INTRODUCTION

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AT THE ONSET OF EXERCISE there is a substantial increase in blood flow to active skeletal muscle. This skeletal muscle hyperemia reflects an increased demand for oxygen in active skeletal muscle. The ability of the sympathetic nervous system to restrict blood flow to these working muscles is controversial. A number of studies have shown that sympathetic blockade has no effect on blood flow in active skeletal muscle (5, 8, 17, 19). On the other hand, other studies have clearly demonstrated the ability of the sympathetic nervous system to restrict blood flow in active skeletal muscle (2, 13, 27, 30, 37). An attenuation of vasoconstriction in the arterial vasculature of skeletal muscle during muscle contraction has been reported by a number of investigators (3, 14, 15, 31, 32, 36). This diminished vascular responsiveness to sympathetic stimulation during muscular contraction was termed "sympatholysis" by Remensnyder et al. (31).

The purpose of this study was to examine exercise-induced alterations in ₁-adrenergic-receptor responsiveness in the vasculature of skeletal muscle. We used an experimental approach that allowed examination of ₁-adrenergic-receptor responsiveness in one hindlimb at rest and during exercise while not affecting systemic hemodynamics in a conscious dog. We hypothesized that ₁-adrenergic-receptor-mediated vasoconstriction to an intra-arterial bolus of a selective ₁-agonist would be attenuated from rest to exercise in an exercise-intensity-dependent manner.

	METHODS AND PROCEDURES

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All experimental procedures were approved by the Institutional Animal Care and Use Committee and were conducted in accordance with the American Physiological Society's Guiding Principles in the Care and Use of Animals. Mongrel dogs were selected for their willingness to run on a motorized treadmill and were instrumented in a series of sterile surgical procedures. Anesthesia was induced with thiopental sodium (15-30 mg/kg; Gensia Pharmaceuticals, Irvine, CA). After intubation with a cuffed endotracheal tube, a surgical level of anesthesia was maintained through mechanical ventilation with 1.5% halothane (Halocarbon Laboratories, River Edge, NJ) and 98.5% oxygen. Antibiotics (cefazolin sodium, Apothecon, Princeton, NJ) and analgesic drugs (buprenorphine hydrochloride, 0.3 mg; Reckitt and Coleman, Kingston-upon-Hull, UK) were given postoperatively. During the first surgical procedure, the carotid arteries were placed in skin tubes in the neck so that they could be cannulated percutaneously to measure arterial blood pressure (23, 24). In the second surgery, all dogs were instrumented with flow probes (4- or 6-mm ultrasonic transit-time flow probes, Transonic Systems, Ithaca, NY) around the external iliac artery to each hindlimb to measure blood flow. The cables were then tunneled under the skin to the back. The dogs were given 2 wk to recover from flow probe implantation. In the final surgery, a heparinized catheter (0.045-in. OD, 0.015-in. ID, 60-cm length, Data Science International, St. Paul, MN) for drug infusion was implanted chronically through a side branch into the femoral artery and tunneled to the back of the dog. To maintain patency, the catheter was flushed daily with saline and filled with a heparin solution (100 IU heparin/ml in 50% dextrose solution). The dogs were given at least 2 days to recover from the final surgery before any experiments were performed.

All experiments were performed in a laboratory in which the temperature was maintained below 20°C. A 20-gauge Teflon catheter (Insyte, Becton-Dickinson, Deseret, Sandy, UT) was inserted retrogradely into the lumen of the carotid artery and attached to a solid-state pressure transducer (Ohmeda, Madison, WI). The flow probes were connected to a transit-time flowmeter (Transonic Systems). In all experiments, the dogs ran on the treadmill at two different intensities: 3 miles/h (4.8 km/h), 0% grade and 6 miles/h (9.7 km/h), 0% grade.

Series 1. Six dogs weighing between 16 and 21 kg received a bolus of 25 µg of phenylephrine, a selective ₁-agonist (American Regent Laboratories, Shirley, NY), into one hindlimb at rest, during steady-state exercise, and postexercise. The dogs received five infusions of an intra-arterial bolus of 25 µg of phenylephrine while sitting quietly in a sling. At least 5 min separated all phenylephrine infusions, which was sufficient time for blood flow to return to baseline level and eliminate any tachyphylaxis to the drug. These data were averaged for determination of ₁-adrenergic-receptor responsiveness at rest. The dogs were then moved to the treadmill and exercised in randomized order at either 3 or 6 miles/h. At 5, 10, and 15 min of exercise, the dogs received an intra-arterial bolus of 25 µg of phenylephrine. Administration of phenylephrine at these time points allowed examination of time-dependent differences in ₁-adrenergic-receptor-mediated reductions in blood flow over a 15-min exercise bout. Two additional infusions were given while the dogs rested quietly in the sling, 5 and 10 min postexercise. The postexercise phenylephrine infusions allowed detection of any postexercise reductions in ₁-adrenergic-receptor-mediated vasoconstriction. The dogs then rested for at least 1 h, after which the phenylephrine infusions were repeated in the same manner at the other exercise intensity.

Series 2. Seven mongrel dogs, weighing between 20 and 23 kg, were used in series 2. In this series, ₁-adrenergic-receptor responsiveness at rest and during exercise was determined in a manner similar to series 1. However, the doses of phenylephrine infused in series 2 were adjusted for the prevailing hindlimb blood flow. We reasoned that because hindlimb blood flow increases during exercise, administration of an identical amount of phenylephrine at rest and exercise (as done in series 1) results in a lower effective concentration of the drug during exercise. Therefore, in series 2, a bolus of 10 µg of phenylephrine was infused into one hindlimb at rest and the effective concentration of the drug (µg of drug/ml of blood flow) was calculated for each individual dog. The same effective concentration was administered during exercise by increasing the dose in proportion to the increase in blood flow (exercise drug dose = resting drug dose × exercise blood flow/resting blood flow). To calculate drug dose during exercise, steady-state blood flow measurements were averaged over 30 s with the MacLab system starting 90 s before the point of drug infusion. Intra-arterial infusions of phenylephrine were made at rest, at 5, 10, and 15 min of exercise, but not postexercise.

	RESULTS

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Intra-arterial infusion of phenylephrine produced a localized vasoconstriction in the experimental limb without corresponding changes in blood flow or conductance in the contralateral limb. Furthermore, intra-arterial infusion of phenylephrine did not affect heart rate or blood pressure in either series of experiments. Figure 1 is an original tracing from an individual dog running on a treadmill at 3 miles/h. This tracing shows reductions in experimental limb blood flow without changes in control limb blood flow or mean arterial pressure.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Original record from a dog exercising on the treadmill at 3 miles/h. Intra-arterial infusion of an 1-agonist phenylephrine (PE; 25 µg) into femoral artery of experimental limb reduced iliac blood flow to the same degree at 5, 10, and 15 min of exercise. Note that there were no changes in blood flow in the control (contralateral) limb.

Table 1 gives baseline hemodynamics for series 1. There were statistically significant increases (P < 0.01) in experimental limb blood flow, control limb blood flow, heart rate, and mean arterial pressure from rest to exercise in an intensity-dependent manner. At rest, intra-arterial infusion of 25 µg of phenylephrine reduced blood flow by 71 ± 8 (SE) ml/min. There were significant differences (P < 0.01) in the percent change in conductance in the experimental limb with infusion of 25 µg of phenylephrine at rest compared with exercise (Fig. 2) because of the diluting effects of higher baseline blood flows (Table 1). There were no significant differences (P > 0.01) in the percent change in conductance in the experimental limb with infusion of 25 µg of phenylephrine at 5, 10, or 15 min of exercise at either workload (Fig. 2). The reproducibility of the response to multiple intra-arterial infusions of phenylephrine during one exercise bout is clearly seen in the original tracing in Fig. 1. The percent change in experimental limb conductance was not different at rest compared with the postexercise infusions of phenylephrine at 5 and 10 min for either exercise intensity (Fig. 2).

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Percent changes in iliac conductance with intra-arterial infusion of 25 µg phenylephrine (series 1). Values are means ± SE. Graphs summarize percent changes from baseline in iliac conductance resulting from intra-arterial phenylephrine infusion at rest, at 5, 10, and 15 min of exercise, and at 5 and 10 min postexercise for the experiments at 3 (A) and 6 miles/h (mph; B). There were significant differences between phenylephrine-induced reductions in iliac conductance at rest and during exercise: * statistically significant difference from rest, P < 0.01. There were no statistically significant differences between resting and postexercise phenylephrine infusions.

Table 2 gives baseline hemodynamics for series 2. Heart rate and hindlimb blood flow increased with exercise in an intensity-dependent manner (P < 0.01). As in series 1, there were no differences in ₁-adrenergic-receptor responsiveness at the three different time points during steady-state exercise. Therefore, the data were pooled for comparison between the two workloads and rest. Proportionally adjusted doses, used to maintain the same effective concentration of phenylephrine from rest to exercise, averaged 10 ± 0, 34 ± 6, and 48 ± 9 µg for rest, 3 miles/h, and 6 miles/h, respectively. Intra-arterial infusion of phenylephrine reduced blood flow from baseline by 114 ± 1, 328 ± 28, and 404 ± 15 ml/min at rest, 3 miles/h, and 6 miles/h, respectively. There were no significant differences in the percent change in experimental limb conductance among any of the three conditions (P > 0.01; Fig. 3).

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Relationship between changes in iliac conductance with the same effective concentration of phenylephrine at rest and during exercise at 3 and 6 miles/h (series 2). Values are means ± SE. There were no statistically significant differences in phenylephrine-induced reductions in iliac conductance among any conditions.

	DISCUSSION

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The major new findings in this study are as follows. 1) There are no differences in ₁-adrenergic-receptor-mediated vasoconstriction in response to an intra-arterial bolus of a selective ₁-agonist at rest or during mild or moderate steady-state exercise. 2) There is no alteration in ₁-adrenergic-receptor-mediated vasoconstriction to an intra-arterial bolus of a selective ₁-agonist between 5 and 15 min of steady-state exercise at a mild or moderate workload. 3) Exogenously activated ₁-adrenergic-receptor-mediated vasoconstriction at rest is not altered by an intervening bout of mild or moderate exercise in the dog.

In the present study, exogenous activation of ₁-adrenergic receptors produced substantial vasoconstriction during exercise. Although there was a decrease in the magnitude of vasoconstriction from rest to exercise with 25 µg of phenylephrine in series 1, we believe this was the result of a decrease in the effective concentration of the agonist from rest to exercise through dilution by higher baseline blood flows during exercise. In series 2, when the same effective concentration of the drug was maintained, phenylephrine induced a similar magnitude of vasoconstriction at rest and during dynamic exercise.

There are several distinct advantages to the experimental protocol used in this study. Small intra-arterial infusions of phenylephrine into one hindlimb allowed examination of vascular reactivity without confounding changes in systemic hemodynamics. In essence, exogenous activation of ₁-adrenergic receptors was examined at rest and during steady-state exercise in a functionally isolated hindlimb. Because there were no changes in heart rate, blood pressure, or blood flow in the contralateral limb, the vasoconstrictor effect of phenylephrine was localized and not of sufficient duration to activate the muscle chemoreflex. Performing this study in a conscious, dynamically exercising animal avoided the confounding effects of anesthesia, allowed natural patterns of muscle recruitment, and permitted a higher intensity of exercise than is achievable in anesthetized animal preparations.

Sympatholysis, manifested as diminished skeletal muscle vasoconstriction to direct stimulation of the sympathetic nerves or administration of exogenous vasoconstrictive substances, has been shown in a number of studies (3, 14, 31, 32, 35, 36). However, the topic is somewhat controversial. Recently, O'Leary and colleagues (27) have provided data suggesting there is an increased sympathetic restraint of blood flow during dynamic exercise compared with rest. On the other hand, we have recently reported that there is an exercise-intensity-dependent decrease in ₁-adrenergic-receptor restraint of blood flow during dynamic exercise (2). The results of the present study indicate that this inverse relationship between exercise intensity and tonic ₁-adrenergic-receptor restraint of blood flow is not a result of a decrease in ₁-adrenergic-receptor responsiveness.

The physiological mechanism responsible for sympatholysis is unclear. We reasoned that a postsynaptic mechanism may account for sympatholysis because sympathetic efferent nerve activity to muscle increases in an exercise-intensity-dependent manner (4, 9) and norepinephrine spillover in active muscle increases during exercise (33). Only one previous study has specifically investigated the involvement of postsynaptic receptors in sympatholysis. Burcher and Garlick (3) demonstrated that there was a reduced vascular responsiveness to norepinephrine, vasopressin, and angiotensin during muscle contractions. However, the same study also provided evidence for a presynaptic mechanism because muscle contractions attenuated the vasoconstriction evoked by sympathetic nerve stimulation more than the vasoconstriction elicited by intra-arterial infusions of norepinephrine (3). To our knowledge, the present study is the first to examine potential changes in responsiveness of vascular ₁-adrenergic receptors during dynamic exercise in conscious animals. We postulated that changes in responsiveness of postsynaptic ₁-adrenergic receptors might be the mechanism of exercise sympatholysis. The data show that when the same effective concentration of the drug was maintained, phenylephrine induced a similar magnitude of vasoconstriction at rest and during dynamic exercise. Thus these findings indicate that there is not an exercise-induced change in responsiveness of postsynaptic ₁-adrenergic receptors in the vasculature of skeletal muscle.

₁-Adrenergic-receptor-mediated vasoconstriction appears to be unaffected by changes in pH (20, 34), hypoxia (20, 34), or ischemia (21). In contrast, postsynaptic ₂-adrenergic receptors, which also contribute to vascular tone in canine skeletal muscles (7, 16), are sensitive to modest reductions in pH (20, 22, 34), hypoxia (20, 34), and ischemia (21). It is possible that the phenomenon of sympatholysis primarily involves metabolic inhibition of ₂-adrenergic receptors. However, it must be noted that the effect of electrically stimulated muscle contractions on ₁- and ₂-adrenergic-receptor-mediated vasoconstriction is contradictory (1, 35). Anderson and Faber (1) showed an attenuation of both ₁- and ₂-adrenergic-receptor-mediated vasoconstriction during muscle contractions, but Thomas et al. (35) reported no attenuation of ₁-adrenergic-receptor-mediated vasoconstriction during muscle contractions. Although ₂-adrenergic-receptor responsiveness during exercise was not assessed in this study, we found no attenuation of ₁-adrenergic-receptor-mediated vasoconstriction during exercise. Potential explanations for differences in susceptibility of ₁- and ₂-adrenergic receptors to metabolic inhibition could include different cellular transduction mechanisms or different distribution of receptors in the skeletal muscle vasculature. Evidence for the latter has been presented by Faber (6), who demonstrated that both ₁- and ₂-adrenergic receptors are present on large arterioles, but only ₂-adrenergic receptors exist on the terminal arterioles. In response to sympathetic stimulation, ₁-adrenergic receptors appear to exert the predominant control over the diameter of the large arterioles, whereas ₂-receptors control the diameter of the terminal arterioles (25). The differential distribution and sensitivity of these receptors may provide a selective means of directing blood flow to areas of high metabolic activity in skeletal muscle during exercise.

Several studies have reported time-dependent changes in the magnitude of sympathetic vasoconstriction during exercise (12, 30, 32). Interruption of tonic sympathetic tone during exercise has been employed by two groups to investigate time-dependent effects of sympathetic activity on blood flow to exercising skeletal muscle. Peterson et al. (30) reported hindlimb sympathectomy did not alter blood flow for the first 2 min of exercise in rats but significantly increased it at 5 and 15 min. In contrast, Buckwalter et al. (2) reported the magnitude of ongoing sympathetic vasoconstriction interrupted with ₁-adrenergic-receptor blockade in active skeletal muscle was not different between 2 and 15 min of exercise in dogs. Rowlands and Donald (32) reported reduced vasoconstriction early in exercise with direct stimulation of the sympathetic nerves to contracting skeletal muscle. However, the magnitude of vasoconstriction returned to resting levels by 30 min. A direct method of inducing smooth muscle contraction in active skeletal muscle was also employed in the present study. However, the vasoconstrictor response to exogenous activation of ₁-adrenergic receptors was similar between 5, 10, and 15 min of steady-state exercise and was not significantly different from rest. Thus we found no alteration in the vasoconstriction produced by exogenous activation of ₁-adrenergic receptors in the skeletal muscle vasculature during the first 15 min of exercise. These results eliminate ₁-adrenergic-receptor responsiveness as a mechanism responsible for the attenuation of sympathetic restraint of blood flow early in exercise reported in previous studies.

A single bout of exercise has been shown to be sufficient to attenuate -adrenergic-receptor-mediated contraction of vascular smooth muscle both in vivo (10) and in vitro (11). However, in the present study, a single bout of mild or moderate exercise did not attenuate the response to intra-arterial infusion of phenylephrine in dogs. We found that intra-arterial phenylephrine produced the same magnitude of vasoconstriction after a single bout of exercise as before exercise. These contradictory results may reflect differences in the predominant muscle fiber type between dogs and rabbits. In addition, the previous studies used a very intense bout of exercise while the present study used a mild and moderate bout of exercise.

The appropriate expression of data concerning vasomotor function has led to some of the controversy in the literature regarding the issue of sympatholysis. Although not consistently used, it is well recognized that vascular conductance is a more appropriate expression of vasomotor function than vascular resistance because of its linear relationship with flow (18, 26). Evidence reported by Kjellmer (14) in support of sympatholysis does not support the concept when the data are expressed as conductance rather than resistance (27-29). Rowlands and Donald (32) noted that, when expressing changes in vascular tone from baseline, the percent change is more appropriate than the absolute change. Despite differing baseline blood flows, a given percent reduction in conductance will always reflect a predictable percent reduction in the radius of the vessel. For example, it can be calculated that a 16% decrease in vessel radius will result in a 50% reduction in conductance and a 50% decrease in vessel radius will result in a 94% reduction in conductance. On the other hand, absolute changes in conductance can vary considerably when identical changes in vessel radius are imposed on differing baseline blood flows. The large differences in baseline flows between exercise and rest make it imperative to express changes in vasomotor tone as a percent rather than an absolute change in conductance. The percent change has a predictable relationship with change in radius of the vessel, whereas the absolute change does not and can lead to inappropriate conclusions. Therefore, if the desire is to compare the degree of vasoconstriction in a vascular bed, which by definition reflects a change in radius, the percent change in conductance more accurately describes these changes. Thus, in the present study, we conclude that intra-arterial infusion of phenylephrine into the arterial vasculature of skeletal muscle produced the same degree of vasoconstriction at rest as during mild and moderate steady-state exercise even though the absolute changes in blood flow and conductance were greater as exercise intensity increased.

Although the present study clearly demonstrates that exogenous activation of ₁-adrenergic receptors can cause vasoconstriction in the arterial vasculature of skeletal muscle during exercise, the existence of sympathetic restraint of blood flow in working skeletal muscle under normal conditions has been controversial. Although there have been numerous studies that indicated no sympathetic restraint of blood flow in working skeletal muscle during exercise (5, 8, 17, 19), the preponderance of the evidence indicates that there is indeed sympathetic restraint of blood flow to active skeletal muscle (2, 13, 27, 30, 37). However the relationship between sympathetic restraint of blood flow to active skeletal muscle and exercise intensity is still unclear.

The results from the present study reveal no attenuation of ₁-adrenergic-receptor mediated vasoconstriction in the skeletal muscle vasculature to an intra-arterial bolus infusion of phenylephrine during or after a mild or moderate bout of exercise in the dog. This study does not support the concept of sympatholysis in relation to ₁-adrenergic-receptor-mediated vasoconstriction.