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The effect of renin and aldosterone inhibition by β-adrenergic blockade on the response to the new diuretic azosemide
European Journal o f Pharmacology, 69 (1981) 439--446
439
© Elsevier/North-Holland Biomedical Press
T H E E F F E C T O F R E N I N A N D A L D O S T E R O N E I N H I B I T I O N BY ~ - A D R E N E R G I C BLOCKADE ON THE RESPONSE TO THE NEW DIURETIC AZOSEMIDE KAREL HORKY, INGEBORG GREGOROVfi-, JANA DVO~..~KOV.~ Laboratory for Endocrinology and Metabolism and the Third Medical Clinic, Charles University Faculty o f Medicine, U nemocnice 1, 128 21 Prague 2, Czechoslovakia
Received 29 July 1980, revised MS received 24 October 1980, accepted 6 November 1980
K. HORK~/, I. GREGOROV/k and J. DVOR.~KOV/~, The effect o f renin and aldosterone inhibition by ~-adrenergic blockade on the response to the new diuretic Azosemide, European J. Pharmacol. 69 (1981) 439--446. The effect of repeated Azosemide infusions (20 mg in 500 ml 5% glucose for one h) on urine volume and electrolyte excretion, and on the activity of the renin-angiotensin-aldosterone system (RAAS) was studied in a group of 15 patients with benign essential hypertension before and during treatment with the ~-adrenergic blocker Trimepranol. Azosemide alone had a marked but short-lasting diuretic and natriuretic effect. Repeated administration on three consecutive days led, however, to a progressive decrease in the natriuretic effectiveness of Azosemide, associated with an increase in plasma renin activity (from 0.413 + 0.123 to 2.631 + 0.421 pmol/1]sec and in plasma aldosterone concentration (from 0.335 +- 0.032 to 1.687 + 0.438 pmol/l). Treatment with Trimepranol 30 rag/day enhanced and prolonged the diuretic and natriuretic response to Azosemide concomitantly with a reduction of its stimulatory effect on RAAS. There results suggest that stimulation of the RAAS might be responsible for the diminishing effectiveness of repeated Azosemide infusions and that the stimulation could be, at least partly, inhibited by a ~-blocker Trimepranol, resulting in a greater diuretic and natriuretic effect of Azosemide. Renin -angiotensin-aldosterone system Diuretics
Azosemide
1. I n t r o d u c t i o n A z o s e m i d e ( p r o d u c t BM 0 2 . 0 0 1 , B o e h ringer M a n n h e i m G m b H , G F R ) is a n e w a n d highly e f f e c t i v e d i u r e t i c , w h o s e s t r u c t u r e , d i u r e t i c e f f e c t a n d a c t i v i t y are similar t o t h o s e o f f u r o s e m i d e (Brater et al., 1 9 7 9 ) . D e s p i t e t h e high a c t i v i t y o f t h e first d o s e o f Azosem i d e , o n r e p e a t e d oral a d m i n i s t r a t i o n it d e c r e a s e d t h e 2 4 h s o d i u m a n d w a t e r excret i o n in h e a l t h y subjects t o t h e c o n t r o l values (Kriick et al., 1 9 7 8 ) . T h e q u e s t i o n w h e t h e r this m a y b e d u e t o t h e a c t i v a t i o n o f t h e m a i n s o d i u m r e t a i n i n g m e c h a n i s m - - t h e renina n g i o t e n s i n ~ a l d o s t e r o n e s y s t e m ( R A A S ) - has n o t y e t b e e n solved. We assessed t h e e f f e c t o n urine a n d e l e c t r o l y t e e x c r e t i o n a n d o n t h e R A A S in p a t i e n t s
Natriuresis
~-Blockers
w i t h b e n i g n essential h y p e r t e n s i o n ( B E H ) o f r e p e a t e d i.v. A z o s e m i d e a d m i n i s t r a t i o n a l o n e and during the simultaneous administration of a ~-adrenergic r e c e p t o r b l o c k i n g agent. T h e o b j e c t i v e o f t h e s t u d y was t o evaluate w h e t h e r t h e ~-blocking a g e n t b y inhibiting t h e R'AAS ( A s s a y k e e n et al., 1 9 7 0 , Bravo et al., 1975) could modify the excretory effect of t h e diuretic.
2. Materials a n d m e t h o d s 2.1. T e s t s u b j e c t s
T h e t e s t subjects w e r e 15 p a t i e n t s t r e a t e d f o r stage I - - I I B E H (WHO criteria) at t h e T h i r d Medical Clinic o f t h e Charles U n i v e r s i t y
440
K. HORKY ET AL.
TABLE 1 Clinical and laboratory characteristics of the patients with benign essential hypertension. All values are means -+ S.E.M. Abbreviations: PNa = plasma sodium concentration, PK = plasma potassium concentration, P c r = plasma creatinine concentration. Group
(A) Azosemide alone
(B) Azosemide + Trimepranol
Diagnosis
Number of patients
Age (years)
Mean blood (kPa)
PNa
PK
(mmol/1)
(mmol/1) (pmol/1)
Pcr
Total
Men
Women
Benign essential hypertension
8
4
4
41.2 + 5.84
17.5 + 0.49
138.5 + 0.71
4.91 -+ 0.12
101.8 + 17.7
Benign essential hypertension
7
4
3
43.6 -+6.12
17.3 + 0.55
139.4 + 0.8
4.31 + 0.10
89.7 + 6.9
Medical Faculty, Prague. A detailed preliminary examination, including electrocardiogram, X-ray examination of the heart and lungs, i.v. urography, evaluation o f renal function and routine biochemical tests excluded the presence o f any serious renal, cardiac and liver disorders o t h e r than mild uncomplicated arterial hypertension. The most i m p o r t a n t clinical and laboratory characteristics of our patients are given in table 1. There were no statistically significant differences in the parameters studied be t w een groups A and B. T h r o u g h o u t the whole study t he patients' diet contained 120--140 m m o l Na/24 h and 60-80 m m o l K / 2 4 h . Fluid intake was not restricted.
2.2. Protocol o f clinical trial Eight patients with BEH (group A) were left untreated for 3 control days and on the 4th, 5th and 6th day, at 9 a.m. were given 20 mg Azosemide in 500 ml of 5% glucose in t h e form o f 1 h infusion. The 7th day was again a control day. Seven patients with BEH (group B) were treated in th e same way as group A, but in addition, t h e y were given the /3-adrenergic
blocker m e t h y p r a n o l (Trimepranol ® Spofa, Czechoslovakia) 10 mg 3 times a day for 5 days prior to and during t he trial Azosemide. Methypranol, an effective ~-receptor blocker, was administered 5 days prior to the actual study to eliminate the possible initial sodium retaining effect of /3-blockers (Nies et al., 1971). The actual study was started when sodium balance had been re-established. The following parameters were studied in all patients: (a) 24 h urine excretion 3 days before and one day after administration of Azosemide. On the days on which Azosemide infusions were given, urine was collected at
6--9, 9--12, 12--15, 15--18, 18--6 h. (b) Urinary sodium and potassium excretion were determined by flame photometry (Flaphokol, Zeiss Jenna), both for the individual sampling periods and as total 24 h excretion. (c) From the plasma and urine creatinine concentration we calculated the endogenous creatinine clearance as the index of glomerular filtration rate (GFR). (d) In plasma we determined sodium and potassium concentration by flame photometry and plasma renin activity (PRA) by radioimmunoassayaccording the method of Haber et al. (1969), using commercialkits (Sorin Ltd., Italy). Plasma aldosterone con-
~-ADRENERGIC BLOCKADE AND THE EFFECT OF A NEW DIURETIC
centration (PAC) was measured b y radioimmunoassay (Malvano et al., 1977), using kits from the same source. Plasma parameters were always studies on the morning of the 3rd control day before breakfast, on the 4th and 6th day before starting the infusion and 2 h
l i nfusi°r I Na/K
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after its completion, and on the morning of the 7th day before breakfast. The results were processed statistically with a Hewlett-Packard computer and the significance of differences between the mean values in group A (given only Azosemide) and
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20 HOURS J # "1'2'1~ "i8 ~, " 9 "1~' 1"5"18 ~" # "1~" i~" 1~"/,' . DAYS13cl 4th I 5th I 6th tC Fig. 1. The effect of repeated infusions of Azosemide (20 mg i.v.) on urine volume (UV), urinary sodium excretion (UNaV), fractional sodium excretion (FENa), potassium excretion (UKV) and urinary sodium to potassium ratio (Na/K) when administered alone (group A, open bars) or simultaneously with a /3-blocker Trimepranol (group B, hatched bars). All values are means +- S.E.M. Statistically significant differences in parameters between group A and B are marked by *
442
K. HORKY ET AL.
g r o u p B ( A z o s e m i d e + T r i m e p r a n o l ) was c o m pared with S t u d e n t ' s t-test. Values with P 0 . 0 5 w e r e r e g a r d e d as s t a t i s t i c a l l y s i g n i f i c a n t .
3. R e s u l t s The mean urine volume, sodium and potassium excretion, fractional sodium excretion at t h e s a m p l i n g p e r i o d s a r e s h o w n in fig. 1. T h e mean urine volume, sodium and potassium e x c r e t i o n in t h e c o n t r o l p e r i o d b e f o r e i n f u sion of the diuretic did not differ significantly b e t w e e n g r o u p s A a n d B. In g r o u p A , A z o s e m i d e r a i s e d t h e u r i n e v o l u m e o n all 3 d a y s d u r i n g w h i c h it was a d m i n i s t e r e d , b u t its e f f e c t was o n l y s h o r t - l a s t i n g a n d d i m i n i s h e d w i t h r e p e a t e d a d m i n i s t r a t i o n . In l a t e r p o s t infusion periods the volume of urine excreted fell b e l o w t h e c o n t r o l values. In g r o u p B, t h e d i u r e t i c e f f e c t o f A z o s e m i d e was e n h a n c e d a n d p r o l o n g e d e s p e c i a l l y d u r i n g t h e first t w o infusions. T h e e f f e c t o n s o d i u m e x c r e t i o n a n d frac-
t i o n a l e x c r e t i o n o f s o d i u m was s i m i l a r . S o d i u m e x c r e t i o n in t h e i n t e r v a l d u r i n g a n d immediately after the infusion of Azosemide w a s g r e a t e r a n d l a s t e d l o n g e r in g r o u p B t h a n in g r o u p A. T h e d e c r e a s e in t h e n a t r i u r e t i c e f f e c t o f r e p e a t e d i n f u s i o n s was m o r e p r o n o u n c e d in g r o u p A t h a n in g r o u p B. In g r o u p A, s o d i u m e x c r e t i o n a f t e r t h e i n f u s i o n also fell s o o n e r b e l o w t h e values seen in t h e control period. The infusion of Azosemide raised potassium excretion during the 9--12 and 12--15 h i n t e r v a l s in b o t h g r o u p A a n d B (fig. 1). When c o m p a r e d to urinary sodium excretion, h o w e v e r , t h e r e l a t i v e i n c r e a s e in p o t a s s i u m e x c r e t i o n w a s l o w e r , a n d a d e f i n i t e i n c r e a s e in urinary N a / K ratio was therefore d e m o n s t r a t e d . In g r o u p B t h e i n c r e a s e in t h e N a / K r a t i o was g r e a t e r a n d o f l o n g e r d u r a t i o n t h a n in g r o u p A. The GFR oscillated around the control values; n o s i g n i f i c a n t i n c r e a s e in t h e G F R was found during infusion of the diuretic. T h e t o t a l 24 h u r i n e v o l u m e , s o d i u m a n d
TABLE 2 The cumulative effect of repeated Azosemide infusions (20 mg i.v.) on the renal excretion of urine, sodium and potassium during 24 h in non-treated (Groups A, n = 8) and Trimepranol-treated (Group B, n = 7) hypertensive patients. All values are means + S.E.M. The underlined values in the group B differ significantly from those of group A. The values during and after infusion of Azosemide which differ significantly from the values in control days are marked ** when P < 0.01 and * when P < 0.05. Abbreviations: UV = diuresis, UNaV = urinary sodium excretion, UKV = urinary potassium excretion.
UV ml/24 h
Group A Group B
UNaV mmol/24 h
Group A Group B
UKV mmol]24 h
Group A Group B
3 control days
4th day (Infusion I)
5th day (Infusion II)
6th day (Infusion III)
7th day post infusion control
1449 -+ 96.2 1339 + 86.2 135.4 + 4.95 119.5 + 7.42 42.9 + 5.81 59.0 + 6.18
2810 + 192.6 ** 3722 -+ 214.5 ** 214.9 + 14.92 ** 390.6 + 28.32 ** 79.6 + 7.87 ** 70.9 + 9.17 **
2042 -+ 185.7 3103 + 206.8 124.3 + 14.63 300.4 + 24.82 61.5 + 6.72 75.9 + 15.57
2107 + 181.6 ** 2147 + 174.5 ** 113.7 -+ 17.83 200.9 + 19.43 * 64.0 + 8.58 73.9 -+ 9.17 *
1229 -+ 217.7 1166 + 164.8 23.7 + 5.96 ** 41.8 + 8.45 ** 41.8 + 4.61 89.3 -+ 14.78 *
* **
**
**
443
/3-ADRENERGIC BLOCKADE AND THE EFFECT OF A NEW DIURETIC
PAC 2"0 PAC 2.0-
•
.modl 1.5t
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•
nmol/I
1.5
,.o4
X"
,-
-
1.0 n
O.5 PRA
prnol/I/s
t/••?•
o S ~
0 3 2-
0 DAYS
Fig. 2. The average values of plasma renin activity (PRA) and plasma aldosterone concentration (PAC) during treatment with Azosemide alone (open bars) and with Azosemide + Trimepranol (hatched bars). The significance between both groups is marked by ** when P < 0.01 and * when P < 0.05.
potassium excretion (table 2) also showed t h a t there was a marked decrease of Azosemide effectiveness during its repeated administration on three consecutive days. The simultaneous administration of Trimepranol enhanced and prolonged the effect of repeated Azosemide infusions, although a slight progressive decrease in urine volume and sodium excretion were again noted. The day following the last infusion of diuretic, a t e n d e n c y to sodium retention --again greater in group A -- was observed. There were no differences between total potassium excretion in group A and B except on the 7th day. Azosemide therapy in the form of repeated infusions was not accompanied by marked changes in plasma creatinine (range in group A from 101.8 + 17.70 to 125.7 + 27.41
~h, •
p
PRA pmol/I/s Fig. 3. Correlation between plasma renin activity (PRA) and plasma aldosterone concentration (PAC) in patients with benign essential hypertension treated with Azosemide alone and Azosemide + Trimepranol.
pmol/1, in group B from 89.7 + 6.19 to 106.2 + 9.48 pmol/1) and potassium concentration (range in group A from 4.46 + 0.12 to 4.10 + 0.05 mmol/1, in group B from 4.35 + 0.18 to 4.18 -+ 0.04 mmol/1), but the plasma concentration of sodium showed a t e n d e n c y to fall after the infusions (in group A from 138.5 + 0.76 to 135.7 +- 0.61 mmol/1, in group B from 139.4 + 0.68 to 135.8 + 1.01 mmol/1). There were no significant differences between the mean PRA and PAC values in groups A and B on the control day and before the first infusion even when the t e n d e n c y to lower levels appeared in group B (fig. 2). The first infusion raised both parameters but significantly so only in group A; after the infusion both PRA and PAC were lower in group B than in group A. However the situation after the third Azosemide infusion obliterated the differences between the groups. In both groups, the changes in PAC were linearly correlated to PRA (fig. 3).
4. Discussion Saluretics are now widely used in the treatment of oedematous states and arterial
444
hypertension. The sodium- and volume depleting effect of their repeated administration mobilizes sodium-retaining mechanisms, in particular the RAAS. The o u t c o m e of this activation may be a decrease in the effectiveness of the diuretics and intensification of some of their undesirable side-effects e.g. potassium depletion (Kassirer and Harrington, 1977). Azosemide is a new diuretic whose effects on renal excretory functions in man are quantitatively and qualitatively similar to those of furosemide (Brater et al., 1979). Its activity is similar to or greater than that of furosemide, depending on the route of administration (Krfick et al., 1978, Brater et al., 1979). In the present study we demonstrated that Azosemide had a marked diuretic and natriuretic effect not only in healthy subjects (Kr~ick et al., 1978), b u t also in patients with BEH. The urine and electrolytes excretory response of our patients with BEH to one h's i.v. infusion of 20 mg of Azosemide was the same as the response o f healthy volunteers. Potassium excretion after Azosemide rose less than would correspond to the increase in sodium excretion and there was thus a marked increase in urinary Na/K ratio during the infusion interval. With reference to the general tendency to potassium depletion after saluretics, this is an advantage. However, longitudinal studies will be needed to examine the effect of the long-term administration of Azosemide on potassium balance. The increase in urine volume and sodium excretion is achieved b y increased fractional sodium excretion and not b y a change in the GFR. This concurs with the findings of Brater (1979) in healthy volunteers subjected to water loading and water restriction. During repeated infusions of Azosemide on three consecutive days, however, its diuretic and natriuretic effectiveness diminished gradually, concomitantly with increasing activity of the RAAS. The stimulatory effect of repeated doses of Azosemide on the RAAS was evident. Both PRA and PAC rose gradually and after the 3rd infusion, attained
K. HORKY ET AL.
several times the initial values (fig. 2). The increase in PRA was presumably due to sodium and volume depletion, as after the administration of furosemide (Fernandez et al., 1976). PAC changes were directly correlated to PRA changes. This is further evidence that also during Azosemide administration in BEH, the renin-angiotensin system is the main mechanism regulating aldosterone secretion (Davis, 1973, Reid and Ganong, 1977) and that the PAC increase is thus largely the outcome of an increase in PRA. A several-fold increase in the activity of the main sodium-retaining mechanism -- the RAAS -- during Azosemide treatment could be one o f the reasons for the diminishing activity and duration of Azosemide effect and for the marked tendency to sodium retention in the post-infusion intervals. Trimepranol potentiated and prolonged the diuretic and natriuretic effect of Azosemide, especially after the first infusion. With repeated administration of the diuretic, the differences between the given parameters in group A (Azosemide alone) and group B (Azosemide + Trimepranol) diminished. All these changes paralleled the effect of Trimepranol on the RAAS. Administration of potent diuretics like furosemide or ethacrynic acid stimulates renin secretion and increases PRA in b o t h animals and in man but the mechanism is not fully understood (Freeman and Davis, 1977; Vander and Luciano, 1967; Winer et al., 1969). The renin stimulating effect of these drugs probably has both a neural and a nonneural component (Zanchetti and Stella, 1975). It seems that the effect of ~-adrenergic blocking agents depends on the dose of furosemide administered and on the volume depletion elicited b y the diuretic (Vander and Carlson, 1969; Naughton et al., 1975). In the cat, when urinary fluid loss was replaced b y saline, the renin-stimulating effect of high doses of furosemide could not be abolished b y propranolol (Johns and Singer, 1973). On the other hand, stimulation of PRA by i.v. furosemide was entirely neural when the
~-ADRENERGIC BLOCKADE AND THE EFFECT OF A NEW DIURETIC
diuretic was given in moderate doses (Stella and Zanchetti, 1977) and could be blunted in man b y ~-blocker administration (Bravo et al., 1972, Leonetti et al., 1975). Furthermore, a definite increase in plasma catecholamines was recently described following acute furosemide infusion in man (Canella et al., 1980). In contrast to the study of Ganong (1972) with dogs and that of Johns and Singer {1973) with cats our clinical trial demonstrated that the stimulating effect of Azosemide on PRA and PAC in hypertensive subjects could be reduced b y a ~-blocker, Trimepranol. The difference could be due to replacement of urinary fluid losses in both animal experiments and to the dose of diuretic used. The blunting b y Trimepranol of the PRA and PAC increase after Azosemide infusions was particularly distinct after the first infusion, less after the second and virtually disappeared after the third. Presumably, this suggest that the strong RAAS stimulating effect of repeated Azosemide infusions overcame the inhibitory effect of the ~-blocker, and/or that non-neural stimuli of renin secretion prevailed. The parallel changes in renal excretory function and in RAAS activity after administration of Azosemide alone and during inhibition of renin secretion with a ~-blocker together with Azosemide, lead us to conclude that stimulation of the RAAS may be one of the factors responsible for the decreasing diuretic and natriuretic effectiveness of repeated Azosemide infusions. Our results demonstrate that i.v. Azosemide had a marked diuretic and natriuretic effect and a relatively small kaliuretic effect in patients with BEH. Azosemide has a distinct stimulant effect on the RAAS, which may play a role in the diminishing activity of repeatedly administered Azosemide. The simultaneous administration of a ~-blocker reduces PRA and PAC, and potentiates and prolongs the natriuretic effect of the diuretic Azosemide.
445
References Assaykeen, T.A., P.L. Clayton, A. Goldfien and W.F. Ganong, 1970, Effect of alpha- and beta-adrenergic blocking agents on the renin response to hypoglycemia and epinephrine in dogs, Endocrinology 87, 1318. Brater, D.C., 1979, Renal sites of action of Azosemide, Clin. Pharmacol. Ther. 25,428. Brater, D.C., S.A. Anderson and S. Strowing, 1979, Azosemide a 'loop' diuretic, and furosemide, Clin. Pharmacol. Ther. 25,435. Bravo, E.L., R.C. Tarazi and H.P. Dustan, 1972, On the mechanism of decreased plasma renin activity (PRA) with beta-adrenergic blockade, Clin. Res. 20,810. Bravo, E.L., R.C. Tarazi and H.P. Dustan, 1975, Betaadrenergic blockade in diuretic treated patients with essential hypertension, New Engl. J. Med. 292, 66. Canella, G., G.B. Picotti, M. Campanini, M.D. Galve, L. Cristinelli, S. De Marinis and R. Maiorca, 1980, Changes in plasma catecholamines and plasma renin activity (PRA) following acute furosemide administration in man. Abstracts of the XVIIth EDTA Congress, Prague, p. 17. Davis, J.O., 1973, The control of renin release, Am. J. Med. 55,333. Fernandez, P.G., D.G. Bryant and M.H. Gault, 1976, Furosemide stimulation of peripheral renin in men and women, New Engl. J. Med. 294, 1182. Freeman, R.H. and J.O. Davis, 1977, The control of renin secretion and metabolism, in: Hypertension, eds. J. Genest et al. (McGraw-Hill Book Co., New York) p. 210. Ganong, W.F., 1972, Sympathetic effects on renin secretion, Mechanism and physiological role, in: Control of Renin Secretion, ed. T.A. Assaykeen (Plenum Press, New York} p. 17. Haber, E., T. Koerner, L.B. Page, B. Kliman and A. Purnode, 1969, Application of a radioimmunoassay for angiotensin I to the physiological measurements of plasma renin activity in normal human subjects, J. Clin. Endocrinol. 29, 1349. Johns, E.J. and B. Singer, 1973, Effect of propranolol and theophylline on renin release caused by furosemide in the cat, European J. Pharmacol. 23, 67. Kassirer, P. and J.T. Harrington, 1977, Diuretics and potassium metabolism: A reassesment of the need, effectiveness and safety of potassium therapy, Kidney Intern. 11,505. Kriick, F., W. Boblok, E. Besenfelder, G. Betzien and B. Kaufmann, 1978, Clinical and pharmacological investigation of the new saluretic Azosemide, European J. Clin. Pharmacol. 14,153. Leonetti, G., G. Mayer, A. Morganti, L. Terzoli, A. Zanchetti, G. Bianchetti, E. Di Salle, P.L. Morselli
446 and C.A. Chidsey, 1975, Hypotensive and renin suppressing activities of propranolol in hypertensive patients, Clin. Sci. Mol. Med. 48,491. Malvano, R., C. Gandolfi, D. Gianessi, P. Gianotti and P. Grosso, 1977, Radioimmunoassay of aldostetone in crude plasma extracts, J. Nucl. Biol. Med. 2O, 213. Naughton, R.J., J.I. Bertoncello and S.L. Skinner, 1975, Abolition of the renin-releasing action of furosemide by acute renal denervation in dogs, Clin. Exp. Pharmacol. Physiol. 2,213. Nies, A.S., J.S. McNeil and R.W. Schrier, 1971, Mechanism of increased sodium reabsorption during propranolol administration, Circulation 44, 456. Reid, I.A. and W.F. Ganong, 1977, Control of aldosterone secretion, in: Hypertension, eds. J. Genest
K. HORK'{/ET AL. et al. (McGraw-Hill Book Co., New York) p. 265. Stella, A. and A. Zanchetti, 1977, Effects of renal denervation on renin release in response to tilting and furosemide, Am. J. Physiol. 232, H500. Vander, A.J. and J. Carlson, 1969, Mechanisms of the effects of furosemide on renin secretion in anesthetized dogs, Circ. Res. 25,145. Vander, A.J. and J.R. Luciano, 1967, Neural and humoral control of renin release in salt depletion, Circ. Res. 20, Suppl. II, 69. Winer, N., D.S. Chokshi, S.Y. Myung and A.D. Freedman A.D., 1969, Adrenergic receptor mediation on renin secretion, J. Clin. Endocrinol. 29, 1168. Zanchetti, A. and A. Stella, 1975, Neural control of renin release, Clin. Sci. Mol. Med. 48, 215s.
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© Elsevier/North-Holland Biomedical Press
T H E E F F E C T O F R E N I N A N D A L D O S T E R O N E I N H I B I T I O N BY ~ - A D R E N E R G I C BLOCKADE ON THE RESPONSE TO THE NEW DIURETIC AZOSEMIDE KAREL HORKY, INGEBORG GREGOROVfi-, JANA DVO~..~KOV.~ Laboratory for Endocrinology and Metabolism and the Third Medical Clinic, Charles University Faculty o f Medicine, U nemocnice 1, 128 21 Prague 2, Czechoslovakia
Received 29 July 1980, revised MS received 24 October 1980, accepted 6 November 1980
K. HORK~/, I. GREGOROV/k and J. DVOR.~KOV/~, The effect o f renin and aldosterone inhibition by ~-adrenergic blockade on the response to the new diuretic Azosemide, European J. Pharmacol. 69 (1981) 439--446. The effect of repeated Azosemide infusions (20 mg in 500 ml 5% glucose for one h) on urine volume and electrolyte excretion, and on the activity of the renin-angiotensin-aldosterone system (RAAS) was studied in a group of 15 patients with benign essential hypertension before and during treatment with the ~-adrenergic blocker Trimepranol. Azosemide alone had a marked but short-lasting diuretic and natriuretic effect. Repeated administration on three consecutive days led, however, to a progressive decrease in the natriuretic effectiveness of Azosemide, associated with an increase in plasma renin activity (from 0.413 + 0.123 to 2.631 + 0.421 pmol/1]sec and in plasma aldosterone concentration (from 0.335 +- 0.032 to 1.687 + 0.438 pmol/l). Treatment with Trimepranol 30 rag/day enhanced and prolonged the diuretic and natriuretic response to Azosemide concomitantly with a reduction of its stimulatory effect on RAAS. There results suggest that stimulation of the RAAS might be responsible for the diminishing effectiveness of repeated Azosemide infusions and that the stimulation could be, at least partly, inhibited by a ~-blocker Trimepranol, resulting in a greater diuretic and natriuretic effect of Azosemide. Renin -angiotensin-aldosterone system Diuretics
Azosemide
1. I n t r o d u c t i o n A z o s e m i d e ( p r o d u c t BM 0 2 . 0 0 1 , B o e h ringer M a n n h e i m G m b H , G F R ) is a n e w a n d highly e f f e c t i v e d i u r e t i c , w h o s e s t r u c t u r e , d i u r e t i c e f f e c t a n d a c t i v i t y are similar t o t h o s e o f f u r o s e m i d e (Brater et al., 1 9 7 9 ) . D e s p i t e t h e high a c t i v i t y o f t h e first d o s e o f Azosem i d e , o n r e p e a t e d oral a d m i n i s t r a t i o n it d e c r e a s e d t h e 2 4 h s o d i u m a n d w a t e r excret i o n in h e a l t h y subjects t o t h e c o n t r o l values (Kriick et al., 1 9 7 8 ) . T h e q u e s t i o n w h e t h e r this m a y b e d u e t o t h e a c t i v a t i o n o f t h e m a i n s o d i u m r e t a i n i n g m e c h a n i s m - - t h e renina n g i o t e n s i n ~ a l d o s t e r o n e s y s t e m ( R A A S ) - has n o t y e t b e e n solved. We assessed t h e e f f e c t o n urine a n d e l e c t r o l y t e e x c r e t i o n a n d o n t h e R A A S in p a t i e n t s
Natriuresis
~-Blockers
w i t h b e n i g n essential h y p e r t e n s i o n ( B E H ) o f r e p e a t e d i.v. A z o s e m i d e a d m i n i s t r a t i o n a l o n e and during the simultaneous administration of a ~-adrenergic r e c e p t o r b l o c k i n g agent. T h e o b j e c t i v e o f t h e s t u d y was t o evaluate w h e t h e r t h e ~-blocking a g e n t b y inhibiting t h e R'AAS ( A s s a y k e e n et al., 1 9 7 0 , Bravo et al., 1975) could modify the excretory effect of t h e diuretic.
2. Materials a n d m e t h o d s 2.1. T e s t s u b j e c t s
T h e t e s t subjects w e r e 15 p a t i e n t s t r e a t e d f o r stage I - - I I B E H (WHO criteria) at t h e T h i r d Medical Clinic o f t h e Charles U n i v e r s i t y
440
K. HORKY ET AL.
TABLE 1 Clinical and laboratory characteristics of the patients with benign essential hypertension. All values are means -+ S.E.M. Abbreviations: PNa = plasma sodium concentration, PK = plasma potassium concentration, P c r = plasma creatinine concentration. Group
(A) Azosemide alone
(B) Azosemide + Trimepranol
Diagnosis
Number of patients
Age (years)
Mean blood (kPa)
PNa
PK
(mmol/1)
(mmol/1) (pmol/1)
Pcr
Total
Men
Women
Benign essential hypertension
8
4
4
41.2 + 5.84
17.5 + 0.49
138.5 + 0.71
4.91 -+ 0.12
101.8 + 17.7
Benign essential hypertension
7
4
3
43.6 -+6.12
17.3 + 0.55
139.4 + 0.8
4.31 + 0.10
89.7 + 6.9
Medical Faculty, Prague. A detailed preliminary examination, including electrocardiogram, X-ray examination of the heart and lungs, i.v. urography, evaluation o f renal function and routine biochemical tests excluded the presence o f any serious renal, cardiac and liver disorders o t h e r than mild uncomplicated arterial hypertension. The most i m p o r t a n t clinical and laboratory characteristics of our patients are given in table 1. There were no statistically significant differences in the parameters studied be t w een groups A and B. T h r o u g h o u t the whole study t he patients' diet contained 120--140 m m o l Na/24 h and 60-80 m m o l K / 2 4 h . Fluid intake was not restricted.
2.2. Protocol o f clinical trial Eight patients with BEH (group A) were left untreated for 3 control days and on the 4th, 5th and 6th day, at 9 a.m. were given 20 mg Azosemide in 500 ml of 5% glucose in t h e form o f 1 h infusion. The 7th day was again a control day. Seven patients with BEH (group B) were treated in th e same way as group A, but in addition, t h e y were given the /3-adrenergic
blocker m e t h y p r a n o l (Trimepranol ® Spofa, Czechoslovakia) 10 mg 3 times a day for 5 days prior to and during t he trial Azosemide. Methypranol, an effective ~-receptor blocker, was administered 5 days prior to the actual study to eliminate the possible initial sodium retaining effect of /3-blockers (Nies et al., 1971). The actual study was started when sodium balance had been re-established. The following parameters were studied in all patients: (a) 24 h urine excretion 3 days before and one day after administration of Azosemide. On the days on which Azosemide infusions were given, urine was collected at
6--9, 9--12, 12--15, 15--18, 18--6 h. (b) Urinary sodium and potassium excretion were determined by flame photometry (Flaphokol, Zeiss Jenna), both for the individual sampling periods and as total 24 h excretion. (c) From the plasma and urine creatinine concentration we calculated the endogenous creatinine clearance as the index of glomerular filtration rate (GFR). (d) In plasma we determined sodium and potassium concentration by flame photometry and plasma renin activity (PRA) by radioimmunoassayaccording the method of Haber et al. (1969), using commercialkits (Sorin Ltd., Italy). Plasma aldosterone con-
~-ADRENERGIC BLOCKADE AND THE EFFECT OF A NEW DIURETIC
centration (PAC) was measured b y radioimmunoassay (Malvano et al., 1977), using kits from the same source. Plasma parameters were always studies on the morning of the 3rd control day before breakfast, on the 4th and 6th day before starting the infusion and 2 h
l i nfusi°r I Na/K
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after its completion, and on the morning of the 7th day before breakfast. The results were processed statistically with a Hewlett-Packard computer and the significance of differences between the mean values in group A (given only Azosemide) and
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20 HOURS J # "1'2'1~ "i8 ~, " 9 "1~' 1"5"18 ~" # "1~" i~" 1~"/,' . DAYS13cl 4th I 5th I 6th tC Fig. 1. The effect of repeated infusions of Azosemide (20 mg i.v.) on urine volume (UV), urinary sodium excretion (UNaV), fractional sodium excretion (FENa), potassium excretion (UKV) and urinary sodium to potassium ratio (Na/K) when administered alone (group A, open bars) or simultaneously with a /3-blocker Trimepranol (group B, hatched bars). All values are means +- S.E.M. Statistically significant differences in parameters between group A and B are marked by *
442
K. HORKY ET AL.
g r o u p B ( A z o s e m i d e + T r i m e p r a n o l ) was c o m pared with S t u d e n t ' s t-test. Values with P 0 . 0 5 w e r e r e g a r d e d as s t a t i s t i c a l l y s i g n i f i c a n t .
3. R e s u l t s The mean urine volume, sodium and potassium excretion, fractional sodium excretion at t h e s a m p l i n g p e r i o d s a r e s h o w n in fig. 1. T h e mean urine volume, sodium and potassium e x c r e t i o n in t h e c o n t r o l p e r i o d b e f o r e i n f u sion of the diuretic did not differ significantly b e t w e e n g r o u p s A a n d B. In g r o u p A , A z o s e m i d e r a i s e d t h e u r i n e v o l u m e o n all 3 d a y s d u r i n g w h i c h it was a d m i n i s t e r e d , b u t its e f f e c t was o n l y s h o r t - l a s t i n g a n d d i m i n i s h e d w i t h r e p e a t e d a d m i n i s t r a t i o n . In l a t e r p o s t infusion periods the volume of urine excreted fell b e l o w t h e c o n t r o l values. In g r o u p B, t h e d i u r e t i c e f f e c t o f A z o s e m i d e was e n h a n c e d a n d p r o l o n g e d e s p e c i a l l y d u r i n g t h e first t w o infusions. T h e e f f e c t o n s o d i u m e x c r e t i o n a n d frac-
t i o n a l e x c r e t i o n o f s o d i u m was s i m i l a r . S o d i u m e x c r e t i o n in t h e i n t e r v a l d u r i n g a n d immediately after the infusion of Azosemide w a s g r e a t e r a n d l a s t e d l o n g e r in g r o u p B t h a n in g r o u p A. T h e d e c r e a s e in t h e n a t r i u r e t i c e f f e c t o f r e p e a t e d i n f u s i o n s was m o r e p r o n o u n c e d in g r o u p A t h a n in g r o u p B. In g r o u p A, s o d i u m e x c r e t i o n a f t e r t h e i n f u s i o n also fell s o o n e r b e l o w t h e values seen in t h e control period. The infusion of Azosemide raised potassium excretion during the 9--12 and 12--15 h i n t e r v a l s in b o t h g r o u p A a n d B (fig. 1). When c o m p a r e d to urinary sodium excretion, h o w e v e r , t h e r e l a t i v e i n c r e a s e in p o t a s s i u m e x c r e t i o n w a s l o w e r , a n d a d e f i n i t e i n c r e a s e in urinary N a / K ratio was therefore d e m o n s t r a t e d . In g r o u p B t h e i n c r e a s e in t h e N a / K r a t i o was g r e a t e r a n d o f l o n g e r d u r a t i o n t h a n in g r o u p A. The GFR oscillated around the control values; n o s i g n i f i c a n t i n c r e a s e in t h e G F R was found during infusion of the diuretic. T h e t o t a l 24 h u r i n e v o l u m e , s o d i u m a n d
TABLE 2 The cumulative effect of repeated Azosemide infusions (20 mg i.v.) on the renal excretion of urine, sodium and potassium during 24 h in non-treated (Groups A, n = 8) and Trimepranol-treated (Group B, n = 7) hypertensive patients. All values are means + S.E.M. The underlined values in the group B differ significantly from those of group A. The values during and after infusion of Azosemide which differ significantly from the values in control days are marked ** when P < 0.01 and * when P < 0.05. Abbreviations: UV = diuresis, UNaV = urinary sodium excretion, UKV = urinary potassium excretion.
UV ml/24 h
Group A Group B
UNaV mmol/24 h
Group A Group B
UKV mmol]24 h
Group A Group B
3 control days
4th day (Infusion I)
5th day (Infusion II)
6th day (Infusion III)
7th day post infusion control
1449 -+ 96.2 1339 + 86.2 135.4 + 4.95 119.5 + 7.42 42.9 + 5.81 59.0 + 6.18
2810 + 192.6 ** 3722 -+ 214.5 ** 214.9 + 14.92 ** 390.6 + 28.32 ** 79.6 + 7.87 ** 70.9 + 9.17 **
2042 -+ 185.7 3103 + 206.8 124.3 + 14.63 300.4 + 24.82 61.5 + 6.72 75.9 + 15.57
2107 + 181.6 ** 2147 + 174.5 ** 113.7 -+ 17.83 200.9 + 19.43 * 64.0 + 8.58 73.9 -+ 9.17 *
1229 -+ 217.7 1166 + 164.8 23.7 + 5.96 ** 41.8 + 8.45 ** 41.8 + 4.61 89.3 -+ 14.78 *
* **
**
**
443
/3-ADRENERGIC BLOCKADE AND THE EFFECT OF A NEW DIURETIC
PAC 2"0 PAC 2.0-
•
.modl 1.5t
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•
nmol/I
1.5
,.o4
X"
,-
-
1.0 n
O.5 PRA
prnol/I/s
t/••?•
o S ~
0 3 2-
0 DAYS
Fig. 2. The average values of plasma renin activity (PRA) and plasma aldosterone concentration (PAC) during treatment with Azosemide alone (open bars) and with Azosemide + Trimepranol (hatched bars). The significance between both groups is marked by ** when P < 0.01 and * when P < 0.05.
potassium excretion (table 2) also showed t h a t there was a marked decrease of Azosemide effectiveness during its repeated administration on three consecutive days. The simultaneous administration of Trimepranol enhanced and prolonged the effect of repeated Azosemide infusions, although a slight progressive decrease in urine volume and sodium excretion were again noted. The day following the last infusion of diuretic, a t e n d e n c y to sodium retention --again greater in group A -- was observed. There were no differences between total potassium excretion in group A and B except on the 7th day. Azosemide therapy in the form of repeated infusions was not accompanied by marked changes in plasma creatinine (range in group A from 101.8 + 17.70 to 125.7 + 27.41
~h, •
p
PRA pmol/I/s Fig. 3. Correlation between plasma renin activity (PRA) and plasma aldosterone concentration (PAC) in patients with benign essential hypertension treated with Azosemide alone and Azosemide + Trimepranol.
pmol/1, in group B from 89.7 + 6.19 to 106.2 + 9.48 pmol/1) and potassium concentration (range in group A from 4.46 + 0.12 to 4.10 + 0.05 mmol/1, in group B from 4.35 + 0.18 to 4.18 -+ 0.04 mmol/1), but the plasma concentration of sodium showed a t e n d e n c y to fall after the infusions (in group A from 138.5 + 0.76 to 135.7 +- 0.61 mmol/1, in group B from 139.4 + 0.68 to 135.8 + 1.01 mmol/1). There were no significant differences between the mean PRA and PAC values in groups A and B on the control day and before the first infusion even when the t e n d e n c y to lower levels appeared in group B (fig. 2). The first infusion raised both parameters but significantly so only in group A; after the infusion both PRA and PAC were lower in group B than in group A. However the situation after the third Azosemide infusion obliterated the differences between the groups. In both groups, the changes in PAC were linearly correlated to PRA (fig. 3).
4. Discussion Saluretics are now widely used in the treatment of oedematous states and arterial
444
hypertension. The sodium- and volume depleting effect of their repeated administration mobilizes sodium-retaining mechanisms, in particular the RAAS. The o u t c o m e of this activation may be a decrease in the effectiveness of the diuretics and intensification of some of their undesirable side-effects e.g. potassium depletion (Kassirer and Harrington, 1977). Azosemide is a new diuretic whose effects on renal excretory functions in man are quantitatively and qualitatively similar to those of furosemide (Brater et al., 1979). Its activity is similar to or greater than that of furosemide, depending on the route of administration (Krfick et al., 1978, Brater et al., 1979). In the present study we demonstrated that Azosemide had a marked diuretic and natriuretic effect not only in healthy subjects (Kr~ick et al., 1978), b u t also in patients with BEH. The urine and electrolytes excretory response of our patients with BEH to one h's i.v. infusion of 20 mg of Azosemide was the same as the response o f healthy volunteers. Potassium excretion after Azosemide rose less than would correspond to the increase in sodium excretion and there was thus a marked increase in urinary Na/K ratio during the infusion interval. With reference to the general tendency to potassium depletion after saluretics, this is an advantage. However, longitudinal studies will be needed to examine the effect of the long-term administration of Azosemide on potassium balance. The increase in urine volume and sodium excretion is achieved b y increased fractional sodium excretion and not b y a change in the GFR. This concurs with the findings of Brater (1979) in healthy volunteers subjected to water loading and water restriction. During repeated infusions of Azosemide on three consecutive days, however, its diuretic and natriuretic effectiveness diminished gradually, concomitantly with increasing activity of the RAAS. The stimulatory effect of repeated doses of Azosemide on the RAAS was evident. Both PRA and PAC rose gradually and after the 3rd infusion, attained
K. HORKY ET AL.
several times the initial values (fig. 2). The increase in PRA was presumably due to sodium and volume depletion, as after the administration of furosemide (Fernandez et al., 1976). PAC changes were directly correlated to PRA changes. This is further evidence that also during Azosemide administration in BEH, the renin-angiotensin system is the main mechanism regulating aldosterone secretion (Davis, 1973, Reid and Ganong, 1977) and that the PAC increase is thus largely the outcome of an increase in PRA. A several-fold increase in the activity of the main sodium-retaining mechanism -- the RAAS -- during Azosemide treatment could be one o f the reasons for the diminishing activity and duration of Azosemide effect and for the marked tendency to sodium retention in the post-infusion intervals. Trimepranol potentiated and prolonged the diuretic and natriuretic effect of Azosemide, especially after the first infusion. With repeated administration of the diuretic, the differences between the given parameters in group A (Azosemide alone) and group B (Azosemide + Trimepranol) diminished. All these changes paralleled the effect of Trimepranol on the RAAS. Administration of potent diuretics like furosemide or ethacrynic acid stimulates renin secretion and increases PRA in b o t h animals and in man but the mechanism is not fully understood (Freeman and Davis, 1977; Vander and Luciano, 1967; Winer et al., 1969). The renin stimulating effect of these drugs probably has both a neural and a nonneural component (Zanchetti and Stella, 1975). It seems that the effect of ~-adrenergic blocking agents depends on the dose of furosemide administered and on the volume depletion elicited b y the diuretic (Vander and Carlson, 1969; Naughton et al., 1975). In the cat, when urinary fluid loss was replaced b y saline, the renin-stimulating effect of high doses of furosemide could not be abolished b y propranolol (Johns and Singer, 1973). On the other hand, stimulation of PRA by i.v. furosemide was entirely neural when the
~-ADRENERGIC BLOCKADE AND THE EFFECT OF A NEW DIURETIC
diuretic was given in moderate doses (Stella and Zanchetti, 1977) and could be blunted in man b y ~-blocker administration (Bravo et al., 1972, Leonetti et al., 1975). Furthermore, a definite increase in plasma catecholamines was recently described following acute furosemide infusion in man (Canella et al., 1980). In contrast to the study of Ganong (1972) with dogs and that of Johns and Singer {1973) with cats our clinical trial demonstrated that the stimulating effect of Azosemide on PRA and PAC in hypertensive subjects could be reduced b y a ~-blocker, Trimepranol. The difference could be due to replacement of urinary fluid losses in both animal experiments and to the dose of diuretic used. The blunting b y Trimepranol of the PRA and PAC increase after Azosemide infusions was particularly distinct after the first infusion, less after the second and virtually disappeared after the third. Presumably, this suggest that the strong RAAS stimulating effect of repeated Azosemide infusions overcame the inhibitory effect of the ~-blocker, and/or that non-neural stimuli of renin secretion prevailed. The parallel changes in renal excretory function and in RAAS activity after administration of Azosemide alone and during inhibition of renin secretion with a ~-blocker together with Azosemide, lead us to conclude that stimulation of the RAAS may be one of the factors responsible for the decreasing diuretic and natriuretic effectiveness of repeated Azosemide infusions. Our results demonstrate that i.v. Azosemide had a marked diuretic and natriuretic effect and a relatively small kaliuretic effect in patients with BEH. Azosemide has a distinct stimulant effect on the RAAS, which may play a role in the diminishing activity of repeatedly administered Azosemide. The simultaneous administration of a ~-blocker reduces PRA and PAC, and potentiates and prolongs the natriuretic effect of the diuretic Azosemide.
445
References Assaykeen, T.A., P.L. Clayton, A. Goldfien and W.F. Ganong, 1970, Effect of alpha- and beta-adrenergic blocking agents on the renin response to hypoglycemia and epinephrine in dogs, Endocrinology 87, 1318. Brater, D.C., 1979, Renal sites of action of Azosemide, Clin. Pharmacol. Ther. 25,428. Brater, D.C., S.A. Anderson and S. Strowing, 1979, Azosemide a 'loop' diuretic, and furosemide, Clin. Pharmacol. Ther. 25,435. Bravo, E.L., R.C. Tarazi and H.P. Dustan, 1972, On the mechanism of decreased plasma renin activity (PRA) with beta-adrenergic blockade, Clin. Res. 20,810. Bravo, E.L., R.C. Tarazi and H.P. Dustan, 1975, Betaadrenergic blockade in diuretic treated patients with essential hypertension, New Engl. J. Med. 292, 66. Canella, G., G.B. Picotti, M. Campanini, M.D. Galve, L. Cristinelli, S. De Marinis and R. Maiorca, 1980, Changes in plasma catecholamines and plasma renin activity (PRA) following acute furosemide administration in man. Abstracts of the XVIIth EDTA Congress, Prague, p. 17. Davis, J.O., 1973, The control of renin release, Am. J. Med. 55,333. Fernandez, P.G., D.G. Bryant and M.H. Gault, 1976, Furosemide stimulation of peripheral renin in men and women, New Engl. J. Med. 294, 1182. Freeman, R.H. and J.O. Davis, 1977, The control of renin secretion and metabolism, in: Hypertension, eds. J. Genest et al. (McGraw-Hill Book Co., New York) p. 210. Ganong, W.F., 1972, Sympathetic effects on renin secretion, Mechanism and physiological role, in: Control of Renin Secretion, ed. T.A. Assaykeen (Plenum Press, New York} p. 17. Haber, E., T. Koerner, L.B. Page, B. Kliman and A. Purnode, 1969, Application of a radioimmunoassay for angiotensin I to the physiological measurements of plasma renin activity in normal human subjects, J. Clin. Endocrinol. 29, 1349. Johns, E.J. and B. Singer, 1973, Effect of propranolol and theophylline on renin release caused by furosemide in the cat, European J. Pharmacol. 23, 67. Kassirer, P. and J.T. Harrington, 1977, Diuretics and potassium metabolism: A reassesment of the need, effectiveness and safety of potassium therapy, Kidney Intern. 11,505. Kriick, F., W. Boblok, E. Besenfelder, G. Betzien and B. Kaufmann, 1978, Clinical and pharmacological investigation of the new saluretic Azosemide, European J. Clin. Pharmacol. 14,153. Leonetti, G., G. Mayer, A. Morganti, L. Terzoli, A. Zanchetti, G. Bianchetti, E. Di Salle, P.L. Morselli
446 and C.A. Chidsey, 1975, Hypotensive and renin suppressing activities of propranolol in hypertensive patients, Clin. Sci. Mol. Med. 48,491. Malvano, R., C. Gandolfi, D. Gianessi, P. Gianotti and P. Grosso, 1977, Radioimmunoassay of aldostetone in crude plasma extracts, J. Nucl. Biol. Med. 2O, 213. Naughton, R.J., J.I. Bertoncello and S.L. Skinner, 1975, Abolition of the renin-releasing action of furosemide by acute renal denervation in dogs, Clin. Exp. Pharmacol. Physiol. 2,213. Nies, A.S., J.S. McNeil and R.W. Schrier, 1971, Mechanism of increased sodium reabsorption during propranolol administration, Circulation 44, 456. Reid, I.A. and W.F. Ganong, 1977, Control of aldosterone secretion, in: Hypertension, eds. J. Genest
K. HORK'{/ET AL. et al. (McGraw-Hill Book Co., New York) p. 265. Stella, A. and A. Zanchetti, 1977, Effects of renal denervation on renin release in response to tilting and furosemide, Am. J. Physiol. 232, H500. Vander, A.J. and J. Carlson, 1969, Mechanisms of the effects of furosemide on renin secretion in anesthetized dogs, Circ. Res. 25,145. Vander, A.J. and J.R. Luciano, 1967, Neural and humoral control of renin release in salt depletion, Circ. Res. 20, Suppl. II, 69. Winer, N., D.S. Chokshi, S.Y. Myung and A.D. Freedman A.D., 1969, Adrenergic receptor mediation on renin secretion, J. Clin. Endocrinol. 29, 1168. Zanchetti, A. and A. Stella, 1975, Neural control of renin release, Clin. Sci. Mol. Med. 48, 215s.