Psoralea corylifolia extracts stimulate cholinergic-like psoralen receptors of tadpole-tail melanophores,leading to skin darkening

The present work was carried out to determine the effects of lyophilized seed extracts of Psoralea corylifolia along with pure psoralen, its active ingredient on the isolated tail-piece melanophores of Bufo melanostictus, a type of disguised smooth muscle cells, which offer excellent in vitro opportunities for studying the effects of pharmacological and pharmaceutical agents. In the present study, it was found that lyophilized extract of P. corylifolia and its active ingredient psoralen induced powerful, dose-dependent, physiologically significant melanin dispersal effects in the isolated tail melanophores of B. melanostictus, which were completely blocked by atropine as well as hyoscine. The per se melanin dispersal effects of lyophilized extracts of P. corylifolia and its active ingredient psoralen were highly potentiated by neostigmine. It appears that the melanin dispersal effects of the extracts of P. corylifolia and psoralen are mediated by cholino-muscarinic or cholino-psoralen like receptors having similar properties that need to be studied further.     

On the action and mechanism of withaferin-A from Withania somnifera,a novel and potent melanin dispersing agent in frog melanophores

The present work was carried out to determine the effects of lyophilized root extracts of Withania somnifera along with pure withaferin-A, on the isolated skin melanophores of frog, Rana tigerina which are disguised type of smooth muscle cells and offer excellent in vitro opportunities for studying the effects of pharmacological and pharmaceutical agents. The lyophilized extract of W. somnifera and its active ingredient withaferin-A induced powerful dose-dependent physiologically significant melanin dispersal effects in the isolated skin melanophores of R. tigerina, which were completely blocked by atropine as well as hyoscine. The per se melanin dispersal effects of lyophilized extracts of W. somnifera and its active ingredient withaferin-A got highly potentiated by neostigmine. It appears that the melanin dispersal effects of the extracts of W. somnifera and withaferin-A is mediated by cholino-muscarinic like receptors having similar properties.     

Effect and mechanism of action of resveratrol: a novel melanolytic compound from the peanut skin of Arachis hypogaea

Purpose: The present work was carried out to determine the effects of ethanolic extracts of Arachis hypogaea and its active ingredient resveratrol on the isolated tail melanophores of the Bufo melanostictus to find the mechanism of skin lightening at the cellular level.

Methods: The tail melanophores of the tadpole B. melanostictus were assayed using the mean melanophore size index and their responses were recorded in presence of various concentrations of the plant extract and its active ingredient along with specific antagonists and potentiator.

Results: Significant skin lightening activity of the extract of A. hypogaea and its active ingredient resveratrol was observed on the tail melanophores of tadpole. The pigment cells responded by distinct aggregation leading to skin lightening, this effect was reversible, as re-immersion in physiological saline made the melanophores return to their normal intermediate state. These melanin aggregating effects were completely blocked by propanolol (beta blocker) and partially blocked by prazosin (alpha blocker) and were also found to be highly potentiated by reserpine.

Conclusion: These studies suggest that the active ingredient of A. hypogaea such as resveratrol can act as a sympathomimetic compound and induce aggregation of melanophores of tadpole B. melanostictus via the induction of beta type of the adrenoceptors. The present study opens new vistas for the use of A. hypogaea and its active ingredient, resveratrol for its clinical application as a nontoxic melanolytic compound for the treatment of hyperpigmentation.     

Subtypes of beta adrenergic receptors mediating pigment dispersion in chromatophores of the medaka, Oryzias latipes

Actions of the adrenergic beta-2 agonists, salbutamol and terbutaline, and the beta-1 antagonists, metoprolol and atenolol, were examined on denervated melanophores and leucophores of a teleost, Oryzias latipes. Beta-2 agonists depressed the pigment-aggregation response of melanophores to norepinephrine, while beta-1 antagonists inhibited the dispersion response of leucophores to isoproterenol but not the melanophore response. These findings suggest that adrenergic receptors mediating pigment dispersion in melanophores are beta-2 and those of leucophores are beta-1. The possible relations between receptor mechanisms and the responses of chromatophores are discussed.     

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.     

Mediation of cholino-piperine like receptors by extracts of Piper nigrum induces melanin dispersion in Rana tigerina tadpole melanophores

Aim: The present study was carried out to determine the effects of lyophilized dried fruit extracts of Piper nigrum and pure piperine on the tadpole melanophores of frog Rana tigerina which offer excellent in vitro opportunities for studying the effects of pharmacological and pharmaceutical agents. The nature of specific cellular receptors present on the neuro-melanophore junction and their involvement in pigmentary responses has been explored.

Material: Effects of lyophilized extracts of P. nigrum and pure piperine were studied on the isolated tail melanophores of tadpoles of the frog R. tigerina as per the modified method.

Results: The extract of P. nigrum and its active ingredient piperine caused significant melanin dispersal responses leading to darkening of the tail melanophores, which were completely antagonized by atropine and hyoscine. These per se melanin dispersal effects were also found to be markedly potentiated by neostigmine an anticholinesterase agent.

Conclusion: It appears that the melanin dispersal effects of the extracts of P. nigrum and pure piperine leading to skin darkening are mediated by cholinergic muscarinic or piperine-like receptors having similar properties.     

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.     

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.     

A proteome map of the pituitary melanotrope cell activated by black‐background adaptation of Xenopus laevis

Upon transfer of Xenopus laevis from a white to a black background, the melanotrope cells in the pituitary pars intermedia secrete α‐melanocyte‐stimulating hormone, which stimulates dispersion of melanin pigment in skin melanophores. This adaptive behavior is under the control of neurotransmitters and neuropeptides of hypothalamic origin. The α‐melanocyte‐stimulating hormone‐producing cells and their hypothalamic control system provide an interesting model to study proteins required for biosynthetic and secretory processes involved in peptide hormone production and for brain–pituitary signaling. We present a 2‐D PAGE‐based proteome map of melanotrope cells from black‐adapted animals, identifying 204 different proteins by MS analysis.     

Volume Changes of Individual Melanosomes Measured by Scanning Force Microscopy

Black pigment cells, melanophores, e.g. located in the epidermis and dermis of frogs, are large flat cells having intracellular black pigment granules, called melanosomes. Due to a large size, high optical contrast, and quick response to drugs, melanophores are attractive as biosensors as well as for model studies of intracellular processes; e.g. organelle transport and G‐protein coupled receptors. The geometry of melanosomes from African clawed toad, Xenopus laevis, has been measured using scanning force microscopy (SFM). Three‐dimensional images from SFM were used to measure height, width, and length of the melanosomes (100 from aggregated cells and 100 from dispersed cells). The volumes of melanosomes isolated from aggregated and dispersed melanophores were significantly different (P<0.05, n=200). The average ellipsoidal volume was 0.14±0.01 (aggregated) and 0.17±0.01 μm³ (dispersed), a difference of 18%. The average major diameter was 810±20 and 880±20 nm for aggregated and dispersed melanosomes, respectively. To our knowledge, this is the first time SFM has been used to study melanosomes. This may provide an alternative non‐destructive technique that may be particularly suitable for studying morphological aspects of various melanin granules.     

On the action and mechanism of withaferin-A from Withania somnifera, a novel and potent melanin dispersing agent in frog melanophores

The present work was carried out to determine the effects of lyophilized root extracts of Withania somnifera along with pure withaferin-A, on the isolated skin melanophores of frog, Rana tigerina which are disguised type of smooth muscle cells and offer excellent in vitro opportunities for studying the effects of pharmacological and pharmaceutical agents. The lyophilized extract of W. somnifera and its active ingredient withaferin-A induced powerful dose-dependent physiologically significant melanin dispersal effects in the isolated skin melanophores of R. tigerina, which were completely blocked by atropine as well as hyoscine. The per se melanin dispersal effects of lyophilized extracts of W. somnifera and its active ingredient withaferin-A got highly potentiated by neostigmine. It appears that the melanin dispersal effects of the extracts of W. somnifera and withaferin-A is mediated by cholino-muscarinic like receptors having similar properties.     

Specific features of the melanophore system in different color morphs of larvae of the common toad (Bufo bufo L.)

In a natural pond among usual black larvae of the common toad (Bufo bufo L.), a few unusual individuals of red-olive coloring were found out. In both morphs we investigated the melanophores of skin using different methods. The ESR-spectrometric analysis has shown the absence of distinctions between morphs by the amount of melanin. Analysis of total preparations of skin has shown the presence of various kinds of melanophore cells both in the derma and in the epidermis. Among typical melanophores, essentially differing cells appeared (atypical cells). In black morph tadpoles, the number of all kinds of melanophores is significantly greater than in red-olive morphs. It is shown that dark coloring is connected with a considerable number of atypical cells in the epidermis imposed on a dense layer of typical dermal melanophores with dispersed melanin.     

Pigment movements in fish melanophores: Morphological and physiological studies

Summary Cyclic adenosine monophosphate (cAMP) has been shown to cause pigment dispersion in amphibian and fish melanophores. Since pigment displacements in melanophores of Pterophyllum scalare are known to be accompanied by assembly and disassembly of microtubules, the effect of cAMP on this process was investigated. Melanophores of isolated scales were treated with cAMP in the presence of vinblastine, a potent antimicrotubular agent. During the initial phase of vinblastine action, cAMP as well as its dibutyryl derivative are capable of counteracting the inhibitory effects of vinblastine on pigment dispersion. In addition, cAMP retains the velocity of pigment dispersion at about the maximum level during 1 hour experiments. Pigment aggregation was unaffected by cAMP. Since pigment dispersion in Pterophyllum-melanophores is accompanied by assembly of microtubules, it is concluded that cAMP influences, at least in part, melanosome dispersion through facilitation of microtubule assembly.     

oca2 regulation of chromatophore differentiation and number is cell type specific in zebrafish

We characterized a zebrafish mutant that displays defects in melanin synthesis and in the differentiation of melanophores and iridophores of the skin and retinal pigment epithelium. Positional cloning and candidate gene sequencing link this mutation to a 410‐kb region on chromosome 6, containing the oculocutaneous albinism 2 (oca2) gene. Quantification of oca2 mutant melanophores shows a reduction in the number of differentiated melanophores compared with wildtype siblings. Consistent with the analysis of mouse Oca2‐deficient melanocytes, zebrafish mutant melanophores have immature melanosomes which are partially rescued following treatment with vacuolar‐type ATPase inhibitor/cytoplasmic pH modifier, bafilomycin A1. Melanophore‐specific gene expression is detected at the correct time and in anticipated locations. While oca2 zebrafish display unpigmented gaps on the head region of mutants 3 days post‐fertilization, melanoblast quantification indicates that oca2 mutants have the correct number of melanoblasts, suggesting a differentiation defect explains the reduced melanophore number. Unlike melanophores, which are reduced in number in oca2 mutants, differentiated iridophores are present at significantly higher numbers. These data suggest distinct mechanisms for oca2 in establishing differentiated chromatophore number in developing zebrafish.     

Maxadilan Activates PAC1 Receptors Expressed in Xenopus laevis Melanophores

Functional interactions between ligands and their cognate receptors can be investigated using the ability of melanophores from Xenopus laevis to disperse or aggregate their pigment granules in response to alterations in the intracellular levels of second messengers. We have examined the response of long‐term lines of cultured melanophores from X. laevis to pituitary adenylate cyclase activating peptide (PACAP), a neuropeptide with vasodilatory activity, and maxadilan, a vasodilatory peptide present in the salivary gland extracts of the blood feeding sand fly. Pituitary adenylate cyclase activating peptide increased the intracellular levels of cyclic adenosine monophosphate (cAMP) and induced pigment dispersion in the pigment cells, confirming that melanophores express an endogenous PACAP receptor. Maxadilan did not induce a response in non‐transfected melanophores. When the melanophores were transfected with complementary DNA (cDNA) from the three different members of the PACAP receptor family, maxadilan induced pigment dispersion specifically and cAMP accumulation in melanophores transfected with the cDNA for PAC1 receptors but not VPAC1 or VPAC2 receptors. A melanophore line was generated that stably expresses the PAC1 receptor.     

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.     

Studies of pigment transfer between Xenopus laevis melanophores and fibroblasts in vitro and in vivo1

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 α‐melanocyte‐stimulating hormone (α‐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.     

The Fugu tyrp1 promoter directs specific GFP expression in zebrafish: tools to study the RPE and the neural crest‐derived melanophores

In vertebrates, pigment cells account for a small percentage of the total cell population and they intermingle with other cell types. This makes it difficult to isolate them for analyzes of their functions in the context of development. To alleviate such difficulty, we generated two stable transgenic zebrafish lines (pt101 and pt102) that express green fluorescent protein (GFP) in melanophores under the control of the 1 kb Fugu tyrp1 promoter. In pt101, GFP is expressed in both retinal pigment epithelium (RPE) cells and the neural crest‐derived melanophores (NCDM), whereas in pt102, GFP is predominately expressed in the NCDM. Our results indicate that the Fugu tyrp1 promoter can direct transgene expression in a cell‐type‐specific manner in zebrafish. In addition, our findings provide evidence supporting differential regulations of melanin‐synthesizing genes in RPE cells and the NCDM in zebrafish. Utilizing the varying GFP expression levels in these fish, we have isolated melanophores via flow cytometry and revealed the capability of sorting the NCDM from RPE cells as well. Thus, these transgenic lines are useful tools to study melanophores in zebrafish.     

Unusual development of light-reflecting pigment cells in intact and regenerating tail in the periodic albino mutant of Xenopus laevis

Unusual light-reflecting pigment cells, “white pigment cells”, specifically appear in the periodic albino mutant (a ^p /a ^p ) of Xenopus laevis and localize in the same place where melanophores normally differentiate in the wild-type. The mechanism responsible for the development of unusual pigment cells is unclear. In this study, white pigment cells in the periodic albino were compared with melanophores in the wild-type, using a cell culture system and a tail-regenerating system. Observations of both intact and cultured cells demonstrate that white pigment cells are unique in (1) showing characteristics of melanophore precursors at various stages of development, (2) accumulating reflecting platelets characteristic of iridophores, and (3) exhibiting pigment dispersion in response to α-melanocyte stimulating hormone (α-MSH) in the same way that melanophores do. When a tadpole tail is amputated, a functionally competent new tail is regenerated. White pigment cells appear in the mutant regenerating tail, whereas melanophores differentiate in the wild-type regenerating tail. White pigment cells in the mutant regenerating tail are essentially similar to melanophores in the wild-type regenerating tail with respect to their localization, number, and response to α-MSH. In addition to white pigment cells, iridophores which are never present in the intact tadpole tail appear specifically in the somites near the amputation level in the mutant regenerating tail. Iridophores are distinct from white pigment cells in size, shape, blue light-induced fluorescence, and response to α-MSH. These findings strongly suggest that white pigment cells in the mutant arise from melanophore precursors and accumulate reflecting platelets characteristic of iridophores.     

Cell Division: Pressure-induced reversal of the antimeiotic effects of heavy water in the oocytes of the starfish,Asterias forbesi

In dermal melanocytes of Rana pipiens, colchicine is known to produce a gradual, dosage-dependent dispersion of melanin granules, irreversible over several hours. This effect is potentiated by a number of chemical agents that normally produce a reversible dispersion of granules. In the present study we examined the effect of high hydrostatic pressure on changes induced in melanocytes by colchicine. In Ringer's solution, samples of skin from a single frog were incubated for 30 minutes at room temperature with or without colchicine, 9 × 10^?5 M. Then two samples, one of which had been pretreated with colchicine, were successively subjected to 12,000 psi for one hour at 25 to 26°C. The degree of dispersion of melanin granules in melanocytes was observed before, during and after the period of pressure. In frog skin pretreated with colchicine, the usually gradual, irreversible dispersion of melanin granules in melanocytes was potentiated. Since high pressure is known to produce solational changes in protoplasm, such changes may accompany dispersion of melanin granules in melanocytes. If this be so, then sol-gel equilibria may be important in the action of dispersing and aggregating agents, many of which are hormones and other physiologically active agents. Finally, the present study supports the hypothesis that colchicine shifts protoplasmic sol-gel equilibria toward a less gelated condition.