Intraperitoneal administration of arginine vasotocin (AVT) induces anorexigenic and anxiogenic actions via the brain V1a receptor-signaling pathway in the tiger puffer, Takifugu rubripes.

Arginine vasotocin (AVT) is produced mainly in the hypothalamus and as a neurohypophyseal hormone peripherally regulates water-mineral balance in sub-mammals. In addition, AVT-containing neurons innervate several areas of the brain, and AVT also acts centrally as both an anorexigenic and anxiogenic factor in goldfish. However, it is unclear whether these central effects operate in fish in general. In the present study, therefore, we investigated AVT-like immunoreactivity in the brain of the tiger puffer, a cultured fish with a high market value in Japan and also a representative marine teleost species, focusing particularly on whether AVT affects food intake and psychomotor activity. AVT-like immunoreactivity was distributed higher in the ventral region of the telencephalon, the hypothalamus and midbrain. Intraperitoneal (IP) administration of AVT at 100 pmol g-1 body weight (BW) increased the immunoreactivity of phosphorylated ribosomal proteinS6 (RPS6), a neuronal activation marker, in the telencephalon and diencephalon, decreased food consumption and enhanced thigmotaxis. AVT-induced anorexigenic and anxiogenic actions were blocked by IP co-injection of a V1a receptor (V1aR) antagonist, Manning compound (MC) at 300 pmol g-1 BW. These results suggest that AVT acts as an anorexigenic and anxiogenic factor via the V1aR-signaling pathway in the tiger puffer brain.

Molecular characterization and distribution of motilin and motilin receptor in the Japanese medaka Oryzias latipes.

Motilin (MLN) is a peptide hormone originally isolated from the mucosa of the porcine intestine. Its orthologs have been identified in various vertebrates. Although MLN regulates gastrointestinal motility in tetrapods from amphibians to mammals, recent studies indicate that MLN is not involved in the regulation of isolated intestinal motility in zebrafish, at least in vitro. To determine the unknown function of MLN in teleosts, we examined the expression of MLN and the MLN receptor (MLNR) at the cellular level in Japanese medaka (Oryzias latipes). Quantitative PCR revealed that mln mRNA was limitedly expressed in the gut, whereas mlnr mRNA was not detected in the gut but was expressed in the brain and kidney. By in situ hybridization and immunohistochemistry, mlnr mRNA was detected in the dopaminergic neurons of the area postrema in the brain and the noradrenaline-producing cells in the interrenal gland of the kidney. Furthermore, we observed efferent projections of mlnr-expressing dopaminergic neurons in the lobus vagi (XL) and nucleus motorius nervi vagi (NXm) of the medulla oblongata by establishing a transgenic medaka expressing the enhanced green fluorescence protein driven by the mlnr promoter. The expression of dopamine receptor mRNAs in the XL and cholinergic neurons in NXm was confirmed by in situ hybridization. These results indicate novel sites of MLN activity other than the gastrointestinal tract. MLN may exert central and peripheral actions through the regulation of catecholamine release in medaka.

Phylogenetic and functional properties of hagfish neurohypophysial hormone receptors distinct from their jawed vertebrate counterparts.

Vertebrate neurohypophysial hormones, i.e., vasopressin- and oxytocin-family peptides, exert versatile physiological actions via distinct G protein-coupled receptors. The neurohypophysial hormone receptor (NHR) family was classically categorized into four subtypes (V1aR, V1bR, V2R and OTR), while recent studies have identified seven subtypes (V1aR, V1bR, V2aR, V2bR, V2cR, V2dR and OTR; V2aR corresponds to the conventional V2R). The vertebrate NHR family were diversified via multiple gene duplication events at different scales. Despite intensive research effort in non-osteichthyes vertebrates such as cartilaginous fish and lamprey, the molecular phylogeny of the NHR family has not been fully understood. In the present study, we focused on the inshore hagfish (Eptatretus burgeri), another group of cyclostomes, and Arctic lamprey (Lethenteron camtschaticum) for comparison. Two putative NHR homologs, which were previously identified only in silico, were cloned from the hagfish and designated as ebV1R and ebV2R. In vitro, ebV1R, as well as two out of five Arctic lamprey NHRs, increased intracellular Ca2+ in response to exogenous neurohypophysial hormones. None of the examined cyclostome NHRs altered intracellular cAMP levels. Transcripts of ebV1R were detected in multiple tissues including the brain and gill, with intense hybridization signals in the hypothalamus and adenohypophysis, while ebV2R was predominantly expressed in the systemic heart. Similarly, Arctic lamprey NHRs showed distinct expression patterns, underscoring the multifunctionality of VT in the cyclostomes as in the gnathostomes. These results and exhaustive gene synteny comparisons provide new insights into the molecular and functional evolution of the neurohypophysial hormone system in vertebrates.

Comparative Observation and Analysis of Preference Behavior Based on Three Types of Taxes and Locomotor Activity in the Goldfish, Carassius auratus.

Psychophysiological studies in vertebrates have focused on taxes as indicators of behavioral change. Actually, a considerable number of studies about anxiety-like and anti-anxiety-like behaviors involving geotaxis, scototaxis, and thigmotaxis have been conducted on fish. However, few analyses considering these behaviors based on taxes in fish have been conducted. Here, using goldfish, we measured the time spent in the bright or dark area of a horizontally long rectangular tank (HLRT), in the upper or lower area of a vertically long rectangular tank (VLRT), and in the central or edge area of a circular tank (CT), respectively, for the first 30 min and the last 30 min in a 3-h period after fish had been introduced to tanks. Dark, lower, and edge preference behaviors were observed for the first 30 min in all tanks. While dark and edge preference behaviors were maintained even for the last 30 min, the lower preference was lost. Swimming distance and the number of area crossings in each tank were also compared between the first 30 min and the last 30 min. Both decreased significantly or tended to decrease in the last 30 min in the HLRT and the CT, but no change was observed in the VLRT. These results suggest that, in goldfish, preference behavior is stable for a short time, and that environmental habituation may depend on the shape of the tank and the elapsed time.

Simultaneous activation of genes encoding urea cycle enzymes and gluconeogenetic enzymes coincides with a corticosterone surge period before metamorphosis in Xenopus laevis.

Amphibian tadpoles are postulated to excrete ammonia as nitrogen metabolites but to shift from ammonotelism to ureotelism during metamorphosis. However, it is unknown whether ureagenesis occurs or plays a functional role before metamorphosis. Here, the mRNA-expression levels of two urea cycle enzymes (carbamoyl phosphate synthetase I [CPSI] and ornithine transcarbamylase [OTC]) were measured beginning with stage-47 Xenopus tadpoles at 5 days post-fertilization (dpf), between the onset of feeding (stage 45, 4 dpf) and metamorphosis (stage 55, 32 dpf). CPSI and OTC expression levels increased significantly from stage 49 (12 dpf). Urea excretion was also detected at stage 47. A transient corticosterone surge peaking at stage 48 was previously reported, supporting the hypothesis that corticosterone can induce CPSI expression in tadpoles, as found in adult frogs and mammals. Stage-46 tadpoles were exposed to a synthetic glucocorticoid, dexamethasone (Dex, 10-500 nM) for 3 days. CPSI mRNA expression was significantly higher in tadpoles exposed to Dex than in tadpoles exposed to the vehicle control. Furthermore, glucocorticoid receptor mRNA expression increased during the pre-metamorphic period. In addition to CPSI and OTC mRNA upregulation, the expression levels of three gluconeogenic enzyme genes (glucose 6-phosphatase, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase 1) increased with the onset of urea synthesis and excretion. These results suggest that simultaneous induction of the urea cycle and gluconeogenic enzymes coincided with a corticosterone surge occurring prior to metamorphosis. These metabolic changes preceding metamorphosis may be closely related to the onset of feeding and nutrient accumulation required for metamorphosis.

Distribution of neuromedin U (NMU)-like immunoreactivity in the goldfish brain, and effect of intracerebroventricular administration of NMU on emotional behavior in goldfish.

Neuromedin U (NMU) is a multifunctional neuropeptide implicated in regulation of smooth muscle contraction in the circulatory and digestive systems, energy homeostasis and the stress response, but especially food intake in vertebrates. Recent studies have indicated the possible involvement of NMU in the regulation of psychomotor activity in rodents. We have identified four cDNAs encoding three putative NMU variants (NMU-21, -25 and -38) from the goldfish brain and intestine. Recently, we have also purified these NMUs and the truncated C-terminal form NMU-9 from these tissues, and demonstrated their anorexigenic action in goldfish. However, there is no information on the brain localization of NMU-like immunoreactivity and the psychophysiological roles of NMU in fish. Here, we investigated the brain distribution of NMU-like immunoreactivity and found that it was localized throughout the fore- and mid-brains. We subsequently examined the effect of intracerebroventricular (ICV) administration of NMU-21, which is abundant only in the brain on psychomotor activity in goldfish. As goldfish prefer the lower to the upper area of a tank, we developed an upper/lower area preference test in a tank for evaluating the psychomotor activity of goldfish using a personal tablet device without an automatic behavior-tracking device. ICV administration of NMU-21 at 10 pmol g-1 body weight (BW) prolonged the time spent in the upper area of the tank, and this action mimicked that of ICV administration of the central-type benzodiazepine receptor (CBR) agonist tofisopam at 100 pmol g-1 BW. These results suggest that NMU-21 potently induces anxiolytic-like action in the goldfish brain.

Oxytocin variation and brain region-specific gene expression in a domesticated avian species.

The Bengalese finch was domesticated more than 250 years ago from the wild white-rumped munia (WRM). Similar to other domesticated species, Bengalese finches show a reduced fear response and have lower corticosterone levels, compared to WRMs. Bengalese finches and munias also have different song types. Since oxytocin (OT) has been found to be involved in stress coping and auditory processing, we tested whether the OT sequence and brain expression pattern and content differ in wild munias and domesticated Bengalese finches. We sequenced the OT from 10 wild munias and 11 Bengalese finches and identified intra-strain variability in both the untranslated and protein-coding regions of the sequence, with all the latter giving rise to synonymous mutations. Several of these changes fall in specific transcription factor-binding sites, and show either a conserved or a relaxed evolutionary trend in the avian lineage, and in vertebrates in general. Although in situ hybridization in several hypothalamic nuclei did not reveal significant differences in the number of cells expressing OT between the two strains, real-time quantitative PCR showed a significantly higher OT mRNA expression in the cerebrum of the Bengalese finches relative to munias, but a significantly lower expression in their diencephalon. Our study thus points to a brain region-specific pattern of neurochemical expression in domesticated and wild avian strains, which could be linked to domestication and the behavioral changes associated with it.

Intracerebroventricular administration of α-melanocyte-stimulating hormone (α-MSH) enhances thigmotaxis and induces anxiety-like behavior in the goldfish Carassius auratus.

α-Melanocyte-stimulating hormone (α-MSH) is a body pigmentation-regulating hormone secreted from the intermediate lobe of the pituitary in vertebrates. It is also produced in the brain, and acts as an anorexigenic neuropeptide involved in feeding regulation. In rodents, intracerebroventricular (ICV) administration of α-MSH has been shown to affect not only feeding behavior, but also psychomotor activity. However, there is still no information regarding the psychophysiological effects of α-MSH on behavior in fish. Therefore, we examined the effect of synthetic α-MSH on psychomotor activity in goldfish. Since this species prefers the edge to the central area of a tank, we used this as a preference test for assessing psychomotor activity. When α-MSH was administered ICV at 1 and 10 pmol g-1 body weight (BW), the time spent in the edge area of a tank was prolonged at 10 pmol g-1 BW. However, α-MSH at these doses did not affect locomotor activity. The action of α-MSH mimicked those of FG-7142 (a central-type benzodiazepine receptor (CBR) inverse agonist with an anxiogenic effect) at 10 pmol g-1 BW and melanotan II (a melanocortin 4 receptor (MC4R) agonist) at 50 pmol g-1 BW, whereas ICV administration of tofisopam (a CBR agonist with an anxiolytic effect) at 10 pmol g-1 BW prolonged the time spent in the central area. The anxiogenic-like effect of α-MSH was abolished by treatment with the MC4R antagonist HS024 at 50 pmol g-1 BW. These data indicate that α-MSH affects psychomotor activity in goldfish, and exerts an anxiogenic-like effect via the MC4R-signaling pathway.

Identification and signaling characterization of four urotensin II receptor subtypes in the western clawed frog, Xenopus tropicalis.

Urotensin II (UII) is involved, via the UII receptor (UTR), in many physiological and pathological processes, including vasoconstriction, locomotion, osmoregulation, immune response, and metabolic syndrome. In silico studies have revealed the presence of four or five distinct UTR (UTR1-UTR5) gene sequences in nonmammalian vertebrates. However, the functionality of these receptor subtypes and their associations to signaling pathways are unclear. In this study, full-length cDNAs encoding four distinct UTR subtypes (UTR1, UTR3, UTR4, and UTR5) were isolated from the western clawed frog (Xenopus tropicalis). In functional analyses, homologous Xenopus UII stimulation of cells expressing UTR1 or UTR5 induced intracellular calcoum mobilization and phosphorylation of extracellular signal-regulated kinase 1/2. Cells expressing UTR3 or UTR4 did not show this response. Furthermore, UII induced the phosphorylation of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) through the UII-UTR1/5 system. However, intracellular cAMP accumulation was not observed, suggesting that UII-induced CREB phosphorylation is caused by a signaling pathway different from that involving Gs protein. In contrast, the administration of UII to cells increased the phosphorylation of guanine nucleotide exchange factor-H1 (GEF-H1) and myosin light chain 2 (MLC2) in all UTR subtypes. These results define four distinct UTR functional subtypes and are consistent with the molecular evolution of UTR subtypes in vertebrates. Further understanding of signaling properties associated with UTR subtypes may help in clarifying the functional roles associated with UII-UTR interactions in nonmammalian vertebrates.

Effect of intracerebroventricular administration of two molecular forms of sulfated CCK octapeptide on anxiety-like behavior in the zebrafish danio rerio.

Cholecystokinin octapeptide with sulfate (CCK-8s) regulates feeding behavior and psychomotor activity. In rodents and goldfish, intracerebroventricular (ICV) injection of CCK-8s decreases food intake and also induces anxiety-like behavior. The zebrafish has several merits for investigating the psychophysiological roles of neuropeptides. However, little is known about the brain localization of CCK and the behavioral action of CCK-8s in this species. Here we investigated the brain localization of CCK-like immunoreactivity and found that it was distributed throughout the brain. As CCK-like immunoreactivity was particularly evident in the ventral habenular nucleus, the interpeduncular nucleus and superior raphe, we subsequently examined the effect of zebrafish (zf) CCK-8s on psychomotor control. Since the zebrafish possesses two molecular forms of zfCCK-8s (zfCCKA-8s and zfCCKB-8s), two synthetic peptides were administered intracerebroventricularly at 1, 5 and 10 pmol g-1 body weight (BW). As the zebrafish shows a greater preference for the lower area of a tank than for to the upper area, we used this preference for assessment of anxiety-like behavior. ICV administration of zfCCKA-8 s or zfCCKB-8s at 10 pmol g-1 BW significantly shortened the time spent in the upper area. The actions of these peptides mimicked that of the central-type benzodiazepine receptor inverse agonist FG-7142 (an anxiogenic agent) at 10 pmol g-1 BW. The anxiogenic-like action of the two peptides was attenuated by treatment with the CCK receptor antagonist proglumide at 200 pmol g-1 BW. These results indicate that zfCCKA-8s and zfCCKB-8s potently induce anxiety-like behavior via the CCK receptor-signaling pathway in the zebrafish brain.

Expression Patterns of PACAP and PAC1R Genes and Anorexigenic Action of PACAP1 and PACAP2 in Zebrafish.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with potent suppressive effects on feeding behavior in rodents, chicken, and goldfish. Teleost fish express two PACAPs (PACAP1, encoded by the adcyap1a gene, and PACAP2, encoded by the adcyap1b gene) and two PACAP receptors (PAC1Rs; PAC1Ra, encoded by the adcyap1r1a gene, and PAC1Rb, encoded by the adcyap1r1b gene). However, the mRNA expression patterns of the two PACAPs and PAC1Rs, and the influence and relationship of the two PACAPs on feeding behavior in teleost fish remains unclear. Therefore, we first examined mRNA expression patterns of PACAP and PAC1R in tissue and brain. All PACAP and PAC1Rs mRNAs were dominantly expressed in the zebrafish brain. However, adcyap1a mRNA was also detected in the gut and testis. In the brain, adcyap1b and adcyap1r1a mRNA levels were greater than that of adcyap1a and adcyap1r1b, respectively. Moreover, adcyap1b and adcyap1r1a mRNA were dominantly expressed in telencephalon and diencephalon. The highest adcyap1a mRNA levels were detected in the brain stem and diencephalon, while the highest levels of adcyap1r1b were detected in the cerebellum. To clarify the relationship between PACAP and feeding behavior in the zebrafish, the effects of zebrafish (zf) PACAP1 or zfPACAP2 intracerebroventricular (ICV) injection were examined on food intake, and changes in PACAP mRNA levels were assessed against feeding status. Food intake was significantly decreased by ICV injection of zfPACAP1 (2 pmol/g body weight), zfPACAP2 (2 or 20 pmol/g body weight), or mammalian PACAP (2 or 20 pmol/g). Meanwhile, the PACAP injection group did not change locomotor activity. Real-time PCR showed adcyap1 mRNA levels were significantly increased at 2 and 3 h after feeding compared with the pre-feeding level, but adcyap1b, adcyap1r1a, and adcyap1r1b mRNA levels did not change after feeding. These results suggest that the expression levels and distribution of duplicated PACAP and PAC1R genes are different in zebrafish, but the anorexigenic effects of PACAP are similar to those seen in other vertebrates.

Intracerebroventricular administration of sulphated cholecystokinin octapeptide induces anxiety-like behaviour in goldfish.

Sulphated cholecystokinin octapeptide (CCK-8s) is involved in feeding regulation as an anorexigenic neuropeptide in vertebrates. In rodents, i.c.v. administration of CCK-8s has been shown to affect not only feeding behaviour, but also psychomotor activity. However, there is still no information available concerning the psychophysiological effects of CCK-8s in goldfish. Therefore, in the present study, we examined the effect of synthetic goldfish (gf) CCK-8s on psychomotor activity in this species. Intracerebroventricular administration of gfCCK-8s at 0.1, 0.5 and 2.5 pmol g-1 body weight (BW) did not affect swimming distance (locomotor activity). Because goldfish prefer the lower to the upper area of a tank, we used this as a preference test (upper/lower test) to assess anxiety-like behaviour. Intracerebroventricular administration of gfCCK-8s at 2.5 pmol g-1 BW shortened the time spent in the upper area. The action of gfCCK-8s mimicked that of FG-7142 (the central-type benzodiazepine receptor inverse agonist, an anxiogenic agent) at 5 and 10 pmol g-1 BW. The anxiogenic-like effect of gfCCK-8s was abolished by treatment with the CCK receptor antagonist proglumide at 50 pmol g-1 BW. We also investigated the localisation of CCK/gastrin-like immunoreactivity in the goldfish brain. CCK/gastrin-like immunoreactivity was observed in the anxiety-related regions (the nucleus habenularis and the interpeduncular nucleus). These data indicate that gfCCK-8s potently affects psychomotor activity in goldfish, and exerts an anxiogenic-like effect via the CCK receptor-signalling pathway.

Distribution of pituitary adenylate cyclase-activating polypeptide 2 in zebrafish brain.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multipotent neuropeptide with an amino acid sequence that is well conserved among vertebrates. In teleosts, including zebrafish, the PACAP gene (adcyap1) has been duplicated to yield adcyap1a (coding PACAP1) and adcyap1b (coding PACAP2). This study aims to determine the distribution of these PACAPs and their mRNAs in zebrafish. We generated a zebrafish PACAP2-specific antibody. Using real-time PCR, we observed that adcyap1b mRNA was primarily localized in the brain, with the highest level in the telencephalon, followed by the diencephalon. Using immunostaining of brain tissue samples, PACAP2 immunoreactivity was observed mainly in the telencephalon, hypothalamus, and cerebellum, and the immunopositive fibers formed a line to the habenula. PACAP2-immunopositive cells were observed in the ventral and dorsal regions of the telencephalon and in the hypothalamic nucleus of the diencephalon in the colchicine-injected brain. This distribution of PACAP2 suggests its involvement in higher brain functions in teleosts, such as learning and cognition, as well as instinctive behaviors such as feeding and emotional regulation.

Neuropeptide Y-Induced Orexigenic Action Is Attenuated by the Orexin Receptor Antagonist in Bullfrog Larvae.

In bullfrog larvae at the pre- and pro-metamorphic stages, feeding behavior is regulated by appetite factors such as orexigenic peptides. In fact, food intake is enhanced by intracerebroventricular (ICV) administration of neuropeptide Y (NPY) and orexin A. Using goldfish, our previous study indicated that the orexigenic action of NPY is mediated by orexin A, suggesting the functional interaction between the two. However, there is little information about whether the action of orexin A mediates the orexigenic action of NPY in bullfrog larvae. Therefore, we examined the effect of the orexin receptor antagonist, SB334867 on the orexigenic action of NPY in larvae. The stimulatory effect of ICV injection of NPY at 10 pmol/g body weight (BW) on food intake was abolished by treatment with SB334867 at 60 pmol/g BW. These results suggest that, in bullfrog larvae, there is a neuronal relationship between the NPY and orexin systems, and that the orexigenic action of NPY is mediated by the orexin A-induced orexigenic action.

Principal function of mineralocorticoid signaling suggested by constitutive knockout of the mineralocorticoid receptor in medaka fish.

As in osmoregulation, mineralocorticoid signaling is implicated in the control of brain-behavior actions. Nevertheless, the understanding of this role is limited, partly due to the mortality of mineralocorticoid receptor (MR)-knockout (KO) mice due to impaired Na+ reabsorption. In teleost fish, a distinct mineralocorticoid system has only been identified recently. Here, we generated a constitutive MR-KO medaka as the first adult-viable MR-KO animal, since MR expression is modest in osmoregulatory organs but high in the brain of adult medaka as for most teleosts. Hyper- and hypo-osmoregulation were normal in MR-KO medaka. When we studied the behavioral phenotypes based on the central MR localization, however, MR-KO medaka failed to track moving dots despite having an increase in acceleration of swimming. These findings reinforce previous results showing a minor role for mineralocorticoid signaling in fish osmoregulation, and provide the first convincing evidence that MR is required for normal locomotor activity in response to visual motion stimuli, but not for the recognition of these stimuli per se. We suggest that MR potentially integrates brain-behavioral and visual responses, which might be a conserved function of mineralocorticoid signaling through vertebrates. Importantly, this fish model allows for the possible identification of novel aspects of mineralocorticoid signaling.

Morphological and molecular investigations of the holocephalan elephant fish nephron: the existence of a countercurrent-like configuration and two separate diluting segments in the distal tubule.

In marine cartilaginous fish, reabsorption of filtered urea by the kidney is essential for retaining a large amount of urea in their body. However, the mechanism for urea reabsorption is poorly understood due to the complexity of the kidney. To address this problem, we focused on elephant fish (Callorhinchus milii) for which a genome database is available, and conducted molecular mapping of membrane transporters along the different segments of the nephron. Basically, the nephron architecture of elephant fish was similar to that described for elasmobranch nephrons, but some unique features were observed. The late distal tubule (LDT), which corresponded to the fourth loop of the nephron, ran straight near the renal corpuscle, while it was convoluted around the tip of the loop. The ascending and descending limbs of the straight portion were closely apposed to each other and were arranged in a countercurrent fashion. The convoluted portion of LDT was tightly packed and enveloped by the larger convolution of the second loop that originated from the same renal corpuscle. In situ hybridization analysis demonstrated that co-localization of Na(+),K(+),2Cl(-) cotransporter 2 and Na(+)/K(+)-ATPase α1 subunit was observed in the early distal tubule and the posterior part of LDT, indicating the existence of two separate diluting segments. The diluting segments most likely facilitate NaCl absorption and thereby water reabsorption to elevate urea concentration in the filtrate, and subsequently contribute to efficient urea reabsorption in the final segment of the nephron, the collecting tubule, where urea transporter-1 was intensely localized.

Urotensin II upregulates migration and cytokine gene expression in leukocytes of the African clawed frog, Xenopus laevis.

Urotensin II (UII) exhibits diverse physiological actions including vasoconstriction, locomotor activity, osmoregulation, and immune response via the UII receptor (UTR) in mammals. However, in amphibians the function of the UII-UTR system remains unknown. In the present study, we investigated the potential immune function of UII using leukocytes isolated from the African clawed frog, Xenopus laevis. Stimulation of male frogs with lipopolysaccharide increased mRNA expression of UII and UTR in leukocytes, suggesting that inflammatory stimuli induce activation of the UII-UTR system. Migration assays showed that both UII and UII-related peptide enhanced migration of leukocytes in a dose-dependent manner, and that UII effect was inhibited by the UTR antagonist urantide. Inhibition of Rho kinase with Y-27632 abolished UII-induced migration, suggesting that it depends on the activation of RhoA/Rho kinase. Treatment of isolated leukocytes with UII increased the expression of several cytokine genes including tumor necrosis factor-α, interleukin-1ß, and macrophage migration inhibitory factor, and the effects were abolished by urantide. These results suggest that in amphibian leukocytes the UII-UTR system is involved in the activation of leukocyte migration and cytokine gene expression in response to inflammatory stimuli.

Cloning and expression of the epithelial sodium channel and its role in osmoregulation of aquatic and estivating African lungfish Protopterus annectens.

The epithelial sodium channel (ENaC) is a sodium (Na(+))-selective aldosterone-stimulated ion channel involved in Na(+) transport homeostasis of tetrapods. We examined full-length cDNA sequences and tissue distributions of ENaCα, ENaCß, and ENaCγ subunits in the African lungfish Protopterus annectens. Protopterus ENaC (pENaC) comprises 3 subunits: pENaCα, pENaCß, and pENaCγ. pENaCα, pENaCß, and pENaCγ subunits are closely related to α, ß, and γ subunits of the Australian lungfish Neoceratodus forsteri ENaC (nENaC), respectively. Three ENaC subunit mRNAs were highly expressed in the gills and moderately expressed in the kidney and rectum of P. annectens. During estivation for 2-4weeks and 2-3months, plasma Na(+) concentration was relatively stable, but plasma urea concentration significantly increased in comparison with the control fish kept in a freshwater environment. Plasma aldosterone concentration and mRNA expression of the ENaCα subunit gradually and significantly decreased in the gills and kidney after 2months of estivation. Thus, aldosterone-dependent Na(+) absorption via ENaC probably exists in the epithelial cells of osmoregulatory organs of lungfish kept in fresh water, whereas plasma Na(+) concentration may be maintained by a mechanism independent of aldosterone-ENaC axis during estivation in lungfish.

Identification, tissue distribution and functional characterization of the ghrelin receptor in West African lungfish, Protopterus annectens.

We identified two ghrelin receptor isoforms, the ghrelin receptor type-1a (GHS-R1a) and its alternative splice form (GHS-R1b) for West African lungfish, Protopterus annectens. Lungfish GHS-R1a and 1b comprised 361 and 281 amino acids, respectively. Lungfish GHS-R1a showed the highest identity to coelacanth GHS-R1a (80.4%). The highest expression of GHS-R1a mRNAs was seen in the brain, liver, ovary, heart, intestine, and gills. GHS-R1b mRNAs were also detected in the same tissues with GHS-R1a, but their expression level was 1/20 that of GHS-R1a. In human embryonic kidney 293 cells transiently expressing lungfish GHS-R1a, rat and bullfrog ghrelin, and two GHS-R1a agonists, GHRP-6 and hexarelin, increased intracellular Ca(2+) concentrations. The intensity of the Ca(2+) increases induced by GHS-R1a agonists was twice when compared to that induced by ghrelin, although the median effective doses (ED50) were similar, suggesting a long-lasting effect of GHS-R1a agonists with similar affinity. We also examined changes in the GHS-R gene expression during an eight-week estivation. Body weight was slightly lowered, but plasma sodium and glucose concentrations decreased; plasma urea concentration increased significantly 4weeks after the start of estivation. Overall, expression of GHS-R1a mRNA decreased, but changes in GHS-R1b mRNA expression were inconsistent with those of GHS-R1a during estivation, suggesting an involvement of GHS-R in energy homeostasis, as seen in mammals. Our results suggest that the ghrelin-GHS-R1a system is present in this lungfish although ghrelin has not yet been found. The structure of GHS-R1a is closer to that of tetrapods than Actinopterygian fish, indicating a process of evolution that follows the Crossopterygii such as coelacanth.

Orexin A enhances food intake in bullfrog larvae.

Orexin is a potent orexigenic peptide implicated in appetite regulation in rodents. However, except for teleost fish, the involvement of orexin in the regulation of feeding in non-mammalian vertebrates has not been well studied. Anuran amphibian larvae feed and grow during the pre- and prometamorphic stages. Therefore, orexigenic factors seem to play important roles in growing larvae. Indeed, our recent studies have demonstrated that neuropeptide Y and ghrelin exert orexigenic actions in bullfrog larvae during the prometamorphic stages. In this study, we examined the effect of intracerebroventricular (ICV) administration of synthetic orexin A on food intake in bullfrog larvae at the prometamorphic stages. Food intake was significantly increased by ICV administration of orexin A (at 6 pmol/g BW) during a 15-min observation period. The orexigenic action of orexin A at 6 pmol/g BW was blocked by treatment with an orexin receptor antagonist, SB334867, at 60 pmol/g BW. These results indicate that orexin A acts as an orexigenic factor in bullfrog larvae.