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Abstract: The natriuretic peptide (NP) family consists of at least seven members; cardiac ANP, BNP and VNP and brain CNPs (CNP1–4). Phylogenetic and comparative genomic analyses showed that CNP4 is the ancestral molecule of the family, from which CNP3 and CNP1/2 were duplicated in this order, and that the three cardiac NPs were generated from CNP3 by tandem duplication. Seven members existed at the divergence of ray-finned fishes and lobe-finned fishes (tetrapods), but some of the NP genes have disappeared during the course of evolution. In ray-finned fishes, all three cardiac NPs exist in chondrostei and some migratory teleost species, but VNP is generally absent and ANP is absent in a group of teleosts (Beloniformes). In tetrapods, ANP and BNP are present in mammals and amphibians, but ANP is usually absent in reptiles and birds. Thus, BNP is a ubiquitous cardiac NP in bony fishes and tetrapods though elasmobranchs and cyclostomes have only CNP3/4 as a cardiac NP. Functional studies indicate that cardiac NPs are essential Na+-extruding hormones throughout vertebrates; they play critical roles in seawater (SW) adaptation in teleosts, while they are important volume-depleting hormones in mammals as water and Na+ are regulated in parallel in terrestrial animals. In mammals, cardiac NPs become prominent in pathological conditions such as heart failure where they are used in diagnosis and treatment. Although the functional role of BNP has not yet been fully elucidated compared with ANP in non-mammalian vertebrates, it appears that BNP plays pivotal roles in the cardiovascular and body fluid regulation as shown in mammals. ANP has previously been recognized as the principal cardiac NP in mammals and teleosts, but comparative studies have revealed that BNP is the only cardiac NP that exists in all tetrapods and teleosts. This is an excellent example showing that comparative studies have created new insights into the molecular and functional evolution of a hormone family. [Copyright &y& Elsevier]

Abstract: The renin–angiotensin system is an enzyme-linked hormonal cascade that plays an important role in body fluid and cardiovascular regulation. The system is initiated by the action of renin on the precursor protein, angiotensinogen (AGT), whose sequence information is scarce because of its high variability among species. In the present study, we cloned AGT in chondrichthyans (elasmobranchs: Triakis scyllium, Dasyatis akajei, Leucoraja erinacea and a holocephalan: Callorhinchus milii). Homology was low among AGTs thus far identified; 25–28% between elasmobranchs and tetrapods and 33–61% even within chondrichthyans. All chondrichthyan angiotensin (ANG) II’s have a unique Pro3 instead of Val3 as seen in all other species. In addition, holocephalan ANG II has an unusual His4 instead of Tyr4. In addition, and the N-terminal amino acid, which is usually Asp1 in tetrapods and Asn1 in fishes, was highly variable (Asp, Asn or Tyr) in chondrichthyans. Molecular phylogenetic analysis showed that chondrichthyan AGT precursors are clustered into a group separated from those of tetrapods and teleosts. The AGT gene was most abundantly expressed in the liver, followed by the kidney, interrenal tissue and rectal gland of Triakis where biological actions of ANG II have been demonstrated. Collectively, we identified diversified AGT genes for the first time in chondrichthyes and showed that their ANG II’s have unique amino acid residues at positions 1, 3 and 4. High variability of ANG II sequences in chondrichthyans is discussed in relation to their unique regulatory mechanisms such as urea-based osmoregulation. [Copyright &y& Elsevier]

The four-spine sculpin Cottus kazika is a euryhaline teleost, in which faster growth in seawater (SW) and freshwater (FW) has been reported. In this study, cDNA clones encoding growth hormone (GH) and insulin-like growth factor I (IGF-I) were isolated from this species to examine the involvement of the GH/IGF-I axis in osmotic adaptation. The amino acid sequence of GH predicted from cDNA was highly similar to those of other fish species, 92% to Sparus aurata, 67% to Paralichthys olivaceus, and 63% to Oncorhynchus keta. The predicted sequence of IGF-I was also exhibited high similarity to those of other fishes, 97% to Myxocephalus scorpius, 95% to P. olivaceus, and 81% to O. keta. Tissue distribution of GH and IGF-I mRNA in fish reared in FW and SW was examined by reverse transcription-polymerase chain reaction (RT-PCR). The GH mRNA was detected only in the pituitary gland. The major site of IGF-I mRNA expression was the liver while minor signals were detected in various tissues including the pituitary, gill, fin, heart, spleen, intestine, and kidney. The expression level of GH mRNA in the pituitary was not different between FW- and SW-reared fish. However, the level of IGF-I mRNA in the liver of SW-reared fish was significantly higher than that of FW-reared fish. These results suggest the possible involvement of hepatic IGF-I in SW adaptation of this species. [Copyright &y& Elsevier]

Natriuretic peptides (NPs) play important roles in osmoregulatory and cardiovascular systems of vertebrates. For functional studies of NPs, rainbow trout (Oncorhynchus mykiss), a euryhaline fish, is an interesting model. The information on homologous NPs of salmonid fish is, however, still incomplete with respect to C-type NP (CNP). In this study, we isolated cDNAs encoding the precursor of CNP from the brain of trout. Predicted mature CNP (CNP-22) sequence was identical to that of killifish Fundulus heteroclitus, and only one amino acid was different from that of the eel Anguilla japonica, demonstrating a greater conservation among different teleost species than is found with atrial NP (ANP) and ventricular NP (VNP). While the preprosegment of trout CNP retained 57% similarity to the eel sequence, similarities were low to those of sharks and tetrapods. The major site of expression identified by RT-PCR was the brain with minor expression in the atrium. The putative mature CNP-22 was synthesized and its biological activity was compared with other trout NPs (ANP and VNP) using trout ventral aorta, efferent branchial and celiacomesenteric arteries and anterior cardinal vein in vitro. Synthetic trout CNP-22 relaxed all pre-contracted vessels with potencies comparable to trout ANP and VNP. [Copyright &y& Elsevier]

Abstract: C-type natriuretic peptide (CNP) cDNA was cloned from the tilapia brain and its inferred mature sequence was chemically synthesized together with previously cloned tilapia A-type and B-type natriuretic peptides (ANP and BNP). The cloned CNP belongs to the CNP-1 type of teleosts. Reverse-transcription polymerase chain reaction showed that the ANP and BNP genes were hardly expressed in the tilapia brain and pituitary, whereas the CNP gene was expressed strongly in the brain and slightly in the pituitary. Effects of homologous natriuretic peptides (100nM each) on growth hormone (GH) and prolactin (PRL) release were examined using dispersed tilapia pituitary cells. Tilapia ANP and BNP stimulated GH and PRL release during 4–8, and 8–24h of incubation. BNP appeared to be more potent than ANP, also stimulating GH and PRL release during 0–4h of incubation. CNP stimulated GH release only during 4–8h of incubation; CNP was without effect on PRL release. All three NPs stimulated GH and PRL mRNA expression in dispersed pituitary cells following 24h of incubation. ANP and BNP significantly elevated intracellular cGMP accumulation in dispersed pituitary cells after 15min of exposure, whereas no effect of CNP was observed. These results indicate a long-lasting stimulation of GH and PRL release by ANP and BNP that is mediated, at least in part, by the guanylyl cyclase-linked NP receptor. [Copyright &y& Elsevier]

Abstract: Guanylyl cyclase C (GC-C) is a single transmembrane receptor for a family of intestinal hormones, guanylins. In the eel, we previously identified three guanylins, whose gene expression was enhanced in the intestine after transfer from fresh water to seawater. However, only limited information is available about the structure and function of their receptor(s). In the present study, we cloned full-length cDNAs encoding two isoforms of GC-C, named GC-C1 and GC-C2, from eel intestine. The predicted GC-C proteins consisted of extracellular ligand-binding domain, membrane-spanning domain, kinase-like domain and cyclase catalytic domain, in which GC-C-specific sequences were largely conserved. Phylogenetic analyses showed that the cloned membrane GCs are grouped with the GC-C of other vertebrates but not with GC-A and GC-B. However, eel GC-Cs appear to have undergone unique structural evolution compared with other GC-Cs. The three eel guanylins (guanylin, uroguanylin and renoguanylin), but not eel atrial natriuretic peptide, stimulated cGMP production dose-dependently in COS cells expressing either of the cloned cDNAs, providing functional support for assignment as eel guanylin receptors. The potency order for cGMP production was uroguanylin>guanylin⩾renoguanylin for GC-C1; guanylin⩾renoguanylin>uroguanylin for GC-C2. The distinctive ligand selectivity was consistent with the low homology (53%) of the extracellular domain of the two GC-Cs compared with that observed for other domains (74–90%). Both GC-C genes were expressed in the alimentary tract (esophagus, stomach and intestine) and kidney, and their expression was higher in the intestine of seawater-adapted eels than that of freshwater eels just as observed with the guanylin genes. However, the expression of the receptor genes was unchanged for 24h after transfer of eels from fresh water to seawater or vice versa, showing slower response of the receptors to salinity changes than their ligands. Collectively, the multiple guanylin—GC-C system may be involved as a paracrine factor in seawater adaptation at the intestine and kidney of the eel. [Copyright &y& Elsevier]

Abstract: Natriuretic peptides (NPs) have diversified from a single NP in cyclostomes and elasmobranchs to multiple NPs in ray-finned fishes where ANP, BNP, VNP, and/or up to four CNPs (CNP-1, 2, 3, and/or 4) have been identified. To trace the evolutionary diversification of NPs in fishes, we analyzed the bichir (Polypterus endlicheri), believed to be the most primitive extant ray-finned fish, for the presence of any NPs by a PCR-based method using primers that amplify all NP cDNAs identified to date. We have cloned cDNAs encoding ANP, BNP, VNP from the heart and three CNPs (CNP-1, 3, and 4) from the brain. An extensive search for CNP-2 from the brain was not successful. The C-terminus of bichir ANP presented an amidation signal as in ray-finned fish ANP. The bichir BNP mRNA had AUUUA repeats in the 3′-untranslated region as observed in all BNP cDNAs of vertebrates. The bichir VNP had a long C-terminal ‘tail’ sequence extending from the intramolecular ring as does teleost VNP. The three bichir CNPs are structurally similar to each teleost counterpart and are grouped after molecular phylogenetic analyses. ANP was most abundantly expressed in the atrium, BNP in the ventricle, and VNP was expressed in both atrium and ventricle. The three CNPs are most abundantly expressed in the brain, and CNP-4 transcripts were found in small amounts in the ventricle and kidney. Taken together, it is clear that all major NPs exist prior to the whole genome duplication that occurred in the teleost lineage. Furthermore, this is the first observation that CNP-3, ANP, BNP, and VNP, whose genes are colocalized in the same chromosome, coexist in a single fish species including teleosts, thereby confirming that CNP-3 is not an ortholog of VNP, and that ANP, BNP, and VNP genes were generated by tandem duplication from the CNP-3 gene. [Copyright &y& Elsevier]