INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS AND PROCEDURES RESULTS DISCUSSION REFERENCES

AT THE ONSET OF EXERCISE, there is an abrupt increase in blood flow to active skeletal muscle. Despite the appeal of a neural mechanism that could account for rapid skeletal muscle vasodilation at the onset of exercise, the preponderance of the evidence does not support the involvement of the autonomic nervous system in exercise hyperemia (2, 4, 6). On the other hand, there is increasing evidence that the sympathetic nervous system constrains blood flow to muscle during steady-state exercise. Dynamic exercise increases postganglionic sympathetic nerve activity to active skeletal muscle (7, 9-11), which elicits vasoconstriction as demonstrated by enhanced steady-state blood flow in contracting muscle after stellate ganglion blockade (12), Bier block (13), acute sympathectomy (18), or local administration of -adrenergic antagonists (1, 5, 16, 22, 23).

Although the aforementioned studies demonstrated sympathetic restraint of blood flow to active skeletal muscle during steady-state exercise, little attention has focused on sympathetic influences on blood flow at the onset of exercise. Two previous studies observed no change in the initial blood flow response to dynamic exercise after sympathectomy (8, 18). In contrast, the greater blood flow response to a single contraction after Bier block (bretylium tosylate) is consistent with sympathetic restraint of blood flow at the onset of exercise (20).

Despite the failure to identify a specific vasodilator metabolite, there is evidence to support the concept that exercise hyperemia is a local phenomenon due to the release of vasodilator agents (15). Several laboratories have described an initial overshoot in blood flow or vascular conductance (2, 6, 8, 18, 19, 21). Although it could be argued that the overshoot in blood flow at the onset of exercise is due to an accumulation of vasodilator metabolites, it has been suggested that the overshoot is attributable to sympathetic vascoconstriction (2, 19, 21), which is evoked by an immediate and sustained increase in sympathetic nerve activity (7, 9, 11). Support for this postulate comes from studies in which autonomic blockade abolished the overshoot in total vascular conductance (19) and femoral vascular conductance (2).

The aim of this study was to examine the influence of sympathetically mediated vasoconstriction on the hyperemic response at the onset of dynamic exercise. Two hypotheses form the basis for this investigation: 1) the initial blood flow response at the onset of exercise is constrained by sympathetically mediated vasoconstriction and 2) the overshoot in blood flow at the onset of exercise is the result of increasing sympathetic vasoconstrictor tone. Both hypotheses were investigated by local administration of -adrenergic antagonists in an exercising hindlimb of chronically instrumented animals.

	METHODS AND PROCEDURES

TOP ABSTRACT INTRODUCTION METHODS AND PROCEDURES RESULTS DISCUSSION REFERENCES

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. Seven mongrel dogs (18-22 kg) were selected for their willingness to run on a motorized treadmill. A series of sterile surgeries was performed for chronic instrumentation of the animals. 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 with 1.5% halothane (Halocarbon Laboratories, River Edge, NJ) and 98.5% oxygen. Postoperatively, animals were given an analgesic for pain management (buprenorphine hydrochlorine, 0.3 mg; Reckitt and Coleman, Kingston-upon-Hull, UK) and treated with antibiotics for 10 days (cefazoline sodium, 1 mg; Apothecon, Princeton, NJ). During the first surgical procedure, the carotid arteries were exposed and placed in skin tubes for percutaneous cannulation and measurement of arterial blood pressure. After a 2-wk recovery period, a second surgery was performed at which time flow probes (4-mm ultrasonic transit-time flow probes; Transonic Systems, Ithaca, NY) were placed around the external iliac artery of each hindlimb to measure hindlimb blood flow. Flow probe cables and connectors were then tunneled under the skin to the back and externalized for access. After a 2-wk recovery period, a final surgery was performed, at which time a heparinized catheter (0.045-in. OD, 0.015-in. ID, 60-cm length; Data Science International, St. Paul, MN) was inserted through a side branch into the femoral artery and tunneled under the skin to the back of the dog to be used for drug infusion. Catheters were flushed daily with saline and filled with a heparin lock (100 IU heparin/ml in 50% dextrose solution) to maintain patency. At least 2 days elapsed between the final surgery and any experimental procedures.

All experiments were performed in a laboratory in which the temperature was maintained below 20°C. On the day of an experiment, animals were brought into the laboratory and placed in a sling where they rested while the flow probes were connected to a transit-time flowmeter (Transonic Systems) and a 20-gauge intravascular catheter (Insyte, Becton-Dickinson, Sandy, UT) was inserted retrogradely into the lumen of the carotid artery and attached to a solid-state pressure transducer (Ohmeda, Madison, WI) for measurement of arterial pressure. After calibration of the pressure transducer and flow probes, the dog was placed on a treadmill.

On separate days, dogs received a bolus intra-arterial infusion of saline (as a control) or combined ₁- and ₂-adrenergic-receptor blockade [100 µg of prazosin (Pfizer, Exton, PA) and 1 mg of rauwolscine (RBI, Natick, MA)], as the dog sat quietly on the treadmill. One minute postinfusion, exercise commenced at 6 miles/h (moderate intensity). The order of the two trials was randomized, and a 48-h wait was imposed after the -blockade experiments to ensure complete elimination of the drugs. Although extensive preliminary experiments have shown these doses to produce effective blockade, this was confirmed in each animal studied. Approximately 2 min into exercise, an intra-arterial bolus of 10 µg of phenylephrine, a selective ₁-agonist (GensiaSicor Pharmaceuticals, Irvine, CA), was administered, followed ~30 s later (when blood flow had returned to baseline) by an intra-arterial bolus of 10 µg of clonidine, a selective ₂-agonist (RBI).

Arterial blood pressure and external iliac blood flow were recorded at 100 Hz directly to a computer (Macintosh G3) with a MacLab system (ADInstruments, Castle Hill, Australia). Data were analyzed off-line with the MacLab software to calculate mean arterial pressure and iliac blood flow. Baseline measurements were averaged over the 10 s immediately before commencement of exercise. During the first 15 s of exercise, all variables were averaged over 1-s intervals (100 consecutive data points) and the highest 1-s average was chosen as the peak blood flow response. Previous investigations in this laboratory have shown that the peak response to dynamic exercise is achieved between 10 and 15 s after commencement of exercise (2, 6). Steady-state blood flows were averaged from 50 to 60 s of exercise. The difference between the peak and steady-state blood flow response was calculated to represent the magnitude of the overshoot.

An  level of P < 0.05 was used to establish statistical significance during all analysis. Statistical analyses of the data were performed with a paired t-test. All data are expressed as means ± SE.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS AND PROCEDURES RESULTS DISCUSSION REFERENCES

Figure 1 presents the ensemble average for hindlimb blood flow for all seven dogs beginning exercise on the treadmill after pretreatment with saline (control) or combined ₁- and ₂-adrenergic blockade. In the control (saline) trial, the commencement of exercise resulted in immediate increases in blood flow in both the control and experimental limbs. Blood flow to both hindlimbs increased rapidly to a peak within the first 10-15 s of exercise followed by a pronounced decline in flow over the remainder of the 60-s bout of exercise. Infusion of the -antagonists prazosin and rauwolscine into the femoral artery of the resting experimental limb increased ipsilateral hindlimb blood flow. Resting blood flow in the experimental limb averaged 135 ± 10 ml/min in the control trial and 563 ± 88 ml/min after -blockade. As in the control trial, commencement of exercise after -blockade induced rapid increases in blood flow in both the control and experimental limbs. The blood flow response in the control limb after -blockade was similar to the hindlimb flow responses observed in the control trial. In contrast, peak blood flow attained at the onset of exercise was enhanced in the experimental limb. In addition, the overshoot in blood flow that was readily apparent in the saline trial and in the control limb during -blockade was not as prominent in the experimental limb under -blockade, exhibiting a much more gradual decline from peak to steady state.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Hindlimb blood flow response to the onset of exercise after an intra-arterial bolus of saline or prazosin + rauwolscine (-blockade) into the experimental limb (n = 7 dogs). Data presented were averaged over 1-s intervals and consist of a 10-s baseline and 60 s of exercise, with arrows indicating the initiation of exercise. For clarity of presentation, error bars are not included. Refer to Fig. 2 for the SEs for the peak and steady-state blood flows. Note the pronounced overshoot in blood flow in both limbs in the saline trial. The overshoot in the experimental limb was markedly attenuated with -blockade.

Figure 2 summarizes the peak and steady-state blood flow values for the experimental limb under both conditions. -Adrenergic blockade before the initiation of exercise resulted in an enhanced peak exercise blood flow (1,227 ± 90 ml/min) compared with the saline condition (933 ± 79 ml/min) (P = 0.01). The steady-state blood flow observed in the experimental limb after -blockade was also significantly elevated (1,058 ± 89 ml/min) compared with the control (629 ± 54 ml/min) (P = 0.002).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Summary of the blood flow responses to treadmill exercise under control (saline) and experimental intervention (-blockade) conditions. Peak represents the highest blood flow attained over a 1-s interval during the first 15 s of exercise. Steady state denotes a 10-s average blood flow taken between 50 and 60 s of exercise. Values are means ± SE. * Significantly different from control, P < 0.01.

The values presented in Fig. 3 depict the magnitude of the blood flow overshoot, which was calculated by subtracting the steady-state blood flow from the peak flow for each condition. It can be readily seen that the magnitude of the overshoot was significantly reduced (P = 0.03) by -adrenergic blockade.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Magnitude of the overshoot in blood flow at the onset of exercise. The overshoot in blood flow was determined as the reduction in blood flow from the peak to the steady-state levels. The magnitude of the overshoot was decreased after -blockade. Values are means ± SE. * Significantly different from control, P < 0.05.

After -blockade at rest, mean arterial blood pressure decreased from 122 ± 6 mmHg in the control trial to 116 ± 6 mmHg after -blockade (P = 0.0035). Mean arterial pressure increased significantly during steady-state exercise and was not different (P = 0.18) between trials (137 ± 8 and 130 ± 5 mmHg for saline and -blockade, respectively).

Efficacy of the blockade was tested with bolus intraarterial infusions of the ₁- and ₂-adrenergic agonists phenylephrine and clonidine, respectively. In every dog, -blockade abolished the reduction in iliac blood flow produced by infusion of phenylephrine (29 ± 6 vs. 2 ± 1%) and clonidine (20 ± 5 vs. 2 ± 2%).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS AND PROCEDURES RESULTS DISCUSSION REFERENCES

There are two major new findings in this study. First, in the absence of sympathetic vasoconstrictor tone, there was an enhanced peak hindlimb blood flow on commencement of treadmill exercise. Second, -adrenergic blockade attenuated the magnitude of the overshoot of hindlimb blood flow at the onset of exercise. These results provide direct evidence that the sympathetic nervous system restrains blood flow at the onset of dynamic exercise.

The sympathetic nervous system is central to the increase and redistribution of cardiac output at the onset of exercise. This redistribution is accomplished by vasoconstriction of inactive tissue, which directs blood flow toward active skeletal muscle to meet the increasing metabolic requirements. In addition to an increase in sympathetic outflow responsible for vasoconstriction in inactive tissues, sympathetic efferent nerve activity to exercising skeletal muscle also increases (7, 9-11). Donald et al. (8) concluded that there was no functional significance of sympathetic outflow on blood flow to active skeletal muscle. In their classic study, hindlimb blood flow was measured during treadmill exercise in dogs before and after surgical sympathectomy. Blood flow responses to exercise were virtually identical in the control and sympathectomized limbs, leading to the conclusion that there is little influence of the sympathetic nervous system on blood flow to active skeletal muscle. These negative results may be attributable to long-term adaptations to the chronic sympathectomy because later investigations employing procedures to acutely abrogate the effects of sympathetic outflow to skeletal muscle have yielded the opposite conclusion. Local anesthetic blockade of the stellate ganglion (12), Bier block (13), or intra-arterial phentolamine (22, 23) elevated blood flow in the exercising human forearm, suggesting that sympathetic outflow can constrain blood flow to active muscle. Similarly, acute lumbar sympathectomy raised steady-state blood flow in rats performing treadmill exercise. In addition, studies using intra-arterial infusion of the selective ₁-adrenergic-receptor antagonist prazosin (5, 16) demonstrated the existence of tonic ₁-adrenergic restraint of blood flow to active skeletal muscle in the dog during steady-state exercise. A follow-up study provided evidence of both ₁- and ₂-adrenergic-receptor-mediated vasoconstriction in exercising skeletal muscle (1). In summary, the above studies provide convincing evidence of sympathetic restraint of skeletal muscle blood flow during steady-state exercise and the present data corroborate these findings.

There have been few studies examining sympathetic control of skeletal muscle blood flow at the onset of exercise. In the study by Donald et al. (8) discussed above, there are no summary data presented, but blood flow curves in Fig. 8 of that study show no systematic differences in the initial blood flow response between intact and sympathectomized limbs. Peterson et al. (18) reported no difference in hindlimb blood flow between intact and sympathectomized rats 30 s after the onset of exercise but significantly higher blood flows in the sympathectomized rats at 5 and 15 min into the exercise bout. It must be noted that this study employed radioactive microspheres, which provide measurements of blood flow at discrete times, thereby limiting the ability to demonstrate temporal changes in blood flow. The results of Shoemaker et al. (20) contrast with the results of these two studies. They measured the forearm blood flow response to a single contraction before and after acute sympathetic blockade (Bier block). Peak forearm blood flow was significantly augmented in the presence of sympathetic blockade. In the present investigation, we produced acute pharmacological sympathectomy by blocking ₁- and ₂-adrenergic receptors, which have been shown previously to mediate vasoconstriction in active skeletal muscle during steady-state exercise (1). Our data reveal an enhanced peak blood flow at the onset of exercise after -adrenergic blockade, indicating that sympathetic vasoconstriction hinders the full expression of metabolic vasodilation at the onset of exercise.

A common observation in our laboratory is the appearance of an overshoot in hindlimb blood flow at the onset of dynamic exercise in the dog. The existence of an overshoot in blood flow and/or conductance also has been shown in previous studies utilizing both animal and human models (2, 6, 8, 18, 19, 21). It is generally believed that the initial vasodilation on commencement of exercise is a local phenomenon due to the release of vasodilator agents in response to the increasing metabolic activity of the muscle. It could be argued that the overshoot in blood flow at the onset of exercise is due to an initial excess accumulation of metabolic vasodilator substances. On the other hand, it has been suggested that the overshoot is attributable to sympathetic vasoconstriction (2, 19, 21). Autonomic blockade, produced by administration of hexamethonium, abolished the overshoot in total vascular conductance (19) and femoral vascular conductance (2). In the present study, we postulated that the overshoot becomes apparent as increasing sympathetic vasoconstriction limits skeletal muscle vasodilation. We reasoned that sympathetic blockade would abolish the overshoot. In fact, -adrenergic blockade significantly attenuated the magnitute of the blood flow overshoot (i.e., the decline from peak to steady-state blood flow). However, our results do not show complete elimination of the overshoot as in the the studies employing ganglionic blockade. The discrepancy between these investigations may be the result of incomplete blockade, although the effectiveness of the -adrenergic block was confirmed in every animal. It is also possible that sympathetic cotransmitters, acting on nonadrenergic receptors (e.g., purinergic or neuopeptide Y), contribute to sympathetic vasoconstriction under these conditions (14, 17). Nevertheless, the present data confirm our hypothesis that the overshoot in blood flow to active muscle at the onset of exercise is, at least in part, a result of an increasing sympathetic vasoconstrictor tone.

There are several advantages to our experimental approach compared with previous investigations. In the present study, we used a conscious animal model in which there are no confounding effects of anesthesia. Second, blood flow to the exercising hindlimbs was measured continuously by using transit-time ultrasound flow probes. Continuous measurement of blood flow allows observation of the overshoot and facilitates detection of transient changes that may be concealed with discrete measurements of blood flow. Investigations that utilize techniques such as radioactive microspheres, plethysmography, or indicator dilution have difficulty resolving an overshoot in blood flow. Furthermore, our experimental model affords the ability to produce acute blockade through intra-arterial infusion of -adrenergic antagonists.

During exercise, there is an increase in conductance and blood flow to active skeletal muscle, which is thought to be the result of local metabolic vasodilation. In addition, there is a immediate and sustained increase in sympathetic nerve activity to exercising skeletal muscle that produces vasoconstriction. This situation, in which there is an elevated blood flow to exercising skeletal muscle in the face of an increased sympathetic nerve activity, represents an apparent paradox (3). It has been asserted that the increase in sympathetic nerve activity and the resulting vasoconstriction in the active skeletal muscle are essential for maintenance of arterial pressure during exercise (16). Because active skeletal muscle receives the largest proportion of cardiac output compared with inactive tissue, active skeletal muscle becomes a primary locus for blood pressure regulation during exercise. Thus the maintenance of muscle perfusion during exercise is ultimately a balance between local vasodilator and sympathetic vasoconstrictor influences.

In conclusion, the results from this investigation show that sympathetic vasoconstriction limits blood flow to active skeletal muscle as evidenced by the elevated peak blood flow at the onset of exercise during sympathetic blockade. In addition, the overshoot in blood flow at the commencement of exercise appears to be the result of increasing sympathetically mediated vasoconstrictor tone of the active muscle.