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1 J Physiol (2012) pp SYMPOSIUM REVIEW Inefficient functional sympatholysis is an overlooked cause of malperfusion in contracting skeletal muscle Bengt Saltin and Stefan P. Mortensen The Copenhagen Muscle Research Centre, Rigshospitalet, Denmark The Journal of Physiology Abstract Contracting skeletal muscle can overcome sympathetic vasoconstrictor activity (functional sympatholysis), which allows for a blood supply that matches the metabolic demand. This ability is thought to be mediated by locally released substances that modulate the effect of noradrenaline (NA) on the α-receptor. Tyramine induces local NA release and can be used in humans to investigate the underlying mechanisms and physiological importance of functional sympatholysis in the muscles of healthy and diseased individuals as well as the impact of the active muscles training status. In sedentary elderly men, functional sympatholysis and muscle blood flow are impaired compared to young men, but regular physical activity can prevent these age related impairments. In young subjects, two weeks of leg immobilization causes a reduced ability for functional sympatholysis, whereas the trained leg maintained this function. Patients with essential hypertension have impaired functional sympatholysis in the forearm, and reduced exercise hyperaemia in the leg, but this can be normalized by aerobic exercise training. The effect of physical activity on the local mechanisms that modulate sympathetic vasoconstriction is clear, but it remains uncertain which locally released substance(s) block the effect of NA and how this is accomplished. NO and ATP have been proposed as important inhibitors of NA mediated vasoconstriction and presently an inhibitory effect of ATP on NA signalling via P2 receptors appears most likely. (Received 16 July 2012; accepted after revision 3 September 2012; first published online 10 September 2012) Corresponding author B. Saltin: The Copenhagen Muscle Research Centre, Rigshospitalet, Section 7652, Blegdamsvej 9, DK-2100 Copenhagen, Denmark. bengt.saltin@rh.regionh.dk Abbreviations MSNA, muscle sympathetic nerve activity; NA, noradrenaline; L-NAME, N G -nitro-l-arginine methyl ester; L-NMMA, N G -monomethyl-l-arginine; ROS, reactive oxygen species; SNA, sympathetic nerve activity. Introduction The onset of exercise induces not only a very rapid increase in cardiac output but also a major redistribution of the blood flow. Sympathetic vasoconstrictor activity impairs perfusion of inactive muscles whereas active muscles abruptly increase their blood flow. Early studies providing evidence for this regulation were published in Bengt Saltin received his education from the medical school at the Karolinska Institute in Stockholm, where he also defended his doctoral thesis in Since then he has spent all his life performing human exercise related research, first in Stockholm, for some years in the USA and then primarily in Copenhagen, Denmark. This work ranges from patient groups and healthy sedentary people to elite-level atheletes of both sexes, focusing on skeletal muscle plasticity and regulation of hyperaemia and heart function. Stefan P. Mortensen (DMSc)earned his master s degree from the University of Copenhagen, was a PhD student in Niels H. Sechers laboratory and received post doctoral training with Bengt Saltin. He is currently leader of the cardiovascular group at the Centre of Inflammation and Metabolism at Rigshospitalet, Demark. His main research interest is in the regulation of the cardiovascular system during exercise and how cardiovascular function is altered in disease states. This report was presented at The Journal of Physiology Symposium on Blood flow regulation: from rest to maximal exercise, which took place at the Main Meeting of The Physiological Society, Edinburgh, UK on 3 July It was commissioned by the Editorial Board and reflects the views of the authors. DOI: /jphysiol

2 6270 B. Saltin and S. P. Mortensen J Physiol and 1962: Rein (1937) showed that an elevated vasomotor outflow caused a reduction in the blood flow to resting muscles, and Remensnyder et al. (1962) confirmed that vasoconstriction is abolished in contracting muscle and termed this phenomenon functional sympatholysis, i.e. the vasoconstrictor effect of the noradrenaline (NA) release was ineffective or blocked. At the time no precise definition was given of functional sympatholysis, but a definition could be that it is a direct action of one or more exercise-produced compound(s) that block the vasoconstrictive effect of NA via its receptor on smooth muscles. In contrast, vasodilatation is the action of a multitude of factors affecting the tonus of smooth muscles including the endothelial cells and upstream vasodilatation (Clifford & Hellsten, 2004; Hellsten et al. 2012). In the following, some old and newer data will be presented to demonstrate the crucial significance of functional sympatholysis for achieving a blood flow that matches the energy demand of human skeletal muscles. However, the reader should be reminded that during intense exercise involving large muscle groups, functional sympatholysis has to be overcome to preserve blood pressure as peak local muscle blood flow can reach ml kg 1 min 1, which would require a cardiac output close to 100 l min 1 (Saltin, 2007). and oxygen delivery to contracting muscles in exercising humans. Tyramine. A limitation to the study of functional sympatholysis in humans has been the lack of interventions to either manipulate the sympathetic outflow or block its action at the receptor site. Tyramine can elevate the NA release from sympathetic nerve terminals and it was first used almost a century ago in studies of the circulation (Tainter, 1929). Infusion of tyramine caused a pressure response in a wide range of species. In a series of experiments in the following decades some of the complex actions of tyramine were evaluated, which revealed a strong α-receptor effect, butalso thata very high dose stimulated the β-receptors, which resulted in some vasodilatation (for references see Brandão et al. (1978) and Trendelenburg (1972)). Compared to NA, tyramine elicited a slightly slower response, which was thought to be attributed to the time for tyramine to act on the sympathetic nerves for the NA release to become increased. Although one study showed that sympatectomy abolished the tyramine effect (Frewin & Whelan, 1968), the belief is that tyramine primarily has its effect on the nerve terminal (Eisenach et al. 2002). Several of the early studies were reproduced in the 1960s in the human forearm (Cohn, Functional sympatholysis Early studies. The capacity to overcome even very high sympathetic nerve activity (SNA) is demonstrated in studies with dynamic one-legged knee-extensor exercise, where a very marked increase in sympathetic activity accomplished by adding forearm ischemic static exercise, did not alter blood flow to the contracting knee extensor muscles, in spite of the 2- to 4-fold higher muscle sympathetic nerve activity (MSNA) (Fig. 1A)(Strange, 1999). Similar findings were reported when SNA was increased by enlarging the muscle mass involved in the exercise (Fig. 1B). One-legged knee-extensor exercise was performed at a given high submaximal power output with measurements of blood flow and NA spillover. When the active muscle mass was increased by adding exercise with the arms and the other leg, NA spillover increased 4-fold above the control exercise, but leg blood flow was unaltered (Savard et al. 1989). In contrast to the study with static forearm contractions and ischaemia, blood pressure was unchanged and so was vascular conductance (whereas the conductance was lowered with the forearm static contraction as it also caused an elevation in blood pressure). These data and other similar observations in the literature (Joyner et al. 1992; Rosenmeier et al. 2004) support the notion of a powerful role of functional sympatholysis in obtaining an appropriate and well-matched blood flow Figure 1. Muscle sympathetic nerve activity during leg exercise with and without static handgrip exercise (SHG) and forearm ischaemia (F I) (A) and noradrenaline spillover and blood flow in the exercising leg with and without additional active muscle groups (B) Modified from Strange 1999 (A) and Savard et al (B). Licence Number

3 J Physiol Muscle blood flow in healthy ageing ; Frewin & Whelan, 1968), but for a long period the regulation of skeletal muscle microcirculatory perfusion became a question of vasodilatation rather than functional sympatholysis. Mediators of functional sympatholysis in humans. To determine the mechanisms of functional sympatholysis, experiments have been conducted to identlify compounds with an effect that could mimic functional sympatholysis, i.e. block the effect of local tyramine infusion or systemic sympathetic stimulation in a similar manner as exercise blocksthevasoconstrictiveeffectofna. Nitric oxide. Nitric oxide (NO) has been suggested to block the α-receptors in rat muscles (Thomas & Victor, 1998; Thomas et al. 2003). Some support for a role of NO in humans can also be found in children with Duchenne muscular dystrophy who are n-nos deficient and have impaired functional sympatholysis during exercise (Sander et al. 2000). Inhibition of systemic NO formation with N G -nitro-l-arginine methyl ester (L-NAME) has been shown to blunt the modulation of sympathetic vasoconstrictor activity in the exercising forearm (Chavoshan et al. 2002), but local inhibition of NO formation with N G -monomethyl-l-arginine (L-NMMA) does not have the same effect (Dinenno & Joyner, 2003). Impaired functional sympatholysis has also been linked to increased levels of reactive oxygen species (ROS) (Fadel et al. 2012). Two important observations appear to rule out an essential role of NO in blocking sympathetic vasoconstriction. Firstly, exogenous NO stimulation of NO availability does not blunt sympathetic vasoconstriction (Rosenmeier et al. 2003). Secondly, inhibition of NO formation does not reduce exercise hyperaemia at the early onset or the steady-state levels even during maximal knee extensions where the SNA is markedly increase (Fig. 2) (Rådegran & Saltin, 1999; Savard et al. 1989). Although these observations point to a non-obligatory role of NO, it could act in synergy with other substances such as prostacyclin (Dinenno & Joyner, 2004) to mediate functional sympatholysis in a similar fashion as the combined role of these two substances in skeletal muscle vasodilatation (Mortensen et al. 2007). ATP. Studies by Rosenmeier et al. (2004, 2008) employed infusion of tyramine and various purines and pyramines as well as exercise to show that UTP and ATP produced a response, which mimicked functional sympatholysis during exercise. In these studies the focus was on nucleotides in plasma, which implies a role of endothelial P2Y receptors to mediate their effect, because arterial ATP does not appear to cross into the interstitial space of the muscle (Mortensen et al. 2009b). We recently found that 2 weeks of leg immobilization markedly lower interstitial ATP concentrations during exercise (S. P. Mortensen, J. H. Svendsen, Y. Hellsten, N. H. Secher & B. Saltin, in review) in association with an impaired functional sympatholysis (Fig. 3A; Mortensen et al. 2012). This opens up the possibility that interstitial ATP interacts directly with the α-receptor possibly via P2Y receptors or via endothelial P2Y receptors (Mortensen et al. 2009a). A likely scenario could be that luminal and interstitial ATP operate synergistically via P2 receptors, which are also markedly affected by the level of physical activity (Fig. 4; Mortensen et al. 2012). The origin of plasma ATP has been proposed to be endothelial and red blood cells (Mortensen et al. 2011), whereas the skeletal muscle is the main source of interstitial ATP (Tu et al. 2010). Functional sympatholysis as a cause of malperfusion with ageing and in disease Ageing. Wahren et al. (1974) first described that leg blood flow during cycling at a given submaximal power output was lower in middle aged individuals as compared with young individuals. More than 25 years later, Proctor et al reported that ageing was associated with an increase in sympathetic vasoconstrictor activity during exercise. Both of these finding have been confirmed in later studies (Koch et al. 2003; Dinenno et al. 2005; Kirby et al. 2011). A link between the reduced exercise hyperaemia and impaired functional sympatholysis remains to be established, but we have recently observed that chronically endurance trained elderly can maintain an intact functional sympatholysis and sufficient blood flow to maintain aerobic metabolism (Fig. 3A; S.P.Mortensen,M.Nyberg,K.Winding&B. Saltin, in review). In contrast, a similar leg blood flow in chronically sedentary elderly was associated with a lower aerobic metabolism, increased lactate release and impaired Figure 2. Leg vascular conductance (VC) at rest and during submaximal and maximal exercise with and without inhibition of nitric oxide formation (L-NMMA) Modified from Rådegran & Saltin (1999).

4 6272 B. Saltin and S. P. Mortensen J Physiol functional sympatholysis. These observations illustrate the need for local determination of muscle perfusion and metabolism to determine the physiological importance of functional sympatholysis and separate the positive effect of long term exercise training on limb blood flow from the age related impairments in blood flow regulation. Despite of a reduced endothelial function (Taddei et al. 1995) and sympatholysis with ageing, the vasodilatory responsiveness and the sympatholytic effect of arterially infused ATP appear to be maintained in the forearm (Kirby et al. 2010, 2011), suggesting that a role of plasma ATP would have to be caused by reduced levels of plasma ATP. Indeed, interstitial ATP concentrations were reduced in the sedentary elderly compared to the active elderly (Fig.5;S.P.Mortensen,M.Nyberg,K.Winding&B. Saltin, in review). Also, skeletal muscle hyperaemia was not increased when NO bioavailability was increased by acute antioxidant infusion, suggesting that NO is not involved in the lowering of exercise hyperaemia in the ageing leg (M. Nyberg, M. Hellsten & S. P. Mortensen, in review). Figure 3. Percentage change in leg blood flow and vascular conductance when tyramine was infused during one-legged knee extensor exercise with a control, detrained and trained leg (A) and in young, sedentary elderly and trained elderly (right panel) A, Different from control leg; P < 0.05, #different from immobilized leg. B, P < 0.05, different from young men, P < 0.05; different from sedentary elderly men, P < Modified from Mortensen et al. 2012a, 2012b. Figure 4. Skeletal muscle interstitial P2Y2 receptor content and localization A, P2Y2 receptor content in vastus lateralis muscle before and after 14 days of immobilization of one leg and 5 weeks of exercise training of the other leg. Different from the control leg, P < 0.05; #different from the immobilized leg, P < B, immunohistochemical localization of purinergic P2Y 2 receptors in human skeletal muscle. Positive staining for P2Y 2 and endothelium superimposed. P2Y 2 purinergic receptors were evident in endothelial cells of capillaries and microvessels (white arrow), and in vascular smooth muscle cells (white arrowhead). Modified from Mortensen et al. 2009a, 2012.

5 J Physiol Muscle blood flow in healthy ageing 6273 Disease. Several disease states have been characterized by a reduced blood flow during exercise (Esposito et al. 2011; Nyberg et al. 2012) and increased levels of sympathetic vasoconstrictor activity (Parati & Esler, 2012), but the role of functional sympatholysis in this apparent malperfusion remains largely unexplored. Recent evidence suggests that functional sympatholysis is impaired in the exercising forearm of middle-aged men and women with essential hypertension (Vongpatanasin et al. 2011). Surprisingly, the forearm blood flow and vascular conductance during exercise were not different between the normotensive and hypertensive subjects, especially considering that the hypertensive subjects also had a higher MSNA response to exercise (Vongpatanasin et al. 2011). In the exercising leg, blood flow is lower in sedentary hypertensive subjects compared to normotensive subjects matched for activity and age (Nyberg et al. 2012). Moreover, 8 weeks of high intensity exercise normalized the blood flow response to exercise (Nyberg et al. 2012). In contrast to the impaired functional sympatholysis in hypertensive subjects, tyramine did not lower moderate intensity exercise hyperaemia in type II diabetics, suggesting that functional sympatholysis may be intact in this patient group (Thaning et al. 2011). However, these patients also had a normal vasodilator response to ACh and it is therefore possible that functional sympatholysis is affected in type 2 diabetics when endothelial dysfunction is also present. With regard to the role of plasma ATP, the sympatholytic properties of ATP appear to be reduced in type 2 diabetics compared to healthy controls and the vasodilator effect of arterially infused ATP is also reduced (Thaning et al. 2010). Taken together, these observations suggest that plasma ATP may not be obligatory in mediating functional sympatholysis during exercise, especially given that in vitro observations suggest that type 2 diabetics also have impaired ATP release from red blood cells (Sprague et al. 2011). Summary and perspective Since the observation by Remensnyder et al. (1962) of the exercise mediated blunting of an elevated sympathetic nerve activity, which they named functional sympatholysis, ample support is now available demonstrating its role in regulating muscle hyperaemia also in exercising humans. Although not yet fully documented, recent findings indicate that ATP may be a principal mediator of this effect via its binding to a P2 receptor (Fig. 6) and both luminal and interstial ATP are in play. This proposal is based on data from studies on human leg muscles (Rosenmeier et al. 2004, 2008; Mortensen et al. 2012). A major future task is to bridge the gap in our knowledge of similarities and dissimilarities in the regulation of the microcirculatory blood flow in legs and arms (Newcomer et al. 2004; Thijssen et al. 2011). This also relates to functional sympatholysis. A loss of an efficient functional sympatholysis contributes to the reduced muscle perfusion observed in ageing muscles, which is less pronounced in physically active elderly, indicating that lack of muscle usage in older individuals is the primary cause rather than ageing per se. In the hamster model, both ageing and SNA impair upstream vasodilatation (Bearden et al. 2004; Haug & Segal, 2005) and it is therefore tempting to propose that functional sympatholysis may involve substances that Figure 5. Muscle interstitial ATP concentrations at rest and during exercise at 12 W and 45% of maximal workload in young, sedentary elderly and active elderly men Different from baseline conditions, P < 0.05; different from young men (same condition), P < 0.05; different from sedentary elderly men (same condition), P < Modified from Mortensen et al. 2012b. Figure 6. Simplified model of functional sympatholysis, tyramine action and potential role of ATP Luminal and interstial ATP can block the vasoconstrictor effect of noradrenaline (NA) released from sympathetic nerve terminals via P2 receptors. Sympathetic vasoconstrctor activity reduces upstream vasodilation by impairing endothelial signaling and functional sympatholysis may also act by restoring upstream vasodilatation (blue arrows). In humans, NA release can be stimulated by arterial infusion of tyramine. See text for further details.

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