0O13-7227/78/O102-0OO2$O2.0O/O Endocrinology Copyright © 1978 by The Endocrine Society

Vol. 102, No. 2

Printed in U.S.A.

Angiotensin II/Aldosterone Dose-Response Curves in the Dog: Effect of Changes in Sodium Balance M. GARY NICHOLLS,* MALCOLM TREE, JEHOIADA J. BROWN, BEN H. DOUGLAS.t ROBERT FRASER, GORDON D. HAY, ANTHONY F. LEVER, JAMES J. MORTON, AND J. IAN S. ROBERTSON* Medical Research Council Blood Pressure Unit and Pathology Department, Western Infirmary, Glasgow, Gil 6NT, Scotland ABSTRACT. The possibility that the responsiveness of plasma aldosterone concentration to angiotensin II alters with changes in sodium balance was investigated in male beagle dogs under conditions of controlled sodium and potassium intake. Angiotensin II was infused at four different rates (usually 3, 6, 12, and 24 ng/kg/min), each for 1 h, 1) after periods of normal sodium diet (32 mEq/day), 2) after moderate sodium depletion (negative cumulative sodium balance 25-58 mEq), 3) after severe sodium depletion (65-116 mEq negative cumulative sodium balance), and 4) after sodium loading (150-212 mEq positive sodium balance), daily potassium intake remaining constant (26 mEq/day)


T WAS suggested by two groups studying man (1, 2) that the sensitivity of the adrenocortical aldosterone response to angiotensin II remains unchanged during sodium depletion. However, contrary findings have been reported from in vivo studies in man (3, 4) and the dog (5, 6) and from in vitro work in the rat (7, 8). An additional, but related controversy has arisen inasmuch as earlier experiments (9), which showed sustained elevation of aldosterone excretion during prolonged infusion of angiotensin II in dogs, have not been confirmed. Other workers have reported minimal or transient elevation of aldosterone levels in the sheep, rat, and dog when angiotensin II is administered for several hours or days (10-12). Interpretation of several of these papers is Received April 6,1977. * Recipient of a Medical Research Council of New Zealand Overseas Research Fellowship. f Visiting Fellow supported by a grant from the American Heart Association. $ Requests for reprints and correspondence should be addressed to: Dr. J. I. S. Robertson, MRC Blood Pressure Unit, Western Infirmary, Glasgow, Gil 6NT, Scotland.

throughout. Angiotensin II/aldosterone dose-response curves after moderate sodium depletion were both elevated and steepened in comparison with those found during normal sodium intake. Severe sodium depletion was associated with even greater elevation of dose-response curves, but individual aldosterone responses to angiotensin II were irregular and unpredictable. Sodium loading significantly diminished aldosterone responsiveness to angiotensin II. Blood pressure increments during angiotensin II infusion were attenuated by sodium depletion. (Endocrinology 102: 485, 1978)

difficult because the portion of the angiotensin II/aldosterone dose-response curve being examined is often uncertain, and because measurement of arterial plasma angiotensin II concentration has rarely been performed. It is clearly desirable to derive as complete doseresponse curves as possible in various states of sodium balance to avoid confusion, and the experimental animal has, in this respect, obvious advantages over man. To help clarify these issues, we have investigated the effect of alterations in sodium balance on angiotensin II/aldosterone dose-response relationships in dogs. Materials and Methods Male pedigreed beagle dogs, 2-3 years of age, were studied. The right carotid artery had previously been exteriorized within a narrow pedicle of skin to facilitate arterial cannulation. For the duration of each experiment the dogs were housed in separate metabolic cages where 24-h collections of urine and feces for electrolyte measurement were made. The diet for any one study was made up from a single batch of ingredients (boiled minced meat and liver with added essential minerals, su-


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crose, fat, and vitamins), and duplicate portions were retained for electrolyte analysis. This basic diet of low sodium (1.6-2.7 mEq/day) and moderate potassium (7.0-11.8 mEq/day) content was supplemented with potassium for all experiments (2 tablets of "Slow K," Ciba, daily; 16 mEq), and with sodium ("Slow Sodium," Ciba, 10 mEq/tablet) as required. Throughout each study, the dogs were fed and weighed once daily. Infusions of angiotensin II were made twice during each experiment: the first after a 4-day period of normal controlled dietary intake (sodium approximately 32 mEq/day, potassium 26 mEq/day), and the second after modification of sodium balance as follows: moderate sodium depletion was induced on five occasions by the iv injection of furosemide (Hoechst), 5 or 25 mg/kg, followed by 4 days of low sodium diet (1.6-2.7 mEq/day), potassium intake being maintained at the previous level (26 mEq/day). Severe sodium depletion was induced in four experiments by an initial intravenous injection of furosemide, 25 mg/kg, followed by up to 12 days of low sodium diet, potassium intake remaining constant at 26 mEq/day. Sodium loading was performed by administration of a diet of high sodium content (400-500 mEq/day), while potassium intake was unchanged, for 5 days (two experiments) or 12 days (one experiment). Between 0800 and 0900 h on the days of angiotensin II infusion, the dogs were placed on an experimental table where body position was restricted to standing or sitting. The exteriorized carotid artery was cannulated (19-gauge butterfly needle, Deseret Pharmaceutical Company) under local anesthesia (1% plain xylocaine) for blood sampling and blood pressure recording, and a fine polythene catheter was inserted into a forelimb vein for infusion. 30- to 45-min control infusion (5% dextrose) was followed by angiotensin II administration at four different incremental rates each lasting 60 min, preliminary studies having shown that this was sufficient time to establish a new steady plateau of plasma angiotensin II and aldosterone. In most cases angiotensin II was infused at 3, 6, 12, and 24 ng/kg/min, but in occasional instances rates as low as 1.5 ng/kg/min and as high as 48 ng/kg/min were used to define fuller doseresponse curves. The angiotensin II (5-isoleucine form, Schwarz-Mann) was infused in 5% dextrose solution. Arterial blood samples for estimation of plasma angiotensin II, aldosterone, cortisol, and electrolyte concentration, and for hematocrit measurement were drawn during the control infusion and just prior to the completion of each of the four

Endo • 1978 Voll02»No2

incremental angiotensin II periods. These samples were centrifuged at 4 C and the plasma stored at —20 C. Mean arterial pressure was recorded throughout using a Devices transducer, and in some cases also with a mercury manometer and kymograph drum. The greatest volume of blood drawn on any single experimental day was 125 ml; the infusion volume was between 82 and 90 ml. In order to assess possible diurnal fluctuations in the several measurements being made, and as a check on the experimental procedure per se, 5% dextrose solution alone was administered iv to three dogs, first after 4 days of normal controlled sodium intake, and again after moderate sodium depletion, the duration of the experiments and the blood sampling procedures being as for the angiotensin II infusion studies. Radioimmunoassay methods were used to measure plasma angiotensin II (13) and plasma aldosterone (14). Plasma cortisol was measured by GLC with electron capture detection (15), and sodium and potassium (in plasma, urine, food, and feces) by flame photometry. To determine the effect of angiotensin II infusion on the metabolic clearance rate of aldosterone, one dog received an incremental angiotensin II infusion, first after 4 days of normal diet and second after moderate sodium depletion (furosemide 5 mg/kg and 4 days of low sodium diet). Before angiotensin II administration on both occasions, an iv bolus of [3H]aldosterone (2 /xCi) was given, then a continuous infusion of the labeled hormone (10 jnCi over 5 h) was maintained throughout the incremental angiotensin II infusion. Arterial samples for measurement of the concentration of labelled aldosterone were drawn at the usual sampling times and at 15min intervals during the final hour of angiotensin II infusion. The [3H]aldosterone was obtained from New England Nuclear Corp., purified by chromatography, and had a final specific activity of 50 Ci/mmol.

Results Detailed results are presented in Table 1. The dogs remained contented and in good health throughout these studies, and no untoward effects were observed during infusion of angiotensin II. Changes induced by variations of sodium status As compared with the periods of normal sodium intake, the following changes were

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-15.5 (-46 to 6) -8.5 (-27 to 6)

181 (150 to 212)

19.2 (11 to 40)

1.7 (-24 to 16)


-96.7 (-65 to-116)

-38.4 (-25 to -58)

-11.2 (-39 to 4)


Cumulative electrolyte balance (mEq)


148 + 2.8

17.3 ± 0.8

143.4 ± 4.2*

146.7 + 3.4

Plasma Na (mEq/liter)

15.4 ± 1.2

15.6 ± 0.9

16.8 ± 1

Body wt (kg)

3.2 ± 0.2*


4.2 ± 0.3**

3.6 ±0.2

Plasma K (mEq/liter)

42.7 ± 0.7

47.1 ±3.3*

46.4 ± 2.3*

43.8 ± 3

Hematocrit (%)


0.5 ± 0.1

2.7 ± 1.3

2.1 ± 2.0

Plasma cortisol Oig/100 ml)


21.7 ± 20.1

218.6 ± 100.5**

67.2 ± 15.1**

30.3 ±

Plasma angiotensin II (pg/ml)

5.3 ± 1.5

123.4 ± 18.6**

28.1 ± 6.9**

5.3 ± 2.1

Plasma aldosterone (ng/100 ml)

119.3 ± 6.8

121.3 ± 3.1

108.4 ± 10.3

116.2 ± 8.9

Mean blood pressure (mm Hg)

Values shown are basal, that is, before angiotensin II infusion. Mean ± 1 SD plotted except for cumulative Na and K balance, where mean and range are given. Levels of statistical significance by t test, comparing each state with normal sodium intake: *P < 0.05, **P < 0.001. " Single value.

Group 1: Normal sodium intake (n = 9) Na, 32 mEq K, 26 mEq Group 2: Moderate sodium depletion (n = 5), furosemide, 5 or 25 mg/kg Na, 2 mEq K, 26 mEq Group 3: Severe sodium depletion (n = 4) Na, 2 mEq, for 12 days K, 26 mEq, for 12 days Group 4: Sodium loaded (n = 3) Na, 400-500 mEq K, 26 mEq

Experimental group, daily electrolyte intake

TABLE 1. Effects of sodium depletion and sodium loading



Endo • 1978 Vol 102 • No 2

observed during alterations in the sodium status of the dogs.

Effect of dextrose infusion and sampling alone

Moderate sodium depletion. This involved a mean cumulative sodium loss of 38.4 mEq associated with a significant fall in body weight. Furosemide induced an initial kaliuresis, but thereafter the low sodium diet was accompanied by a positive daily external potassium balance. Cumulative potassium balance was positive in all cases. Plasma sodium concentration fell, whereas plasma potassium concentration rose significantly and there was a significant rise in the hematocrit (Table 1). Plasma cortisol levels were not significantly altered.

Detailed results are given in Table 2. Neither in normal sodium status nor after moderate sodium depletion were dextrose infusion and blood taking accompanied by systematic or significant changes in plasma sodium, potassium, angiotensin II, or aldosterone concentrations.

Severe sodium depletion. This caused changes which were similar in direction, but generally more severe, than those induced by moderate sodium depletion. However, cumulative potassium balance was negative or only just positive. Plasma cortisol was again not altered.

Angiotensin II/aldosterone relationships before angiotensin II infusion As compared with the period of normal sodium intake, moderate sodium depletion induced significant increases in the basal arterial plasma concentrations of angiotensin II and aldosterone, and these values showed further marked elevations in severe sodium depletion (Tables 1 and 2 and Fig. 1). Sodium loading depressed plasma angiotensin II in all but one instance, although plasma aldosterone concentrations were unchanged.

Sodium loading Loading resulted in a positive cumulative sodium balance with a rise in body weight and a variable change in potassium balance. Plasma potassium concentration fell significantly. Plasma cortisol, measured in one experiment only, was within the range seen during the normal sodium diet.

Angiotensin curves



In all four states of sodium balance studied, the graded infusions of angiotensin II produced progressive increases in plasma aldosterone concentration in parallel with the increments of arterial plasma angiotensin II,

TABLE 2. Effects on plasma electrolytes, plasma hormone levels, and blood pressure of dextrose infusion with repeated blood sampling, during normal controlled diet, and after sodium depletion Infusion of 5% dextrose

Plasma sodium (mEq/liter) Normal sodium Sodium depletion Plasma potassium (mEq/liter) Normal sodium Sodium depletion Plasma aldosterone (ng/100 ml) Normal sodium Sodium depletion Plasma angiotensin II (pg/ml) Normal sodium Sodium depletion Mean blood pressure (mm Hg) Normal sodium Sodium depletion



147.5 ± 2.5 145 ± 1.5

148 ± 2 144.5 ± 1.5

147 ± 2.5 144.5 ± 1

147 ± 2 146 ± 2.5

3.4 ± 0.5 3.9 ± 0.1

3.4 ± 0.4 3.8 ± 0.2

3.5 ± 0.4 3.8 ± 0.2

3.4 ± 0.2 3.7 ± 0.1

3.4 ± 0.4 3.7 ± 0.2

3.3 ± 0.2 3.6 ± 0.2

4.3 ± 1.5 28.7 ± 3*

3.8 ± 1.1 31.5 ± 9*

4.8 ± 1.2 31.1 ± 2.5**

4.8 ± 1.2 27 ± 9.6*

5.3 ± 2.4 26.3 ± 5.6*

5.1 ± 2.5 32.1 ± 7*

22 ± 4.6 48.7 ± 24

17.3 ± 7.5 58.5 ± 15*

21.7 ± 13.6 52.7 ± 6*

21.3 ± 13.1 58.3 ± 19.2

20 ± 9.5 55.7 ± 23.2

20 ± 7 55.7 ± 18.9*

123.4 ± 8.4 104.2 ± 12

124.7 ± 11 102.3 ± 18.4

121.7 ± 9.5 105 ± 15.5

120.3 ± 13.6 98.3 ± 15.2*

114 ± 17.6 96 ± 18.8



4h 147 ± 3 146.5 ± 2

5h 147.5 ± 2.5 145.5 ± 1.5

110.3 ± 14 102 ± 15.6

Three animals were studied in both sodium-depleted and normal states. Means ± 1 SD shown. Comparisons by paired t test: *p < 0.05; **p < 0.001. Significant changes seen only on comparison of normal with sodium-depleted state. No measurement changed significantly in either state on repeated blood sampling.

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tion was very significantly steepened, as well as being shifted upwards (Fig. 2). The regression line for severe sodium depletion, although shifted even further upwards, was only slightly steeper than that of moderate sodium depletion. This regression line also was significantly steeper than that obtained in normal sodium status. By contrast, the regression line for the sodium-loaded dogs was significantly flattened as compared with that of the dogs on normal sodium diet (Fig. 2). To ensure that the augmented aldosterone responsiveness to angiotensin II observed in sodium depletion was not related to the sequence of dietary variations (i.e., normal so200


r-O 5b



< if)

I 100


1O 1OO 1OOO Arterial Plasma Angiotensin II pg/ml

FIG. 1. Individual




sponse curves during normal sodium intake (•); after moderate sodium depletion (O); after severe sodium depletion (x); and after sodium loading (•). In the severe sodium depletion group, closed triangles (A) denote basal (pre-angiotensin II infusion) values. Abscissa scale is logarithmic.

and the positive correlations were highly significant (Fig. 2). On semi-logarithmic plots, the closest approximations to sigmoid curves were seen during normal sodium balance and in moderate sodium depletion (Fig. 1). In severe sodium depletion, with very marked elevation of basal arterial plasma angiotensin II and aldosterone concentrations, angiotensin II infusions evoked often large but rather irregular aldosterone responses (Fig. 1). As compared with the angiotensin II/aldosterone regression line in normal sodium balance, that of moderate sodium deple-



8O F-8O8 p

aldosterone dose-response curves in the dog: effect of changes in sodium balance.

0O13-7227/78/O102-0OO2$O2.0O/O Endocrinology Copyright © 1978 by The Endocrine Society Vol. 102, No. 2 Printed in U.S.A. Angiotensin II/Aldosterone...
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