Kanta Mizusawa

Green light stimulates somatic growth in the barfin flounder Verasper moseri.

We examined the effects of different light wavelengths-blue, green, and red-on the somatic growth of the barfin flounder Verasper moseri, a flatfish. The light sources used were fluorescent lamps and a combination of daylight and fluorescent lamps that produced ambient light. These light sources were filtered using blue, green, or red filters. During the experiments, the fish were reared in indoor tanks with running seawater of natural temperature and fed with commercial pellets twice daily until satiety. The tanks were white in color. Fish were exposed to constant light emitted from the fluorescent lamps (9:15, light:dark; 08:00-17:00, light) for 14 weeks from October or September to January or to ambient light with a 14-week natural photoperiod from September to December. The wavelengths that were filtered from the fluorescent lamp light modified the growth of the fish, i.e., fish reared under green or blue light exhibited a greater total length (TL; P<0.01) and body weight (BW; P<0.01) than those reared under red light. In contrast, in the case of fish exposed to filtered ambient light, fish reared under green light exhibited a greater TL (P<0.01) and BW (P<0.01) than fish exposed to other wavelengths-blue-, red-, and nonfiltered ambient light. Our results indicate that flounder growth was modified by certain wavelengths, namely, green and red light, which had growth-stimulating and growth-inhibiting effects, respectively.

Possible paracrine function of α-melanocyte-stimulating hormone and inhibition of its melanin-dispersing activity by N-terminal acetylation in the skin of the barfin flounder, Verasper moseri

Melanocyte-stimulating hormone (MSH) is generated from a precursor protein, proopiomelanocortin (POMC), mainly in the pituitary. The barfin flounder, Verasper moseri, expresses three different POMC genes (Pomc), among which Pomc-c is also expressed in the skin. Herein, we characterized the biological significance of POMC and MSH produced in barfin flounder skin. The reverse transcription polymerase chain reaction showed the expression of Pomc-c in isolated non-chromatophoric dermal cells. Mass spectrometry analyses of fractions of skin extract separated by high-performance liquid chromatography revealed the presence of a peptide with a molecular mass corresponding to Des-acetyl (Ac)-alpha-MSH-C derived from POMC-C. These results indicate that, in addition to endocrine functions, MSH in barfin flounder is associated with skin pigmentation via paracrine mechanisms. On the other hand, in vitro studies showed that Des-Ac-alpha-MSH-C dispersed pigments in both melanophores and xanthophores. These functions are similar to those of Des-Ac-alpha-MSH, which differs from Des-Ac-alpha-MSH-C only at the C-terminus, generated from POMC-A and -B. Alpha-MSH, which has an acetyl group at the N-terminus, led to pigment dispersion in xanthophores, but showed no effect in melanophores. A series of bioassays indicated that acetylation enhances MSH activity in xanthophores, but inhibits it in melanophores, suggesting that receptors for MSHs expressed in xanthophores and melanophores are different from each other.

Photic regulation of arylalkylamine N-acetyltransferase 1 mRNA in trout retina.

Melatonin production in the pineal organ and retina is controlled by both light-dark cycles and a circadian clock via the oscillating activity of arylalkylamine N-acetyltransferase (AANAT) in most vertebrates. However, this clock regulation is absent in the rainbow trout (Oncorhynchus mykiss) pineal organ: the trout has two different AANAT genes (AANATI and AANAT2), and AANAT2 mRNA levels in the pineal organ did not exhibit circadian oscillation In this study, we confirmed by RT-PCR analysis that AANATI is expressed only in the retina, while AANAT2 is expressed in the pineal organ and brain. Real-time quantitative PCR analysis demonstrated that AANATI mRNA levels in the retina exhibited daily variations with high levels during the dark phase under light-dark cycles, but kept high and low titers under constant darkness and constant light, respectively. Thus, AANATI gene expression in the trout retina is regulated not by a circadian clock but by lighting conditions.

Retina-Type Rhodopsin Gene Expressed in the Brain of a Teleost, Ayu (Plecoglossus altivelis)

Abstract Ayu (Plecoglossus altivelis) is a teleost whose gonadal development is stimulated by shortened daylength and is a useful model to study the mechanism of photoperiodism. However, localization and characteristics of the photoreceptor that mediates photoperiodism in gonadal development remain to be determined. To identify the photoreceptive molecule that regulates photoperiodic responses, in the present study, we have cloned and characterized the cDNA encoding an opsin gene expressed in the ayu brain, a putative site of the photoreceptor for photoperiodism. The identified opsin was rhodopsin that is identical to the rhodopsin expressed in the retina. Phylogenetic analysis demonstrated that this rhodopsin belongs to the retina-type but not to the pineal-specific rhodopsin group. Genomic polymerase chain reaction (PCR) demonstrated that the ayu rhodopsin gene is intron-less. Southern and Northern blots and reverse-transcription PCR analyses indicate that the same rhodopsin gene is expressed in the retina and the brain but not in the pineal organ of ayu. These results indicate that the rhodospin gene is expressed in the retina and brain and mediates not only visual but also nonvisual functions such as photoperiodism and entrainment of the circadian clock.

Molecular cloning and expression of two melanin-concentrating hormone receptors in goldfish.

Melanin-concentrating hormone (MCH) is a neurohypophysial hormone and induces melanin aggregation in the skin in teleosts. MCH also has multiple roles in the central regulation of food intake in teleosts and mammals. MCH receptors (MCH-R) are among type I G-protein-coupled receptors. Here, we cloned two MCH receptors from goldfish, Carassius auratus. The amino acid sequence of goldfish MCH-R1 had 57-88% homology with fish MCH-R1 and 49-50% homology with mammalian MCH-R1, while the amino acid sequence of goldfish MCH-R2 had 72-92% homology with fish MCH-R2 and 32% homology with human MCH-R2. Phylogenetic analysis showed that these two MCH-Rs are orthologous to the respective mammalian MCH-Rs. The common amino acid residues for ligand binding, signal transduction, and receptor conformation were well conserved in these receptors, although some intracellular basic-amino-acid-rich domains, which have been shown to exist in human MCH-R1 and MCH-R2, were absent in goldfish MCH-R2. When stably expressed in HEK293 cells, both goldfish MCH-R1 and MCH-R2 displayed a strong, dose-dependent, transient elevation of intracellular calcium in response to salmon MCH (EC(50)=0.8nM and 31.8nM, respectively). In contrast to goldfish MCH-R2, goldfish MCH-R1 signaling is not sensitive to pertussis toxin, suggesting an exclusive Galphaq coupling of goldfish MCH-R1 in the mammalian cell-based assay. Reverse transcriptase PCR revealed that both MCH-R1 and MCH-R2 mRNA are distributed in various tissues in goldfish. The various tissues including the brain and skin express both MCH-R1 and MCH-R2. These results suggest that these functional receptors mediate multiple effects of MCH in goldfish.

Inhibiting roles of melanin-concentrating hormone for skin pigment dispersion in barfin flounder, Verasper moseri.

Barfin flounders change their surface color pattern to match their background. We have reported evidence of the association between hormones and body color changes in this fish. First, bolus intraperitoneal injection with melanin-concentrating hormone (MCH) immediately turned the skin color pale, while injection with melanocyte-stimulating hormone (MSH) did not change the skin color. Second, gene expression levels of MCH change in response to background color, while those of MSH do not. We also reported the expression of an MCH receptor gene (Mch-r2) in the skin of this fish. In this study, we aimed to further evaluate the roles of MCH in skin color change. First, long-term adaptation of adult barfin flounder to black or white background colors induced significantly different pigment migration patterns in both melanophores and xanthophores (P<0.05). However, continuous intraperitoneal injection with MCH did not influence chromatophore proliferation. Then, using in vitro experiments, we found that MCH aggregates both melanophores and xanthophores, and inhibits the pigment-dispersing activity of MSH in a similar manner. Finally, we identified transcripts of Mch-r2 in cells isolated from both melanophores and xanthophores. Taken together, the evidence suggests that MCH aggregates pigments via MCH-R2 in concert with the nervous system by overcoming the melanin-dispersing activities of MSH in barfin flounder.

In vitro photic entrainment of the circadian rhythm in melatonin release from the pineal organ of a teleost, ayu (Plecoglossus altivelis) in flow-through culture

The effects of light on the circadian rhythm in melatonin release from the pineal organ of a teleost, ayu (Plecoglossus altivelis) were investigated in flow-through culture. Under the reversed light-dark (LD) cycle, the melatonin rhythm phase shifted as compared with those under the normal LD cycle. This phase shift persisted even under constant darkness (DD). Single 6-h light pulses starting at six different circadian phases under DD acutely suppressed melatonin release. Phase-dependent phase shifts in the circadian rhythm of melatonin release were also observed. The phase response curve to light pulses in the ayu pineal organ is typical of that found in many circadian systems. Thus, the ayu pineal organ should provide a useful model for analyzing the physiological and molecular basis of the entrainment mechanism of vertebrate circadian system.

Posttranslational Modifications of Proopiomelanocortin in Vertebrates and Their Biological Significance

Proopiomelanocortin (POMC) is the precursor of several peptide hormones generated in the pituitary gland. After biosynthesis, POMC undergoes several posttranslational modifications, including proteolytic cleavage, acetylation, amidation, phosphorylation, glycosylation, and disulfide linkage formation, which generate mature POMC-derived peptides. Therefore, POMC is a useful model for the investigation of posttranslational modifications. These processes have been extensively investigated in mammals, primarily in rodents. In addition, over the last decade, much information has been obtained about the posttranslational processing of POMC in non-mammalian animals such as fish, amphibians, reptiles, and birds through sequencing and peptide identification by mass spectrometry. One POMC modification, acetylation, is known to modulate the biological activities of POMC-derived α-melanocyte-stimulating hormone (α-MSH) having an acetyl group at N-terminal through potentiation or inhibition. This bidirectional regulation depends on its intrinsic roles in the tissue or cell; for example, α-MSH, as well as desacetyl (Des-Ac)-α-MSH, stimulates pigment dispersion in the xanthophores of a flounder. In contrast, α-MSH does not stimulate pigment dispersion in the melanophores of the same species, whereas Des-Ac-α-MSH does. Regulation of pigment-dispersing activities may be associated with the subtle balance in the expression of receptor genes. In this review, we consider the posttranslational modifications of POMC in vertebrates from an evolutionary aspect, with a focus on the relationship between acetylation and the biological activities of α-MSH as an important consequence of posttranslational modification.

Pigment-dispersing activities and cortisol-releasing activities of melanocortins and their receptors in xanthophores and head kidneys of the goldfish Carassius auratus

The five subtypes of melanocortin receptors (MCRs) mediate the functions of α-melanocyte-stimulating hormone (α-MSH) and adrenocorticotropic hormone (ACTH). In fish, these hormones are involved in pigment dispersion and cortisol release, respectively. α-MSH-related peptides exhibit ACTH-like activity in certain fishes. We recently found that multiple Mcr transcripts are expressed in some cell types in the barfin flounder, which is related to regulation of α-MSH activities. Similar results were also observed for the cortisol-releasing activity of α-MSH-related peptides in the head kidney. The present study was undertaken to assess relationship between the expression of multiply expressed Mcrs and α-MSH activities using goldfish. We also determined if α-MSH-related peptides exhibit ACTH-like activity in goldfish. The transcripts of Mc1r, but not those of other subtypes, were observed in xanthophores. α-MSH, which has an acetyl group at the N-terminus, was found to disperse pigment in a dose-dependent manner in xanthophores. This potency was found to be slightly greater than that of desacetyl-α-MSH. These results support our findings that MCR has a higher affinity for α-MSH when single Mcr subtype is expressed. On the other hand, transcripts of Mc2r, but not those of other subtypes, were observed in the head kidney. ACTH(1-24)-stimulated cortisol release was observed in a dose-dependent manner, while α-MSH-related peptides showed no activity. It therefore appears that MC2R also acts as an ACTH-specific receptor in goldfish and that association of α-MSH-related peptides upon release of cortisol is uncommon in fishes.

Food Deprivation Increases the Expression of the Prepro-Orexin Gene in the Hypothalamus of the Barfin Flounder, Verasper moseri

Orexins (orexin-A and -B) are involved in the regulation of food intake in mammals. In the barfin flounder, Verasper moseri, we previously reported that orexin-A-like-immunoreactive (ir) cell bodies are localized in the hypothalamus, which is a possible orexigenic center in fish. However, the physiological roles of orexin in the barfin flounder remain unclear. Here, we cloned prepro-orexin cDNA and examined the effects of feeding status on orexin gene expression in the barfin flounder to obtain a better insight into the roles of orexins in feeding regulation. A molecular cloning study showed that barfin flounder prepro-orexin cDNA encodes a 145 amino acid (aa) polypeptide containing orexin-A (43 aa) and orexin-B (28 aa). Prepro-orexin gene transcripts were detected in the hypothalamus, pituitary, and several peripheral organs such as the eyeball, gills, head kidney, body kidney, spleen, testis, and the skin on the eye-side of the flounders body. Furthermore, the mean prepro-orexin mRNA expression level in the hypothalamus was significantly higher in fasted than in fed fish. These results show that fasting regulates orexin mRNA in the hypothalamus and suggest that orexin is involved in feeding regulation in barfin flounder.