Departments of 1 Physiology and 2 Pharmacology and Toxicology, Biocenter Oulu, University of Oulu, FIN-90014 Oulu; and 3 Department of Biology, University of Turku, FIN-20014 Turku, Finland
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ABSTRACT |
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The present study tested the hypothesis that salmon cardiac peptide (sCP), a new member of the family of natriuretic peptides, has an important role in the regulation of fluid balance and cardiovascular function. Intra-arterial administration of sCP increased urine output in salmon. It had a diuretic effect in rat as well, but the potency was lower. sCP increased the sodium excretion in proportion to the increased urine flow. Blood pressure was not affected by sCP in either species. Acute volume expansion elevated the plasma level of sCP in salmon, and an acute transfer of salmon from fresh to sea water decreased the circulating sCP level. Cardiac immunoreactive sCP or sCP mRNA levels were not affected by transfer to sea water. These results indicate that sCP has an important physiological role in defending salmon against volume overload but that it does not appear to contribute to the short-term regulation of blood pressure. sCP provides an excellent model of the general mechanisms of regulation of the A-type (atrial) natriuretic peptide system.
osmoregulation; diuresis; natriuresis; volume overload; hyperosmotic environment
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INTRODUCTION |
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CONTROL OF WATER AND SALT BALANCE involves a complex interplay of regulatory mechanisms. The cardiac natriuretic peptides play an important part in this regulation (18, 21). The first member of the peptide family, atrial (A-type) natriuretic peptide (ANP) (7) has been shown to be a multifunctional hormone protecting volume and pressure homeostasis (18, 25). In mammals, the release of ANP from the heart is enhanced by stretch of cardiac myocytes caused, for example, by an increase in blood volume. ANP reduces the cardiac load by decreasing the systemic blood pressure and intravascular volume. The volume-depleting action is a result of increased excretion of water and electrolytes in the kidney and inhibition of the renin-angiotensin-aldosterone system (18, 21).
Although the biology of the natriuretic peptides has been studied extensively, the cellular and molecular elements important for the physiological actions are not yet well understood. We used salmon cardiac peptide (sCP), a recently discovered teleost cardiac hormone related to the natriuretic peptides (32, 34) as a model in our attempt to clarify these mechanisms. As judged by the cDNA and gene sequence, sCP represents a new subgroup of the natriuretic peptide family (20, 32). However, the storage, processing, and molecular sizes of the stored and circulating forms resemble those of ANP (13, 35). In addition, the release from isolated ventricle is similarly sensitive to mechanical load and utilizes the regulated pathway, as does ANP secretion from the mammalian cardiac atria (13). Finally, the basal gene expression of ANP and sCP appears to be regulated by similar elements (20). Thus sCP could be used to elucidate the conserved features important for the actions and regulation of the ANP-like peptide hormones. We have now examined the biological profile of sCP, with special emphasis on its potential role as a volume regulating hormone.
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MATERIALS AND METHODS |
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Experimental animals.
Salmon (Salmo salar) of both sexes, aged 4-5 yr and
weighing 640 ± 120 g, were obtained from the Finnish Game
and Fisheries Research Institute (Taivalkoski, Finland, 65°6' lat.),
where they were reared in fresh water under natural temperature and
photoperiod. The fish were transported to the Department of Physiology,
University of Oulu, in closed plastic packages filled with oxygenated
water and were released into water tanks containing unchlorinated,
recirculating tap water. The water quality was maintained by biological
filtration and biweekly water changes of ~20%. The salmon were
stocked in a 1,500-l tank at a density of 16 fish per tank. The
experiments were performed in November and January, when the water
temperature was 0.2°C and the seasonal photoperiod was 4 h light
and 20 h dark. In the laboratory, the maintenance and experimental
ambient temperature was 6°C and the light-dark cycle was 8:16 h.
Dissolved oxygen levels in the experimental tanks were >80% oxygen saturation.
Bolus injection of synthetic sCP into salmon.
For the administration of sCP, measurement of arterial blood pressure,
and collection of blood samples, the salmon were cannulated via the
dorsal aorta as described previously (29). The fish were
anesthetized in well aerated tricaine solution (100 mg/l, MS-222, Sigma
Chemical, St. Louis, MO), the pH of which was adjusted to 7.0 with
sodium carbonate. The anesthesia took place at the temperature of the
water in the tank. When the animals became unresponsive to handling,
they were weighed, placed on the operating table, and covered with a
cold wet towel. The gills were not irrigated during the operation. The
dorsal aorta was cannulated near its origin via the buccal cavity with
PE-50 tubing (0.96 mm OD, 0.58 mm ID; Portex, Hythe, UK) with the help
of a stainless steel wire. The cannula was pretreated with a small
amount of heparin (5,000 IU/ml; Lövens, Ballenrup, Denmark). The
catheter was exteriorized through the snout via a short PE-10 tube
(1.57 mm OD, 1.14 mm ID, Portex), which pierced the upper jaw through
the snout. The cannula was filled with heparinized saline (100 IU/ml)
and plugged with a stainless steel pin. The urinary bladder was
catheterized through the urinary pore with PE-50 tubing filled with
saline and worked ~4 cm into the animal. This cannula was anchored
with a ligature (3-0 coated polyester fiber; Deknalon, Hamburg,
Germany) to the anal fin. The whole procedure, from immersing the fish into the tricaine solution to reviving the fish, took 15 min or less.
The fish were placed in individual black plastic tubes floating in the
holding tank and were allowed to recover overnight. On the next day,
the dorsal aortic cannula was flushed with heparinized saline and
attached to a three-way stopcock (Codan, Espergaerde, Denmark), one arm
of which was connected to a pressure transducer (Micron Instruments,
Simi Valley, CA) and a Grass polygraph (Grass Instruments, Quincy, MA).
Zero pressure was adjusted to the level of the water surface, and the
calibration was carried out using a tube filled with water. The
experiment was started by monitoring the baseline dorsal aortic
pressure for 20 min, before which a blood sample (1 ml) was withdrawn
from the arterial cannula for the measurement of basal plasma
immunoreactive (ir)-sCP and the amino-terminal (NT) fragment of pro-sCP
(NT-pro-sCP). The blood sample was immediately centrifuged in a
microfuge (1 min, 13,000 rpm; Heraeus Instruments, Osterode, Germany).
The plasma was removed into a chilled Eppendorff tube on ice, and the
blood cells were gently resuspended in 0.5 ml of cold salmon Ringer
solution (in mmol/l: 140 NaCl, 10 NaHCO3, 2 NaH2PO4, 1 MgSO4, 1 CaCl2, 4 KCl, pH 7.8) and injected back into the fish. This
injection lasted 1 min and was followed by a 0.2-ml flush of
heparinized saline (100 IU/ml). Synthetic sCP (32) or
vehicle (salmon Ringer solution) was injected into the dorsal aorta as
a single bolus. An sCP dose of 60 pmol/kg was delivered in volumes
ranging from 0.3 to 0.5 ml, and the peptide injection was followed by a
0.1-ml saline flush. Arterial blood samples of 0.5 ml were taken at 30, 60, 90, 120, and 150 min after the injections. The plasma, obtained from centrifugation in a microfuge (2 min, 13,000 rpm), was placed on
ice. These blood samples were replaced with an equal volume of salmon
Ringer solution. The urine catheter emptied into polyethylene tubes
that were placed below the water surface outside the holding tank to
ensure constant urine flow from the bladder. Fractions of 30 min were
collected using a fraction collector for the measurement of volume,
sodium, and potassium. The ion concentrations were measured from urine
with an ion selective analyzer (Kone Instruments, Espoo, Finland) and
the osmolality with an osmometer (Advanced Wide-Range Osmometer,
Advanced Instruments, Needham Heights, MA). After the experiments, the
fish were killed with a sharp blow on the head. All plasma and urine
samples were stored at 70°C.
Infusion of synthetic sCP into rats. The physiological actions of mammalian ANP and B-type, or brain, natriuretic peptide (BNP) are mediated via the ANP receptors (NPR-A). Because mature sCP structurally resembles mammalian cardiac natriuretic peptides, we were interested in whether characteristics important for ligand recognition by the mammalian natriuretic peptide receptors, specifically NPR-A, are present in sCP. Thus the effectiveness of synthetic sCP was studied in rat. The surgical preparation and experimental setup have been described previously (16). Briefly, under anesthesia [1 part Hypnorm (Janssen Pharmaceutical, Beerse, Belgium), 1 part Dormicum (Roche, Espoo, Finland), 2 parts sterile water (3.3 ml/kg ip)], a PE-60 catheter (0.76 mm ID, 1.22 mm OD; Intramedic, Becton-Dickinson, Sparks, MD) was placed into the abdominal aorta through the left femoral artery, and a PE-50 catheter (0.58 mm ID, 0.965 mm OD, Intramedic) was inserted into the femoral vein. All of the catheters were exteriorized behind the neck, filled with heparinized saline (500 IU/ml) solution, and plugged with a stainless steel pin. After the operation, the rats were housed individually in cages and had free access to food and water. A day after the operation, the rats were placed individually in metabolic cages without food and water. The arterial catheter was attached to a pressure transducer (Micron Instruments, Los Angeles, CA), and the mean arterial pressure (MAP) and heart rate (HR) were recorded with Ponemah Physiology Platform software (Gould Instrument Systems, Valley View, OH). The venous catheter was connected to a syringe infusion pump (Braun Perfusor ED, Braun Melsungen, Melsungen, Germany) for administration of peptide or its vehicle (0.9% NaCl). Rats were left undisturbed for 30 min before the recording of hemodynamic variables and collecting of urine.
The experiment was started by recording the MAP and HR for 25 min before 1.0 ml blood was drawn from the arterial catheter for the measurement of basal plasma ir-NT-pro-ANP and ir-BNP levels. Because the plasma sample size did not allow measurement of both ir-ANP and ir-BNP, the NT-pro-ANP analysis was chosen for the estimation of secretion of pro-ANP-derived peptides. When blood pressure, HR, and right atrial pressure were stabilized near to control values (in ~5 min), synthetic sCP was administered intravenously at a dose of 660 pmol · kgAcute volume overload in salmon.
Salmon were given an intra-arterial infusion of saline to find out
whether acute blood volume expansion affects the release of sCP.
Cannulation was performed as described for bolus injection of synthetic
sCP into salmon. The acute volume overload was caused by intra-arterial
administration of precooled 0.9% NaCl (10 ml/kg body wt during
2-3 min). For the measurement of the plasma levels of ir-sCP and
NT-pro-sCP, arterial blood samples of 0.5 ml were drawn in chilled
tubes containing 1.5 mg K2-EDTA/ml blood at 10, 20, and 30 min and at 1, 2, and 3 h and centrifuged at +4°C to separate the
plasma. The plasma samples were stored at 70°C until assayed. All
of the blood samples were replaced with an equal volume of saline.
After the experiments, the fish were killed with a sharp blow on the head.
Transfer of salmon from fresh water to sea water.
We used an acute transfer of cannulated salmon from fresh water to sea
water to study the effect of exposure to a hyperosmotic environment on
the regulation of the sCP system. The dorsal aorta and the urinary
bladder were cannulated as described, except that the urinary pore was
sealed with cyanoacrylate adhesive and a small piece of vinyl. The fish
were placed in individual plastic tubes floating in a 300-l
experimental tank. Two to three tubes were placed in each tank
containing well aerated, filtered, recirculating tap water. The water
quality was followed daily with measurements of dissolved oxygen
saturation (OxyGuard International, Birkerød, Denmark) and weekly with
measurement of nitrite (Aqua Vital Multisticks; Aquarium Münster,
Telgte, Germany). The next day, a 0.7-ml blood sample was drawn via the
arterial cannula for the measurement of basal plasma ir-sCP,
ir-NT-pro-sCP, Na+, Cl, K+, and
osmolality. Urine was collected via the catheter as two 60-min
fractions by use of an LKB Redirac fraction collector (LKB, Bromma,
Sweden) to determine the volume and NT-pro-sCP, Na+, and
K+ concentrations. The fish were randomly divided into two
groups, which were acutely transferred into 300-l tanks containing
either fresh water or artificial sea water. The transfer was not
associated with any mortalities, but two fish showed slight
restlessness for a few minutes after the transfer. Blood samples of 0.7 ml were drawn at 30, 60, and 120 min and at 24, 48, and 72 h after the transfer. The plasma was separated by centrifugation at +4°C and
stored at
70°C. The blood cells were gently resuspended in 0.5 ml
of chilled salmon Ringer solution and injected back into the fish. In
the fish kept in fresh water, the urine was collected in two 60-min
fractions starting from the transfer, as described. In the fish
transferred to sea water, the urine flow had decreased to very low
levels within 2 h after the transfer. To obtain a volume large
enough for determination of the electrolytes, osmolality, and
ir-NT-pro-sCP, the urine was collected in 24-h fractions. After the
experiments, the fish were killed with a sharp blow on the head. The
heart was removed and the atrium and ventricle separated, weighed, and
frozen in liquid N2. The plasma and tissue samples were
stored at
70°C.
Determination of sCP mRNA by quantitative RT-PCR.
Total RNA was extracted from salmon cardiac tissue by means of the
acid-phenol method (6). Aliquots of the guanidine
isothiocyanate homogenates were stored at 70°C for use in
radioimmunoassay. The cDNA first strand was synthesized using Moloney
murine leukemia virus reverse transcriptase. The quantitative PCR
reactions were performed with the ABI 7700 Sequence Detection System
using the TaqMan chemistry and the primers and the bifunctional probes
for sCP and r18S RNA described previously (20).
Radioimmunoassays. The assays for rat BNP (19), rat NT-pro-ANP (39), sCP (35), and NT-pro-sCP (38) were performed as described previously. For the direct NT-pro-sCP assay, salmon plasma samples were diluted 1:50. The dilution was 1:2.5 for rat plasma samples in the direct NT-pro-ANP assay. The salmon and rat plasma samples and salmon urine samples were extracted with SepPak C18 cartridges (Waters, Milford, MA) before the sCP and BNP radioimmunoassays. Aliquots of the homogenates obtained during RNA extraction (see Determination of sCP mRNA by quantitative RT-PCR) were used to measure ir-sCP in the tissue samples. No more than 1 µl of the guanidine isothiocyanate extract was used directly in the radioimmunoassay because of the interference caused by the strongly chaotropic buffer. The urine samples were extracted as described for plasma samples for use in the NT-pro-sCP radioimmunoassay.
Statistical analyses. Results are expressed as means ± SE unless otherwise indicated. An unpaired Student's t-test was used for comparison between treated and control groups. The results from repeated measures were analyzed using one-way analysis of variance followed by the Newman-Keuls test. The level P < 0.05 was considered statistically significant.
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RESULTS |
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To find out the acute effects of sCP, a bolus injection (60 pmol/kg) was given into the dorsal aorta of chronically cannulated salmon, and a constant infusion of sCP (660 pmol · kg1 · min
1 for 150 min) was given intravenously to conscious Sprague-Dawley rats. We first
determined the circulating levels of sCP. The bolus injection elevated
the salmon plasma level of ir-sCP from 125 ± 8 to 285 ± 21 pmol/l within 30 min (P < 0.001, n = 11; Fig. 1). Plasma ir-sCP returned to a
level indistinguishable from the control level by 60 min after the
injection. In control fish, the circulating ir-sCP concentration did
not change significantly during the study period from the basal level
of 126 ± 4 pmol/l (n = 9). Circulating NT-pro-sCP
was not affected by the bolus injection of sCP (Fig. 1). As expected
from the cross-reactivity profile of our sCP antiserum
(34), ir-sCP was undetectable in rat plasma in the basal
conditions. The infusion of synthetic sCP resulted in the very high
plasma ir-sCP concentration of ~25 nmol/l (Fig.
2A). sCP infusion did not have
any significant effect on the endogenous levels of rat plasma
NT-pro-ANP or BNP (Fig. 2, B and C).
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The renal effects of sCP were studied by collection of urine and
analysis of the excreted volume and electrolyte concentrations. The
bolus injection of synthetic sCP to salmon resulted in a fourfold increase in the urine flow (P < 0.01, n = 7; Fig.
3A). The maximal increase from
0.4 ± 0.1 to 1.7 ± 0.3 ml · kg1 · h
1 was detected
during the first 30 min after the sCP bolus, and the significant
diuretic effect lasted for
90 min. The urine flow remained constant
in control fish injected with salmon Ringer solution (0.5 ml · kg
1 · h
1). The
administration of sCP did not cause any significant changes in the
urine Na+ concentration or osmolality, but the total amount
of Na+ and osmolytes excreted was increased in proportion
to the diuretic effect (Fig. 3, B and C). The
renal effects of sCP in the rat were evaluated during a constant
infusion of sCP over a 2-h period. Although there was no significant
effect on the urine flow at any of the time points, the total amount of
urine excreted during the whole study period was significantly higher
in rats with sCP infusion compared with the control rats
(P < 0.01, n = 8 for both groups; Fig.
4A). sCP did not have any
influence on rat urine Na+ concentration or osmolality. The
total electrolyte excretion was, however, increased in proportion to
the diuretic effect (Fig. 4, B and C).
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In contrast to the remarkable effects on renal function, synthetic sCP did not appear to have any significant influence on the cardiovascular parameters measured. The mean blood pressure was not affected by the sCP administration in salmon or rats. In the control group of salmon, the resting baseline dorsal aortic pressure (PDA) was 26.5 ± 2 mmHg and in the experimental group 25.5 ± 1.3 mmHg. At the end of the experimental period, PDA in the control group was 21.5 ± 1.9 mmHg (n = 5) and in the experimental group 21.4 ± 1.5 mmHg (n = 8). In the rats, the MAP varied between 119 ± 4 and 121 ± 5 mmHg during the infusion in the control and experimental groups, without any significant differences between the groups or time points. In the rats, the HR did not show any significant changes as a result of infusion of sCP, averaging 386 ± 7 beats/min in the control group and 403 ± 3 beats/min in the experimental group during the 150-min study period.
Mechanical load enhances the secretion of sCP from isolated perfused
salmon ventricle (13, 32). To study whether loading the
heart by acute volume expansion influences the plasma levels of sCP in
vivo, salmon were given an intra-arterial bolus of saline. Theoretically, the bolus of 10 ml/kg body wt saline increases the blood
volume by ~25%. The circulating level of ir-sCP increased on average
1.7-fold (from 153 ± 37 to 256 ± 79 pmol/l;
n = 7) within 10 min after the administration
of the saline bolus (P < 0.05; Fig.
5A). The sCP plasma
level had returned to the baseline value within 60 min after the volume
expansion. Plasma NT-pro-sCP concentration did not change
significantly, varying between 840 ± 99 and 1,030 ± 115 pmol/l (n = 7; Fig. 5B).
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Increased osmolality has been shown to stimulate the cardiac release of
ANP from isolated rat atria (3). Teleosts dehydrate after
transfer to sea water (8), and their plasma ionic
concentrations are higher in sea water than in fresh water
(10). To study whether the increased plasma osmolality and
ionic concentration have any effects on the circulating sCP or the
cardiac peptide and sCP mRNA levels, fresh-water salmon were acutely
transferred to sea water. Cannulated salmon were used to study the
effect of dehydration and increased plasma osmolality, resulting from
the hyperosmotic environment, on the levels of circulating and cardiac
sCP as well as those of cardiac sCP mRNA. The plasma osmolality was
significantly increased to 330 ± 1 mosmol/kgH2O
(n = 5) 2 h after the transfer to sea water
(P < 0.05) compared with that found in the fresh-water controls (313 ± 6 mosmol/kgH2O; n = 5) and continued to increase throughout the experiment (Fig.
6A). The baseline plasma
concentrations of Na+, Cl, and K+
were 159.6 ± 1.7, 132.6 ± 1.8, and 2.2 ± 0.06 mmol/l,
respectively (n = 10), in accord with previous studies
(27, 31). The plasma Na+ and Cl
concentrations were significantly elevated, starting at 24 h after
the transfer of salmon to sea water, compared with the salmon kept in
fresh water (Fig. 6, B and C; P < 0.001, n = 5). In contrast to Na+ and
Cl
ion plasma concentrations, the amount of
K+ in the plasma did not show any major changes (Fig.
6D). The plasma ir-sCP level was significantly lower on
day 3 in salmon transferred to sea water compared with those
kept in fresh water (P < 0.01, n = 5;
Fig. 7A). On the other hand,
plasma ir-NT-pro-sCP did not differ significantly between the
fresh-water and sea-water groups or among any of the time points (Fig.
7B).
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The tissue ir-sCP and sCP mRNA of fresh-water and sea-water salmon were studied to find out the influence of dehydration and increased plasma osmolality on the storage of sCP. The atrial and ventricular ir-sCP concentrations did not show any significant differences between the fresh-water and sea-water groups. The average atrial and ventricular ir-sCP concentrations in the samples from fresh water and sea water salmon were 11.3 ± 1.5 and 1.9 ± 0.1 nmol/g, respectively. The differences in atrial and ventricular sCP mRNA levels between the fresh-water and sea-water groups were not statistically significant either, although the levels tended to be lower in the latter.
In the present study, the urine flow, urine osmolality, and
Na+ concentration were examined to evaluate the amount of
renally removed volume and sodium in salmon in hypo- and hyperosmotic environments. In addition, the presence of NT-pro-sCP in urine was
studied by radioimmunoassay. The urine flow rate in cannulated adult
salmon kept in fresh water was 2.7 ± 0.6 ml · kg1 · h
1 during the
experiment. The transfer of salmon from fresh to sea water decreased
the urine flow almost immediately to very low levels (Fig.
8A; P < 0.05, n = 3). The minimum urine flow rate of 0.2 ± 0.1 ml · kg
1 · h
1, which was
maintained to the end of the experiment, was reached within 2 h
after the transfer to sea water. The baseline (fresh water) urine
osmolarity was 54 ± 10 mosmol/kgH2O and the
Na+ concentration 22.3 ± 3.4 mmol/l (Fig. 8,
B and C; n = 6). During the 3-day
experiment, the urine osmotic concentration remained practically
unchanged in salmon kept in fresh water. On the other hand, the urine
osmolality increased significantly within 24 h after the transfer
in the salmon transferred to sea water (Fig. 8B;
P < 0.05, n = 3). Because of the very
small urine volume, it was not possible to analyze the urine osmolality
at 2 h after the transfer to sea water. The urine Na+
concentration was significantly higher 24 h after the transfer to
sea water (Fig. 8C; P < 0.001, n = 3) compared with the salmon staying in fresh water.
ir-NT-pro-sCP was detectable in solid-phase extracted urine samples. In
fresh-water salmon, the levels were 9.3 ± 2 pmol/l
(n = 7). No statistically significant differences were
detected in the urine ir-NT-pro-sCP levels between the experimental groups or among the time points.
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DISCUSSION |
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We recently discovered a novel cardiac peptide hormone, sCP, a teleost representative of the family of natriuretic peptides (20, 32, 34). The natriuretic peptides have previously been found to have direct and indirect diuretic, natriuretic, and hypotensive effects (21, 22, 41). Now, we wanted to test whether the biological profile of sCP is consistent with the presumed central role of the natriuretic peptides in the regulation of teleost fluid balance and cardiovascular function.
A potent diuretic effect is one of the most characteristic features of
the natriuretic peptides (18, 21). In teleosts, homologous
heart extract and heterologous ANP have been found to cause diuresis in
toadfish (15), and eel and rat ANP has been reported to
exert diuretic effects in trout (23). In the present
study, intravenous administration of synthetic sCP increased urine
ouput in both salmon and rats. In salmon, a single dose of 60 pmol/kg
caused an impressive diuretic response. The urine flow was still
significantly elevated at 90 min after the bolus, even though the
plasma sCP concentration had returned to the baseline level within 60 min. In rats, infusion of a fairly large amount of sCP (330 pmol · kg1 · min
1) was
required to induce the diuretic effect. It does, however, show that the
structural features of sCP and those of ANP and BNP are sufficiently
conserved, so that rat NPR-A receptor can bind sCP. The relatively
prolonged diuretic action of sCP in salmon is consistent with previous
results on ANP in mammals (2, 5) showing that the cellular
response, and thereby the biological effect, outlasts the changes in
plasma peptide concentrations. On the other hand, sCP infusion did not
affect the endogenous levels of rat plasma NT-pro-ANP, despite our
previous findings that ANP can directly inhibit its own release via
NPR-A receptors in rats (17). Nor did sCP have any
significant effect on the circulating levels of BNP, as might have been
expected on the basis of earlier findings in a genetic model of ANP
deficiency that increased synthesis of BNP can compensate for the
decreased concentration of ANP (30).
In mammals, besides diuresis, ANP causes an increased excretion of
electrolytes, especially Na+ and Cl
(18, 21). In toadfish, homologous heart extracts and
mammalian ANP have been found to increase the excretion of
Na+ but not of Mg2+, Ca2+, or
K+ (15). In fresh-water trout, eel ANP has
been reported to increase the urine osmolarity (23). In
those studies, a statistically significant natriuretic response
exceeding the diuresis was, however, obtained only with very high doses
of natriuretic peptides (20 µg of mammalian ANP per toad or 10 µg/kg body wt of eel ANP per trout). In the present study, sCP caused
a significant natriuretic response in both salmon and rat, but the
response was proportionate to the diuretic effect. Accordingly, the
urine osmolality did not change significantly in response to sCP
administration. Thus our present results indicate that sCP participates
in teleost volume regulation by its potent capability to increase urine
output and sodium excretion. It should be remembered, however, that, in
contrast to the domination of the kidney in terrestrial vertebrates, both the kidneys and the gills are important sites of water and electrolyte excretion in fishes (10). The potential role
of the gill as a target organ to sCP should be carefully examined in
future studies.
Another key characteristic of the natriuretic peptides is their hypotensive action (18, 25). However, the decrease of blood pressure in normotensive experimental animals has usually been achieved only with high doses of peptides. It appears that the decrease of the blood pressure and the concomitant increase of the heart rate require doses of ANP that exceed those causing diuresis and natriuresis (2, 15, 23, 28, 40). In fresh-water eels, administration of homologous ANP with a dose of ~50 pmol/kg has been found to induce a decrease in the mean arterial pressure (24). In the present study, a bolus intra-arterial injection of sCP into salmon, causing brisk and prolonged diuresis, did not affect the mean arterial pressure. Moreover, the mean arterial pressure as well as the heart rate were unchanged in rats in which sCP was infused at doses resulting in very high plasma levels and significant diuresis. Thus, in the biological profile of sCP, the effects on the fluid and electrolyte balance appear to predominate over those on the cardiovascular system.
Teleosts are hyperosmotic to fresh water but hyposmotic to sea water. Thus they have chronic problems with the salt and water balance because of the ionic and osmotic gradients between the epithelia and the environment. Fresh-water fish face a threat of volume overload and salt loss, whereas marine teleosts tend to lose water and gain salt (4, 10). Euryhaline fish such as migrating salmon face both kinds of challenges and must therefore be able to modulate the level and type of osmoregulation. Thus they provide an ideal model for studies on osmo- and ionoregulatory mechanisms. The cardiac natriuretic peptides have been shown to be important for the regulation of the water and salt balance in both physiological and pathophysiological conditions in mammals; therefore, peptide hormones belonging to this family have been proposed to have an essential role in fish osmoregulation as well (9, 10). In mammals, volemic stimuli are potent regulators of the cardiac natriuretic peptide release (25, 36), whereas in the eel an increase in plasma osmolality rather than blood volume has been proposed to be a major regulator of eel atrial and ventricular natriuretic peptides (12). In the present study, we examined the effect of increased plasma volume and dehydration and enhanced osmolality on sCP in salmon.
In mammals, atrial stretch is the most widely recognized stimulus for ANP secretion (25, 36). The increase of plasma volume, resulting in heart wall stretch, has been shown to elevate the circulating ANP concentration in a large number of animal species (1, 12, 14, 26, 37). In the present study, acute volume overload of fresh-water salmon with saline (25% of estimated blood volume) induced a 1.7-fold increase of the circulating sCP concentration. In recent studies (13, 32), we have shown that sCP secretion from isolated salmon ventricle is rapidly and dose-dependently increased by mechanical load. It may be argued that the response to volume expansion in intact salmon was modest, considering the brisk increase of sCP secretion that loading causes in perfused salmon ventricle. However, the exact magnitude of the atrial and ventricular pressure increase caused by the volume expansion is not known. Moreover, isotonic saline injected into the dorsal aorta is prone to diffuse rapidly into the interstitial compartment. On the other hand, fresh-water salmon are facing the threat of volume overload to begin with, and the response to plasma volume expansion probably would have been more prominent in salmon with reduced circulating volume. Nevertheless, considering the biological effects of sCP (see RESULTS), we propose that the volume expansion-induced elevation of circulating sCP levels is an important homeostatic defense mechanism.
In contrast to the stimulating effect of plasma volume expansion on circulating sCP, increased plasma osmolality appears to be associated with a decreased level of sCP. Plasma ir-sCP was significantly lower in salmon kept in sea water for 3 days compared with the control fish remaining in fresh water. On the other hand, the exposure to sea water did not have any significant effect on the levels of ir-sCP or sCP mRNA in cardiac tissue, apparently the sole site of sCP production (20, 32, 35).
The plasma concentration of NT-pro-sCP remained unchanged, or even tended to increase, on transfer of salmon from fresh water to sea water. This is surprising, because NT-pro-sCP is formed by cardiomyocytes from the same precursor as sCP and released into the bloodstream in equimolar concentrations with sCP (35). The elimination pathways of the two peptides are, however, completely different. In analogy with mammalian ANP and BNP, sCP is rapidly eliminated, presumably by binding to receptors both with and without guanyl cyclase activity and by neutral endopeptidase-mediated degradation. On the other hand, NT-pro-sCP, a biologically inert peptide, probably lacks specific elimination pathways and is slowly excreted to the urine. In the present study, we demonstrated by homologous radioimmunoassay the presence of NT-pro-sCP in salmon urine. Previously NT-pro-ANP has been found in the urine in mammals (11). Thus the divergent responses of circulating levels of sCP and NT-pro-sCP may be explained by two mechanisms. Transfer to sea water could enhance the elimination of sCP, most probably by increasing the amounts of clearance receptors or by enhancing neutral peptidase activity. This would result in decreased plasma sCP levels and stable NT-pro-sCP levels without any change in the cardiac secretion rate of pro-sCP-derived peptides. In a recent study (33), we found strong evidence that modulation of the elimination rate explains the alterations of sCP plasma levels caused by varying ambient temperature. On the other hand, the presumably slower elimination, due to the greatly decreased urine output, would counteract the reduced production of NT-pro-sCP and could thus mask the decrease of plasma NT-pro-sCP levels. Further studies are required to find out their relative importance.
In mammals, the physiological function of ANP is thought to be the protection of the heart against volume and pressure overload (25). The secretion of ANP is stimulated by cardiac myocyte stretch. It causes diuresis, natriuresis, vasorelaxation, fluid shift from the intravascular to the interstitial compartment, and the inhibition of volume-conserving hormonal systems. Our present results suggest that sCP has an equivalent homeostatic role in teleosts to ANP in mammals in the regulation of volume and electrolyte balance. We demonstrated in the present study that sCP possesses potent diuretic and natriuretic activity. Its release from the heart is stimulated by increased extracellular volume in vivo, as found in the present study, and by increased mechanical load in vitro, as reported in our previous studies (13, 32). The similar physiological actions of mammalian and salmon cardiac natriuretic peptides, as well as the ability of rat natriuretic peptide receptors to recognize sCP, indicate high functional conservation of the natriuretic peptide system, spanning the large phylogenetic distance between teleosts and mammals. Thus sCP provides an excellent model for studies on the basic mechanisms of regulation of the A-type natriuretic peptides.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the staff of the Game and Fisheries Institute in Taivalkoski for providing the salmon used in this study. We are grateful to Eero Kouvalainen for help with the statistical analyses. We thank Helka Koisti, Pirjo Korpi, Tuula Lumijärvi, Tuula Taskinen, and Alpo Vanhala for expert technical assistance.
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FOOTNOTES |
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This study was supported by grants from the Academy of Finland, the Sigrid Jusélius Foundation, the Finnish Cultural Foundation, and the Farmos Research and Science Foundation.
Address for reprint requests and other correspondence: O. Vuolteenaho, Dept. of Physiology, POB 5000, FIN-90014 Univ. of Oulu, Finland (E-mail: olli.vuolteenaho{at}oulu.fi).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
April 16, 2002;10.1152/ajpendo.00321.2001
Received 24 July 2001; accepted in final form 1 April 2002.
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