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Journal of Physiology (2002), 540.1, pp. 377-386
© Copyright 2002 The Physiological Society
DOI: 10.1113/jphysiol.2001.013153
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ABSTRACT |
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Nitric oxide (NO) attenuates-adrenergic vasoconstriction in contracting rodent skeletal muscle, but it is unclear if NO plays a similar role in human muscle. We therefore hypothesized that in humans, NO produced in exercising skeletal muscle blunts the vasoconstrictor response to sympathetic activation. We assessed vasoconstrictor responses in the microcirculation of human forearm muscle using near-infrared spectroscopy to measure decreases in muscle oxygenation during reflex sympathetic activation evoked by lower body negative pressure (LBNP). Experiments were performed before and after NO synthase inhibition produced by systemic infusion of NG-nitro-L-arginine methyl ester (L-NAME). Before L-NAME, LBNP at -20 mmHg decreased muscle oxygenation by 20 ± 2 % in resting forearm and by 2 ± 3 % in exercising forearm (n = 20), demonstrating metabolic modulation of sympathetic vasoconstriction. As expected, L-NAME increased mean arterial pressure by 17 ± 3 mmHg, leading to baroreflex-mediated supression of baseline muscle sympathetic nerve activity (SNA). The increment in muscle SNA in response to LBNP at -20 mmHg also was attenuated after L-NAME (before, +14 ± 2; after, +8 ± 1 bursts min-1; n = 6), but this effect of L-NAME was counteracted by increasing LBNP to -40 mmHg (+19 ± 2 bursts min-1). After L-NAME, LBNP at -20 mmHg decreased muscle oxygenation similarly in resting (-11 ± 3 %) and exercising (-10 ± 2 %) forearm (n = 12). Likewise, LBNP at -40 mmHg decreased muscle oxygenation both in resting (-19 ± 4 %) and exercising (-21 ± 5 %) forearm (n = 8). These data advance the hypothesis that NO plays an important role in modulating sympathetic vasoconstriction in the microcirculation of exercising muscle, because such modulation is abrogated by NO synthase inhibition with L-NAME.
(Received 15 August 2001; accepted after revision 14 December 2001)
Corresponding author G. D. Thomas: University of Texas Southwestern Medical Center, Hypertension Division, 5323 Harry Hines Boulevard, Dallas, TX 75390-8586, USA. Email: gail.thomas{at}utsouthwestern.edu
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INTRODUCTION |
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Activation of the sympathetic nervous system plays an essential role in the co-ordinated cardiovascular response to dynamic exercise, producing increases in heart rate and contractility and causing regional vasoconstriction in non-skeletal muscle beds such as the renal and splanchnic circulations. Exercise-induced increases in sympathetic outflow also are targeted to skeletal muscle (Hansen et al. 1994). In non-exercising muscle, sympathetic activation causes vasoconstriction, which helps to redistribute cardiac output to the active muscles. In exercising muscle, however, the vasoconstrictor response to sympathetic activation is blunted, in part by local metabolic products of contraction (Anderson & Faber, 1991; Hansen et al. 1996; Thomas & Victor, 1998). Such metabolic modulation is particularly evident in the small resistance arterioles of the muscle microcirculation (Anderson & Faber, 1991), which may serve to optimize blood flow distribution within the exercising muscles.
Of the numerous vasoactive substances produced in the exercising muscles, recent studies suggest that nitric oxide (NO) may play a key role in the modulation of sympathetic vasoconstriction (Thomas et al. 1998; Thomas & Victor, 1998; Sander et al. 2000). Multiple sources of NO exist in skeletal muscle, including the vascular endothelium which expresses endothelial NO synthase (eNOS) and the skeletal myocytes which express neuronal NOS (nNOS) (Nakane et al. 1993; Kobzik et al. 1994, 1995). Pharmacological NOS inhibition impairs the modulation of -adrenergic vasoconstriction in the contracting hindlimb of anaesthetized rats and wild-type mice (Thomas et al. 1998; Thomas & Victor, 1998). A similar impairment is observed in the untreated contracting hindlimb of two different genetic mouse models with nNOS-deficient skeletal muscle, the nNOS knockout mouse and the mdx mouse (Thomas et al. 1998). The latter is a model of Duchenne muscular dystrophy (DMD), in which a primary deficiency of the cytoskeletal protein dystrophin results in a secondary reduction of skeletal muscle nNOS (Brenman et al. 1995; Chang et al. 1996).
Like mdx mice, boys with DMD also exhibit defective modulation of sympathetic vasoconstriction in exercising muscle, providing the only evidence to date that NO is involved in the contraction-induced attenuation of sympathetic vasoconstriction in humans (Sander et al. 2000). However, dystrophic muscle is characterized by changes in the expression and function of numerous proteins (Straub & Campbell, 1997), raising the possibility that the abnormal phenotype observed in DMD patients is not related specifically to NOS deficiency. We therefore sought to determine if we could extend the results of these previous studies in anaesthetized rodents and conscious DMD patients to normal, healthy subjects. To test the hypothesis that NO produced in exercising skeletal muscle blunts the vasoconstrictor response to sympathetic activation, we used near-infrared (NIR) spectroscopy to measure changes in tissue oxygenation induced by reflex sympathetic activation in the exercising forearm of healthy subjects before and after infusion of the potent NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME). In addition, because NOS inhibition potentially could affect numerous processes in skeletal muscle including mitochondrial respiration (King et al. 1994; Shen et al. 2000), we first performed experiments in anaesthetized rats to ensure that the close linear relationship between sympathetically mediated decreases in muscle blood flow and muscle oxygenation was not altered by L-NAME.
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METHODS |
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Anaesthetized animals
Protocols for the animal studies were reviewed and approved by the Institutional Animal Care and Research Advisory Committee at the University of Texas Southwestern Medical Center.
Experimental preparation. Five female Sprague-Dawley rats (240-310 g; Charles River, Kingston, MA, USA) were anaesthetized with ketamine (80 mg kg-1, I.P.) and atropine sulphate (0.5 mg kg-1, S.C.) followed by -chloralose (60 mg kg-1, I.V. supplemented with 10 mg kg-1 h-1, I.V.). Rats were mechanically ventilated via a tracheal cannula with room air and supplemental O2. Core temperature was maintained at 37 °C with an external heat source. A jugular vein and carotid artery were cannulated for drug infusions and arterial pressure measurements, respectively. A Doppler flow probe was placed around the left femoral artery to measure changes in blood flow velocity by recording the pulsatile and mean Doppler shifts in kilohertz using a VF-1 pulsed Doppler flow system (Crystal Biotech, Holliston, MA, USA). Femoral vascular conductance (kHz mmHg-1) was calculated as the quotient of mean Doppler shift and mean arterial pressure. The left lumbar sympathetic chain was isolated, placed on bipolar platinum electrodes and covered with dental silicone (S4i, Bisico, Bielefeld, Germany). Haemodynamic data were collected, digitally stored, and analysed using a computer-based data acquisition system (MacLab, ADInstruments, Milford, MA, USA).
Skeletal muscle oxygenation by NIR spectroscopy. NIR spectroscopy was used as previously described to measure changes in tissue concentrations of oxygenated haemoglobin and myoglobin (HbO2 + MbO2) (Hansen et al. 1996). To monitor the tissue absorption of NIR light, two fibre optical bundles were placed directly on the exposed medial surface of the left gastrocnemius muscle. NIR signals at four different wavelengths (775, 810, 870 and 904 nm) were sequentially sampled at a rate of 1 Hz s-1, converted to optical densities using established algorithms (Piantadosi, 1989), and stored digitally for analysis using a custom-made NIR spectrophotometer (Duke University, Durham, NC, USA). Responses to lumbar sympathetic nerve stimulation were determined by calculating the difference between 20 s (i.e. the average of 20 consecutive NIR samples) of baseline immediately preceding the onset of stimulation and the last 20 s of stimulation. Changes in the NIR signals in response to interventions are expressed as a percentage of the total labile signal (TLS), which was defined in each experiment as the maximal decrease in HbO2 + MbO2 produced by complete mechanical occlusion of the terminal aorta.
Conscious humans
Protocols for the human studies were reviewed and approved by the Institutional Review Board at the University of Texas Southwestern Medical Center. All procedures conformed to the standards set by the Declaration of Helsinki. Thirty-one healthy volunteer subjects (22 male, 9 female) age 19 to 33 years (mean, 26.0 ± 0.6 years) participated in this study. Informed, written, consent was obtained from each subject prior to this study. Several of the subjects participated in more than one protocol on separate occasions. An Investigational New Drug number was obtained from the US Food and Drug Administration for the use of L-NAME in human subjects. All experimental data were recorded continuously using a computer-based data acquisition system (MacLab).
Haemodynamic measurements. Subjects were studied in the supine position. An intravenous catheter was inserted into a forearm vein for the administration of L-NAME. Heart rate was measured continuously by electrocardiography and arterial pressure by automated oscillometric sphygmomanometry (Welch Allyn, Skaneateles Falls, NY, USA). Resting forearm blood flow was measured by venous occlusion plethysmography and expressed as mililitres per 100 ml forearm volume (Siggaard-Andersen, 1970). Forearm vascular conductance was calculated as the quotient of forearm blood flow and mean arterial pressure.
Drug administration. To inhibit local sympathetic neurotransmission, bretylium tosylate (1 mg kg-1; Astra Scientific Intl., Westborough, MA, USA) was administered to one forearm by a regional intravenous technique (Bier-block) as previously described (Hansen et al. 1996). To inhibit NOS activity, L-NAME (4 mg kg-1; Clinalfa, Läufelfingen, Switzerland) was infused intravenously over a one hour period.
Muscle sympathetic nerve activity (SNA). Multiunit postganglionic SNA was recorded with tungsten microelectrodes inserted selectively into muscle nerve fascicles of the peroneal nerve by microneurography (Vallbo et al. 1979). The neural signals were amplified (by 95.5 
103), filtered (bandwidth, 700-2000 Hz), rectified, and integrated (time constant, 0.1 s) to obtain a mean voltage neurogram. A recording of muscle SNA was considered acceptable when the neurogram revealed spontaneous, pulse-synchronous bursts of neural activity, with a minimum signal-to-noise ratio of 3:1, that increased during phases II and III of the Valsalva manoeuvre, but not during arousal stimuli (loud noise, skin pinch). SNA was expressed as: (a) the number of bursts of sympathetic activity per minute or per 100 heart beats and (b) the number of bursts per minute multiplied by the mean burst amplitude in that minute (total activity).
Lower body negative pressure (LBNP). The subject's lower body was enclosed to the level of the iliac crest in a negative pressure chamber. Pressure inside the chamber was altered by suction and measured continuously by a Statham transducer. LBNP at -20 or -40 mmHg was applied for 3 min to reflexly increase muscle SNA. Application of LBNP causes highly reproducible reflex increases in muscle SNA before, during and after handgrip exercise (Scherrer et al. 1988; Hansen et al. 1996).
Skeletal muscle oxygenation by NIR spectroscopy. For the human studies, two fibre optical bundles were placed directly on the skin over the left flexor digitorum profundus muscle, which is the main muscle recruited during handgrip exercise. NIR signals at four different wavelengths (775, 825, 850 and 905 nm) were sequentially sampled at a rate of 1 Hz s-1 and converted to optical densities according to established algorithms using a NIR spectrophotometer (NIRO 500, Hamamatsu Phototonics, Hamamatsu, Japan). The converted signals from the NIR spectrophotometer were then output to a personal computer where they were stored digitally for later analysis using MacLab software. LBNP responses were obtained by calculating the difference between 20 s (i.e. the average of 20 consecutive NIR samples) of baseline immediately preceding the onset of LBNP and the last 20 s of LBNP. NIR responses were expressed as a percentage of the total labile signal (TLS) which was determined in each experiment as the difference in HbO2 + MbO2 in the forearm at rest and during sustained circulatory arrest (inflation of a pneumatic cuff on the upper arm to 280 mmHg).
Handgrip exercise. Handgrip was performed with a custom-made handgrip dynamometer connected to a force transducer (Interface MFG, Scottsdale, AZ, USA). Force was displayed on an oscilloscope to provide the subject with visual feedback. Before each experiment, the subject's maximal voluntary contraction (MVC) was determined. Subjects performed intermittent, isometric handgrip (20 handgrips min-1, 50 % duty cycle) at 20 % MVC for 7-8 min. Previous studies have shown that this mild level of handgrip exercise alone does not activate muscle SNA (Batman et al. 1994; Ray et al. 1994; Hansen et al. 1996).
Experimental protocols
Protocol 1. Effect of NOS inhibition on decreases in muscle blood flow and oxygenation in response to direct activation of sympathetic nerves in resting rat hindlimb (n = 5 rats). As NO may independently influence skeletal muscle blood flow and metabolism (Shen et al. 2000), we first sought to determine if NOS inhibition would alter the relationship between decreases in flow and muscle oxygenation in response to direct activation of sympathetic nerves. Blood pressure, femoral blood flow velocity, and NIR signals were recorded simultaneously in response to direct electrical stimulation of the lumbar sympathetic chain for 3 min with 1 ms pulses of 5 V at a frequency of 0.25, 0.5, 1, 1.5 or 2 Hz. Rats were then treated with L-NAME (4 mg kg-1, I.V.) and the protocol was repeated. At the end of the experiments, the rats were killed with sodium pentobarbitone (150 mg kg-1).
Protocol 2. Effect of NOS inhibition on reflex activation of muscle sympathetic nerves and subsequent vasoconstrictor responses in resting human forearm (n = 6 subjects). Systemic NOS inhibition with L-NAME increases blood pressure by ~20 mmHg in humans (Sander, 1999), which would be expected to activate arterial baroreflexes. The purpose of this protocol was to determine the effect of L-NAME-induced hypertension on baseline muscle SNA and forearm blood flow, and on the reflex activation of muscle SNA in response to LBNP. Blood pressure, heart rate and muscle SNA were recorded in response to 3 min of LBNP at -20 mmHg before L-NAME and in response to LBNP at -20 and -40 mmHg after L-NAME(4 mg kg-1). In a subset of subjects (n = 4), forearm blood flow responses to LBNP also were measured before and after L-NAME.
Protocol 3. Effect of NOS inhibition on the attenuation of sympathetically mediated decreases in muscle oxygenation in exercising human forearm (n = 27 subjects). The purpose of this protocol was to test our main hypothesis that NOS inhibition would impair the normal ability of forearm exercise to attenuate sympathetically mediated decreases in muscle oxygenation. To test this, blood pressure, heart rate, handgrip force and NIR signals were recorded in response to 3 min of LBNP applied at rest and during min 4-6 of the handgrip exercise before and after L-NAME (4 mg kg-1, I.V.). In one group of subjects (n = 12), a constant level of LBNP (-20 mmHg) was used before and after L-NAME. In a second group of subjects (n = 8), the level of LBNP was increased from -20 mmHg before L-NAME to -40 mmHg after L-NAME. L-NAME infusion was not randomized between the two bouts of handgrip exercise because of its long half-life. To control for a potential order effect, in a third group of subjects (n = 9) responses to LBNP at -20 mmHg were measured in the absence of L-NAME during two bouts of handgrip exercise performed one hour apart. In a fourth group of subjects (n = 4), NIR responses to LBNP at -20 and -40 mmHg were measured in resting forearm, before and after local inhibition of forearm sympathetic neurotransmission to evaluate the effect of LBNP-induced decreases in arterial pressure alone on muscle oxygenation.
Statistical analysis
Data are expressed as means ± S.E.M. Statistical analysis was performed using repeated measures of analysis with Scheffe's post hoc test. A P value less than 0.05 was considered significant.
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RESULTS |
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In anaesthetized rats, NOS inhibition does not alter the relationship between sympathetically mediated decreases in blood flow and oxygenation in skeletal muscle
Direct electrical stimulation of the lumbar sympathetic nerves produced frequency-dependent decreases in femoral blood flow velocity and gastrocnemius muscle oxygenation in resting rat hindlimb (Fig. 1). Before L-NAME, the sympathetically mediated decreases in flow and oxygenation were highly correlated in each rat (correlation coefficients ranged from 0.97 to 0.98). Systemic infusion of L-NAME in these same rats produced expected increases in mean arterial pressure (before, 94 ± 7; after, 136 ± 5 mmHg; P < 0.05), but did not affect femoral blood flow (before, 0.89 ± 0.09; after, 0.86 ± 0.18 kHz; P > 0.05). L-NAME also did not alter the relationship between sympathetically mediated decreases in muscle blood flow and oxygenation (composite slope before L-NAME, 2.37; after L-NAME, 2.62; P > 0.05; Fig. 1B).
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Figure 1. Effect of NOS inhibition on the relationship between sympathetically mediated decreases in blood flow and muscle oxygenation in resting hindlimb of anaesthetized rats A, segments of an original record showing the arterial blood pressure (BP), femoral blood flow and gastrocnemius muscle oxygenation (HbO2 + MbO2) responses to sympathetic nerve stimulation (SNS) before and after L-NAME. Terminal aortic occlusion (Occl.) was used to determine the total labile signal (TLS). OD, optical density. B, summary data (n = 5) showing that sympathetically mediated decreases in hindlimb blood flow and muscle oxygenation are described by a linear relationship that is not significantly altered by L-NAME. | ||
In conscious humans, NOS inhibition causes baroreflex-mediated suppression of muscle SNA, but does not alter the forearm vasoconstrictor response to a given increment in muscle SNA
Before L-NAME, LBNP at -20 mmHg produced robust increases in muscle SNA (+14 ± 2 bursts min-1) and decreases in forearm blood flow (-28 ± 8 %) and vascular conductance (-29 ± 7 %; Fig. 2). As expected, systemic infusion of L-NAME increased blood pressure by 17 ± 3 mmHg, resulting in baroreflex-mediated inhibition of both heart rate and muscle SNA (Table 1). As a result, LBNP at -20 mmHg elicited smaller increases in muscle SNA (+8 ± 1 bursts min-1) and correspondingly smaller decreases in forearm blood flow (-19 ± 9 %) and vascular conductance (-17 ± 7 %; Fig. 2). However, this effect of L-NAME was overcome by increasing LBNP to -40 mmHg, resulting in increases in muscle SNA (+19 ± 2 bursts min-1) and decreases in forearm blood flow (-33 ± 6 %) and vascular conductance (-31 ± 4 %) that were equivalent to the responses to LBNP at -20 mmHg before L-NAME (Fig. 2).
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Figure 2. Effect of NOS inhibition on the muscle sympathetic nerve activity (SNA) and forearm vascular conductance responses to lower body negative pressure (LBNP) in humans Before L-NAME, LBNP at -20 mmHg produced robust increases in muscle SNA and decreases in forearm vascular conductance. After L-NAME, the responses to this same level of LBNP were attenuated significantly, probably because of baroreflex-mediated suppression of baseline muscle SNA. However, responses were fully restored when LBNP was increased to -40 mmHg. Muscle SNA, n = 6; forearm vascular conductance, n = 4. | ||
NOS inhibition impairs the modulation of sympathetically mediated decreases in muscle oxygenation in contracting forearm
Before L-NAME, LBNP at -20 mmHg reproducibly decreased muscle oxygenation in resting forearm by 20 ± 2 % (Fig. 3A and Fig. 4). Handgrip alone at 20 % MVC decreased muscle oxygenation by 33 ± 3 % to a new steady state within the first minute of exercise. When LBNP was superimposed during steady-state handgrip, muscle oxygenation decreased by only 2 ± 3 %, responses that were attenuated compared with those in resting forearm (P < 0.05; Fig. 3A and Fig. 4).
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Figure 3. Effect of NOS inhibition on muscle oxygenation responses to reflex sympathetic activation in resting and exercising forearm A, before L-NAME, the decrease in muscle oxygenation (HbO2 + MbO2) in resting forearm elicited by lower body negative pressure (LBNP) at -20 mmHg was greatly attenuated during rhythmic handgrip at 20 % of maximal voluntary contraction. B, in this same subject after L-NAME, LBNP at -40 mmHg elicited similar decreases in muscle oxygenation in resting and exercising forearm. Shaded areas, provided to aid visual comparison, indicate the total decreases in muscle oxygenation in response to LBNP beginning with the onset of the stimulus and ending with the return of muscle oxygenation to its pre-LBNP baseline. OD, optical density; TLS, total labile signal. | ||
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Figure 4. Effect of NOS inhibition on sympathetically mediated decreases in muscle oxygenation in resting and exercising forearm Before L-NAME (open bars), the decreases in muscle oxygenation (HbO2 + MbO2) evoked by lower body negative pressure (LBNP) at -20 mmHg in resting forearm were attenuated during handgrip (HG) exercise (n = 20). After L-NAME (filled bars), LBNP at -20 mmHg (n = 12) or -40 mmHg (n = 8) evoked decreases in oxygenation that were similar in resting and exercising forearm. TLS, total labile signal. | ||
Systemic infusion of L-NAME markedly increased blood pressure and decreased heart rate in these healthy subjects (Table 2). After L-NAME, the decreases in muscle oxygenation in response to LBNP at -20 mmHg were reduced in resting forearm (-11 ± 3 %), reflecting the attenuated muscle SNA responses to LBNP. In contrast to the effect of handgrip before L-NAME, LBNP-induced decreases in muscle oxygenation were not attenuated during handgrip after L-NAME (-10 ± 2 %; Fig. 4). In some subjects, LBNP was increased to -40 mmHg to match the decreases in muscle oxygenation observed in resting forearm before L-NAME (-19 ± 4 %). Under these conditions as well, LBNP-induced decreases in muscle oxygenation were not attenuated during handgrip (-21 ± 5 %; Fig. 3B and Fig. 4). L-NAME did not affect the decreases in muscle oxygenation in response to handgrip alone in any of the subjects (-26 ± 3 %). In control subjects not treated with L-NAME, LBNP-induced decreases in muscle oxygenation were similarly attenuated during two consecutive bouts of handgrip (1st bout: rest, -16 ± 2 %, handgrip, -4 ± 4 %, P < 0.05 vs. rest; 2nd bout: rest, -16 ± 3 %, handgrip, -7 ± 2 %, P < 0.05 vs. rest). Additional control experiments indicated that the decreases in muscle oxygenation evoked by LBNP at -20 or -40 mmHg were abolished when forearm sympathetic neurotransmission was blocked with bretylium despite greater LBNP-induced decreases in blood pressure post-bretylium (Table 3).
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DISCUSSION |
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We have reported previously that metabolic modulation of sympathetic vasoconstriction is impaired in contracting skeletal muscle of rats and mice after pharmacological NOS inhibition with L-NAME, and in the nNOS-deficient muscle of nNOS knockout mice, mdx mice, and DMD patients (Thomas et al. 1998; Thomas & Victor, 1998; Sander et al. 2000). We now report that a similar impairment is produced in conscious, healthy humans after systemic infusion of L-NAME. Using NIR spectroscopic measurements of changes in tissue oxygenation to estimate sympathetic vasoconstrictor responses in the skeletal muscle microcirculation, we observed a robust attenuation of sympathetically mediated decreases in tissue oxygenation in the exercising forearms of our subjects before, but not after, treatment with L-NAME. Taken together with our previous studies in experimental animal models and in DMD patients, the present study in healthy subjects further substantiates a role for NO in the modulation of sympathetic neural control of skeletal muscle blood flow and oxygenation during exercise.
In this study, we used NIR spectroscopy to measure changes in tissue oxygenation to assess the degree of local metabolic modulation of sympathetic vasoconstriction in the microcirculation of exercising human skeletal muscle. This technique exploits the principle that NIR light easily penetrates tissue and is absorbed by the iron centres of haemoglobin and myoglobin, providing a continuous non-invasive measurement of tissue oxygen sufficiency (Piantadosi, 1989; Mancini et al. 1994). Previous studies have indicated that the contribution of myoglobin to the NIR signal is minor in skeletal muscle, and that the majority of the NIR absorbance is due to the presence of haemoglobin, which normally is confined to the vascular space (Seiyama et al. 1988). Furthermore, because large blood vessels maximally absorb photons, changes in the NIR signals are caused by light absorption in the small arterioles, capillaries and venules of the microcirculation (Mancini et al. 1994). Therefore, during steady-state conditions when oxygen utilization is constant, changes in the NIR signals should primarily reflect changes in oxygen delivery (i.e. blood flow). Based on this logic, we previously have shown that reflex sympathetic activation produced by non-hypotensive levels of LBNP causes robust, reproducible decreases in muscle blood flow and muscle oxygenation, both of which are abolished by local forearm-sympathetic blockade (Hansen et al. 1996; Sander et al. 2000). In the present study we found it necessary to use a higher level of LBNP that produced small but significant decreases in blood pressure which conceivably could reduce muscle perfusion, leading to decreased muscle oxygenation and overestimation of the sympathetic vasoconstrictor response as measured by NIR spectroscopy. However, our data showing that LBNP-induced decreases in blood pressure do not affect muscle oxygenation when forearm sympathetic neurotransmission is blocked, strongly argue against this possibility. Taken together, these observations suggest that under the appropriate experimental conditions tissue oxygenation responses measured by NIR spectroscopy can be used to provide a sensitive and reliable index of sympathetic vasoconstriction in human skeletal muscle.
In order to confidently use NIR spectroscopy to evaluate the modulation of vasomotor responses by NO in the present study, we first had to ensure that the relationship between sympathetically mediated decreases in muscle blood flow and muscle oxygenation was not altered by NOS inhibition. This was a concern because NO is proposed to have numerous effects in skeletal muscle, including vasodilatation, stimulation of glucose uptake, inhibition of mitochondrial respiration and inhibition of contractile function (Stamler & Meissner, 2001). We chose to use an anaesthetized rat model for these experiments for a number of reasons. First, we can obtain continuous, simultaneous measurements of hindlimb blood flow by Doppler velocimetry and muscle oxygenation by NIR spectroscopy. Second, by electrically stimulating sympathetic nerves we can study responses over a wide frequency range that encompasses the changes in muscle oxygenation that we typically observe in our human experiments. Third, systemic NOS inhibition with L-NAME produces many of the same haemodynamic changes in rats and humans including large increases in blood pressure with minimal effects on resting muscle blood flow. Fourth, systemic NOS inhibition does not interfere with direct stimulation of efferent sympathetic nerves in the rats, unlike its profound effects to suppress baseline muscle SNA and LBNP-induced reflex increases in SNA in the human subjects. Our finding that L-NAME did not alter the close linear relationship between sympathetically mediated decreases in blood flow and muscle oxygenation in the quiescent rat hindlimb provided the experimental rationale for using NIR spectroscopy to evaluate the effect of L-NAME on the muscle oxygenation responses to reflex sympathetic activation in resting and exercising human forearm.
Based on our previous work, we know that a 4 mg kg-1 dose of L-NAME given intravenously to young normotensive subjects would cause sustained increases in arterial pressure of approximately 20 mmHg (Sander et al. 1999). We anticipated that this L-NAME-induced hypertension would be a potent stimulus to activate arterial baroreceptors, resulting in a reflex suppression of muscle SNA. Despite this effect on baseline nerve traffic, we were still able to evoke reflex increases in muscle SNA by applying LBNP. Although the sympathetic response to LBNP at -20 mmHg was reduced by 50 % after L-NAME, this effect was easily overcome by increasing the level of LBNP to -40 mmHg, thereby providing an equivalent vasoconstrictor stimulus, before and after NOS inhibition. Regardless of the protocol used (constant or increased LBNP), the key finding was that sympathetically mediated decreases in muscle oxygenation were attenuated in the exercising forearms of our subjects before, but not after, L-NAME. Based on the results of the rat experiments showing that L-NAME did not alter the sensitivity of NIR spectroscopy to detect decreases in muscle blood flow, these data provide evidence that L-NAME impaired the modulation of sympathetic control of oxygenation and presumably blood flow in the microvasculature of exercising human muscle.
Previously, we and others have reported an effect of NO to antagonize noradrenergic vasoconstriction in skeletal muscle (Ohyanagi et al. 1992; Patil et al. 1993; Lau et al. 1998, 2000; Thomas et al. 1998; Thomas & Victor, 1998; Sander et al. 2000; Grange et al. 2001). In conscious humans performing exercise, this interaction between NO and the sympathetic nervous system could occur at multiple sites. Clearly NO can act directly on vascular smooth muscle to reduce its responsiveness to vasoconstrictor stimuli as evidenced by numerous studies in isolated blood vessels (Martin et al. 1986; Topouzis et al. 1991). The present study in humans, as well as our previous studies in rats and mice provide experimental support for the idea that NO interferes with noradrenergic signalling at the level of the vascular smooth muscle (Thomas et al. 1998; Thomas & Victor, 1998). However, NO may also oppose sympathetic vasoconstriction by inhibiting release of noradrenaline from post-ganglionic sympathetic nerve terminals (Greenberg et al. 1990; Schwarz et al. 1995; Costa et al. 2001). Additionally, NO could act centrally to reduce sympathetic outflow during exercise, perhaps via an inhibitory effect on sympathoexcitatory neurons in the brainstem (Zanzinger, 1999).
Our use of the non-selective NOS inhibitor L-NAME precludes any definitive conclusions regarding the specific source of NO that attenuates sympathetic vasoconstriction in exercising muscle. Endothelial NOS, which was originally identified in the vascular endothelium also is expressed diffusely at low levels in the cytosol of skeletal muscle fibres (Kobzik et al. 1995). Neuronal NOS, which is highly expressed at the sarcolemma of skeletal muscle fibres (Nakane et al. 1993; Kobzik et al. 1994), is also found in peripheral nitroxidergic nerves (Yoshida et al. 1993) and in vascular smooth muscle cells (Segal et al. 1999). Our previous studies in various animal (nNOS knockout mice, mdx mice) and human (boys with DMD) models of nNOS-deficient skeletal muscle advanced the hypothesis that skeletal muscle-derived NO is an important modulator of sympathetic control of oxygenation and blood flow in exercising muscle (Thomas et al. 1998; Thomas & Victor, 1998; Sander et al. 2000). Recent studies in isolated mouse skeletal muscles lacking either nNOS or eNOS substantiate an important role for nNOS as a predominate source of NO production and subsequent cGMP formation in contracting muscle (Lau et al. 2000; Grange et al. 2001). Collectively, these studies suggest that some of the NO produced by sarcolemmal nNOS in the contracting skeletal muscle fibres diffuses to adjacent microvessels where it activates the vasodilatatory guanylyl cyclase-cGMP cascade, thereby modulating vasomotor tone.
Although a number of previous human studies have investigated the effect of NO on vascular regulation in exercising muscle, our study is unique for several reasons. First, we examined the interaction between NO and noradrenergic vasoconstriction, rather than the effect of NO on exercise hyperaemia per se. In the only prior study in healthy humans to focus on this interaction, intraarterial infusion of noradrenaline into the exercising forearm produced similar decreases in blood flow before and after local NOS inhibition (Wilson & Kapoor, 1993), suggesting that NO does not modulate -adrenergic vasoconstriction during exercise. However, in that study venous occlusion plethysmography was used to measure whole-limb blood flow during transient suspension of the exercise, in contrast to our study in which NIR spectroscopy permitted us to measure microvascular responses in exercising forearm muscle. Although NO has been shown to modulate vasomotor responses at all levels of the arterial circulation, it may be particularly efficacious for inhibition of vasoconstriction mediated by 2-adrenergic receptors (Ohyanagi et al. 1992), which control the small, distal arterioles of the microcirculation (Faber, 1988).
Second, we chose to use L-NAME to inhibit NOS because of its greater potency and more sustained effect compared to NG-monomethyl-L-arginine (L-NMMA), which is more commonly used in human studies. Although we did not evaluate the extent of NOS inhibition in our study, Frandsen et al. (2001) recently reported that NOS activity in biopsies from the vastus lateralis was reduced by 67 % after systemic infusion of 4 mg kg-1 L-NAME in humans, the same dose used in our study. Despite markedly reduced NOS activity, L-NAME did not alter leg blood flow as measured by thermodilution during knee extensor exercise (Frandsen et al. 2001), suggesting that NO does not play an essential role in blood flow regulation in exercising muscle. However, the possibility that an effect of L-NAME to reduce muscle blood flow was masked by the concomitant suppression of sympathetic outflow caused by the L-NAME-induced increase in blood pressure was not explored in that study. This confounding effect of NOS inhibition to reduce one vasodilator stimulus (NO) while simultaneously increasing another (sympathetic withdrawal) was shown recently to conceal a substantial contribution of NO to locomotor-induced vasodilatation in dogs (Sheriff et al. 2000).
In summary, the present study provides evidence to suggest that NO is involved in the modulation of sympathetic control of oxygenation and blood flow in the microvasculature of exercising human skeletal muscle. Such modulation may play a role in the co-ordinated regulation of skeletal muscle blood flow during exercise because muscle contraction, in addition to its well-known effect to reflexly increase sympathetic neural outflow (Mitchell et al. 1983), also activates NOS and increases NO production (Balon & Nadler, 1994; Hirschfield et al. 2000). We speculate that chronic disruption of the NO pathway, either by reduced production or increased inactivation of NO, may contribute to enhanced sympathetic vasoconstriction in exercising skeletal muscle in such diverse disease conditions as Duchenne muscular dystrophy, heart failure, or renal failure.
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Acknowledgements
This work was funded by the National Institutes of Health Grant HL06296 (R.G.V.). B.C. was supported by an American College of Cardiology Merck Research Fellowship Award and the National Institutes of Health Training Grant HL07360. M.S. was supported by the Danish Heart Foundation and the Michaelsen Foundation. T.S. was supported by a UT Southwestern Medical Student Research Fellowship. J.H. was supported by the Danish Heart Foundation and the Danish Research Council. G.D.T. was supported by an American Heart Association, Texas Affiliate Grant-in-Aid BG98-R-064.
Author's present address
Mikael Sander: Copenhagen Muscle Research Centre, Rigshospitalet, DK-2200, Copenhagen N, Denmark.
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