Role of nitric oxide in exercise sympatholysis

J Appl Physiol 97: 417 423, 2004. First published March 12, 2004; 10.1152/japplphysiol.01181.2003. HIGHLIGHTED TOPIC Skeletal and Cardiac Muscle Blood Flow Role of nitric oxide in exercise sympatholysis John B. Buckwalter, Jessica C. Taylor, Jason J. Hamann, and Philip S. Clifford Departments of Anesthesiology and Physiology, Medical College of Wisconsin and Veterans Affairs Medical Center, Milwaukee, Wisconsin 53295 Submitted 4 November 2003; accepted in final form 10 March 2004 Buckwalter, John B., Jessica C. Taylor, Jason J. Hamann, and Philip S. Clifford. Role of nitric oxide in exercise sympatholysis. J Appl Physiol 97: 417 423, 2004. First published March 12, 2004; 10.1152/japplphysiol.01181.2003. The production of nitric oxide is the putative mechanism for the attenuation of sympathetic vasoconstriction (sympatholysis) in working muscles during exercise. We hypothesized that nitric oxide synthase blockade would eliminate the reduction in -adrenergic-receptor responsiveness in exercising skeletal muscle. Ten mongrel dogs were instrumented chronically with flow probes on the external iliac arteries of both hindlimbs and a catheter in one femoral artery. The selective 1-adrenergic agonist (phenylephrine) or the selective 2-adrenergic agonist (clonidine) was infused as a bolus into the femoral artery catheter at rest and during mild and heavy exercise. Before nitric oxide synthase inhibition with N G -nitro-l-arginine methyl ester (), intra-arterial infusions of phenylephrine elicited reductions in vascular conductance of 91 3, 80 5, and 75 6% (means SE) at rest, 3 miles/h, and 6 miles/h and 10% grade, respectively. Intra-arterial clonidine reduced vascular conductance by 65 6, 39 4, and 30 3%. After, intra-arterial infusions of phenylephrine elicited reductions in vascular conductance of 85 5, 85 5, and 84 5%, whereas clonidine reduced vascular conductance by 67 5, 45 3, and 35 3%, at rest, 3 miles/h, and 6 miles/h and 10% grade. 1-Adrenergic-receptor responsiveness was attenuated during heavy exercise. In contrast, 2-adrenergic-receptor responsiveness was attenuated even at a mild exercise intensity. Whereas the inhibition of nitric oxide production eliminated the exercise-induced attenuation of 1-adrenergic-receptor responsiveness, the attenuation of 2-adrenergic-receptor responsiveness was unaffected. These results suggest that the mechanism of exercise sympatholysis is not entirely mediated by the production of nitric oxide. blood flow; autonomic nervous system; dogs; vasoconstriction WITH THE INITIATION OF EXERCISE, there is a substantial increase in oxygen demand in exercising skeletal muscle. To supply the additional oxygen required by the exercising muscle, there is an increase in blood flow to active skeletal muscle. Concomitant with the increase in blood flow to contracting skeletal muscle, there is also an increase in sympathetic nerve activity to skeletal muscle (11). A number of studies have clearly demonstrated that the sympathetic nervous system restrains skeletal muscle hyperemia during exercise (2, 5, 16, 26, 27, 37). However, the relationship between sympathetic vasoconstriction in exercising skeletal muscle and exercise intensity is an issue of continued debate. The preponderance of recent evidence suggests that sympathetic vasoconstriction is attenuated during exercise compared with rest (7, 10, 12, 29, 30, 33, 36). The notion that sympathetic constriction in the arterial vasculature of exercising skeletal muscle is blunted is not a new concept. Indeed, over 40 years ago, a diminished vascular responsiveness to sympathetic stimulation during muscular contraction was termed functional sympatholysis by Remensnyder et al. (28). Although there are some data suggesting the attenuation of 1 -adrenergic-receptor-mediated vasoconstriction in exercising skeletal muscle (7), recent evidence has emerged suggesting that the 2 -adrenergic receptor plays the more prominent role in exercise sympatholysis (1, 33). Although 2 -adrenergic receptors were traditionally described as autoregulatory receptors located on the presynaptic nerve terminal, subsequent studies demonstrated the existence of postsynaptic 2 -receptors in vascular smooth muscle (13). There is convincing evidence that 2 -adrenergic receptors contribute to the neurally mediated tone in the skeletal muscle vasculature at rest (15, 17) and during exercise (2). Although 2 -adrenergic receptors produce vasoconstriction in skeletal muscle during exercise, this vasoconstriction appears to be substantially blunted by even modest amounts of exercise. Recently, studies by Thomas and colleagues (10, 31, 34, 35) have provided evidence that the mechanism by which sympathetic vasoconstriction is attenuated in contracting skeletal muscle is related to the production of nitric oxide. The studies from their laboratory in humans (10, 31) and animals (34, 35) suggest that the production of nitric oxide is a crucial link in sympatholysis. The purpose of this study was to determine in the exercising dog model whether exercise-induced alterations in 1 - and 2 -adrenergic-receptor responsiveness in the vasculature of skeletal muscle are related to the production of nitric oxide. We employed a previously used experimental approach in conscious dogs that allows the examination of -adrenergicreceptor responsiveness in the vasculature of one hindlimb at rest and during exercise. This experimental design employs intra-arterial infusion of small doses of vasoactive drugs in the vasculature of skeletal muscle and has been previously used to demonstrate attenuation of 1 - and 2 - adrenergic-receptor responsiveness at rest and during exercise (7). We hypothesized that inhibition of nitric oxide production with N G -nitro-l-arginine methyl ester () would abolish the attenuation in -adrenergic-receptor responsiveness from rest to exercise. Address for reprint requests and other correspondence: J. B. Buckwalter, Anesthesia Research 151, VA Medical Center, Milwaukee, WI 53295 (E-mail: jbuckwal@mcw.edu). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. http://www.jap.org 417

418 NITRIC OXIDE AND -ADRENERGIC-RECEPTOR RESPONSIVENESS MATERIALS AND METHODS All experimental methods and procedures were approved by the Institutional Animal Care and Use Committee. Mongrel dogs (n 10), weighing 17 24 kg, were selected for their willingness to run on a motorized treadmill. The dogs were instrumented in a series of three sterile surgeries. In the first surgery, the carotid arteries were exteriorized and enclosed in a skin flap (23, 24). Two incisions on the neck, 8 cm in length, were made bilaterally, the first 1 cm lateral of the midline and the other 2.5 cm lateral to the first incision. The subcutaneous tissue was dissected away from the skin between the incisions to create a flap, effectively denervating the skin and eliminating the need for topical anesthesia when catheterizing the artery. After separation of the strap muscles, each carotid artery was dissected from the carotid sheath and wrapped in a skin flap by suturing the edges together with an interrupted stitch using a 3-0 monofilament suture. The strap muscles were approximated with an absorbable suture, and the skin on the neck approximated with an interrupted stitch using a 3-0 monofilament suture. The dogs were allowed to recover from this procedure for at least 10 days before undergoing any other surgeries. In the second surgery, 4-mm ultrasonic transit-time flow probes (Transonic Systems, Ithaca, NY) were placed around the external iliac arteries. A midline incision was made in the abdomen, and the rectus abdominus was retracted to reach the arteries. The external iliac arteries were then separated from the surrounding tissue, and the flow probes were placed around them. The space between the arteries and the probes was filled with a surgical sponge (Merocel, Mystic, CN) to cushion the probe and prevent adipose tissue from infiltrating the probe window, which would interfere with the ultrasonic signal. The incision was closed, and the probe connectors were then tunneled under the skin, the length of the back. The connectors were then exteriorized just distal to the scapulae and the opening was closed around them. The animals were allowed to heal for at least 2 wk before undergoing the final surgery. This provides an adequate amount of time for the tissue to grow around the probe and displace air to allow for the measurement of an adequate acoustic signal. Finally, in one hindlimb, a heparinized catheter (0.045-in. OD, 0.015-in. ID, 60-cm length, Data Science International, St. Paul, MN) was inserted retrogradely into a branch of the femoral artery and tunneled under the skin to the back. The limb with the catheter is referred to as the experimental limb while the contralateral limb is designated the control limb. After the final surgery, the dogs were allowed to recover at least 2 days before participating in an experiment. For all surgeries, the dogs were anesthetized with thiopental sodium (15 30 mg/kg; Gensia Pharmaceuticals, Irvine, CA) and intubated with a cuffed endotracheal cuff. Anesthesia was maintained at a surgical level by mechanically ventilating the dogs with a mixture of 1.5% halothane (Halocarbon Laboratories, River Edge, NJ) and 98.5% oxygen. Antibiotics (cefazolin, Apothecon, Princeton, NJ) and analgesic drugs (buprenorphine hydrochloride, 0.3 mg; Reckitt and Coleman, Kingston-upon-Hull, UK) were given postoperatively. To maintain the patency of the catheter, it was flushed daily with saline and filled with a heparin solution (100 IU heparin/ml in 50% dextrose solution). On the day of the experiment, the dogs were brought to the laboratory, which was maintained at 20 C. The dogs were acutely instrumented with a 20-gauge intravascular catheter (Insyte, Becton Dickinson, Deseret, Sandy, UT) inserted retrogradely into one of the carotid arteries. The catheter was then attached to a solid-state pressure transducer (Ohmeda, Madison, WI) for the purpose of measuring blood pressure, and the flow probes were attached to a transittime flowmeter (Transonic Systems). A sling was used to restrain the dogs during instrumentation and administration of drugs at rest. To examine -adrenergic-receptor responsiveness in the arterial vasculature of skeletal muscle at rest and during exercise, the selective 1-agonist phenylephrine (American Regent Laboratories, Shirley, NY) and the selective 2-agonist clonidine (RBI, Natick, MA) were administered separately via intra-arterial boluses into the experimental hindlimb. These agonists were chosen for their selectivity for the appropriate -adrenergic-receptor subtype and their ability to be dissolved in an aqueous solution. Each drug was given intra-arterially, at a quantity of 5 g/bolus while the dog rested quietly in the sling. After 5 min of rest (sufficient time for experimental limb blood flow to return to baseline), a second infusion of the same drug was given. This protocol was then repeated using the other -agonist. Four total infusions were administered at rest: two infusions of phenylephrine and two of clonidine. The dog was then moved to the treadmill to begin an exercise protocol at either a mild intensity of 3 miles/h (4.8 km/h) or a high intensity of 6 miles/h (9.7 km/h) at a 10% grade. The quantity of drug given during exercise was adjusted from rest to compensate for the elevated steady-state blood flow during exercise. For each individual dog, the dose given during exercise was increased in proportion to the increase in blood flow from rest according to the following formula: dose during exercise 5 g (exercise blood flow/resting blood flow). Two bolus infusions of either phenylephrine or clonidine were administered at each exercise intensity. After 15 20 min of rest, the infusions were repeated at a different exercise intensity. The dog was then allowed to rest for 2 h, after which the exercise protocols were repeated using the other agonist. Each exercise bout lasted 15 min with the first infusion occurring 5 min into the bout, the two infusions separated by 5 min, and the dog completing the protocol 5 min after the second infusion. On a separate day, the above experiments were repeated in the presence of the nitric oxide synthase inhibitor (15 mg/kg iv, Sigma Laboratories, St. Louis, MO). Effective nitric oxide synthase blocked was inferred from a rise in resting mean arterial pressure. To determine whether attenuation in -adrenergic receptor responsiveness was related to a general reduction in the ability of the arterial vasculature of skeletal muscle to constrict during exercise, a separate set of experiments was performed in five dogs. In these experiments, vasoconstriction was produced with intra-arterial infusions of vasopressin (American Regent Laboratories) at rest and during exercise at 3 miles/h (4.8 km/h) and 6 miles/h (9.7 km/h) and 10% grade. As with the other agonist infusions, the dose of the vasopressin was adjusted during exercise for the increase in baseline blood flow. The protocol for these experiments was the same as those described above. The dose of vasopressin administered in this set of experiments was 0.05 U/100 ml of experimental limb blood flow (1 unit 2.5 g). Blood pressure and control and experimental limb blood flow were recorded to a computer (Mac G3 Power PC) using a MacLab system at 100 Hz (ADInstruments, Castle Hill, Australia). Data were analyzed offline, and mean arterial pressure, heart rate, iliac blood flow and vascular conductance (blood flow/mean arterial pressure) were calculated using the MacLab software. Vascular conductance was chosen over vascular resistance because it has been shown that conductance provides a better reflection of a change in vascular tone that is primarily manifested as a change in blood flow rather than pressure (18). Baseline values were averaged over 30 s before infusion, and the response to each infusion was averaged over 1 s (100 consecutive data points) with the peak response chosen for analysis. The average response to the two infusions for each -adrenergic agonist under each condition was used for analysis. Analysis of the data was performed separately for each drug with a repeated-measures analysis of variance. An -level of P 0.05 was used to establish statistical significance. Where significant F ratios were found, a Tukey s post hoc test was performed. All data are expressed as means SE.

NITRIC OXIDE AND -ADRENERGIC-RECEPTOR RESPONSIVENESS Table 1. Hemodynamic measurements at each workload before and after intra-arterial infusion of phenylephrine with and without treatment 419 HR, beats/min MAP, mmhg Limb Blood Blood Pre Post Pre Post Pre Post Pre Post Rest 87 7 85 9 106 4 105 4 148 22 148 24 135 18 11 5* 3 miles/h 165 15 166 19 109 3 113 4 384 30 369 35 328 33 73 26* 6 miles/h and 10% grade 234 19 228 19 120 5 131 6* 1,019 66 1,029 72 892 64 246 72* Rest 49 3 44 4 123 5 123 5 90 17 90 13 72 12 8 3* 3 miles/h 130 11 129 11 124 3 129 5 312 10 301 19 271 22 40 15* 6 miles/h and 10% grade 217 15 220 17 133 4 145 6* 898 38 894 44 722 38 124 41* Values are means SE. Pre, before infusion; Post; after infusion;, N G -nitro-l-arginine methyl ester. There were increases in heart rate (HR), mean arterial pressure (MAP), and blood flow as exercise intensity increased (P 0.05). After infusion, MAP was significantly elevated compared with control, P 0.05. *Significantly different from Pre value, P 0.05. RESULTS Table 1 provides hemodynamic values for phenylephrine infusions under control conditions and after nitric oxide synthase inhibition at rest and during exercise. Exercise produced intensity-dependent increases in heart rate, mean arterial pressure, and limb blood flow (P 0.05). Intravenous administration of increased mean arterial pressure, reduced heart rate, and reduced limb blood flow at rest and during exercise compared with control (P 0.05). Under control conditions and after, intra-arterial infusion of phenylephrine did not alter heart rate or blood flow in the control limb. In addition, phenylephrine infusion did not alter blood pressure except at the highest exercise intensity. However, at rest and at each exercise intensity, intra-arterial infusion of phenylephrine produced significant (P 0.05) reductions in blood flow and conductance to skeletal muscle in the experimental limb. The absolute changes in experimental limb blood flow and conductance produced by phenylephrine under control conditions and after nitric oxide synthase inhibition with are shown in Table 2. Although absolute changes in blood flow and conductance provide information in regard to oxygen delivery and blood pressure regulation, determination of the amount of vasoconstriction (vascular responsiveness) produced by intra-arterial infusion of phenylephrine requires that the data be expressed as a percent change in vascular conductance from baseline (3). Accordingly, Fig. 1 shows vascular responsiveness to 1 -receptor stimulation at rest and during exercise with and without the inhibition of nitric oxide production. Under control conditions, there was an attenuation of 1 -receptor responsiveness during exercise at 6 miles/h and 10% compared with rest. After nitric oxide synthase blockade with, there was no attenuation of 1 -receptor responsiveness during exercise. Table 3 provides hemodynamic values for clonidine infusions under control conditions and after nitric oxide synthase inhibition at rest and during exercise. Exercise produced intensity-dependent increases in heart rate, mean arterial pressure, and limb blood flow (P 0.05). Intravenous administration of increased mean arterial pressure and reduced heart rate at rest and during exercise compared with control (P 0.05). Under control conditions and after, intraarterial infusion of clonidine did not alter heart rate, mean arterial pressure, or blood flow in the control limb. However, at rest and during exercise, intra-arterial infusion of clonidine produced substantial reductions in blood flow and conductance in the experimental limb (Table 4). Vascular responsiveness to 2 -receptor stimulation at rest and during exercise with and without the inhibition of nitric oxide production is illustrated in Fig. 2. Under control conditions, there was an attenuation of Table 2. Absolute changes in blood flow and conductance after intra-arterial infusion of phenylephrine Blood Conductance, ml min 1 mmhg 1 Rest 123 16 1.20 0.19 3 miles/h 254 10 2.30 0.08 6 miles/h and 10% grade 646 62 5.65 0.59 Rest 63 11 0.52 0.09 3 miles/h 230 22 1.85 0.17 6 miles/h and 10% grade 597 35 4.53 0.27 Values are means SE. Fig. 1. Percent changes from baseline in iliac conductance resulting from intra-arterial infusion of the selective 1-agonist phenylephrine under control conditions and after the administration of N G -nitro-l-arginine methyl ester (). Values are means SE. Under control conditions, there was a significant difference (P 0.05) between the amount of constriction produced at rest and during exercise at 6 miles/h and 10% grade. After the administration of, there were no significant differences (P 0.05) in the amount of constriction produced at rest and during exercise.

420 NITRIC OXIDE AND -ADRENERGIC-RECEPTOR RESPONSIVENESS Table 3. Hemodynamic measurements at each workload before and after intra-arterial infusion of clonidine with and without treatment HR, beats/min MAP, mmhg Limb Blood Blood Pre Post Pre Post Pre Post Pre Post Rest 77 5 76 7 105 4 102 6 135 14 104 13 96 11 36 9* 3 miles/h 162 12 159 16 113 4 116 4 362 24 361 22 312 28 187 24* 6 miles/h and 10% grade 234 20 229 15 125 5 127 5 948 52 958 43 840 56 598 77* Rest 52 3 50 4 119 5 119 5 86 10 85 16 73 8 24 5* 3 miles/h 137 7 141 13 130 3 133 4 340 21 340 24 288 26 161 24* 6 miles/h and 10% grade 212 10 217 13 132 4 135 4 907 34 905 27 779 61 519 57* Values are means SE. There were increases in HR, MAP, and blood flow as exercise intensity increased (P 0.05). After infusion, MAP was significantly elevated compared with control, P 0.05. *Significantly different from Pre value, P 0.05. 2 -receptor responsiveness at both exercise intensities compared with rest. This attenuation was not altered (P 0.05) after nitric oxide synthase blockade with. As shown in Fig. 3, intra-arterial infusions of vasopressin produced substantial vasoconstriction at rest and during exercise. There were no statistically significant differences (P 0.05) in the magnitude of the vasoconstriction produced at rest compared with exercise (Fig. 3). Vasopressin reduced experimental limb blood flow by 39 5, 235 22, and 342 40 ml/min and reduced conductance by 0.39 0.02, 2.21 0.18, and 3.26 0.23 ml min 1 mmhg 1 at rest, 3 miles/h, and 6 miles/h and 10% grade, respectively. DISCUSSION This study tested the hypothesis that the exercise-induced attenuation in 1 - and 2 -adrenergic-receptor responsiveness in the vasculature of skeletal muscle is due to the production of nitric oxide. There are three notable findings. First, inhibition of nitric oxide production with eliminated the attenuation in 1 -adrenergic-receptor responsiveness during heavy exercise compared with rest. Second, inhibition of nitric oxide production did not affect the attenuation of 2 -adrenergicreceptor responsiveness. 2 -Adrenergic-receptor responsiveness remained attenuated from rest to exercise in an exercise intensity-dependent manner even after administration. Third, there was not a general reduction in the ability of the arterial vasculature of skeletal muscle to constrict during exercise. Compared with rest, the vasoconstrictor response to intra-arterial infusions of vasopressin was maintained even during heavy exercise. Exercise sympatholysis or a diminished sympathetic vasoconstriction in exercising skeletal muscle has been shown by a number of different laboratories in both animal and human subjects (7, 10, 12, 29, 30, 33, 36). Although the findings from the present study are consistent with the concept of sympatholysis, the physiological mechanism by which sympatholysis occurs remains unclear. -Adrenergic-receptor responsiveness (particularly 2 ) has been shown to be attenuated by a number of factors. In addition to muscle contractions, 2 -adrenergicreceptor-mediated vasoconstriction is attenuated by factors such as reductions in ph (20, 22, 32), hypoxia (20, 32), and ischemia (21). It is possible that all of these conditions, including muscle contraction, act by a similar mechanism. A number of studies by Thomas and colleagues (10, 31, 34, 35) have provided evidence that the mechanism by which sympathetic vasoconstriction is attenuated in contracting skeletal muscle is related to the production of nitric oxide. This evi- Table 4. Absolute changes in blood flow and conductance after intra-arterial infusion of clonidine Blood Conductance, ml min 1 mmhg 1 Rest 59 6 0.55 0.06 3 miles/h 124 15 0.95 0.15 6 miles/h and 10% grade 241 20 1.75 0.21 Rest 48 5 0.34 0.05 3 miles/h 126 8 0.98 0.09 6 miles/h and 10% grade 259 21 2.04 0.18 Values are means SE. Fig. 2. Percent changes from baseline in iliac conductance resulting from intra-arterial infusion of the selective 2-agonist clonidine under control conditions and after the administration of. Values are means SE. Under control conditions, 2-adrenergic-receptor responsiveness was attenuated from rest in an exercise intensity-dependent manner. There were significant differences (P 0.05) in the amount of vasoconstriction produced at rest compared with exercise at 3 miles/h or 6 miles/h and 10% grade. The administration of did not alter this relationship. After, there remained significant differences (P 0.05) in the amount of vasoconstriction produced at rest compared with exercise at 3 miles/h or 6 miles/h and 10% grade.

Fig. 3. Percent changes from baseline in iliac conductance resulting from intra-arterial infusion of vasopressin. Values are means SE. There were no significant differences (P 0.05) in the amount of vasoconstriction produced at rest compared with exercise at 3 miles/h or 6 miles/h and 10% grade. NITRIC OXIDE AND -ADRENERGIC-RECEPTOR RESPONSIVENESS 421 dence comes from carefully conducted experiments in both humans (10, 31) and animals (34, 35). It was on the basis of these results that we hypothesized that a reduction in 1 - and 2 -adrenergic-receptor responsiveness in the vasculature of skeletal muscle was due to the production of nitric oxide. Indeed, we did find that the attenuation in 1 -adrenergicreceptor responsiveness was abolished after the inhibition of nitric oxide synthase production with. However, the attenuation of 2 -adrenergic-receptor responsiveness from rest to exercise was unaffected by nitric oxide inhibition. After the administration of, 2 -adrenergic-receptor responsiveness was attenuated from rest in an exercise intensity-dependent manner just as it was under control conditions. Recently, Dinenno and Joyner (12) showed similar results to those found in the present study. In human subjects, the attenuated constrictor response to clonidine in the exercising skeletal muscle vasculature was unaffected by the inhibition of nitric oxide production. The authors concluded that the production of nitric oxide is not obligatory for exercise sympatholysis in contracting skeletal muscles in humans. This appears to be true in the dog as well for sympatholysis involving the 2 -adrenergic receptor. Taken together, the present results and those from Dinenno and Joyner indicate that the production of nitric oxide may play less of a role in sympatholysis than previously thought. It would appear that nitric oxide may not be the only mechanism by which sympatholysis occurs. Therefore, although the present study does not discount a role for nitric oxide in sympatholysis, an alternative mechanism(s) may also play a role in exercise sympatholysis. The functional significance of exercise sympatholysis is a topic for debate. However, there is reason to believe that sympatholysis may play an important role in optimizing perfusion within the active muscle. The manner in which sympatholysis influences blood flow distribution within the exercising skeletal muscle may be related to the anatomic distribution of the -adrenergic receptors in the skeletal muscle vasculature and the prominent attenuation of 2 -adrenergic-receptor-mediated vasoconstriction by muscle contraction. Faber (14) demonstrated that both 1 - and 2 -adrenergic receptors are present on large arterioles, but only 2 -receptors exist on the terminal arterioles. Dynamic exercise has been shown to produce large increases in sympathetic nerve activity to the exercising skeletal muscle (11). It appears that this increase in sympathetic nerve activity does not produce skeletal muscle vasodilation (4, 8) but is constrictive in nature (2, 5, 16, 26). If it is assumed that the substance(s) that attenuates sympathetic vasoconstriction is being released from the active muscle, terminal arterioles (populated by 2 -adrenergic receptors) in close proximity to metabolically active fibers would be most susceptible to sympatholysis. Thus a large increase in vasoconstrictor sympathetic nerve activity to skeletal muscle with the initiation of muscle contraction would produce vasoconstriction in areas of low metabolic activity, but the vasoconstriction would be attenuated in areas of vigorous contraction, thus shunting blood flow within the muscle to the areas where oxygen demand is highest. During exercise, blood flow to active skeletal muscle is substantially elevated compared with the blood flow levels seen at rest. It could be argued that the diminished response to sympathetic stimulation seen during exercise is a nonspecific result of the elevation in blood flow rather than being distinctly related to exercise. Although we did not specifically investigate this possibility in the present study, a number of laboratories have addressed this concern (12, 33, 36). These groups have shown that, in the absence of muscle contraction, pharmacologically induced increases in blood flow to levels similar to those seen during exercise do not produce a similar attenuation in vasoconstriction as does exercise. It could also be argued that muscle contraction produces a nonspecific change in the ability of the arterial vascular smooth muscle to contract. To examine this possibility, additional experiments were performed in which vasoconstriction was produced by a mechanism not involving the autonomic nervous system. Data from the present study showing that the vasoconstrictor response to intra-arterial infusions of vasopressin was well maintained even during heavy exercise argue against a general reduction in the responsiveness of the arterial vasculature. It is interesting that the vasoconstrictor response to intra-arterial vasopressin was well maintained during exercise in the present study. In contrast to phenylephrine and clondine, vasopressin does not mimic sympathetic vasoconstriction. It is possible that only sympathetic vasoconstriction of the arterial vasculature of skeletal muscle is attenuated during exercise and not vasoconstriction produced by other endogenous vasoconstrictor substances. Previously, Burcher and Garlick (9) examined the vasoconstrictor response of vasopressin and angiotensin in the hindlimb of pentobarbital-anesthetized dog under constant-flow conditions. Although these authors reported a diminished vasoconstrictor effect with these agonists, the experimental model is less than ideal because delivering a constant flow during electrically stimulated muscle contractions undoubtedly produces hindlimb ischemia. Indeed, both pentobarbital and ischemia have been shown to attenuate vasoconstriction independent of exercise (19, 21). In the present study, the vasoconstrictor response to vasopressin was measured in a conscious dynamically exercising dog without the confounding effects of anesthesia. The vasoconstrictor response was well maintained during exercise compared with rest, indicating a continued ability of the vascular smooth muscle to contract. It is clear that exogenous activation of 1 - and 2 -adrenergic receptors produced vasoconstriction during exercise. However, one difficulty in using exogenous infusions is ensuring the same vasoconstrictor stimulus across variable baseline blood

422 NITRIC OXIDE AND -ADRENERGIC-RECEPTOR RESPONSIVENESS flows. We reasoned that intra-arterial infusions of a constant dose of drug at rest and during exercise results in a decrease in the effective concentration of the agonist through dilution by higher baseline blood flows during exercise. This dilution of the agonist would result in less vasoconstriction as baseline blood flow increased during exercise. Indeed, in a previous publication from our laboratory (6), phenylephrine infusions that were not adjusted for changes in baseline blood flow showed less vasoconstriction during exercise compared with rest. However, when the dose of phenylephrine was adjusted from the increase in baseline flow during exercise, it produced the same amount of vasoconstriction as at rest (6). Another potential complication in the present study involves the presence of prejunctional 2 -adrenergic receptors on sympathetic nerve terminals that act in an autoregulatory manner such that stimulation by norepinephrine released into the synapse inhibits further release of norepinephrine. One might expect that intra-arterial infusions of clonidine would stimulate prejunctional 2 -receptors, inhibiting the release of norepinephrine and reducing vasoconstriction in the arterial vasculature of skeletal muscle. However, we believe the primary effect of clonidine infused intra-arterially is to stimulate postjunctional 2 -receptors because our laboratory has previously shown that intra-arterial infusions of clonidine cause similar magnitudes of vasoconstriction in intact and sympathectomized limbs (7). The appropriate expression of data concerning vasomotor function has led to some of the controversy in the literature regarding the issue of sympatholysis. Indeed in some instances, opposite conclusions can be made if resistance instead of conductance is used to express changes in vasomotor function (25). Because of the linear relationship between vascular conductance and flow, conductance should be used to express vasomotor tone in these types of studies (18, 25). In addition, changes in vasomotor function from baseline should be expressed as percent change in conductance if the desire is to compare the degree of vasoconstriction in a vascular bed across differing baseline levels (3). Therefore, in the present study, when comparing the magnitude of vasoconstriction to the agonist infusions at rest and during exercise, the data are expressed as a percent change in conductance. The results from the present study reveal that 2 -adrenergicreceptor responsiveness in the arterial vasculature of skeletal muscle is attenuated from rest to exercise in an exercise intensity-dependent manner. However, 1 -adrenergic receptors in the arterial vasculature of skeletal muscle are more resistant to inhibition, and there is attenuation of 1 -adrenergic-receptor-mediated vasoconstriction only during heavy exercise compared with rest. Inhibition of nitric oxide production abolished the attenuation in 1 -adrenergic-receptor responsiveness during heavy exercise. In contrast, the attenuation of 2 -adrenergic-receptor-mediated vasoconstriction was unaffected by nitric oxide synthase inhibition. These data indicate that the production of nitric oxide is not responsible for sympatholysis involving the 2 -adrenergic-receptor responsiveness. There was no attenuation in vascular responsiveness to intra-arterial infusions of vasopressin during exercise, indicating that the ability of the arterial vasculature to constrict during exercise is well maintained. The functional importance of exercise sympatholysis may be to provide a selective means of shunting blood flow within the contracting muscle to areas of high metabolic activity. ACKNOWLEDGMENTS The authors acknowledge the valuable technical assistance of Paul Kovac and Kelly Allbee. GRANTS This project was supported by the Medical Research Service of the Department of Veterans Affairs and the National Heart, Lung, and Blood Institute. REFERENCES 1. Anderson KM and Faber JE. Differential sensitivity of arteriolar 1- and 2-adrenoceptor constriction to metabolic inhibition during rat skeletal muscle contraction. Circ Res 69: 174 184, 1991. 2. Buckwalter JB and Clifford PS. -Adrenergic vasoconstriction in active skeletal muscles during dynamic exercise. Am J Physiol Heart Circ Physiol 277: H33 H39, 1999. 3. Buckwalter JB and Clifford PS. The paradox of sympathetic vasoconstriction in exercising skeletal muscle. Exerc Sport Sci Rev 29: 159 163, 2001. 4. 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