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. 2001 Feb 15;531(Pt 1):257–264. doi: 10.1111/j.1469-7793.2001.0257j.x

Exercise-induced hyperaemia and leg oxygen uptake are not altered during effective inhibition of nitric oxide synthase with NG-nitro-l-arginine methyl ester in humans

U Frandsen 1, J Bangsbo 1, M Sander 1, L Höffner 1, A Betak 1, B Saltin 1, Y Hellsten 1
PMCID: PMC2278445  PMID: 11179408

Abstract

  1. In the present study the highly potent nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine methyl ester (l-NAME) was intravenously infused and examined for its efficacy in inhibiting NOS activity and in altering blood flow and oxygen uptake in human skeletal muscle.

  2. The plasma concentrations of l-NAME and its active metabolite NG-nitro-l-arginine (l-NA), and the activity of NOS in skeletal muscle were measured in healthy male subjects (n = 6) before (control) and after 60 min of intravenous infusion of l-NAME (4 mg kg−1). In another group of healthy males (n = 8), the physiological effects of l-NAME were studied at rest, and during submaximal and exhaustive knee extensor exercise before (control) and 30 min after l-NAME infusion (4 mg kg−1).

  3. The plasma concentrations of l-NAME and l-NA were highest (8.4 ± 1.6 and 8.3 ± 0.8 μmol l−1) after 60 min of l-NAME infusion. Ninety minutes later mainly l-NA remained in plasma (5.1 ± 0.4 μmol l−1). Thirty minutes after l-NAME infusion, the muscle l-NA content was 38 ± 4 μmol (kg dry wt)−1 and muscle NOS activity was reduced by 67 ± 8 % (P < 0.05).

  4. Leg blood flow and leg oxygen uptake during submaximal and exhaustive exercise were similar (P > 0.05) following l-NAME infusion and in control. Blood flow during recovery was lower in the l-NAME condition (P < 0.05).

  5. In conclusion, the present study shows for the first time that systemic infusion of l-NAME in humans causes a marked reduction in skeletal muscle NOS activity. Despite this attenuated NOS activity, exercise-induced hyperaemia and oxygen uptake were unaltered. Thus, the data strongly suggest that NO is not essential for the regulation of blood flow or oxygen uptake in contracting human skeletal muscle.

Nitric oxide (NO) is a diffusible molecular messenger that mediates the relaxation of vascular smooth muscle and thus vasodilatation (Palmer et al. 1987). The enzyme responsible for NO synthesis, NO synthase (NOS), is located in human skeletal muscle in vascular endothelium (eNOS) as well as in skeletal muscle cells (nNOS; Frandsen et al. 1996), and there is evidence that NO synthesis in skeletal muscle is elevated in response to muscle contraction (Balon & Nadler, 1994). NO could therefore be of importance for the marked vasodilatation observed in contracting human skeletal muscle (Andersen & Saltin, 1985).

By use of the inhibitor of NO synthase NG-monomethyl-l-arginine (l-NMMA) it has been demonstrated that NO contributes to the control of skeletal muscle blood flow at rest and during recovery from exercise (Vallance et al. 1989; Rådegran & Saltin, 1999), but the importance of NO for the physiological control of muscle blood flow during dynamic exercise in humans is controversial. Findings in previous studies have supported (Gilligan et al. 1994; Dyke et al. 1995; Katz et al. 1996) as well as rejected (Wilson & Kapoor, 1993; Shoemaker et al. 1997; Rådegran & Saltin, 1999) a role of NO in skeletal muscle vasodilatation during dynamic exercise. The discrepancy in these findings could in part be explained by the fact that several studies have measured blood flow with venous occlusion plethysmography, a technique that requires the termination of exercise and thus in actuality measures flow in early recovery.

The reason for the effectiveness of l-NMMA in reducing muscle blood flow at rest and during recovery, but not during exercise, is not clear; however, one potential explanation is that l-NMMA mainly inhibits eNOS, and thus only affects basal tone. Recently, another competitive inhibitor of NO synthase, NG-nitro-l-arginine methyl ester (l-NAME), has become available for use in humans. l-NAME is hydrolysed by esterases, in the blood as well as in tissues, to NG-nitro-l-arginine (l-NA), which then becomes the active component for NOS inhibition (Pfeifer et al. 1996). l-NA is a more potent inhibitor of constitutive NOS than l-NMMA (Vargas et al. 1991; Sander et al. 1999). In contrast to l-NMMA, l-NA is not metabolized to l-citrulline by NOS (Griffith et al. 1996) and transport over the cell membrane occurs via the amino acid transporter system for l-leucine (L-system; Schmidt et al. 1993). Based on these properties it has been proposed that continuous l-NAME administration results in an intracellular accumulation of l-NA and a consequent progressive inhibition of NOS with time (Griffith et al. 1996). Thus, it is possible that systemic infusion of l-NAME would provide a more potent inhibition of NOS, including nNOS, in skeletal muscle cells. Use of l-NAME in vivo in combination with direct determination of l-NA concentration and NOS activity in the muscle tissue would, therefore, shed further light on the importance of NO for the physiological control of skeletal muscle vasodilatation during exercise.

In addition to the effect on muscle blood flow, there is evidence in the literature that NO participates in the regulation of mitochondrial respiration through reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain (Cleeter et al. 1994; Brown, 1995). In vivo support for this hypothesis has been provided by Shen and co-workers (Shen et al. 1995) who demonstrated that oxygen consumption was elevated in active dog skeletal muscle upon inhibition of NO. Whether the rate of muscle oxygen uptake in contracting human skeletal muscle is modulated by inhibition of NO synthesis with l-NAME has not been investigated.

In the present study it was hypothesized that systemic infusion of the potent NOS inhibitor l-NAME in healthy human subjects would result in a substantial inhibition of muscle NOS activity with a consequent lowering of muscle blood flow and an increase in muscle oxygen uptake during dynamic exercise.

METHODS

Subjects

Fourteen healthy male subjects (age, 25 ± 2 years (range, 21-32 years); height, 182 ± 2 cm (177-190 cm); body mass, 75 ± 4 kg (62-95 kg)) took part in the study. Six of the subjects participated in protocol I and eight of the subjects in protocol II. All subjects were informed of the experimental procedures, the potential risks and discomfort, and that they could withdraw from the study at any time. All subjects gave their written informed consent. The experiments were carried out with the approval of the local ethics committee of Copenhagen and Fredriksberg and conformed with the Declaration of Helsinki. The subjects did not ingest caffeine, tea or nicotine for 48 h prior to the experiment.

protocol i: measurement of the potency and specificity of l-NAME

Subjects (n = 6) rested for 30 min. A catheter was then placed in the brachial vein and l-NAME (4 mg (kg body mass)−1; Clinalfa, Läufelfingen, Switzerland) was infused over 60 min. In five of the subjects, biopsies were obtained with a Bergstrüm needle from the vastus lateralis muscle under local anaesthesia prior to l-NAME infusion and 30 min after completion of the l-NAME infusion. Sixty minutes after completion of the l-NAME infusion, l-arginine (200 mg (kg body mass)−1), was infused over 15 min. Blood pressure was measured with an automated sphygmanometer (Dinamap, Critikon) and heart rate was continuously monitored.

protocol ii: physiological effects of l-NAME infusion

Eight subjects were familiarized with the one-legged knee extensor exercise model (Andersen et al. 1985) several days prior to the experiment. On the day of the experiment, the subjects rested for 30 min and catheters were then placed 1-2 cm distal to the inguinal ligament in the femoral artery and femoral vein under local anaesthesia. A thermistor was inserted through the femoral venous catheter and was advanced 8-10 cm proximal to the tip. Microdialysis dialysis probes (CMA 60; Carnegie Medicine, Stockholm, Sweden) were positioned in the vastus lateralis muscle and perfused as previously described (Hellsten et al. 1998). Thereafter, the subjects performed 30 min of one-legged knee-extensor exercise on a modified Krogh ergometer at an intensity of 30 W and with a frequency of 60 extensions per minute. Five of the subjects also performed a graded knee-extensor exercise protocol until exhaustion beginning at 50 W for 2 min and with a 10 W increase every minute up to the peak load of 90 ± 4 W (range, 70-100 W). After a 30 min recovery period, l-NAME (4 mg (kg body mass)−1 in 30 ml saline) was infused intravenously over 1 h. Thirty minutes after completion of the l-NAME infusion, the subjects repeated the sub-maximal (n = 8) and the graded (n = 5) exercise protocols. Blood samples (2 ml) were drawn simultaneously from the femoral artery and vein at rest, after 10 and 20 min of sub-maximal exercise, and at 1 and 2 min after exercise, both without and with prior l-NAME infusion. During the graded exercise protocol, arterial and venous blood samples were drawn just before exhaustion. Blood flow was measured with the thermodilution technique (Andersen & Saltin, 1985) immediately after blood sampling. An occlusion cuff placed just below the knee was inflated (220 mmHg) during blood sampling and blood flow measurements. Dialysate from the microdialysis probes was collected for 30 min at rest in control and for 30 min from the termination of l-NAME infusion. Heart rate and intra-arterial blood pressure (Simonsen & Weel Medico Teknik, Denmark) were continuously monitored.

Measurements and analysis

The concentration of l-NA in muscle biopsies was determined in neutralized perchloric acid extracts with reverse-phase HPLC as previously described (Tabrizi-Fard & Fung, 1996). The recovery of l-NA in muscle extracts and plasma was 98 ± 4 and 95 ± 3 %, respectively. The concentrations of l-NAME and l-NA in the microdialysate and plasma were determined with a reverse-phase HPLC method (Tullson et al. 1990). Microdialysates were injected without prior treatment whereas plasma samples were diluted 1:2 with water and were then ultrafiltered through 0.45 μm filters (Spin X, Costar, Cambridge, MA, USA) prior to injection.

Muscle NOS activity was determined with the l-citrulline assay (Bredt & Snyder, 1990) in freeze-dried muscle biopsy material dissected free of blood, fat and connective tissue. As l-NA is competitive with l-arginine, the proportion of l-arginine to l-NA was kept constant by addition of l-NA to the muscle homogenate in a concentration corresponding to the relative increase in l-arginine due to the addition of l-[14C]arginine. The endogenous l-arginine content was assumed to be 2.2 mmol (kg dry wt)−1 (MacLean et al. 1991) whereas the l-NA content was based on measurements on muscle biopsies in the present study. The enzyme activity is expressed as the number of picomoles of l-citrulline produced per minute per milligram dry weight of muscle (pmol min−1 (mg dry wt)−1).

The arterial and venous blood samples were analysed for haemoglobin (Hb), O2 saturation and blood PO2, PCO2 and pH (ABL 510, Radiometer, Denmark). Haematocrit was determined in triplicate by microcentrifugation.

Calculations

Leg oxygen uptake was calculated as the product of the difference in femoral arterial and venous oxygen content and leg blood flow according to Ficks's principle. Mean arterial blood pressure (MAP) was calculated as diastolic pressure plus one-third of the pulse pressure.

Statistics

All values are presented as means ±s.e.m. Significant differences of means for the physiological effects of l-NAME infusion were determined by using ANOVA with Fisher's post hoc test. Muscle NOS activity in l-NAME and control was compared with Student's t test. A P value of less than 0.05 was regarded as being statistically significant.

RESULTS

Distribution and efficacy of l-NAME and l-NA

l-NA and l-NAME concentrations in plasma, interstitial fluid and muscle

The l-NAME and l-NA concentrations in plasma increased from below detection levels to 8.4 ± 1.6 and 8.3 ± 0.8 μmol l−1, respectively, during the 60 min of l-NAME infusion (Fig. 1). After infusion, the l-NAME levels fell rapidly whereas the concentration of l-NA remained elevated and was still high (5.1 ± 0.4 μmol l−1) 90 min after the end of infusion. Before infusion of l-NAME, l-NAME and l-NA were not detectable in the muscle dialysate. Dialysate collected during the 30 min period from the termination of l-NAME infusion contained 0.96 ± 0.08 and 0.63 ± 0.09 μmol l−1l-NAME and l-NA, respectively. The muscle l-NA content 30 min after l-NAME infusion was 38 ± 4 μmol (kg dry wt)−1 (n = 5).

Figure 1. Concentrations of l-NAME and l-NA in plasma.

Figure 1

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Concentrations of l-NAME (▪) and l-NA (□) in human plasma before, during and after 60 min of venous infusion of 4 mg (kg body mass)−1l-NAME (n = 6). Values are means ±s.e.m.

Skeletal muscle NOS activity

The activity of NOS was 57 ± 5 % lower (P < 0.05) in muscle biopsies obtained after l-NAME infusion than in control biopsies (2.65 ± 0.23 vs. 6.23 ± 0.29 pmol min−1 (mg dry wt)−1). When compensating for the relative increase in l-arginine due to the addition of 14C-labelled l-arginine to the reaction buffer, the NOS activity following l-NAME infusion was 67 ± 8 % lower (P < 0.05; 2.24 ± 0.4 pmol min−1 (mg dry wt)−1) than that in control biopsies (n = 5).

Systemic responses to l-NAME and l-arginine

MAP and heart rate

At rest, 1 h infusion of l-NAME (4 mg (kg body mass)−1) resulted in a sustained increase in blood pressure and a decrease in heart rate that remained 30 min after termination of the l-NAME infusion (Table 1; n = 6). Fifteen minutes of infusion of l-arginine (200 mg (kg body mass)−1) reversed the blood pressure and heart rate responses to l-NAME back to baseline levels (Table 1; n = 6).

Table 1.

Haemodynamic responses at baseline, during and after l-NAME infusion and after l-arginine infusion

l-NAME (4 mg kg-1) l-NAME recovery + l-Arginine (200 mg kg-1)
Parameter Baseline 30 min 60 min 30 min 15 min
Protocol I (n = 6)
Systolic blood pressure (mmHg) 125 ± 3 135 ± 2* 138 ± 3* 141 ± 3* 128 ± 5
Diastolic blood pressure (mmHg) 67 ± 3 82 ± 2* 85 ± 2* 85 ± 2* 67 ± 4
Mean arterial blood pressure (mmHg) 87 ± 4 100 ± 3* 103 ± 3* 103 ± 2* 88 ± 4
Heart rate (beats min-1) 56 ± 3 44 ± 2* 41 ± 1* 41 ± 2* 53 ± 4
Protocol II (n = 8)
Systolic blood pressure (mmHg) 116 ± 3 135 ± 3* 143 ± 3* 147 ± 3* —
Diastolic blood pressure (mmHg) 65 ± 1 86 ± 1* 88 ± 2* 89 ± 2* —
Mean arterial blood pressure (mmHg) 78 ± 2 100 ± 2* 104 ± 2* 108 ± 2* —
Heart rate (beats min-1) 57 ± 2 41 ± 1* 40 ± 2* 41 ± 2* —
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*

Significant difference from baseline (P < 0.05).

During and after sub-maximal exercise, MAP (Fig. 2) was higher (P < 0.05) and heart rate (Fig. 3) lower (P < 0.05) in the l-NAME condition than in control. At the end of the exhaustive exercise, MAP was similar in the two conditions whereas heart rate was lower (P < 0.05) after l-NAME infusion than in control.

Figure 2.

Figure 2

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Mean arterial blood pressure

Mean arterial blood pressure without (control; □) and with prior l-NAME infusion (l-NAME; ▪) at rest (Rest), during sub-maximal exercise at 30 W (Submax; n = 8), and 1 and 2 min after exercise (Recovery; n = 8) as well as during peak exercise at 90 ± 4 W (Peak; n = 5). Values are means ±s.e.m.* Significant difference from control value (P < 0.05).

Figure 3.

Figure 3

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Heart rate

Heart rate without (control; □) and with prior l-NAME infusion (l-NAME; ▪) at rest (Rest), during sub-maximal exercise at 30 W (Submax; n = 8), and 1 and 2 min after exercise (Recovery; n = 8) as well as during peak exercise at 90 ± 4 W (Peak; n = 5). Values are means ±s.e.m.* Significant difference from control value (P < 0.05).

Local responses to l-NAME

Leg blood flow

Leg blood flow was similar in the l-NAME and control conditions during sub-maximal exercise (20 min: 3.5 ± 0.3 vs. 3.5 ± 0.2 l min−1, respectively; P > 0.05). After sub-maximal exercise, leg blood flow was lower (P < 0.05) after l-NAME infusion than in control (Fig. 4A). At the end of exhaustive exercise no difference (P > 0.05) was observed in leg blood flow (6.9 ± 1.3 vs. 6.3 ± 1.0 l min−1) between the l-NAME and control conditions.

Figure 4.

Figure 4

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Leg blood flow and vascular conductance

Leg blood flow (A) and vascular conductance (B) without (control; □) and with prior l-NAME infusion (l-NAME; ▪) during sub-maximal exercise at 30 W (Submax; n = 8), and 1 and 2 min after exercise (Recovery: n = 8) as well as during peak exercise at 90 ± 4 W (Peak; n = 5). Values are means ±s.e.m.* Significant difference from control value (P < 0.05).

Leg vascular conductance

Leg vascular conductance during sub-maximal exercise was lower (P < 0.05) after l-NAME infusion than in control during sub-maximal exercise (20 min: 30.4 ± 1.5 vs. 33.5 ± 1.6 ml min−1 mmHg−1) and after sub-maximal exercise, but was not different (P > 0.05) at the end of exhaustive exercise (46.3 ± 3.9 vs. 42.6 ± 3.8 ml min−1 mmHg−1; Fig. 4B).

Leg oxygen extraction and uptake

At rest prior to exercise, leg arterial-venous O2 difference (O2 extraction) was higher (P < 0.05) following l-NAME infusion than in control (Fig. 5A). Leg O2 extraction and O2 uptake during sub-maximal exercise were similar (P > 0.05) in the l-NAME and control conditions (Fig. 5A and B). Leg oxygen extraction during recovery from sub-maximal exercise was higher (P < 0.05) after l-NAME infusion than in control, but leg O2 uptake was similar (P > 0.05). At the end of the exhaustive exercise there was no difference between the l-NAME condition and control in leg O2 extraction and leg O2 uptake.

Figure 5.

Figure 5

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Leg oxygen extraction and uptake

Leg oxygen extraction (A) and uptake (B) without (control; □) and with prior l-NAME infusion (l-NAME; ▪) at rest (Rest), during sub-maximal exercise at 30 W (Submax; n = 8), and 1 and 2 min after exercise (Recovery; n = 8) as well as during peak exercise at 90 ± 4 W (Peak; n = 5). Values are means ±s.e.m.* Significant difference from control value (P < 0.05).

DISCUSSION

The present study demonstrates that systemic infusion of the NOS inhibitor l-NAME in humans results in an accumulation of l-NA in skeletal muscle tissue and an approximately 70 % reduction in NOS activity. Despite the efficacy in inhibiting muscle NOS, l-NAME had no effect on exercise-induced muscle hyperaemia or oxygen uptake. In combination, these data strongly suggest that NO is not essential for the regulation of exercise-induced hyperaemia or oxygenation in humans. Furthermore, the data show that, at rest, the increase in blood pressure after l-NAME infusion was due to the inhibition of NOS as verified by the reversibility with l-arginine.

The degree of skeletal muscle NOS inhibition with systemic infusion of NOS blockers has never before been documented in humans. In the present study we therefore posed the question of whether previous observations of a lack of effect of l-NMMA (Wilson & Kapoor, 1993; Shoemaker et al. 1997; Rådegran & Saltin 1999) on exercise-induced hyperaemia could be explained by insufficient inhibition of NOS in skeletal muscle and, in particular, ineffective inhibition of nNOS, which may be important for exercise-induced hyperaemia. With infusion of the more potent NOS inhibitor l-NAME in the present study, we obtained close to 70 % inhibition of skeletal muscle NOS activity, thus both eNOS and nNOS should have been inhibited. Despite this effective inhibition, we observed no effect of l-NAME on the rate of blood flow during exercise. The results demonstrate that previous observations of a lack of effect of NOS inhibitors on exercise-induced hyperaemia in humans cannot necessarily be explained by an insufficient inhibition of muscle NO formation. The data therefore support the notion that NO is not essential for skeletal muscle vasodilatation during exercise.

The present observation of the efficacy of l-NAME in reducing blood flow in recovery from exercise is in agreement with previous studies showing lower blood flow rates both at rest and during recovery from exercise after l-NMMA infusion (Wilson & Kapoor, 1993; Shoemaker et al. 1997; Rådegran & Saltin, 1999). These observations remain to be explained in the light of the lack of effect of NOS inhibition during exercise. The effects of l-NMMA and l-NAME on haemodynamics at rest are clearly related to NOS inhibition as evidenced by the reversal of the haemodynamic effects of NOS inhibition with infusion of large doses of l-arginine in the present and a previous human study (Sander et al. 1999) and by a reduction of the haemodynamic response to acetylcholine after l-NMMA infusion (Rådegran & Saltin, 1999). One possibility is, therefore, that vascular tone at rest and during recovery is regulated by the continuous formation of NO by eNOS in the vascular endothelium, whereas the regulation of exercise-induced hyperaemia mainly occurs via the action of other vasodilators. Several locally formed vasodilators, such as potassium, prostacyclin and adenosine, have been suggested to play a role in exercise-induced hyperaemia and it is likely that it is not one but a combination of vasodilators that act together to produce the precise match of muscle blood flow to oxygen demand (Andersen & Saltin, 1985).

l-NAME administration in the present study resulted in an l-NA concentration in the vastus lateralis muscle of 38 μmol (kg dry wt)−1, corresponding to approximately 10 μmol l−1 tissue fluid. This value appears reasonable since in plasma the combined concentrations of l-NAME and l-NA peaked at approximately 17 μmol l−1 after 60 min of l-NAME infusion. The infused l-NAME was rapidly metabolized to l-NA in plasma and not all of the l-NA had been taken up by the tissues at the time when the biopsy was taken, 30 min after the end of infusion, as evidenced by the relatively high levels of circulating l-NA in the blood at that time. A relatively slow uptake of l-NA from plasma was also indicated by the fact that the concentration of l-NA in the muscle interstitium at the time of the biopsy was only a fraction of that in plasma.

The observation in the present study that, after l-NAME infusion, blood pressure was higher and heart rate lower at rest, and during and after sub-maximal exercise, demonstrates that NO is involved in the regulation of systemic vascular resistance and thereby blood pressure and heart rate in humans not only at rest as previously reported (Stamler et al. 1994; Sander et al. 1999) but also during exercise and recovery from exercise. The effect of l-NAME on blood pressure has primarily been attributed to the inhibition of NO-mediated vasodilatation, but also to a delayed involvement of a sympathetic component. The latter mechanism was demonstrated in a recent study in which systemic α-adrenergic receptor blockade was shown to have little effect on the blood pressure response to 1 h of l-NAME infusion, whereas it eliminated the additional increase in blood pressure observed over the next 2 h (Sander et al. 1999).

An explanation for the finding that heart rate decreases in response to the inhibition of NO synthesis could be that the elevated blood pressure initiates a baroreflex that results in the withdrawal of sympathetic efferent activity and the augmentation of vagal activity to the heart. In this context it should also be mentioned that a possible withdrawal of sympathetic activity induced by the increased blood pressure in the l-NAME condition could have reduced the vasoconstrictor tone in the muscle during sub-maximal exercise, thus masking the potential vasoconstriction due to the reduced level of NO formation. However, the finding that during exhaustive exercise, blood pressure was similar in control and after l-NAME infusion, whereas there was no difference in blood flow between the two conditions, strongly argues against this possibility. Furthermore, the likelihood of the observed higher blood pressure (10-15 mmHg) during sub-maximal exercise with l-NAME fully cancelling out a reduction in flow due to NOS inhibition appears small.

It should also be pointed out that a decrease in the calculated leg vascular conductance during sub-maximal exercise was observed in the l-NAME trial. This observation could suggest that l-NAME infusion induced vasoconstriction in the exercising muscle and that blood flow was unaltered due to the increased perfusion pressure. However, the lower conductance may simply have reflected vasoconstriction in vascular beds of non-active tissues of the limb. This notion is further supported by the observation that conductance was similar in control and following infusion of l-NAME during exhaustive exercise when more tissue was activated. Moreover, oxygen consumption during exercise was similar in the control and l-NAME conditions, thus the normal matching between blood flow and oxygen uptake was maintained despite NOS inhibition.

In the present study we examined whether NO inhibition affected oxygen uptake in human skeletal muscle and found no difference in leg oxygen extraction or leg oxygen uptake during or in recovery from exercise with and without prior l-NAME infusion. This observation contradicts findings in animal studies in which it has been observed that oxygen uptake is elevated in vivo in resting and contracting dog skeletal muscle (Shen et al. 1994, 1995) and resting rabbit skeletal muscle (King et al. 1994) when NO synthesis is inhibited with l-NA (30 mg (kg body mass)−1) and l-NAME (20 mg (kg body mass)−1), respectively. The explanation for this discrepancy is not clear, but it appears that there could be a species difference, possibly related to a difference in the physiological functions of NO. Nevertheless, the present data suggest that, in human skeletal muscle, NO is not a modulator of mitochondrial respiration.

In conclusion, the present study shows for the first time that NO inhibition in humans in vivo with l-NAME causes a marked reduction in skeletal muscle NOS activity without affecting exercise-induced hyperaemia or oxygen uptake in skeletal muscle. The data strongly suggest that NO is not essential for the regulation of blood flow or oxygen uptake in contracting human skeletal muscle in vivo.

Acknowledgments

The authors wish to acknowledge the excellent technical assistance of Merete Vannby, Inge-Lise Kring and Karina Olsen. The project was funded by The Danish National Research Foundation (Jnr. 504-14).

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