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. 2004 Oct 21;561(Pt 3):893–901. doi: 10.1113/jphysiol.2004.073619

Augmented sympathetic vasoconstriction in exercising forearms of postmenopausal women is reversed by oestrogen therapy

Paul J Fadel 1, Zhongyun Wang 1, Hitoshi Watanabe 1, Debbie Arbique 1, Wanpen Vongpatanasin 1, Gail D Thomas 1
PMCID: PMC1665388  PMID: 15498809

Abstract

Sympathetic vasoconstriction is normally attenuated in exercising muscles of young men and women. Recent evidence indicates that such modulation, termed functional sympatholysis, may be impaired in older men. Whether a similar impairment occurs in older women, and what role oestrogen deficiency might play in this impairment, are not known. Based on the strong positive correlation between circulating oestrogen levels and functional sympatholysis previously reported in female rats, we hypothesized that sympatholysis would be impaired in oestrogen-deficient postmenopausal women, and that this impairment would be reversed by oestrogen replacement. To test these hypotheses, we measured vasoconstrictor responses in the forearms of pre- and postmenopausal women using near infrared spectroscopy to detect decreases in muscle oxygenation in response to reflex activation of sympathetic nerves evoked by lower body negative pressure (LBNP). In eight premenopausal women, LBNP decreased muscle oxygenation by 20 ± 1% in resting forearm, but only by 3 ± 2% in exercising forearm (P < 0.05). In contrast, in eight postmenopausal women, LBNP decreased muscle oxygenation by 15 ± 3% in resting forearm, and by 12 ± 4% in exercising forearm (P > 0.05). After 1 month of transdermal oestradiol replacement in these women, the normal effect of exercise to blunt sympathetic vasoconstriction was restored (rest, −19 ± 3%; exercise, −2 ± 3%; P < 0.05). These data indicate that functional sympatholysis is impaired in oestrogen-deficient postmenopausal women. The effect of short-term unopposed oestrogen replacement to correct this impairment implicates a role for oestrogen in the sympathetic neural control of muscle haemodynamics during exercise.

To sustain physical activity, increases in skeletal muscle blood flow must meet the metabolic demands of contracting muscle. Activation of the sympathetic nervous system plays an important role in the redistribution of blood flow during exercise by causing vasoconstriction in nonexercising muscles and visceral organs, which redirects cardiac output to the active muscles. At the same time, the normal ability of sympathetic activation to cause vasoconstriction is attenuated in the active muscles, in part due to an effect of muscle metabolites to diminish the vasoconstrictor response to α-adrenergic receptor activation (Anderson & Faber, 1991; Thomas et al. 1998; Thomas & Victor, 1998). Such modulation, termed functional sympatholysis (Remensnyder et al. 1962), may constitute a protective mechanism that optimizes muscle blood flow in the face of the increased sympathetic vasoconstrictor drive that occurs during exercise.

In healthy older men, vasoconstrictor responses to acute sympathetic activation induced by a cold pressor test are reportedly augmented in the leg muscles during cycling exercise, resulting in reduced vascular conductance in the active muscles (Koch et al. 2003). These data suggest that ageing may impair functional sympatholysis in men. Whether sympatholysis also might be impaired in older women is not known, although previous studies have shown that limb vasodilatation is reduced, and that systemic vascular resistance and blood pressure are augmented during dynamic exercise in older women (Ogawa et al. 1992; Fleg et al. 1995; Green et al. 2002; Proctor et al. 2003). One unique aspect of ageing in women that was not addressed in these previous studies, but could potentially contribute to impaired vasoregulation during exercise in older females, is the decline in endogenous oestrogen that occurs following menopause.

In this regard, we recently reported that the vasoconstrictor response to sympathetic activation was enhanced in contracting muscles of anaesthetized ovariectomized rats, indicating that functional sympatholysis was impaired in these animals (Fadel et al. 2003). This impairment was prevented by short-term treatment with 17β-oestradiol. In contrast, it was not affected by either short-term treatment with progesterone or acute infusion of 17β-oestradiol. Taken together, these findings strongly implicated oestrogen-deficiency as the underlying cause of the enhanced sympathetic vasoconstriction in the contracting muscles of the ovariectomized rats. We now want to know whether these findings in young anaesthetized rats, which were oestrogen-deficient due to surgical removal of the ovaries, can be translated to the clinical setting of postmenopausal women.

Therefore, in the present study we hypothesized that functional sympatholysis would be impaired in oestrogen-deficient postmenopausal women, and that this impairment would be reversed by short-term oestrogen-replacement therapy (ORT). To test these hypotheses, we measured vasoconstrictor responses in the microcirculation of resting and exercising forearms of pre- and postmenopausal women using near infrared spectroscopy to detect decreases in muscle oxygenation evoked by the reflex activation of sympathetic nerves. To evaluate the role of oestrogen deficiency in the postmenopausal women, studies were repeated after 4 weeks of transdermal ORT.

Methods

Subjects

Eight premenopausal women and eight healthy postmenopausal women participated in the study after providing informed, written consent. Protocols were approved by the University of Texas Southwestern Medical Center Institutional Review Board, and were conducted in accordance with the Declaration of Helsinki. The premenopausal women were not using oral or subcutaneous contraceptives, and were studied during the follicular phase of their ovarian cycle. Prior to enrollment in the study, five of the postmenopausal women had been on ORT for durations ranging from 6 months to 5.5 years (n = 4, oral oestrogen; n = 1, transdermal oestradiol), while three of the women were not on ORT. After discontinuing ORT for 4 weeks, serum oestradiol levels were <40 pg ml−1 at the start of the study in all of the postmenopausal women. All of the subjects were normotensive, had no history of cardiovascular disease, and were not taking any medications. On experimental days, subjects were asked to abstain from caffeinated beverages for at least 8 h and from strenuous activity for 24 h before arriving at the laboratory.

Measurements

Subjects were studied in the supine position. Blood samples were taken and centrifuged, and the plasma was stored at −20°C for subsequent analysis of 17β-oestradiol using 125I-labelled radioimmunoassay kits (Mayo Clinic). Heart rate (HR) was recorded continuously from the electrocardiogram, and blood pressure was measured by automated oscillometric sphygmomanometry (CE0050, Welch Allyn, Skaneateles Falls, NY, USA).

Skeletal muscle oxygenation

Near infrared (NIR) spectroscopy was used to measure changes in tissue concentrations of oxygenated haemoglobin and myoglobin (HbO2 + MbO2) in the forearm, as previously described (Hansen et al. 1996; Chavoshan et al. 2002; Fadel et al. 2004). To monitor NIR light absorption, two fibre-optic bundles spaced 2 cm apart were placed over the flexor digitorum profundus muscle, which is the main muscle recruited during handgrip. NIR signals were sequentially sampled at a rate of 1 Hz, converted to optical density values using established algorithms, output to a personal computer, and digitally stored for later analysis. Changes in the NIR signals were quantified as a percentage of the total labile signal (TLS), which was defined in each experiment as the maximal decrease in HbO2 + MbO2 achieved during inflation of a pneumatic cuff on the upper arm to 220 mmHg for 2 min, beginning just prior to the end of exercise. Compared to Doppler measures of forearm blood flow, changes in tissue oxygenation provide a reliable measure of sympathetic vasoconstriction in resting and exercising muscle (Fadel et al. 2004).

Reflex activation of sympathetic nerves

Lower body negative pressure (LBNP) was used to produce reflex sympathetic vasoconstriction in the forearm. The subject's lower body was enclosed to the level of the iliac crest in a negative pressure chamber. LBNP at −20 mmHg primarily unloads the cardiopulmonary baroreceptors, evoking reproducible increases in muscle sympathetic nerve activity (MSNA) (Hansen et al. 1996). Multiunit recordings of MSNA were obtained with unipolar tungsten microelectrodes inserted into muscle fascicles of the peroneal nerve by microneurography (Vallbo et al. 1979). Neural signals were amplified, filtered (bandwidth 700–2000 Hz), rectified and integrated (time constant, 0.1 s) to obtain mean voltage neurograms. MSNA was expressed as burst frequency (bursts min−1) and total activity (burst frequency × mean burst amplitude).

Handgrip exercise

Maximal voluntary contraction (MVC) for each subject was designated as the greatest of at least three maximal squeezes of a handgrip dynamometer (Stoelting, Chicago, IL, USA). Subjects performed intermittent handgrip to the rhythm of a metronome (20 handgrips min−1; 50% duty cycle) at 30% MVC for 6 min. Force production was displayed on an oscilloscope to provide subjects with visual feedback. This level of handgrip alone does not increase MSNA (Hansen et al. 1996).

Experimental protocols

Blood pressure, HR, and forearm muscle oxygenation were measured in response to 2 min of LBNP at −20 mmHg applied at rest and during min 3–5 of handgrip. This protocol was performed once by all of the subjects, and was repeated in the postmenopausal subjects after 4 weeks of transdermal 17β-oestradiol at the clinically recommended dose of 100 μg day−1, which would be expected to restore circulating oestradiol to premenopausal levels. A time course of 4 weeks was chosen to parallel our recent animal study in which ovariectomized rats were treated with subcutaneous 17β-oestradiol for 3–4 weeks (Fadel et al. 2003). Subsets of the postmenopausal subjects performed three additional protocols. First, to examine the reversibility of the effects of oestrogen, the main protocol was repeated 4 weeks after termination of ORT. Second, to assess the reproducibility of the LBNP-induced changes in tissue oxygenation within each phase of the study (i.e. before and during ORT), subjects were studied on two separate days. Third, to evaluate the effect of oestrogen on LBNP-induced sympathoexcitation, MSNA responses to LBNP were measured at rest and during handgrip both before and during ORT.

Data and statistical analysis

Power calculations were performed to determine the sample sizes needed at the 0.05 level of significance for varying effect sizes (expected differences) based on the results of previous studies using the same experimental techniques and biological endpoints (Hansen et al. 1996; Chavoshan et al. 2002; Fadel et al. 2004). These historical data indicated that a sample size of four provides 90% power to detect robust functional sympatholysis, which is defined as an attenuation of the LBNP-induced decrease in muscle oxygenation by 80% or more in exercising versus resting forearm. To detect an impairment in functional sympatholysis of 60% or more, the sample size is increased to nine for 90% power or to seven for 80% power.

Data were acquired and analysed using PowerLab hardware and software (ADInstruments, Milford, MA, USA). Blood pressure, HR and muscle oxygenation responses to LBNP were determined by calculating the difference between 20 s of baseline immediately preceding LBNP and the last 20 s during LBNP. Because of the intermittent bursting pattern of MSNA, 60 s of data at baseline and during LBNP were analysed to obtain accurate measurements. For this protocol, the blood pressure, HR and muscle oxygenation responses to LBNP also were calculated using 60 s intervals.

Statistical analyses were performed using paired t tests or repeated measures ANOVA followed by Bonferroni post-hoc tests. Day-to-day reproducibility of functional sympatholysis was assessed by using Bland–Altman method of comparison. P < 0.05 was considered significant. Data are presented as means ± s.e.m.

Results

Subject characteristics are shown in Table 1. Premenopausal women were studied during the follicular phase of their ovarian cycle (8 ± 1 days after the onset of menses), and postmenopausal women were studied 8 ± 2 years after their last menstrual period. In the postmenopausal women, ORT increased serum oestradiol to premenopausal levels (Table 1). ORT also decreased systolic blood pressure (117 ± 3 to 113 ± 4 mmHg; P > 0.05), diastolic blood pressure (74 ± 2 to 70 ± 3 mmHg, P < 0.05), and mean arterial pressure (MAP) (Table 2), but did not affect HR (Table 2). MAP and HR responses to LBNP or rhythmic handgrip, performed alone and in combination, were similar before and during ORT (Table 2).

Table 1.

Subject characteristics

Premenopausal Postmenopausal
Follicular phase Before ORT During ORT
Age (years) 25 ± 2 54 ± 2 —
Serum oestradiol (pg ml−1) 144 ± 58 22 ± 5 136 ± 22*
BMI (kg m−2) 24.0 ± 1.6 27.5 ± 1.1 27.4 ± 1.1
Maximal handgrip (kg) 32 ± 1 29 ± 2 28 ± 2
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Values are mean ± s.e.m. BMI, body mass index.

*

P < 0.05 versus before oestrogen-replacement therapy (ORT)

Table 2.

Baseline values and responses to lower body negative pressure (LBNP) and rhythmic handgrip performed alone and in combination

Premenopausal Postmenopausal
Follicular phase Before ORT During ORT
MAP (mmHg) 77 ± 2 88 ± 3 85 ± 3*
ΔLBNP −1 ± 1 −1 ± 1 +1 ± 1
ΔHandgrip +5 ± 1 +9 ± 2 +8 ± 2
ΔLBNP + handgrip +2 ± 1 +2 ± 1 +2 ± 1
HR (beats min−1) 58 ± 2 65 ± 3 64 ± 3
ΔLBNP +1 ± 1 +1 ± 2 +1 ± 1
ΔHandgrip +12 ± 1 +7 ± 1 +8 ± 1
ΔLBNP + handgrip +1 ± 2 +3 ± 1 −1 ± 2
MSNA (bursts min−1) — 31 ± 5 25 ± 3
ΔLBNP — +9 ± 1 +11 ± 2
ΔHandgrip — +2 ± 4 +2 ± 2
ΔLBNP + handgrip — +8 ± 2 +12 ± 2
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Values are means ± s.e.m. Mean arterial pressure (MAP) and heart rate (HR), n = 8; muscle sympathetic nerve activity (MSNA), n = 4.

*

P < 0.05 versus before ORT.

Functional sympatholysis is impaired in oestrogen-deficient postmenopausal women

LBNP evoked substantial decreases in muscle oxygenation in the resting forearms of both premenopausal (−20 ± 1%) and postmenopausal (−15 ± 3%) subjects, without altering either MAP or HR (Figs 1 and 2, Table 2). Handgrip alone increased MAP (+5 ± 1, +9 ± 2 mmHg) and HR (+12 ± 1, +7 ± 1 beats min−1), and decreased muscle oxygenation (−29 ± 5, −25 ± 7%) in the premenopausal and postmenopausal subjects, respectively. When superimposed during handgrip, LBNP evoked only a small decrease in muscle oxygenation in the exercising forearms of the premenopausal subjects (−3 ± 2%; P < 0.05 versus response at rest), signifying robust functional sympatholysis. In contrast, LBNP produced a large decrease in muscle oxygenation in the exercising forearms of the postmenopausal subjects (−12 ± 4%; P > 0.05 versus response at rest), indicating impaired sympatholysis (Figs 1 and 2).

Figure 1. Recordings from a premenopausal woman and an oestrogen-deficient postmenopausal woman.

Figure 1

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In the premenopausal subject, the decrease in muscle oxygenation (oxygenated haemoglobin and myoglobin, HbO2 + MbO2) evoked by lower body negative pressure (LBNP) at rest was attenuated during forearm exercise. This exercise-induced attenuation was impaired in the postmenopausal subject. OD, optical density; TLS, total labile signal.

Figure 2. Muscle oxygenation responses to LBNP in resting and exercising forearm in premenopausal women and in postmenopausal women before and during ORT.

Figure 2

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Group means are shown as solid symbols. HG, handgrip; *P < 0.05 versus rest.

Functional sympatholysis is restored in postmenopausal women by ORT

After 4 weeks of ORT in the postmenopausal subjects, LBNP evoked decreases in muscle oxygenation in resting forearm (−19 ± 3%) that were similar to the responses before ORT (Fig. 2). Handgrip alone also produced increases in MAP (+8 ± 2 mmHg) and HR (+8 ± 1 beats min−1) and decreases in muscle oxygenation (−24 ± 8%) that were similar to the responses before ORT (Table 2). However, in contrast to the response before ORT, LBNP superimposed during handgrip evoked only a small decrease in muscle oxygenation in the exercising forearm (−2 ± 3%, P < 0.05 versus response at rest) (Fig. 2). This effect of ORT to restore functional sympatholysis was fully reversed in three of the postmenopausal subjects who were restudied 4 weeks after termination of ORT (Fig. 3).

Figure 3. Muscle oxygenation responses to LBNP in resting and exercising forearm in three postmenopausal women studied before, during and after (4 week washout) ORT.

Figure 3

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The impairment in the normal attenuation of sympathetic vasoconstriction in exercising forearm that was present in untreated postmenopausal women was reversed by ORT, and recurred after cessation of ORT. *P < 0.05 versus rest.

In an effort to better understand the effect of ORT to restore functional sympatholysis, several additional control experiments were performed in the postmenopausal subjects. First, to separate potential effects of random variation or subject familiarization from those of ORT, the reproducibility of the muscle oxygenation response to LBNP at rest and during handgrip was evaluated by repeating studies in six of the subjects on two separate days. These experiments revealed little day-to-day variability in the impairment of functional sympatholysis before ORT, or in the restoration of sympatholysis during ORT (Fig. 4). The bias of the difference in functional sympatholysis between day 1 and day 2 was −0.17 (with 0 indicating complete agreement), and all data points were within the Bland–Altman limits of agreement (±2 s.d.).

Figure 4. Reproducibility of the exercise-induced modulation of the LBNP-induced decreases in muscle oxygenation in six postmenopausal women studied on two separate days, either before or during ORT.

Figure 4

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Second, to determine if the improvement in functional sympatholysis observed during ORT might be explained by a change in the sympathetic vasoconstrictor stimulus rather than a change in the vascular response, MSNA was recorded in four of the postmenopausal women who consented to the microneurography procedure. This sample size is sufficient to provide 90% power at the 0.05 level of significance to detect the estimated 70% decrease in the MSNA response to LBNP that would be required to explain the profoundly attenuated (≈90%) muscle oxygenation response to LBNP in the exercising forearms of the postmenopausal women during ORT (Vissing et al. 1994; Fadel et al. 2004). In these subjects, the increases in MSNA evoked by LBNP were similar at rest and during handgrip, both before and during ORT (Table 2 and Fig. 5). In contrast, the decreases in muscle oxygenation evoked by LBNP were similar in resting and exercising forearms before ORT, but were markedly attenuated in the exercising forearms during ORT. Muscle oxygenation responses to LBNP for this protocol were calculated using 60 s data intervals to match the time frame used for the analysis of MSNA (see Methods). To confirm that these results did not differ from those of the previous protocols that were based on 20 s intervals, we recalculated the muscle oxygenation responses using the shorter interval. Results were similar using either 20 s (before ORT: rest, −13 ± 1% and handgrip, −8 ± 4%; during ORT: rest, −18 ± 4% and handgrip, −1 ± 5%) or 60 s intervals (Fig. 5).

Figure 5. Changes in forearm muscle oxygenation and muscle sympathetic nerve activity (MSNA) in response to LBNP at rest and during handgrip in four postmenopausal women studied before and during ORT.

Figure 5

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*P < 0.05 versus rest.

Discussion

The major new findings of our study are twofold. First, we report that the blunting of sympathetic vasoconstriction that normally occurs in exercising muscle (i.e. functional sympatholysis) is impaired in healthy postmenopausal women. Second, this impairment was reversed by 1 month of unopposed transdermal ORT. The present data extend our recent study of ovariectomized rats to postmenopausal women, indicating that sympathetic responsiveness is enhanced in the exercising muscles of oestrogen-deficient females and that short-term oestrogen replacement is an effective countermeasure to restore the normal attenuation of sympathetic vasoconstriction in the active muscles.

This is the first study to compare the effect of exercise on the responsiveness of the skeletal muscle vasculature to sympathetic activation in younger and older women. Consistent with our previous studies, the vasoconstrictor response to reflex sympathetic activation was attenuated by 50% or more during a moderate level of forearm exercise in all of the younger premenopausal subjects (Hansen et al. 1996; Sander et al. 2000; Chavoshan et al. 2002; Fadel et al. 2004). In contrast, such robust attenuation was lacking in the majority of the older untreated postmenopausal subjects. It is unlikely that this impairment in functional sympatholysis in the older women was due to an exaggerated sympathetic vasoconstrictor stimulus. Although MSNA was measured in only half of the older women, the increases evoked by LBNP were well within the range of responses typically observed in younger subjects in our laboratory (Hansen et al. 1996; Chavoshan et al. 2002), and were similar to the responses reported by Davy et al. (1998) in both younger and older men. The enhanced sympathetic vasoconstriction in the exercising forearms of the older women also probably cannot be explained by a general age-related increase in vascular reactivity because α-adrenergic vasoconstrictor responses in resting muscle are reported to decrease, rather than increase, with advancing age (Hogikyan & Supiano, 1994; Dinenno et al. 2002).

Instead, our data implicate oestrogen deficiency as the underlying cause of the enhanced sympathetic responsiveness in the exercising muscles of the untreated postmenopausal women. When serum oestradiol was restored to premenopausal levels by a short course of ORT in the older women, functional sympatholysis also was restored. These results were remarkably similar to our recent finding of impaired functional sympatholysis in young ovariectomized rats that was prevented by short-term (3–4 week) treatment with oestradiol, but not with progesterone (Fadel et al. 2003). The consistent findings of our studies in older women and in younger female rats suggest that the defective functional sympatholysis may be more closely related to oestrogen deficiency than to biological age. However, we cannot completely exclude an effect of age because the vasoconstrictor response to sympathetic activation also is reported to be greater in the exercising legs of healthy older men compared to younger men (Koch et al. 2003). This raises the unexplored possibility that the mechanism increasing sympathetic vasoconstriction in exercising muscle may differ in older men and women.

Although we do not know the precise mechanism by which ORT restored functional sympatholysis in the postmenopausal women in our study, the results do help us to narrow the list of potential explanations. ORT had no effect on the blood pressure or HR responses to LBNP or handgrip, whether performed alone or in combination, making it unlikely that changes in these variables contributed to the improvement in sympatholysis. ORT also had no effect on the MSNA responses to LBNP at rest or during handgrip, indicating that there was no substantial change in the sympathetic vasoconstrictor stimulus. Although microneurography was performed in only half of the postmenopausal subjects, this small sample size provided sufficient statistical power to detect the estimated 70% attenuation in the MSNA response to LBNP that would be required to explain the profoundly attenuated (≈90%) muscle oxygenation response to LBNP in the exercising forearms of the postmenopausal women during ORT (Vissing et al. 1994; Fadel et al. 2004). Taken together, these results suggest that the effect of ORT to improve functional sympatholysis is mediated by a change in the vascular response to MSNA in the exercising muscles.

Studies in a variety of experimental preparations, including cultured smooth muscle cells, isolated blood vessels, and intact animals and humans, have identified a number of potential mechanisms by which oestrogen might diminish sympathetic vasoconstriction. These include decreases in α-adrenergic receptor expression (Zhang & Davidge, 1999), sympathetic innervation (Zoubina & Smith, 2001), protein kinase C activity (Kanashiro & Khalil, 2001), and intracellular Ca2+ (Zhang et al. 1994), as well as increases in endothelium-dependent vasodilatation (Gilligan et al. 1994). However, in our study, ORT was associated with attenuated sympathetic vasoconstrictor responses in exercising muscle, but not in resting muscle. Because this change in sympathetic responsiveness was observed only in exercising muscle, it probably cannot be explained by a general effect of oestrogen to reduce vascular tone. A more plausible explanation is that oestrogen upregulates one or more of the mechanisms that are normally engaged to oppose sympathetic vasoconstriction in exercising muscle.

In this regard, nitric oxide (NO) has been identified as one of the potential mediators of functional sympatholysis in a number of previous animal and human studies (Thomas et al. 1998; Thomas & Victor, 1998; Sander et al. 2000; Chavoshan et al. 2002; Fadel et al. 2003; Buckwalter et al. 2004), although its precise role in humans remains controversial (Dinenno & Joyner, 2003; Rosenmeier et al. 2003). In skeletal muscle, NO is produced by endothelial NO synthase (eNOS), which is highly expressed in the vascular endothelium (Kobzik et al. 1995), and by neuronal NOS (nNOS), which is highly expressed in the skeletal muscle cells (Nakane et al. 1993; Kobzik et al. 1994). Decreased activity or expression of NOS, particularly nNOS, results in defective functional sympatholysis (Thomas et al. 1998; Thomas & Victor, 1998; Sander et al. 2000; Chavoshan et al. 2002; Fadel et al. 2003). We recently reported that nNOS, but not eNOS, is decreased in skeletal muscle of ovariectomized rats, and that this decrease was associated with enhanced sympathetic responsiveness in contracting muscle (Fadel et al. 2003). These effects of ovariectomy were prevented by oestrogen replacement. Based on these previous findings, we speculate that one way in which ORT restored functional sympatholysis in the postmenopausal women in our study may have been by upregulating skeletal muscle nNOS. Interestingly, 4 months of transdermal ORT at half the dose used in our study (50 μg day−1) was reported to increase nNOS expression in neutrophils (Garcia-Duran et al. 1999), suggesting that nNOS is sensitive to changes in the circulating level of oestrogen in humans.

Oestrogen also is reported to activate K+ channels, including the metabolically regulated ATP-sensitive potassium (KATP) channel (Ranki et al. 2002; Lee et al. 2003). Studies in both animals and humans have advanced the hypothesis that sympathetic vasoconstriction in exercising muscle is attenuated in part by the activation of vascular KATP channels (Thomas et al. 1997; Keller et al. 2004), providing another potential mechanism by which ORT could improve functional sympatholysis in postmenopausal women. In addition, both ageing and oestrogen deficiency are associated with increased production of oxygen free radicals in the vasculature (Wassmann et al. 2001; Solhaug, 2003). We previously have shown that oxidative stress impairs functional sympatholysis in an animal model of heart failure (Thomas et al. 2001), raising the possibility that the antioxidant effects of oestrogen may play a role in the beneficial effect of ORT on functional sympatholysis in the present study.

Whether the conclusions of our study based on responses in a small muscle mass performing rhythmic contractions can be extended to larger muscle masses performing dynamic exercise remains to be determined. However, previous studies have shown that systemic vascular resistance and blood pressure increase more during dynamic exercise in older women than in older men or younger women (Ogawa et al. 1992; Fleg et al. 1995; Green et al. 2002). Recently, Proctor et al. (2003) reported that the increases in leg vascular conductance during graded cycling exercise were attenuated in older women who were not taking any form of hormone therapy compared to younger women, and suggested that increased vasoconstriction in the exercising muscles of the older women may have contributed to this difference. The results of our study support this concept, and lead us to speculate that ORT might attenuate the vascular resistance and blood pressure responses to dynamic exercise in postmenopausal women by reducing sympathetic vasoconstriction in the active muscles.

Transdermal ORT has previously been reported to result in small decreases in basal blood pressure (Cagnacci et al. 1999; Seely et al. 1999; Vongpatanasin et al. 2001), which may be mediated in part by decreases of 25–30% in basal MSNA (Vongpatanasin et al. 2001; Weitz et al. 2001). Consistent with these findings, in our study diastolic and mean blood pressures were significantly reduced, and basal MSNA was decreased by 19% in the postmenopausal women during ORT, although the latter result was not statistically significant (P = 0.098). However, we would advise caution in the interpretation of these data because our study was neither designed nor appropriately powered to detect these rather modest decreases in basal blood pressure and MSNA. Direct comparison of the results also are complicated by differences in the dosage and duration of ORT used in these studies.

Also, our study was not designed to directly compare sympathetic vasoconstriction in younger and older women. Although the impaired functional sympatholysis in the older untreated women suggests that sympathetic vasoconstriction in exercising muscle is greater in older than in younger women, additional studies are required to answer this question. These would entail careful measurement and matching of the vasoconstrictor stimulus (e.g. MSNA, noradrenaline spillover) in younger and older subjects in order to determine if the absolute level of sympathetic vasoconstriction in exercising muscle increases with ageing.

In summary, the results of our study indicate that functional sympatholysis is less robustly expressed in the forearm muscles of postmenopausal women than in premenopausal women. The effect of ORT to fully restore functional sympatholysis in the postmenopausal women suggests a key role for oestrogen deficiency, although the underlying mechanism remains to be determined. Furthermore, the effect of ORT was apparent after only 1 month of transdermal oestradiol at a clinically relevant dose of 100 μg day−1. Whether this effect persists with more prolonged exposure to oestrogen or with concomitant progesterone replacement are important unresolved questions in light of the results of large prospective studies indicating that long-term hormone replacement therapy may not provide overall cardiovascular benefit (Hulley et al. 1998; Rossouw et al. 2002). We speculate that a future challenge may be to develop more refined interventions that can elicit the potential positive benefits of oestrogen on cardiovascular function while avoiding the apparent detrimental prothrombotic effects.

Acknowledgments

We thank Dr Robert Haley and Beverly Huet for their expert assistance in the statistical analyses. This work was supported by NIH grants HL64784 and HL06296 (G.D.T). P.J.F. was supported by NIH training grant HL07360 and individual NRSA HL69648.

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