Sympathetic Vasoconstriction in Skeletal Muscle: Adaptations to Exercise Training

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1 ARTICLE Sympathetic Vasoconstriction in Skeletal Muscle: Adaptations to Exercise Training Timothy P. Just, Ian R. Cooper, and Darren S. DeLorey Faculty of Physical Education and Recreation, University of Alberta, Edmonton, Alberta, Canada JUST, T.P., I.R. COOPER, and D.S. DELOREY. Sympathetic vasoconstriction in skeletal muscle: adaptations to exercise training. Exerc. Sport Sci. Rev., Vol. 44, No. 4, pp , Sympathetic vasoconstriction in the skeletal muscle vascular bed is essential for the regulation of vascular resistance and therefore control of blood pressure and muscle blood flow at rest and during exercise. In this article, we address the hypothesis that aerobic exercise training alters sympathetic vasoconstrictor responsiveness and enhances contraction-mediated inhibition of sympathetic vasoconstriction (functional sympatholysis) through a nitric oxide dependent mechanism. Key Words: nitric oxide, sympatholysis, autonomic nervous system, adrenergic receptor, vasoconstriction Key Points The sympathetic nervous system is integral to the control of blood pressure and skeletal muscle blood flow at rest and during exercise. Aerobic exercise training modulates sympathetic vasoconstrictor responsiveness and enhances functional sympatholysis through a nitric oxide dependent mechanism. Exercise training may be effective in the treatment and prevention of chronic diseases characterized by elevated sympathetic nerve activity and vascular dysfunction. INTRODUCTION The sympathetic nervous system is of primary importance in the regulation of peripheral vascular resistance and is therefore integral to the control of systemic blood pressure and the distribution of cardiac output to vital organs and tissues (23). Skeletal muscle is one of the largest vascular beds in the body; as such, it receives a considerable portion of cardiac output at rest and during exercise and substantially contributes to the regulation of total peripheral resistance. In response to exercise, the cardiovascular system must satisfy the competing demands of adequately perfusing active skeletal muscle while simultaneously maintaining systemic blood pressure (23). Cardiac output increases, and local vasodilation in Address for correspondence: Darren S. DeLorey, Ph.D., Faculty of Physical Education and Recreation, University of Alberta, Van Vliet Complex, Edmonton, Alberta, T6G 2H9 Canada ( ddelorey@ualberta.ca). Accepted for publication: June 30, Associate Editor: Philip S. Clifford, Ph.D., FACSM /4404/ Exercise and Sport Sciences Reviews DOI: /JES Copyright 2016 by the American College of Sports Medicine active skeletal muscle facilitates the matching of local O 2 delivery to local O 2 demand (23). Efferent sympathetic nerve activity increases concomitantly and constricts blood vessels in nonactive tissue to direct blood toward exercising muscle, whereas in active skeletal muscle, vasoconstriction balances the robust local vasodilatation to prevent a profound decrease in peripheral vascular resistanceandadeclinein blood pressure. In contracting skeletal muscle, the vasoconstrictor response to sympathetic nerve activity is blunted, and this contractionmediated inhibition of vasoconstriction has been termed functional sympatholysis (22). Our present understanding is that, in response to exercise, the active skeletal muscle and/or endothelium-release molecules that inhibit sympathetic neurotransmitter release and/or blunt the responsiveness of postsynaptic sympathetic receptor (33,34). The local blunting of sympathetic vasoconstrictor responsiveness in active muscle allows sympathetic nerve activity to constrict inactive tissue, while simultaneously optimizing perfusion of active muscle and the maintenance of blood pressure. Although there is no definitive evidence that any one molecule is required for sympatholysis, the vasodilator nitric oxide (NO) is produced by both the endothelium and skeletal muscle in response to exercise and has been shown to inhibit sympathetic vasoconstriction in resting and contracting skeletal muscle (9,10,32,34). Aerobic exercise training has been shown to reduce blood pressure and enhance skeletal muscle vascular function and the control of muscle blood flow at rest and during exercise (30). The basic physiological mechanisms that underlie exercise training mediated improvements in vascular function have not been elucidated fully. However, exercise training has consistently been shown to increase NO bioavailability and enhance NO-dependent vascular function in experimental models of endothelial dysfunction (30). In healthy people and animals, training adaptations in endothelial function seem to 137

2 be dependent on the limb, muscle, vascular segment investigated, and training paradigm used (10,11,19,28). In this article, we address the novel hypothesis that exercise training alters sympathetic vasoconstrictor responsiveness and contraction-mediated inhibition of sympathetic vasoconstriction through an NO-dependent mechanism (Fig. 1). Advancing our understanding of the fundamental, mechanistic effects of exercise training on sympathetic vascular control may be particularly important in the treatment and prevention of chronic disease conditions that are characterized by elevated sympathetic nerve activity and vascular dysfunction. EXERCISE TRAINING AND EFFERENT SYMPATHETIC NERVE ACTIVITY Inputs from several brain stem nuclei that regulate the cardiovascular system, neurotransmitters, signal molecules (e.g., glutamate, angiotensin II, NO), and peripheral reflexes (e.g., baroreceptors, chemoreceptors) are integrated to determine the level of efferent muscle sympathetic nerve activity (MSNA) at rest and during exercise (14). Exercise training does not seem to alter resting MSNA in healthy people (2). During exercise, the direct measurement of MSNA to contracting muscle is technically challenging and therefore, our understanding of how exercise training impacts sympathetic activity during exercise is based largely on measures of plasma norepinephrine (NE). Although the available evidence is limited and plasma catecholamines may not be reflective of efferent sympathetic activity directed to active muscle (4), the increase in plasma NE in response to exercise seems to be reduced after exercise training in humans (21,25). In healthy animals and animal models of disease characterized by sympatho-excitation (e.g., hypertension, heart failure, myocardial infarction), aerobic exercise training has been shown to reduce resting efferent sympathetic nerve activity (typically measured as renal sympathetic nerve activity) (5,18). Altered central autonomic signaling by glutamate, angiotensin, NO, as well as altered neural reflex function seems to underlie the inhibitory effects of exercise training on sympathetic nerve activity in animal models (5,18). Consistent with findings in animals, exercise training also has been shown to reduce resting MSNA in patient populations (e.g., heart failure, hypertension, metabolic syndrome) with elevated sympathetic activity (2). In summary, aerobic exercise training has been shown to blunt efferent MSNA at rest and in response to exercise, particularly in conditions characterized by elevated sympathetic nerve activity. Further studies are necessary to fully characterize the effects of exercise training on MSNA, particularly during exercise, and to advance our understanding of the doseresponse relation between exercise training and the regulation of efferent sympathetic nerve activity. EXERCISE TRAINING AND SYMPATHETIC VASOCONSTRICTION The functional consequence of MSNA in the skeletal muscle vascular bed is vasoconstriction. The neurotransmitters NE, adenosine triphosphate (ATP), and neuropeptide Y (NPY) bind to postsynaptic α 1 -andα 2 -adrenoreceptors, purinergic (P2X), and NPY-Y1 receptors on vascular smooth muscle to produce vasoconstriction (Fig. 1). The magnitude of vasoconstriction in a vascular bed is a function of the amount of efferent nerve activity, the quantity of neurotransmitter released per Figure 1. A conceptual model illustrating our hypothesis that exercise training modulates sympathetic vasoconstrictor responsiveness and enhances contractionmediated inhibition of sympathetic vasoconstriction through a nitric oxide (NO)-dependent mechanism. The schematic depicts the release of the neurotransmitters norepinephrine (NE), adenosine triphosphate (ATP), and neuropeptide Y (NPY) that bind to α-adrenoreceptors (α 1 and α 2 ), P2X, and NPY-Y1 receptors, respectively, to constrict arterioles in skeletal muscle. NO derived from endothelial nitric oxide synthase (enos) and neuronal NOS (nnos) inhibits sympathetic vasoconstriction. Training adaptations in sympathetic vasoconstrictor responsiveness and NO-mediated inhibition of vasoconstriction are intensity dependent and may occur in response to small volumes of exercise. Dashed arrows indicate areas where there is a particular need for further research. 138 Exercise and Sport Sciences Reviews

3 nerve impulse, and the responsiveness of postsynaptic sympathetic receptors. Our premise is that chronic aerobic exercise training alters the magnitude of the vasoconstrictor response to sympathetic nerve activity (sympathetic vasoconstrictor responsiveness) through effects on postsynaptic sympathetic receptors (Fig. 1). In vitro studies of isolated vascular segments have demonstrated that postsynaptic sympathetic receptors are sensitive to acute changes in their environment (e.g., ph, O 2 ) (29). Chronic exercise training also has been shown to alter the responsiveness of postsynaptic receptors to sympathetic stimuli (13,28,35). In miniature swine, 7 days of treadmill exercise (2 h/d at 3.5 miles/h) augmented the vasoconstrictor response of femoral and brachial arteries to NE (13). In contrast, the vasoconstrictor response to NE was decreased in cremaster arterioles after 6 wk of swim training (1 h/d, 7d/wk) (35) and unchanged in arterioles from gracilis and soleus muscle after 4 wk of mild-intensity (22 28 m/min, 1% 2% grade, min/d, 5 d/wk) and wk of heavy-intensity treadmill exercise training (30 m/min, 15% grade, 1 h/d, 5 d/wk), respectively (6,28). Collectively, the data from studies in isolated blood vessels have demonstrated that exercise training can alter postsynaptic sympathetic receptor responsiveness; however, the effects of exercise training seem to be dependent on the muscle and vascular segment investigated and the exercise training stimulus. Furthermore, in vitro studies of blood vessels may not be reflective of the effects of exercise training on the integrated regulation of the skeletal muscle resistance vasculature in vivo, and this approach does not permit assessment during muscular contraction. Recently, our laboratory has used treadmill exercise training and an in vivo anesthetized rat preparation to investigate the effect of aerobic exercise training on evoked sympathetic vasoconstrictor responsiveness in resting and contracting skeletal muscle. After 4 wk of moderate- (30 m/min, 5 degrees grade, 30 min/d, 5 d/wk) and heavy-intensity (40 m/min, 5 degrees grade, 15 min/d, 5 d/wk) exercise training, sympathetic vasoconstrictor responsiveness in resting skeletal muscle was augmented in a training intensity dependent manner (Fig. 2A) (7,10). Exercise training also enhanced endothelium-dependent vasodilation (EDD), and the improvement in EDD was correlated with the increase in resting sympathetic vasoconstrictor responsiveness (Fig. 2B). This relation suggests that increased sympathetic vasoconstrictor responsiveness may be necessary to oppose an increase in local vasodilator signaling (7). In agreement, aerobic exercise training has been shown to produce parallel increases in EDD and tonic sympathetic vasoconstriction at rest in middle-aged men (27). To explore the mechanism underlying the augmented sympathetic vasoconstrictor responsiveness in resting skeletal muscle after exercise training, we used selective α-adrenoreceptor antagonists to dissect the contributions of α 1 -andα 2 -adrenoreceptors to evoked vasoconstriction in sedentary and exercise-trained rats. Both α 1 -andα 2 -adrenoreceptor mediated vasoconstrictor responses were augmented in resting skeletal muscle of exercisetrained compared with sedentary rats (9,12). Selective α 2 - adrenoreceptor blockade did not alter evoked vasoconstriction in sedentary rats but significantly blunted vasoconstrictor responses in mild- and heavy-intensity-exercise trained rats, suggesting that α 2 -adrenoreceptors contribute to evoked vasoconstrictor Figure 2. A. Augmented resting sympathetic vasoconstrictor responsiveness (expressed as the percentage change of femoral vascular conductance (FVC) in response to sympathetic stimulation) after mild- and heavy-intensity exercise training. B. The relation between the magnitude of sympathetic vasoconstriction and acetylcholine-mediated vasodilation in sedentary, mild-intensity-, and heavy-intensity-exercise trained rats. [Adapted from (7). Copyright 2012 The American Physiological Society. Used with permission.] responses in resting muscle in the exercise-trained state (9). Selective α 1 -adrenoreceptor antagonism blunted evoked vasoconstrictor responses at rest in both sedentary and exercise-trained rats (12). However, α 1 -adrenoreceptor blockade abolished the difference in resting sympathetic vasoconstrictor responsiveness between sedentary and exercise-trained rats, suggesting that α 1 -adrenoreceptor mediated vasoconstriction in resting skeletal muscle was augmented after exercise training (12). EXERCISE TRAINING AND SYMPATHOLYSIS In 1962, Remensnyder et al. (22) demonstrated that direct stimulation of sympathetic nerves produced a smaller vasoconstriction in contracting, compared with resting, skeletal muscle of dogs. This contraction-mediated inhibition of vasoconstriction has been demonstrated repeatedly in animals and humans and has been termed functional sympatholysis. Despite considerable research, the mechanism(s) responsible for sympatholysis has not been identified. However, our present understanding is that a vasoactive molecule released from the endothelium, skeletal muscle, or possibly red blood cells may inhibit the release of sympathetic neurotransmitters or the responsiveness of postsynaptic sympathetic receptors (16,20,34). Recent studies have demonstrated that aerobic exercise training enhances sympatholysis in rodents (10,11,15) and Volume 44 Number 4 October 2016 Exercise Training and Vasoconstriction 139

4 Figure 3. The magnitude of sympatholysis calculated as the difference between the percentage change in femoral vascular conductance (FVC) in response to sympathetic stimulation delivered at 2 Hz (left panel) and 5 Hz (right panel) at rest and during contraction at 30% and 60% maximal contractile force (MCF) in sedentary (open), mild-intensity (light gray), and heavy-intensity trained rats (dark gray) before and after nitric oxide synthase (NOS) blockade with L-NAME (hatched bars; 5 mg kg 1 IV). Values are mean (SD). *Significant difference between all groups at specified contractile state (P <0.05). Significant difference from the sedentary group at specified contractile state (P < 0.05). Significant difference from mild-intensity trained group at specified contractile state (P < 0.05). **Significant difference between control and L-NAME conditions within the same training group (P < 0.05). [Adapted from (10). Copyright 2013 John Wiley and Sons. Used with permission.] humans (16). Indeed, studies from our laboratory have shown that aerobic exercise training in rats enhances sympatholysis in a training intensity dependent manner (Fig. 3) (9 12). Furthermore, our studies suggest that the mechanistic basis for enhanced sympatholysis after exercise training was augmented blunting of α-adrenoreceptor mediated vasoconstriction and enhanced NO-dependent inhibition of vasoconstriction. Selective α 2 -adrenoreceptor blockade did not alter sympatholysis in sedentary and mild-intensityexercise trained rats but modestly reduced sympatholysis in heavy-intensity-exercise trained rats (9). The decline in sympatholysis in the presence of selective α 2 -adrenoreceptor blockade suggests that the enhanced sympatholysis after heavy-intensity exercise training was partly attributable to an increased ability to inhibit α 2 -adrenoreceptor mediated vasoconstriction (9). However, sympatholysis remained greater in exercise-trained compared with sedentary rats in the presence of selective α 2 -adrenoreceptor blockade indicating that exercise training also may have augmented contraction-mediated inhibition of α 1 -adrenoreceptor or nonadrenoreceptormediated vasoconstriction (9). In a subsequent study, selective α 1 -adrenoreceptor blockade abolished exercise training induced improvements in sympatholysis, demonstrating that exercise training augmented contraction mediated blunting of α 1 -adrenoreceptor vasoconstriction (12). Collectively, our data suggest that the regulation of α-adrenoreceptor mediated vasoconstriction in the skeletal muscle vasculature becomes more complex after exercise training. The relative contributions of α 1 -andα 2 -adrenoreceptors to evoked vasoconstrictor responses are altered, and α-adrenoreceptors become more susceptible to inhibition during contraction after aerobic exercise training. These adaptations to training may enhance local control of vasoconstriction and the distribution of blood flow between and within skeletal muscles at rest and during exercise. Consistent with this notion, the distribution of muscle blood flow has been shown to change in response to exercise training. NO MEDIATED SYMPATHOLYSIS NO is synthesized through the conversion of L-arginine to L-citrulline in a reaction catalyzed by the enzyme NO synthase (NOS). NO is a potent vasodilator and several studies have demonstrated that NO contributes to, but is not required for, the control of skeletal muscle blood flow at rest and in response to exercise (3,8,10,24,27,34). NO also has been shown to inhibit sympathetic vasoconstriction in resting and contracting skeletal muscle of animals and humans (3,10,34). As described earlier, our laboratory has reported that aerobic exercise training enhanced sympatholysis in a training intensity dependent manner. Exercise training has been shown to increase NO bioavailability and improve NO-mediated vascular function in human and animal experimental models (10,11,15), making NO an attractive potential mediator of enhanced sympatholysis after exercise training. Pharmacological blockade of NOS reduced sympatholysis in exercise-trained rats, demonstrating that exercise training augmented NO-mediated 140 Exercise and Sport Sciences Reviews

5 sympatholysis (Fig. 3) (10). Consistent with our findings, exercise training also has been shown to enhance sympatholysis in hypertensive rats and NOS inhibition ameliorated the augmented sympatholysis (15). To our knowledge, the effect of exercise training on NO-mediated sympatholysis in humans has not been investigated. However, lifelong physical activity seems to maintain functional sympatholysis and NO bioavailability in older adults (17,19). The cellular mechanism of NO-mediated sympatholysis has not been identified; however, NO has been shown to inhibit both α 1 -and a 2 -adrenoreceptor mediated vasoconstriction in isolated arteries (20). To determine if increased NO-mediated inhibition of α 1 -or α 2 -adrenoreceptor vasoconstriction underlies enhanced NO-dependent sympatholysis after exercise training, we investigated the effects of combined pharmacological blockade of NO production and α 1 -orα 2 -adrenoreceptors on sympathetic vasoconstriction (9,12). In the presence of selective blockade of α 2 -adrenoreceptors, NOS inhibition reduced the magnitude of sympatholysis in heavy-intensityexercise trained rats, suggesting that exercise training enhanced NO-mediated inhibition of α 1 -adrenoreceptor- or nonadrenoreceptor vasoconstriction (9). Consistent with that interpretation, NOS inhibition in the presence of selective of α 1 -adrenoreceptor blockade did not alter sympatholysis in sedentary and exercise-trained rats, suggesting that functional α 1 -adrenoreceptors are required for NO-dependent sympatholysis (12). Similar findings have been reported in untrained dogs, where NOS inhibition abolished contraction-mediated inhibition of α 1 -adrenoreceptors but did not change the blunting of α 2 -adrenoreceptor mediated vasoconstriction during exercise (1). Taken together, the data from these studies indicate that the mechanism responsible for enhanced sympatholysis after exercise training was primarily augmented NO-dependent inhibition of α 1 -adrenoreceptor mediated vasoconstriction. Furthermore, the data suggest that NO is not required to inhibit α 2 -adrenoreceptor and nonadrenoreceptor mediated vasoconstriction in contracting skeletal muscle. WHAT IS THE SOURCE OF NO? Three isoforms of the NOS enzyme exist: endothelial (enos), neuronal (nnos), and inducible (inos) (26). The enos and nnos isoforms are expressed in skeletal muscle, and the endothelium and both enos and nnos have been implicated in the regulation of skeletal muscle blood flow at rest and in response to exercise (26). During exercise, endothelial shear stress increases enos-mediated NO production and an increase in skeletal muscle cytosolic [Ca 2+ ] stimulates NO production by nnos resulting in an increase in NO bioavailability (26). Investigation of the NOS isoform-specific contributions to the inhibition of sympathetic vasoconstriction is important to advance our understanding of the mechanistic regulation of skeletal muscle vascular resistance but also is of clinical relevance. Several chronic diseases (e.g., heart failure, hypertension, metabolic syndrome) are characterized by elevated sympathetic outflow, diminished vascular function, decreased NO bioavailability, and exercise intolerance (5,15,24,32). Elucidation of NOS isoform-specific regulation of vascular tone will help to identify therapeutic targets in the treatment of vascular dysfunction and exercise intolerance. Indeed, a reduced ability to inhibit sympathetic vasoconstriction during muscular contraction has been reported in humans and rodents with diminished nnos expression (24,32), suggesting that NO derived from nnos is essential for sympatholysis. Whether findings from disease populations/models reflect NOS isoform-specific contributions to the inhibition of sympathetic vasoconstriction in health has not been investigated. Our laboratory recently used isoform-specific blockade of nnos followed by nonselective NOS blockade to partition the relative contributions of NO derived from nnos and enos to the inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle (8,11). The rationale for the experimental approach was that an increase in sympathetic vasoconstrictor responsiveness in the presence of selective nnos blockade would be reflective of nnos-mediated inhibition of sympathetic vasoconstriction, and the difference between the constrictor response in the selective nnos and nonselective NOS blockade would reflect enosmediated inhibition of sympathetic vasoconstriction. This approach also allowed determination of the fractional contribution of NO derived from nnos to total NO-mediated inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle. In resting skeletal muscle, NO derived from both enos and nnos inhibited sympathetic vasoconstriction; however, NO derived from enos was particularly important for the inhibition of sympathetic vasoconstriction under resting conditions and was responsible for approximately 70% of total NO-mediated inhibition (8). During exercise, both enos and nnos continued to inhibit vasoconstriction; however, nnos made a proportionally larger contribution to the inhibition of vasoconstriction in contracting skeletal muscle (~50%). These data suggest that nnos-mediated NO production increases markedly in response to exercise and newly derived NO from nnos is critical to inhibit vasoconstriction in contracting skeletal muscle (8,11). We also have investigated the effect of aerobic exercise training on NOS isoform-specific inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle (11). In agreement with our previous study (8), NO derived from both enos and nnos inhibited vasoconstriction in resting skeletal muscle (11). The relative contributions of NO derived from enos (~65%) and nnos (~35%) to total NO inhibition were similar to our prior study and were not different between sedentary and exercise-trained rats, indicating that exercise training does not alter NOS isoform-specific blunting of vasoconstriction under resting conditions. In contracting skeletal muscle, nnos made a proportionally larger contribution to the inhibition of vasoconstriction in both sedentary and exercise-trained rats in agreement with our earlier findings. However, the fractional contribution of nnos-derived NO to total NO inhibition of vasoconstriction in skeletal muscle was substantially larger in exercise-trained (~75%) compared with sedentary (~50%) rats (11). Indeed, selective nnos blockade abolished the enhanced sympatholysis in exercise-trained rats, and subsequent nonselective NOS blockade did not further reduce sympatholysis (Fig. 4) (11). These data demonstrate that the exercise training induced enhancement of NO-dependent sympatholysis was attributable to augmented nnos-dependent sympatholysis. Consistent with enhanced nnos-mediated vascular function, skeletal muscle nnos expression was greater in exercise-trained compared with sedentary rats (Fig. 5) (11). Volume 44 Number 4 October 2016 Exercise Training and Vasoconstriction 141

6 required to establish the role of skeletal muscle fiber types in training-induced adaptations in sympatholysis. In summary, the accumulated evidence from our recent studies suggests that exercise training may be beneficial in the treatment of conditions characterized by reduced nnos expression (assuming the absence of a genetic mutation impacting nnos expression) and NO-dependent vascular dysfunction. Figure 4. The magnitude of sympatholysis calculated as the difference between the percentage change in femoral vascular conductance (FVC) in response to sympathetic stimulation delivered at 2 Hz (upper panel) and 5 Hz (lower panel) at rest and during contraction at 60% maximal contractile force (MCF) in sedentary and exercise-trained rats in control, selective neuronal nitric oxide synthase (nnos) blockade (SMTC; 0.6 mg kg 1 IV) and nonselective NOS blockade conditions (L-NAME 5 mg kg 1 IV). *Indicates a significant difference (P < 0.05) between groups. ^Indicates a significant difference (P < 0.05) from control conditions within a group. [Adapted from (11). Copyright 2014 John Wiley and Sons. Used with permission.] The training-induced increase in nnos expression seemed to occur in muscle with a high proportion of Type II, fast-twitch, glycolytic muscle fibers. It has been argued that vasoconstriction is more readily inhibited during contraction of glycolytic compared with oxidative skeletal muscle (31). Thus, although speculative, it is possible that aerobic exercise training produces adaptations in the fibers or blood vessels of glycolytic muscles that enhance NO-mediated sympatholysis. Further research is SUMMARY AND FUTURE DIRECTIONS The sympathetic nervous system is an important regulator of the cardiovascular system. Sympathetic activation at the onset of exercise is critical to increase and redistribute cardiac output to exercising skeletal muscles. In the active skeletal muscle, sympathetic vasoconstriction and local vasodilation function in an integrative manner to regulate vascular resistance and optimize the perfusion of active muscle/fibers. In this article, we have presented evidence that the sympathetic nervous system is highly responsive to aerobic exercise training and that there is considerable plasticity in the control of sympathetic vasoconstriction in the skeletal muscle vascular bed. Exercise training altered vascular reactivity to sympathetic stimulation and the relative contributions of postsynaptic α- adrenoreceptors to evoked vasoconstriction in resting and contracting skeletal muscle. Exercise training also enhanced functional sympatholysis in a training-intensity dependent manner through an NO-dependent mechanism. The augmented NO-dependent sympatholysis in the exercisetrained state was attributable to enhanced nnos-mediated inhibition of vasoconstriction and augmented blunting of α 1 -adrenoreceptor mediated vasoconstriction (Fig. 1). Further research is required in several areas to advance our mechanistic and integrative understanding of the effects of aerobic exercise training on the regulation of sympathetic vasoconstriction at rest and during exercise (Fig. 1). Knowledge of the effect of exercise training on central and neural reflex control of MSNA during exercise would advance our understanding of how adaptations in the control of efferent sympathetic Figure 5. The endothelial nitric oxide synthase (enos) (A), inducible (inos) (B), and neuronal (nnos) (C) expression from medial gastrocnemius (MG; white), lateral gastrocnemius (LG; light gray), and soleus (Sol; dark gray) muscles in sedentary (S) and exercise-trained (ET) groups. NOS expression was normalized to α-tubulin protein levels. Values are mean (SD). #Indicates a main effect of exercise training on muscle nnos expression. *Indicates a significant difference (main effect) in NOS expression between muscles. P < 0.05 was considered statistically significant. [Adapted from (11). Copyright 2014 John Wiley and Sons. Used with permission.] 142 Exercise and Sport Sciences Reviews

7 outflow are integrated with adaptations in sympathetic vasoconstrictor responsiveness in the control of skeletal muscle vascular resistance and blood pressure. Investigation of the effects of exercise training on the expression and distribution of postsynaptic sympathetic receptors and the cellular mechanisms responsible for exercise training induced alterations in sympathetic vasoconstrictor responsiveness is required to fully elucidate the effects of exercise training on sympathetic vasoconstriction (Fig. 1). Finally, there is no consensus on the optimal dose of exercise training to improve vascular health and function (Fig. 1). We (7,9,10) and others (13,35) have shown that vascular adaptations are sensitive to the intensity and volume of exercise training. However, further knowledge of how the vasculature responds to each component of an exercise training paradigm (frequency, intensity, duration, and mode) as well as the time course of adaptations would inform the development of exercise prescriptions for the treatment and prevention of vascular dysfunction and exercise intolerance. Acknowledgments The authors thank Nicholas Jendzjowsky for his contributions to the projects presented in this article. This study was supported by grants from the Natural Sciences and Engineering Research Council of Canada and the Canadian Foundation for Innovation. References 1. Buckwalter JB, Taylor JC, Hamann JJ, Clifford PS. Role of nitric oxide in exercise sympatholysis. J. Appl. Physiol. 2004; 97: discussion Carter JR, Ray CA. Sympathetic neural adaptations to exercise training in humans. Auton. Neurosci. 2015; 188: Chavoshan B, Sander M, Sybert TE, Hansen J, Victor RG, Thomas GD. 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