Download Effects of Volume Change on Circulating Immunoreactive Atrial

Effects of Volume Change
on Circulating Immunoreactive
Atrial Natriuretic Factor in Rats
MASAKAZU K O H N O , KERRY B. CLEGG, AND MOHINDER P. SAMBHI
Downloaded from http://hyper.ahajournals.org/ by guest on August 2, 2017
SUMMARY The mammalian atrial hormone atrial natriuretic factor (ANF) has been shown to have
potent natriuretic and diuretic actions as well as vasodilator effects when released into the circulation.
To investigate how the levels of the circulating form of this peptide c
cular fluid volume, we measured immunoreactive ANF in the plasm
load, acute furosemide treatment, and chronic water restriction. Cii
ANF increased significantly ( p < 0.001) 1 minute after acute saline 1
within 5 minutes. Volume contraction induced by furosemide treat
significantly reduced the circulating immunoreactive ANF. These
expansion causes an immediate release of immunoreactive ANF intt
volume contraction results in a decline of circulating levels of imm
tained during chronic volume contraction. These results suggest t
intravascular fluid volume and respond by changing the levels of A
and thereby participate in the regulation of body fluids and, perl
(Hypertension 10: 171-175, 1987)
KEY WORDS • atrial natriuretic factor
treatment • acute saline load
• chronic water restriction
I
• acute furosemide
ANF (irANF) into the perfusate,8 and a positive correlation of ANF release with right atrial and pulmonary
wedge pressure in humans has been reported.9 These
data imply that ANF may be secreted from cardiac
atria by stimulation of atrial stretch or baroreceptors
and may play a role in the regulation of body fluids and
blood pressure. Therefore, we investigated whether
the circulating levels of ANF change with expansion as
well as contraction of intravascular fluid volume. The
circulating concentration of irANF in Wistar rats following acute saline load, acute furosemide administration, and chronic water restriction was quantitated by
radioimmunoassay.
NTRAVENOUS injection of rats with extracts of
the atria, but not the ventricles, results in pronounced natriuretic and diuretic responses.1
Mammalian cardiac atria have been shown to contain a
mixture of biologically active peptides, atrial natriuretic factors (ANFs), of heterogeneous molecular
weights.2"5 Recently, the major circulating form in the
rat has been characterized as the 28 amino acid a-atrial
natriuretic peptide.6 All of the mechanisms controlling
the secretion of ANF have yet to be determined. Mechanical distention of the left atrium, by obstructing
the mitral orifice, has been shown to result in an increase in urine flow and sodium excretion.7 Similarly,
an increase in perfusion pressure on isolated hearts
resulted in an increased release of immunoreactive
Materials and Methods
Determination of Immunoreactive ANF and
Plasma Renin Activity
Immunoreactive ANF was extracted from 2 ml of
plasma by binding to Sep-Pak C-18 cartridges (Millipore-Waters, Milford, MA, USA) using the procedure
of Lang et al. 8 The extracted peptides were dried by
vacuum centrifugation (Savant, Hicksville, NY, USA)
and dissolved in the assay buffer (0.01 M sodium
phosphate, pH 7.4, 0.05 M NaCl, 0.1% bovine serum
From the Hypertension Division, Department of Medicine, Veterans Administration Medical Center, Sepulveda, and the Department of Medicine, University of California at Los Angeles, Los
Angeles, California.
Supported by research funds of the Veterans Administration.
Address for reprints: Mohinder P. Sambhi, M.D., Ph.D., Sepulveda VA Medical Center, 16111 Plummer Street (111-H), Sepulveda, CA 91343.
Received February 15, 1986; accepted March 4, 1987.
171
172
HYPERTENSION
Downloaded from http://hyper.ahajournals.org/ by guest on August 2, 2017
albumin, 0.1% Nonidet P40, 0.01% NaN3). The recovery of ANF by extraction was calculated by adding
known quantities of a-rat ANF (1-28), 100 to 200
pg/ml, to plasma previously treated with dextran-coated charcoal to remove endogenous ANF. Recovery
was 63.9 ±3.6%.
Radioimmunoassays were performed using antibody and [I25I]ANF from Peninsula Laboratories (Belmont, CA, USA) directed against a-human ANF added to 100 (JLI of extract or plasma as previously
described.10 Standard curves were prepared with 100
(x.1 of a-rat ANF, 50 to 5000 pg/ml, in assay buffer. At
the end of the incubation, 100 /x\ each of normal rabbit
serum diluted 1:50 in assay buffer and goat anti-rabbit
IgG serum diluted 1:10 in assay buffer were added and
allowed to precipitate for 24 hours. The samples were
diluted with an additional 0.5 ml of assay buffer, and
the precipitate was collected by centrifugation at 1700
g for 30 minutes. The unbound counts were aspirated,
and the precipitates counted in a Micromedic gamma
counter (Horsham, PA, USA). The effective range of
the standard curve was between 5 and 200 pg of ANF,
and 50% displacement was obtained with 22 pg of
ANF. The interassay variation was 13.5% (n = 10),
and the intra-assay variation was 5.0% (n = 20). Plasma renin activity (PRA) was measured by procedures
previously reported."
All assays were performed in duplicate, and statistical significance was determined by paired and unpaired Student's t tests.
Acute Saline Load
Twenty male Wistar rats (weight, 300-400 g; Simonsen Labs, Gilroy, CA, USA) that had been maintained on ad libitum food and water were divided into
four groups. The rats were anesthetized intraperitoneally with sodium pentobarbital (6 mg/100 g), and the
left jugular vein and left carotid artery cannulated with
polyethylene tubing (PE-50, Clay Adams, Parsippany,
NJ, USA). The trachea was also cannulated with a
catheter (PE-240). Thirty minutes after operation, a 5ml sample of blood was withdrawn from the carotid
artery of Control Group 1 (n = 5). The remaining animals were infused with isotonic saline, 2 ml/100 g,
over a period of 15 seconds into the jugular vein. After
infusion, 5-ml blood samples were withdrawn at 1
minute in Group 2 (n = 5); 3 minutes in Group 3
(n = 5), and 5 minutes in Group 4 (n = 5). An additional 20 animals underwent sham operation and samples were taken as described.
All samples were immediately placed into ice-cold
tubes containing 10 fi\ of aprotinin (250 KIU; Sigma
Chemical, St. Louis, MO, USA) and heparin, 10
IU/ml of blood. Plasma was separated by a 10-minute
centrifugation at 1700 g, 4°C and immediately frozen
at — 20 °C for subsequent radioimmunoassay of ANF.
Assays were performed within 24 hours of collecting
the samples, and prolonged storage was avoided.
Acute Furosemide Administration
Furosemide (1 mg/100 g) was administered intraperitoneally to seven male Wistar rats (weight,
VOL 10, No 2, AUGUST 1987
300-400 g), and the rats were placed in metabolic
cages. After 5 hours, the rats were removed from the
cages and weighed and the urine volume was measured. The animals were immediately killed by decapitation, and the blood collected as described. Circulating irANF, PRA, and hematocrit were measured.
Blood samples from seven control rats (weight,
300-400 g) without furosemide administration were
collected and assayed in parallel.
Chronic Water Restriction
Female Wistar rats (weight, 210-300 g) were maintained on a normal sodium diet. The rats were randomly assigned to a water-restricted group (n = 9) or a
normal control group (n = 7) given free access to water. The water-restricted group was allowed a total
water intake of 1 ml/100 g body weight for 4 days and
then no water for 24 hours before death. The rats were
weighed at the beginning and end of the experiment.
The rats were placed in a metabolic cage during the last
24 hours. Urine was collected, and the volume was
measured. The rats were killed by decapitation, and
the blood was collected. Plasma was prepared as already described for radioimmunoassay of ANF and
PRA.
Results
Acute Saline Load
Table 1 shows the concentration of circulating
irANF in groups of rats before and 1,3, and 5 minutes
after saline load. The irANF was measured after extraction of the plasma with C-18 silica. The concentration of irANF at 1 minute was significantly higher
compared with control level (/?<0.001). Although
circulating irANF declined at 3 minutes compared with
that at 1 minute (p<0.02), it was still significantly
higher than the control level (p<0.05). Five minutes
after infusion, the concentration of irANF (115 ± 18
pg/ml) approached the basal level (92 ± 13 pg/ml). In
sham-operated rats the circulating level of irANF did
not vary significantly over the course of the experiment
and remained near the control level, thus demonstrating that the withdrawal-replacement sampling technique did not severely alter the ANF level.
Volume Depletion by Furosemide
Table 2 shows the effect of furosemide administration on body weight, urine volume, PRA, hematocrit,
and plasma irANF. The irANF concentration was decreased significantly (p< 0.005) from the control level, while the PRA and hematocrit were significantly
increased (p<0.05). Initially, the average body
weight of the control and experimental animals was
identical. However, 5 hours after furosemide treatment, body weight significantly declined (p<0.0l)
and was accompanied by increased urine volume.
Chronic Water Restriction
Table 3 shows the body weight, 24-hour urine volume/100 g body weight, PRA, and concentration of
irANF in control animals (n = 7) and animals with
173
CIRCULATING ANF AND VOLUME CHANGE/Kohno et al.
TABLE 1. Effect ofAcute Saline Load on Plasma Immunoreactive
ANF
Plasma irANF (pg/ml)
1 min
3 min
Control
5 min
Treatment
Saline infusion 94+11 253 ± 39 *
175±36t 115 ±18
91 ±17
89 ±14
93 ±21
Sham operation 92±13
Values are means ± SD. irANF = immunoreactive ANF.
*p< 0.001, compared with control values.
tp<0.05, compared with control and 1-minute values.
Downloaded from http://hyper.ahajournals.org/ by guest on August 2, 2017
TABLE 2. Effect of Furosemide Administration on Immunoreactive ANF
Furosemide
Control
Parameter
(n = 7)
(« = 7)
Body weight (g)
354±12
354±10
Before administration
346+11
328 ±7*
After administration
4.3 ±0.6
Urine volume (ml/5 hr)
16.2±2.1t
36.3±0.7
44.6±2.4$
Hematocrit (%)
5.6+1.7
16.9±8.9t
PRA (ng ANG I/ml/hr)
1%±33
13O±26§
irANF (pg/ml)
Values are means ± SD. irANF = immunoreactive ANF; ANG
I = angiotensin I.
*p<0.01, tp<0.001, tp<0.02, §p<0.005, compared with
control values.
chronic water restriction (n = 9). The circulating
irANF concentration was significantly greater in the
control animals than in the water-restricted group
(p < 0.001) for both extracted and unextracted plasma.
Initially, the average body weight of the control and
experimental rats was identical. However, severe water restriction significantly reduced the body weights
of the experimental group (p<0.001). As expected,
the 24-hour urine volume was reduced in the volumedepleted group (p<0.00\) and the PRA was significantly elevated (p < 0.001) as compared with the control group.
Effect of Decapitation and Anesthesia on
Immunoreactive ANF
Figure 1 shows the effect of the different methods of
collecting blood samples on the basal level of extracted
irANF measured in control rats. The control level of
irANF in plasma prepared from blood collected
following decapitation was significantly higher
(p<0.001) than that in plasma obtained from carotid
artery cannulation with the rats under pentobarbital
anesthesia. The plasma level of irANF in another
group of control animals, anesthetized with pentobarbital 30 minutes before decapitation, was intermediate
between die values found with decapitation or anesthesia alone.
Discussion
Mammalian cardiac atria contain biologically active
peptides, ANFs, that are capable of producing diuresis, natriuresis, and vasodilation.1-l2 The amino acid
sequence of ANF peptides has been determined, peptides that mimic the actions of endogenous factors have
been synthesized,2"5 and the structure and sequence of
the ANF gene have been determined.13 Further, ANF
has been shown to be synthesized by cardiocytes in
vitro and secreted as the high molecular weight 126
amino acid proANF,14 which is subsequently converted to a 28 amino acid circulating form of these peptides6 in the presence of serum. ANF release by the
atria may play a role in the regulation of body fluids
and blood pressure. Lang et al.8 have demonstrated
that acute volume load serves as a trigger for the release of ANF into the circulation. Available evidence
is currently compatible with the hypothesis that volume expansion causes an increase in ANF; however,
evidence that volume contraction, particularly chronic
contraction, causes a decrease in circulating ANF has
been limited." We have presented data here that demonstrates the effect of both acute volume expansion
and acute and chronic volume contraction on the increase and decrease, respectively, of circulating ANF.
The effect of rapid volume expansion induced by
saline infusion on the irANF level in extracted plasma
from groups of rats at various time points was determined. The basal level of irANF (92 pg/ml) in extracted samples increased within 1 minute after the infusion
by 140 to 150 pg/ml. This result indicates that acute
volume expansion, induced through bolus injection of
normal saline, causes the immediate release of ANF
into the general circulation. Circulating levels of
irANF were decreased 3 minutes after infusion but had
returned to basal levels 5 minutes after infusion.
The rapid increase and subsequent decrease of
irANF during volume load are consistent with the findings of Lang et al.,8 in which increased atrial perfusion
pressure in vitro increased ANF release. The rapid
clearance of irANF and subsequent return to control
levels is indicative of a circulating half-life of approximately 2 minutes for the released ANF. This rate of
TABLE 3. Effect of Chronic Water Restriction on Plasma Immunoreactive ANF
Group
Weight (g)
5 days
0 days
249 + 32
249 + 29
24-hour
urine volume
(ml/100 g)
5.9 + 0.8
PRA
(ng ANG 1/
ml/hr)
6.0±1.4
Control (n = 7)
Water restricted
255 ±33
216±33*
12.2 + 3.4*
(n = 9)
1.5 + 0.2*
Values are means + SD. ANG I = angiotensin I; irANF = immunoreactive ANF.
*p<0.001, compared with control values.
Plasma irANF
(pg/ml)
190±30
112±12*
174
FIGURE 1. Anesthesia- induced variation in extracted concentration of immunoreactive ANF. Plasma
immunoreactive ANF was measured in blood samples
recovered by (A) immediate decapitation; (B) decapitation 30 minutes after pentobarbital anesthesia (6
mglkg i.p.); (C) carotid artery cannulation 30 minutes after pentobarbital anesthesia. Single asterisk
indicates significant difference compared with B or C
value (p<0.001). Double asterisk indicates significant difference compared with C value (p<0.01).
HYPERTENSION
VOL
10,
No
2,
AUGUST
1987
250
10
a
200
150
c
o
u
u.
z
X
n-8
T
n-10
100
50
Downloaded from http://hyper.ahajournals.org/ by guest on August 2, 2017
B
clearance is in agreement with previous volume expansion data.8 The mechanism of ANF clearance has yet to
be determined; however, ANF has been shown to be
very sensitive to inactivation by several proteolytic
enzymes.16 Whether degradation occurs at the tissue
level or in the general circulation is not certain, but the
recent data of Crozier et al.17 indicate that circulating
levels of ANF are reduced approximately 50% by passage through major organs. The data of Bloch et al.14
further indicate that the low molecular weight ANF
peptide is reasonably stable in serum for the duration
of proANF cleavage (60 minutes at 21 °C; 30 minutes
at 37 °C) and thus suggests that tissue degradation or
binding is responsible for the loss of irANF from the
circulation.
The basal levels of irANF in the acute saline load
experiments were'lower than control values observed
for furosemide administration or chronic water restriction. In the latter experiments blood samples were collected by decapitation, whereas blood samples in the
former experiments were withdrawn from the carotid
artery with the rats under anesthesia. The observed
differences in the controls may be explained by two
mechanisms. First, pentobarbital anesthesia is associated strongly with a decline in circulating irANF.18
Second, decapitation and the associated tachycardia
induced by the procedure and resulting hemodynamic
changes may raise the level of irANF. This contention
is supported by preliminary data (see Figure 1) comparing decapitation and anesthesia on the basal level of
extracted irANF in rats. Decapitation in the presence
of pentobarbital anesthesia resulted in irANF values
intermediate (111 ± 12 pg/ml; n = 8) between those
found with decapitation (193 ± 19 pg/ml; n = 10) or
pentobarbital alone (93 ± 12 pg/ml; n = 10). Further
studies will be necessary to elucidate the mechanism of
ANF release under these experimental conditions.
The effects of acute and chronic volume depletion
on the irANF levels were examined by rapid diuresis
and chronic water deprivation. The administration of
furosemide resulted in significant volume depletion, as
indicated by weight loss, higher PRA, and increased
hematocrit. These data suggest that the amount of ANF
released by the atria in response to volume depletion is
reduced. This lower concentration of ANF is consistent with the apparent physiological role of ANF and
indicates that the atria respond to the decrease in fluid
volume by reducing the rate of ANF release. In agreement with this acute response, chronic water deprivation, which also resulted in a severe volume contraction (a 14% weight loss and a 200% increase in PRA),
was similarly associated with a decreased level of circulating irANF. Consistent with our observation, atrial
intracellular granule concentrations have been shown
to be higher in rats after water deprivation,"1 x which
has led to the suggestion that these granules are the
storage site for ANF.21 How the atria regulate the production and secretion of ANF remains to be determined; however, the observed increase in cellular
granularity and lower circulating ANF concentrations
can be interpreted as a retention of ANF by the atria.
The retention of ANF by the atria during water deprivation is accompanied by a reduction in atrial ANF
messenger RNA,15 suggesting that the tissue level of
ANF is more dependent on the rate of release than on
the rate of biosynthesis.
Consistent with the role of ANF in diuresis, urine
volumes were also significantly lower in water-restricted rats compared with the values in control rats.
Many factors, including a reduction in the putative
CIRCULATING ANF AND VOLUME CHANGE/Kohno et al.
hypothalamic natriuretic hormone22 and increased activity of the renin-angiotensin-aldosterone system and
vasopressin, may be associated with the reduction of
urine volume after water restriction. However, a decline in the circulating level of ANF may also play a
major role in the reduction of urine volume.
In the present study, we demonstrated that the circulating concentration of irANF increases after volume
expansion and decreases after volume contraction.
Since the atria are in an ideal location to sense changes
in the intravascular fluid volume, the atria may respond to chronic as well as acute changes in intravascular fluid volume by altering the level of ANF release, thereby participating in the regulation of body
fluids and, possibly, blood pressure.
9.
10.
11.
12.
13.
14.
Downloaded from http://hyper.ahajournals.org/ by guest on August 2, 2017
1.
2.
3.
4.
5.
6.
7.
8.
References
De Bold AJ, Borenstein HB, Veress AT, Sonnenberg H. A
rapid and potent natriuretic response to intravenous injection of
atrial myocardial extract in rats. Life Sci 1981;28:89-94
Kanagawa K, Matuo H. Purification and complete amino acid
sequence of a-human atrial natriuretic polypeptide (a-hanp).
Biochem Biophys Res Commun 1984;118:131—139
Atlas SA, Kleinert HD, Camargo MJ, et al. Purification, sequencing and synthesis of natriuretic and vasoactive rat atrial
peptide. Nature 1984;309:717-719
Currie MG, Geller DM, Cole BR, et al. Purification and sequence analysis of bioactive atrial peptides (atriopeptins). Science 1984;223:67-69
Geller DM, Currie MG, Wakitani K, et al. Atriopeptins: a
family of potent biologically active peptides derived from
mammalian atria. Biochem Biophys Res Commun 1984;120:
333-338
Schwartz D, Geller DM, Manning PT, et al. Ser-Leu-Arg-ArgAtriopeptin III: the major circulating form of atrial peptide.
Science 1985;229:397-400
Henry JP, Gauer OH, Reeves JL. Evidence of the atrial location of receptors influencing urine flow. Circ Res 1956;4:
85-90
Lang RE, Tholken H, Ganten D, Luft FC, Ruskoako H, Unger
15.
16.
17.
18.
19.
20.
21.
22.
175
TH. Atrial natriuretic factor: a circulating hormone stimulated
by volume loading. Nature 1985;314:264-266
Rodeheffer RJ, Tanaka I, ImadaT, Hollister AS, Robertson D,
Inagami T. Atrial pressure and secretion of atrial natriuretic
factor into the human central circulation. J Am Coll Cardiol
1986;8:18-26
Kohno M, Takaori K, Matsuura T, Murakawa K, Kanayama
Y, Takeda T. Atrial natriuretic polypeptide in atria and plasma
in experimental hyperthyroidism and hypothyroidism. Biochem Biophys Res Commun 1986;134:178—183
Barrett JD, Eggena P, Sambhi MP. Influence of angiotensinase
inhibitors on the enzymatic activity of renin. Biochem Med
1976;16:157-168
Currie M, Geller DM, Cole BR, et al. Bioactive cardiac substances: potent vasorelaxant activity in mammalian atria. Science 1983;222:71-73
Seidman CE, Bloch KD, Klein KA, Smith JA, Seidman JG.
Nucleotide sequences of the human and mouse atrial natriuretic
factor genes. Science 1984;226:1206-1209
Bloch KD, Scott JA, Zisfein JB, et al. Biosynthesis and secretion of proatrial natriuretic factor by cultured rat cardiocytes.
Science 1985 ;230:1168-1171
Takayanagi R, Tanaka I, Maki M, Inagami T. Effects of
changes in water-sodium balance on levels of atrial natriuretic
factor messenger RNA and peptide in rats. Life Sci 1985;
36:1843-1848
Thibault G, Garcia R, Cantin M, Genest J. Atrial natriuretic
factor: characterization and partial purification. Hypertension
1983;5(suppl I):I-75-I-80
Crozier IG, Nicholls MG, Ikram H, Espiner EA, Yandle TG,
Jans S. Atrial natriuretic peptide in humans: production and
clearance by various tissues. Hypertension 1986;8(suppl II):II11-11-15
Hork'y K, Gutkowska J, Garcia R, Thibault G, Genest J,
Cantin M. Effect of different anesthetics on immunoreactive
atrial natriuretic factor concentrations in rat plasma. Biochem
Biophys Res Commun 1985;129:651-657
Garcia R, Gantin M, Thibault G, Ong H, Genest J. Relationship of specific granules to the natriuretic and diuretic activity
of rat atria. Experientia 1982;38:1071-1073
De Bold AJ. Heart atria granularity effects of changes in waterelectrolyte balance. Proc Soc Exp Biol Med 1979; 161:508-511
De Bold AJ. Tissue fractionation studies on the relationship
between an atrial natriuretric factor and specific atrial granules.
Can J Physiol Pharmacol 1982;60:324-330
Haddy FJ. Natriuretic hormone: the missing link in low renin
hypertension? Biochem Pharmacol 1982;31:3159—3161
Effects of volume change on circulating immunoreactive atrial natriuretic factor in
rats.
M Kohno, K B Clegg and M P Sambhi
Downloaded from http://hyper.ahajournals.org/ by guest on August 2, 2017
Hypertension. 1987;10:171-175
doi: 10.1161/01.HYP.10.2.171
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1987 American Heart Association, Inc. All rights reserved.
Print ISSN: 0194-911X. Online ISSN: 1524-4563
The online version of this article, along with updated information and services, is located
on the World Wide Web at:
http://hyper.ahajournals.org/content/10/2/171
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally
published in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center,
not the Editorial Office. Once the online version of the published article for which permission is being
requested is located, click Request Permissions in the middle column of the Web page under Services.
Further information about this process is available in the Permissions and Rights Question and Answer
document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Hypertension is online at:
http://hyper.ahajournals.org//subscriptions/