Departments of 1 Physiology and 2 Pharmacology and Toxicology, Biocenter Oulu, University of Oulu, Finland, Oulu FIN-90014
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ABSTRACT |
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We recently characterized a novel heart-specific hormone from salmon (salmon cardiac peptide, sCP). We have now prepared a recombinant plasmid expressing the NH2-terminal fragment of pro-sCP (NT-pro-sCP) and used it to set up a specific RIA for the peptide. Because of the sensitivity of the assay and the high circulating levels, NT-pro-sCP can be measured from as little as 2 µl of serum. This enables repeated sampling from the same animal in different experimental setups. Mechanical load increased the release of NT-pro-sCP from isolated perfused salmon ventricle, in parallel with sCP. Bolus injection of human endothelin-1 (ET-1; 1 µg) in the dorsal aorta of salmon resulted in an extensive increase of serum NT-pro-sCP (from 0.99 ± 0.11 to 4.6 ± 1.5 nmol/l). The response was abolished by pretreatment with a specific type A ET (ETA) receptor antagonist (BQ-123) but not with a type B ET receptor antagonist (BQ-788).The NT-pro-sCP levels had a good correlation with those of sCP (r2 = 0.75). Our results demonstrate the practical usefulness of circulating NT-pro-sCP as a marker of the endocrine function of salmon heart. They also suggest that ET-1 has an important role in regulating sCP release from teleost heart by an ETA receptor-mediated mechanism.
natriuretic peptide; endothelin; recombinant protein; radioimmunoassay; study in vivo
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INTRODUCTION |
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NATRIURETIC PEPTIDES are cardiac hormones with an important role in regulating cardiovascular and fluid homeostasis (2). The release of A- and B-type natriuretic peptides (ANP and BNP) from the heart is stimulated by increased load (11, 14, 18) and by various paracrine factors, such as endothelin-1 (3, 16, 30). They reduce the cardiac load by causing natriuresis, diuresis, and relaxation of the vascular smooth muscle and by inhibiting the renin-angiotensin-aldosterone system (21). Although blood volume expansion is an important stimulus for ANP secretion in mammals, in nonmammalian eel, ANP secretion has been reported to be more sensitive to osmotic than volemic stimuli. In this teleost, ANP causes excretion of Na+, thereby promoting adaptation to seawater (9).
In mammals, ANP is stored in the secretory granules of atrial myocytes as a large-molecular-weight prohormone (proANP). It is processed to low-molecular-weight ANP during exocytosis (33), presumably by the membrane-bound serine protease corin (36). The products are the 28-amino-acid biologically active ANP and the inert 98-amino-acid NH2-terminal fragment NT-proANP (8, 31). The elimination of ANP from the circulation is very rapid, resulting in low and labile plasma concentrations. The half-life of rat NT-proANP in rat circulation has been reported to be eight times longer than that of ANP (32). Therefore, and because its plasma concentrations are much higher, the measurement of NT-proANP is preferred over that of ANP in the assessment of cardiac function, e.g., in patients with congestive heart failure (22). The same appears to apply for NT-proBNP and BNP (20).
We have recently cloned and characterized from salmon a novel peptide hormone, salmon cardiac peptide (sCP), structurally and functionally related to mammalian ANP and BNP (27). sCP appears to be produced exclusively in the heart, and it has turned out to be a very useful model for defining the general features of natriuretic peptide gene regulation and promoter function. For example, despite the very small overall sequence homology caused by the extensive phylogenetic distance, its promoter is as active as that of rat ANP in driving expression in neonatal rat atrial myocytes (17). We have previously set up a specific RIA for sCP and used it to study the distribution of the peptide hormone and to characterize its secretion pattern (13, 28). Mechanical load and endothelin-1 were found to be potent inducers of sCP release from salmon ventricle in vitro (13, 27). Because of the lower concentration and lability of sCP, an extraction step appears to be mandatory for the reliable measurement of sCP from the plasma or serum. The sample volume (at least 0.2 ml) required for the sCP RIA sets a limit to the number of serial samples that can be drawn from the same animal. To circumvent these problems, we hypothesized that the 97-amino-acid NT-pro-sCP (17) could be used to assess the release of sCP and thus the endocrine function of salmon heart analogously with the utilization of NT-proANP and NT-proBNP measurements in mammals. We report here the preparation of a recombinant plasmid expressing NT-pro-sCP, the purification of the recombinant protein, and development of a specific RIA for NT-pro-sCP suitable for the direct measurement of NT-pro-sCP in salmon serum. Mechanical load increased the release of NT-pro-sCP from isolated perfused salmon ventricles, in parallel with sCP. We also demonstrate that endothelin-1, via the endothelin type A (ETA) receptor, is a potent secretagogue for pro-sCP peptides in vivo.
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MATERIALS AND METHODS |
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Expression and purification of GST-NT-pro-sCP fusion protein.
Full-length cDNA (27) was used as the template to amplify
the sequence encoding the 97-amino-acid NT-pro-sCP
[pro-sCP-(1-97); see Ref. 17]. The PCR reaction
used the following 5' and 3' primers containing BamHI and
EcoRI linkers, respectively: 5'-GCGGATCCCATGTGTTGGGCAGACC-3' and 3'-GCGAATTCTCATCTGGTGGCCATGAGC-5'. The 3' primer included a
termination codon (TGA) right after the COOH-terminal Arg of the
peptide sequence. Thirty cycles of amplification were run with the
following denaturation, annealing, and extension temperatures: 94, 63, and 72°C, respectively. The gel-purified PCR product was cloned into
the EcoRI/BamHI site of pGEX-4T-1 (Amersham
Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK). The
authenticity of the construct structure was confirmed by sequencing
with an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster
City, CA). The GST-NT-pro-sCP fusion protein was expressed in
Escherichia coli BL21. An overnight culture of E. coli, transformed with the recombinant plasmid, was grown at
37°C until an optical density at 660 nm reached 0.6. Isopropyl
-D-thiogalactopyranoside was added to a final
concentration of 0.1 mM, and the growth was continued for 1-2 h.
The bacterial cells were harvested by centrifugation at 7,000 g for 10 min at +4°C, resuspended in PBS (50 µl/ml
culture), and sonicated. The cell lysate was cleared by centrifugation, and the supernatant was applied to a glutathione-Sepharose 4B column (Amersham Pharmacia Biotech). The column was washed three times
with a 10× column bed volume of PBS. The fusion protein was eluted
with 10 mM glutathione in 50 mM Tris · HCl, pH 8.0, and stored
in aliquots at
20°C. Products were analyzed by SDS-PAGE using 12%
polyacrylamide gel and Coomassie brilliant blue staining.
Release and purification of recombinant NT-pro-sCP. Thrombin digestion (1) was used to cleave GST from NT-pro-sCP. The thrombin digestion (1 U/100 µg) was done at room temperature for 1 h in the elution buffer (10 mM glutathione in 50 mM Tris · HCl, pH 8.0). Final purification of the peptide was accomplished by reverse-phase HPLC (RP-HPLC) using a 1 × 25-cm Vydac C4 column eluted with a linear 40-min gradient from 10 to 40% acetonitrile in aqueous trifluoroacetic acid (TFA). The elution rate was 2 ml/min, and the peptide was collected by hand on the basis of the 220-nm ultraviolet absorbance trace. The size and quantity of the recombinant NT-pro-sCP was determined by gel-filtration HPLC using a Waters ProteinPak-125 column (3.9 × 300 mm) eluted with 40% acetonitrile in aqueous 0.1% TFA with the flow rate of 1 ml/min. The effluent was monitored for absorbance at 220 nm. The column was calibrated with BSA (void volume), rat proANP-(1-126) (14 kDa), synthetic sCP-29 (3 kDa), and 125I (total volume). The identity of the recombinant protein was confirmed with 10 cycles of NH2-terminal sequence analysis using an ABI 477A gas phase sequencer (Applied Biosystems).
Preparation of antiserum to NT-pro-sCP. Recombinant GST-NT-pro-sCP fusion protein (1 mg in 1 ml of saline) was emulsified with an equal volume of Freund's complete adjuvant (Difco Laboratories, Detroit, MI) and injected at multiple sites on the back of a goat. Two booster injections with one-half the amount of antigen emulsified in incomplete adjuvant were given at monthly intervals.
Radioiodination of NT-pro-sCP.
Recombinant NT-pro-sCP was radioiodinated with the chloramine-T
technique (4) and purified by desalting in a 2-ml Sephadex G-25 M (Amersham Pharmacia Biotech) column, followed by final purification by RP-HPLC using a 4.6 × 250-mm Vydac
C18 column (Separations Group, Hesperia, CA) eluted with a
40-min 10-50% acetonitrile gradient in aqueous 0.1% TFA. The
flow rate was 1 ml/min. Fractions of 1 ml were collected and monitored
for radioactivity in a Multi-Gamma gamma counter (Wallac, Turku,
Finland). When stored at 20°C, the radioiodinated peptide was
usable for ~6 wk.
NT-pro-sCP RIA procedure. Recombinant NT-pro-sCP ranging from 5 to 1,250 pg/tube was pipetted in duplicates of 100 µl for use as standard. The serum samples were diluted 1:50 with assay buffer, and duplicates of 100 µl were used directly in NT-pro-sCP RIA. Goat anti-NT-pro-sCP serum (1:75,000 final dilution) was added (100 µl), and the tubes were incubated overnight at +4°C. Finally, 125I-labeled NT-pro-sCP (100 µl) was added, and the tubes were incubated for another 16-20 h at +4°C. The bound and free fractions were separated by precipitation with 0.5 ml of 8% polyethylene glycol 6000 containing donkey anti-goat IgG antiserum (Scantibodies Laboratory, Santee, CA) and normal goat serum (1 µl) as carrier. After centrifugation (2,000 g for 15 min at +8°C) and decantation of the supernatants, the precipitates were counted in a Wallac Clinigamma 1272 gamma counter.
Other RIAs. Serum samples were extracted with Sep-Pak C18 cartridges (Waters, Milford, MA) for use in sCP RIA. The assay procedure has been described in detail previously (28). Human endothelin-1 RIA were performed as described earlier (34).
HPLC analysis of serum and cardiac tissue samples. A salmon serum sample (300 µl) was mixed with 200 µl 0.25% TFA in acetonitrile. The precipitate was removed by centrifugation at 10,000 g for 15 min, and the supernatant was passed through a 0.45-µm Millipore filter. The clear sample was applied through a Rheodyne loop injector to a Waters Proteinpak 125 HPLC gel filtration column (3.9 × 300 mm), eluted with 40% acetonitrile in aqueous 0.1% TFA at the flow rate of 1 ml/min. Fractions of 0.5 ml were collected and dried in a SpeedVac concentrator (Savant Instruments, Hicksville, NY). The dried residues were dissolved in 0.5 ml of assay buffer for use in NT-pro-sCP RIA. The tissue samples were pulverized in liquid N2 and homogenized in 4.5 vol of boiling H2O and 4.5 vol of 2 M acetic acid/40 mM HCl. The homogenates were centrifuged (15,000 g, 30 min at 4°C), and the supernatants were lyophilized and dissolved in 300 µl of 40% acetonitrile in aqueous TFA for use in chromatography.
Effect of mechanical load on the secretion of immunoreactive
NT-pro-sCP and sCP from isolated perfused salmon ventricle.
Mature salmon of both sexes weighing 703 ± 31.2 g
(n = 17) were held in 350-liter tanks with circulated
and aerated water at 8°C during the autumn and at 3°C during the
winter. The photoperiod mimicked the natural conditions at the time of
the experiments, with a 12:12-h light-dark cycle during autumn and a
9:15-h light-dark cycle during the winter. The salmon ventricle
perfusion system has been described in detail previously
(13). Briefly, salmon were killed, and the beating
ventricle was removed and mounted in an organ bath. The ventricles,
weighing 0.829 ± 0.045 g (n = 17), were paced by
a focal stimulus (1 ms, 11/min) and perfused with a flow rate of 3.8 ml/min. The perfusate was collected in 1-min fractions using a fraction
collector. The ventricles were first perfused without mechanical load
(basal level) for 5 min. Then mechanical load was applied by raising
the outflow height for 20 min, after which the load was returned to the
basal level for 5 min. Loads of 4, 8, and 13 cm were tested. The
fractions were stored at 20°C for use in the RIAs. The ratio of
immunoreactive sCP or NT-pro-sCP in each fraction to the mean level in
fractions 1-5 (basal) was calculated to obtain a
numerical estimate of the effect of the load. For statistical analysis,
the mean NT-pro-sCP level between 16 and 20 min (the peak load
response, see Ref. 13) was compared between the loads of
0, 4, 8, and 13 cmH2O.
Effect of human endothelin-1 on the release of pro-sCP-derived peptides in vivo. The experiments were approved by the Animal Care and Use Committee of the University of Oulu. Mature salmon (Salmo salar) of either sex, weighing 576 ± 47 g (mean ± SE, n = 10), were purchased from the Finnish Game and Fisheries Institute at Taivalkoski. They were kept in a 1,500-liter tank circulated with aerated tap water at 6°C under a light-dark cycle appropriate for the season (15 h light, 9 h dark). The fish were kept in the tank for up to 7 days, during which time they were not fed.
The salmon were anesthetized before the surgery by tricaine (ethyl m-aminobenzoate methanesulfonate, MS-222; 100 mg/l; Sigma Chemical) buffered with sodium carbonate. The dorsal aorta was cannulated through the roof of the buccal cavity using polyethylene tubing (25). The cannula was filled with heparinized saline (100 USP units/ml heparin in 9.0 g/l NaCl). The fish were then revived and placed in plastic cylinders (length 72 cm, diameter 20 cm) immersed in the tank. The experiments were conducted 48 h after the surgery. Basal blood samples of 0.8 ml were drawn through the cannula before the injection of endothelin-1 or vehicle. A bolus dose of 1 µg (1.7 µg/kg body wt) of human endothelin-1 (Peninsula Laboratories Europe, Merseyside, UK) was injected in the dorsal aorta followed by a 0.1-ml heparinized saline flush. The injection lasted 5 s. Blood samples of 0.3 ml (for the NT-pro-sCP and endothelin-1 assays) were drawn at 7, 12, and 30 min after the administration of endothelin-1, and samples of 0.8 ml (for the sCP assay) were drawn at 20 and 40 min. Each blood sample was replaced with the same volume of saline, and the cannula was filled with heparinized saline. Control experiments were performed identically, except that vehicle (saline) was administered instead of endothelin-1. The blood samples were centrifuged at 2,000 g for 15 min. The serum samples were stored atEffect of endothelin-1 receptor antagonists in vivo. Mature salmon of either sex, weighing 448 ± 19 g (mean ± SE, n = 17), were used and kept under the conditions described above, with the exception that the light-dark cycle was 24 h light and 0 h dark, appropriate to the season when these experiments were performed. A 25-fold molar excess (relative to endothelin-1) of either BQ-123 (a specific ETA receptor antagonist; Sigma) or BQ-788 [endothelin type B (ETB) antagonist; Sigma] was administered as a bolus injection to the dorsal aorta of six fish 5 min (15) before the administration of human endothelin-1 (1 µg, 2.2 µg/kg body wt) via the same route. Five fish served as vehicle controls receiving only endothelin-1.
Statistical analysis. Results are expressed as means ± SE. ANOVA for repeated measures followed by the Newman-Keul's post hoc test was used for statistical analysis of the results. Correlation coefficients were calculated by linear regression analysis. A value of P < 0.05 was considered significant.
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RESULTS |
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Preparation and purification of recombinant GST-NT-pro-sCP fusion
protein and NT-pro-sCP.
A recombinant expression plasmid was constructed, containing in tandem
the sequences encoding GST and pro-sCP-(1-97) (NT-pro-sCP) followed by a stop codon. Expression of the plasmid in E. coli yielded 2.4 mg of soluble GST-NT-pro-sCP fusion protein/l
bacterial culture. Figure 1A
shows SDS-PAGE analysis of GST-NT-pro-sCP, purified by
glutathione-Sepharose affinity chromatography. The fusion protein was
used as such as an immunogen to raise the anti-NT-pro-sCP antiserum
(see below). GST was removed from the fusion protein by thrombin
digestion, and NT-pro-sCP was purified to homogeneity by RP-HPLC (Fig.
1B). The yield of pure recombinant NT-pro-sCP was 0.40 mg/l
culture. Because of the method of production, the final recombinant
NT-pro-sCP product contains an extra Gly-Ser dipeptide sequence at the
NH2 terminus, originating from GST. The apparent molecular
weight (10,000-15,000), as assessed by gel filtration
HPLC (data not shown), was consistent with the expected molecular
weight (11,026) of NT-pro-sCP. The authenticity of
recombinant NT-pro-sCP was further confirmed by 10 cycles of NH2-terminal sequence analysis. It yielded the sequence
Gly-Ser-His-Val-Leu-Gly-Arg-Pro-Tyr-Pro, which is exactly as expected
from sCP cDNA, with the exception of the GST-derived Gly-Ser dipeptide
at the extreme NH2 terminus.
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Development and characterization of NT-pro-sCP RIA.
The goat anti-NT-pro-sCP antiserum was specific for the NT-pro-sCP
sequence and did not cross-react (molar cross-reaction <0.1%) with
synthetic sCP, human ANP, human BNP, human C-type natriuretic peptide,
eel ANP, rainbow trout ventricular natriuretic peptide (VNP) or human
endothelin-1, or with recombinant human NT-proANP or human
NT-pro-BNP. In the RIA, the least-detectable dose varied from
0.500 to 1.400 fmol/tube, which corresponds to circulating
concentrations of 0.250-0.700 nmol/l when serum was assayed
according to the incubation protocol described in
MATERIALS AND METHODS. The high
sensitivity resulted in the following two advantages: 1)
serum NT-pro-sCP could be assayed without extraction, and 2)
only a very small amount of serum (2 × 2 µl) was required for
the assay. Serial dilutions of salmon serum diluted in parallel with
the NT-pro-sCP standard (Fig. 2), and
thus the assay fulfills the basic requirement for use in quantification
of NT-pro-sCP. The recovery of NT-pro-sCP added to salmon serum
(99.8 ± 3.2%) was linear in the concentration range 1-11
nmol/l (y = 1.06x 0.38, r2 = 0.999). The intra- and interassay
coefficients of variation were 7 (n = 10) and 14%
(n = 7), respectively.
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Effect of bolus injection of human endothelin-1 on circulating NT-pro-sCP and sCP. Our previous studies have shown that human endothelin-1 increases the release of sCP in the isolated perfused salmon ventricle preparation (13). Because of the potentially important role of endothelin-1 in regulating the natriuretic peptide system, as found earlier in mammals (12, 35), we were interested in finding out the effects of endothelin-1 on the sCP system in vivo. This served also as a test of the usefulness of our novel NT-pro-sCP assay for monitoring the endocrine function of salmon heart. sCP was assayed simultaneously to serve as a reference. However, because of the need for a larger sample volume and extraction, sCP could only be assayed in a subset of the samples. Human endothelin-1 was assayed from the salmon serum samples to determine the actual input of the experiments.
As expected, in the control experiments, where only vehicle was injected, serum endothelin-1 levels were all below the detection limit of the human endothelin-1 assay (0.165 nmol/l) at 1:50 dilution of the samples (Fig. 5A). Administration of the endothelin-1 bolus (1 µg) caused a rapid increase in the serum levels of human endothelin-1, with the peak (1.16 ± 0.244 nmol/l) at 7 min after the injection. Thereafter, the levels decreased slowly to 0.402 ± 0.108 nmol/l at 40 min after the injection. Serum levels of immunoreactive NT-pro-sCP remained relatively stable during the control experiments (Fig. 5B), varying between 0.552 ± 0.038 and 0.506 ± 0.040 nmol/l (n = 3). Endothelin-1 administration caused a relatively slow but large increase of serum NT-pro-sCP levels from 0.987 ± 0.112 nmol/l at 0 min to 4.61 ± 1.49 nmol/l at the end of the experiment (n = 7). A parallel increase of serum sCP was observed in response to the endothelin-1 bolus (Fig. 5C), with the levels increasing from 0.063 ± 0.009 to 0.949 ± 0.459 nmol/l (n = 7). In control experiments (n = 3), serum levels of immunoreactive sCP remained stable throughout the experiment, varying from 0.042 ± 0.001 to 0.053 ± 0.005 nmol/l.
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DISCUSSION |
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In this study, we prepared a recombinant plasmid expressing NT-pro-sCP as a fusion protein with GST and purified the fusion protein and NT-pro-sCP. We used the recombinant protein to set up a specific RIA suitable for measuring circulating NT-pro-sCP levels from minute serum samples. We demonstrated with the RIA that serum NT-pro-sCP levels can be used to estimate the levels of the biologically active peptide sCP both in vitro and in vivo. Our results showed that mechanical load dose dependently increases the release of NT-pro-sCP from isolated perfused salmon ventricle, in parallel with sCP. We also showed in salmon that endothelin-1 is a potent secretagogue of the sCP system in vivo and that the effect of endothelin-1 is mediated by ETA-like receptors. Our results suggest that endothelin-1 has a paracrine role in regulating the endocrine activity of teleost heart.
When starting the present project, we had made available an RIA for sCP (28) usable for both in vitro and in vivo studies. However, because the clearance of circulating sCP is rapid (28) and may vary considerably in different environmental conditions, the secretion rate of pro-sCP-derived peptides is difficult to assess from the serum levels of sCP. In addition, the lability of sCP necessitates an extraction step, resulting in a relatively large sample volume. We wanted to study the physiology of the sCP system also in in vivo models where repeated serum sampling was required. Therefore, the first objective of the present study was to set up, with the assays of the NH2-terminal fragments of mammalian ANP (26) and BNP (7) as models, an RIA for NT-pro-sCP that could be used with minute amounts of unextracted serum.
We developed a specific RIA for the measurement of circulating levels of NT-pro-sCP, by utilizing the GST-NT-pro-sCP fusion protein for immunization and the purified NT-pro-sCP for radioiodination and standardization. The sensitivity of the assay, combined with the high circulating levels, made it possible to measure NT-pro-sCP levels in very small sample volumes (2 µl serum). In actual experiments, this means that a practically unlimited number of serial samples can be drawn from the same animal without any significant volume loss. Parallel measurements, both in basal conditions and when stimulated by endothelin-1, showed a good correlation between serum NT-pro-sCP and sCP, demonstrating that circulating NT-pro-sCP can be used as an indicator of serum sCP activity. Moreover, in the isolated perfused salmon ventricle preparation, mechanical load caused a parallel increase of both sCP and NT-pro-sCP. Thus, in this sense, NT-pro-sCP resembles mammalian NT-proANP, widely used as a useful and practical marker of plasma ANP activity because of its stability in the circulation (e.g., see Refs. 5, 10, and 23).
Our gel filtration studies showed that immunoreactive NT-pro-sCP in serum has a molecular weight in between those of the biologically active sCP [pro-sCP-(98-126), relative molecular weight (Mr) 3,004] and the full-length pro-sCP-(1-26) (Mr 14,030), a result consistent with the sequence of NT-pro-sCP being pro-sCP-(1-97). We have previously found that the form stored in cardiac myocytes is the intact pro-sCP (17, 13). Therefore, it appears that salmon ventricle produces sCP in a manner resembling the production of ANP in mammalian atrium. ProANP is processed during the exocytosis of the secretory granules (33), evidently catalyzed by the membrane-bound serine protease corin (36). Hence, as is the case with ANP and NT-proANP, NT-pro-sCP appears to be cosecreted from salmon cardiomyocytes in equimolar quantities with sCP. As a result, the circulating levels of NT-pro-sCP can be used to estimate the rate of secretion of sCP from salmon heart. The fact that we found the basal serum levels of NT-pro-sCP to be more than five times higher than those of sCP, on a molar basis, suggests that the elimination of NT-pro-sCP differs markedly from that of sCP. It appears that the fates of circulating sCP and NT-pro-sCP resemble those of mammalian ANP and NT-proANP. ANP is removed quickly from the circulation by neutral endopeptidase-mediated proteolysis and by binding to the ANP and C-type natriuretic peptide receptors (21), whereas NT-proANP appears to be eliminated only slowly via the kidneys (32).
We had previously found that endothelin-1 increases significantly the release of sCP from isolated perfused salmon ventricle (13). In the present study, we wanted to test the efficacy of endothelin-1 in vivo. We monitored the secretion of pro-sCP-derived peptides with the measurement of NT-pro-sCP in unextracted serum samples. We also determined, after solid-phase extraction, the level of immunoreactive sCP in a subset of serum samples for comparative purposes. According to our present results, human endothelin-1 is also a powerful secretagogue of pro-sCP peptides in vivo. Because we have previously found that endothelin stimulates sCP release from salmon ventricle in vitro (13), the mechanism of action appears to be at least partly direct. The potent hemodynamic effects of endothelin-1, such as increase in blood pressure (37, 35), could also be important in vivo. In previous studies with the teleost rainbow trout, endothelin-1 has been found to cause several short-term oscillations in blood pressure followed by a sustained hypertensive period (19, 15, 6). In mammals, it has been found that locally induced vasoactive agents, such as endothelin-1, mediate, in a paracrine fashion, part of the stretch-induced ANP secretion (29). Combined inhibition of endothelin and ANG II receptors has been reported to almost completely abolish the volume load-induced ANP release in vivo in rats (16).
Previous studies in mammals have shown that endothelin-1 stimulates ANP and BNP secretion by an ETA-mediated mechanism (30). In the present study, a specific ETA antagonist, but not an ETB antagonist, was capable of inhibiting the endothelin-1-induced activation of the sCP system in vivo. Neither of the antagonists had any significant effect on the basal levels of the sCP peptides. The results demonstrate that endothelin-1 has similar potent effects and a mechanism of action on the cardiac natriuretic peptides in such phylogenetically distant species as rat and salmon. This indicates a fundamental role for endothelin-1 in regulating the endocrine function of the heart.
Thus endothelin-1 appears to be a regulator of cardiac endocrine function, conserved through a very long period of evolution. This appears to apply for the overall regulation of the natriuretic peptides as well. We recently found that, despite the very low general homology between the nucleotide sequences, the promoter of sCP is as effective as mammalian ANP promoters in driving expression in cultured rat cardiomyocytes (17). The promoter elements required for activation by endothelin-1 are only partially known. Therefore, analysis of the sCP system could help in defining the phylogenetically conserved function of the natriuretic peptide promoters and specifically the regulation of the mammalian ANP and BNP promoters by mechanical and humoral stimuli.
To determine the actual input of our in vivo experiments, we measured, by a specific RIA, the level of human endothelin-1 in salmon circulation after injection of the peptide in the dorsal aorta. According to the results, endothelin-1 has a very prolonged half-life in teleost circulation, after the initial distribution phase. After 40 min from the bolus injection of 1 µg of human endothelin-1, the peptide levels had steadied to an apparent plateau of 0.402 ± 0.108 nmol/l. As a comparison, the basal levels in human circulation are ~0.003-0.004 nmol/l (34). Therefore, caution is warranted when planning experiments in teleost fish with the use of constant infusion of endothelin because of the apparent risk of accumulation of the peptide in the circulation. On the basis of our present findings, the actual circulating endothelin levels should be measured for proper interpretation of the results, especially if a steady-state system is aimed at.
In conclusion, we prepared a recombinant protein corresponding to the NH2-terminal sequence of pro-sCP-(1-97). We developed and characterized, using the recombinant protein, a sensitive and specific RIA that is suitable for measuring NT-pro-sCP concentrations in microliter samples of unextracted salmon serum. The assay enables monitoring of the endocrine function of salmon heart in vivo, with practically unlimited serial sampling. This decreases the experimental variation and should reduce the number of animals consumed. It should also make feasible experiments that were not possible with previous methods, for example, studies on the short-term dynamics of cardiac hormone secretion. In the present study, we used the method to find out the effects of endothelin-1 on salmon cardiac endocrine function in vivo. Endothelin-1 caused a marked and long-lasting elevation in circulating NT-pro-sCP and sCP. Further experiments demonstrated that this effect is mediated through ETA receptors. Although indirect hemodynamic effects most likely contribute to the elevation of circulating sCP levels, the present results, together with our previous in vitro findings, indicate that endothelin-1 is an important natriuretic peptide secretagogue in teleost fish. This, and the fact that the mechanism of action of endothelin-1 appears to be the same, indicates that the regulation of the cardiac endocrine function is conserved over the great phylogenetic distance between fish and mammals. In future studies, it would be interesting to find out whether mechanical load-induced secretion of pro-sCP peptides is mediated or modulated by paracrine release of endothelin-1, as is the case with mammalian ANP.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the expert technical assistance of Helka Koisti, Tuula Lumijärvi, and Alpo Vanhala. We thank Eero Kouvalainen for help with statistical methods. We are also grateful to the personnel of the Game and Fisheries Research Institute at Taivalkoski.
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FOOTNOTES |
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This study was supported by grants from the Academy of Finland, the Sigrid Jusélius Foundation, and the Finnish Foundation of Cardiovascular Research.
Address for reprint requests and other correspondence: O. Vuolteenaho, Dept. of Physiology, Univ. of Oulu, 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.
10.1152/ajpendo.00102.2001
Received 7 March 2001; accepted in final form 16 October 2001.
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