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. 2013 Mar 18;591(Pt 11):2885–2896. doi: 10.1113/jphysiol.2013.251082

Muscle fibre-type dependence of neuronal nitric oxide synthase-mediated vascular control in the rat during high speed treadmill running

Steven W Copp 1, Clark T Holdsworth 1, Scott K Ferguson 1, Daniel M Hirai 1, David C Poole 1, Timothy I Musch 1
PMCID: PMC3690692  PMID: 23507879

Abstract

We have recently shown that nitric oxide (NO) derived from neuronal NO synthase (nNOS) does not contribute to the hyperaemic response within rat hindlimb skeletal muscle during low-speed treadmill running. This may be attributed to low exercise intensities recruiting primarily oxidative muscle and that vascular effects of nNOS-derived NO are manifest principally within glycolytic muscle. We tested the hypothesis that selective nNOS inhibition via S-methyl-l-thiocitrulline (SMTC) would reduce rat hindlimb skeletal muscle blood flow and vascular conductance (VC) during high-speed treadmill running above critical speed (asymptote of the hyperbolic speed versus time-to-exhaustion relationship for high-speed running and an important glycolytic fast-twitch fibre recruitment boundary in the rat) principally within glycolytic fast-twitch muscle. Six rats performed three high-speed treadmill runs to exhaustion to determine critical speed. Subsequently, hindlimb skeletal muscle blood flow (radiolabelled microspheres) and VC (blood flow/mean arterial pressure) were determined during supra-critical speed treadmill running (critical speed + 15%, 52.5 ± 1.3 m min−1) before (control) and after selective nNOS inhibition with 0.56 mg kg−1 SMTC. SMTC reduced total hindlimb skeletal muscle blood flow (control: 241 ± 23, SMTC: 204 ± 13 ml min−1 (100 g)−1, P < 0.05) and VC (control: 1.88 ± 0.20, SMTC: 1.48 ± 0.13 ml min−1 (100 g)−1 mmHg−1, P < 0.05) during high-speed running. The relative reductions in blood flow and VC were greater in the highly glycolytic muscles and muscle parts consisting of 100% type IIb+d/x fibres compared to the highly oxidative muscles and muscle parts consisting of ≤35% type IIb+d/x muscle fibres (P < 0.05). These results extend our understanding of vascular control during exercise by identifying fibre-type-selective peripheral vascular effects of nNOS-derived NO during high-speed treadmill running.

Key points

  • Neuronal nitric oxide (NO) synthase (nNOS) inhibition does not impact skeletal muscle blood flow or vascular conductance (VC) during low-speed (20 m min−1) treadmill running.

  • This may be due to the fact that low exercise intensities recruit primarily oxidative muscle and that nNOS-derived NO contributes to vascular control primarily within glycolytic muscle.

  • Rats ran in the severe-intensity domain at 15% above critical speed (an important glycolytic fast-twitch fibre recruitment boundary in the rat) before and after selective nNOS inhibition with S-methyl-l-thiocitrulline (SMTC).

  • SMTC reduced blood flow and VC during supra-critical speed treadmill running (52.5 ± 1.3 m min−1) with the greatest proportional reductions observed in glycolytic fast-twitch compared to oxidative slow- and fast-twitch muscle. There were no effects of SMTC on muscle blood flow or VC during low-speed running (20 m min−1).

  • The present data reveal important fibre-type- and exercise intensity-dependent peripheral vascular effects of nNOS-derived NO during whole-body exercise.

Introduction

The ability of the cardiovascular system to increase skeletal muscle blood flow and, therefore, O2 delivery during exercise is accomplished via neurohumoral activation (to increase cardiac output and initiate blood flow redistribution) and local mechanical and vasomotor control mechanisms (reviewed by Joyner & Wilkins, 2007). The robust active muscle blood flow response supports sustained exercise performance whereas many disease processes are hallmarked by O2 delivery impairment and exercise intolerance (e.g. heart failure, reviewed by Poole et al. 2012).

NO is an important cardiovascular signalling molecule that has been reported to play an integral role in promoting exercise hyperaemia in animals (Hirai et al. 1994; King et al. 1994) and humans (Schrage et al. 2004). In healthy subjects NO is synthesized enzymatically via nNOS (type I) or endothelial nitric oxide synthase (eNOS; type III). The vasodilatory contributions of eNOS-derived NO secondary, at least in part, to vascular endothelial shear stress during exercise are well known (reviewed by Green et al. 1996). The peripheral vascular effects of nNOS-derived NO, particularly during dynamic exercise, remain controversial. For example, our laboratory reported recently that acute administration of the selective nNOS inhibitor SMTC reduced rat hindlimb skeletal muscle blood flow at rest but not during low-speed treadmill running (Copp et al. 2010b). Conversely, nNOS-derived NO attenuates skeletal muscle sympathetic vasoconstriction during muscle contractions in both rats (Thomas et al. 1998, 2003) and humans (Sander et al. 2000). Importantly, this ‘functional sympatholysis’ may occur within glycolytic but not oxidative skeletal muscle and at high contraction intensities only (Thomas et al. 1994). The fibre-type- and contraction-intensity-dependent vascular effects of nNOS-derived NO may account for the unchanged skeletal muscle blood flow following nNOS inhibition during low-speed treadmill running (Copp et al. 2010b) which recruits primarily oxidative slow- and fast-twitch muscle (Gollnick et al. 1974; Laughlin & Armstrong, 1982). It is possible that high-speed treadmill running which additively recruits highly glycolytic fast-twitch muscle may unmask obligatory nNOS-derived NO vasomotor control during dynamic exercise. This information would have important implications for diseased (e.g. heart failure, Thomas et al. 2001; Copp et al. 2012) and aged (Hirai et al. 2012) populations which have been associated with impaired nNOS-mediated function.

Our laboratory has demonstrated recently that critical speed (the asymptote of the hyperbolic speed versus time-to-exhaustion relationship for high-speed running) can be determined empirically in the rat and that running above, relative to below, critical speed recruits glycolytic fast-twitch muscle to a greater extent than oxidative slow- and fast-twitch muscle (Copp et al. 2010a). In this regard, the ability to accurately measure blood flow distribution among and within active skeletal muscles during high-speed running (Armstrong & Laughlin, 1985; Musch et al. 2001) specifically above critical speed (Copp et al. 2010a) makes the rat an ideal model to examine the fibre-type dependency of nNOS-mediated vascular control during exercise. Therefore, the present investigation tested the hypothesis that selective nNOS inhibition with SMTC would reduce rat hindlimb skeletal muscle blood flow and VC during treadmill running above critical speed whereas there would be no effects of SMTC during low-speed treadmill running. Furthermore, we hypothesized that during supra-critical speed running the greatest relative reductions in blood flow and VC would occur principally within the muscles and muscle parts composed of highly glycolytic type IIb and IId/x fibres.

Methods

Ethical approval

The experimental procedures described in the present investigation were approved by the Institutional Animal Care and Use Committee of Kansas State University and were conducted according the animal use guidelines mandated by The Journal of Physiology (Drummond, 2009).

A total of 17 male Sprague–Dawley rats (∼4 months old, body mass 387 ± 13 g, Charles River Laboratories, Wilmington, MA, USA) were used in the present investigation. Rats were housed two per cage in accredited facilities (Association for the Assessment and Accreditation of Laboratory Animal Care) with food and water provided ad libitum. Twelve rats were assigned initially to either the high-speed (n= 6) or low-speed (n= 6) group and acclimatized to running on a custom-built motor-driven treadmill over 1 week (5–6 days up to 5 min day−1). The treadmill was set to a 5% incline which was maintained throughout the experimental protocol.

Determination of critical speed

Rats in the high-speed group performed a peak O2 uptake (Inline graphic) test as described previously (Musch et al. 1988). Specifically, rats underwent a progressive ramp-style treadmill test inside a metabolic chamber which was placed on the treadmill belt. Ambient air was drawn through the chamber via a vacuum pump (Neptune-Dyna model 4K, Dover, NJ, USA), through drierite (anhydrous CaSO4), through a flowmeter (Fischer-Porter model 10A1378, Burr Ridge, IL, USA) and delivered subsequently to inline O2 and CO2 analysers (O2: model S-3A/I, CO2: model CD-3A AEI Technologies, Pittsburgh, PA, USA). The speed of the treadmill and corresponding Inline graphic and carbon dioxide production (Inline graphic) were monitored and recorded continuously throughout the test. The test was terminated when the rat was unable or unwilling to keep pace with the treadmill belt.

Critical speed was determined as described previously (Copp et al. 2010a). Rats in the high-speed group performed at least three runs to exhaustion, in random order, at a range of speeds corresponding to 90–115% of the speed that elicited Inline graphic. For each constant-speed test rats were given a 2 min warm-up at 20–25 m min−1 which was followed by 1 min of quiet resting. The treadmill speed was then increased rapidly over an ∼10 s period to the predetermined speed. Rats were encouraged to run by applying manual bursts of high pressure air at the hindlimbs whenever they drifted towards the back of the treadmill lane. Tests were terminated when the rat could not maintain pace with the treadmill belt despite obvious exertion. Time-to-exhaustion was measured to the nearest tenth of a second. The following criteria constituted a successful constant-speed test: (1) the ability of the rat to quickly settle into a normal running gait at the beginning of the test, (2) a noticeable change in gait (i.e. lowering of the hindquarters and a rising of the snout) preceding exhaustion, and (3) marked attenuation of the righting reflex when placed in the supine position. A minimum of 24 h rest was given between consecutive exercise tests. Following completion of the constant-speed tests, the individual critical speeds were determined by (1) the hyperbolic speed–time model (time =D′/(speed – critical speed)), where the asymptote of the hyperbolic curve is the critical speed and the curvature constant is D′, and (2) the linear 1/time model (speed =D′× 1/time + critical speed), where speed is plotted as a function of the inverse of time (s)-to-exhaustion, the intercept of the regression line is the critical speed, and the slope is D′(Gaesser et al. 1995). For both models, D′ (vertical distance in metres) was multiplied by the rats’ average weight in kilograms for the constant-speed tests to determine m kg, then converted to W′ in joules (J) using the relationship: 1 J = 0.103155 m kg. Both the hyperbolic and linear 1/time models were used to ensure accuracy and robustness of the critical speed estimation.

Measurement of hindlimb skeletal muscle blood flow

On the final experimental day anaesthesia was induced with a 5% isoflurane–O2 mixture with maintenance on 2–3% isoflurane–O2. Subsequently, one catheter (PE-10 connected to PE-50, Clay Adams Brand, Sparks, MD, USA) was placed in the ascending aorta via the right carotid artery and a second catheter was placed in the caudal (tail) artery. The catheters were tunnelled subcutaneously to the dorsal cervical region and exteriorized through a puncture wound in the skin. Anaesthesia was terminated and the animal was given ≥1 h to recover prior to blood flow measurement.

Following recovery, each rat (high-speed group) was placed on the treadmill and the tail artery catheter was connected to a 1 ml plastic syringe and a Harvard infusion/withdrawal pump while the carotid artery catheter was connected to a pressure transducer (Gould Statham P23ID, Valley View, OH, USA) and chart recorder for continuous monitoring of mean arterial pressure (MAP) and heart rate (HR). Exercise was then initiated and each rat ran at a speed of 25 m min−1 for 1 min. At the 1 min mark the speed of the treadmill was increased to the speed equalling 15% above each individual rat's critical speed determined from the linear 1/time model (selected for the slightly, but not significantly, higher average critical speed estimation from this model, see Results). After 1.5 min of running at this speed (2.5 min of total exercise time) blood withdrawal from the tail artery catheter was initiated at a rate of 0.25 ml min−1 while MAP and HR were measured and recorded simultaneously via the carotid artery catheter. The carotid artery catheter was then disconnected from the pressure transducer and ∼0.6 × 106 microspheres (85Sr or 57Co in random order, Perkin Elmer, Waltham, MA, USA) were injected via the carotid catheter into the aortic arch. Following microsphere injection and while the rat was still running a 0.3 ml blood sample was taken from the carotid artery catheter for determination of arterial pH, blood gases and [lactate]. Exercise was then terminated and blood withdrawal from the caudal artery catheter (which was occurring simultaneously with microsphere injection) was stopped. The reference blood sample was collected and rats were then given 1 h to recover.

After the 1 h recovery period, 0.56 mg kg−1 (dissolved in 0.5 ml of heparinized saline) of the selective nNOS inhibitor SMTC (Furfine et al. 1994; Narayanan & Griffith, 1994) was infused as a bolus into the caudal artery catheter (see below and Discussion for details regarding assessment of the efficacy and selectivity of nNOS inhibition). The tail catheter was then connected to a 1 ml syringe which was placed in the Harvard pump for blood withdrawal. Two minutes following SMTC infusion a second bout of exercise, microsphere injection (differently labelled from the first run), and blood sample collection were performed exactly as described above for the control condition. Approximately 10–15 min following exercise cessation the non-selective NOS inhibitor NG-nitro-l-arginine-methyl-ester (l-NAME) was administered and MAP and HR were monitored and recorded for an additional ∼10 min (see below for more details).

Low-speed group

For rats in the low-speed group MAP, HR and skeletal muscle, kidney and splanchnic organ blood flow were measured before and after nNOS inhibition with SMTC exactly as described above for the high-speed group except the treadmill speed was set to 20 m min−1 for both the control and SMTC exercise bouts (i.e. same experimental protocol as Copp et al. 2010b).

Assessment of efficacy and selectivity of nNOS inhibition with SMTC

To confirm that SMTC was mediating effects via nNOS inhibition (as opposed to some unknown/unexpected non-specific effect), MAP was measured in five additional rats (instrumented as described above) in which drug administration order was reversed such that non-selective NOS inhibition with l-NAME preceded SMTC. We reasoned that if SMTC was indeed acting via nNOS inhibition there would be no additional SMTC-induced MAP increase in the presence of prior l-NAME administration. In the rats from the exercise groups the selectivity of SMTC for nNOS versus eNOS was assessed via measurement of the hypotensive responses to rapid ACh infusions (10 μg kg−1 in 0.2 ml saline into the tail artery catheter) while the rats sat quietly on the treadmill belt under control, SMTC and l-NAME conditions. The magnitude and recovery time of the hypotensive response to ACh were used as an index of eNOS-mediated function. The absence of any blunting of the hypotensive response to ACh following SMTC was considered consistent with the notion that SMTC did not impact eNOS-mediated function (see Discussion for more details).

Determination of blood flow and VC

Following the experimental protocol each animal was killed by pentobarbital overdose (≥100 mg kg−1) administered via the carotid artery catheter. The thorax was opened, and placement of the carotid artery catheter into the aortic arch was confirmed by anatomical dissection. Organs of the splanchnic region, the kidneys, diaphragm, intercostal muscles and the 28 individual muscles and muscle portions of the rat hindlimb were identified and removed. The tissues were blotted, weighed, and placed immediately into counting vials.

The radioactivity of each tissue was determined on a γ scintillation counter (Packard Auto Gamma Spectrometer model 5230, Downers Grove, IL, USA). Accounting for cross-talk between isotopes, blood flows to each tissue were determined using the reference sample method (Musch & Terrell, 1992) and expressed as millilitres per minute per 100 g of tissue (ml min−1 (100 g)−1). Adequate microsphere mixing was verified for each injection by the demonstration of a <15% difference between blood flow to the right and left kidneys and/or to the right and left hindlimb musculature. Blood flows were normalized to the MAP measured immediately preceding microsphere injection and expressed as VC (ml min−1 (100 g)−1 mmHg−1).

Data analyses

All data are presented as mean ± SEM. MAP, HR and ACh responses were compared using ANOVAs with Student–Newman–Keuls post hoc tests where appropriate. Blood flow, VC and blood sample variables were compared with paired Student's t tests. z tests were used to determine when reductions in blood flow and VC were different from zero. Significance was accepted at P < 0.05.

Results

High-speed group

Inline graphic and critical speed estimation

The average Inline graphicwas 86 ± 1 ml kg−1 min−1 (respiratory exchange ratio = 1.07 ± 0.03) with a peak speed of 54.4 ± 2.4 m min−1. The times to exhaustion at 90, 100 and 115% of peak speed and the hyperbolic and linear 1/time model fits and estimated critical speeds for a representative rat are shown in Fig. 1. The coefficient of determination (r2, hyperbolic: 0.96 ± 0.03, linear 1/time: 0.96 ± 0.02, P > 0.05), W′ (hyperbolic: 162 ± 67, linear: 139 ± 66 J, P > 0.05) and estimated critical speed (hyperbolic: 44.0 ± 1.8, linear 1/time: 45.6 ± 1.2 m min−1, P > 0.05) were not different between models. The average running speed at which blood flow measurements were performed was 52.5 ± 1.3 m min−1 (15% above critical speed from the 1/time model).

Figure 1.

Figure 1

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Estimation of critical speed (represented by the horizontal dashed lines) via the hyperbolic and linear 1/time models for a representative rat.

Effects of SMTC on MAP, HR and blood sample variables

At rest, SMTC increased MAP and reduced HR compared to control. During exercise, SMTC increased MAP whereas HR was not different between conditions (Table 1). Exercising arterial blood pH (control: 7.39 ± 0.02, SMTC: 7.40 ± 0.02), Inline graphic (control: 85.2 ± 2.1, SMTC: 88.3 ± 3.3 mmHg), and [lactate] (control: 6.1 ± 0.4, SMTC: 6.7 ± 0.8 mmol l−1) were not different (P > 0.05 for all) between conditions. SMTC significantly reduced Inline graphic (control: 21.7 ± 0.8, SMTC: 19.8 ± 0.8 mmHg, P < 0.05).

Table 1.

Effects of SMTC on MAP and HR at rest and during high-speed treadmill running (15% above critical speed, 52.5±1.3 m min−1)

Control SMTC
Rest
 MAP (mmHg) 125 ± 4 137 ± 5*
 HR (beats min−1) 410 ± 9 393 ± 11*
Exercise
 MAP (mmHg) 130 ± 4 139 ± 4*
 HR (beats min−1) 538 ± 6† 555 ± 11†
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Data are mean ± SEM, *P < 0.05 versus control. †P < 0.05 versus rest.

Effects of SMTC on hindlimb skeletal muscle blood flow and VC

In marked contrast to low-speed running (see below and also Copp et al. 2010b), SMTC reduced total hindlimb skeletal muscle blood flow and VC during high-speed running above critical speed (Fig. 2). Specifically, blood flow was reduced in 14, and VC in 22, of the 28 individual muscles or muscle parts of the rat hindlimb (Table 2). Reductions in blood flow and VC were found in some muscles across the spectrum of oxidative capacities and muscle fibre-type compositions. However, the relative reductions in blood flow and VC following SMTC were greater in the highly glycolytic (100% type IIb+d/x fibres, n= 5) compared to the highly oxidative (≤35% type IIb+d/x fibres, n= 5, Fig. 3) muscles and muscle portions. Moreover, note in Fig. 4 that within representative individual muscles containing distinct red and white portions the relative reductions in blood flow and VC following SMTC were greater in the glycolytic white versus the oxidative red portions of those muscles.

Figure 2.

Figure 2

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Effects of nNOS inhibition with SMTC (0.56 mg kg−1) on hindlimb skeletal muscle blood flow and vascular conductance (VC) during treadmill running at 20 m min−1 (from present low-speed group) and 15% above critical speed (CS+15%, 52.5 ± 1.3 m min−1). *P < 0.05 versus control.

Table 2.

Effects of SMTC on individual hindlimb muscle or muscle part blood flow (ml min−1 (100 g)−1) and vascular conductance (VC, ml min−1 (100 g)−1 mmHg−1) during high-speed treadmill running (15% above critical speed, 52.5±1.3 m min−1)

Blood flow VC
Control SMTC Control SMTC
Ankle extensors
 Soleus (21.3, 9%) 366 ± 37 309 ± 14 2.87 ± 0.37 2.23 ± 0.11*
 Plantaris (21.8, 80%) 394 ± 29 382 ± 16 3.04 ± 0.20 2.75 ± 0.11
 Gastrocnemius, red (36.2, 14%) 566 ± 48 533 ± 22 4.40 ± 0.42 3.85 ± 0.22*
 Gastrocnemius, white (8.1, 100%) 141 ± 24 99 ± 12* 1.10 ± 0.19 0.71 ± 0.09*
 Gastrocnemius, mixed (25.7, 91%) 284 ± 25 255 ± 6 2.21 ± 0.22 1.84 ± 0.09*
 Tibialis posterior (18.3, 73%) 347 ± 41 334 ± 26 2.70 ± 0.33 2.41 ± 0.21
 Flexor digitorum longus (10.6, 68%) 187 ± 47 145 ± 20 1.46 ± 0.37 1.06 ± 0.17
 Flexor halicus longus (12.3, 71%) 183 ± 18 153 ± 9* 1.42 ± 0.14 1.10 ± 0.07*
Ankle flexors
 Tibialis anterior, red (39.3, 63%) 572 ± 79 429 ± 44* 4.50 ± 0.76 3.12 ± 0.40*
 Tibialis anterior, white (18.4, 80%) 222 ± 42 156 ± 22* 1.75 ± 0.40 1.14 ± 0.19*
 Extensor digitorum longus (21.6, 76%) 107 ± 19 90 ± 18 0.84 ± 0.16 0.66 ± 0.15*
 Peroneals (20.3, 67%) 222 ± 26 143 ± 15* 1.73 ± 0.21 1.05 ± 0.13*
Knee extensors
 Vastus intermedius (33.2, 4%) 585 ± 50 520 ± 33 4.52 ± 0.34 3.76 ± 0.28*
 Vastus medialis (20.2, 82%) 424 ± 36 383 ± 12 3.27 ± 0.21 2.76 ± 0.09*
 Vastus lateralis, red (42.3, 35%) 482 ± 99 476 ± 72 3.61 ± 0.67 3.39 ± 0.50
 Vastus lateralis, white (8.3, 100%) 188 ± 24 144 ± 19* 1.47 ± 0.21 1.05 ± 0.16*
 Vastus lateralis, mixed (19.2, 89%) 336 ± 29 325 ± 13 2.60 ± 0.22 2.35 ± 0.14
 Rectus femoris, red (25.6, 66%) 458 ± 50 399 ± 33 3.51 ± 0.32 2.89 ± 0.28*
 Rectus femoris, white (15.1, 100%) 267 ± 29 218 ± 18* 2.06 ± 0.20 1.58 ± 0.15*
Knee flexors
 Biceps femoris, anterior (8.4, 100%) 127 ± 26 95 ± 19* 1.01 ± 0.23 0.70 ± 0.16*
 Biceps femoris, posterior (14.0, 92%) 191 ± 23 150 ± 11* 1.50 ± 0.21 1.09 ± 0.11*
 Semitendinosus (12.6, 83%) 88 ± 15 55 ± 7* 0.69 ± 0.13 0.40 ± 0.06*
 Semimembranosus, red (20.6, 72%) 316 ± 46 259 ± 21 2.47 ± 0.40 1.88 ± 0.17*
 Semimembranosus, white (10.2, 100%) 135 ± 32 95 ± 17* 1.06 ± 0.27 0.69 ± 0.13
Thigh adductors
 Adductor longus (18.9, 5%)a 378 ± 70 269 ± 44* 2.98 ± 0.64 1.94 ± 0.31*
 Adductor magnus and brevis (18.5, 89%) 204 ± 38 158 ± 22* 1.60 ± 0.33 1.14 ± 0.16*
 Gracilis (15.4, 77%) 89 ± 25 53 ± 9* 0.69 ± 0.19 0.38 ± 0.07*
 Pectinius (20.2, 69%) 176 ± 73 136 ± 81 1.43 ± 0.66 1.04 ± 0.65*
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Data are mean ± SEM. Numbers in parentheses represent the citrate synthase activity (μmol min−1 g−1) and percentage sum of type IIb+d/x muscle fibres, respectively, as reported previously by Delp & Duan (1996). aSee text for comments regarding contrasting report of the adductor longus muscle fibre-type composition by Eng et al. (2008). *P < 0.05 versus control.

Figure 3. Relative changes in blood flow (ΔBlood flow) and VC (ΔVC) following nNOS inhibition with SMTC (0.56 mg kg−1) during high-speed treadmill running in the highly oxidative and highly glycolytic muscles and muscle parts.

Figure 3

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Oxidative muscles (n= 5, adductor longus, red portion of the vastus lateralis, vastus intermedius, soleus, and red portion of the gastrocnemius) are composed of ≤35% type IIb+d/x fibres and have 3-fold higher citrate synthase activity versus glycolytic muscles (Delp & Duan, 1996). Glycolytic muscles (n= 5, anterior portion of the biceps femoris, white portions of the semimembranosus, rectus femoris, gastrocnemius, and vastus lateralis) are composed of 100% type IIb+d/x fibres (Delp & Duan, 1996). *P < 0.05 versus oxidative, †P < 0.05 versus zero.

Figure 4. Relative changes in blood flow (ΔBlood flow) and vascular conductance (ΔVC) following nNOS inhibition with SMTC (0.56 mg kg−1) during high-speed treadmill running for oxidative red and glycolytic white portions of representative individual muscles.

Figure 4

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Oxidative red (dashed lines and filled circles): 47 ± 14% type IIb+d/x muscle fibres and 3-fold higher citrate synthase activity versus glycolytic white portions (Delp & Duan, 1996). Glycolytic white (continuous lines and open circles): all 100% type IIb+d/x muscle fibres (Delp & Duan, 1996). VL, vastus lateralis; Semimem, semimembranosus; RF, rectus femoris; Gastroc, gastrocnemius. SEM bars are omitted for clarity (range across all muscles 4–12%). *P < 0.05 versus control.

Effects of SMTC on respiratory skeletal muscle, kidney and splanchnic organ blood flow and VC

Blood flow and VC were reduced in the diaphragm and intercostal muscles following SMTC compared to control (Table 3, P < 0.05). Blood flow and VC to the kidneys and all organs of the splanchnic region were not different between control and SMTC conditions (Table 3, P > 0.05 for all).

Table 3.

Effects of SMTC on respiratory skeletal muscle, kidney and splanchnic organ blood flow (ml min−1 (100 g)−1) and vascular conductance (VC, ml min−1 (100 g)−1 mmHg−1) during high-speed treadmill running (15% above critical speed, 52.5±1.3 m min−1)

Blood flow VC
Control SMTC Control SMTC
Diaphragm (39.1, 50%) 355 ± 55 267 ± 27* 2.76 ± 0.43 1.94 ± 0.23*
Intercostals (12.0, ∼82%) 60 ± 7 47 ± 4* 0.47 ± 0.06 0.34 ± 0.03*
Kidney 318 ± 65 255 ± 57 2.46 ± 0.48 1.81 ± 0.38
Stomach 35 ± 5 29 ± 4 0.28 ± 0.05 0.20 ± 0.03
Adrenals 227 ± 51 158 ± 28 1.77 ± 0.40 1.13 ± 0.19
Spleen 19 ± 5 24 ± 6 0.15 ± 0.04 0.17 ± 0.04
Pancreas 59 ± 10 42 ± 4 0.47 ± 0.09 0.30 ± 0.02
Small intestine 164 ± 32 141 ± 16 1.29 ± 0.27 1.00 ± 0.10
Large intestine 97 ± 17 86 ± 12 0.76 ± 0.14 0.61 ± 0.08
Liverb 18 ± 4 21 ± 4 0.14 ± 0.03 0.15 ± 0.03
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Data are mean ± SEM. Numbers in parentheses represent the citrate synthase activity (μmol min−1 g−1) and percentage sum of type IIb+d/x muscle fibres, respectively, as reported previously by Delp & Duan (1996). bDenotes arterial, not portal, blood flow and VC. *P < 0.05 versus control.

Low-speed group

Similar to both the high-speed group and our previous report (Copp et al. 2010b), SMTC increased resting MAP (↑11 ± 2 mmHg, P < 0.05) and reduced HR (↓38 ± 16 beats min−1) compared to control. In marked contrast to the high-speed group, during treadmill running at 20 m min−1 SMTC had no effects on MAP, HR, or blood flow and VC to the total hindlimb musculature (Fig. 2) or any individual muscle or muscle part compared to control (P > 0.05 for all). Similarly, diaphragm and intercostal muscle blood flow and VC were not different between conditions (P > 0.05). SMTC reduced blood flow and VC to the kidneys, adrenals, stomach and small intestine (P < 0.05 for all). As these data replicated closely those published previously (see Copp et al. 2010b), in the interests of brevity and clarity the reader is referred to that publication.

Efficacy and selectivity of nNOS inhibition with SMTC

SMTC followed by l-NAME administration produced a step-like increase in MAP above control values (Fig. 5A, left panel). Conversely, when the drug administration order was reversed, SMTC administered in the presence of prior l-NAME infusion produced no further MAP increase (Fig. 5A, right panel). The magnitude (ΔMAP, Fig. 5B, left panel) and recovery speed (time to 50% MAP recovery, Fig. 5B, right panel) of the hypotensive response to ACh infusions were not different between control and SMTC conditions whereas they were blunted significantly following l-NAME.

Figure 5. Resting MAP data demonstrating the efficacy and selectivity of nNOS inhibition with SMTC.

Figure 5

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A, the step-like MAP increases when SMTC preceded l-NAME (left panel, high-speed group, n= 6) and the lack of any further MAP increase when SMTC followed l-NAME (right panel, n= 5) confirm that SMTC mediated its effects via NOS inhibition. B, the lack of effects of SMTC on the magnitude (left panel) and recovery speed (right panel) of the hypotensive response to ACh is consistent with intact eNOS-mediated function (high-speed group, n= 6). *P < 0.05 versus control, †P < 0.05 versus SMTC.

Discussion

Consistent with our hypothesis, nNOS inhibition with SMTC reduced rat hindlimb skeletal muscle blood flow and VC during high-speed treadmill running (52.5 ± 1.3 m min−1) above critical speed. Reductions in blood flow and VC were found in some muscles and muscle portions across the range of oxidative capacities and fibre-type compositions; however, the greatest relative reductions were found predominantly within glycolytic muscles composed primarily of type IIb+d/x fibres. The SMTC-induced reductions in skeletal muscle blood flow and VC during high-speed running contrast markedly with the lack of effects of nNOS inhibition with SMTC on blood flow or VC in any individual muscle or muscle part during low-speed (20 m min−1) running. The present data reveal important fibre-type- and exercise-intensity-dependent peripheral vascular effects of nNOS-derived NO during whole-body locomotory exercise.

Relationship with the literature

Low-intensity exercise recruits primarily motor units innervating oxidative slow- and fast-twitch muscle fibres whereas motor units innervating glycolytic fast-twitch muscle fibres are recruited additively as exercise intensity increases (Gollnick et al. 1974; Laughlin & Armstrong, 1982). For high-intensity exercise the critical speed (or power) represents an important metabolic rate where exercise performed above critical speed/power systematically drives Inline graphic toward its maximum value (Poole et al. 1988; Broxterman et al. 2013). In the rat, supra-critical speed treadmill running elicits marked preferential type IIb and d/x fast-twitch muscle fibre recruitment (Copp et al. 2010a). That nNOS inhibition reduced relative blood flow and VC during supra-critical speed running to the greatest extent in glycolytic fast-twitch muscles in the present investigation is a crucial extension of the previous findings that nNOS-derived NO attenuates sympathetic vasoconstriction in glycolytic but not oxidative muscles during electrically induced rat muscle contractions (Thomas et al. 1994, 1998, 2003). Moreover, artificial simultaneous recruitment of all motor units may explain why nNOS inhibition reduced skeletal muscle blood flow and VC during electrically induced contractions of the mixed muscle-fibre-type rat spinotrapezius muscle (Copp et al. 2011). A fibre-type-selective vascular effect of nNOS-derived NO is consistent with the identification of greater nNOS activity in predominantly type II, compared to type I, rat muscles (Kobzik et al. 1994) although human oxidative type I muscle may actually contain more nNOS than type II muscle (Frandsen et al. 1996). The mechanisms of nNOS-mediated vasomotor control within glycolytic type II muscle during high-speed running probably involves attenuation of the exercise-induced augmented sympathetic vasoconstrictor signal (i.e. functional sympatholysis) mediated via α2-adrenergic receptors (Thomas et al. 1994, 1998). However, a more direct vasodilatory role for nNOS-derived NO has also been identified in rat fast-twitch muscle (Grange et al. 2001), acting presumably via alterations in intra-smooth muscle cell cyclic guanosine monophospate (cGMP) concentration.

In the present investigation the absolute magnitude of SMTC-induced reductions in blood flow and VC were similar between some oxidative and glycolytic muscles. However, those reductions were not consistent or statistically significant in the majority of the oxidative muscles. A noted exception is the adductor longus which is only 5% type IIb+d/x muscle fibres according to the histochemical analysis of Delp & Duan (1996). Interestingly, using immunological methods, Eng and colleagues (2008) reported that the adductor longus is composed of ∼97% type IIb+d/x fibres. Thus, our present conclusion of a selective, but not exclusive, influence of nNOS inhibition within glycolytic fast-twitch muscles during high-speed running (as depicted, for example, in Fig. 3) may actually be an underestimation. Furthermore, emphasis is placed on the relative reductions in blood flow and VC following SMTC in this study because this designates the magnitude of the nNOS-derived NO signal relative to other vasodilator candidates within the individual muscles. Any pathological disruption in nNOS-mediated function (and resultant impairments in O2 delivery) would lower the microvascular Inline graphic in proportion to its relative vasomotor contribution. Thus, dysfunctional nNOS signalling would reduce microvascular Inline graphic to the greatest extent in glycolytic muscles. This would presumably incur major negative consequences for capillary–myocyte O2 flux, metabolic control, and contractile performance within those fibres (Stary & Hogan, 1999), especially considering that glycolytic muscles evidence lower microvascular Inline graphic values compared to oxidative muscles in the control condition (McDonough et al. 2005).

In addition to muscle metabolic and contractile characteristics as reported here, it is likely that other factors, for example, muscle contraction intensity (Thomas et al. 1994), metabolic rate, and/or muscle function also influence the presence and extent of nNOS-derived NO vasomotor control. In support of the latter, nNOS inhibition with SMTC reduced blood flow and VC within predominantly oxidative rat hindlimb muscles at rest where muscle function (i.e. postural recruitment) may dictate nNOS-mediated vascular control. It is also noteworthy that blood flow and VC were reduced following SMTC in the intercostals and highly oxidative diaphragm (although composed of 50% type IIb+d/x muscle fibres) muscles during high-speed but not low-speed running. Admittedly, force generation by these respiratory muscles was not necessarily matched before and after SMTC (as it was for the hindlimb muscles, a key feature of the current experimental design). Force generation/recruitment was, if anything, enhanced given the well-characterized inhibitory effects of nNOS-derived NO on skeletal muscle contractile function (Kobzik et al. 1994) and the lower arterial Inline graphic which indicates a greater exercise-induced hyperventilation, most likely increasing respiratory muscle work. Therefore, the lower blood flow and VC following nNOS inhibition within the diaphragm and intercostals may well reflect a particularly important vasodilatory role for nNOS-derived NO in those muscles.

In the present investigation nNOS inhibition with SMTC did not reduce blood flow or VC to the kidneys or any splanchnic organs during high-speed running. This contrasts with the obligatory nNOS-derived NO renal vascular control reported by our laboratory (present data, Copp et al. 2010b) and others (Ichihara et al. 1998; Wakefield et al. 2003). However, high exercise intensities elicit marked sympathetically induced vasoconstriction within non-muscular tissues and inactive muscle thereby promoting cardiac output redistribution towards active skeletal muscle. This is evidenced by the lower kidney and splanchnic organ blood flows and VCs compared to that found in the rat at rest and during low-speed treadmill running (Copp et al. 2010b). At high exercise intensities obligatory nNOS-derived NO kidney and splanchnic organ vascular control may be obviated in order to facilitate this blood flow redistribution.

nNOS has been identified within nerves, skeletal muscle and key cardiovascular control centres in the brain (Patel et al. 2001; Stamler & Meissner, 2001). Importantly, an identical systemic SMTC dose as used here does not impact lumbar or renal sympathetic nerve discharge (Copp et al. 2013). This supports the view that the effects of SMTC observed here reflect peripheral nNOS-derived NO vascular control specifically.

Efficacy and selectivity of nNOS inhibition with SMTC

The efficacy and selectivity of nNOS inhibition shown here is supported by: (1) the lack of further MAP increase with SMTC in the presence of prior l-NAME administration (Fig. 5A, right panel), (2) the previously reported 17-fold selectivity of SMTC for nNOS over eNOS in rat tissue in vivo (Furfine et al. 1994), (3) a comprehensive SMTC dose–response analysis indicating marked selectivity of SMTC for nNOS over eNOS at low doses (i.e. <1.0 mg kg−1, Wakefield et al. 2003), and (4) the present demonstration that 0.56 mg kg−1 of SMTC did not alter the hypotensive response to ACh whereas it was blunted significantly following non-selective NOS inhibition with l-NAME. Importantly, higher SMTC doses also blunt the hypotensive response to ACh (Komers et al. 2000; Wakefield et al. 2003) thus supporting the sensitivity of this assessment as an indicator of nNOS selectivity. Analysis of the hypotensive response to ACh has been utilized extensively to assess the selectivity of SMTC for nNOS in conscious rats (Wakefield et al. 2003; Copp et al. 2010b) and humans (Seddon et al. 2008, 2009), although we acknowledge that systemic ACh infusion may not necessarily impact eNOS exclusively. However, it is also compelling that non-selective NOS inhibition with l-NAME in the rat running at low (Hirai et al. 1994) and high (Musch et al. 2001) speeds markedly reduces blood flow and VC with the greatest effects observed in highly oxidative muscles. In direct contrast, 0.56 mg kg−1 of SMTC did not impact blood flow or VC whatsoever during low-speed running and the greatest relative reductions during high-speed running were found primarily within glycolytic fast-twitch muscles. Had the SMTC dose used here been high enough to inhibit eNOS at least some effects would have been anticipated during low-speed running. Therefore, we believe that SMTC as used here did not impact eNOS-mediated function.

Conclusions

In the present investigation, selective nNOS inhibition with SMTC reduced skeletal muscle blood flow and VC principally within highly glycolytic fast-twitch muscle during high-speed treadmill running above critical speed. These pronounced effects of nNOS inhibition during supra-critical speed exercise differ dramatically from the lack of effects observed at low running speeds (present data; Copp et al. 2010b). This identifies, for the first time, that nNOS-derived NO is an integral controller of skeletal muscle blood flow; acting in a fibre-type- and exercise-intensity-dependent manner during whole-body locomotory exercise that contrasts markedly with eNOS-derived NO. Specifically, eNOS (plus nNOS) blockade with l-NAME induces the greatest blood flow and VC decrements in the most highly oxidative fibres and, as such, is manifested at both low and high running speeds. The present investigation has important implications for cardiovascular disorders associated with reduced NO bioavailability and blood flow impairments during exercise and diseases associated specifically with altered nNOS structure and/or function.

Acknowledgments

The authors would like to thank Ms K. Sue Hageman for excellent technical assistance. S.W.C. is supported by an American Heart Association (AHA) Midwest Affiliate Pre-doctoral Fellowship. Experiments were funded by a Kansas State University SMILE (to T.I.M.), NIH (HL-108328, to D.C.P.), and AHA Midwest Affiliate Grant (10GRNT4350011, to D.C.P.) awards.

Glossary

eNOS

endothelial nitric oxide synthase

HR

heart rate

l-NAME

NG-nitro-l-arginine-methyl-ester

MAP

mean arterial pressure

nNOS

neuronal nitric oxide synthase

NO

nitric oxide

NOS

nitric oxide synthase

SMTC

S-methyl-l-thiocitrulline

VC

vascular conductance

Author contributions

Conception and design of the experiments: S.W.C., T.I.M. and D.C.P. Data collection and analysis: S.W.C., C.T.H., S.K.F., D.M.H., T.I.M. and D.C.P. Manuscript preparation and revision: S.W.C., C.T.H., S.K..F., D.M.H., T.I.M. and D.C.P. All authors have approved the final version of the manuscript.

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