studies suggest that estrogen
replacement may increase stimulated NO release in the
arterial vasculature of postmenopausal women. Estrogen
replacement has been shown to augment endothelial
dependent flow-mediated vasodilatation to reactive hyperemia in
the brachial artery (
), and brachial artery infusion of
17bestradiol increases endothelium-dependent vasodilatation to
Changes in vascular NO activity may be an important
mechanism mediating the detrimental effect of the
menopause and male gender on cardiovascular mortality and the
benefits of estrogen and premenopausal status in reducing
cardiovascular risk. However, no data exist on vascular
responses in age-matched pre- and postmenopausal women or
on gender effects in middle age. The aims of the present
study were to determine the effects of menopausal status,
gender, and estrogen replacement on arterial NO activity,
NO sensitivity, and stimulated NO release.
Subjects and Methods
For the menopause and gender studies, healthy female and male
subjects, aged 45–55 yr, were studied: 15 regularly menstruating women
(mean 6 sd age, 48 6 2 yr) with serum FSH levels below 30 IU/L, 12
postmenopausal women (51 6 3 yr) with at least a 12-months history of
amenorrhea and serum FSH levels above 30 IU/L, and 14 male subjects
(51 6 3 yr). The effects of estrogen replacement were studied in a
separate group of 20 healthy postmenopausal subjects, aged 45– 65 yr or
otherwise described as above 60 6 5 yr.
All subjects were nonsmokers, were taking no regular medication,
and had normal history, examination (blood pressure, ,150/90 mm
Hg), 12-lead resting electrocardiogram, blood count, glucose, and
electrolytes. For the menopause and gender studies, all subjects had a
nonfasting cholesterol of less than 6.5 mmol/L. Written informed
consent was obtained from the subjects, and studies were approved by the
Newcastle joint ethics committee.
All study drugs needing reconstitution were dissolved in sterile 0.9%
saline. The following drugs were administered: norepinephrine (Sanofi
Pharmaceuticals, Inc., Winthrop, UK), used as a control vasoconstrictor
not affecting NO release; l-NG-monomethyl-arginine
(CalbiochemNovabiochem, Nottingham, UK), a nitric oxide synthase inhibitor,
used to measure basal NO release; oral conjugated estrogens (Wyeth
Laboratories, Maidenhead, UK); serotonin hydrochloride
(CalbiochemNovabiochem), used as an endothelium-dependent NO-dependent
vasodilator; and glyceryl trinitrate (David Bull Laboratories, Inc.,
Warwick, UK), used as a NO donor.
Studies were performed in a quiet, temperature-controlled laboratory
(25–27 C) between 1000 –1300 h. Subjects refrained from caffeine and
alcohol and consumed no food for at least 2 h before the studies and
rested supine for 30 min before commencement. Premenopausal studies
were performed between 7–12 days from the start of the last menstrual
period. After rest, a 27-gauge cannula (Cooper’s Needle Works,
Birmingham, UK) was inserted into the brachial artery of the nondominant
arm after administration of 1% lignocaine. Drugs or physiological saline
were infused continuously at 1.0 mL/min. Forearm blood flow (FBF)
was measured at 10-min intervals during 30 min of saline infusion to
establish baseline FBF values. For constrictor studies, norepinephrine
was infused at three doses (40, 80, and 160 ng/min), each for 5 min.
Saline was then reinfused until baseline blood flows were reestablished,
and then l-NG-monomethyl-arginine (L-NMMA) was infused at 3 doses
(250, 500, and 1000 mg/min), each for 5 min. For both infusions, blood
flows were measured during the last 2 min of each infusion period. The
dilator studies were performed in 12 pre- and 10 postmenopausal
women who had participated in the constrictor studies, at least 7 days
after the initial studies. The above protocol was used with FBF responses
measured to the infusion of 3 doses of glyceryl trinitrate (GTN) (250, 500,
and 1000 ng/min), each for 5 min, followed by saline until baseline blood
flows were reestablished, and then 3 doses of serotonin (18, 60, and 180
ng/min), each for 5 min. During serotonin infusion, FBF responses were
measured during the first 2 min of infusion. Similarly, for the gender
studies, the above protocols for constrictor and dilator studies were used
on 14 men. Cross-over study designs were not used for either constrictor
or dilator studies because of persisting vasoconstriction and
vasodilatation with, respectively, L-NMMA and serotonin, whereas the effects
of norepinephrine and GTN reversed within 20 min after continuing
Initial studies of constrictor responses to norepinephrine used doses
of 10, 20, and 40 ng/min in 3 pre- and 4 postmenopausal subjects.
However, responses were consistently lower than that seen at the lowest
dose of L-NMMA. To obtain constrictor responses to norepinephrine
comparable to those seen with L-NMMA, the subsequent 12 pre- and 8
postmenopausal and male subjects were studied at doses of 40, 80, and
160 ng/min. Data for these groups are presented.
Estrogen replacement studies were performed on 2 separate
occasions. FBF constrictor responses to norepinephrine and L-NMMA were
determined in 10 women, and dilator responses to serotonin and GTN
were determined in a separate group of 10 women, before and after 14
days of treatment with oral conjugated estrogen (625 mg daily) using the
FBF (milliliters per 100 mL/min) was measured simultaneously in
both arms by venous occlusion plethysmography, according to the
method of Whitney (
), using galidinium in SILASTIC brand (Dow
Corning Corp., Midland, MI) strain gauges. During recording, the hands
were excluded from the circulation by inflation of the wrist cuffs to 200 mm
Hg. The upper arm cuffs were then inflated to 50 mm Hg for 10 s in each
15-s cycle. Data were recorded directly onto computer using a MacLab3
system (AD Instruments Pty. Ltd., Castlehill, Australia) with on-line slope
analysis to determine FBF. The average of five consecutive measurements
for each measurement period was derived to determine FBF.
Forearm vascular resistance was derived from mean arterial pressure
and baseline FBF. Differences in baseline heart rate, blood pressure, FBF,
and forearm vascular resistance between and within groups were
compared by Student’s t test. Within-subject differences in FBF in the control
and infused arms were assessed using two separate repeated measures
ANOVA. Further analysis was undertaken if ANOVA suggested a
statistically significant change in FBF over time. FBF responses were
expressed as the percentage of FBF during baseline infusion of saline. The
overall drug response in each subject was assessed by the maximal
response (percentage) and a summary response, calculated as the
summation of the percentage of constrictor or dilator responses for the three
doses of the infused drug (arbitrary units). Data are expressed as the
mean 6 sem. Between-group comparisons were undertaken using initial
repeated measures of ANOVA. Within-group comparisons were
undertaken using paired Student’s t test. P , 0.05 was considered
Menopause and gender studies
Subject details are shown in Table 1. Mean age was higher
in postmenopausal subjects, but blood pressure, weight,
body mass index, cholesterol, and baseline FBF were similar.
Men were taller, but had similar body mass indexes as
women. There was no significant change in FBF in the control
arm during the course of the studies. Constriction in response
to L-NMMA (Fig. 1A) was increased in premenopausal
women (maximum, 47 6 3%; summary, 118 6 10; mean 6
sem) compared to that in postmenopausal women
[maximum, 33 6 6% (P , 0.05); summary, 82 6 14 (P , 0.05)] and
males (maximum, 37 6 2%; summary, 89 6 6; P , 0.05), with
no difference between the latter two groups. In contrast,
constrictor responses to norepinephrine (Fig. 1B) were
increased in males (maximum, 47 6 6%; summary, 125 6 13)
compared to those in both premenopausal [maximum, 30 6
3% (P , 0.05); summary, 81 6 8 (P , 0.05)] and
postmenopausal [maximum, 31 6 6% (P , 0.05); summary, 88 6 16
(P , 0.05)] groups, with no difference between the latter two
Responses to GTN and serotonin (Fig. 2, A and B) were not
significantly different in the three groups (GTN:
premenopausal: maximum, 142 6 18%; summary, 296 6 37;
postmenopausal: maximum, 162 6 29%; summary, 363 6 63;
male: maximum, 129 6 21%; summary, 281 6 31; serotonin:
premenopausal: maximum, 99 6 11%; summary, 210 6 15;
postmenopausal: maximum, 82 6 11%; summary, 190 6 23;
male: maximum, 67 6 11%; summary, 160 6 23).
Estrogen replacement study
Subject details are shown in Table 2. Cholesterol decreased
after estrogen treatment. FBF did not change in the control
arm during the course of the studies. Constriction to
LNMMA increased after estrogen [maximum, 54 6 3% vs. 40 6
5% (P , 0.05); summary, 132 6 17 vs. 89 6 14 (P , 0.05); Fig.
3A]. The constriction response to norepinephrine was
unchanged [maximum, 45 6 6% vs. 38 6 6% (P 5 0.28);
summary, 117 6 14 vs. 97 6 14 (P 5 0.27); Fig. 3B]. Dilatation
responses to GTN and serotonin were unchanged after
estrogen therapy [GTN: maximum, 96 6 18% vs. 101 6 14%
(P 5 0.80); summary, 214 6 38 vs. 219 6 29 (P 5 0.88);
serotonin: maximum, 92 6 26% vs. 74 6 12% (P 5 0.17);
summary, 199 6 26 vs. 162 6 25 (P 5 0.19)].
(n 5 10)
This study is the first to show reduced constrictor
responses to an NO synthase inhibitor in postmenopausal
women and males compared to age-matched premenopausal
peers, indicating a reduction in basal NO activity associated
with the menopause. This change appears to be specific, with
no change in the responsiveness of smooth muscle to
norepinephrine in postmenopausal women. Two weeks of
estrogen replacement therapy in postmenopausal women
restores basal vascular NO activity to levels seen in
premenopausal women. However, in our studies we found
no alteration in stimulated NO release or smooth muscle
sensitivity in association with the menopause or with
estrogen replacement. We have also demonstrated men have basal
vascular NO activity comparable to that in age-matched
postmenopausal women, but have enhanced responsiveness
Effects of the menopause and estrogen replacement on vascular NO activity
Animal studies suggest that basal vascular NO activity
may be reduced after the menopause (
), and that estrogen
increases agonist-dependent NO activity (
). A study of NO
metabolites in postmenopausal women receiving hormone
replacement therapy observed a 50% increase in nitrite levels
after 1 yr of estrogen therapy, but no increase with the
estrogen/progesterone combination, suggesting that
unopposed estrogen increases NOS activity (
Sorenson et al. recently reported no increase in
flow-mediated brachial artery dilatation in postmenopausal women
taking combined estradiol and northisterone replacement
). These findings suggest that progesterone may
attenuate the beneficial effects of unopposed estrogen on
endothelial vascular function. Some studies suggest that the
menopause and estrogen replacement may alter stimulated
NO release in response to endothelium-dependent
vasodilators, with reduced vasodilatation to acetylcholine at
around the age of the menopause described (
infusion of estradiol in postmenopausal women increases the
vasodilator response to acetylcholine (
). The effects of
chronic oral and transdermal estrogen administration on
stimulated NO release are unclear. Lieberman et al. reported
an increased flow-mediated response to reactive hyperemia
after 9 weeks of estrogen therapy in postmenopausal women
). In contrast, two other studies found no change in
response to acetylcholine (
). Vasodilator responses to
acetylcholine are not solely mediated via NO, and alterations
in the release of bradykinin or other mediators may have
confounded interpretation of these studies. One small study
has examined the effects of estrogen therapy on basal NO
activity, showing an increased response to L-NMMA, a NO
synthase inhibitor, in six perimenopausal women after 8
weeks of therapy (21). Responses to a control vasoconstrictor
were not performed in this study. Our observations suggest
that the L-NMMA effect is specific, and it is not due to a
generalized increase in smooth muscle responsiveness to
constrictor agents. The estrogen replacement group we
studied were older and had greater cholesterol concentrations
than the postmenopausal group studied in the menopause/
gender studies. It is possible the effects of estrogen
replacement therapy would differ in a younger group if increased
age or hypercholesterolemia was associated with altered
responsiveness of the vessel wall to estrogen. The absence of
any significant difference in baseline L-NMMA responses
between the two postmenopausal groups suggests that basal
NO activity was not appreciably affected by increasing age
or hypercholesterolemia. Despite the changes in basal NO
activity observed with the menopause and after estrogen
replacement, no difference was observed in basal blood flow,
perhaps suggesting that vasoconstrictor influences are
decreased after estrogen withdrawal at the menopause.
However, many studies describe reduced endothelin plasma
levels or activity after estrogen replacement, suggesting that a
reduction in endothelin activity after the menopause is
Effects of gender on vascular NO activity
Our finding of reduced vascular NO activity in
middleaged men compared to premenopausal female peers
contrasts with observations in younger subjects, in whom no
significant gender difference was observed in FBF responses
to L-NMMA in healthy subjects in their twenties (
gender difference in L-NMMA responses observed in our
studies could reflect a preferential age-associated decline in
vascular NO activity in males, perhaps due to
estrogenmediated preservation of vascular NO activity in women
before the menopause. Alternatively, there may be gender
differences in age-associated changes in structural
differences in forearm vasculature that could influence the
response to vasoconstrictors. Interestingly, studies of NO
production as determined by conversion of l-arginine to nitrite,
demonstrate reduced whole body NO production in young
to middle-aged healthy males compared to premenopausal
). A number of animal studies have found gender
differences in vascular NO activity. Basal release of NO from
aortic rings is greater in female rabbits, and ovariectomy
reduces basal NO release to male levels (
Pressureinduced myogenic constriction of rat gracilis muscle
arterioles is less pronounced in female rats because of enhanced
NO release, mediated through estrogen increasing NO
release in response to vascular wall shear stress (
observations have been made in rat coronary arteries (29).
Effect of gender on a-adrenergically mediated responses
Our observation of enhanced vasoconstriction in response
to norepinephrine, a nonselective a-adrenoceptor agonist, in
middle-aged males, has been reported in healthy young
). This most likely reflects enhanced a-adrenergic
vascular sensitivity in males. Differences in a-adrenergic
responsiveness with gender and after ovariectomy have been
described in some animal vascular beds (
observations suggest that circulating sex hormones may
modulate vascular adrenergic responsiveness. Norepinephrine
acts through both a1- and a2-adrenoceptors in the forearm to
produce vasoconstriction. Norepinephrine may also act on
endothelial cell a2-adrenoceptors to release NO, which could
attenuate the vasoconstriction produced by norepinephrine
). Gender differences in NO release stimulated by
norepinephrine could account for the increased responsiveness
of males to norepinephrine. Further studies examining
vascular responses to selective a1- and a2-adrenoceptors in the
presence and absence of NO inhibition would be of interest
in establishing the underlying mechanisms accounting for
these gender differences. Although increased NO activity
could blunt norepinephrine responses in premenopausal
women, this would not account for the difference observed
in our studies between postmenopausal females and males
and the lack of any difference between pre- and
Interpretation of study findings
The increased response to L-NMMA in premenopausal
women could be due to either increased NO synthesis or
increased sensitivity of arterial smooth muscle to released
NO. Our finding that vasodilator responses to GTN, a NO
donor, are not altered after the menopause or after systemic
estrogen replacement confirms the results of previous
12, 19 –21
). In one of these studies a small increase in the
vasodilator response to nitroprusside was observed after
local intraarterial infusion of estradiol, but not after 3 weeks
of transdermal estrogen replacement (20). Thus, the changes
we found in NO activity at the menopause probably reflect
a decrease in NO activity after the menopause, rather than a
change in smooth muscle sensitivity to NO, which is reversed
with estrogen therapy. The additional finding that there is an
enhanced constrictor response to noradrenaline in males
most likely relates to enhanced a-adrenergic responsiveness,
but could be due to a generalized increase in vasoconstrictor
responsiveness. However, the latter is unlikely, as there was
no difference in the response to L-NMMA between males
and postmenopausal women.
Mechanisms underlying change in NO activity
The mechanism responsible for increased NO activity in
premenopausal women compared to that in postmenopausal
women and males and after estrogen replacement is most
likely increased endothelial NO synthase activity secondary
to higher levels of circulating 17b-estradiol. 17b-Estradiol
increases constitutive NO synthase activity in endothelial
cells from human umbilical veins and bovine aortas (
pregnancy, a hyperestrogenic state, an increase in the
expression of uterine artery NOS has also been found (
addition to the estrogenic effects on NOS, other mechanisms
may account for the present observations. The phenomenon
of increased NO release due to increased flow is well
). Interestingly, Sudhir et al. reported an increase in
basal FBF after estrogen replacement (
). In contrast, we
found no change in FBF with estrogen or the menopause,
suggesting that alterations in flow do not contribute to the
observed changes in NO activity. Blood pressure changes at
the menopause or after estrogen replacement could affect
basal NO activity (
). However, most studies have found no
independent effect of the menopause or estrogen therapy on
blood pressure, and our results confirm this (
estrogen typically increases high density lipoprotein
cholesterol by 10% and reduces low density lipoprotein cholesterol
by 15% (
). Changes in cholesterol and lipoprotein levels
may also modify basal vascular NO activity (
there was only a small (,5%) reduction in total serum
cholesterol levels after estrogen therapy, it is possible that
changes in high density lipoprotein and low density
lipoprotein cholesterol may have indirectly contributed to the
increased vascular NO activity, independent of any direct
effect of estrogen on NO synthesis or release. Two weeks of
estrogen treatment would not be expected to produce final
steady state changes in serum lipid concentrations, and
longer term estrogen treatment could result in greater
increases in vascular NO activity through such changes.
Insulin resistance may influence endothelial function. We did
not determine insulin resistance in subjects. However, body
mass index and blood pressure were similar in all groups.
Clinical relevance of alterations in vascular NO activity to cardiovascular risk
Alterations in basal vascular NO activity may account for
some of the changes in cardiovascular risk seen with the
menopause and estrogen replacement. Reduced vascular NO
activity has been found in conditions associated with an
increased risk of cardiovascular disease, such as
), hypercholesterolemia (
), smoking (
). Treatment of these conditions reverses the decline
in NO activity. Estrogen has direct beneficial vascular effects
with reduction in platelet aggregation, inhibition of smooth
muscle proliferation, and reduction in cholesterol influx (
and these changes may be mediated through the enhanced
production of NO. This may therefore be an important
mechanism in any cardioprotective role of estrogen, in addition to
its effect on lipids. Our findings in the forearm circulation
may not directly mirror changes in the coronary and
cerebrovascular arterial vascular beds, which are of primary
relevance to cardiovascular risk, although increased vascular
NO release in response to acetylcholine after intracoronary
estradiol infusion has been described in postmenopausal
women undergoing cardiac catheterization (
The present studies demonstrate that premenopausal
status is associated with increased basal vascular NO activity in
the human forearm arterial vasculature compared to that in
postmenopausal female and male peers. Vascular NO
activity is increased to premenopausal levels after 2 weeks of
estrogen replacement. Males demonstrate increased
sensitivity to norepinephrine compared to both pre- and
postmenopausal female peers. These changes in NO activity may
contribute to the increase in cardiovascular risk seen after the
menopause and any cardioprotective effect of estrogen
replacement therapy. Reduced vascular NO activity and
increased a-adrenergic sensitivity may contribute to the
increased cardiovascular risk in males compared to that in
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