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Division of Cardiology, Department of Medicine, David Geffen School of Medicine, University of California, Los Angelos, California 90095
Submitted 5 April 2004 ; accepted in final form 25 June 2004
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ABSTRACT |
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sympathetic nerve activity; exercise; prostaglandins; adenosine
Evidence in healthy animals and humans is accumulating that the muscle mechanoreceptors also play an important role in mediating sympathetic activation during exercise, especially rhythmic exercise (3, 12). Furthermore, animal data suggest that the group III mechanosensory neurons are actually polymodal and that mechanosensitive neurons may be sensitized to mechanical stimuli by ischemic metabolites (1, 21). In anesthetized cats, Rotto and Kaufman (21) found that indomethacin significantly inhibited sympathetic activation during rhythmic muscle contraction. In studies in healthy humans, Batman and colleagues (3) used low-level rhythmic handgrip (RHG) to specifically engage the muscle mechanoreceptors without engaging the metaboreceptors. They found a progressive increase in muscle sympathetic nerve activity (MSNA), beginning after the first few minutes of exercise, and postulated that the mechanosensitive nerve endings had been sensitized by low levels of accumulating ischemic metabolites. Herr and colleagues (12) used signal-averaging techniques during dynamic exercise in healthy humans to demonstrate that muscle mechanoreceptor activation increased MSNA. MSNA responses increased after an onset latency of 4–6 s of exercise, consistent with the concept that these receptors were sensitized by chemical products of muscle contraction. No attempt was made in these studies in humans to identify the responsible metabolic by-product(s), but prostaglandins, lactic acid, potassium ions, adenosine, and others are potential candidates (1, 3, 12, 21).
On the basis of the controversy surrounding which ischemic metabolites activate the muscle metaboreceptors and this additional finding that muscle mechanoreceptors may also be influenced by ischemic metabolites, we thought it would be important to begin sorting out which compounds sensitize muscle mechanoreceptors in healthy humans. From the available animal data and the availability of pharmacological compounds that could be used in humans to interfere with these ischemic metabolites, we chose to focus on two metabolites: prostaglandins and adenosine. The major new finding of this report is that inhibition of cyclooxygenase during exercise dramatically and virtually completely eliminates the reflex sympathetic activation during muscle mechanoreceptor activation in healthy humans. Inhibition of adenosine receptors with aminophylline had no effect on the sympathetic activation during muscle mechanoreceptor activation.
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METHODS |
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After written informed consent on University of California, Los Angelos, Internal Review Board-approved informed consent forms was obtained, 34 normal volunteers (22 men and 12 women, mean age 33.3 ± 2.1 yr) participated in these studies. All subjects were healthy nonsmokers and not taking any medications, including over-the-counter medications such as aspirin.
Microneurography
With the use of the technique of microneurography, MSNA was recorded from the peroneal nerve (8, 26). Briefly, multiunit postganglionic muscle sympathetic nerve recordings were made using a tungsten microelectrode. Signals were amplified by a factor of 50,000–100,000 and band-pass filtered (700–2,000 Hz). Nerve activity was rectified and integrated (time constant 0.1 s) to obtain a mean voltage display of sympathetic nerve activity that was recorded on paper. All recordings of MSNA met previously established and described criteria (8, 26). Muscle sympathetic bursts were identified by visual inspection by a single investigator (H. R. Middlekauff) and expressed as burst frequency (bursts/min) and total activity (arbitrary units/min). Total activity per minute was determined by the sum of the heights of individual bursts per minute. The interobserver and intraobserver variability in identifying bursts is <10% and <5%, respectively (3, 19).
Rhythmic Handgrip Exercise
Low-level rhythmic exercise was used to engage principally the muscle mechanoreceptors without simultaneously activating the metaboreceptors. Maximum voluntary capacity (MVC) was determined by having the subject briefly squeeze a handgrip dynamometer (Stoelting) at maximal levels. The greatest of two maximal contractions was selected as the MVC. Rhythmic handgrip exercise was performed to 20% of the subject's MVC at a rate of 30 contractions/ min for 3 min. A metronome was used to optimize uniformity of contraction rate.
Posthandgrip Circulatory Arrest
Posthandgrip circulatory arrest was performed to assess whether the muscle metaboreceptors had been inadvertently engaged. Five seconds before the conclusion of the exercise, a blood pressure cuff was rapidly inflated to suprasystolic blood pressure levels (220 mmHg) using an automatic inflation device (University of Iowa). Exercise was then terminated so that muscle mechanoreceptors and central command were no longer engaged. This maneuver is a well-established means to trap ischemic metabolites in the exercising muscle bed, thereby isolating the muscle metaboreceptor contribution to activation of MSNA. The cuff was deflated after 2 min.
Instrumentation
An intra-arterial catheter was placed in the brachial artery for drug infusion. The skin over the brachial artery was anesthetized with 1% lidocaine, and a 3-Fr catheter was inserted with the use of sterile techniques.
Pharmacological Inhibition of Ischemic Metabolites
Intrabrachial arterial indomethacin was used to inhibit forearm production of prostaglandins during exercise. The dose (0.3 mg/100 ml forearm volume infused over 20 min immediately before exercise) has been shown to virtually eliminate forearm prostaglandin release during static handgrip exercise in healthy humans (29).
Intrabrachial arterial aminophylline, a nonselective adenosine receptor antagonist, was administered at a rate of 1 µg·ml forearm volume–1·min–1 beginning 10 min before exercise and continuing through recovery. This dose has been shown to significantly blunt the increase in MSNA during static handgrip exercise in healthy humans (5).
Miscellaneous
Blood pressure was monitored noninvasively with an automatic blood pressure cuff (Press-Mate 8800, Colin Medical Instrument; San Antonio, TX). Systolic, diastolic, and mean blood pressure was measured every 20–30 s at baseline and during handgrip exercise. Heart rate (HR) was monitored continuously through lead II of the ECG.
Experimental Protocols
Two different drugs were tested in two different groups of normal subjects without overlap. A timeline of the protocol is shown in Fig. 1. After an unblinded saline infusion administered in the same volume and rate as the study material, the exercise protocol (as described in Rhythmic Handgrip Exercise) was performed. After a 45-min rest period for recovery, study material (either drug or saline) was administered in a double-blind, randomized fashion, and the exercise protocol was repeated. Subjects were invited to return to repeat the entire protocol on a different day, during which the study material that they did not receive the first day was administered in a double-blind fashion the second day (e.g., blinded aminophylline on day 1 and blinded saline on day 2). With this design, unblinded saline served as a control for the drug effect. The double-blind saline served as a control for an order or time effect. The half-life of the drugs prohibited a design in which both saline and drug were only administered in a double-blind manner on the same day, because a subject receiving drug during the first exercise session of the day may have a spillover effect during the second exercise session.
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Data are presented as means ± SE. Repeated-measures ANOVA was used for comparison of between and within-group means and to compute the P values. Paired t-test, which is a special case of repeated-measures ANOVA when there are only two time periods, was also used in some of the analyses. P values <0.05 were considered significant.
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RESULTS |
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In this study, 16 subjects were enrolled: 13 subjects completed the blinded indomethacin protocol, 12 subjects completed the blinded saline protocol, and 9 subjects completed both protocols.
Hemodynamic responses. Resting mean arterial pressure (MAP) and HR are shown in Table 1. MAP and HR increased similarly during exercise during saline and during blinded saline runs (Table 2). Intra-arterial indomethacin had no effect on resting MAP or HR (Table 1). The increases in MAP and HR during exercise were not blunted following indomethacin infusion (Table 2).
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In this study 18, subjects were enrolled: 17 subjects completed the blinded aminophylline protocol, 13 subjects completed the blinded saline protocol, and 12 subjects completed both protocols.
Hemodynamic responses. Resting MAP and HR are shown in Table 1. MAP and HR increased similarly during exercise during saline and during blinded saline runs (Table 2). Intra-arterial aminophylline had no effect on resting MAP or HR (Table 1). The increases in MAP and HR during exercise were not blunted following aminophylline infusion (Table 2).
Muscle sympathetic nerve activity. After unblinded saline control and blinded saline infusion, MSNA increased significantly and similarly during RHG (drug, P = NS; time, P = 0.0001; drug-time interaction, P = NS) (Fig. 2). Thus there was no order effect. MSNA measured as total activity increased from baseline beginning with the second minute of exercise (2,690 ± 505 vs. 3,403 ± 885 total activity, P = 0.01). After blinded aminophylline, this increase of MSNA was not blunted (Fig. 2) (drug, P = NS; time, P = 0.04; drug-time interaction, P = NS).
Once again, to investigate whether the muscle metaboreceptors were activated during exercise, PHG-CA was performed (2). During saline infusion, MSNA levels during PHG-CA were not elevated compared with those during recovery (Fig. 3), eliminating the possibility that sympathetic activation was mediated by muscle metaboreceptors.
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DISCUSSION |
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In these in vivo studies in humans it was not possible to directly record muscle mechanoreceptor activity. Thus the first point of discussion must be the evidence that the low-level dynamic exercise used in these studies solely activated the muscle mechanoreceptors. Second, because central command was certainly engaged during exercise, albeit at a low level, we must address the possibility that central command and/or muscle metaboreceptors mediated the sympathoexcitation during exercise. To address the first issue, let us turn to animal studies in which muscle mechanoreceptor activity can be directly recorded. Pickar and colleagues (20) used low-intensity dynamic exercise in decerebrate cats and found that group III mechanosensitive afferent nerve fibers were extremely sensitive to small changes in muscle tension. In fact, muscle mechanoreceptors were observed to fire synchronously with each muscle contraction. Several other studies in animals have confirmed these findings as well (1, 28). Victor and colleagues (28) found that rhythmic activation of group III afferent mechanosensitive fibers caused a synchronous increase in reflex renal sympathetic nerve activity in anesthetized cats. In decerebrate cats, Adreani and Kaufman (1) reported that group III nerve fibers fired during low-level dynamic exercise and, interestingly, fired more when dynamic exercise was accompanied by arterial occlusion compared with the freely perfused state.
Just as important that low-level dynamic exercise in our study does engage mechanoreceptors is the evidence that it does not engage metaboreceptors. The strongest evidence that the muscle metaboreceptors were not engaged by this exercise paradigm is found in the PHG-CA protocol (2). During PHG-CA, ischemic metabolites are trapped in the muscle bed at the conclusion of exercise, and the arm is relaxed, thus isolating the muscle metaboreceptors from muscle mechanoreceptors and central command. During PHG-CA in these studies, sympathetic nerve levels were not elevated compared with the recovery period, excluding the possibility that the muscle metaboreceptors were engaged. Finally, it is unlikely that the decline in sympathetic activation during indomethacin compared with saline was mediated by a decrease in central command because the level of exercise, and thus the level of effort, was the same during both exercise sessions. Furthermore, when the subject was randomized to saline instead of indomethacin, there was no diminution in the sympathetic response during exercise; there was no order effect.
Role of Prostaglandins in Sensitizing Muscle Mechanoreceptors
In our study, MSNA tended to increase in only the third minute of rhythmic exercise. This onset latency is consistent with other reports (3, 9) and may reflect the time necessary for low levels of metabolic by-products to accumulate and sensitize muscle mechanoreceptors. The cyclooxygenase inhibitor indomethacin administered into the brachial artery markedly and definitively eliminated the increase in MSNA during low-level dynamic exercise. This was not an order or time effect because in subjects randomized to saline, the increase in MSNA was unchanged.
Data are accumulating that prostaglandins play a principle role in sensitizing many types of afferent neurons in both health and disease. In studies of enhanced peripheral nociception, or "hyperalgesia," prostaglandins have been implicated in sensitizing, rather than directly stimulating, nociceptors (4). The cellular electrophysiological mechanisms of this enhanced sensitivity include increasing intracellular cAMP, thereby decreasing the threshold of activation for tetrodotoxin-resistant sodium channels, and/or eliminating slow postspike afterhyperpolarizations, or by shifting the voltage threshold of nonselective cation current channels, thereby allowing an influx of charge (4, 10). Each of these ion channels described on nociceptors is also present on group III mechanosensitive neurons as well.
Data in animal studies support the concept that prostaglandins sensitize muscle mechanoreceptors. In the anesthetized cat, arachidonic acid, a precursor of prostaglandins, was found to stimulate group III mechanosensitive afferent nerve fibers, and this effect was blocked by pretreatment with indomethacin (21). Prostaglandin levels have been shown to increase during exercise in healthy humans (29). During more strenuous contractions or fatiguing exercise, data both support (11, 24) and refute (7, 9) a role for prostaglandins in contributing to cardiovascular reflexes during exercise, presumably mediated by muscle metaborecptors. The focus of our study of low-level exercise was the effect of cyclooxygenase products on muscle mechanoreceptors and not metaboreceptors. This is the first study in humans demonstrating that cyclooxygenase products, likely prostaglandins, sensitize muscle mechanoreceptor control of MSNA in healthy humans.
Role of Adenosine in Sensitizing Muscle Mechanoreceptors
In our study, we saw no evidence that blockade of adenosine receptors with aminophylline influenced the MSNA, HR, or MAP responses during exercise. The dose of aminophylline was identical to that shown to inhibit sympathetic activation during static exercise, presumably mediated by muscle metaboreceptors (5).
The role of adenosine in mediating sympathetic excitation during exercise in healthy humans is very controversial. Costa and Biaggioni (5) administered intra-brachial adenosine in healthy humans and found an early increase in MSNA. Furthermore, they reported that intra-arterial aminophylline blunted the sympathetic activation during exercise in healthy humans. Adenosine levels, measured by the microdialysis method, increase during exercise (6). On the other hand, MacLean and colleagues (15) found that intra-arterial adenosine administered into the femoral artery resulted in a delayed increase in MSNA, which was prevented by preventing systemic spillover of adenosine from the leg. These investigators concluded that the sympathoexcitatory effect of adenosine was not attributable to stimulation on limb afferent neurons but to stimulation of neurons elsewhere in the body, such as the arterial chemoreceptors. In animal studies, 2-chloroadenosine, an adenosine analog, was not found to stimulate type III and IV muscle afferents (21). In cellular electrophysiology studies using vagal afferent cell bodies cells from healthy rabbits, we found that adenosine did not directly stimulate neurons but did sensitize them to other stimuli by eliminating slow, postspike afterhyperpolarizations (17). However, these current studies in healthy humans do not lend further support to the concept that adenosine plays an important role in sensitizing muscle mechanoreceptors.
Limitations
The greatest limitation of this study is the lack of information on the levels of these ischemic metabolites in the interstitium where the afferent nerve terminals are located. Effluent venous prostaglandin levels assume a steady state in metabolism and transport, which is likely not the case in these short (3 min) exercise paradigms. At best, venous levels are a crude estimate of the interstitial concentrations. Adenosine has such a short half-life in blood that venous levels are meaningless. The microdialysis method, in which several probes are placed into the exercising muscle bed, are reflective of an interstitial metabolite concentration. However, this technique takes more than 3 min to collect and thus is not technically feasible in these short exercise protocols in the small muscle bed of the forearm.
Only one cyclooxygenase inhibitor indomethacin was used in these studies. A chemical effect of indomethacin that is distinct from its effect on cyclooxygenase has not been ruled out. Future studies utilizing a chemically dissimilar cyclooxygenase inhibitor will resolve this point.
Although we did not detect a role for adenosine in sensitizing the muscle mechanoreceptors in this study in healthy humans, it seems premature to completely eliminate adenosine as having a role in this process. Cyclooxygenase products clearly have a very early and robust role in the acute sensitization of muscle mechanoreceptors during mild exercise. Nonetheless, it is conceivable that during a longer bout of exercise or a more strenuous exercise protocol, one or more additional metabolic by-products, including lactic acid, adenosine, and possibly others, may be necessary to sensitize the muscle mechanoreceptors.
Significance
Two prior studies support the notion that muscle mechanoreceptors are important mediators of MSNA during exercise, especially rhythmic exercise, in healthy humans. Their role in disease may be of more importance; muscle mechanoreceptors may the principle mediators of MSNA activation during exercise in patients with heart failure. Sterns and colleagues (25) were the first to report that muscle metaboreceptor control of MSNA was blunted in heart failure, and they hypothesized that sympathoexcitation during exercise may be mediated by muscle mechanoreceptors instead. Middlekauff et al. (18) reported that the muscle metaboreceptor control of renal blood flow was blunted and that muscle mechanoreceptor control was augmented in patients with heart failure. Smith and colleagues (23) reported enhanced muscle mechanoreceptor activity in a rat model of heart failure. Interestingly, Scott and colleagues (22) measured a series of ischemic metabolites in peripheral blood following exercise in patients with heart failure and concluded that only prostaglandins levels correlated with the enhanced, ergoreceptor-mediated ventilation during exercise. These correlative studies that rely on venous metabolite levels have limitations as outlined above but open the door for further studies using pharmacological blockade to investigate the role of prostaglandins in mediating the abnormal reflex responses in heart failure.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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REFERENCES |
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MSNA) during exercise. A: in the indomethacin protocol after saline and blinded saline infusions, MSNA increased similarly with exercise [drug effect, P = not significant (NS); time effect, P = 0.001; and time-drug interaction, P = NS]. B: after the blinded indomethacin infusion, the increase in MSNA during exercise was eliminated (drug effect, P = < 0.0001; time effect, P = 0.03; and time-drug interaction, P = 0.03). *3-min saline vs. 3-min indomethacin, P < 0.0001. C: in the aminophylline protocol after saline and blinded saline infusions, MSNA increased similarly with exercise (drug effect, P = NS; time effect, P = 0.0001; and time-drug interaction, P = NS). D: after blinded aminophylline infusion, the increase in MSNA during exercise was not blunted compared with the increase during saline (drug effect, P = NS; time effect, P = 0.04; and time-drug interaction, P = NS).