Shigenori Nobata

Area postrema, a brain circumventricular organ, is the site of antidipsogenic action of circulating atrial natriuretic peptide in eels

SUMMARY 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.

Potent cardiovascular effects of homologous urotensin II (UII)-related peptide and UII in unanesthetized eels after peripheral and central injections

We cloned cDNAs encoding urotensin II (UII)-related peptide (URP) and UII in Japanese eel, Anguilla japonica, the former being the first such cloning in teleost fishes. Unlike the exclusive expression of UII in the urophysis, the URP gene was expressed most abundantly in the brain (medulla oblongata) followed by the urophysis. Peripheral injections of URP into eels increased blood pressure by 16.1 ± 0.8 mmHg at 0.1 nmol/kg in ventral aortic blood pressure (P(VA)) and with similar potency and efficacy to that of UII (relative potency of URP to UII = 0.83). URP/UII and ANG II preferentially acted on the branchial and systemic circulations, respectively, and the duration of effect was distinct among the three peptides in the order of UII (60 min) >URP (30 min) >ANG II (14 min) in P(VA). Urantide, a mammalian UII receptor antagonist, inhibited the URP effect (-63.6 ± 5.2%) to a greater extent than for UII (-39.9 ± 5.0%). URP and UII constricted isolated eel branchial and systemic arteries, showing their direct actions on the vascular smooth muscle. Central injection of URP increased blood pressure by 12.3 ± 0.8 mmHg at 50 pmol/eel in P(VA) and with similar efficacy but less potency (relative potency = 0.47) and shorter duration compared with UII. The central actions of URP/UII were more potent on the branchial circulation than on the systemic circulation, again opposite the effects of ANG II. The similar responses to peripheral and central injections suggest that peripheral hormones may act on the brain. Taken together, in eels, URP and UII are potent cardiovascular hormones like ANG II, acting directly on the peripheral vasculature, as well as a central vasomotor site, and their actions are mediated to different degrees by the UII receptor.

Diversified cardiovascular actions of six homologous natriuretic peptides (ANP, BNP, VNP, CNP1, CNP3, and CNP4) in conscious eels

The natriuretic peptide (NP) family consists of seven paralogs [atrial NP (ANP), brain NP (BNP), ventricular NP (VNP), and C-type NP 1-4 (CNP1-4)] in teleosts, but relative biological activity of the seven NPs has not been comprehensively examined using homologous peptides. In this study, we newly identified CNP3 and CNP4 in eels to use homologous peptides, but the CNP2 gene may have been silenced in this species. The CNP4 gene was expressed exclusively in the brain as CNP1, but the CNP3 gene, from which cardiac ANP, BNP, and VNP were generated by tandem duplication, was most abundantly expressed in the pituitary, suggesting its local action. All NPs induced hypotension dose dependently after intra-arterial injection with a potency order of ANP > VNP > BNP > CNP4 > CNP1 = CNP3. The degree of hypotension was similar at the ventral and dorsal aorta, indicating similar actions on the branchial and systemic circulation. The hypotension induced by cardiac NPs was longer lasting than CNPs, probably because of the difference in preferential receptors. Among cardiac NPs, the hypotensive effect of VNP lasted much longer than those of ANP and BNP, even though VNP disappeared from the blood more quickly than ANP. To analyze the unique effect of VNP, we examined possible involvement of the autonomic nervous system using ANP, VNP, and CNP3. Beta-adrenergic blockade diminished hypotensive effects of all three NPs, but alpha-adrenergic and cholinergic blockade enhanced only the effect of VNP, suggesting a specific mechanism for the VNP action. The NP-induced tachycardia was diminished by all blockers examined. Furthermore, the cardiovascular action of VNP was not impaired by a blocker of NP receptor, HS-142-1. Taken together, the homologous NPs exhibit diverse cardiovascular actions in eels partially through the autonomic nervous system, and the unique VNP action may be mediated by a novel receptor that has not been identified in teleosts.

Potent cardiovascular actions of homologous adrenomedullins in eels

Adrenomedullin (AM), known as a multifunctional hormone in mammals, forms a unique family of five paralogous peptides in teleost fish. To examine their cardiovascular effects using homologous AMs in eels, we isolated cDNAs encoding four eel AMs, and named AM1 (ortholog of mammalian AM), AM2, AM3 (paralog of AM2 generated only in teleost lineage), and AM5 according to the known teleost AM sequences. Unlike pufferfish, not only AM1 but AM2/3 and AM5 were expressed ubiquitously in various eel tissues. Synthetic mature AM1, AM2, and AM5 exhibited vasodepressor effects after intra-arterial injections, and the effects were more potent at dorsal aorta than at ventral aorta. This indicates that AMs preferentially act on peripheral resistance vessels rather than on branchial arterioles. The potency was in the order of AM2 = AM5 >> AM1 in both freshwater (FW) and seawater (SW) eels, which is different from the result of mammals in which AM1 is as potent as, or more potent than, AM2 when injected peripherally. The minimum effective dose of AM2 and AM5 in eels was 1/10 that of AM1 in mammals. The hypotension reached 50% at 1.0 nmol/kg of AM2 and AM5, which is much greater than atrial natriuretic peptide (20%), another potent vasodepressor hormone. Even with such hypotension, AMs did not change heart rate in eels. In addition, AM1 increased blood pressure at ventral aorta and dorsal aorta immediately after an initial hypotension at 5.0 nmol/kg, but not with AM2 and AM5. These data strongly suggest that specific receptors for AM2 and AM5 exist in eels, which differ from the AM1 receptors identified in mammals.

The area postrema in hindbrain is a central player for regulation of drinking behavior in Japanese eels

It is recognized that fish will drink the surrounding water by reflex swallowing without a thirst sensation. We evaluated the role of the area postrema (AP), a sensory circumventricular organ (CVO) in the medulla oblongata, in the regulation of drinking behavior of seawater (SW) eels. The antidipsogenic effects of ghrelin and atrial natriuretic peptide and hypervolemia and hyperosmolemia (1 M sucrose or 10% NaCl) as well as the dipsogenic effects of angiotensin II and hypovolemia (hemorrhage) were profoundly diminished after AP lesion (APx) in eels compared with sham controls. However, the antidipsogenic effect of urotensin II was not influenced by APx, possibly due to the direct baroreflex inhibition on the swallowing center in eels. When ingested water was drained via an esophageal fistula, water intake increased 30-fold in sham controls but only fivefold in APx eels, suggesting a role for the AP in continuous regulation of drinking by SW eels. After transfer from freshwater to SW, APx eels responded normally with an immediate burst of drinking, but after 4 wk these animals showed a much greater increase in plasma osmolality than controls, suggesting that the AP is involved in acclimation to SW by fine tuning of the drinking rate. Taken together, the AP in the hindbrain of eels plays an integral role in SW acclimation, acting as a conduit of information from plasma for the regulation of drinking, probably without a thirst sensation. This differs from mammals in which sensory CVOs in the forebrain play pivotal roles in thirst regulation.

Potent osmoregulatory actions of homologous adrenomedullins administered peripherally and centrally in eels

The teleost adrenomedullin (AM) family consists of three groups, AM1/AM4, AM2/AM3, and AM5. In the present study, we examined the effects of homologous AM1, AM2, and AM5 on drinking and renal function after peripheral or central administration in conscious freshwater eels. AM2 and AM5, but not AM1, exhibited dose-dependent (0.01-1 nmol/kg) dipsogenic and antidiuretic effects after intra-arterial bolus injection. The antidiuretic effect was significantly correlated with the degree of associated hypotension. To avoid the potential indirect osmoregulatory effects of AM-induced hypotension, infusion of AMs was also performed at nondepressor doses. Drinking was enhanced dose-dependently at 0.1-3 pmol.kg(-1).min(-1) of AM2 and AM5, matching the potency and efficacy of angiotensin II (ANG II), the most potent dipsogenic hormone known thus far. AM2 and AM5 infusion also induced mild antidiuresis, while AM1 caused antinatriuresis. Additionally, AMs were injected into the third and fourth ventricles of conscious eels to assess their site of dipsogenic action. However, none of the AMs at 0.05-0.5 nmol induced drinking, while ANG II was highly dipsogenic. AM2 and ANG II injected into the third ventricle increased arterial pressure while AM5 decreased it in a dose-dependent manner, and both AM2 and AM5 decreased blood pressure when injected into the fourth ventricle. These data suggest that circulating AM2 and AM5 act on a target site in the brain that lacks the blood-brain barrier. Collectively, the present study showed that AM2 and AM5 are potent osmoregulatory hormones in the eel, and their actions imply involvement in seawater adaptation of this euryhaline species.

Hormonal control of drinking behavior in teleost fishes; insights from studies using eels

Marine teleost fishes drink environmental seawater to compensate for osmotic water loss, and the amount of water intake is precisely regulated to prevent dehydration or hypernatremia. Unlike terrestrial animals in which thirst motivates a series of drinking behaviors, aquatic fishes can drink environmental water by reflex swallowing without searching for water. Hormones are key effectors for the regulation of drinking. In particular, angiotensin II and atrial natriuretic peptide are likely candidates for physiological regulators because of their potent dipsogenic and antidipsogenic activities, respectively. In the eel, these hormones act on the area postrema in the medulla oblongata, a circumventricular structure without blood-brain barrier, which then regulates the activity of the glossopharyngeal-vagal motor complex. These motor neurons in the hindbrain innervate the upper esophageal sphincter muscle and other swallowing-related muscles in the pharynx and esophagus for regulation of drinking. Thus, the neural circuitry for drinking in fishes appears to be confined within the hindbrain. This simple mechanism is much different from that of terrestrial animals in which thirst sensation is induced through hormonal actions on the subfornical organ and organum vasculosum of the lamina terminalis that are located in the forebrain. It seems that the neural and hormonal mechanism that regulates drinking behavior has evolved from fishes depending on the availability of water in their natural habitats.

Relative antidipsogenic potencies of six homologous natriuretic peptides in eels

Atrial natriuretic peptide (ANP) exhibits a potent antidipsogenic effect in seawater (SW) eels to limit excess Na+ uptake, thereby effectively promoting SW adaptation. Recently, cardiac ANP, BNP and VNP and brain CNP1, 3 and 4, have been identified in eels. We examined the antidipsogenic effect of all homologous NPs using conscious, cannulated eels in both FW and SW together with parameters that affect drinking. A dose-response study (0.01–1 nmol/kg) in SW eels showed the relative potency of the antidipsogenic effect was in the order ANP ≥ VNP > BNP = CNP3 > CNP1 ≥ CNP4, while the order was ANP = VNP = BNP > CNP3 = CNP1 = CNP4 for the vasodepressor effect. The minimum effective dose of ANP for the antidipsogenic effect is much lower than that in mammals. ANP, BNP and VNP at 0.3 nmol/kg decreased drinking, plasma Na+ concentration and aortic pressure and increased hematocrit in SW eels. The cardiac NPs induced similar changes in drinking, aortic pressure and hematocrit in FW eels, but aside from BNP no change in plasma Na+ concentration. CNPs had no effect on drinking, plasma Na+ concentration and hematocrit but induced mild hypotension in both FW and SW eels, except for CNP3 that inhibited drinking in SW eels. These results show that ANP, BNP and VNP are potent antidipsogenic hormones in eels in spite of other regulatory factors working to induce drinking, and that CNPs are without effects on drinking except for the ancestor of the cardiac NPs, CNP3.

Natriuretic Peptides in Developing Medaka Embryos: Implications in Cardiac Development by Loss-of-Function Studies

Cardiac natriuretic peptides (NPs), atrial NP (ANP) and B-type NP (BNP), and their receptor, guanylyl cyclase (GC)-A have attracted attention of many basic and clinical researchers because of their potent renal and cardiovascular actions. In this study, we used medaka, Oryzias latipes, as a model species to pursue the physiological functions of NPs because it is a suitable model for developmental analyses. Medaka has two ligands, BNP and C-type NP3 (CNP3) (but not ANP), that have greater affinity for the two O. latipes GC-A receptors (OLGC), OLGC7 and OLGC2, respectively. CNP3 is the ancestral molecule of cardiac NPs. Initially, we examined developmental expression of cardiac NP/receptor combinations, BNP/OLGC7 and CNP3/OLGC2, using quantitative real-time PCR and in situ hybridization. BNP and CNP3 mRNA increased at stages 25 (onset of ventricular formation) and 22 (appearance of heart anlage), respectively, whereas both receptor mRNAs increased at as early as stage 12. BNP/OLGC7 transcripts were found in arterial/ventricular tissues and CNP3/OLGC2 transcripts in venous/atrial tissues by in situ hybridization. Thus, BNP and CNP3 can act locally on cardiac myocytes in a paracrine/autocrine fashion. Double knockdown of BNP/OLGC7 genes impaired ventricular development by causing hypoplasia of ventricular myocytes as evidenced by reduced bromodeoxyuridine incorporation. CNP3 knockdown induced hypertrophy of atria and activated the renin-angiotensin system. Collectively, it appears that BNP is important for normal ventricular, whereas CNP3 is important for normal atrial development and performance, a role usually taken by ANP in other vertebrates. The current study provides new insights into the role of cardiac NPs in cardiac development in vertebrates.

Exploring New CGRP Family Peptides and their Receptors in Vertebrates

Vertebrates have expanded their habitats from aquatic to terrestrial environments, which has accompanied the evolution of cardiovascular and osmoregulatory hormones. Specifically, mammals have developed mechanisms to maintain high blood pressure and blood volume, while extant fishes have developed hypotensive and Na-extruding mechanisms to adapt to the marine environment where they underwent a vast diversification. The CGRP family is one of the hormone systems that decrease blood pressure and blood volume. Within the CGRP family of teleost fishes, we found that adrenomedullins (AMs) have diversified and five paralogs (AM1-5) form an independent subfamily. Based on this discovery in fishes, we found AM2 and AM5 in mammals. In mammalian species that have AM2 and/or AM5, the peptides assume greater importance in the case of pathophysiological disturbances in pressure and fluid balance such as hypertension and cardiac and renal failure. In addition, novel functions of AM peptides have been suggested by the discovery of AM2 and AM5 in mammals. Current research on the CGRP family is focused on the identification of new receptors for AM2/AM5 and the establishment of AM2 knockout mice, which will enable new developments in the basic and clinical research on this intriguing hormone family. Importantly, comparative fish studies can contribute to new developments in our understanding of the function of the AM peptides.