Pigment Dispersion In Melanophores Research Articles

A new insight into the pigmentation of the three-spined stickleback exposed to oxidative stress: day and night study

The diverse and changing pigmentation of the skin allows fish to adapt to environmental conditions for survival and communication with conspecifics. However, various physical and chemical environmental factors, including pollutants, affect fish coloration. Therefore, the implementation of an analysis of skin pigmentation has been considered in fish well-being and ecotoxicological studies. A physiological color change is achieved by the motility of melanin-containing organelles: they aggregate into the perikaryon or disperse throughout the cytoplasm of melanophores in response to various stimuli. In our study, we addressed the issue of implementing the analysis of pigment dispersion in melanophores in stickleback skin to assess the response of fish to oxidative stress. We examined pigment dispersion in day and night skin samples collected from the dorsal, lateral and ventral regions. The degree of pigment dispersion we assessed by the melanophore index. The total number of melanophores counted in the defined skin area was significantly higher in the night samples than in the day samples. Only in day samples of dorsal skin we observed the significant changes in pigment dispersion after exposure to stress: melanin was predominantly in the aggregated state. In the night samples, we did not report any response to stress in any part of the skin. Examination of pigment dispersion in melanophores in stickleback skin can be useful for assessing the welfare of fish and detecting toxic agents in the environment, but under specified conditions: in sticklebacks, it is analysis of dorsal skin during the day.

Melatonin and cortisol as components of the cutaneous stress response system in fish: Response to oxidative stress

The skin being a passive biological barrier that defends the organism against harmful external factors is also a site of action of the system responding to stress. It appears that melatonin (Mel) and its biologically active metabolite AFMK (N1-acetyl-N2-formyl-5-methoxykynuramine), both known as effective antioxidants, together with cortisol, set up a local (cutaneous) stress response system (CSRS) of fish, similar to that of mammals. Herein we comment on recent studies on CSRS in fish and show the response of three-spined stickleback skin to oxidative stress induced by potassium dichromate. Our study indicates that exposure of the three-spined stickleback to K2Cr2O7 affects Mel and cortisol levels and pigment dispersion in melanophores in the skin. In our opinion, an increased concentration of Mel and cortisol in the skin may be the strategy to cope with oxidative stress, where both components act locally to prevent damage caused by active oxygen molecules. Furthermore, the pigment dispersion may be a valuable, easy-to-observe mark of oxidative stress, useful in the evaluation of fish welfare.

Further evidence on acetylation-induced inhibition of the pigment-dispersing activity of α-melanocyte-stimulating hormone

Our previous studies showed that in barfin flounder, α-melanocyte-stimulating hormone (α-MSH) stimulates pigment dispersion in xanthophores, while it shows negligible effects in melanophores. The present study was undertaken to evaluate whether these results are limited to barfin flounder by using Japanese flounder. Three subtypes of proopiomelanocortin gene encoding melanocortins (MCs) were expressed in the Japanese flounder pituitary, one of which was also expressed in the skin. Expression of melanocortin 5 receptor gene (Mc5r) was observed in isolated xanthophores, while that of Mc1r and Mc5r was found in melanophores. In the xanthophores of Japanese flounder skin, α-MSH as well as desacetyl (Des-Ac)-α-MSH and diacetyl (Di-Ac)-α-MSH exhibited dose-dependent pigment-dispersing activities, indicating that the signals of α-MSH-related peptides were mediated by MC5R. On the other hand, α-MSH did not stimulate pigment dispersion in melanophores, while Des-Ac-α-MSH and Di-Ac-α-MSH did, thus indicating that the expression of two different types of Mcr is related to the decrease in α-MSH activity. Thus, the molecular repertoire in MC system observed in Japanese flounder is similar to that in barfin flounder. Moreover, the relationship between the pigment-dispersing activities of α-MSH-related peptides and the expression of Mcr subtypes in xanthophores and melanophores were also similar between Japanese flounder and barfin flounder. Consequently, we hypothesize that inhibition of α-MSH activity could be due to the formation of heterodimers comprising MC1R and MC5R, often observed in G-protein-coupled receptors.

Identification of novel hexapeptide agonists at the Xenopus laevis melanophore melanocortin receptor

We used a combinatorial chemical approach to identify novel agonists for the endogenous melanocortin receptor expressed in Xenopus laevis melanophores. A random one-bead one-compound hexapeptide library was screened to detect new molecules able to induce pigment dispersion in melanophores. Our approach led to the discovery of seven related novel peptides able to stimulate pigment dispersion with EC50 in the range of 0.1–10 μM. Their action was inhibited by the amphibian melanocortin receptor antagonist dWRL. These novel peptides share no significant sequence homology with known melanocortins. This study may aid in the understanding of the chemical interaction between the melanocortin receptors and their ligands.

Molecular mechanisms of pigment transport in melanophores.

We present an overview of the research on intracellular transport in pigment cells, with emphasis on the most recent discoveries. Pigment cells of lower vertebrates have been traditionally used as a model for studies of intracellular transport mechanisms, because these cells transport pigment organelles to the center or to the periphery of the cell in a highly co-ordinated fashion. It is now well established that both aggregation and dispersion of pigment in melanophores require two elements of the cytoskeleton: microtubules and actin filaments. Melanosomes are moved along these cytoskeletal tracks by motor proteins. Recent studies have identified the motors responsible for pigment dispersion and aggregation in melanophores. We propose a model for the possible roles of the two cytoskeletal transport systems and how they might interact. We also discuss the putative mechanisms of regulation of pigment transport, especially phosphorylation. Last, we suggest areas of research that will receive attention in the future in order to elucidate the mechanisms of organelle transport.

Melatonin induces dispersion of pigment in melanophores of some teleostean species

Melatonin induces dispersion of pigment in melanophores of some teleostean species

Functional analysis by imaging of melanophore pigment dispersion of chimeric receptors constructed by recombinant polymerase chain reaction

Analysis of functional aspects of the molecular structure of proteins often requires a means to selectively alter structure and subsequently analyze function. We have adapted a method of overlap extension polymerase chain reaction (PCR) to generate multiple domain replacements in G-protein coupled receptors. The examples described herein are β 2-adrenergic receptors whose G-protein coupling domains have been replaced by homologous domains of olfactory receptors, but the procedure has also been used to produce constructs with mutations, deletions, and fusions of two complete open reading frames. The chimeric olfactory-adrenergic receptors were assayed by functional expression in clonal lines of Xenopus melanophores. The ability of G-protein coupled second messenger pathways to cause translocation of pigment organelles within melanophores allows the use of video microscopy to assay the function of the chimeric receptors. Digital automation of microscope stage, camera, and image processing allows multiple parallel experiments to be performed. Melanophores allow responses mediated by the Gs, Gq and Gi pathways to be assayed with equal efficiency and the specificity of the coupling between chimera (or receptor) and G-protein subtypes can be rapidly determined.

Melanophore pigment dispersion responses to agonists show two patterns of sensitivity to inhibitors of cAMP-dependent protein kinase and protein kinase C.

Melanophore pigment dispersion is a sensitive bioassay for activation of the adenylyl cyclase and phospholipase C second-messenger pathways. The necessity of protein kinase activation in causing pigment dispersion was confirmed for eight agonists of endogenous melanophore receptors and for two transfected receptors. All agonists and receptors previously shown to elevate intracellular cAMP in melanophores--melanocyte stimulating hormone, light, (-) norepinephrine, 5-hydroxytrptamine, and the beta2-adrenergic receptor--were able to stimulate pigment dispersion in the presence of Ro31-8220, a potent inhibitor of protein kinase C, but were blocked in the presence of H89, an inhibitor of cAMP-dependent protein kinase. The bombesin receptor, which elevates intracellular IP3 in melanophores, was unable to stimulate pigment dispersion in the presence of Ro31-8220 or H89. Agonists whose mechanism of activation of pigment dispersion are unknown were also tested. Endothelin 3 responses were blocked by both H89 and Ro31-8220, predicting coupling to phospholipase C. Vasoactive intestinal polypeptide, oxytocin, and calcitonin gene-related peptide beta responses were blocked only by H89, predicting coupling to adenylyl cyclase.

Melanophore pigment dispersion responses to agonists show two patterns of sensitivity to inhibitors of cAMP‐dependent protein kinase and protein kinase C

Melanophore pigment dispersion responses to agonists show two patterns of sensitivity to inhibitors of cAMP‐dependent protein kinase and protein kinase C

Novel receptors for melatonin that mediate pigment dispersion are present in some melanophores of the pencil fish ( Nannostomus)

1. Comparing the daytime and the night-time pigmentary patterns of the skin of the pencil fish, Nannostomus beckfordi, we noticed that specific regions of dark spots that were part of the night-time pattern became pale during the day. 2. Microscopic observations revealed that melanosomes in the melanophores in those regions were aggregated during the day but became dispersed at night. 3. These melanophores responded to melatonin by dispersal of melanosomes while the cells on other parts of the body responded to melatonin by aggregation of the pigment in the normal way. 4. The melanophores that responded to melatonin by pigment dispersion responded normally to other hormones and neurotransmitters, as did those on other parts of the skin. 5. The results indicate that, in addition to the known melatonin receptor that mediates the aggregation of melanosomes, there also exists an unusual receptor which mediates the dispersion of pigment in melanophores. We have tentatively designated this receptor the ‘beta-melatonin receptor’.

Pigment-dispersing hormone-immunoreactive neurons and their relation to serotonergic neurons in the blowfly and cockroach visual system

The pigment-dispersing hormone (PDH) family of neuropeptides comprises a series of closely related octadecapeptides, isolated from different species of crustaceans and insects, which can be demonstrated immunocytochemically in neurons in the central nervous system and optic lobes of some representatives of these groups (Rao and Riehm 1989). In this investigation we have extended these immunocytochemical studies to include the blowfly Phormia terraenovae and the cockroach Leucophaea maderae. In the former species tissue extracts were also tested in a bioassay: extracts of blowfly brains exhibited PDH-like biological activity, causing melanophore pigment dispersion in destalked (eyestalkless) specimens of the fiddler crab Uca pugilator. using standard immunocytochemical techniques, we could demonstrate a small number of pigment-dispersing hormone-immunoreactive (PDH-IR) neurons innervating optic lobe neuropil in the blowfly and the cockroadh. In the blowfly the cell bodies of these neurons are located at the anterior base of the medulla. At least eight PDH-IR cell bodies of two size classes can be distinguished: 4 larger and 4 smaller. Branching immunoreactive fibers invade three layers in the medulla neuropil, and one stratum distal and one proximal to the lamina synaptic layer. A few fibers can also be seen invading the basal lobula and the lobula plate. The fibers distal to the lamina appear to be derived from two of the large PDH-IR cell bodies which also send processes into the medulla. These neurons share many features in their lamina-medulla morphology with the serotonin immunoreactive neurons LBO-5HT described earlier (see Nässel 1988). It could be demonstrated by immunocytochemical double labeling that the serotonin and PDH immunoreactivities are located in two separate sets of neurons. In the cockroach optic lobe PDH-IR processes were found to invade the lamina synaptic region and form a diffuse distribution in the medulla. The numerous cell bodies of the lamina-medulla cells in the cockroach are located basal to the lamina in two clusters. Additional PDH-IR cell bodies could be found at the anterior base of the medulla. The distribution and morphology of serotonin-immunoreactive neurons in the cockroach lamina was found to be very similar to the PDH-IR ones. It is hence tempting to speculate that in both species the PDH- and serotonin-immunoreactive neurons are functionally coupled with common follower neurons. These neurons may be candidates for regulating large numbers of units in the visual system.(ABSTRACT TRUNCATED AT 400 WORDS)

Morphology of the pars intermedia and the melanophore-stimulating cells in Xenopus laevis in relation to background adaptation

The melanophore-stimulating hormone (MSH) cells of the amphibian pars intermedia secrete the peptide α-melanophore-stimulating hormone (α-MSH), which induces pigment dispersion in dermal melanophores. The purpose of the present study was to determine (1) whether prolonged activation of the secretory activity of the pars intermedia is associated with hypertrophy, hyperplasia, or both and (2) whether the MSH cells function as a homogeneous or heterogeneous population in meeting the physiological demand for MSH. The demand for MSH was manipulated by adapting animals for at least 3 weeks to white, two shades of grey, or black backgrounds. Morphometric analysis showed that the intermediate lobe volume was positively correlated with the degree of pigment dispersion in the melanophores. The number of MSH cells per lobe was not affected by the degree of pigment dispersion. Therefore, we conclude that enlargement of the tissue associated with MSH cell activation involves hypertrophy rather than hyperplasia. Ultrastructural examination indicated that the majority of MSH cells in black-adapted animals are biosynthetically active, whereas the cells of white-adapted animals are relatively inactive and show granule storage. The pars intermedia of grey-adapted toads contained both active and inactive cells, indicating that MSH cells respond as a heterogeneous cell population in meeting the endocrine demand imposed by background.

C-terminal deletion analogs of a crustacean pigment-dispersing hormone

This study deals with the effect of deamidation and C-terminal truncation on the potency of an octadecapeptide pigment-dispersing hormone (PDH: Asn- Ser-Gly-Met-Ile-Asn-Ser-Ile-Leu-Gly-Ile-Pro-Arg-Val-Met-Thr-Glu-Ala-NH 2), first described as light-adapting distal retinal pigment hormone (DRPH) from Pandalus borealis. Bioassay of synthetic analogs for melanophore pigment dispersion in destalked fiddler crabs ( Uca pugilator) showed that deamidation causes a 300-fold decrease in potency. The analogs 1–17-NH 2 and 1–16-NH 2 were about 3 times more potent than 1–18-OH. Further truncation led to decreases in potency, with the peptide 1–9-NH 2 being the smallest C-terminal deletion analog to display activity (0.001% potency). Smaller analogs (1–8-NH 2, 1–6-NH 2 and 1–4-NH 2) were inactive when tested in doses as high as 500 nmoles/crab. On the basis of our earlier work on N-terminal deletion analogs and the present findings the residues 6 to 9 seem to be important for PDH action.

Characterization of a pigment-dispersing hormone in eyestalks of the fiddler crab Uca pugilator

A pigment-dispersing hormone (PDH) from eyestalks of the fiddler crab Uca pugilator has been purified by gel filtration, ion-exchange chromatography, partition chromatography, and reversed-phase liquid chromatography. Based on automated gas-phase sequencing and subsequent identification of carboxyl-terminal amide, we have assigned the primary structure of this peptide as Asn-Ser-Glu-Leu-Ile-Asn-Ser-Ile-Leu-Gly-Leu-Pro-Lys-Val-Met-Asn-Asp-Ala-NH (2). We have confirmed the sequence by synthesizing this peptide and demonstrating that the synthetic PDH and the native PDH display identical chromatographic behavior and biological activity. This hormone is a member of a family of invertebrate neuropeptides that includes a light-adapting/pigment-dispersing octadecapeptide hormone from the prawn Pandalus borealis. In assays for melanophore pigment dispersion in destalked fiddler crabs, Uca PDH was 21-fold more potent than Pandalus PDH. These two hormones share a hexapeptide core sequence (residues 5-10: -Ile-Asn-Ser-Ile-Leu-Gly-) as well as the amino- and carboxyl-terminal residues but differ at positions 3, 4, 11, 13, 16, and 17. These results point to speciesrelated or group-specific structural differences among crustacean PDHs.

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

1. 1. 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. 2. 2. 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. 3. 3. These findings suggest that adrenergic receptors mediating pigment dispersion in melanophores are beta-2 and those of leucophores are beta-1. 4. 4. The possible relations between receptor mechanisms and the responses of chromatophores are discussed.

The development of the pars intermedia and its role in the regulation of dermal melanophores in the larvae of the amphibian Xenopus laevis

The ontogenesis of biosynthesis of pro-opiomelanocortin (POMC)-related peptides in the pars intermedia of Xenopus laevis tadpoles was studied. The results were related to the capacity of the animal to adapt to background color through regulation of pigment dispersion in dermal melanophores. Using immunocytochemical techniques with antisera to α-melanophore-stimulating hormone (α-MSH), it was revealed that this peptide first appeared at developmental stage 37 38 , just prior to the animal's ability to adapt to background. It was shown that pigment dispersion in melanophores between stages 33 and 39 was not dependent on melanotropins of pituitary origin. Using in vitro biosynthetic studies it was possible to follow POMC biosynthetic activity, its processing and the release of peptides from stage 48 onward. Among the newly synthesized peptides observed were a γ 3-MSH-like peptide, des- N-α-acetyl-α-MSH, α-MSH, and two endorphin-like peptides. By stage 57 a biosynthetic pattern almost identical to that of the adult pars intermedia had evolved. It was concluded that stage 39 40 is a critical stage in the simultaneous development of a number of the components involved in the neuroendocrine control of background adaptation.

Calcium requirement for alpha-MSH action on melanophores: studies with forskolin.

alpha-MSH-induced pigment dispersion in melanophores shows an absolute requirement for extracellular Ca2+. To localize Ca2+ sites involved in the mechanism of action of alpha-MSH we studied the effects of Ca2+ deprivation on alpha-MSH and forskolin-induced melanophore responses. In an in vitro melanophore system employing ventral tailfins of Xenopus tadpoles, melanophore responses were assayed in terms of pigment dispersion and the phosphorylation state of a 53 kDa melanophore-specific protein. In the same melanophore system alpha-MSH has been shown to specifically increase the phosphorylation of this 53 kDa protein. Forskolin induces a dose-dependent pigment dispersion (EC50 7 X 10(-7) M). In contrast to the dispersion induced by alpha-MSH forskolin-induced dispersion does not require extracellular Ca2+. Moreover, in a Ca2+-free medium melanophores with permanently activated MSH-receptors aggregate, but can be redispersed by the addition of forskolin. Forskolin increases 53 kDa phosphorylation in a dose-dependent manner. Maximal stimulation with forskolin (10(-5) M) is four-fold and equals maximal 53 kDa phosphorylation obtainable with alpha-MSH. The MSH-induced increase in 53 kDa phosphorylation is inhibited by Ca2+ deprivation, whereas the forakolin-induced increase is unaffected. Our results suggest that alpha-MSH and forskolin stimulate melanophores through a common pathway and confirm that cAMP is a second messenger in alpha-MSH action in this system. We conclude that the Ca2+ sites in the mechanism of alpha-MSH action on melanophores precede adenylate cyclase activation.

Structure-activity relationships of peptides derived from ACTH, β-LPH and MSH with regard to avoidance behavior in rats

Decades ago it was recognized that the pharmacological profile of melanocortin ligands that stimulated grooming behavior in rats was strikingly similar to that of Xenopus laevis melanophore pigment dispersion. After cloning of the melanocortin MC1 receptor, expressed in melanocytes, and the melanocortin MC4 receptor, expressed mainly in brain, the pharmacological profiles of these receptors appeared to be very similar and it was demonstrated that these receptors mediate melanocortin-induced pigmentation and grooming respectively. Grooming is a low priority behavior that is concerned with care of body surface. Activation of central melanocortin MC4 receptors is also associated with meal termination, and continued postprandial stimulation of melanocortin MC4 receptors may stimulate natural postprandial grooming behavior as part of the behavioral satiety sequence. Indeed, melanocortins fail to suppress food intake or induce grooming behavior in melanocortin MC4 receptor-deficient rats. This review will focus on how melanocortins affect grooming behavior through the melanocortin MC4 receptor, and how melanocortin MC4 receptors mediate feeding behavior. This review also illustrates how melanocortins were the most likely candidates to mediate grooming and feeding based on the natural behaviors they induced.

Studies on the mechanism of action of a pigment dispersing chromatophorotropin in the fiddler crab, Uca pugilator

1. 1. A23187 induces pigment dispersion and potentiates hormone-induced pigment dispersion in isolated legs of Uca pugilator. 2. 2. cAMP and Bt 2AMP induce pigment dispersion in melanophores, leucophores, and erythrophores, and both potentiate hormone-induced pigment dispersion in melanophores and leucophores of U. pugilator. 3. 3. cGMP induces pigment dispersion in all three chromatophore types and both potentiates and inhibits hormone-induced dispersion in a dose dependent manner in isolated legs of U. pugilator. 4. 4. Colchicine and Cytochalasin-B inhibit hormone-induced pigment dispersion in melanophores, leucophores, and erythrophores of U. pugilator.

The purification and partial characterization of the melanophore-dispersing hormone of Uca pugilator

Fractionation of 1000 Uca pugilator eyestalks by a combination of gel filtration on Sephadex G-25 and successive ion exchange chromatography on CM- and DEAE-cellulose resulted in the isolation of two major peaks of melanophore-dispersing activity which were designated MDH-1 and MDH-2. These hormones coeluted on Sephadex G-25 superfine, but showed distinct elution profiles on CM-cellulose. The homogeneity of MDH-1 was determined in two different paper chromatography and paper electrophoresis systems. In these systems a single fluorescamine-positive spot comigrated with MDH activity. N-Terminal analysis of MDH-1 by the dansyl-Cl procedure resulted in the identification of aspartic acid at this position. MDH-1 stimulated pigment dispersion in melanophores and leucophores in an identical manner, and in both systems a dose of 30 ng/μl caused a half-maximal response.