Plasma Met-enkephalin and catecholamine to intense exercise in humans JAMES B. BOONE, JR., TERRY SHERRADEN, KRYSTYNA ROLAND0 BERGER, AND GLEN R. VAN LOON

responses

PIERZCHALA,

Division of Endocrinology and Metabolism, Department of Medicine, University of Kentucky, and Veterans Administration Medical Center, Lexington, Kentucky 40536; and Applied Physiology Laboratory, Department of Exercise and Sport Science, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-8700 BOONE, JAMES B., JR., TERRY SHERRADEN, KRYSTYNA PIERZCHALA, ROLANDO BERGER, AND GLEN R. VAN LOON. Hasma Met-enkephalin and catecholamine responses to intense exercise in humans. J. Appl. Physiol. 73(l): 388-392, 1992.Native and cryptic Met-enkephalin and catecholamines are coreleasedin responseto stress. However, it is not known whether Met-enkephalin and catecholaminesexhibit concurrent temporal relationships in responseto exercise. The purposeof this investigation wasto examine the coreleaseof catecholaminesand Met-enkephalin in endurance-trained (n = 6) and untrained (n = 6) male subjects during a 6-min bout of exercise:4 min at 70% of maximal 0, uptake (TO, m,) followed by 2 min at 120% VO, mm.Peak catecholamine levels were found at 1 min of recovery. In trained subjects,native Met-enkephalin peaked during exercise at 70% VO, max, declined during exerciseat 120%TO, -, and returned to basallevels by 1 min of recovery. In the untrained subjects,native Met-enkephalin peakedat 120%vo2 max(6 min) and returned to baseline by 5 min of recovery. In both groups, cryptic Met-enkephalin peakedat 70%60, max and returned to basal levels during exerciseat 120%VO, mEu( . These data demonstratethat during exercisethere is a temporal dissociationin plasmalevels of Met-enkephalin and catecholamines. plasma opioid peptides; epinephrine; norepinephrine; proenkephalin peptides

ENKEPHALIN IMMUNOREACTIVE PEPTIDES and opioid-

like products are found in high concentrations within the adrenal medulla chromaffin cells, central nervous system, gastrointestinal tract, pituitary, sympathetic nerve endings, and ganglia (6,7,18). Enkephalins and partially processed precursors are costored with catecholamines in the adrenal chromaffin vesicles and in sympathetic nerve terminals and ganglia (6). The costorage of enkephalins and catecholamines in the adrenal gland and sympathetic ganglia may have physiological significance, because they are coreleased in vitro (18), in vivo (ll), during splanchnic nerve stimulation (6, 7), and during restraint stress (2). The corelease of enkephalins and catecholamines may allow enkephalins to modulate the adrenergic responses at the effector tissue (ll), perhaps by binding to catecholamines (22). In a number of tissues, including adrenal medulla and brain, enkephalin-containing peptides are converted to 388

native Met-enkephalin via a series of enzymatic reactions that involve processing by carboxypeptidase B- and trypsin-like enzymes (27). The Met-enkephalin sequence in such peptides has been referred to as cryptic Met-enkephalin. The physiological relevance of cryptic Met-enkephalin remains unclear. Recently it has been reported that plasma cryptic Met-enkephalin increased in response to restraint stress in rats (20). The catecholamine response during exercise in both trained and untrained subjects is well documented; however, the pattern of the enkephalin response to exercise has yet to be clearly defined. Plasma native Met-enkephalin levels did not change after a Nordic ski race (19). Similarly, no significant increases in Leu-enkephalinlike radio-receptor activity were reported after a lo-mile race in trained male runners (9) or in response to submaximal exercise (10). A significant increase in native Met-enkephalin levels was reported in female subjects during submaximal exercise; the increase was attenuated by 8 wk of training (13). Conversely, the native Met-enkephalin response to maximal exercise was twofold higher in trained than in untrained male subjects (8). Several factors may contribute to these conflicting findings, such as time of sampling, times of testing, native Met-enkephalin assay conditions, exercise intensity and duration, and the sex of the subjects. In trained endurance athletes, an inverse relationship between peptide F immunoreactivity (proenkephalin 107-140) and epinephrine concentration in plasma in response to maximal exercise has been reported (17). Recently, it has been demonstrated in fit but not trained men that the peptide F and catecholamine responses to exercise were similar (16). Whereas peptide F contains native Met-enkephalin sequences and may be cleaved in peripheral circulation, the biological relevance of peptide F is unknown. Currently, no reported investigations have examined both the native Met-enkephalin and catecholamine responses to exercise. Therefore we have examined in humans the plasma responses of cryptic and native Met-enkephalin to exercise and whether the time course of the cryptic and native Met-enkephalin responses are similar to the catecholamine responses. Furthermore, because conflicting results exist regarding the effects of training, we have evaluated the responses in trained and untrained subjects.

0161-7567/92 $2.00 Copyright 0 1992 the American Physiological Society

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MET-ENKEPHALIN

TABLE

RESPONSE

389

TO EXERCISE

1. Physical characteristics and basal hormone ZeveZs 0%IllaX

ml kg-’

Subjs

l

Trained Untrained

l

min-’

58.0t2.3* 38.2k2.7

Weight, ka

Age,

Cryptic ME, nM

ME,

Yr

PM

EPI, nM

NE, nM

79.Ok4.5 85.7t4.7

26.2k3.2 24.8t1.6

3.6t0.9 5.5t1.6

11.2k3.0 8.7k1.9

0.26t.08 0.22~03

2.5k0.4 1.9kO.5

EPI, epinephrine;

NE, norepinephrine.

Values are means t SE of 6 subjs per group. - - ME, Met-enkephalin; groups, P < 0.05.

METHODS Subjects. Twelve healthy college-age males volunteered for the study. The endurance-trained subjects (n = 6) were currently engaged in aerobic exercise training, and the untrained subjects (n = 6) had not been involved in exercise training for 22 yr. Cycle ergometer maximal 0, uptake (VO, m,) was 58.0 t 2.3 and 38.2 t 2.7 ml. kg-’ min-l for trained and untrained subjects, respectively. All testing was performed at 0800 h after an 8-h fast. Subjects gave their written consent to participate after the risks of the investigation were explained to them verbally and in writing. This study was approved by the Human Investigations and Studies Committee at the University of Kentucky. Descriptive data and basal hormone levels for these individuals are presented in Table .1. vo ,-pretest. One week before the experimental trial the subjects reported for a Vo2maxtest on a Monark 688 bicycle ergometer. The initial work load was 100 W for 3 min. The work load was increased 50 W every 3 min thereafter, until the subjects could no longer maintain the pedal cadence of 80 rpm. The criteria that vo2,,, showed no further increase or a decrease with increasing work loads and respiratory exchange ratio >l.l5 were used to terminate the test. Expired air was analyzed for volume and for 0, and CO, concentration with a Beckman metabolic measurement cart. Exercise work loads for the subsequent exercise trial were determined by regression analysis from the VO, maxpretest. Experimental protocol and blood analysis. Subjects entered the laboratory at 0700, and a 21-gauge indwelling catheter was placed in an antecubital vein of the right forearm. Heparinized saline (50 U/ml) was infused periodically to keep the line patent. After 1 h of seated rest, a blood sample was drawn. The subjects were then escorted to the cycle ergometer. The duration of the cycle protocol was 6 min: 4 min at a resistance equal to ~70% . vo 2 maxfollowed by 2 min at m 120% Vo2 max. The supramaximal work load of 120% 1702max was calculated by multiplying the final resistance during the VO, maxtest by 1.2. Pedal cadence was maintained with a metronome set at 80 rpm. Immediately after exercise, subjects were given a 2-min cool-down ride with zero resistance. Subjects remained seated on the bicycle ergometer throughout a 200min recovery period. Collection of blood. The basal or resting blood sample was drawn after 1 h of seated rest. The exercise samples were drawn at 4 and 6 min (end of exercise) and at 1,3,5, 7, and 20 min of recovery. For all blood sampling, the catheter line was flushed with heparinized saline, a l-ml sample was drawn and discarded, and then a 5-ml sample was drawn with a new ice-cold syringe. The sample was l

* Significantly

different between

then divided into two equal aliquots, with one aliquot placed in an ice-cold polypropylene tube that contained 0.5% citrate, 0.1% EDTA, and aprotinin (200 kIU/ml of blood) final concentrations as described previously for enkephalin (20). Blood was centrifuged at 40,000 g for 30 min at 4”C, and then plasma was acidified with 0.5 N HCl final concentration and stored at -80°C until assay. The other aliquot was placed in an ice-cold polypropylene tube containing 1.0% dithiothreitol and 0.1% ethylene glycol-bis (p-aminoethyl ether)-N,N,N’,N’-tetracetic acid centrifuged at 30,000 g for 10 min. The deproteinized plasma was stored at -8OOC until assayed in duplicate for determination of epinephrine and norepinephrine by a single-isotope radioenzymatic assay procedure (24). Pre- and postexercise aliquots of blood were obtained from a finger prick for determination of hematocrit, hemoglobin, and total protein (10). Extraction of native Met-enkephalin from plasma. Acidified plasma was neutralized with 60 mM phosphate buffer, and then pH was adjusted to 10.2 with 10 N NaOH. Samples were applied to Porapak Q columns (Waters), and native Met-enkephalin was eluted with 3 ml of absolute ethanol. After lyophilization, native Metenkephalin was reconstituted in 50 mM phosphate buffer (pH 6.5) containing 0.2% bovine serum albumin and assayed by radioimmunoassay with Met-enkephalin antiserum (Immuno Nuclear), 1251-Met-enkephalin (New England Nuclear), and Met-enkephalin standard (Peninsula Laboratories) as previously described (20). The native Met-enkephalin antiserum showed cross-reactivities of ~1.0% against Met-enkephalin-Arg-Phe, Met-enkephalin-Arg-Gly-Leu, and ,&endorphin, 2.0% against Leu-enkephalin, and 20% against peptide F. Recovery of Met-enkephalin standard in the radioimmunoassay was 95%. Intra- and interassay coefficients of variation were 7 and 11%, respectively. Quantitation of totaL peptidase-derivable Met-enkephaZin in plasma. Circulating cryptic Met-enkephalin may be

derived from larger precursor peptides of molecular mass ~30 kDa, which are derived from proenkephalin, or from other proteins unrelated to proenkephalin but containing the Tyr-Gly-Gly-Phe-Met sequence or bound to plasma protein carriers. Cryptic Met-enkephalin was measured after peptidase digestion by use of the incubation conditions previously described for plasma (20). Briefly, cryptic Met-enkephalin was derived by digestion of 100 ml acidified plasma with TPCK-treated trypsin (100 mg) in a water bath at 37OC for 30 min, followed by further incubation with carboxypeptidase B (5 mg) and chicken egg white trypsin inhibitor (250 mg) for 15 min. Enzymatic hydrolysis is stopped by addition of 250 ml ice-cold tris (hydroxymethyl) aminomethane-HCl (pH 7.7) and placing samples on ice. Samples were processed

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390

MET-ENKEPHALIN

RESPONSE

as described above for native Met-enkephalin. All samples were assayed in duplicate in a single assay for each hormone. All chemicals were purchased from Sigma Chemical. Statistical analysis. The data were analyzed by a twoway analysis of variance with repeated measures and Scheffe’s post hoc test (12). Student’s t test was used to compare descriptive data. Significance was set at the 0.05 level. All data are reported as means t SE.

TO EXERCISE

. A

2.5 2.0 1.5 I-E L., cpx Q) E 1.0 .-c8 0.5

. B

I’

RESULTS

There were no significant differences in basal levels of all hormones (P > 0.05; Table 1). Catecholamines increased significantly above resting values at 70% and 120% ire 2 maxin both groups (Fig. 1, A and B). Peak values for norepinephrine and epinephrine occurred at 1 min postexercise (P < 0.01). Norepinephrine levels returned to baseline by 20 min in both groups. Plasma epinephrine returned to basal levels by 7 min of recovery in both groups. Trained subjects exhibited a significantly greater epinephrine and norepinephrine response than untrained subjects at 1 min of recovery (P < 0.05). In contrast to the catecholamine responses, native Met-enkephalin levels in the trained group peaked at 70% V02max (4 min of exercise; p < 0.05), were significantly lower by 6 min (120% VO, max), and were the same as basal levels by 1 min of recovery. In the untrained group, native Met-enkephalin levels peaked at 7O%iro,,,, were the same at 120% Vo2 m8x, and did not return to basal levels until 5 min of recovery. The peak response was not significantly different between groups (Fig. lc). In both groups, cryptic Met-enkephalin levels peaked at 70% V02mm (P < 0.05) and returned to basal levels during exercise at 120% Vo2 max(Fig. 10). There were no significant differences between trained and untrained subjects in cryptic Met-enkephalin levels at any time point (P < 0.05). During the exercise, hematocrit decreased slightly (45?3 t 1.9 to 44.3 t 2.2%), hemoglobin decreased (16.5 t 1.7 to 15.8 t 1.9 g/100 ml), and total plasma proteins increased (8.1 t 0.9 to 8.4 t 1.1 g/100 ml). None of these changes was significant, suggesting no significant changes in blood volume. DISCUSSION

The major finding of this investigation is the differences in the plasma levels of catecholamines and Metenkephalin during intense exercise. On the basis of the corelease of catecholamines and native Met-enkephalin during splanchnic nerve stimulation (6, 7) and restraint stress (20), we had hypothesized that catecholamines and native Met-enkephalin would exhibit similar responses to exercise. As expected, the catecholamine concentration increased about four- to eightfold pith the increase in exercise intensity from 70 to 120% VO, max(15, 17). However, despite the increase in exercise intensity and catecholamine levels, the native and cryptic Met-enkephalin levels in the trained subjects declined during supramaximal exercise. The magnitude of the catecholamine response was dependent on the intensity of exercise. The norepinephrine

- L

-*

+

I

II

+-trained -b-untrained

0 1

0’

’

basal

461

I

35

I+ exercis+-

I

I

I

7

28

recouery*~

TIME

(mid

1. Plasma epinephrine (A), norepinephrine (B), native Metenkephalin (C), and cryptic Met-enkephalin (0) responses (means + SE; n = 6 subjs/group) to a 6-min bout of exercise: 4 min at 70% of maximal O2 uptake (vo2 ma=)and 2 min at 120% 90~ -. *Significantly different from rest, P < 0.05. There were no significant differences between groups in basal hormone levels. FIG.

response was significantly greater than the epinephrine response, as previously reported (15,17). Because of the greater absolute exercise intensity, the only difference between groups in catecholamine levels occurred after supramaximal exercise (17). This is at odds with a previous report (15), in which epinephrine levels were greater in the trained than in the untrained subjects at 60,100, and 110% v02max. The finding of an increase in enkephalins during the 70% Vo2mm exercise agrees with a previous report (13). The basal and exercise levels of native Met-enkephalin in the present investigation are severalfold lower than previously reported levels (8,13,19), which makes it difficult to compare results. Using the radio-receptor assay, Farrell et al. (10) reported basal levels of Leu-enkephalin that were similar to the basal levels of native Met-enkephalin in the present study. However, he also reported levels that were about twofold higher (9). Because the ratio of Met- to Leu-enkephalin in proenkephalin is 4:1,

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MET-ENKEPHALIN

RESPONSE

theoretically basal levels of Met-enkephalin should be about fourfold higher. The radio-receptor assay detects ligands that are “free” to bind to the opioid receptor. Differences in basal levels may be related to the blood collection techniques utilized. Significant degradation of the pentapeptide occurs when blood is collection in EDTA; however, the addition of citrate prevents the degradation (3). The addition of aprotinin prevents degradation of larger precursor peptides into native Met-enkephalin (3, 20). Furthermore, the acidification of plasma is an important step in maintaining the stability of native Met-enkephalin in plasma (3, 20). Because none of the previous investigations (8-10, 13, 19) have reported the use of both citrate and aprotinin in the collection of the blood samples and acidification of the plasma, the discrepancy in basal and exercise levels of native Met- or Leu-enkephalin may be related to the blood collection methodology (3, 20). The native Met-enkephalin responses differed between trained and untrained subjects. In trained subjects, native Met-enkephalin peaked during exercise at 70% vozrmu (4 min) and returned to basal levels by 1 min of recovery. In the untrained subjects, native Met-enkephalin levels peaked at 4 min, remained elevated during exercise at 120% VO, mU, and returned to baseline at 5 min of recovery. In both groups, cryptic Met-enkephalin peaked at 4 min of exercise and returned to basal levels at 6 min of exercise. This suggests that training may alter the native Met-enkephalin response to exercise while having no effect on cryptic Met-enkephalin. Similarly, 1 wk of daily exposure to restraint stress attenuated the native Met-enkephalin response to stress but had no effect on the cryptic Met-enkephalin response to stress (20). It has been demonstrated that untrained subjects metabolize enkephalins at a faster rate than trained subjects (14). The results from this investigation demonstrate that plasma levels of native Met-enkephalin decreased faster in the trained than in the untrained subjects. However, plasma levels are affected by rates of appearance and disappearance. Because Met-enkephalin has a short half-life in plasma (0.5-1.0 min), the rapid decline in Met-enkephalin levels in trained subjects may reflect an early reduction in appearance rate rather than differences in metabolism (27). The effect of exercise on enkephalin metabolism is unclear. A possible explanation for the decline in enkephalins during supramaximal exercise may be a depletion of enkephalin stores. In response to restraint stress, Met-enkephalin peaks early in the stress and returns to basal levels by 7 min of stress (20), possibly because of a depletion of enkephalin stores in response to the initial stress. In this first report of the cryptic Met-enkephalin response to exercise, the cryptic Met-enkephalin response was markedly different from the catecholamine response. The decreasing levels of cryptic Met-enkephalin during supramaximal exercise, when catecholamines are increasing, suggest a temporal dissociation of plasma hormone levels during intense exercise. The finding of a decrease in cryptic Met-enkephalin with an increase in catecholamines is similar to the peptide F and catecholamine response during graded exercise (17). Further-

TO

EXERCISE

391

more there are some similarities between the cryptic Met-enkephalin response to exercise in humans and the response to restraint stress in rats. Peak levels of Metenkephalin occurred early in the stress (0.5 min) and declined to basal levels by 7 min, despite the continuation of the stress and the sustained elevated levels of catecholamines (20). The significance of the increase in cryptic Met-enkephalin in response to exercise or stress is unknown. It is possible that cryptic Met-enkephalin is cleaved in peripheral circulation to a bioactive peptide. Peptide hydrolysis of large proteins and peptides in plasma yields several bioactive peptides (5, 25). Proteolytic cleavage from circulating precursors may represent a physiological mechanism for increasing the concentration of circulating bioactive peptides. Trypsin-like enzymes may cleave cryptic Met-enkephalin to the pentapeptide in a manner similar to kallikrein, an enzyme that processes kininogen to bradykinin-related peptide (21). In the untrained subjects, the possibility exists that the sustained levels of native Met-enkephalin during supramaximal exercise resulted from the cleavage of cryptic Met-enkephalin. However, in the trained subjects this seems unlikely, because native and cryptic Met-enkephalin levels declined in a similar time frame. The dissociation of plasma levels of Met-enkephalin and catecholamines during supramaximal exercise was unexpected, because the hormones are thought to be cosecreted in response to a variety of stressors (1,6). The mechanisms that mediated this temporal dissociation cannot be explained by the present data; however, there are several possibilities. One mechanism would be an increase in clearance rate. Another possibility would be an increase in the rate of degradation that is exercise-intensity related. Although this possibility cannot be eliminated, an increase in degradation that is exercise-intensity dependent has not been demonstrated for enkephalins to date. Furthermore, maximal exercise appears to have no effect on enkephalin hydrolysis activity (14). Another possibility would be a depletion of native Metenkephalin stores. It has been demonstrated that a second peak in cryptic and native Met-enkephalin occurs at 30 min during restraint stress (20). The second peak may represent a new synthesis and release of the peptide after depletion in response to the initial stress. Another possible mechanism would involve different-sized pools of adrenal and sympathetic granules containing various amounts of native Met-enkephalin and catecholamines. Immunoreactivity studies. demonstrate the existence of immature granules that contain partially processed precursors that are released in response to an increase in K+ but not to splanchnic nerve stimulation or mild stimuli (6, 7, 23). This suggests that immature granules are released in response to intense stimuli. Finally, there may a source of native Met-enkephalin that does not contain catecholamines. This seems plausible because, in rats, chemical sympathectomy in combination with adrenal demedullation had no effect on basal native Met-enkephalin levels or the increase in response to restraint stress (1). Furthermore the combination of chemical sympathectomy and adrenal demedullation resulted in an increase in basal plasma levels of native Met-enkephalin;

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392

MET-ENKEPHALIN

RESPONSE

this indicates that in rats neither the adrenergic sympathetic nerves nor the adrenal medulla is the major source of basal native Met-enkephalin (1). However, adrenal Met-enkephalin levels in the rat are severalfold lower than in humans (27). The physiological significance of the temporal dissociation in plasma levels of Met-enkephalin and catecholamines during exercise is unknown. Although Met-enkephalin has been implicated in a wide variety of centrally mediated processes, its function as a peripherally circulating hormone is unclear. However, recently, Caffrey et al. (4) suggested that exogenous Met-enkephalin decreases vascular resistance in dog hindlimb via a peripheral site in the sympathetic ganglia. Furthermore, recent studies suggest that Met-enkephalin attenuates catecholamine activity (26), possibly by binding to catecholamines (22). We thank Laura Brown and Francis Bobbitt for technical assistance, Judy Kiritsy-Roy, Lesley Marson, and Barbara Barron for reviewing the manuscript, and Nicole Jones for editorial comments. Address for reprint requests: J. B. Boone, Jr., Applied Physiology Laboratory, Dept. of Exercise and Sport Sciences, University of North Carolina-Chapel Hill, Fetzer Gymnasium, CB 8700, Chapel Hill, NC 275994700. Received 21 January 1992; accepted in final form 4 May 1992. REFERENCES B. A., K. PIEFUCHALA, AND G. R. VAN LOON. Source of stress-induced increase in plasma Met-enkephalin in rats: contribution of adrenal medulla and/or sympathetic nerves. J. Neuroen-

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