JOURNAL
Vol.
OF
AHWED
41, No. 1, July
hYMOLOGY
1976.
Printed
in U.S.A.
Plasma renin, angiotensin II, and plasma and urinarv aldosterone in running exercise u
K. J. KOSUNEN AND A, J. PAKARINEN Department of Clinical Chemistry, University
of Helsinki,
KOSUNEN, K. J., AND A. J. PAKARINEN. Plasma renin, angiotensin II, and plasma and urinary aldosterone in running exercise. J. Appl. Physiol. 41(l): 26-29. 1976. -Plasma renin activity (PRA), renin concentration (PRC), angiotensin II and plasma and urinary aldosterone of four male athletes were investigated before and after a running exercise of 3 x 300 m. After the exercise, there were marked increases in all these parameters. The maximal increases (of the means and the ranges), found in the samples taken 30 min after the exercise, were: 108% (27-230%, P < 0.05) in PRA, 490% (240800%, P < 0.01) in PRC, 830% (400-1,970%, P < 0.025) in plasma angiotensin II and 1,600% (160-3,920%, P < 0.02) in plasma aldosterone. The increase in the urinary excretion of aldosterone was 120% (42-180%, P < 0.025). This study demonstrates that intense physical exercise may cause marked changes in all the three main components of the renin-angiotensin-aldosterone system. The significance of these changes for the physiological function of the human organism in physical stress needs further investigation.
renin activity; total protein; cise
renin concentration; serum electrolytes; serum renin-angiotensin-aldosterone system in exer-
INFORMATION concerning the components of ‘the renin-angiotensin-aldosterone system during physical exercise is scanty. It has been reported that physical exercise may increase the plasma renin activity (PRA) (1, 3-5, 7, 10, ll), and also the plasma aldosterone (19). However, we could not find reports, in which PRA, plasma angiotensin II, and aldosterone have been measured simultaneously during physical stress, Consequently we decided ‘to study each component of the renin-angiotensin-aldosterone system measuring PRA, plasma renin concentration (PRC), plasma angiotensin II, and plasma and urinary aldosterone of four male athletes in relation to running. MATERIALS
AND
METHODS
The test subjects were four Zl- to 23-yr-old male runners on an unrestricted salt diet. The exercise was 3 x 300 m run in August 1974 at 11-12 AM with 5-min rest between the first and the second 300 m and 3 min between the second and the third runs. The whole exercise lasted about 10 min. The present study was a part of a larger experiment and the exercise performed is described in greater detail elsewhere (H. Ntiveri, S. Rehunen, K. Kuoppasalmi, I. Tulikoura, and M. Htirkijnen; in preparation).
Hospital, Helsinki, Finland
The venous blood samples were taken before the run, immediately (less than 30 s) after the exercise, then 30 min, 1, 3, and 6 h, and 1 and 3 days after the run. All blood samples were taken with the subjects in the supine position. The blood samples for PRA, PRC, and angiotensin II were collected in prechilled tubes containing 0,15 ml 6% solution of Na,-EDTA per 10 ml of blood, The samples for aldosterone were collected in heparinized glass tubes. The blood samples were kept on ice and centrifuged in the cold. The plasma was immediately refrigerated and stored at -WC. The urine excreted in 2 h was collected just before and 2 h after the run. The urine samples were stored at -20°C. PRA and PRC were measured by the angiotensin I radioimmunoassay kit of CEA-IRE-SORIN, Paris, France. PRC was determined using EDTA-plasma from a nephrectcmized man as a source of the renin substrate. This renin-free plasma was added to the samples to saturate the angiotensin I generation reaction with the renin substrate. The criteria for reliability of the PRA assay were the following: sensitivity 0.21 pg angiotensin I 1-l h-l, intra-assay precision 10.4% (coeficient of variation, CV), and interassay precision 13.1% (CV). All samples of each test subject were measured in one assay. Angiotensin II was determined according to the method of Gocke et al. (8). 2,3-Dimercaptopropanol(O.16 pg/ml plasma) was added to the plasma samples to inhibit the converting enzyme and 8-hydroxyquinoline sulfate (0.66 mg/ml plasma) to inhibit the angiotensinases. lz51-labeled angiotensin II was made by iodinating [Aspl-Ile5]angiotensin II (Medical Research Council, London, England) with Na-lz51 (Atomenergi, Studsvik, Sweden) as described by Greenwood & Hunter (9). 12% labeled angiotensin II was separated from the other reaction products according to Nielsen et al. (12). Angiotensin II antiserum was purchased from CEA, Paris, France. Although described to be specific for angiotensin II, it may also bind degradation products of angiotensin II in the venous plasma (14). The incubations were carried out in 25 mmolll Tris buffer, pH 7.4. Merthiolate (0.25 mg/ml) and lysozyme (2.5 mg/ml) were added to the btier to prevent bacterial growth. The unbound 125 I-labeled angiotensin II was separated by a charcoal-dextran suspension (720 mg charcoal, Norit A from AMEND, Drug & Chemical Co., Inc., New York City and 72 mg Dextran T 70 from Pharmacia, Uppsala, Sweden, in 50 ml of Tris buffer) and the antiserumbound angiotensin II was counted for radioactivity. Senl
THE
Meilahti
l
26
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EXERCISE
AND
RENIN-ANGIOTENSIN-ALDOSTERONE
27
SYSTEM
sitivity of the method was 2 pg, intra-assay precision 10.0% (CV) and interassay precision 14.7% (CV). All samples were analyzed in one assay. Plasma and urine aldosterone were determined by the radioimmunological method developed in our laboratory by Pakarinen et al. (13). The aldosterone antibody was kindly supplied by National Institute of Arthritis and Digestive Diseases, Bethesda, Md. Sensitivity of the plasma method was 10 pg, intra-assay variation 10.2% (CV) and interassay variation 13.0% (CV). The respective values of the urine method were: Sensitivity 10 pg, intra-assay variation 7.3% (CV) and interassay variation 9.2% (CV). All samples of each test subject were analyzed in one assay. Serum sodium and potassium were determined by flame photometry (2), and chloride by a potentiometric method (6). Serum total protein concentration was measured as described by Reinhold (15). PRA and PRC are expressed as micrograms of angiotensin I generated per liter of plasma, per hour of incubation. Plasma angiotensin II concentration is expressed as ng *l-l, plasma aldosterone concentration as pmol +I and urinary aldosterone excretion as nmol h-l. The statistical analyses were carried out by the matched-pair Student t-test, and the significances were calculated from the absolute values.
values) was 98 t 69 (SE) pmol 1-l. After the exercise the four test subjects had increased values, with a high individual variation (Fig. 3). The maximal aldosterone concentration was found immediately or 30 min after the run. The mean increase at 30 min was 1,600% (range 157-3,919%; P < 0.02). The aldosterone values then decreased relatively rapidly, and within 3 h a plateau was reached. The urinary excretion of aldosterone was measured from the urine excreted in 2 h before and 2 h after the l
TABLE 1, PRA, PRC, plasma angiotensin aldosterone after intense running exercise Sample
PRAke;~i;y..-
Taken
PRC, PIS antiotensin .l-‘.h-’
.1-l .h-I
Before running Just aRer 30 min aRer 1 h aRer 3 h aRer 6 h aRer 1 day afier 3 days aRer
2.42 4.12 4.66 4.29 3.81 2.87 2.67 1.88
2 + + k 5 -t 2 +
0.31 0.24 0.39 0.22 0.15 0.07 0.37 0.23
I
3.78 5 0.82 19.90 5 2.83
Angiotensin ng. 1-l 22.2 93.3 145.5 81.9 66.4 36.7 35.0 21.6
+ t 2 2 * + 2 c
II, and
II,
8.5 14.4 33.3 21.4 14.4 7.9 15.0 7.7
Aldosterone, pmol-1-l 97.8 326.2 582.5 261.0 124.1 252.8 350.1 304.7
k f 2 2 + k 2 k
68.6 157.9 104.8 101.9 59.2 131.2 79.6 42.6
l
Values
are expressed
as means
2 SE for four test subjects.
RESULTS
Plasma renin activity and renin concentration. The mean PRA of the test subjects before the run (control values) was 2.4 t 0.3 (SE) pg angiotensin I *l-l h-l (Table 1). Immediately after the exercise the PRA values were significantly (P < 0.02) higher than the controls (Fig. 1). The maximal increases were found in the samples taken immediately or 30 min after the run, the increase (expressed as the mean and the ranges) being 108 percent (27-231%; P < 0.05) at 30 min. One hour after the run the values were still above the controls (P < 0.05), and the control values were reached 6-24 h after the exercise. To find out, whether the increase in PRA was due to an increased plasma renin concentration, PRC was determined in the control samples and in the samples taken 30 min after the run. The control value of PRC was 3,8 +- 0 . 8 (SE) pg angiotensin I 41-l h-l (Table 1). At 30 min the mean increase in PRC .was 486% (range 243-799%; P < 0.01). Plasma angiotensin II. Compared with the controls (22 t 9 SE ng *l-l), significant (P < 0.005) increases in plasma angiotensin II values were found immediately after the run, the mean increase being 382% (range 95823%) (Fig. 2). As in the case of the PRA, the maximal increases in angiotensin II were found in the samples taken immediately or 30 min after the exercise, At 30 min the mean increase was 827% (range 405-1,965%; P < 0.025). Six hours after the run the angiotensin II values were still above the controls Cp < 0.05), and within 24 h they reached the control level. Plasma and urinary aldosterone. Before the exercise the mean plasma aldosterone concentration (control l
HMRunnlng
I ,
/“---+-0
05
1
3 TIME AFTER
6 hours
1
3 days
RUNNING
FIG. 1. Changes in plasma renin activity (PRA) after an intense running exercise. Control values (= 100%) were 2.4 + 0.3 (SE) pg angiotensin I * 1-l. h-l. Various symbols represent four test subjects.
l
100I
I
I
0 05
I
I
1
3 TIME
FIG.
intense ng.l-‘.
AFTER
1 1 -6 hours 1
3&w
RUNNING
2. Changes in plasma angiotensin II concentration running exercise. Control values (= 100%) were Various svmbols renresent four test subjects.
after an 22 2 9 (SE)
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28
K.
J. KOSUNEN
AND
A. J. PAKARINEN
not obtained until several hours after the exercise. There may be several reasons for the increased PRA found in the present study: several investigators have accumulated evidence indicating that increased sympathetic activity, and high circulating catecholamine concentrations are perhaps the most important factors increasing PFU during physical exercise (3-5, 10, 11). It
FIG. 3. Changes in plasma aldosterone concentration intense running exercise. Control values (= 100%) were pmol .l+. Various symbols represent four test subjects.
after an 98 k 69 (SE)
exercise. The mean urinary excretion of aldosterone before the run was 1.8 -+ 0.5 (SE) nmol*h+ and the respective value after the exercise 3.7 2 0.5 nmol 9h-l e 0 A A (Fig. 4). This increase (120%) was statistically signifiFIG. 4. Urinary excretion rate of aldosterone after an intense cant (P < 0.025). running exercise. Aldosterone excretion rate was determined from the urine excreted in 2 h before (white columns) and 2 h after the Serum total protein concentration was measured from exercise (black columns). Various symbols represent four test subblood samples to evaluate possible changes in plasma jects. volume. Compared with the control values, there was a mean increase of 11% in the total protein concentration TABLE 2. Serum electrdytes and total protein of the samples taken immediately after the run, while after intense running exercise 30 min later the values were at the control level again (Table 2). Total Protein, Sample Taken K’, mmol-1.’ Na+, mmol.1 I Cl-, mmol.1 I g.1 ’ Serum electrolytes. The results of serum sodium, poBefore running 142.5 * 1.2 3.90 k 0.13 104.0 -+ 0.7 66.5 5 2.4 tassium, and chloride determinations are shown in Taafter 150.0 ? 1.7 3.60 k 0.16 106.3 1 1.4 73.8 * 0.9 ble 2. Compared with the starting values, there was a Just 30 min aRer 143.5 k 1.3 4.00 f 0.07 103.3 4 0.8 67.0 5 1.5 small but significant increase of about 5% in the serum 1 h aRer 141.8 k 1.7 4.28 k 0.03 104.5 2 1.3 65.8 4 3.2 3 h aRer 141.3 k 1.0 3.78 2 0.06 106.8 * 0.8 64.7 -t 3.8 sodium concentration immediately after the run, while 6 h aRer 140.8 + 2.0 3.73 1: 0.17 104.5 f 1.2 62.3 k 2.5 30 minutes later serum sodium was at the control level 1 day aRer 140.0 + 1.3 3.85 f 0.09 106.3 IF 0.8 60.0 k 2.7 3 days aRer 139.5 t 1.0 3.95 -t 0.21 104.5 I T 0.5 59.3 It 1.7 again. Before the exercise the concentration of serum potassium was 3.90 k 0.13 (SE) mmol T1 and immediValues are expressed as means -+ SE for four test subjects. ately after the exercise 3.60 ? 0.16 (SE) mmol 1-l 4 However, this difference was not statistically signifi2600r T cant (P > 0.3). There were no marked changes in serum PRA M PRC o---o chloride concentration during the experiment. ANGIOTENSIN II l
ALDOSTERONE
-
DISCUSSION
After the relatively short, very intense running exercise the test subjects had a marked increase in all the measured components of the renin-angiotensin-aldosterone system (see Fig. 5 for a summary). The magnitude of the increase was far greater than could be explained by the hemoconcentration, since only a small increase (maximally 11% immediately after the run) was found in serum total protein concentration. This conclusion is supported by the results of Naveri et al. (unpublished observations) which show that the blood hematocrit values of these test subjects just after the run were only 7% higher than those before the run. In the case of PRA, plasma angiotensin II, and plasma aldosterone the maximums of the mean values were found in the samples taken 30 min after the run, and the control levels of PRA and antiotensin II were
a
kf 500 ! ’ 1 6hours
1
0
0.5
1
3 TIME
AFTER
I1
3 days
RUNNING
FIG. 5. Summary of changes in plasma renin activity (PRA), plasma renin concentration (PRC), plasma angiotensin II, and aldosterone concentrations after intense running exercise. Each point is the mean + SE of values for four test subjects. Control values (= 100%): PRA 2.4 2 0,3 (SE) pg angiotensin IT1 ‘h-l, PRC 3.8 4 0.8 (SE) PA-g angiotensin IF *h-l, angiotensin II 22 -+ 9 (SE) ng Y, and aldosterone 98 2 69 (SE) nmol . l?
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EXERCISE
AND
RENIN-ANGIOTENSIN-ALDOSTERONE
29
SYSTEM
has also been shown that renal arterial infusion of cyclic AMP may increase renin secretion (20, 21). In connection with the exercise of the present study, plasma cyclic AMP was studied and markedly increased concentrations were found in the plasma samples taken immediately after the run (Nfiveri et al., unpublished observations). Fasola et al. (7) have reported that, in healthy subjects, muscular exercise does not increase the plasma renin substrate concentration. However, the significance of renin substrate concentration in the PRA changes found in the present study is unclear. Although increased PRC (PRA after saturating the renin reaction with the substrate) after the exercise was also found, its relationships to PRA and to the plasma renin substrate concentration under physiological conditions, e.g., in physical stress, remains uncertain. - The increase in plasma angiotensin II concentration may be explained by the increased PRA. The parallel changes in PRA and angiotensin II are in accordance with the generally accepted view that renin increases the amount of angiotensin II in the blood. It is evident that the increase found in plasma aldosterone values was, at least partly, due to a stimulatory effect of high plasma angiotensin II concentrations on the secretion of aldosterone from the adrenal cortex. When calculated on the basis of serum total protein concentration, there was slight decrease in plasma volume just after the exercise. It is possible that this change contributed to the high plasma aldosterone after the run. On the other hand, the present results could not demonstrate any distinct relationship between serum potassium concentration and the changes found in plasma aldosterone, since the differences in the mean potassium values before and after the exercise were not
statistically significant. Renin and aldosterone are metabolized by the liver. Thus, it is possible that a decreased hepatic elimination of renin and aldosterone has contributed to the increases in PRA and aldosterone found after the run. This possibility is supported by the findings that hepatic blood flow may be markedly decreased during muscular exercise ( 16, 17) , and the metabolism of renin and aldosterone in the li ver can be directly correlated wi th the hepatic blood flow (18). It is possible that the marked changes found in each component of the blood pressure regulating renin-angiotensin-aldosterone system may have important hemodynamic effects. Although the duration of the exercise was quite short, the increases in PRA and angiotensin II concentration lasted a relatively long time. Thus, it is possible that in runners, who train two or more times a day, the PRA and angiotensin II concentration may remain exceptionally high. The extent to which these marked changes in the renin-angiotensin-aldosterone system are causally related to the hemodynamic changes found during exercise are of fund amental importance and need further investigations. We thank Mrs. Aila Heikkinen for her skillful technical assistance. We are also indebted to Prof. Matti Htirkijnen, MD, and Prof. Herman Adlercreutz, MD, for their help in organizing the experimental conditions, and for the useful discussions during this work. The good advice of Pertti M&sky, MSc, is also acknowledged. An abstract of this study was presented at the 9th International Congress on Clinical Chemistry, 13-18 July 1975, Toronto, Canada. This work was partly supported by the Finnish Ministry of Education. Received
for publication
9 July
1975.
REFERENCES 1, AURELL, M,, AND P. VIKGREN. Plasma renin activity in supine muscular exercise. J. Appl. PhysioL. 31: 839-841, 1971. 2. BENOTTI, J. Sodium and potassium by flame photometry. Std. Methods CZin. Chem. 1: 102-112, 1953. 3. Bozovre, I,., AND 5. CASTENFORS. Effect of dihydralazine on plasma renin activity and renal function during supine exercise in normal subjects. Acta Physiol. Stand. 70: 281-289, 1967. 4. BOZOVIC, L., AND 3. CASTENFORS. Effect of ganglionic blocking on plasma renin activity in exercising and painstressed rats. Acta PhysioL. Stand.. 70: 290-292, 1967. 5. CASTENFORS, J. Renal function during exercise. Acta Physiol. Stand. Suppl. 293: l-44, 1967. 6. COTTLOVE, E. Chloride. Std. Methods Clilz. Chem. 3: 81-92, 1961. 7. FASOLA, A. F., B. L. MARTZ, AND 0. M. HELMER. Plasma renin activity during supine exercise in offspring of hypertensive parents. J. Appl. Physiol. 25: 410-415, 1968. 8. GOCKE, D. J., J. GERTEN, L. M. SHERWOOD, AND J. H. LARAGH. Physiological and pathological variations of plasma angiotensin II in man. Circulation Res. 24-25, Suppl. 1: 131-147, 1969. 9. HUNTER, W. M., AND F. C. GREENWOOD. Preparation of iodine131 labelled human growth hormone of high specific activity. Nature 194: 495-496, 1962. 10. KOTCHEN, T. A., L. H. HARTLEY, E. I-I. MOUGEY, L. G. JONES, AND J. W. MASON. Renin, norepinephrine, and epinephrine responses to graded exercise. J. AppZ. Physiol. 31: 178-184, 1971. 11. LEON, A. S., W. A. PETTINGER, AND M. A. SAVIANO. Enhancement of serum renin activity by exercise in the rat. hfed. Sci. Sports 5: 40-43, 1973. 12. NIELSEN, M. D., M. JORGENSEN, AND J. GIE~E. lZ%labelling of angiotensin I and II. Acta &~&rinoL. 67: 104-116, 1971.
13. PAKARINEN, A. J., L. KOSKINEN, AND H. ADLERCREUTZ. Determination of plasma and urine aldosterone by radioimmunoassay. Stand. J. Clin. Lab. Invest. In press. 14. POULSEN, K. Measurements of renin-angiotensin-aldosterone. Pharmacol. Rev. 25: 249-257, 1973. 15. REINHOLD, J. G. Total protein, albumin, and globulin. Std; Methods Clin. Chem. 1: 88-97, 1953. 16. ROWELL, L. B., J. R. BLACKMON, AND R. A. BRUCE. Indocyanine green clearance and estimated hepatic blood flow during mild to maximal exercise in upright man. J. Clin. Invest. 43: 1677-1690, 1964. 17. ROWELL, L. B., J. R. BLACKMON, R. H. MARTIN, J. A. MAZZAREI,LA, AND R. A. BRUCE. Hepatic clearance of indocyanine green in man under thermal and exercise stresses. J. Apple. Physiol. 20: 384-394, 1965. 18. SCHNEIDER, E. G., J. 0. DAVIS, J. S. BAUMBER, AND J. A. JOHNSON. The hepatic metabolism of renin and aldosterone. CircuZation Res. 26-27, Suppl. 1: 175-183, 1970. 19. SUNDSFJORD, 5. A., S. B. STROMME, I-3. E. REFSUM, AND A. AAKVAAG. Plasma aldosterone (PA), plasma renin activity (PRA), and cortisol (PF) in exercise. Acta Endocrinol. Suppl. 177: 184, 1973. 20. WINER, N., D. S. CHOKSHI, AND W. G. WALKENHORST. Effects of cyclic AMP, sympathomimetic amines, and adrenergic receptor antagonists on renin secretion. Circulation Res. 29: 239-248, 1971. 21. YAMAMOTO, K., T. OKAHARA, Y. ABE, J. UEDA, T. KISHIMOTO, AND S. MORIMOTO. Effects of cyclic AMP and dibutyryl cyclic AMP on renin release in vivo and in vitro. Japan. Circulation J. 37: 1271-1276, 1973.
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Vol.
OF
AHWED
41, No. 1, July
hYMOLOGY
1976.
Printed
in U.S.A.
Plasma renin, angiotensin II, and plasma and urinarv aldosterone in running exercise u
K. J. KOSUNEN AND A, J. PAKARINEN Department of Clinical Chemistry, University
of Helsinki,
KOSUNEN, K. J., AND A. J. PAKARINEN. Plasma renin, angiotensin II, and plasma and urinary aldosterone in running exercise. J. Appl. Physiol. 41(l): 26-29. 1976. -Plasma renin activity (PRA), renin concentration (PRC), angiotensin II and plasma and urinary aldosterone of four male athletes were investigated before and after a running exercise of 3 x 300 m. After the exercise, there were marked increases in all these parameters. The maximal increases (of the means and the ranges), found in the samples taken 30 min after the exercise, were: 108% (27-230%, P < 0.05) in PRA, 490% (240800%, P < 0.01) in PRC, 830% (400-1,970%, P < 0.025) in plasma angiotensin II and 1,600% (160-3,920%, P < 0.02) in plasma aldosterone. The increase in the urinary excretion of aldosterone was 120% (42-180%, P < 0.025). This study demonstrates that intense physical exercise may cause marked changes in all the three main components of the renin-angiotensin-aldosterone system. The significance of these changes for the physiological function of the human organism in physical stress needs further investigation.
renin activity; total protein; cise
renin concentration; serum electrolytes; serum renin-angiotensin-aldosterone system in exer-
INFORMATION concerning the components of ‘the renin-angiotensin-aldosterone system during physical exercise is scanty. It has been reported that physical exercise may increase the plasma renin activity (PRA) (1, 3-5, 7, 10, ll), and also the plasma aldosterone (19). However, we could not find reports, in which PRA, plasma angiotensin II, and aldosterone have been measured simultaneously during physical stress, Consequently we decided ‘to study each component of the renin-angiotensin-aldosterone system measuring PRA, plasma renin concentration (PRC), plasma angiotensin II, and plasma and urinary aldosterone of four male athletes in relation to running. MATERIALS
AND
METHODS
The test subjects were four Zl- to 23-yr-old male runners on an unrestricted salt diet. The exercise was 3 x 300 m run in August 1974 at 11-12 AM with 5-min rest between the first and the second 300 m and 3 min between the second and the third runs. The whole exercise lasted about 10 min. The present study was a part of a larger experiment and the exercise performed is described in greater detail elsewhere (H. Ntiveri, S. Rehunen, K. Kuoppasalmi, I. Tulikoura, and M. Htirkijnen; in preparation).
Hospital, Helsinki, Finland
The venous blood samples were taken before the run, immediately (less than 30 s) after the exercise, then 30 min, 1, 3, and 6 h, and 1 and 3 days after the run. All blood samples were taken with the subjects in the supine position. The blood samples for PRA, PRC, and angiotensin II were collected in prechilled tubes containing 0,15 ml 6% solution of Na,-EDTA per 10 ml of blood, The samples for aldosterone were collected in heparinized glass tubes. The blood samples were kept on ice and centrifuged in the cold. The plasma was immediately refrigerated and stored at -WC. The urine excreted in 2 h was collected just before and 2 h after the run. The urine samples were stored at -20°C. PRA and PRC were measured by the angiotensin I radioimmunoassay kit of CEA-IRE-SORIN, Paris, France. PRC was determined using EDTA-plasma from a nephrectcmized man as a source of the renin substrate. This renin-free plasma was added to the samples to saturate the angiotensin I generation reaction with the renin substrate. The criteria for reliability of the PRA assay were the following: sensitivity 0.21 pg angiotensin I 1-l h-l, intra-assay precision 10.4% (coeficient of variation, CV), and interassay precision 13.1% (CV). All samples of each test subject were measured in one assay. Angiotensin II was determined according to the method of Gocke et al. (8). 2,3-Dimercaptopropanol(O.16 pg/ml plasma) was added to the plasma samples to inhibit the converting enzyme and 8-hydroxyquinoline sulfate (0.66 mg/ml plasma) to inhibit the angiotensinases. lz51-labeled angiotensin II was made by iodinating [Aspl-Ile5]angiotensin II (Medical Research Council, London, England) with Na-lz51 (Atomenergi, Studsvik, Sweden) as described by Greenwood & Hunter (9). 12% labeled angiotensin II was separated from the other reaction products according to Nielsen et al. (12). Angiotensin II antiserum was purchased from CEA, Paris, France. Although described to be specific for angiotensin II, it may also bind degradation products of angiotensin II in the venous plasma (14). The incubations were carried out in 25 mmolll Tris buffer, pH 7.4. Merthiolate (0.25 mg/ml) and lysozyme (2.5 mg/ml) were added to the btier to prevent bacterial growth. The unbound 125 I-labeled angiotensin II was separated by a charcoal-dextran suspension (720 mg charcoal, Norit A from AMEND, Drug & Chemical Co., Inc., New York City and 72 mg Dextran T 70 from Pharmacia, Uppsala, Sweden, in 50 ml of Tris buffer) and the antiserumbound angiotensin II was counted for radioactivity. Senl
THE
Meilahti
l
26
Downloaded from www.physiology.org/journal/jappl by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on September 3, 2018. Copyright © 1976 American Physiological Society. All rights reserved.
EXERCISE
AND
RENIN-ANGIOTENSIN-ALDOSTERONE
27
SYSTEM
sitivity of the method was 2 pg, intra-assay precision 10.0% (CV) and interassay precision 14.7% (CV). All samples were analyzed in one assay. Plasma and urine aldosterone were determined by the radioimmunological method developed in our laboratory by Pakarinen et al. (13). The aldosterone antibody was kindly supplied by National Institute of Arthritis and Digestive Diseases, Bethesda, Md. Sensitivity of the plasma method was 10 pg, intra-assay variation 10.2% (CV) and interassay variation 13.0% (CV). The respective values of the urine method were: Sensitivity 10 pg, intra-assay variation 7.3% (CV) and interassay variation 9.2% (CV). All samples of each test subject were analyzed in one assay. Serum sodium and potassium were determined by flame photometry (2), and chloride by a potentiometric method (6). Serum total protein concentration was measured as described by Reinhold (15). PRA and PRC are expressed as micrograms of angiotensin I generated per liter of plasma, per hour of incubation. Plasma angiotensin II concentration is expressed as ng *l-l, plasma aldosterone concentration as pmol +I and urinary aldosterone excretion as nmol h-l. The statistical analyses were carried out by the matched-pair Student t-test, and the significances were calculated from the absolute values.
values) was 98 t 69 (SE) pmol 1-l. After the exercise the four test subjects had increased values, with a high individual variation (Fig. 3). The maximal aldosterone concentration was found immediately or 30 min after the run. The mean increase at 30 min was 1,600% (range 157-3,919%; P < 0.02). The aldosterone values then decreased relatively rapidly, and within 3 h a plateau was reached. The urinary excretion of aldosterone was measured from the urine excreted in 2 h before and 2 h after the l
TABLE 1, PRA, PRC, plasma angiotensin aldosterone after intense running exercise Sample
PRAke;~i;y..-
Taken
PRC, PIS antiotensin .l-‘.h-’
.1-l .h-I
Before running Just aRer 30 min aRer 1 h aRer 3 h aRer 6 h aRer 1 day afier 3 days aRer
2.42 4.12 4.66 4.29 3.81 2.87 2.67 1.88
2 + + k 5 -t 2 +
0.31 0.24 0.39 0.22 0.15 0.07 0.37 0.23
I
3.78 5 0.82 19.90 5 2.83
Angiotensin ng. 1-l 22.2 93.3 145.5 81.9 66.4 36.7 35.0 21.6
+ t 2 2 * + 2 c
II, and
II,
8.5 14.4 33.3 21.4 14.4 7.9 15.0 7.7
Aldosterone, pmol-1-l 97.8 326.2 582.5 261.0 124.1 252.8 350.1 304.7
k f 2 2 + k 2 k
68.6 157.9 104.8 101.9 59.2 131.2 79.6 42.6
l
Values
are expressed
as means
2 SE for four test subjects.
RESULTS
Plasma renin activity and renin concentration. The mean PRA of the test subjects before the run (control values) was 2.4 t 0.3 (SE) pg angiotensin I *l-l h-l (Table 1). Immediately after the exercise the PRA values were significantly (P < 0.02) higher than the controls (Fig. 1). The maximal increases were found in the samples taken immediately or 30 min after the run, the increase (expressed as the mean and the ranges) being 108 percent (27-231%; P < 0.05) at 30 min. One hour after the run the values were still above the controls (P < 0.05), and the control values were reached 6-24 h after the exercise. To find out, whether the increase in PRA was due to an increased plasma renin concentration, PRC was determined in the control samples and in the samples taken 30 min after the run. The control value of PRC was 3,8 +- 0 . 8 (SE) pg angiotensin I 41-l h-l (Table 1). At 30 min the mean increase in PRC .was 486% (range 243-799%; P < 0.01). Plasma angiotensin II. Compared with the controls (22 t 9 SE ng *l-l), significant (P < 0.005) increases in plasma angiotensin II values were found immediately after the run, the mean increase being 382% (range 95823%) (Fig. 2). As in the case of the PRA, the maximal increases in angiotensin II were found in the samples taken immediately or 30 min after the exercise, At 30 min the mean increase was 827% (range 405-1,965%; P < 0.025). Six hours after the run the angiotensin II values were still above the controls Cp < 0.05), and within 24 h they reached the control level. Plasma and urinary aldosterone. Before the exercise the mean plasma aldosterone concentration (control l
HMRunnlng
I ,
/“---+-0
05
1
3 TIME AFTER
6 hours
1
3 days
RUNNING
FIG. 1. Changes in plasma renin activity (PRA) after an intense running exercise. Control values (= 100%) were 2.4 + 0.3 (SE) pg angiotensin I * 1-l. h-l. Various symbols represent four test subjects.
l
100I
I
I
0 05
I
I
1
3 TIME
FIG.
intense ng.l-‘.
AFTER
1 1 -6 hours 1
3&w
RUNNING
2. Changes in plasma angiotensin II concentration running exercise. Control values (= 100%) were Various svmbols renresent four test subjects.
after an 22 2 9 (SE)
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28
K.
J. KOSUNEN
AND
A. J. PAKARINEN
not obtained until several hours after the exercise. There may be several reasons for the increased PRA found in the present study: several investigators have accumulated evidence indicating that increased sympathetic activity, and high circulating catecholamine concentrations are perhaps the most important factors increasing PFU during physical exercise (3-5, 10, 11). It
FIG. 3. Changes in plasma aldosterone concentration intense running exercise. Control values (= 100%) were pmol .l+. Various symbols represent four test subjects.
after an 98 k 69 (SE)
exercise. The mean urinary excretion of aldosterone before the run was 1.8 -+ 0.5 (SE) nmol*h+ and the respective value after the exercise 3.7 2 0.5 nmol 9h-l e 0 A A (Fig. 4). This increase (120%) was statistically signifiFIG. 4. Urinary excretion rate of aldosterone after an intense cant (P < 0.025). running exercise. Aldosterone excretion rate was determined from the urine excreted in 2 h before (white columns) and 2 h after the Serum total protein concentration was measured from exercise (black columns). Various symbols represent four test subblood samples to evaluate possible changes in plasma jects. volume. Compared with the control values, there was a mean increase of 11% in the total protein concentration TABLE 2. Serum electrdytes and total protein of the samples taken immediately after the run, while after intense running exercise 30 min later the values were at the control level again (Table 2). Total Protein, Sample Taken K’, mmol-1.’ Na+, mmol.1 I Cl-, mmol.1 I g.1 ’ Serum electrolytes. The results of serum sodium, poBefore running 142.5 * 1.2 3.90 k 0.13 104.0 -+ 0.7 66.5 5 2.4 tassium, and chloride determinations are shown in Taafter 150.0 ? 1.7 3.60 k 0.16 106.3 1 1.4 73.8 * 0.9 ble 2. Compared with the starting values, there was a Just 30 min aRer 143.5 k 1.3 4.00 f 0.07 103.3 4 0.8 67.0 5 1.5 small but significant increase of about 5% in the serum 1 h aRer 141.8 k 1.7 4.28 k 0.03 104.5 2 1.3 65.8 4 3.2 3 h aRer 141.3 k 1.0 3.78 2 0.06 106.8 * 0.8 64.7 -t 3.8 sodium concentration immediately after the run, while 6 h aRer 140.8 + 2.0 3.73 1: 0.17 104.5 f 1.2 62.3 k 2.5 30 minutes later serum sodium was at the control level 1 day aRer 140.0 + 1.3 3.85 f 0.09 106.3 IF 0.8 60.0 k 2.7 3 days aRer 139.5 t 1.0 3.95 -t 0.21 104.5 I T 0.5 59.3 It 1.7 again. Before the exercise the concentration of serum potassium was 3.90 k 0.13 (SE) mmol T1 and immediValues are expressed as means -+ SE for four test subjects. ately after the exercise 3.60 ? 0.16 (SE) mmol 1-l 4 However, this difference was not statistically signifi2600r T cant (P > 0.3). There were no marked changes in serum PRA M PRC o---o chloride concentration during the experiment. ANGIOTENSIN II l
ALDOSTERONE
-
DISCUSSION
After the relatively short, very intense running exercise the test subjects had a marked increase in all the measured components of the renin-angiotensin-aldosterone system (see Fig. 5 for a summary). The magnitude of the increase was far greater than could be explained by the hemoconcentration, since only a small increase (maximally 11% immediately after the run) was found in serum total protein concentration. This conclusion is supported by the results of Naveri et al. (unpublished observations) which show that the blood hematocrit values of these test subjects just after the run were only 7% higher than those before the run. In the case of PRA, plasma angiotensin II, and plasma aldosterone the maximums of the mean values were found in the samples taken 30 min after the run, and the control levels of PRA and antiotensin II were
a
kf 500 ! ’ 1 6hours
1
0
0.5
1
3 TIME
AFTER
I1
3 days
RUNNING
FIG. 5. Summary of changes in plasma renin activity (PRA), plasma renin concentration (PRC), plasma angiotensin II, and aldosterone concentrations after intense running exercise. Each point is the mean + SE of values for four test subjects. Control values (= 100%): PRA 2.4 2 0,3 (SE) pg angiotensin IT1 ‘h-l, PRC 3.8 4 0.8 (SE) PA-g angiotensin IF *h-l, angiotensin II 22 -+ 9 (SE) ng Y, and aldosterone 98 2 69 (SE) nmol . l?
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EXERCISE
AND
RENIN-ANGIOTENSIN-ALDOSTERONE
29
SYSTEM
has also been shown that renal arterial infusion of cyclic AMP may increase renin secretion (20, 21). In connection with the exercise of the present study, plasma cyclic AMP was studied and markedly increased concentrations were found in the plasma samples taken immediately after the run (Nfiveri et al., unpublished observations). Fasola et al. (7) have reported that, in healthy subjects, muscular exercise does not increase the plasma renin substrate concentration. However, the significance of renin substrate concentration in the PRA changes found in the present study is unclear. Although increased PRC (PRA after saturating the renin reaction with the substrate) after the exercise was also found, its relationships to PRA and to the plasma renin substrate concentration under physiological conditions, e.g., in physical stress, remains uncertain. - The increase in plasma angiotensin II concentration may be explained by the increased PRA. The parallel changes in PRA and angiotensin II are in accordance with the generally accepted view that renin increases the amount of angiotensin II in the blood. It is evident that the increase found in plasma aldosterone values was, at least partly, due to a stimulatory effect of high plasma angiotensin II concentrations on the secretion of aldosterone from the adrenal cortex. When calculated on the basis of serum total protein concentration, there was slight decrease in plasma volume just after the exercise. It is possible that this change contributed to the high plasma aldosterone after the run. On the other hand, the present results could not demonstrate any distinct relationship between serum potassium concentration and the changes found in plasma aldosterone, since the differences in the mean potassium values before and after the exercise were not
statistically significant. Renin and aldosterone are metabolized by the liver. Thus, it is possible that a decreased hepatic elimination of renin and aldosterone has contributed to the increases in PRA and aldosterone found after the run. This possibility is supported by the findings that hepatic blood flow may be markedly decreased during muscular exercise ( 16, 17) , and the metabolism of renin and aldosterone in the li ver can be directly correlated wi th the hepatic blood flow (18). It is possible that the marked changes found in each component of the blood pressure regulating renin-angiotensin-aldosterone system may have important hemodynamic effects. Although the duration of the exercise was quite short, the increases in PRA and angiotensin II concentration lasted a relatively long time. Thus, it is possible that in runners, who train two or more times a day, the PRA and angiotensin II concentration may remain exceptionally high. The extent to which these marked changes in the renin-angiotensin-aldosterone system are causally related to the hemodynamic changes found during exercise are of fund amental importance and need further investigations. We thank Mrs. Aila Heikkinen for her skillful technical assistance. We are also indebted to Prof. Matti Htirkijnen, MD, and Prof. Herman Adlercreutz, MD, for their help in organizing the experimental conditions, and for the useful discussions during this work. The good advice of Pertti M&sky, MSc, is also acknowledged. An abstract of this study was presented at the 9th International Congress on Clinical Chemistry, 13-18 July 1975, Toronto, Canada. This work was partly supported by the Finnish Ministry of Education. Received
for publication
9 July
1975.
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13. PAKARINEN, A. J., L. KOSKINEN, AND H. ADLERCREUTZ. Determination of plasma and urine aldosterone by radioimmunoassay. Stand. J. Clin. Lab. Invest. In press. 14. POULSEN, K. Measurements of renin-angiotensin-aldosterone. Pharmacol. Rev. 25: 249-257, 1973. 15. REINHOLD, J. G. Total protein, albumin, and globulin. Std; Methods Clin. Chem. 1: 88-97, 1953. 16. ROWELL, L. B., J. R. BLACKMON, AND R. A. BRUCE. Indocyanine green clearance and estimated hepatic blood flow during mild to maximal exercise in upright man. J. Clin. Invest. 43: 1677-1690, 1964. 17. ROWELL, L. B., J. R. BLACKMON, R. H. MARTIN, J. A. MAZZAREI,LA, AND R. A. BRUCE. Hepatic clearance of indocyanine green in man under thermal and exercise stresses. J. Apple. Physiol. 20: 384-394, 1965. 18. SCHNEIDER, E. G., J. 0. DAVIS, J. S. BAUMBER, AND J. A. JOHNSON. The hepatic metabolism of renin and aldosterone. CircuZation Res. 26-27, Suppl. 1: 175-183, 1970. 19. SUNDSFJORD, 5. A., S. B. STROMME, I-3. E. REFSUM, AND A. AAKVAAG. Plasma aldosterone (PA), plasma renin activity (PRA), and cortisol (PF) in exercise. Acta Endocrinol. Suppl. 177: 184, 1973. 20. WINER, N., D. S. CHOKSHI, AND W. G. WALKENHORST. Effects of cyclic AMP, sympathomimetic amines, and adrenergic receptor antagonists on renin secretion. Circulation Res. 29: 239-248, 1971. 21. YAMAMOTO, K., T. OKAHARA, Y. ABE, J. UEDA, T. KISHIMOTO, AND S. MORIMOTO. Effects of cyclic AMP and dibutyryl cyclic AMP on renin release in vivo and in vitro. Japan. Circulation J. 37: 1271-1276, 1973.
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