INTRODUCTION

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A VARIETY OF MECHANISMS are thought to explain the large increases in blood flow seen in exercising muscles (12, 16). These include factors released from the active muscles and mechanical interactions between the contracting muscles and the vessels they surround through the "muscle pump" (12, 16, 17). Recently, Segal and Kurjiaka (15) and Welsh and Segal (22) have proposed that acetylcholine (ACh) spillover from motor nerves causes vasodilation in resistance vessels in close proximity to the active muscle fibers. In the latter study, stimulation of motor nerves to hamster retractor muscles in the presence of a postsynaptic neuromuscular-blocking drug was associated with atropine-sensitive vasodilation of resistance vessels in the absence of muscle contractions (22). This dilation caused resistance vessels with baseline diameters between ~20 and 60 µm to increase their diameters by ~10 µm. Such marked dilation should cause large increases in total muscle flow. Additionally, the dilation seen in paralyzed muscle was ~40% of that seen during electrically stimulated contractions at 40% maximum force. By contrast, past studies have demonstrated that motor nerve stimulation during acute muscle paralysis has little impact on whole limb blood flow (1).

With this information as background, the purpose of the present study was to test the hypothesis that activation of motor nerves in the presence of neuromuscular-blocking drugs can cause marked increases in skeletal muscle blood flow in humans. To test this hypothesis, we selectively administered the long-acting neuromuscular- blocking drug pipecuronium to one forearm and asked subjects to attempt to perform handgripping with the paralyzed forearm while forearm blood flow (FBF) was measured (11, 19). The results of our study indicate that attempts to contract acutely paralyzed forearm muscles do not cause marked hyperemic responses in the human forearm.

	MATERIALS AND METHODS

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Subjects

Experimental Procedures

Subject monitoring. Heart rate (HR) and blood pressure (BP) were measured with an automated sphygmomanometer placed around the lower calf. Systemic oxygen saturation was measured continuously by using pulse oximetry.

Electromyography (EMG). To assess electrical activity in the "paralyzed" forearm muscles, two surface EMG electrodes were placed 5 cm apart on the experimental forearm. The raw signal was filtered, amplified, and recorded as previously described (14). An auditory version of the EMG signal was also monitored.

Pipecuronium. Pipecuronium bromide, a long-acting nondepolarizing neuromuscular-blocking drug, was used to eliminate EMG activity and force production in the experimental forearm (11). This drug competes with ACh for the nicotinic receptor at the motor end plate, thereby preventing depolarization and thus muscle contraction. Pipecuronium does not release histamine, block the autonomic nervous system, or bind to muscarinic cholinergic receptors (11).

Regional intravenous drug administration. This technique was employed to administer pipecuronium and provide profound temporary muscle paralysis in the experimental (nondominant) forearm in a way that would cause minimal systemic effects and allow normal force production in the control forearm (19). An 18-gauge catheter was placed in a hand vein of the experimental forearm. The arm was then raised above heart level to empty the forearm veins. This maneuver was followed by inflation of an arm cuff to 250 mmHg. After inflation of the arm cuff, 0.5-1.0 mg pipecuronium dissolved in 40-50 ml normal saline was infused into the isolated forearm. Because of individual variability, the dose and volume of the administered drug differed among the subjects. Cuff inflation was maintained for 10 min to allow time for the drug to diffuse throughout the forearm and bind to the nicotinic receptors. After 10 min, cuff pressure was slowly released and digital compression of the brachial artery was maintained for 5 min to prevent reactive hyperemia in the forearm from flushing the drug into the systemic circulation (3). If the initial dose of pipecuronium did not abolish forearm force production or EMG activity, a second dose was given until sufficient paralysis occurred. After the experimental trial, the subjects were monitored until contraction in the experimental forearm returned to at least 60% of maximum voluntary contraction (MVC).

Experimental Protocol

Data Analysis

	RESULTS

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Effects of Regional Pipecuronium Administration on Muscle Force

Fifteen minutes after neuromuscular blockade was complete, MVC in the experimental forearm was still undetectable in all subjects and unchanged (i.e., ~40 kg) in the control forearm. Thirty minutes after the block, no muscle force could be generated in the experimental forearms of three of the five subjects. Force was barely detectable in one subject and was 40% of MVC in the fifth subject. Sixty minutes after the block, the subjects could generate between 69 and 85% of their maximum force with the experimental forearm.

Blood flow responses. FBF in subjects at rest was 3.4 ± 0.8 ml · 100 ml¹ · min¹. When the subjects were asked to attempt to contract their paralyzed forearm as vigorously as possible, FBF rose to a peak of 4.8 ± 1.2 ml · 100 ml¹ · min¹ (P < 0.05 vs. control). Individual responses are shown in Fig. 1, top. In four of the five subjects, the peak FBF response to attempted contractions was seen during the first minute of effort. During the peak blood flow response, FVR fell from 34.6 ± 7.1 units at rest to 28.6 ± 7.9 units during the peak blood flow response with attempted contractions (P < 0.05 vs. control). In general, the peak response did not occur immediately and was not sustained.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Individual and mean responses for forearm blood flow (FBF; top), heart rate (HR; middle), and mean arterial pressure (MAP; bottom) at baseline and during attempts to contract an acutely paralyzed forearm. Top: modest increases in FBF responses were seen in 4 of 5 subjects. Middle: HR increased in 3 of 5 subjects, with little change or a slight decrease in 2 subjects observed. Bottom: MAP increased markedly in 2 subjects, modestly in 2 subjects, and minimally in 1 subject. FBF data indicate that attempts to maximally contract paralyzed forearm muscles have little impact on muscle blood flow. HR and MAP data indicate that, at least in some subjects, "central command signal" normally associated with contractions was present. Numbers allow indentification of FBF, HR, and MAP responses in each subject. * P < 0.05 for mean values, attempted contractions vs. baseline.

HR and BP responses. In an effort to determine whether attempted contractions caused a "normal" rise in BP we compared the HR and MAP responses during attempted contractions to a 2-min handgrip at 30% MVC performed by the control forearm. In subjects at rest, HR was 62 ± 4 beats/min and MAP was 101 ± 6 mmHg. During the second minute of exercise at 30% of MVC, HR was 69 ± 4 beats/min and MAP was 112 ± 9 mmHg (both P < 0.05 vs. control). Before attempted handgripping with the paralyzed forearm, HR was 63 ± 3 beats/min and MAP was 102 ± 6 mmHg. During attempted contractions, peak HR and MAP responses during attempts to contract the paralyzed forearm were 73 ± 8 beats/min and 109 ± 9 mmHg, respectively. Only the increase in MAP was significant (P < 0.05 vs. baseline). Three of the five subjects showed obvious increases in HR, and two subjects showed little or no effect. The individual HR and BP changes are shown in Fig. 1, middle and bottom, respectively. Two subjects also underwent a cold pressor test, which consisted of placing the nonparalyzed arm in ice-cold water. In these subjects, marked (30-50%) vasoconstriction was seen in the paralyzed forearm.

	DISCUSSION

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The major finding of this study is that attempted contractions of paralyzed human muscles cause only modest and transient changes in FBF. This finding indicates that ACh spillover from motor nerves is not a major vasodilating or hyperemic mechanism in exercising muscle. The technical limitations and physiological implications of this finding are discussed below.

Selective administration of the postsynaptic neuromuscular-blocking drug pipecuronium into the experimental forearm completely eliminated detectable skeletal muscular activity (as demonstrated by an absence of EMG activity and force) during a maximal-effort handgrip attempt by each subject with the paralyzed forearm. However, the normal vasoconstrictor responses to a cold pressor test were seen after pipecuronium administration, confirming that the autonomic innervation of the forearm was intact. This experimental model enabled us to determine whether efferent motor activity without contractions can cause FBF to rise during attempted handgrip exercise.

Issues Related to Our Experimental Paradigm

Three of the five subjects also showed HR or BP responses to attempted contractions, indicating that a "central command" signal normally associated with muscle contractions had occurred (21). Two of the subjects showed an increase in HR of 20 or more beats per minute and an increase in MAP of ~15 mmHg during attempted contractions. In one of the aforementioned subjects, FBF increased transiently during attempted contraction from 4.5 at rest to 5.8 ml · 100 ml¹ · min¹. In the subject, FVR fell from 23 to 20 units. In the other subject, FBF increased transiently from 5.5 to 8.2 ml · 100 ml¹ · min¹ with attempted contraction, and FVR fell from 21 to 16 units. In the above two subjects it is possible a forearm vasodilator response similar to that seen during mental stress occurred (6). This dilation is caused primarily by a rise in BP in combination with transient sympathetic withdrawal to the forearm. These factors then appear to evoke local release of nitric oxide (6, 8).

HR and BP responses seen in our subjects are consistent with the observations of Iwamoto and colleagues (9). They used partial neuromuscular blockade with curare and studied the contribution of central command and afferent feedback from the contracting muscle to HR and BP responses to exercise. They concluded that although the initial increases in both HR and BP are dependent on central command signals (cortical irradiation) that were independent of the muscle force generated, the overall magnitude of the response is dependent, in part, on the size of the active muscle mass and force generated (9). Similar observations were also made by Victor et al. (21). By contrast, in the present study, no muscle force was produced, so the contributions of afferent feedback from the active muscles would have been absent. This may explain why the BP and HR responses in some of the subjects were modest compared with what might have been expected on the basis of the previous studies that used partial neuromuscular blockade so that muscle force could still be generated while central command was augmented (9, 21).

Evidence for ACh Spillover During Exercise Hyperemia

Unfortunately, Welsh and Segal (22) did not report total muscle blood flow for their preparations, but if dilation of the magnitude they observed in the paralyzed hamster muscle microvessels had been seen in the paralyzed human forearm, one would have expected large increases in forearm flow. In this context, rhythmic forearm exercise at 40% of maximum usually evokes a muscle blood flow value of ~20 ml · 100 ml¹ · min¹, and with heavier exercise values of ~40 ml · 100 ml¹ · min¹ can been seen in many subjects (9).

Finally, if ACh spillover from motor nerves plays an important role in exercise hyperemia, then infusion of atropine into blood vessels that perfuse active skeletal muscle should cause transient reductions in skeletal muscle blood flow. In this context, when atropine is infused into the contracting hindlimbs of conscious dogs or rats running on the treadmill, there is no change in blood flow (2, 4). Additionally, when atropine is infused into the brachial artery that perfuses a contracting forearm muscle, no change in forearm flow is noted (18). These observations suggest that ACh from motor nerves (or any source) is not essential for observation of the normal vasodilator response in exercising skeletal muscles.

In summary, our data and studies that have infused atropine into vascular beds perfusing active muscle indicate that ACh released in association with efferent motor activity is unlikely to be a major mediator of skeletal muscle vasodilation and increases in exercising muscle blood flow in humans. The small transient changes in flow we saw during attempts to maximally contract a forearm temporarily paralyzed with the neuromuscular-blocking drug pipecuronium in some subjects are minimal compared with the vast increases in flow possible in exercising muscles (2, 12). These findings highlight the continued uncertainty about "the" factor or factors that are responsible for exercise hyperemia. Along these lines it is more likely that substances released from contracting muscles in conjunction with the muscle pump play a major role in causing exercise hyperemia (2, 12, 16, 17).