Maria Aparecida Visconti

Physiological Color Change in the Bullfrog, Rana Catesbeiana

Adults of Rana catesbeiana maintained for 4 days in 12:12 light/dark regimen exhibited a rhythmic color change of 24 hr. Under constant light, however, the rhythm disappeared, and the reflectance values gradually became greater, that is the animals became lighter. Under constant darkness, the rhythm was also abolished, but the animals tended to a darker color. On black background the skin darkening proceeded at a faster rate as compared to the skin lightening of animals adapting to a white background. The difference in color change rate suggests that the darkening responses are probably mediated by an increase in a circulating hormone, whereas skin lightening probably results from the serum level decrease of the same hormone. Most certainly, this hormone is alpha-MSH, as the in vitro assays demonstrated its high potency as a full darkening agonist (EC50 = 9 x 10(-10) M). Prolactin (EC50 = 7.7 x 10(-8) M) and endothelins 2 (EC50 = 1.3 x 10(-6) M) and 3 (EC50 = 4.8 x 10(-7) M) were also full agonists, but 100- to 1000-fold less potent than alpha-MSH. Isoproterenol, in the absence or presence of dibenamine, and endothelin-1 also elicited darkening responses in a dose-related manner, but reaching only 23% and 35% of the maximal darkening, respectively. Isoproterenol darkening effect was completely blocked by propranolol, confirming its action through beta-adrenoceptors. These results, taken together with the lack of lightening activity of norepinephrine on alpha-MSH-darkened skins, suggest that R. catesbeiana melanophores do not possess very active beta-adrenoceptors and lack alpha-adrenoceptors. On the other hand, the lightening agonist melatonin elicited only half-maximal dose-dependent reversal of MSH-induced darkening. Our results suggest that the chromatic rhythm is not endogenous, and most likely is determined by the light/dark cycle effect on alpha-MSH secretion.

Melatonin biological activity and binding sites in human melanoma cells

Abstract:  The effects of melatonin, N‐acetylserotonin and serotonin on the growth and tyrosinase activity of SK‐Mel 23 and SK‐Mel 28 human melanoma cell lines were investigated. Binding assays were also performed to establish the nature of the binding site. SK‐Mel 28 cells were responsive to melatonin and its precursors, exhibiting a decrease in growth and an increase in tyrosinase activity after a 72 hr treatment. N‐acetylserotonin was as potent as melatonin, the minimal effective concentration (MEC, which is defined as the smallest concentration that elicits a measurable biological response, significantly different from control) being 10−8 m. Serotonin was the least potent (MEC = 10−6 m). Both melatonin antagonists, prazosin and luzindole, exhibited no effect per se and reversed both responses to melatonin. SK‐Mel 23 cells, however, showed no significant responses to the indoleamines. Competition binding assays in SK‐Mel 28 cells demonstrated the presence of binding sites to 2‐[125 I]‐iodomelatonin, which was displaced by the unlabelled hormone, by both antagonists, and by N‐acetylserotonin. The curve adjustment of the displacement values with melatonin suggests the existence of two binding sites, with the following Ki values: 1.0 × 10−10 m and 6.5 × 10−6 m. Ki values for acetylserotonin, prazosin and luzindole were, respectively, 3.8 × 10−8 m, 1.2 × 10−8 m, and 8.3 × 10−6 m. Surprisingly, in SK‐Mel 23 cells, melatonin and luzindole were able to compete with the radioligand, with Ki values of 3.1 × 10−8 and 2.4 × 10−8 m, respectively. Our data suggest that SK‐Mel 28 cells probably possess high affinity binding sites to melatonin and, in addition, MT3 low affinity binding sites, because N‐acetylserotonin was as effective as the native hormone, and prazosin effectively blocked the actions of melatonin. Both sites are functional as demonstrated by the blockade promoted by both luzindole and prazosin on the proliferative and melanogenic responses. Although growth and tyrosinase activity of SK‐Mel 23 cells were not affected by melatonin or its precursors, this cell line possesses high affinity binding sites, which may be non‐functional, or trigger responses other than the ones herein investigated.

Differential structural requirements for the MSH and MCH activities of melanin concentrating hormone

H-Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val-OH , melanin concentrating hormone (MCH), exhibits both melanin granule concentrating and dispersing (MSH-like) activities. Fragment analogues of MCH were synthesized as described herein and the melanotropic activities of the peptides were determined. In the frog (Rana pipiens) and lizard (Anolis carolinensis) skin bioassays, the 5-17 and 5-14 fragments of MCH were inactive (at concentrations of 10(-5)M or less), whereas the 1-14 sequence exhibited minimal (about 10%) MSH-like activity compared to MCH, which, as reported previously, was about 600 times less active than alpha-MSH. In the teleost (fish) skin bioassay, the MCH5-17 analogue was equipotent to MCH, whereas the 1-14 analogue was 10-30 times and the cyclic N- and C- terminal truncated analogue, MCH5-14, was about 300 times less active than MCH. These results suggest that the N-terminal sequence is particularly critical to MSH-like activity in the tetrapod species studied, whereas other structural regions of MCH, particularly in the C-terminal, are more related to MCH activity in teleosts.

Comparative EPR and fluorescence conformational studies of fully active spin-labeled melanotropic peptides

Similar to melanocyte stimulating hormone (α‐MSH), its potent and long‐acting analogue, [Nle4, D‐Phe7]α‐MSH, when labeled with the paramagnetic amino acid probe 2,2,6,6‐tetramethylpiperidine‐N‐oxyl‐4‐amino‐4‐carboxylic acid (Toac), maintains its full biological potency, thus validating any comparative structural investigations between the two labeled peptides. Correlation times, calculated from the electron paramagnetic resonance signal of Toac bound to the peptides, and Toac–Trp distances, estimated from the Toac fluorescence quenching of the Trp residue present in the peptides, indicate a more rigid and folded structure for the potent analogue as compared to the hormone, in aqueous medium.

Adrenoceptors in normal and malignant human melanocytes.

Catecholamines are potent melanophore agonists in poikilothermic vertebrates, but little is known about their action on mammalian melanocytes. Stimulation of α1-adrenoceptors by norepinephrine does not affect melanogenesis or dendricity [1], whereas metaproterenol increases cell proliferation, tyrosinase activity and TRP-1 expression [2] in human melanocytes. Catecholamine levels in plasma and urine have been correlated with the occurrence of vitiligo [3–6], and with the control of calcium uptake in human keratinocytes and melanoma cells [7]. There have been few reports on the pharmacologic characterization of adrenoceptors in epidermal cells. β2Adrenoceptors are present in human keratinocytes [8, 9], A-375 melanoma cells [10] and B16 murine melanoma cells [11, 12]. It has been reported that human melanocytes lack adrenoceptors unless the cells are previously treated with epinephrine or norepinephrine for 24 h [1]. The aim of this work was to determine the adrenoceptor subtypes in normal human melanocytes and in human melanoma SK-Mel 23 cells using competitive binding assays. SK-Mel 23 melanoma cells (a kind gift from Dr. A. Albino, Sloan Kettering Memorial Hospital) were kept in Ham’s F-10 medium supplemented with 10% fetal calf serum (Cultilab, Brazil), 2 g/l NaHCO3 and 15 mM Hepes, pH 7.4, in the absence of antibiotics, at 37°C in an atmosphere containing 5% CO2. Human melanocytes were obtained from neonatal foreskins and were maintained in MCDB-153 medium supplemented with 20% fetal calf serum, 1 μg/ml TPA, 2 μg/ml cholera toxin and 2% penicillin/streptomycin (Sigma Chemical Co.) at 37°C in an atmosphere containing 5% CO2. Fibroblasts were eliminated from melanocyte cultures by treatment with 100 μg/ml geneticin (Sigma) for 48 h. Cholera toxin and TPA were removed from the medium 24 h before the assays. Cells were seeded in 12-well plates, and after 24–48 h they were rinsed with Earle’s solution (2 × 1 ml) [13], and preincubated with increasing concentrations of cold ligAna Cristina Scarparo · Maria Aparecida Visconti · Alice Reis de Oliveira · Ana Maria de Lauro Castrucci

Melanopsin in chicken melanocytes and retina

Abstract The vertebrate pigment cell, with the exception of mammals and birds, is able to provide the animal with rapid colour changes, which involve dispersion and aggregation of pigment granules in response to hormonal and neuronal agents, and in some cases as a direct response to light. The search for the mechanisms through which Xenopus leavis melanophores respond to light led to the discovery of a new photopigment, melanopsin, with a different spectral sensitivity to that of rhodopsin. This photopigment was also found in mammalian retinal ganglion cells that project to the suprachiasmatic nucleus and other non-visual retinorecipient areas. Herein we demonstrate (by RT-PCR, cloning and sequencing) for the first time that chick melanocytes express melanopsin, and confirmed the presence of the protein by immunocytochemistry. In the chicken retina, we revealed by immunocytochemistry that ganglion cells express melanopsin, but the highest density of immunopositive cells was found in the inner nuclear layer. Quantitative PCR showed that the retina of animals kept in 6 h light: 18 h dark possessed three-fold higher melanopsin mRNA content than animals kept in longer photoperiod, thus demonstrating that light modulates melanopsin expression in chickens.

Photoperiod and Testosterone Modulate Growth and Melanogenesis of S91 Murine Melanoma

In vivo and in vitro assays were performed with S91 murine melanoma cells aiming to investigate the effects of testosterone and photoperiod on tumor growth and melanogenesis (tyrosinase activity). In vivo assays were performed by inducing melanoma tumors in castrated mice receiving increasing concentrations of testosterone and submitted to varying photoperiod regimens. The results demonstrated that the increase of melanin content was higher in animals submitted to the longest days, thus demonstrating the importance of photoperiod length in melanin synthesis. Increase in tumor growth and protein content was observed in testosterone-treated animals submitted to 12L:12D; in testosterone-treated animals submitted to 4L:20D and 20L:4D tumor growth was significantly smaller. In S91 cultured cells, testosterone increased cell proliferation and reduced tyrosinase activity in a dose-dependent manner. Radioactive binding assays demonstrated that the hormone was acting through low affinity testosterone receptors, since the presence of aromatase inhibitor did not affect the binding assay in a statistically significant way, and all the in vitro experiments were performed in the presence of the inhibitor. Our in vivo data added to the in vitro results corroborate the hypothesis that S91 melanoma cells directly respond to testosterone and that this effect is modulated by light.

The expression of melanopsin and clock genes in Xenopus laevis melanophores and their modulation by melatonin

Vertebrates have a central clock and also several peripheral clocks. Light responses might result from the integration of light signals by these clocks. The dermal melanophores of Xenopus laevis have a photoreceptor molecule denominated melanopsin (OPN4x). The mechanisms of the circadian clock involve positive and negative feedback. We hypothesize that these dermal melanophores also present peripheral clock characteristics. Using quantitative PCR, we analyzed the pattern of temporal expression of Opn4x and the clock genes Per1, Per2, Bmal1, and Clock in these cells subjected to a 14-h light:10-h dark (14L:10D) regime or constant darkness (DD). Also, in view of the physiological role of melatonin in the dermal melanophores of X. laevis, we determined whether melatonin modulates the expression of these clock genes. These genes show a time-dependent expression pattern when these cells are exposed to 14L:10D, which differs from the pattern observed under DD. Cells kept in DD for 5 days exhibited overall increased mRNA expression for Opn4x and Clock, and a lower expression for Per1, Per2, and Bmal1. When the cells were kept in DD for 5 days and treated with melatonin for 1 h, 24 h before extraction, the mRNA levels tended to decrease for Opn4x and Clock, did not change for Bmal1, and increased for Per1 and Per2 at different Zeitgeber times (ZT). Although these data are limited to one-day data collection, and therefore preliminary, we suggest that the dermal melanophores of X. laevis might have some characteristics of a peripheral clock, and that melatonin modulates, to a certain extent, melanopsin and clock gene expression.

Ionic requirements for PCH-induced pigment aggregation in the freshwater shrimp, Macrobrachium potiuna, erythrophores☆

Abstract The effects of either cation removal or ionic channel blockade were determined on the dose-response curve (DRC) to PCH (pigment-concentrating hormone) in Macrobrachium potiuna erythrophores. In sodium-, potassium- and calcium-free salines, the pigment-aggregating responses to PCH were depressed; in the former condition, maximal aggregation was not achieved and the slope of the regression curve determined from the DRC was significantly different from control. Tetrodotoxin, verapamil or tetraethylammonium (TEA) treatments also diminished the pigment-aggregating responses to PCH, and the slopes of the regression curves were different from control in the presence of 10 −6 M verapamil or 10 −6 M TEA. Interestingly, the DRC determined in the absence of both sodium and calcium ions was not significantly different from control. When verapamil was applied in sodium-free conditions, maximal aggregation was prevented. The erythrophore resting membrane potential ranged from −62 mV to −78 mV and did not vary during PCH-induced pigment aggregation as compared to the control. Our results suggest that transient modifications of potassium equilibrium potential may interfere with PCH signal transduction, revealing a more relevant role of potassium in the process, and that a sodium influx and an intracellular calcium mobilization are necessary to maintain a cytosolic balance between the ions for normality of PCH-induced responses.

Mechanisms of pigmentation loss in subterranean fishes

Organismos exclusivamente subterrâneos (troglobios) usualmente exibem, entre as apomorfias relacionadas a evolucao em isolamento nesse ambiente (troglomorfismos), a reducao, ate perda total, das estruturas visuais e da pigmentacao melânica. Os mecanismos de regressao ocular em troglobios tem sido intensivamente estudados, sobretudo em peixes como os lambaris cegos mexicanos do genero Astyanax, e salamandras como Proteus anguinus. Por outro lado, poucos sao os trabalhos abordando a perda da pigmentacao nesses organismos. A ictiofauna subterrânea brasileira destaca-se nao so pela riqueza de especies (23 conhecidas ate o momento) como tambem pelas diferencas no seu grau de troglomorfismo, sem uma correlacao taxonomica. O presente estudo abordou as especies brasileiras totalmente desprovidas de qualquer traco de pigmentacao melânica: o caraciforme Stygichthys typhlops (Characidae) e os siluriformes Ancistrus formoso (Loricariidae), Rhamdiopsis sp.1 (Heptapteridae; cavernas da Chapada Diamantina, Bahia) e Rhamdiopsis sp. 2 (caverna de Campo Formoso, Bahia). Com a finalidade de investigar se essa despigmentacao e resultado de bloqueio em algum passo da cadeia de sintese de melanina, foram feitos testes in vitro, utilizando-se fragmentos da nadadeira caudal extraidos de exemplares mantidos vivos, de reacao a administracao de L-DOPA. Com excecao dos de Rhamdiopsis sp. 2, os exemplares estudados revelaram-se DOPA(+), i.e., houve producao de melanina apos a administracao de L-DOPA, o que indica que sua despigmentacao e devida a uma disfuncao da tirosinase, enzima responsavel pela transformacao de tirosina em L-DOPA nos melanoforos, os quais, portanto, ainda existem nesses peixes. Ja os exemplares Rhamdiopsis sp. 2, assim como o terco conspicuamente despigmentado da populacao de Trichomycterus itacarambiensis, especie estudada anteriormente sob esse aspecto, sao DOPA(-), seja porque o bloqueio na sintese de melanina ocorre em um passo a jusante da producao de L-DOPA, aparentemente o caso de T. itacarambiensis (heranca monogenica em vista da descontinuidade fenotipica), ou porque houve perda total dos melanoforos. A perda fisiologica da capacidade de sintetizar melanina, aparentemente causada por diferentes mecanismos nas populacoes DOPA(+) e naquelas DOPA(-), pode co-existir com a reducao no numero e tamanho dos melanoforos, como observado nos dois tercos pigmentados de T. itacarambiensis, provavelmente determinada por heranca poligenica, que produz uma distribuicao continua de fenotipos.