Pharmacological induction of skin pigmentation unveils the neuroendocrine circuit regulated by light

Light‐regulated skin colour change is an important physiological process in invertebrates and lower vertebrates, and includes daily circadian variation and camouflage (i.e. background adaptation). The photoactivation of melanopsin‐expressing retinal ganglion cells (mRGCs) in the eye initiates an uncharacterized neuroendocrine circuit that regulates melanin dispersion/aggregation through the secretion of alpha‐melanocyte‐stimulating hormone (α‐MSH). We developed experimental models of normal or enucleated Xenopus embryos, as well as in situ cultures of skin of isolated dorsal head and tails, to analyse pharmacological induction of skin pigmentation and α‐MSH synthesis. Both processes are triggered by a melanopsin inhibitor, AA92593, as well as chloride channel modulators. The AA9253 effect is eye‐dependent, while functional data in vivo point to GABA_A receptors expressed on pituitary melanotrope cells as the chloride channel blocker target. Based on the pharmacological data, we suggest a neuroendocrine circuit linking mRGCs with α‐MSH secretion, which is used normally during background adaptation.     

Structural changes in the pars intermedia of the cichlid teleost Sarotherodon mossambicus as a result of background adaptation and illumination

Summary The MSH producing cells in the pars intermedia of Sarotherodon mossambicus have been shown to be involved in background adaptation processes. Reflected light received by the eyes affects the activity of these cells. In the present study the hypothesis has been tested that also the pineal organ, as a second photoreceptor, is involved in regulation of the metabolic activity of the MSH cells. The pineal organ appears to contain photoreceptor cells and is considered to be capable of transferring information about light conditions to the animal. Removal of the pineal organ of fish kept on a black background has no effect on activity of MSH cells, whereas the activity of these cells in fish kept in darkness is increased. Thus it seems that the pineal organ exercises its influence on MSH cells only in darkness and that this influence results in a reduced activity of these cells. It is therefore concluded that the metabolic activity of MSH cells is inhibited not only by reflected light received by the eyes, but also by the action of the pineal organ as a result of the absence of illumination.No structural signs of secretory activity can be observed in the pineal, which might indicate synthesis or release of substances like melatonin. However, administration of melatonin reduces the activity of MSH cells. Neither pinealectomy nor treatment with melatonin has any influence on the second cell type of the pars intermedia, the PAS positive cells.     

Two light‐activated neuroendocrine circuits arising in the eye trigger physiological and morphological pigmentation

Two biological processes regulate light‐induced skin colour change. A fast ‘physiological pigmentation change’ (i.e. circadian variations or camouflage) involves alterations in the distribution of pigment containing granules in the cytoplasm of chromatophores, while a slower ‘morphological pigmentation change’ (i.e. seasonal variations) entails changes in the number of pigment cells or pigment type. Although linked processes, the neuroendocrine coordination triggering each response remains largely obscure. By evaluating both events in Xenopus laevis embryos, we show that morphological pigmentation initiates by inhibiting the activity of the classical retinal ganglion cells. Morphological pigmentation is always accompanied by physiological pigmentation, and a melatonin receptor antagonist prevents both responses. Physiological pigmentation also initiates in the eye, but with repression of melanopsin‐expressing retinal ganglion cell activity that leads to secretion of alpha‐melanocyte‐stimulating hormone (α‐MSH). Our findings suggest a model in which eye photoperception links physiological and morphological pigmentation by altering α‐MSH and melatonin production, respectively.     

Seeing the light to change colour: An evolutionary perspective on the role of melanopsin in neuroendocrine circuits regulating light‐mediated skin pigmentation

Melanopsin photopigments, Opn4x and Opn4m, were evolutionary selected to “see the light” in systems that regulate skin colour change. In this review, we analyse the roles of melanopsins, and how critical evolutionary developments, including the requirement for thermoregulation and ultraviolet protection, the emergence of a background adaptation mechanism in land‐dwelling amphibian ancestors and the loss of a photosensitive pineal gland in mammals, may have helped sculpt the mechanisms that regulate light‐controlled skin pigmentation. These mechanisms include melanopsin in skin pigment cells directly inducing skin darkening for thermoregulation/ultraviolet protection; melanopsin‐expressing eye cells controlling neuroendocrine circuits to mediate background adaptation in amphibians in response to surface‐reflected light; and pineal gland secretion of melatonin phased to environmental illuminance to regulate circadian and seasonal variation in skin colour, a process initiated by melanopsin‐expressing eye cells in mammals, and by as yet unknown non‐visual opsins in the pineal gland of non‐mammals.     

Retinally perceived light is not essential for photic regulation of pineal melatonin rhythms in the pigeon: studies with microdialysis

Using in vivo microdialysis, effects of retinally perceived light on pineal melatonin release and its rhythmicity was examined in the pigeon. In the first experiment, light-induced suppression of pineal melatonin release was studied. Although light given to the whole body during the dark strongly suppressed pineal melatonin release to a daytime level, light exclusively delivered to the eyes did not remarkably inhibit melatonin release. In the second experiment, in order to determine whether retinally perceived light has phase-shifting effects on pineal melatonin rhythms, pigeons were given a single light pulse of 2 h at circadian time (CT) 18 and the phases of the second cycle after the light pulse were compared with those of control pigeons without the light pulse. In this experiment, phase advances of pineal melatonin rhythms were observed when the light was given to the whole body but not when only the eyes were illuminated. In a third experiment, after entrainment to light-dark 12:12 (LD 12:12) cycles, birds whose heads were covered with black tapes were transferred into constant light (LL) conditions and only the eyes were exposed to new LD cycles for 7 days (the phase was advanced by 6 h from the previous cycles) using a patching protocol. This procedure, however, could not entrain pineal melatonin rhythms to the retinal LD cycles. These results indicate that the eyes are not essential for photic regulation of pineal melatonin release and its rhythmicity in the pigeon.Abbreviations CT circadian time - LD light-dark - LL constant light - SCN suprachiasmatic nucleus - LLdim constant dim light - NE norepinephrine - SCG superior cervical ganglia - WB whole body - E eye - EX extraretina - C control     

A retro‐inverso α‐melanocyte stimulating hormone analog with MC1R‐binding selectivity

α‐melanocyte stimulating hormone (α‐MSH) is a tridecapeptide fragment of pro‐opiomelanocortin (POMC) with broad effects on appetite, skin pigmentation, hormonal regulation, and potential roles in both inflammation and autoimmunity. The use of this peptide as an anti‐inflammatory agent is limited by its low selectivity between the melanocortin receptors, susceptibility to proteolytic degradation, and rapid clearance from circulation. A retro‐inverso (RI) sequence of α‐MSH was characterized for receptor activity and resistance to protease. This peptide demonstrated surprisingly high selectivity for binding the melanocortin receptor 1 (MC1R). However, RI‐α‐MSH exhibited a diminished binding affinity for MC1R compared to α‐MSH. Mapping of the residues critical for agonist activity, receptor binding, and selectivity by alanine scanning, identified the same critical core tetrapeptide required for the native peptide. Modest improvements in affinity were obtained by conservative changes employing non‐natural amino acids and substitution of the C‐terminal sequence with a portion of a MC1R ligand peptide previously identified by phage display. Recombination of these elements yielded a peptide with an identical K_i as α‐MSH at MC1R and a lower EC₅₀ in Mel‐624 melanoma cells. A number of other structural modifications of the RI peptide were found to differ in effect from those reported for the L ‐form α‐MSH, suggesting a significantly altered interaction with the MC1R. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Effects of Melanogenesis‐Inducing Nitric Oxide and Histamine on the Production of Eumelanin and Pheomelanin in Cultured Human Melanocytes

Melanin pigments produced in human melanocytes are classified into two categories; black coloured eumelanin and reddish‐yellow pheomelanin. Stimulation of melanocytes with α‐melanocyte‐stimulating hormone (α‐MSH), one of several melanogenic factors, has been reported to enhance eumelanogenesis to a greater degree than pheomelanogenesis, which contributes to hyperpigmentation in skin. Nitric oxide (NO) and histamine are also melanogenesis‐stimulating factors that are released from cells surrounding melanocytes following ultraviolet (UV) irradiation. In this study, the effects of NO and histamine on the ratio of eumelanin and pheomelanin were examined in human melanocytes, and then compared with that of α‐MSH. The amounts of eumelanin and pheomelanin were quantified using high‐performance liquid chromatography analysis after oxidation and hydrolysis of melanin. Melanogenesis was induced by the addition of α‐MSH, NO, or histamine to melanocytes. The amount of eumelanin production significantly increased with independent stimulation by these melanogenic factors, especially histamine, while that of pheomelanin significantly increased with α‐MSH and NO, but only slightly with histamine. As a result, the ratio of eumelanin and pheomelanin increased significantly with the addition of NO or histamine. These results suggest that NO and histamine, as in the case of α‐MSH, may contribute to UV‐induced hyperpigmentation by enhancing eumelanogenesis.     

α‐MSH tripeptide analogs activate the melanocortin 1 receptor and reduce UV‐induced DNA damage in human melanocytes

One skin cancer prevention strategy that we are developing is based on synthesizing and testing melanocortin analogs that reduce and repair DNA damage resulting from exposure to solar ultraviolet (UV) radiation, in addition to stimulating pigmentation. Previously, we reported the effects of tetrapeptide analogs of α‐melanocortin (α‐MSH) that were more potent and stable than the physiological α‐MSH, and mimicked its photoprotective effects against UV‐induced DNA damage in human melanocytes. Here, we report on a panel of tripeptide analogs consisting of a modified α‐MSH core His⁶‐d ‐Phe⁷‐Arg⁸, which contained different N‐capping groups, C‐terminal modifications, or arginine mimics. The most potent tripeptides in activating cAMP formation and tyrosinase of human melanocytes were three analogs with C‐terminal modifications. The most effective C‐terminal tripeptide mimicked α‐MSH in reducing hydrogen peroxide generation and enhancing nucleotide excision repair following UV irradiation. The effects of these three analogs required functional MC1R, as they were absent in human melanocytes that expressed non‐functional receptor. These results demonstrate activation of the MC1R by tripeptide melanocortin analogs. Designing small analogs for topical delivery should prove practical and efficacious for skin cancer prevention.     

Melanopsin photoreception in the eye regulates light‐induced skin colour changes through the production of α‐MSH in the pituitary gland

How skin colour adjusts to circadian light/dark cycles is poorly understood. Melanopsin (Opn4) is expressed in melanophores, where in vitro studies suggest it regulates skin pigmentation through a ‘primary colour response’ in which light photosensitivity is translated directly into pigment movement. However, the entrainment of the circadian rhythm is regulated by a population of melanopsin‐expressing retinal ganglion cells (mRGCs) in the eye. Therefore, in vivo, melanopsin may trigger a ‘secondary colour response’ initiated in the eye and controlled by the neuro‐endocrine system. We analysed the expression of opn4m and opn4x and melanin aggregation induced by light (background adaptation) in Xenopus laevis embryos. While opn4m and opn4x are expressed at early developmental times, light‐induced pigment aggregation requires the eye to become functional. Pharmacological inhibition of melanopsin suggests a model whereby mRGC activation lightens skin pigmentation via a secondary response involving negative regulation of alpha‐melanocyte‐stimulating hormone (α‐MSH) secretion by the pituitary.     

Daily Oscillation in Melatonin Synthesis in The Turkey Pineal Gland and Retina: Diurnal and Circadian Rhythms

The aim of the present study was to examine arylalkylamine N‐acetyltransferase (AANAT) activity and melatonin content in the pineal gland and retina as well as the melatonin concentration in plasma of the turkey (Meleagris gallopavo), an avian species in which several physiological processes, including reproduction, are controlled by day length. In order to investigate whether the analyzed parameters display diurnal or circadian rhythmicity, we measured these variables in tissues isolated at regular time intervals from birds kept either under a regular light‐dark (LD) cycle or under constant darkness (DD). The pineal gland and retina of the turkey rhythmically produced melatonin. In birds kept under a daily LD cycle, melatonin levels in the pineal gland and retina were high during the dark phase and low during the light phase. Rhythmic oscillations in melatonin, with high night‐time concentrations, were also found in the plasma. The pineal and retinal melatonin rhythms mirrored oscillations in the activity of AANAT, the penultimate enzyme in the melatonin biosynthetic pathway. Rhythmic oscillations in AANAT activity in the turkey pineal gland and retina were circadian in nature, as they persisted under conditions of constant darkness (DD). Transferring birds from LD into DD, however, resulted in a potent decline in the amplitude of the AANAT rhythm from the first day of DD. On the sixth day of DD, pineal AANAT activity was still markedly higher during the subjective dark than during the subjective light phase; whereas, AANAT activity in the retina did not exhibit significant oscillations. The results indicate that melatonin rhythmicity in the turkey pineal gland and retina is regulated both by light and the endogenous circadian clock. The findings suggest that environmental light may be of primary importance in the maintenance of the high‐amplitude melatonin rhythms in the turkey.     

Structural changes in the pars intermedia of the cichlid teleost Sarotherodon mossambicus as a result of background adaptation and illumination

Summary The pars intermedia of Sarotherodon mossambicus (Tilapia mossambica) contains two cell types which can be differentiated at both the light and electron microscopic level. The predominant cell type is lead haematoxyline positive, and has been shown to be the MSH producing cell type by means of immunocytochemical staining at the ultrastructural level. The changes in cellular and nuclear volume, as well as the results of stereological measurements on the cytoplasmic organelles, show that the activity of MSH cells is high on a black background and low on a white background or in total darkness. In blinded fish under a normal day-night regime the activity of the MSH cell is as high as that in black adapted fish, whereas the activity is low when the blinded fish are kept in total darkness. From the observed differences in activity of the MSH cells between the experimental groups, it is concluded that the MSH cells are not activated by the absence of reflected light, but by a high ratio between direct and reflected light. A second light-sensitive organ, supposedly the pineal gland, is also involved in the background response of the MSH producing cells.     

Light intensities required to suppress nocturnal melatonin secretion in albino and pigmented rats

Sprague-Dawley albino rats or Long-Evans pigmented rats were exposed during the dark phase of the daily light:dark cycle to various intensities of a sunlight-stimulating white fluorescent light (0.022, 0.044, 0.110, 0.220, 0.440 or 2.200 μW/cm²) for 30 min; pineal glands and trunk blood samples were then collected and assayed for melatonin by radioimmunoassay. Albino rats exposed to irradiances of 0.110 μW/cm² or less had pineal melatonin levels that were not significantly different from those of unexposed animals; higher irradiances significantly (P < 0.001) reduced melatonin levels. In contrast, as little as 0.022 μW/cm² significantly (P < 0.02) reduced pineal and serum melatonin levels in the pigmented rats. These results suggest that something other than the simple presence or absence of eye pigmentation is the critical factor in determining the sensitivity of the rat's pineal to retinal-mediated photic suppression of melatonin synthesis.     

The Avian Pineal Gland

The pineal gland plays a key role in the control of the daily and seasonal rhythms in most vertebrate species. In mammals, rhythmic melatonin (MT) release from the pineal gland is controlled by the suprachiasmatic nucleus via the sympathetic nervous system. In most non‐mammalian species, including birds, the pineal gland contains a self‐sustained circadian oscillator and several input channels to synchronize the clock, including direct light sensitivity. Avian pineal glands maintain rhythmic activity for days under in vitro conditions. Several physical (light, temperature, and magnetic field) and biochemical (Vasoactive intestinal polypeptide (VIP), norepinephrine, PACAP, etc.) input channels, influencing release of melatonin are also functional in vitro, rendering the explanted avian pineal an excellent model to study the circadian biological clock. Using a perifusion system, we here report that the phase of the circadian melatonin rhythm of the explanted chicken pineal gland can be entrained easily to photoperiods whose length approximates 24 h, even if the light period is extremely short, i.e., 3L:21D. When the length of the photoperiod significantly differs from 24 h, the endogenous MT rhythm becomes distorted and does not follow the light‐dark cycle. When explanted chicken pineal fragments were exposed to various drugs targeting specific components of intracellular signal transduction cascades, only those affecting the cAMP‐protein kinase‐A system modified the MT release temporarily without phase‐shifting the rhythm in MT release. The potential role of cGMP remains to be investigated.     

Rhythmic melatonin secretion in different teleost species: an in vitro study

The rhythmic production of melatonin is governed by intrapineal oscillators in all fish species so far investigated except the rainbow trout. To determine whether the latter represents an exception among fish, we measured in vitro melatonin secretion in pineal organs of nine wild freshwater and six marine teleost species cultured at constant temperature and under different photic conditions. The results demonstrate that pineal organs of all species maintain a rhythmic secretion of melatonin under light:dark cycles and complete darkness, and strongly suggest that most fish possess endogenous intrapineal oscillators driving the rhythm of melatonin production, with the exception of the rainbow trout.Abbreviations LD light:dark - DD dark:dark - NAT N-acetyltransferase - RIA radioimmunoassay     

Pleiotropy in the melanocortin system: expression levels of this system are associated with melanogenesis and pigmentation in the tawny owl (Strix aluco)

The adaptive function of melanin‐based coloration is a long‐standing debate. A recent genetic model suggested that pleiotropy could account for covariations between pigmentation, behaviour, morphology, physiology and life history traits. We explored whether the expression levels of genes belonging to the melanocortin system (MC1R, POMC, PC1/3, PC2 and the antagonist ASIP), which have many pleiotropic effects, are associated with melanogenesis (through variation in the expression of the genes MITF, SLC7A11, TYR, TYRP1) and in turn melanin‐based coloration. We considered the tawny owl (Strix aluco) because individuals vary continuously from light to dark reddish, and thus, colour variation is likely to stem from differences in the levels of gene expression. We measured gene expression in feather bases collected in nestlings at the time of melanin production. As expected, the melanocortin system was associated with the expression of melanogenic genes and pigmentation. Offspring of darker reddish fathers expressed PC1/3 to lower levels but tended to express PC2 to higher levels. The convertase enzyme PC1/3 cleaves the POMC prohormone to obtain ACTH, while the convertase enzyme PC2 cleaves ACTH to produce α‐melanin‐stimulating hormone (α‐MSH). ACTH regulates glucocorticoids, hormones that modulate stress responses, while α‐MSH induces eumelanogenesis. We therefore conclude that the melanocortin system, through the convertase enzymes PC1/3 and PC2, may account for part of the interindividual variation in melanin‐based coloration in nestling tawny owls. Pleiotropy may thus account for the covariation between phenotypic traits involved in social interactions (here pigmentation) and life history, morphology, behaviour and physiology.     

Neuroendocrine effects of light

The light/dark cycle to which animals, and possibly humans, are exposed has a major impact on their physiology. The mechanisms whereby specific tissues respond to the light/dark cycle involve the pineal hormone melatonin. The pineal gland, an end organ of the visual system in mammals, produces the hormone melatonin only at night, at which time it is released into the blood. The duration of elevated nightly melatonin provides every tissue with information about the time of day and time of year (in animals that are kept under naturally changing photoperiods). Besides its release in a circadian mode, melatonin is also discharged in a pulsatile manner; the physiological significance, if any, of pulsatile melatonin release remains unknown. The exposure of animals including man to light at night rapidly depresses pineal melatonin synthesis and, therefore, blood melatonin levels drop precipitously. The brightness of light at night required to depress melatonin production is highly species specific. In general, the pineal gland of nocturnally active mammals, which possess rod-dominated retinas, is more sensitive to inhibition by light than is the pineal gland of diurnally active animals (with cone-dominated retinas). Because of the ability of the light/dark cycle to determine melatonin production, the photoperiod is capable of influencing the function of a variety of endocrine and non-endocrine organs. Indeed, melatonin is a ubiquitously acting pineal hormone with its effects on the neuroendocrine system having been most thoroughly investigated. Thus, in nonhuman photoperiodic mammals melatonin regulates seasonal reproduction; in humans also, the indole has been implicated in the control of reproductive physiology.Summary of a Plenary Lecture presented by the author in Vienna, August, 1990     

Plasticity for colour adaptation in vertebrates explained by the evolution of the genes pomc,pmch and pmchl

Different camouflages work best with some background matching colour. Our understanding of the evolution of skin colour is based mainly on the genetics of pigmentation (“background matching”), with little known about the evolution of the neuroendocrine systems that facilitate “background adaptation” through colour phenotypic plasticity. To address the latter, we studied the evolution in vertebrates of three genes, pomc, pmch and pmchl, that code for α‐MSH and two melanin‐concentrating hormones (MCH and MCHL). These hormones induce either dispersion/aggregation or the synthesis of pigments. We find that α‐MSH is highly conserved during evolution, as is its role in dispersing/synthesizing pigments. Also conserved is the three‐exon pmch gene that encodes MCH, which participates in feeding behaviours. In contrast, pmchl (known previously as pmch), is a teleost‐specific intron‐less gene. Our data indicate that in zebrafish, pmchl‐expressing neurons extend axons to the pituitary, supportive of an MCHL hormonal role, whereas zebrafish and Xenopus pmch+ neurons send axons dorsally in the brain. The evolution of these genes and acquisition of hormonal status for MCHL explain different mechanisms used by vertebrates to background‐adapt.