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.

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.

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.

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.

Classification of Participants Based on Increase-Decrease Rate Model of Reaction Time to Personality Trait Words.

In this experiment, we tried to measure personality by reaction time (RT) to stimuli of personality trait words. There were interindividual and intraindividual differences in the factors that caused the reaction time to fluctuate. The intraindividual differences for personality trait words were caused by changes due to circumstances for the same participant. The increased stimulus reaction time (sRT) model for simple reaction time was used as an index to indicate personality traits. As a result, participants could be classified into two major hierarchical clusters. The participants in Cluster 1 showed innovative dominance. The participants in Cluster 2 were obedient and conservative. The independent variable was measured by the physiological index using sRT for classify the participants. Participants in Cluster 2 had a reduced stress response to the experiment and showed a tendency to be compliant. Moreover, immediately after the RT measurement session with a laptop computer started, participants in Cluster 1 showed decreased HEG and increased amylase values and had a somewhat negative attitude. The physiological dependent variable were measured by using salivary amylase and hemoencephalography (HEG). And, the psychological dependent variable was the Big Five personality inventory. All of them ware using to verify the participant's classification. Participants in Cluster 2 had significantly higher conscientiousness than those in Cluster 1. Therefore, we suggest that it is possible to classify personality traits from RT by using sRT based on intraindividual differences.

Light-at-night exposure affects brain development through pineal allopregnanolone-dependent mechanisms.

The molecular mechanisms by which environmental light conditions affect cerebellar development are incompletely understood. We showed that circadian disruption by light-at-night induced Purkinje cell death through pineal allopregnanolone (ALLO) activity during early life in chicks. Light-at-night caused the loss of diurnal variation of pineal ALLO synthesis during early life and led to cerebellar Purkinje cell death, which was suppressed by a daily injection of ALLO. The loss of diurnal variation of pineal ALLO synthesis induced not only reduction in pituitary adenylate cyclase-activating polypeptide (PACAP), a neuroprotective hormone, but also transcriptional repression of the cerebellar Adcyap1 gene that produces PACAP, with subsequent Purkinje cell death. Taken together, pineal ALLO mediated the effect of light on early cerebellar development in chicks.

Purification and identification of native forms of goldfish neuromedin U from brain and gut.

Neuromedin U (NMU) plays important roles in energy homeostasis in rodents and birds. Previously, our group has isolated four cDNAs encoding precursor proteins of NMU from the goldfish brain and gut, and it was assumed that these transcripts are produced by alternative splicing. We have also demonstrated that intracerebroventricular (ICV) injection of putative goldfish NMU inhibits food intake. However, as native goldfish NMU has not yet been identified, we attempted to purify it from goldfish brain and gut extracts. To assess NMU activity in fractions at each purification step, we measured changes in the intracellular concentrations of Ca2+ using HEK293 cells expressing goldfish NMU-R1 or -R2. We isolated a 25-amino-acid peptide (NMU-25) from the brain and gut and found that its primary structure is similar to that of mammalian NMU. Another 21-amino-acid peptide (NMU-21) was purified from the brain, but not from the gut. Furthermore, a 9-amino-acid peptide (NMU-9) identical to the C-terminus of NMU-21 and -25 was also isolated from the brain and gut. Treatment with synthetic NMU-9, -21 and -25 dose-dependently increased the intracellular Ca2+ concentration in mammalian cells expressing goldfish NMU-R1 and -R2. We also examined the effect of ICV-administered synthetic goldfish NMUs on goldfish food intake. NMU-25 inhibited food intake to the same degree as NMU-21. However, the inhibitory effect of NMU-9 was slightly weaker than those of NMU-21 and -25. These results indicate that several molecular forms of NMU exist in the goldfish brain and gut, and that all of them play physiological roles via NMU-R1 and NMU-R2.

Melatonin is a potential drug for the prevention of bone loss during space flight.

Astronauts experience osteoporosis-like loss of bone mass because of microgravity conditions during space flight. To prevent bone loss, they need a riskless and antiresorptive drug. Melatonin is reported to suppress osteoclast function. However, no studies have examined the effects of melatonin on bone metabolism under microgravity conditions. We used goldfish scales as a bone model of coexisting osteoclasts and osteoblasts and demonstrated that mRNA expression level of acetylserotonin O-methyltransferase, an enzyme essential for melatonin synthesis, decreased significantly under microgravity. During space flight, microgravity stimulated osteoclastic activity and significantly increased gene expression for osteoclast differentiation and activation. Melatonin treatment significantly stimulated Calcitonin (an osteoclast-inhibiting hormone) mRNA expression and decreased the mRNA expression of receptor activator of nuclear factor κB ligand (a promoter of osteoclastogenesis), which coincided with suppressed gene expression levels for osteoclast functions. This is the first study to report the inhibitory effect of melatonin on osteoclastic activation by microgravity. We also observed a novel action pathway of melatonin on osteoclasts via an increase in CALCITONIN secretion. Melatonin could be the source of a potential novel drug to prevent bone loss during space flight.

Intracerebroventricular administration of arginine vasotocin (AVT) induces anorexigenesis and anxiety-like behavior in goldfish.

Arginine vasotocin (AVT) is known as a neurohypophyseal hormone that regulates water- and mineral-balance in non-mammalian vertebrates. Recent studies revealed that AVT also exerts central effects on behavior. The goldfish has several merits for evaluation of behavioral changes. However, there is few information on the behavioral action of AVT in this species. Here we examined the effects of AVT on food intake and psychomotor activity. AVT was administered intracerebroventricularly at 1, 5 and 10 pmol g-1 body weight (BW). Intracerebroventricular (ICV) administration of AVT at 5 and 10 pmol g-1 BW significantly decreased food intake during 30 min after injection and recovery from anesthesia. The AVT-induced anorexigenic action was attenuated by treatment with the AVT receptor V1aR antagonist Manning compound (MC) at 50 pmol g-1 BW. As the goldfish tends to prefer the lower to the upper area of a tank, we used this preference behavior for assessing psychomotor activity during a 30-min observation period. ICV administration of AVT at 1, 5 and 10 pmol g-1 BW significantly prolonged the time spent in the lower area, but did not affect locomotor activity in the tank at any dose. The action of AVT was similar to that of the central-type benzodiazepine receptor inverse agonist FG-7142 at 10 pmol g-1 BW. AVT-induced anxiety-like behavior was blocked by treatment with MC at 50 pmol g-1 BW. These results indicate that AVT affects food intake and psychophysiological status, and also induces anorexigenic- and anxiogenic-like actions via the V1aR-signaling pathway in the goldfish 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.

A pilot study of change in cerebral activity during personality rating by questionnaire and personal computer.

The purpose of this study was to examine cerebral blood flow in the frontal cortex area during personality self-rating tasks. Our two hypotheses were (1) cerebral blood flow varies based on personality rating condition and (2) cerebral blood flow varies based on the personality traits. This experiment measured cerebral blood flow under 3 personal computer rating conditions and 2 questionnaire conditions. Comparing the rating conditions, the results of the t-test indicated that cerebral blood flow was higher in the questionnaire condition than it was in the personal computer condition. With respect to the Big Five, the result of the correlation coefficient, that is, cerebral blood flow during a personality rating task, changed according to the trait for agreeableness. The results of the analysis of the 5-cluster on individual differences indicated that certain personality traits were related to the factors that increased or decreased cerebral blood flow. An analysis of variance indicated that openness to experience and Behavioural Activation System-drive was significant given that participants with high intellectual curiosity were motivated in this experiment, thus, their cerebral blood flow may have increased. The significance of this experiment was that by employing certain performance measures we could examine differences in physical changes based on personality traits.

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.

Imorin: a sexual attractiveness pheromone in female red-bellied newts (Cynops pyrrhogaster).

The male red-bellied newt (Cynops pyrrhogaster) approaches the female's cloaca prior to performing any courtship behaviour, as if he is using some released substance to gauge whether she is sexually receptive. Therefore, we investigated whether such a female sexual attractiveness pheromone exists. We found that a tripeptide with amino acid sequence Ala-Glu-Phe is secreted by the ciliary cells in the epithelium of the proximal portion of the oviduct of sexually developed newts and confirmed that this is the major active substance in water in which sexually developed female newts have been kept. This substance only attracted sexually developed male newts and acted by stimulating the vomeronasal epithelial cells. This is the first female sexual attractiveness peptide pheromone to be identified in a vertebrate.

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.

Two zebrafish G2A homologs activate multiple intracellular signaling pathways in acidic environment.

Human G2A is activated by various stimuli such as lysophosphatidylcholine (LPC), 9-hydroxyoctadecadienoic acid (9-HODE), and protons. The receptor is coupled to multiple intracellular signaling pathways, including the Gs-protein/cAMP/CRE, G12/13-protein/Rho/SRE, and Gq-protein/phospholipase C/NFAT pathways. In the present study, we examined whether zebrafish G2A homologs (zG2A-a and zG2A-b) could respond to these stimuli and activate multiple intracellular signaling pathways. We also examined whether histidine residue and basic amino acid residue in the N-terminus of the homologs also play roles similar to those played by human G2A residues if the homologs sense protons. We found that the zG2A-a showed the high CRE, SRE, and NFAT activities, however, zG2A-b showed only the high SRE activity under a pH of 8.0. Extracellular acidification from pH 7.4 to 6.3 ameliorated these activities in zG2A-a-expressing cells. On the other hand, acidification ameliorated the SRE activity but not the CRE and NFAT activities in zG2A-b-expressing cells. LPC or 9-HODE did not modify any activity of either homolog. The substitution of histidine residue at the 174(th) position from the N-terminus of zG2A-a to asparagine residue attenuated proton-induced CRE and NFAT activities but not SRE activity. The substitution of arginine residue at the 32nd position from the N-terminus of zG2A-a to the alanine residue also attenuated its high and the proton-induced CRE and NFAT activities. On the contrary, the substitution did not attenuate SRE activity. The substitution of the arginine residue at the 10th position from the N-terminus of zG2A-b to the alanine residue also did not attenuate its high or the proton-induced SRE activity. These results indicate that zebrafish G2A homologs were activated by protons but not by LPC and 9-HODE, and the activation mechanisms of the homologs were similar to those of human G2A.

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.