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Original Articles: Hormones and Reproductive Health |
-Adrenergic Responses in Resistance Vessels during the Menstrual Cycle in Healthy Women1
Center for Clinical Pharmacology (N.N.C., R.J.M., P.V., A.D.H.) and EURODIAB (N.N.C., H.M.C.), Department of Epidemiology and Public Health, University College London, London, United Kingdom WC1E 6BT
Address all correspondence and requests for reprints to: Dr. N. N. Chan, EURODIAB, University College London, 1–19 Torrington Place, London, United Kingdom WC1E 6BT. E-mail: nnkachan{at}aol.com
Abstract
During the menstrual cycle, changes in endothelium-dependent
vasodilatation have been demonstrated in conduit vessels in
vivo, but responses in resistance vessels have not been
studied. The aim of this study was to examine endothelium-dependent
vasodilatation, the effects of local nitric oxide synthesis, and
-adrenergic constriction in resistance vessels during the menstrual
cycle in 15 healthy female volunteers (mean age, 28.07 ± 2.1 yr).
Forearm blood flow in response to intrabrachial infusion of bradykinin
(10, 30, and 100 pmol/min; endothelium-dependent vasodilator), glyceryl
trinitrate (4, 8, and 16 nmol/min; endothelium-independent
vasodilator), noradrenaline (60, 120, and 240 pmol/min; -adrenergic
receptor agonist), and
NG-monomethyl-L-arginine (1, 2,
and 4 µmol/min; nitric oxide synthase inhibitor) was assessed by
venous occlusion plethysmography. All subjects were studied in early
menstrual phase (days 1–4) and midcycle (days 10–13). Vasodilator
response to bradykinin, expressed as the within-subject mean difference
in the area under the dose-response curve between phases, was
significantly increased at midcycle compared with that in the early
menstrual phase (486.5 ± 165.0; P = 0.01),
whereas there was no significant difference in response to glyceryl
trinitrate (185.8 ± 239.0; P = 0.45). The
vasoconstrictor response to noradrenaline was significantly greater at
midcycle (97.1 ± 39.4; P = 0.027), but the
response to
NG-monomethyl-L-arginine was not
significantly different (17.5 ± 35.2; P =
0.63). Serum estradiol was approximately 3-fold higher at midcycle,
with a mean difference of 252.3 ± 56.0 pmol/L
(P = 0.0005). Progesterone concentrations were not
significantly different (-0.11 ± 0.1 nmol/L;
P = 0.28). Differences in endogenous estrogen
levels between menstrual phases may underlie changes in bradykinin and
noradrenaline responses. If exogenous estrogens have similar effects,
the balance of these two opposing actions may determine whether
estrogen replacement in postmenopausal women has beneficial or harmful
effects on the vasculature.
THE RISK OF developing coronary heart disease is lower in premenopausal women than in men of similar age (1). It has been assumed that estrogen contributes to this cardiovascular protection in women. This concept is supported by findings from observational studies that estrogen replacement therapy is associated with a lower incidence of coronary heart disease and slower progression of coronary artery lesions in postmenopausal women (2). However, the beneficial effect of oral combined hormone replacement therapy was not confirmed by the Heart and Estrogen/Progestin Replacement Study, a large randomized secondary prevention trial (3).
Estrogens exert multiple effects on the blood vessel, on both the
endothelium and vascular smooth muscle cells (4). Some of
these effects might be beneficial, and others harmful. The vascular
endothelium releases a variety of vasoactive substances, including
nitric oxide (NO), that promote vasodilatation and inhibit
atherogenesis and thrombosis. Experimental evidence suggests that
estrogen may alter vascular reactivity both acutely and chronically via
modulation of NO production by the vascular endothelium
(4). Physiological concentrations of estrogen cause a
rapid release of NO in cultured bovine and human endothelial cells
(5, 6), whereas long-term administration of estrogen
enhances endothelium-dependent vasodilatation (7, 8).
Acute effects may be mediated in part by activation of preformed
endothelial NO synthase protein (9) and chronic effects
through increased transcription of endothelial NO synthase
(10). Estrogens also exert a number of other potentially
important effects on blood vessels, including effects on
cyclooxygenase, prostaglandins, and adrenoreceptors
(11, 12, 13). Some of these actions (such as stimulation of
vasodilatory prostacyclin) (12) might be protective to the
vasculature, whereas other actions (such as an increase in
2-adrenergic receptor-mediated platelet
hyperreactivity to noradrenaline) (11) might be
potentially detrimental.
The estrogen level fluctuates during the menstrual cycle, with the
lowest level seen during menses, followed by a rise in the follicular
phase, reaching a peak at midcycle. Hence, in vivo vascular
studies of women at different phases of the menstrual cycle should
enable a physiological assessment of the effects of endogenous
estrogen. Previous studies of women during the menstrual cycle have
demonstrated variability in cardiovascular responses, including
flow-mediated dilatation (14, 15, 16), arterial distensibility
(17), sympathetic outflow (18), and
2-adrenergic responses to agonists
(19), between different phases of the menstrual cycle. The
aim of the present study was to test the hypothesis that
agonist-stimulated and basal endothelium-dependent NO-mediated vascular
responses in resistance vessels differ in the phase with the peak
estrogen level (midcycle) and the phase with the lowest estrogen level
(early menstrual phase) of the menstrual cycle.
Subjects and Methods
Subjects
Fifteen healthy female volunteers were recruited, including 10
Caucasians, 2 Afro-Caribbeans, 2 Indians, and 1 Chinese. All subjects
were between 19–47 yr of age and had had regular menstrual cycles
(26–34 days) for at least 3 months before the study. All were
nonsmokers, had no past medical history of note, and were not taking
any form of medication. No subject had been taking the oral
contraceptive pill in the 6 months before the study. Apart from 1
subject who previously had a termination of pregnancy, all other
subjects were nulliparous. Other subject characteristics are summarized
in Table 1
. Each volunteer was given a
detailed written information sheet before enrolment in the study.
Informed consent was obtained from each participant before the study.
The study was approved by the local ethics committee.
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Forearm blood flow studies were performed on 2 occasions in each volunteer, 1 study after the onset of menses (EMP) on days 1–4 and the other at midcycle on days 10–13. Studies were performed either in the morning (3 EMP and 4 midcycle studies) or in the afternoon (12 EMP and 10 midcycle studies), with the exception of 1 subject who was studied in the evening during midcycle. All subjects were advised to avoid drinks containing caffeine 24 h before the study. After the subjects had relaxed for 5 min, blood pressure was taken in the right arm using an automated device (Omron 705CP, OMRON Health Europe B.V., The Netherlands) with the subject seated. Nonfasting blood samples were taken and centrifuged immediately for 15 min, and serum was stored at -70 C. Sex hormone levels and the lipid profile of all subjects were measured after completion of the study. Serum estradiol and progesterone concentrations were measured by a sensitive RIA (DiaSorin, Inc., Woking, Berks), and serum lipids were measured enzymatically. In 12 subjects the EMP study was performed first.
Study protocol
Studies were performed in a quiet temperature-controlled (24–27 C) laboratory. With participants supine, a 27-guage stainless steel needle (Cooper’s Needle Works, Birmingham, UK) was inserted into the brachial artery of the nondominant arm under local anesthesia with 1 mL 1% lignocaine. Drugs were dissolved in 0.9% sodium chloride solution (normal saline) and were infused at 0.5 mL/min. Forearm blood flow was recorded simultaneously in both arms by venous occlusion plethysmography (20) calibrated to measure absolute blood flow with temperature-compensated strain gauges attached to the forearms. During measurements, upper arm congesting cuffs were inflated to 40 mm Hg for 10 of every 15 s, and circulation to the hands was excluded by inflating the wrist cuffs to 200 mm Hg.
After 15 min of normal saline infusion, basal blood flow was measured for 2 min. Infusions of bradykinin (CLINALFA, Laufelfingen, Switzerland; 10, 30, and 100 pmol/min, each dose for 3 min), glyceryl trinitrate (GTN; David Bull Laboratories, Inc., Warwick, UK; 4, 8, and 16 nmol/min, each dose for 5 min), noradrenaline (Levophed, Sanofi Pharmaceuticals, Inc., Guildford, UK; 60, 120, and 240 pmol/min, each dose for 5 min), and NG-monomethyl-L-arginine (L-NMMA; CLINALFA, Laufelfingen, Switzerland; 1, 2, and 4 µmol/min, each dose for 5 min) were performed, with responses to each drug infusion separated by a 10-min saline washout period. Forearm blood flow was measured for the final minute of each infusion period for each dose of each drug. Flow was recorded for approximately 10 s in every 15 s, and the mean of the four measurements of each recording period was used for data analysis. Baseline blood flow was expressed as milliliters of blood per 100 mL forearm vol/min, and dose-response curves were constructed for all four vasoactive agents.
Statistical analysis
The ratio of blood flow in the infused forearm to that in the noninfused forearm was calculated for each measurement period. Responses to drugs were expressed as the percent change in the forearm blood flow ratio (infused arm/control arm) relative to the immediately preceding baseline flow ratio (infused arm/control arm). This controls for effects of external influences, such as state of arousal, sympathetic activation, and temperature, during the experiment. We tested whether there was an interaction between drugs and vascular response to drugs in the two phases of menstrual cycle using repeated measures ANOVA. The area under the dose-response curve (AUC) was calculated for each drug as a summary measure of drug response to allow quantitative comparison between the two phases of menstrual cycle. The difference in AUC and subject characteristics at midcycle and early menstrual phase was compared using the paired t test. The SEM was used as an index of dispersion. P < 0.05 was considered statistically significant.
Results
Baseline variables
There was no significant difference in basal blood flow, blood
pressure, glucose, or lipid profile between early menstrual phase and
midcycle measurements (Table 1
).
Dilator studies
Bradykinin and GTN caused dose-dependent increases in blood flow
in both phases of the menstrual cycle. The increase in forearm blood
flow in response to bradykinin at midcycle was significantly greater
than that during the early menstrual phase, as determined by AUC
(1349.7 ± 180.2 vs. 863.1 ± 112.6; Fig. 1). The mean within-person difference in
AUC between phases was 486.5 ± 165.0 (P = 0.01;
Table 2). The response to GTN during the
midcycle, however, was not significantly different from that during the
early menstrual phase (AUC, 1565.5 ± 195.27 vs.
1379.7 ± 235.35; Fig. 2), and the
mean difference in AUC was 185.8 ± 239.0 (P =
0.45).
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A decrease in blood flow in response to both noradrenaline and
L-NMMA was observed in both phases of the menstrual cycle.
The reduction in FBF in response to noradrenaline during the midcycle
was significantly greater than that during the early menstrual phase
(AUC, 307.9 ± 37.9 vs. 210.9 ± 25.4; Fig. 3). The mean difference in AUC was
97.1 ± 39.4 (P = 0.027; Table 2). No significant
difference in the response to L-NMMA was detected
(AUC, 348.89 ± 36.6 vs. 366.4 ± 26.2; Fig. 4). The mean difference in AUC was
17.7 ± 35.2 (P = 0.63). As shown in Table 2,
serum estradiol was higher at midcycle than in the early menstrual
phase (midcycle, 376.1 ± 62.2 pmol/L; range, 166-1082; early
menstrual phase, 123.8 ± 18.2 pmol/L; range, 58–324 pmol/L),
with a mean difference in AUC of 252.3 ± 56.0 (P
= 0.0005), but progesterone concentrations were not significantly
different (midcycle, 1.01 ± 0.12 nmol/L; range, 0.45–2.1 nmol/L;
early menstrual phase, 1.15 ± 0.13 nmol/L; range, 0.35–2.0
nmol/L), with a mean difference in AUC of -0.11 ± 0.1 nmol/L
(P = 0.3).
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Discussion
The results of our study indicate that vascular responses in resistance vessels change during the menstrual cycle in women. Specifically, at a time of high estrogen levels, the responses to the endothelium-dependent dilator bradykinin were enhanced, with no change in basal NO-mediated dilatation or vascular smooth muscle responsiveness to an exogenous NO donor. However, changes in vascular reactivity were not confined to endothelium-dependent responses, and noradrenaline-induced vasoconstriction was also enhanced during the high estrogen phase of the cycle. Progesterone, blood pressure, and lipid profile did not change during the two menstrual phases in our study. As progesterone may attenuate the actions of estradiol on endothelium-dependent vasodilatation (21), we chose to study our subjects during phases of the menstrual cycle in which progesterone levels are lowest to minimize its effect on vascular reactivity. Thus, it is likely that changes in endogenous estrogen level contribute to this vascular variability in forearm resistance vessels. Although estrogen is the most likely factor to account for changes in vascular reactivity, it remains possible that changes in vascular reactivity may also be due to cyclical changes in factors not measured in this study. Nevertheless, these findings have important implications for understanding the physiology of vascular changes during menstrual phases as well as interpreting previous vascular studies in the literature.
Agonist-stimulated endothelium-dependent vasodilatation
Bradykinin, one of the major endogenous regulators of vascular tone, produces endothelium-dependent vasodilatation via the bradykinin B2 receptor. Its effects in the forearm circulation are mediated in part through stimulation of NO release (22). In the present study in 15 healthy women, we found that bradykinin-induced vasodilatation was significantly enhanced during the midcycle compared with that during the early menstrual phase, whereas vasodilatation in response to GTN (endothelium-independent vasodilatation) was not significantly different between the two phases. This is consistent with previous findings in conduit vessels that vasodilatation in response to increased flow, another endothelium-dependent dilator stimulus, was greatest during midcycle compared with those in the follicular and luteal phases (15) and that responses in the follicular phase were greater than those in the luteal phase (16). Similarly, sex hormone deprivation after ovariectomy (for uterine leiomyoma) in women has been associated with a significant reduction in acetylcholine-induced vasodilatation in resistance vessels, which was restored 3 months after estrogen replacement therapy (8). Together these studies and ours indicate that endogenous estrogen significantly enhances agonist-stimulated endothelium-dependent vasodilatation. Indeed, the degree of variation in bradykinin response throughout the cycle was comparable to the difference reported between health and cardiovascular disease in previous studies of endothelial function (23).
Effect of NO synthase inhibition
Despite the clear changes in endothelium-dependent vasodilatation, we found no difference in the degree of vasoconstriction in response to L-NMMA between the two phases of the menstrual cycle, suggesting that basal NO-mediated dilatation in the forearm circulation is not altered by different menstrual phases. Previously, it has been found that peak expiratory NO in healthy women is markedly increased at midcycle (24), and that total NO production, as assessed by nitrite/nitrate concentrations, peaks at midcycle in premenstrual women (25) and is significantly increased in postmenopausal women after estrogen replacement therapy (26). The reasons for the differences between these studies and ours are not clear. However, the response to L-NMMA in our study offers a functional assessment of the basal NO:cGMP pathway, whereas measurements of NO or nitrite/nitrate in other studies (25, 26) reflect purely quantitative NO synthesis. Furthermore, NO may arise from any of the three NO synthase isoforms and from multiple cellular/tissue sites; therefore, changes in total body NO production may not give clear insight into endothelial NO generation. The present study had 80% power to detect a 30% change in the AUC of the L-NMMA dose-response curve at a significance level of P = 0.05. Although it is possible that smaller differences in the L-NMMA response occur during the menstrual cycle, the simplest explanation of our finding is that basal NO-mediated dilatation does not significantly change with physiological changes in the estrogen level, at least in the forearm resistance vessels.
In many situations, alterations in basal NO release (as assessed by L-NMMA constriction) and stimulated NO release (as assessed by dilatation to ACh or bradykinin) occur together. However, this is not always the case (27). In the present study where enhanced bradykinin-stimulated vasodilatation was not accompanied by any change in the L-NMMA response, it is possible that estrogen up-regulates signal transduction mechanisms that link B2 receptor activation to NO synthesis without altering basal NO synthesis. An alternative explanation is that as the vascular effect of bradykinin is only partially NO mediated, the enhanced vasodilatation may be due to augmentation of other endothelium-derived mediators during the high estrogen phase. Indeed, it has recently been suggested that endothelium-derived hyperpolarizing factor might be the predominant mediator in bradykinin-induced vasodilatation in human forearm resistance vessels (28). The findings of enhanced bradykinin-induced vasodilatation in our study may reflect modulation of endothelium-derived hyperpolarizing factor release/activity during the menstrual cycle. Further studies would be required to test this hypothesis directly.
Changes in -adrenergic receptor-mediated vasoconstriction
Although much recent interest has focused on the effects of
estrogen on endothelial function, there is also evidence that hormonal
changes during the menstrual cycle may modulate the sympathetic nervous
system, with effects on noradrenaline synthesis (29) and
-adrenergic receptor number (30) and sensitivity
(31). Increased urinary and plasma noradrenaline
(29, 32) have been reported during the luteal phase of the
cycle; the free plasma noradrenaline concentration is higher in women
than in men and correlates with the plasma estradiol concentration
(29). Estrogen also up-regulates
2-adrenergic receptor in myometrium in animals
and humans (30, 33), and several lines of evidence suggest
that estrogen may modulate -adrenergic receptor sensitivity in the
vasculature. In ovariectomized rats, estrogen enhances vasoconstriction
induced by vascular smooth muscle cell
2-adrenergic receptor activation in isolated
mesenteric arteries (13). In humans, vasoconstriction in
response to noradrenaline is greater in men than in premenstrual women
(studied during the first 14 days of the menstrual cycle)
(31), and 1-adrenergic
vasoconstriction in forearm resistance vessels is significantly greater
during the luteal phase than the follicular phase of the menstrual
cycle (19). In contrast,
2-adrenergic vasoconstriction was
significantly increased during the follicular phase in white (but not
black) women (19). Thus, there is a growing body of
evidence to support the concept that estrogens modulate sympathetic
activity, but the effects are complex, and the magnitude and direction
of these changes have differed from study to study. In our study we
examined the effects of changes in estradiol concentrations within the
physiological range. We found a relatively shallow dose response to
noradrenaline, as reported previously in women (31), and
found that vasoconstriction in response to noradrenaline was
significantly enhanced during midcycle compared with that in the early
menstrual phase. These findings are consistent with data from animal
studies and may reflect estrogen-mediated alterations in -adrenergic
receptor sensitivity. Although the mechanisms involved in the menstrual
phase-dependent changes in -adrenergic receptor sensitivity are
unclear, the change in -adrenergic response has important
implications. Firstly, this estrogen-mediated increase in
-adrenergic vasoconstriction may counteract the enhanced
vasodilatory effects of other mediators. Secondly, noradrenaline has
been widely used as a comparative control agent for L-NMMA
in vascular studies, and it is now clear that the menstrual phase of
women should be taken into account during data interpretation.
Study limitations
Although the simplest interpretation of our findings is that high concentrations of estrogens alter vascular reactivity to bradykinin and noradrenaline, there are limitations that warrant discussion. Firstly, all subjects were studied in the nonfasting state (1–2 h after breakfast or lunch); hence, there may have been variations in the lipid concentrations. Although triglyceride concentration will vary with nonfasting state, there is no evidence that acute hypertriglyceridemia alters vascular reactivity (34), and triglyceride concentrations were comparable in the two phases. Further analysis showed that the within-subject difference in triglyceride concentrations between menstrual phases does not account for changes in bradykinin and noradrenaline responses (P = 0.434 and P = 0.644, respectively). Secondly, other factors, such as body weight, exercise, diet, and physical activity, were not measured, but the fact that each subject serves as her own control should minimize these differences.
Conclusion
In conclusion, there is enhanced endothelium-dependent
vasodilatation and enhanced -adrenergic vasoconstriction of
resistance vessels in women at midcycle compared with the early
menstrual phase measurements. Differences in estradiol levels between
cycle phases may underlie changes in bradykinin and noradrenaline
responses. If these are estrogenic effects, and if exogenous estrogens
have similar actions, the balance of these two opposing actions may
determine whether estrogen replacement therapy has beneficial or
harmful effects on the vasculature.
Acknowledgments
We thank Dr. G. S. Conway for his endocrine advice and D. Lambrou for his statistical guidance.
Footnotes
1 This work was supported by the British Heart Foundation. 
Received August 18, 2000.
Revised December 7, 2000.
Revised February 12, 2001.
Accepted February 17, 2001.
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receptor subclasses by gonadal steroids in human myometrium. J
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