Naloxone augments muscle sympathetic during isometric exercise in humans

nerve activity

PETER A. FARRELL, THOMAS J. EBERT, AND JOHN P. KAMPINE Departments of Physiology and Anesthesiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226; and Laboratory for Human Performance Research, The Pennsylvania State University, University Park, Pennsylvania 16802

FARRELL,~ETER A., THOMAS J. EBERT,ANDJOHNP. KAMPINE. Naloxone augments muscle sympathetic nerve activity during isometric exercise in humans. Am. J. Physiol. 260 (Endocrinol. Metab. 23): E379-E388, 1991.-The influence of an endogenous opioid peptide (EOP) antagonist (naloxone, 1.2 mg iv bolus) on muscle sympathetic nerve activity (MSNA, microneurography) was studied on 19 young male and female volunteers. Isometric handgrip, cold pressor test, and acute baroreceptor unloading with sodium nitroprusside (autonomic stresses) were carried out under two conditions, one group (n = 11) before (control responses) and after naloxone and another group (n = 8) before and after placebo saline. Monitored cardiovascular variables included heart rate, central venous pressure (jugular vein catheter), arterial blood pressure (radial artery catheter), circulating catecholamines, and forearm blood flow. At rest, cardiovascular variables and MSNA were not affected by either naloxone or saline. MSNA (total activity = burst frequency x burst amplitude/100 cardiac cycles) increased during isometric handgrip to a greater extent (30 t 6 vs. 16 t 5 arbitrary units) after naloxone compared with control trials (P < 0.05). After naloxone, arterial systolic and diastolic blood pressures were higher during handgrip exercise. These augmented arterial pressures and MSNA responses were not evident during either the cold pressor test or the sodium nitroprusside stress. These data suggest that isometric muscle contraction elicits a sympathetic neural response that may be modified by EOP. This interaction is not evident during two other stresses, when sympathetic responses are equal to or greater than those provoked by isometric handgrip exercise. endogenous opioids; catecholamines; pressor test; baroreceptor function

muscle

afferents;

cold

OF the sympathetic nervous system and heightened release of several hormones during circulatory stress contributes to the ability of an organism to meet the metabolic and circulatory demands of that stress. It is probable that these responses are modulated to prevent excessive activation beyond that required to meet stress demands. One proposed modulator of stress responses is the endogenous opioid peptide system. Several lines of evidence suggest that endogenous opioids (13, 14, 19) and exogenous opiates (1) modify sympathetic neural activity in the periphery. We (9) and others (10) have extended these previous in vitro and in situ studies to the whole body level by demonstrating that the increase in circulating catecholamines during dynamic exercise in humans is enhanced when opioid peptide receptors are antagonized. THE ACTIVATION

The interpretation of circulating norepinephrine (NE) as an index of sympathetic neural activity is complicated by several facts. Circulating concentrations of NE represent spillover from synapses, therefore differences in regional blood flow or reuptake mechanisms for NE can influence the final amount of NE that reaches the peripheral blood. These and other well-documented concerns (3) suggest caution in interpreting a change in circulating NE concentration as a change in sympathetic neural activity. Direct recordings (33) from peripheral sympathetic nerves that innervate skeletal muscle blood vessels [muscle sympathetic nerve activity (MSNA)] in humans permit a more direct evaluation of efferent sympathetic responses to stressful stimuli. Consistent increases in MSNA and plasma NE concentrations occur during a cold pressor test (35), isometric handgrip exercise (IHE) (29), and hypotension produced by infusions of sodium nitroprusside (7,24). Whether the endogenous opioid peptides normally play a modulatory role during these stresses is not clear. Cardiovascular adjustments to IHE are partially regulated by afferents from contracting muscle (22). Whether these afferents are important to endogenous opioid peptide activation during exercise has not been established. However, a recent study in humans by Kjaer et al. (18) demonstrated that, when sensory nervous activity was blocked by epidural anesthesia, the exerciseinduced increase in circulating ,8-endorphin was abolished. Thus it may be that afferents activated by muscle contraction rather than general sympathetic activation are necessary for opioid-sympathetic interactions to be evident. In the present study, cold pressor, IHE, and acute hypotensive stresses were utilized to activate the sympathetic nervous system. Cardiovascular, MSNA, and circulating NE responses to these stressesunder placebocontrolled conditions were compared with stress responses elicited after opioid receptor antagonism with naloxone. Our findings demonstrate that naloxone administration augments the sympathetic nervous system responses to IHE, suggesting that the endogenous opioid peptide system has a role in modulating these responses in conscious humans. METHODS

Nineteen healthy volunteers (aged 20-40 yr) were studied. Each provided informed consent after approval E379

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of all experimental procedures by the Zablocki Veterans Medical Center Research Review Committee. Subjects were instructed to abstain from nicotine, caffeine, and theophylline-containing substances for 8 h before the study. Subjects were randomly assigned to receive either intravenous naloxone (n = 11) or placebo (isotonic saline; n = 8). Subjects were instrumented and studied while in the supine position. Heart rate was monitored from lead II of the electrocardiogram. A 20-gauge radial artery catheter (right arm) was placed for determination of arterial blood pressure, and an 18-gauge catheter was inserted through the external jugular vein and advanced to an intrathoracic location for monitoring central vein pressure and for blood sampling. A 20-gauge catheter was inserted into a forearm vein (right arm) and used for injection of vasoactive drugs as well as for infusion of naloxone or placebo. A small bellows connected to a pressure transducer was strapped to the abdomen to record respiratory excursions. Respiratory excursions were used to determine whether the subject altered his/ her breathing pattern during the autonomic stresses. Either breath-holding or a Valsalva maneuver during the autonomic stresses would significantly alter MSNA (5). The left arm was elevated and supported above the level of the heart, and a mercury-in-Silastic strain gauge connected to a temperature-compensating saddle was strapped around the forearm. Forearm blood flow was determined by standard venous occlusion techniques (8). Instruments for the measurement of blood flow and pressure were calibrated immediately before and after data collection. Each subject underwent a trial run of three autonomic stresses to become familiar with the procedures. Isometric exercise was performed using the right hand by gripping a Stoelting dynamometer for a 3-min period at 25% of each individual’s previously determined maximum voluntary contraction. Subjects were instructed to maintain relaxed quiet respiration throughout each handgrip procedure. Compliance with this instruction was monitored by observing respiratory excursions on an oscilloscope. The cold pressor test consisted of placing the right hand, up to the level of the wrist, in a bucket of ice water for 60 s. Baroreceptor activation testing consisted of injecting 100 pg of sodium nitroprusside through the intravenous catheter. Approximately 1 min later, during peak hypotension (unloading of baroreceptors), a lOO-pg bolus of phenylephrine was injected to restore blood pressure and to elevate it slightly above baseline for a period of l-2 min. This last protocol permitted quantitative analyses of the baroreceptor reflex control of the heart and peripheral sympathetic nervous system across a broad range of arterial blood pressure perturbations. No data were collected during the trial run. Peroneal Nerve Recordings After completion of the trial runs of the autonomic tests, the right leg was cushioned and supported. The bony prominence of the proximal head of the fibula on the lateral aspect of the leg was identified and marked. Brief electrical impulses (1 Hz, 30-40 V, 150 mA) were

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delivered below this mark to identify the location of the peroneal nerve. The skin was then cleansed, and two small epoxy-coated Tungsten needles (TM1 Electronics, Iowa City, IA) were inserted. One needle was advanced to an area just outside the peroneal nerve. A second needle was advanced into the peroneal nerve. The location of the nerve was identified by applying brief (1 Hz, 0.3 V, 150 mA) electrical impulses to the needle. When a muscle nerve fascicle within the peroneal nerve was entered, a distinct muscular contraction in the distribution of the deep or superficial peroneal nerve was observed. The stimulation was halted, and signals from both needles were passed to a preamplifier (which employed common mode rejection to reduce noise), amplifier, and integrator (total gain = 100,000). Characteristic bursts of sympathetic efferent activity were sought by fine manipulations of the needle within the muscle nerve fascicle. The identity of these bursts and their distinction from neural activity in skin sympathetic efferent nerve fibers has been described in detail elsewhere (6, 33). Procedures Once an acceptable nerve site was obtained, a lo-min quiet rest period was observed, followed by 5 min of resting hemodynamic measurements and sympathetic nerve recordings. A blood sample (8 ml for each sample) was obtained from the central venous catheter, centrifuged (2,000 revolutions/min, 4°C) within 5 min of the blood draw, and the plasma was removed and frozen at -70°C for determination of plasma catecholamine concentrations. The IHE test, cold pressor test, and baroreceptor testing were sequentially performed always in the same order. Jugular venous blood was sampled immediately after the completion of both the handgrip and cold pressor tests. Responses during these nondrug trials represent control responses to which the responses after placebo (saline) or naloxone could be compared. Each autonomic stress was separated by a 5-min recovery period. After this first series of the three autonomic tests (control responses) with no drug infusion, the subjects rested for 10 min, and then naloxone (1.2 mg, 1 group of 11 subjects) or placebo (another group of 8 different subjects) was given intravenously over a period of 2 min. Subjects and test administrators were blind to the nature of the drug used in any trial. One attending physician had knowledge of the drug used. The dose of naloxone was less than one-fourth of that previously demonstrated to alter the circulating catecholamine response to IHE (20) or the cold pressor test (2) in humans. Additionally, this dose (per kg body wt), given intrathecally to dogs (23)) significantly blocks morphine-induced reduction of arterial blood pressure responses to exercise. Hemodynamic and MSNA data were again collected, and 13 t 1 (SE) min after drug administration a blood sample was again obtained for determination of plasma catecholamines. The autonomic tests and blood sampling were then repeated in identical fashion to those performed before naloxone or placebo administration. The total elapsed time was 40 min for one sequence of the three autonomic tests. This period includes rest intervals between each test and the 13-min rest period after naloxone

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or placebo infusion. The effects of naloxone occur within 2 min of intravenous administration and last at least 1 h depending on the dose. We judged the 13-min period between naloxone administration and the performance of the second series of three autonomic stresses to be appropriate in terms of the expected duration of effectiveness of naloxone (4, 37). Mechanistic insights concerning a possible role for central command in the MSNA response to IHE can be gained by occluding blood flow from the active muscle during recovery from exercise. In three subjects, additional data were collected after each handgrip trial. In these subjects, circulation to the exercising arm was arrested for 2 min during recovery with a tourniquet (300 mmHg). This was applied immediately before completion of the handgrip exercise. Hemodynamic data and sympathetic outflow were averaged during each of the 2min periods of arm ischemia. These data were obtained in identical fashion during the second handgrip exercise performed after naloxone infusion. Biochemical Measurements Blood samples were collected into chilled tubes containing ethylene glycol-bis (P-aminoethyl ether)N,N,N’,N’-tetraacetic acid (EGTA) and reduced glutathione. Circulating catecholamines were measured in duplicate using high-performance liquid chromatography coupled to electrochemical detection (12). A known amount of catecholamines and 3,4-dihydroxybenzylamine (DHBA) added to 2 ml of phosphate-buffered saline were included with each subject’s chromatogram and served as internal standards. After addition of DHBA to each subject’s plasma and the internal standards, activated alumina was used to extract the amines. The lower limits of detection were 20 and 13 pg/ml for NE and epinephrine (E), respectively. The within-assay coefficient of variation was 8%, and all samples for one subject were analyzed on the same day. Samples from subjects who received naloxone were analyzed on the same day as samples from subjects who received saline. Statistical Analyses Between-group comparisons. A two-group design was chosen to eliminate the variability in MSNA recordings inherent in the measurement when made on the same subjects yet on separate days. This two-group design allowed for a stable MSNA recording with and without the infusion of an opioid antagonist. Cardiovascular and MSNA measures at rest and during the autonomic stresseswere compared between groups using analysis of variance. Within-group comparisons. The most important comparisons were those that evaluated cardiovascular and MSNA responses within the same group. Baseline (before stress) hemodynamic and MSNA measurements and consecutive 1-min responses to the 3-min handgrip exercise were compared (repeated measures ANOVA) with identical time points during handgrip exercise performed after naloxone or placebo administration. When significant F ratios were identified, specific time points were

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E381

compared using the Student-Newman-Keuls procedure. Catecholamine, hemodynamic, and MSNA responses to the cold pressor test and baroreceptor tests were analyzed with Student’s t tests. Derived P values co.05 were considered to be significant. Quantification of responses to pharmacological manipulations of blood pressure employed least-squares regression analyses. R-R interval and sympathetic nerve activity were averaged for each 2-mmHg decrease of blood pressure. Then the sigmoid relationship between average decreases in systolic pressure and R-R interval were plotted, and the linear portion of these curves was used to derive a slope and correlation coefficient. A similar process was applied to the relationship between average decreases in diastolic pressure and muscle sympathetic nerve activity. In most cases, this response was linear (not sigmoidal). The method of averaging R-R interval and MSNA data in 2-mmHg increments before applying regression analyses has been previously described by Ebert (7). This process lessens the influence of random non-baroreceptor-mediated moment-to-moment variations in R-R interval and MSNA, which occur due to respiration and descending neural outflow from higher cortical centers. Derived slopes were compared before and after naloxone or placebo trials. Unless otherwise noted, values are presented as means t SE. RESULTS

Resting Conditions None of the subjects demonstrated hyperreactivity to the instrumentation employed in this study. Resting values for all cardiovascular or endocrine variables were representative of normal resting subjects in the supine position (6, 9, 24). Control and naloxone groups did not differ significantly (P > 0.05). Naloxone had no significant effect on any cardiovascular or endocrine variable when measured in the resting state (Table 1). The frequency of MSNA during resting conditions was not affected by naloxone (before, 11.3 t 1 bursts/min vs. after, 10.2 t 2 bursts/min). Similar results were found when bursts were expressed per 100 cardiac cycles and when burst amplitudes were compared (data not shown). Hemodynamic and MSNA measures returned to baseline by the end of the recovery (5 min) period that separated each autonomic stress. Autonomic Stresses The hemodynamic, hormonal, and MSNA responses to autonomic stress maneuvers are shown in Tables 2 and 3 and Figs. l-4. Analysis of variance for betweengroup comparisons failed to reveal significant differences in the response of any variable during the three autonomic stresses. Each autonomic stress provoked sympathetic activation (P < 0.05). In general, acute hypotension elicited the greatest increase in MSNA, whereas the cold pressor test elicited the least activation; however, these differences were not statistically significant. IHE All subjects completed the two trials of IHE. Prehandgrip baseline hemodynamics and MSNA were un-

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1. Resting cardiovascular, muscle sympathetic nerve activity, and plasma catecholamine levels

TABLE

Placebo

Cardiovascular R-R interval, ms SP, mmHg DP, mmHg CVP, mmHg MSNA, bursts/min MSNA, bursts/100 cardiac MSNA (total activity) Hormones Norepinephrine, pg/ml Epinephrine, pg/ml

cycles

Values are means k SE; n, no. of subjects. CVP, central venous pressure; bursts/min, cycles (units are arbitrary).

Naloxone group PI Nal Placebo group PI P2

(m)

P2

(n = 11)

1,015+50 133t4 63k2 6t2 15.2*2 24.6t4 21.9t4

1,008*53 140t4 66t3 6t2 14.6t3 23.5&4 24.0t5

1,044+93 144t4 67k2 7k4 11.3&l l&3&2 l&3&4

1 ,031t87 140t4 66t2 6t4 10.2*2 15.9t2 14.823

240t21 26t4

260t26 28t4

246t17 40t6

sympathetic nerve of nerve activity;

activity; MSNA

265k30 48t13

SP, systolic blood pressure; DP, diastolic blood total activity, burst frequency x amplitude/100

15.9t2

-1,208+200

0.97t0.01

16.422

-1,341+265

0.98t0.01

26.4t4 22.1t4

-2,635+659 -2,150+497

0.98t0.01 0.97_to.o1

vs. DP

-4.6ltl.O -5.33tl.3

324t74

-0.95t0.02

37Ok9-96

-0.96:O.Ol

-3.82tl.O

264t73 329t149

-0.98t0.01 -0.98_tO.O1

Values are means t SD. R-R interval (ms); SP, systolic blood pressure (mmHg); MSNA, muscle sympathetic nerve activity (total burst activity); DP, diastolic blood pressure; PI, acute hypotension (1st trial with no drug administration); Nal, acute hypotension (2nd trial with naloxone administration); P2, acute hypotension (2nd trial with saline administration). Linear equation was RR = m(SP) t constant.

changed after naloxone (Fig. 1 and Table 1). Group responses to IHE are provided in Figs. 2 and 3. This stress elicited significant decreases in R-R interval and significant increases in arterial blood pressures (systolic and diastolic) and MSNA (P < 0.01; Figs. 2 and 3). The control group showed changes in these measures of cardiovascular stress after saline infusion identical to those found during previous trials without saline. There were no significant changes in central venous pressure or forearm vascular resistance during IHE (data not shown). The responses to IHE were highly reproducible in subjects receiving placebo (Figs. 2 and 3), and no significant difference between control and placebo trials existed for any variable in this group. However, naloxone administration resulted in augmented (P < 0.05) MSNA and arterial blood pressure responses during IHE (Figs. 2 and 3, naloxone group). The augmented response in MSNA (bursts/min) was observed in 10 of 11 subjects.

pressure; cardiac

3. Cardiac R-R interval and MSNA responses to IHE and to 2 min of posthandgrip ischemia for three subjects TABLE

r

vs. SP

-4.59t1.9

Group

Postinfusion

R-R interval

MSNA Naloxone group PI Nal Placebo group PI

Naloxone Preinfusion

MSNA, muscle burst frequency

y-Intercept (constant)

(n = 8) Postinfusion

TABLE 2. Slopes and intercepts for relationship between R-R interval and systolic blood pressure and MSNA vs. diastolic blood pressure during acute hypotension Slope

Group

Preinfusion

Heart rate, ms Control Naloxone MSNA (total activity) Control Naloxone

IHE min)

Ischemia

Pre-lHE

(3rd

871t75

740+84

934klOO

928-+96

868t65

714760

90lt96

920*92

19.6&5 16.6t5

46k12 54t16

1 min

2 min

62t35

48225

52t21

49t21

Values are means -t- SE. Control, 1st handgrip trial; naloxone, 2nd handgrip trial after intravenous naloxone. MSNA, muscle sympathetic nerve activity (burst frequency x amplitude/100 cardiac cycles); IHE, isometric handgrip exercise. Statistical analyses were not applied.

The one exceptional subject demonstrated nearly identical responses after naloxone when compared with the control tests. Central venous blood concentrations of catecholamines are shown in Fig. 4. Although the NE increase was large during control trials in the placebo group, the increase was not statistically significant (P > 0.05). The increases in NE during handgrip trials after naloxone appeared larger than increases during control handgrip trials, but again this augmentation was not significant. Figure 4 shows that circulating concentrations of E increased after IHE (P < 0.05) and that this increase in absolute concentration was greater during the naloxone trials (P < 0.05). Cold Pressor Test Cold stress provided significant (P < 0.01) increases in arterial blood pressure, forearm vascular resistance, and MSNA (Fig. 5). Neither placebo nor naloxone influenced these responses to cold stress. Central venous pressure (data not shown) and R-R interval remained unchanged. It should be noted that MSNA response to cold tended to be higher after naloxone, although the difference was not statistically significant (P = 0.1). An augmented response in MSNA (bursts/min) after naloxone was observed in 4 of 11 subjects. Circulating (central venous blood) NE concentrations

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NALOXONE

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NERVES

A Before

Naloxone

IN

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B Administration R-R ink629 msec SBP=180 mmHg DBP=78 mmHg

R-R int=772 msec SBP=144 mmHg DBP=70 mmHg

After

Naloxone

Administration

. . . .. ...- ... ... tLh

R-R int=751 msec SBP=151 mmHg DBP=70 mmHg Integrated Muscle Sympathetic Nerve Activity w>

R-R int=620 msec SBP=194 mmHg DBy=82 mmHg

-

5*o

2*5 oo l

S

mmu

FIG. 1. Representative tracing of muscle sympathetic (peak, B) isometric handgrip exercise. R-R int, R-R pressure.

nerve interval;

activity SBP,

increased during cold stress from 246 & 17 to 322 t 65 pg/ml in the control trial of the naloxone group. Similar responses were found for the saline group (data not shown). Cold stress after naloxone treatment caused a greater response (265 t 30 to 386 t 43 pg/ml), but this increase was not statistically greater than that observed during the control trial. Saline treatment did not affect the NE response in the placebo group (data not shown). Circulating concentrations of E increased during cold stress from 40 t 6 to 59 t 9 pg/ml during control trials in the naloxone group. After naloxone, a significantly greater increase in E was observed (48 k 13 to 112 t 28 pg/ml; P < 0.05). E responses in the saline-treated group were not different between control responses (26 & 4 to 58 + 8 pg/ml) and responses after saline (28 t 4 to 63 t 11 ig/ml). Sodium Nitroprusside

Stress

During acute hypotension, mean arterial pressure decreased -10 mmHg before phenylephrine was used to counteract the effect of this agent. The decrease in MAP was similar for both trials in both groups. Baroreceptormediated tachycardia and increases in MSNA occurred in response to acute hypotension; however, these responses were not affected by naloxone. Cardiac baroreceptor sensitivity expressed as the linear segment of the sigmoid relationship between R-R interval and systolic pressure is provided in Table 2. Naloxone did not significantly affect the slope of the relationship. Additionally, the slope of the relationship between MSNA and diastolic pressure also was not significantly altered by naloxone (Table 2). Posthandgrip

E383

Ischemia

Because of the small number of subjects (n = 3) who had this additional manipulation, no statistical evalua-

from 1 subject systolic blood

before (baseline, pressure; DBP,

A) and during diastolic blood

tion was performed. Heart rate returned to prehandgrip values immediately when IHE was stopped (Table 3). MSNA remained elevated for the 2 min post-IHE, when blood flow from the exercised muscle was occluded. Treatment with naloxone did not influence the sustained elevation in MSNA during posthandgrip ischemia. DISCUSSION

The major finding in this study is that the responses to IHE, which include activation of peripheral sympathetic outflow, are enhanced after antagonism of the endogenous opioid peptides. To our knowledge, this in vivo demonstration of a direct interaction between naloxone and sympathetic nerves in humans has not been reported previously. This enhanced sympathoexcitation during handgrip exercise after naloxone administration may partially account for higher systolic and diastolic blood pressures as well as slightly, but not significantly, higher circulating NE concentrations and elevations in heart rate. Seals and Enoka (28) have demonstrated that prior fatiguing IHE can significantly enhance the MSNA and electromyographic response to subsequent IHE trials. Three considerations suggest that the enhanced MSNA we observed after naloxone was not caused by test-retest phenomena. 1) The group that received saline demonstrated identical MSNA responses during the second trial; 2) in contrast to the protocol used by Seals and Enoka (28), IHE in this study was nonfatiguing; and 3) approximately 40 min elapsed between IHE trials. This time between IHE trials was four (36) and two (28) times longer than previous studies that have noted either no effect of prior IHE (36) or elevated MSNA (28) due to prior exercise. These considerations suggest that the augmented MSNA during naloxone trials were due to

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r

0

II

e--

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control response response after naloxone

A

R-R interval, msec

-100 -200

60

60

A

40

40

systolic pressure,

20

T

mmHg oJ

,

20

/I * +

0 I

I

I

J

FIG. 2. R-R interval and blood pressure responses to isometric handgrip exercise for group that received a saline infusion (left) and group that received naloxone (right) during a second set of autonomic stresses. Error bars represent means t SE. * Naloxone caused a significant elevation in response (P C 0.05).

30

A

diastolic pressure, mmHg

2o ,o

20

0

0

-10

IO

I

1.0

I

2.0

1

3.0

minutes

of handgrip

exercise

the antagonist and not caused by residual effects of prior exercise. Prior work in both humans (2, 9, 20) and animals (15) demonstrated that increases in circulating NE concentrations during various stresses tend to be greater after endorphin antagonism; yet, these increases were not significant. The more direct measure of sympathoexcitation (MSNA) used in this study suggests a significant inhibitory role for endorphins on muscle sympathetic nerve activity. This interaction between naloxone and MSNA was more apparent during IHE than during cold stress or acute hypotension. Because these other stresses also cause sympathoexcitation, it may be that muscle contraction-induced mechanisms are unique to the MSNA-naloxone interaction we observed. Cold stress resulted in significant increases in MSNA, NE, forearm vascular resistance, and arterial blood pressure, yet these responses were not significantly altered by either placebo or naloxone. It should be noted that MSNA and NE responses during the naloxone coldstress trial tended to be higher [not significant (NS)] than during the control trial. This finding complements

an earlier report by Bouloux et al. (2), which demonstrated enhanced (but not significantly) NE responses during a cold pressor test after naloxone administration. Thus endorphins may partially regulate sympathoexcitation during cold stress but to a lesser extent than that demonstrated during IHE. Baroreflex-mediated responses to acute hypotension were also not significantly influenced by naloxone. Several lines of evidence suggest that endogenous opioid peptides partially regulate baroreceptor function (25). Rubin et al. (26) found that naloxone significantly increased baroreflex sensitivity [R-R interval/mean arterial pressure (R-R/MAP)] during sodium nitroprussideinduced hypotension. However, this effect was not evident until 3 h after naloxone infusion. Our findings are compatible with that study, since baroreflex sensitivity was not altered by naloxone during short-duration hypotension. Some animal studies have demonstrated a significant role for endorphins in blood pressure control during isometric contractions or acute hypotension (23, 31), and this interaction may be mediated in either the central nervous system or periphery (39). Our data in

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r

A MSNA, bursts/ minute

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E385

40

40 30

SYMPATHETIC

I --

G

Q--

G

control response response after placebo

control response response after naloxone

30 1 --O---

20 10

0 -10

30 A MSNA, bursts/ 100 cardiac cycles

FIG. 3. Muscle sympathetic nerve activity (MSNA) responses during isometric handgrip exercise in group that received saline infusion (Left) and group that received naloxone (right). Error bars represent means t 1 SE. * Naloxone caused a significant elevation in response (P < 0.05).

20 10 -

60

60

7

A MSNA, freq x amplitude/ 100 cardiac cycles

I

1.0

I

2.0

minutes .

1

I

(

3.0

of

1.0

handgrip

conscious humans do not support a role for endorphins in reflex cardiovascular adjustments to acute short-duration hypotension. MSNA increases provoked by the three stresses were not significantly different during the control (nondrug) trials for either group. Therefore, the apparent selective affect of naloxone on the inhibition of MSNA during IHE is probably not explained by a greater perceived effort or stress of IHE compared with cold stress or acute hypertension. In contrast to IHE (16), cold stress and acute hypotension (34) probably do not activate muscle mechano- or chemoreceptor afferents. Our data do not provide definitive results in terms of which afferents are involved in or are necessary for an endorphin-sympathoexcitation interaction to be evident. An obvious difference between the stresses was that IHE but not the other stresses required muscle contraction and activated different afferents (21, 27, 38). The stimuli to these afferents may be mechanical or chemical changes in the contracting muscle, which lead to activation of a mechano- (30) or chemoreflex (38).

2.0

3.0

exercise Chemoreceptor activation has been suggested as necessary for appropriate cardiovascular adjustment to static exercise (21). Muscle afferents may also contribute to activation of endogenous opioid peptides during dynamic exercise. Kjaer et al. (18) demonstrated that increases in circulating ,&endorphin (an endogenous opioid peptide) during exercise are abolished by epidural blockade. Carefully controlled epidural anesthesia is thought to selectively block afferent signals from unmyelinated (C-fiber) or thinly myelinated (a-6) afferents, which should be activated by the level of IHE used in our study. Although the endorphin responsible for altering MSNA responses to IHE is unknown, the work by Kjaer et al. (18) suggests that circulating ,&endorphin may be a candidate for this role. Although this interaction is possible, it is also clear that many other mechanisms could explain our observation. As one example, naloxone also directly inhibits the actions of enkephalins that are known to inhibit sympathetic nerve activity in situ (14). Further complicating the interpretation of our data is the fact that enkephalins are found in noradrenergic neurons in several areas of the body (see Ref. 13 for review) and that a

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NALOXONE 0

q

baseline peak response

EFFECTS

ON

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NERVES

IN

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to handgrip

l-

norepinephrine, Pg/ml

300

200

control

epinephrine,

response

response

after

placebo

50

50

25

25

control

response

response

after

naloxone

control

response

response

after

naloxone

FIG. 4. Jugular vein concentration of catecholamines before (open bars) and after (hatched bars) isometric handgrip exercise (IHE). Placebo values (left) are means ~fl SE for group (n = 8) that received saline during second IHE. Naloxone values (right) are for group (n = 11) that received naloxone during second IHE. * Significant increase after IHE. ** Response after IHE was higher during naloxone trial (P < 0.05).

Pm1

control

response

response

after

placebo

direct intraneuronal mechanism could be postulated to explain the inhibition we observed. Extensive literature (40) exists, which indicates that opioids in the spinal cord and on afferent nerves innervating the spinal cord are important modulators of neural transmission. Our finding of an enhanced MSNA due to naloxone is compatible in some respects with several previous studies that have investigated a role of spinal opioids in the pressor response to exercise in animals. Hill and Kaufman (11) recently demonstrated that an enkephalin analogue ( [D-Ala2]Met-enkephalinamide, given intrathetally) significantly reduced the pressor response to static muscle contraction in chloralose-anesthetized cats. However, that study further showed that this opioid inhibition of the pressure response was probably not due to reduced sympathetic outflow. Because naloxone reversed the effect of the analogue in that study (11) in a compatible direction to that reported here, it is possible that opioids interact with ascending messagesto the central nervous system, which eventually are transmitted back down to the periphery by efferent sympathetic nerves (MSNA). This endorphin-ascending messageinteraction could occur in the spinal cord. Pomeroy et al. (23) have demonstrated that intrathetally administered naloxone significantly reverses morphine-induced reductions in the pressor responses to dynamic exercise in conscious dogs. Again, this direction of change in pressure due to an exogenous opiate is compatible with our results. It should be noted that naloxone administered alone did not consistently alter the blood pressure response to dynamic exercise in dogs. This finding is not compatible with our results in conscious humans. In addition to direct peripheral interactions between endorphins and neural transmission perhaps located in the spinal cord, since naloxone crosses the blood-brain barrier (32), a mechanism located in the central nervous system is also a possible explanation for our observations. Endorphins are located and have important functions

in the central nervous system. These areas include centers that have previously been shown to coordinate cardiovascular responses to exercise. The augmentation of MSNA by naloxone may represent a modulation of functions that have been described as “central command.” An endorphin involvement in central command could be inhibitory or stimulatory, and our data cannot provide definitive insights into which effect predominates. As an example, Mark et al. (21) have suggested that central command is inhibitory to MSNA responses to IHE. Our data are consistent with an inhibitory role for endorphins, because when endorphin receptors were blocked, MSNA responses were enhanced. However, Victor et al. (36) have recently shown that central command is stimulatory rather than inhibitory to MSNA during IHE. If endorphins have a role in this stimulation and are acting centrally, the mechanism may involve a disinhibition process. As recently reviewed by Illes (l3), this mode of action would not be uncommon for endogenous opioids. More research, perhaps using opioid antagonists that are restricted from the central nervous system, is needed to clarify a possible role for endorphins in modulating efferent neural outflow from the brain. In accordance with previous work (2l), heart rate fell rapidly and MSNA remained elevated during the posthandgrip period, when blood flow from the active arm was occluded immediately after exercise. Naloxone did not alter this pattern of response, which cast doubt on the possibility that endorphins are involved with chemoreceptor-mediated muscle afferent activation after IHE. A greater number of subjects and other experimental manipulations must be studied before the exact nature of the endorphin-muslce afferent interaction is resolved. Circulating E responses to IHE were also higher after naloxone. This finding is similar to our previous work using dynamic exercise to cause increases in circulating E. This confirmation supports our previous interpretation that adrenal medullary responses to stress are par-

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NALOXONE

l la A R-R interval, msec

EFFECTS

ON

SYMPATHETIC

NERVES

IN

HUMANS

E387

control responses response after placebo or naloxone

A forearm vascular 10 resistance, mm Hg mI/min/l OOml

1 T 0

-100

40

A systolic pressure, mm Hg

A diastolic pressure, mm Hg

20

A MSNA, total activity

13

0

IO

50

26

A f-N4 bursts/ minute

FIG. 5. Cardiodynamic and muscle sympathetic nerve activity (MSNA) responses to cold pressor test. T Pretest values for MSNA were lower for naloxone group compared with placebo group. Error bars represent means & 1 SE.

placebo group

25

naloxone group

tially modulated by endogenous opioid peptides (9). Resting hemodynamics and MSNA were not affected by naloxone. This is compatible with several reports showing no difference in circulating catecholamine concentrations due to endogenous opioid peptide antagonism, when animals (15) or humans (2, 9, 10, 26) are studied at rest. This consistency across different studies and models strengthens the interpretation that endorphins do not exert tonic control over the sympathetic nervous system. When the organism is stressed, both the endorphin and the sympathetic nervous systems are activated, and one purpose of endorphin activation is to modulate sympathoexcitation. Our data suggest that not all stressesthat activate the sympathetic nervous system necessarily reveal an endorphin-sympathetic interaction. Thus acute hypotension using sodium nitroprusside required the greatest efferent activation to smooth muscle (in the naloxone group) in our study, yet naloxone did not affect this response. Further work is required to

0

placebo group

naloxone group

identify why some stresses require an endorphin-sympathetic interaction, whereas other stresses do not. In summary, this study demonstrates that the inhibitory influence of endogenous opioid peptides on sympathetic neural activity is evident when direct MSNA is used as the index of sympathoactivation. This interaction was more apparent with an autonomic stress (IHE) that required muscle contraction rather than with other stresses that also resulted in increased MSNA yet did not involve muscle contractions. Further study is required to determine whether this interaction is caused by muscle afferents that activate central nervous system endorphins (spinal and/or brain), which then modulate (inhibit) the sympathoadrenal response to IHE. We thank Monica Bleck, Toni Denahan, Deanne Tryzinko, and Marlin Druckenmiller for their expert technical assistance during these studies. We also thank Becky M. Maurer for excellent clerical skills during the preparation of the manuscript. This work was conducted at the Zablocki Veterans Administration

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E388

NALOXONE

EFFECTS

Medical Center, Milwaukee, WI 53226. Address for reprint requests: P. A. Farrell, Laboratory Performance Research, The Pennsylvania State University, Park, PA 16802. Received

9 July

1990; accepted

in final

form

7 November

ON

SYMPATHETIC

for Human University 1990.

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