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Am J Physiol Regul Integr Comp Physiol 280: R1727-R1735, 2001;
0363-6119/01 $5.00
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Vol. 280, Issue 6, R1727-R1735, June 2001

Enhanced expression and release of C-type natriuretic peptide in freshwater eels

Yoshio Takei1, Koji Inoue1, Kenji Ando2, Tsuyoshi Ihara3, Takeshi Katafuchi3, Masahide Kashiwagi3, and Shigehisa Hirose3

1 Ocean Research Institute, the University of Tokyo, Nakano-ku, Tokyo 164 - 8639; 2 Nippon Paint Company Limited, Shinagawa-ku, Tokyo 140 - 8675; and 3 Department of Biological Sciences, Tokyo Institute of Technology, Midori-ku, Yokohama 226 - 8501, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

C-type natriuretic peptide (CNP) is recognized as a paracrine factor acting locally in the brain and periphery. To assess the role of CNP in teleost fish, a cDNA encoding a CNP precursor was initially cloned from the eel brain. CNP message subsequently detected by ribonuclease protection assay, using the cDNA as probe, was most abundant in the brain followed by liver, gut, gills, and heart. Expression was generally higher in freshwater (FW) than in seawater (SW) eels, but not in the brain. Plasma CNP concentration measured by a newly developed homologous radioimmunoassay for eel CNP was higher in FW than in SW eels. The CNP concentration was also higher in the heart of FW eels but not in the brain. These results show that CNP is abundantly synthesized in peripheral tissues of FW eels and secreted constitutively into the circulation. Therefore, CNP is a circulating hormone as well as a paracrine factor in eels. Together with our previous demonstration that CNP-specific receptor expression is enhanced in FW eels, it appears that CNP is a hormone important for FW adaptation. Because atrial NP (ANP) promotes SW adaptation in eels, CNP and ANP, despite high sequence identity, appear to have opposite effects on environmental adaptation of the euryhaline fish.

cDNA cloning of eel CNP; natriuretic peptide family; tissue distribution of CNP mRNA; radioimmunoassay of eel CNP; osmoregulation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

C-TYPE NATRIURETIC PEPTIDE (CNP) belongs to a family of peptides that shares a unique 17-amino acid ring structure within the molecule. Among the family members, atrial NP (ANP) and brain NP (BNP) are circulating hormones secreted from the heart and play important roles in cardiovascular and body fluid homeostasis (2, 33). By contrast, CNP is principally an autocrine/paracrine factor whose function has not yet been fully elucidated. CNP was first identified in 1990 in the brain of mammals and fish as a third member of the NP family (22, 30, 36) and was initially thought to be a neuropeptide. Since then, its presence and gene expression have been demonstrated in a number of peripheral tissues including the adrenal medulla, intestine, kidney, vascular endothelium, and chondrocytes (19). Because CNP concentration in plasma is extremely low in mammals (4, 7), CNP may function as a paracrine factor in these peripheral organs.

Comparative biochemical studies of the NP family members have been carried out extensively in a variety of vertebrate species, and their structures have been determined. Comparison of the amino acid sequences revealed that, of the NP family, CNP is most highly conserved among the members, suggesting its important function throughout vertebrates (5, 33). In cartilaginous fishes that diverged earlier from the main trunk of vertebrate evolution than did bony fishes, only CNP has been identified in the heart and brain (25, 31, 32). Thus CNP may be an ancestral molecule of the NP family. Unlike ANP and BNP genes that are localized in tandem on the same chromosome, the CNP gene is on a different chromosome (39). It is likely, therefore, that the ANP (or BNP) gene was duplicated from the CNP gene after the divergence of cartilaginous fishes, and the duplication of ANP/BNP gene occurred later in vertebrate phylogeny. For these reasons, CNP may be a good material in which to pursue the original function of the NP family, but its function is the least understood among the family members.

Except in mammals, the components of the NP system including the receptors have been well characterized only in a teleostean species, the eel. Currently, three ligands [ANP, ventricular NP (VNP), and CNP] and four NP receptors (NPR-A, NPR-B, NPR-C, and NPR-D) have been identified in this species (33). With respect to the ligands, ANP, CNP, and VNP were isolated from the atrium, brain, and ventricle, respectively, and cDNA clones were obtained for ANP and VNP (37, 38). However, cDNA for eel CNP has not yet been cloned. The biological action of ANP has been well characterized in eels; it has been revealed that ANP is important for seawater (SW) adaptation (33). However, an osmoregulatory role of CNP has not yet been examined in the eel except that the expression of NPR-B, a specific receptor for CNP (16), is augmented in the osmoregulatory organs of freshwater (FW) eels (14).

In the present study, we initially isolated a cDNA clone encoding eel CNP from the eel brain and characterized it. Using the cDNA as a probe, we examined the expression of CNP mRNA in various tissues of FW and SW eels by RNase protection assay and RT-PCR. Based on the sequence information, mature eel CNP-22 was synthesized and a homologous radioimmunoassay for eel CNP was established. Using the radioimmunoassay, we measured CNP concentrations in plasma and in major CNP-producing tissues in both FW and SW eels. The results showed that CNP synthesis is strongly enhanced in FW eels and that a large amount of CNP is circulating in the blood of FW eels, suggesting that CNP may be involved in adaptation of the euryhaline fish to FW environments.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

Cultured, immature eels, Anguilla japonica, were purchased from a local dealer. They were maintained without feeding in a 1-ton FW tank or a half-ton SW tank for at least 2 wk before use. For the experiments comparing FW and SW eels, eels were purchased at the same time and acclimated for the same period to either medium. Water in the tank was continuously circulated, aerated, and regulated at 18°C.

cDNA Cloning of Eel CNP

Materials. Restriction enzymes, DNA ligation kit, and random primer DNA labeling kit were obtained from Takara; the plasmid vector pBluescript II and RNA transcription kit from Stratagene; nitrocellulose filters (BA 85, 0.45 µm) from Schleicher and Schuell; Magnagraph from Micron Separations; RNA size markers and RNase A from Boehringer Mannheim; RNase T1 from Gibco; 32P-labeled nucleotides from New England Nuclear Research Division; X-Omat AR X-ray film from Kodak; and 5'-end labeling kit from Amersham.

Screening. A previously constructed eel brain cDNA library (13) was used for screening of eel CNP cDNA. The probe for screening was 50-mer oligonucleotide, GGCTGGAACCGAGGCTGCTTTGGCCTCAAGCTGGATCGGATTGGCTCCCT, synthesized based on the amino acid sequence of eel CNP (36); the codons were chosen based on the codon usage frequency. Approximately 7 × 105 plaques from the eel brain cDNA library were plated at a density of 30,000 plaques/15-cm plate. Duplicate plaque lifts were prepared with nitrocellulose filters. The filters were prehybridized for 2 h and then hybridized for 16 h at 37°C in the presence of probes labeled with 32P by 5'-end labeling kit. Prehybridization and hybridization buffer contained 6× NaCl/Cit (0.15 M NaCl and 15 mM sodium citrate, pH 7.0), 5× Denhardt's solution (0.1% each of bovine serum albumin, polyvinylpyrrolidone, and Ficoll), and 0.1% SDS. After hybridization, filters were washed twice with 2× NaCl/Cit containing 0.05% SDS at room temperature for 5 min, once with 2× NaCl/Cit at 37°C for 1 h, and then with 1× NaCl/Cit at 37°C for 1 h. After autoradiography, double positive plaques were rescreened until a cDNA clone was isolated. DNA sequences were determined by the dideoxynucleotide chain-termination method (26) using a DNA sequencer (4000LS Long ReadIRTM).

Northern blot analysis. Total RNA (20 µg) obtained from eel brain was separated by electrophoresis with 1% agarose gel containing 2.2 M formamide, 20 mM MOPS, 8 mM sodium acetate, and 1 mM EDTA, pH 7.0. After electrophoresis, the separated RNA was transferred to a nylon membrane and chemically cross-linked with ultraviolet light. The membrane was prehybridized for 3 h at 42°C in 50% formamide, 5× SSPE (0.15 M NaCl, 15 mM NaH2PO4, and 1 mM EDTA, pH 7.0), 5× Denhardt's solution, 100 µg/ml denatured herring sperm DNA, and 1% SDS and then hybridized with 1× 106 cpm/ml of 5'-end 32P-labeled probe for 16 h. The membrane was washed twice with 2× NaCl/Cit containing 0.05% SDS at room temperature for 5 min and once with 1× NaCl/Cit containing 0.1% SDS at 60°C for 1 h. The membrane was then exposed to an imaging plate for 10 h, which was observed in a FLA 2000 imaging analyzer (Fuji Film).

Detection of CNP mRNA Expression

RNase protection analysis. After anesthesia in 0.1% (wt/vol) tricaine methanesulphonate, the brain, gill, cardiac atrium, ventricle, liver, esophagus, stomach, intestine, kidney, and head kidney including interrenal tissues were immediately dissected out from three FW and three SW eels. Total RNA was extracted from pooled tissues by the acid guanidinium thiocyanate/phenol/chloroform method (3). For the synthesis of antisense cRNA probe, template DNA was prepared by subcloning the 169-bp SalI-HincII fragment of the eel CNP cDNA into pBluescript II and linearizing it with SalI. Antisense cRNA probe was transcribed in vitro using T3 RNA polymerase and alpha -[32P]UTP. After the transcription reaction, the template DNA was removed by digestion with RNase-free DNase I. The radioactive cRNA probe (1 × 105 cpm) was annealed with 10 µg of RNA for 12 h at 45°C in 80% formamide, 40 mM PIPES, 5 mM EDTA, and 400 mM NaCl, pH 6.4. Nonannealing nucleotides were then digested with RNases A and T1 at final concentrations of 40 and 2 µg, respectively, in a buffer containing 10 mM Tris · HCl, 300 mM NaCl, and 5 mM EDTA, pH 7.4, at 30°C for 1 h. The protected fragment was purified by phenol/chloroform extraction, precipitated with ethanol, and electrophoresed in a 5% polyacrylamide gel containing 7 M urea. The gels were dried and exposed to X-Omat AR films with an intensifying screen at -80°C.

Analysis by RT-PCR. Total RNA was extracted using Isogen (Nippon Gene) from various tissues of three FW and three SW eels. The cartilaginous tissue (gill arch) and arterial tissue (dorsal aorta) were added, because local synthesis and action of CNP have been demonstrated in these tissues of mammals (6, 9). RT was performed using Superscript First-Strand Synthesis System for RT-PCR (Life Technologies, Takara). The reaction mixture containing 50 U of Superscript II reverse transcriptase, 20 mM Tris · HCl (pH 8.4), 50 mM KCl, 5 mM MgCl2, 10 mM dithiothreitol, 0.5 mM dNTPs, 0.5 µg of oligo(dT)12-18 primer, and 5 µg RNA was incubated at 42°C for 50 min and then at 70°C for 15 min. For detection of CNP gene transcripts, 25 µl of Ex Taq buffer containing 1 µl reverse-transcribed mixture (Takara), 0.2 mM dNTPs, 0.4 µM sense and antisense primers, and 0.625 U of Ex Taq (Takara) was prepared on ice, and 28 cycles of thermal sequential steps, 94°C for 30 s, 57°C for 30 s, and 72°C for 45 s, were started immediately. Primers used were eel CNP-(82-101), 5'-GAGCTAGACCAGAGACAACA-3', and eel CNP-(795-813), 5'-GGACCTTCACACTGCACTT-3'. Furthermore, to determine the source of CNP secreted into the circulation in FW eel, we collected the heart (atrium plus ventricle), anterior intestine (including esophagus and stomach), and gills from six FW eels (body weight: 185.0 ± 3.1 g) and six SW eels (body weight: 182.3 ± 7.1 g) to compare the CNP expression, because these tissues expressed more CNP message in FW than in SW fish in the above experiment in spite of high variability of data. In this experiment, cycles for thermal sequential steps were increased to 30 to enhance the sensitivity of detection. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene transcripts were used as an internal standard. GAPDH is expressed ubiquitously in most tissues and used as an internal control for specific gene expression (41). A partial cDNA sequence for eel GAPDH (deposited to DNA Data Bank of Japan: AB049458) was obtained by RT-PCR using consensus primers designed from corresponding sequences of other fish GAPDH. From the obtained eel sequence, a sense primer, 5'-CATTGGTCGCCTGGTCCTCA-3', and an antisense primer, 5'-ATGCCCGTCAGCTTCCCGTT-3', were designed. RT-PCR for detection of GAPDH gene transcripts was carried out by the same protocol as for CNP except for annealing temperature (62°C). PCR products were electrophoresed onto 1.5% agarose gels and stained with ethidium bromide; the signal intensity was quantified on a Fuji FLA-2000 imaging analyzer.

Radioimmunoassay for Eel CNP

Antibody production. Synthetic eel CNP-22 was conjugated with bovine thyroglobulin and injected alone or in combination with an equal volume of Freund's complete adjuvant (Difco Laboratories) into 10 Japanese White rabbits. To raise specific antisera that do not cross-react with eel ANP and VNP that have >60% sequence identity to CNP, we initially injected the antigen at low doses (30 µg) with the complete adjuvant and then left the animals uninjected for 1 mo to select the clone(s) in vivo; then the titer of the selected clone(s) was increased gradually by increasing the dose of antigen over 8 mo as described previously (34). However, only two of 10 rabbits raised antisera that are usable for radioimmunoassay in terms of specificity, affinity, and titer.

Iodination of ligand. Eel CNP-22 with a tyrosine residue added to the NH2 terminus (eel [Tyr0]CNP) was synthesized and iodinated by the lactoperoxidase method as described previously (34). Eel 125I-labeled [Tyr0]CNP was purified by reverse-phase HPLC in an ODS-120T column with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid from 15 to 60% for 60 min. This HPLC condition can separate monoiodinated CNP from noniodinated CNP as well as from di-iodinated CNP. The purified monoiodinated CNP was dissolved in assay buffer (10 nM phosphate buffer, pH 7.4, containing 140 mM NaCl, 10 mM 2K-EDTA, 20 mM glycine, 10 mM epsilon -aminocapronic acid, 1 mM sodium azide, and 0.1% Triton X-100) containing 5% BSA (RIA grade, Sigma) as a quencher and stored in aliquots at -20°C until use. The labeled ligands were used within 2 mo of iodination. The specific activity was calculated as 2,160 Ci/mmol on the day of iodination.

Procedures for radioimmunoassay. The radioimmunoassay was performed by a double antibody method as reported previously (34). Briefly, samples were mixed with anti-CNP serum (#1, 1:5,000 dilution) and preincubated for 20 h at 4°C. Eel 125I-[Tyr0]CNP (5,000 cpm) was then added, incubated further for 24 h at 4°C, and bound radioligands were separated from unbound forms after the addition of goat anti-rabbit IgG serum and polyethylene glycol 8000 (Sigma). The amount of CNP in the sample was read on the standard curve for eel CNP-22 after fitting to a logistic curve. The nonspecific binding of radioligand to the tube was 1.4 ± 0.1% (n = 3), and the radioligand bound to the antibody without cold ligands was 17.3 ± 1.4% (n = 3). The recovery of eel CNP added to the plasma was 93.5 ± 2.9% (n = 3).

Preparation of plasma and tissue samples. Six FW and eight SW eels were anesthetized as above, and 2-2.5 ml of blood was collected from the caudal vein into the chilled syringe containing 1.2 mg 2K-EDTA/ml blood. Subsequently, the brain and the heart were dissected out, the latter being separated into atrium and ventricle and frozen on dry ice. The blood was centrifuged at 2,200 g for 15 min, and plasma was stored at -20°C until assayed. The tissues were boiled for 5 min in 1 ml of 0.1 M acetic acid, homogenized at 4°C for 90 s in Polytron homogenizer at the maximum speed, and centrifuged at 250,000 g for 15 min. The supernatant was lyophilized and reconstituted in 1 ml assay buffer for radioimmunoassay.

Statistical analysis. Differences in CNP concentrations between FW and SW eels were compared with Student's t-test. The values are expressed as means ± SE.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation of Eel CNP cDNA

By screening 7 × 105 plaques, we detected 32 positives, 14 of which were plaque purified and sequenced. Sequencing of these clones revealed that a clone named lambda eCnp1.6 contained an insert of 1.6 kb covering the entire CNP coding region (Fig. 1). The prepro-CNP consisted of 131 amino acid residues with two Met residues at the translation initiation site. A signal sequence consisting of 20 amino acid residues, assessed by analogy to shark pro-CNP (31), was present at the NH2 terminus of prepro-CNP and the mature CNP-22 was present at the COOH terminus. In the 3'-untranslated region, six repetitions of ATTTA sequence and two polyadenylation signals were detected. Except for the CNP-22 sequence in which only four amino acid residues are different between eel and mammals, eel CNP sequence exhibited <15% identity with other CNPs identified to date (Fig. 2). The Northern blot analysis showed that the predominant mRNA expressed in the eel brain had a size of ~1.6 kb (Fig. 3).


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  		Fig. 1.   Restriction map of lambda Cnp1.6 clone (A) and cDNA and deduced amino acid sequences of eel C-type natriuretic peptide (CNP) (B). The open box in the restriction map indicates the coding region. Numbers at right correspond to those of nucleotides and amino acids at the right end of the sequence. CNP-22 sequence is underlined. ATTTA signals identified in the 3'-noncoding region are shaded. Putative polyadenylation signals are boxed. The nucleotide sequence reported in this paper has been deposited in the GenBank with Accession Number D88022.



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  		Fig. 2.   Amino acid sequence comparison of CNP precursors among various vertebrate species. Residues identical to the eel sequence across species are reversed. Arrow, potential cleavage site of signal peptide predicted from the NH2-terminal sequence of shark pro-CNP. The mature CNP-22 regions are boxed. *Conserved Cys residues in CNP-22. GenBank Accession Numbers are D88022 (eel), X59991 (shark), D17413 (frog I), D17414 (frog II), and D28874 (human).



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  		Fig. 3.   Northern blot analysis of eel CNP mRNA. Total RNA (20 µg) from the brain was used. The positions of size markers are shown at right.

RNase Protection Assay and RT-PCR

The RNase protection analysis showed that high levels of transcripts of eel CNP gene were detected in the brain of both FW and SW eels (Fig. 4). Low levels were found in the atrium, ventricle, and liver of FW eels but not in SW eels. No apparent signals were detected in other tissues examined with this method. However, RT-PCR detected more extensive expression of CNP signals in various tissues such as the gills, digestive tract, and kidney (Fig. 5). The expression was generally higher in FW than in SW eels. Quantitative analysis using eel GAPDH as an internal control showed that CNP message was highly expressed in the brain and liver and moderately in the gills, heart, and digestive tract (Fig. 6). The high expression in the liver is due to the low relative expression of GAPDH with the same amount of mRNA. The level of expression did not differ in the brain and liver of FW and SW eels but appeared higher in the gill and heart of FW eels than of SW eels. Statistical comparisons using six fish in each group showed that the difference was significant in the heart (1.80 ± 0.10 in FW vs. 1.46 ± 0.10 in SW by the ratio of CNP-GAPDH) and digestive tract (0.89 ± 0.03 in FW vs. 0.63 ± 0.07 in SW), but not in the gill.


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  		Fig. 4.   CNP mRNA levels measured by ribonuclease protection analysis in various tissues from 3 freshwater (FW) eels (A) and 3 seawater (SW) eels (B). Some small tissues were pooled from 3 eels for RNA extraction because at least 10 µg of total RNA was necessary for each assay. The protected fragments were analyzed by fluorography after electrophoresis.



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  		Fig. 5.   CNP mRNA levels determined by RT-PCR in various tissues of an FW eel (A) and an SW eel (B). (RT-), the result without reverse transcription, demonstrating the absence of genomic DNA contamination; (RT+), the result with reverse transcription. GAPDH, the result of amplification of glyceraldehyde-3-phosphate dehydrogenase used as an internal control.



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  		Fig. 6.   Quantification of CNP mRNA levels in various tissues of 3 FW eels (shaded bars) and 3 SW eels (open bars). The CNP signals are corrected by respective GAPDH signals and expressed as percentages. Values are means ± SE.

Measurement of Plasma and Tissue CNP Concentrations

The antiserum used in the radioimmunoassay for eel CNP (#1) had ~1% cross-reactivity with human CNP and <0.1% cross-reactivity with dogfish CNP, human ANP, and human BNP. More importantly, the antiserum had <0.1% cross-reactivity with eel ANP and VNP. The minimum detectable quantity of eel CNP was 0.63 fmol/tube, and half-maximal effective dose of the standard curve was 4.8 fmol/tube (Fig. 7). Because of the high sensitivity, plasma CNP concentration could be measured directly with less than 0.1 ml of plasma without extraction. Serial dilutions of eel plasma with assay buffer produced curves parallel to the standard curve for synthetic eel CNP-22 (Fig. 7).


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  		Fig. 7.   A standard curve for radioimmunoassay of eel CNP (open circle ) and a serial dilution curve of eel plasma (). The dilution curve is approximately parallel to the standard curve.

Plasma CNP concentration measured by the homologous radioimmunoassay was more than fivefold higher in eels adapted to FW than those adapted to SW (Fig. 8A). Both atrial and ventricular concentrations of CNP were also higher in FW eels than in SW eels; however, CNP concentration in the brain did not differ between the two groups (Fig. 8B).


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  		Fig. 8.   CNP concentrations in plasma (A) and tissues (B) of FW eels (filled bars) and SW eels (open bars). *P < 0.05 compared with the value of FW eels. Values are means ± SE (n = 6 FW eels; n = 8 SW eels).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

cDNA Cloning and mRNA Expression of Eel CNP

The amino acid sequence of CNP has been determined in a variety of vertebrate species ranging from cartilaginous fish to mammals. However, its cDNA has been cloned only from the shark (25) and frog (15) in nonmammalian species except for venom-specific CNP from the snake Bothrops jararaca (21). In the present study, we isolated and sequenced a cDNA of eel CNP, thus adding a teleostean species to the list of CNP cDNAs. Comparing the prepro-CNP sequences known to date, we find that only mature CNP-22 sequence is conserved across different species. The high variability of the pro-CNP sequence is consistent with the fact that CNP is an ancestral molecule of the NP family and thus has the longest history of evolution among the family members (33). Conversely, the high conservation of mature CNP-22 sequence indicates its important physiological function throughout vertebrate species, probably through the restraint imposed by the interaction with its specific receptor, NPR-B.

The synthesis and storage of CNP have been demonstrated in a number of tissues and its local regulatory function has been suggested in mammals (19). In the brain, CNP is dipsogenic (24) and inhibits vasopressin secretion (28). CNP has been established as an endothelium-derived relaxing factor and growth-regulating factor involved in vascular remodeling (9). CNP is also involved locally in the proliferation and growth of chondrocytes (6) and osteoblasts (29). The presence of CNP and its local action are likely in the kidney (40) and intestine (10). The current study showed that CNP message was abundantly expressed in the brain, followed by the liver, heart, intestine, and gills of the eel. However, biological actions of CNP in these tissues have not been examined yet in eels, except for the observation of a weak inhibitory action on intestinal NaCl absorption (17). Compared with mammals, the expression of CNP gene was low in the blood vessel, cartilage, and kidney of eels.

CNP as a Circulating Hormone in Eels

In mammals, ANP and BNP are circulating hormones secreted from the heart in response to an increase in blood volume (23). However, CNP is principally an autocrine/paracrine factor, and its plasma concentration is extremely low compared with ANP and BNP (4, 7). In eels, ANP and VNP are cardiac hormones circulating in the blood whose secretion is regulated by plasma osmolality rather than blood volume (12). Because CNP is isolated from the brain in the eel as well (36), CNP may be a paracrine factor in the brain of this fish. However, the present study showed that CNP circulates at high concentrations in the blood of FW eels. CNP is also a circulating hormone as well as a paracrine factor in the dogfish Triakis scyllia (32). It seems, therefore, that CNP, which still retains the ancestral nature of the NP family, is both a paracrine factor and a circulating hormone in phylogenetically ancient vertebrates. After gene duplication, however, ANP and BNP (VNP) took over the endocrine role and have been synthesized and secreted from the heart. Consequently, the role of CNP has been limited to a local paracrine action in higher vertebrates.

One of the major sources of plasma CNP in the eel may be the heart, as demonstrated in the shark (32). The supportive data are that both atrium and ventricle synthesize and store substantial amounts of CNP and that enhanced synthesis of cardiac CNP is correlated well with increased plasma CNP concentration as observed in FW eels. The brain synthesizes and stores CNP most abundantly, but there were no differences between FW and SW eels. Compared with the high ANP content in the heart, the cardiac CNP content seems insufficient to be a source of plasma CNP. However, it is shown that ANP is stored in the granules and secreted in response to osmotic or volemic stimuli (2, 33), whereas cardiac CNP may be released constitutively into the circulation without being stored in the granules. A rather low level of CNP mRNA in the heart compared with the plasma CNP level may be due to its quick turnover as suggested by the presence of repetitive AUUUA sequences in the 3'-noncoding region (27). The mRNA of BNP also has similar repetitive sequences, and its product is secreted constitutively into the circulation (20).

Plasma ANP concentration increases transiently for a few hours after FW eels are transferred to SW and returns to an FW level after adaptation to SW (11). However, plasma CNP concentration was higher in eels adapted to FW than those in SW as shown in this study. The difference in the response to environmental salinity also supports the constitutive nature of CNP secretion. ANP is stored in the atrial granules and secreted via a regulatory pathway in response to an increase in plasma osmolality in eels (12). However, because ANP disappears quickly from the circulation (the half-life of eel ANP is ~90 s, H. Kaiya and Y. Takei, unpublished data), the increase in plasma ANP concentration is only transient. By contrast, high plasma CNP concentration is maintained in FW eels, probably because elevated constitutive secretion continues in association with the enhanced expression of CNP gene in FW environments.

Physiological Function of CNP in Eels

As discussed above, CNP synthesis and secretion are augmented in peripheral tissues of FW eels, so that significant amounts of CNP are circulating in the blood of FW eels. In a previous study, we showed that the CNP-specific receptor NPR-B is more abundantly expressed in osmoregulatory organs of FW eels than in those of SW eels (14). Thus a CNP/NPR-B system may play a role in adaptation of eels to FW environments. The way in which CNP promotes FW adaptation is not fully understood; however, our recent data show that the infusion of CNP into FW eels increases plasma Na+ concentration and enhances radioactive 22Na uptake from the environment (Y. Takei and J. C. Rankin, unpublished data). These results indicate that CNP increases Na+ uptake by the gill and promotes FW adaptation. CNP may act in a paracrine fashion as well as from the blood in the gills, because the gills synthesize appreciable amounts of CNP as observed in the present study.

Perspectives

Although ANP and CNP are members of the same hormone family, ANP appears to be involved in SW adaptation of eels (33), whereas CNP may be involved in FW adaptation as suggested in this study. Therefore, although ANP and CNP share high sequence similarity (71.4%), they promote euryhaline eels' adaptation to opposite osmotic environments, indicating that the NP family is important for the excellent osmotic adaptability of eels. This dissociation of the effects can be achieved by the distinct specific receptors [ANP for NPR-A or guanylyl cyclase (GC)-A and CNP for NPR-B or GC-B]. The eel has another member of the NP family, VNP, that also shares high sequence identity with ANP and CNP. Because VNP has considerably high affinity to NPR-B as well as NPR-A in eels (14), it is likely that VNP assists ANP and CNP to promote the euryhaline fish's adaptation to SW and FW, respectively.

ANP and CNP are oligopeptide hormones that are secreted immediately in response to changes in environmental salinity. It is generally thought that these fast-acting hormones stimulate the secretion of slow-acting hormones that are important for long-term adaptation to a new environment. Long-acting osmoregulatory hormones in teleost fish that have been suggested thus far are prolactin for FW adaptation (8) and cortisol and growth hormone for SW adaptation (1, 18). Interestingly, plasma prolactin concentration is maintained at a high level when euryhaline fish are in FW as is CNP, whereas plasma cortisol and growth hormone concentrations increase only transiently after the transfer of the fish from FW to SW as observed with ANP (11). Therefore, it is likely that ANP and CNP stimulate the release of these long-acting hormones for SW and FW adaptation, respectively. In fact, eel ANP stimulates cortisol secretion in SW eels but not in FW eels (35). Therefore, it is interesting to examine whether ANP stimulates growth hormone secretion and whether CNP stimulates prolactin secretion in the euryhaline teleost species.


    ACKNOWLEDGEMENTS

We thank Drs. S. Hyodo and H. Sakaguchi of Ocean Research Institute and Dr. M. Itakura of Tokyo Institute of Technology for discussion. We also thank S. Hasegawa of Ocean Research Institute and S. Satoh and K. Tanaka of Tokyo Institute of Technology for excellent technical assistance.


    FOOTNOTES

This work was supported by grants-in-aid for Specially Promoted Grant (01902008) and for Creative Basic Research (12NP0201) from the Ministry of Education, Science, Sports and Culture of Japan; by a research grant for Cardiovascular Diseases from the Ministry of Health and Welfare of Japan; and by an SRF Grant for Biomedical Research.

Address for reprint requests and other correspondence: Y. Takei, Div. of Physiology, Dept. of Marine Bioscience, Ocean Research Institute, Univ. of Tokyo, 1-15-1 Minamidai, Nakano-ku, Tokyo 164-8639, Japan (E-mail: takei{at}ori.u-tokyo.ac.jp).

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.

Received 18 September 2000; accepted in final form 9 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Regul Integr Comp Physiol 280(6):R1727-R1735
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