Cardiovascular and Sympathetic Effects of Nitric Oxide Inhibition at Rest and During Static Exercise in Humans
Background Nitric oxide (NO) regulates vascular tone and blood pressure, and studies in animals suggest that it does so, at least in part, by modulating sympathetic neural outflow. Loss of NO-induced vasodilator tone and restraint on sympathetic vasoconstrictor outflow could lead to exaggerated vasoconstrictor and pressor responses to physical stress in humans.
Methods and Results To determine the role of NO in the modulation of central sympathetic outflow and vascular tone at rest and during a physical stress, we tested effects of systemic inhibition of NO synthase by NG-monomethyl-l-arginine (L-NMMA) infusion (a stereospecific inhibitor of NO synthase) on sympathetic nerve activity (microneurography), regional vascular resistance, and blood pressure at rest and during static handgrip. The major new findings are that (1) under resting conditions, L-NMMA infusion, which increased mean arterial pressure by ≈10%, did not have any detectable effect on muscle sympathetic nerve activity, whereas a similar increase in arterial pressure evoked by phenylephrine infusion (an NO-independent vasoconstrictor) decreased the rate of sympathetic nerve firing by ≈50%; (2) during static handgrip, the exercise-induced sympathetic nerve responses were preserved during L-NMMA infusion but markedly attenuated during phenylephrine infusion; and (3) the L-NMMA–induced loss of vasodilator tone did not result in exaggerated exercise-induced pressor and calf vasoconstrictor responses.
Conclusions These findings indicate that NO is involved in the central regulation of sympathetic outflow in humans and suggest that both neuronal and endothelial NO synthesis may contribute to the regulation of vasomotor tone.
Over the past few years, nitric oxide (NO),1 which is synthesized from the amino acid l-arginine by the enzyme NO synthase, has emerged as an important regulatory mechanism of vasomotor tone and arterial pressure in both animals and humans.2–4 Studies in humans using NG-monomethyl-l-arginine (L-NMMA), which by its action as a competitive stereospecific inhibitor of NO synthase inhibits both basal and stimulated NO synthesis, indicate that NO plays an important role in the regulation of basal vasomotor tone5 and in the mediation of vasodilatory responses to a variety of physiological6,7 and pharmacological5,8 stimuli. Studies in several animal species suggest that the pressor effects observed during systemic L-NMMA infusion are caused, at least in part, by alterations in sympathetic vasoconstrictor tone.2,9–15 In the clinical setting, an impairment in NO synthesis and/or release has been demonstrated in hypercholesterolemia,16,17 atherosclerosis,18,19 heart failure,20,21 and hypertension.22,23 Such a loss of NO-induced vasodilator tone (and sympathoinhibition) could lead to exaggerated vasoconstrictor and pressor responses to mental and physical stress.24
We examined effects of systemic inhibition of NO synthase by L-NMMA infusion on sympathetic nerve activity, regional vascular resistance, and arterial pressure both at rest and during static exercise, an intervention that evokes large increases in arterial pressure that are mediated by decreases in parasympathetic and increases in sympathetic efferent activity,25 and compared these effects with those of phenylephrine infusion (an NO-independent vasoconstrictor).
We studied 15 lean, healthy male volunteers (weight, 71.2±7.4 kg; height, 180±4 cm; body mass index, 22.1±1.9 kg/m2; age, 25±7 years, mean±SD). All subjects were normotensive, were taking no medications, and had no evidence of metabolic or cardiovascular disease. All the studies were performed in the morning after an overnight fast. The subjects were asked to abstain from alcohol, caffeine, and tobacco for at least 24 hours before each study. The experimental protocol was approved by the Institutional Review Board on Human Investigation, and all subjects provided written informed consent.
The subjects were studied in the supine position. Heart rate (ECG), respiratory excursions (pneumobelt), force of muscle contraction (Harpenden handgrip dynamometer, British Indicators Ltd), blood flow in the resting forearm and calf (venous occlusion plethysmography),26 and efferent muscle sympathetic nerve activity (MSNA) were recorded continuously on an electrostatic recorder and a tape recorder (R71, TEAC Corp). Respiratory excursions were monitored to detect inadvertent performance of a Valsalva maneuver or prolonged expiration, because these respiratory maneuvers can markedly stimulate sympathetic outflow.27 Blood pressure was measured with an automated sphygmomanometric system (Datascope Accutorr, Datascope Corp). Intravenous catheters were inserted into a right and a left antecubital vein, one for drug infusion and the other for blood sampling.
Recording of Sympathetic Nerve Activity
Multiunit recordings of postganglionic sympathetic nerve activity were obtained with unipolar tungsten microelectrodes inserted selectively into muscle nerve fasciculi of the peroneal nerve posterior to the fibular head by the microneurographic technique of Vallbo et al.28 The neural signals were amplified 20 000 to 50 000 times, filtered (bandwidth, 700 to 2000 Hz), rectified, and integrated (time constant, 0.1 second) to obtain a mean voltage display of sympathetic activity. A recording of sympathetic activity was considered acceptable when it revealed spontaneous, pulse synchronous bursts of neural activity, with the largest bursts showing a minimal signal-to-noise ratio of 3:1. In each study, we documented that we were recording sympathetic outflow to skeletal muscle by demonstrating that the neural activity did not respond to arousal stimuli (loud noise) or a pinch of the skin but showed a characteristic biphasic response to the Valsalva maneuver.29
For analysis, printed filtered and mean voltage neurograms were visually inspected to identify bursts of sympathetic nerve discharge. The recordings were all analyzed by the same observer who was blinded to the pharmacological intervention assigned to the subject. The intraobserver and interobserver coefficients of variation of the mean in identifying bursts are <6% and <9%, respectively.30 Nerve traffic was expressed both as number of bursts per minute, an index of the frequency of the activity, and as bursts per minute times mean burst amplitude, an index of integrated (total) activity. For quantitative analysis, data were normalized by transcribing nerve recordings from FM tape to a hard copy in such a way that mean burst amplitudes were comparable in all subjects.
Measurement of Muscle Blood Flow
While recording sympathetic outflow to calf muscles in one leg, we simultaneously measured blood flow in the contralateral leg and forearm by venous occlusion plethysmography using mercury-in-Silastic strain gauges. The forearm and the calf were elevated 10 to 15 cm above the level of the right atrium to collapse the veins. Circulation to the hand and foot was arrested by inflating a cuff around the wrist and the ankle during blood flow determinations, which were performed at 15-second intervals over a 5-minute period.
At the beginning of each experiment, the subject’s maximal voluntary contraction was determined using a handgrip dynamometer. Subjects performed static handgrip at 33% maximal voluntary contraction for 2 minutes, aided by a visual feedback of the force output.
Drugs were dissolved in physiological saline immediately before use. L-NMMA and l-arginine were obtained from Clinalfa, phenylephrine from Winthrop Pharmaceuticals, and sodium nitroprusside from Roche.
Protocol 1: Cardiovascular and Sympathetic Effects of L-NMMA Infusion at Rest
Nine subjects participated in this protocol. After instrumentation, the subjects rested quietly for 30 minutes. They then received sequential infusions of normal saline (1 mL/min) for 30 minutes, L-NMMA (50 μg · kg−1 · min−1) for 60 minutes, and l-arginine (50 mg · kg−1 · min−1) for 10 minutes. Hemodynamic measurements and sympathetic nerve activity were recorded for 5 minutes of each 15-minute period during the 30 minutes of saline and the 60 minutes of L-NMMA infusion and during the last 5 minutes of l-arginine infusion.
To determine the effects of inhibitory baroreflexes (activated by L-NMMA–induced increases in arterial pressure) on L-NMMA–induced sympathetic responses, we performed additional experiments in five of the nine subjects. The subjects received sequential infusions of normal saline for 30 minutes, phenylephrine (titrated at a rate to match the increase in arterial pressure produced by L-NMMA infusion) for 20 minutes, normal saline for 30 minutes, L-NMMA (50 μg · kg−1 · min−1) for 45 minutes, and concomitant L-NMMA and sodium nitroprusside (titrated at a rate to offset the L-NMMA–induced increase in arterial pressure) for 15 minutes. Hemodynamic measurements and sympathetic nerve activity were recorded for 5 minutes of each 15-minutes period during the 30-minute baseline period and during the last 10 minutes of phenylephrine, L-NMMA, and L-NMMA plus sodium nitroprusside infusion.
In three additional subjects, phenylephrine was infused over 60 minutes at a rate to mimic the temporal profile of the increase in blood pressure observed during the 60 minutes of L-NMMA infusion.
To examine the effects of time on sympathetic nerve activity, in five subjects we infused saline (1 mL/min) over 2 hours. This infusion did not have any detectable effect on sympathetic nerve activity; values for MSNA burst frequency (total activity) were 15±2 bursts per minute (159±15 U) before the start of infusion and 14±1 bursts per minute (138±13 U) and 15±1 bursts per minute (156±14 U) 60 and 120 minutes after the start of the saline infusion, respectively.
Protocol 2: Cardiovascular and Sympathetic Effects of L-NMMA Infusion During Static Exercise
Eight subjects participated in this protocol. In each subject, we measured hemodynamic and sympathetic responses during 2 minutes of static handgrip: one performed during saline infusion, and one performed 45 minutes after the start of L-NMMA infusion (50 μg · kg−1 · min−1).
To determine the effects of inhibitory baroreflexes (activated by L-NMMA–induced increases in arterial pressure) on exercise-induced sympathetic responses, we performed additional experiments in five more subjects. In each subject, we measured hemodynamic and sympathetic responses during three 2-minute bouts of static handgrip: one performed during saline infusion, one performed during phenylephrine infusion (titrated at a rate to match the increase in baseline arterial pressure produced by L-NMMA infusion), and one performed during L-NMMA (50 μg · kg−1 · min−1) infusion.
Protocol 3: Cardiovascular and Sympathetic Effects of High-Dose L-NMMA Infusion
To determine whether the differences between the findings of the present study and those of an earlier report31 were related to the much higher dose of L-NMMA infused, we performed additional experiments in three subjects in whom L-NMMA was infused at the rate (500 μg · kg−1 · min−1 for 15 minutes) used by Hansen et al.31 Hemodynamic measurements and sympathetic nerve activity were recorded for 5 minutes of each 15-minute period during the 30 minutes of baseline and during the entire 15-minute period of L-NMMA infusion.
Protocol 4: Cardiovascular Effects of l-Arginine Infusion in the Absence of L-NMMA
To exclude the possibility that in protocol 1, l-arginine was acting as a nonspecific vasodilator (rather than as a stereospecific substrate for NO synthase), in six subjects we examined effects of l-arginine infusion (50 mg · kg−1 · min−1 for 10 minutes) alone on arterial pressure and heart rate.
Protocol 5: Sympathetic Effects of Concomitant Infusion of L-NMMA and Nitroprusside From Baseline
To examine whether MSNA increases not only during the rapid reduction of blood pressure to its baseline values induced by adding nitroprusside to L-NMMA (protocol 1) but also when the increase in blood pressure is prevented, in five subjects we coinfused nitroprusside and L-NMMA from baseline. Particular care was taken not to lower blood pressure below the resting baseline level.
Mean arterial pressure was calculated as diastolic pressure plus one third of the pulse pressure. Vascular resistance in the forearm and calf was determined as mean arterial pressure in millimeters of mercury divided by blood flow in milliliters per minute per 100 millimeters of tissue; it was expressed in units.
The measurements of MSNA, blood flow in the forearm and the calf, blood pressure, and heart rate that were collected over 5-minute periods were averaged to a single value.
Statistical analysis was performed with paired two-tailed t tests and ANOVA with repeated measures followed by Fisher’s post hoc test. A value of P<.05 was considered significant. Data are given as mean±SE unless stated otherwise.
Cardiovascular and Sympathetic Effects of L-NMMA Infusion at Rest
L-NMMA infusion, which increased mean arterial pressure progressively by 7±1 mm Hg (P<.001) and decreased heart rate by 3±1 bpm (P=.003), did not have any detectable sympathetic effect throughout the entire infusion period. Mean arterial pressure increased progressively from 78±2 mm Hg at baseline to 81±2, 82±2, and 85±2 mm Hg 15, 30, and 60 minutes, respectively, after the start of L-NMMA infusion, whereas MSNA burst frequency (total activity) remained unchanged: 22±2 bursts per minute (350±38 U) during saline infusion and 21±2, 19±2, and 23±3 bursts per minute (348±49, 321±34, and 371±45 U) during L-NMMA infusion. l-Arginine infusion rapidly reversed the L-NMMA–induced changes in arterial pressure and heart rate without altering MSNA (Tables 1⇓ and 2⇓ and Figs 1⇓ and 2⇓).
Phenylephrine infusion and L-NMMA infusion, which increased mean arterial pressure comparably (by 8±1 and 7±1 mm Hg, respectively), evoked directionally opposite effects on MSNA; during phenylephrine infusion, MSNA burst frequency decreased by 8±1 bursts per minute, whereas it increased by 2±2 bursts per minute during L-NMMA infusion (P=.016). When phenylephrine was infused over 60 minutes to mimic the temporal profile of the increase in blood pressure during L-NMMA infusion, MSNA burst frequency decreased from 20±1 bursts per minute (362±40 U) at baseline to 10±2, 8±1, and 8±1 bursts per minute (138±9, 113±7 and 108±22 U) 15, 30, and 60 minutes, respectively, after the start of the phenylephrine infusion.
When during L-NMMA infusion sodium nitroprusside was coinfused to offset the L-NMMA–induced increase in arterial pressure, the MSNA burst frequency was roughly twofold higher than during saline infusion (37±5 versus18±4 bursts per minute, P=.04). Similarly, when sodium nitroprusside and L-NMMA were coinfused from baseline, the L-NMMA–induced increase in arterial pressure was prevented (mean arterial pressure was 84±5 mm Hg at baseline and 84±5 and 83±6 mm Hg after 30 and 60 minutes, respectively, of L-NMMA/nitroprusside infusion), whereas MSNA burst frequency (and total activity) now increased more than twofold (P<.001) from 11±2 bursts per minute (270±60 U) at baseline to 25±5 bursts per minute (659±90 U) and 29±4 bursts per minute (756±176 U) after 30 and 60 minutes, respectively, of L-NMMA/sodium nitroprusside infusion.
Cardiovascular and Sympathetic Effects of L-NMMA Infusion During Static Exercise
Resting mean arterial pressure was higher during systemic L-NMMA infusion than during saline infusion (93±3 versus 84±3 mm Hg, P=.02), whereas resting MSNA burst frequency was similar under both conditions. Static handgrip evoked increases in mean arterial pressure, heart rate, and MSNA that were comparable in both time course and magnitude during saline infusion and L-NMMA infusion. L-NMMA infusion attenuated the decrease in resting forearm vascular resistance during the first minute of handgrip; it decreased by 24.7±7.4% during saline infusion but by only 9.5±6.6% during L-NMMA infusion (P=.049) (Table 3⇓ and Figs 3⇓ and 4⇓).
Phenylephrine and L-NMMA infusion increased baseline mean arterial pressure similarly by 9±2 mm Hg (P=.016 versus saline infusion) and 9±1 mm Hg (P=.002 versus saline infusion) above the values observed during saline infusion (Fig 3⇑). Static handgrip evoked increases in mean arterial pressure that were similar during infusion of phenylephrine (22±5 mm Hg), L-NMMA (24±7 mm Hg), and saline (25±6 mm Hg). In contrast, the exercise-induced sympathetic activation was markedly attenuated during phenylephrine infusion; during the second minute of handgrip, MSNA burst frequency increased by 18±1 bursts per minute during saline infusion, by 19±3 bursts per minute during L-NMMA infusion, but by only 3±1 bursts per minute (73±20 U) during phenylephrine infusion (F=40.4, P<.0001 versus saline infusion and F=18.9, P<.001 versus L-NMMA infusion).
High-Dose L-NMMA Infusion
In contrast to low-dose L-NMMA infusion (protocols 1 and 2), high-dose L-NMMA infusion rapidly and markedly increased mean arterial pressure and decreased MSNA and heart rate. All three variables had reached a new plateau within 5 minutes of the start of infusion; at the end of the 15 minutes of L-NMMA infusion, mean arterial pressure had increased from 80±3 to 90±2 mm Hg (P=.007), heart rate had decreased from 53±4 to 43±4 bpm (P=.01), MSNA burst frequency had decreased from 22±2 to 7±1 bursts per minute (P=.01), and MSNA total activity had decreased from 302±42 to 95±9 U (P=.01).
Cardiovascular Effects of l-Arginine Infusion in the Absence of L-NMMA
l-Arginine infusion alone (in contrast to infusion after L-NMMA; protocol 1) did not have any detectable effect on mean arterial pressure (72±3 mm Hg at baseline and 71±2 mm Hg at the end of the 10-minute infusion) or heart rate (55±4 and 54±5 bpm, respectively).
The major new findings are that (1) under resting conditions, systemic inhibition of NO synthase by L-NMMA infusion, which increased mean arterial pressure by ≈10%, did not have any detectable effect on MSNA, whereas a similar increase in arterial pressure evoked by phenylephrine infusion decreased the rate of sympathetic nerve firing by roughly 50 percent; (2) during static handgrip, the exercise-induced sympathetic nerve responses were preserved during L-NMMA infusion but markedly attenuated during phenylephrine infusion; (3) the L-NMMA–induced loss of vasodilator tone did not result in exaggerated exercise-induced pressor and calf vasoconstrictor responses; and (4) L-NMMA infusion attenuated the exercise-induced vasodilation in the resting forearm at the beginning of isometric exercise. These results indicate that systemic inhibition of NO synthase has sympathoexcitatory effects in humans and suggest that NO, in addition to its direct vasodilator action, plays a role in the neural regulation of vascular tone. Moreover, these data suggest that in healthy subjects, inhibition of nitric oxide release does not lead to exaggerated vasoconstrictor responses during static exercise.
Studies in animals have provided evidence that neuronal NO is involved in the central regulation of sympathetic outflow.2,9–15 Here, we provide the first such evidence in humans. This interpretation is based on the following observations. First, during L-NMMA infusion, the progressive increase in arterial pressure—in marked contrast to phenylephrine infusion—was not accompanied by any detectable decrease in MSNA, indicating a sympathoexcitatory effect of NO synthase inhibition (as further evidenced by the handgrip studies). Second, when the L-NMMA–induced increase in arterial pressure was either reversed or prevented by concomitant nitroprusside infusion, the rate of sympathetic nerve firing was roughly twofold higher than that observed at the same arterial pressure at baseline. This finding indicates that normally the L-NMMA–induced sympathetic activation is masked by an inhibitory effect of arterial baroreflexes (whose function is preserved32 or even augmented33 during acute NO synthase inhibition), an interpretation that is consistent with observations made in anesthetized animals.10 Third, when L-NMMA infusion was followed by l-arginine infusion, the l-arginine–induced decrease in arterial pressure (an effect caused specifically by restoration of NO synthesis as evidenced by protocol 4) did not trigger any detectable increase in MSNA. This observation further strengthens the interpretation that NO is involved in the tonic restraint of sympathetic outflow.2,9–15
The vascular actions of nitric oxide have been the focus of much interest, and it has been postulated that impaired production and release of NO could lead to exaggerated and paradoxical vasoconstrictor responses to physical stress.25 Our findings challenge this concept and demonstrate that even though baseline arterial pressure was significantly higher during NO synthase inhibition, the exercise-induced increases in blood pressure (and calf vascular resistance) were comparable, not larger than those evoked by handgrip alone. Moreover, our data indicate that the lack of an exaggerated exercise-induced pressor response during L-NMMA infusion cannot be explained on the basis of an attenuated sympathetic nerve response to this form of exercise, because MSNA increased comparably during handgrip performed alone and performed during L-NMMA infusion. The latter finding contrasts with the markedly attenuated exercise-induced sympathetic responses observed during phenylephrine infusion and provides further evidence for a sympathoexcitatory effect of NO synthase inhibition in humans. This observation is consistent with findings in ex vivo and anesthetized in vivo animal preparations2,9–15 but differs from those of a recent study in humans that provided no evidence for a sympathoexcitatory effect of L-NMMA infusion.33 This difference appears to be related to the infusion in this earlier study of much larger (up to nine times) doses of L-NMMA over a short period of time (as evidenced by our high-dose L-NMMA infusion studies), a finding that is expected to have important implications on the design of future studies examining the role of NO in the regulation of sympathetic neural outflow in humans. The underlying mechanism(s) for the differential sympathetic and hemodynamic effects of low- and high-dose L-NMMA infusion are not clear but could include different ED50 doses for L-NMMA–induced blockade of neural versus endothelial NO synthesis. Alternatively, the tonic central neural restraint on sympathetic outflow by NO may be to limited, for its withdrawal to counterbalance the baroreflex-mediated sympathoinhibition evoked by the higher blood pressure rise during high-dose L-NMMA infusion.
In the resting forearm, blood flow increases and vascular resistance decreases during the first minute of static handgrip, an observation that has been attributed to stimulation of cholinergic vasodilator mechanisms.34 The present findings suggest that stimulation of NO release through activation of cholinergic35 and/or nonadrenergic noncholinergic36 pathways plays an important role in the mediation of the exercise-induced vasodilation in the resting forearm.
This study shows that NO is involved in the central regulation of sympathetic outflow in humans and suggests that both neuronal and endothelial NO synthesis may contribute to the regulation of vasomotor tone. In these short-term experiments in healthy subjects, inhibition of NO synthase had sympathoexcitatory effects that were masked by an inhibitory effect of the baroreflexes. It is possible that in disease states such as heart failure that are characterized by an impairment in both baroreflex function and NO synthesis and release, the conjunction of these two defects could be one of the factors contributing to sustained sympathetic activation.
This work was supported by grants from the Swiss National Science Foundation (32-36280.92 and 32-46797.96), the International Olympic Committee, the Emma Muschamp Foundation, and the Placide Nicod Foundation. We are indebted to Vincent Pichot for help with some of the final studies.
Presented in part at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 14–17, 1994, and at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13–16, 1995, and published in abstract form (Circulation. 1994;90[suppl I]:I-316, and Circulation. 1995;92[suppl I]:I-12).
- Received April 30, 1997.
- Revision received August 4, 1997.
- Accepted August 20, 2010.
- Copyright © 1997 by American Heart Association
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