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Thirst has evolved for vertebrate terrestrial adaptation. We previously showed that buccal drying induced a series of drinking behaviours (migration to water-taking water into the mouth-swallowing) in the amphibious mudskipper goby, thereby discovering thirst in ray-finned fish. However, roles of dipsogenic/antidipsogenic hormones, which act on the thirst center in terrestrial tetrapods, have remained unclear in the mudskipper thirst. Here we examined the hormonal effects on the mudskipper drinking behaviours, particularly the antagonistic interaction between angiotensin II (AngII) and atrial natriuretic peptide (ANP) which is important for thirst regulation in mammalian 'forebrain'. Expectedly, intracerebroventricular injection of ANP in mudskippers reduced AngII-increased drinking rate. ANP also suppressed the neural activity at the 'hindbrain' region for the swallowing reflex, and the maintenance of buccopharyngeal water due to the swallowing inhibition may attenuate the motivation to move to water. Thus, the hormonal molecules involved in drinking regulation, as well as the influence of buccopharyngeal water, appear to be conserved in distantly related species to solve osmoregulatory problems, whereas hormonal control of thirst at the forebrain might have been acquired only in tetrapod lineage during evolution.

Atrial and B-type natriuretic peptides (ANP and BNP) are cardiac hormones important for cardiovascular and body fluid regulation. In some teleost species, an additional member of the natriuretic peptide family, ventricular NP (VNP), has been identified. In this study, we examine tissue distribution of these three NPs in the eel heart. Quantitative real-time PCR showed that anp is almost exclusively expressed in atria, bnp equally in atria and ventricles and vnp three-fold more in ventricles than in atria. The amount of bnp transcript overall in the heart was 1/10 those of anp and vnp. There was no difference in transcript levels between freshwater and seawater-acclimated fishes. Immunohistochemistry using specific antisera and in situ hybridization using gene-specific probes showed that NP signals were detected in most atrial and ventricular myocytes with some regional differences in density. Because of high sequence similarity of the three NPs, each of the three NP antisera individually was pre-incubated with 10 -8  M of the other two non-targeted cardiac NPs to increase the specificity. A few atrial myocytes contained all three NPs in the same cell. Immuno-electron microscopy identified many dense-core vesicles containing ANP in atria and VNP in ventricles and some vesicles contained both ANP and VNP as demonstrated using pre-absorbed antisera. Based on these data and those of previous studies, we suggest that in eels ANP is secreted from atria in a regulatory pathway and VNP from ventricles in a constitutive pathway. In addition, VNP, not BNP, is the principal ventricular hormone in eels.

An animal-borne blood sampler with data-logging functions was developed for phocid seals, which collected two blood samples for the comparison of endocrinological/biochemical parameters under two different conditions. The sampler can be triggered by preset hydrostatic pressure, acceleration (descending or ascending), temperature, and time, and also manually by light. The sampling was reliable with 39/50 (78%) successful attempts to collect blood samples. Contamination of fluids in the tubing to the next blood sample was <1%, following the prior clearance of the tubing to a waste syringe. In captive harbor seals ( Phoca vitulina ), the automated blood-sampling method was less stressful than direct blood withdrawal, as evidenced by lower levels of stress hormones ( P < 0.05 for ACTH and P = 0.078 for cortisol). HPLC analyses showed that both cortisol and cortisone were circulating in seal blood. Using the sampler, plasma levels of cardiovascular hormones, atrial natriuretic peptide (ANP), AVP, and ANG II were compared in grey seals ( Halichoerus grypus ), between samples collected when the animals were on land and in the water. HPLC analyses determined that [Met 12 ] ANP (1-28) and various forms of angiotensins (ANG II, III, and IV) were circulating in seal blood. Although water immersion profoundly changes the plasma levels of cardiovascular hormones in terrestrial mammals, there were only tendencies toward an increase in ANP ( P = 0.069) and a decrease in AVP ( P = 0.074) in the seals. These results suggest that cardiovascular regulation in phocid seals may have undergone adaptation during evolution of the carnivore to a semiaquatic lifestyle., (Copyright © 2016 the American Physiological Society.)

The natriuretic peptide (NP) family consists of at least seven members; cardiac ANP, BNP and VNP and brain CNPs (CNP1-4). Phylogenetic and comparative genomic analyses showed that CNP4 is the ancestral molecule of the family, from which CNP3 and CNP1/2 were duplicated in this order, and that the three cardiac NPs were generated from CNP3 by tandem duplication. Seven members existed at the divergence of ray-finned fishes and lobe-finned fishes (tetrapods), but some of the NP genes have disappeared during the course of evolution. In ray-finned fishes, all three cardiac NPs exist in chondrostei and some migratory teleost species, but VNP is generally absent and ANP is absent in a group of teleosts (Beloniformes). In tetrapods, ANP and BNP are present in mammals and amphibians, but ANP is usually absent in reptiles and birds. Thus, BNP is a ubiquitous cardiac NP in bony fishes and tetrapods though elasmobranchs and cyclostomes have only CNP3/4 as a cardiac NP. Functional studies indicate that cardiac NPs are essential Na(+)-extruding hormones throughout vertebrates; they play critical roles in seawater (SW) adaptation in teleosts, while they are important volume-depleting hormones in mammals as water and Na(+) are regulated in parallel in terrestrial animals. In mammals, cardiac NPs become prominent in pathological conditions such as heart failure where they are used in diagnosis and treatment. Although the functional role of BNP has not yet been fully elucidated compared with ANP in non-mammalian vertebrates, it appears that BNP plays pivotal roles in the cardiovascular and body fluid regulation as shown in mammals. ANP has previously been recognized as the principal cardiac NP in mammals and teleosts, but comparative studies have revealed that BNP is the only cardiac NP that exists in all tetrapods and teleosts. This is an excellent example showing that comparative studies have created new insights into the molecular and functional evolution of a hormone family., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Accumulating evidence indicates that circulating atrial natriuretic peptide (ANP) potently reduces excess drinking to ameliorate hypernatremia in seawater (SW) eels. However, the cerebral mechanism underlying the antidipsogenic effect is largely unknown. To localize the ANP target site in the brain, we examined the distribution of ANP receptors (NPR-A) in eel brain immunohistochemically using an antiserum specific for eel NPR-A. The immunoreactive NPR-A was localized in the capillaries of various brain regions. In addition, immunoreactive neurons were observed mostly in the medulla oblongata, including the reticular formation, glossopharyngeal-vagal motor complex, commissural nucleus of Cajal, and area postrema (AP). Trypan Blue, which binds serum albumin and does not cross the blood-brain barrier, was injected peripherally and stained the neurons in the AP but not other NPR-A immunopositive neurons. These histological data indicate that circulating ANP acts on the AP, which was further confirmed by physiological experiments. To this end, the AP in SW eels was topically destroyed by electric cauterization or were by chemical lesion of its neurons by kainic acid, and ANP (100 pmol kg(-1)) was then injected into the circulation. Both heat-coagulative and chemical lesions to the AP greatly reduced an antidipsogenic effect of ANP, but the ANP effect was retained in sham-operated eels and in those with lesions outside the AP. These results strongly suggest that the AP, a circumventricular organ without a blood-brain barrier, serves as a functional window of access for the circulating ANP to inhibit drinking in eels.

Dolphins are aquatic animals free from gravity, and this may have imposed significant changes in their cardiovascular status and its hormonal regulation compared with terrestrial animals. This study molecularly characterized two major cardiovascular hormones, atrial and B-type natriuretic peptides (ANP and BNP) and measured their changes in dolphin plasma concentrations in relation to the cardiovascular status of the animal. We initially identified ANP and BNP in three species of dolphins (Lagenorhynchus obliquidens, Phocoenoides dalli and Tursiops truncatus). ANP precursors are highly conserved in most mammals, but dolphin BNP precursors were more variable. In molecular phylogenetic analyses, dolphin ANP and BNP precursors grouped with those of artiodactyls, particularly to the camel peptides. The chromatographic characterization of tissue and plasma molecular forms using specific radioimmunoassays showed that the predominant ANP and BNP in the atrium are prohormone and mature peptide, respectively, whereas mature ANP and BNP are circulating in the dolphin blood. A mass spectrometric analysis showed that atrial BNP consists of 26 amino acids, rather than the 32-amino-acid form detected in other mammals. Finally, changes in plasma ANP and BNP concentrations were examined in captive bottlenose dolphins (Tursiops truncatus) after their pool was drained. Plasma ANP and BNP concentrations did not change after landing, unlike terrestrial mammals. Plasma angiotensin II and cortisol concentrations did not change either, showing minor stress after landing. Since landed dolphins show a different cardiovascular status on land than terrestrial mammals, plasma ANP and BNP concentrations seem to reflect the cardiovascular status characteristic of dolphins.

Atrial natriuretic peptide (ANP) reduces plasma Na+ concentration and promotes seawater (SW) adaptation in SW eels. However, little is known about the mechanisms for the hyponatremic effect of ANP. In order to evaluate the role of ANP in the whole-body Na+ homeostasis of marine teleost, we reviewed previous in vivo experiments using exogenously administered ANP and present additional experiments to assess the role of endogenous ANP in Na+ homeostasis in conscious SW eels. The Na+ influx and efflux rate across the body surfaces including the gills measured with isotopic 22Na were not altered by the hyponatremic dose (5 pmol kg-1 min-1) of ANP infusion in SW eels. ANP infusion also had no effect on renal Na+ excretion in SW eels. In contrast, ANP strongly inhibited drinking, and the inhibition was quantitatively correlated with the hyponatremic effect of ANP. Further, intestinal absorption of Na+ was inhibited by ANP as examined in situ using intestinal sac in conscious SW eels. The combined inhibitory actions of ANP on drinking and intestinal absorption were sufficient to explain the decrease in plasma Na+ concentration. In addition, removal of endogenous circulating ANP by immunoneutralization increased plasma Na+ concentration with a concomitant increase in drinking rate in SW eels. These results strongly suggest that endogenous ANP is involved in the hyponatremic regulation through actions on drinking, and probably on intestine, in SW eels.

Atrial natriuretic peptide (ANP) decreases plasma Na+ concentration and promtes seawater (SW) adaptation in eels. The hyponatremia may most probably be caused by increased branchial extrusion of Na+, but the mechanism has not been determined yet. The present study examined initially the effects of ANP on branchial Na+ efflux in vivo using isotopic 22Na. However, the efflux rate was not altered by infusion of a hyponatremic dose of ANP (5 pmol.kg(-1).min(-1)). Therefore, we sought to examine whether the ANP-mediated hyponatremia is caused by a decrease in the uptake of Na+ from the environment. Since a decrease in drinking was highly correlated with a degree of hyponatremia, conscious SW eels were infused with dilute SW into the stomach at a normal drinking rate to offset the antidipsogenic effect of ANP. Under this regimen, the hyponatremic effect of ANP was abolished. Then, we examined the site of Na+ absorption in the alimentary tract by measuring the changes in ion composition of intraluminal fluid along the tract. Since Na+ was absorbed at the esophagus and anterior/middle intestine, a sac was prepared at each site and the effects of ANP were examined in situ in conscious SW eels. ANP infusion did not alter Na+ absorption at the esophagus, but it profoundly reduced the absorption at the intestine. Together with our previous finding that ANP does not alter renal Na+ excretion, we propose that ANP reduces plasma Na+ concentration in SW eels by inhibiting drinking and subsequent absorption of Na+ by the intestine.

Atrial and B-type natriuretic peptide (ANP and BNP) are cardiac hormones synthesized and secreted by the myoendocrine cells of the heart. They exert potent actions on body fluid balance. Since various body organs including the heart are under high physiological stress during water and food deprivation in the desert nomads, we intended to perform molecular biological and histological studies of ANP in the heart of the dromedary camel Camelus dromedarius. Initially, we isolated cDNAs encoding ANP from the atrium and BNP from the atrium and ventricle of the dromedary camel. Putative mature ANP, deduced from the cDNA sequence, was identical to that of human and pig ANP, but the putative mature BNP was more diverse and was most similar to pig BNP (94% identity). Thus, we used antisera raised against human ANP that did not cross-react with pig BNP in the subsequent immunohistochemical studies. The ANP-expressing myoendocrine cells are most concentrated in the right atrium, to a lesser extent in the left atrium, and almost absent in the left ventricle. The immuno-positive cells are scattered uniformly in each region and are characterized by the presence of immunoreactive granular deposits around the nucleus. The left atrium comprises some ramifications of conductive cells (Purkinje fibers), some of which also contained ANP-immunoreactive granules. At the electron microscopic level, myoendocrine cells possessed secretory granules primarily in the perinuclear zone and a well-developed Golgi apparatus. The present study is the first comprehensive report dealing with the molecular cloning and immunohistochemical localization of ANP in the heart of a desert dwelling mammal.

In teleost fish and tetrapods, the natriuretic peptide (NP) family consists of ANP (atrial natriuretic peptide), BNP (brain natriuretic peptide) and VNP (ventricular natriuretic peptide) that are secreted from the heart, and C-type natriuretic peptide (CNP) that is found in the brain. However, CNP is the only NP identified in the heart and brain of elasmobranchs, suggesting that it is the ancestral type of the NP family and that ANP, BNP and VNP appeared later in the vertebrate phylogeny. To delineate more clearly the molecular evolution of this hormone family, we determined the sequence of NP molecule(s) in evolutionarily the oldest vertebrate group, the cyclostomes. We have cloned a novel NP cDNA from the heart and brain of hagfish, Eptatretus burgeri, using the RACE method and degenerate primers that amplify all known types of NP cDNAs. The novel NP, named EbuNP after the scientific name of this hagfish, appears to be the only NP in the heart and brain, as no other NP cDNAs were amplified even after specific removal of the cloned EbuNP mRNA from the mRNA pool, except for a minor alternatively spliced EbuNP cDNA with a truncated 3'-untranslated sequence. The EbuNP was equally similar to known NPs but was not considered to be a CNP because of the presence of a C-terminal tail sequence. The EbuNP gene was abundantly expressed in the cardiac atrium, ventricle, portal heart and brain but scarcely in the intestine; no expression was observed in the gill and kidney. Mass spectrometry of affinity-purified EbuNP in plasma, heart and brain revealed a 68 amino acid peptide circulating in the blood and stored in the heart, which is cleaved at the typical cleavage signal of a processing enzyme, furin, as observed in mammalian BNP. The C-terminal Gly residue was used for amidation as is the case in eel ANP. The immunoreactive EbuNP was not detected in the brain, suggesting the presence of a different processing form in the brain. These results show that the molecular evolution of the NP family in vertebrates is more complex than previously thought.

The effects of three eel natriuretic peptides (NPs), i.e., ANP, VNP, and CNP on plasma cortisol levels were investigated in conscious freshwater (FW)- and seawater (SW)-adapted eels with permanent arterial catheter. The experiment was performed between 9:00 and 15:00 of the day, when the natural plasma cortisol level was relatively stable. After a single intra-arterial injection of ANP, VNP, or CNP at 100 pmol/kg, only CNP, but not ANP or VNP, increased plasma cortisol concentration in FW eels. In SW eels, however, only ANP at the same dose increased plasma cortisol concentration. The effect of CNP in FW eels and that of ANP in SW eels were dose-dependent between 10 and 1000 pmol/kg. On the other hand, ANP and VNP were equally effective, but CNP was ineffective, in increasing hematocrit in both FW and SW eels. These results show that the effect of NPs on plasma cortisol level is dependent on the ambient salinity in the eel. Since cortisol plays a pivotal role in environmental adaptation of fishes, the results suggest a possible involvement of ANP and CNP in the adaptation to SW and FW, respectively. Furthermore, this is the first evidence showing that ANP and VNP exert different effects in fish despite they share the same receptor, NPR-A., (Copyright 2003 Elsevier Science (USA))

The effects of angiotensin II (ANG II) and natriuretic peptides (NPs) of the eel (ANP, atrial natriuretic peptide; CNP, C-type natriuretic peptide; and VNP, ventricular natriuretic peptide) on prolactin (PRL(188) and PRL(177)) and growth hormone (GH) release from the organ-cultured tilapia pituitary were examined. Eel ANG II at concentrations greater than 1 nM stimulated the release of PRL(188) and PRL(177) in a dose-related manner during the first hour of incubation. Significant stimulation by 100 nM ANG II on PRL(177) release was observed until 4h of incubation, and on PRL(188) release until 12 h. No effect of ANG II was seen on GH release. None of the NPs altered the release of PRLs at any time point. On the other hand, eel VNP at concentrations greater than 1 nM stimulated GH release in a dose-related manner after 4 h, and significant stimulation was observed until 48 h. Eel CNP was less effective than eel VNP; significant stimulation of GH release was observed at 1 and 10 nM during 24-48 h of incubation. No significant effect of eel ANP on GH release was seen at any concentration. ANG II had no effect on GH release at any time point. There was no change in mRNA levels of PRLs or GH in the pituitaries incubated with ANG II for 8 h or those incubated with the NPs for 48 h. These results indicate rapid and short-lasting stimulation by ANG II on PRL release and slow and long-lasting stimulation by VNP and CNP on GH release from the tilapia pituitary., (Copyright 2003 Elsevier Science (USA))

The natriuretic peptide system of a euryhaline teleost, the Japanese eel (Anguilla japonica), consists of three types of hormones [atrial natriuretic peptide (ANP), ventricular natriuretic peptide (VNP), and C-type natriuretic peptide (CNP)] and four types of receptors [natriuretic peptide receptors (NPR)-A, -B, -C, and -D]. Although ANP is recognized as a volume-regulating hormone that extrudes both Na(+) and water in mammals, ANP more specifically extrudes Na(+) in eels. Accumulating evidence shows that ANP is secreted in response to hypernatremia and acts to inhibit the uptake and to stimulate the excretion of Na(+) but not water, thereby promoting seawater (SW) adaptation. In fact, ANP is secreted immediately after transfer of eels to SW and ameliorates sudden increases in plasma Na(+) concentration through inhibition of drinking and intestinal absorption of NaCl. ANP also stimulates the secretion of cortisol, a long-acting hormone for SW adaptation, whereas ANP itself disappears quickly from the circulation. Thus ANP is a primary hormone responsible for the initial phase of SW adaptation. By contrast, CNP appears to be a hormone involved in freshwater (FW) adaptation. Recent data show that the gene expression of CNP and its specific receptor, NPR-B, is much enhanced in FW eels. In fact, CNP infusion increases (22)Na uptake from the environment in FW eels. These results show that ANP and CNP, despite high sequence identity, have opposite effects on salinity adaptation in eels. This difference apparently originates from the difference in their specific receptors, ANP for NPR-A and CNP for NPR-B. VNP may compensate the effects of ANP and CNP for adaptation to respective media, because it has high affinity to both receptors. On the basis of these data, the authors suggest that the natriuretic peptide system is a key endocrine system that allows this euryhaline fish to adapt to diverse osmotic environments, particularly in the initial phase of adaptation.

Evidence has accumulated that atrial natriuretic peptide (ANP) plays important roles in sea-water adaptation in eels. However, the roles of the other two natriuretic peptides (CNP and VNP) in osmoregulation have not been examined yet. In the present study, the effects of homologous ANP, CNP and VNP were compared on plasma Na+ concentration (an indicator of plasma osmolality), hematocrit (an approximate indicator of blood volume) and drinking rate in freshwater- and seawater-adapted eels. In seawater eels, ANP and VNP, but not CNP, infused at 5 pmol/kg/min decreased plasma Na+ concentration and drinking rate and increased hematocrit. In freshwater eels, ANP and VNP failed to decrease plasma Na+ concentration but increased hematocrit to the same extent as in seawater eels. Inhibition of drinking was not detectable in freshwater eels because of little drinking before NP infusions. These results show that the effects of NPs on plasma Na+ concentration, drinking rate and hematocrit are mediated by NPR-A, since only ANP and VNP that bind with higher affinity to NPR-A are effective in seawater eels. The mechanisms of regulation of plasma Na+ concentration and hematocrit are unknown, but NPR-A is present in the responsible tissues for regulation of hematocrit in both freshwater and seawater eels. However, NPR-A may be absent in the tissues of freshwater eels that are responsible for regulation of plasma Na+ concentration.

Natriuretic peptides (NPs) have been implicated in cardiovascular regulation in rainbow trout (Oncorhyncus mykiss), and it has been observed that the vasorelaxant activity of distinct trout and human NPs is similar in isolated trout arteries. This study characterizes the response of a variety of vessels from rainbow trout and other salmonids to different NPs. The effects of heterologous (rat atrial and human atrial) and homologous (rainbow trout atrial and rainbow trout ventricular) NPs were examined in precontracted efferent branchial arteries from rainbow trout (O. mykiss, Kamloops strain), lake whitefish (Coregonus clupeaformis), and in rainbow trout celiacomesenteric arteries and anterior cardinal veins. The response to mammalian NPs was also examined in efferent branchial arteries from the steelhead (O. mykiss, Skamania strain), coho salmon (Oncorhyncus kisutch), brook trout (Salvelinus fontinalis), and brown trout (Salmo trutta). In general, there were relatively few differences that were species, peptide, or vessel specific. There was no difference in the sensitivity (concentration producing a half-maximal response EC(50)) or efficacy (percent relaxation, i.e., E(max)) of trout or whitefish efferent branchial arteries to any NP, except human NP, which was significantly less effective (greater EC(50) and lower E(max)) in whitefish arteries. There were no differences in E(max) of mammalian NPs in efferent branchial arteries from any species, and only coho and brook trout had significantly different EC(50)'s (coho, 1.0+/-0.2 nM; brook trout, 4. 2+/-0.6 nM; and other species, from 1.9 to 3.5 nM). Rainbow and coho anterior cardinal veins were less sensitive than arteries to mammalian NPs (EC(50)'s; 8.8+/-2.0, 2.0+/-0.1 vs. 3.0+/-0.9, 1.0+/-0. 2, respectively), whereas brown trout veins were more sensitive (1. 0+/-0.2, 3.5+/-1.3, respectively). Sodium nitroprusside (SNP), which activates soluble guanylate cyclase, was vasodilatory, albeit significantly less potent than all NPs, in efferent branchial arteries of all species. SNP was significantly more potent in trout than whitefish efferent branchial arteries, whereas it was equally efficacious in these vessels. These results demonstrate that multiple vessels from various salmonids are similarly responsive to the vasorelaxant effects of a variety of NPs and that the salmonid NP receptor has relatively little ability to discriminate between homologous and heterologous peptides. We conclude that the vascular NP receptor complex is highly conserved among salmonids. Further, salmonids utilize cyclic guanosine monophosphate (cGMP) elevations for reductions of vascular tonus by both particulate and soluble guanylate cyclase pathways.

The effects of eel atrial natriuretic peptide (ANP) on drinking were investigated in eels adapted to freshwater (FW) or seawater (SW) or in FW eels whose drinking was stimulated by a 2-ml hemorrhage. An intra-arterial infusion of ANP (0.3-3.0 pmol . kg-1 . min-1), which increased plasma ANP level 1.5- to 20-fold, inhibited drinking dose dependently in all groups of eels. The drinking rate recovered to the level before ANP infusion within 2 h after infusate was replaced by saline. The inhibition at 3.0 pmol . kg-1 . min-1 was profound in FW eels and hemorrhaged FW eels, whereas significant drinking still remained after inhibition in SW eels. Plasma ANG II concentration also decreased dose dependently during ANP infusion and recovered to the initial level after saline infusion in all groups of eels. The decrease at 3.0 pmol . kg-1 . min-1 was large in FW eels and hemorrhaged FW eels compared with that of SW eels. Thus the changes in drinking rate and plasma ANG II level were parallel during ANP infusion. Plasma sodium concentration and osmolality decreased during ANP infusion in SW and FW eels, and they were restored after saline infusion. In hemorrhaged FW eels, however, ANP infusion did not alter plasma sodium concentration and osmolality. Hematocrit did not change during ANP infusion in any group of eels. Collectively, ANP infusion at physiological doses decreased drinking rate and plasma ANG II concentration in parallel in both FW and SW eels. It remains undetermined whether the inhibition of drinking is caused by direct action of ANP or through inhibition of ANG II, which is known as a potent dipsogen in all vertebrate species, including eels.

A complementary DNA (cDNA) encoding eel atrial natriuretic peptide (ANP) precursor was specifically amplified from eel atrial mRNAs by rapid-amplification polymerase chain reaction. The sequence analysis of the cDNA using multiple clones revealed that the preproANP consists of 140 amino acid residues carrying a signal sequence at its N-terminus and a mature ANP at its C-terminus. An additional glycine residue was attached to the C-terminus of previously isolated eel ANP. The glycine residue may be used for amidation of the C-terminus or removed after processing. The cleavage site of a signal peptide with 22 amino acid residues was confirmed by isolation of proANP protein from eel atria. The proANP sequence deduced from the cDNA was also confirmed for 71% of the isolated protein. Sequence comparison with other natriuretic peptides revealed that eel ANP is more similar to mammalian ANP than to B-type natriuretic peptide (BNP) at both amino acid and nucleotide sequence levels. The eel ANP gene was a single copy gene as shown by Southern blot analysis. Northern blot analysis showed that eel ANP mRNA is approximately 0.8 kb in size and exclusively detected in the atrium. Thus, eel ANP is a true atrial hormone judging from both the sequence and the site of production. However, reverse transcription-polymerase chain reaction detected ANP message in the brain, gill, cardiac ventricle, red body of swim bladder (rete mirabilis), intestine, head kidney (including interrenal and chromaffin tissues) and kidney. Most of these tissues are involved in ion and/or gas exchange in fishes.

The cardiovascular effects of homologous natriuretic peptides (fNPs) were examined in the bullfrog, Rana catesbeiana. Synthetic bullfrog atrial natriuretic (fANP), brain natriuretic (fBNP) and C-type natriuretic peptides (fCNP I and fCNP II), were tested in vitro and compared under the same experimental conditions. All frog NPs produced a significant, and concentration-dependent, reduction in tension (relaxant effect) in the isolated dorsal aorta. Frog CNP II exhibited similar vasorelaxation profile as that of fANP, while fBNP and fCNP I had lower activity than fANP. Frog NPs inhibited norepinephrine induced contraction and fCNP II was most potent. In isolated preparations of atrium and ventricle, fCNP I and II produced a significant, and concentration-dependent, reduction in tension, but neither fANP nor fBNP produced any significant effects. Frog CNP II is most potent among fNPs in relation to reduce the cardiac output. Chronotropic responses of the heart to administrations of NPs were insignificant. The present results for the first time showed that fNPs play roles in the control of cardiovascular system, both blood vessels and heart, in the bullfrog.

The intestine is a major osmoregulatory organ in euryhaline fishes which allows them to survive in the sea, and natriuretic peptides have been implicated in regulation of transmural transport. Atrial (ANP) and ventricular natriuretic peptide (VNP) were identified in eel intestine. Elution profiles of ANP and VNP from high-performance liquid chromatography (HPLC) were determined by radioimmunoassay using highly specific antisera. Elution times of immunoreactive ANP and VNP in HPLC were identical to those of authentic peptide standards and were consistent with the relative molecular masses of these peptides. Tissue localization of ANP and VNP was accomplished by fluorescence immunohistochemistry. Immunoreactive cells were observed in the epithelium of anterior, middle, and posterior regions of intestine. Reverse transcription of mRNA isolated from intestine and subsequent polymerase chain reaction amplification yielded appropriate-size products consistent with ANP and VNP expression. Together, these data show that natriuretic peptides are synthesized locally in eel intestine, rather than trapped from the circulation.

Atrial and brain natriuretic peptides (ANP and BNP, respectively) are two cardiac natriuretic peptides (NPs) found in tetrapods from amphibians to mammals, whereas ANP and ventricular NP (VNP) have been identified in eel hearts. Because VNP has also been found in the rainbow trout ventricle, we attempted to isolate NP from trout cardiac atria in order to determine whether ANP and VNP are common cardiac NPs in teleosts. In the present experiments, we isolated VNP and a novel atrial NP consisting of 29 amino acid residues from the atria. This new trout NP exhibited similar sequence identity to mammalian ANP and BNP (50-60%). Its homology to eel ANP was low (52%) compared with high homology of trout and eel VNP (78%). Based on yield, the content of this new NP in trout atria may be even smaller than that of VNP. The new trout atrial NP exhibited low relaxant activity in the chick rectum (only 1/10 of that of trout VNP), and extremely low vasorelaxant activity in the rat aortic strip (only 1/400 of that of human ANP). However, the new trout NP was equipotent with trout VNP and human ANP in relaxing trout epibranchial artery. Based on the sequence similarity with other NPs and on atrial content, the new NP isolated from trout atria cannot yet be assigned to a known member of the NP family.

In eels, plasma osmolality rather than blood volume is a major regulator of atrial and ventricular natriuretic peptide (ANP and VNP) secretion. The present study examined the effects of changes in blood volume on ANP and VNP secretion stimulated by an increase in plasma osmolality in freshwater-adapted eels. Basal plasma ANP and VNP levels were decreased by 2 ml of blood withdrawal (28% of total blood volume), but not changed by blood volume expansion with 2 ml of 0.9% NaCl solution containing 2% dextran. The blood loss suppressed the increased plasma ANP level caused by an injection of 2.5 ml/kg of 1.7 M NaCl solution at 60 min (120.5 +/- 31.0 fmol/ml, n = 5) compared with controls without blood volume manipulation (586. 6 +/- 43.6 fmol/ml, n = 5), but the plasma ANP level transiently increased in bled fish immediately after osmotic stimulus, probably due to the release of ANP stored in the cardiac tissues after the blood loss. Changes in plasma VNP were not so evident as those of ANP. In contrast, blood volume expansion augmented the increase in plasma ANP and VNP levels within 60 min after osmotic stimulus compared with controls. The recovery of plasma VNP level was quicker than that of plasma ANP. Increases in plasma Na, Cl concentrations, and osmolality were not different among hypovolemic, normovolemic, and hypervolemic eels after osmotic stimulation. It is concluded that volume itself is a minor regulator for ANP and VNP secretion compared with osmotic stimulus, but it plays a modulatory role in osmotically induced ANP and VNP secretion in eels.

Active venous regulation of cardiovascular function is well known in mammals but has not been demonstrated in fish. In the present studies, the natriuretic peptides (NP) rat atrial natriuretic peptide (ANP) and trout ventricular natriuretic peptide (VNP), clearance receptor inhibitor SC-46542, and sodium nitroprusside (SNP) were infused into unanesthetized trout fitted with pressure cannulas in the ventral aorta, dorsal aorta, and ductus Cuvier, and a ventral aorta (VA) flow probe was used to measure cardiac output (CO). In another group, in vivo vascular (venous) capacitance curves were obtained during ANP or SNP infusion. The in vitro effects of NP on vessels and the heart were also examined. ANP, VNP, and SC-46542 decreased central venous pressure (PVen), CO, stroke volume (SV), and gill resistance (RG), whereas systemic resistance (RS) and heart rate (HR) increased. Dorsal aortic pressure (PDA) transiently increased and then fell even though RS remained elevated. ANP decreased mean circulatory filling pressure (MCFP), increased vascular compliance at all blood volumes, and increased unstressed volume in hypovolemic fish. ANP had no direct effect on the heart. ANP responses in vivo were not altered in trout made hypotensive by prior treatment with the angiotensin-converting enzyme inhibitor lisinopril. SNP reduced ventral aortic pressure (PVA), PDA, and RS, increased CO and HR, but did not affect PVen, SV, or RG. SNP slightly decreased MCFP but did not affect compliance or unstressed volume. In vitro, large systemic arteries were more responsive than veins to NP, whereas SNP relaxed both. These results show that, in vivo, NP decrease venous compliance, thereby decreasing venous return, CO, and arterial pressure. Conversely, SNP hypotension is due to decreased RS. This is the first evidence for active regulation of venous capacitance in fish, which probably occurs in small veins or venules. The presence of venous baroreceptors is also suggested.

Changes in plasma atrial and ventricular natriuretic peptide (ANP and VNP) concentrations were examined in conscious eels, Anguilla japonica, after transfer from freshwater (FW) to seawater (SW) or vice versa. These changes were compared in ventral and dorsal aortic blood since clearance receptors are present in the gills. Plasma Na concentration and osmolality in the ventral aortic blood rose linearly after transfer of FW eels to SW. Six hours after transfer, plasma ANP levels increased from 28.1 +/- 2.9 to 44.8 +/- 5.4 fmol/ml (n = 6) (P < 0.05) and plasma VNP levels also increased from 63.6 +/- 3.8 to 91.2 +/- 6.2 fmol/ml (n = 6) (P < 0.05). However, the levels gradually returned to the FW levels despite plasma Na concentration and osmolality remaining elevated. Dorsal aotic plasma ANP and VNP levels were unchanged after transfer of FW eels to SW, possibly due to extraction of secreted peptides by the branchial clearance receptors. After transfer of SW-adapted eels to FW, plasma ANP and VNP levels were unchanged, while both plasma Na concentration and osmolality decreased. These results indicate that plasma ANP and VNP levels are increased by an increase in plasma Na concentration or osmolality after transfer of FW eels to SW. However, the sustained increase in plasma Na concentration or osmolality no longer stimulated ANP and VNP secretion, and plasma ANP and VNP levels gradually returned to the FW levels gradually on adaptation to SW.

125I-labeled eel atrial natriuretic peptide (ANP) was administered into the ventral or dorsal aorta of freshwater (FW) and seawater (SW) eels, Anguilla japonica, and the major target organs were explored by whole body autoradiography. Localization of the ANP binding in the target organs was also examined at tissue and cell levels by microautoradiography using tissue sections. Whole body autoradiography revealed that the specific label was accumulated predominantly in the gill, with lesser amounts in the atrium, kidney, liver, and urinary bladder. Autoradiographic grains were most dense in the secondary lamellae of the gill, particularly on the side of the efferent filamental artery. Other binding sites in target tissues were the glomerulus of the kidney, epicardium and endocardium of the atrium, bile duct/blood vessels of the liver, and interrenal cells of the head kidney. There was no difference in the distribution and density of grains between injections into the ventral aorta and dorsal aorta, although, in the former, injected 125I-labeled eel ANP passes through the gill before reaching peripheral target tissues. There was a tendency for downregulation of ANP binding sites in SW eels, especially in the gill. These results show that specific ANP binding sites are present in organs that are implicated in osmoregulation and cardiovascular regulation in eels and further suggest that the number of ANP binding sites varies according to changes in the environmental salinity.

The effects of acute manipulation of plasma osmolality and blood volume on plasma atrial and ventricular natriuretic peptide (ANP and VNP) levels were examined in conscious freshwater eels, Anguilla japonica. A bolus injection of hypertonic NaCl (0.85 M and 1.7 M, 2.5 ml/kg body weight) through a catheter into the ventral aorta produced increases in plasma Na concentration and osmolality with parallel concentration-dependent, transient increases in plasma ANP and VNP levels. Plasma ANP and VNP levels also increased after injection of 1.7 M mannitol solution which produced an increase in plasma osmolality but a decrease in plasma Na concentration. However, injection of a 2.0 M solution of urea, which does not cause cellular dehydration in mammals, produced only small increases in plasma ANP and VNP levels, although plasma osmolality increased. A bolus injection of 10 or 25 ml/kg isotonic saline supplemented with 2% dextran for colloidal osmotic pressure, which theoretically increased blood volume by 29% or 71%, produced volume-dependent, transient increases in plasma ANP and VNP levels without changes in plasma Na concentration and osmolality. Similar volume expansion with dialysed eel plasma caused greater increases than with dextran-saline. However, these increases were much smaller than those after osmotic stimuli. These results indicate that secretion of ANP and VNP is regulated by two receptor mechanisms: osmo-receptors activated by cellular dehydration, not specifically by hypernatraemia, and volume or stretch receptors activated by hypervolaemia. The relative importance of the osmoreceptive mechanism is greater in eels than in mammals where volaemic regulation dominates over osmotic regulation for ANP secretion.

Plasma levels of atrial and ventricular natriuretic peptide (ANP and VNP) were measured in freshwater (FW)- and seawater (SW)-adapted eels using homologous radioimmunoassays. Anesthesia with tricaine (MS222) and exposure of the pericardium for blood sampling from the ventral aorta affected plasma ANP and VNP levels. ANP and VNP levels in the ventral aorta were higher than those in the dorsal aorta when blood was collected simultaneously from both aortae of cannulated conscious FW eels, perhaps reflecting branchial extraction of the peptides. This phenomenon was not observed in SW eels. Based on these results, blood was sampled from conscious eels through a catheter implanted in the ventral aorta because it appears to reflect most accurately ANP secretion. Plasma ANP and VNP levels measured in more than 100 conscious FW and SW eels displayed large variations at each month despite acclimation to laboratory conditions at 180 for more than 1 week in FW eels or for more than 2 weeks in SW eels. However, concentrations of ANP and VNP averaged from all data were not different between FW and SW eels: 68.1 +/- 2.9 (FW: n = 155) vs 60.7 +/- 3.6 (SW: n = 104) for ANP, 51.6 +/- 3.0 (FW: n = 69) vs 55.0 +/- 3.7 (SW: n = 54) for VNP (fmol/ml in each case). Plasma Na concentration and osmolality were significantly higher in SW eels than in FW eels. Plasma ANP and VNP levels thus do not seem to be affected by chronic changes in plasma Na concentration and osmolality.

Ventricular natriuretic peptide (VNP), a possibly new type of natriuretic peptide with an extended C-terminal tail, has been isolated from eel cardiac ventricles. We investigated the effects of eel VNP on the kidney and cardiovascular system and compared these results with those of mammalian peptides in dogs. Eel VNP, human (dog) ANP, human and dog BNPs were infused into the renal artery at non-hypotensive doses. All peptides produced similar diuresis and natriuresis, but cardiac output and the left and right ventricular stroke work were decreased by BNPs, but ANP and eel VNP did not change these parameters. Systemic vascular resistance was increased by BNPs, but unaffected by other peptides. These results show that eel VNP has renal effects similar to ANP and BNP, but it elicits responses in the heart different from those of BNPs in anesthetized dogs.

Ventricular natriuretic peptide (VNP), a newly discovered type of cardiac natriuretic peptide identified in eels, has a unique amino acid sequence and biological activity compared with other members of the natriuretic peptide family. We have cloned a cDNA encoding the eel VNP precursor from a cDNA library of eel ventricles and determined its sequence. Sequence analysis showed that the preproVNP consists of 150 amino acid residues containing a signal sequence of 22 amino acid residues at its N terminus and mature VNP(1-36) at its C terminus. Comparison with two types of mammalian cardiac natriuretic peptides (A and B type natriuretic peptides; ANP and BNP) revealed that VNP showed greater overall sequence identity to ANP than to BNP at both the cDNA and amino acid sequence levels. Since VNP cDNA lacks the repetitive ATTTA sequence in the 3' non-coding region, which is a characteristic common to all BNP cDNAs sequenced to date, VNP may differ from BNP. While mRNA for eel ANP was detected only in the atrium, that for eel VNP was more abundant in the ventricle than in the atrium. Thus VNP is regarded as the first truly ventricular hormone in terms of synthesis and storage. Southern blot analysis indicated that VNP is also present in the quail, which belongs to a taxon where BNP has already been identified.

Ventricular natriuretic peptide (VNP) is a new type of cardiac natriuretic peptide initially isolated from the eel ventricle. VNP has been isolated from cardiac ventricles of the rainbow trout, Oncorhynchus mykiss, and found to consist of 35 amino acid residues carrying a C-terminal tail sequence with 14 amino acid residues. Thus, the long C-terminal sequence characteristic of VNP was also conserved in the trout VNP. A VNP with 4 amino acid residues truncated from the C-terminus was also isolated from trout ventricles and sequenced. Sequence identity of trout VNP to eel VNP was 77%; while it was 56% to eel A-type natriuretic peptide (ANP). Trout VNP caused characteristic biphasic vasopressor/depressor effects in the trout similar to those produced by rat and eel ANP. Trout VNP and human ANP were almost equipotent in their vasopressor and depressor activity in trout. Unlike eel VNP, therefore, homologous VNP did not exhibit greater activity in the trout. In the rat, however, trout VNP was more potent than eel peptide and was almost equipotent to human ANP for both vasodepressor and natriuretic effects. The high potency of trout VNP appears to be due in part to its longer-lasting effect compared to human ANP.

C-type natriuretic peptides (CNP) of varying length were isolated from the atrium or ventricle of a shark, Lamna ditropis and their amino acid sequences were determined. Although the sequence of Lamna CNP was highly homologous to those of other CNPs sequenced to date, the Lamna CNP-41, the longest CNP identified in this study, has one amino acid replacement from those of Triakis scyllia and Scyliorhinus canicula, and three amino acid replacements from that of Squalus acanthias. The degree of similarity of CNP molecules coincides well with their systematic positions in the cladogram of elasmobranchs; Lamna, Triakis and Scyliorhinus belong to the same order, but Lamna and Squalus belong to different orders. The facts that Lamna and Triakis are in different suborders but Triakis and Scyliorhinus are in the same suborder and have identical CNP-41, also support this evolutionary implication.

Ventricular natriuretic peptide (VNP) with 25 amino acid residues was isolated from the low molecular weight fraction of acid extracts of eel cardiac ventricles. No other short forms of VNP were recovered from the fraction. This peptide was named eel VNP(1-25) because it was a C-terminally truncated form of the previously isolated eel VNP(1-36). As observed before with eel VNP(1-36), eel VNP(1-25) had a much higher (146-fold) vasodepressor activity than human atrial natriuretic peptide (ANP) in eels, but was a third to a half as active in rats with respect to vasodepressor and natriuretic activities. Eel VNP(1-25) was generally less potent than eel VNP(1-36) for vasodepressor and natriuretic effects. A specific radioimmunoassay (RIA) has been developed for the measurement of eel VNP. The antiserum, raised against eel VNP(1-36), was highly specific and did not exhibit significant cross-reactivity with eel ANP and C-type natriuretic peptide, even though their amino acid sequences have more than 60% homology with that of eel VNP. The sensitivity of assay was 0.5 fmol/tube for eel VNP(1-36) with more than 99% confidence. Such high sensitivity permitted direct assaying of VNP with only a few microliters of plasma. In fresh water eels, the concentration of VNP in the cardiac ventricle was higher than those in the atrium or brain and that of ANP in the ventricle. Thus, VNP seems to be a ventricular hormone. Although ANP is a major circulating hormone in mammals, the plasma concentration of VNP was threefold higher than that of ANP.(ABSTRACT TRUNCATED AT 250 WORDS)

A highly specific and sensitive radioimmunoassay has been developed for the measurement of eel atrial natriuretic peptide (ANP). The antiserum, raised against eel ANP-(1-27) did not cross-react with two other eel natriuretic peptides, i.e. eel ventricular natriuretic peptide and C-type natriuretic peptide (CNP), or with any mammalian ANPs, CNPs or brain natriuretic peptides so far identified. The minimal detectable amount was 0.39 fmol (0.90 pg)/tube with more than 99% confidence. Because of its high sensitivity, the radioimmunoassay makes it possible to measure eel ANP directly with only a few microlitres of plasma without extraction. Using the radioimmunoassay we found high levels of ANP in the atrium (11 +/- 2 pmol/mg wet tissue, n = 8), and much lower levels in the ventricle (56 +/- 8 fmol/mg, n = 8) and the brain (22 +/- 1 fmol/mg, n = 8) of eels. Eel plasma contained a large amount of ANP (247 +/- 66 fmol/ml, n = 8) compared with the levels reported in mammals, although atrial levels are similar between eels and mammals. Gel-permeation chromography revealed that a major form of ANP stored in the eel atrium, ventricle and brain has a molecular mass of approximately 14 kDa but low molecular forms of about 3 kDa are predominant in eel plasma. A detailed analysis with reverse-phase high-performance liquid chromatography showed that a major molecular form circulating in eel plasma is ANP-(1-27). ANP-(1-27) was also detected in small amounts in the eel atrium, ventricle and brain.

Ventricular natriuretic peptide (VNP) appears to be a new member of the ANP family, as judged by its uniquely long C-terminal 'tail' sequence and by its cDNA sequence. The eel VNP causes the entire spectra of actions known to be characteristic of the ANP family, and its vascular and renal effects are more potent than other eel natriuretic peptides in both eels and rats. Since VNP is a hormone originating from ventricles, it is possible that VNP is secreted more quickly and profoundly than atrium-originated ANP, in response to an increase in afterload or stenosis of the coronary artery, resulting in recovery of ventricular function. Therefore, identification of VNP in mammals is awaited for the development of a new drug which counteracts against heart failure.

Eel atrial natriuretic peptide inhibited the serosa-negative transepithelial potential difference and short-circuit current, accompanied by a decrease in NaCl and water absorption across the seawater eel intestine. Similar effects were obtained after treatment with N-terminally truncated eel atrial natriuretic peptide (5-27), indicating that N-terminal amino acids are not essential for the action of eel atrial natriuretic peptide. Although mammalian atrial natriuretic peptides also inhibited the short-circuit current, a 100-fold higher concentration was required to obtain the same effect as with eel atrial natriuretic peptide, indicating that eel atrial natriuretic peptide is 100 times as potent in eel intestine as the mammalian atrial natriuretic peptides. Similarly, in mammalian atrial natriuretic peptide, the four N-terminal amino acids had no significant effects. However, when the C-terminal tyrosine was removed, the potency of rat atrial natriuretic peptide was lowered. Compared with the effects of acetylcholine, serotonin and histamine, eel atrial natriuretic peptide was the most potent inhibitor, with 100% inhibition at 10(-7) M; 50% inhibition was obtained at 10(-2) M in acetylcholine, and 30% inhibition in serotonin (10(-5) M) and histamine (10(-3) M). These inhibitory effects of eel atrial natriuretic peptide were not diminished even in the presence of tetrodotoxin, and were mimicked by 8-bromoguanosine 3',5'-cyclic monophosphate. Based on these results, structure-activity relationships of eel atrial natriuretic peptide and a possible mechanism of action of eel atrial natriuretic peptide are discussed.

A new natriuretic peptide, which exhibits the entire spectrum of actions known to be characteristic of atrial and brain natriuretic peptides (ANP and BNP), was isolated from eel cardiac ventricles and has been named ventricular natriuretic peptide (VNP). The primary structure of eel VNP is characterized by its uniquely long C-terminal 'tail' that extends from the second half-cystine. Thus, eel VNP appears to be a novel natriuretic peptide of a type not found in mammals. With respect to natriuretic (rat) and vasodepressor (rat and eel) activities, eel VNP is much more potent than human ANP in eels and almost equipotent in rats. Strong tachyphylaxis is observed for the vasodepressor effect in both rats and eels, whereas it is not observed for the natriuretic effect in rats.

A high-molecular-weight form of C-type natriuretic peptide (CNP) was isolated from both cardiac atria and ventricles of European dogfish, Scyliorhinus canicula, and its primary structure was determined. The peptide consists of 115 amino acid residues, in which the C-terminal 22 residues show high homology to CNPs identified to date. This is the first direct evidence for the presence of natriuretic peptide in the cartilaginous fish, and for the presence of CNP in an organ other than the brain.

A peptide exhibiting natriuretic and vasodepressor activity was isolated from eel brains. Its amino acid sequence was found to be similar to, but distinct from, that of the eel atrial natriuretic peptide (ANP), and it is characterized by the absence of the C-terminal sequence that follows the second-half cystine. The extent of sequence homology of this peptide to known mammalian brain natriuretic peptides (BNPs) is greater than to ANPs. Therefore, we have named this peptide "eel BNP-like peptide". The isolation of this peptide provides the first evidence for the presence of two different molecular types of natriuretic peptide in a single non-mammalian species. The potency of the eel BNP-like peptide relative to that of human ANP, in terms of its vasodepressor activity, was 117 in the eel, 1.7 in the quail, and 0.08 in the rat. Thus, eels exhibit a high degree of sensitivity to this native peptide just as they do to eel ANP.

A peptide exhibiting vasodepressor and natriuretic activities in rats was isolated from eel atria, and its primary structure was determined as H-Ser-Lys-Ser-Ser-Ser-Pro-Cys-Phe-Gly-Gly-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Tyr-Ser- Gly-Leu-Gly-Cys-Asn-Ser-Arg-Lys-OH. This peptide, termed eel atrial natriuretic peptide (ANP), has sequence homology of 59% to mammalian (human or rat) ANP, 52% to fowl ANP, and 46% to frog ANP. When the biological activity of synthetic eel ANP was compared with that of human ANP, the eel peptide was 110 times more potent for the vasodepressor activity in eels, nearly equipotent for the vasodepressor activity in quails, and 20 times less potent for the vasodepressor and natriuretic activity in rats.