An increase in extracellular Ca(2+) concentration induces pigment aggregation in teleostean melanophores

An increase in the concentration of Ca(2+) ions in the external medium ([Ca(2+)](o)) induced pigment aggregation in melanophores of three species of freshwater teleosts examined. Denervated melanophores were refractory to elevations of [Ca(2+)](o). The pigment-aggregating action was inhibited by the sympathetic blocking agents, phentolamine, prazosin and yohimbine. Bretylium, an agent known to block the release of the neurotransmitter, interfered with the response effectively. Ca(2+) blockers, such as Mn(2+), verapamil and gallopamil, also inhibited the response, possibly by inhibiting Ca(2+) entry into the presynaptic elements of melanosome-aggregating fibers. The conclusion is that the increase in [Ca(2+)](o) may induce membrane depolarization of presynaptic nervous elements around the melanophores, which open the voltage-dependent Ca(2+) channels there. The liberation of adrenergic neurotransmitter follows, which induces the aggregation of pigment in melanophores.     

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.     

Long-term cultivation of amphibian melanophores. In vitro ageing and spontaneous transformation to a continuous cell line

Pure melanophore populations isolated from the tail skin of the tadpole, Rana catesbeiana, were mass cultured for a period of 2-3 years. All cell lines of amphibian melanophores studied exhibited growth crisis (in vitro ageing) followed by spontaneous transformation to a continuous cell line, as shown by changes in growth characteristics in mass culture and in clone culture, by the appearance of the cells, and by measurements of cell volumes. Even after becoming a continuous cell line, amphibian melanophores continued to have a diploid chromosome number (2n = 26) in three of four cell lines examined. The chromosome mode in one cell line, however, changed to thirty. Measurement of melanin dispersion after the addition of alpha-melanocyte-stimulating hormone suggested that the mechanism for melanin dispersion in melanophores changed during in vitro ageing.     

beta-Adrenergic receptor subtypes in melanophores of the marine gobies Tridentiger trigonocephalus and Chasmichthys gulosus.

The subtype of beta-adrenergic receptors in melanophores of the marine gobies Tridentiger trigonocephalus and Chasmichthys gulosus was studied. Pigment of denervated melanophores in isolated, split caudal fins was preliminarily aggregated by incubating the specimens in a physiological saline containing 10 microM phentolamine and 30-100 microM verapamil or 2-10 nM melatonin, and the responses of the melanophores to a beta-adrenergic agonist added to the incubating medium were recorded photoelectrically. The beta-adrenergic agonists noradrenaline, adrenaline, isoproterenol, salbutamol and, dobutamine were all effective in evoking a dispersion of melanophore pigment in the presence of phentolamine and verapamil or melatonin. The pigment-dispersing effect of noradrenaline (beta 1-selective agonist) was inhibited by metoprolol (beta 1-selective antagonist), propranolol,- and butoxamine. Whereas, the effect of salbutamol (beta 2-selective agonist) was hardly inhibited by metoprolol, though it was considerably inhibited by propranolol and ICI-118551. It was estimated that beta 1- and beta 2-adrenergic receptors coexist at ratios of 8.6:91.4, in the melanophore of Tridentiger trigonocephalus, and 25:75, in the melanophore of Chasmichthys gulosus, through the analyses of Hofstee plots of the effects of the beta-adrenergic drugs. It was suggested that the relation between the pigment-dispersing effect of a beta-adrenergic agonist on the melanophores and the concentration of the drug follows mass action kinetics, when the effect is mainly caused by the activation of beta 2-adrenergic receptors of the melanophores. However, when it is mainly caused by the activation of beta 1-adrenergic receptors of the melanophores, the relation does not follow mass action kinetics.     

Fibronectin-induced migration of melanophores in vitro in scales of medaka,Oryzias latipes

Summary The effects of fibronectin on melanophores were examined in two mutant strains of medaka, Oryzias latipes: mm (BmmR), which has condensed melanophores and normal dendritic melanophores; and cm (BcmR), which has condensed melanophores. When medaka scales were cultured in the presence of fibronectin, melanophores of the wild type and dendritic melanophores of the mm mutant changed their shape and migrated, whereas melanophore migration was rarely seen in the absence of fibronectin. Melanophores of the cm mutant and condensed melanophores of the mm mutant did not migrate even in the presence of fibronectin. When melanophores of the wild type and mm mutant were condensed by adrenalin, they did not migrate. On the other hand, when melanophores of the cm mutant were dispersed by theophylline, they were able to migrate. These results indicate that fibronectin induces the migration of melanophores and that dispersion of melanin granules may be requisite for such migration.     

Berberine-induced pigment dispersion in Bufo melanostictus melanophores by stimulation of beta-2 adrenergic receptors

Abstract

Reduced production of melanin by decreased or the absence of melanocytes leads to various hypopigmentation disorders, and the development of melanogenetic agents for photoprotection and hypopigmentation disorders is one of the top priority areas of research. Hence, the present study was carried out to elucidate the ability of berberine, a principal active ingredient present in the roots of the herb Berberis vulgaris to stimulate pigment dispersion in the isolated skin melanophores of the toad Bufo melanostictus. In the present study, mean melanophore size index of the isolated skin melanophores of B. melanostictus was assayed after treating with various concentrations of berberine. A marked melanin dispersion response leading to skin darkening was observed in the isolated melanophores of toad in response to berberine, which was found to be mediated through beta-2 adrenergic receptors. The physiologically significant dose-related melanin dispersion effects of berberine per se were found to be completely abolished by propranolol, which is a specific beta-2 adrenergic receptor blocker. These per se melanin dispersal effects were also found to be markedly potentiated by isoprenaline, which is a specific beta-adrenoceptor agonist. The results indicate that berberine causes a tremendous, dose-dependent, physiologically significant pigment dispersing in the isolated skin melanophores of B. melanostictus.     

Effect of naphthalene on color changes of the sand fiddler crab, Uca pugilator

The polycyclic aromatic hydrocarbon, naphthalene, inhibited the melanin dispersion, which normally occurs in the fiddler crab, Uca pugilator, during the dark phase of its circadian rhythm of color change. Naphthalene produced this inhibition no matter whether the melanin was fully aggregated or more or less dispersed at the time of initial exposure. Inhibition of the circadian melanin dispersion does not appear to be due to a direct action of naphthalene on the melanophores. This inhibition was concentration-dependent with acute daily exposure. Naphthalene does not, however, inhibit melanin dispersion in response to a black background, indicating thereby that naphthalene-exposed crabs continue to synthesize melanin-dispersing hormone and norepinephrine.     

Cellular Aspects of the Control of Physiological Color Changes in Amphibians

SYNOPSIS. The present paper is concerned mainly with the melanin-dispersingeffect of melanocyte-stimulating hormones (MSH's) on the skinmelanophores of amphibians. In addition, some of the more recentevidence for the unihumoral theory of the control of color changeis reviewed. The mechanism of dispersion of melanin is stillunknown, but evidence is accumulating that the action of MSHmay be mediated by an increase in the melanophoric content ofadenosine 3', 5'-monophosphate (cyclic AMP). For example, cyclicAMP has a specific, reversible melanin-dispersing effect onthe melanophores of the isolated skin of R. pipiens and Xenopuslaevis. It also has a reversible "melanophore—expanding"effect on the tissue—cultured embryonic melanophores ofthe spotted salamander, Ambystoma maculatum. The effect of cyclicAMP on melanophores of R. pipiens does not require sodium butis inhibited by hypertonicity. Finally, new evidence is presented that confirms that the melanin-dispersingeffect of catecholamines on melanophores of X. laevis is mediatedby beta adrenergic receptors,because it is blocked by the highlyspecific ß—blocking agent, propranolol. On theother hand, the melanin-aggregating effect of catecholamineson amphibian melanophores appears to be mediated by alpha adrenergicreceptors. There is even a possibility that the effects of catecholaminesare also mediated through a control of cyclic AMP levels inmelanophores, with beta adrenergic stimulation producing anincrease in cyclic AMP levels, followed by dispersion of melanin,and alpha adrenergic stimulation producing a decrease in cyclicAMP levels, followed by aggregation of melanin.     

Biosensing of opioids using frog melanophores

Spectacular color changes of fishes, frogs and other lower vertebrates are due to the motile activities of specialized pigment containing cells. Pigment cells are interesting for biosensing purposes since they provide an easily monitored physiological phenomenon. Melanophores, containing dark brown melanin pigment granules, constitute an important class of chromatophores. Their melanin-filled pigment granules may be stimulated to undergo rapid dispersion throughout the melanophores (cells appear dark), or aggregation to the center of the melanophores (cells appear light). This simple physiological response can easily be measured in a photometer. Selected G protein coupled receptors can be functionally expressed in cultured frog melanophores. Here, we demonstrate the use of recombinant frog melanophores as a biosensor for the detection of opioids. Melanophores were transfected with the human opioid receptor 3 and used for opiate detection. The response to the opioid receptor agonist morphine and a synthetic opioid peptide was analyzed by absorbance readings in an aggregation assay. It was shown that both agonists caused aggregation of pigment granules in the melanophores, and the cells appeared lighter. The pharmacology of the expressed receptors was very similar to its mammalian counterpart, as evidenced by competitive inhibition by increasing concentrations of the opioid receptor inhibitor naloxone. Transfection of melanophores with selected receptors enables the creation of numerous melanophore biosensors, which respond selectively to certain substances. The melanophore biosensor has potential use for measurement of substances in body fluids such as saliva, blood plasma and urine.     

Responses of isolated scale melanophores of a fresh water carp,Cirrhinus mrigala (Ham.) to zidovudine

Zidovudine (AZT) induced concentration related aggregation in C. mrigala melanophores. Denervated melanophores failed to respond to AZT. Specific and nonspecific alpha adrenoceptor antagonists completely blocked the responses of fish melanophores to AZT. Histamine and prostaglandin antagonists also inhibited aggregation of the melanophores induced by AZT. The results suggest that AZT may release a mixture of neurotransmitter like substances, which cause the aggregation of this fish melanophores.     

Proliferation in vitro of melanophores from Xenopus laevis

Melanophores of wild-type and periodic albino mutants of Xenopus laevis were successfully cultured in vitro. They proliferated in the presence of alpha-melanocyte-stimulating hormone (alpha-MSH or cyclic adenosine monophosphate (cAMP) at a doubling time of 8-10 days. These proliferating melanophores retained their phenotypes, ability to synthesize melanin, and melanin-dispersing response to MSH stimulation. Neither depigmentation nor selective cell death of periodic albino melanophores was observed for at least 4 months during the cultivation.     

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.     

Effect of cyclic AMP and cytochalasin B on tissue cultured melanophores of Xenopus laevis

The effects of cytochalasin B or low concentrations of adenosine 3′,5′-monophosphate (cyclic AMP) were tested on melanophores in hanging drop preparations of neural fold explants from Xenopus laevis embryos in Barths' solution. After one week in culture, the melanophores were punctate in this medium. Cyclic AMP at 5 mM consistently caused reversible morphological transformation of these cells to the stellate state, whether they were situated within an epithelial outgrowth or isolated on the surface of the coverglass. Only the isolated melanophores consistently responded to 1 mM cyclic AMP. Cytochalasin B at 1–10 μg/ml caused aggregation of melanin granules in stellate cells, but left long, narrow cell branches containing some melanosomes. Its effect was at least partially reversible and appeared to be dose dependent. At 1% concentration, dimethyl sulfoxide caused melanin dispersion.     

Control of a teleost social signal

Territorial male Haplochromis burtoni (Teleostei; Cichlidae) have a dark facial stripe, the 'eyebar', which can appear and disappear within seconds, independently of other coloration patterns. It is used to signal territory ownership and aggressive intent. Some males, called 'barless', have functional melanophores in the eyebar region but never display this pattern, because melanin in eyebar pigment cells is never dispersed. The eyebar melanophores are controlled by a specialized branch of the maxillary nerve. Lesioning the 'eyebar nerve' resulted in immediate melanin dispersion and consequent darkening of the eyebar pattern, and it abolished the normal paling response in all behavioral situations. Nerve lesion produced similar results in both barred and barless males, except that the coloration of the denervated eyebar in barless males was more similar to camouflage markings than to the conspicuous black eyebar used as a social signal. Electrical stimulation of the maxillary nerve produced melanin aggregation. Photoelectric recordings of this paling response revealed no differences between barred and barless males, or between the eyebar and other facial chromatophores that do not function as visual displays. Thus, the difference in the physiological state of eyebar melanophores in intact barred and barless males cannot be explained by differences in peripheral nerve anatomy or physiology.     

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.     

Studies of pigment transfer between Xenopus laevis melanophores and fibroblasts in vitro and in vivo

Frog melanophores rapidly change colour by dispersion or aggregation of melanosomes. A long-term colour change exists where melanosomes are released from melanophores and transferred to surrounding skin cells. No in vitro model for pigment transfer exists for lower vertebrates. Frog melanophores of different morphology exist both in epidermis where keratinocytes are present and in dermis where fibroblasts dominate. We have examined whether release and transfer of melanosomes can be studied in a melanophore-fibroblast co-culture, as no frog keratinocyte cell line exists. Xenopus laevis melanophores are normally cultured in conditioned medium from fibroblasts and fibroblast-derived factors may be important for melanophore morphology. Melanin was exocytosed as membrane-enclosed melanosomes in a process that was upregulated by alpha-melanocyte-stimulating hormone (alpha-MSH), and melanosomes where taken up by fibroblasts. Melanosome membrane-proteins seemed to be of importance, as the cluster-like uptake pattern of pigment granules was distinct from that of latex beads. In vivo results confirmed the ability of dermal fibroblasts to engulf melanosomes. Our results show that cultured frog melanophores can not only be used for studies of rapid colour change, but also as a model system for long-term colour changes and for studies of factors that affect pigmentation.     

Observations on skin colour changes in juvenile lumpsuckers

This study reports on an experimental investigation of colour change in young lumpsuckers Cyclopterus lumpus , employing Munsell colour charts to quantify skin hue, colour value and chroma. Juvenile lumpsuckers can live pelagically close to the sea surface, or can attach to floating seaweed and debris with their suckers. When swimming they adopt a cryptic default light green coloration, with the dorsum being darker than the belly (countershading). This default colour is also seen in complete darkness. When attached to objects they take a colour stimulus from below and to the sides and change colour. They can achieve good colour matches (noticeable in 3 min; complete within 15 min) with substrata that are green-yellow in colour, especially the brown alga Ascophyllum nodosum . Colour matching is largely dependent upon changes in colour value (i.e. lightness/darkness) produced by melanin dispersal or concentration in the melanophores. Thus, on artificial backgrounds outside the range of achievable hue matching, they still achieve good matchings of colour value. Slight hue changes were recorded (probably reflecting interaction of basic skin colour with melanin dispersal state), but chroma (brightness) variations were negligible. Lumpsucker colour depends upon light intensity, allowing sustained colour matching as weed illumination changes.     

Effects of lithium on pigmentation in the embryonic zebrafish (Brachydanio rerio)

Pigment cell precursors of the embryonic zebrafish give rise to melanophores, xanthophores and/or iridophores. Cell signaling mechanisms related to the development of pigmentation remain obscure. In order to examine the mechanisms involved in pigment cell signaling, we treated zebrafish embryos with various activators and inhibitors of signaling pathways. Among those chemicals tested, LiCl and LiCl/forskolin had a stimulatory effect on pigmentation, most notable in the melanophore population. We propose that the inositol phosphate (IP) pathway, is involved in pigment pattern formation in zebrafish through its involvement in the: (1) differentiation/proliferation of melanophores; (2) dispersion of melanosomes; and/or (3) synthesis/deposition of melanin. To discern at what level pigmentation was being effected we: (1) counted the number of melanophores in control and experimental animals 5 days after treatment; (2) measured tyrosinase activity and melanin content; and (3) employed immunoblotting techniques with anti-tyrosine-related protein-2 and anti-melanocyte-specific gene-1 as melanophore-specific markers. Although gross pigmentation increased dramatically in LiCl- and LiCl/forskolin treated embryos, the effect on pigmentation was not due to an increase in the proliferation of melanophores, but was possibly through an increase in melanin synthesis and/or deposition. Collectively, results from these studies suggest the involvement of an IP-signaling pathway in the stimulation of pigmentation in embryonic zebrafish through the synthesis/deposition of melanin within the neural crest-derived melanophores.     

Physiological Color Changes in Reptiles

SYNOPSIS. The physiological regulation of color changes in reptilesas studied in the lizard, Anolis carolinensis, is discussed.In Anolis, the ability to adapt to a background is dependentupon the level of circulating MSH, therelease of which is dependenton information received through the eyes. Blinded (or intact)lizards are brown under conditions of strong illumination andgreen under conditions of lower light intensities, and, again,these color changes are regulated by MSH. According to Kleinholz,color changes in the blinded lizard are regulated by dermalphotoreceptors. High or low temperatures directly affect thecolor of Anolis skins and alter the rate at which skins respondto hormones. Aggregationof melanin granules within Anolis melanophoresin response to sympathomimetic stimulation is regulated throughalpha adrenergic receptors whereas dispersion of melanin granulesin response to such stimulation is controlled through beta adrenergicreceptorspossessed by the melanophores. Most Anolis melanophores possessboth alpha and beta adrenergic receptors, but some melanophorespossess only beta adrenergic receptors. In the normal physiologyof the lizard, under conditions of stress, stimulation of alphaadrenergic receptors by catecholamines leads to an "excitement—pallor"followedby an "excitement—darkening" resulting from stimulationof beta adrenergic receptors which causes dispersion of melaningranules within localized populations of melanophores. Thus,in Anolis, dispersion of melanin granules within melanophoresis regulated by both MSH and by catecholamines. Evidence ispresented that the intracellular level of cyclic 3', 5'-AMPwithin melanophores may be responsible for the regulation ofmovement of melanin granules.     

β-Adrenergic Receptor Subtypes in Melanophores of the Marine Gobies Tridentiger trigonocephalus and Chasmichthys gulosus

The subtype of β-adrenergic receptors in melanophores of the marine gobies Tridentiger trigonocephalus and Chasmichthys gulosus was studied. Pigment of denervated melanophores in isolated, split caudal fins was preliminarily aggregated by incubating the specimens in a physiological saline containing 10 μM phentolamine and 30–100 μM verapamil or 2–10 nM melatonin, and the responses of the melanophores to a β-adrenergic agonist added to the incubating medium were recorded photoelectrically. The β-adrenergic agonists noradrenaline, adrenaline, isoproterenol, salbutamol and, dobutamine were all effective in evoking a dispersion of melanophore pigment in the presence of phentolamine and verapamil or melatonin. The pigment-dispersing effect of noradrenaline (β₁-selective agonist) was inhibited by metoprolol (β₁-selective antagonist), propranolol, and butoxamine. Whereas, the effect of salbutamol (β₂-selective agonist) was hardly inhibited by metoprolol, though it was considerably inhibited by propranolol and ICI-118551. It was estimated that β₁- and β₂-adrenergic receptors coexist at ratios of 8.6:91.4, in the melanophore of Tridentiger trigonocephalus, and 25:75, in the melanophore of Chasmichthys gulosus, through the analyses of Hofstee plots of the effects of the β-adrenergic drugs. It was suggested that the relation between the pigment-dispersing effect of a β-adrenergic agonist on the melanophores and the concentration of the drug follows mass action kinetics, when the effect is mainly caused by the activation of β₂-adrenergic receptors of the melanophores. However, when it is mainly caused by the activation of β₁-adrenergic receptors of the melanophores, the relation does not follow mass action kinetics.