Masazumi Sugimoto

Bhc‐cNMPs as either Water‐Soluble or Membrane‐Permeant Photoreleasable Cyclic Nucleotides for both One‐ and Two‐Photon Excitation

Cyclic nucleoside monophosphates (cNMPs) play key roles in many cellular regulatory processes, such as growth, differentiation, motility, and gene expression. Caged derivatives that can be activated by irradiation could be powerful tools for studying such diverse functions of intracellular second messengers, since the spatiotemporal dynamics of these molecules can be controlled by irradiation with appropriately focused light. Here we report the synthesis, photochemistry, and biological testing of 6‐bromo‐7‐hydroxycoumarin‐4‐ylmethyl esters of cNMP (Bhc‐cNMP) and their acetyl derivatives (Bhc‐cNMP/Ac) as new caged second messengers. Irradiation of Bhc‐cNMPs quantitatively produced the parent cNMPs with one‐photon uncaging efficiencies (Φε) of up to one order of magnitude better than those of 2‐nitrophenethyl (NPE) cNMPs. In addition, two‐photon induced photochemical release of cNMP from Bhc‐cNMPs (7 and 8) can be observed with the two‐photon uncaging action cross‐sections (δu) of up to 2.28 GM (1 GM=10−50 cm4 s photon−1), which is the largest value among those of the reported Bhc‐caged compounds. The wavelength dependence of the δu values of 7 revealed that the peak wavelength was twice that of the one‐photon absorption maximum. Bhc‐cNMPs showed practically useful water solubility (nearly 500 μM), whereas 7‐acetylated derivatives (Bhc‐cNMPs/Ac) were expected to have a certain membrane permeability. Their advantages were demonstrated in two types of biological systems: the opening of cAMP‐mediated transduction channels in newt olfactory receptor cells and cAMP‐mediated motility responses in epidermal melanophores in scales from medaka fish. Both examples showed that Bhc and Bhc/Ac caged compounds have great potential for use in many cell biological applications.

Ultrastructure of the Dermal Chromatophores in a Lizard (Scincidae: Plestiodon latiscutatus) with Conspicuous Body and Tail Coloration

Abstract Microscopic observation of the skin of Plestiodon lizards, which have body stripes and blue tail coloration, identified epidermal melanophores and three types of dermal chromatophores: xanthophores, iridophores, and melanophores. There was a vertical combination of these pigment cells, with xanthophores in the uppermost layer, iridophores in the intermediate layer, and melanophores in the basal layer, which varied according to the skin coloration. Skin with yellowish-white or brown coloration had an identical vertical order of xanthophores, iridophores, and melanophores, but yellowish-white skin had a thicker layer of iridophores and a thinner layer of melanophores than did brown skin. The thickness of the iridophore layer was proportional to the number of reflecting platelets within each iridophore. Skin showing green coloration also had three layers of dermal chromatophores, but the vertical order of xanthophores and iridophores was frequently reversed. Skin showing blue color had iridophores above the melanophores. In addition, the thickness of reflecting platelets in the blue tail was less than in yellowish-white or brown areas of the body. Skin with black coloration had only melanophores.

Apoptosis in skin pigment cells of the medaka, Oryzias latipes (Teleostei), during long-term chromatic adaptation: the role of sympathetic innervation

Abstract. Many teleost fish can adapt their body color to a background color by changing the morphology and density of their skin pigment cells. Melanophore density in fish skin decreases during long-term adaptation to a white background. Although cell death, especially apoptosis, is thought to be involved in these morphological changes, there are no data clearly supporting this mechanism. Using medaka fish, Oryzias latipes, we observed that, on a white background, melanophore size was reduced first and this was followed by a decrease in melanophore density caused by gradual cell death. The process of cell death included loss of cell activity, cell fragmentation, phagocytosis of the fragments, and clearance via the epidermis. Apoptosis was assessed by the appearance of phosphatidylserine on the cell surface of melanophores that had lost motile activity, and DNA fragments involved in cell fragmentation were detected by the TUNEL (TDT-mediated dUTP-biotin nick end-labeling) assay. However, when chemically denervated fish were used, although melanophore size was reduced as expected, cell death was suppressed even on a white background. In skin tissue culture, apoptosis in melanophores was stimulated significantly by norepinephrine, but not by melanin-concentrating hormone. These results indicate that melanophore density decreases by apoptosis, and suggest that sympathetic innervation has an important role in the regulation of apoptosis in melanophores. In analogous fashion, leucophores showed a significant decrease in density with an increase of cell death on a black background. We suggest that apoptosis regulates the balance of pigment cells in the skin of medaka fish to adapt their body color to a particular background.

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 microM) induced pigment aggregation, and the aggregated state was maintained in the presence of MCH. However, at higher MCH concentrations (such as 1 and 10 microM), 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 Ca(2+) ions. By contrast, medaka melanophores responded to MCH only by pigment aggregation, even at the highest concentration employed (10 microM). Since it is known that medaka melanophores possess specific receptors for alpha-melanophore-stimulating hormone (alpha-MSH), the possibility that interaction between MSH receptors and MCH at high doses in the presence of Ca(2+) 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 microM 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.

Integumental reddish‐violet coloration owing to novel dichromatic chromatophores in the teleost fish, Pseudochromis diadema

In the reddish‐violet parts of the skin of the diadema pseudochromis Pseudochromis diadema, we found novel dichromatic chromatophores with a reddish pigment and reflecting platelets. We named these novel cells ‘erythro‐iridophores’. In standard physiological solution, erythro‐iridophores displayed two hues, red and dark violet when viewed with an optical microscope under ordinary transmission light and epi‐illumination optics, respectively. Under transmission electron microscopy, however, we observed no typical red chromatosomes, i.e., erythrosomes, in the cytoplasm. High‐performance thin‐layer chromatography (HPTLC) analysis of the pigment eluted from the erythro‐iridophores indicated that carotenoid is the main pigment generating the reddish color. Furthermore, when the irrigating medium was a K+‐rich saline solution, the color reflected from the erythro‐iridophores changed from dark violet to sky blue, but the red coloration remained. The motile activities of the erythro‐iridophores may participate in the changes in the reddish‐violet shades of the pseudochromis fish.

The involvement of calmodulin in motile activities of fish chromatophores

Abstract 1. Effects of W-7 and W-5, calmodulin antagonists, on the pigment aggregation within melanophores and coloring response of iridophores were examined in the blue damselfish, Chrysiptera cyanea . 2. W-7 was found to antagonize norepinephrine-induced responses of the chromatophores, whereas W-5 had only a slight effect on inhibition of the responses. 3. H-7, a specific antagonist of protein kinase C, did not arrest the responses of melanophores and iridophores at all. 4. The chromatophores responded normally to norepinephrine in Ca 2+ , Mg 2+ -free saline solution. 5. These results indicate that it is a Ca 2+ /calmodulin-regulated enzyme and not protein kinase C that is involved in motile activities of fish chromatophores. Ca 2+ may be supplied from an intracellular store.

Pigment cell mechanisms underlying dorsal color-pattern polymorphism in the Japanese four-lined snake.

To provide histological foundation for studying the genetic mechanisms of color‐pattern polymorphisms, we examined light reflectance profiles and cellular architectures of pigment cells that produced striped, nonstriped, and melanistic color patterns in the snake Elaphe quadrivirgata. Both, striped and nonstriped morphs, possessed the same set of epidermal melanophores and three types of dermal pigment cells (yellow xanthophores, iridescent iridophores, and black melanophores), but spatial variations in the densities of epidermal and dermal melanophores produced individual variations in stripe vividness. The densities of epidermal and dermal melanophores were two or three times higher in the dark‐brown‐stripe region than in the yellow background in the striped morph. However, the densities of epidermal and dermal melanophores between the striped and background regions were similar in the nonstriped morph. The melanistic morph had only epidermal and dermal melanophores and neither xanthophores nor iridophores were detected. Ghost stripes in the shed skin of some melanistic morphs suggested that stripe pattern formation and melanism were controlled independently. We proposed complete‐ and incomplete‐dominance heredity models for the stripe‐melanistic variation and striped, pale‐striped, and nonstriped polymorphisms, respectively, according to the differences in pigment‐cell composition and its spatial architecture. J. Morphol. 274:1353–1364, 2013.

Novel Dichromatic Chromatophores in the Integument of the Mandarin Fish Synchiropus splendidus

The bluish coloration of the integument in teleosts is usually generated by light-reflecting chromatophores, called iridophores, that contain very thin light-reflecting platelets in the cytoplasm. We previously reported, however, that novel light-absorbing chromatophores named “cyanophores” display a bluish hue in the integument of the mandarin fish Synchiropus splendidus and the psychedelic fish S. picturatus. In the present report, we found novel light-absorbing dichromatic chromatophores in limited regions of the integument of the mandarin fish. Light microscopy under transmission optics revealed that the novel dichromatic chromatophores in the dermis around the margins of the bluish regions display both bluish and reddish hues. In the cytoplasm, both erythrosomes and cyanosomes were observed under transmission electron microscopy. The morphological features of the cyanosomes contained in the novel dichromatic chromatophores did not differ from those in the cyanophores in blue regions. Pharmacological treatment with norepinephrine induced aggregation of both the erythrosomes and cyanosomes of the novel dichromatic chromatophores, suggesting that the dichromatic chromatophores possess motile activity, the same as other lightabsorbing chromatophores, which contributes to the role of their color changes observed in response to various environmental signals. The variety of colors, patterns, and spectacular color changes the animals can exhibit is a subject of much interest. The generation of color and color changes is important to many species because it provides protection and assists in survival in their habitats. The delicate changes in hues and patterns can also be used to communicate with conspecifics.

REGULATION OF MELANOPHORE RESPONSIVENESS IN THE BACKGROUND-ADAPTED MEDAKA,ORYZIAS LATIPES : CHANGE IN THE INTRACELLULAR SIGNALING SYSTEM

The responsiveness of melanophores of the medaka fish (wild type, Oryzias latipes) to a neurotransmitter and hormones is changed differentially after long-term adaptation to a black or white background. In the present study, we further examined whether this phenomenon involved some change in the intracellular signaling system. Using a permeabilized melanophore model, in which pigment granules could be dispersed by exogenously applied cAMP, the requirement of cAMP for pigment-dispersing reaction was revealed to be higher in melanophores of fish adapted to a black background (B cells) than in those of white background-adapted fish (W cells). Specific inhibitors of cAMP-dependent protein kinase (PKA) and cyclic nucleotide phosphodiesterase did not reduce the difference in the pigment dispersion level between B and W cells. A similar result was obtained with the free catalytic subunit of PKA. In contrast, the inhibition of protein phosphatase activity by okadaic acid diminished the difference in the responsiveness between B and W cells. These results suggest that the activity of protein phosphatase in B cell is higher than that in W cells, and that the change in the melanophore responsiveness by long-term chromatic adaptation to a background involves the change in the enzyme activity in the intracellular signaling system.

Effects of Androgens on the Development of Nuptial Coloration and Chromatophores in the Bitterling Rhodeus ocellatus ocellatus

Sexually mature male bitterlings, Rhodeus ocellatus ocellatus, exhibit distinct nuptial color, whereas females maintain a body color similar to that of juveniles. In the present study, body color and chromatophores were compared between male and female bitterlings, and the effects of androgens on body color and chromatophore densities were examined in females to clarify the role of androgen in the development of nuptial coloration and chromatophores. Males showed green, blue, and red color in specific regions of their skin and red color on the dorsal and caudal fins; females showed a subdued silver body color. For chromatophores, small greenish-type iridophores were observed in the green color region in the skin of males, whereas females had large spindle-shaped silvery-type iridophores in corresponding regions. Many erythrophores were observed in males in blue and red color regions in the skin and red color regions in the fins, but females possessed xanthophores in corresponding regions. The melanophore density of the skin was not different between males and females, but the distribution of melanophores in the fins was different between them. Treatment with 11-ketotestosterone or methyltestosterone induced male-type nuptial coloration in the female skin and fins. The distribution of chromatophores in androgen-treated females was similar to that in sexually mature males: an increase in the number of greenish-type iridophores and erythrophores was also observed in the skin. These results indicate that androgen induces male-type nuptial coloration in the bitterling and that the responses of chromatophores to androgen differ with the type and distribution site of the chromatophores.