A ‘reverse’ phylogenetic approach for identification of novel osmoregulatory and cardiovascular hormones in vertebrates

www.elsevier.com/locate/yfrne

Frontiers inNeuroendocrinology

Frontiers in Neuroendocrinology 28 (2007) 143–160

Review

A ‘reverse’ phylogenetic approach for identificationof novel osmoregulatory and cardiovascular hormones in vertebrates

Yoshio Takei *, Maho Ogoshi, Koji Inoue

Laboratory of Physiology, Ocean Research Institute, University of Tokyo, 1-15-1 Minamidai, Nakano, Tokyo 164-8639, Japan

Available online 26 May 2007

Abstract

Vertebrates expanded their habitats from aquatic to terrestrial environments during the course of evolution. In parallel, osmoregu-latory and cardiovascular systems evolved to counter the problems of desiccation and gravity on land. In our physiological studies onbody fluid and blood pressure regulation in various vertebrate species, we found that osmoregulatory and cardiovascular hormones havechanged their structure and function during the transition from aquatic to terrestrial life. In fact, Na+-regulating and vasodepressor hor-mones play essential roles in fishes, while water-regulating and vasopressor hormones are dominant in tetrapods. Accordingly, Na+-reg-ulating and vasodepressor hormones, such as natriuretic peptide (NP) and adrenomedullin (AM), are much diversified in teleost fishescompared with mammals. Based on this finding, new NPs and AMs were identified in mammals and other tetrapods. These hormoneshave only minor roles in the maintenance of normal blood volume and pressure in mammals, but their importance seems to increasewhen homeostasis is disrupted. Therefore, such hormones can be used for diagnosis and treatment of body fluid and cardiovascular dis-orders such as cardiac/renal failure and hypertension. In this review, we introduce a new approach for identification of novel Na+-reg-ulating and vasodepressor hormones in mammals based on fish studies. Until recently, new hormones were first discovered in mammals,and then identified and applied in fishes. However, chances are increasing in recent years to identify new hormones first in fishes then inmammals, based on the difference in the regulatory systems between fishes and tetrapods. As the direction is opposite from the traditionalphylogenetic approach, we added ‘reverse’ to its name. The ‘reverse’ phylogenetic approach offers a typical example of how comparativefish studies can contribute to the general and clinical endocrinology.� 2007 Elsevier Inc. All rights reserved.

Keywords: Environmental adaptation; Aquatic fishes; Terrestrial tetrapods; Cardiovascular regulation; Body fluid regulation; Natriuretic peptide family;Calcitonin gene-related peptide family; Comparative genomics; Molecular evolution

1. Introduction

Life originated in ancient seas some 3.8–4 billion yearsago [46]. Since then, organisms have increased their com-plexity in form and function and expanded their habitatsduring evolution. Vertebrates are considered to haveevolved from invertebrate chordates in salt water [20], thensubsequently entered fresh water prior to returning to theseas or moving onto land [106,118]. During the transitionfrom aquatic to terrestrial life, animals encountered prob-lems that had not been experienced in water, namely desic-

0091-3022/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.yfrne.2007.05.001

* Corresponding author. Fax: +81 3 5351 6463.E-mail address: [email protected] (Y. Takei).

cation and gravity. Thus, terrestrial animals developedmechanisms to conserve body water [10,111,124] andacquired powerful hearts and high blood pressure to main-tain the circulation in the face of gravity. These changes inbody fluid and blood pressure regulation have drivenchanges in osmotic and cardiovascular regulatory systems.The endocrine system has evolved to play a key role in theadaptational changes in body fluid and blood pressure reg-ulation required for life on land, since it is now known thatmany osmoregulatory and cardiovascular hormones haveevolved in their structure and function to counter theeffects of desiccation and gravity [10].

We have been investigating the evolution of body fluidand blood pressure regulation in vertebrates with specialreference to hormonal regulation. During the course of

mailto:[email protected]

Fig. 1. A schema illustrating the difference in body fluid regulation amongtetrapods, freshwater (FW) fishes and seawater (SW) fishes. Terrestrialtetrapods usually retain both water and NaCl in the body. However, FWfish excrete water and retain NaCl, and SW fish regulate them oppositely.In addition, aquatic fish escape from gravity by buoyancy of water, whichprofoundly affected the cardiovascular system. For details, see text.

144 Y. Takei et al. / Frontiers in Neuroendocrinology 28 (2007) 143–160

these studies, we found that the regulatory system and therole of hormones in the system differ greatly between fishand tetrapods [124,129]. Furthermore, due to the availabil-ity of genome databases in various vertebrate species, wefound that some osmoregulatory and cardiovascular hor-mones are diversified and form unique families of paralo-gous peptides in different vertebrate groups. As will bediscussed in detail below, ion-regulating hormones are par-ticularly diversified in teleost fish, probably to maintain ionbalance in hypotonic fresh water (FW) and/or hypertonicseawater (SW) environments. Similarly, vasodepressor(hypotensive) hormones have several additional paralogsin teleost fish, which is probably because of the low bloodpressure in aquatic species. These hormones include natri-uretic peptide (NP), adrenomedullin (AM) and guanylin,which were discovered recently in mammals. By contrast,water-retaining and vasopressor hormones such as antidi-uretic hormone (vasopressin) and angiotensin II have beenknown in mammals for almost a century because of theirimportant roles in water and salt retention and blood pres-sure regulation in terrestrial animals. Thus, the majorosmoregulatory and cardiovascular hormones have chan-ged during evolution according to their relative importancein different habitats.

By a comparative genomic approach, we can estimatethe chromosomal location of a fish gene in humans andother mammals. Using this technique, we attempted tolocate the ion-regulating and vasodepressor hormone genesin mammals that are diversified in teleost fish, and foundthat the orthologs of some of these genes also exist in mam-mals. In this review, therefore, we introduce a new phylo-genetic approach for the identification of new hormonesin mammals and other tetrapods based on the differencein the body fluid and blood pressure regulation betweenaquatic and terrestrial animals.

2. Evolution of the regulatory system in vertebrates

2.1. Difference in body fluid regulation between fishes andtetrapods

The mechanisms of body fluid regulation differ greatlyamong animals in different habitats. Among the aquaticvertebrates, marine species that have plasma osmolalityca. one third of SW (teleost fish, reptiles, birds and mam-mals) are constantly faced with the problem of dehydrationand excess ions entering the body. The condition is espe-cially severe in marine teleosts in which thin and extensiverespiratory surfaces of the gill serve as a route for the lossof water and the gain of ions [76]. However, they can com-pensate for the loss of water by drinking environmental SWand absorbing water together with Na+ and Cl�. Theexcess Na+ and Cl� absorbed with water are excretedthrough mitochondria-rich (chloride) cells of the gill.Therefore, marine fish do not suffer from dehydration eventhough the hyperosmotic marine environment is morestressful than the terrestrial environment in terms of water

retention. This shows that the mechanisms for Na+ extru-sion, not water retention, are the most important for sur-vival in SW (Fig. 1).

By contrast, FW fish are constantly exposed to the prob-lems of over-hydration and hyponatremia; water enters thebody by osmosis and Na+ and Cl� leave the body downconcentration gradients in the gill [76]. Thus, FW fish drinklittle water and excrete copious amounts of dilute urine tocope with over-hydration, while they take up Na+ in thegill in exchange for protons [152]. Therefore, the mecha-nism for Na+ retention, rather than water extrusion, isessential for fish to survive in FW (Fig. 1).

In contrast to aquatic species, terrestrial animals had todevelop mechanisms to increase the gain and decrease theloss of water [10,111]. Accordingly, drinking (thirst) isstimulated by multiple mechanisms such as cellular andextracellular dehydration and angiotensin II [37], and mostof the filtered water in the primary urine is reabsorbed bythe kidney [10]. Furthermore, land is generally a Na+-defi-cient environment and terrestrial animals, particularly gra-nivores and herbivores whose food is deficient of Na+, havedeveloped mechanisms to retain Na+ in the body; e.g.,induction of sodium appetite and reabsorption of Na+ bythe kidney [124]. An important role of Na+ for retentionof blood volume is illustrated by the fact that drinking ofplain water after hypovolemia decreases plasma osmolalityand thus vasopressin secretion, resulting in the urinaryexcretion of ingested water, but drinking of isotonic salinerestores blood volume. Thus, terrestrial animals have toretain both water and Na+ to maintain body fluid balance,although water retention is of primary importance to sur-vival on the land (Fig. 1). Further, water and Na+ are reg-ulated in the same direction (for retention) in tetrapods,

Y. Takei et al. / Frontiers in Neuroendocrinology 28 (2007) 143–160 145

but they are regulated in the opposite direction in fishes;water is retained and Na+ is extruded in SW fish andNa+ is retained and water is extruded in FW fish. There-fore, the mechanisms for regulating body fluid balance dif-fer greatly between animals in different habitats.

2.2. Osmoregulatory hormones

Osmoregulatory or body fluid-regulating hormones aregenerally categorized into two groups depending on thetime course of their actions [81,129]. The fast- and short-acting hormones are secreted quickly in response tochanges in body fluid balance and disappear from the cir-culation in seconds to minutes. These hormones act onthe brain to modulate thirst and sodium appetite, and alsoon a variety of osmoregulatory organs to change theactivity of existing transporters/channels in the transportepithelia. These hormones are usually oligopeptide hor-mones such as angiotensin II, vasopressin/vasotocin,ANP, and guanylin. The slow- and long-acting hormonesare secreted slowly in response to changes in body fluid bal-ance and stay in the circulation for hours to days. Theychange the structure and function of osmoregulatoryorgans by de novo synthesis of transporters/channels andadhesion proteins for long-term adaptation to a new osmo-tic environment. After transfer of euryhaline fish from FWto SW, for instance, branchial chloride cells change theirlocation (from primary to secondary lamella), morphology(development of tubular system) and function (fromabsorption to secretion of Na+ and Cl�). Slow- and long-acting hormones are high-molecular-weight proteins or ste-roids such as growth hormone, prolactin and mineralocor-ticoids. It is worth noting that the fast- and short-actinghormones often alter the secretion of other long-acting hor-mones before disappearing from the circulation. For exam-ple, angiotensin II and ANP modulate anterior pituitaryhormone and mineralocorticoid secretion in fishes and tet-rapods [6,32,40,69].

Reflecting the differences in body fluid regulation, majorosmoregulatory hormones are different between fishes andtetrapods [10,124]. In tetrapods, water-retaining hormonessuch as vasopressin/vasotocin and angiotensin II playessential roles. Vasopressin/vasotocin is a powerful antidi-uretic hormone that acts on the renal tubule in truly terres-trial animals (mammals, birds and reptiles), and on theurinary bladder in semi-aquatic amphibians [9]. It also pro-motes water uptake by the skin of amphibians [1]. Angio-tensin II is a potent dipsogenic hormone in all tetrapodspecies [37], and it also acts on the frog skin to promotewater and Na+ absorption [68]. In addition, aldosteroneis a long-acting hormone that increases Na+ absorptionin the renal tubules of terrestrial species, which indirectlypromotes water retention [117]. These water- and Na+-retaining hormones were discovered in the early 20th cen-tury, as an excess or deficiency of such hormones resultedin severe fluid and pressure imbalances such as diabetesinsipidus, hypertension, and hyperaldosteronism

[12,45,78]. Further, manipulation of the vasopressin gene[44] and the genes of the renin–angiotensin system [30] inmice induces similar fluid and pressure imbalance thatleads to death, if the balance is not restored by an interven-tion. In contrast, water- and Na+-extruding hormones havenot been found until the presence of a potent natriureticand diuretic factor was reported in the heart in 1981 [29].The factor, later known as ANP, was not discovered earlierprobably because of its minor role in the maintenance ofnormal water and salt balance. In fact, disruption of theANP gene in mice did not alter normal blood volumeand plasma osmolality [83]. However, the regulatory roleof ANP is highlighted when normal water balance is dis-rupted or in the case of cardiac and renal failure [107].For example, mice lacking the ANP gene that are fed ahigh Na+ diet have severer hypertension than their normallittermates [83]. More recently, AM and guanylin havebeen discovered and found to be water and Na+ extrudinghormones [39,140]. Thus, in mammals, water- and Na+-retaining hormones have a longer history of research thanextruding hormones.

In contrast to terrestrial animals, ion-regulating hor-mones appear to be more important in fishes [81]. Forinstance, vasotocin, a vasopressin homolog, is not an anti-diuretic hormone in fish [9]; its exogenous administrationusually causes diuresis because of its potent vasopressoreffect, and it is only weakly antidiuretic through the glo-merular effect when blood pressure is kept constant in thetrunk preparation of trout [3]. Angiotensin II inducesdrinking in fish, but the potency is much lower than thatin mammals and birds [126]. Angiotensin II acts on the gillto increase Na+,K+-ATPase activity in SW eels [77]. In tel-eost fish, aldosterone synthesis is negligible, so that cortisolacts not only as a glucocorticoid but also as a mineralo-cortocoid and is involved in both SW and FW adaptation[149]. In addition, growth hormone and insulin-like growthfactor I (IGF-I) are involved in SW adaptation in salmo-nids and other species [81,133]. Cortisol and growth hor-mone/IGF-I act in concert to increase the number ofSW-type chloride cells in the gill that excrete Na+ andCl� [80]. Prolactin is an important hormone for FW adap-tation in all teleost species thus far examined [81,133]. Pro-lactin decreases osmotic permeability to water in varioustransport epithelia [50], but its action on Na+ and Cl�

transport has not been examined yet. It seems that majorosmoregulatory hormones in teleosts have their primaryactions on Na+-regulation, not water-regulation asobserved in tetrapods.

Compared with Na+-retaining hormones, Na+-extrud-ing hormones seem to be predominant in teleost fish. Atypical example is ANP, which is a major Na+-extrudinghormone in eels and promotes adaptation to SW. ANP issecreted in response to the transfer of eels from FW toSW and it acts to eliminate Na+, but not water, from thebody (Fig. 2) [129]. ANP inhibits drinking and subsequentabsorption of Na+ by the intestine in SW eels at a low dosethat is without vascular effects [145]. In the kidney, ANP

Fig. 2. Comparison of biological actions of atrial natriuretic peptide(ANP) in seawater (SW)-adapted eels (a) and mammals (b). The majordifferences are stimulus for ANP secretion (volume vs. osmotic) and renaleffects (diuretic vs. antidiuretic). The effect on mineralocorticoid secretionis also opposite. However, ANP stimulate Na+ excretion in both cases, asaldosterone retains but cortisol extrudes Na+ by activation of Na+, K+-ATPase. Cortisol and growth hormone are known as long-acting, SW-adapting hormones in teleost fish. For details, see text.


increases urinary Na+ concentration but decreases urinevolume [132]. Therefore, ANP is antidiuretic in the eel,but not natriuretic because of the decreased urine volume.ANP does not act directly on the gill to increase Na+,K+-ATPase activity, but it stimulates the secretion of cortisoland growth hormone, thereby increasing the number ofexcretory type chloride cells that function in long-termSW adaptation [40,69]. Therefore, ANP maintains plasmaNa+ concentration in SW by inhibiting drinking and intes-tinal Na+ absorption, which results in the facilitation ofSW adaptation. In fact, removal of circulating ANP inSW eels by immunoneutralization increases drinking andplasma Na+ concentration [145]. In contrast to teleost fish,ANP is a hormone that decreases blood volume in mam-mals by excreting both water and Na+ from the body(Fig. 2). This indicates that ANP is originally a Na+-extruding hormone in fish but becomes a volume-depletinghormone in terrestrial animals as water and Na+ are regu-lated in the same direction (Fig. 1). The parallel movementof water and Na+ may be due to the development of waterchannels on the transport epithelia in tetrapods.

Guanylin is recognized as a volume-depleting hormonein mammals because of its potent action on fluid secretioninto the intestinal lumen, which is induced by Cl� secretionthrough the CFTR Cl� channels [39]. In eel, guanylin alsoseems to induce Cl� secretion into the lumen throughCFTR [157]. However, the secreted Cl� may be used tomobilize absorptive-type Na+–K+–2Cl�-cotransportersthat are located on the apical membrane of intestinal epi-thelial cells, resulting in increased fluid absorption. Thus,guanylin may also be a SW-adapting hormone in teleostfish.

Although several Na+-extruding hormones are knownin teleost fish, a major Na+-retaining hormone that pro-motes FW adaptation has not yet been identified. The mostimportant FW-adapting hormone, prolactin, may possiblyincrease Na+ uptake from the low ion environments via thegill, but no data are yet available in teleost fish though sucheffect is suggested in the rat nephron [17]. We recentlyfound that CNP, a member of the NP family, stimulates22Na uptake by the gills and increases plasma Na+ concen-tration in FW eels [145], but more hormones that take upNa+ from environments should exist in teleost fish. Asmentioned earlier, the evolution of early fishes occurredin FW in the early Paleozoic era [106,118]. Subsequently,teleost fish re-entered SW in the Mesozoic era after theyobtained an ability to extrude excess Na+ via mitochon-dria-rich cells that developed in the gills. As there were onlya small number of competitors, teleost fish experienced anexplosive species diversification in SW. Thus, teleost fishalone comprise more than half of the total vertebrate spe-cies [94]. The species diversification in SW may be relatedto the fact that Na+-extruding hormones are much diversi-fied in teleost fish.

2.3. Differences in cardiovascular regulation in fishes and

tetrapods

All animals are exposed to the effects of gravity on theirbody fluids. Thus, terrestrial animals have developed a car-diovascular system that circulates blood against the gravi-tational force, in which the heart plays a central role(Fig. 1). Among cardiac tissues, the ventricle is responsiblefor the maintenance of powerful circulation, which isensured by thick and dense cardiac muscles and a constantsupply of oxygenated blood to them via the coronary arter-ies [52]. Accordingly, high cardiac performance and higharterial pressure are characteristics of the heart of terres-trial animals.

In contrast, the effect of gravity on body fluids is almostnullified by the buoyancy produced by the high specificgravity of water in aquatic animals (Fig. 1). Therefore,all body fluids are shifted to the central part of the body,and the heart can easily pump blood to supply the organs[33]. Arterial blood pressure of fishes is generally low com-pared to mammals, 20–30 mm Hg in average at the dorsalaorta, except for some active swimmers like tuna [15]. Theventricular muscles of teleost fish are generally loose, and

Orthodox com

parative study

Fishes

Tetrapods

New hormoneNew function

‘Reverse’phylogenetic approach

Discovery ofdiversified hormones

Importantfunction

Genomedatabase

Fig. 3. A schema illustrating the concept of ‘reverse’ phylogeneticapproach. Previously, hormones were first discovered in mammals andapplied to fishes. Recently, however, some new hormones are discoveredfirst in fish then in mammals. In particular, new Na+-extruding andvasodepressor hormones are identified in tetrapods as paralogs ofdiversified fish hormones. Further, new and essential function of knownhormones can be extracted from fish studies. This approach was named‘reverse’ phylogenetic approach as it is opposite from the previousdirection.


blood cells can directly contact each myocyte to supplyoxygen to the cell. Therefore, ischemic heart failure isimprobable in fish even with a poorly developed coronarysystem [35]. Furthermore, circulating blood volume of tel-eost fish (3–4% of body weight) is much smaller than thatof terrestrial animals [49].

2.4. Cardiovascular hormones

Consistent with the differences in the cardiovascular sys-tem, the major cardiovascular hormones differ betweenaquatic fishes and terrestrial tetrapods, particularly endo-thermic species. In mammals and birds, vasopressor andcardiotropic hormones such as angiotensin II, vasopres-sin/vasotocin, endothelins, and urotensin II play essentialroles in the maintenance of high cardiac performance andarterial pressure [10]. Vasopressin and angiotensin II areparticularly important as knockout of these genes resultedin a hypotensive phenotype in mice as mentioned above.On the other hand, vasodepressor hormones do not seemto be important for the maintenance of blood pressure.For instance, disruption of the ANP gene did not affectarterial pressure in mice with normal hydromineral balance[83], although knockout of other vasodepressor genes suchas AM are sometimes lethal for other reasons during theearly development [19]. These findings suggest that vaso-pressor hormones are more important than vasodepressorhormones for the maintenance of high arterial pressure interrestrial tetrapods.

In fishes, angiotensin II, vasotocin, endothelin and uro-tensin II are all potent vasopressor hormones [133]. How-ever, the role of these hormones in the maintenance ofarterial pressure may be minimal in teleost fish, althoughthe effect of disruption of their genes has not yet beenexamined. On the other hand, vasodepressor hormonessuch as NP and AM are highly potent in fish [133]. Fur-thermore, these vasodepressor hormones are much diversi-fied and form unique families of paralogous peptides inteleost fish. This suggests that vasodepressor hormonesare more important than vasopressor hormones for themaintenance of low arterial pressure in aquatic fishes.

3. A ‘reverse’ phylogenetic approach

In comparative endocrinology, studies of the structuresand functions of hormones across phylogeny can revealnew insights into the fundamental biology of hormones.The power of this comparative approach derives from thegreat variety of species of different phylogenetic status,and their occupancy of diverse environmental and ecologi-cal niches. Historically, the discovery of non-mammalianhormones followed, and was guided by, earlier knowledgein mammals of homologous hormones to which clinical sig-nificance was often attached (Fig. 3). Recently, and in con-trast, the discovery of hormones in fishes has led to theidentification of homologs in mammals. This is due to ourexpanding knowledge about the basic biology of fishes

[28,57,76,94,133], and to the recent establishment ofgenome databases for a growing number of fish species.There are several excellent reviews that summarize the ben-efits of the phylogenetic approach using vertebrates andinvertebrates, and that have contributed to our knowledgeabout the structure and function of hormones e.g.,[18,22,27,54,74,86,114,115]. In this review, the focus is onosmoregulatory and cardiovascular hormones in fishesand the linkage to hormone discovery in mammals, withemphasis on differences between aquatic and terrestrial ani-mals in body fluid and blood pressure regulation (Fig. 3).

3.1. From fish to mammals

There seem to be two major routes for the discovery ofnovel hormones in mammals based on fish studies. The firstof these depends on the identification of new hormones infish-specific endocrine organs such as the caudal neurose-cretory system and corpuscles of Stannius [121]. The neuro-peptides urotensins I and II were isolated from theurophysis, a neurohemal organ of the caudal neurosecre-tory system located at the distal end of the spinal cord ofbony fishes [102]. Urotensin I shares sequence similarityand an evolutionary origin with hypothalamic corticotro-phin-releasing hormone (CRH), and stimulates the hypo-thalamus-pituitary-interrenal axis [73]. Urotenisn II isstructurally similar to somatostatin and increases arterialpressure through a direct action on the vascular smoothmuscle [8]. Stanniocalcin was isolated from the corpuscleof Stannius, a fish-specific organ that is buried in the renaltissue, and it exerts potent hypocalcemic action [148]. The


orthologs of urotensins and stanniocalcin have now beenidentified in mammals and been found to play importantroles in mammals also [123]. Furthermore, urotensin Iacquired new functions in mammals and the gene dupli-cated to produce paralogous hormones perhaps associatedwith adaptation to terrestrial life. These paralogous hor-mones have different names in mammals, being urocortin(urotensin-like corticotropic hormone) and stresscopin (tocope with stress) [123]. Urotensin II is the most potentvasoconstrictor known in the human, even more potentthan endothelin [4]. Stanniocalcin is expressed in the renaltubules of mammals and appears to be involved in phos-phate transport [99]. A paralog, named stanniocalcin 2,was discovered in mammals [21], which leads back to thediscovery of the ortholog in teleost fish [75]. In additionto the fish-specific endocrine organs, Kawauchi andcoworkers have discovered two new pituitary hormonesin salmonids, somatolactin [100] and melanin concentrat-ing hormone (MCH) [66]. Somatolactin belongs to thegrowth hormone family that is composed of growth hor-mone (somatotropin) and prolactin, and is named afterthese two hormones. However, somatolactin has not beenidentified in tetrapods including amphibians. MCH is, asits name indicates, an albinic hormone secreted from theneurointermediate lobe of the pituitary in teleost fish [92].MCH is now identified in the hypothalamus of mammalsand found to be a major appetite-inducing hormone[144]. In fact, knockout of the MCH gene results in a leanphenotype [116].

The second of the two routes for the discovery of newhormones is the identification in mammals of paralogs ofdiversified fish hormones. Some examples will be intro-duced below. Hsu and colleagues have taken a similar com-parative approach in combination with the reversepharmacologic approach and have identified several newhormones such as intermedin and relaxin 3 [54,105]. Themain feature of our approach is to target osmoregulatoryand cardiovascular hormones in the context of habitat dif-ferences between fishes and tetrapods.

3.2. History and benefits of ‘reverse’ phylogenetic approach

We have been seeking an essential hormone for SWadaptation in teleost fish. Although the SW environmentis even harsher than on land in terms of water retention,hormones that are indispensable for SW adaptation arenot known. However, we found that Na+-extruding hor-mones are more important than water-retaining hormonesfor SW adaptation, since SW fish can easily replenish inter-nal water by drinking and excretion of excess Na+ from thebody. Furthermore, we found that the major Na+-extrud-ing hormones (NPs, AMs and guanylins) identified in tele-ost fish are also vasodepressor and are diversified in teleostfish compared with mammals [55,98,156]. The duplicatedgenes acquired new functions and may have been retainedin teleost fish. Due to the recent technical advances in com-parative genomics and bioinformatics, we can now track a

teleost gene to a chromosome in mammals and determinethe fate of that gene [57]. Using this technique, we identi-fied a number of new hormones in mammals [97,131].Some of the duplicated teleost hormones seem to have dis-appeared in mammals, but they still have bioactivity inmammals probably because the hormone receptor gene stillexists and is expressed. Therefore, this research flow, i.e.,identification of diversified hormones in fish and subse-quent discovery of the orthologs in mammals, appears tobe promising as a new approach in endocrinology.

In recent years, many G-protein coupled receptors witha seven membrane spanning domain have been identified inthe human genome database because of their conservedsequence. Some of these receptors are most likely to be hor-mone receptors, but they have ‘orphan receptor’ statusbecause their ligands are unknown. Using the orphanreceptors, new ligand hormones have been discovered.Since orthodox research direction is from the hormone toits receptor, this approach is called ‘reverse’ pharmacology[25]. Similarly, since the phylogenetic approach we suggesthere, from fish to mammals, is opposite to the earlier direc-tion in comparative studies, we suggest here that the newapproach be referred to as ‘reverse’ phylogenetic (Fig. 3).In the following sections, we will introduce some examplesof the identification of new hormones in teleost fish andtheir subsequent discovery in mammals, using thisapproach.

4. The natriuretic peptide family

The first member of the NP family, ANP, was isolatedfrom the rat atria as a diuretic/natriuretic and hypotensivefactor [59], which was followed by the discovery of brainand C-type NP (BNP and CNP) [125]. The basic structureof NP consists of an intramolecular ring formed by a disul-fide bond and N-terminal and C-terminal ‘tail’ sequences;CNP lacks the C-terminal ‘tail’. Cardiac ANP and BNPare not only potent natriuretic/diuretic hormones[51], butthey are also important cardioprotective hormones thatdecrease cardiac preload and afterload [84]. Thus, bothpeptides are widely used for diagnosis and treatment of car-diac failure. In the brain, ANP antagonizes various aspectsof angiotensin II actions on thirst and sodium appetite,vasopressin secretion, and sympathetic nerve activity[14,125]. CNP is a true neuropeptide that is abundantlyexpressed and stored in the brain, but its central action isstill obscure [127].

4.1. Non-mammalian natriuretic peptides (NPs)

ANP, BNP and CNP are common members of the NPfamily identified in mammals. In other tetrapods, BNPand CNP are isolated from the chicken, and ANP, BNP,and two CNPs (CNP-I and CNP-II) are found in amphib-ians [125,143]. In teleost fishes, ANP, CNP, and a new NPisolated from the cardiac ventricle, named VNP, were iden-tified in eel and trout [72,134]. ANP, BNP and VNP are


cardiac hormones, while CNP is a brain hormone in birds,amphibians and teleosts (see [143]). In elasmobranchs(sharks and rays), only CNP has been detected in the heartand brain [72,125]. Among NPs, CNP is the most highlyconserved with identity of 85% even between mammalsand elasmobranchs. The sequence identity of ANP is highin mammals but low between mammals and teleosts. BNPis highly variable within mammalian species, but more con-served than ANP between mammals and teleosts. Althoughtetrapod BNP and teleost VNP have only a low identity,they are thought to be homologous peptides [125]. Judgingfrom the presence in elasmobranchs and the high sequenceidentity, CNP is thought to be an ancestral molecule of theNP family, from which cardiac ANP and BNP/VNP aregenerated. ANP and BNP seem to have been producedby tandem duplication as these two genes are located onthe same chromosome in human [136].

In the genome database of pufferfish, four CNP genes(CNP-1, -2, -3 and -4) and a BNP-like gene were detected[55]. The four CNP genes are localized on different chro-

M. glutinosa NP

E. burgeri NP

PufferfishCNP-4

Pufferfish CNP-3

Medaka CNP-3

Pufferfish CNP-1

Medaka CNP-1

Medaka BNPPufferfi

MedakaCNP-4

100

10095

99

100

96

62

CNP-4

CNP-1

CNP-3

BNP

57

Hagfish NP

Fig. 4. Phylogenetic tree of cyclostome natriuretic peptides (NPs) and seven temethod. The numbers on the interior nodes are bootstrap values in percent.

mosomes, and the ANP and BNP gene are found withthe CNP-3 gene on the same chromosome. Therefore, itseems that the ANP and BNP genes are generated by tan-dem duplication from the CNP-3 gene. In the eel and trout,BNP cDNA was isolated in addition to ANP and VNP[56]. Therefore, the NP family consists of seven members(ANP, BNP, VNP, and CNP-1 through -4) in teleosts,although some of the NP genes seem to have disappearedin some species. Cardiac NPs (ANP, BNP, and VNP) havehigh affinity to A-type NP receptor (NPR-A), while brain-specific NPs (CNP-1 and CNP-2) have high affinity toNPR-B [129]. CNP-3 and CNP-4 are expressed in bothbrain and heart and exhibit affinity to both NPR-A andNPR-B in medaka [55].

Synteny analyses and phylogenetic analyses of NP precur-sors showed that mammalian CNP is an ortholog of teleostCNP-4, avian CNP is CNP-3, amphibian CNPs are CNP-3and CNP-4, and elasmobranch CNP is CNP-3 [55]. Interest-ingly, teleost CNP-1, not CNP-3 and CNP-4, is most similarto mammalian CNP-4, avian CNP-3, amphibian CNP-3 and

sh BNP

Trout VNP

Eel VNP

Tilapia ANP

PufferfishANP

Pufferfish CNP-2

Medaka CNP-2

G. australis CNP

L. japonica CNP

99100

100

64

100

100

51

CNP-2

ANP

VNP

Lamprey CNP

leost NPs depicted on the precursor sequences using maximum likelihood


elasmobranch CNP-3 at the mature sequence, although theprosegment is dissimilar between different CNP species. Thismay exemplify convergent evolution due to the binding toNPR-B. The exon–intron structure is identical between theCNP-1 and CNP-2 genes and between the CNP-3 andCNP-4 genes [55]. Furthermore, the CNP-1, CNP-2 andCNP-3 genes are linked to the three different enolase genes,respectively. These data indicate that the CNP-1 and CNP-2 genes and the CNP-3 and CNP-4 genes are duplicated ineach combination, and that the CNP-1 or CNP-2 gene is pro-duced by block duplication from the CNP-3 gene after link-age with the enolase gene.

4.2. Early evolution of the NP gene

It appears that CNP-4 may be the ancestral molecule ofthe NP family, as its gene is not liked to the enolase gene.To assess the ancestral gene further, we searched for NP in

Amphibian Reptileb

Lobe-finnedbony fish

C2

C3

C4

C1A

B

V

C2

C3

C4

C1A

B

V

Ancestral NP(CNP-4)

565 Myr ago

528 M

AC2 C1C3 VC4

CartilaginousfishCyclostome Ray-f

bony

a

Fig. 5. (a) Evolutionary history of the seven members of the natriuretic peptid(C3), and CNP4 (C4) in vertebrates. The process of diversification is shown by li(b) A detailed picture showing the fate of seven NPs in tetrapods. The X mark

the most primitive extant vertebrates, cyclostomes (lam-prey and hagfish). A CNP cDNA was cloned from threelamprey species in different genera [64], and a NP cDNAthat possessed a C-terminal ‘tail’ sequence was cloned fromthree hagfish species in different genera [65]. Both cyclo-stome NPs were abundantly expressed in the heart andbrain. Phylogenetic analyses showed that both are clus-tered with teleost CNP-4 (Fig. 4). Thus, it is most likelythat the CNP-4 gene is the ancestral gene, from whichthe CNP-3 gene is first generated and linked to the enolasegene.

It is known that the whole genome duplication occurredthree times during vertebrate evolution; the first round(1R) at the transition from chordates to vertebrates, thesecond round (2R) at the transition from agnathans tognathostomes, and the third round (3R) after divergenceof the teleost lineage. As CNP-1 and CNP-2 were identifiedonly in teleosts, one of the genes may be duplicated from

Bird Mammal

C2

C3

C4

C1A

B

V

C2

C3

C4

C1A

B

V

yr ago

450 Myr ago

C4B C4BAC1 VTetrapod

inned fish

C3 C3

e family, ANP (A), BNP (B), VNP (V), CNP-1 (C1), CNP-2 (C2), CNP-3nes. The numbers below the tree show the approximate time of divergence.on the NP shows ‘undetectable’. The question mark shows ‘not examined’.


the CNP-3 gene at the 3R (Fig. 5). However, four CNPcDNAs were identified in the primitive ray-finned fish,sturgeon (Acipenser transmontanus) and bichir (Polypterus

endlicheri), which had not experienced the 3R [63,147].Furthermore, as multiple enolase genes exist in elasmo-branchs, the CNP-1 and CNP-2 genes may have been pro-duced by block duplication from the CNP-3 gene beforethe divergence of ray-finned fish.

4.3. Evolution of cardiac NP genes

ANP, BNP and VNP are three cardiac NPs originatedfrom CNP-3 (Fig. 5). As the ANP, BNP and VNP genesare expressed almost exclusively in the heart [63,147], theseNPs are circulating hormones specialized in body fluid andcardiovascular regulation. The relative importance for car-diac protection may be increased in tetrapods, in which theheart must maintain high performance to circulate bloodagainst gravity. In mammals, ANP and BNP stimulatethe same receptor but have some different roles; whileANP is secreted from atria via a regulatory pathway inresponse to an increased blood volume, BNP is secretedvia a constitutive pathway mainly from the ventricle inmammals [154]. These multiple cardiac NP genes may havesurvived during evolution by gaining different regulatoryand functional roles.

Among 10 teleost species examined, only eel and salmo-nids had VNP in the heart. As VNP was detected in themore primitive ray-finned fish, bichir and sturgeon, butnot in the more advanced group, the VNP gene may be lostduring teleost evolution [56]. In addition, ANP was unde-tectable in the heart and the genome of Japanese medaka.Thus, the only cardiac NP common to all teleost species isBNP. In tetrapods, all mammalian and amphibian speciesthus far examined possess both ANP and BNP in the heart.In birds, however, only BNP was detectable in the heartof chicken (Fig. 5). In reptiles, only BNP cDNA wasisolated from the heart of crocodile, Crocodylus porosus(S. Trajanovska, J.A. Donald, unpublished data). Thus,BNP is the common cardiac NP in tetrapods. Since allvertebrate species that have heart-specific NPs have BNP,it is possible that BNP, but not ANP as previously thought,is the basic cardiac NP in vertebrates.

Linkage analysis in the rainbow trout that possessesall cardiac NPs, ANP, BNP and VNP, showed that thesethree genes are located in tandem on the same chromo-some [56]. A recent study to determine the order of thesegenes using genomic DNA of eels revealed that the VNPgene is localized upstream of the BNP gene in the sameorientation (K. Inoue, unpublished data). Therefore, theNP genes are located in the order of CNP-3, VNP,BNP and ANP on the chromosome of eel. The orderof duplication of ANP, BNP and VNP from the CNP-3 gene is not known. To summarize the cardiac NPs, itis now known that (1) BNP is retained in all vertebratespecies thus far examined, (2) ANP may be lost in some

teleosts, reptiles and birds, and (3) VNP is retained onlyin primitive ray-finned fish.

4.4. New NPs identified in tetrapods

It seems that four CNP genes and three cardiac NP geneshad existed before the divergence of ray-finned fish and lobe-finned fish (coelacanth and lungfish) from which tetrapodswere derived. Accordingly, some of these genes may still existin the tetrapod lineage. Thus far, CNP-4 is identified in mam-mals and amphibians, and CNP-3 in birds and amphibians,but CNP-1 and CNP-2 have never been identified in the tet-rapod lineage [55]. Concerning cardiac NPs, BNP is identi-fied in all tetrapod species and ANP is found in mammalsand amphibians, but VNP has not been identified in the tet-rapod lineage (Fig. 5). In the genome database of chicken,Gallus gallus, where new data are gradually accumulating,we recently identified two new NP genes (S. Trajanovska,K. Inoue, Y. Takei, J.A. Donald, unpublished data). Thefirst gene is located between the CNP-3 and BNP genesand is most probably the VNP gene as judged by its locationon the chromosome. The gene was highly expressed in thekidney but only weakly in the heart of chicken. The secondis the CNP-1 gene as judged by the synteny analysis of neigh-boring genes and by the phylogenetic analysis of precursors(Fig. 5). Thus, it appears that the orthologs of CNP-1 andVNP still exist in the tetrapod lineage, although we couldnot detect these genes in human, mouse and Xenopus inwhich genome data are available.

Although extensive searches for the VNP gene in thehuman genome database have not been successful thus far,there is circumstantial evidence suggesting the presence ofa third cardiac NP in the human. A unique NP, namedDNP, has been isolated from the venom gland of the snake,Dendroaspis angusticeps, which is structurally similar toVNP in terms of long C-terminal ‘tail’ sequence [112]. Sincethe venom gland often expresses organ-specific genes that arenot expressed in other tissues, DNP is most likely to beunique to the venom of this poisonous snake. However, spe-cific antisera raised against DNP measured DNP-like immu-noreactivity in the human heart, and its plasma levelincreased in patients with cardiac failure [104]. Since the anti-serum does not cross-react with human ANP, BNP andCNP, the presence of new NP-like peptide is suggested inthe human heart. An extensive search for a new NP sequenceon human chromosome 1, on which VNP should exist withANP and BNP, has not yet been successful probably becauseof changes in critical amino acid residues that are conservedin all NPs thus far identified. Therefore, there remains a pos-sibility that a novel cardiac NP exists in mammals.

4.5. Evolutionary history of the natriuretic peptide family

Fig. 5 summarizes the evolutionary history of the NPfamily in vertebrates. The ancestral NP is most likely tobe CNP-4 type, but it may have a long C-terminal ‘tail’sequence like hagfish NP. The ancestral CNP-4 gene may

AM

Brain

Blood vessel

Skin

Kidney

Lung

Heart

• vasodilation• oxidative damage• angiogenesis

• contractility• heart rate

antimicrobic

• diuresis• natriuresis

• sympathetic tone• food intake• water intake• sodium appetitebronchodilation

Pituitary

• oxytocin• prolactin• ACTH

Fig. 6. Biological actions of adrenomedullin (AM) thus far reported inmammals. In terms of osmoregulatory and cardiovascular actions, AM isa Na+-extruding and vasodepressor hormone. For details, see text.

CLR

CGRP AM

CTR

AMY CT CRSP

1

2

3

ligand

RAMP

receptor

AM2

1

33

Fig. 7. Ligand selectivity of the CGRP family peptides to receptor andreceptor activity-modifying protein (RAMP) complex. AM, adrenomed-ullin; AMY, amylin; CGRP, calcitonin gene-related peptide; CRSP,calcitonin receptor-stimulating peptide; CT, calcitonin; CTR, calcitoninreceptor; CLR, calcitonin receptor-like receptor.


have been duplicated to generate the CNP-3 gene at the 2Rwhole genome duplication that occurred at the transitionto the jawed vertebrates. The CNP-4 gene seems to havebeen silenced in elasmobranchs. It is not known whetherthe heart-specific NP genes already existed in the cartilagi-nous fish, but abundant expression of the CNP-3 gene inthe heart indicates that CNP-3 acted as a cardiac NPbefore generation of the ANP, BNP and VNP genes. How-ever, three cardiac NPs and four CNPs existed before the3R whole genome duplication. This means that all sevengenes were further duplicated once in the teleost lineage,but the duplicated counterparts disappeared during thetime span of 320 million years after the 3R [146]. Two sub-types of NP receptor, NPR-A, were cloned in medaka andthey are shown to be generated by the 3R [153]. Therefore,the history of multiplication of the NP system appears todiffer between ligands and receptors.

It seems that several NP genes have disappeared in tet-rapods, particularly CNP genes, but cardiac NP genes suchas ANP and BNP are retained. The retention of the cardiacNP genes in tetrapods may be related to the protection ofthe heart, as ANP and BNP are known as important car-dioprotective hormones. However, all cardiac NPs, ANP,BNP and VNP, exist in primitive ray-finned fish includingbichir, sturgeon and some primitive teleost species (eel andsalmonids). These aquatic species need not have a powerfulheart to pump blood against gravity. The sturgeon, eel andsalmon are migratory species that experience both FW andSW during their life span. Thus, the cardiac NPs may havemajor functions in body fluid regulation in these fishes, butthey obtained a new function as cardioprotective hormonesin tetrapods. Since three cardiac NPs and four CNPs existin the eel and trout, comparison of their osmoregulatoryand cardiovascular effects may elucidate why such multipleNPs are retained in these teleost species.

5. The calcitonin gene-related peptide family

The calcitonin (CT) gene-related peptide (CGRP) familyis composed of the structurally conserved peptides, CGRP,AM, amylin (AMY) has important functions in mammals[87,88]. Recently identified CT receptor-stimulating peptide(CRSP) is also included in this family [62]. All of these pep-tides have an intramolecular ring of 6 amino acid residuesformed by a disulfide bond and an amidated C-terminus.Two CGRP genes have been identified in primates androdents, each coding for CGRP-a and CGRP-b that arealmost identical at the mature peptide region. CGRP wasfirst isolated from the rat brain [2], and later shown toact on the brain vasculature to modulate pain production[7,11]. In the periphery, CGRP is a powerful vasodilatorand thus decreases arterial pressure [5]. CT is another bio-active peptide produced by alternative splicing from theCGRP gene [2]. CT is secreted in response to an increasein plasma Ca2+ concentration, and acts to promote reab-sorption of Ca2+ to the bone and urinary excretion ofCa2+ [28], and decreases pain perception [79]. AM was first

isolated from the culture medium of human pheochromo-cytoma [67] and exerts various functions in many tissuesincluding vasodepressor and diuretic/natriuretic effects(Fig. 6) [71]. In addition, AM exerts multiple centralactions such as stimulation of sympathetic tone [108], mod-ulation of behaviors [90,109,137] and pituitary hormonesecretion [113,141]. AMY, or islet amyloid polypeptide,was first isolated from the amyloid of the islet of Langer-hans [151]. AMY is secreted from pancreatic b-cellstogether with insulin in response to an increase of plasmaglucose level [36]. CRSP was isolated from the porcinebrain [61]. CRSP is presumed to be the cardinal ligand ofthe encephalic CT receptor (CTR) because CT mRNA isnot expressed in the brain.

The CGRP family of peptides exhibit biological actionsthrough two types of receptors, CTR and CTR-like recep-tor (CLR), coupled with one of the receptor activity-mod-ifying proteins (RAMPs) (Fig. 7) [82]. CGRP binds to theCLR and RAMP1 complex (CLR-RAMP1) with highaffinity, and AM to CLR-RAMP2 or CLR-RAMP3[103]. AMY has high affinity to CTR-RAMP1 or CTR-RAMP3 [89]. Both CT and CRSP stimulate CTR alone,with CRSP exhibiting higher affinity [60].


5.1. New adrenomedullin family in teleost fish

AM is more potent than CGRP for osmoregulatoryactions including inhibition of thirst/sodium appetite andnatriuresis/diuresis, but both peptides are nearly equipo-tent in their cardiovascular actions [13]. Thus, we assumedthat AM is also an important osmoregulatory hormone inteleost fish. We searched for AM in the genome database oftiger pufferfish, and found five distinct AM-like sequences(Fig. 8). As all five AM-like genes were expressed asmRNA in the pufferfish, they were named AM1, 2, 3, 4and 5 [98]. While mature pufferfish AM1 and AM4 are con-nected to the C-terminal peptide by several arginine resi-dues as is the case of mammalian AM, mature AM2 andAM3 are localized at the C-terminus of the precursor.The AM5 precursor has a single arginine residue at theC-terminus. In terms of the site of production, the AM1and AM4 genes were expressed ubiquitously in various tis-sues as is mammalian AM. However, the AM2 and AM3genes were expressed almost exclusively in the brain, andthe AM5 gene was expressed in immune-related tissues

me

puffer-1

medaka-1puffer-4

medaka-4

dog-1

cow-1human-1

pig-1

mouse-1

rat-1

dog-2 cow-2human-2m

60 4981

61

100

567

96

90

96 49

82

AM1/AM4

Fig. 8. Phylogenetic tree of the adrenomedullin (AM) family peptides thus famethod. Bootstrap value is given at each node in percent.

such as the spleen, thymus, and head kidney (equivalentto bone marrow in mammals). Five AMs exist in all teleostspecies thus far examined including green pufferfish (Tetra-

odon nigroviridis), medaka, and zebrafish (Danio rerio)[130]. The five teleost AMs seem to be divided into threegroups, AM1/4, AM2/3, and AM5, with respect to the sim-ilarity of mRNA sequence, exon–intron organization, andtissue distribution. Synteny analyses showed that humanAM is an ortholog of pufferfish AM1 (and AM4). Phyloge-netic analysis of all CGRP family members in teleosts andmammals showed that the five AMs are clustered as a dis-tinct group from those of CGRP and AMY [98]. Thus, thefive AMs seem to form an independent subfamily in teleostfishes (Fig. 8).

Consistent with the multiple AM peptides in teleost fish,receptors for the CGRP family are also diversified in tele-ost fish; three CLRs (CLR1, 2 and 3) and six RAMPs(RAMP1, 2a, 2b, 3, 4 and 5) were identified in the puffer-fish, Takifugu obscurus [91]. The suffix number ofRAMP1-3 largely coincides with the similarity to the mam-malian counterpart as shown by the phylogenetic analysis.

human-5

dog-5

pig-5

cow-5

puffer-5

daka-5

ouse-2rat-2puffer-3

puffer-2

medaka-2

medaka-3

77

100

98

99

56

87

46

97

8

AM5

AM2/AM3

8691

r identified in mammals and teleost fish depicted using neighbor-joining


The affinity to each member of the CGRP family has beenexamined in CLR and RAMP complexes transientlyexpressed in COS cells. High affinity was demonstrated ineach combination of ligand and receptor complex; CGRPand CLR1-RAMP1/4, AM1 and CLR1-RAMP2/3/5 orCLR2-RAMP2, AM2/AM5 and CLR1-RAMP3 (seeFig. 7). Since both CLR1 and RAMP2 are expressed inthe gills and in the renal proximal tubules of the pufferfish,AM1 may have some actions on these osmoregulatory tis-sues [91].

The CGRP family members have been identified also innon-mammalian species. CT has been isolated from thestingray [135], bullfrog [155], a few species of reptiles[119], and chicken [96]. CGRP is isolated as a peptide orcDNA in birds [85], amphibians [26], and teleost fish[120]. Two distinct CGRP genes exist in the genome dat-abases of pufferfish, medaka, and zebrafish. Four CTs iso-lated from the salmon may be due to the tetraploidy of thisspecies [58,95]. Teleost CT has much greater hypocalcemiceffect than homologous CT in mammals [28], but has onlya weak hypocalcemic effect in teleost fish [110]. In additionto mammals and teleost fish, AM(1) was identified in thechicken [158]. The chicken AM has high sequence identityto human AM at the mature peptide region (72%). TheAMY cDNA was cloned in the chicken [34], and its geneis detectable in the genome database of teleost and a partialAMY sequence was determined in the sculpin [150]. Thephysiological role of AMY in non-mammalian vertebratesremains to be investigated.

5.2. Novel adrenomedullin identified in mammals

As discussed above, five teleost AMs can be divided intothree groups, AM1/4, AM2/3 and AM5, and mammalianAM is an ortholog of teleost AM1/4. Therefore, wehypothesized that AM2/3 and AM5 may also exist in mam-mals. We searched for an ortholog of AM2/3 in the gen-ome database of mammals using a newly developedsearch program and identified novel AM2 in the human,mouse and rat [131]. The AM2 gene is expressed abun-dantly in the kidney, digestive tracts, immune organs(spleen and thymus), and submaxillary gland of mice.However, the expression of the AM2 gene in the brain isnot so pronounced in mice as in pufferfish. Synteny analysisshowed that the mammalian AM2 gene is orthologous topufferfish AM2/3. Phylogenetic analysis and the exon–intron organization of mammalian AM2 also support itsorthologous relationship to teleost AM2 (Fig. 8). Rohet al. [105] also found the AM2 gene in the cDNA libraryof human pituitary and named it intermedin (IMD) as itis stored abundantly in the intermediate lobe of pituitary.Therefore, this peptide is now called AM2/IMD [128].

AM2 exerts potent cardiovascular actions in mice whenadministrated intravenously [131], and natriuretic anddiuretic actions in rats when infused into the renal artery[42]. The peripheral cardiovascular effects were analyzedin some detail in the rat and sheep [16,23,31,41,101]. The

intracerebroventricular injection of AM2 resulted in neuro-genic hypertension and tachycardia through sympatheticactivation in rats [138]. The central effect of AM2 is morepotent than that of AM, although the potency is reversedin the periphery. AM2 activates the renin–angiotensin–aldosterone system and the NP system in sheep [23] andincreases the release of anterior and posterior pituitary hor-mones in rats [47,70,139,142]. Immunoreactive AM2 waslocalized in the oxytocin and vasopressin producing neu-rons of human brain [122]. In addition, centrally adminis-tered AM2 inhibits motivational behavior such as foodintake and water intake in the rat [138]. Further, AM2ameliorates oxidative injury of the endothelial cells in thecerebral vasculature, thereby maintaining blood–brain bar-rier function [24].

The effect of AM2 on cAMP production was examinedin cultured cells expressing CLR and RAMP complexes[105,131]. AM2 exhibited moderate but smaller affinity toCLR-RAMP3 compared with AM. AM2 had negligibleaffinity to CLR-RAMP2 to which AM exhibits high affin-ity. This result coincides with the lower potency of AM2than AM in its actions on the cardiovascular system andkidney [42]. Since the central effects of AM2 are muchgreater than AM and the central AM2 effects are only par-tially blocked by AM antagonists, there should be a AM2-specific receptor in the brain [48,138]. Since CLR andRAMP are more diversified in the pufferfish, the ‘reverse’phylogenetic approach may be useful for identification ofnew AM2 receptors in mammals [38].

In addition to AM2, another member of the teleost AMfamily, AM5, was identified very recently in mammals andXenopus [97]. Synteny analysis clearly showed that thesemammalian and frog genes are orthologous to the teleostAM5 gene (Fig. 8). The AM5 gene is detected in the data-bases of human, chimpanzee, dog, pig and cow, and thecDNA was cloned from the spleen of the pig (unpublisheddata). The porcine AM5 gene was exclusively expressed inthe immune-related organs such as thymus and spleen,which coincide well with the AM5-producing tissues inteleost fish [98]. The effects of AM5 on immune functionare now under investigation in mammals and in teleost fish.The AM5 genes in the human and chimpanzee are dis-rupted by the deletion of two nucleotides in the codingregion, but their sequences are still highly conserved withother mammalian AM5 genes (Fig. 8) [97]. Therefore, theprimate AM5 gene may be expressed as a different proteinwith some important functions.

5.3. Evolutionary history of the CGRP family

As novel AM2 and AM5 were identified in mammals,we attempted to trace the process of diversification of theCGRP family in vertebrates by the comparative genomicapproach [97]. We found that the CGRP family consistsof two CGRPs (CGRP1 and 2), five AMs (AM1-5), anda single AMY in teleosts, and two CGRPs (CGRP-a and-b), three AMs (AM1, AM2 and AM5), and a single


AMY in mammals. In pufferfish, medaka and zebrafish,the AM1 and CGRP1 genes, the AM2, AM4, and CGRP2genes, and the AM3 and AMY genes are localized on thesame chromosomes, respectively (Fig. 9). Jaillon et al.[57] proposed a new concept for genome evolution in ver-tebrates based on their studies in the pufferfish (Tetraodon).According to these authors, 12 proto-chromosomes thathad existed in the ancestral bony fish were duplicated ca.320–350 Myr ago in the teleost lineage (3R), and the dupli-cated chromosomes were fused and re-arranged into 21chromosomes in Tetraodon. Similar chromosomal re-arrangements have also been reported in medaka [93]. Inmedaka, chromosome 3 on which the AM1 and CGRP1genes exist, originated from type E proto-chromosomeand chromosome 23 on which the AM3 and AMY genesexist originated from type F proto-chromosome. Further-more, chromosome 6 on which the AM2, AM4, andCGRP2 genes exist was formed by fusion of the two proto-chromosomes. Thus, it is probable that the combination ofAM1/4-CGRP1/2 genes and AM2/3-AMY genes were,respectively, duplicated at the teleost-specific 3R wholegenome duplication, and one of the duplicated AMY geneshas disappeared (Fig. 9). The same results were obtainedwith the Tetraodon CGRP family genes, but we could notlocate the AM2 and AMY genes on Tetraodon chromo-some 19 on which they should exist. The AM5 gene is

Teleost-specific 3R whole genome duplication

Chromosomalrearrangement

AM1CGRP1

AM2

CGRP2AM4

AMYAM3

AM5

Proto-chro

medaka

AM5

19 3 6 23

E ty

Fig. 9. Evolutionary history of the CGRP family genes inferred

located on a different proto-chromosome, and the dupli-cated AM5 gene may have been silenced after the 3R(Fig. 9). There are several genes around the AM5 genewhose paralogs are linked with the AM1 and AM2 genes,suggesting that the AM5 gene shares the ancestral genewith the AM1 and AM2 genes. Thus, all CGRP familypeptides may have evolved from a single ancestral gene.

In human, the AM(1) and CGRP genes are linked onchromosome11, but the AM2 and AMY genes are on dif-ferent chromosomes, probably as a result of chromosomalre-arrangement. Consistently, the ancestral chromosome ofthe region around the AM2 gene on chromosome 22 andthe AMY gene on chromosome 12 are originated fromthe same proto-chromosome F in humans [57]. It is furthershown that the chromosomal regions of humans where theAM1-CGRP genes and the AM2-AMY genes are locatedare evolutionarily related to each other [53]. The twoCGRP genes of mammals, CGRP-a and CGRP-b, are clo-sely located on the same chromosome, showing that thetwo genes are the product of tandem duplication (Fig. 9).Thus, the origin of the two CGRP genes is differentbetween teleosts (whole genome duplication) and mammals(gene duplication). In fact, only the CGRP-a gene pro-duces CT mRNA by alterative splicing in mammals, whileboth CGRP1 and CGRP2 genes produce CT in teleosts.Additionally, the CRSP gene is localized in the vicinity of

AM1CGRP

AMYAM2

mosomes

AM1CGRP-αCGRP-β(CRSP)

AMY

AM2

TranslocationTandem duplication

human

AM5

19 11 12 22

pe F type

by the comparative genomic analyses. For details, see text.


the CGRP gene in all mammalian species (ungulates andcarnivores) in which its presence has been demonstrated(Fig. 9), suggesting that the CRSP gene is the product oftandem duplication of the CGRP gene.

The ancestral gene of the CGRP family is not known. Itis probable that both the AM1-CGRP cluster and theAM2-AMY cluster had existed on proto-chromosomes inancestral bony vertebrates (Fig. 9). Thus, the two clusterswere most probably generated by the 2R whole genomeduplication that occurred at the transition from agnathansto gnathostomes or by the 1R whole genome duplication atthe transition from chordates to early vertebrates. Thus, itis necessary to determine which clusters had existed in thegenome of extant agnathans and invertebrate chordates.More anciently, the gene coding for CT-like peptide hasbeen found in the tunicate, Ciona intestinalis [43]. This dis-covery suggests that the ancestral gene may be a CGRP-like gene, but the tunicate gene does not seem to transcribea CGRP-like mRNA by alternative splicing. Thus, thereremains a possibility that other genes of the CGRP familystill exist in chordates. Further investigation is required toclarify the ancestral gene of the CGRP family.

6. Conclusions

In vertebrates, all functional genes in the DNA havebeen exposed to selection pressure during the evolutionfrom aquatic to terrestrial habitat, and the proteins codedby the genes are subject to change according to its relativeimportance in the new environment. During the course ofour studies on osmoregulation and cardiovascular regula-tion in various vertebrate species, we found that severalhormone genes have undergone profound evolutionarychanges in their structure and function that are adaptiveto survival in the new environment. In physiological studieson the NP system, we found that ANP is a principal Na+-extruding hormone that promotes SW adaptation in teleostfish. This is in contrast to mammals where ANP is a vol-ume-depleting hormone that extrudes both Na+ and water.We demonstrated that the essential function of ANP isNa+ extrusion, but it changes to volume depletion in mam-mals as water and Na+ are always regulated in the samedirection in terrestrial animals. We also found that Na+-extruding and vasodepressor hormones are more diversi-fied in teleost fishes than in mammals as exemplified bythe NP family and the CGRP family of peptides are exam-ples. Based on these findings, we then discovered new NPs(CNP1 and VNP) in birds and new AMs (AM2 and AM5)in mammals and other tetrapods. We recognize that thecurrent approach has been used narrowly to identify para-logs of diversified fish hormones in tetrapods. However,this approach can be expanded to pursue understandingof various regulatory systems than body fluid and cardio-vascular regulation, and to important differences in verte-brates other than between aquatic and terrestrial animals,such as between ectothermic and endothermic animals,and oviparous and viviparous animals to name two exam-

ples. There are other phylogenetic approaches that lead tothe discovery of new hormones (see above), but the currentapproach is novel in that the context and focus stem fromdifferences in the physiological systems among vertebrategroups.

Finally, we would like to emphasize the increasingopportunity for comparative endocrinologist to contributeto mammalian and clinical endocrinology by utilizing ourknowledge about the functional and molecular evolutionof hormones in diverse vertebrate and invertebrate species.To this end, our findings reported here demonstrate andhighlight the strength and merit of comparative studies.The ‘reverse’ phylogenetic approach illustrated here is anexample of just how fish studies can contribute to mamma-lian endocrinology. The idea will be further developed inthe future to be used in other physiological systems andin other animal groups including invertebrates.

Acknowledgments

The authors thank Dr. John A. Donald of Deakin Uni-versity for editing English of this manuscript. The idea of‘reverse’ phylogenetic approach has been inspired by manycollaborators, particularly through collaboration with Drs.Tetsuya Hirano, Shigehisa Hirose, Akiyoashi Takahashi,Susumu Hyodo, Christopher A. Loretz, John A. Donald,Tes Toop, Richard J. Balment, Neil Hazon, and KennethR. Olson, which is deeply appreciated.

References

[1] R. Acher, J. Chauvet, Y. Rouille, Adaptive evolution of waterhomeostasis regulation in amphibians: vasotocin and hydrins, Biol.Cell 89 (1997) 283–291.

[2] S.G. Amara, J.L. Arriza, S.E. Leff, L.W. Swanson, R.M. Evans,M.G. Rosenfeld, Expression in brain of a messenger RNA encodinga novel neuropeptide homologous to calcitonin gene-related peptide,Science 22 (1985) 1094–1097.

[3] S. Amer, J.A. Brown, Glomerular actions of arginine vasotocin inthe in-situ perfused trout kidney, Am. J. Physiol. 269 (1995) R775–R780.

[4] R.S. Ames, H.M. Sarau, J.K. Chambers, R.N. Willette, N.V. Alyar,A.M. Romanic, C.S. Louden, J.J. Foley, C.F. Sauermelch, R.W.Coatney, Z.H. Ao, J. Disa, S.D. Holmes, J.M. Stadel, J.D. Martin,W.S. Liu, G.I. Glover, S. Wilson, D.E. McNelty, C.E. Ellis, N.A.Elshourbagy, U. Shabon, J.J. Trill, D.W.P. Hay, E.H. Ohlstein, D.J.Bergsma, S.A. Douglas, Human urotensin-II is a potent vasocon-strictor and agonist for the orphan receptor GPR14, Nature 401(1999) 282–286.

[5] K. Ando, Y. Ito, E. Ogata, T. Fujita, Vasodilating actions ofcalcitonin gene-related peptide in normal man: comparison withatrial natriuretic peptide, Am. Heart J. 123 (1992) 111–116.

[6] D.E. Arnold-Reed, R.J. Balment, Atrial natriuretic factor stimulatesin-vivo and in0vitro secretion of cortisol in teleosts, J. Endocrinol.128 (1991) R17–R20.

[7] U. Arulumani, A. Maassenvandenbrink, C.M. Villalon, P.R. Sax-ena, Calcitonin gene-related peptide and its role in migrainepathophysiology, Eur. J. Pharmacol. 500 (2004) 315–330.

[8] N. Ashton, Renal and vascular actions of urotensin II, Kidney Int.70 (2006) 624–629.

[9] J. Balment, W. Lu, E. Weybourne, J.M. Warne, Arginine vasotocina key hormone in fish physiology and behaviour: a review with


insights from mammalian models, Gen. Comp. Endocrinol. 147(2006) 9–16.

[10] P.J. Bentley, Endocrines and Osmoregulation, Springer, Verlag,Berlin, 1971, pp. 1–300.

[11] G.C. Bird, J.S. Han, Y. Fu, H. Adwanikar, W.D. Willis, V.Neugebauer, Pain-related synaptic plasticity in spinal dorsal hornneurons: role of CGRP, Mol. Pain 2 (2006) 31–50.

[12] B. Bohus, D. de Wied, The vasopressin deficient Brattleboro rats: anatural knockout model used in the search for CNS effects ofvasopressin, Prog. Brain Res. 119 (1998) 555–573.

[13] S.D. Brain, A.D. Grant, Vascular actions of calcitonin gene-relatedpeptide and adrenomedullin, Physiol. Rev. 84 (2004) 903–934.

[14] B.M. Brenner, B.J. Ballermann, M.E. Gunning, M.L. Zeidel,Diverse biological actions of atrial natriuretic peptide, Physiol.Rev. 70 (1990) 665–699.

[15] R.W. Brill, P.G. Bushnell, The cardiovascular system of tunas, in:B.A. Block, E.D. Stevens (Eds.), Tuna—Physiology, Ecology, andEvolution, Academic Press, San Diego, 2001, pp. 79–120.

[16] H. Burk Kandilici, B. Gumsel, A. Wasserman, N. Lippton,Intermedin/adrenomedullin-2 dilates the rat pulmonary vascularbed: dependence on CGRP receptors and nitric oxide release,Peptides 27 (2006) 1390–1396.

[17] L. Bussieres, K. Laborde, M. Dechaux, C. Sachs, Effects of prolactinon Na–K–ATPase activity along the rat nephron, Pflu. Arch. 409(1987) 182–187.

[18] J.C.R. Cordoso, V.C. Pinto, F.A. Vieira, M.S. Clark, D.M. Power,Evolution of secretin family GPCR members in the metazoan, BMCEvol. Biol. 6 (2006) 108.

[19] K.M. Caron, O. Smithies, Extreme hydrops fetalis and cardiovas-cular abnormalities in mice lacking a functional adrenomedullingene, Proc. Natl. Acad. Sci. USA 98 (2001) 615–619.

[20] R.L. Carroll, in: Vertebrate Paleontology and Evolution, W.H.Freeman and Company, New York, 1998, pp. 16–25.

[21] A.C.M. Chang, R.R. Reddel, Identification of a second stanniocal-cin cDNA in mouse and human: Stanniocalcin 2, Mol. Cell.Endocrinol. 141 (1998) 95–99.

[22] C.L. Chang, S.Y.T. Hsu, Ancient evolution of stress-regulatingpeptides in vertebrates, Peptides (2004) 1681–1688.

[23] C.J. Charles, M.T. Rademaker, A.M. Richards, Hemodynamic,hormonal, and renal actions of adrenomedullin-2 in normalconscious sheep, Endocrinology 147 (2006) 1871–1877.

[24] L. Chen, B. Kis, H. Hashimoto, D.W. Busija, Y. Takei, H.Yamashita, Y. Ueta, Adrenomedullin 2 protects rat cerebralendothelial cells from oxidative damage in vitro, Brain Res. 1086(2006) 42–49.

[25] O. Civelli, Y. Saito, Z. Wang, H.P. Nothacker, R.K. Reinscheid,Orphan GPCRs and their ligands, Pharmacol. Ther. 110 (2006) 525–532.

[26] J.M. Conlon, M.C. Tonon, H. Vaudry, Isolation and structuralcharacterization of calcitonin gene-related peptide from the brainand intestine of the frog, Rana ridibunda, Peptides 14 (1993) 581–586.

[27] J.M. Conlon, D. Larhammar, The evolution of neuroendocrinepeptides, Gen. Comp. Endocrinol. 142 (2005) 53–59.

[28] D.H. Copp, Calciton, in: discovery, development, and clinicalapplication, Clin. Invest. Med. 17 (1994) 268–277.

[29] A.J. de Bold, H.B. Borenstein, A.T. Veress, H. Sonnenberg, A rapidand potent natriuretic response to intravenous injection of atrialmyocardial extract in rats, Life Sci. 28 (1981) 89–94.

[30] T.M. Doan, N. Gletsu, J. Cole, K.E. Bernstein, Genetic manipula-tion of the renin–angiotensin system, Curr. Opin. Nephrol. Hyper-tens. 10 (2001) 483–491.

[31] F. Dong, M.M. Taylor, W.K. Samson, J. Ren, Intermedian(adrenomedullin-2) enhances cardiac contractile function via aprotein kinase C- and protein kinase A-dependent pathway inmurine ventricular myocytes, J. Appl. Physiol. 101 (2006) 778–784.

[32] S.M. Eckert, T. Hirano, T.A. Leedom, Y. Takei, E.G. Grau, Effectsof angiotensin II and natriuretic peptides of the eel on prolactin and

growth hormone release in the tilapia, Oreochromis mossambicus,Gen. Comp. Endocrinol. 130 (2003) 333–339.

[33] M. Epstein, Renal effects of head-out water immersion in humans: a15-year update, Physiol. Rev. 72 (1992) 563–621.

[34] L. Fan, G. Westermark, S.J. Chan, D.F. Steiner, Altered genestructure and tissue expression of islet amyloid polypeptide in thechicken, Mol. Endocrinol. 8 (1994) 713–721.

[35] A.P. Farrell, D.R. Jones, The heart, in: Fish Physiology, in: W.W.Hoar, D.J. Randall, A.P. Farrell (Eds.), The Cardiovascular System,vol. 12A, Academic Press, San Diego, 1992, pp. 1–88.

[36] H.C. Fehmann, V. Weber, R. Goke, B. Goke, R. Arnold, Cosecre-tion of amylin and insulin from isolated rat pancreas, FEBS Lett.262 (1990) 279–281.

[37] J.T. Fitzsimons, Angiotensin, thirst, and sodium appetite, Physiol.Rev. 78 (1998) 583–686.

[38] S.M. Foord, S.D. Topp, M. Abramo, J.D. Holbrook, New methodsfor researching accessory proteins, J. Mol. Neurosci. 26 (2005) 265–276.

[39] L.R. Forte, R.M. London, W.J. Krause, R.H. Freeman, Mecha-nisms of guanylin action via cyclic GMP in the kidney, Annu. Rev.Physiol. 62 (2000) 673–695.

[40] B.K. Fox, T. Naka, K. Inoue, Y. Takei, T. Hirano, G.E. Grau, Invitro effects of homologous natriuretic peptides on growth hormoneand prolactin release in the tilapia, Oreochromis mossambicus, Gen.Comp. Endocrinol. 150 (2007) 270–277.

[41] Y. Fujisawa, Y. Nagai, A. Miyatake, T. Shokoji, A. Nishiyama, S.Kimura, Y. Abe, Roles of adrenomedullin 2 in regulating thecardiovascular and sympathetic nervous systems in conscious rats,Am. J. Physiol. 290 (2006) H1120–H1127.

[42] Y. Fujisawa, Y. Nagai, A. Miyatake, Y. Takei, K. Miura, T.Shoukouji, A. Nishiyama, S. Kimura, Y. Abe, Renal effects of a newmember of adrenomedullin family, adrenomedullin 2, in rats, Eur. J.Pharmacol. 497 (2004) 75–80.

[43] N. Fujiwara, T. Sekiguchi, T. Kawada, T. Sakai, T. Amakawa, N.Satoh, H. Satake, Identification of Ciona intestinalis calcitonin andits receptor, Zool. Sci. 22 (2005) 1498.

[44] T.M. Fujiwara, D.G. Bichet, Molecular biology of hereditarydiabetes insipidus, J. Am. Soc. Nephrol. 16 (2005) 2836–2846.

[45] R.D. Gordon, J.H. Laragh, J.V. Funder, Low renin hypertensivestates: perspectives, unsolved problems, future research, TrendsEndocrinol. Metab. 16 (2005) 108–113.

[46] S.J. Gould, The Book of Life: An Illustrated History of theEvolution of Life on Earth, W.W. Norton and Co. Inc, 2001.

[47] H. Hashimoto, S. Hyodo, M. Kawasaki, T. Mera, L. Chen, A. Soya,T. Saito, H. Fujihara, T. Higuchi, Y. Takei, Y. Ueta, Centrallyadministered adrenomedullin-2 activates hypothalamic oxytocin-secreting neurons, causing elevated plasma oxytocin level in rats,Am. J. Physiol. 289 (2005) E753–E761.

[48] H. Hashimoto, S. Hyodo, M. Kawasaki, M. Shibata, T. Saito, H.Suzuki, H. Ohtsubo, T. Yokoyama, H. Fujihara, T. Higuchi, Y.Takei, Y. Ueta, Adrennomedullin 2 (AM2)/intermedin is a morepotent activator of hypothalamic oxytocin-secreting neurons thanAM possibly through an unidentified receptor in rats, Peptides 28(2007) 1104–1112.

[49] I.W. Henderson, N. Hazon, K. Hughes, Hormones, ionic regulationand kidney function in fishes, Symp. Soc. Exp. Biol. 39 (1985) 245–265.

[50] T. Hirano, T. Ogasawara, J.P. Bolton, N.L. Collie, S. Hasegawa, M.Iwata, Osmoregulatory role of prolactin in lower vertebrates, in: R.Kirsch, B. Lahlou (Eds.), Comparative Physiology of EnvironmentalAdaptation, Karger, Basek, 1987, pp. 112–124.

[51] J.R. Hirsch, M. Meyer, W.-G. Forssmann, ANP and its role in theregulation of renal tubular transport processes, in: A.J. Kastin (Ed.),Handbook of Biologically Active Peptides, Academic Press, SanDiego, 2006, pp. 1251–1256.

[52] J.I. Hoffman, J.A. Spaan, Pressure-flow relations in coronarycirculation, Physiol. Rev. 70 (1990) 331–390.

[53] J.M.N. Hoovers, E. Redeker, F. Speleman, J.W.M. Hoppener, S.Bhola, J. Bliek, N. van Roy, N.J. Leschot, A. Westerveld, M.


Mannens, High-resolution chromosomal localization of the humancalcitonin/CGRP/IAPP gene family members, Genomics 15 (1993)525–529.

[54] S.Y.T. Hsu, New insights into the evolution of the relaxin-LGRsignaling system, Trends Endocrinol. Metab. 14 (2003) 303–309.

[55] K. Inoue, K. Naruse, S. Yamagami, H. Mitani, N. Suzuki, Y. Takei,Four functionally distinct C-type natriuretic peptides found in fishreveal new evolutionary history of the natriuretic system, Proc. Natl.Acad. Sci. USA 100 (2003) 10079–10084.

[56] K. Inoue, T. Sakamoto, S. Yuge, H. Iwatani, S. Yamagami, M.Tsutsumi, H. Hori, M.C. Cerra, B. Tota, N. Suzuki, N. Okamoto,Y. Takei, Structural and functional evolution of three cardiacnatriuretic peptides, Mol. Biol. Evol. 22 (2005) 2428–2434.

[57] O. Jaillon, J.M. Aury, F. Brunet, J.L. Petit, N. Stange-Thomann,E. Mauceli, L. Bouneau, C. Fischer, C. Ozouf-Costaz, A. Bernot,S. Nicaud, D. Jaffe, S. Fisher, G. Lutfalla, C. Dossat, B. Segurens,C. Dasilva, M. Salanoubat, M. Levy, N. Boudet, S. Castellano, V.Anthouard, C. Jubin, V. Castelli, M. Katinka, B. Vacherie, C.Biemont, Z. Skalli, L. Cattolico1, J. Poulain, V. de Berardinis, C.Cruaud, S. Duprat, P. Brottier, J.P. Coutanceau, J. Gouzy, G.Parra, G. Lardier, C. Chapple, K.J. McKernan, P. McEwan, S.Bosak, M. Kellis, J.N. Volff, R. Guigo, M.C. Zody, J. Mesirov, K.Lindblad-Toh, B. Birren, C. Nusbaum, D. Kahn, M. Robinson-Rechavi, V. Laudet, V. Schachter, F. Quetier, W. Saurin, C.Scarpelli, P. Wincker, E.S. Lander, J. Weissenbach, H.R. Crollius,Genome duplication in the teleost fish Tetraodon nigroviridis

reveals the early vertebrate proto-karyotype, Nature 431 (2004)946–957.

[58] H. Jansz, K. Martial, J. Zandberg, G. Milhaud, A.A. Benson, A.Julienne, M.S. Moukhtar, M. Cressent, Identification of a newcalcitonin gene in the salmon Oncorhynchus gorbuscha, Proc. Natl.Acad. Sci. USA 93 (1996) 12344–12348.

[59] K. Kangawa, H. Matsuo, Purification and complete amino acidsequence of a-human atrial natriuretic polypeptide (a-hANP),Biochem. Biophys. Res. Commun. 118 (1984) 131–139.

[60] T. Katafuchi, K. Hamano, N. Minamino, Identification, structuraldetermination, and biological activity of bovine and canine calcito-nin receptor-stimulating peptides, Biochem. Biophys. Res. Commun.313 (2004) 74–79.

[61] T. Katafuchi, K. Kikumoto, K. Hamano, K. Kangawa, H. Matsuo,N. Minamino, Calcitonin receptor-stimulating peptide, a newmember of the calcitonin gene-related peptide family. Its isolationfrom porcine brain, structure, tissue distribution, and biologicalactivity, J. Biol. Chem. 278 (2003) 12046–12054.

[62] T. Katafuchi, N. Minamino, Structure and biological properties ofthree calcitonin receptor-stimulating peptides, novel members of thecalcitonin gene-related peptide family, Peptides 25 (2004) 2039–2045.

[63] A. Kawakoshi, S. Hyodo, K. Inoue, Y. Kobayashi, Y. Takei, Fournatriuretic peptides (ANP, BNP, VNP and CNP) coexist in thesturgeon: new evidence for the presence of BNP in fish lineage, J.Mol. Endocrinol. 32 (2004) 547–555.

[64] A. Kawakoshi, S. Hyodo, M. Nozaki, Y. Takei, Identification of asingle natriuretic peptide (NP) in cyclostomes, lamprey and hagfish:CNP-4 is an ancestral gene of the NP family, Gen. Comp.Endocrinol. 148 (2006) 41–47.

[65] A. Kawakoshi, S. Hyodo, A. Yasuda, Y. Takei, A novel natriureticpeptide that is exclusively expressed in the heart and brain of themost primitive extant vertebrate, the hagfish (Eptatretus burgeri), J.Mol. Endocrinol. 31 (2003) 209–220.

[66] H. Kawauchi, I. Kawazoe, M. Tsubokawa, M. Kishida, B.I. Baker,characterization of melanin-concentrating hormone in the chumsalmon pituitaries, Nature 305 (1983) 321–323.

[67] K. Kitamura, K. Kangawa, M. Kawamoto, Y. Ichiki, S. Nakamura,H. Matsuo, T. Eto, Adrenomedullin: a novel hypotensive peptideisolated from human pheochromocytoma, Biochem. Biophys. Res.Commun. 192 (1993) 553–560.

[68] H. Kobayashi, Y. Takei, The Renin–Angiotensin System. Compar-ative Aspects, Springer, Berlin, 1996, pp. 1–245.

[69] Y.-Y. Li, Y. Takei, Ambient salinity-dependent effects of homolo-gous natriuretic peptides (ANP, VNP and CNP) on plasma cortisollevels in the eel, Gen. Comp. Endocrinol. 130 (2003) 317–323.

[70] C. Lin Chang, J. Roh, J. Park, C. Klein, N. Cushman, R.V.Haberberger, S.Y.T. Hsu, Intermedin functions as a pituitaryparacrine factor regulating prolactin release, Mol. Endocrinol. 19(2005) 2824–2838.

[71] J. Lopez, A. Martınez, Cell and molecular biology of the multi-functional peptide, adrenomedullin, Int. Rev. Cytol. 221 (2001) 1–92.

[72] C.A. Loretz, C. Pollina, Natriuretic peptides in fish physiology,Comp. Biochem. Physiol. 125A (2000) 169–187.

[73] D.A. Lovejoy, R.J. Balment, Evolution and physiology of thecorticotrophin-releasing factor (CRF) family of neuropeptides invertebrates, Gen. Comp. Endocrinol. 115 (1999) 1–22.

[74] D.A. Lovejoy, A.A. Chawaf, M.Z.A. Cadinouche, Teneurin C-terminal associated peptides: an enigmatic family of neuropeptideswith structural similarity to the corticotrophin-releasing factor andcalcitonin families of peptides, Gen. Comp. Endocrinol. 148 (2006)299–305.

[75] C.W. Luo, M.D. Pisarska, A.J.W. Hsueh, Identification of astanniocalcin paralogs, stanniocalcin-2, in fish and the paracrineactions of stanniocalcin-2 in the mammalian ovary, Endocrinology146 (2005) 469–476.

[76] W.S. Marshall, Grosell, Ion transport, osmoregulation, and acid-base balance in homeostasis and reproduction, in: D.H. Evans, J.B.Claiborne (Eds.), The Physiology of Fishes, 3rd ed., CRC Press,Boca Raton, 2005, pp. 177–230.

[77] S. Marsigliante, A. Muscella, G.P. Vinson, C. Storelli, AngiotensinII receptors in the gill of sea water- and freshwater-adapted eel, J.Mol. Endocrinol. 18 (1997) 67–76.

[78] J.B. Marteau, M. Zaiou, G. Siest, S. Visvikis-Siest, Geneticdeterminants of blood pressure regulation, J. Hypertens. 23 (2005)2127–2143.

[79] M.I. Martın, C. Goicoechea, M.I. Colado, M.J. Alfaro, Analgesiceffect of salmon-calcitonin administered by two routes, effect onmorphine analgesia, Eur. J. Pharmacol. 224 (1992) 77–82.

[80] S.D. McCormick, Hormonal control of gill Na+,K+-ATPase andchloride cell function, in: C.M. Wood, T.J. Shuttleworth (Eds.),Cellular and Molecular Approaches to Fish Ionic Regulation,Academic Press, Dan Diego, 1995, pp. 285–315.

[81] S.D. McCormick, Endocrine control of osmoregulation in teleostfish, Am. Zool. 41 (2001) 781–794.

[82] L.M. McLatchie, N.J. Fraser, M.J. Main, A. Wise, J. Brown, N.Thompson, R. Solari, M.G. Lee, S.M. Foord, RAMPs regulate thetransport and ligand specificity of the calcitonin-receptor-likereceptor, Nature 393 (1998) 333–339.

[83] L.G. Melo, M.E. Steinhelper, S.C. Pang, Y. Tsu, U. Ackermann,ANP in regulation of arterial pressure and fluid-electrolyte balance:lessons from genetic mouse models, Physiol. Genomics 3 (2000) 45–58.

[84] N. Minamino, T. Horio, T. Nishikimi, Natriuretic peptides in thecardiovascular system, in: A.J. Kastin (Ed.), Handbook of Biolog-ically Active Peptides, Academic Press, San Diego, 2006, pp. 1199–1207.

[85] S. Minvielle, M. Cressent, M.C. Delehaye, N. Segond, G. Milhaud,A. Jullienne, M.S. Moukhtar, F. Lasmoles, Sequence and expressionof the chicken calcitonin gene, FEBS Lett. 233 (1987) 63–68.

[86] K. Morgan, R.P. Millar, Evolution of GnRH ligand precursors andGnRH receptors in protochordate and vertebrate species, Gen.Comp. Endocrinol. 139 (2004) 191–197.

[87] R. Muff, W. Born, J.A. Fischer, Calcitonin, calcitonin gene-relatedpeptide, adrenomedullin and amylin: homologous peptides, separatereceptors and overlapping biological actions, Eur. J. Endocrinol. 133(1995) 17–20.

[88] R. Muff, W. Born, T.A. Lutz, J.A. Fischer, Biological importance ofthe peptides of the calcitonin family as revealed by disruption andtransfer of corresponding genes, Peptides 25 (2004) 2027–2038.


[89] R. Muff, N. Buhlmann, J.A. Fischer, W. Born, An amylin receptor isrevealed following co-transfection of a calcitonin receptor withreceptor activity modifying proteins-1 or –3, Endocrinology 140(1999) 2924–2927.

[90] T.C. Murphy, W.K. Samson, The novel vasoactive hormone,adrenomedullin, inhibits water drinking in the rat, Endocrinology136 (1995) 2459–2463.

[91] K. Nag, A. Kato, T. Nakada, K. Hoshijima, A.C. Mistry, Y. Takei,S. Hirose, Molecular and functional characterization of adrenomed-ullin receptors in pufferfish, Am. J. Physiol. 290 (2006) R467–R478.

[92] N. Naito, Y. Nakai, H. Kawauchi, Y. Hayashi, Immunocytochem-ical identification of melanin-concentrating hormone in the brainand pituitary of the teleost fishes Oncorhynchus keta and Salmo

gairdneri, Cell Tissue Res. 242 (1985) 41–48.[93] K. Naruse, M. Tanaka, K. Mita, A. Shima, J. Postlethwait, H.

Mitani, A medaka gene map: the trace of ancestral vertebrate proto-chromosomes revealed by comparative gene mapping, Genome Res.14 (2004) 820–828.

[94] J.S. Nelson, Fishes of the World, 4th ed., John Wiley and Sons, Inc.,Hoboken, 2006, pp. 1–14.

[95] H.D. Niall, H.T. Keutmann, D.H. Copp, J.T. Potts Jr., Amino acidsequence of salmon ultimobranchial calcitonin, Proc. Natl. Acad.Sci. USA 64 (1969) 771–778.

[96] A. Nieto, F. Moya, J.L. Candela, Isolation and properties of twocalcitonins from chicken ultimobranchial glands, Biochim. Biophys.Acta 322 (1973) 383–391.

[97] M. Ogoshi, K. Inoue, K. Naruse, Y. Takei, Evolutionary history ofthe calcitonin gene-related peptide family in vertebrates revealed bycomparative genomic analyses, Peptides 27 (2006) 3154–3164.

[98] M. Ogoshi, K. Inoue, Y. Takei, Identification of a novel adreno-medullin gene family in teleost fish, Biochem. Biophys. Res.Commun. 311 (2003) 1072–1077.

[99] H.S. Olsen, M.A. Cepeda, Q.Q. Zhang, C.A. Rosen, B.L. Vozzolo,G.F. Wagner, Human stanniocalcin: A possible hormonal regulatorof mineral metabolism, Proc. Natl. Acad. Sci. USA 93 (1996) 1792–1796.

[100] M. Ono, Y. Takayama, M. Randweaver, S. Sakata, T. Yasunaga, T.Noso, H. Kawauchi, cDNA cloning of somatolactin, a pituitaryprotein related to growth hormone and prolactin, Proc. Natl. Acad.Sci. USA 87 (1990) 4330–4334.

[101] C. Pan, J. Yang, D. Cai, J. Zhao, H. Gerns, J. Yang, J. Chang, C.Tang, Y. Qi, Cardiovascular effects of newly discovered peptideintermedin/adrenomedullin 2, Peptides 26 (2005) 1640–1646.

[102] C. Parmentier, J. Taxi, R. Balment, G. Nicolas, A. Calas, Caudalneurosecretory system of the zebrafish: ultrastructural organizationand immunocytochemical detection of urotensins, Cell Tissue Res.325 (2006) 111–124.

[103] D.R. Poyner, P.M. Sexton, I. Marshall, D.M. Smith, R. Quirion, W.Born, R. Muff, J.A. Fischer, S.M. Foord, International union ofpharmacology. XXXII. The mammalian calcitonin gene-relatedpeptides, adrenomedullin, amylin, and calcitonin receptors, Phar-macol. Rev. 54 (2002) 233–246.

[104] A.M. Richards, J.G. Lainchbury, M.G. Nicholls, A.V. Cameron,T.G. Yandle, Dendroaspis natriuretic peptide: endogenous ordubious? Lancet 359 (2002) 5–6.

[105] J. Roh, C.L. Chang, A. Bhalla, C. Klein, S.Y.T. Hsu, Intermedin is acalcitonin/calcitonin gene-related peptide family peptide actingthrough the calcitonin receptor-like receptor/receptor activity-mod-ifying protein receptor complexes, J. Biol. Chem. 279 (2004) 7264–7274.

[106] A.S. Romer, B.H. Grove, Environment of the early vertebrates, Am.Midland Nat. 16 (1935) 805–856.

[107] H. Ruskoaho, Atrial natriuretic peptide: synthesis, release, andmetabolism, Pharmacol. Rev. 44 (1992) 479–602.

[108] M. Saita, A. Shimokawa, T. Kunitake, K. Kato, T. Hanamori, K.Kitamura, T. Eto, H. Kannan, Central actions of adrenomedullin oncardiovascular parameters and sympathetic outflow in consciousrats, Am. J. Physiol. 274 (1998) R979–R984.

[109] W.K. Samson, T.C. Murphy, Adrenomedullin inhibits salt appetite,Endocrinology 138 (1997) 613–616.

[110] Y. Sasayama, Y. Takei, S. Hasegawa, D. Suzuki, Direct rises inblood Ca levels by infusing a high-Ca solution into the blood streamaccelerate the secretion of calcitonin from the ultimobranchial glandin eels, Zool. Sci. 19 (2002) 1039–1043.

[111] K. Schmidt-Nielsen, Desert Animals—Physiological Problems ofHeat and Water, Dover Publication Inc., New York, 1979.

[112] H. Schweitz, P. Vigne, D. Moinier, C. Frelin, M. Lazdunski, A newmember of the natriuretic peptide family is present in the venom ofthe green mamba (Dendroaspis angusticeps), J. Biol. Chem. 267(1992) 13928–13932.

[113] R. Serino, Y. Ueta, Y. Hara, M. Nomura, Y. Yamamoto, I.Shibuya, Y. Hattori, K. Kitamura, K. Kangawa, J.A. Russell, H.Yamashita, Centrally administered adrenomedullin increases plasmaoxytocin level with induction of c-fos messenger ribonucleic acid inthe paraventricular and supraoptic nuclei of the rat, Endocrinology140 (1999) 2334–2342.

[114] N.M. Sherwood, S.L. Krueckl, J.E. McRory, The origin andfunction of the pituitary adenylate cyclase-activating polypeptide(PACAP)/glucagon superfamily, Endocr. Rev. 21 (2000) 619–670.

[115] N.M. Sherwood, J.A. Tello, G.J. Roch, Neuroendocrinology ofprotochordates: insights from Ciona genomics, Comp. Biochem.Physiol. A 144 (2006) 254–271.

[116] M. Shimada, N.A. Tritos, B.B. Lowell, L.S. Flier, E. Maratos-Flier,Mice lacking melanin-concentrating hormone are hypophagic andlean, Nature 396 (1998) 670–674.

[117] E. Skadhauge, Steroids and adaptation to salinity changes in highervertebrates, in: R. Kirsch, B. Lahlou (Eds.), Comparative Physiol-ogy of Environmental Adaptations, Karger, Basel, 1987, pp. 103–111.

[118] H.W. Smith, Water regulation and its evolution in the fishes, Quart.Rev. Biol. 7 (1932) 1–26.

[119] N. Suzuki, C. Eguchi, T. Hirai, Y. Sasayama, Nucleotide sequencesof reptile calcitonins: their high homology to chicken calcitonin,Zool. Sci. 14 (1997) 833–836.

[120] N. Suzuki, T. Suzuki, T. Kurokawa, Cloning of a calcitonin gene-related peptide from genomic DNA and its mRNA expression inflounder, Paralichthys olivaceus, Peptides 22 (2001) 1435–1438.

[121] K. Takahashi, Fish peptides, in: A.J. Kastin (Ed.), Handbook ofBiologically Active Peptides, Academic Press, San Diego, 2006, pp.1515–1519.

[122] K. Takahashi, K. Kikuchi, Y. Maruyama, T. Urabe, K. Nakajima,H. Sasano, Y. Imai, O. Murakami, K. Totsune, Immunocytochem-ical localization of adrenomedullin 2/intermedin-like immunoreac-tivity in human hypothalamus, heart and kidney, Peptides 27 (2006)1383–1389.

[123] K. Takahashi, K. Totsune, Urotensin and its related peptides, in:A.J. Kastin (Ed.), Handbook of Biologically Active Peptides,Academic Press, San Diego, 2006, pp. 1209–1213.

[124] Y. Takei, Comparative physiology of body fluid regulation invertebrates with special reference to thirst regulation, Jpn. J. Physiol.50 (2000) 171–186.

[125] Y. Takei, Structural and functional evolution of the natriureticpeptide system in vertebrates, Int. Rev. Cytol. 194 (2000) 1–66.

[126] Y. Takei, Hormonal control of drinking in eels: an evolutionaryapproach, in: N. Hazon, G. Flik (Eds.), Osmoregulation andDrinking in Vertebrates, BIOS Scientific Publishers Inc., Oxford,2002, pp. 61–82.

[127] Y. Takei, Brain (B-type) natriuretic peptide and C-type natriureticpeptide, in: A.J. Kastin (Ed.), Handbook of Biologically ActivePeptides, Academic Press, San Diego, 2006, pp. 825–832.

[128] Y. Takei, Adrenomedulin 2/intermedin, in: A.J. Kastin (Ed.),Handbook of Biologically Active Peptides, Academic Press, SanDiego, 2006, pp. 1263–1268.

[129] Y. Takei, S. Hirose, The natriuretic peptide system in eel: a keyendocrine system for euryhalinity? Am. J. Physiol. 282 (2002) R940–R951.


[130] Y. Takei, S. Hyodo, T. Katafuchi, N. Minamino, Novel fish-derivedadrenomedullin in mammals: structure and possible function,Peptides 25 (2004) 1643–1655.

[131] Y. Takei, K. Inoue, M. Ogoshi, T. Kawahara, H. Bannai, S.Miyano, Mammalian homolog of fish adrenomedullin 2: identifica-tion of a novel cardiovascular and renal regulator, FEBS Lett. 556(2004) 53–58.

[132] Y. Takei, H. Kaiya, Antidiuretic effect of eel ANP infused atphysiological doses in seawater-adapted eels, Anguilla japonica,Zool. Sci. 15 (1998) 399–404.

[133] Y. Takei, C.A. Loretz, Endocrinology, in: D.H. Evans, J.B.Claiborne (Eds.), The Physiology of Fishes, 2nd ed., CRC Press,Boca Raton, 2005, pp. 271–318.

[134] Y. Takei, A. Takahashi, T.X. Watanabe, K. Nakajima, S. Sakaki-bara, A novel natriuretic peptide isolated from eel cardiac ventricles,FEBS Lett. 282 (1991) 317–320.

[135] Y. Takei, A. Takahashi, T.X. Watanabe, K. Nakajima, S. Sakaki-bara, N. Suzuki, Y. Sasayama, C. Oguro, New calcitonin isolatedfrom the ray, Dasyatis akajei, Biol. Bull. 180 (1991) 485–488.

[136] N. Tamura, Y. Ogawa, A. Yasoda, H. Itoh, Y. Saito, K. Nakao,Two cardiac peptide genes (atrial natriuretic peptide and brainnatriuretic peptide) are organized in tandem in the mouse andhuman genomes, J. Mol. Cell. Cardiol. 28 (1996) 1811–1815.

[137] G.M. Taylor, K. Meeran, D. O’shea, D.M. Smith, M.A. Ghatei,S.R. Bloom, Adrenomedullin inhibits feeding in the rat by amechanism involving calcitonin gene-related peptide receptors,Endocrinology 137 (1996) 3260–3264.

[138] M.M. Taylor, S.L. Bagley, W.K. Samson, Intermedin/adrenomed-ullin-2 acts within central nervous system to elevate blood pressureand inhibit food and water intake, Am. J. Physiol. 288 (2005) R919–R927.

[139] M.M. Taylor, S.L. Bagley, W.K. Samson, Intermedin/adrenomed-ullin-2 inhibits growth hormone release from cultured, primaryanterior pituitary cells, Endocrinology 147 (2006) 859–864.

[140] M.M. Taylor, W.K. Samson, Adrenomedullin and the integrativephysiology of fluid and electrolyte balance, Microscopy Res. Tech.57 (2002) 105–109.

[141] M.M. Taylor, W.K. Samson, A possible mechanism for the action ofadrenomedullin in brain to stimulate stress hormone secretion,Endocrinology 145 (2004) 4890–4896.

[142] M.M. Taylor, W.K. Samson, Stress hormone secretion is altered bycentral administration of intermedin/adrenomedullin-2, Brain Res.1045 (2005) 199–205.

[143] T. Toop, J.A. Donald, Comparative aspects of natriuretic peptidephysiology in non-mammalian species: a review, J. Comp. Physiol. B174 (2004) 189–204.

[144] N.A. Tritos, D. Vicent, J. Gillette, D.S. Ludwig, E.S. Flier, E.Maratos-Flier, Functional interactions between melaini-concentrat-

ing hormone, neuropeptide Y, and anorectic neuropeptides in the rathypothalamus, Diabetes 47 (1998) 1687–1692.

[145] T. Tsukada, Y. Takei, Integrative approach to osmoregulatoryaction of atrial natriuretic peptide in seawater eels, Gen. Comp.Endocrinol. 147 (2006) 31–38.

[146] K. Vandepoele, W. De Vos, J.S. Taylor, A. Meyer, Y. Van de Peer,Major events in the genome evolution of vertebrates: Paranome ageand size differ considerably between ray-finned fishes and landvertebrates, Proc. Natl. Acad. Sci. USA 101 (2004) 1638–1643.

[147] A. Ventura, A. Kawakoshi, K. Inoue, Y. Takei, Multiple natriureticpeptides coexist in the most primitive extant ray-finned fish, bichirPolypterus endlicheri, Gen. Comp. Endocrinol. 146 (2006) 251–256.

[148] G.F. Wagner, G.E. Dimattia, The stanniocalcin family of proteins,J. Exp. Zool. A 305 (2006) 269–280.

[149] S.E. Wendelaar Bonga, The stress response in fish, Physiol. Rev. 77(1997) 591–625.

[150] G.T. Westermark, S. Falkmer, D.F. Steiner, S.J. Chan, U.Engstrom, P. Westermark, Islet amyloid polypeptide is expressedin the pancreatic islet parenchyma of the teleostean fish, Myoxo-

cephalus scorpius, Comp. Biochem. Physiol. 133 (2002) 119–125.[151] P. Westermark, C. Wernstedt, T.D. Obrien, D.W. Hyden, K.H.

Johnson, Islet amyloid in type-2 human diabetes-millitus and adultdiabetic cats contains a novel putative polypeptide hormone, Am. J.Pathol. 127 (1987) 414–417.

[152] J.M. Wilson, P. Laurent, B.L. Tufts, D.J. Benos, M. Donositz, A.W.Vogl, D.J. Randall, NaCl uptake by the branchial epithelium infreshwater teleost fish: an immunological approach to ion-transportprotein localization, J. Exp. Biol. 203 (2000) 2279–2296.

[153] S. Yamagami, N. Suzuki, Diverse forms of guanylyl cyclases inmedaka fish—their genomic structure and phylogenetic relationshipsto those in vertebrates and invertebrates, Zool. Sci. 22 (2005) 819–835.

[154] H. Yasue, M. Yoshimura, H. Sumida, K. Kikuta, K. Kugiyama, M.Jougasaki, H. Ogawa, K. Okumura, M. Mukoyama, K. Nakao,Localization and mechanism of secretion of B-type natriureticpeptide in comparison with those of A-type natriuretic peptide innormal subjects and patients with heart failure, Circulation 90 (1994)195–203.

[155] A. Yoshida, H. Kaiya, Y. Takei, T.X. Watanabe, K. Nakajima, N.Suzuki, Y. Sasayama, Primary structure of bullfrog calcitonin, Gen.Comp. Endocrinol. 107 (1997) 147–152.

[156] S. Yuge, K. Inoue, S. Hyodo, Y. Takei, A novel guanylin family(guanylin, uroguanylin and renoguanylin) in eels: possible osmoreg-ulatory hormones in intestine and kidney, J. Biol. Chem. 278 (2003)22726–22733.

[157] S. Yuge, Y. Takei, Regulation of ion transport in eel intestine by thehomologous guanylin family of peptides, Zool. Sci. (in press).

[158] E. Zudaire, N. Cuesta, A. Martınez, F. Cuttitta, Characterization ofadrenomedullin in birds, Gen. Comp. Endocrinol. 143 (2005) 10–20.

Documents

A ‘reverse’ phylogenetic approach for identification of novel osmoregulatory and cardiovascular hormones in vertebrates