General and Comparative Endocrinology 171 (2011) 258–266

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General and Comparative Endocrinology

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Review

B-type natriuretic peptide (BNP), not ANP, is the principal cardiac natriureticpeptide in vertebrates as revealed by comparative studies

Yoshio Takei a,⇑, Koji Inoue b, Sofie Trajanovska c, John A. Donald c

a Laboratory of Physiology, Atmosphere and Ocean Research Institute, University of Tokyo, Chiba 277-8564, Japanb Laboratory of Molecular Marine Biology, Atmosphere and Ocean Research Institute, University of Tokyo, Chiba 277-8564, Japanc School of Life and Environmental Sciences, Deakin University, Geelong, Vic. 3217, Australia

a r t i c l e i n f o

Article history:Received 25 December 2010Revised 8 February 2011Accepted 20 February 2011Available online 22 March 2011

Keywords:Natriuretic peptide familyMolecular evolutionFunctional evolutionComparative genomics

0016-6480/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.ygcen.2011.02.021

⇑ Corresponding author. Address: Atmosphere andUniversity of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Ch4 7136 6201.

E-mail address: takei@aori.u-tokyo.ac.jp (Y. Takei)

a b s t r a c t

The natriuretic peptide (NP) family consists of at least seven members; cardiac ANP, BNP and VNP andbrain CNPs (CNP1–4). Phylogenetic and comparative genomic analyses showed that CNP4 is the ancestralmolecule of the family, from which CNP3 and CNP1/2 were duplicated in this order, and that the threecardiac NPs were generated from CNP3 by tandem duplication. Seven members existed at the divergenceof ray-finned fishes and lobe-finned fishes (tetrapods), but some of the NP genes have disappeared duringthe course of evolution. In ray-finned fishes, all three cardiac NPs exist in chondrostei and some migratoryteleost species, but VNP is generally absent and ANP is absent in a group of teleosts (Beloniformes). Intetrapods, ANP and BNP are present in mammals and amphibians, but ANP is usually absent in reptilesand birds. Thus, BNP is a ubiquitous cardiac NP in bony fishes and tetrapods though elasmobranchsand cyclostomes have only CNP3/4 as a cardiac NP.

Functional studies indicate that cardiac NPs are essential Na+-extruding hormones throughoutvertebrates; they play critical roles in seawater (SW) adaptation in teleosts, while they are important vol-ume-depleting hormones in mammals as water and Na+ are regulated in parallel in terrestrial animals. Inmammals, cardiac NPs become prominent in pathological conditions such as heart failure where they areused in diagnosis and treatment. Although the functional role of BNP has not yet been fully elucidatedcompared with ANP in non-mammalian vertebrates, it appears that BNP plays pivotal roles in the cardio-vascular and body fluid regulation as shown in mammals. ANP has previously been recognized as theprincipal cardiac NP in mammals and teleosts, but comparative studies have revealed that BNP is the onlycardiac NP that exists in all tetrapods and teleosts. This is an excellent example showing that comparativestudies have created new insights into the molecular and functional evolution of a hormone family.

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1. Introduction

It has been suggested that vertebrates first evolved from chor-dates in the coastal area where rivers flow into the seas [11]. Theancient fishes entered inland fresh waters and then expanded theirhabitats further into the sea and onto the land. In parallel with eco-logical evolution, the physiological systems such as osmoregula-tory and cardiovascular systems have undergone evolution [66].In terms of osmoregulation, fishes regulate water and ions, partic-ularly Na+ and Cl�, in the opposite direction depending on environ-mental salinities; retention of water and extrusion of ions inseawater (SW) and retention of ions and extrusion of water in freshwater (FW). However, terrestrial tetrapods regulate them in the

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same direction, retention of both water and ions, as the land is gen-erally a dry and salt-deficient environment. In terms of cardiovas-cular regulation, tetrapods maintain high arterial pressure tocirculate blood against gravity, whilst fishes have lower arterialpressure as they are almost free from gravitational force due tothe buoyancy of water.

Reflecting such differences in body fluid and pressure regula-tion, Na+/water-retaining and vasopressor hormones such asangiotensin II, vasopressin and aldosterone play critical roles inbody fluid and pressure homeostasis in terrestrial tetrapods. Incontrast, Na+-extruding and vasodepressor hormones such asnatriuretic peptides (NPs) and adrenomedullins are more impor-tant and potent in fishes, particularly in teleost fishes that maintainplasma Na+ concentration ca. one third of SW and have low bloodpressure (ca. 20 mmHg). Teleosts have flourished after re-enteringthe seas in the Jurassic era (ca. 160 Myr ago) where they acquiredan ability to extrude excess ions from the mitochondria-rich cells,which may explain why Na+-extruding hormones are predominant

Fig. 1. Phylogenetic distribution of natriuretic peptides in different classes ofvertebrates. Presence and absence are determined by isolation of peptides andcDNAs and by genome and EST database searches. It has not yet been determinedwhether cyclostomes have both CNP4 and CNP3 or CNP4 alone. +, present; �,absent; ?, not determined. (a) Absent in pufferfish, medaka and zebrafish genomes;(b) absent in medaka genome; (c) CNP1/2 exists in X. tropicalis genome; (d) presentin tortoise. Red square shows linkage with the enolase gene. Asterisk shows theoccurrence of an intron shift.

Y. Takei et al. / General and Comparative Endocrinology 171 (2011) 258–266 259

in extant teleost fishes. By contrast, the role of Na+-extruding,vasodepressor hormones is less obvious in mammals, which prob-ably delayed their discovery.

Atrial natriuretic peptide (ANP) was first identified as a natri-uretic/diuretic and hypotensive factor in the rat atria [12], andwas isolated and sequenced in rats and humans in 1983 [15,30].Subsequently, brain (B-type, BNP) and C-type NP (CNP) were iso-lated from the porcine brain in 1988 and 1990, respectively[59,60]. In fishes, ANP was isolated from eel atria in 1989 [68], ven-tricular NP (VNP) from eel ventricles in 1991 [69], and CNP fromeel brains in 1990 [70]. In fact, CNP was first isolated in the killifishin early 1990 prior to the report in mammals because of a muchhigher content in the teleost brain [49]. Although VNP was isolatedfrom the eel ventricle, it was initially thought to be an ortholog ofBNP, as BNP is secreted abundantly from the ventricle of the failingheart in mammals [47].

After the completion of genome projects in the pufferfish, Taki-fugu rubripes, we searched for NPs in the database and found fourCNPs (CNP1�4) [26]. We termed them CNPs because they lackthe C-terminal ‘tail’ sequence that extends from the intramolecularring flanked by a disulfide bond. Furthermore, we identified ANPand BNP in the teleost genome and EST databases. We also identi-fied BNP in the eel; the failure to identify BNP in the initial peptideisolation is due to its low activity in the assay used during purifica-tion. Therefore, it is now generally accepted that the NP family con-sists of at least seven members, ANP, BNP, VNP and four CNPs.

The cardiac NPs and CNPs bind guanylyl cyclase-linked receptortype A (NPR-A) and type B (NPR-B), respectively, and transmit theirsignals intracellularly via cGMP [24,40,44]. All NPs also bind NPR-Cand teleost-specific NPR-D that lack intracellular guanylyl cyclasedomain [31]. The NPR-C was thought to be a clearance receptorthat regulates plasma and local extracellular levels of NPs, butnew physiological functions have been suggested in mammals asNPR-C activation causes inhibition of cAMP production and/or acti-vation of phospholipase C [50]. However, comparative studies ofNP receptors are still in their infancy and beyond the scope of thisreview.

In this short review, we first summarize the molecular evolu-tion of the NP family in vertebrates with emphasis on the historyof diversification revealed by comparative genomic analyses. Then,the NP members are described in some detail in fishes and tetra-pods, in particular cardiac ANP, BNP and VNP that play pivotal rolesin body fluid regulation. We will introduce mammalian NPs in aseparate section as cardiac NPs are now important targets in clin-ical medicine. Finally, the difference in molecular diversificationand physiological function of NPs among vertebrate groups willbe discussed in relation to the habitat-related body fluid regulation(land, FW and SW).

2. Evolutionary history of diversification in the natriureticpeptide family

2.1. The ancestral NP

There are several reports suggesting the presence of ANP ininvertebrates and even in unicellular Paramecium [81] and inplants [82] using mammalian ANP antisera, but it is far from beingestablished [63]. In fact, NPs have not been isolated as a peptide orcDNA in invertebrates. We have searched the genome databases ofchordates (ascidians, Ciona intestinalis and C. savignyi, and amphi-oxus, Branchiostoma floridae) for the ancestor of the NP gene butto no avail. The most ancient animal in which an NP gene has beenidentified is hagfish (Cyclostomata). Thus, it seems that the first NPsystem appeared during early evolution of vertebrates before thedivergence of agnathans and gnathostomes. The hagfish NP was

discovered by cDNA analysis in Eptatretus burgeri and was the onlyNP found in hagfish [35]. The existence of hagfish NP in the plasma,heart, and brain was also confirmed by a mass spectrometry. Inter-estingly, hagfish NP has a C-terminal tail, whose terminus is ami-dated like teleost ANP, but its sequence similarity to ANP is nothigher than that to BNP and VNP. Thus, it was difficult to categorizeit to any known subtype of NPs at that time. The hagfish NP cDNAwas also isolated from other hagfish species [33].

In another group of cyclostomes, lamprey, a single NP was alsoidentified. It was categorized as CNP due to the absence of the C-terminal tail [33]. The phylogenetic position of the lamprey CNPwas analyzed against the four CNPs discovered in teleosts, usingthe whole precursor sequence deduced from the cDNA. As a result,it was most similar to CNP4. Interestingly, hagfish NP is also in-cluded in the clade of lamprey CNP and teleost CNP4. This resultwas supported by the existence of the processing signal RXXR atthe N-terminus of the inferred mature peptide that is conservedamong hagfish NP, lamprey CNP, and CNP4 of tetrapods and tele-osts. Thus, it was suggested that the first NP to appear in the ances-tral vertebrate lineage was CNP4-like (Fig. 1), although whether ithad a C-terminal tail or not is unknown.

2.2. Divergence of four CNP genes

It is thought that the CNP1, CNP2, and CNP3 genes were gener-ated from the CNP4-like ancestral NP gene by chromosome or geneduplications before the divergence of chondrichthyans and oste-ichthyans [26]. The three CNP genes are localized on different chro-mosomes having linkages to three paralogs of enolase genes, whichare suggested to have triplicated before the chondrichthyan andosteichthyian divergence [72]. Although it is difficult to presumethe order of generation of the three CNPs by phylogenetic analysesusing either CNP precursor sequences or enolase sequences, theCNP1 and CNP2 genes and the CNP3 and CNP4 genes, respectively,isolated from pufferfish have the same exon-intron arrangements.The intron specific to the CNP3 and CNP4 genes exists in the hag-fish NP gene [26], which suggests that the CNP3 gene was dupli-cated first. Thus, it is concluded that four CNPs were generated in

260 Y. Takei et al. / General and Comparative Endocrinology 171 (2011) 258–266

the following order: (i) the ancestral CNP gene was duplicated intoCNP3 and CNP4 genes, (ii) linkage was formed between the CNP3gene and the ancestral enolase gene, (iii) the CNP3 gene and theancestral gene of the CNP1 and CNP2 genes diverged via chromo-somal duplication, (iv) change in the exon-intron arrangement oc-curred in the ancestral gene of CNP1/2 gene, and (v) the CNP1 andCNP2 genes were generated by another chromosomal duplication(Fig. 1).

2.3. Generation of ANP, BNP, and VNP genes by tandem duplication ofCNP3 gene

The ANP and BNP genes are thought to be generated by tandemduplication of the CNP3 gene because the three genes are tandemlylocalized in the green pufferfish genome [26]. The synteny aroundthe three genes is conserved between mammals and ray-finnedfishes although the CNP3 gene is absent in mammals. Thus, it isevident that tandem duplications of the ANP and BNP genes oc-curred before the divergence of ray-finned fishes and lobe-finnedfishes (Fig. 1).

Concerning the VNP gene, it has been difficult to locate the po-sition from genome databases because it was identified in rela-tively primitive ray-finned fishes, the bichir, sturgeon, eel, andsalmon [27,32,80], for which whole genome databases are notavailable. Inoue et al. [27] revealed, by linkage mapping in rainbowtrout, that the VNP gene is also in the neighborhood of the ANP andBNP genes. In addition, we found that the VNP gene is localizedapproximately 1.5 kb upstream of the BNP gene in the eel (Inoueet al., unpublished). Thus, the VNP gene may also be generatedthrough a tandem gene duplication event similar to those of ANPand BNP (Fig. 1).

It is evident that the generation of the ANP and BNP genes oc-curred before the divergence of ray-finned fishes and lobe-finnedfishes because ANP and BNP exist in both teleosts (teleostei) andtetrapods. However, at present, it remains unclear when the VNPgene was generated. Trajanovska et al. [75] found a new NP gene

Fig. 2. An alignment of mammalian ANP, BNP and CNP mature peptide sequences. Maspecies are shaded. Disulphide bonds are connected by brackets. Asterisks at the C-tersequence.

in the chicken genome database, which was named Renal NP(RNP) because of remarkable expression in the kidney. RNP is pre-sumed to be the tetrapod ortholog of VNP because the position ofits gene relative to other cardiac NP genes is the same as that of eelVNP. In addition, the deduced peptide sequence has a long C-termi-nal tail containing a cluster of basic amino acids as other VNPs.Thus, generation of the VNP gene may also be before the ray-finnedfishes and lobe-finned fishes divergence. In addition, ANP, BNP, andVNP genes are not detected in the vicinity of CNP1 and CNP2 genesin teleosts, suggesting that the ANP, BNP, and VNP genes wereduplicated from the CNP3 gene after generation of the ancestralCNP1/2 gene.

2.4. Specific loss of NP genes in each phylogenetic lineage

The history of generation of the seven NP genes described abovesuggests that the ANP, BNP, VNP, and four CNPs existed before thedivergence of ray-finned fish and lobe-finned fish (tetrapod) lin-eages. In fact, all seven members have been discovered from theprimitive ray-finned fishes. However, some of the cardiac NPscould not be identified in specific teleost groups; VNP is absentin most of the advanced groups and ANP in one group, Belonifor-mes. By contrast, four CNPs are retained in most teleost groups; de-tails will be described in Section 5. The molecular data of the NPfamily are not available in the lobe-finned fishes.

In contrast to the ray-finned fish lineage, loss of the NP subtypesin the tetrapod lineage is more remarkable and variable (Fig. 1). Aswill be discussed in more detail in Section 4, ANP, BNP, CNP3 andCNP4 are retained in amphibians, BNP, CNP1, CNP3 and possiblyVNP in birds, and ANP, BNP and CNP4 in mammals. Although theloss of CNP2 may have occurred in the early phase of tetrapod evo-lution, the loss of VNP in amphibians, that of ANP and CNP4 inbirds, and that of VNP, CNP1, and CNP3 in mammals are obviouslyindependent events. Collectively, NP subtypes have been lost ineach vertebrate class in a lineage-specific manner, but BNP isretained in all bony fishes and tetrapods as a cardiac hormone.

mmalian CNP is categorized as CNP4. The amino acids common in more than halfminal end of ANP and BNP show that an intron starts from there in the genome

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2.5. NP evolution and the third round whole genome duplication inteleosts

It is known that two rounds of whole genome duplications oc-curred in ancestral vertebrates, and it is likely that these duplica-tions are involved in the generation of the four types of CNPs. It isalso known that an additional whole genome duplication (3R) oc-curred in the early stage of ray-finned fish evolution [53]. This im-plies that all the seven NP genes were once duplicated into 14genes because the 7 NPs already existed before the divergence ofray-finned fishes and lobe-finned fishes. However, we have notfound any evidence of redundant NP genes from fish genome dat-abases except for the second CNP2 that is found in the zebrafish gen-ome database (see below), but whose position on the genome hasnot been assigned. It is probable that the redundant genes have beenlost, as has been suggested for many other duplicated genes [53]. Forexample, in medaka we found two copies of the ENO2 gene on chro-mosomes 11 and 16, respectively, which are sibling chromosomesduplicated by 3R. However, the CNP1 gene is found only on chromo-some 16, which suggests that it has been lost on chromosome 11; thesame phenomenon is also found in the stickleback genome.

3. Natriuretic peptides in mammals

3.1. Molecular studies

As mentioned above, mammals have two cardiac NPs (ANP andBNP) and a CNP that is an ortholog of CNP4 [26]. Comparing the ma-ture peptide sequences in mammals, it is obvious that CNP is mostconserved; among 21 species examined, the sequence is identical in18 species and only one amino acid is altered in 2 species (Fig. 2).We found a unique CNP in the little brown bat (Myotis lucifugus),which is similar to teleost CNP4 in sequence. The second most con-served NP is ANP; among 25 species examined, the sequence isidentical in 13 species, 1 amino acid is different in 7 species, 2 ami-no acids different in 4 species, and 3 amino acids different in onespecies (Fig. 2). In opossum (Monodelphis domestica) and platypus(Ornithorhynchus anatinus), one amino acid insertion has occurredin the N-terminal region, but in general the mature sequences arehighly similar to other ANPs. By contrast, BNP sequences are highlyvariable among species (Fig. 2). For example, 1 amino acid is alteredeven between human and gorilla BNP and the sequence identity be-

Fig. 3. An alignment of ANP, BNP and CNP mature peptide sequences in non-mammalianscientific names of species, see text. Disulphide bonds are connected by brackets.

tween human and rat BNP is only 50%. The high conservation of theCNP and ANP sequences indicates important common functionsamong different species, while the high sequence variability ofBNP suggests a species-specific role.

3.2. Functional studies

Since its discovery, ANP has attracted the attention of clinicalresearchers with respect to its role as an anti-hypertensive agentas it is the most potent and efficacious vasodepressor hormonethus far known [56]. In addition, ANP is the most potent natriuretichormone to counter hypertension caused by excessive salt intake.Later studies have shown that ANP is closely related to heart fail-ure; plasma ANP concentration increases in proportion to theseverity of heart failure [2], and its administration improves theindices of the failing heart [51]. The plasma of normal subjects con-tains only mature ANP, but the failing heart secretes significantamounts of proANP. As plasma ANP concentration increases in pa-tients with no apparent symptoms of heart failure, it has been rou-tinely measured as a useful biomarker to predict future occurrenceof heart failure.

More recently, BNP and its N-terminal fragment of proBNP with76 amino acids, named NT-proBNP, were recognized as bettermarkers for diagnosis of heart failure. The heart failure is in facta ventricular failure and BNP is expressed abundantly in the ventri-cle [10]. The plasma BNP levels are also used for prognosis of pa-tients with a failing heart because they are highly predictive ofsubsequent morbidity of congestive and ischemic heart failure.Accordingly, BNP has attracted greater attention of clinicalresearchers than ANP in recent years, which has resulted in a hugenumber of clinical papers that deal with BNP. BNP administrationis also a better treatment than ANP [85] partly because of its longerplasma half life (ca. 22 min) than ANP (2–3 min). Thus, recombi-nant BNP, named nesiritide, was clinically used to improve the car-diac indices [7].

4. Natriuretic peptides in non-mammalian tetrapods

4.1. Molecular studies

Amphibian and chelonian ANP are characterized by an IGAQSGsequence within the cystine ring, which is similar to mammalian

tetrapods. Amino acid residues common to more than half species are shaded. For

Fig. 4. Northern blot analysis of 10 lg of heart RNA from selected reptilian andavian species after hybridization with a 32P-labelled tortoise ANP cDNA probe. Theposition and sizes of the RNA markers are shown on the left. Equal loading of RNA asrepresented by the rRNA bands is shown in the corresponding ethidium bromidestained gel.

262 Y. Takei et al. / General and Comparative Endocrinology 171 (2011) 258–266

ANP and distinct from BNP and CNP (Fig. 3). In amphibians, ANPpeptide was first isolated from two Rana species [41,52]. Subse-quently, ANP mRNAs have been sequenced in Bufo marinus [13],

Fig. 5. An alignment of teleostean and chondrostean ANP, BNP, VNP (a) and 4 types of Cresidues common in more than half species are shaded. Disulphide bonds are connected

Hyla japonica [68], and two Xenopus species [36]. Interestingly,the predicted amino acid sequence of B. marinus and H. japonicaANP has an extended C-terminal tail segment, which can be ex-plained by a single nucleotide mutation. Two isoforms of ANP wererecently identified in X. laevis and found to share 91% amino acididentity [73]; one of the ANP cDNAs is identical to the publishedX. laevis ANP sequence [57]. In the X. tropicalis genome, only oneANP gene is tandemly located in synteny with the BNP and CNP3genes [73]. It remains to be determined whether the two ANPmRNAs in X. laevis are products of tetraploidy. ESTs that encodeANP are available for Rana picrica and two urodele species,Notophthalmus viridescens and Ambystoma tigrinum (Fig. 3). Inreptiles and birds, ANP was only present in atria of tortoise(Chelodina longicollis), but was not expressed in lizard (Tiliquascincoides), snake (Pseudonaja textilis), crocodile (Crocodylusporosus), pigeon (Columba livia) and magpie (Cracticus tibicen)[73,74]. Furthermore, northern analysis of heart mRNA from thesame species with a tortoise ANP cDNA probe could not revealany ANP transcripts using low stringency hybridization (Fig. 4),and ANP is not found in the genome and/or EST databases of greenanole (Anolis carolinensis), chicken, turkey (Meleagris gallopavo) andzebrafinch (Taeniopygia guttata). Thus, ANP may have been lost inthe evolution of non-chelonian reptiles and birds.

NP (b) mature peptide sequences. Zebrafish has two types of CNP2. The amino acidby brackets.

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BNP appears to be ubiquitous in amphibians, reptiles and birds,and is characterized by a sequence of CFGRR in the cystine ring. Likeall BNP mRNAs, ATTTA repeats exist in the 30 untranslated region. Inamphibians, BNP was first isolated in R. catesbeiana [16] and thensubsequently cloned from X. laevis [58], B. marinus [13], H. japonica[73], and X. tropicalis [73]. Furthermore, BNP from two urodele spe-cies is present in the EST database as is ANP but only the predictedmature peptide is available for newt. Remarkably, newt BNP isnearly identical to that of birds and reptiles but has lower identitywith X. laevis and B. marinus (Fig. 3). In chicken, a 29 amino acid NPwas originally isolated from the heart [46] and its cDNA cloned [1],which was concluded to be BNP after initially being assigned asANP. Molecular cloning showed that BNP is expressed in the atriaof tortoise, lizard, snake, crocodile, pigeon and magpie [73,74].Analysis of the turkey and zebrafinch genomes and/or EST dat-abases shows that BNP genes are present (Fig. 3), but ANP wasnot located. Thus, it seems that the primary cardiac peptide in rep-tiles and birds, with the exception of chelonians, is BNP. The struc-ture of mature BNP has been strongly conserved during theevolution of reptiles and birds. For example, mature BNP of chicken,crocodile, tortoise and turkey is identical, while only a single aminoacid substitution is present in the tail region of pigeon and magpieBNP (Fig. 3). Therefore, unlike mammals, there has been high selec-tive pressure to conserve the sequence of BNP, especially if the ANPgene has been lost. Furthermore, given that newt BNP is nearlyidentical to reptilian and avian BNP, it is possible that the newt se-quence is very close to the ancestral BNP form that has remainedvirtually unchanged in the evolution of reptiles and birds.

In amphibians, CNP was first isolated from the brain of R.catesbeiana [84], and then two different CNP mRNAs (CNP I andCNP II) were cloned [39], which are in fact CNP3 and CNP4 [26](Fig. 3). Interestingly, three distinct CNPs are present in the X. trop-icalis genome and/or EST databases, two of which were identifiedas orthologs of CNP3 and CNP4, respectively, using phylogeneticand chromosomal synteny analysis [73]. The third CNP gene couldnot be assigned to a particular CNP subtype using synteny as itcould not be located in the X. tropicalis genome [73]. In X. laevis,only two CNPs can be found in the EST database, one of which isCNP4. The second X. laevis CNP shares a 95% sequence identity tothe third X. tropicalis CNP. A CNP3 ortholog was not found in theX. laevis EST database. In birds, a 22 amino acid CNP was isolatedfrom chicken brain [4] and its mRNA sequence predicted in silicofrom the chicken genome [25]. A second CNP is also found in thechicken, turkey and zebrafinch genomes. The two avian CNPs areorthologs of teleost CNP1 and CNP3 [75]. CNP2 was not identifiedin non-mammalian tetrapods.

In addition, a novel NP, named RNP as mentioned above, was iden-tified in the chicken genome and cloned [75]; a putative RNP orthologcan also be found in the turkey genome but not in zebrafinch. Thechicken RNP gene is located between the CNP3 and BNP genes onchromosome 21 and shares conserved linkage with the VNP, CNP3and BNP genes in Japanese eel. Therefore, it is possible that the chick-en RNP gene is orthologous to the VNP gene. Collectively, BNP ap-pears to be the ubiquitous cardiac NP in non-mammalian tetrapods.

4.2. Functional studies

The physiological role of NPs in non-mammalian tetrapods hasbeen reviewed previously [13,71] and the following is a brief re-view of the role of cardiac NPs. In amphibians, the plasma levelof ANP and BNP has not been determined in any setting of volumeor osmotic manipulation, which is a key gap in our knowledge ofNP function in this group. Like other NPs, homologous BNP is adilator of blood vessels but has no effect on heart rate or force ofcontraction in R. catesbeiana [78] and B. marinus [13]. In the skin

of R. catesbeiana, BNP has no direct effect on transport across theskin but did inhibit the effects of AVT [79].

Since chicken BNP was isolated shortly after the discovery ofNPs, the role of BNP in non-mammalian tetrapods has been mostcomprehensively investigated in birds using chicken BNP [17]. Induck (Anas platyrhynchos), the plasma BNP level was elevated inhypervolemia and depressed in hemorrhaged or water-deprivedbirds [18,22], which indicates volemic regulation of BNP secretion.BNP acts on the duck kidney to elicit diuresis and natriuresis[18,19,55]. In chicken, BNP is a dilator of isolated aorta and, notsurprisingly, caused a significant hypotension in vivo [73,75]. Incontrast, BNP did not significantly affect mean arterial blood pres-sure or heart rate in duck [55]. BNP can also modulate endocrinesystems such as the renin–angiotensin–aldosterone system andAVT, which cause salt and water retention [20]. BNP inhibited aldo-sterone release in duck [22] but, in contrast, stimulated aldoste-rone production in turkey [38]. In lizard, Sceloporus undulates,BNP inhibited aldosterone secretion from dispersed adrenocorticalcells [6]. BNP has also been shown to be a significant stimulant ofsalt gland secretion in duck [54] and crocodile (Crocodylus porosus)[9], and BNP immunoneutralization inhibits stimulated salt glandsecretion in the duck [21].

5. Natriuretic peptides in fishes

5.1. Molecular studies

As mentioned above, cyclostomes (hagfish and lamprey) havean NP that is classified into the CNP4 group by molecular phyloge-netic analysis [33], whereas cartilaginous fishes also have a singleCNP that is in the CNP3 group [26,34,61]. The CNPs are producedabundantly in both heart and brain of these animals and thus alsoact as circulating hormones [33,62].

In ray-finned fishes, NPs have diversified and their roles appearto be differentiated into circulating hormone secreted from theheart (ANP, BNP, VNP) and paracrine factors in the brain and othertissues (CNPs) (Fig. 5). Four CNP genes have been retained in mostteleost species. The VNP gene was lost after divergence of salmo-nids, and the ANP gene was specifically lost in Beloniformes thatincludes medaka [27]. Thus, five or more NP subtypes are retainedin ray-finned fishes. This implies that most of the duplicated genesat 3R have been lost from the chromosomes during teleost evolu-tion. Among cardiac NPs, therefore, BNP is the only one that existsacross all species thus far examined (>30). As mentioned, no infor-mation is available on the NPs in lobe-finned fishes.

Comparing cardiac NP sequences within ray-finned fishes, BNPis more conserved than ANP whereas the number of VNP sequencesis too small for comparison (Fig. 5). The consecutive dibasic (RR) se-quence just after conserved CFG in the intramolecular ring is foundin teleost BNP as in tetrapods. Among CNPs, it is apparent that CNP1is most conserved and readily identifiable in the EST databases. Bycontrast, CNP2 is not as conserved and is the least identifiable CNPin the database. In fact, intensive efforts to isolate CNP2 cDNA wereunsuccessful in the bichir [80] and eel [48]. As CNP1 and CNP2 areexpressed almost exclusively in the CNS in the medaka and bothhave high affinity only to the B-type NP receptor [27], both mayshare similar functions. CNP3 is unique in that D is generally re-placed by E in the conserved DRI sequence within the ring(Fig. 5). The mature CNP4 sequence differs considerably from mam-malian CNP and the C-terminal ‘tail’ extending from the ring occursin some teleost species like zebrafish as noted in hagfish CNP4.

5.2. Functional studies

Consistent with the presence of a diversified NP family, NPs playpivotal roles in body fluid and pressure homeostasis in teleost

264 Y. Takei et al. / General and Comparative Endocrinology 171 (2011) 258–266

fishes. ANP is an important SW-adapting hormone in the eel[64,65], and is secreted soon after transfer from FW to SW in re-sponse to increased plasma osmolality [29]. ANP ameliorates ex-cess increases in plasma osmolality by limiting drinking ofenvironmental SW and inhibiting absorption of Na+ from ingestedSW [3,43,76]. Thus, plasma Na+ concentration decreases duringANP infusion [67]. Conversely, removal of circulating ANP by anANP antiserum (immunoneutralization) increases drinking rateand plasma Na+ concentration in SW eels [77]. Interestingly, ANPincreases urine Na+ concentration but decreases urine volume;thus, there is no difference in net Na+ excretion [67]. Furthermore,ANP increases cortisol secretion only in SW eels in vivo [42] and en-hances steroidogenic action of ACTH in the interrenal tissue of SWeels in vitro (Ventura et al., unpublished data). A steroidogenic ac-tion of ANP has been demonstrated from interrenal tissue of otherteleost species [5,37]. All the data mentioned above support thenotion that ANP is a SW-adapting hormone that limits excess Na+

uptake from the environment. In the rainbow trout, however, themajor stimulus for ANP secretion and synthesis is an increase inblood volume [8,28], and ANP induces diuresis and natriuresis[14] as observed in mammals. The difference between the two spe-cies may be due to their origin; the eel is originally a SW speciesexposed to osmotic challenges, while the trout is basically a FWfish exposed to volemic challenges.

In contrast to ANP, CNP is a FW-adapting hormone in the eel.The first CNP isolated from the teleost brain was CNP1. The CNP1gene is up-regulated in FW eels and infusion of CNP1 into FW eelsincreases 22Na uptake from the environment, resulting in increasedplasma Na+ concentration (Takei et al., unpublished data). These ef-fects were demonstrated only in FW eels. Furthermore, CNP1 in-creases plasma cortisol concentration only in FW eels [42], andmore recent data showed that CNP1 and CNP4, but not ANP, BNPand VNP, enhances steroidogenic action of ACTH in isolated inter-renal tissue from FW eels (Ventura et al., unpublished data). Corti-sol is known as a long-acting hormone that promotes adaptation toboth FW and SW environments [83]. Together with the promotingeffect of ANP on SW adaptation, the NP family appears to governthe euryhalinity of the eel by promoting adaptation to both FWand SW by acting directly or by promoting the secretion of long-acting hormones such as cortisol [65].

6. Evolutionary considerations

6.1. Molecular evolution

Comparative studies have revealed the history of diversificationof the NP family during vertebrate evolution. Before the genomedatabases were available in teleosts, we believed that CNP wasthe ancestral NP of the family as only CNP was found in elasmo-branchs and CNP was more conserved in each vertebrate classand among different classes (mammals, birds, amphibians, teleostsand elasmobranchs) than ANP and BNP. However, it was laterfound that these conserved CNPs are of different types; CNP3 inelasmobranchs, CNP1 in teleosts, CNP3 in amphibians, CNP1 inbirds and CNP4 in mammal (Figs. 2, 3 and 5). Interestingly, theseconserved CNPs are the major CNP in each species, and these CNPsof different types are more similar to each other than to the sameCNP type of the species; e.g., mammalian CNP4 is more similar toteleost CNP1 (86.4%) than to teleost CNP4 (59.1%). In particular,the sequence within the intramolecular ring is identical betweenmammalian CNP4 and teleost CNP1, suggesting convergent evolu-tion or functional restriction of the mature sequence. However,such conservation applies only to the mature sequence, and molec-ular phylogenetic analyses of the precursor sequence classified theCNPs of the same type (1–4) into a cluster.

It was shown that CNP3 is the precursor of cardiac NPs as a re-sult of tandem duplication. This observation coincides well withelasmobranch CNP3, which is synthesized profoundly in both car-diac atria and ventricles and secreted into the blood as a circulatinghormone [62]. In medaka, CNP3 has higher affinity to one of thetwo types of NPR-A than to NPR-B, thus sharing a common charac-teristic to ANP and BNP [27]. Medaka BNP binds a second NPR-Awith high affinity, while other CNPs bind only to NPR-B. In fact,the CNP3 gene is more abundantly expressed in the kidney andheart than in the brain of medaka [27]. It seems that an ancestralCNP4-like peptide was expressed in both brain and periphery(heart) as evidenced in hagfish and lamprey, followed by CNP3 incartilaginous fishes. However, ANP, BNP and VNP took over the roleof cardiac hormones after they were duplicated from CNP3, andCNP3 may have lost its function and disappeared in mammals.

Previously, ANP was thought to be the primary cardiac NP invertebrates, as ANP is much more conserved than BNP in mam-mals, and BNP was not yet discovered in teleosts. However, com-parative genomic analyses identified BNP in various teleostspecies, and BNP was identified in eels and salmonids in additionto VNP. Furthermore, it was found that chicken NP isolated fromthe heart was BNP as judged from its mature sequence and phylo-genetic analysis of its precursor, although it was initially thoughtto be ANP. Later studies showed that BNP is the only cardiac NPidentifiable in all species of bony fishes and tetrapods thus farexamined, as ANP is absent in most birds and reptiles and in someteleost species. Intriguingly, mature BNP sequences are more con-served than ANP sequences in non-mammalian tetrapods and tel-eost fishes, which is in contrast to the situation in mammals.Judging from the molecular data from various vertebrate species,it is most likely that BNP is the primary cardiac NP in vertebrates.

6.2. Functional evolution

It appears that the primary action of cardiac NPs in vertebratesis to decrease total Na+ content in the body, which leads to lower-ing arterial pressure with a concomitant vasodilatory action [66].This idea stems from the actions of ANP in SW eels, where ANP actsdirectly to limit Na+ uptake (inhibition of drinking and intestinalNa+ absorption) and indirectly to promote Na+ excretion throughenhanced differentiation of SW-type chloride cells by stimulationof cortisol and growth hormone secretion [65]. Previously, ANPwas thought to be a volume-depleting hormone that is secretedin response to hypervolemia and acts to decrease both water andNa+ to restore normal blood volume. However, comparative fishstudies have revealed that the primary action of ANP is not onwater but on Na+. Then, why did ANP become a volume regulatinghormone in mammals or tetrapods? As discussed in the Introduc-tion, tetrapods usually regulate water and Na+ in the same direc-tion, as water moves together with ions across absorptiveepithelia of the intestine and renal tubules through abundantly ex-pressed water channels. However, the abundance of water chan-nels is limited in fishes that regulate water and Na+ in theopposite direction. For instance, the SW eel esophagus absorbsNa+ and Cl� but is almost impermeable to water [23].

From the functional aspect, the role of BNP in cardiovascularand body fluid regulation is scarce compared with ANP in non-mammalian species, except in the chicken where BNP is the onlycardiac NP (see Section 4.2). We compared vasodepressor andosmoregulatory actions of homologous ANP, VNP and BNP in theeel and found that ANP and VNP are more potent and efficaciousthan BNP [45,48]. We do not know the effect of BNP in medaka fishthat has only BNP as a cardiac hormone. It seems that the relativepotency of cardiac NPs is species dependent when there are multi-ple NPs.

Y. Takei et al. / General and Comparative Endocrinology 171 (2011) 258–266 265

In summary, it is obvious that comparative studies are benefi-cial not only to trace the evolutionary history of diversification ofa hormone family such as the NP family, but also to extract theessential action of a hormone by comparing their actions in ani-mals living in different habitats and utilizing diverse pressureand body fluid regulation.

Acknowledgments

We thank Dr. Robert Dores and Dr. Ian Henderson, Editors-in-Chief of General and Comparative Endocrinology, for giving us anopportunity to contribute to the 50th Anniversary issue. We alsothank the staff and current and former graduate students of Atmo-sphere and Ocean Research Institute, University of Tokyo and Dr.Tess Toop of Deakin University for their inspiration.

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