Eur J Clin Pharmacol (1990) 38:223-227
© Springer-Verlag 1990
D O P A decarboxylase inhibition does not influence the diuretic and natriuretic response to exogenous s-atrial natriuretic peptide in man S. K a g e y a m a 1, J. B r o w n 2, R. C a u s o n 1, M. O ' F l y n n 1, and V. A b e r 1 Royal Postgraduate Medical School, London, UK 2 Physiological Laboratory, University of Cambridge, Downing Street, Cambridge, UK Received: April 12, 1989/Accepted in revised form: October 3,1989
Summary. The role of dopamine synthesis in the renal actions of h u m a n a-atrial natriuretic peptide (c~ANP) was investigated in six dehydrated volunteers using the D O P A decarboxylase inhibitor carbidopa. Each subject received oral placebo or carbidopa (100 rag) followed by an infusion of c~ANP 10 pmol. kg 1 m i n - 1for 1 h. The responses to placebo alone and to carbidopa alone were investigated on separate occasions. a A N P produced a similar increase in plasma immunoreactive o~ANP whether placebo or carbidopa p r e t r e a t m e n t had been given. Urinary dopamine excretion was increased by a A N E Carbidopa p r e t r e a t m e n t substantially attenuated this increase without affecting the natriuretic or water-diuretic response to a A N R Carbidopa also failed to alter the change in filtration fraction produced by c~ANR The results suggest that increased synthesis of intrarenal dopamine is not required for the renal effects of c~ANP in man. Key words: atrial natriuretic peptide, dopamine; renal response
H u m a n o~-atrial natriuretic peptide (o~ANP) is a h o r m o n e originating f r o m the cardiac atria. Its physiological and pathophysiological actions are still being investigated, but m a n y of the effects of ~ A N P and its close pharmacological analogues are similar to those of dopamine, including increasing sodium and water excretion, suppressing renin and aldosterone release, and relaxing vascular smooth muscle [1-3]. In b o t h m a n and the dog, urinary dopamine excretion is increased by a saline infusion [4-6]. The extracerebral D O P A - d e c a r b o x y l a s e inhibitor carbidopa blocks the natriuresis normally caused by saline infusion in dogs [5-6], suggesting that endogenous dopamine m a y sometimes mediate sodium excretion. Therefore, dopamine may also be involved in the renal action of A N R Indeed, almost all studies in anaesthetised rats have shown that DA1 dopamine receptor antagonists readily abolish the natriuretic and diuretic responses to these peptides [710]. However, in a detailed study of the anaesthetised dog, the DA~ antagonist S C H 23390 failed to inhibit the na-
triuresis of A N P [11]. A recent study in the unanaesthetised rat failed to show an inhibitory effect of DA~ antagonists [12]. In man, partial attenuation of the renal response to a high dose of ~ A N P has b e e n reported after p r e t r e a t m e n t with carbidopa, while the DA1 antagonist (+)-sulpiride failed to attenuate the renal effects of otANP [13,14]. The present investigation examined the effects of carbidopa on the renal actions of infusions of ctANP which raised the circulating concentration to within the pathophysiological range for man.
Materials and methods Studies were conducted with Ethical Committee approval, and with the informed consent of each volunteer. Six healthy male volunteers (mean age 33 y, range 25-40 y), who had been taking their standard diets, were studied on 4 days, after dehydration for 12 h, in random order, and at least i week apart. On two days, subjects ingested 50 mg carbidopa (Merck, Sharp & Dohme, U. K.) at 19.00 h and again at 07.00 h, before receiving an intravenousinfusionofl0pmol.kg-l.min i c~ANP (BissendorfPeptides, FRG), between 10.30-11.30 h on one day, or its vehicle (0.9% NaC140 ml plus human serum albumin 4 ml) between 10.30-11.30 h on the other day. On a further two days, subjects ingested placebo ta"blets at 19.00 h and 07.00 h before receiving the same infusion of c~ANP or vehicle. At 08.30 h on each study day, the subject received an intravenous loading dose of 50 mg. kg- i inulin (Laevosan-Gesellschaft, Austria) and 8 mg. kg ~sodium para-aminohippurate (PAH; Merck, Sharp & Dohme, USA). Inulin was then infused at 33 mg. min- ~and PAH at 13 mg. min 1until 12.30 h. On the two days with aANR subjects passed urine at 09.3010.30 h (basal), 10.30-11.00 h and 11.00.11.30 h (during c~ANPinfusion) and 11.30-12.30 h (post infusion). Similar collections were made on the two days on which the vehicle alone was infused, except that only one sample spanned the infusion (10.30-11.30 h), because a lower urinary flow was anticipated. Urine 3 ml for dopamine assay was immediately treated with 0.1% w/v sodium metabisulphite 100 gl and snap frozen. On each day, 20 ml venous blood was collected and at 09.25, 10.25, 11.25 and 12.25 h appropriately anticoagulated for measurement of plasma electrolytes, inulin, PAH and plasma renin activity (PRA). Blood 5 ml was also taken at those times into prechilled tubes containing 5 mg Na2-EDTA and 250 gl aprotinin (20,000 kallikrein inactivator units/ml, Bayer, FRG). Plasma was separated immediately by centrifugation at 4 °C for measurement of plasma c~ANRInulin and PAH plasma blanks were measured in 5 ml venous
S. Kageyama et al.: DOPA decarboxylase inhibition and ANP
224
Table 1. Urinary dopamine excretion (~g/h) Vehicle infusion day 0-60 min 60-120 min
Pretreatment
ANP infusion day 120-180 min
Placebo 17.0 (4.2) 18.0 (4.0) 18.6 (3.7) Carbidopa 1.3 (0.4) 1.4 (0.2) 1.9 (0.5) *: p < 0.01 for quadratic trends over time within each day
0-60 min
60-90 min
90-120 min
120-180 min
17.4 (3.9) 1.5 (0.2)
21.7 (4.9) 2.0 (0.4)
27.3 (5.5) 3.2 (0.8)
22.9 (5.2)* 2.5 (0.8)*
Table 2. Urine osmolality (mosml/kg) Pretreatment
Vehicle infusion day 0-60 min
60-120 min
ANP infusion day 120-180 min
Placebo 867 (55) 835 (60) 856 (51) Carbidopa 766 (42) 685 (90) 724 (74) *: p < 0.01, **: p < 0.001 for cubic trends over time within each day blood taken just before the loading doses were given. Plasma and urine for measurement of PAH, inulin, dopamine, PRA and c~ANP were snap frozen and stored in liquid nitrogen. Plasma and urine were assayed for electrolytes by flame photometry, for osmolality by freezing-point depression, for inulin by a resorcinol method [15], and for PAH by a diazo-dye method [16]. PRA was measured by radioimmunoassay (Cis Ltd, UK). Intra-assay and inter-assay variation of the PRA assay were 7.5% and 7.7%, respectively. Plasma aANP was measured by radioimmunoassay (Amersham, UK) after extraction using C-8 Bond Elut columns [17]. Intraassay and inter-assay variation of the c~ANP assay were 8.7% and 11.1%, respectively. Urinary free dopamine was measured by a previously described radioenzymatic assay [18]. Arterial pressure and pulse were recorded automatically (Dinamap, Criticon Inc., USA) in triplicate, every 30 min from 08.50 h each day. Results are mean and (SEM). Statistical analysis was done by repeated measures of analysis of variance.
0-60 min
60 90 rain
90-120 min
120-180 min
880 (49) 785 (82)
698 (97) 580 (141)
306 (107) 327 (133)
617 (74)** 601 (79)*
about 90% of the urinary dopamine excretion on both days with c~ANP and with vehicle alone (Table 1). Despite this, the average sodium excretion during the hour of o~ANP infusion was increased by 62% above its baseline on the day with placebo and by 66% after carbidopa. Moreover, neither the trend with time nor the peak rate of sodium excretion was significantly different during a A N P infusion with and without carbidopa (Fig. 1). c~ANP also increased urinary flow and decreased urinary osmolality, and, again, the trend with time and the final magnitude of the responses was not significantly altered by carbidopa (Fig. 1; Table 2). Baseline levels on all days, and time trends on days with the vehicle infusion, were not significantly different for sodium excretion, urinary flow and urinary osmolality, whether or not carbidopa was given. Mean arterial pressure did not change significantly on any day, but infusions of ~ANP caused a tachycardia, which was not significantly altered by carbidopa (Table 3). P R A fell with c~ANP infusion from 1.7 (0.3) to 0.9 (0.1) ng. ml -~. h -~ after placebo pretreatment, and from
Results c~ANP significantly increased urinary dopamine excretion at the same time as it increased sodium excretion; vehicle alone had no effect. Carbidopa pretreatment prevented
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4O 50C
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60 i 120
120 i 180
=.. ¢-
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0 60
6,0 120
120 180
O
400
Fig. 1. Urinary flow, sodium excretion and potassium excretion. Upper panel: vehicle infusion. Lower panel: c~ANP infusion, Open circles show vehicle infusion without carbidopa, and closed circles show vehicle infusion with carbidopa. Open triangles show c~ANP infusion without carbidopa and dosed triangles show c~ANP infusion with carbidopa. *: p < 0.01 for cubic trends over time within each day. **: p < 0.01, and ***: p < 0.001 for quadratic trends over time within each day
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Yime(min)
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Time(min)
40
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120 120 180
Time(min)
S. Kageyama et al.: DOPA decarboxylase inhibition and ANP
225
Table 3. Mean arterial pressure and pulse rate Pretreatment
60-120 min
81 (1) 81 (3)
80 (1) 82 (3)
Pulse Placebo 64 (2) 64 (2) beats.min ~ Carbidopa 63 (2) 63 (2) *: p < 0.001 for linear trends over time within each day
MAP mmHg
ANP infusion day
Vehicle infusion day 0-60 min
Placebo Carbidopa
120-180 rain
0-60 rain
60-90 min
90-120 min
120-180 rain
80 (2) 82 (2)
78 (2) 82 (2)
76 (2) 82 (2)
74 (1) 79 (2)
75 (3) 80 (3)
64 (2) 63 (2)
62 (2) 59 (2)
65 (2) 64 (2)
71 (2) 64 (3)
71 (2)* 67 (2)*
1.5 (0.2) to 1.0 (0.2) rig. m1-1. h-1 after carbidopa (Fig. 2). Vehicle infusion had no effect on PRA. The filtration fraction was increased significantly by a A N R because effective renal plasma flow tended to fall whilst the G F R was relatively constant. None of these trends were significantly affected by carbidopa (Fig. 3). The levels of plasma immunoreactive a A N P achieved by infusion were also not significantly affected by carbidopa (Fig. 4). Discussion
The infusion of a A N P given here raised the plasma level of the peptide to approximately 250 pM, similar to levels seen in disease [19]. Confirming previous reports, the treatment was natriuretic, increased the flow of a more dilute urine, and tended to reduce effective renal plasma flow, with no change or a small increase in inulin clearance, and an increase in the filtration fraction [19-22]. The renal mechanism of action of c~ANP is still controversial, but studies in the rat have suggested that renal dopamine is involved [7-10]. The majority of renal dopamine is synthesized locally and it probably accounts for most of the dopamine excreted in the urine [2, 23]. In the present study, (~ANP increased urinary dopamine excretion. A previous human study found no such increase, but
600 t-.
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13_ r'r" w 300 (3
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90
i
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120 i
180
'- 120 E 110 -~ 100 ~- 90
ANP Infusion
--,3.0
shorter infusions of ~ANP were used [24]. The increased urinary dopamine excretion found here would be consistent with dopaminergic mediation of the renal effects of ~ A N E However, carbidopa substantially inhibited urinary dopamine excretion both before and during peptide infusion, without significantly affecting any of the renal changes caused by a A N E By contrast, carbidopa attenuated both the increase in urinary dopamine excretion and the natriuresis caused by saline infusions in the dog [5, 6]. Thus, the situation with ~ANP in man resembles that for frusemide; carbidopa restricts the increase in urinary dopamine excretion but not the change in P R A or sodium excretion caused by frusemide [25].
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© Springer-Verlag 1990
D O P A decarboxylase inhibition does not influence the diuretic and natriuretic response to exogenous s-atrial natriuretic peptide in man S. K a g e y a m a 1, J. B r o w n 2, R. C a u s o n 1, M. O ' F l y n n 1, and V. A b e r 1 Royal Postgraduate Medical School, London, UK 2 Physiological Laboratory, University of Cambridge, Downing Street, Cambridge, UK Received: April 12, 1989/Accepted in revised form: October 3,1989
Summary. The role of dopamine synthesis in the renal actions of h u m a n a-atrial natriuretic peptide (c~ANP) was investigated in six dehydrated volunteers using the D O P A decarboxylase inhibitor carbidopa. Each subject received oral placebo or carbidopa (100 rag) followed by an infusion of c~ANP 10 pmol. kg 1 m i n - 1for 1 h. The responses to placebo alone and to carbidopa alone were investigated on separate occasions. a A N P produced a similar increase in plasma immunoreactive o~ANP whether placebo or carbidopa p r e t r e a t m e n t had been given. Urinary dopamine excretion was increased by a A N E Carbidopa p r e t r e a t m e n t substantially attenuated this increase without affecting the natriuretic or water-diuretic response to a A N R Carbidopa also failed to alter the change in filtration fraction produced by c~ANR The results suggest that increased synthesis of intrarenal dopamine is not required for the renal effects of c~ANP in man. Key words: atrial natriuretic peptide, dopamine; renal response
H u m a n o~-atrial natriuretic peptide (o~ANP) is a h o r m o n e originating f r o m the cardiac atria. Its physiological and pathophysiological actions are still being investigated, but m a n y of the effects of ~ A N P and its close pharmacological analogues are similar to those of dopamine, including increasing sodium and water excretion, suppressing renin and aldosterone release, and relaxing vascular smooth muscle [1-3]. In b o t h m a n and the dog, urinary dopamine excretion is increased by a saline infusion [4-6]. The extracerebral D O P A - d e c a r b o x y l a s e inhibitor carbidopa blocks the natriuresis normally caused by saline infusion in dogs [5-6], suggesting that endogenous dopamine m a y sometimes mediate sodium excretion. Therefore, dopamine may also be involved in the renal action of A N R Indeed, almost all studies in anaesthetised rats have shown that DA1 dopamine receptor antagonists readily abolish the natriuretic and diuretic responses to these peptides [710]. However, in a detailed study of the anaesthetised dog, the DA~ antagonist S C H 23390 failed to inhibit the na-
triuresis of A N P [11]. A recent study in the unanaesthetised rat failed to show an inhibitory effect of DA~ antagonists [12]. In man, partial attenuation of the renal response to a high dose of ~ A N P has b e e n reported after p r e t r e a t m e n t with carbidopa, while the DA1 antagonist (+)-sulpiride failed to attenuate the renal effects of otANP [13,14]. The present investigation examined the effects of carbidopa on the renal actions of infusions of ctANP which raised the circulating concentration to within the pathophysiological range for man.
Materials and methods Studies were conducted with Ethical Committee approval, and with the informed consent of each volunteer. Six healthy male volunteers (mean age 33 y, range 25-40 y), who had been taking their standard diets, were studied on 4 days, after dehydration for 12 h, in random order, and at least i week apart. On two days, subjects ingested 50 mg carbidopa (Merck, Sharp & Dohme, U. K.) at 19.00 h and again at 07.00 h, before receiving an intravenousinfusionofl0pmol.kg-l.min i c~ANP (BissendorfPeptides, FRG), between 10.30-11.30 h on one day, or its vehicle (0.9% NaC140 ml plus human serum albumin 4 ml) between 10.30-11.30 h on the other day. On a further two days, subjects ingested placebo ta"blets at 19.00 h and 07.00 h before receiving the same infusion of c~ANP or vehicle. At 08.30 h on each study day, the subject received an intravenous loading dose of 50 mg. kg- i inulin (Laevosan-Gesellschaft, Austria) and 8 mg. kg ~sodium para-aminohippurate (PAH; Merck, Sharp & Dohme, USA). Inulin was then infused at 33 mg. min- ~and PAH at 13 mg. min 1until 12.30 h. On the two days with aANR subjects passed urine at 09.3010.30 h (basal), 10.30-11.00 h and 11.00.11.30 h (during c~ANPinfusion) and 11.30-12.30 h (post infusion). Similar collections were made on the two days on which the vehicle alone was infused, except that only one sample spanned the infusion (10.30-11.30 h), because a lower urinary flow was anticipated. Urine 3 ml for dopamine assay was immediately treated with 0.1% w/v sodium metabisulphite 100 gl and snap frozen. On each day, 20 ml venous blood was collected and at 09.25, 10.25, 11.25 and 12.25 h appropriately anticoagulated for measurement of plasma electrolytes, inulin, PAH and plasma renin activity (PRA). Blood 5 ml was also taken at those times into prechilled tubes containing 5 mg Na2-EDTA and 250 gl aprotinin (20,000 kallikrein inactivator units/ml, Bayer, FRG). Plasma was separated immediately by centrifugation at 4 °C for measurement of plasma c~ANRInulin and PAH plasma blanks were measured in 5 ml venous
S. Kageyama et al.: DOPA decarboxylase inhibition and ANP
224
Table 1. Urinary dopamine excretion (~g/h) Vehicle infusion day 0-60 min 60-120 min
Pretreatment
ANP infusion day 120-180 min
Placebo 17.0 (4.2) 18.0 (4.0) 18.6 (3.7) Carbidopa 1.3 (0.4) 1.4 (0.2) 1.9 (0.5) *: p < 0.01 for quadratic trends over time within each day
0-60 min
60-90 min
90-120 min
120-180 min
17.4 (3.9) 1.5 (0.2)
21.7 (4.9) 2.0 (0.4)
27.3 (5.5) 3.2 (0.8)
22.9 (5.2)* 2.5 (0.8)*
Table 2. Urine osmolality (mosml/kg) Pretreatment
Vehicle infusion day 0-60 min
60-120 min
ANP infusion day 120-180 min
Placebo 867 (55) 835 (60) 856 (51) Carbidopa 766 (42) 685 (90) 724 (74) *: p < 0.01, **: p < 0.001 for cubic trends over time within each day blood taken just before the loading doses were given. Plasma and urine for measurement of PAH, inulin, dopamine, PRA and c~ANP were snap frozen and stored in liquid nitrogen. Plasma and urine were assayed for electrolytes by flame photometry, for osmolality by freezing-point depression, for inulin by a resorcinol method [15], and for PAH by a diazo-dye method [16]. PRA was measured by radioimmunoassay (Cis Ltd, UK). Intra-assay and inter-assay variation of the PRA assay were 7.5% and 7.7%, respectively. Plasma aANP was measured by radioimmunoassay (Amersham, UK) after extraction using C-8 Bond Elut columns [17]. Intraassay and inter-assay variation of the c~ANP assay were 8.7% and 11.1%, respectively. Urinary free dopamine was measured by a previously described radioenzymatic assay [18]. Arterial pressure and pulse were recorded automatically (Dinamap, Criticon Inc., USA) in triplicate, every 30 min from 08.50 h each day. Results are mean and (SEM). Statistical analysis was done by repeated measures of analysis of variance.
0-60 min
60 90 rain
90-120 min
120-180 min
880 (49) 785 (82)
698 (97) 580 (141)
306 (107) 327 (133)
617 (74)** 601 (79)*
about 90% of the urinary dopamine excretion on both days with c~ANP and with vehicle alone (Table 1). Despite this, the average sodium excretion during the hour of o~ANP infusion was increased by 62% above its baseline on the day with placebo and by 66% after carbidopa. Moreover, neither the trend with time nor the peak rate of sodium excretion was significantly different during a A N P infusion with and without carbidopa (Fig. 1). c~ANP also increased urinary flow and decreased urinary osmolality, and, again, the trend with time and the final magnitude of the responses was not significantly altered by carbidopa (Fig. 1; Table 2). Baseline levels on all days, and time trends on days with the vehicle infusion, were not significantly different for sodium excretion, urinary flow and urinary osmolality, whether or not carbidopa was given. Mean arterial pressure did not change significantly on any day, but infusions of ~ANP caused a tachycardia, which was not significantly altered by carbidopa (Table 3). P R A fell with c~ANP infusion from 1.7 (0.3) to 0.9 (0.1) ng. ml -~. h -~ after placebo pretreatment, and from
Results c~ANP significantly increased urinary dopamine excretion at the same time as it increased sodium excretion; vehicle alone had no effect. Carbidopa pretreatment prevented
IO0t 2O0 I
°°f
60
4O 50C
E- I00~ i 0i 60
60 i 120
120 i 180
=.. ¢-
-~
"°f
40
-5 E ::::L
E ._~ 500
0 60
6,0 120
120 180
O
400
Fig. 1. Urinary flow, sodium excretion and potassium excretion. Upper panel: vehicle infusion. Lower panel: c~ANP infusion, Open circles show vehicle infusion without carbidopa, and closed circles show vehicle infusion with carbidopa. Open triangles show c~ANP infusion without carbidopa and dosed triangles show c~ANP infusion with carbidopa. *: p < 0.01 for cubic trends over time within each day. **: p < 0.01, and ***: p < 0.001 for quadratic trends over time within each day
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300
o Q_
200 E T,_
~200
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100 I
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Yime(min)
6'o 9'o 60 1½0 120 180
Time(min)
40
o i
60
9'o I
i
i
120 120 180
Time(min)
S. Kageyama et al.: DOPA decarboxylase inhibition and ANP
225
Table 3. Mean arterial pressure and pulse rate Pretreatment
60-120 min
81 (1) 81 (3)
80 (1) 82 (3)
Pulse Placebo 64 (2) 64 (2) beats.min ~ Carbidopa 63 (2) 63 (2) *: p < 0.001 for linear trends over time within each day
MAP mmHg
ANP infusion day
Vehicle infusion day 0-60 min
Placebo Carbidopa
120-180 rain
0-60 rain
60-90 min
90-120 min
120-180 rain
80 (2) 82 (2)
78 (2) 82 (2)
76 (2) 82 (2)
74 (1) 79 (2)
75 (3) 80 (3)
64 (2) 63 (2)
62 (2) 59 (2)
65 (2) 64 (2)
71 (2) 64 (3)
71 (2)* 67 (2)*
1.5 (0.2) to 1.0 (0.2) rig. m1-1. h-1 after carbidopa (Fig. 2). Vehicle infusion had no effect on PRA. The filtration fraction was increased significantly by a A N R because effective renal plasma flow tended to fall whilst the G F R was relatively constant. None of these trends were significantly affected by carbidopa (Fig. 3). The levels of plasma immunoreactive a A N P achieved by infusion were also not significantly affected by carbidopa (Fig. 4). Discussion
The infusion of a A N P given here raised the plasma level of the peptide to approximately 250 pM, similar to levels seen in disease [19]. Confirming previous reports, the treatment was natriuretic, increased the flow of a more dilute urine, and tended to reduce effective renal plasma flow, with no change or a small increase in inulin clearance, and an increase in the filtration fraction [19-22]. The renal mechanism of action of c~ANP is still controversial, but studies in the rat have suggested that renal dopamine is involved [7-10]. The majority of renal dopamine is synthesized locally and it probably accounts for most of the dopamine excreted in the urine [2, 23]. In the present study, (~ANP increased urinary dopamine excretion. A previous human study found no such increase, but
600 t-.
E 500 E 4o0
13_ r'r" w 300 (3
6t0
60
90
i
6'0 i 120
910 i 120
120 i
180
'- 120 E 110 -~ 100 ~- 90
ANP Infusion
--,3.0
shorter infusions of ~ANP were used [24]. The increased urinary dopamine excretion found here would be consistent with dopaminergic mediation of the renal effects of ~ A N E However, carbidopa substantially inhibited urinary dopamine excretion both before and during peptide infusion, without significantly affecting any of the renal changes caused by a A N E By contrast, carbidopa attenuated both the increase in urinary dopamine excretion and the natriuresis caused by saline infusions in the dog [5, 6]. Thus, the situation with ~ANP in man resembles that for frusemide; carbidopa restricts the increase in urinary dopamine excretion but not the change in P R A or sodium excretion caused by frusemide [25].
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