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. 2005 Aug 4;568(Pt 3):1021–1033. doi: 10.1113/jphysiol.2005.090068

Effects of nitric oxide synthase inhibition by l-NAME on oxygen uptake kinetics in isolated canine muscle in situ

Bruno Grassi 1, Michael C Hogan 2, Kevin M Kelley 3, Richard A Howlett 2, L Bruce Gladden 3
PMCID: PMC1464191  PMID: 16081490

Abstract

Nitric oxide (NO) has an inhibitory action on O2 uptake (V˙O2) at the level of the mitochondrial respiratory chain. The aim of this study was to evaluate the effects of NO synthase (NOS) inhibition on muscle V˙O2 kinetics. Isolated canine gastrocnemius muscles in situ (n= 6) were studied during transitions from rest to 4-min of electrically stimulated contractions corresponding to ∼60% of the muscle peak V˙O2. Two conditions were compared: (i) Control (CTRL) and (ii) l-NAME, in which the NOS inhibitor l-NAME (20 mg kg−1) was administered. In both conditions the muscle was pump-perfused with constantly elevated blood flow (Q˙), at a level measured during a preliminary contraction trial with spontaneous self-perfused Q˙. A vasodilatory drug was also infused. Arterial and venous O2 concentrations were determined at rest and at 5–7 s intervals during the transition. V˙O2 was calculated by Fick's principle. Muscle biopsies were obtained at rest and during contractions. Muscle force was measured continuously. Phosphocreatine hydrolysis and the calculated substrate level phosphorylation were slightly (but not significantly) lower in l-NAME than in CTRL. Significantly (P < 0.05) less fatigue was found in l-NAME versus CTRL. The time delay (TDf) and the time constant (τf) of the ‘fundamental’ component of V˙O2 kinetics were not significantly different between CTRL (TDf 7.2 ± 1.2 s; and τf 10.6 ± 1.3, ±s.e.m.) and l-NAME (TDf 9.3 ± 0.6; and τf 10.4 ± 1.0). Contrary to our hypothesis, NOS inhibition did not accelerate muscle V˙O2 kinetics. The down-regulation of mitochondrial respiration by NO does not limit the kinetics of adjustment of oxidative metabolism at exercise onset.

Experiments conducted by our group on the isolated dog gastrocnemius in situ provided evidence in favour of the concept that the factors controlling the kinetics of muscle O2 uptake (V˙O2) during metabolic transitions are mainly related to an intrinsically slow adaptation of oxidative metabolism to adjust to the new metabolic requirement (Grassi et al. 1998a; b; 2000); this slow adjustment to a new metabolic rate does not seem related to pyruvate dehydrogenase activation status (Grassi et al. 2002).

Nitric oxide (NO) produced by the endothelial form of NO synthase (eNOS) causes vasodilation, which increases muscle blood flow and O2 delivery at the onset of contractions (Joyner & Dietz, 1997). The neuronal isoform of NOS (nNOS), present in skeletal muscle, may mediate the metabolic inhibition of sympathetic vasoconstriction (‘functional sympatholysis’) that is inherent to resting skeletal muscle (Thomas & Victor, 1998), and may contribute to the hyperemic response to muscle contraction. On the other hand, intramyocyte NO (produced by different NOS isoforms) binds to the O2-binding site of cytochrome c oxidase, the terminal enzyme in the electron transport chain. NO has a high affinity for the O2-binding site when the latter is reduced, and therefore competes with O2 for the site. At physiological levels NO specifically and reversibly inhibits cytochrome c oxidase, and thereby mitochondrial respiration (see reviews by: Brown, 2000; and Stamler & Meissner, 2001). Whereas the effects of NO on cytochrome c oxidase are those best characterized, several other enzymes related to energy transduction can be inhibited by NO (Brown, 2000; Stamler & Meissner, 2001). Through its combined effects of vasodilation and V˙O2 inhibition, NO may serve as part of a feedback mechanism aimed at increasing O2 delivery and reducing the reliance on O2 extraction to meet the increase in muscle O2 needs; by this mechanism, NO would work in the direction of maintaining higher intramyocyte PO2 levels during exercise (Shen et al. 2000).

Thus, NO could be responsible, at least in part, for the intrinsically slow adaptation of oxidative metabolism at exercise onset. Inhibition of NOS by administration of the l-arginine analog Nω-nitro-l-arginine methyl ester (l-NAME) elicited a slightly faster ‘phase 2’ (see Whipp et al. 2002) pulmonary V˙O2 kinetics in exercising horses, both during heavy (Kindig et al. 2001) and moderate intensity exercise (Kindig et al. 2002), as well as in humans, during moderate (Jones et al. 2003), heavy (Jones et al. 2004) and supramaximal (Wilkerson et al. 2004) exercise. In all these studies, however, for methodological reasons the potentially negative effects of NOS inhibition on vasodilation and O2 delivery to muscle could not be determined. A restricted muscle O2 delivery would work in the direction of a slower V˙O2 kinetics (e.g. see Hughson et al. 1996), thereby masking the effects of NOS inhibition on mitochondrial respiration. The isolated gastrocnemius preparation in situ allows pump-perfusion of the muscle with constantly elevated blood flows during metabolic transitions (Grassi et al. 1998a, b; 2000). This would prevent any reduction of O2 delivery due to NOS inhibition. Any effect of the latter on mitochondrial respiration could therefore be fully manifest, potentially becoming more pronounced than those observed in the studies mentioned above (Kindig et al. 2001, 2002; Jones et al. 2003, 2004; Wilkerson et al. 2004).

Additionally, all previous studies evaluated pulmonary V˙O2 kinetics, with specific attention paid to the metabolically relevant, or ‘fundamental’, phase 2 (see Whipp et al. 2002). The different phases of pulmonary V˙O2 kinetics, however, cannot be considered as absolutely separate entities. As elegantly demonstrated in the modelling study by Barstow et al. (1990), when slower cardiac output kinetics are paired with unchanged muscle V˙O2 kinetics, the results are a lengthening of the ‘cardiodynamic’ phase (phase 1, see Whipp et al. 2002) and a faster phase 2 of pulmonary V˙O2 kinetics. Thus, a restricted muscle O2 delivery could engender a faster pulmonary V˙O2 kinetics, even in the presence of an unchanged muscle V˙O2 kinetics. Thus, the need to confirm the previously mentioned observations (Kindig et al. 2001, 2002; Jones et al. 2003, 2004; Wilkerson et al. 2004) in terms of skeletal muscle V˙O2 kinetics appears obvious.

Therefore, the hypothesis of the present study was that NOS inhibition by l-NAME, through relief of NO inhibition of cytochrome c oxidase, would speed V˙O2 kinetics in a skeletal muscle preparation in situ, in which the negative effects of NOS inhibition on vasodilation and O2 delivery can be prevented by pump-perfusing the muscle with high blood flows and by the administration of a vasodilator. A faster V˙O2 kinetics after l-NAME administration would indicate that the down-regulation of mitochondrial respiration by NO is an essential determinant of the relatively slow adjustment of intramuscular oxidative metabolism to the new metabolic requirement at exercise onset.

Methods

The study was conducted with approval of the Institutional Animal Care and Use Committee of Auburn University, Auburn, Alabama, where the experiments were performed.

Six adult mongrel dogs of either sex (body mass 24.3 ± 1.4 kg, mean ±s.e.m.) were anaesthetized with pentobarbital sodium (30 mg kg−1), with maintenance doses given as required to maintain a deep plane of surgical anaesthesia as indicated by a complete absence of pedal, palpebral and corneal reflexes. The dogs were intubated with an endotracheal tube and ventilated with a respirator (Model 613, Harvard, Holliston, MA, USA). The rectal temperature was maintained at ∼37°C with a heating pad and a heating lamp. After surgical preparation the animals were treated with heparin (2000 U kg−1). Ventilation was maintained at a level that produced normal arterial PO2 and PCO2 values.

Surgical preparation

The gastrocnemius-plantaris-flexor digitorum superficialis muscle complex (for convenience referred to as ‘gastrocnemius’) was isolated as previously described (Stainsby & Welch, 1966). Briefly, a medial incision was made through the skin of the left hindlimb from midthigh to the ankle. The insertion tendons of the sartorius, gracilis, semitendinosus and semimembranosus muscles were cut to allow these muscles to be folded back to expose the gastrocnemius. To isolate the venous outflow from the gastrocnemius, all the vessels draining into the popliteal vein, except those from the gastrocnemius, were ligated. The popliteal vein was cannulated, and flow (Q˙) was measured with a flow-through-type transit-time ultrasound flow probe (6NRB440, Transonic Systems, Ithaca, NY, USA). Venous outflow was returned to the animal via a reservoir attached to a cannula in the left jugular vein. The arterial circulation to the gastrocnemius was isolated by ligating all vessels from the femoral and popliteal artery that did not enter the gastrocnemius. The right femoral artery was also isolated and cannulated. Blood from this artery was passed through tubing to a roller pump (Model no. 7520–25, Head Model no. 7016–20, Cole-Palmer Masterflex, Vernon Hills, IL, USA) and then through another cannula into the contralateral, isolated popliteal artery supplying the gastrocnemius. Y-connectors positioned before and after the pump allowed either spontaneous perfusion of the gastrocnemius, at the animal's own blood pressure, or controlled flow at any desired level by adjusting the pump setting. A T-connector in the tubing to the gastrocnemius was connected to a pressure transducer (Model RP-1500, Narco Biosystems, Houston, TX, USA) for measurement of muscle perfusion pressure. Arterial blood samples were taken from another T-connector in the cannula exiting the right femoral artery prior to the roller pump.

A portion of the calcaneus, with the two tendons from the gastrocnemius attached, was cut away at the heel and clamped around a metal rod for connection to an isometric myograph via a load cell (SM-250, Interface, Scottsdale, AZ, USA) and a universal joint coupler. The universal joint allowed the muscle to always pull directly in line with the load cell, and thus prevented the application of torque to the cell. The other end of the muscle was left attached to its origin; both the femur and the tibia were fixed to the base of the myograph by bone nails. A turnbuckle strut was placed parallel to the muscle between the tibial bone nail and the arm of the myograph to minimize flexing of the myograph.

The sciatic nerve, which innervates the gastrocnemius, was exposed and isolated near the muscle, doubly ligated and cut between the ties. The distal stump of the nerve, ∼1.5–3.0 cm in length, was pulled through a small epoxy electrode containing two wire loops for stimulation. The muscle was covered with saline-soaked gauze and a thin plastic sheet to prevent drying and cooling.

Experimental design

To evoke muscle contractions, the nerve was stimulated by supramaximal square pulses of 4.0–6.0 V amplitude and 0.2 ms duration (Grass S48 stimulator, West Warwick, RI, USA), isolated from ground by a stimulus isolator (Grass SIU8TB). Before each experiment, the muscle was set at optimal length by progressively lengthening the muscle as it was stimulated at a rate of 0.2 Hz, until a peak in developed tension (total tension minus resting tension) was obtained. For the experiments, isometric tetanic contractions were triggered by stimulation with trains of stimuli (4–6 V, 200 ms pulses at 50 Hz) at a rate of 0.5 Hz for a 4-min period. Based on studies of peak V˙O2 in this model (Kelley et al. 1996; Ameredes et al. 1998), this stimulation pattern elicits ∼60% of peak metabolic rate for this muscle.

Tetanic contractions were chosen in order to allow a rapid attainment of a steady-state of developed force. Peak force was in fact reached from the very first contraction. For the purposes of the study it was critical to obtain truly ‘rectangular’ increases of the forcing function, represented by the developed force. Each isometric tetanic contraction lasted 200 ms, and was separated from the following by 1.8 s, during which the muscle was relaxing or relaxed.

For each dog, the experiment consisted of two contraction periods of 4-min duration, preceded by a resting baseline. The contraction periods were separated by at least 45 min of rest. The investigated metabolic transition was therefore a transition from rest to submaximal contraction. Two conditions were compared as follows.

First, a control condition (CTRL), in which the muscle was pump-perfused at a constant Q˙, adjusted 15–30 s before the start of the contraction to a level corresponding to the steady-state level during contractions, as determined in a preliminary trial with spontaneous adjustment of Q˙. When the blood supply to the gastrocnemius was switched from self-perfused to pump-perfused, at least 15 min were allowed for the haemodynamic parameters to stabilize. In this CTRL condition, 10 ml of physiological saline solution were also infused intravenously over ∼3 min before contraction onset.

Secondly, a treatment condition (l-NAME), in which 20 mg of l-NAME per kg of body mass, diluted in 10 ml of physiological saline, were infused intravenously over ∼3 min before contraction onset. It has been previously demonstrated, in the same experimental model, that this dosage of l-NAME produces effective NOS inhibition for at least 1 h (King et al. 1994). Also in this l-NAME condition, the muscle was pump-perfused at the constant Q˙ utilized in the CTRL condition.

In both the above experimental conditions, to prevent vasoconstriction and inordinate pressure increases with the elevated Q˙, 1–2 ml min−1 of a 10−2m adenosine solution (in physiological saline) was also infused intra-arterially using a pump, beginning 15–30 s before the onset of contractions. The adenosine infusion was then continued throughout the contraction period. This dosage of the drug was previously shown to be effective in obtaining a significant vasodilation at the muscle level without causing significant metabolic effects (such as changes in resting V˙O2, in V˙O2 at the same submaximal level of contraction, or in V˙O2max and acid–base status) (Kurdak et al. 1994; Grassi et al. 1998a, 2000). As plasma concentrations of the active metabolite of l-NAME, Nω-nitro-l-arginine, remain elevated for hours after l-NAME infusion (Frandsen et al. 2001), the order of treatments could not be randomized, and CTRL was always performed before l-NAME.

In two of the dogs, the vascular effects of l-NAME were tested using the acetylcholine (ACh) bolus method (King et al. 1994; Shen et al. 2000). ACh is known to activate muscarinic receptors on endothelial cells, to enhance the biosynthesis and release of NO and, consequently, to induce an endothelium-dependent vascular relaxation. A bolus of ACh (0.3 μg kg−1 of estimated muscle weight) was infused in the artery feeding the gastrocnemius several minutes before contraction onset, in both CTRL and l-NAME conditions. As observed previously (King et al. 1994; Shen et al. 2000), the blood pressure drop observed after the ACh bolus infusion was ∼50% lower with l-NAME (average value of −22 mmHg) than in the CTRL (−48 mmHg), confirming the presence of significant vascular effects of the drug.

At the end of the experiments the dogs were killed with an overdose of pentobarbital. The gastrocnemius was excised and weighed, and the weight was utilized to normalize variables per unit of muscle mass, when appropriate.

Measurements

Output from the pressure transducer was recorded on a strip chart recorder while outputs from the load cell and flowmeter (T206, Transonic Systems) were fed through strain gauge and transducer couplers, respectively, into a computerized (PowerComputing PowerBase 240 Macintosh clone, Power Computing, Austin, TX, USA) data acquisition system (SuperScope II and instruNet Model 100B D/A input/output system, GW Instruments, Somerville, MA, USA). The load cell reaches 90% of full response within 1 ms while the flowmeter was set to a pulsatile cutoff frequency of 100 Hz; both signals were sampled at a rate of 100 Hz by the computerized data acquisition system. The load cell was calibrated with known weights prior to each experiment. The flowmeter was calibrated with a graduated cylinder and clock during and after each experiment. Vascular resistance was calculated as muscle perfusion pressure (BPm) divided by Q˙.

Samples of arterial blood entering the muscle and of venous blood from the popliteal vein were drawn anaerobically in heparinized syringes. As the arterial values varied only slightly throughout each experiment, arterial samples were taken at rest, before the contractions and immediately after the contraction periods. A polyethylene tube (internal diameter 0.8 mm, length 37 cm; total volume including luer hub 0.25 ml) was threaded into the popliteal vein cannula to the point where the vein exited the gastrocnemius. This allowed collection of venous blood immediately draining from the muscle. Venous samples were taken at rest (∼10 s before the onset of contractions), every 5–7 s during the first 75 s of contractions, and every 30–45 s thereafter until the end of the contraction period. The precise time of each venous sample was recorded.

Blood samples were immediately stored in ice and analysed within 30 min of collection. Both arterial and venous blood samples were analysed at 37°C for PO2, PCO2 and pH using a blood gas, pH analyser (IL 1304, Instrumentation Laboratories), and for haemoglobin concentration ([Hb]) and percentage saturation of Hb (SO2,%) with a CO-Oximeter (IL 282, Instrumentation Laboratories, Lexington, MA, USA), set for dog blood. These instruments were calibrated before and during each experiment. Dissolved O2 was also accounted for in the calculation of blood O2 concentration.

The V˙O2 of the gastrocnemius was calculated by Fick's principle as V˙O2 = Q˙ × Ca − vO2, where Ca − vO2 is the difference in O2 concentration between arterial blood (CaO2) and venous blood (CvO2). The V˙O2 was calculated at discrete time intervals corresponding to the timing of the blood samples.

Muscle biopsies were obtained by superficial excision of muscle pieces with a scalpel during both experimental conditions (CTRL and l-NAME): at rest, after 1 min of contractions and during the last 15 s of the contraction period. Biopsy samples were immediately frozen in liquid nitrogen. Subsequently, the samples were freeze-dried and dissected from visible connective tissue and blood. The freeze-dried samples (4–8 mg) were placed into tubes and extracted with 0.5 m HClO4 (containing 1 mm EDTA) and neutralized with 2.2 m KHCO3. This extract was used for determination of adenosine triphosphate (ATP), phosphocreatine (PCr), creatine (Cr) and lactate concentrations using enzymatic spectrophotometric assays (Beckman DU 640B) (Bergmeyer, 1974). Muscle metabolite concentrations were expressed in mmol per kg of dry mass (mmol (kg DM)−1). Free adenosine diphosphate concentration ([ADP]), expressed in μmol (kg DM)−1, was calculated as previously described (Dudley et al. 1987) by assuming equilibrium of the creatine kinase reaction using the measured ATP, PCr and creatine content, and cellular pH estimated from the muscle lactate content according to the regression equation of Sahlin et al. (1976). Substrate level phosphorylation (SLP), that is total ‘anaerobic’ ATP yield, during contractions was estimated (in mmol (kg DM)−1) as: Δ[PCr]+ (1.5 Δ[lactate]) + 2 Δ[ATP] (Greenhaff et al. 1994), in which Δ indicates the difference between concentrations at rest and at 4 min during contractions (for ATP and PCr) or between concentrations at 4 min and at rest (for lactate). In order to differentiate the role of SLP in the transition phase and at steady-state, the calculation presented above was also carried out twice: (i) from rest to 1 min of contractions, and (ii) from 1 to 4 min of contractions.

Kinetics analysis

The V˙O2 data were fitted by two equations. Equation (1) was of the form:

graphic file with name tjp0568-1021-m1.jpg (1)

In this equation, yBAS indicates the baseline value obtained at rest before contraction onset, Af indicates the amplitude between yBAS and the steady-state value at the end of the contraction period, TDf the time delay and τf the time constant of the function. The subscript f indicates that these parameters relate to the ‘fundamental’ component of the V˙O2 kinetics (Whipp et al. 2002).

Equation (2) was of the form:

graphic file with name tjp0568-1021-m2.jpg (2)

In this equation, As, TDs and τs indicate, respectively, the amplitude, the time delay and the time constant of the ‘slow’ component of the kinetics (Whipp et al. 2002). The equation that best fitted the experimental data was determined using the F-test (see below). That is to say, when eqn (2) provided a better fit of data, a slow component of the V˙O2 kinetics was present, superimposed on the fundamental component.

To facilitate a comparison with the results reported by previous studies (Grassi et al. 1996; 1998a, b; 2000, 2002), eqn (1) or eqn (2) was solved to calculate the time necessary to reach 50% (t50%, corresponding to the ‘half-time’ of the overall response) and 63% (t63%, corresponding to the ‘mean response time’ of the overall response) of the differences between the resting baseline and steady-state value obtained towards the end of contractions.

Statistical analysis

Values were expressed as means ±s.e.m. To determine the statistical significance of differences between two means, a paired Student's t test (2-tailed) was performed. To determine the statistical significance of differences among more than two means, a repeated-measures analysis of variance was performed. A Tukey's post hoc test was utilized to discriminate where significant differences occurred. Data fitting by exponential functions was performed using an iterative least-squares approach. Comparisons between fittings with different exponential models was carried out with an F-test. The level of significance was set at P < 0.05. Data fitting and statistical analyses were carried out by utilizing a commercially available software package (GraphPad Prism, GraphPad Software Inc., San Diego, CA, USA).

Results

The mass of the gastrocnemius muscles was 95 ± 8 g. Rectal temperature was not affected by l-NAME administration.

Resting values of the main variables pertinent to O2 transport and utilization, acid-base status and haemodynamics are shown in Table 1. No significant differences were observed between the two experimental conditions, with the exception of Ca − vO2, O2 extraction and V˙O2, which were significantly higher in l-NAME than in CTRL.

Table 1.

Resting values of the main variables pertinent to O2 transport and utilization, acid-base status and haemodynamics, in the CTRL and l-NAME conditions

CTRL l-NAME
[Hb]a (g (100 ml)−1) 14.0 ± 0.8 14.5 ± 1.1
PaO2 (Torr) 108 ± 5 117 ± 8
PvO2 (Torr) 85 ± 3 87 ± 13
PaCO2 (Torr) 35.2 ± 1.2 34.0 ± 2.0
pHa 7.405 ± 0.013 7.398 ± 0.012
SaO2 (%) 93.6 ± 0.5 93.8 ± 0.5
CaO2 (ml (100 ml)−1) 18.5 ± 1.0 19.3 ± 1.4
Q˙ (ml (100 g)−1 min−1) 108.0 ± 8.4 108.1 ± 8.4
Q˙ × CaO2 (ml (100 g)−1 min−1) 19.5 ± 0.7 20.4 ± 0.9
Ca−vO2 (ml (100 ml)−1) 0.4 ± 0.1 1.7 ± 0.2*
V˙O2 (ml (100 g)−1 min−1) 0.4 ± 0.1 1.8 ± 0.2*
O2 extraction (%) 2 ± 0 9 ± 1*
BPm (mmHg) 158 ± 16 190 ± 27
Vascular resistance (mmHg (100 g) min ml−1) 1.5 ± 0.2 1.8 ± 0.4
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Values are means ±s.e.m.; n= 6. [Hb]a, arterial blood haemoglobin concentration; PaO2, arterial blood PO2; PaCO2, arterial blood PCO2; pHa, arterial blood pH; SaO2, arterial blood percentage Hb saturation with O2; CaO2, arterial blood O2 content; Q˙, muscle blood flow; Q˙ × aO2, O2 delivery to muscle; Ca − vO2, arterio-venous O2 concentration difference; V˙O2, muscle O2 uptake; BPm, muscle perfusion pressure; Vascular resistance = BPm/Q˙. See text for further details. *P < 0.01 compared with CTRL.

Steady-state values during contractions for the same variables as those of Table 1 are shown in Table 2. No significant differences were observed between the two experimental conditions. Ca − vO2 and V˙O2 were slightly but not significantly higher in l-NAME than in CTRL.

Table 2.

Steady-state values during contractions of the main variables pertinent to O2 transport and utilization, acid-base status and haemodynamics, in the CTRL and l-NAME conditions

CTRL l-NAME
[Hb]a (g (100 ml)−1) 14.0 ± 0.8 14.5 ± 1.1
PaO2 (Torr) 108 ± 5 119 ± 8
PvO2 (Torr) 31 ± 2 31 ± 3
PaCO2 (Torr) 35.3 ± 1.2 34.0 ± 2.1
pHa 7.405 ± 0.013 7.395 ± 0.012
SaO2 (%) 93.6 ± 0.5 93.9 ± 0.5
CaO2 (ml (100 ml)−1) 18.9 ± 1.1 19.4 ± 1.5
Q˙ (ml (100 g)−1 min−1) 108.0 ± 8.4 108.1 ± 8.4
Q˙ × CaO2 (ml (100 g)−1 min−1) 20.0 ± 0.7 20.5 ± 0.9
Ca − vO2 (ml (100 ml)−1) 10.5 ± 1.4 11.4 ± 1.6
Q˙ (ml (100 g)−1 min−1) 10.8 ± 0.6 11.8 ± 0.8
O2 extraction (%) 55 ± 4 58 ± 4
BPm (mmHg) 131 ± 8 135 ± 9
Vascular resistance (mmHg (100 g) min ml−1) 1.3 ± 0.2 1.3 ± 0.2
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Values are means ±s.e.m.; n= 6.

Force production at the beginning of the contraction period (mean values calculated over five contractions, using kilopond units: 1 kp = 9.80665 N) was 41.0 ± 5.6 kp (100 g of muscle)−1 in CTRL and 41.1 ± 5.9 kp (100 g)−1 in l-NAME (no significant difference). After 30 s and 1 min of contractions force values were, respectively, 40.0 ± 5.5 and 39.1 ± 5.4 kp (100 g)−1 in CTRL, and 41.1 ± 5.9 and 41.0 ± 5.9 kp (100 g)−1 in l-NAME (both these l-NAME values were significantly higher than those in CTRL). Thus, during the metabolic transition, force production was not exactly ‘rectangular’ in CTRL, although, when expressed as a percentage of the initial force, force decreases at 30 s and at 1 min of contractions are minor (2% and 5%, respectively). Individual and mean values of the fatigue index (mean values of force calculated over five contractions at the end of each minute/initial force) are shown in Fig. 1. The fatigue index was higher (i.e. the muscles showed less fatigue) in l-NAME than in CTRL. A statistically significant difference between the values obtained in l-NAME and in CTRL was observed at all times after time zero.

Figure 1. The fatigue index.

Figure 1

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Individual (broken lines) and mean (±s.e.m.) values (symbols and solid lines) of the fatigue index (mean values of force calculated over 5 contractions at the end of each minute/initial force) in the CTRL andl-NAME conditions. *P < 0.05 compared with the corresponding CTRL value.

Muscle metabolite (ATP, free ADP, PCr, Cr, lactate) concentrations at rest and after 1 and 4 min of contractions are shown in Table 3. No significant differences were observed between the values obtained in CTRL and in l-NAME at any time or for any variable. In both conditions, [ATP] remained constant at rest and during contractions, whereas [PCr] decreased and free [ADP], [Cr] and [lactate] increased significantly from rest to contractions. Free [ADP] was slightly higher (no significant difference) in l-NAME than in CTRL at rest and after 1 min of exercise, whereas the opposite was true at 4 min of exercise. [PCr] decrease from rest to 4 min of exercise were slightly less (no significant difference) in l-NAME versus CTRL. Estimates of substrate level phosphorylation (SLP, expressed in mmol of ATP (kg DM)−1), calculated between rest and 4 min of exercise, were slightly but not significantly lower in l-NAME (19.0 ± 7.4 mmol (kg DM)−1) than in CTRL (49.3 ± 16.8 mmol (kg DM)−1). This tendency towards lower SLP in l-NAME could not be attributed to the transition phase: between rest and 1 min of exercise, indeed, SLP was 15.4 ± 2.9 mmol (kg DM)−1 in CTRL and 17.8 ± 4.4 mmol (kg DM)−1 in l-NAME, whereas between 1 and 4 min of exercise (that is, at steady-state) SLP was 33.9 ± 18.0 mmol (kg DM)−1 in CTRL and 1.1 ± 5.1 mmol (kg DM)−1 in l-NAME. Even this difference did not reach statistical difference (P= 0.12), due to variability among animals and to the fact that in one of the dogs SLP was actually slightly higher in l-NAME than in CTRL.

Table 3.

Muscle metabolites concentrations at rest and after 1 and 4 min of contractions in the CTRL and l-NAME conditions

CTRL l-NAME
Rest 1-min 4-min Rest 1-min 4-min
[ATP] 26.5 ± 1.0 26.9 ± 1.3 25.6 ± 1.1 26.0 ± 1.1 26.4 ± 1.3 26.3 ± 1.1
Free [ADP] 77.8 ± 2.6 109.3 ± 2.7 141.2 ± 14.9 85.8 ± 7.0 117.9 ± 10.3 123.2 ± 10.6
[PCr] 99.8 ± 3.0 87.9 ± 3.3 74.3 ± 6.9 95.7 ± 3.4 84.0 ± 2.8 82.4 ± 3.3
[Creatine] 44.8 ± 1.7 57.0 ± 2.0 70.2 ± 5.9 48.7 ± 2.5 60.4 ± 3.2 62.1 ± 4.6
[Lactate] 5.8 ± 1.3 8.7 ± 1.1 20.5 ± 7.5 7.7 ± 2.5 12.2 ± 2.7 11.9 ± 2.8
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Values are means ±s.e.m.; n = 6. Brackets denote concentrations. All data are expressed as mmol (kg DM)−1, with the exception of free [ADP], which is expressed as μmol (kg DM)−1. ATP, adenosine triphosphate; ADP, adenosine diphosphate; PCr, phosphocreatine. See text for further details.

The V˙O2 values for individual animals are presented in Fig. 2, together with the curves obtained by fitting eqn (1) or eqn (2). In two dogs (numbers 5 and 6) eqn (2) provided a better data-fit than eqn (1), both in CTRL and in l-NAME, indicating the presence of a slow component of V˙O2 kinetics. In all animals resting V˙O2 values were higher in l-NAME than in CTRL. In 4 animals out of 6 (exceptions being dogs 4 and 6) V˙O2 values were also higher in l-NAME than in CTRL at the end of the contraction period. The V˙O2 kinetics did not appear to be substantially different in the two experimental conditions in any of the dogs. In Fig. 3, V˙O2 (Fig. 3A) and ΔV˙O2 (V˙O2 increase with respect to rest Fig. 3B) values are shown V˙O2 in l-NAME was higher than in CTRL at rest, and tended to be higher throughout the transition and at the end of the contraction period, whereas the kinetics appeared to be very similar in the two conditions. On the other hand, ΔV˙O2 values were slightly lower in l-NAME than in CTRL, suggesting that the higher V˙O2 observed in l-NAME, in Fig. 3A, was mainly a consequence of an upward shift of the baseline values. Values of TDf, τf and mean response times (MRTf= TDf+τf) for the fundamental component of V˙O2 kinetics, obtained by fitting either eqn (1) or eqn (2) (see above), are presented in Fig. 4. The TDf was slightly higher in l-NAME than in CTRL, the difference being at the limit of statistical significance (P= 0.06). No difference between the two conditions was observed for τf, whereas MRTf was slightly but significantly higher in l-NAME. The Af was 9.8 ± 0.6 (mlO2 (100 g)−1 min−1) in CTRL and 9.6 ± 0.6 in l-NAME (no significant difference). TDs values were 95.1 and 80.0 s in CTRL (dogs 5 and 6, respectively), and 39.8 and 80.6 s in l-NAME. ‘As’ values (mlO2 (100 g)−1 min−1) were 1.6 and 1.4 in CTRL (dogs 5 and 6, respectively), and 2.9 and 1.3 in l-NAME. ‘As’ corresponded to ∼15–20% of the total amplitude of the V˙O2 response. Values of t50% were 14.7 ± 0.7 s in CTRL and 17.4 ± 0.8 s in l-NAME (P < 0.01); t63% were 18.0 ± 1.0 s in CTRL, and 21.0 ± 1.2 s in l-NAME (P < 0.05).

Figure 2. O2 uptake (V˙O2) kinetics in six individual animals.

Figure 2

Figure 2

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O2 uptake (V˙O2) values for individual animals, together with the curves obtained by fitting eqn (1) or eqn (2) (see Methods), in the CTRL and l-NAME conditions. In two dogs (i.e. numbers 5 and 6) eqn (2) provided a better fit of data compared to eqn (1), in both CTRL and l-NAME conditions, indicating the presence of a slow component of V˙O2 kinetics. Vertical broken lines indicate the onset of contractions at time zero.

Figure 3. V˙O2 and Δ V˙O2 at rest, and during 4-min of contractions.

Figure 3

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Mean (±s.e.m.) V˙O2 (panel A) and ΔV˙O2 (V˙O2 increases with respect to rest, panel B) values in the CTRL and l-NAME conditions.

Figure 4. Time delay, time constant, and mean response times for the ‘fundamental’ component of V˙O2 kinetics.

Figure 4

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Mean (±s.e.m.) values of time delay (TDf), time constant (τf) and mean response times (MRTf= TDf+τf) for the ‘fundamental’ component of V˙O2 kinetics, obtained by fitting either eqn (1) or eqn (2) (see Methods) in the CTRL and l-NAME conditions. *P < 0.05 compared with corresponding CTRL.

Discussion

The main finding of the present study was that, contrary to our hypothesis, NOS inhibition by l-NAME did not elicit faster V˙O2 kinetics in the isolated dog gastrocnemius in situ. Thus, according to our results, inhibition of cytochrome c oxidase (and possibly of other enzymes) by NO does not appear to limit the speed of adjustment of oxidative metabolism at exercise onset, or, at least, inhibition of cytochrome c oxidase by NO is not an essential constituent of this relatively slow response. A complex and vital system such as oxidative metabolism is in fact probably characterized by a redundancy of regulatory mechanisms, and it cannot be excluded that a regulatory role by one factor, when this factor is eliminated, can be taken up by others.

In our experimental model, muscle blood flow and convective O2 delivery to muscles were kept constantly elevated, at rest and during the transition, by pump-perfusing the muscle at high blood flows and by the administration of a vasodilatory drug. These interventions counteracted any possible reduction of muscle blood flow deriving from NOS inhibition. Such reduction was described by previous authors both in the isolated dog gastrocnemius in situ (King-VanVlack et al. 2002) and in running dogs (Shen et al. 2000), as well as in other experimental animal models (Hirai et al. 1994). The results obtained in exercising humans appear more contradictory (Joyner & Dietz, 1997; Frandsen et al. 2001; Boushel et al. 2002). Our main postulate was that elimination of a possible reduction in convective O2 delivery, obtained by pump-perfusing the muscle at high blood flows and by the vasodilatory drug, would allow any effect of NOS inhibition on mitochondrial respiration to become fully manifest, and therefore more pronounced (in terms of a faster V˙O2 kinetics) than those observed by previous authors in horses (Kindig et al. 2001, 2002) and in humans (Jones et al. 2003, 2004; Wilkerson et al. 2004).

Effects on parameters of V˙O2 kinetics

Unexpectedly, we did not observe any speeding of the V˙O2 kinetics. If anything, some of the kinetics parameters we determined (TDf, MRTf, t50%, and t63%, see Methods) suggest slightly slower V˙O2 kinetics with l-NAME. The main difference was actually related to the slightly longer TDf values observed in l-NAME. It appears as if the higher resting V˙O2 in l-NAME, in the presence of a substantially similar requirement for oxidative phosphorylation in the two conditions, somehow slightly delayed the further V˙O2 increase due to contractions onset. Once it occurred, however, this further V˙O2 increase showed the same kinetics in the two experimental conditions, as demonstrated by the almost identical τf values. In any case, all parameters which necessarily incorporate TDf (i.e. MRTf, t50%, and t63%) were slightly higher (indicating slower kinetics) in l-NAME.

Two dogs showed a slow component of V˙O2 kinetics in both experimental conditions. This small number does not of course allow any statistical comparison.

Comparisons with previous studies

In the present study NOS inhibition by l-NAME did not speed muscle V˙O2 kinetics. How can this unexpected finding be explained in view of the faster pulmonary V˙O2 kinetics observed by previous authors in horses (Kindig et al. 2001, 2002) and in humans (Jones et al. 2003, 2004; Wilkerson et al. 2004)?

The first aspect to consider is that the variables we determined concerned muscle V˙O2 kinetics, whereas those determined by the previously mentioned authors (Kindig et al. 2001, 2002; Jones et al. 2003, 2004; Wilkerson et al. 2004) concerned pulmonary V˙O2 kinetics. Even though the ‘fundamental component’ (or phase 2) of pulmonary V˙O2 kinetics is usually considered to reflect rather closely that of muscle V˙O2 (Whipp et al. 2002; Barstow et al. 1990; Grassi et al. 1996), the issue of interposition (and possible changes) of O2 stores between the sites of gas exchange at the skeletal muscle level and those at the lungs is real, and it could have influenced the results of the studies mentioned above, particularly in the presence of relatively small differences in V˙O2 kinetics between experimental conditions. As Jones et al. (2003) stated in the discussion concerning their finding of a faster pulmonary V˙O2 kinetics after l-NAME in humans: ‘Further studies involving direct measures of muscle blood flow and O2 extraction across a working muscle are required to confirm this’. Hypothetically, NOS inhibition could cause some vasoconstriction in venular vascular beds during the transition, and could ‘squeeze’ some venous blood relatively poor in O2 towards the heart. This would elicit, even in the presence of unchanged muscle V˙O2 kinetics, faster pulmonary V˙O2 kinetics, independently from the effects of NOS inhibition on mitochondrial respiration. Moreover, the fundamental component of pulmonary V˙O2 kinetics can be influenced by the duration of the ‘cardiodynamic component’ (Whipp et al. 2002), so that different phases of pulmonary V˙O2 kinetics cannot be considered as absolutely separate entities. As elegantly demonstrated in the modelling study by Barstow et al. (1990), when slower cardiac output kinetics are paired with unchanged muscle V˙O2 kinetics, the results are a lengthening of the ‘cardiodynamic’ phase (phase 1, see Whipp et al. 2002) and a faster phase 2 of pulmonary V˙O2 kinetics. Thus, a restricted muscle O2 delivery could determine faster pulmonary V˙O2 kinetics, even in the presence of unchanged muscle V˙O2 kinetics. A slightly longer cardiodynamic phase was described after l-NAME administration in the studies by Kindig et al. (2001), Jones et al. (2003), and Wilkerson et al. (2004), and it could well be explained by a reduced cardiac output or muscle blood flow adjustment attributable to the drug (e.g. see Kindig et al. 2000). A longer cardiodynamic phase, then, could explain, at least in part, the slightly faster fundamental component of pulmonary V˙O2 kinetics described in those studies, irrespective of effects of the drug on muscle V˙O2 kinetics. As mentioned above, these confounding factors could play a significant role in the presence of relatively small actual differences of muscle V˙O2 kinetics among conditions.

As for the two studies conducted on horses (Kindig et al. 2001, 2002), it should be noted that these animals' locomotory muscles, although characterized by a high oxidative capacity, are composed of 80–90% fast-twitch fibres (see, e.g. the studies mentioned by Kindig et al. 2002), whereas the dog gastrocnemius used in the present study is characterized by a high percentage of oxidative fibres (Maxwell et al. 1977). Although we are unaware of studies demonstrating a fibre-type specificity of the metabolic effects of NOS inhibition, such specificity appears conceivable. Different effects of NOS inhibition in different fibre types could explain, at least in part, the different results obtained by the present study compared to those by Kindig et al. (2001, 2002). Also in the study by Kindig et al. (2001) the effects of l-NAME on the fundamental component of the pulmonary V˙O2 kinetics were rather small (see, e.g. the top panel of their Fig. 1), although the effects on τf were statistically significant. In that study, moreover, the exercise protocol was a moderate-to-heavy exercise transition (from ∼50% to ∼80% of the animals' V˙O2peak), and the different type of transition could make comparisons with the present as well as with other studies more difficult.

Metabolic effects of NOS inhibition

Even though, in the present study, NOS inhibition by l-NAME did not speed up muscle V˙O2 kinetics, it did exert significant metabolic effects on other variables. By putatively relieving the down-regulation of mitochondrial function due to NO, NOS inhibition enhanced O2 extraction and V˙O2 at rest. NOS inhibition also caused a tendency towards higher V˙O2 values throughout the contraction period, even though net V˙O2 increase compared to rest was slightly lower in l-NAME than in CTRL. Considered to be one of the main controllers of oxidative phosphorylation, free [ADP] was slightly (although not significantly) higher in l-NAME than in CTRL at rest. This could explain, at least in part, the higher resting V˙O2 values obtained in l-NAME. On the other hand, at 4 min of exercise the free [ADP] was slightly lower in l-NAME than in CTRL. This could explain, at least in part, the lower ΔV˙O2 observed in l-NAME during contractions. In l-NAME we also observed less muscle fatigue, and trends toward less PCr hydrolysis and a lower contribution of substrate level phosphorylation. The significant increase in O2 extraction and V˙O2 at rest, and the trend towards higher O2 extraction and V˙O2 during contractions after NOS inhibition appear to be in agreement with several previous studies (King et al. 1994; O'Leary et al. 1994; Shen et al. 1995, 2000; King-Vanvlack et al. 2002). In those studies NOS inhibition decreased blood flow and increased O2 extraction, with the result being either an unchanged or an increased V˙O2. In our study NOS inhibition was accompanied by an experimentally elevated muscle blood flow, so that the increased muscle O2 extraction resulted in a higher V˙O2. Higher V˙O2 after the administration of the NOS inhibitor Nω-monomethyl-l-arginine was also demonstrated in healthy human volunteers by Gilligan et al. (1994) during handgrip exercise, and by Endo et al. (1994) during static exercise.

The reduced muscle fatigue described in the present study appears in accordance with literature data. It has been reported that l-NAME may increase force production during twitch contractions, demonstrating that NO (or related molecules) can attenuate force production of limb muscle in situ (King-VanVlack et al. 1995). The presence of an nNOS isoform with force-inhibitory activity has been confirmed in a variety of muscle preparations in animals (Stamler & Meissner, 2001). Previous work has also demonstrated the ability of endogenous and exogenous NO to accelerate the time-dependent decline in force production (fatigue) over 4 h, in both diaphragm and gastrocnemius muscle (Gath et al. 1996). Reactive nitrogen and oxygen species are increased in exercising and fatiguing muscle, and NOS inhibition protects against a decline in function over time (Reid, 1996). In the present study NOS inhibition did not increase isometric force production at the beginning of the contraction period, but significantly counteracted the decline in force during the 4 min of contractions.

The question could be asked whether the reduced fatigue (i.e. the less pronounced decrease in force production) after NOS inhibition is accompanied by increased energy utilization, or if changes in efficiency of muscle contraction should be hypothesized. An overall evaluation of energy expenditure in the two experimental conditions is needed to answer this question. Mean ±s.e.m. ΔV˙O2 values (i.e. net V˙O2 increases with respect to the resting baseline) at the end of the contraction period were 10.5 ± 0.5 ml (100 g)−1 min−1 in CTRL and 9.9 ± 0.7 ml (100 g)−1 min−1 in l-NAME. Estimates of substrate level phosphorylation (SLP) (see Methods) between rest and the fourth minute of exercise were 49.3 ± 16.8 mmol of ATP (kg DM)−1 in CTRL and 19.0 ± 7.4 mmol of ATP (kg DM)−1 in l-NAME. Assuming a P: O ratio of 6, and the equivalence of 22.4 ml O2 for each mmol of O2, it can be calculated that, once expressed in ml O2 (100 g ‘wet’ muscle)−1 min−1, SLP was 1.15 ml O2 (100 g)−1 min−1 in CTRL and 0.44 O2 (100 g)−1 min−1 in l-NAME. These values correspond, respectively, to ∼11% and ∼4% of the ‘aerobic’ energy yield (ΔV˙O2 at steady-state) in the two conditions. If we add SLP to ΔV˙O2, and divide the obtained sum by force production (expressed in kp (100 g)−1), we obtain values of energy expenditure per unit of force production, which can be taken as an index of efficiency of muscle contraction. These values corresponded to 0.43 ± 0.13 ml O2 min−1 kp−1 in CTRL and to 0.35 ± 0.10 ml O2 min−1 kp−1 in l-NAME (P= 0.03); the lower value in l-NAME suggests an increased efficiency. That is to say, the less pronounced decrease in force production (i.e. the higher overall force production) in l-NAME seems to be associated with an increased efficiency of muscle contraction. Such increased efficiency could be either mechanical or biochemical in nature. However, it should be noted that if instead of ΔV˙O2, we utilize V˙O2 values for the calculation, the difference in efficiency does not reach statistical significance (energy expenditure per unit of force production: 0.44 ± 0.13 ml O2 min−1 kp−1 in CTRL, versus 0.41 ± 0.12 ml O2 min−1 kp−1 in l-NAME; P= 0.09).

Methodological considerations

As discussed above, even though in the present study we did not directly measure NOS activity, it has been shown that the l-NAME dosage we utilized produces effective NOS inhibition for at least 1 h (King et al. 1994). Moreover, the presence of significant vascular effects by the drug was demonstrated in some of the dogs in the present study by the ACh bolus infusion test (King et al. 1994; Shen et al. 2000) (see Methods).

It must be recognized that the experimental model we utilized presents some disadvantages compared to other more ‘physiological’ models, like running horses or cycling humans. These disadvantages have been discussed at length in previous papers by our group (Grassi et al. 1998a, b; 2000, 2002), and mainly refer to the intrinsic invasiveness of the preparation and to the unphysiological stimulation pattern (synchronous tetanic contractions). The ‘contraction protocol’, moreover, is not precisely a square-wave, as the muscles fatigued to different levels in the two experimental conditions). However, analysis of V˙O2 kinetics mainly refers to the first minute of the contraction periods, during which the effects of fatigue are limited.

Conclusions

NOS inhibition by l-NAME did not speed V˙O2 kinetics in the isolated dog gastrocnemius in situ, where possible negative effects of l-NAME on convective O2 delivery were prevented by pump-perfusing the muscle with high blood flows throughout the transition from rest to contractions. Several factors (see above) could explain, at least in part, the conflicting results compared to those of previous studies conducted on pulmonary V˙O2 kinetics in horses (Kindig et al. 2001, 2002) and in humans (Jones et al. 2003, 2004; Wilkerson et al. 2004). Although further studies are needed to clarify the issue, the present study suggests that the inhibition of mitochondrial respiration by NO does not limit the kinetics of adjustment of oxidative metabolism at exercise onset.

Acknowledgments

The authors are grateful to Dr Jerzy Zoladz and Dr Bernard Korzeniewski for constructive criticism. Technical assistance by Dr Robin E. Pattillo is acknowledged. Financial support by NIH AR40155, by NATO Collaborative Linkage Grant LST.CLG 979220 and by institutional funds (FIRST) from the University of Milano is also acknowledged.

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