Effect of acute dietary nitrate supplementation on sympathetic vasoconstriction at rest and during exercise

Abstract

Dietary nitrate ($\text{N}{\text{O}}_3^{-}$) supplementation has been shown to reduce resting blood pressure. However, the mechanism responsible for the reduction in blood pressure has not been identified. Dietary $\text{N}{\text{O}}_3^{-}$ supplementation may increase nitric oxide (NO) bioavailability, and NO has been shown to inhibit sympathetic vasoconstriction in resting and contracting skeletal muscle. Therefore, the purpose of this study was to investigate the hypothesis that acute dietary $\text{N}{\text{O}}_3^{-}$ supplementation would attenuate sympathetic vasoconstrictor responsiveness at rest and during exercise. In a double-blind randomized crossover design, 12 men (23 ± 5 yr) performed a cold-pressor test (CPT) at rest and during moderate- and heavy-intensity alternate-leg knee-extension exercise after consumption of $\text{N}{\text{O}}_3^{-}$ rich beetroot juice (~12.9 mmol $\text{N}{\text{O}}_3^{-}$) or a $\text{N}{\text{O}}_3^{-}$-depleted placebo (~0.13 mmol $\text{N}{\text{O}}_3^{-}$). Venous blood was sampled before and 2.5 h after the consumption of beetroot juice for the measurement of total plasma nitrite/$\text{N}{\text{O}}_3^{-}$ [NO_x]. Beat-by-beat blood pressure was measured by Finometer. Leg blood flow was measured at the femoral artery via Doppler ultrasound, and leg vascular conductance (LVC) was calculated. Sympathetic vasoconstrictor responsiveness was calculated as the percentage decrease in LVC in response to the CPT. Total plasma [NO_x] was greater (P < 0.001) in the $\text{N}{\text{O}}_3^{-}$ (285 ± 120 µM) compared with the placebo (65 ± 30 µM) condition. However, mean arterial blood pressure and plasma catecholamines were not different (P > 0.05) between $\text{N}{\text{O}}_3^{-}$ and placebo conditions at rest or during moderate- and heavy-intensity exercise. Sympathetic vasoconstrictor responsiveness (Δ% LVC) was not different (P > 0.05) between $\text{N}{\text{O}}_3^{-}$ and placebo conditions at rest ($\text{N}{\text{O}}_3^{-}$: −33 ± 10%; placebo: −35 ± 11%) or during moderate ($\text{N}{\text{O}}_3^{-}$: −18 ± 8%; placebo: −20 ± 10%)- and heavy ($\text{N}{\text{O}}_3^{-}$: −12 ± 8%; placebo: −11 ± 9%)-intensity exercise. These data demonstrate that acute dietary $\text{N}{\text{O}}_3^{-}$ supplementation does not alter sympathetic vasoconstrictor responsiveness at rest or during exercise in young healthy males.

NEW & NOTEWORTHY Dietary nitrate may increase nitric oxide bioavailability, and nitric oxide has been shown to attenuate sympathetic vasoconstriction in resting and contracting skeletal muscle and enhance functional sympatholysis. However, the effect of dietary nitrate on sympathetic vasoconstrictor responsiveness is unknown. Acute dietary nitrate supplementation did not alter blood pressure or sympathetic vasoconstrictor responsiveness at rest or during exercise in young healthy males.

INTRODUCTION

Dietary nitrate ($\text{N}{\text{O}}_3^{-}$) supplementation with beetroot juice has been shown to reduce resting blood pressure (BP) in sedentary and exercise-trained men and women (26, 35, 66, 67), and in patient populations (36, 56). Dietary $\text{N}{\text{O}}_3^{-}$ appears to increase nitric oxide (NO) bioavailability and enhance brachial artery flow-mediated dilation (5, 24, 67, 70). NO has also been shown to inhibit sympathetic vasoconstriction in resting and contracting skeletal muscle (sympatholysis) (30–32, 34, 59, 63). Therefore, dietary $\text{N}{\text{O}}_3^{-}$ supplementation may attenuate sympathetic vasoconstriction at rest and during exercise and alter sympathetic control of BP. Consistent with this notion, our laboratory has recently demonstrated that acute treatment with the NO cofactor tetrahydrobiopterin attenuated sympathetic vasoconstrictor responsiveness at rest and during exercise in rats (33).

An increase in NO bioavailability may also alter efferent sympathetic outflow. Indeed, Notay et al. (46) recently reported that acute dietary $\text{N}{\text{O}}_3^{-}$ supplementation decreased muscle sympathetic nerve activity (MSNA) at rest and blunted the MSNA response to sympathoexcitation via static handgrip exercise in young adults. However, the effect of dietary $\text{N}{\text{O}}_3^{-}$ supplementation on sympathetic nervous system activity during large muscle mass dynamic exercise has not been investigated.

Therefore, the purpose of this study was to investigate the effect of acute dietary $\text{N}{\text{O}}_3^{-}$ supplementation on sympathetic vasoconstrictor responsiveness, BP, and plasma norepinephrine (NE) and epinephrine (Epi) (an index of sympathetic outflow) at rest and during large muscle mass dynamic exercise in humans. It was hypothesized that acute dietary $\text{N}{\text{O}}_3^{-}$ supplementation would: 1) attenuate sympathetic vasoconstrictor responsiveness at rest and during exercise and enhance sympatholysis; 2) reduce plasma catecholamines at rest and during dynamic large-muscle mass exercise and in response to sympathoexcitation, and 3) reduce BP at rest and blunt the BP response to exercise and sympathoexcitation.

METHODS

Subjects.

Twelve healthy young males (23 ± 5 yr) volunteered and provided written informed consent to participate in the study. All subjects were nonobese nonsmokers and did not have respiratory, cardiovascular, metabolic, neurological, or musculoskeletal disease. Subjects were not taking any medications known to alter the cardiovascular, respiratory, or metabolic response to exercise. Subjects were recreationally active but were not engaged in an exercise-training program during the project. The study was approved by the University of Alberta Health Research Ethics Board and was conducted according to the Declaration of Helsinki (2008).

Experimental protocol.

Subjects reported to the Integrative Human Exercise Physiology Laboratory at the University of Alberta on four separate occasions. Subjects reported to the laboratory in a rested state (no exercise 24 h prior), after having consumed a light meal 2 h before testing and having abstained from caffeine, alcohol, and ibuprofen for 12 h before testing. Subjects also refrained from using antibacterial mouthwash, toothpaste, or chewing gum before testing.

On day 1, subjects completed an incremental exercise test to volitional exhaustion (V̇o_2peak) on a cycle ergometer (Ergoselect 200 K; Ergoline, Bitz, Germany) for determination of maximal aerobic capacity. Testing began with 2 min of resting data collection, after which the work rate was progressively incremented (30 W/min) in a ramp-like fashion. Criteria used to establish a maximal test included a plateau in O₂ consumption (V̇o₂) despite an increase in work rate, a respiratory exchange ratio >1.10, achievement of >90% of age-predicted maximal heart rate (HR), and volitional exhaustion.

On day 2, subjects performed an alternate-leg incremental exercise test to volitional exhaustion on a knee-extensor ergometer, as previously described (2, 11, 42). Briefly, subject legs were attached to padded bars attached to a lever arm on a Monark cycle ergometer (model 814E) to allow for alternating two-legged knee extension. Following a resting baseline, knee-extension exercise was performed at a cadence of 30 contractions/min for each leg in alternating pattern. Exercise was initiated from a baseline work rate of 18 watts and was increased by 3 watts every minute until volitional exhaustion or the subject was unable to maintain the required cadence. Criteria used to establish a maximal test included a plateau in V̇o₂ despite an increase in work rate, a respiratory exchange ratio >1.10, and volitional exhaustion. The maximum work rate achieved was used to calculate the work rates for constant-load knee-extensor exercise on testing days 3 and 4.

Testing on days 3 and 4 was performed in a randomized double-blind crossover manner and separated by at least 48 h. Subjects were asked to consume the same meal 12 h before and the same breakfast and snacks before testing on days 3 and 4. Subjects were also provided with a list of foods containing $\text{N}{\text{O}}_3^{-}$ and were asked to avoid eating the foods on the list 48 h before testing. Following arrival at the laboratory at 8:00 AM on each day, an indwelling venous catheter was inserted in the antecubital vein of their right arm. Venous blood (10 ml) was drawn for the measurement of resting total plasma [$\text{N}{\text{O}}_3^{-}$/nitrite ($\text{N}{\text{O}}_2^{-}$)] (NOx) levels. Subjects then consumed either a placebo $\text{N}{\text{O}}_3^{-}$-depleted beetroot juice (~0.13 mmol $\text{N}{\text{O}}_3^{-}$: Beet It, James White Drinks, Ipswich, UK) or a $\text{N}{\text{O}}_3^{-}$-rich beetroot juice (~12.9 mmol $\text{N}{\text{O}}_3^{-}$). Following 2.5 h of rest, the subject was seated and rested quietly on the alternate-leg knee-extension ergometer, and another venous blood sample was withdrawn. Data were collected under resting conditions for 7 min, subjects then performed a cold-pressor test (CPT) where they submerged their hand in −4°C ice water for 3 min, and another venous blood sample was withdrawn at the midway point of the CPT.

Following a recovery period, subjects performed 10 min of constant-load alternate-leg knee extension at 30% (24 ± 6 watts) and at 60% (47 ± 9 watts) of the peak work rate achieved on day 2. Knee-extension exercise was performed over a 2-s duty cycle (1 s contraction, 1 s relaxation), resulting in 30 contractions/min for each leg in an alternating pattern. Following 6 min of exercise, subjects submerged their hand in −4°C ice water for 3 min. Venous blood was sampled during exercise and the CPT. The timeline for testing and blood samples is shown in Fig. 1. Blood draws were attempted during each CPT; however, because of venoconstriction, it was either not possible to withdraw an adequate amount of blood or the blood sample was hemolyzed and unusable. Therefore, catecholamines could not be measured in response to the CPT. Blood samples for four subjects were mishandled postcollection; therefore, the sample size for catecholamine analysis at all other time points is n = 8. Rest and exercise trials were separated by a recovery period of ~30 min.

Fig. 1.

Timeline of testing on days 3 and 4. Following a resting blood sample, beetroot juice (BR) containing dietary nitrate ($\text{N}{\text{O}}_3^{-}$) or placebo was consumed (time 0). After 2.5 h, a resting blood sample was taken while the subject was seated in the alternate-leg knee-extension ergometer. The resting trial (Rest) and moderate (MOD)- and heavy (HVY)-intensity exercise trials were separated by ~30 min of recovery. Subjects performed a 3-min cold pressor test (CPT) at rest and during each exercise bout. Blood samples were taken at rest, during exercise, and during each CPT and are denoted by the arrows.

Measurements.

Oxygen consumption, CO₂ production, and ventilation were measured breath-by-breath using a mass flow sensor and metabolic cart (Vmax 229d; Viasys; Healthcare, Palm Springs, CA). The ECG was continuously recorded in a three-lead configuration (Power Laboratory 16/30; AD instruments, Colorado Springs, CO), and HR was derived from the ECG waveform. Beat-by-beat arterial BP was measured by finger photoplethysmography (Finometer, Amsterdam, the Netherlands). BP was also measured by sphygmomanometer, and Finometer BP was corrected to manually measured pressures when pressure differences were observed. Cardiac output (Q) and stroke volume (SV) were derived from the BP waveform via Modelflow. Modelflow estimates stroke volume from a three-component model that includes aortic impedance, arterial compliance, and vascular resistance (68). Studies that have compared echocardiography, dye-densitometry, and thermodilution measures of cardiac output with Modelflow estimates have reported that Modelflow reliably tracks changes in cardiac output in response to exercise and sympathoexcitation (13, 23, 27, 43, 51, 55, 57).

Femoral artery mean blood velocity (MBV) was measured in the right leg using pulsed-Doppler ultrasonography (Vivid I; GE, Waukesha, WI). Data were acquired continuously with a 7.5-MHz probe with a 45° angle of insonation placed on the skin surface 2–3 cm distal to the inguinal ligament. The diameter of the common femoral artery was measured during diastole in triplicate at rest. Previous studies have reported that the common femoral artery does not dilate during exercise; therefore, resting diameters were used to calculate blood flow at rest and during exercise (50, 53). Mean blood velocity was measured on a beat-by-beat basis. Leg blood flow (LBF) was calculated as LBF (ml/min) = MBV (cm/s)·πr²·60, where r is the radius of the femoral artery. Leg vascular conductance (LVC) was calculated as LBF/mean arterial pressure (MAP) (l·min⁻¹·mmHg⁻¹). The percentage change in LVC in response to the CPT was used to assess sympathetic vasoconstrictor responsiveness at rest and during exercise.

After withdrawal, venous blood samples were immediately mixed with EDTA and centrifuged at 2,500 revolutions/min for 10 min. Plasma was separated into aliquots, placed in microcentrifuge tubes, and frozen for subsequent analysis of plasma NE ([NE]) and Epi ([Epi]) concentration and plasma $\text{N}{\text{O}}_3^{-}$ ([$\text{N}{\text{O}}_3^{-}$]) and $\text{N}{\text{O}}_2^{-}$ concentration ([$\text{N}{\text{O}}_2^{-}$]). All samples from the same subjects were run in the same assay, and in duplicate. $\text{N}{\text{O}}_3^{-}$ and $\text{N}{\text{O}}_2^{-}$ were analyzed in plasma, using a commercially available colorimetric assay kit (Nitrate/Nitrite Colorimetric Assay Kit ab65328; Abcam, Toronto, Ontario), according to the procedures provided by the manufacturer. Total $\text{N}{\text{O}}_3^{-}$/$\text{N}{\text{O}}_2^{-}$ was measured after the addition of $\text{N}{\text{O}}_3^{-}$ reductase cofactor to each diluted sample, and the mixture was incubated for 3 h to allow for the full conversion of $\text{N}{\text{O}}_3^{-}$ to $\text{N}{\text{O}}_2^{-}$. Greiss reagent was then added, which converts $\text{N}{\text{O}}_2^{-}$ to a deep purple azo compound. The absorbance was measured at 540 nm, and quantification of each sample was performed with a calibration curve. The intra-assay coefficient of variation for the duplicate samples was 2.51%. Samples and standard curve were also prepared in the absence of $\text{N}{\text{O}}_3^{-}$ reductase for measurement of plasma $\text{N}{\text{O}}_2^{-}$. Plasma $\text{N}{\text{O}}_3^{-}$ is then calculated as the difference between total plasma $\text{N}{\text{O}}_3^{-}$/$\text{N}{\text{O}}_2^{-}$ and plasma $\text{N}{\text{O}}_2^{-}$ . Unfortunately, plasma $\text{N}{\text{O}}_2^{-}$ in our samples is below the detection limit of the assay kit. Therefore, we report total $\text{N}{\text{O}}_3^{-}$/$\text{N}{\text{O}}_2^{-}$ (NOx).

Plasma [NE] and [Epi] were measured using a commercially available enzyme-linked immunoassay kit using the manufacturer’s recommended procedures (2-CAT Plasma ELISA High Sensitive Enzyme Immunoassay; Rocky Mountain Diagnostics, Colorado Springs, CO). Briefly, NE and Epi were extracted by using a cis-diol-specific affinity gel, acylated, and then modified enzymatically. The absorbance was measured at 540 nm, and quantification of each sample was performed with a calibration curve. The intra-assay coefficient of variation for the duplicate samples was 15.34 and 9.33% for [NE] and [Epi], respectively.

Data analysis.

Data were recorded using a PowerLab 16/30 system and Chart 7 data acquisition software (AD Instruments) at a sampling frequency of 100 Hz.

Absolute values of HR, MAP, systolic BP (SBP), diastolic BP (DBP), LBF, and LVC were measured at rest and during exercise. Changes in HR, MAP, SBP, DBP, LBF, and LVC in response to the CPT at rest and during exercise were calculated. For each variable, the difference between the peak/nadir response (20 s average) and preceding baseline was calculated and was expressed as an absolute and percentage change. The percentage change of LVC (∆%LVC) in response to sympathoexcitation is the accepted metric to assess the magnitude of sympathetic vasoconstrictor responsiveness, since it accurately reflects the change in resistance vessel radius across different baseline levels of vascular conductance (7, 61). The magnitude of functional sympatholysis was calculated as the difference between the vasoconstrictor response at rest and during exercise (∆%LVC).

Statistical analysis.

All data are reported as means ± SD. The effect of dietary $\text{N}{\text{O}}_3^{-}$ supplementation on HR, MAP, SBP, DBP, LBF, LVC, and V̇o₂ at rest and during moderate- and heavy-intensity exercise was analyzed by two-way (exercise intensity × treatment) repeated-measures ANOVA. The effect of dietary $\text{N}{\text{O}}_3^{-}$ supplementation on the response (absolute change) of HR, MAP, SBP, DBP, LBF, LVC, and V̇o₂ to the CPT at rest and during exercise was analyzed by two-way (exercise intensity × treatment) repeated-measures ANOVA. The effect of dietary $\text{N}{\text{O}}_3^{-}$ supplementation on sympatholysis and sympathetic vasoconstrictor responsiveness (∆%LVC) at rest and during exercise was also analyzed by two-way (exercise intensity × treatment) repeated-measures ANOVA. When significant main effects and/or interactions were identified, Student-Newman-Keuls post hoc analyses were performed (SigmaPlot version 13.0; Systat Software, San Jose, CA). A P value <0.05 was considered statistically significant.

RESULTS

Subject characteristics.

Subject age, height, weight, body mass index, absolute and relative V̇o_2peak from the maximal cycling test, and maximal work rate and V̇o_2peak achieved during the alternate-leg knee-extension test are reported in Table 1.

Table 1.

Subject characteristics

Age, yr	23 ± 5.0
Height, cm	178 ± 8
Weight, kg	73.0 ± 9.5
Body mass index	23 ± 1.6
Absolute V̇o2peak, l/min	3.34 ± 1.08
Relative V̇o2peak, ml·kg−1·min−1	44.8 ± 11.7
Peak KE work rate, watts	77 ± 15
Peak KE V̇o2, l/min	1.78 ± 0.08

Values are means ± SD. KE, knee extension.

Effect of $\text{N}{\text{O}}_3^{-}$ supplementation at rest.

$\text{N}{\text{O}}_3^{-}$-rich beetroot juice elevated (P < 0.05) total plasma No_x concentration ([NO_x]) levels, whereas the $\text{N}{\text{O}}_3^{-}$-depleted beetroot juice did not alter (P > 0.05) total plasma [NO_x] (Fig. 2). Resting HR, cardiac output, MAP, SBP, DBP, LBF, and LVC were not different between placebo and $\text{N}{\text{O}}_3^{-}$-supplemented conditions (Table 2) (P > 0.05). Dietary $\text{N}{\text{O}}_3^{-}$ did not alter resting plasma [NE] or [Epi] (Fig. 3) (P > 0.05).

Fig. 2.

Total plasma $\text{N}{\text{O}}_3^{-}$/nitrite ($\text{N}{\text{O}}_2^{-}$) at rest and 2.5 h following consumption of beetroot juice containing $\text{N}{\text{O}}_3^{-}$ (n = 12) or $\text{N}{\text{O}}_3^{-}$-depleted (n = 12) beetroot juice. [NO_x], total $\text{N}{\text{O}}_3^{-}$/$\text{N}{\text{O}}_2^{-}$ concentration. Values are means ± SD. *Significant difference from placebo trial, P < 0.001.

Table 2.

Effect of dietary $\text{N}{\text{O}}_3^{-}$ at rest and during moderate- and heavy-intensity alternate-leg knee-extension exercise

	Rest		Moderate-Intensity Exercise		Heavy-Intensity Exercise
	Placebo	$\text{N}{\text{O}}_3^{-}$	Placebo	$\text{N}{\text{O}}_3^{-}$	Placebo	$\text{N}{\text{O}}_3^{-}$
Heart rate, beats/min	63 ± 10	61 ± 9	88 ± 11*	85 ± 7*	108 ± 12*#	105 ± 10*#
Stroke volume, ml/beat	103 ± 10	97 ± 16	127 ± 21*	125 ± 26*	125 ± 22*	131 ± 29*
Cardiac output, l/min	6.5 ± 1.0	5.9 ± 0.9	11.1 ± 1.5*	10.5 ± 2.1*	13.3 ± 2.0*#	14.1 ± 2.6*#
Mean arterial pressure, mmHg	84 ± 8	82 ± 5	97 ± 9*	98 ± 7*	116 ± 13*#	110 ± 5*#
Systolic blood pressure, mmHg	121 ± 12	117 ± 7	152 ± 14	151 ± 13	180 ± 18*#	169 ± 15*#
Diastolic blood pressure, mmHg	66 ± 6	64 ± 6	69 ± 9*	73 ± 6*	84 ± 11*#	81 ± 4*#
Leg blood flow, l/min	0.346 ± 0.086	0.324 ± 0.054	1.676 ± 0.365*	1.722 ± 0.310*	2.158 ± 0.366*#	2.266 ± 0.377*#
Leg vascular conductance, l·min−1·mmHg−1	0.004 ± 0.001	0.004 ± 0.001	0.017 ± 0.004*	0.018 ± 0.003*	0.019 ± 0.004*#	0.021 ± 0.003*#
Oxygen consumption, l/min	0.257 ± 0.022	0.270 ± 0.031	0.802 ± 0.130*	0.788 ± 0.086*	1.161 ± 0.146*#	1.169 ± 0.138*#

Values are means ± SD. A significant (P < 0.05) main effect of exercise intensity was observed.

^*Significant post hoc test difference from rest.

^#Significant post hoc test difference from moderate-intensity exercise.

Fig. 3.

Plasma norepinephrine ([norepinephrine]) and epinephrine ([epinephrine]) concentration at rest and during moderate- and heavy-intensity exercise in the placebo (n = 8) and $\text{N}{\text{O}}_3^{-}$ (n = 8) conditions. Values are means ± SD. A main effect of exercise intensity was observed. P < 0.05, significant post hoc test difference from rest (*) and significant post hoc test difference from moderate-intensity exercise (#).

Effect of $\text{N}{\text{O}}_3^{-}$ supplementation during exercise.

MAP, SBP, DBP, HR, Q, LVC, V̇o₂, and LBF increased (P < 0.05) in response to exercise and were not different between the placebo and $\text{N}{\text{O}}_3^{-}$ supplementation conditions during moderate- and heavy-intensity exercise (Table 2) (P > 0.05). A main effect of exercise intensity (P < 0.05) on plasma [NE] and [Epi] was observed. Post hoc testing demonstrated that plasma [NE] was greater (P < 0.05) during heavy-intensity exercise compared with rest, and [Epi] was greater (P < 0.05) during heavy-intensity exercise compared with rest and moderate-intensity exercise (Fig. 3). Plasma [NE] or [Epi] were not different (P > 0.05) between placebo (n = 8) and $\text{N}{\text{O}}_3^{-}$-supplemented (n = 8) conditions during moderate- and heavy-intensity exercise (Fig. 3).

Effect of $\text{N}{\text{O}}_3^{-}$ supplementation on the response to the CPT.

The responses of HR, SV, Q, MAP, SBP, DBP, LBF, LVC, and V̇o₂ to the CPT at rest and during moderate- and heavy-intensity exercise are reported in Table 3. Sympathetic vasoconstrictor responsiveness (decrease in LVC in response to CPT) was not different (P > 0.05) between the $\text{N}{\text{O}}_3^{-}$ supplementation and placebo conditions at rest or during moderate- and heavy-intensity exercise (Fig. 4). The magnitude of sympatholysis was also not different (P > 0.05) in the $\text{N}{\text{O}}_3^{-}$ and placebo conditions (Fig. 5).

Table 3.

Effect of dietary $\text{N}{\text{O}}_3^{-}$ on the response to cold-pressor test at rest, and during moderate- and heavy-intensity knee-extension exercise

	Rest		Moderate Exercise		Heavy Exercise
	Placebo	$\text{N}{\text{O}}_3^{-}$	Placebo	$\text{N}{\text{O}}_3^{-}$	Placebo	$\text{N}{\text{O}}_3^{-}$
ΔHeart rate, beats/min	5 ± 8	6 ± 10	−2 ± 6*	−4 ± 7*	−6 ± 6*	−5 ± 5*
ΔStroke volume, ml/beat	10 ± 11	15 ± 17	2 ± 7*	5 ± 7*	1 ± 6*	−3 ± 14*
ΔCardiac output, l/min	1.14 ± 0.8	1.63 ± 1.3	0.43 ± 0.8*	0.85 ± 0.9*	0.80 ± 0.8*	0.33 ± 1.6*
ΔMean arterial pressure, mmHg	22 ± 8	23 ± 11	17 ± 10*	18 ± 8*	9 ± 10*#	10 ± 8*#
ΔSystolic blood pressure, mmHg	29 ± 10	23 ± 11	23 ± 14	25 ± 10	14 ± 16*#	18 ± 14*#
ΔDiastolic blood pressure, mmHg	19 ± 7	19 ± 9	15 ± 9*	15 ± 7*	6 ± 7*#	6 ± 6*#
ΔLeg blood flow, l/min	−0.063 ± 0.06	−0.044 ± 0.03	−0.115 ± 0.14	−0.079 ± 0.13	−0.107 ± 0.12	−0.144 ± 0.10
ΔLeg vascular conductance, l·min−1·mmHg−1	−0.0015 ± 0.001	−0.0013 ± 0.001	−0.0037 ± 0.0028*	−0.0033 ± 0.0028*	−0.0023 ± 0.003*	−0.0029 ± 0.002*
ΔOxygen consumption, l/min	0.053 ± 0.06	0.061 ± 0.10	−0.020 ± 0.07*	−0.006 ± 0.07*	0.006 ± 0.05	0.027 ± 0.10

Values are means ± SD. No effect of $\text{N}{\text{O}}_3^{-}$ was observed on any variable at any exercise intensity. A significant (P < 0.05) main effect of exercise intensity was observed for all variables except leg blood flow.

^*Significant post hoc test difference from rest.

^#Significant post hoc test difference from moderate intensity.

Fig. 4.

Percentage change in leg vascular conductance (LVC) at rest and during moderate- and heavy-intensity exercise in the placebo (n = 12) and $\text{N}{\text{O}}_3^{-}$ (n = 12) conditions in response to a cold pressor test. Values are means ± SD. *Significant difference across all exercise conditions, P < 0.001.

Fig. 5.

Magnitude of sympatholysis during moderate- and heavy-intensity exercise in the placebo (n = 12) and $\text{N}{\text{O}}_3^{-}$ (n = 12) trial. LVC, leg vascular conductance. Values are means ± SD. *Significant difference from moderate-intensity exercise, P < 0.01.

DISCUSSION

The purpose of the present study was to investigate the effect of acute dietary $\text{N}{\text{O}}_3^{-}$ supplementation on the control of sympathetic vasoconstriction at rest and in response to exercise. Consistent with previous studies, acute consumption of $\text{N}{\text{O}}_3^{-}$-rich beetroot juice increased total plasma [NO_x], suggesting that NO bioavailability was enhanced following consumption of beetroot juice (26, 35, 41, 66, 67, 69). However, BP, plasma catecholamines, and sympathetic vasoconstrictor responsiveness were not different between placebo and $\text{N}{\text{O}}_3^{-}$ supplementation conditions at rest or during exercise, indicating that dietary $\text{N}{\text{O}}_3^{-}$ supplementation did not alter sympathetic nervous system-mediated vascular control at rest or during exercise in young healthy men.

Effect of $\text{N}{\text{O}}_3^{-}$ supplementation on sympathetic vasoconstrictor responsiveness.

Dietary $\text{N}{\text{O}}_3^{-}$ supplementation did not alter sympathetic vasoconstrictor responsiveness at rest or during moderate- or heavy-intensity exercise in the present study. Several studies in humans and rodents have reported that NO inhibits sympathetic vasoconstriction at rest (21, 44, 58) and during muscle contraction (9, 15, 21, 22, 31, 44, 47, 49, 54, 60, 63). Indeed, NO derived from both endothelial nitric oxide synthase (29, 47) and neuronal nitric oxide synthase (29, 54, 60, 62) has been shown to inhibit sympathetic vasoconstriction in human and animal preparations. In contrast, Dinenno and Joyner (12) reported that NOS inhibition did not alter tyramine-evoked vasoconstrictor responses in the forearm at rest or during handgrip exercise. Although the collective results from these studies are inconclusive, overall the accumulated evidence indicates that inhibition of NO production impairs the inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle of humans and rodents.

Fewer studies have investigated the effect of upregulating NO bioavailability on the inhibition of sympathetic vasoconstriction. The present findings suggest that enhanced NO bioavailability via beetroot juice supplementation does not alter sympathetic vasoconstrictor responsiveness to sympathoexcitation at rest or during exercise. Consistent with the present findings, oral sodium $\text{N}{\text{O}}_3^{-}$ did not alter sympatholysis measured by near-infrared spectroscopy during small-muscle-mass mild-intensity (20% maximal voluntary contractile force) intermittent isometric handgrip exercise (45). Rosenmeier et al. (52) have also reported that infusion of the NO donor sodium nitroprusside did not alter vasoconstrictor responsiveness to tyramine or selective α-adrenergic receptor agonists. In contrast, our laboratory has shown that acute supplementation with the NO cofactor tetrahydrobiopterin reduced sympathetic vasoconstrictor responsiveness in resting and contracting skeletal muscle of rats (33).

Although the available scientific evidence is not conclusive, the present findings and other studies in humans indicate that enhanced NO bioavailability does not alter sympathetic vasoconstrictor responsiveness to sympathoexcitation at rest or during exercise.

An increase in NO bioavailability following dietary $\text{N}{\text{O}}_3^{-}$ supplementation may augment the scavenging of reactive O₂ species (ROS). Several studies have reported that treatment with superoxide scavenging anions enhanced the inhibition of sympathetic vasoconstriction in resting and contracting muscle (14, 17, 28, 64). In contrast, treatment with exogenous antioxidants has been shown to impair vasodilation (65). Although ROS were not measured in the present study, the lack of difference in sympathetic vasoconstrictor responsiveness and skeletal muscle blood flow between dietary $\text{N}{\text{O}}_3^{-}$and placebo conditions suggests that ROS levels were not different between placebo and $\text{N}{\text{O}}_3^{-}$ conditions.

Effect of $\text{N}{\text{O}}_3^{-}$ on blood pressure at rest and during exercise.

Resting MAP, SBP, and DBP were not different between placebo and $\text{N}{\text{O}}_3^{-}$ supplementation conditions in the present study. Consistent with the present findings, other laboratories have also reported no effect of acute dietary $\text{N}{\text{O}}_3^{-}$ supplementation on resting BP in young normotensive adults (8, 37). In contrast, several studies have reported a reduction of resting BP following acute dietary $\text{N}{\text{O}}_3^{-}$ supplementation with beetroot juice (4, 26, 35, 38, 41, 66, 67, 69). The dose of dietary $\text{N}{\text{O}}_3^{-}$ used in present study (12.9 mmol) was similar or above the amount of dietary $\text{N}{\text{O}}_3^{-}$ used in other studies (26, 69), suggesting that the lack of effect of dietary $\text{N}{\text{O}}_3^{-}$ on BP in the present study was not related to the dose of dietary $\text{N}{\text{O}}_3^{-}$. Subjects in the present study also had V̇o_2peak values (~45 ± 12 ml·kg⁻¹·min⁻¹) similar to those in other studies that have reported reduced BP following acute dietary $\text{N}{\text{O}}_3^{-}$ supplementation (38, 41, 66, 69), suggesting that the lack of effect of dietary $\text{N}{\text{O}}_3^{-}$ on resting BP in the present study was not a function of aerobic fitness. Ghosh et al. (19) have reported reductions in SBP and DBP following acute dietary $\text{N}{\text{O}}_3^{-}$ supplementation in hypertensive adults, suggesting that resting BP may influence the effect of dietary $\text{N}{\text{O}}_3^{-}$ on BP. However, chronic dietary $\text{N}{\text{O}}_3^{-}$ supplementation has been shown to have minimal effects on resting BP in hypertensive diabetics (20). Furthermore, Lansley et al. (38) and Vanhatalo et al. (66) have reported reduced SBP following $\text{N}{\text{O}}_3^{-}$ supplementation in subjects with BPs similar to those in the present study. Collectively, these studies suggest that the effect of dietary $\text{N}{\text{O}}_3^{-}$ supplementation on the control of BP is not a function of resting BP. Further investigation is required to fully understand the effect of dietary $\text{N}{\text{O}}_3^{-}$ on the regulation of BP.

Effect of $\text{N}{\text{O}}_3^{-}$ supplementation on plasma catecholamines.

Plasma catecholamines were not different between the placebo or $\text{N}{\text{O}}_3^{-}$ condition at rest or during exercise in the present study. Local brain stem inhibition of NOS has been shown to increase sympathetic nerve activity, and studies have reported a decrease in BP following injection of NO donors in the brain stem, suggesting that brain stem NO bioavailability may inhibit efferent sympathetic nerve activity (6, 25, 48). Whether dietary $\text{N}{\text{O}}_3^{-}$ treatment results in increased NO bioavailability in the cardiovascular control centers of the brain stem has not been established. However, in a group of healthy young African-American females, Bond et al. (4) reported increased heart rate variability with an unchanged low- to high-frequency spectral power ratio following acute dietary $\text{N}{\text{O}}_3^{-}$ supplementation, suggesting that $\text{N}{\text{O}}_3^{-}$ supplementation may alter autonomic control of heart rate. Moreover, Notay et al. (46) recently reported that acute dietary $\text{N}{\text{O}}_3^{-}$ supplementation decreased resting MSNA and increased MSNA (burst incidence) in response to static handgrip exercise. Interestingly, these changes in MSNA occurred despite dietary $\text{N}{\text{O}}_3^{-}$ supplementation having no effect on resting blood pressure and the blood pressure response to handgrip exercise. Differences in the mode and intensity of exercise, muscle mass involved in the exercise, method of sympathoexcitation, and index used to assess sympathetic outflow between these studies make it difficult to determine the reason for these divergent findings. Furthermore, the inability to measure catecholamines in response to the CPT in the present study limits our ability to compare responses to sympathoexcitation. We would not, however, expect acute dietary $\text{N}{\text{O}}_3^{-}$ supplementation to alter the expression of postsynaptic α-adrenergic receptors; therefore, similar levels of circulating catecholamines appear consistent with similar vasoconstrictor responsiveness at rest and during exercise in the present study.

Effect of dietary $\text{N}{\text{O}}_3^{-}$ on leg blood flow and V̇o₂ at rest and during exercise.

Consistent with previous studies of forearm blood flow (8, 16, 37), acute dietary $\text{N}{\text{O}}_3^{-}$ supplementation did not alter resting leg blood flow in the present study. Ferguson et al. (16) also reported no difference in resting hindlimb muscle blood flow between rats that received 5 days of dietary $\text{N}{\text{O}}_3^{-}$ supplementation and controls. Collectively, the results of these studies suggest that enhanced NO bioavailability via dietary $\text{N}{\text{O}}_3^{-}$ does not alter the regulation of resting skeletal muscle blood flow.

Dietary $\text{N}{\text{O}}_3^{-}$ did not alter blood flow during exercise in the present study. Consistent with the present findings, several other studies have also reported no difference in blood flow during handgrip exercise in dietary $\text{N}{\text{O}}_3^{-}$ and placebo conditions (8, 10, 44). In contrast, Ferguson et al. (16) reported increased total hindlimb blood flow during exercise in rats given 5 days of supplemental dietary $\text{N}{\text{O}}_3^{-}$. The findings of Ferguson et al. (16) may be specific to rats and may also reflect a difference between chronic versus acute $\text{N}{\text{O}}_3^{-}$ supplementation. Furthermore, muscle blood flow was primarily increased to fast-twitch muscles of the rat hindlimb, suggesting that the effect of dietary $\text{N}{\text{O}}_3^{-}$ on vascular control may be fiber-type specific (16).

Acute dietary $\text{N}{\text{O}}_3^{-}$ supplementation did not alter V̇o₂ during moderate- or heavy-intensity knee extension exercise in the present study. Other studies have also reported no effect of acute dietary $\text{N}{\text{O}}_3^{-}$ supplementation on the O₂ cost of exercise (3, 18). In contrast, chronic (3–5 days) dietary $\text{N}{\text{O}}_3^{-}$ supplementation has been shown to reduce the O₂ cost of exercise (1, 39, 40), suggesting that chronic $\text{N}{\text{O}}_3^{-}$ supplementation may be necessary to alter metabolic efficiency.

In conclusion, acute dietary $\text{N}{\text{O}}_3^{-}$ supplementation increased total plasma [NO_x], suggesting that NO bioavailability was enhanced. However, blood pressure, blood flow, plasma catecholamines, and sympathetic vasoconstrictor responsiveness were not different between placebo and $\text{N}{\text{O}}_3^{-}$ supplementation conditions at rest or during exercise. These results suggest that enhanced NO bioavailability following dietary $\text{N}{\text{O}}_3^{-}$ supplementation did not alter sympathetic nervous system-mediated vascular control at rest or during exercise in young healthy males.

GRANTS

This project was supported by grants from the Natural Sciences and Engineering Research Council of Canada, the Canadian Foundation for Innovation, and the Sport Science Association of Alberta.

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

C.J.d.V. and D.S.D. conceived and designed research; C.J.d.V. and D.S.D. performed experiments; C.J.d.V. and D.S.D. analyzed data; C.J.d.V. and D.S.D. interpreted results of experiments; C.J.d.V. and D.S.D. prepared figures; C.J.d.V. and D.S.D. drafted manuscript; C.J.d.V. and D.S.D. edited and revised manuscript; C.J.d.V. and D.S.D. approved final version of manuscript.