Four-Hour Infusions of Synthetic Atrial
Natriuretic Peptide in Normal Volunteers
JEROME BIOLLAZ, JURG NUSSBERGER, MARINETTE PORCHET, FRANQOISE BRUNNER-FERBER,
ELIZABETH S. OTTERBEIN, HECTOR GOMEZ, BERNARD WAEBER, AND H A N S R.
BRUNNER
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SUMMARY Two doses of synthetic atrial natriuretic peptide (0.5 and 5.0 /xg/min) and its vehicle
were infused intravenously for 4 hours in eight salt-loaded normal volunteers, arid the effect on blood
pressure, heart rate, renal hemodynamics, solute excretion, and secretion of vasoactive hormones was
studied. The 0.5 /oig/min infusion did not alter blood pressure or heart rate, whereas the 5.0 /xg/min
infusion significantly reduced the mean pressure by 20/9 mm Hg after 2.5 to 3 hours and increased the
heart rate slightly. Inulin clearance was not significantly changed, but the mean p-aminohippurate
clearance fell by 13 and 32% with the lower and higher doses, respectively. Urinary excretion of
sodium and chloride increased slightly with the lower dose. With the higher dose, a marked increase in
urinary excretion of sodium, chloride, and calcium was observed, reaching a peak during the second
hour of the infusion. Potassium and phosphate excretion did not change significantly. A brisk increase
in urine flow rate and fractional water excretion was seen only during the first hour of the high-dose
infusion. Signs and symptoms of hypotension were observed in two subjects. No change in plasma
renin activity, angiotensih II, or aldosterone was observed during either infusion, but a marked
increase occurred after discontinuation of the high-dose infusion. In conclusion, the 5 /xg/min infusion
induced a transient diuretic effect, delayed maximal natriuretic activity, and a late fall in blood
pressure, with no change in inulin clearance but a dose-related decrease in p-aminohippurate clearance. Despite large amounts of sodium excreted arid blood pressure reduction, no coiinterregulatory
changes were observed in the renin-angiotensin-aldosterone system or plasma vasopressin levels
during the infusion. (Hypertension 8 [Suppl II]: II-96-II-105, 1986)
KEY WORDS • glomerular filtration rate • effective renal plasma flow • filtration
fraction * natriuresis • blood pressure • renin-angiotensih-aldosterone system •
arginine vasopressin
injections followed by a short infusion.4 In these studies, the blood pressure effects as well as solute excretion were monitored. The peptides elicited natriuresis
and diuresis4- 8-9 and induced a transient increase in
creatinine clearance,9 while slightly decreasing blood
pressure.8
Although the triggering mechanisms and the daily
profile of ANP secretion in humans are not yet known,
it seems likely that a continuous infusion would yield
more information on its physiological role in blood
pressure regulation and renal handling of solutes than a
bolus injection. We have recently evaluated the dosereponse relationship for blood pressure, heart rate, and
skin blood flow during an ANP infusion lasting several
hours.10 The aim of the present study was to assess the
pharmacological effect of a 4-hour infusion of synthetic ANP, at two different doses, or its vehicle on renal
hemodynamics, urinary solute excretion, and plasma
levels of various hormones in healthy volunteers under
sodium-loaded conditions.
A TRIAL natriuretic peptides (ANPs) have recent/ \
ly been added to the list of circulating horJL \ . mones that seem to play a part in cardiovascular, volume, and sodium homeostasis. These peptides
are synthesized and secreted by the atria1'2 and can be
found in the blood stream.3"7 The availability of synthetic ANPs has made it possible to study their effects
in normal humans, providing a basis for the rational
use of these substances to treat various diseases.
Atrial natriuretic peptides have been administered to
normal volunteers as bolus injections only8-9 or bolus
From the Department, of Medicine, University Hospital, Lausanne, Switzerland (J. Bioilaz, J. Nussberger, M. Porchet, B.
Waeber, H.R. Brunner), and Merck Sharp and Dohme Research
Laboratories, Zurich, Switzerland, arid Rahway, New Jersey
(F. BninnerrFerber, E. Otterbein, H. Gomez).
Supported by grants from Merck Sharp and Dohme Research
Laboratories, the Cardiovascular Research Foundation, and the
Swiss National Science Foundation.
Address for reprints: Hans R. Brunner, M.D., Department of
Medicine, C.H.U.V., 1011 Lausanne, Switzerland.
11-96
FOUR-HOUR INFUSION OF ATRIAL NATRIURETIC PEPTIDE/fliollaz et al.
Subjects and Methods
Eight male volunteers, aged 23 to 28 years (mean,
25.4 years) and weighing between 62 and 79 kg
(mean, 68.8 kg), participated in the study. Each volunteer gave a medical history and underwent a complete
physical examination. Routine laboratory tests were
done before and after the administration of the drug.
The nature and the purpose of the study had previously
been explained, and written informed consent obtained. The protocol was approved by the Hospital
Ethics Committee.
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Design of the Study
The subjects were allowed free dietary sodium intake and given 5 g/day of NaCl in the form of salt
capsules during the 5 days preceding each study day.
Urine samples were collected during the 24 hours of
the fifth day to estimate electrolyte excretion. On the
study day, the volunteers came to the hospital metabolic unit at 0700 after an overnight fast. They had been
instructed to drink 250 ml of tap water after rising.
Upon arrival, they were weighed and placed on a bed.
They remained supine, except for voiding, and fasted
throughout the study procedure. Three intravenous
catheters were inserted into antecubital veins: two in
the same forearm for infusions of inulin, /j-aminohippurate (PAH), and the experimental drug, and the third
catheter in the contralateral forearm for blood drawing.
Two doses of synthetic atrial natriuretic Ile12(3-28)eicosahexapeptide and its vehicle were infused
and compared at weekly intervals in a single blind
three-way crossover study. The study period consisted
of a 2-hour baseline period and a 4-hour treatment
period, followed by a 2-hour recovery phase. After a
priming dose, infusion of inulin and PAH in 0.9%
saline in amounts calculated to provide plasma concentrations of approximately 400 and 20 /u.g/ml, respectively, were started at a rate of 2 ml/min, to measure
the glomerular filtration rate (GFR) as the clearance of
inulin and the effective renal plasma flow (ERPF) as
the clearance of PAH. Two hours later, an infusion of
the vehicle (0.9% saline) or of ANP was started and set
to deliver 0, 0.5, or 5.0 /ug/min of ANP.
Blood pressure and heart rate were measured at 30minute intervals. Twelve-lead electrocardiograms
were recorded before and 0.5, 4, and 6 hours after the
start of the experimental infusions. Blood samples for
the measurement of electrolytes, inulin, and PAH were
drawn at hourly intervals. Blood samples for the measurement of osmolality, plasma renin activity, immunoreactive angiotensin II, aldosterone, arginine vasopressin, venous pH, and bicarbonate content were
drawn at - 2 , - 0 . 5 , 0.5, 1, 4, and 6 hours from the
start of the infusion. Urine samples were collected at
hourly intervals. To ensure adequate urine flow, the
subjects were asked to drink 200 ml of water per hour.
Drug
ANP L-364,343 is a synthetic polypeptide of 26
amino acids. It has been synthesized at Merck Sharp
and Dohme Research Laboratories (Rahway, NJ,
USA)." 1 2 The sequence corresponds to a-human
11-97
ANP,13-M except for the substitution of isoleucine for
methionine at position 12 and deletion of the two
N-terminal amino acids serine and leucine. Its biological activity is equivalent to the naturally occurring
hormone.
Analytical Methods
The methods used to measure plasma renin activity"
(Nussberger et al., unpublished data), plasma angiotensin II," aldosterone," and arginine vasopressin18
have been reported previously.
Inulin and PAH concentrations in plasma and urine
were measured by a microadaptation of a diphenylamine procedure" and by a microadaptation of a
Bratton-Marshall procedure,20 respectively. Osmolality was determined as the freezing point depression
with a Roebling Automatik Mikroosmometer (Berlin,
Germany). Sodium and potassium were analyzed by
direct flame photometry; calcium, phosphate, and
plasma chloride were quantified photometrically
(Greiner Electronics Selective Analyser G300; Langenthal, Switzerland); and urinary chloride was measured with a Corning chloride meter (Halstead, England). A 105 hand-held digital pH-meter in automatic
temperature compensation mode (Coming, Halstead,
Essex, England) was used for the determination of
urinary pH.
Constant infusions were carried out with a syringe
pump (Perfusor ED 1-300; B. Braun, Melsungen, Germany) for ANP and a volumetric infusion and transfusion pump (Doltron PIM 717; Uster, Switzerland) for
inulin and PAH.
Renal Parameters and Statistical Evaluation
Clearances were calculated by the traditional method, using the formula Q = U, X V/Px, with C representing clearance of substance x, U% and fx urine and
plasma concentrations of substance x, and V the rate of
urine flow in milliliters per minute. Fractional excretion was calculated as the clearance of substance x
divided by the clearance of inulin (C/GFR). Plasma
concentrations used for the clearance calculations were
the average of the values obtained at the beginning and
at the end of each clearance period. The filtration fraction was calculated as the ratio between inulin and
PAH clearances (GFR/ERPF). Free water clearance
was
(CH,O)
calculated as the urinary flow rate (V)
minus osmolar clearance ( Q ^ ) : C Hj0 = V — C^,,,.
The statistical significance of differences was evaluated by a two-way analysis of variance, with replications followed by the Fisher least significant difference
test; a p value of less than 0.05 was considered the
minimum level of significance.21 Of the eight subjects
enrolled in the study, only six received all three scheduled treatments, and only data for these six subjects
have been used for the statistical evaluation. Results
are expressed as means ± SEM.
Results
Blood Pressure and Heart Rate
Figure 1 depicts the time course of blood pressure
and heart rate. No change was observed with the vehi-
11-98
1985 BLOOD PRESSURE COUNCIL
SUPPL
II
HYPERTENSION, VOL
8, No 6,
JUNE
1986
A D Vehicle
n-8
• • ANP, O5yg/min n-8
• • A N P , 5.0jjg/m)n n-6
• Mean blood pressure
FIGURE 1. Time course of blood pressure
and heart rate before, during, and after 4-hour
infusions of 0 (vehicle), 0.5, and 5 ftg/min of
atrial natriuretic peptide of the rat sequence
(ANPr) in sodium-loaded normal volunteers.
120
n 100
E BO
e
"i
1 60
•a
40
m
20
°
70 *
e
-OS
Mean ± SEM
0.5
1
2JS 3
15
Tims hours
4.5
P < 0.01 n
p < oooi
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cle infusion and the ANP infusion at 0.5 /xg/min.
However, the higher dose (5.0 /xg/min) significantly
reduced diastolic and systolic blood pressure, from
115/76 to 95/67 mm Hg after a latency period of 2.5 to
3 hours. After the higher dose had been stopped, blood
pressure increased slowly, and systolic pressure was
still significantly reduced 1 hour later. The heart rate
did not change during the vehicle and low-dose ANP
infusions but increased slightly with the high-dose
infusion.
Renal Function
The effect of ANP infusion on renal function is
summarized in Table 1. GFR was not affected by
ANP, although a trend toward an increase in GFR
during the first hour (120% of the baseline value) was
apparent with the 5.0 /Ag/min infusion (Figure 2, upper
panel). ERPF was slightly reduced by the 0.5 /ig/min
infusion (p<0.05 vs vehicle at 3 hours) but was markedly reduced from 2 to 4 hours by the 5.0 /xg/min
infusion (78.4% [p<0.02], 71.6% [p<0.001], and
61.8% [/><0.001] of the baseline value at 2, 3, and 4
hours, respectively), slowly returning toward control
values during the recovery phase (see Figure 2, middle
panel). The filtration fraction therefore rose from the
preinfusion value of 20.9% to 25.1% during the 0.5
/xg/min infusion (p<0.05; p<0.0l vs vehicle) and
from 19.6 to 29.9% during the 5.0 /Ltg/min infusion
(p<0.001) (see Figure 2, lower panel).
Electrolytes and Water Excretion
Data on electrolyte excretion are summarized in
Table 2.
Sodium and Chloride
Mean sodium excretion during the 24 hours preceding the experimental days was 269, 281, and 207
mmol for the vehicle, 0.5 /u,g/min, and 5.0 /xg/min
infusion phases, respectively. This corresponds to a
daily salt intake of 12 to 16 g. The amount of sodium
administered as the result of the infusion of inulin and
PAH was approximately 250 fimoVmin, to which the
50
OP < 0.02
OOP < 0.01 n l s - O . !
ooo P < 0.0O1
infusion of ANP or its vehicle added 77 /i.mol/min.
Sodium removed by the blood sampling process has
not been considered.
At time 0, mean sodium excretion was around 265
/imol/min. During and after the vehicle infusion, mean
sodium excretion remained stable at around 350 fimoU
min. During the infusion of 0.5 /xg/min of ANP, the
maximal sodium excretion rate increased to 472 /xmol/
min during the second hour and remained above control values during the third and fourth hours (/?<0.05
vs vehicle); during the first hour of the recovery period
it returned to the value during the vehicle infusion and
dropped below that value during the second hour of
recovery (Figure 3, upper panel). During the infusion
of 5.0 /Ag/min of ANP, marked natriuresis was observed, with a peak during the second hour of the
infusion (968 /xmol/min, p<0.001). From the third
hour on, the natriuresis, although still twice the value
during the vehicle infusion (732 /xmol/min), began to
fall, reaching 488 /xmol/min during the fourth hour.
Recovery phase values were well below those observed during the vehicle infusion (187 and 108 /xmol/
min at 5 and 6 hours, respectively). The chloride excretion rate was similar to that of sodium (see Figure 3,
middle panel).
Mean fractional sodium excretion was 1.8% at time
0 and remained between 2.2 and 2.4% during the saline infusion. After 2 hours of infusion of 0.5 and 5.0
/xg/min of ANP, it had increased to 3.0 and 5.6%,
respectively. At the same time, mean fractional chloride excretion, which was stable at around 2.6 to 3.0%
with the vehicle infusion, increased to 4.5 and 7.5%
with the low- and high-dose infusion of ANP, respectively (see Table 1). Plasma sodium and chloride concentrations did not change significantly in response to
ANP.
Potassium
Potassium excretion did not increase but actually
decreased during the three study periods, including the
period of vehicle administration. The largest reduction
occurred during the high-dose infusion of ANP. Mean
11-99
FOUR-HOUR INFUSION OF ATRIAL NATRIURETIC PEPTIDE/fi/o/Zaz et al.
TABLE 1. Effect of a 4-Hour Infusion of Atrial Natriuretic Peptide on Renal Function in Salt-Loaded Volunteers
Renal
function
(jig/min)
0
GFR
(ml/min)
0.5
5.0
0
ERPF
(ml/min)
0.5
5.0
0
FF(%)
0.5
5.0
0
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
FEN.(%)
0.5
5.0
0
FEc(%)
Hours
ANP
0.5
5.0
113±3
111±5
108 ± 6
113±4
113±7
130±4
113±4
113 + 5
118±8
115 + 6
114±4
118 + 4
105 ±3
114±5
124 + 6
109±3
110±2
104±6
6
110 + 5
109 + 3
107 + 8
570 ±8
531 ±22
554 ±32
569±13
494 ±31
533 ±21
569 ±17
468 + 28
434±26§
570 + 29
462 ± 26§
397 + 23t^
538+16
461 ±26
342 + 65+H
536 ± 20
484+19
468±43*
553+16
504 + 27
494 + 32
19.8±0.5
20.9 ±0.4
19.6±0.6
19.8±0.5
23.1 ±0.9
24.6±1.4+'||
20.5 ±0.5
25.0±1.2*§
29.9±1.8+"fl
19.6±0.4
25.1±1.1*'||
29.7±1.9 + H
20.2 ±0.4
22.8±0.7
23.0±2.2*
19.9 + 0.5
21.8 + 0.6
21.6+1.4
1.8±0.1
1.6±0.1
1.9 + 0.2
2.2 + 0.2
2.2±0.2
3.9±0.5 + U
2.2±0.2
2.2±0.2
2.3±0.2
3.0±0.3 + §
5.6±0.6 + H
3.0±0.3t§
4.4±0.5+fl
2.8±0.3t
2.4 + 0.2
2.1 ±0.1
2.1 ±0.3
3.0±0.2
2.7±0.2
4.9±0.7+>1
2.8±0.3
3.7±0.4t§
7.5±0.9t'U
2.6 + 0.3
4.5±l.lt§
5.6±0.6 + H
0
1
2
20.0 ±0.5
24.3 + 1.0
27.3±1.5t-1
3
20.2±2.7
16.4±2.5
21.6 + 2.9
20.9 ±1.9
18.2±2.7
15.7±3.0
17.7±2.6
19.2 + 2.2
13.7±2.1§
5.0
18.2±2.4
18.4±1.9
9.0±1.3*'§
Values are means ± SEM. ANP = atrial natriuretic peptide; GFR = glomerular filtration
filtration fraction; FE = fractional excretion.
%p < 0.001 vs time 0.
\p < 0.01 vs vehicle,
*p < 0.05 vs time 0.
§p < 0.05 vs vehicle.
Sp < 0.001 vs vehicle.
tp < 0.01 vs time 0.
0
FEK(%)
0.5
fractional potassium excretion fell from 21.6 to
11.5%, from 19.2 to 9.7%, and from 18.2 to a low of
9% after the vehicle, low-dose, and high-dose infusions, respectively (see Table 2).
Calcium
Mean urinary calcium excretion followed a pattern
similar to that of sodium (see Figure 3, lower panel). It
did not change appreciably during the vehicle infusion
and increased only slightly with the 0.5 /xg/min infusion, from 4.6 to a maximum of 6.3 timol/min at 2
hours. It increased significantly with the 5.0 /xg/min
infusion, from 4.3 to 11.5 /nmol/min at 1 hour and to
15.3 timol/min at 2 hours; it decreased to 11.5 and 7.9
/xmol/min during the third and fourth hours of the
infusion, respectively, and fell below control values to
2.8 and 1.4 /imol/min (/?<0.05 vs vehicle), during
the 2-hour recovery phase.
Phosphates
Mean phosphate excretion remained between 18.7
and 20.5 /imol/min during the vehicle phase. No significant change was observed with the low-dose infusion, but a lower excretion tended to occur during the
recovery phase. Although mean phosphate excretion
increased somewhat with the high-dose ANP infusion
(from 18.5 to 27.0 /imol/min at 1 hour) and tended to
4
5
2.4 + 0.2*
1.9 + 0.2
3.3 + 0.3t
2.3±0.2
2.4±0.3*
l.l±0.2||
0.7±0.2tH
2.7±0.2
3.4±0.4t
4.1±0.5t§
2.7±0.3
2.8 + 0.4
1.3±0.2||
2.8 + 0.3
2.2±0.3
0.8+0.3M!
13.5 + 2.3*
11.5±1.8t
11.4±2.3
9.7 + 0.9*
12.8±4.2*
10.8 + 0.8*
rate; ERPF = effective renal plasma flow; FF =
15.9 + 2.3*
12.9±1.9
9.0±1.7*
decrease during the recovery phase, these changes
were not statistically significant.
Diuresis, Fractional Excretion of Water, and Free
Water Clearance
All relevant data are summarized in Table 3. Urine
flow (Figure 4, upper panel) and the fraction of filtered
water excreted (V/GFR) remained stable during the
vehicle infusion and the low-dose ANP infusion, although during the recovery phase of the latter infusion
a significant decrease could be observed. A brisk increase in mean urine flow, from 7.3 to 15.1 ml/min,
occurred during the first hour of the 5.0 tig/min infusion, but by the fourth hour, urine flow had fallen
below values observed during the vehicle infusion.
The mean fractional excretion of water almost doubled
during the first hour of the high-dose ANP infusion,
from 6.9 to 11.6%, then immediately returned to values that were not statistically different from those obtained with the vehicle infusion.
The tonicity of the urine remained stable during the
entire study period with the vehicle and low-dose ANP
infusions. During the first hour of the high-dose infusion, the urine became slightly more hypotonic, and
the free water clearance reached a high positive value
(see Figure 4, middle panel). Urine became hypertonic
during the recovery phase. The high-dose ANP infu-
II-100
1985 BLOOD PRESSURE COUNCIL
sion produced a twofold increase in osmolar clearance
at 2 hours (see Figure 4, lower panel), and a 2.5-fold
increase in mean free water clearance occurred at 1
hour.
Urinary pH was the same during the vehicle and
low-dose infusions. It did not change at peak diuresis
ANP, oi Vehicle Infusion
SUPPL
II
HYPERTENSION, VOL
8, No 6,
JUNE
1986
during the high-dose infusion but fell significantly
below vehicle infusion values ( — 0.7 and —0.8 pH
units, p < 0.01) during the recovery period. The plasma bicarbonate concentration was not significantly altered during any of the study periods.
Plasma Hormones
As a result of high sodium intake during the five
days preceding each experimental phase, baseline values of plasma renin activity, angiotensin II, and aldosterone were very low.
c WO
120
ANP, or v»«d» Infusion
80
5 40
Downloaded from http://hyper.ahajournals.org/ by guest on June 18, 2017
-
0'
ANPf or Vehicle Infmron
600
n
| 500
E
o
oc
ANPr or Vehicle Infusion
400
1200
u
c
I 200
u
< 100
1
2
3
4
HOURS
5
6
ANPf or Vehicle Infusion
1
20
2
3
4
II
S
S
ANP, or whtcW Infusion
10
12
Mean ± SEM
FIGURE 2. Effects of 4-hour infusions of atrial natriuretic
peptide of the rat sequence (ANPr) at 0.5 ftglmin (dark gray
columns) and 5 ftg/min (light gray columns) and its vehicle
(blank columns) on inulin clearance (upperpanel), p-aminohippurate (PAH) clearance (middle panel), and filtration fraction
(lower panel) in sodium-loaded normal volunteers. The significance of differences is represented by open circles, for comparisons with time 0, and by asterisks, for comparisons with the
vehicle infusion, as follows: p<0.05 (I sign), p<0.01 (2
signs), and p<0.001 (3 signs).
8
4
Mean ± SEM
Hours
FIGURE 3. Influence of 4-hour infusions of atrial natriuretic
peptide (ANP) at two doses and its vehicle on the urinary excretion rate of sodium (upper panel), chloride (middle panel), and
calcium (lower panel) in sodium-loaded normal volunteers.
Symbols are explained in the legend for Figure 2.
U-101
FOUR-HOUR INFUSION OF ATRIAL NATRIURETIC PEPTIDE/BioIlaz et al.
TABLE 2. Effect of a 4-Hour Infusion
Electrolyte
excretion
(itmol/
ANP
min)
i>g/min)
0
0
276±18
253 ±14
0.5
UN. x V
5.0
276 ±28
of Atrial Natriuretic Peptide on Electrolyte Excretion in Salt-Loaded Volunteers
2
Hours
3
351 ±32
472±54t
968±155tH
354 ±30
474±39t§
732 + 91+-1
338 ± 26
447±42*§
488±116*
344±27
327 ±33
665±101 + 1
319±36
443 ±55*
942±158 + 'f
202 ±31
515±105t-||
688 + 85+H
296 + 26
391 ±42
452 ±109*
294±34
315 + 30
161 ±37
315±28
242 + 21
79±28§
62.7 + 7.8
54.3 + 11.5
37.4±12.2*
54.8±8.1*
50.0±10.3
50.0±12.7
47.0±5.4t
36.8±6.1*
46.7±6.6
4.5 ±0.6
4.4±0.7
2.8±0.3
4.9±0.5
3.3±0.8
1.4±0.4§
0
0.5
5.0
267 ±18
242 ±15
234 ±28
0
0.5
5.0
96.2± 11.5
87.4± 12.0
8O.0±11.5
95.6±8.4
87.9± 15.8
97.2± 12.8
86.4 ±10.9
81.1 ±13.9
61.0±11.3
69.3±9.1
69.3± 14.3
39.8±7.1*§
Uc. x V
0
0.5
5.0
4.2±0.6
4.6±1.4
4.3±0.9
4.5±0.5
5.4±1.1
11.5±1.3 + H
4.5±0.5
6.3±1.2
15.3±2.2+'U
4.5±0.5
6.0±1.0
11.5±1.1 + H
Up x V
0
0.5
5.0
19.9±2.2
20.2 ±2.6
18.5±2.1
18.9±2.1
20.4±2.8
27.0±3.2
18.7 + 2.1
19.1±2.1
25.1 ±3.5
20.5±2.1
19.3±2.0
24.0±2.8
Ua x V
UK x V
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357±21
351+35
729±102 + H
5
343 ±34
360 + 43
187±35§
1
4
4.1 ±0.6
5.1 ± 1.0
7.9±2.0t||
20.1 ±1.9
17.9±2.5
21.8 + 5.0
6
368 ±33
255±41§
108±33.4
18.7+1.8
13.7±1.9
13.6±2.7
19.4±2.3
16.0±0.9
16.5 i t 1.1
Values are means ± SEM. ANP = atrial natriuretic peptide. Other abbreviations denote urinary excretion of sodium (U NB X V), chloride
(U a x V), potassium (UK x V), calcium (UQ, X V), phosphate (Up x V).
*p < 0.05 vs time 0.
tp < 0.001 vs time 0.
\\p < 0.01 vs vehicle,
§p < 0.05 vs vehicle.
\p < 0.001 vs vehicle.
tp < 0.01 vs time 0.
TABLE 3. Effect of a 4-Hour Infusion of Atrial Natriuretic Peptide on Water and Osmolality in Salt-Loaded Volunteers
Water/
osmolality
ANP
(Mg/ min)
Hours
4
0.5
5.0
0
7.3±1.5
7.8±1.2
7.3+1.0
1
5.9±0.8
6.9±0.9
15.1±1.4 + ^
6.5+0.9
7.7±1.0
9.7+1.3
6.6+1.2
6.5 ±0.9
8.0±1.0
6.8 ±1.0
6.3±0.8
4.5 ±1.1
6.0 ±0.9
3.5 + 0.7*
1.8±O.3+?
6
6.5±0.8
5.1 + 1.0*
l.8±0.4 + 1I
FEHZOW
0
0.5
5.0
6.6 ±1.4
7.2+1.2
6.9±1.1
5.4±0.8
5.9±0.8
11.6±0.9 + 1
5.8±0.8
7.1 ±0.9
8.2±0.8
5.9+1.1
5.8±0.9
6.8±0.8
6.4 + 0.9
5.5±0.8
4.3±0.6§
5.7±0.9
3.3 + 0.7t
1.6±0.2 + ^
6.1 ±0.8
4.7 + 0.9*
1.6±0.4+-H
U™ x V
0
0.5
5.0
1236±76
1126 + 50
1146±67
1308±41
1262+101
1893±245+D
1275 ±85
1493±125*
2349 ±341 Ml
1183 + 64
1382±136
1794 + 258t4
1161+56
1346±88
1342 ±363
1183 ±107
990±115
7O4 + 8O*§
1175 ±63
977 ±40
586 + 77t4
C ^ (ml/min)
0
0.5
5.0
4.3+0.3
4.0±0.2
4.0±0.2
4.6±0.1
4.5 ±0.4
6.6±0.9t§
4.5±0.3
5.3+0.4
8.2±1.2+-<'
4.2±0.2
4.9 + 0.5
6.3±0.9t4
4.1 ±0.2
4.8±0.3
4.8±1.3
4.2±0.4
3.5 ±0.4
2.0 ±0.4
4.1+0.2
3.5±0.1
2.6±0.8
C H2O (ml/min)
0
0.5
5.0
3.0±1.4
3.8±1.2
3.7±1.2
1.3±0.8
2.2±0.8
9.1+0.6M
2.0±0.8
2.7+0.9
2.7±0.7
2.4±1.0
1.6±0.8
2.4±0.9
2.7±0.9
1.5 + 0.7*
1.6±1.0
1.9 + 0.8
0.0±0.6t
-0.4±0.2t§
2.4±0.7
1.6 + 0.9*
-0.3±0.4t§
0
V (ml/min)
(^tosm/min)
2
3
Values are means ± SEM. ANP = atrial natriuretic peptide; V = urinary flow rate;
load; C ^ = osmolar clearance; CHv> = free water clearance.
tp < OTOOI vs time 0.
\p < 0.01 vs vehicle.
*p < 0.05 vs time 0.
§p < 0.05 vs vehicle. ty> < 0.001 vs vehicle.
t/7 < 0.01 vs time 0.
5
= fractional excretion of water, Umm X V = osmolar
n-102
1985 BLOOD PRESSURE COUNCIL
SUPPL
ANP. or Vehicle Infulion
II HYPERTENSION, VOL 8, No 6, JUNE 1986
14
I"
a
10
2
ANPr or Vehicle Inlusion
-03
as
1
4
ANPr or vehicle infusion
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5
6
-2
-0.5
0.5
1
4
ANP. or Vehicle Inlution
FIGURE 5. Effects of 4-hour infusions of atrial natriuretic
peptide (ANP) at two doses and its vehicle on the plasma levels
of immunoreactive angiotensin II and aldosterone in sodiumloaded normal volunteers. Symbols are explained in the legend
for Figure 2.
10 i
Mean * SEM
Time (Hours)
FIGURE 4. Urine flow rate, clearance of free water (CHlO),
and osmolar clearance as influenced by 4-hour infusions of
atrial natriuretic peptide (ANP) at two doses and its vehicle in
sodium-loaded normal volunteers. Symbols are explained in the
legend for Figure 2.
Figure 5 depicts the angiotensin II and aldosterone
values measured during the three experimental infusions. Angiotensin II remained at very low levels until
the end of the infusions at 4 hours. Two hours after
discontinuation of the high-dose ANP infusion, angiotensin II concentrations were markedly increased
(/?<0.001 vs vehicle). A tendency toward enhanced
angiotensin II levels was already apparent after 4
hours, but this was mostly due to changes observed in
the two volunteers who developed signs and symptoms
of marked hypotension (see below). Aldosterone followed the same pattern.
The results of plasma renin activity and arginine
vasopressin measurements are shown in Figure 6.
Plasma renin activity exhibited the same pattern as that
described for angiotensin II and aldosterone, but arginine vasopressin did not change with either of the ANP
doses.
Tolerance
During the fourth hour of the high-dose infusion of
ANP, one volunteer felt lightheaded when he got up to
void. After 2 hours of the high-dose infusions, another
volunteer had a sudden fall in blood pressure in the
supine position, accompanied by bradycardia, nausea,
and diaphoresis. The infusion was immediately
stopped, and all the signs and symptoms disappeared
within 5 minutes. After this incident, the 5.0 /xg/min
dose was no longer administered. No significant
changes in electrocardiographic tracings occurred during any of the studies.
Discussion
In these studies it was found that ANP had various
blood pressure, renal, and hormonal effects in normal
volunteers. At the high dose, it decreased blood pressure and increased the heart rate. It also reduced ERPF
FOUR-HOUR INFUSION OF ATRIAL NATRIURETIC PEPTIDE/fi/o//az et at.
ANP, Of vhlcte Infusion
*•*
ANP, <x vahlcto infusion
1.2
1.0
i
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L
0 8
| 1
1I
0.6
ri
1 1 1 1
no!
-2
Mean ± SEM
-0.5
ttS
1
Hours
FIGURE 6. Effects of 4-hour infusions of atrial natriuretic
peptide (ANP) at two doses and its vehicle on plasma renin
activity (PRA) and plasma levels ofarginine vasopressin (AVP)
in sodium-loaded volunteers. Symbols are explained in the legend for Figure 2.
in a dose-dependent manner, without changing GFR,
thus raising the filtration fraction. It enhanced the urinary excretion of sodium, chloride, and calcium but
altered neither kaliuresis nor phosphaturia. At the high
dose, it induced a marked but transient diuresis with an
increase in the free water clearance. It did not change
the already low levels of plasma renin activity, angiotensin II, and aldosterone, but after discontinuation of
ANP, all these hormones increased.
The effects of ANP on blood pressure and heart rate
were seen only at an infusion rate of 5.0 /i.g/min and
mostly with a lag time of more than 2 hours (i.e., after
infusion of a cumulative dose of more than 600 /ig).
This confirms our previous observations.10 A puzzling
feature of ANP pharmacology is the dissociation between the short half-life4-9 and the relatively long lagtime and duration of action on blood pressure after
either a bolus injection8 or short infusions.4-l0 The direct vasodilator action of the compound is not sufficient to reduce blood pressure, and other factors, such
as natriuresis, intracellular shifts of electrolytes, the
lack of response of the renin-angiotensin-aldosterone
system, or the effect of ANP on the autonomic nervous
system, may have played a part.
ANP has been shown in experimental studies either
not to change GFR22-23 or to markedly increase it.24"28
II-103
Although a trend toward an increase in GFR was transiently observed during the high-dose infusion, no statistically significant change in GFR was noted in our
experiments. Clearance periods of 1 hour may be too
long to reflect short-lived variations in GFR. Nevertheless, GFR alterations, if they occur, must be transient, and they do not seem to explain fully the renal
effects of ANP infusions. Besides a too-long integration time, other factors might explain the absence of
GFR changes in our study, such as species differences,
use of synthetic instead of natural atrial extracts, use of
infusions instead of bolus injections, or differences in
doses administered (we used small doses, compared
with those used in experiments showing increases in
GFR).
ANP has been shown to decrease,23-29 to increase,25- 29~32 or not to alter26"28 renal plasma flow, depending on the experimental conditions.33 However, it
appears that ANP does not increase ERPF in a sustained manner in normal intact animals. The dosedependent fall in PAH clearance observed with ANP
infusions in our study may reflect either a fall in ERPF
or diminished tubular PAH secretion. The present data
do not permit us to distinguish between the two possibilities. The distinction between an alteration in ERPF
and a change in PAH extraction is relative, since it has
been shown that the extraction of PAH is reduced
during acute vasodilation and increased during vasoconstriction.34 Furthermore, a direct effect of ANP on
PAH secretion cannot be ruled out, since it has been
shown that a low-molecular-weight substance in the
plasma of salt-loaded animals inhibits the uptake of
PAH by rabbit kidney cortical slices.35 Finally, the
decrease in renal plasma flow may partially be explained by the increased hematocrit.
The decrease in ERPF observed during infusion of
ANP has two possible explanations. One is the vasodilating effect of ANP, which has been shown to be
comparable in nature to that of sodium nitroprusside36
and may reduce the venous return and consequently the
cardiac output, a determinant of renal blood flow. Another possibility is a shift of renal blood flow away
from the cortex.25- ^ 3 2 - 3 7 Since PAH clearance reflects
only cortical plasma flow, this shift could alter the
extraction of PAH, therefore affecting the accuracy of
an ERPF estimation based on PAH clearance. However, such a shift in renal blood flow between cortex and medulla due to any vasoactive agent is still
controversial.38
The ensuing rise in the filtration fraction has important consequences for renal solute excretion. Since a
rise in the fraction increases the efferent arteriolar protein concentration and hence oncotic pressure, an increase in proximal solute reabsorption will result.39
This mechanism would tend to counteract the diuretic
effect of ANP and could possibly explain the bellshaped natriuresis curve observed in the present study,
as well as in animal studies with the same synthetic
ANP (E. H. Blaine, personal communication, 1985).
The ANP-induced increase in the filtration fraction is a
general finding, since it has been observed in isolated
n-i04
1985 BLOOD PRESSURE COUNCIL
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perfused rat kidney,2329 in intact dogs,2627 and in
rats.28
That ANP promotes sodium and calcium excretion
with chloride as the principal anion is accepted by all
investigators. However, the effect of ANP on potassium and phosphate excretion is still debated, though
in healthy volunteers, no marked change in phosphate
excretion has so far been reported. The qualitative
nature of solute excretion is important, since it points
to the mechanism (or mechanisms) of action underlying ANP. Experimental studies have suggested either
that the increase in renal solute excretion is due primarily to hemodynamic changes23-16- ^ N or that this effect
is mediated not by changes in GFR22n-32-4(M3 but rather by redistribution of blood flow,32 by inhibition of
proximal tubular reabsorption,44-43 by loop reabsorption,32-43 or by altered collecting duct reabsorption.22 40~42 Our study was not designed to examine this
issue. Nevertheless, the absence of parallel changes in
GFR and natriuresis and the absence of marked
changes in phosphate excretion, a known marker of
proximal tubular reabsorption, in association with unmodified potassium excretion, are indirect arguments
against a primary hemodynamic mechanism and a
proximal site of action of ANP under the present experimental conditions. Indeed, increased distal delivery of sodium due either to an increase in the filtered
load or to an inhibition of proximalreabsorptionwould
increase potassium excretion, unless distal reabsorption of sodium were simultaneously impaired. In any
case, a distal tubular effect must be present to explain
our findings.
The high-dose ANP infusion induced transient water diuresis (positive free water clearance) in the absence of osmotic hemodilution. Since no change was
observed in arginine vasopressin levels and since ANP
does not seem to interact with arginine vasopressin
receptors (M. A. Napier, personal communication,
1985), the transient nature of the increased water diuresis cannot be attributed to arginine vasopressin. A
more likely explanation is either increased delivery of
filtrate out of the proximal tubule (see above discussion of the trend toward an increase in GFR at the high
dose) or diminished free water loss secondary to sodium transport inhibition.
In the absence of substantial changes in plasma bicarbonate concentrations, the changes in urinary pH
seen after discontinuation of the high-dose ANP infusion suggest no disturbance in buffer capacity but rather an increase in acid secretion. Since therateof hydrogen-ion secretion is modulated by aldosterone, it is not
surprising to find this pH modification coupled with an
increase in potassium excretion and paralleling the
surge in the plasma aldosterone level, once the inhibitory effect of ANP has vanished.
In spite of marked natriuresis and a fall in blood
pressure, we observed no significant effect of ANP on
levels of plasma renin activity, angiotensin II, aldosterone, or arginine vasopressin — observations similar to those of Richards et al .8 and Tikkanen et al .4 This
may conflict with reports of a depressor effect of ANP
SUPPL
II HYPERTENSION, VOL 8, No 6, JUNE 1986
on these hormones. 262746 '' 7 However, there is probably no contradiction, since in the present study, plasma renin activity, angiotensin II, and aldosterone levels were extremely low to start with, because of salt
loading, and a depressor effect of ANP could not have
been manifested. Interestingly, a significant increase
in these hormones was observed during the recovery
phase, suggesting that ANP nevertheless had an inhibitory effect on their secretion.
Since ANP is expected to have a short half-life and
therefore steady state conditions should be achieved
rapidly with infusion, the relatively slow development
of the renal response, which was maximal only during
the second hour of continuous infusion, raises some
interesting questions about its mechanism of action.
The in vitro inhibition of basal and stimulated aldosteronereleaseby atrial extracts43 suggests that a decrease
in aldosterone levels could contribute to the natriuresis. The time course of the natriuresis observed in our
study could support this view. However, the absence
of any significant change in aldosterone during our
study does not support such a view, though the absence
of kaliuresis in the face of marked natruiresis points to
a similar site of action for ANP. Alternatively, ANP
may have to induce protein synthesis in the tubular
cells to become active.
In summary, 4-hour infusions of two doses of synthetic ANP were administered to normal volunteers,
and their effects were compared with those of vehicle
administration. The high dose (5 /xg/min) induced a
transient diuretic effect, delayed maximal natriuretic
activity, and a late fall in blood pressure. No change in
GFR occurred, but a dose-dependent decrease in ERPF
and a consequential rise in the filtration fraction were
observed. Despite large amounts of sodium excreted
and blood pressure reduction, ANP administration was
largely unaccompanied by counterregulatory changes
in therenin-angiotensin-aldosteronesystem and in arginine vasopressin levels.
Note added in proof: The unpublished data of
Nussberger et al. mentioned in the text (see Analytical
Methods) will be published in 1986.^
Acknowledgments
We thank Monique Appenzeller and Gisele Maridor for technical
assistance, and Anne-Francoise Staid and Anne Campiche for secretarial assistance.
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Hypertension. 1986;8:II96
doi: 10.1161/01.HYP.8.6_Pt_2.II96
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1986 American Heart Association, Inc. All rights reserved.
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