Klinische
W°Chesnchrift
Klin. Wschr. 56, 3 7 - 4 1 (1978)
© Springer-Verlag 1978
Effect of Prolonged Low-Dose Infusions of IleS-Angiotensin II on Blood Pressure, Aldosterone and Electrolyte Excretion in Sodium Replete Man W. Oelkers, M. Sch6nesh6fer, G. Schultze and B. Bauer Division of Endocrinology, Department of Internal Medicine, Klinikum Steglitz, Freie Universit/it Berlin, Germany
EinfluB einer niedrig dosierten Langzeit-Infusion yon IleS-Angiotensin II auf Blutdruck, Aldosteron und Elektrolytausscheidung beim Menschen Zusammenfassung. Zwei gesunde junge Mfinner, die eine normal gesalzene Di/it al3en (135 raM/Tag), erhielten ftir 132 heine Infusion mit 3 ng/kg/min Angiotensin II. Die Plasma-Angiotensin II-Konzentration stieg dadurch in einen Bereich wie bei mfiBiger Natriumverarmung. - Die Plasma-Aldosteron-Konzentration und die Ausscheidung von Aldosteron-18Glucuronid waren w/ihrend der gesamten Infusionsperiode deutlich erh6ht. Ein leichter Abfall beider MeBgr6gen in der zweiten HNfte der Infusionsperiode ist wahrscheinlich durch Natriumretention und Abfall yon Plasma-(K ÷) bedingt. Der circadiane Rhythmus des Plasma-Aldosterons und -Cortisols blieb w/ihrend der Infusion erhalten. - Der Blutdruck stieg mit zunehmender Natrium-Retention yon zun/ichst + 8 auf + 15 - 20 mm Hg an. Die Ergebnisse stehen partiell im Widerspruch zu Befunden anderer Autoren an Hunden und Schafen. Sie best/itigen die Annahme, dab Angiotensin II beim Menschen die Aldosteronsekretion chronisch stimulieren kann und nicht nur eine ,,permissive" Funktion hat. Sehliisselwiirter: Angiotensin-Infusion - Aldosteron - Cortisol - Blutdruck. Summary. Two healthy young males on a constant normal sodium diet (135 raM/day) were infused for 132 h with 3 ng/kg/min of angiotensin II. Plasma angiotensin II levels were thereby raised to the range * Supported by a grant of the Deutsche Forschungsgemeinschaft ** Presented in part to the 22rid symposium of the Deutsche Gesellschaft ffir Endokrinologie, Travemtinde, 1977 Offprint requests to: Prof. Dr. W. Oelkers, Klinikum Steglitz, Division of Endocrinology, Department of Internal Medicine, Hindenburgdamm 30, D-1000 Berlin 45
of moderate sodium depletion. Plasma aldosterone and the urinary excretion rate of aldosterone-18-glucuronide were markedly increased during the whole infusion period and returned to control levels after the infusion was stopped. A slight tendency of aldosterone secretion to decrease towards the end of infusion was probably due to sodium retention (appr. 200 mM and 350 mM respectively) and to a fall in plasma potassium by approximately 0.5mM/1. Plasma aldosterone during infusion, maintained circadian variations similar to those of cortisol. Plasma cortisol patterns were unaffected by angiotensin II. Blood pressure increased gradually during angiotensin II infusion, reflecting changes in sodium balance. The results, differing from those of studies in dog and sheep, support the assumption that angiotensin II is an important regulator of aldosterone secretion in man rather than a merely permissive factor.
Key words: Angiotensin infusion - Aldosterone Cortisol - Blood pressure.
It is controversial whether prolonged angiotensin II infusions may stimulate aldosterone secretion in a sustained manner in animals and man. The question is of importance, since a transient effect of angiotensin II on aldosterone secretion as observed by Blair-West etal.[3,4] in the sheep and by Mc C a a e t a l . [11] in the dog would lead to the conclusion that elevated plasma angiotension II levels in states of secondary hyperaldosteronism may be a permissive, but not an important regulating factor of aldosterone secretion in those conditions. Ames et al. [1], infusing rather large dosos of angiotensin II into normal man, described a sustained stimulation of aldosterone secretion, yet the dose of the peptide infused had to be gradually reduced due to large rises in blood pressure.
38
W. Oelkers et al.: Effect of Prolonged Low-Dose Infusion
3ng/Kg/min Jle5-AT I[ intraveneously Average
110 -
Mean mm HgBP
100 l
1
90
Plasma Cortisot pg 100mt
20 L
10 -
Plasma Aldo 40 ng 100 mt 30 . 20-
AER
4
tJg/h
3 2 1
Plasma
AT II
•
120
121
PRA
80 1 20 60 40 %0
"
20
[t
~
n
n
n
""-
Plasma-Na* 14~ 4~,5K÷ K+30 ~ - ] Excretion 20 1 in6h 10 (m Mol) BODY WEIGHT (Kg): 65,5 Na-Balance (m Mol):
in 6h
(mMol)
66,0 +98
67,5 e184
67,0 +165
66,9 .214
67,5 .180
65~
60 40
. . . . . .
-c ....... i . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20 Day 1 2 3 4 ] 5 I 6 ] 7 1 8 I 9 I 10 [ 11 I 12 Hour 3 91521 3 91521 3 91521 3 91521 3 91521 3 91521 3 91521 3 9
We report on the effect of a constant low-dose infusion of IleS-angiotensin II in two normal males on a normal sodium diet which lead to an increase in plasma angiotensin II levels to the range observed in moderate sodium depletion.
Methods Experimental Protocol Two normotensive male students (subject 1 : 24 years of age, 65 kg of weight; subject 2:23 years of age, 75 kg of weight) consented to participate in the study, after the protocol had been carefully explained to them. The protocol comprised a preparatory, a control and an infusion period. The same diet containing 10-20 mM of sodium and approximately 70 mM of potassium daily plus a daily supplement of 7 g MaC1 (mean daily sodium intake: 135 mM) was given throughout the study of 11 days duration. During the preparatory period (4 days), the experimental subjects followed their normal activities and collected 24 h urine samples for the measurement of volume and the excretion rates of sodium, potassium
Fig. 1. Results in subject 1 : BP blood pressure; A T H angiotensin It ; Aldo aldosterone; AER excretion rate of aldosterone-18-glucuronide; PRA plasma renin activity. Average sodium-intake and electrolyte-excretion rates are given for 6 hour-periods
and aldosterone-I8-glucuronide. The experimental subjects were admitted to the metabolic ward in the evening of the 4th preparatory day. They were asked to remain strictly recumbent for at least I h betbre blood samples were collected and for at least 5 rain before blood pressure was measured every two hours daring the day by sphygmomanometer. For the rest of the day, they were predominantly recumbent or sitting in bed, but they were allowed to get up for toilet needs, for cleaning and for weighing. At 3 a.m. of the 5th day, the experimental subjects emptied their bladders, and from now on, urine was collected in 6 h periods until 9 a.m. of day 12. Blood samples for measuring plasma aldosterone and cortisol were collected by venipuncture at 9 a.m., 3 p.m. and 9 p.m. every day, those for measuring plasma angiotensin II concentration at 9 a.m. and 9 p.m., samples for estimating plasma sodium, potassium and renin activity at 9 a.m. only. Total blood loss from day 5 to 12 amounted to approximately 500 ml in each subject. Blood pressure was measured throughout the experiment from 7 a.m. to 9 a.m. every second hour. Day 5 was the "control period". At 9 p.m. on day 5, a thin intravenous catheter was passed through the left anticubital vein up to the axillary vein, and 3 ml of 5% glucose in water per hour was infused with the aid of a BraunMelsungen perfusor up to 3 a.m. of day 6. The infusion was then
W. Oelkers et al. : Effect of Prolonged Low-Dose Infusion
39
Average
110 ~]
BpMean
lO0 1
mm Hg
90 1 J
Plasma Cortisol
l~Sml Plasma Aide
n~_ 100ml
AER ug!h
Plasma-ATII pg
Plasma -Na* 14 (mMol/l) 1~ K*-
Excretion
in6h (mMol)
4 ,2 2
BODY WEIGHJ'(Kg) :75,2[ Na*-~
Excretion in 6h (mMo[)
Fig. 2, Results in subject 2 : see legend to Figure 1
Na-BALANCE: 80 -] (mMol)
0
60 4 ['7
I
40 ~1 I
Day 1 2 3 4 t 5 Hour
changed into a solution of IleS-angiotensin II (Schwartz-Mann) in 5% glucose, which had been passed through a Millex-millipore disposable filter unit (0,22 gm). The angiotensin II concentration was made up individually for both subjects to provide an infusion rate of 3 ng angiotensin II/kg/min with an infusion volume of 3 ml/h. This infusion was continually appIied until 3 p.m. of day 11 with an interruption for 1 0 - 1 5 min every morning between 9 a.m, and 10 a.m,, while the subjects took a shower and went to the toilet. Fresh angiotensin II solutions were made up every day. Angiotensin II was infused for 51/2 days or 132h altogether. The experiment was completed after a second control period of t8 h duration at 9 a.m. on day 12.
Analytical Methods Sodium and potassium in serum and urine were measured by standard flame photometry. Plasma renin activity (PRA) was measured by our own modification [12] of the method of ttaber et al [9]. Plasma angiotensin II concentration (P-AT II) was estimated according to Dfisterdieck and Mc Elwee [6], and the excretion rate of aldosterone-18-glucuronide (AER) by the radioimmunoassay method of Vecsei et al. [I7]. Plasma aldosterone (P-AI) and cortisol were determined by radioimmunoassay after separation of the steroids by paper chromatography [15].
F ~
[
76,5
.94
77,5
4176
77,4 .258
78,2 .305
78,2 .349
IF-l
76,1
['~] [ I ]
Na* Intakein 6h
[-7
d_J__[ .......
[
1 6 I 7 8 I 9 I 10 I 11 112 3 9 15213 9 1521 3 9 1521 3 9 1521 3 9 15213 9 1521 3 9 1521 3 9
Results All results are illustrated in Figures 1 and 2. In both experimental subjects, sodium intake roughly equalled the intake during the 4 preparatory days. During the control day in the ward (day 5), sodium excretion was smaller during the night than the day, and PRA, P-AT II, AER, P-A1 and plasma cortisol remained in the normal range. During angiotensin II infusion, P-AT II was constantly increased to 35-80 pg/ml. PAT II fell to control levels after the infusion had been stopped. In both subjects PRA was markedly suppressed during angiotensin II infusion. Subject 1 (Fig. 1) retained about 180 mM of sodium during the first two days of angiotensin II infusion. Thereafter, cumulative sodium balance fluctuated around +200 mM, corresponding to a weight-gain of 2 kg. Subject 2 (Fig. 2) retained sodium up to the last day of angiotensin II infusion, when the cumulative sodium balance was +350 mM, and the weight had increased by 3 kg.
40 In the latter subject, natriuresis after topping the infusion was much higher than in the first one. Potassium excretion was slightly greater during angiotensin II infusion than in the combined preparatory and control periods, but the increase was not statistically significant (p > 0.05) according to an unpaired t-test with the data of both subjects combined. Plasma sodium concentration remained unchanged, but plasma potassium concentration fell by about 0.5 mM/1 in both subjects on the 4th day of angiotensin infusion. P-A1 was markedly elevated during the whole infusion period, but on the average, P-At seems to be slightly lower towards the end of infusion than during the first two days. In subject 1, P-AI exhibited a clear-cut circadian rhythm during infusion which was almost synchronous to that of plasma cortisol. The rhythm of P-A1 was less regular in subject 2 in contrast to that of cortisol. AER was markedly elevated in subject 1 from the beginning until the end of angiotensin II infusion. In subject 2, the initial increase in AER was still more impressive than in subject 1, but AER tended to fall during the last three days of infusion to about the same level that prevailed during the whole infusion period in subject 1. In both subjects P-A1 and AER fell substantially when the infusion had been stopped. Average mean blood pressure (diastolic BP+½ amplitude) was 8 mm Hg above control on the first day of infusion in both subjects. On the 6th day of infusion, mean BP was 16 and 21mm Hg above control in subjects 1 and 2 respectively. The increase in mean blood pressure during the 6 days of angiotensin II infusion seems to reflect the different patterns in cumulative sodium retention in the two subjects studied.
Discussion
Previous collaborative studies with the M R C Blood Pressure Unit group in Glasgow showed that P-AT II levels during moderate sodium depletion in man are in the order of 60 pg/ml [5, 13]. The P-AT II levels prevailing during prolonged infusion of 3 ng/kg/min of IleS-Angiotensin II, which is the human form of the peptide [2], are in the same order of magnitude. The present studies are therefore of physiological interest. It is apparent that P-A1 and AER were permanently elevated during the angiotensin II infusion period of 51/2 days duration. The slight tendency of P-At to decrease during prolonged angiotensin II infusion, and the more pronounced decrease of AER in subject 2 towards the end of infusion are probably attributable to a decreasing sensitivity of the adrenal zona glomerulosa against angiotensin II because of
W. Oelkers et al. : Effect of Prolonged Low-DoseInfusion sodium retention. Previous studies [10, 13, 14] have shown that sodium depletion in man increases the sensitivity of the adrenal gland against angiotensin II. It is thus likely that sodium loading causes the opposite shift in sensitivity. The fact is also noteworthy that plasma potassium levels decreased after the third day of angiotensin II infusion. Since a fall in plasma potassium concentration p e r see may reduce aldosterone secretion [7], this late effect of angiotensin II infusion is also likely to contribute to the slight reduction of P-A1 and AER in our experiment towards the end of the infusion period. However, inspite of sodium retention and a fall in plasma potassium concentration, no doubt exists that P-A1 and AER were significantly elevated thoughout the whole angiotensin II infusion period. This is definitely proved by a marked fall of P-A1 and AER in the postinfusion period. The elevation of both P-A1 and AER during angiotensin II infusion, which were both measured by specific radioimmunoassays after chromatographic purification, almost certainly excludes the possibility that major changes in aldosterone metabolism rather than secretion are involved. Furthermore, during infusion of the same dosage of angiotensin II in man, Genest et al. [8] observed a decrease in the metabolic clearance rate of aldosterone by 20% only, while in one of our previous studies [13], 6 ng/kg/min of angiotensin II did not alter the plasma disappearance curve of 3H-aldosterone in a normal subject. The circadian rhythm of P-A1, being almost synchronous to that of cortisol in subject 1, suggests that ACTH is a modulating factor of aldosterone secretion in the condition studied, while the overall secretion level is determined by the contrived elevation in plasma angiotensin II concentration. Some circadian variation of AER is also apparent, as described previously [14], but the maxima in AER lay slightly behind the peaks in P-A1. The gradual increase in mean blood pressure is probably due to increasing angiotensin II sensitivity of resistance blood vessels with increasing sodium retention. This observation is in agreement with those of Ames et al. [1]. The finding of a sustained stimulation of aldosterone secretion by prolonged angiotension II infusion confirms results of Ames et al. [1] in man and of Urquhart et al. [16] in the dog, although both studies used much higher doses of angiotensin II. This fact probably produced plasma angiotensin II levels outside the range observed in states of sodium deficiency and in most instances of secondary hyperaldosteronism. Our findings are at variance, however, with those of Blair-West et al. [3, 4] in the sheep and of Mc Caa et al. [11] in dogs. Blair-West et al. [3] infused 1 lag/ min of angiotensin II for 4 days intravenously into
W. Oelkers et al. : Effect of Prolonged Low-Dose Infusion
sheep. Related to weight, this dose corresponds to about 15 to 20 ng/kg/min. After 1 day of angiotensin II infusion, the initially raised aldosterone secretion rate had returned to the control niveau. Mc Caa et al. [1 I] infused 5 ng/kg/min ofangiotensin II into sodium replete dogs. Plasma aldosterone levels were markedly elevated during the first hours of infusion but had returned to control levels on the second day of angiotensin II infusion. In both studies, no comments are made on change in sodium balance. It is not possible to explain our different results in man with important differences in the experimental set-up. The question remains therefore open whether so marked species differences may exist in this respect. The results described in the present paper are in agreement with the assumption that angiotensin II is not merely a permissive factor, but potentially a long-term regulator of aldosterone secretion in man, the effectiveness of which is modified by sodium balance, plasma potassium concentration and ACTH. We gratefully acknowledge the expert technical assistance of Mrs. G. Wagner, Mrs. H. Kutschke and Mrs. H. Harendt in hormone analyses and the generous provision of angiotensin II-, cortisoland aldosterone-antibodies by Dr. A.F. Lever, Glasgow, Dr. P. Vecsei, Heidelberg, and the N.I.H. Bethesda, Md., USA.
References 1. Ames, R.P., Borkowski, H.J., Sicinski, A.M., Laragh, J.H.: Prolonged infusions of angiotensin II and norepinephrine and blood pressure, electrolyte balance, and aldosterone and cortisol secretion in normal man and in cirrhosis with ascites. J. clin. Invest. 44, 1171 (1965) 2. Arakawa, K., Nakatani, M., Minohara, A., Nakamura, M.: Isolation and amino acid composition of human angiotensin I. Biochem. J. 104, 900 (1967) 3. Blair-West, J.R., Cain, M., Catt, K., Coghlan~ J.P., Denton, D.A.° Funder, J.W., Scoggins, B.A., Wright, R.D. : Aldosterone regulation and biosynthesis. In: Proc. 3rd Int. Congr. Endo~ crin., Int. Congr. Series 184, Excerpta Med. (Amst.) 276 (1969) 4. Blair-West, J.R., Coghlan, J.P., Denton, D.A., Funder, J.W., Scoggins, B.A. : Role of the renin-angiotensin system in control of aldosterone secretion. Adv. Exp. Biol. Med. 17, 167 (1972) 5. Brown, J.J., Fraser, R., Lever, A.F., Morton, J.J., Oelkers, W., Robertson, J.I.S., Young, J.: Further observations on the relationship between plasma angiotensin II and aldosterone during sodium deprivation. In: Mechanisms of hypertension (M.P. Sambhi ed.), Internat. Congr. Series 302, Excerpta Med. (Amst.) I48 (1973)
41 6. Dfisterdieck, G., Mc Elwee, G.: Estimation of angiotensin II concentration in human plasma by radioimmunoassay. Some applications to physiological and clinical states. Europ. J. clin. Invest. 2, 32 (1971) 7. Funder, J.W., Blair-West, J.R., Coghlan, J.P., Denton, D.A., Scoggins, B.A., Wright, R.D.: Effect of plasma (K-) on the secretion of aldosterone. Endocrinology 85, 381 (1969) 8. Genest, J., Nowaczynski, W., KiicheI, O., Boucher, R., RojoOrtega, J.M., Constantopoulos, G., Ganten, D., Messerli, F. : The adrenal cortex and essential hypertension. Rec. Prog. Hormone Res. 32, 377 (1976) 9. Haber, E., Koerner, T.~ Page, L.B., Kliman, B., Purnode, A.: Application of a radioimmunoassay for angiotensin I to the physiologic measurements of plasma renin activity in normal human subjects. J. Clin. Endocrinol. Metab. 29, 1349 (1969) 10. Hollenberg, N.K., Chenitz, W.R., Adams, D,F., Williams, G.H. : Reciprocal influence of salt intake on adrenal glomerulosa and renal vascular responses to angiotensin II in normal man. J. clin. Invest. 54, 34 (1974) 11. Mc Caa, R.E., Mc Caa, C.S., Guyton, A.C. : Role of angiotensin II and potassium in the long-term regulation of aldosterone secretion in intact conscious dogs. Circ. Res. suppl. I to vols. 36+37, 57 (1975) 12. Oelkers, W., Sch6nesh6fer, M., Bl/imel, A. : Effects of progesterone and four synthetic progestagens on sodium balance and the renin-aldosterone system in man. J. Clin. Endocrinol. Metab. 39, 882 (1974) 13. Oelkers, W., Brown, J.J., Fraser, R., Lever, A.F., Morton, J.J., Robertson, J.I.S.: Sensitization of the adrenal cortex to angiotensin II in sodium depleted man. Circ. Res. 34, 69 (1974) 14. Oelkers, W., Sch6nesh6fer, M., Schuttze, G., Brown, J.J., Fraser, R., Morton, J.J., Lever, A.F,, Robertson, J.I.S. : Effect of prolonged low-dose angiotensin II infusion on the sensitivity of adrenal cortex in man. Circ. Res. suppl. I to vols. 36+37, 49 (1975) 15. Sch6nesh6fer, M. : Simultaneous determination of eight adrenal steroids in human serum by radioimmunoassay. J. Steroid Biochem. accepted 16. Urquhart, J., Davis, J.O., Higgins, J.T. jr. : Effects of prolonged infusion of angiotensin II in normal dogs. Amer. J. Physiol. 205, 1241 (1963) 17. Vescei, P., Penke, B., Joumaah, A.: Radioimmunoassay of atdosterone and of its 18-oxo-glucuronide in human urine. Experientia (Basel) 28, 622 (1972)
Prof. Dr. W. Oelkers Klinikum Steglitz Division of Endocrinology Department of Internal Medicine Hindenburgdamm 30 D-1000 Berlin 45
W°Chesnchrift
Klin. Wschr. 56, 3 7 - 4 1 (1978)
© Springer-Verlag 1978
Effect of Prolonged Low-Dose Infusions of IleS-Angiotensin II on Blood Pressure, Aldosterone and Electrolyte Excretion in Sodium Replete Man W. Oelkers, M. Sch6nesh6fer, G. Schultze and B. Bauer Division of Endocrinology, Department of Internal Medicine, Klinikum Steglitz, Freie Universit/it Berlin, Germany
EinfluB einer niedrig dosierten Langzeit-Infusion yon IleS-Angiotensin II auf Blutdruck, Aldosteron und Elektrolytausscheidung beim Menschen Zusammenfassung. Zwei gesunde junge Mfinner, die eine normal gesalzene Di/it al3en (135 raM/Tag), erhielten ftir 132 heine Infusion mit 3 ng/kg/min Angiotensin II. Die Plasma-Angiotensin II-Konzentration stieg dadurch in einen Bereich wie bei mfiBiger Natriumverarmung. - Die Plasma-Aldosteron-Konzentration und die Ausscheidung von Aldosteron-18Glucuronid waren w/ihrend der gesamten Infusionsperiode deutlich erh6ht. Ein leichter Abfall beider MeBgr6gen in der zweiten HNfte der Infusionsperiode ist wahrscheinlich durch Natriumretention und Abfall yon Plasma-(K ÷) bedingt. Der circadiane Rhythmus des Plasma-Aldosterons und -Cortisols blieb w/ihrend der Infusion erhalten. - Der Blutdruck stieg mit zunehmender Natrium-Retention yon zun/ichst + 8 auf + 15 - 20 mm Hg an. Die Ergebnisse stehen partiell im Widerspruch zu Befunden anderer Autoren an Hunden und Schafen. Sie best/itigen die Annahme, dab Angiotensin II beim Menschen die Aldosteronsekretion chronisch stimulieren kann und nicht nur eine ,,permissive" Funktion hat. Sehliisselwiirter: Angiotensin-Infusion - Aldosteron - Cortisol - Blutdruck. Summary. Two healthy young males on a constant normal sodium diet (135 raM/day) were infused for 132 h with 3 ng/kg/min of angiotensin II. Plasma angiotensin II levels were thereby raised to the range * Supported by a grant of the Deutsche Forschungsgemeinschaft ** Presented in part to the 22rid symposium of the Deutsche Gesellschaft ffir Endokrinologie, Travemtinde, 1977 Offprint requests to: Prof. Dr. W. Oelkers, Klinikum Steglitz, Division of Endocrinology, Department of Internal Medicine, Hindenburgdamm 30, D-1000 Berlin 45
of moderate sodium depletion. Plasma aldosterone and the urinary excretion rate of aldosterone-18-glucuronide were markedly increased during the whole infusion period and returned to control levels after the infusion was stopped. A slight tendency of aldosterone secretion to decrease towards the end of infusion was probably due to sodium retention (appr. 200 mM and 350 mM respectively) and to a fall in plasma potassium by approximately 0.5mM/1. Plasma aldosterone during infusion, maintained circadian variations similar to those of cortisol. Plasma cortisol patterns were unaffected by angiotensin II. Blood pressure increased gradually during angiotensin II infusion, reflecting changes in sodium balance. The results, differing from those of studies in dog and sheep, support the assumption that angiotensin II is an important regulator of aldosterone secretion in man rather than a merely permissive factor.
Key words: Angiotensin infusion - Aldosterone Cortisol - Blood pressure.
It is controversial whether prolonged angiotensin II infusions may stimulate aldosterone secretion in a sustained manner in animals and man. The question is of importance, since a transient effect of angiotensin II on aldosterone secretion as observed by Blair-West etal.[3,4] in the sheep and by Mc C a a e t a l . [11] in the dog would lead to the conclusion that elevated plasma angiotension II levels in states of secondary hyperaldosteronism may be a permissive, but not an important regulating factor of aldosterone secretion in those conditions. Ames et al. [1], infusing rather large dosos of angiotensin II into normal man, described a sustained stimulation of aldosterone secretion, yet the dose of the peptide infused had to be gradually reduced due to large rises in blood pressure.
38
W. Oelkers et al.: Effect of Prolonged Low-Dose Infusion
3ng/Kg/min Jle5-AT I[ intraveneously Average
110 -
Mean mm HgBP
100 l
1
90
Plasma Cortisot pg 100mt
20 L
10 -
Plasma Aldo 40 ng 100 mt 30 . 20-
AER
4
tJg/h
3 2 1
Plasma
AT II
•
120
121
PRA
80 1 20 60 40 %0
"
20
[t
~
n
n
n
""-
Plasma-Na* 14~ 4~,5K÷ K+30 ~ - ] Excretion 20 1 in6h 10 (m Mol) BODY WEIGHT (Kg): 65,5 Na-Balance (m Mol):
in 6h
(mMol)
66,0 +98
67,5 e184
67,0 +165
66,9 .214
67,5 .180
65~
60 40
. . . . . .
-c ....... i . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20 Day 1 2 3 4 ] 5 I 6 ] 7 1 8 I 9 I 10 [ 11 I 12 Hour 3 91521 3 91521 3 91521 3 91521 3 91521 3 91521 3 91521 3 9
We report on the effect of a constant low-dose infusion of IleS-angiotensin II in two normal males on a normal sodium diet which lead to an increase in plasma angiotensin II levels to the range observed in moderate sodium depletion.
Methods Experimental Protocol Two normotensive male students (subject 1 : 24 years of age, 65 kg of weight; subject 2:23 years of age, 75 kg of weight) consented to participate in the study, after the protocol had been carefully explained to them. The protocol comprised a preparatory, a control and an infusion period. The same diet containing 10-20 mM of sodium and approximately 70 mM of potassium daily plus a daily supplement of 7 g MaC1 (mean daily sodium intake: 135 mM) was given throughout the study of 11 days duration. During the preparatory period (4 days), the experimental subjects followed their normal activities and collected 24 h urine samples for the measurement of volume and the excretion rates of sodium, potassium
Fig. 1. Results in subject 1 : BP blood pressure; A T H angiotensin It ; Aldo aldosterone; AER excretion rate of aldosterone-18-glucuronide; PRA plasma renin activity. Average sodium-intake and electrolyte-excretion rates are given for 6 hour-periods
and aldosterone-I8-glucuronide. The experimental subjects were admitted to the metabolic ward in the evening of the 4th preparatory day. They were asked to remain strictly recumbent for at least I h betbre blood samples were collected and for at least 5 rain before blood pressure was measured every two hours daring the day by sphygmomanometer. For the rest of the day, they were predominantly recumbent or sitting in bed, but they were allowed to get up for toilet needs, for cleaning and for weighing. At 3 a.m. of the 5th day, the experimental subjects emptied their bladders, and from now on, urine was collected in 6 h periods until 9 a.m. of day 12. Blood samples for measuring plasma aldosterone and cortisol were collected by venipuncture at 9 a.m., 3 p.m. and 9 p.m. every day, those for measuring plasma angiotensin II concentration at 9 a.m. and 9 p.m., samples for estimating plasma sodium, potassium and renin activity at 9 a.m. only. Total blood loss from day 5 to 12 amounted to approximately 500 ml in each subject. Blood pressure was measured throughout the experiment from 7 a.m. to 9 a.m. every second hour. Day 5 was the "control period". At 9 p.m. on day 5, a thin intravenous catheter was passed through the left anticubital vein up to the axillary vein, and 3 ml of 5% glucose in water per hour was infused with the aid of a BraunMelsungen perfusor up to 3 a.m. of day 6. The infusion was then
W. Oelkers et al. : Effect of Prolonged Low-Dose Infusion
39
Average
110 ~]
BpMean
lO0 1
mm Hg
90 1 J
Plasma Cortisol
l~Sml Plasma Aide
n~_ 100ml
AER ug!h
Plasma-ATII pg
Plasma -Na* 14 (mMol/l) 1~ K*-
Excretion
in6h (mMol)
4 ,2 2
BODY WEIGHJ'(Kg) :75,2[ Na*-~
Excretion in 6h (mMo[)
Fig. 2, Results in subject 2 : see legend to Figure 1
Na-BALANCE: 80 -] (mMol)
0
60 4 ['7
I
40 ~1 I
Day 1 2 3 4 t 5 Hour
changed into a solution of IleS-angiotensin II (Schwartz-Mann) in 5% glucose, which had been passed through a Millex-millipore disposable filter unit (0,22 gm). The angiotensin II concentration was made up individually for both subjects to provide an infusion rate of 3 ng angiotensin II/kg/min with an infusion volume of 3 ml/h. This infusion was continually appIied until 3 p.m. of day 11 with an interruption for 1 0 - 1 5 min every morning between 9 a.m, and 10 a.m,, while the subjects took a shower and went to the toilet. Fresh angiotensin II solutions were made up every day. Angiotensin II was infused for 51/2 days or 132h altogether. The experiment was completed after a second control period of t8 h duration at 9 a.m. on day 12.
Analytical Methods Sodium and potassium in serum and urine were measured by standard flame photometry. Plasma renin activity (PRA) was measured by our own modification [12] of the method of ttaber et al [9]. Plasma angiotensin II concentration (P-AT II) was estimated according to Dfisterdieck and Mc Elwee [6], and the excretion rate of aldosterone-18-glucuronide (AER) by the radioimmunoassay method of Vecsei et al. [I7]. Plasma aldosterone (P-AI) and cortisol were determined by radioimmunoassay after separation of the steroids by paper chromatography [15].
F ~
[
76,5
.94
77,5
4176
77,4 .258
78,2 .305
78,2 .349
IF-l
76,1
['~] [ I ]
Na* Intakein 6h
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Results All results are illustrated in Figures 1 and 2. In both experimental subjects, sodium intake roughly equalled the intake during the 4 preparatory days. During the control day in the ward (day 5), sodium excretion was smaller during the night than the day, and PRA, P-AT II, AER, P-A1 and plasma cortisol remained in the normal range. During angiotensin II infusion, P-AT II was constantly increased to 35-80 pg/ml. PAT II fell to control levels after the infusion had been stopped. In both subjects PRA was markedly suppressed during angiotensin II infusion. Subject 1 (Fig. 1) retained about 180 mM of sodium during the first two days of angiotensin II infusion. Thereafter, cumulative sodium balance fluctuated around +200 mM, corresponding to a weight-gain of 2 kg. Subject 2 (Fig. 2) retained sodium up to the last day of angiotensin II infusion, when the cumulative sodium balance was +350 mM, and the weight had increased by 3 kg.
40 In the latter subject, natriuresis after topping the infusion was much higher than in the first one. Potassium excretion was slightly greater during angiotensin II infusion than in the combined preparatory and control periods, but the increase was not statistically significant (p > 0.05) according to an unpaired t-test with the data of both subjects combined. Plasma sodium concentration remained unchanged, but plasma potassium concentration fell by about 0.5 mM/1 in both subjects on the 4th day of angiotensin infusion. P-A1 was markedly elevated during the whole infusion period, but on the average, P-At seems to be slightly lower towards the end of infusion than during the first two days. In subject 1, P-AI exhibited a clear-cut circadian rhythm during infusion which was almost synchronous to that of plasma cortisol. The rhythm of P-A1 was less regular in subject 2 in contrast to that of cortisol. AER was markedly elevated in subject 1 from the beginning until the end of angiotensin II infusion. In subject 2, the initial increase in AER was still more impressive than in subject 1, but AER tended to fall during the last three days of infusion to about the same level that prevailed during the whole infusion period in subject 1. In both subjects P-A1 and AER fell substantially when the infusion had been stopped. Average mean blood pressure (diastolic BP+½ amplitude) was 8 mm Hg above control on the first day of infusion in both subjects. On the 6th day of infusion, mean BP was 16 and 21mm Hg above control in subjects 1 and 2 respectively. The increase in mean blood pressure during the 6 days of angiotensin II infusion seems to reflect the different patterns in cumulative sodium retention in the two subjects studied.
Discussion
Previous collaborative studies with the M R C Blood Pressure Unit group in Glasgow showed that P-AT II levels during moderate sodium depletion in man are in the order of 60 pg/ml [5, 13]. The P-AT II levels prevailing during prolonged infusion of 3 ng/kg/min of IleS-Angiotensin II, which is the human form of the peptide [2], are in the same order of magnitude. The present studies are therefore of physiological interest. It is apparent that P-A1 and AER were permanently elevated during the angiotensin II infusion period of 51/2 days duration. The slight tendency of P-At to decrease during prolonged angiotensin II infusion, and the more pronounced decrease of AER in subject 2 towards the end of infusion are probably attributable to a decreasing sensitivity of the adrenal zona glomerulosa against angiotensin II because of
W. Oelkers et al. : Effect of Prolonged Low-DoseInfusion sodium retention. Previous studies [10, 13, 14] have shown that sodium depletion in man increases the sensitivity of the adrenal gland against angiotensin II. It is thus likely that sodium loading causes the opposite shift in sensitivity. The fact is also noteworthy that plasma potassium levels decreased after the third day of angiotensin II infusion. Since a fall in plasma potassium concentration p e r see may reduce aldosterone secretion [7], this late effect of angiotensin II infusion is also likely to contribute to the slight reduction of P-A1 and AER in our experiment towards the end of the infusion period. However, inspite of sodium retention and a fall in plasma potassium concentration, no doubt exists that P-A1 and AER were significantly elevated thoughout the whole angiotensin II infusion period. This is definitely proved by a marked fall of P-A1 and AER in the postinfusion period. The elevation of both P-A1 and AER during angiotensin II infusion, which were both measured by specific radioimmunoassays after chromatographic purification, almost certainly excludes the possibility that major changes in aldosterone metabolism rather than secretion are involved. Furthermore, during infusion of the same dosage of angiotensin II in man, Genest et al. [8] observed a decrease in the metabolic clearance rate of aldosterone by 20% only, while in one of our previous studies [13], 6 ng/kg/min of angiotensin II did not alter the plasma disappearance curve of 3H-aldosterone in a normal subject. The circadian rhythm of P-A1, being almost synchronous to that of cortisol in subject 1, suggests that ACTH is a modulating factor of aldosterone secretion in the condition studied, while the overall secretion level is determined by the contrived elevation in plasma angiotensin II concentration. Some circadian variation of AER is also apparent, as described previously [14], but the maxima in AER lay slightly behind the peaks in P-A1. The gradual increase in mean blood pressure is probably due to increasing angiotensin II sensitivity of resistance blood vessels with increasing sodium retention. This observation is in agreement with those of Ames et al. [1]. The finding of a sustained stimulation of aldosterone secretion by prolonged angiotension II infusion confirms results of Ames et al. [1] in man and of Urquhart et al. [16] in the dog, although both studies used much higher doses of angiotensin II. This fact probably produced plasma angiotensin II levels outside the range observed in states of sodium deficiency and in most instances of secondary hyperaldosteronism. Our findings are at variance, however, with those of Blair-West et al. [3, 4] in the sheep and of Mc Caa et al. [11] in dogs. Blair-West et al. [3] infused 1 lag/ min of angiotensin II for 4 days intravenously into
W. Oelkers et al. : Effect of Prolonged Low-Dose Infusion
sheep. Related to weight, this dose corresponds to about 15 to 20 ng/kg/min. After 1 day of angiotensin II infusion, the initially raised aldosterone secretion rate had returned to the control niveau. Mc Caa et al. [1 I] infused 5 ng/kg/min ofangiotensin II into sodium replete dogs. Plasma aldosterone levels were markedly elevated during the first hours of infusion but had returned to control levels on the second day of angiotensin II infusion. In both studies, no comments are made on change in sodium balance. It is not possible to explain our different results in man with important differences in the experimental set-up. The question remains therefore open whether so marked species differences may exist in this respect. The results described in the present paper are in agreement with the assumption that angiotensin II is not merely a permissive factor, but potentially a long-term regulator of aldosterone secretion in man, the effectiveness of which is modified by sodium balance, plasma potassium concentration and ACTH. We gratefully acknowledge the expert technical assistance of Mrs. G. Wagner, Mrs. H. Kutschke and Mrs. H. Harendt in hormone analyses and the generous provision of angiotensin II-, cortisoland aldosterone-antibodies by Dr. A.F. Lever, Glasgow, Dr. P. Vecsei, Heidelberg, and the N.I.H. Bethesda, Md., USA.
References 1. Ames, R.P., Borkowski, H.J., Sicinski, A.M., Laragh, J.H.: Prolonged infusions of angiotensin II and norepinephrine and blood pressure, electrolyte balance, and aldosterone and cortisol secretion in normal man and in cirrhosis with ascites. J. clin. Invest. 44, 1171 (1965) 2. Arakawa, K., Nakatani, M., Minohara, A., Nakamura, M.: Isolation and amino acid composition of human angiotensin I. Biochem. J. 104, 900 (1967) 3. Blair-West, J.R., Cain, M., Catt, K., Coghlan~ J.P., Denton, D.A.° Funder, J.W., Scoggins, B.A., Wright, R.D. : Aldosterone regulation and biosynthesis. In: Proc. 3rd Int. Congr. Endo~ crin., Int. Congr. Series 184, Excerpta Med. (Amst.) 276 (1969) 4. Blair-West, J.R., Coghlan, J.P., Denton, D.A., Funder, J.W., Scoggins, B.A. : Role of the renin-angiotensin system in control of aldosterone secretion. Adv. Exp. Biol. Med. 17, 167 (1972) 5. Brown, J.J., Fraser, R., Lever, A.F., Morton, J.J., Oelkers, W., Robertson, J.I.S., Young, J.: Further observations on the relationship between plasma angiotensin II and aldosterone during sodium deprivation. In: Mechanisms of hypertension (M.P. Sambhi ed.), Internat. Congr. Series 302, Excerpta Med. (Amst.) I48 (1973)
41 6. Dfisterdieck, G., Mc Elwee, G.: Estimation of angiotensin II concentration in human plasma by radioimmunoassay. Some applications to physiological and clinical states. Europ. J. clin. Invest. 2, 32 (1971) 7. Funder, J.W., Blair-West, J.R., Coghlan, J.P., Denton, D.A., Scoggins, B.A., Wright, R.D.: Effect of plasma (K-) on the secretion of aldosterone. Endocrinology 85, 381 (1969) 8. Genest, J., Nowaczynski, W., KiicheI, O., Boucher, R., RojoOrtega, J.M., Constantopoulos, G., Ganten, D., Messerli, F. : The adrenal cortex and essential hypertension. Rec. Prog. Hormone Res. 32, 377 (1976) 9. Haber, E., Koerner, T.~ Page, L.B., Kliman, B., Purnode, A.: Application of a radioimmunoassay for angiotensin I to the physiologic measurements of plasma renin activity in normal human subjects. J. Clin. Endocrinol. Metab. 29, 1349 (1969) 10. Hollenberg, N.K., Chenitz, W.R., Adams, D,F., Williams, G.H. : Reciprocal influence of salt intake on adrenal glomerulosa and renal vascular responses to angiotensin II in normal man. J. clin. Invest. 54, 34 (1974) 11. Mc Caa, R.E., Mc Caa, C.S., Guyton, A.C. : Role of angiotensin II and potassium in the long-term regulation of aldosterone secretion in intact conscious dogs. Circ. Res. suppl. I to vols. 36+37, 57 (1975) 12. Oelkers, W., Sch6nesh6fer, M., Bl/imel, A. : Effects of progesterone and four synthetic progestagens on sodium balance and the renin-aldosterone system in man. J. Clin. Endocrinol. Metab. 39, 882 (1974) 13. Oelkers, W., Brown, J.J., Fraser, R., Lever, A.F., Morton, J.J., Robertson, J.I.S.: Sensitization of the adrenal cortex to angiotensin II in sodium depleted man. Circ. Res. 34, 69 (1974) 14. Oelkers, W., Sch6nesh6fer, M., Schuttze, G., Brown, J.J., Fraser, R., Morton, J.J., Lever, A.F,, Robertson, J.I.S. : Effect of prolonged low-dose angiotensin II infusion on the sensitivity of adrenal cortex in man. Circ. Res. suppl. I to vols. 36+37, 49 (1975) 15. Sch6nesh6fer, M. : Simultaneous determination of eight adrenal steroids in human serum by radioimmunoassay. J. Steroid Biochem. accepted 16. Urquhart, J., Davis, J.O., Higgins, J.T. jr. : Effects of prolonged infusion of angiotensin II in normal dogs. Amer. J. Physiol. 205, 1241 (1963) 17. Vescei, P., Penke, B., Joumaah, A.: Radioimmunoassay of atdosterone and of its 18-oxo-glucuronide in human urine. Experientia (Basel) 28, 622 (1972)
Prof. Dr. W. Oelkers Klinikum Steglitz Division of Endocrinology Department of Internal Medicine Hindenburgdamm 30 D-1000 Berlin 45
