Synthesis and biological actions of melanin concentrating hormone

A melanin (melanosome) concentrating hormone, MCH, was synthesized and the methodology for its synthesis is detailed. This heptadecapeptide, H-Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val-OH , stimulated melanosome concentration (centripetal aggregation) within melanophores of all species of teleost fishes studied. Melanosome aggregation in response to MCH was not blocked by Dibenamine as was the response to norepinephrine (NE), demonstrating that melanosome aggregating responses to MCH and NE are mediated through separate receptors. Melanosome aggregation in response to MCH was reversed by an equimolar concentration of alpha-melanocyte stimulating hormone (alpha-MSH). In contrast, MCH stimulated melanosome dispersion (centrifugal movement) within melanophores of a frog (Rana pipiens) and a lizard (Anolis carolinensis). Therefore, MCH exhibits both melanosome concentrating and dispersing actions depending upon the species studied.     

Ionic requirements for melanin concentrating hormone (MCH) actions on teleost Poecilia reticulata melanophores

Melanin concentrating hormone (MCH) is a cyclic heptadecapeptide, Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, synthesized in the hypothalamus and released by the neurohypophysis of teleost fish. This hormone is a potent lightening agent of fish skin. This lightening results from the stimulation of a centripetal melanosome (melanin granule) migration to a perinuclear position within integumental melanophores. MCH and related fragment analogues, MCH5-17 and MCH1-14 were used to investigate the ionic requirements for receptor activation by MCH on dermal melanophores of the fish Poecilia reticulata. In calcium-free saline, the sensitivity of the melanophores to MCH and MCH1-14 increased, whereas the sensitivity of the cells to MCH5-17 decreased. Verapamil diminished the sensitivity to MCH5-17, but did not affect melanophore responses to MCH or MCH1-14. The melanosome aggregating response to MCH was not affected in the presence of tetrodotoxin or in sodium- or potassium-free (choline-substituted) saline. These results suggest that neither TTX-sensitive sodium channels nor extracellular sodium or potassium ions play a role in MCH-induced melanosome aggregation. It is known that MCH and MCH1-14 also exhibit MSH-like melanosome dispersion within melanophores, skin darkening activity on fish melanophores whereas MCH5-17 lacks this characteristic. Since the darkening activity of MCH and MCH1-14 requires calcium, these analogues exhibited a diminished lightening (MCH-like) activity in the presence of the divalent cation. In the absence of the N-terminal tetrapeptide sequence (necessary for the expression of MSH-like activity), a role for calcium on melanosome aggregation became evident. These results demonstrate a bifunctional role of calcium on melanosome movements.     

Melanin concentrating hormone (MCH) effects on teleost (Chrysiptera cyanea) melanophores

The in vitro biological actions of synthetic chum salmon melanin concentrating hormone (MCH) on melanophores of the blue damselfish (a teleost), Chrysiptera cyanea, were studied. This cyclic heptadecapeptide stimulated melanosome (melanin granule) aggregation (centripetal migration) within melanophores at a threshold concentration of about 10(-10) M. The action of this putative hormone was not blocked by alpha- or beta-adrenoceptor antagonists. It was concluded that the effects of MCH were direct and were not mediated indirectly through the actions of adrenergic neurotransmitters released from nerve terminals. Further evidence for this view comes from the observation that, unlike the case of neurotransmitter release, melanosome aggregation in response to MCH proceeded in the absence of calcium. The possible role of MCH in the control of color change of teleost fishes is discussed.     

Melanin concentrating hormone (MCH): structure-function aspects of its melanocyte stimulating hormone-like (MSH-like) activity

Melanin concentrating hormone (MCH) is a heptadecapeptide, Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, synthesized in the brain and secreted from the pars nervosa of teleost fish. This hormone stimulates melanosome (melanin granule) aggregation within integumental melanocytes of fishes but, in contrast, stimulates melanosome dispersion within tetrapod (frog and lizard) melanocytes. We determined the message sequence of the primary structure of MCH which is responsible for its MSH-like component of activity. Removal of the N-terminal amino acid results in an almost total loss of MSH-like activity. The C-terminal amino acid is also essential for full MSH-like activity since the analogue, MCH(1-16), is about 100 times less active than MCH. Therefore, the entire heptadecapeptide sequence of MCH appears to contribute to the MSH-like activity of MCH. Ring-contracted analogues (e.g., [Ala5, Cys10]MCH) of MCH are almost devoid of any melanosome aggregating (MCH-like) activity but generally possess considerable or as great an MSH-like activity as MCH. Racemization of MCH by heat-alkali treatment drastically reduces the MCH-like activity of MCH, but does not enhance the MSH-like activity of the hormone.     

Comparative physiology of the renin-angiotensin system.

Renin or a renin-like substance is found in the kidneys of many vertebrate species. It is absent from the kidneys of cyclostomes and elasmobranchs and first appears in holosteans and the bony fishes as well as in all higher vertebrate species. Juxtaglomerular cell granules also appear first in holosteans and the bony fishes while the macula densa first appears in amphibians. In telecost fishes, the corpuscles of Stannius contain Bowie-stainable granules and a renin-like pressor substance. Among classes and, in some cases, species of vertebrates, specificity in the reaction of renin with a substrate has been demonstrated. There is also some species and class variation in the angiotensin molecule since angiotensins of fishes, amphibians, reptiles and birds have chemical characteristics different from each other and from those of ammmals. A role for renin in stimulating interrenal gland steroid biosynthesis and in influencing water and ion regulation in nonmammalian vertebrates is discussed.     

The effects of background colouration and α-MSH treatment on melanophore frequency in winter flounder,Pleuronectes americanus

Winter flounder, Pleuronectes americanus, adapting to black or white backgrounds display significant increase and decline respectively in the number of visible epidermal melanophores over periods up to 8 weeks or longer. This contrasts with a stability in the number of visible dermal melanophores during the same periods of exposure to each background. Flounders treated with -melanophore stimulating hormone exhibited an enhanced rate of increase in number of visible epidermal melanophores when the background was changed from white to black, whereas white-adapted flounder treated with -melanophore stimulating hormone without background change did not manifest any such increase in number of epidermal melanophores. Flounder treated with -melanophore stimulating hormone after transfer from black to white displayed a similar initial decline in visible epidermal melanophore number as in control fish, but the final decline was significantly attenuated. Thus -melanophore stimulating hormone, which has no apparent influence on melanosome dispersion in this species, may have a limited morphological melanophore regulatory role which is discussed in relation to possible antagonistic and synergistic factors that could influence melanogenesis and visible melanophore numbers.Abbreviations DMI dermal melanophore index - EMI epidermal melanophore index - LSD least significant difference - MCH melanosome concentrating hormone - MIF melanogenesis inhibiting factor - MSF melanogenesis stimulating factor - MSH melanophore stimulating hormone     

Regulation of eye and jaw colouration in three‐spined stickleback Gasterosteus aculeatus

Fish can change their skin and eye colour for background matching and signalling. Males of Gasterosteus aculeatus develop ornamental blue eyes and a red jaw during the reproductive season, colours that are further enhanced during courtship. Here, the effects of different hormones on physiological colour changes in the eyes and jaws of male and female G. aculeatus were investigated in vitro. In an in vivo experiment, G. aculeatus were injected with a receptor blocker of a pivotal hormone (noradrenaline) that controls colour change. In males, noradrenaline had aggregating effects on melanophore and erythrophore pigments resulting in blue eyes and a pale jaw, whereas melanocyte‐concentrating hormone (MCH) and melatonin resulted in a pale jaw only. When noradrenalin was combined with melanocyte stimulating hormone (MSH) or prolactin, the jaw became red, while the eyes remained blue. In vivo injection of yohimbine, an alpha‐2 adrenoreceptor blocker, resulted in dispersion of melanophore pigment in the eyes and inhibited the blue colouration. Altogether, the data suggest that noradrenalin has a pivotal role in the short‐term enhancement of the ornamental colouration of male G. aculeatus, potentially together with MSH or prolactin. This study also found a sex difference in the response to MCH, prolactin and melatonin, which may result from different appearance strategies in males, versus the more cryptic females.     

The structure and evolution of the melanocortin and MCH receptors in fish and mammals

Zebrafish are an excellent genetic model system for studying developmental and physiological processes. Pigment patterns in zebrafish are affected by mutations in three types of chromatophores. The behavior of these cells is influenced by alpha-melanocyte-stimulating hormone (alphaMSH) and melanin-concentrating hormone (MCH). Mammals have five alphaMSH receptors (melanocortin receptors) and one or two MCH receptors. We have identified the full complement of melanocortin and MCH receptors in both zebrafish and the pufferfish, Fugu. Zebrafish have six melanocortin receptors, including two MC5R orthologues, while Fugu, lacking MC3R, has only four. We also demonstrate that Fugu and zebrafish have two and three MCHR genes, respectively. MC2R and MC5R are physically linked in all species examined. Unlike other species, we find the Fugu genes contain introns, one of which is in a conserved location and is probably ancestral. We also detail the differential expression of the zebrafish genes throughout development.     

Tris buffer effects on melanophore aggregating responses

The adverse effects of tris (hydroxymethyl)-aminomethane (Tris) are for the first time reported on melanophore responses to agonists and potassium chloride. Melanophore responses in Tris- or bicarbonate-buffered solutions were compared. In the presence of Tris, the cumulative dose-response curve to norepinephrine was significantly shifted to the left, whereas methoxamine dose-response curves were similar in both buffers. The percentage aggregation in response to synthetic MCH (melanin concentrating hormone) was not affected by Tris in the bathing medium. The cumulative dose-response curve to potassium chloride was leftward shifted (one log case) in Tris-buffered solution. These results suggest that in fish melanophore preparations Tris might exert its actions on the presynaptic membrane and/or on the synaptic cleft enzyme COMT, drawing on a greater availability of neurotransmitters at the melanophore membrane receptors.     

Melanocyte stimulating hormone and melanin concentration hormone may be structurally and evolutionarily related

Two melanotropic peptides, melanin concentration hormone (MCH) and alpha-melanocyte stimulating hormone (alpha-MSH), exert opposing actions on melanosome (melanin granule) movements within teleost pigment cells, melanocytes (melanophores). MCH stimulates melanosome aggregation to the cell center whereas alpha-MSH stimulates pigment organelle dispersion out into the dendritic processes of the melanocytes. The actions of alpha-MSH are dependent upon extracellular calcium (Ca2+), whereas those of MCH are actually enhanced in the absence of the cation. At high concentrations (10(-5)-10(-8) M) MCH also exhibits MSH-like activity (autoantagonism), an effect which is abolished in the absence of Ca2+. Therefore, MCH exhibits MCH-like as well as MSH-like activity depending on the presence or absence of extracellular Ca2+. An analogue of MCH, [Ala5, Cys10]MCH, has been synthesized which is totally devoid of MCH activity but still exhibits MSH-like activity. These results suggest that the two melanotropic peptides share some component of structural similarity and may be evolutionarily related.     

Pertussis toxin blocks melatonin-induced pigment aggregation inXenopus dermal melanophores

Summary The molecular mechanism of action for the pineal hormone melatonin was explored by testing melatonin interaction with the components of the hormone-sensitive adenylate cyclase complex in aXenopus dermal melanophore bioassay. Forskolin was employed to stimulate melanosome dispersion. The ability of melatonin to reverse forskolin-stimulated pigment dispersion was assessed, as was the effect of pertussis toxin on the ability of melatonin to aggregate dispersed pigment.Forskolin elicited dispersal of melanosomes in a dose dependent manner (EC₅₀=12 nM) in meninges from stage 52–56 tadpoles ofXenopus laevis. Maximal pigment dispersion was obtained with 100 nM forskolin. Melatonin reversed this effect of forskolin (EC₅₀=1.5 nM), causing pigment aggregation. Pertussis toxin blocked the melatonin-induced aggregation (EC₅₀=358 ng/ml).Prior treatment of the melanophore containing meningeal explants with pertussis toxin results in blockade of melatonin induced pigment aggregation. A 41 kDa pertussis toxin substrate is found in explant homogenates treated with³²P-NAD and pertussis toxin. The availability of this substrate is reduced by prior treatment of intact explants with pertussis toxin and depletion of melatonin responsiveness corresponds to depletion of the 41 kDa substrate. Together, these data suggest that melatonin action upon amphibian dermal melanosomes is mediated by a system requiring a protein similar to the regulatory protein Ni used by mammalian cells to mediate the action of hormones which inhibit adenylate cyclase through a cell surface receptor.Abbreviations MI melanophore index - MSH melanocyte stimulating hormone - FCS fetal calf serum     

Control of chromatophore movements in dermal chromatic units of blue damselfish--I. The melanophore

Mechanisms controlling pigment movements in the melanophore of the blue damselfish, Chrysiptera cyanea, were studied. Histological observations revealed that the melanophore had three-dimensionally developed processes to envelop overlying small iridophores, and thus participated in the construction of a simple dermal chromatophore unit. Nervous stimulation, catecholamines and melatonin brought about melanosome aggregation in the melanophore. The actions of the nervous stimulation and catecholamines were antagonized by alpha adrenolytic agents. A beta adrenergic agonist, metaproterenol, adenosine and adenine nucleotides, and alpha-MSH acted as pigment-dispersing agents. These results indicate that the melanophore of the present material is controlled quite orthodoxly by adrenergic nerves and endocrines, notwithstanding the fact that it has quite a unique morphology among fish species, and that its motile rate is remarkably high.     

Melanin concentrating hormone. III. Melanin concentrating hormone (MCH): the message sequence

Melanin concentrating hormone (MCH) is a heptadecapeptide synthesized by the hypothalamus and secreted by the neurohypophysis of the teleost pituitary gland. MCH stimulates melanosome aggregation within teleost melanocytes but also exhibits MSH-like (melanosome dispersing) activity on tetrapod (frog and lizard) melanocytes. We have synthesized a number of MCH analogues to determine the essential features of the primary structure necessary to stimulate either melanosome aggregation or dispersion in fish or tetrapod melanocytes, respectively. An analysis of the potencies and actions of these analogues on vertebrate melanocytes is provided and demonstrates that the two activities have different structural requirements.     

Comparative analyses of the pigment-aggregating and -dispersing actions of MCH on fish chromatophores

In melanophores of the peppered catfish and the Nile tilapia, melanin-concentrating hormone (MCH) at low doses (<1 μM) induced pigment aggregation, and the aggregated state was maintained in the presence of MCH. However, at higher MCH concentrations (such as 1 and 10 μM), pigment aggregation was immediately followed by some re-dispersion, even in the continued presence of MCH, which led to an apparent decrease in aggregation. This pigment-dispersing activity at higher concentrations of MCH required extracellular Ca²⁺ ions. By contrast, medaka melanophores responded to MCH only by pigment aggregation, even at the highest concentration employed (10 μM). Since it is known that medaka melanophores possess specific receptors for α-melanophore-stimulating hormone (α-MSH), the possibility that interaction between MSH receptors and MCH at high doses in the presence of Ca²⁺ might cause pigment dispersion is ruled out. Cyclic MCH analogs, MCH (1–14) and MCH (5–17), failed to induce pigment dispersion, whereas they induced aggregation of melanin granules. These results suggest that another type of MCH receptor that mediates pigment dispersion is present in catfish and tilapia melanophores, and that intact MCH may be the only molecule that can bind to these receptors. Determinations of cAMP content in melanophores, which were isolated from the skin of three fish species and treated with 10 nM or 10 μM MCH, indicate that MCH receptors mediating aggregation may be coupled with Gi protein, whereas MCH receptors that mediate dispersion may be linked to Gs. The response of erythrophores, xanthophores and leucophores to MCH at various concentrations was also examined, and the results suggest that the distribution patterns of the two types of MCH receptors may differ among fish species and among types of chromatophore in the same fish.     

The relative in vitro responsiveness of melanophores of winter flounder to α-MSH and MCH

Melanin-concentrating hormone (MCH) was more potent than the antagonistic melanophore stimulating hormone (α-MSH) for the winter flounder Pleuronectes americanus when mixtures of the two were tested on melanophore preparations. Melanophores from individual pattern components displayed different relative sensitivities to the two hormones. These differences indicate that there are pattern-related variations in MCH and α-MSH receptor density and affinity suggesting that these hormones may have modulatory roles on the pattern-related differential neural regulation of the melanophores.     

Protein-kinase C mediates MCH signal transduction in teleost, synbranchus marmoratus, melanocytes

MCH (melanin concentrating hormone) is a heptadecapeptide, Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, which stimulates melanosome (melanin granule) aggregation to a perinuclear position within teleost fish integumental melanocytes, resulting in lightening of the skin. The mechanisms of action of MCH are unknown. Drugs that affect the diacylglycerol/inositol triphosphate pathway were used to investigate the possible roles of this pathway in the mechanisms of action of MCH on Synbranchus marmoratus (teleost) melanocytes. The shift of the dose-response curve to MCH in the presence of various concentrations of 4-bromophenacyl bromide and neomycin sulphate, phospholipase C inhibitors, suggests that phospholipase C is stimulated after MCH receptor activation. Low concentrations (10(-9) to 10(-8) M) of the phorbol ester TPA exhibited MCH-like activity, eliciting a dose-dependent melanosome aggregation. Higher doses, however, displaced to the right the dose-response curve to MCH, as did the protein kinase C inhibitors, dibucaine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7). These results support the assumption that protein kinase C mediates the pigment aggregating activity of MCH. Both MCH and norepinephrine lightening actions were abolished by beta-glycerophosphate, a phosphatase inhibitor, suggesting that a protein dephosphorylation occurs during melanosome aggregation, and is, therefore, a common event triggered by MCH and norepinephrine, although both agonists act through separate receptors and exhibit different transduction mechanisms.     

The role of calcium in MSH stimulated melanosome dispersion

We have examined the role of the calcium ion in the response of the melanophores of the lizard, Anolis carolinensis to alpha-melanocyte stimulating hormone (alpha-MSH) by a rate method which allows kinetic analysis of melanosome dispersion. Dose-response curves to alpha-MSH were compared in the presence of different calcium concentrations, and Schild regression plots were constructed. An avidly binding analogue of alpha-MSH, Nle4-D-Phe7-alpha-MSH, was used to demonstrate kinetically the involvement of calcium in the melanophore response both for MSH receptor binding and the subsequent transduction of the MSH signal across the melanophore membrane.     

A Rapid Quantitative Bioassay for Evaluating the Effects of Ligands Upon Receptors That Modulate cAMP Levels in a Melanophore Cell Line

A new method for rapidly evaluating the effects of drugs on receptors that regulate intracellular cAMP in a cell line derived from Xenopus laevis melanophores has been developed. Melanophores were plated into sterile 96 well microtiter plates, and 3 days later the cells were treated with melatonin for 30 min to induce melanosome aggregation. Subsequent exposure to MSH or adrenergic agonists caused dose dependent pigment dispersion that peaked within 30 min. The cumulative pigment displacement from cells could be quantitated by using a microplate reader to measure changes in transmittance of light through the wells. The acquired data enabled detailed and reproducible dose response curves and time course analyses to be generated. In addition, the assay followed for the rapid characterization of the effects of antagonists upon the (β adrenergic receptor (β AR). The assay has the potential to test the effects of ligands upon any receptor capable of mediating pigment translocation in the melanophore cell line.     

Molecular components and mechanism of adrenergic signal transduction in mammalian pineal gland: regulation of melatonin synthesis

Rhythmic neural outputs from the hypothalamic suprachiasmatic nucleus (SCN), which programme the rhythmic release of norepinephrine (NE) from intrapineal nerve fibers, regulate circadian rhythm of melatonin synthesis. Increased secretion of NE with the onset of darkness during the first half of night stimulates melatonin synthesis by several folds. NE binds to both alpha1- and beta-adrenergic receptors present on the pinealocyte membrane and initiates adrenergic signal transduction via cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) generating pathways. The NE-induced adrenergic signal transduction switches 'on' melatonin synthesis during the early hours of night by stimulating expression of the rate-limiting enzyme of melatonin synthesis, N-acetyltransferase (AA-NAT) via cAMP-protein kinase A (PKA)-cAMP response element binding protein (CREB)-cAMP response element (CRE) pathway as well as by increasing AA-NAT activity via cAMP-PKA-14-3-3 protein pathway. Simultaneously, adrenergically-induced expression of inducible cAMP early repressor (ICER) negatively regulates aa-nat gene expression and controls the amplitude of melatonin rhythm. In the second half of night, increased release of acetylcholine from central pinealopetal projections, inhibition of NE secretion by SCN, withdrawal of adrenergic inputs and reversal of events that took place in the first half lead to switching 'off' of melatonin synthesis. Adrenergic signal transduction via cGMP-protein kinase G (PKG)-mitogen activated protein kinase (MAPK)-ribosomal S6 kinase (RSK) pathway also seems to be fully functional, but its role in modulation of melatonin synthesis remains unexplored. This article gives a critical review of information available on various components of the adrenergic signal transduction cascades involved in the regulation of melatonin synthesis.     

Comparative study on pattern recognition receptors in non-teleost ray-finned fishes and their evolutionary significance in primitive vertebrates

Pattern recognition receptors(PRRs) play important roles in innate immunity system and trigger the specific pathogen recognition by detecting the pathogen-associated molecular patterns. The main four PRRs components including Toll-like receptors(TLRs), RIG-I-like receptors(RLRs), NOD-like receptors(NLRs) and C-type lectin receptors(CLRs) were surveyed in the five genomes of non-teleost ray-finned fishes(NTR) including bichir(Polypterus senegalus), American paddlefish(Polyodon spathula), alligator gar(Atractosteus spatula), spotted gar(Lepisosteus oculatus) and bowfin(Amia calva), representing all the four major basal groups of ray-finned fishes. The result indicates that all the four PRRs components have been well established in these NTR fishes. In the RLR-MAVS signal pathway, which detects intracellular RNA ligands to induce production of type I interferons(IFNs), the MAVS was lost in bichir particularly. Also, the essential genes of recognition of Lipopolysaccharide(LPS) commonly in mammals like MD2, LY96 and LBP could not be identified in NTR fishes. It is speculated that TLR4 in NTR fishes may act as a cooperator with other PRRs and has a different pathway of recognizing LPS compared with that in mammals. In addition, we provide a survey of NLR and CLR in NTR fishes. The CLRs results suggest that Group V receptors are absent in fishes and Group II and VI receptors are well established in the early vertebrate evolution. Our comprehensive research of PRRs involving NTR fishes provides a new insight into PRR evolution in primitive vertebrate.