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1 Faculty of Medicine2 Faculty of Kinesiology, University of Calgary, Calgary, Alberta, Canada 3 Faculty of Medicine, University of California San Diego, CA, USA
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Abstract |
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-nitro-L-arginine methyl ester (L-NAME; 1 mM). Adenosine is a vasodilator that can act through both NO-dependent and -independent pathways; the NO-independent vasodilatory action of adenosine allowed us to match the perfusion rate and convective O2 delivery in this L-NAME group to those of the other groups. In the second group the perfusate was treated with adenosine only (Ado). In the third group the perfusate received no treatment and served as a control (Con). Oxygen consumption (
) was on average 26 and 14% lower during the contraction bout in L-NAME and Ado, respectively, versus Con. In Ado, lactate efflux was similar to Con and force was reduced in proportion to 
versus Con, whereas L-NAME was associated with a 32% lower lactate efflux and similar force to Con. Therefore, the lower 
:force development ratio in the L-NAME group demonstrates that the O2 cost of force development is reduced by NOS inhibition independent of convective O2 delivery.
(Received 26 July 2005;
accepted after revision 23 August 2005; first published online 25 August 2005)
Corresponding author R. T. Hepple: Faculty of Kinesiology, University of Calgary, 2500 University Dr. NW, Calgary, Alberta, Canada T2N 1 N4. Email: hepple{at}ucalgary.ca
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Introduction |
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Numerous studies have been conducted using inhibition of nitric oxide synthase (NOS) to ascertain the effects of NO on skeletal muscle aerobic metabolism. Past data, however, have been difficult to interpret and compare owing to differences in experimental models used. Specifically, studies investigating the effects of NOS inhibition on skeletal muscle function have been conducted in ‘whole body’ protocols (Endo et al. 1994; Kindig et al. 2001, 2002), and self-perfused isolated muscle preparations (Ameredes & Provenzano, 1999; King-VanVlack et al. 2002; Hillig et al. 2003). As O2 consumption (
) is determined by an interaction between numerous variables, including blood flow, blood O2 carrying capacity, diffusion of O2 from blood to tissues, ATP demands and O2 utilization by mitochondria, it is clear that the net result of altering NO production on 
depends upon the balance between frequently opposing effects at different levels in this sequence of events. As a result, in ‘whole body’ and in many in situ experimental protocols, the identification of effects that are specific to skeletal muscle is confounded by the aforementioned multiple sites of action for NO. As a case in point, reductions of skeletal muscle blood flow and, subsequently, convective oxygen delivery that are often associated with the impaired vasodilatory action observed with NOS inhibitors, such as N-nitro-L-arginine methyl ester (L-NAME), confound interpretation of previous results regarding the specific role of NO within the contracting muscles (e.g. Shen et al. 2000; Frandsenn et al. 2001; King-VanVlack et al. 2002). At the mitochondrial level, however, it is well known that NO binds to the cytochrome oxidase complex in the electron transport chain. This binding of NO to cytochrome oxidase reversibly inhibits mitochondrial respiration in skeletal muscle (Brown, 1995; Giulivi, 1998). Therefore, a reduction in this inhibition should, theoretically, augment aerobic function. In support of this hypothesis, a number of studies have shown faster onset of aerobic metabolism in the transition to steady-state exercise (Kindig et al. 2001, 2002; Jones et al. 2003). Alternatively, two recent studies report a lower 
for a given contractile demand with NOS inhibition (Hillig et al. 2003; King-VanVlack et al. 2002), and it remains unclear whether this lower 
represents a response to reduced O2 delivery or an effect of NOS inhibition within the contracting myocytes per se. Therefore, to help clarify the role of NOS inhibition on aerobic metabolic function in contracting skeletal muscles, the present study used a pump-perfused rat hindlimb preparation that allowed for matched rates of skeletal muscle convective oxygen delivery, with and without NOS inhibition, to test the hypothesis that NOS inhibition would improve skeletal muscle aerobic metabolic performance.
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Methods |
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All experiments were conducted with approval from the University of Calgary Animal Care Committee. Adult male Sprague–Dawley rats obtained from our in-house colony were maintained at the University of Calgary's Heritage Medical Research Building's animal resource centre vivarium under a 12 h:12 h light:dark cycle at 22°C and fed Purina rat chow and water ad libitum. An in situ single perfused rat hindlimb preparation (Gorski et al. 1986; Hepple et al. 2002) was used to permit control of muscle blood flow during experiments. After anaesthetizing the animal with 70–75 mg kg–1 sodium pentobarbitone (I.P.), the left hindlimb was prepared by removing the skin and isolating the sciatic nerve. The sciatic nerve was then ligated and cut proximally for placement of a platinum nerve hook electrode connected to an electrical stimulator (Grass S48). As previously, the gluteal nerve was severed to avoid stimulation of the upper hindlimb musculature (Gorski et al. 1986; Hepple et al. 2002). Following preparation of the sciatic nerve, the Achilles tendon was severed with a portion of the calcaneus intact and the gastrocnemius–plantaris–soleus muscle group was secured with 1/0 (metric 6.0) non-compliant silk thread in preparation for attachment to a force transducer (FT-10, Grass Instruments). The contralateral iliac artery and vein were ligated and the right gastrocnemius–plantaris–soleus muscle group was excised, trimmed of fat, and weighed. Following surgery, the left hindlimb was secured to an aluminium base-plate by a bone clamp placed around the proximal femur and a cable tie placed around the ankle, to minimize movement artefact during muscle contractions. Catheters (22 gauge in the artery and 20 gauge in the vein) were then inserted into the left iliac artery and vein and advanced into the femoral artery and vein, respectively, to initiate flow to and from the hindlimb. The animal was killed with an intracardiac injection of 25 mg sodium pentobarbitone. All exposed tissues of the experimental hindlimb were wrapped in warm saline-soaked gauze, Saran wrap and aluminium foil to avoid moisture and heat loss throughout the experiment. Muscle and perfusate temperatures were maintained at 37°C.
Perfusion medium
Bovine whole blood was collected weekly from a local abattoir. The erythrocytes were ‘washed’ in three changes of Krebs–Henseleit buffer by centrifugation at 5000 g(10 mins each wash), with aspiration of the supernatant and buffy coat between washes. The washed erythrocytes were then stored at 4°C in Krebs–Henseleit buffer containing 5 mM glucose and used within 3 days of collection. The standard perfusion medium for all three groups consisted of a Krebs–Henseleit bicarbonate buffer (pH 7.4) containing 4.5% bovine serum albumin (Sigma; dialysed 48 h), bovine erythrocytes (haematocrit 43%, verified by direct observation in centrifuged capillary tubes), 5 mM glucose, 100 mU ml–1 insulin, 1000 mU ml–1 heparin and 0.15 mM pyruvate. During experiments, the erythrocyte-containing perfusion medium (
400 ml) was recirculated after discarding the first 50 ml of venous effluent.
Experimental protocol
Animals were divided into three groups. In one group, N–nitro-L-arginine methyl ester (L-NAME; 1 mM, Sigma) and adenosine (0.01 mM, Sigma Chemicals) were added to the perfusate (L-NAME group; n
= 13). L-NAME is a well-known NOS inhibitor (e.g. King-VanVlack et al. 1995; Ameredes & Provenzano, 1999; Kindig et al. 2001) and adenosine is a potent vasodilator that acts through both NO-dependent and -independent pathways (Wei & Kontos, 1990; Ishizaka et al. 1991; Li et al. 1995). Note that the dosage of L-NAME was estimated from previous studies showing that 20–25 mg (kg body mass)–1 provides effective NOS inhibition in vivo (Kindig et al. 2001; King-VanVlack et al. 2002), and was validated in our lab (see Determination of NOS inhibition). Since the in situ pump-perfused model relies on NO-dependent, flow-induced vasodilatation (vascular conductance is markedly compromised with L-NAME alone in this model, Fig. 1), Adenosine was added to the L-NAME group to permit vasodilatation in the absence of NO, and thus make it possible to achieve similar perfusion rates and muscle convective oxygen delivery among groups. A second group was treated with adenosine only (0.01 mM; n
= 14; Ado group) to ascertain whether adenosine alone was responsible for any effects observed in the L-NAME group. A third group (n
= 6), receiving no interventions, served as a control (Con group). It should be noted that control animals are the same set as described in a previous study (F60 group) (Hepple et al. 2003).
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15% of total hindlimb flow was perfusing the gastrocnemius–plantaris–soleus muscle group; Hepple et al. 2003).
Muscle length and stimulation voltage (7 V) were adjusted to yield maximal tension, and tetanic muscle contractions (200 ms trains at 100 pulses s–1, each 0.2 ms in duration) at a frequency of 60 tetani min–1 were evoked by electrical stimulation for 4 min. Blood samples were drawn anaerobically from the arterial catheter prior to contractions and from venous effluent every 30 s for 4 min during muscle contractions and analysed for [haemoglobin] ([Hb]), O2 saturation (SO2), PO2, and [lactate] by a blood gas analyser (Nova Biomedical Stat Profile M). Arterial and venous O2 content was determined using the formula: [O2]
= 1.39[Hb]SO2
+ 0.003PO2.
of the hindlimb was calculated by multiplying the arteriovenous oxygen content difference by the hindlimb perfusate flow. A number of normalization calculations were performed to obtain muscle mass-specific 
for this preparation. Previous microsphere experiments showed that all of the hindlimb muscles, with the exception of the gluteals, were perfused in this preparation (R. T. Hepple, unpublished results; Gorski et al. 1986). As performed previously (Hepple et al. 2003), 
was normalized to the mass of the contracting muscles (gastrocnemius, plantaris, soleus, tibialis anterior and remaining deep tibial muscles) after subtracting the 
contributed by the non-contracting muscles (assumed to be 71% of resting 
). Lactate efflux was determined from the product of perfusate flow and the arteriovenous [lactate] difference. The result was then normalized to contracting muscle mass as for 
. Perfusate samples were also drawn for 4 min following contractions in a subset of animals in each group (n
= 6 in Con, n
= 6 in Ado and n
= 5 in L-NAME) to monitor lactate efflux during recovery. By measuring the tubing volume between the hindlimb vasculature and venous sampling port, combined with pump speed, we were able to calculate the time delay in erythrocyte movement from the hindlimb vasculature to the venous sampling port (Hepple et al. 2003). This allowed for retrospective time alignment of effluent O2 content with its corresponding force development. Time-integrated force development was calculated for each 30 s period as the product of tension, the frequency of contractions (contractions per second) and tetanic duration to yield units of newtons per second.
Determination of NOS inhibition
To confirm that NOS was being inhibited in our experiments, one animal received 1 mM L-NAME only, while another received only 1 mM
N-nitro-D-arginine methyl ester (D-NAME), the inactive enantiomer of L-NAME. Perfusion rate was held constant at 5 ml min–1 and vascular conductance (the quotient of perfusate flow and net perfusion pressure) was monitored for 15 min.
Perfusate flow
Muscle perfusate flow distribution was determined for a subset of animals from each group (n = 5 in Con, n = 4 in Ado and n = 4 in L-NAME) using coloured microspheres (Hepple et al. 2002). Briefly, 1 ml (approximately 290 000 microspheres) of a known concentration of microspheres (15.5 µm diameter, Dye Trak, Triton Technology, San Diego, CA) were slowly infused into a side-port in the tubing proximal to the arterial catheter. A 6 ml syringe of saline was infused following the microspheres to ensure that all spheres reached the hindlimb vasculature. During microsphere and saline infusions, perfusate flow was reduced on the pump proportionally to the rate of infusion to minimize perfusion pressure fluctuations. The gastrocnemius–plantaris–soleus muscle group was then excised from the rat and each muscle was individually separated, trimmed of fat, weighed, and heated in a 60°C water bath in centrifuge tubes containing 4 M KOH until each muscle was fully digested. Once digested, the contents of each tube were filtered through 8 µm membranes (Whatman Nucleopore) to isolate the spheres. These membranes were then placed into microcentrifuge tubes containing 1 ml of N,N-dimethyl formamide to dissolve the microsphere shells and release their colour. The absorbance of the contents was then measured after 10 min using a spectrophotometer (Biochrom Ultrospec 2100 Pro) at a wavelength of 448 nm (yellow microspheres) and 594 nm (violet microspheres), and the number of microspheres trapped in each muscle sample was calculated from the regression equations provided by the manufacturer. As done previously (Hepple et al. 2002), the perfusate flow to the gastrocnemius–plantaris–soleus muscle group was considered representative of the total contracting muscle mass (comprising the gastrocnemius–plantaris–soleus muscle group, tibialis anterior muscle and remaining tibial muscles (Gorski et al. 1986). Thus, mass-specific skeletal muscle convective O2 delivery to the contracting muscles was estimated as the product of arterial O2 content and the mass-specific perfusate flow for the gastrocnemius–plantaris–soleus muscle group (Hepple et al. 2002).
Data analysis
Values are presented as means ± S.E.M. To test for differences in muscle perfusate flow between groups, a two-way ANOVA (muscle x treatment) and a Holm-Sidak post hoc test was used. A two-way ANOVA with a Holm-Sidak post hoc test was also used for any variable studied as a function of time (treatment x time). Differences between the remaining variables were assessed with a one-way AVOVA with a Holm-Sidak post hoc test. The Holm-Sidak multiple comparison test applies a ‘step-down’ critical P value approach in determining significance to maximize statistical power without compromising the risk of making a type I error. As such, critical P values are determined by the number of multiple comparisons (more comparisons mean that a higher critical P value is required for significance) and are stepped down for each subsequent multiple comparison test after the uncorrected P values are ordered from smallest to largest (Glantz, 2002).
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Results |
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Descriptive data for the animals are found in Table 1. There were no significant differences in body mass, mass of the gastrocnemius–plantaris–soleus muscle group or total contracting muscle mass between groups. The perfusion pressure and perfusate flows (Table 2) in the control group were similar to those seen previously in our laboratory with this preparation (Hepple et al. 2002). Owing to the vasoconstrictive effects of hyperoxia on vascular smooth muscle, perfusion pressures may have been slightly elevated from those expected at more modest oxygen deliveries. Note also that vascular conductance (the quotient of perfusate flow and net perfusion pressure) was not different between groups (data not shown). There were no between group differences in the distribution of perfusate flow to the gastrocnemius–plantaris–soleus muscle group as a whole or as individual muscles (Fig. 2). Although microsphere data were not collected on the remaining force-producing muscles (35% total contracting mass), the assumption was made that, like the gastrocnemius, plantaris and soleus, there would be no between-group differences. On average, the gastrocnemius–plantaris–soleus muscle group received 15.6 ± 0.3% of the total hindlimb perfusate flow, and was not different between groups. Mass-specific skeletal muscle convective O2 delivery was effectively matched between groups with averages of 513 ± 24 µmol O2 min–1 (100 g)–1 for Con, 546 ± 18 µmol O2 min–1 (100 g)–1 for Ado and 490 ± 22 µmol O2 min–1 (100 g)–1 for the L-NAME group. Noteworthy is the fact that when vascular conductance was plotted over several minutes in an animal receiving 1 mM L-NAME alone (no adenosine) at a flow of 5 ml min–1, no steady state was reached, with conductance falling steadily until termination of the experiment owing to extremely high perfusion pressures (Fig. 1). In contrast, an animal receiving only 1 mM D-NAME did reach a plateau under the same perfusion conditions (Fig. 1). Since control experiments show a relatively constant vascular conductance despite increases in flow to 
10 ml min–1 (R. T. Hepple, personal observation), these results are consistent with effective NOS inhibition in our experiments.
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Resting 
was not different between groups (Table 2). The initial tension produced was similar in Con (12.6 ± 0.7 N g–1), Ado (11.6 ± 0.9 N g–1) and L-NAME groups (13.4 ± 0.9 N g–1). Similarly, no difference was observed in the fatigue responses when the decline in tension throughout the contraction bout was considered between Con (68 ± 1% reduction from peak), Ado (70 ± 1%), and L-NAME groups (69 ± 2%; Fig. 3). However, it is important to note that there was a significant main effect difference, indicating lower force production throughout the 4 min contraction bout, in the Ado group (mean force production throughout the 4 min contraction bout 4.6 ± 0.1 N g–1) compared to both the Con (5.2 ± 0.2 N g–1) and L-NAME groups (5.4 ± 0.1 N g–1). 
during contractions was lower in both the L-NAME and Ado groups versus Con; this effect was larger in the L-NAME group (Fig. 4).
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and lactate efflux were very similar in terms of the differences observed between groups (see insets in Figs 3, 4 and 5). This was also true of the lactate efflux responses during muscle contractions for the subset of animals that were followed for 4 min into recovery (Fig. 5, inset A).
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Using the quotient of 
and time-integrated force development normalized to the mass of the gastrocnemius–plantaris–soleus muscle group as an index of muscle contractile economy (Fig. 6), it is clear that although 
and force were reduced in Ado versus Con, these effects were proportional (i.e. 
/time-integrated force development was not different from Con). In contrast, the 
/time-integrated tension was on average 26% lower in the L-NAME group, indicating a reduced O2 cost of force development in this group.
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Discussion |
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and tension development and no difference in lactate efflux during a 4 min contraction bout versus control conditions. In contrast, muscles treated with L-NAME (to inhibit NOS) and adenosine (to permit vasodilatation in the absence of NO in our model) in combination (L-NAME group) had similar force production coupled with lower 
and lower lactate efflux during contractions versus Con. Thus, L-NAME treatment yielded a lower 
/time-integrated force development throughout the contraction bout, indicating a reduced O2 cost of force development with NOS inhibition. Pump-perfused hindlimb preparation
To put our results in context with other studies using similar preparations, peak tension development from the gastrocnemius–plantaris–soleus muscle group (13 N g–1) was similar to an earlier study (13–15 N g–1) using similar stimulation parameters (Robinson et al. 1994). Perfusate flow data in the present study (160 ml min–1 (100 g)–1 for the soleus, 95 ml min–1 (100 g)–1 for the plantaris and 51 ml min–1 (100 g)–1 for the gastrocnemius muscles in control conditions) are consistent with previous studies using the pump-perfused rat hindlimb preparation (160–190, 50–100 and 50–80 ml min–1 (100 g)–1 for the soleus, plantaris and gastrocnemius muscles, respectively; Gorski et al. 1986; McAllister & Terjung, 1990). Noteworthy is the fact that a previous study using the same strain of rat demonstrated that blood flows for these specific muscles can reach significantly higher values during exercise in vivo (225, 225 and 160 ml min–1 (100 g)–1 for the soleus, plantaris and gastrocnemius muscles, respectively; Laughlin & Armstrong, 1982). Control values for peak 
under these contraction conditions in the present study (23 ± 1 µmol min–1) are also consistent with control values from other studies using this model under similar stimulation conditions (20–25 µmol min–1 (Hood et al. 1986; Hepple et al. 2002, 2003). Note that the strength of using this model for the present investigation was the ability to achieve similar rates of muscle mass-specific perfusate flow and convective O2 delivery between groups. Note also that data from the microsphere infusion studies indicate that neither L-NAME nor adenosine alters the perfusate flow distribution among individual muscles of the gastrocnemius–plantaris–soleus muscle group from control conditions in this model. Thus, the differences in 
, force and lactate efflux observed between groups are not confounded by differences in muscle perfusate flow, distribution or convective O2 delivery. It should be noted, however, that the present data do not rule out redistribution of perfusate flow within the microvasculature of individual muscles. Another significant observation was that vascular conductance did not reach steady state with L-NAME alone in the perfusate but did so with D-NAME alone (Fig. 1). Myogenic vasoconstriction in response to elevated transmural pressure will lead to further increases in blood pressure by increasing peripheral resistance unless opposed by other mechanisms (de Wit et al. 1997). Shear stress-induced augmentation of endothelial NO release provides one such mechanism (Juncos et al. 1995). Therefore, the inability of the vasculature to overcome this myogenic response with L-NAME alone demonstrates that endothelial NO release was inhibited.
Effects of NO on contractile function
Although NO depresses submaximal force development (Kobzik et al. 1994; Reid et al. 1998; Reid, 1998), past studies have shown that NO has no effect on maximal force production (Kobzik et al. 1994; Morrison et al. 1996; Albertini et al. 1997). In this regard, a previous study in rat diaphragm skeletal muscle suggested that the effects of NO on tension were most evident at 40 Hz and began to converge with control values once the stimulation frequency increased beyond 100 Hz (Reid et al. 1998). Since the frequency of pulses in the tetanic trains used in the present study was 100 Hz, any effect of NOS inhibition on force would be expected to be small. This is consistent with what was seen in the L-NAME group, since there was no difference in either initial force or the rate of decline in force versus Con. In contrast, the lower force development observed throughout contractions in the Ado group versus Con and L-NAME groups suggests a depressed contractile response with adenosine treatment. Prior studies of the effects of adenosine on skeletal muscle contractile function have not reported this effect. Indeed, Reading & Barclay (2001) observed a reduced fatigue in isolated mouse soleus and extensor digitorum longus with adenosine treatment. The contrasting results could be due to the lower stimulation frequency (50 Hz), lower adenosine concentration (10–5 mM, versus 10–2 mM in the present study), or other methodological differences (e.g. mouse muscle) in the study of Reading & Barclay (2001) versus the present study.
Effect of NOS inhibition on aerobic metabolism in contracting muscle: a comparison of models
Given the wide variety of models used previously to study the effect of NOS inhibition on skeletal muscle function, it is important to consider how differences between studies impact the conclusions drawn. As 
approaches maximal values, blood flow becomes a modulating influence. Thus, a limitation to evaluating the effect of NOS inhibition on skeletal muscle 
from previous investigations is that experimental protocols, both in vivo and in situ, have not permitted control over blood flow. Specifically, the majority of past studies have reported reductions in blood flow with NOS inhibition. For example, in chronically instrumented dogs running at both intermediate and high intensity, a systemic infusion of nitro-L-arginine (35 mg ml–1, I.V.), markedly reduced blood flow (Shen et al. 2000). These results were confirmed in humans both when forearm blood flow during static hand-gripping (4–5 kg for 3 min) was reduced following an intrabrachial artery infusion of the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) at 4 µmol min–1 for 5 min (Endo et al. 1994) and when L-NMMA (infused at a constant rate of 5 mg min–1 (l thigh volume)–1 for the first 5 min, and thereafter at a rate of 1 mg min–1(l thigh volume)–1 for the following 15 min at rest and 6 min during exercise) reduced blood flow in the isolated human quadriceps muscle during knee extensor exercise at a work rate of 30 W (Hillig et al. 2003). It should be noted, however, that in at least one study conducted in humans there was no change in blood flow during either submaximal or exhaustive knee extensor exercise with systemic infusion of L-NAME (4 mg kg–1; Frandsenn et al. 2001). To add further confusion, there seems to be no clear relationship between blood flow and 
with NOS inhibition. Specifically, one study which reported a reduced blood flow with NOS inhibition (4 µmol min–1 for 5 min) during static hand gripping (4–5 kg for 3 min, frequency not reported) reported no change in 
(Endo et al. 1994). Failure to alter 
in this study could indicate that the reduction in blood flow was insufficient to limit O2 flux to the mitochondria (e.g. submaximal contractile conditions) and/or that the degree of NOS inhibition was incomplete (e.g. due to a short exposure time of 5 min to the NOS inhibitor prior to exercise). In contrast, another investigation demonstrated that the reduced blood flow seen with NOS inhibition (in chronically instrumented dogs running at intermediate and high intensity after NOS inhibition) was associated with an augmented 
(Shen et al. 2000). Noteworthy is that the increased 
with NOS inhibition shown in this latter study was associated with relatively large increases in perfusion pressure and/or reduced vascular conductance (Shen et al. 2000), which could complicate the interpretation (Ward & Hussain, 1994). In support of augmented aerobic function with NOS inhibition, horses running progressively from low to high intensity on a treadmill demonstrated faster onset of aerobic metabolism (i.e. faster 
on-kinetics) in the transition to steady-state exercise following infusion of L-NAME (20 mg kg–1; Kindig et al. 2001; 2002,). It should be noted that in the studies by Kindig et al. (2001, 2002) 
was measured at the mouth and therefore may not reflect a true ‘muscle effect’, since other tissues in the body are also consuming oxygen. Similar results were reported in humans cycling at intensities progressing from low to moderately high during infusion of L-NAME (4 mg kg–1 in 50 ml saline over 60 min), where 
was also measured at the mouth (Jones et al. 2003).
In an attempt to provide tighter control of experimental variables while investigating the effects of NOS inhibition on muscle function and aerobic metabolism, a recent study used an in situ preparation in which oxygen consumption was measured across electrically stimulated, self-perfused dog gastrocnemius muscle (King-VanVlack et al. 2002). In this study, a decrease in 
during intense twitch contractions (4 twitches s–1) was accompanied by a maintained tension development with L-NAME (20 mg kg–1
I.V.), such that the 
:tension ratio was reduced. This change was interpreted as evidence of a limitation in O2 supply consequent to the reduction in blood flow with L-NAME. However, the authors acknowledged that alterations in contractile economy due to NOS inhibition could not be ruled out, since other criteria for O2 supply limitation were lacking (no change in muscle venous effluent pH observed, no data for lactate available; King-VanVlack et al. 2002). Support for increased contractile economy with NOS inhibition was provided when a study by Hillig et al. (2003) reported a reduced 
during constant workload (30 W) knee-extensor exercise in humans with the use of the NOS inhibitor, L-NMMA, in conjunction with a blocker of cytochrome P450 2C9. Although leg blood flow was reduced in this experiment, the lower muscle lactate efflux and submaximal O2 extraction led the authors to conclude that the contractions were more economical (Hillig et al. 2003). Interestingly, this previous study by Hillig et al. (2003) noted that neither NOS blockade nor inhibition of cytochrome P450 2C9 alone altered 
and suggested that the combined effects of NOS inhibition and cytochrome P450 2C9 inhibition must yield a different effect from NOS inhibition alone. Since the present results are at odds with this conclusion (more economical force development with L-NAME did not require inhibition of cytochrome P450 2C9 in our experiments), insight into the basis of these discrepancies requires further study.
The issues mentioned above prompted us to employ a pump-perfused rat hindlimb model that would allow us to study the effect of NOS inhibition on skeletal muscle contractile and aerobic performance independent of blood flow and convective oxygen delivery. Under conditions of matched skeletal muscle convective O2 delivery, we observed that NOS inhibition yielded a lower 
despite very similar force production compared to the control group. Since a reduced 
could be compensated for by increasing anaerobic glycolysis, we also followed lactate production during contractions and for 4 min into recovery. In this regard, not only was lactate efflux lower with L-NAME, but the decline in lactate efflux in recovery from contractions followed a similar temporal pattern to the control group, suggesting no impairment of lactate transport with L-NAME. As such, we have interpreted these findings as evidence of a reduced O2 cost of force development consequent to NOS inhibition. However, future assessment of the intramuscular glycogen, phosphocreatine and ATP concentrations following contractions with NOS inhibition are needed to distinguish whether the O2 cost of contractions is reduced owing to a mitochondrial effect (e.g. increased P:O ratio (ratio of ATP produced to the number of oxygen atoms converted to water)), versus an altered ATP cost of contraction where a sparing of glycogen, phosphocreatine and ATP might be expected.
Conclusions
In summary, our results demonstrate that NOS inhibition, using L-NAME, results in a reduction in 
and lactate efflux, but similar force production versus that seen in control conditions at similar rates of skeletal muscle convective O2 delivery. As such, the resulting lower 
/time-integrated force development with L-NAME treatment is consistent with the notion that NOS inhibition improves contractile economy by reducing the O2 cost of contractions.
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; n
= 1) and L-NAME (•; n
= 1) only in perfusate.
; n
= 14) and L-NAME-treated groups (
; n
= 13) 
versus time during a 4 min contraction bout (60 tetani min–1) in control (•; n
= 6), adenosine- (
in the Ado and L-NAME groups versus Con during the contraction bout. Inset represents data for a subset of animals that had microsphere determination of muscle blood flow (i.e. the animals whose blood flow responses are depicted in 
and time-integrated force development) during a 4 min contraction bout (60 tetani min–1) in control (•; n
= 6), adenosine- (