RESUMO
The non-visual opsin, melanopsin, expressed in the mammalian retina, is considered a circadian photopigment because it is responsible to entrain the endogenous biological clock. This photopigment is also present in the melanophores of Xenopus laevis, where it was first described, but its role in these cells is not fully understood. X. laevis melanophores respond to light with melanin granule dispersion, the maximal response being achieved at the wavelength of melanopsin maximal excitation. Pigment dispersion can also be triggered by endothelin-3 (ET-3). Here we show that melanin translocation is greater when a blue light pulse was applied in the presence of ET-3. In addition, we demonstrated that mRNA levels of the melanopsins Opn4x and Opn4m exhibit temporal variation in melanophores under light/dark (LD) cycles or constant darkness, suggesting that this variation is clock-driven. Moreover, under LD cycles the oscillations of both melanopsins show a circadian profile suggesting a role for these opsins in the photoentrainment mechanism. Blue-light pulse decreased Opn4x expression, but had no effect on Opn4m. ET-3 abolishes the circadian rhythm of expression of both opsins; in addition the hormone increases Opn4x expression in a dose-, circadian time- and light-dependent way. ET-3 also increases the expression of its own receptor, in a dose-dependent manner. The variation of melanopsin levels may represent an adaptive mechanism to ensure greater melanophore sensitivity in response to environmental light conditions with ideal magnitude in terms of melanin granule dispersion, and consequently color change.
Assuntos
Ritmo Circadiano/efeitos dos fármacos , Ritmo Circadiano/fisiologia , Endotelinas/farmacologia , Opsinas de Bastonetes/metabolismo , Animais , Ritmo Circadiano/efeitos da radiação , Luz , Melaninas/metabolismo , Melanóforos/citologia , Melanóforos/efeitos dos fármacos , Melanóforos/metabolismo , Melanóforos/efeitos da radiação , Fotoperíodo , Receptores de Endotelina/genética , Receptores de Endotelina/metabolismo , Xenopus laevisRESUMO
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.
Assuntos
Animais , Proteínas CLOCK/metabolismo , Melanóforos/fisiologia , Melatonina/farmacologia , Opsinas de Bastonetes/metabolismo , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Proteínas CLOCK/genética , Relógios Circadianos/efeitos dos fármacos , Relógios Circadianos/genética , Relógios Circadianos/fisiologia , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Melanóforos/efeitos dos fármacos , Reação em Cadeia da Polimerase , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , RNA Mensageiro , Opsinas de Bastonetes/efeitos dos fármacos , Xenopus laevis , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismoRESUMO
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.
Assuntos
Proteínas CLOCK/metabolismo , Melanóforos/fisiologia , Melatonina/farmacologia , Opsinas de Bastonetes/metabolismo , Fatores de Transcrição ARNTL/genética , Fatores de Transcrição ARNTL/metabolismo , Animais , Proteínas CLOCK/genética , Relógios Circadianos/efeitos dos fármacos , Relógios Circadianos/genética , Relógios Circadianos/fisiologia , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Melanóforos/efeitos dos fármacos , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro , Opsinas de Bastonetes/efeitos dos fármacos , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
The ability of UV radiation to stimulate color change in vertebrates is well known; however, the signaling pathway involved is not fully explained. Since nitric oxide (NO) is among the candidates for this role, in this study the participation of NO signaling in the pigment migration induced by UV radiation in melanophores of the crab Chasmagnathus granulatus was investigated. When the NO donor, SIN-1, was incubated with pieces of epidermis, there was an induction of a dose-dependent pigment dispersion (in vitro assays). When male adults were exposed to different doses of UVA and UVB, N(G)-nitro-l-arginine-methyl-ester, an NO synthase (NOS) blocker produced a decrease of the pigment dispersion induced by UV (in vivo assays). However, in similar assays, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, an NO scavenger, decreased only the pigment dispersion induced by UVA. Interestingly, buthionine sulfoximine did not produce any change in pigment dispersion induced by UVA (in vivo assays) and SIN-1 (in vitro assays). Our results using NADPH-diaphorase histochemistry and immunocytochemistry against nNOS indicated the production of NO by epidermal cells. In conclusion, we suggest that NO is a key molecule for the induction of pigment dispersion in the melanophores of Chasmagnthus granulatus, and also that NOS activation is a fundamental step for this process.
Assuntos
Braquiúros/efeitos da radiação , Melanóforos/efeitos da radiação , Óxido Nítrico/fisiologia , Pigmentação/efeitos da radiação , Raios Ultravioleta , Animais , Braquiúros/efeitos dos fármacos , Braquiúros/fisiologia , Brasil , Relação Dose-Resposta a Droga , Masculino , Melanóforos/efeitos dos fármacos , Melanóforos/metabolismo , Molsidomina/análogos & derivados , Molsidomina/farmacologia , Doadores de Óxido Nítrico/farmacologia , Pigmentação/efeitos dos fármacosRESUMO
The biological activity of endothelins (ETs) in non-innervated Synbranchus marmoratus melanophores was demonstrated. These peptides induced a dose-dependent pigment aggregation (lightening skin) in these cells. However, they presented EC50's (effective concentration required to produce 50% of response) 26, 106 and 35 times higher than, respectively, the melanin concentrating hormone (MCH) EC50, and exhibited a characteristic temporal and dose-dependent autodessensibilization of the aggregative effect on the melanophores of this fish. The receptor characterization suggested the presence of the ET(B) subtype, since BQ-788 (selective antagonist of ET(B)) but not BQ-485 (selective antagonist of ET(A)) blocked the aggregative effect of the hormones. Confirming these data, sarafotoxin (SRTX) S6c, a toxin selective for ET(B), induced maximal aggregation of pigment granules. S6c presented an EC50 6.8 times higher than the MCH EC50, and 3.9, 15.6 and 5.1 times lower than the EC50's ETs, respectively. The melanotropic effect of SRTX S6b and vasoactive intestinal contractor (VIC) were demonstrated for the first time in this work. SRTX S6b induced a dose-dependent pigment aggregation and presented an EC50 2.54 and 17.2 times higher than the S6c and MCH EC50's, respectively. Compared to the ETs it was 1.53, 6.19 and 2.03 times lower, respectively.
Assuntos
Endotelinas/metabolismo , Proteínas de Peixes/metabolismo , Melanóforos/metabolismo , Smegmamorpha/metabolismo , Animais , Cor , Endotelinas/classificação , Melanóforos/efeitos dos fármacos , Fatores de Tempo , Vasoconstritores/farmacologia , Venenos de Víboras/farmacologiaRESUMO
Melatonin is a weak dose-independent lightening agonist in fish skin, a moderate dose-dependent lightening agonist in toad skin and a potent lightening agent in frog and lizard skins (reversing in a dose-dependent manner the darkening caused by alpha-melanocyte-stimulating hormone). In frog skins, previous exposure to melatonin reduced further lightening actions of the indoleamine, and in toad skins, increasing concentrations of melatonin elicited decreasing lightening responses, suggesting an autodesensitizing action of the hormone. Various concentrations of melatonin diminished the responses to the lightening agonist melanin-concentrating hormone (MCH) in fish skins and to the darkening agonists alpha-MSH in toad, frog and lizard skins and isoproterenol in frog skins. In vitro inhibitory actions of melatonin are mimicked in the absence of the hormone in skin preparations from toads kept in continuous darkness for 48 hr. The lipophylic nature of the indoleamine associated with the results herein described suggests intracellular actions of melatonin on vertebrate pigment cells.
Assuntos
Hormônios Hipotalâmicos/farmacologia , Isoproterenol/farmacologia , Melaninas/farmacologia , Melanóforos/efeitos dos fármacos , Melatonina/farmacologia , Fotoperíodo , Hormônios Hipofisários/farmacologia , alfa-MSH/farmacologia , Animais , Anuros , Relação Dose-Resposta a Droga , Técnicas In Vitro , Lagartos , Rana pipiens , Pele/citologia , Pele/efeitos dos fármacosRESUMO
Norepinephrine (NE) and phenylephrine (Phe) were employed to study the ionic requirements for alpha adrenoceptor activation in the teleost Poecilia reticulata melanophores. As expected the beta adrenoceptor blocker, propranolol, increased the sensitivity of the preparation to NE (5.8 times), and was therefore employed in all the experimental procedures. Neither cocaine (a neuronal uptake blocker) nor dexamethasone (an extraneuronal uptake blocker) enhanced the sensitivity of the preparation to NE, suggesting that these inactivating mechanisms would not play a role in P. reticulata pigmentary system. However, in the absence of calcium, the dose-response curve (DRC) to NE was displaced to the left about 3.5 times, whereas the DRC to Phe was not affected. These results indicate that a neuronal uptake is active, but was not demonstrated by the classical pharmacological tools, probably due to an assymmetric display of the nervous endings. The DRC to NE was rightward displaced (14.1 times) in the presence of the calcium channel blocker Verapamil, whereas the DRC to Phe was not affected. These data suggest that P. reticulata melanophores possess a mixed population of alpha 1 and alpha 2 adrenoceptors, the activation of the latter eliciting an extracellular calcium influx. In sodium-free saline, the DRC to NE was rightward shifted (6.6 times) and the response to Phe was impaired in such a way that the maximal response was not achieved. The DRC to both NE and Phe were rightward displaced (7.9 and 2.7 times respectively) in the presence of the sodium channel blocker tetrodotoxin (TTX) 10(-7)M.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Catecolaminas/fisiologia , Íons , Melanóforos/fisiologia , Poecilia/fisiologia , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Cocaína/farmacologia , Dexametasona/farmacologia , Relação Dose-Resposta a Droga , Feminino , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/fisiologia , Melanóforos/efeitos dos fármacos , Melanóforos/ultraestrutura , Norepinefrina/farmacologia , Fenilefrina/farmacologia , Propranolol/farmacologia , Receptores Adrenérgicos alfa/efeitos dos fármacos , Receptores Adrenérgicos alfa/fisiologia , Tetrodotoxina/farmacologia , Verapamil/farmacologiaRESUMO
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.
Assuntos
Cálcio/fisiologia , Hormônios Hipotalâmicos , Melaninas/farmacologia , Melanóforos/efeitos dos fármacos , Hormônios Hipofisários/farmacologia , Sódio/fisiologia , Animais , Cálcio/farmacologia , Agregação Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Melanóforos/citologia , Concentração Osmolar , Poecilia , Sódio/farmacologiaRESUMO
1. The darkening actions of MCH (melanin concentrating hormone), alpha-MSH and the synthetic analog [Nle4, D-Phe7]-alpha-MSH on the toad, Bufo ictericus ictericus, melanophores were studied regarding the role of calcium in the hormone receptor coupling, signal transduction and intracellular pigment translocation. 2. In the absence of external calcium, MCH and both melanotropins still elicit maximal skin darkening. 3. Verapamil, a calcium-channel blocker, completely abolishes the alpha-MSH-induced response and partially inhibits MCH-induced darkening, although the calcium carrier, ionophore A23187, was unable to promote any pigment translocation. 4. Since darkening responses promoted by cyclic nucleotides proceeded normally in the presence of verapamil and extracellular calcium was not necessary for melanotropin dispersing action, it is suggested that the blocking activity obtained with verapamil is probably due to an impairment of the Ca2+-dependent adenylate cyclase activity. 5. Reversal of melanotropin-induced darkening could be obtained with melatonin, in both normal and Ca2+-free Ringer, whereas MCH darkening is reversed by melatonin only in the absence of calcium. 6. The results seem to indicate that calcium is not required for hormone receptor binding and pigment migration, whereas it is specifically needed for signal transduction.