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.
- intracerebroventricular administration
- teleost fish
teleost fish maintain their body fluid osmolality at one-third of seawater, whether they are in freshwater or in seawater. To achieve body fluid homeostasis, fish regulate water and ion transport in osmoregulatory organs such as the gill, kidney, and digestive tract. Marine teleost fish suffer from water efflux and ion influx as a result of the difference in tonicity between extracellular fluid and environmental seawater, while the opposite fluxes occur in freshwater fish. For adaptation to hyperosmotic seawater, therefore, it is essential for fish to excrete excess ions and to absorb water from the environment. To this end, marine teleosts drink a large volume of seawater, which is absorbed through the digestive tract, and ions are actively excreted by the gill and kidney. Several hormones are known to be involved in the regulation of water intake in teleosts. Angiotensin II (ANG II) is a well-established dipsogenic hormone throughout vertebrates including fish (8, 38). In contrast, atrial natriuretic peptide (ANP) suppresses drinking rate (41) and also has an antidiuretic effect in teleost fish (37). In addition to these hormones, growth hormone (GH) and cortisol are known to be involved in adaptation to and survival in seawater (26). On the other hand, prolactin contributes to freshwater survival by promoting ion uptake and inducing cell proliferation (29). Despite the identification of such key osmoregulatory hormones, it is evident that fish osmoregulation involves other factors.
Adrenomedullin (AM) is a potent cardiovascular regulator in mammals. AM is ubiquitously expressed in various tissues and exhibits multiple effects (16). It was initially isolated from human pheochromocytoma cells and reported to be potently hypotensive (12). AM is expressed abundantly in vascular endothelial cells and in vascular smooth muscle cells, so that it is considered to play an important role in mammalian cardiovascular regulation in both a paracrine and an endocrine fashion (11). Mammalian AM effects include others related to body fluid regulation such as renal diuresis and natriuresis (9), inhibition of drinking and sodium appetite when administered centrally (20, 30), and the regulation of pituitary arginine vasopressin and oxytocin secretion (32). Among nonmammalian species, teleost fish are the only group in which this hormone has been studied thus far. Previously, our group identified five AMs in several teleost species (AM1 through AM5) and found that three ancestral types of AM (AM1, AM2, and AM5) existed before the divergence of ray-finned fish from lobe-finned fish (24, 25). This discovery led us to the further discovery of novel AM2 and AM5 in mammals (36, 25). Mammalian AM2 has effects on cardiovascular and body fluid regulation as observed with AM. AM2 decreases blood pressure in the rat, but its effect is less potent than AM (5). In rat and sheep, AM2 promotes diuresis when administered peripherally (2, 5), but high doses cause antidiuresis as a result of the accompanying hypotension (36). The major effects of AM2 in mammals known to date are on cardiovascular and body fluid regulation, similar to AM. The recently identified AM5 also induces hypotension after peripheral injection and hypertension after intracerebroventricular administration in mammals, but the peripheral effects are less potent than AM (35).
Since the discovery of the teleost AM family in 2003, physiological actions of the diverse AM peptides have not yet been well investigated in fish. Accordingly, in the present work we used the eel Anguilla japonica, exploiting established surgical techniques to perform in vivo experiments in conscious animals (42). Using this preparation, Nobata et al. (22) recently examined the cardiovascular effect of homologous AM peptides in the eel and reported that AM2 and AM5 were potently hypotensive. Since body fluid regulation in fish is closely related to cardiovascular function, the AM family may also play a significant part in body fluid homeostasis in teleost fish. In the present study, therefore, three major peptides of the teleost AM family, AM1, AM2, and AM5, were chosen to examine their effects on body fluid regulation. These three AMs existed before ray-finned fish had diverged from lobe-finned fish, and they were further duplicated in the teleost lineage at the third-round whole genome duplication (25). However, the counterpart of duplicated AM5 appears to have been silenced during teleost evolution. Sequence comparisons have shown that eel AM1 and AM2 are more similar to mammalian AM and AM2 than eel AM4 and AM3, respectively, and thus appear to retain the original sequence characteristics. As parameters for body fluid regulation, we measured drinking rate, urine volume, and urinary Na+ concentration. Because preliminary experiments indicated that AMs were dipsogenic and antidiuretic, we used freshwater eels in the present study. In seawater eels, drinking rate is already enhanced and urine volume is suppressed, so it may be difficult to observe further effects of AMs on these parameters. The effects with AMs are compared with those of other potent osmoregulatory hormones, ANG II and ANP. Additionally, to explore the potential site of action of AMs, we have compared the effects of peripherally and centrally administered AMs. The present study is the first to report osmoregulatory actions of homologous AMs in a nonmammalian species.
MATERIALS AND METHODS
Cultured male A. japonica (body wt 180.9 ± 15.0 g) were purchased from a local dealer. All were kept in freshwater without feeding for more than a week before use. Water in the tank was filtered, aerated, and maintained at 18°C. All animal experiments in this study were approved by the Animal Experiment Committee of the University of Tokyo and performed in accordance with the “Manual for Animal Experiments”.
Surgical Procedures: Peripheral Administration
Eels were anesthetized in 0.1% (wt/vol) tricaine methanesulfonate for 15 min and placed on an operation board for surgery. The dorsal aorta was cannulated with polyethylene tube (0.5-mm ID, 0.8-mm OD) for injections of hormones and measurement of blood pressure as described previously (42). Plastic tubes were inserted into the esophagus and stomach to measure drinking rate and into the urinary bladder to measure urine flow. After surgery, eels were placed in a plastic trough through which aerated water was circulated at 18°C. After overnight recovery from anesthesia and surgery, the catheter of the dorsal aorta was connected via a three-way stopcock to a pressure transducer (Nihon Koden, Tokyo, Japan) for continuous monitoring of blood pressure. The signal was amplified by a carrier amplifier (7903, NEC San-ei, Tokyo, Japan) and recorded by a pen recorder (Rikadenki, Tokyo, Japan). The esophageal and bladder catheters were connected to drop counters for continuous measurement of drinking and urine flow rates, respectively. The stomach catheter was connected to a pulse injector synchronized with the esophageal drop counter for reintroduction of ingested water.
Eel AM1, AM2, AM5, and ANP were synthesized by the Peptide Institute (Osaka, Japan) with a solid-phase method. Human AM2 and teleost-type [Asn1, Val5]ANG II were purchased from the Peptide Institute.
Experimental Protocol: Bolus Injection
Six eels were used for this experiment. After recovery from surgery, eel AM1, AM2, and AM5, human AM2, and eel ANP were injected as a bolus at doses of 0.01, 0.1, and 1 nmol/kg in 0.05 ml of 0.9% NaCl containing 0.01% Triton X-100 (Nakarai, Kyoto, Japan), which prevents adsorption of peptides to the walls of the tube and syringe. The hormones were administered via the dorsal aorta in random order. Each injection was followed by an injection of 0.08 ml of saline to flush the dead volume of the tubes. Blood pressure was monitored throughout the experiment. The drinking rate and urine flow rate were measured by counting each drop from the catheter (1 drop was ∼15 μl). The effects on blood pressure were evaluated in terms of maximal changes after injection of hormones.
Experimental Protocol: Infusion
Six eels were used for this experiment. The infused hormones were eel AM1, AM2, AM5, and ANG II. The schedule of infusion was initiated with saline (control vehicle) for 1 h, followed by hormones at 0.1, 0.3, 1.0, and 3.0 pmol·kg−1·min−1 for a period of 1 h each, and finally 2 h of saline delivered at a rate of 0.4 ml/h. To nullify the increase in blood volume from the infusion, 0.4 ml of blood was collected at the end of each hour. Drinking rate, urine flow rate, and blood pressure were monitored throughout the experiment as for the bolus injection series. Urine was collected during each phase, and Na+ concentration was measured by an atomic absorption spectrophotometer (Z5300, Hitachi, Japan).
Surgical and Experimental Protocol: Central Administration
Eels were anesthetized in 0.1% (wt/vol) tricaine methanesulfonate, and catheters were inserted into the dorsal aorta, esophagus, and stomach as described above. Cephalic skin was then cut, and muscle was removed to reveal the skull. A hole (∼1 mm in diameter) was made by dental drill at 5 mm posterior from the caudal verge of the eye under a KOM 300 stereomicroscope (Konan, Hyogo, Japan). The dura mater was carefully punctured with sharp forceps to avoid bleeding in order to make a passage for the cannula. The head of the eel was then fixed in a stereotaxic apparatus (Narishige, Tokyo, Japan). A stainless steel guide cannula (0.35-mm ID, 0.6-mm OD, 1-cm length) was inserted to a depth of 1.8 mm from the skull surface and fixed with dental cement (Fig. 1). The tip of the cannula was aimed at a position 1.5 mm above the bottom of the third ventricle. For the injection into the fourth ventricle, a hole was made 1 cm posterior from the caudal verge of the eye and the guide cannula was inserted to a depth of 1.1 mm from the skull surface. The implanted guide cannula was plugged with a stainless steel rod (0.3-mm OD) except for the time of injection. To confirm that the cannula tip was in the third ventricle, 0.5 μl of 1 mM ANG II was injected before the experiment and only animals that started drinking were used for further experimentation (n = 10). The tip of the cannula for the fourth ventricle was aimed 1 mm posterior to the cerebellum (n = 10). The intracerebroventricularly administered doses of eel AM1, AM2, AM5, or ANG II were 0.1, 0.3, and 1 mmol/l in 0.5 μl of 0.9% NaCl containing 0.01% Triton X-100. Blood pressure and drinking rate were monitored throughout the experiment.
Changes in arterial blood pressure were expressed in terms of percentage of maximal change compared with the preinjection level and analyzed by Student's t-test. The drinking rate and urine flow rate were measured every 5 min after bolus injection of hormones, and values were compared with the preinjection measures by one-way ANOVA and following multiple comparison test (Dunnett test). In the infusion experiment, the values measured during hormone infusion were compared with those measured during the initial saline infusion phase and evaluated by Student's t-test. Significance was determined at P < 0.05.
Effect of Peripheral Administration of AMs
Effects of bolus injections.
Drinking rate did not change over the 30 min after saline injection. Time course changes in drinking rate after bolus injections of eel AM1, AM2, and AM5 are shown in Fig. 2, A, C, and E, respectively. Injection of AM2 and AM5 increased drinking rate, which was significant after 10 min at 1 nmol/kg. The enhanced drinking rate continued longer (for >30 min) after injection of AM5 than after injection of AM2. In contrast, AM1 significantly decreased water intake 5 min after injection of 1 nmol/kg. Although the dipsogenic responses were accompanied by a decrease in arterial pressure, the decreases in blood pressure and increases in drinking rate were not significantly correlated after injection of either AM2 or AM5 (Fig. 3A). ANP was administered as a positive control to confirm the responsiveness of the experimental system, and it suppressed drinking at 1 nmol/kg as previously reported (44) (Fig. 2G). Human AM2 increased the drinking rate as potently as eel AM2 and AM5 at 1 nmol/kg (Fig. 2G).
Urine flow rate did not change over the 30 min after saline injection. AM1 did not alter urine flow rate (Fig. 2B). AM2 induced dose-related decreases in urine flow rate, and the decrease was significant at 1 nmol/kg (Fig. 2D). AM5 was less potent than AM2, with no significant decrease at 0.1 nmol/kg (Fig. 2F). However, AM5 induced profound antidiuresis at 1 nmol/kg evident 5 min after injection, and the decrease in urine flow rate continued >30 min. There was a significant correlation between the decreases in blood pressure and urine flow rates over the 30 min after injection of AM2 and AM5 (Fig. 3B).
Effects of infusions.
Infusion of AM2 and AM5 at doses between 0.1 and 3 pmol·kg−1·min−1 induced drinking in a dose-related manner (Fig. 4). The enhanced drinking returned to control level quickly after infusate was changed from AM2 to saline, whereas the high drinking rate was maintained longer after cessation of AM5 infusion. Infusion of ANG II also induced drinking, which appeared equipotent to AM2 and AM5 effects. In contrast, AM1 tended to suppress water intake, and a significant reduction was detected at 1 pmol·kg−1·min−1 (Fig. 4).
Changes in Na+ excretion calculated from the volume and Na+ concentration of urine are shown in Fig. 5. Infusion of AM1 did not change urine flow rate significantly even at 3 pmol·kg−1·min−1, but it dose-dependently decreased Na+ excretion and the decrease continued for >2 h after cessation of AM1 infusion. AM2 significantly reduced both urine volume and Na+ excretion in a dose-dependent manner (Fig. 5). The antidiuresis and antinatriuresis gradually diminished after the infusate was changed back to saline (Fig. 5). The renal effects of AM5 were much smaller than for AM2, and significant antidiuresis was detected only for 1 pmol·kg−1·min−1 AM5 infusion (Fig. 5). Blood pressure did not change during infusion of any of the hormones (Fig. 5).
Effect of Central Injection of AMs
Injection into the third ventricle.
Injection of ANG II into the third ventricle at 500 pmol induced rapid elevation in blood pressure, which was used for the validation of a successful intracerebroventricular injection through the implanted cannula. The effect of ANG II was dose dependent at 50–500 pmol; the increase was already significant at 50 pmol (Fig. 6A). AM1 significantly decreased arterial pressure after injection to the third ventricle only at 500 pmol (Fig. 6B). In contrast to the peripheral injection, AM2 significantly elevated blood pressure dose-dependently after central injection (Fig. 6A). In contrast to the effect of AM2, intracerebroventricular injection of AM5 depressed blood pressure dose-dependently, with the effect becoming significant at 500 pmol. However, the decrease was much smaller compared with that after peripheral injections of equivalent doses of AM5 (data not shown). AM2 significantly decreased, whereas AM5 increased, heart rate at intracerebroventricular injection of 500 pmol (Table 1). ANG II and AM1 did not change heart rate after injection into the third ventricle.
As observed after peripheral infusion, ANG II accelerated drinking rate immediately after injection into the third ventricle at 500 pmol (Fig. 6C). However, no significant effect on drinking was detected after third ventricle administration of any of the AMs including AM2 and AM5 at 500 pmol, although equivalent doses of AM2 and AM5 exhibited potent dipsogenic effects when administered peripherally (Fig. 4).
Injection into the fourth ventricle.
The intracerebroventricular injection of ANG II into the fourth ventricle induced elevation in blood pressure as it did when administered into the third ventricle. In contrast to the hypertensive responses following injection into the third ventricle, AM2 decreased blood pressure after injection into the fourth ventricle. Vasodepressor effects of central AM2 were less potent than for peripheral AM2 injection. Blood pressure also decreased after injections of AM1 and AM5 (Fig. 6B). AM1 significantly increased heart rate, whereas the other hormones injected into the fourth ventricle had no effect on heart rate (Table 1). In contrast to the dipsogenic response to injection into the third ventricle, ANG II did not induce drinking after injection into the fourth ventricle. AM peptides also did not alter drinking rate as observed after injections into the third ventricle (Fig. 6D).
The present study revealed that AM2 and AM5 have potent dipsogenic effects by both bolus injection and infusion into the circulation of the eel. Drinking of environmental seawater plays a direct and critical part in maintenance of body fluid homeostasis of marine teleosts. In hyperosmotic seawater, oral ingestion is the only way in which water can be taken into the body. Therefore, the dipsogenic actions of AM2 and AM5 suggest that they may be involved in seawater adaptation. The effects of these AMs were as potent as those of the other established dipsogenic hormone, ANG II. In general, blood pressure and blood volume are closely related, i.e., a decrease in blood volume causes hypotension. This fact suggests that the dipsogenic effect of AMs could possibly be an indirect compensatory response to a perceived decrease in blood volume as blood pressure fell. However, there were no significant correlations between the decrease in blood pressure and the increase in drinking rate observed after bolus injections of AM2 and AM5. Furthermore, AM2 and AM5 induced water intake during infusion when no hypotension was observed. Thus AM2 and AM5 accelerate water intake at least in part independently of their effects on blood pressure.
Although both AM2 and AM5 enhanced drinking after peripheral infusion, the pattern of their effects was different. Drinking returned to the preinfusion level shortly after the cessation of AM2 infusion, but it remained elevated for a few hours after cessation of the infusion of AM5. AM5 may have a longer half-life, or may induce a release of secondary dipsogenic factor(s). There is a possibility that the dipsogenic effects of AM2 and AM5 are indirect, because AM2 increases plasma renin activity and thus ANG II concentration in conscious sheep (3). In the present study, ANG II induced drinking and the increase also lasted for hours after the ANG II infusion was stopped. Because ANG II may have a much shorter half-life than AM, as shown in mammals (1), the sustained increase may be caused by some unknown factor(s).
In contrast to the dipsogenic effect, the antidiuretic responses to bolus AM2/AM5 injections may be caused by the potent accompanying hypotension (22), because significant correlations were detected between the decreases in urine volume and blood pressure. In teleost fish, urine flow rate is regulated principally by glomerular filtration rate (GFR) rather than tubular reabsorption. Furthermore, systemic blood pressure profoundly influences GFR in teleost fish, which differs from the autoregulated mammalian kidney, in which tubular reabsorption primarily determines urine volume. Thus in the present study the observed reduction in urine flow after bolus injection of AM2 and AM5 is likely to be due to the decrease in hydrostatic pressure at the glomerulus caused by the induced hypotension. In the rat, AM2 directly affects renal tubule water and Na+ reabsorption without altering GFR, resulting in diuresis and natriuresis (6).
The infusion protocol was considered to provide a more physiologically relevant challenge than bolus injection, since it likely mimics more closely changes in plasma endogenous hormone concentration. Infusion of AM1 decreased only Na+ excretion, and AM2 decreased both urine flow and Na+ excretion while blood pressure was maintained at normal levels. In contrast to these data, most of the previous studies in mammals report that AM and AM2 induce diuresis and natriuresis (5, 41). However, there is one conflicting report that AM increased Na+ reabsorption in the rabbit kidney tubule in vitro (14). The antinatriuretic effect of AM1 observed in the present study may be caused by an increase in renal tubule Na+ reabsorption because no antidiuresis was observed, which might be expected if the primary response was to reduce GFR. To clarify the mechanisms of this tubular effect, further investigations using in vitro systems are required.
Centrally administered AMs exhibited effects quite different from those evoked by peripheral administration. AM2 increased blood pressure when injected into the third ventricle but decreased it when injected into the fourth ventricle. AM2 decreased heart rate after injection into the third ventricle, which may be a compensatory effect following the induced hypertension. These effects could be mediated by the activation or suppression of the sympathetic or parasympathetic nervous system. In mammals, intracerebroventricular administration of AM or AM2 produced a hypertensive effect through sympathetic activation (28, 39). Therefore, the increased blood pressure in eel after AM2 seems to be caused by sympathetic activation, while the decrease is likely to be mediated by parasympathetic activation and sympathetic suppression. Together, these data suggest that peripherally administered AM2 acts directly on blood vessels to decrease blood pressure in the eel, independent of any central effect. Moreover, a convulsion-like behavior was observed in the eels when AM2 was administered to the third ventricle. This observation suggests that AM2 may also have an effect on the motor system in the brain. Calcitonin receptor-like receptor (CLR) and calcitonin receptor, possible receptors of AM2, are related to motor activity and pain in the mammalian central nervous system (CNS) (3, 15, 31). Thus there is a possibility that AM2 also has some functions other than body fluid regulation in the CNS of fish.
Despite potent dipsogenic effects of AM2 and AM5 observed after peripheral administration, drinking rate was not altered by intracerebroventricular injections of AMs. Kozaka et al. (13) previously compared the dipsogenic potency of various hormones injected into the fourth ventricle of the eel and showed that ANG II is the most potent. In the present study, ANG II promoted rapid and potent drinking when injected into the third ventricle. However, ANG II did not significantly induce drinking when administered into the fourth ventricle, which does not accord with the previous report. This discrepancy may be due to differences in dose and injection volume. Kozaka's group administered 10 μl of fluid, which seems likely to have diffused throughout the intracranial space (eel brain weight is ∼60 mg). The volume injected in the present study (0.5 μl) may have a more local action restricted to regions close to the site of administration.
The brain parenchyma is generally isolated from the blood by the blood-brain barrier (BBB), but there are several regions called circumventricular organs (CVOs) that lack the BBB (17). The brain tissue is also protected from the substances in the cerebrospinal fluid (CSF) by the brain-CSF barrier (BCSFB) (34). In mammals, ANG II stimulates water intake through receptors distributed in CVOs such as the subfornical organ (SFO) and organum vasculosus of the lamina terminalis (OVLT) (4, 10). Although SFO and OVLT have not been identified in the eel brain (19), ANG II is likely to have acted on the equivalent sites around the third ventricle to induce drinking in the present eel experiment. In contrast, centrally administered AM2 and AM5 did not induce drinking, although they were as dipsogenic as ANG II when given peripherally. Therefore, it is likely that AM2 and AM5 act from the blood side on an unidentified CVO in the third ventricle or area postrema (AP) in the fourth ventricle, as they may cannot cross the BCSFB because of their larger size (∼7,000 Da) compared with ANG II (∼1,000 Da). In fact, it has been shown that peripheral administration of AM activates neurons in the AP and in related nuclei of the rat (45, 33). In the eel, peripherally administered ANP has been shown to act on the AP to inhibit drinking (43).
It is noteworthy that AM2 and AM5 were more potent than AM1 in both cardiovascular and osmoregulatory actions in the eel. In our previous study (22), AM2 and AM5 caused hypotension but AM1 was much less potent (<1/100) in the eel. The efficacy of the two hormones far exceeds that of ANP, one of the most hypotensive hormones known to date. In mammals, however, AM(1) has more potent cardiovascular and renal effects than AM2 (5). If all three AMs investigated in the present study act on the same receptor, the receptor-binding affinity of AM2 and AM5 should be higher than that of AM1 in the eel. However, in another teleost species, Takifugu obscurus, the order of ligand affinity was different from the potency order shown in the present physiological study (21). The receptor for AM is comprised of CLR and receptor activity-modifying protein (RAMP) (18). Nag et al. (21) reported that Takifugu AM1 was more potent than AM2 and AM5 for cAMP production in cell systems with any combination of the diversified three CLRs and five RAMPs, as shown for mammalian AM(1) with CLR and RAMP2/3 combinations (27, 35). Therefore, it seems likely that unknown receptor(s) specific for AM2 and AM5 may exist in teleosts. The existence of an AM2-specific receptor has also been predicted in mammals. AM2 is considered to act on both AM receptor (CLR + RAMP2 or 3) and CGRP receptor (CLR + RAMP1) (2). However, oxytocin release induced by centrally administered AM2 was not completely blocked even by coadministration of receptor antagonists, AM22-52 and CGRP8-37, in rats (7). Additionally, AM2, but not AM(1), inhibits GH-releasing hormone-stimulated GH release in the rat (40) and AM2 increases plasma aldosterone level while AM(1) decreases it in sheep and rats (3, 23).
Perspectives and Significance
The present study has shown for the first time that AMs are highly potent osmoregulatory hormones in teleost fish. This builds on our group's previous report of the cardiovascular actions of AMs in fish (22). Furthermore, AM2 and AM5 are more potent than AM1 in the eel, in contrast to mammals, in which AM(1) is the most potent. These findings suggest the presence of a novel AM receptor in teleosts and mammals. AM2 and AM5 induced opposite cardiovascular responses in the present study after central administration, suggesting that AM2 and AM5 may act on different receptors. Thus there may be at least two different receptors for AM2 and AM5 in teleosts. To identify these novel receptors, the eel will be an excellent model for future studies. If these receptors are identified in the eel, this may lead to their subsequent identification in mammals or even throughout vertebrates. From a physiological perspective, comparative fish studies should also lead to identification of currently unknown functions of AM2 and AM5 in mammals. Although AM1 has little cardiovascular and osmoregulatory effect in the eel, it may play an important part in other homeostatic mechanisms. In teleost fish, AM1/AM4 and AM2/AM3 were duplicated at the third-round whole genome duplication (24). AM1/AM4 sequences diverged during subsequent teleost evolution, while AM2/AM3 sequences are highly conserved (90% identity in the eel; Ref. 22). Therefore, it is of interest to identify the cause of such great differences in sequence conservation for the two groups of hormones by examining their physiological actions in the eel. These studies may provide new insight into the functional role of the AM family throughout vertebrate groups.
This work was supported by a Grant-in-Aid for Scientific Research (A) to Y. Takei (13304063 and 16207004) and a Research Fellowship for Young Scientists to M. Ogoshi from the Japan Society for the Promotion of Science.
We thank Prof. Richard J. Balment of the University of Manchester for polishing the manuscript, Dr. Susumu Hyodo for valuable comments, and Sanae Hasegawa for expert technical assistance.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2008 the American Physiological Society