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The discovery of melanocortins in 1916 has resulted in more than 100 years of research focused on these peptides. Extensive studies have elucidated well-established functions of melanocortins mediated by cell surface receptors, including MSHR (melanocyte-stimulating hormone receptor) and ACTHR (adrenocorticotropin receptor). Subsequently, three additional melanocortin receptors (MCRs) were identified. Among these five MCRs, MC3R and MC4R are expressed primarily in the central nervous system, and are therefore referred to as the neural MCRs. Since the central melanocortin system plays important roles in regulating energy homeostasis, targeting neural MCRs is emerging as a therapeutic approach for treating metabolic conditions such as obesity and cachexia. Early efforts modifying endogenous ligands resulted in the development of many potent and selective ligands. This review focuses on the ligands for neural MCRs, including classical ligands (MSH and agouti-related peptide), nonclassical ligands (lipocalin 2, β-defensin, small molecules, and pharmacoperones), and clinically approved ligands (ACTH, setmelanotide, bremelanotide, and several repurposed drugs).

The genetic origin of human skin pigmentation remains an open question in biology. Several skin disorders and diseases originate from mutations in conserved pigmentation genes, including albinism, vitiligo, and melanoma. Teleosts possess the capacity to modify their pigmentation to adapt to their environmental background to avoid predators. This background adaptation occurs through melanosome aggregation (white background) or dispersion (black background) in melanocytes. These mechanisms are largely regulated by melanin-concentrating hormone (MCH) and α-melanocyte-stimulating hormone (α-MSH), two hypothalamic neuropeptides also involved in mammalian skin pigmentation. Despite evidence that the exogenous application of MCH peptides induces melanosome aggregation, it is not known if the MCH system is physiologically responsible for background adaptation. In zebrafish, we identify that MCH neurons target the pituitary gland-blood vessel portal and that endogenous MCH peptide expression regulates melanin concentration for background adaptation. We demonstrate that this effect is mediated by MCH receptor 2 (Mchr2) but not Mchr1a/b. mchr2 knock-out fish cannot adapt to a white background, providing the first genetic demonstration that MCH signaling is physiologically required to control skin pigmentation. mchr2 phenotype can be rescued in adult fish by knocking-out pomc, the gene coding for the precursor of α-MSH, demonstrating the relevance of the antagonistic activity between MCH and α-MSH in the control of melanosome organization. Interestingly, MCH receptor is also expressed in human melanocytes, thus a similar antagonistic activity regulating skin pigmentation may be conserved during evolution, and the dysregulation of these pathways is significant to our understanding of human skin disorders and cancers., Competing Interests: The authors have declared that no competing interests exist.

The clinical hallmarks of infections caused by critical respiratory viruses consist of pneumonia, which can progress to acute lung injury (ALI), and systemic manifestations including hypercoagulopathy, vascular dysfunction, and endotheliitis. The disease outcome largely depends on the immune response produced by the host. The bio-molecular mechanisms underlying certain dire consequences of the infection partly arise from an aberrant production of inflammatory molecules, an event denoted as "cytokine storm". Therefore, in addition to antiviral therapies, molecules able to prevent the injury caused by cytokine excess are under investigation. In this perspective, taking advantage of melanocortin peptides and their receptors, components of an endogenous modulatory system that exerts marked anti-inflammatory and immunomodulatory influences, could be an effective therapeutic strategy to control disease evolution. Exploiting the melanocortin system using natural or synthetic ligands can form a realistic basis to counteract certain deleterious effects of respiratory virus infections. The central and peripheral protective actions exerted following melanocortin receptor activation could allow dampening the harmful events that trigger the cytokine storm and endothelial dysfunction while sustaining the beneficial signals required to elicit repair mechanisms. The long standing evidence for melanocortin safety encourages this approach., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Lonati, Gatti and Catania.)

The melanocortin peptides have an important role in regulating body weight and appetite. Mice that lack the desacetyl-α-MSH and α-MSH peptides (Pomc tm1/tm1 ) develop obesity. This effect is exacerbated by a high fat diet (HFD). However, development of obesity in female Pomc tm1/tm1 mice during chronic HFD conditions is not fully accounted for by the increased energy intake. We hypothesized that the protection against chronic HFD-induced obesity imparted by MSH peptides in females is mediated by sex-specific alterations in the gut structure and gut microbiota. We determined that female WT mice had reduced jejunum villus length and increased crypt depth in response to chronic HFD. WT males and Pomc tm1/tm1 mice lacked this adaptation to a chronic HFD. Both Pomc tm1/tm1 genotype and chronic HFD were significantly associated with gut microbiota composition. Sex-specific associations between Pomc tm1/tm1 genotype and gut microbiota were observed in the presence of a chronic HFD. Pomc tm1/tm1 females had significantly reduced fecal acetate and propionate concentrations when compared to WT females. We conclude that MSH peptides influence jejunum villus length, crypt depth and the structure of the gut microbiota. These effects favor reduced nutrient absorption and occur in addition to the recognized roles of desacetyl-α-MSH and α-MSH peptides in appetite control.

Overcoming adverse effects and selectively delivering drug to target cells are two major challenges in the treatment of ulcerative colitis (UC). Lysine-proline-valine (KPV), a naturally occurring tripeptide, has been shown to attenuate the inflammatory responses of colonic cells. Here, we loaded KPV into hyaluronic acid (HA)-functionalized polymeric nanoparticles (NPs). The resultant HA-KPV-NPs had a desirable particle size (∼272.3 nm) and a slightly negative zeta potential (∼-5.3 mV). These NPs successfully mediated the targeted delivery of KPV to key UC therapy-related cells (colonic epithelial cells and macrophages). In addition, these KPV-loaded NPs appear to be nontoxic and biocompatible with intestinal cells. Intriguingly, we found that HA-KPV-NPs exert combined effects against UC by both accelerating mucosal healing and alleviating inflammation. Oral administration of HA-KPV-NPs encapsulated in a hydrogel (chitosan/alginate) exhibited a much stronger capacity to prevent mucosa damage and downregulate TNF-α, thus they showed a much better therapeutic efficacy against UC in a mouse model, compared with a KPV-NP/hydrogel system. These results collectively demonstrate that our HA-KPV-NP/hydrogel system has the capacity to release HA-KPV-NPs in the colonic lumen and that these NPs subsequently penetrate into colitis tissues and enable KPV to be internalized into target cells, thereby alleviating UC., (Copyright © 2016 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

The estimated heritability of human BMI is close to 75%, but identified genetic variants explain only a small fraction of interindividual body-weight variation. Inherited epigenetic variants identified in mouse models named "metastable epialleles" could in principle explain this "missing heritability." We provide evidence that methylation in a variably methylated region (VMR) in the pro-opiomelanocortin gene (POMC), particularly in postmortem human laser-microdissected melanocyte-stimulating hormone (MSH)-positive neurons, is strongly associated with individual BMI. Using cohorts from different ethnic backgrounds, including a Gambian cohort, we found evidence suggesting that methylation of the POMC VMR is established in the early embryo and that offspring methylation correlates with the paternal somatic methylation pattern. Furthermore, it is associated with levels of maternal one-carbon metabolites at conception and stable during postnatal life. Together, these data suggest that the POMC VMR may be a human metastable epiallele that influences body-weight regulation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The suprachiasmatic nucleus (SCN) and arcuate nucleus (ARC) have reciprocal connections; catabolic metabolic information activates the ARC and inhibits SCN neuronal activity. Little is known about the influence of the SCN on the ARC. Here, we investigated whether the SCN modulated the sensitivity of the ARC to catabolic metabolic conditions. ARC neuronal activity, as determined by c-Fos immunoreactivity, was increased after a hypoglycemic stimulus by 2-deoxyglucose (2DG). The highest ARC neuronal activity after 2DG was found at the end of the light period (zeitgeber 11, ZT11) with a lower activity in the beginning of the light period (zeitgeber 2, ZT2), suggesting the involvement of the SCN. The higher activation of ARC neurons after 2DG at ZT11 was associated with higher 2DG induced blood glucose levels as compared with ZT2. Unilateral SCN-lesioned animals, gave a mainly ipsilateral activation of ARC neurons at the lesioned side, suggesting an inhibitory role of the SCN on ARC neurons. The 2DG-induced counterregulatory glucose response correlated with increased ARC neuronal activity and was significantly higher in unilateral SCN-lesioned animals. Finally, the ARC as site where 2DG may, at least partly, induce a counterregulatory response was confirmed by local microdialysis of 2DG. 2DG administration in the ARC produced a higher increase in circulating glucose compared with 2DG administration in surrounding areas such as the ventromedial nucleus of the hypothalamus (VMH). We conclude that the SCN uses neuronal pathways to the ARC to gate sensory metabolic information to the brain, regulating ARC glucose sensitivity and counterregulatory responses to hypoglycemic conditions.

Drug discovery in skin pharmacotherapy is an enormous, continually expanding field. Researchers are developing novel and sensitive pharmaceutical products and drugs that target specific receptors to elicit concerted and appropriate responses. The pigment-bearing cells called melanophores have a significant contribution to make in this field. Melanophores, which contain the dark brown or black pigment melanin, constitute an important class of chromatophores. They are highly specialized in the bidirectional and coordinated translocation of pigment granules when given an appropriate stimulus. The pigment granules can be stimulated to undergo rapid dispersion throughout the melanophores, making the cell appear dark, or to aggregate at the center, making the cell appear light. The major signals involved in pigment transport within the melanophores are dependent on a special class of cell surface receptors called G-protein-coupled receptors (GPCRs). Many of these receptors of adrenaline, acetylcholine, histamine, serotonin, endothelin and melatonin have been found on melanophores. They are believed to have clinical relevance to skin-related ailments and therefore have become targets for high throughput screening projects. The selective screening of these receptors requires the recognition of particular ligands, agonists and antagonists and the characterization of their effects on pigment motility within the cells. The mechanism of skin pigmentation is incredibly intricate, but it would be a considerable step forward to unravel its underlying physiological mechanism. This would provide an experimental basis for new pharmacotherapies for dermatological anomalies. The discernible stimuli that can trigger a variety of intracellular signals affecting pigment granule movement primarily include neurotransmitters and hormones. This review focuses on the role of the hormone and neurotransmitter signals involved in pigment movement in terms of the pharmacology of the specific receptors.

Cyclic AMP (cAMP) is a ubiquitous second messenger that regulates a variety of biological processes. The magnitude and duration of cAMP expression are regulated by both production and hydrolysis. Melanocyte-stimulating hormone (MSH) plays a crucial role in pigment cell differentiation via cAMP-regulated expression of the master transcription factor MITF. We report the identification of phosphodiesterase 4D3 as a direct target of the MSH/cAMP/MITF pathway. This creates a negative feedback loop that induces refractoriness to chronic stimulation of the cAMP pathway in melanocytes. This homeostatic pathway highlights a potent mechanism controlling melanocyte differentiation that may be amenable to pharmacologic manipulation for skin cancer prevention.

Medetomidine is a new antifouling agent, and its effects in non-target aquatic organisms have been investigated. Earlier short-term studies in fish have shown a skin lightening response to medetomidine, but effects after chronic exposure have not been studied. In fish, the dark pigment melanin is contained within specialized cells, melanophores. Medetomidine binds to the melanophore alpha2-adrenoceptor, which stimulates pigment aggregation resulting in the light appearance. In the present study, rainbow trout (Oncorhynchus mykiss) was long-term exposed to 0.5 and 5.0 nM of medetomidine via water for 54 d. The fish were then photographed for paleness quantification and the images were analyzed using ImageJ analysis software. Additionally, scales were removed and used for in vitro function studies of the melanophores, monitoring the response to melanophore stimulating hormone (MSH) and subsequent medetomidine addition. The number of melanophores was also investigated. As a result of the medetomidine exposure, fish from the 5 nM treatment were significantly paler than control fish and the melanophores from these fishes were also more aggregated. Melanophores from all the treatments were functional, responding to MSH by dispersion and to subsequent medetomidine by aggregation. However, the results indicate a difference in sensitivity among treatments. The number of melanophores in the scales did not change significantly after long term exposure to medetomidine. These results suggest that the observed paleness may be reversible, even after chronic exposure., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Background & Aims: One of the challenges to treating inflammatory bowel disease (IBD) is to target the site of inflammation. We engineered nanoparticles (NPs) to deliver an anti-inflammatory tripeptide Lys-Pro-Val (KPV) to the colon and assessed its therapeutic efficacy in a mouse model of colitis., Methods: NPs were synthesized by double-emulsion/solvent evaporation. KPV was loaded into the NPs during the first emulsion of the synthesis process. To target KPV to the colon, loaded NPs (NP-KPV) were encapsulated into a polysaccharide gel containing 2 polymers: alginate and chitosan. The effect of KPV-loaded NPs on inflammatory parameters was determined in vitro as well as in the dextran sodium sulfate-induced colitis mouse model., Results: NPs (400 nm) did not affect cell viability or barrier functions. A swelling degree study showed that alginate-chitosan hydrogel containing dextran-fluorescein isothiocyanate-labeled NPs collapsed in the colon. Once delivered, NPs quickly released KPV on or within the closed area of colonocytes. The inflammatory responses to lipopolysaccharide were reduced in Caco2-BBE (brush border enterocyte) cells exposed to NP-KPV compared with those exposed to NPs alone, in a dose-dependent fashion. Mice given dextran sodium sulfate (DSS) followed by NP-KPV were protected against inflammatory and histologic parameters, compared with mice given only DSS., Conclusions: Nanoparticles are a versatile drug delivery system that can overcome physiologic barriers and target anti-inflammatory agents such as the peptide KPV to inflamed areas. By using NPs, KPV can be delivered at a concentration that is 12,000-fold lower than that of KPV in free solution, but with similar therapeutic efficacy. Administration of encapsulated drug-loaded NPs is a novel therapeutic approach for IBD., (Copyright 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Objective: The medial hypothalamus mediates leptin-induced glucose uptake in peripheral tissues, and brain melanocortin receptors (MCRs) mediate certain central effects of leptin. However, the contributions of the leptin receptor and MCRs in individual medial hypothalamic nuclei to regulation of peripheral glucose uptake have remained unclear. We examined the effects of an injection of leptin and the MCR agonist MT-II into medial hypothalamic nuclei on glucose uptake in peripheral tissues., Research Design and Methods: Leptin or MT-II was injected into the ventromedial (VMH), dorsomedial (DMH), arcuate nucleus (ARC), or paraventricular (PVH) hypothalamus or the lateral ventricle (intracerebroventricularly) in freely moving mice. The MCR antagonist SHU9119 was injected intracerebroventricularly. Glucose uptake was measured by the 2-[(3)H]deoxy-d-glucose method., Results: Leptin injection into the VMH increased glucose uptake in skeletal muscle, brown adipose tissue (BAT), and heart, whereas that into the ARC increased glucose uptake in BAT, and that into the DMH or PVH had no effect. SHU9119 abolished these effects of leptin injected into the VMH. Injection of MT-II either into the VMH or intracerebroventricularly increased glucose uptake in skeletal muscle, BAT, and heart, whereas that into the PVH increased glucose uptake in BAT, and that into the DMH or ARC had no effect., Conclusions: The VMH mediates leptin- and MT-II-induced glucose uptake in skeletal muscle, BAT, and heart. These effects of leptin are dependent on MCR activation. The leptin receptor in the ARC and MCR in the PVH regulate glucose uptake in BAT. Medial hypothalamic nuclei thus play distinct roles in leptin- and MT-II-induced glucose uptake in peripheral tissues.

The anorexigenic neuromodulator alpha-melanocyte-stimulating hormone (alpha-MSH; referred to here as alpha-MSH1-13) undergoes extensive posttranslational processing, and its in vivo activity is short lived due to rapid inactivation. The enzymatic control of alpha-MSH1-13 maturation and inactivation is incompletely understood. Here we have provided insight into alpha-MSH1-13 inactivation through the generation and analysis of a subcongenic mouse strain with reduced body fat compared with controls. Using positional cloning, we identified a maximum of 6 coding genes, including that encoding prolylcarboxypeptidase (PRCP), in the donor region. Real-time PCR revealed a marked genotype effect on Prcp mRNA expression in brain tissue. Biochemical studies using recombinant PRCP demonstrated that PRCP removes the C-terminal amino acid of alpha-MSH1-13, producing alpha-MSH1-12, which is not neuroactive. We found that Prcp was expressed in the hypothalamus in neuronal populations that send efferents to areas where alpha-MSH1-13 is released from axon terminals. The inhibition of PRCP activity by small molecule protease inhibitors administered peripherally or centrally decreased food intake in both wild-type and obese mice. Furthermore, Prcp-null mice had elevated levels of alpha-MSH1-13 in the hypothalamus and were leaner and shorter than the wild-type controls on a regular chow diet; they were also resistant to high-fat diet-induced obesity. Our results suggest that PRCP is an important component of melanocortin signaling and weight maintenance via control of active alpha-MSH1-13 levels.

The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor mainly expressed in the central nervous system of vertebrates. Activation of the MC4R leads to a decrease in food intake, whereas inactivating mutations are a genetic cause of obesity. The binding of agouti-related protein (AGRP) reduces not only agonist-stimulated cAMP production (competitive antagonist) but also the basal activity of the receptor, as an inverse agonist. Transgenic zebrafish overexpressing AGRP display increased food intake and linear growth, indicative of a physiological role for the melanocortin system in the control of the energy balance in fish. We report on the cloning, pharmacological characterization, tissue distribution, and detailed brain mapping of a sea bass (Dicentrarchus labrax) MC4R ortholog. Sea bass MC4R is profusely expressed within food intake-controlling pathways of the fish brain. However, the activity of the melanocortin system during progressive fasting does not depend on the hypothalamic/pituitary proopiomelanocortin (POMC) and MC4R expression, which suggests that sea bass MC4R is constitutively activated and regulated by AGRP binding. We demonstrate that AGRP acts as competitive antagonist and reduces MTII-induced cAMP production. AGRP also decreases the basal activity of the receptor as an inverse agonist. This observation suggests that MC4R is constitutively active and supports the evolutionary conservation of the AGRP/MC4R interactions. The inverse agonism, but not the competitive antagonism, depends on the presence of a phosphodiesterase inhibitor (IBMX). This suggests that inverse agonism and competitive antagonism operate through different intracellular signaling pathways, a view that opens up new targets for the treatment of melanocortin-induced metabolic syndrome.

Background Aims: The non-exudative form of age-related macular degeneration (ARMD) is characterized by a progressive decay of retinal pigment epithelium cells at the posterior pole of the eye. As mesenchymal stromal cells (MSC) have been shown to differentiate into various cell types from the mesodermal and ectodermal lineages, we investigated whether we can induce a phenotype displaying retinal pigment epithelium (RPE) characteristics., Methods: The differentiation of human lipo-aspirate-derived MSC toward the RPE lineage was triggered by exposure to conditioned medium from either human or porcine RPE cells. In a second approach we tested whether adding vasoactive intestinal peptide (VIP) is capable of further modifying differentiation processes. Resulting cell populations were assessed for expression of RPE-specific markers by immunofluorescence, quantitative real time (RT)-polymerase chain reaction (PCR) and Western blotting. The potential for pigment synthesis was assessed by the response to melanocyte-stimulating hormone (MSH)., Results: Following culture of undifferentiated MSC with RPE-conditioned medium and/or VIP, expression of typical RPE markers bestrophin, cytokeratins 8 and 18 and RPE 65 was induced. MSH induced the formation of pigmented granula in differentiated MSC., Conclusions: MSC are shown to express RPE markers upon induction with either RPE-conditioned medium and/or VIP. The gain of basic functional features of RPE cells was indicated by melanin synthesis. This alludes to a differentiation potential of MSC into the neuroectodermal lineage, yielding cells with phenotypic characteristics of RPE cells.

Disruptions of the melanocortin signaling system have been linked to obesity. We investigated a possible role of the central nervous melanocortin system (CNS-Mcr) in the control of adiposity through effects on nutrient partitioning and cellular lipid metabolism independent of nutrient intake. We report that pharmacological inhibition of melanocortin receptors (Mcr) in rats and genetic disruption of Mc4r in mice directly and potently promoted lipid uptake, triglyceride synthesis, and fat accumulation in white adipose tissue (WAT), while increased CNS-Mcr signaling triggered lipid mobilization. These effects were independent of food intake and preceded changes in adiposity. In addition, decreased CNS-Mcr signaling promoted increased insulin sensitivity and glucose uptake in WAT while decreasing glucose utilization in muscle and brown adipose tissue. Such CNS control of peripheral nutrient partitioning depended on sympathetic nervous system function and was enhanced by synergistic effects on liver triglyceride synthesis. Our findings offer an explanation for enhanced adiposity resulting from decreased melanocortin signaling, even in the absence of hyperphagia, and are consistent with feeding-independent changes in substrate utilization as reflected by respiratory quotient, which is increased with chronic Mcr blockade in rodents and in humans with loss-of-function mutations in MC4R. We also reveal molecular underpinnings for direct control of the CNS-Mcr over lipid metabolism. These results suggest ways to design more efficient pharmacological methods for controlling adiposity.

We have characterized the biochemical function of the melanocortin 1 receptor (MC1R), a critical regulator of melanin synthesis, from 9 phylogenetically diverse primate species with varying coat colors. There is substantial diversity in melanocyte-stimulating hormone (MSH) binding affinity and basal levels of activity in the cloned MC1Rs. MSH binding was lost independently in lemur and New World monkey lineages, whereas high basal levels of MC1R activity occur in lemurs and some New World monkeys and Old World monkeys. Highest levels of basal activity were found in the MC1R of ruffed lemurs, which have the E94K mutation that leads to constitutive activation in other species. In 3 species (2 lemurs and the howler monkey), we report the novel finding that binding and inhibition of MC1R by agouti signaling protein (ASIP) can occur when MSH binding has been lost, thus enabling continuing regulation of the melanin type via ASIP expression. Together, these findings can explain the previous paradox of a predominantly pheomelanic coat in the red ruffed lemur (Varecia rubra). The presence of a functional, MSH-responsive MC1R in orangutan demonstrates that the mechanism of red hair generation in this ape is different from the prevalent mechanism in European human populations. Overall, we have found unexpected diversity in MC1R function among primates and show that the evolution of the regulatory control of MC1R activity occurs by independent variation of 3 distinct mechanisms: basal MC1R activity, MSH binding and activation, and ASIP binding and inhibition. This diversity of function is broadly associated with primate phylogeny and does not have a simple relation to coat color phenotype within primate clades.

Ultraviolet-light (UV)-induced tanning is defective in numerous 'fair-skinned' individuals, many of whom contain functional disruption of the melanocortin 1 receptor (MC1R). Although this suggested a critical role for the MC1R ligand melanocyte stimulating hormone (MSH) in this response, a genetically controlled system has been lacking in which to determine the precise role of MSH-MC1R. Here we show that ultraviolet light potently induces expression of MSH in keratinocytes, but fails to stimulate pigmentation in the absence of functional MC1R in red/blonde-haired Mc1r(e/e) mice. However, pigmentation could be rescued by topical application of the cyclic AMP agonist forskolin, without the need for ultraviolet light, demonstrating that the pigmentation machinery is available despite the absence of functional MC1R. This chemically induced pigmentation was protective against ultraviolet-light-induced cutaneous DNA damage and tumorigenesis when tested in the cancer-prone, xeroderma-pigmentosum-complementation-group-C-deficient genetic background. These data emphasize the essential role of intercellular MSH signalling in the tanning response, and suggest a clinical strategy for topical small-molecule manipulation of pigmentation.

Leptin and insulin share some hypothalamic signaling molecules, but their central administration induces different effects on hepatic glucose fluxes. Acute insulin infusion in the third cerebral ventricle inhibits endogenous glucose production (GP), whereas acute leptin infusion stimulates gluconeogenesis but does not alter GP because of a compensatory decrease in glycogenolysis. Because melanocortin agonists also stimulate hepatic gluconeogenesis, here we examined whether central melanocortin blockade modifies the acute effects of leptin on GP, on gluconeogenesis, on glycogenolysis, and/or on the hepatic expression of the gluconeogenic enzymes glucose-6-phosphatase (Glc-6-Pase) and phosphoenolpyruvate carboxykinase (PEPCK). Systemic or central administration of leptin alone did not alter GP, despite increasing both the rate of gluconeogenesis and the expression of Glc-6-Pase and PEPCK. When activation of the central melanocortin pathway was prevented, the effects of leptin on gluconeogenesis, Glc-6-Pase, and PEPCK were abolished, and a marked suppression of glycogenolysis resulted in decreased GP. We conclude that leptin regulates hepatic glucose fluxes through a melanocortin-dependent pathway leading to stimulation of gluconeogenesis and a melanocortin-independent pathway causing inhibition of GP and glycogenolysis.

The sensory neuron-specific G protein coupled receptors (SNSRs) have been described as a family of receptors whose expression in small diameter sensory neurons in the trigeminal and dorsal root ganglia suggests an implication in nociception. To date, the physiological function(s) of SNSRs remain unknown. Hence, the aim of the present study was to determine the effects of rat SNSR1 activation on nociception in rats. The pharmacological characterization of rat SNSR1 was initially performed in vitro to identify a specific ligand, which could be used subsequently in the rat for physiological testing. Among all ligands tested, gamma2-MSH was the most potent at activating rat SNSR1. Structure-activity relationship studies revealed that the active moiety recognized by rat SNSR1 was the C-terminal part of gamma2-MSH. The radiolabeled C-terminal part of gamma2-MSH, gamma2-MSH-6-12, bound with high affinity to membranes derived from rat skin and spinal cord, demonstrating the presence of receptor protein at both the proximal and distal terminals of dorsal root ganglia. To investigate the physiological role of SNSR, specific ligands to rat SNSR1 were tested in behavioral assays of pain sensitivity in rats. Selective rat SNSR1 agonists produced spontaneous pain behavior, enhanced heat and mechanical sensitivity when injected intradermally, and heat hypersensitivity when injected centrally, consistent with the localization of rat SNSR1 protein at central and peripheral sites. Together, these results clearly indicate that the SNSR1 plays a role in nociception and may provide novel therapeutic opportunities for analgesia.

Background: Plasminogen-deficient mice exhibit behavioral differences in response to stress, including a markedly reduced acoustic startle reflex response compared with wild-type (WT) littermates. The acoustic startle reflex activates the hypothalamic-pituitary axis and is modulated by these hormones., Objectives: The purpose of this study was to investigate whether plasminogen plays a role in the processing of hormones in the hypothalamic-pituitary axis., Methods: In this study the concentration of plasma, pituitary, and brain hypothalamic-pituitary axis hormones and precursor processing was examined in WT and plasminogen deficient (Plg-/-) mice before and after acoustic startle reflex testing., Results: Plasma adrenocorticotropic hormone (ACTH), beta-endorphin and alpha-melanocyte stimulating hormone were elevated after acoustic startle reflex testing in both WT and (Plg-/-) mice. However, in the Plg-/- mice, beta-endorphin values were 43, 35, and 45% lower in the plasma, pituitary, and whole brain, respectively, compared with the WT mice. Plasmin readily degraded precursor peptides, the 23-kDa precursor, beta-lipotropin, and ACTH, when presented as purified proteins or as the secretory products of mouse pituitary cells (AtT-20). The precursor peptide, 23 kDa, for beta-endorphin and alpha-melanocyte stimulating hormone was reduced in the pituitaries from the Plg-/- mice, and the mRNA for Plg was found in pituitaries from WT mice. Infusion of beta-endorphin and alpha-melanocyte stimulating hormone into the brain of Plg-/- mice increased acoustic startle reflex., Conclusions: The results of this study show that plasmin is involved in the processing of hormones derived from the pro-opiomelanocortin precursor in the intermediate pituitary. A deficiency of plasminogen reduces processing of beta-endorphin and alpha-melanocyte stimulating hormone, and interferes with normal brain function.

alpha-MSH signals by binding to the melanocortin-1 receptor (MC-1R) and elevating cyclic AMP in several different cells. The anti-inflammatory properties of this peptide are also believed to be cyclic AMP dependent. The carboxyl terminal tripeptides of alpha-MSH (KPV / KP-D-V) are the smallest minimal sequences reported to prevent inflammation but it is not known if they operate via MC-1R or cyclic AMP. The aim of this study was to examine the intracellular signalling of key MSH and ACTH peptides in human keratinotocytes. No elevation in cyclic AMP was detected in either HaCaT or normal human keratinocytes in response to alpha-MSH, KPV or ACTH peptides. Rapid and acute intracellular calcium, however, were observed in HaCaT keratinocytes in response to alpha-MSH (10(-15)-10(-7) M), KPV (10(-15)-10(-7) M), KP-D-V (10(-15)-10(-7) M) and ACTH (10(-15)-10(-7) M), but only in the presence of PIA, an adenosine agonist that inhibits the cyclic AMP pathway. Normal keratinocytes responded to all the above peptides but in addition responded to ACTH 1-17 (10(-13)-10(-7) M) in contrast to the HaCaT keratinocytes. Stable transfection of Chinese hamster ovary cells with the MC-1 receptor showed that alpha-MSH and the KPV peptides elevated intracellular calcium.

We have prepared fresh pituitary gland slices from adult and, for the first time, from newborn mice to assess modulation of secretory activity via voltage-activated Ca(2+) channels (VACCs). Currents through VACCs and membrane capacitance have been measured with the whole-cell patch-clamp technique. Melanotrophs in newborns were significantly larger than in adults. In both newborn and adult melanotrophs activation of VACCs triggered exocytosis. All pharmacologically isolated VACC types contributed equally to the secretory activity. However, the relative proportion of VACCs differed between newborns and adults. In newborn cells L-type channels dominated and, in addition, an exclusive expression of a toxin-resistant R-type-like current was found. The expression of L-type VACCs was up-regulated by the increased oestrogen levels observed in females, and was even more emphasized in the cells of pregnant females and oestrogen-treated adult male mice. We suggest a general mechanism modulating endocrine secretion in the presence of oestrogen and particularly higher sensitivity to treatments with L-type channel blockers during high oestrogen physiological states.

Background: The authors recently demonstrated that administration of the melanocortin-4 receptor antagonist SHU9119 decreased neuropathic pain symptoms in rats with a sciatic chronic constriction injury. The authors hypothesised that there is a balance between tonic pronociceptive effects of the spinal melanocortin system and tonic antinociceptive effects of the spinal opioid system. Therefore, they investigated a possible interaction between these two systems and tested whether opioid effectiveness could be increased through modulation of the spinal melanocortin system activity., Methods: In chronic constriction injury rats, melanocortin and opioid receptor ligands were administered through a lumbar spinal catheter, and their effects on mechanical allodynia were assessed by von Frey probing., Results: Naloxone (10-100 microg) dose-dependently increased allodynia (percent of maximum possible effect of -67 +/- 9%), which is in agreement with a tonic antinociceptive effect of the opioid system. SHU9119 decreased allodynia (percent of maximum possible effect of 60 +/- 13%), and this effect could be blocked by a low dose of naloxone (0.1 microg), which by itself had no effect on withdrawal thresholds. Morphine (1-10 microg) dose-dependently decreased allodynia (percent of maximum possible effect of 73 +/- 14% with the highest dose tested). When 0.5 microg SHU9119 (percent of maximum possible effect of 47 +/- 14%) was given 15 min before morphine, there was an additive antiallodynic effect of both compounds., Conclusions: Together, these data confirm that there is an interaction between the spinal melanocortin and opioid systems and that combined treatment with melanocortin-4 receptor antagonists and opioids might possibly contribute to the treatment of neuropathic pain.

We used the cell-attached mode of patch-clamp technique to measure discrete attofarad steps in membrane capacitance (C(m)), reporting area changes in the plasma membrane due to unitary exocytic and endocytic events. To investigate the role of the actin cytoskeleton in elementary exocytic and endocytic events, neuroendocrine rat melanotrophs were treated with Clostridium spiroforme toxin (CST), which specifically depolymerises F-actin. The average amplitude of exocytic events was not significantly different in control and in CST-treated cells. However, the amplitude of endocytic events was significantly smaller in CST-treated cells as compared to controls. The frequency of exocytic events increased by 2-fold in CST-treated cells relative to controls. In control cells the average frequency of exocytic events (upsilon;(exo)) was lower than the frequency of endocytic events (upsilon;(endo)) with a ratio upsilon;(exo)/upsilon;(endo) < 1. In the toxin treated cells, the predominant process was exocytosis with a ratio (upsilon;(exo)/upsilon;(endo) > 1). To study the coupling between the two processes, the slopes of regression lines relating upsilon;(exo) and upsilon;(endo) in a given patch of membrane were studied. The slopes of regression lines were similar, whereas the line intercepts with the y-axis were significantly different. The increased frequency of unitary exocytic events in CST-treated cells is consistent with the view, that the actin cytoskeleton acts as a barrier for exocytosis. While the disassembly of the actin cytoskeleton diminishes the size of unitary endocytic events, suggesting an important role of the actin cytoskeleton in determining the size of endocytic vesicles, the coupling between exocytosis and endocytosis in a given patch of membrane was independent of the state of the actin cytoskeleton.

Like leptin, the pancreatic hormone insulin is an important adiposity signal to the brain. We report that the hypothalamic melanocortin system is an important target of the actions of insulin to regulate food intake and body weight. Hypothalamic neurons expressing insulin receptors were found to coexpress the melanocortin precursor molecule pro-opiomelanocortin (POMC), and administration of insulin into the third cerebral ventricle of fasted rats increased expression of POMC mRNA. Finally, a subthreshold dose of the melanocortin antagonist SHU-9119 prevented the reduction in food intake caused by third-ventricular insulin administration. These data suggest that the hypothalamic melanocortin system mediates the anorexic effects of central insulin, as well as of leptin.

The melanocortin receptors, MC3R and MC4R, are G protein-coupled receptors that are involved in regulating energy homeostasis. Using a luciferase reporter gene under the transcriptional control of a cAMP- responsive element (CRE), the coupling efficiency of the MC4R and MC3R to G-proteins was previously shown to be different. MC4R exhibited only 30-50% of the maximum activity induced by MC3R. To assess the role of the different MC3R and MC4R domains in G-protein coupling, several chimeric MC3R/MC4R receptors were constructed. The relative luciferase activities, which were assessed after transfecting the chimeric receptors into HEK 293T cells, showed that the i3 (3rd intracellular) loop domain has an essential role in the differential signaling of MC3R and MC4R. To reveal which amino acid residue was involved in the MC4R-specific signaling in the i3 loop, a series of mutant MC4Rs was constructed. Reporter gene analysis showed that single mutations of Arg(220) to Ala and Thr(232) to either Val or Ala increased the relative luciferase activities, which suggests that these specific amino acids, Arg(220) and Thr(232), in the i3 loop of MC4R play crucial roles in G-protein coupling and the subtype-specific signaling pathways. An examination of the inositol phosphate (IP) levels in the cells transfected with either MC3R or MC4R after being exposed to the melanocortin peptides revealed significant stimulation of IP production by MC3R but no detectable increase in IP production was observed by MC4R. Furthermore, none of the MC4R mutants displayed melanocortin peptide-stimulated IP production. Overall, this study demonstrated that MC3R and MC4R have distinct signaling in either the cAMP- or the inositol phospholipid-mediated pathway with different conformational requirements.

In the lamprey, adrenocorticotropin (ACTH) and melanotropins (MSHs) are produced from two distinct precursors, proopiocortin (POC) and proopiomelanotropin (POM). Both POC and POM have been suggested to be glycoproteins. The present study aimed to demonstrate glycoconjugates in ACTH and MSH cells in the pituitary of adult sea lampreys (Petromyzon marinus) by means of a lectin histochemistry. A total of 19 kinds of lectins were tested. ACTH cells were distributed in both the rostral pars distalis and the proximal pars distalis, and were stained positively with N-acetylglucosamine binding lectins (i.e., succinylated wheat germ agglutinin), N-acetylgalactosamine binding lectins (i.e., soybean agglutinin), D-mannose binding lectins (i.e., Lens culinaris agglutinin), and D-galactose binding lectins (i.e., Erythrina cristagall lectin). MSH cells were distributed in the pars intermedia, and were stained with N-acetylgalactosamine binding lectins (i.e., Dolichos biflorus agglutinin), D-mannose binding lectin (Pisum sativum agglutinin) and D-galactose binding lectins (i.e., peanut agglutinin). These results suggested that ACTH and MSH cells produce different types of glycoconjugates which may be attributed to the difference in glycoconjugate moieties between the precursor proteins, POC and POM.

Leptin and insulin are distinct adiposity signals that regulate food intake and body weight. Because recent evidence suggests that the central catabolic action of each is mediated by the hypothalamic melanocortin system, we tested the hypothesis that leptin and insulin interact within the brain, either additively or synergistically, to suppress food intake and reduce body weight. Using a within-subjects design, we co-administered leptin and insulin into the 3rd cerebral ventricle (i.c.v.) over a wide range of doses, and compared the combined effects to what occurred following the administration of each peptide alone. The data suggest that leptin and insulin interact sub-additively to regulate food intake and body weight over the first few hours. That is, the ability of combinations of leptin and insulin to reduce food intake and body weight was less than what would be predicted by the sum of their independent actions. Over 24 hours, however, the combined doses fit a strictly additive model. These data therefore imply a redundancy in the functional intracellular pathways or neuronal circuits that leptin and insulin utilize in the acute regulation of food intake and body weight, and they further imply that over time, the redundancy dissipates.

The published data for six melanocortin peptides binding to wild-type and chimeric melanocortin MC(1)/MC(3) receptors were analysed using the novel proteo-chemometrics modelling approach. The chimeric receptors and the peptides were coded using binary descriptors and used to correlate with the experimental data for affinity or selectivity for peptides binding to receptors. Correlations were achieved using partial least squares projection to latent structures (PLS) and statistically valid models were obtained. The models were further improved by adding cross-terms and applying orthogonal signal correction. The models were validated using external prediction, with half of the data being excluded from the modelling. Interpretation of the results using PLS coefficient plots revealed that the binding pocket for the melanocortins is located between the first, second, third, sixth and seventh transmembrane regions of the melanocortin receptors, in good agreement with previous three-dimensional models for the interactions of melanocortins with melanocortin receptors. Further, analysis of cross-terms between peptide descriptors indicated that the proteo-chemometrics modelling is able to distinguish between differences in the conformational space of the peptides that affect binding affinity and selectivity.

Melanocortins play a critical role in appetite and body weight regulation, because manipulations of this pathway can lead to the development of obesity in several animal models. The purpose of this study was to use a melanocortin receptor agonist and antagonist to evaluate the involvement of melanocortins in feeding, energy metabolism, and body weight regulation in lean and obese Zucker rats. Central administration of a melanocortin receptor antagonist (SHU9119) elevated food intake and body weight of lean Zucker rats but had little effect in obese Zucker rats. In contrast, the melanocortin receptor agonist MTII reduced food intake in both lean and obese rats but was more potent in the obese Zucker rats. These data indicate the existence of functional melanocortin receptors in both lean and obese Zucker rats but suggest that obese Zucker rats have reduced endogenous melanocortin tone. In addition to its effects on food intake, MTII infusion elevated oxygen consumption and decreased respiratory quotient dose dependently during the light cycle. Our data suggest that a melanocortin receptor agonist can induce weight loss by increasing energy expenditure and promoting body fat utilization in addition to its inhibitory effects on food intake in both obese and lean Zucker rats.

The native hormone alpha-melanocyte-stimulating hormone (alpha-MSH) and its more potent analog [Nle(4),D-Phe(7)]alpha-MSH (NDP-alpha MSH), labeled at the amino terminal with the fluorescent aminobenzoic acid (Abz) isomers, were examined by fluorescence methods. We observed energy transfer between the tryptophan(9) residue acting as donor and Abz as acceptor, the transfer being more pronounced to the ortho-form of the acceptor. Within the hypothesis that different peptide conformations coexist in equilibrium during the fluorescence decay, we supposed that the intensity decay was modulated by an acceptor-donor distance distribution function f(r). From the time-resolved fluorescence experimental data, we recovered the distance distribution between Abz and Trp(9), using the CONTIN program, within the framework of the Förster resonance energy transfer model. The methodology proved to be useful to provide quantitative information about conformational dynamics of melanotropins and its dependency on the solvent. In aqueous medium, alpha-MSH has a broad Abz-Trp(9) distance distribution, reflecting the structural flexibility of the peptide. Three different distance populations could be identified in the labeled analog NDP-alpha MSH in water, indicating distinct conformational states for the synthetic peptide, compared with the native hormone. Measurements in trifluoroethanol resulted in the recovery of two Abz-Trp(9) distance populations, both for the native and the analog hormones, reflecting the decrease, induced by the solvent, of the conformational states available to the peptides.

The adrenal gland requires stimuli from peptides derived from the ACTH precursor, pro-opiomelanocortin (POMC), to maintain its tonic state. Studies have proposed that a specific postsecretional cleavage of the nonmitogenic N-terminal 16 kDa fragment, also known as pro-gamma-melanotropin (pro-gamma-MSH), is required, releasing shorter fragments that promote adrenal growth. Here, we provide evidence for this hypothesis by the cloning and characterization of a serine protease that is upregulated during growth of the adrenal cortex. It is expressed exclusively in the outer adrenal cortex, the site of cell proliferation, and in the Y1 adrenal cell line. We also show that it is required for growth of Y1 cells, remains bound to the cell surface, and cleaves its substrate, pro-gamma-MSH, at a specific bond.

Nitric oxide (NO) is implicated in the regulation of various endocrine functions, but the effect of NO on GABA(A) receptor transmission has never been reported in endocrine cells. In the present study, we have investigated the effects of various agents acting on the NO transduction pathway on GABA(A) receptor function in frog pituitary melanotrophs. Histochemical studies using the NADPH-diaphorase reaction and immunohistochemical labeling with antibodies against neuronal NO synthase (nNOS) revealed that nNOS is expressed in the intermediate lobe of the pituitary and in cultured melanotrophs. Whole-cell patch-clamp recordings showed that the specific substrate of NOS L-arginine (L-Arg, 10(-4) M) or the NO donor sodium nitroprusside (10(-5) M) provoked a long-lasting inhibition of the current evoked by GABA (5 x 10(-6) M). The NOS inhibitor L-nitroarginine (10(-5) M) produced a biphasic effect, i.e. a transient decrease followed by a delayed increase of the GABA-evoked current amplitude. Similarly, the specific nNOS inhibitor 7-nitroindazole and the specific inducible NOS (iNOS) inhibitor aminoguanidine (10(-5) M each) provoked a transient depression of the current followed by a sustained potentiation. Formation of cGMP in neurointermediate lobes was enhanced by L-Arg (10(-4) M) and by the calcium-releasing agent caffeine (10(-4) M), and inhibited by the calmodulin (CaM)/Ca2+ complex blocker W7 (10(-5) M). The GABA-evoked current was potentiated by the guanylyl cyclase inhibitor ODQ (10(-8)-10(-7) M) and inhibited by the protein kinase G (PKG) activator 8pCPT-cGMP (3 x 10(-7)-3 x 10(-5) M). The present data indicate that NO, produced by a CaM/Ca2+-dependent NOS in frog melanotrophs, exerts an autocrine inhibitory effect on the GABA-evoked current. The action of NO on the GABA(A) receptor function is mediated through activation of the cGMP/PKG pathway.

Chloride redistribution during type A gamma-aminobutyric acid (GABA(A)) currents (I(GABA)) has been investigated in cultured frog pituitary melanotrophs with imposed intracellular chloride concentration ([Cl(-)](i)) in the whole cell configuration or with unaltered [Cl(-)](i) using the gramicidin-perforated patch approach. Prolonged GABA exposures elicited reproducible decaying currents. The decay of I(GABA) was associated with both a transient fall of conductance (g(GABA)) and shift of current reversal potential (E(GABA)). The shift of E(GABA) appeared to be time and driving force dependent. In the gramicidin-perforated patch configuration, repeated GABA exposures induced currents that gradually vanished. The fading of I(GABA) was due to persistent shifts of E(GABA) as a result of g(GABA) recovering from one GABA application to another. In cells alternatively clamped at potentials closely flanking resting potential and submitted to a train of brief GABA pulses, a reversal of I(GABA) was observed after 150 s recording. It is demonstrated that, in intact frog melanotrophs, shifts of E(GABA) combine with genuine receptor desensitization to depress I(GABA). These findings strongly suggest that shifts of E(GABA) may act as a negative feedback, reducing the bioelectrical and secretory responses induced by an intense release of GABA in vivo.

Melanocortin peptides regulate a variety of physiological processes. Five melanocortin receptors (MC-R) have been cloned and the MC3R and MC4R are the main brain MC receptors. The aim of this study was to identify structural requirements in both ligand and receptor that determine gamma-melanocyte-stimulating hormone (MSH) selectivity for the MC3R versus the MC4R. Substitution of Asp10 in [Nle4]Lys-gamma2-MSH for Gly10 from [Nle4]alpha-MSH, increased both activity and affinity for the MC4R while the MC3R remained unaffected. Analysis of chimeric MC3R/MC4Rs and mutant MC4Rs showed that Tyr268 of the MC4R mainly determined the low affinity for [Nle4]Lys-gamma2-MSH. The data demonstrate that Asp10 determines selectivity for the MC3R, however, not through direct side chain interactions, but probably by influencing how the melanocortin core sequence is presented to the receptor-binding pocket. This is supported by mutagenesis of Tyr268 to Ile in the MC4R which increased affinity and activity for [Nle4]Lys-gamma2-MSH, but decreased affinity for two peptides with constrained cyclic structure of the melanocortin core sequence, MT-II and [D-Tyr4]MT-II, that also displayed lower affinity for the MC3R. This study provides a general concept for peptide receptor selectivity, in which the major determinant for a selective receptor interaction is the conformational presentation of the core sequence in related peptides to the receptor-binding pocket.

Immunohistochemical staining of human skin specimen showed the stronger localization of proopiomelanocortin peptides near the suprabasal layer of the epidermis, where keratinocytes are mostly differentiated. To test the possibilities of whether the production of proopiomelanocortin peptides or their receptor-binding activity or both is increased during differentiation of keratinocytes, we treated the cells in culture with Ca2+ to induce their differentiation. The production of proopiomelanocortin peptides and its gene expression were not induced significantly, but the binding ability of melanocortin receptor, as well as its gene expression were stimulated by Ca2+. Ultraviolet B irradiation, an inducer of differentiation, stimulated both proopiomelanocortin production and melanocortin receptor expression. These data show that normal human keratinocytes express melanocortin receptor similar to melanocytes, and that it is induced during differentiation.

1. We designed and synthesized several novel cyclic MSH analogues and tested their affinities for cells expressing the MC1, MC3, MC4 and MC5 receptors. 2. One of the substances HS028 (cyclic [AcCys11, dichloro-D-phenylalanine14, Cys18, Asp-NH2(22)]-beta-MSH11-22) showed high affinity (Ki of 0.95nM) and high (80 fold) MC4 receptor selectivity over the MC3 receptor. HS028 thus shows both higher affinity and higher selectivity for the MC4 receptor compared to the earlier first described MC4 receptor selective substance HS014. 3. HS028 antagonised a alpha-MSH induced increase in cyclic AMP production in transfected cells expressing the MC3 and MC4 receptors, whereas it seemed to be a partial agonist for the MC1 and MC5 receptors. 4. Chronic intracerebroventricularly (i.c.v.) administration of HS028 by osmotic minipumps significantly increased both food intake and body weight in a dose dependent manner without tachyphylaxis for a period of 7 days. 5. This is the first report demonstrating that an MC4 receptor antagonist can increase food intake and body weight during chronic administration providing further evidence that the MC4 receptor is an important mediator of long term weight homeostasis.

Inflammatory processes contribute to neurodegenerative disease, stroke, encephalitis, and other central nervous system (CNS) disorders. Activated microglia are a source of cytokines and other inflammatory agents within the CNS and it is therefore important to control glial function in order to preserve neural cells. Melanocortin peptides are pro-opiomelanocortin-derived amino acid sequences that include alpha-melanocyte-stimulating hormone (alpha-MSH) and adrenocorticotropic hormone (ACTH). These peptides have potent and broad anti-inflammatory effects. We tested effects of alpha-MSH (1-13), alpha-MSH (11-13), and ACTH (1-24) on production of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and nitric oxide (NO) in a cultured murine microglial cell line (N9) stimulated with lipopolysaccharide (LPS) plus interferon gamma (IFN-gamma). Melanocortin peptides inhibited production of these cytokines and NO in a concentration-related fashion, probably by increasing intracellular cAMP. When stimulated with LPS + IFN-gamma, microglia increased release of alpha-MSH. Production of TNF-alpha, IL-6, and NO was greater in activated microglia after innmunoneutralization of endogenous alpha-MSH. The results suggest that alpha-MSH is an autocrine factor in microglia. Because melanocortin peptides inhibit production of pro-inflammatory mediators by activated microglia they might be useful in treatment of inflammatory/degenerative brain disorders.

1. We synthesized a novel series of cyclic melanocyte stimulating hormone (MSH) analogues and tested their binding properties on cells transiently expressing the human melanocortin1 (MC1), MC3, MC4 and MC5 receptors. 2. We discovered that compounds with 26 membered rings of [Cys4,D-Nal7,Cys11]alpha-MSH(4-11) displayed specific MC4 receptor selectivity. The preference order of the different MC receptor subtypes for the novel [Cys4D-Nal7Cys11]alpha-MSH(4-11) analogues are distinct from all other known MSH analogues, particularly as they bind the MC4 receptor with high and the MC1 receptor with low relative affinities. 3. HS964 and HS014 have 12 and 17 fold MC4/MC3 receptor selectivity, respectively, which is much higher than for the previously described cyclic lactam and [Cys4,Cys10]alpha-MSH analogues SHU9119 and HS9510. 4. HS964 is the first substance showing higher affinity for the MC5 receptor than the MC1 receptor. 5. HS014, which was the most potent and selective MC4 receptor ligand (Ki 3.2 nM, which is approximately 300 fold higher affinity than for alpha-MSH), was also demonstrated to antagonize alpha-MSH stimulation of cyclic AMP in MC4 receptor transfected cells. 6. We found that a compound with a 29 membered ring of [Cys3,Nle10,D-Nal7,Cys11]alpha-MSH(3-11) (HS010) had the highest affinity for the MC3 receptor. 7. This is the first study to describe ligands that are truly MC4 selective and a ligand having a high affinity for the MC3 receptor. The novel compounds may be of use in clarifying the physiological roles of the MC3, MC4 and MC5 receptors.

1. In frog pituitary melanotrophs, GABA induces a transient stimulation followed by prolonged inhibition of hormone secretion. This biphasic effect is inconsistent with the elevation of cytosolic calcium and the inhibition of electrical activity also provoked by GABA in single melanotrophs. In the present study, standard patch-clamp configurations and gramicidin-perforated patches were used to investigate the physiological GABAA receptor-mediated response and intracellular chloride concentration ([Cl-]i) in cultured frog melanotrophs. 2. In the gramicidin-perforated patch configuration, 1 microM GABA caused a depolarization associated with an action potential discharge and a slight fall of membrane resistance. In contrast, at a higher concentration (10 microM) GABA elicited a depolarization accompanied by a transient volley of action potentials, followed by a sustained inhibitory plateau and a marked fall of membrane resistance. Isoguvacine mimicked the GABA-evoked responses, indicating a mediation by GABAA receptors. 3. In gramicidin-perforated cells, the depolarizing excitatory effect of 1 microM GABA was converted into a depolarizing inhibitory action when 0.4 microM allopregnanolone was added to the bath solution. 4. After gaining the whole-cell configuration, the amplitude and/or direction of the GABA-evoked current (IGABA) rapidly changed before stabilizing. After stabilization, the reversal potential of IGABA followed the values predicted by the Nernst equation for chloride ions when [Cl-]i was varied. 5. In gramicidin-perforated cells, the steady-state I-V relationships of 10 microM GABA- or isoguvacine-evoked currents yielded reversal potentials of -37.5 +/- 1.6 (n = 17) and -38.6 +/- 2.0 mV (n = 8), respectively. These values were close to those obtained by using a voltage-ramp protocol in the presence of Na+, K+ and Ca2+ channel blockers. The current evoked by 1 microM GABA also reversed at these potentials. 6. We conclude that, in frog pituitary melanotrophs, chloride is the exclusive charge carrier of IGABA. In intact cells, the reversal potential of IGABA is positive to the resting potential because of a relatively high [Cl-]i (26.5 mM). Under these conditions, GABA induces a chloride efflux responsible for a depolarization triggering action potentials. However, GABA at a high concentration or in the presence of the potentiating steroid allopregnanolone exerts a concomitant shunting effect leading to a rapid inhibition of the spontaneous firing.

The non-homologous N-terminal regions of four human melanocortin (MC) receptors were truncated in order to investigate their putative participation in ligand binding. Eleven constructs were made, where different numbers of residues from the N terminus were deleted. These constructs were used for transient expression experiments in COS cells and analysed by ligand binding. The results show that 27, 25, 28, and 20 amino acids could be deleted from the N terminus of the human MC1, MC3, MC4 and MC5 receptors, respectively, including all potential N-terminal glycosylation sites in the MC1 and the MC4 receptors, without affecting ligand binding or expression levels. The results indicate that the N-terminal regions of the human MC1, MC3, MC4 and MC5 receptors, do not play an important role for the ligand binding properties of these receptors.

Cell heterogeneity designates the phenomenon by which a particular cell type is composed of morphologically and physiologically distinct cell subpopulations. We have previously isolated two subsets of melanotrope cells in the intermediate lobe of the frog pituitary by means of a separation procedure based on a Percoll density gradient High density (HD) melanotrope cells were found to exhibit a more granulated cytoplasm and a lower secretory rate than low density (LD) cells. In the present study, we have investigated the biochemical and functional characteristics of each melanotrope cell subpopulation by using various approaches, including chromatographic analysis for the measurement of the proportion of acetylated alpha MSH, microfluorimetric measurement of the cytosolic free calcium concentration ([Ca2+]i) and in situ hybridization for quantification of POMC messenger RNA (mRNA). Under basal conditions, LD melanotrope cells showed higher secretory activity, acetylation rate, [Ca2+]i, and POMC mRNA content compared to HD cells. Incubation of the cells with 100 nM TRH for 2 h induced a more pronounced activation of alpha MSH secretion, [Ca2+]i mobilization, and POMC mRNA accumulation in LD than in HD melanotrope cells. Conversely, TRH increased the rate of acetylation of alpha MSH in HD cells, but did not affect acetylation in LD cells. Taken together, these results demonstrate that the frog intermediate lobe is composed of two subsets of endocrine cells with distinct biochemical and functional characteristics. The coexistence of two cell subpopulations in the frog pars intermedia is consistent with the idea of a cell secretory cycle, in which each melanotrope subset represents a specific state of cellular activity.

We developed two solid-phase reagent systems for microscopic visualization and characterization of melanocyte-stimulating hormone (MSH) receptors of melanoma cells. Multiple copies of [Nle-4,D-Phe-7]-alpha-MSH, a potent analog of alpha-MSH, were conjugated to microspheres (latex beads) or macrospheres (polyamide beads) through a thioether or disulfide bond. Binding between the beads and mouse and human melanoma cells was examined by scanning electron microscopy and by light microscopy. Each mouse and human melanoma cell (of all cell lines) evinced binding to the beads. Binding of the melanotropin conjugates was not restricted to any one phase of the cell cycle. Specificity of binding was demonstrated by several studies. Negative controls included cell types of nonmelanocyte origin (e.g., mammary cancer cells) and beads that lacked the melanotropic ligand or had other attached ligands. Beads with a disulfide-linked melanotropin analog served as a direct control. Treatment of these beads with DTT during or before incubation of the beads with melanoma cells (resulting in release of the MSH analog from the beads) eliminated binding of the beads to melanoma cells. Binding interactions between melanoma cells and melanotropin-bound beads also could be abolished by prior incubation with unconjugated MSH analog. During these experiments, certain membrane receptor-hormone associated phenomena, such as capping (aggregation) of the receptor-ligand complex, also were observed. These results provide visual evidence that MSH receptors are a property common to melanoma cells. Normal human epidermal melanocytes and keratinocytes were also shown to express melanotropin receptors by the same criteria established for melanoma cells.

The release of POMC-derived peptides from the melanotrope cells in the pituitary pars intermedia of Xenopus laevis is regulated by multiple inhibitory and stimulatory neurochemical messengers. In this study we examined the role of acetylcholine (ACh) in regulating melanotrope cell activity. The origin of ACh was studied in in vitro biosynthesis experiments, where it was demonstrated that dissociated melanotropes can synthesize ACh. In addition, immunocytochemical studies showed the presence of the ACh-synthesizing enzyme choline acetyltransferase in the cytoplasm of melanotrope cells. Oscillations in the concentration of intracellular free calcium ions are thought to be the driving force for the secretion of POMC-derived peptides from Xenopus melanotrope cells. A technique was developed to study simultaneously calcium oscillations and peptide secretion from the same population of single melanotrope cells in vitro. ACh concentration-dependently raises the intracellular free calcium concentration by increasing both the frequency and amplitude of the calcium oscillations as well as the release of POMC-derived peptides. The actions of ACh on the melanotrope cell were mimicked by muscarine, but not by nicotine, indicating the exclusive presence of a muscarinic cholinergic receptor. Furthermore, selective muscarinic receptor antagonists showed that the actions of ACh are mediated through an M1-subtype muscarinic receptor. Immunofluorescence cytochemistry in combination with confocal laser scanning microscopy visualized muscarinic receptors on the surface of melanotrope cells. It is concluded that ACh stimulates the release of POMC-derived peptides from the Xenopus melanotrope cell in an autocrine way, acting via a muscarinic M1 receptor on the intracellular free calcium concentration.

Rat hypothalamic neurons and endocrine cells from the intermediate lobe of the pituitary were grown in dissociated coculture. Neurons positively stained with an antibody against glutamate decarboxylase established apparent contacts with the alpha-melanocyte-stimulating hormone-positive endocrine cells. These sites of contact were intensely labeled with an antibody against the synaptic protein synapsin I and displayed ultrastructural features characteristic of synapses. Using patch-clamp recordings, we have demonstrated that these contacts correspond to functional GABAergic synapses. The synaptic currents were blocked reversibly by bicuculline (5 microM) and SR95531 (5 microM), two competitive antagonists of the GABAA receptor. At a holding potential of -60 mV, spontaneously occurring IPSCs (s-IPSCs) had small amplitudes (10-100 pA), whereas electrically evoked IPSCs (ee-IPSCs) had amplitudes up to 1 nA. The rise times of both types of IPSCs were fast ( < or = 1 msec), and their decaying phases were fitted in most cases with a single exponential function (time constant 50 msec). The amplitude distribution of s-IPSCs did not reveal clear, equally spaced peaks and was little affected by tetrodotoxin, suggesting that most s-IPSCs were miniature IPSCs. Reduction of extracellular calcium concentration to 0.3 mM induced a marked decrease in s-IPSC frequency and revealed a single amplitude peak at 10 pA, suggesting that a single quantum of GABA activates 8-10 GABAA channels. Thus, our preparation might be an interesting model to study different aspects of synapse formation between a central neuron and its target as well as the fundamental mechanisms of synaptic transmission at central synapses.

The human melanocortin-1 (MC1) receptor was stably expressed in the amelanotic mouse melanoma cell clone B16-G4F which does not express its own (mouse) MC1 receptor and hence is unresponsive to alpha melanocyte stimulating hormone (alpha MSH). From several stable transfectant cell lines expressing the human MC1 receptor in relatively high numbers, three melanin producing clones (G4F-12, 14, and 15) and one amelanotic clone (G4F-7) were further analyzed in competition binding experiments and in cAMP and melanin assays. The dissociation constants (KD) for [Nle4, D-Phe7]-alpha MSH in all four clones ranged from 0.187 to 0.705 nmol/l, thus corresponding to the KD observed with the different human melanoma cell lines so far studied. Intracellular cAMP content was 3- to 5-fold higher than that of control cells, and alpha MSH induced an additional 1.5- to 1.7-fold increase. G4F-15 cells secreted melanin into the medium whereas the other clones did not secrete melanin. The extent of melanin secretion was similar to that of fully alpha MSH-stimulated B16-F1 mouse melanoma cells but the onset of secretion was delayed. alpha MSH induced an additional dose-related increase (up to 1.3-fold) in melanin production which could be suppressed by the addition of specific alpha MSH antibodies without altering the constitutive part of melanogenesis. Human and mouse agouti proteins, which inhibit basal and alpha MSH-induced melanogenesis in B16-F1 cells, both reduced alpha MSH-induced melanin production in G4F-15 cells but did not affect the constitutive melanogenesis. These results indicate that human MC1 receptor expressed in mouse B16-G4F cells induces constitutive activation of the signalling pathway controlling melanogenesis, most likely by tightly coupling to Gs alpha, in a similar manner to that reported for constitutively active receptor mutants in other systems.

Secretogranin III (SgIII) is an acidic protein of unknown function that is present in the storage vesicles of many neuroendocrine cells. It is coexpressed with the prohormone proopiomelanocortin in the intermediate pituitary of Xenopus laevis. We developed an antiserum to investigate the biosynthesis of SgIII in pulse-chase incubated Xenopus neurointermediate lobes. SgIII was synthesized as a 61- or 63-kDa (N-glycosylated) protein and processed to a 48-kDa form which, in turn, was partially cleaved to fragments of 28 and 20 kDa. The 48-, 28-, and 20-kDa cleavage products, but not their precursors, were secreted. This secretion is regulated and can be blocked in parallel with that of proopiomelanocortin-derived peptides by the hypothalamic factors dopamine, gamma-aminobutyric acid, and neuropeptide Y. Coexpression of Xenopus SgIII with prohormone convertase (PC)1 or PC2 in transfected fibroblasts was sufficient to reconstitute the processing events observed in the neurointermediate lobes. Site-directed mutagenesis revealed that Xenopus SgIII is cleaved at two dibasic sites, namely Lys68-Arg69 and Arg237-Arg238. Pulse-chase incubations of lobes with Na2[35S]SO4 showed that SgIII is sulfated in the trans-Golgi network before it is processed. Finally, SgIII processing was found in several neuroendocrine cell types from various species. We conclude that SgIII is a precursor protein and that the intact molecule can only have an intracellular function, whereas an extracellular role can only be attributed to its cleavage products.

It has been known for many years that adipocytes express high affinity ACTH and alpha-melanocyte stimulating hormone (MSH) binding sites, and that ACTH, alpha-MSH, and beta-lipotropin are potent lipolytic hormones. We show here that the adipocyte response to the melanocortin peptides results from the expression of both the MC2 (ACTH) receptor as well as the newly discovered MC5 receptor. Using RT-PCR and Northern blot hybridization, high levels of MC2 receptor messenger RNA (mRNA) were found in all adipose tissues examined in the mouse, whereas MC5 receptor mRNA was found in a subset of these. Both receptors mRNAs were also found in the 3T3-L1 cell line but only after the cells had been induced to differentiate into adipocytes. This cell line was then used to characterize the pharmacological properties of the MC2 and MC5 receptor sites in situ. The MC2 receptor exhibits properties similar to the ACTH receptor characterized in adrenocortical cells, coupling to activation of adenylyl cyclase with an EC50 of approximately 1 nM. An MSH binding site characterized in these cells is presumably the MC5 receptor, based on the observation that this is the only other melanocortin receptor mRNA detected in these cells. The MC5 receptor in the 3T3-L1 adipocyte activated adenylyl cyclase in response to alpha-MSH stimulation. Interestingly, Nle4, D-Phe7-alpha-MSH (NDP-MSH), a commonly used synthetic alpha-MSH agonist, was a potent antagonist of the MC5 receptor expressed in the 3T3-L1 cell line. Although the agouti signaling peptide is a potent antagonist of NDP-MSH binding to the MC1 and MC4 melanocortin receptors, agouti was unable to block NDP-MSH binding in the 3T3-L1 adipocyte.