Endogenous nitric oxide in the control of skeletal muscle oxygen extraction during exercise

Our previous studies uncovered an inhibitory effect of nitric oxide (NO) on leg skeletal muscle respiration in dogs at rest. The role of NO in the modulation of O2 consumption and O2 extraction in hindlimb muscle during elevated metabolic states was investigated in chronically instrumented dogs while walking and at three exercise intensities which markedly increased hindlimb blood flow. Walking resulted in increased O2 consumption by 17 +/- 4 mL min-1 and O2 extraction from 24 +/- 1 to 37 +/- 8%, with no alteration in hindlimb blood flow (BFLeg) and vascular resistance (VRLeg). Running at the highest speed (9.1 mph) resulted in an increase in BFLeg from 0.67 +/- 0.05 to 2.2 +/- 0.1 L min-1, a reduction of VRLeg and elevation of hindlimb O2 consumption from 33 +/- 3 to 226 +/- 21 mL min-1 and O2 extraction from 29 +/- 2 to 61 +/- 5%, with a decrease in leg venous PO2 from 38 +/- 1 to 25 +/- 1 mmHg. After nitro-L-arginine (NLA) (35 mg kg-1, i.v.) to inhibit endogenous NO synthesis, walking caused greater increases in hindlimb O2 consumption (29 +/- 5 mL min-1) and O2 extraction (43 +/- 1 to 60 +/- 3%) (both P < 0.05), with no significant change in BFLeg. During running at the highest speed, BFLeg was 1.9 +/- 0.1 L min-1 (P < 0. 05) and VRLeg was higher, accompanied by increases in hindlimb O2 consumption from 49 +/- 7 to 318 +/- 24 mL min-1 and O2 extraction from 41 +/- 2 to 79 +/- 4% (both P < 0.05), with a greater decrease in leg venous PO2 from 33 +/- 1 to 20 +/- 1 mmHg (P < 0.05). Similar results were found for intermediate levels of exercise. Our results indicate that NO modulates hindlimb skeletal muscle O2 extraction and O2 usage whether blood flow increased or not during exercise.     

Lactate concentrations in human skeletal muscle biopsy, microdialysate and venous blood during dynamic exercise under blood flow restriction

The intramuscular microdialysate lactate concentration during dynamic exercise with various degrees of blood flow restriction and its relation to lactate concentration in skeletal muscle biopsy and venous blood were studied. Nine healthy males performed three one-legged knee extension exercises (Ex 1–3). Blood flow was restricted stepwise by applying supra-atmospheric pressure over the working leg. Microdialysate mean (range) lactate concentrations at the end of the exercise periods were 3.2 (0.5–6.6), 4.4 (1.1–9.8) and 7.9 (1.1–11.6) mmol·l^–1 during unrestricted, moderately restricted and severely restricted blood flow respectively. There was a significant correlation between microdialysate and venous lactate concentrations at the end of all three exercise periods. Microdialysate lactate concentration correlated significantly to skeletal muscle biopsy lactate concentration at the end of Ex 1. In conclusion, microdialysate lactate concentration in the working muscle increased step-wise with increasing blood flow restriction. It showed a better correlation to venous than to muscle biopsy lactate, which is possibly partly explained by the characteristics of diffusion between body compartments and differences in time resolution between the methods used.An erratum to this article can be found at     

Adenosine and nitric oxide in exercise-induced human skeletal muscle vasodilatation

The vasoactive substances adenosine and nitric oxide (NO) are credible candidates in the local regulation of skeletal muscle blood flow. Adenosine and NO have both been shown to increase in skeletal muscle cells and interstitial fluid during exercise and the enzymes responsible for their formation, AMP 5'-nucleotidase and NO synthase (NOS), have been shown to be activated upon muscle contraction. In vitro as well as in vivo evidence suggest that the contraction-induced increase in interstitial adenosine concentration largely stems from extracellular formation via the membrane-bound ecto-form of AMP 5'-nucleotidase. It remains unclear whether the exercise-induced NO formation in muscle originates from endothelial NOS in the microvascular endothelium, or from neuronal NOS (nNOS) in nerve cells and muscle fibres. Functional evidence for the role of adenosine in muscle blood flow control stems from studies using adenosine receptor agonists and antagonists, adenosine deaminase or adenosine uptake inhibitors. The majority of these studies have been performed on laboratory animals and, although the results show some discrepancy, the majority of studies indicate that adenosine does participate in the regulation of muscle blood flow. In humans, evidence is lacking. The role of NO in the regulation of skeletal muscle blood flow has mainly been studied using NOS inhibitors. Despite a large number of studies in this area, the role of NO for the contraction-induced increase in skeletal muscle blood flow is uncertain. The majority, but not all, human and animal studies show that, whereas blockade of NOS reduces muscle blood flow at rest and in recovery from exercise, there is no effect on the exercise-induced increase in muscle perfusion. Conclusive evidence for the mechanisms underlying the precise regulation of the multiphased increase in skeletal muscle blood flow during exercise and the role and potency of various vasoactive substances, remain missing.     

Quantitative assessment of nitric oxide in rat skeletal muscle and plasma after exercise

Nitric oxide (NO), a short-lived vasoactive substance that has multiple physiological functions, is also involved in skeletal muscle physiology. This work examines the levels of nitrate (the metabolic end-product of NO) in muscle and plasma after different exercise protocols: namely acute, eccentric, cardiac stress and training. Plasma nitrate levels were augmented after strenuous exercise and did not change after training. The vastus intermedius and the gastrocnemius, both oxidative muscles, showed the highest concentrations of cytosolic nitrate after strenuous exercise. NO levels varied, depending on the fibre type, and this may correlate well with the specific contractile function performed. Electronic Publication     

Kinetics of estimated human muscle capillary blood flow during recovery from exercise

The kinetic characteristics of muscle capillary blood flow (Qcap) during recovery from exercise are controversial (e.g. one versus two phases). Furthermore, it is not clear how the overall Qcap kinetics are temporally associated with muscle oxygen uptake (VO2m) kinetics. To address these issues, we examined the kinetics of Qcap estimated from the rearrangement of the Fick equation (Qcap=VO2m/C(a-v)O2) using the kinetics of pulmonary VO2 (VO2p, primary component) and deoxy-haemoglobin concentration ([HHb]) as indices of VO2m and C(a - v)O2 (arterio-venous oxygen difference) kinetics, respectively. VO2p (l min-1) was measured breath by breath and [HHb] (microm) was measured by near infrared spectroscopy during moderate (M; below lactate threshold, LT) and heavy exercise (H, above LT) in nine subjects. The kinetics of Qcap were biphasic, with an initial fast phase (tauI; M=9.3+/-4.9 s and H=6.0+/-3.8 s) followed by a slower phase 2 (tauP; M=29.9+/-8.6 s and H=47.7+/-26.0 s). For moderate exercise, the overall kinetics of Qcap (mean response time [MRT], 36.1+/-8.6 s) were significantly slower than the kinetics of VO2p (tauP; 27.8+/-5.3 s) and [HHb] (MRT for [HHb]; 16.2+/-6.3 s). However, for heavy exercise, there was no significant difference between MRT-[HHb] (34.7+/-10.4 s) and tauP for VO2p (32.3+/-6.7 s), while MRT for Qcap (48.7+/-21.8 s) was significantly slower than MRT for [HHb] and tauP for VO2p. In conclusion, during recovery from exercise the estimated Qcap kinetics were biphasic, showing an early rapid decrease in blood flow. In addition, the overall kinetics of Qcap were slower than the estimated VO2m kinetics.     

Physiology of nitric oxide in skeletal muscle

In the past five years, skeletal muscle has emerged as a paradigm of "nitric oxide" (NO) function and redox-related signaling in biology. All major nitric oxide synthase (NOS) isoforms, including a muscle-specific splice variant of neuronal-type (n) NOS, are expressed in skeletal muscles of all mammals. Expression and localization of NOS isoforms are dependent on age and developmental stage, innervation and activity, history of exposure to cytokines and growth factors, and muscle fiber type and species. nNOS in particular may show a fast-twitch muscle predominance. Muscle NOS localization and activity are regulated by a number of protein-protein interactions and co- and/or posttranslational modifications. Subcellular compartmentalization of the NOSs enables distinct functions that are mediated by increases in cGMP and by S-nitrosylation of proteins such as the ryanodine receptor-calcium release channel. Skeletal muscle functions regulated by NO or related molecules include force production (excitation-contraction coupling), autoregulation of blood flow, myocyte differentiation, respiration, and glucose homeostasis. These studies provide new insights into fundamental aspects of muscle physiology, cell biology, ion channel physiology, calcium homeostasis, signal transduction, and the biochemistry of redox-related systems.     

NADH in human skeletal muscle during short-term intense exercise

The influence of high-intensity bicycle exercise on the redox level and lactate accumulation in skeletal muscle (m. quadriceps femoris) of man has been investigated. Six subjects exercised to exhaustion at a load corresponding to 100% VO2max. Muscle content of NADH, determined by the bioluminescence technique, increased from (means +/- SEM) 0.089 +/- 0.007 mmol/kg dry wt. at rest to 0.190 +/- 0.031 after 2 min of exercise (P less than 0.05) and to 0.213 +/- 0.021 at exhaustion (P less than 0.05). Values after 2 min exercise and at exhaustion were not statistically different (P greater than 0.05). Muscle lactate was increased 13-fold after 2 min of exercise and 22-fold at exhaustion as compared to the resting value. After 10 min recovery NADH was restored back to the pre-exercise level whereas muscle lactate was still elevated. The increase of muscle NADH during exercise is in contrast to earlier studies on isolated animal muscles, where an oxidation of NADH was observed during contractions. The difference might be due to the experimental model (isolated muscle vs. in vivo) or to the analytical method (qualitative data by reflectance fluorimetri from the surface of intact muscle vs. quantitative data from muscle extracts). Calculations of the cytosolic NADH concentration from the lactate dehydrogenase equilibrium show that 95% or more of the NADH is confined to the mitochondrial compartment. The observed increase of muscle NADH therefore imply that the redox potential of the mitochondria is decreased during intense exercise.(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of nitric oxide on in vivo human skeletal muscle properties

We have investigated the action of exogenous nitric oxide (NO) on the strength and contractile properties of human skeletal muscle working in vivo. Maximum isometric voluntary contraction force (MVC) of the quadriceps was measured and superimposed electrical stimulation was used to estimate the level of activation and 'true maximum force' (TMF). Force-frequency relationships were determined to assess changes in contractile properties of the muscle. Subjects in the experimental group (E, n=10) were measured before and during two separate periods of treatment with different doses of glyceryl trinitrate, a NO donor, delivering 100 (GTN100) or 200 (GTN200) microg h-1 as a trans-dermal patch. A control group (C, n=6) was measured during two similar periods whilst taking an oral placebo. There was a significant increase in strength with GTN200 (MVC: +5. 15%; TMF: +3.87%). There was no change in the strength of group C. There was a trend towards reduced forces at submaximal frequencies with GTN administration but the most notable change was a decline in twitch force (approximately 12%, P < 0.05) with GTN100 treatment and this remained depressed throughout the study. No changes were seen in the contractile properties of the control group C. The present results show that GTN treatment increased maximum voluntary strength but decreased twitch tension. The time course and dose-response characteristics indicate that these are two separate actions of NO on human muscle working in vivo.     

Increased nitric oxide synthase activity and Hsp90 association in skeletal muscle following chronic exercise

Exercise training results in dynamic changes in skeletal muscle blood flow and metabolism. Nitric oxide (NO) influences blood flow, oxidative stress, and glucose metabolism. Hsp90 interacts directly with nitric oxide synthases (NOS), increasing NOS activity and altering the balance of superoxide versus NO production. In addition, Hsp90 expression increases in various tissues following exercise. Therefore, we tested the hypothesis that exercise training increases Hsp90 expression as well as Hsp90/NOS association and NOS activity in skeletal muscle. Male, Sprague–Dawley rats were assigned to either a sedentary or exercise trained group (n = 10/group). Exercise training consisted of running on a motorized treadmill for 10 weeks at 30 m/min, 5% grade for 1 h. Western blotting revealed that exercise training resulted in a 1.9 ± 0.1-fold increase in Hsp90 expression in the soleus muscle but no increase in neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase, or endothelial nitric oxide synthase (eNOS). Exercise training also resulted in a 3.4 ± 1.0-fold increase in Hsp90 association with nNOS, a 2.3 ± 0.4-fold increase association with eNOS measured by immunoprecipitation as well as a 1.5 ± 0.3-fold increase in eNOS phosphorylation at Ser-1179. Total NOS activity measured by the rate of conversion of L-[¹⁴C]arginine to L-[¹⁴C]citrulline was increased by 1.42 ± 0.9 fold in soleus muscle following exercise training compared to controls. In summary, a 10-week treadmill training program in rats results in a significant increase in total NOS activity in the soleus which may be due, in part, to increased NOS interaction with Hsp90 and phosphorylation. This interaction may play a role in altering muscle blood flow and skeletal muscle redox status.     

Effects of exhaustive stretch-shortening cycle exercise on muscle blood flow during exercise

Aim: The influence of exhaustive stretch‐shortening cycle exercise (SSC) on skeletal muscle blood flow (BF) during exercise is currently unknown. Methods: Quadriceps femoris (QF) BF was measured in eight healthy men using positron emission tomography before and 3 days after exhaustive SSC exercise. The SSC protocol consisted of maximal and submaximal drop jumps with one leg. Needle biopsies of the vastus lateralis muscles were taken immediately and 2 days after SSC for muscle endothelial nitric oxide synthase (eNOS) and interleukin‐1‐beta (IL‐1β) mRNA level determinations. Results: All subjects reported subjective muscle soreness after SSC (P < 0.001), which was well in line with a decrease in maximal isometric contraction force (MVC) and increase in serum creatine kinase activity (CK) (P = 0.018). After SSC muscle BF was 25% higher in entire QF (P = 0.043) and in its deep and superficial muscle regions, whereas oxygen uptake remained unchanged (P = 0.893). Muscle biopsies revealed increased IL‐1β (30 min: 152 ± 75%, P = 0.012 and 2 days: 108 ± 203%, P = 0.036) but decreased or unchanged eNOS (30 min; ?21 ± 57%, P = 0.050 and 2 days: +101 ± 204%, P = 0.779) mRNA levels after SSC. Conclusion: It was concluded that fatiguing SSC exercise induces increased muscle BF during exercise, which is likely to be associated with pro‐inflammatory processes in the exercised muscle.     

Adenosine and nitric oxide in exercise‐induced human skeletal muscle vasodilatation

The vasoactive substances adenosine and nitric oxide (NO) are credible candidates in the local regulation of skeletal muscle blood flow. Adenosine and NO have both been shown to increase in skeletal muscle cells and interstitial fluid during exercise and the enzymes responsible for their formation, AMP 5′‐nucleotidase and NO synthase (NOS), have been shown to be activated upon muscle contraction. In vitro as well as in vivo evidence suggest that the contraction‐induced increase in interstitial adenosine concentration largely stems from extracellular formation via the membrane‐bound ecto‐form of AMP 5′‐nucleotidase. It remains unclear whether the exercise‐induced NO formation in muscle originates from endothelial NOS in the microvascular endothelium, or from neuronal NOS (nNOS) in nerve cells and muscle fibres. Functional evidence for the role of adenosine in muscle blood flow control stems from studies using adenosine receptor agonists and antagonsits, adenosine deaminase or adenosine uptake inhibitors. The majority of these studies have been performed on laboratory animals and, although the results show some discrepancy, the majority of studies indicate that adenosine does participate in the regulation of muscle blood flow. In humans, evidence is lacking. The role of NO in the regulation of skeletal muscle blood flow has mainly been studied using NOS inhibitors. Despite a large number of studies in this area, the role of NO for the contraction‐induced increase in skeletal muscle blood flow is uncertain. The majority, but not all, human and animal studies show that, whereas blockade of NOS reduces muscle blood flow at rest and in recovery from exercise, there is no effect on the exercise‐induced increase in muscle perfusion. Conclusive evidence for the mechanisms underlying the precise regulation of the multiphased increase in skeletal muscle blood flow during exercise and the role and potency of various vasoactive substances, remain missing.     

Calprotectin is released from human skeletal muscle tissue during exercise

Skeletal muscle has been identified as a secretory organ. We hypothesized that IL-6, a cytokine secreted from skeletal muscle during exercise, could induce production of other secreted factors in skeletal muscle. IL-6 was infused for 3 h into healthy young males ( n = 7) and muscle biopsies obtained at time points 0, 3 and 6 h in these individuals and in resting controls. Affymetrix microarray analysis of gene expression changes in skeletal muscle biopsies identified a small set of genes changed by IL-6 infusion. RT-PCR validation confirmed that S100A8 and S100A9 mRNA were up-regulated 3-fold in skeletal muscle following IL-6 infusion compared to controls. Furthermore, S100A8 and S100A9 mRNA levels were up-regulated 5-fold in human skeletal muscle following cycle ergometer exercise for 3 h at ∼60% of     in young healthy males ( n = 8). S100A8 and S100A9 form calprotectin, which is known as an acute phase reactant. Plasma calprotectin increased 5-fold following acute cycle ergometer exercise in humans, but not following IL-6 infusion. To identify the source of calprotectin, healthy males ( n = 7) performed two-legged dynamic knee extensor exercise for 3 h with a work load of ∼50% of peak power output and arterial–femoral venous differences were obtained. Arterial plasma concentrations for calprotectin increased 2-fold compared to rest and there was a net release of calprotectin from the working muscle. In conclusion, IL-6 infusion and muscle contractions induce expression of S100A8 and S100A9 in skeletal muscle. However, IL-6 alone is not a sufficient stimulus to facilitate release of calprotectin from skeletal muscle.     

Proton efflux in human skeletal muscle during recovery from exercise

In recovery from exercise, phosphocreatine resynthesis results in the net generation of protons, while the net efflux of protons restores pH?to resting values. Because proton efflux rate declines as pH?increases, it appears to have an approximately linear pH-dependence. We set out to examine this in detail using recovery data from human calf muscle. Proton efflux rates were calculated from changes in pH?and phosphocreatine concentration, measured by ³¹P magnetic resonance spectroscopy, after incremental dynamic exercise to exhaustion. Results were collected post hoc into five groups on the basis of end-exercise pH. Proton efflux rates declined approximately exponentially with time. These were rather similar in all groups, even when pH?changes were small, so that the apparent rate constant (the ratio of efflux rate to pH?change) varied widely. However, all groups showed a consistent pattern of decrease with time; the halftimes of both proton efflux rate and the apparent rate constant were longer at lower pH. At each time-point, proton efflux rates showed a?significant pH-dependence [slope 17 (3)?mmol?·?l^?1?· min^?1?·?pH?unit^?1 at the start of recovery, mean (SEM)], but also a significant intercept at resting pH?[16?(3)?mmol?·?l^?1?·?min^?1 at the start of recovery]. The intercept and the slope both decreased with time, with halftimes of 0.37?(0.06) and 1.4 (0.4)?min, respectively. We conclude that over a wide range of end-exercise pH, net proton efflux during recovery comprises pH-dependent and pH-independent components, both of which decline with time. Comparison with other data in the literature suggests that lactate/proton cotransport can be only a small component of this initial recovery proton efflux.     

The role of nitric oxide during healing of trauma to the skeletal muscle

Introduction  

The role of NO in muscle injury is not clear.     

Utilization of long-chain fatty acids in human skeletal muscle during exercise

Long-chain fatty acids (LCFA) are important sources of energy in contracting skeletal muscle: during the course of endurance exercise the contribution of LCFA in energy metabolism increases whereas when the intensity of exercise increases, the energy need is covered more and more by carbohydrates. Although this has been known for nearly 100 years, the mechanisms controlling fatty acid uptake and oxidation during various exercise modes are still not completely elucidated. Besides passive diffusion, data suggest that both membrane-associated and cytosolic fatty acid binding proteins are involved in the uptake of LCFA into skeletal muscle. However, data from human studies suggest that the regulation of fatty acid utilization in skeletal muscle during exercise lies mainly within the entrance into the mitochondria or metabolism within the mitochondria. Although possible compartmentalization within the cell makes definitive conclusions difficult, available evidence suggests that changes in malonyl CoA concentration in muscle do not play a major regulatory role in controlling LCFA oxidation during exercise in man. In contrast, it is suggested that the availability of free carnitine may play a major regulatory role in oxidation of LCFA during exercise.     

Skeletal muscle blood flow in humans and its regulation during exercise

Regional limb blood flow has been measured with dilution techniques (cardio-green or thermodilution) and ultrasound Doppler. When applied to the femoral artery and vein at rest and during dynamical exercise these methods give similar reproducible results. The blood flow in the femoral artery is ～0.3 L min^?1 at rest and increases linearly with dynamical knee-extensor exercise as a function of the power output to 6–10 L min^?1 (Q = 1.94 + 0.07 load). Considering the size of the knee-extensor muscles, perfusion during peak effort may amount to 2–3 L kg^?1 min^?1, i.e. ～100-fold elevation from rest. The onset of hyperaemia is very fast at the start of exercise with T_½ of 2–10 s related to the power output with the muscle pump bringing about the very first increase in blood flow. A steady level is reached within ～10–150 s of exercise. At all exercise intensities the blood flow fluctuates primarily due to the variation in intramuscular pressure, resulting in a phase shift with the pulse pressure as a superimposed minor influence. Among the many vasoactive compounds likely to contribute to the vasodilation after the first contraction adenosine is a primary candidate as it can be demonstrated to (1) cause a change in limb blood flow when infused i.a., that is similar in time and magnitude as observed in exercise, and (2) become elevated in the interstitial space (microdialysis technique) during exercise to levels inducing vasodilation. NO appears less likely since NOS blockade with L -NMMA causing a reduced blood flow at rest and during recovery, it has no effect during exercise. Muscle contraction causes with some delay (60 s) an elevation in muscle sympathetic nerve activity (MSNA), related to the exercise intensity. The compounds produced in the contracting muscle activating the group III–IV sensory nerves (the muscle reflex) are unknown. In small muscle group exercise an elevation in MSNA may not cause vasoconstriction (functional sympatholysis). The mechanism for functional sympatholysis is still unknown. However, when engaging a large fraction of the muscle mass more intensely during exercise, the MSNA has an important functional role in maintaining blood pressure by limiting blood flow also to exercising muscles.