α-Adrenergic control of blood flow during exercise: effect of sex and menstrual phase

Abstract

Sex differences exist in autonomic control of the cardiovascular system. This study was designed to directly test sex or female menstrual phase-related differences in α-adrenergic control of blood flow during exercise. We hypothesized that women would exhibit reduced α-adrenergic vasoconstriction compared with men during exercise; in addition, women would constrict less during the early luteal than the early follicular phase of the female menses. Young men (n = 10) were studied once and women (n = 9) studied twice, once during the early follicular phase and once during the early luteal phase of female menses. We measured forearm blood flow (FBF; Doppler ultrasound of the brachial artery) during rest and steady-state dynamic exercise (15 and 30% of maximal voluntary contraction, 20 contractions/min). A brachial artery catheter was inserted for the local administration of α-adrenergic agonists [phenylephrine (PE; α₁) or clonidine (CL; α₂)]. Blood flow responses to exercise [forearm vascular conductance (FVC)] were similar between all groups. At rest, infusion of PE or CL decreased FVC in all groups (40–60% reduction). Vasoconstriction to PE was abolished in all groups at 15 and 30% exercise intensity. Vasoconstriction to CL was reduced at 15% and abolished at 30% intensity in all groups; women had less CL-induced constriction during the early luteal than early follicular phase (P < 0.017, 15% intensity). These results indicate that vasodilator responses to forearm exercise are comparable between men and women and are achieved through similar paths of α-adrenergic vascular control at moderate intensities; this control may differ at low intensities specific to the female menstrual phase.

the understanding of vascular control mechanisms responsible for skeletal muscle blood flow during exercise stems from predominantly male participants. Given the potential for sex-specific differences in physiological control during exercise, these results are limited in application. Evidence from both animal and human studies indicates that women demonstrate greater blood flow to exercising muscles compared with men (19, 25, 28). The prominent role of the sympathetic nervous system in the integrated exercise response (29, 37) provides support that sex differences in sympathetic control have the potential to influence muscle blood flow responses.

At rest, women exhibit reduced vasoconstrictor responses to sympathetic stimulation in both the forearm and calf compared with men (16, 18). One potential mechanism may be related to the vasodilatory effect of estrogen and its ability to inhibit α-adrenoceptor binding (32, 33). After acute exposure to increased levels of estrogen, perimenopausal women exhibit blunted norepinephrine (NE) responsiveness (33). Possible sex differences in sympathetic control during exercise remain unknown.

Despite sympathetic activation during exercise, blood flow and oxygen delivery increase proportionally to meet the metabolic demand of the contracting muscle. Muscle contractions and subsequent metabolites reduce responsiveness to NE in the vascular beds of contracting skeletal muscle, and blunted sympathetic vasoconstriction, termed functional sympatholysis, is observed (26). As such, at higher exercise intensities, NE-mediated vasoconstriction is limited further (1, 26).

Female hormones appear to modulate functional sympatholysis. Specifically, impaired functional sympatholysis (enhanced adrenergic vasoconstriction) has been observed in the contracting muscles of ovariectomized rats (9) and postmenopausal women (8). After treatment with 17β-estradiol (9) or estrogen replacement therapy (8), this impairment was attenuated; that is, adrenergic vasoconstriction was reduced during exercise with the presence of exogenous estrogen. The role of progesterone is less clear; currently, the vascular effects of progesterone alone and in combination with estrogen are inconclusive (10, 12, 24, 40).

Given recent reports of blunted α-adrenergic responses in young women compared with men at rest and greater muscle blood flow during exercise, we hypothesized that functional sympatholysis would be greater in women than men; that is, women would exhibit less α-adrenergic-mediated vasoconstriction than men during exercise. Additionally, female hormones are associated with reduced sympathetic-mediated vasoconstriction; thus we hypothesized that women would exhibit enhanced sympatholysis during the early luteal phase compared with the early follicular phase of the menstrual cycle, when hormone levels are high. Important to study design, all women studied were naturally cycling, and their hormone levels were not artificially controlled by supplements or contraceptives.

MATERIALS AND METHODS

Subjects.

Young healthy men (n = 10) and women (n = 9) participated in the study. All subjects completed a screening process in which physical activity and personal health history were assessed. All subjects were recreationally active, nonsmokers, free from overt cardiovascular disease, and not taking any cardiovascular medications. Female subjects were excluded if pregnant or using hormonal birth control. Women had a regular menstrual cycle and were studied once during the early follicular (EF) phase and once during the early luteal (EL) phase of the menstrual cycle. For logistical and safety purposes, female subjects completed the EF visit first, and ≥6 wk separated the two visits. Men visited the laboratory one single time.

Subjects were instructed to refrain from exercise, aspirin, NSAIDs, alcohol, and caffeine for 24 h prior to the study day. Written, informed consent was obtained from all subjects. All procedures were approved by the Institutional Review Board at the University of Wisconsin-Madison and conformed to the standards set by the Declaration of Helsinki.

Measurements.

Weight and height measurements were performed, and body mass index was calculated as weight in kilograms divided by height in squared meters (kg/m²). Heart rate was monitored continuously by a three-lead ECG. Blood pressure was measured continuously from a pressure transducer connected to the indwelling arterial catheter. Forearm volume was determined using water displacement.

Brachial artery catheterization.

Subjects laid supine for insertion of the catheter. Under aseptic conditions and after local anesthesia (2% lidocaine), a 20-gauge, 5-cm catheter was placed in the brachial artery of the nondominant forearm in the antecubital fossa (1 male subject was studied in the dominant arm due to an anatomic anomaly in the nondominant arm). The catheter was used for local administration of vasoactive drugs and for blood sampling (30, 41). The catheter was flushed continuously at 3 ml/h with heparinized saline.

Blood sampling.

Thirty minutes after catheter placement and prior to exercise or drug infusions, whole blood was drawn from the arterial catheter. Blood samples were centrifuged and plasma was frozen at −20°C. Samples were later analyzed for hormone levels (estradiol, progesterone) using radioimmunoassay and enzyme immunoassay.

FBF.

Blood flow (artery diameter, blood velocity) was measured with Doppler Ultrasound (Vivid 7; General Electric). A 12-MHz linear array probe was placed approximately midway between the antecubital and axillary regions medial to the biceps brachii muscle. The ultrasound probe operator continuously adjusted the probe position to maintain a fixed insonation angle of 60°, with the sample volume adjusted to cover the width of the brachial artery, compensating for small movements during exercise (21, 30, 31).

Dynamic forearm exercise.

Each subject laid supine with the nondominant arm extended to the side at ∼90°. Dynamic and rhythmic forearm exercise required that participants squeeze and release two handles together 4–5 cm to raise and lower a weight over a pulley at a rate of 20 times/min (at a duty cycle of 1 s contraction-2 s relaxation) (21, 30, 41). Functional sympatholysis is graded with the level of exercise intensity; therefore, forearm exercise was completed at two workloads. Forearm maximal voluntary contraction (MVC) of the nondominant arm was determined as the average of the highest two measurements from five consistent trials using a hand dynamometer; forearm exercise was completed at 15 and 30% of MVC. This forearm exercise model is identical to that used in several laboratories (17, 31, 41).

Intra-arterial drug infusions.

All drugs [phenylephrine (Baxter Healthcare, Deerfield, IL), clonidine (Xanodyne Pharmaceuticals, Newport, KY), and sodium nitroprusside (Hospira, Lake Forest, IL)] were infused via the brachial artery catheter and were mixed specifically for each study visit to standard concentrations. Phenylephrine (PE; 0.03125 μg·dl forearm volume⁻¹·min⁻¹ normalized to blood flow) is a selective α₁-adrenergic agonist. Clonidine (CL; 0.15 μg·dl forearm volume⁻¹·min⁻¹ normalized to blood flow) is a selective α₂-adrenergic agonist that acts primarily postjunctionally (3, 4). Both were infused to determine postjunctional α-adrenergic vasoconstrictor responsiveness (4, 29). By administering all drugs locally (dose/l forearm volume), we were able to minimize systemic blood pressure changes and activation of other counterregulatory systems. All drug infusions were also adjusted for the blood flow conditions on the basis of steady-state FBF and forearm volume in an effort to normalize the concentrations of each drug in the blood perfusing the forearm across conditions where blood flow might differ within and between groups (7, 29, 41).

As a separate control condition, we assessed the effect of passive vasodilation on α-adrenergic vasoconstrictor responsiveness. To elevate resting FBF to similar levels observed during exercise, we infused sodium nitroprusside (NTP; 2 μg·dl forearm volume⁻¹·min⁻¹ normalized to blood flow) (37). Nitroprusside releases nitric oxide to directly relax vascular smooth muscle.

Study protocols.

All testing was performed in the supine position. A total of seven study conditions were randomized and counterbalanced between subjects with, a 10-min rest period after each trial (Fig. 1A). NTP was infused as a high-flow control and was followed by PE infusion. For each additional drug infusion (PE, CL), we assessed three levels of exertion: 1) rest, 2) forearm exercise at 15% of MVC, and 3) forearm exercise at 30% of MVC. Each trial was 7 min in length, with PE or CL infused during the final 3 min (Fig. 1B). Beat-to-beat heart rate, blood pressure, and brachial artery blood velocity measurements were obtained throughout each trial. This model is similar to those published previously (7, 17, 29, 37, 41).

Fig. 1.

Experimental design. A: study protocol; this study required 7 study conditions involving rest, forearm exercise, or nitroprusside (NTP) infusion. Drug orders were randomized and counterbalanced. Each condition was followed by a 10-min rest period (R). B: breakdown by condition; the NTP trial required NTP infusion for 7 min, with phenylephrine (PE) infused during the final 3 min (left). Exertion trials were 7 min in length, with PE or clonidine (CL) infused during the final 3 min (right). Black boxes signify steady-state measurements of blood velocity (followed by vessel diameter; ↓) used in data analysis. MVC, maximal voluntary contraction.

Data acquisition.

Data were collected using similar methods published previously (21). Heart rate (HR) was derived from the ECG, and values were confirmed with blood pressure tracings. Blood flow (FBF) was determined as the product of mean blood velocity (MBV; cm/s) and vessel cross-sectional area (CSA; radius in cm²) and was reported in milliliters per minute [FBF = (MBV)(CSA)(60 s/min)]. Arterial blood velocity was assessed continuously throughout each study condition (except during intermittent artery diameter measurements). Reported pulse-wave velocities were measured beat to beat at the last 30 s of rest, steady-state exercise, and drug infusion to reduce contraction-to-contraction-induced variability in blood flow (Fig. 1B). Diameter measurements typically resulted in loss of pulse wave signal for 15 s. To determine vessel CSA, artery diameters were taken as an average of five measurements in late diastole. Arterial diameter was measured on B-mode images in the part of the artery running perpendicular to the ultrasound beam and was identified by strong wall signals in the longitudinal section of the artery in each image. All measurements were obtained from video images taken at rest, after 3 min of each condition, and after 3 min of vasoconstrictor infusion (PE, CL) and were assessed offline by a well-trained operator.

A commercial interface unit (Multigon Industries) processed the angle-corrected, intensity-weighted Doppler audio information from the GE Vivid ultrasound system into a flow velocity signal via fast Fourier transform; this method has been validated previously in our laboratory by measuring volumetric flow through a tube of known diameter. In addition, the transfer of Doppler audio signal to PowerLab has been validated (2). This signal was sampled in real time with signal-processing software (PowerLab; ADInstruments). All hemodynamic data were digitized, stored on a computer at 400 Hz, and analyzed offline using PowerLab; post-processing using PowerLab's Chart 5 application package yielded mean blood velocities, blood pressures, and HRs.

Data analysis.

The primary analysis was to test whether vasoconstriction resulting from infusion of α-adrenergic agonists was different between groups (men vs. EF women vs. EL women). The main dependent variables were forearm vascular conductance (FVC) normalized to forearm limb volume and the percent changes in FVC after infusion of α-adrenergic agonists. To determine FVC at relative workloads, FBF measurements (ml/min) were normalized for blood pressure and forearm volume under each specific condition; FVC was reported as ml·min⁻¹·100 mmHg⁻¹·100 ml⁻¹. FVC measurements are most appropriate for sex comparisons because women exhibit lower blood pressure and forearm volume compared with men (Tables 1 and 2). A change in FVC from rest was calculated as [FVC during exercise] − [FVC at rest]. We used percentage reduction in FVC as our standard index to compare vasoconstrictor responses to agonists across conditions; this method has emerged as the most appropriate way to compare vasoconstrictor responsiveness under conditions where marked differences exist in steady-state blood flow (3, 34, 37). Percent reduction in FVC (%change FVC) after vasoconstrictor administration was calculated as [(FVC post) − (FVC pre) / (FVC pre)] × 100.

Table 1.

Subject demographics

	Men (n = 10)	Women (n = 9)
Age, yr	25 ± 2	26 ± 2
Height, cm*	183.8 ± 2.4	167.1 ± 2.5
Weight, kg*	80.8 ± 2.9	64.9 ± 3.4
BMI, kg/m2	24.0 ± 0.9	23.2 ± 1.1
Forearm volume, ml*	1,161 ± 52	811 ± 46
Maximal voluntary contraction, kg*	47 ± 3	31 ± 1
Exercise workload, kg
15%*	7.1 ± 0.5	4.6 ± 0.2
30%*	14.2 ± 1.0	9.2 ± 0.4

Values are means ± SE. BMI, body mass index.

^*P < 0.05.

Table 2.

Forearm hemodynamics with PE infusion

	Men		EF Women		EL Women
	SS	PE	SS	PE	SS	PE
Diameter, cm
Rest	0.46 ± 0.01*†	0.47 ± 0.01*†	0.38 ± 0.02	0.37 ± 0.01	0.39 ± 0.01	0.39 ± 0.01
15%	0.47 ± 0.01*†‡	0.48 ± 0.01*†	0.37 ± 0.02	0.38 ± 0.02§	0.39 ± 0.01	0.39 ± 0.01
30%	0.48 ± 0.01*†‡	0.49 ± 0.01†*§	0.40 ± 0.02‡	0.40 ± 0.02	0.41 ± 0.01‡	0.41 ± 0.01
Heart rate, beats/min
Rest	52 ± 2	52 ± 3	56 ± 2	57 ± 3	59 ± 2	58 ± 2
15%	55 ± 3	58 ± 4	62 ± 3‡	62 ± 2	66 ± 3	70 ± 5
30%	56 ± 4‡	60 ± 3§	63 ± 3	69 ± 3	69 ± 4	69 ± 5
Mean blood pressure, mmHg
Rest	92 ± 2	93 ± 3	85 ± 3	87 ± 4	86 ± 4	87 ± 4
15%	97 ± 2*†‡	100 ± 3	87 ± 3	90 ± 4	87 ± 3	88 ± 4
30%	99 ± 2*‡	104 ± 3*†§	87 ± 3	91 ± 3§	88 ± 4	91 ± 3
Blood flow, ml·min−1·100 ml−1
Rest	13.0 ± 2.9	7.0 ± 1.4§	9.8 ± 1.5	4.9 ± 0.8§	6.5 ± 1.2	4.6 ± 1.0§
15%	34.3 ± 3.6‡	31.8 ± 3.0	28.7 ± 3.6‡	27.4 ± 3.3	24.6 ± 2.7‡	23.1 ± 2.2
30%	45.9 ± 4.0‡	52.0 ± 4.6§	44.2 ± 4.4‡	44.4 ± 5.3	37.1 ± 2.8‡	39.3 ± 3.9
Vascular conductance, ml·min·100 mmHg−1·100 ml−1
Rest	14.3 ± 3.1	7.7 ± 1.6§	11.7 ± 1.9	5.8 ± 1.1§	7.7 ± 1.5	5.4 ± 1.3§
15%	35.6 ± 4.0‡	32.3 ± 3.2	33.3 ± 4.5‡	30.9 ± 4.0	28.7 ± 3.3‡	26.9 ± 3.0
30%	46.3 ± 3.9‡	50.1 ± 4.4	52.3 ± 6.8‡	49.7 ± 7.4	43.4 ± 4.5‡	43.6 ± 4.4

Values are means ± SE. PE, phenylephrine; SS, steady-state; EF, early follicular; EL, early luteal. SS values before and after drug infusion (PE).

^*Difference between EF men and women (P < 0.017);

^†difference between EL men and women (P < 0.017);

^‡significant change from rest to exercise within groups (P < 0.05);

^§significant change from rest to PE infusion within groups (P < 0.05).

Statistics.

All statistics were done with the assistance of a biostatistician. Differences between rest/exercise as well as steady-state/drug infusion were assessed for normality (Shapiro-Wilk) and were compared using a Student's t-test approach to determine whether the parameter of interest was different from zero within a group (sex/menstrual phase) at each workload. In case of nonnormal data, the equivalent nonparametric version, Wilcoxon signed rank, was used. Groups were compared using ANOVA at each workload to determine the significance of sex/menstrual phase on various parameters of interest. All data are presented as means ± SE. Significant main effects (P < 0.05) were followed by a Bonferroni adjustment for multiple comparisons; thus, P values <0.017 were considered significant when three comparisons were made (men vs. EF women, men vs. EL women, and EF vs. EL women). All P values reported were two-sided; analyses were performed using SAS statistical software version 9.2 (SAS Institute, Cary, NC).

RESULTS

Subject characteristics.

Ten men and nine women completed the study. Subject characteristics are summarized in Table 1. There were no significant differences between groups in regard to age and body mass index [P = not significant (NS)]. As expected, men had greater height, weight, MVC, and forearm volume than women (P < 0.05). Each female participant was followed for a minimum of 3 mo to confirm a regular menstrual cycle before participation (cycle length 28 ± 1 days); EF visits were on day 3 ± 0.3, and EL visits were on day 15 ± 0.8 of the female menstrual cycle. Blood plasma was collected from each study participant; however, the majority of samples were lost in a freezer malfunction. The viable samples were from a subset of women studied (n = 3). Each subject exhibited higher plasma [estradiol] and [progesterone] during the EL visit (105 ± 34 and 3,738 ± 3,015 pg/ml, respectively) compared with the EF visit (36 ± 4 and 590 ± 85 pg/ml, respectively), although increases were not statistically significant (P = 0.06, P = 0.18).

Systemic responses to exercise.

HR was not statistically different between groups at rest; HR significantly increased 4–9 beats/min with exercise within groups (see Tables 2 and 3). Brachial artery diameter was greater in men at each condition (rest and exercise) compared with women during either phase (P < 0.017); diameter increased ∼0.02 cm in each group with exercise, although changes were not significant for all trials (Tables 2 and 3). Because of differences in blood pressure between men and women (Table 2), all blood flow measurements were normalized for perfusion pressure and are reported as FVC. In addition, small but significant changes in arterial blood pressure (3–7 mmHg) occurred between rest and exercise within some trials (Tables 2 and 3).

Table 3.

Forearm hemodynamics with CL infusion

	Men		EF Women		EL Women
	SS	CL	SS	CL	SS	CL
Diameter, cm
Rest	0.47 ± 0.01*†	0.47 ± 0.01*†	0.37 ± 0.02	0.38 ± 0.01	0.38 ± 0.02	0.38 ± 0.02
15%	0.48 ± 0.01*†‡	0.48 ± 0.01*†	0.38 ± 0.02	0.38 ± 0.02	0.39 ± 0.01‡	0.39 ± 0.01
30%	0.49 ± 0.01*†‡	0.49 ± 0.01*†	0.39 ± 0.02‡	0.40 ± 0.02	0.40 ± 0.01‡	0.40 ± 0.01
Heart rate, beats/min
Rest	53 ± 3	52 ± 3	59 ± 3	59 ± 3	59 ± 2	57 ± 2
15%	54 ± 3	58 ± 3§	63 ± 3‡	62 ± 3	62 ± 3	63 ± 3
30%	60 ± 3‡	63 ± 3	68 ± 3‡	70 ± 3	67 ± 3‡	69 ± 3
Mean blood pressure, mmHg
Rest	95 ± 1*	97 ± 2*	88 ± 2	88 ± 2	90 ± 3	90 ± 3
15%	96 ± 2†	96 ± 2†	90 ± 2	90 ± 2	86 ± 3‡	85 ± 3
30%	98 ± 2‡	101 ± 2	91 ± 2‡	93 ± 2	90 ± 3	93 ± 3§
Blood flow, ml·min−1·100 ml−1
Rest	12.5 ± 2.2	4.9 ± 0.9§	10.9 ± 1.7	4.6 ± 1.1§	6.9 ± 1.4	2.9 ± 0.5§
15%	33.8 ± 3.7‡	29.0 ± 2.6§	29.6 ± 2.6‡	23.8 ± 2.2§	22.4 ± 2.6‡	21.3 ± 2.8
30%	48.4 ± 3.6†‡	46.1 ± 2.3†	40.5 ± 1.9‡	39.5 ± 2.8	35.4 ± 2.5‡	34.2 ± 3.2
Vascular conductance, ml·min−1·100 mmHg−1·100 ml−1
Rest	13.3 ± 2.3	5.1 ± 0.9§	12.5 ± 2.0	5.1 ± 1.2§	7.9 ± 1.7	3.2 ± 0.5§
15%	35.3 ± 4.0‡	30.3 ± 2.8§	33.1 ± 2.8‡	26.7 ± 2.5§	26.8 ± 3.7‡	25.5 ± 3.7
30%	49.3 ± 3.9‡	45.5 ± 2.2	44.5 ± 2.4‡	42.6 ± 2.8	40.1 ± 3.5‡	36.9 ± 3.5

Values are means ± SE. CL, clonidine. SS values before and after drug infusion (CL).

^*Difference between EF men and women (P < 0.017);

^†difference between EL men and women (P < 0.017);

^‡significant change from rest to exercise within groups (P < 0.05);

^§significant change from rest to CL infusion within groups (P < 0.05).

Systemic responses to drug infusions.

Brachial artery diameter, HR, and arterial pressure with drug infusion are summarized in Tables 2 and 3. Small but significant within-group changes in brachial artery diameter, HR, and arterial blood pressure (∼5 mmHg) occurred with PE infusion (P < 0.05). In addition, small but significant within-group changes in HR and blood pressure were seen with CL infusion (P < 0.05).

Vasodilatory responses to exercise.

Steady-state FVC (prior to drug infusion) was similar between PE and CL trials (P = NS) and is therefore presented as an average of the two trials (Fig. 2). Resting FVC was greater in men than in women during the EL phase (P < 0.017). Both exercise intensities (15 and 30% MVC) increased blood flow in all groups from rest in an intensity-specific manner. FVC responses to exercise were similar between groups (Fig. 2A). When FVC was normalized for differences in resting measures, the change in FVC from resting values was similar between groups (P = NS; Fig. 2B).

Fig. 2.

Forearm vascular hemodynamics at rest and steady-state exercise (means ± SE) A: forearm vascular conductance (FVC) in men, early follicular (EF) phase women, and early luteal (EL) phase women at 3 levels of exertion (rest, 15% MVC, and 30% MVC). FVC was greater in men than in women during the EL phase at rest. ‡P < 0.017, men vs. EL women. B: a change in FVC from rest in men, EF women, and EL women during forearm exercise at 15 and 30% MVC. Values were similar between groups.

α₁-Adrenergic vasoconstriction during exercise.

Percent reductions in FVC after PE infusion are summarized in Fig. 3. The vasoconstrictor responses to PE during exercise were significantly blunted compared with responses during rest (P < 0.017). At both intensities, exercise abolished any reduction in FVC due to vasoconstriction with exogenous PE. This response was similar between groups (P = NS).

Fig. 3.

α₁-Adrenergic vasoconstriction to PE (means ± SE). Constriction was calculated as a %change in FVC from steady-state to postinfusion of PE; the reduction in FVC observed at rest was abolished with exercise at 15 and 30% intensity.

α₂-Adrenergic vasoconstriction during exercise.

Percent reductions in FVC after CL infusion are summarized in Fig. 4. Infusion of CL decreased FVC by ∼60% in all groups at rest. The vasoconstrictor responses to CL during 15% exercise intensity in men and EF women were significantly blunted compared with responses during rest (P < 0.017); this vasoconstrictor response was abolished in women during the EL phase (P < 0.017). Exercise abolished any change in FVC during 30% intensity similarly between groups (P = NS).

Fig. 4.

α₂-Adrenergic vasoconstriction to CL (means ± SE). Constriction was calculated as a %change in FVC from steady-state to postinfusion of CL; the reduction in FVC observed at rest was abolished with exercise at 30% intensity. Values were different between women during the EF and EL phases during 15% exercise. †P < 0.017, EF women vs. EL women.

Vasodilatory responses to NTP.

NTP infusion increased FVC (men 58 ± 9, EF 47 ± 5, EL 55 ± 7 ml·min⁻¹·100 mmHg⁻¹·100 ml⁻¹), and steady-state levels of FVC were comparable with those seen during exercise at 30% MVC; these values were similar between groups (P = NS; data not shown). Infusion of PE decreased FVC similarly between groups (men −37 ± 7, EF −44 ± 8, EL −36 ± 3%, P = NS) to a level comparable with those seen with infusion of PE at rest (men −36 ± 9, EF −45 ± 10, EL −32 ± 9%).

DISCUSSION

Current understanding of mechanisms behind the control of exercise blood flow stems from predominantly male participants. Given the potential for sex-specific differences in physiological control, it is important to determine whether adrenergic control mechanisms are dependent upon sex or menstrual phase. This study directly examined sex and menstrual phase-related differences in adrenergic-mediated vasoconstriction both at rest and during exercise in normally cycling women. The novel findings of this study include the following: 1) exogenous α₁-adrenergic vasoconstriction is abolished in both men and women at 15 and 30% exercise intensity, regardless of menstrual phase (Fig. 3 and Table 2); 2) during the early luteal phase of the female menses, women respond to an exogenous α₂-adrenergic agonist with less vasoconstriction during exercise at 15% effort (Fig. 4 and Table 3); and 3) exogenous α₂-adrenergic vasoconstriction is abolished similarly in men and women at 30% exercise intensity, regardless of menstrual phase (Fig. 4 and Table 3). Findings from this study suggest that blood flow responses to forearm exercise are not different between men and women and are achieved through a similar degree of functional sympatholysis at moderate intensities; however, α₂-adrenergic vasoconstriction may differ at lower intensities relative to female menstrual phase.

Sex-specific differences in exercise blood flow.

Recent evidence both supports (19, 25) and refutes (14) the notion that women respond to exercise with greater blood flow to exercising muscle compared with men. When conductance was expressed in relative units (ml·min⁻¹·100 mmHg⁻¹·100 ml⁻¹) at relative workloads (%MVC), no differences in the steady-state exercise responses were observed between men and women (Fig. 2B). Similarly, when vascular conductance was expressed in absolute units (ml·min⁻¹·100 mmHg⁻¹) on a continuum of absolute workloads (kg), no sex-specific differences were observed (data not shown). Results from the current study support the theory that forearm vasodilator response to forearm exercise is similar between men and women, regardless of menstrual phase. Discrepancies with other study results may be due to limb- (19, 27, 36), exercise intensity- (20, 27), or exercise modality-specific differences in vascular control of blood flow (14). Gonzales et al. (14) reported similar forearm blood flows between women and men in response to dynamic handgrip exercises; however, exercise was performed at gradually increasing intensities (ramping) to exhaustion, making it difficult to compare directly with our moderate steady-state exercise. During single-leg knee extension exercise, Parker et al. (25) found that the hyperemic response to exercise was greater in young women compared with men at workloads >40% of maximal effort. Subjects in the current study performed forearm exercise at workloads ≤30% of maximal effort; because of the research design, workloads >40% of maximal effort in the arm may be confounded by the development of muscle fatigue, increases in muscle sympathetic nerve activity, or other systemic responses (37). Similarly, performing this complex design in the leg can increase systemic responses in addition to increasing risk to human participants. Taken together, our data clearly exhibit similar exercise vasodilatory responses between men and women at intensities ≤30% of MVC. Future invasive studies of this nature will need to address this research question at higher intensities in both limbs.

Functional sympatholysis.

The current study locally infused NTP to increase forearm blood flow to levels similar to those observed during exercise (29). We observed no differences in endothelium-independent vasodilation between groups (data not shown). In addition, combined infusion of NTP with an α₁-adrenergic agonist exhibited an ∼40% reduction in vascular hemodynamics similarly between groups (data not shown). Using NTP as a measure of high blood flow, these data indicate that passive vasodilation does not impact α-adrenergic vasoconstrictor responsiveness in resting muscle. Thus any differences observed in vascular responses to adrenergic infusions during exercise are specific to the working muscle (functional sympatholysis).

In human forearms, the degree of sympatholysis in men has been shown to be similar between α₁- and α₂-adrenergic receptor subtypes during low-intensity (10–15% MVC) exercise (29). The current data confirm this finding and extend results both to women and to moderate-intensity exercise; our findings suggest that, at low to moderate intensities, exogenous α₁- (15 and 30% MVC) and α₂-adrenergic vasoconstriction (30% MVC) in all groups is not only blunted but abolished (Figs. 3 and 4).

Complete elimination of exogenous α-adrenergic vasoconstriction has previously been observed during leg extension exercise in humans (42). However, results in the exercising leg suggest that α₂-adrenoceptors are more sensitive to metabolic inhibition than α₁-adrenergic receptors (42). Similar results have been observed in animal models; functional sympatholysis at lower workloads may be due primarily to blunting of postjunctional α₂-adrenergic receptor-mediated vasoconstriction, and α₁-mediated responses are preserved until heavy exercise (3, 34). Taken together, these observations suggest that the functional distribution of α-adrenoceptors is not the same in the arm compared with leg circulation (Refs. 17 and 29 and the current study). Species and limb differences in adrenergic receptor density, distribution, and responsiveness may exist. In addition, relative exercise intensities and differences in pharmacological agonists may explain conflicting results.

Sex differences in α-adrenergic control.

At rest, women have been shown to exhibit blunted vasoconstrictor responses to sympathetic stimulation in both the forearm and calf compared with male subjects (16, 18). This was not observed in the current study; vasoconstrictor responses to adrenergic agonists were similar between groups at rest (P = NS). Discrepancies may be due to differences in experimental approach; a strength of our study was the direct assessment of alterations in α-adrenergic vasoconstrictor responses by local infusion of receptor-specific pharmacological agonists directed at α₁- and α₂-adrenergic receptors.

Previous studies have used nonspecific pharmacological agonists (18) or cold pressor test (16) to elicit sympathetic responses. Infusing exogenous NE or tyramine into the brachial artery (18, 29) limits the ability to discriminate between specific α-adrenergic receptors. In addition, NE may also bind β-adrenergic receptors. It has been shown that stimulation of β₂-adrenergic receptors causes greater forearm vasodilation in women at midmenstrual cycle than it does in men (15, 18). Therefore, any group differences observed with NE infusion could be due to differences in α-adrenergic vasoconstriction, β₂-adrenergic vasodilation, or both. By directly testing vasoconstrictor responses to specific adrenergic agonists, we clearly present that α-adrenergic vasoconstriction is largely similar in the forearm circulation of men and women.

Considering that adrenergic responsiveness may not be consistent between rest and exercise (5), it was important to systematically address sex-specific differences that may exist specifically during exercise. Contrary to our hypothesis, responsiveness to the α₁-adrenergic agonist PE was similar between men and women both at rest and during exercise (Fig. 3). In addition, our results suggest responsiveness to the α₂-adrenergic agonist CL to be similar between men and women (Fig. 4).

Menstrual phase differences in functional sympatholysis.

To our knowledge, this is the first study to directly assess differences in vascular responsiveness to α-adrenergic stimulation during natural hormone fluxes in the female menstrual cycle (no hormonal therapy or hormone contraceptives were used in these subjects). Impaired functional sympatholysis has been observed in both human (postmenopausal) and animal (ovariectomized) models exhibiting low levels of female hormones; this impairment was attenuated with estrogen therapy (8, 9). In the current study, we observed exogenous α₁-adrenergic responses to be similar between groups when studying young women with regular menstrual cycles. In addition, α₂-adrenoceptor sympathetic vasoconstrictor responses were blunted during exercise in women during the EL phase compared with the EF phase at low exercise intensity (15% MVC). Whereas this altered sympatholysis will not likely affect systemic blood pressure regulation at these low exercise intensities, our data suggest that there is a difference in vascular control with changes in the menstrual cycle.

Acute changes in circulating estrogen levels may play a role in the observed difference; potential mechanisms include decreased NE binding to receptors (32), suppressed receptor expression (44), altered sympathetic innervation (45), and altered gene transcription of vasoactive metabolites (22) with increases in plasma estrogen concentration. Increased estrogen levels have been shown to upregulate nitric oxide that normally opposes sympathetic vasoconstriction. However, this idea may be limited given that nitric oxide appears to mediate sympatholysis in rats (9, 35) but is not obligatory in humans (6, 29).

Additionally, it is important to consider that women during the EL phase of the menstrual cycle will exhibit increased levels of estrogen in addition to rises in progesterone; currently, the vascular effects of estrogen and progesterone alone and in combination are inconclusive (40). Previous research suggests that the favorable vascular effects of estrogen are attenuated (10) or maintained (12, 24) in the presence of progesterone.

Taken together, our results indicate that menstrual phase-related differences in adrenergic responsiveness are not observed with exogenous α₁-adrenergic stimulation and are specific to α₂-stimulation. Along these lines, Freedman and Girgis (11) suggest that, at rest, women are less responsive to exogenous α₂-stimulation during the luteal phase than the follicular phase of female menses. Research in rabbits suggests that estrogen will depress α₂- but not α₁-adrenergic responsiveness, possibly because of a reduced density of α₂-receptors (13); this has yet to be shown in humans. In contrast to our results, Freedman and Girgis (11) also observed greater responsiveness to exogenous α₁-stimulation at rest during the luteal phase than during the follicular phase. The relationship between α₁- and α₂-adrenergic vasoconstriction appears to be complex and may differ under a variety of hormonal and exercise conditions.

Our findings also suggest that women achieve similar vasodilatory responses to exercise (Fig. 2B), with lower α₂-adrenergic vasoconstriction (Fig. 4) during the EL phase compared with women during the EF phase. However, resting sympathetic nerve activity has been shown to be higher during the EL phase (23), which might offset any reduction observed in α-adrenergic vasoconstriction. Thus, acute changes in estrogen in normally cycling women may lead to reduced α-adrenergic responsiveness during conditions of higher circulating NE. The combined effect may result in similar FVC measurements between menstrual phases. Future research is needed to better understand the effect of estrogen with or without progesterone on adrenergic responsiveness, the role this plays during different phases of a normal female menses, and its influence at higher exercise intensities.

Experimental considerations.

Recent evidence in aging men suggests that the level of sympatholysis is dependent upon whether responses are compared at relative or absolute workloads (43). In the current study, women exhibited lower MVC than male participants (Table 1), and thus, at a given relative workload, women completed lower absolute work. In a subset of participants, we were able to compare a similar absolute workload (∼8.5 kg) at different relative intensities (15% for men and 30% for women). Results indicate the level of sympatholysis to be similar between sexes (Fig. 5); the lack of sex differences in forearm adrenergic vasoconstriction does not appear to be dependent on whether responsiveness is assessed at relative or absolute intensities in this population. However, it is important to consider that these data are from only a subset of participants; future studies should include multiple exercise intensities to assess differences on the basis of intensity, because the appropriateness of measuring blood flow levels at absolute and relative intensities is debatable (43).

Fig. 5.

Adrenergic vasoconstriction at an average absolute workload (means ± SE). Data are reported at an average absolute workload (men, 8.5 ± 0.5 kg; EF women, 8.5 ± 0.4 kg) in a subset of participants (men, n = 4; EF women, n = 5). Constriction was calculated as a %change in FVC from steady-state to postdrug infusion. A: %FVC in men and EF women at rest and exercise at ∼8.5 kg with infusion of PE. The reduction in FVC was blunted with exercise. B: %FVC in men and EF women at rest and exercise at ∼8.5 kg with infusion of CL. The reduction in FVC was blunted with exercise. Values were similar between groups.

A potential limitation of the present study is the lack of plasma catecholamine and hormone measurements at rest and during exercise. However, catecholamine measurements have been collected previously using a similar study design (41), and responses were similar between men and women during dynamic forearm exercise at 20% MVC. Given that the small muscle mass and mild to moderate exercise intensities used in this study have been shown previously to maintain basal sympathetic nerve activity (38, 39), we likely avoided changes in muscle sympathetic nerve activity and activation of other counterregulatory systems.

We also lacked plasma hormone measurement in several subjects due to sample loss in a freezer malfunction. Thus we cannot state explicitly that each female subject exhibited elevated estrogen and/or progesterone. However, we recorded the menstrual cycle length (28 ± 1 days) of each female participant for ∼3 mo before enrollment in the study, we studied women on days 3 ± 0.3 and 15 ± 0.8 to correspond to EF and EL phases, respectively, of the female menses, and we obtained plasma hormone levels in a subset of women studied. In women studied (n = 3), each exhibited higher plasma [estradiol] during the EL than during the EF phase (P = 0.06). Taken together, we are confident that women were studied during two distinct phases of the menstrual cycle and that differences observed in adrenergic responsiveness were likely due to increases in female hormone levels. However, our design does not allow us to differentiate between effects due to estradiol and/or progesterone, since both were elevated. This elevation is similar to that seen during the majority of female menses and thus holds physiological relevance.

Last, we collected blood velocity data throughout exercise plus drug infusion; however, only the last 30 s of data were presented. This decision was made based on assessment of steady-state data collected in our laboratory and results from previous studies (7, 29, 41). However, if this analysis missed an initial nadir response to an adrenergic agonist, our results may overestimate sympatholysis. Whereas post hoc analysis in a subset of participants does not change the interpretation of our results (data not shown), researchers should consider a study design that would allow for this type of analysis in the future.

Conclusion.

We assessed whether sex or menstrual cycle phase alters α-adrenergic vasoconstrictor responses in human skeletal muscle during exercise. Our results indicate that sex differences do not exist in forearm blood flow during mild to moderate exercise; however, women exhibit reduced α₂-adrenergic vasoconstriction during exercise at 15% effort during the EL compared with the EF phase of the female menses. Furthermore, exogenous α₁- and α₂-adrenergic constriction is abolished during exercise at 30% effort similarly between men and women, regardless of menstrual phase. This suggests that blood flow responses to forearm exercise are similar between men and women and are achieved through similar adrenergic vascular control mechanisms at moderate intensities; however, these mechanisms in the forearm may differ at lower intensities specific to menstrual phase.

GRANTS

This study was supported by American Heart Association predoctoral award no. 0815622G (J. K. Limberg), the University of Wisconsin-Madison Virginia Horne Henry Foundation (J. K. Limberg, W. G. Schrage), and Grant 1ULRR025011 from the Clinical and Translational Science Award program of the National Center for Research, National Institutes of Health.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.