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Background: Melanotan II (MT II) is a synthetic analogue of α-melanocyte-stimulating hormone that, via interaction with the melanocortin 1 receptor, induces skin hyperpigmentation. The unregulated acquisition of MT II injections via the internet and other outlets has become popular over the last decades in order to exploit its properties for use as a tanning agent. Due to the covert nature of MT II use, it is difficult to assess the extent of its use among the general population and to characterise any associated side effects. Objectives: The aim of this study was to qualitatively examine MT II use, as portrayed on online forums, and to explore the motivations for its use and side effect profile. Methods: Data were extracted retrospectively from UK and Ireland online chatrooms and forums from January 2016 to October 2017. Inclusion criteria were active MT II chatrooms and forums considered to be within the public domain. An inductive thematic analysis identified themes within discussion threads. Results: A total of 623 discussion entries were extracted; 205 participants contributed to these entries. Emergent themes included motivation for MT II use, misinformation in the context of using an unregulated product, product preparation and administration, dosing regimens, sunbed use, side effects and concerning practices associated with MT II use. Conclusion: Motivations for MT II use included the pursuit of a tanned appearance, often in anticipation of sun holidays and fitness/body building competitions. Clinicians should be aware not only of the potential risks in relation to pigmented skin lesions, but also remain cognisant of the other medical hazards associated with the use of this substance, namely transmission of infectious diseases, use of potentially contaminated products, polypharmacy, and sunbed exposure.

Background: Deregulation of orexigenic and anorexigenic pathways occurs among adolescents with obesity. Alpha-melanocyte-stimulating hormone (α-MSH) is a key catabolic mediator of energy homeostasis and an important anorexigenic neuropeptide in the control of energy balance and thermogenesis. However, it was not well explored if α-MSH can modulate long-term weight loss therapy responses in a dependent manner according to its concentration. Our hypothesis is that a high α-MSH concentration at baseline promotes better modulation of anorexigenic/orexigenic pathways in obese adolescents. Methods: One hundred ten post-pubertal obese adolescents (body mass index >95th percentile) were submitted to 1 year of interdisciplinary therapy (clinical, nutritional, psychological, physical exercise, and physiotherapy support). Body composition and plasma levels of α-MSH, neuropeptide Y (NPY), melanin-concentrating hormone, and agouti-related peptide (AgRP) were measured before and after therapy. The volunteers were grouped on the basis of Tertiles of α-MSH concentration: Low (1.57 ng/mL). Significance was set as p < 0.05. Results: The treatment promoted a significant improvement in body adiposity and fat free mass for all groups. It is important to note that only in the high α-MSH group, a significant increase of the α-MSH/NPY ratio and decrease NPY/AgRP ratio post treatment were observed. Conclusion: The high α-MSH concentration promotes better modulation of anorexigenic/orexigenic pathways in obese adolescents following long-term weight loss therapy and this is important in clinical practice.

Background: Ultraviolet (UV) irradiation is well known to promote inflammation and pigmentation of skin. UVB mainly affects dermatitis and pigmentation. Coffee contains a number of polyphenols, such as caffeic acid (CA) and chlorogenic acid (CGA) but their in vivo bioactivity for photobiology remains unclear. Methods: C57BL/6j male mice were irradiated with UVB (1.0 kJ/m2/day) for 3 days. Five days after the final session of UVB irradiation, the dorsal skin, ear epidermis, and blood samples were analyzed to investigate the inflammatory factors, melanogenesis factors and related hormones. Results: After the oral administration of CA (100 mg/day) or CGA (100 mg/day) for 8 days, only CA was found to inhibit dermatitis and pigmentation. The pathway by which CA inhibits dermatitis is related to the mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK)1/2/cAMP response element binding protein (CREB) pathway. Otherwise, the pathway by which CA inhibits pigmentation is related to the activation of the β-endorphin-μ-opioid receptor and suppresses the cAMP-microphthalmia-associated transcription factor (MITF) pathway. Conclusion: It is suggested that the oral administration of CA prevented dermatitis and pigmentation after UVB irradiation in mice.

Background: Unlicensed use of melanotan-II (MT-II) to promote skin pigmentation has become prevalent amongst young people attending fitness centres. We present a case where the melanocyte stimulation of MT-II in combination with the use of sun tanning beds coincided with cutaneous melanoma. Observation: A 20-year-old woman with Fitzpatrick skin type II was referred to a dermatology clinic. Clinical examination revealed a suspicious black melanocytic lesion in her left gluteal region. Furthermore, her skin was universally intensely pigmented. The melanocytic lesion was excised, and histology confirmed the diagnosis of melanoma. Three months prior to the excision the patient had conducted a 3- to 4-week course of self-injections with MT-II, intending an augmentation of sunbed tanning. Conclusions and Relevance: This observation brings attention to the potential risks related to the use of the cyclic α-melanocyte-stimulating hormone analogue MT-II. There are several hazardous aspects of the possible widespread use of MT-II. As the drug is unlicensed and incompletely tested, the extent and types of adverse effects are unknown. Clinicians are advised to be aware of the problem, and counsel their at-risk patients regarding the potential hazards related to the use of MT-II.

Background/Aims: Leptin restores gonadotropic function in lean hypogonadotropic animals by an unknown mechanism. We aimed to test the hypothesis that restoration of gonadotropic function is a result of an upregulation of central acetylated melanocortin production. Methods and Results: Lean ovariectomised (OVX) ewes received intracerebroventricular (i.c.v.) infusions of leptin (or vehicle) for 3 days, which upregulated proopiomelanocortin (POMC) mRNA and restored pulsatile luteinizing hormone (LH) secretion. A melanocortin agonist (MTII), but not naloxone treatment, reinstated pulsatile LH secretion in lean OVX ewes. We treated (i.c.v.) lean OVX ewes with leptin (or vehicle) and measured peptide levels and post-translational modification in the arcuate nucleus (ARC). Levels of β-endorphin (β-END) were lower in lean animals, with no effect of leptin treatment. Desacetyl-α-MSH was the predominant form of α-melanocyte-stimulating hormone (α-MSH) in the ARC and levels were similar in all groups. In another group of lean and normal-weight OVX ewes, we measured the different forms of α-MSH in ARC, hypothalamus (ARC-removed) and the preoptic area (POA). Acetylated α-MSH levels were lower in lean animals in the terminal beds of the hypothalamus and POA but not the ARC. Conclusions: Leptin corrects the hypogonadotropic state in the lean condition by upregulation of POMC gene expression, and may increase transport and acetylation of melanocortins to target cells in the brain. Melanocortin treatment restores LH secretion in lean animals.

Objective: There is evidence that brain death has detrimental effects on peripheral organs. Clinical and experimental studies on organ donors showed marked inflammation in tissue samples of livers and kidneys collected during brain death. The inflammatory reaction is characterized by release of cytokines and inflammatory cell infiltration. Because melanocortins and their receptors are significant modulators of inflammation, we hypothesized that downregulation of melanocortin receptors during brain death could contribute to enhance inflammation. Methods: Using real-time polymerase chain reaction (PCR) analysis, we determined expression of melanocortin receptors in liver biopsies obtained from brain-dead organ donors before cold ischemia and in normal liver tissue during resection of benign focal lesions of the liver. Tissue biopsies were also analyzed for expression of intercellular adhesion molecule-1 (ICAM-1), which has a central function in inflammatory cell migration. Results: Expression of melanocortin-1 receptor (MC1R) mRNA was markedly reduced in liver samples obtained from brain-dead organ donors compared to hepatic tissue collected during resection of benign focal lesions of the liver. Conversely, expression of the adhesion molecule ICAM-1 was significantly increased in livers of brain-dead organ donors. Conclusions: Disruption of the endogenous anti-inflammatory circuit based on MC1R could contribute to tissue damage during brain death.

Resistin is a new adipokine expressed in mouse, rat and human adipose tissue. Resistin may be an important link between obesity and insulin resistance, though this controversial view is complicated by the discovery of multiple sites of resistin expression, including human macrophages, placenta and pancreas. In previous studies we demonstrated that the mouse hypothalamo-pituitary system was also a site of resistin production. Pituitary resistin is developmentally regulated, reduced in the ob/ob mouse and severely down-regulated by food deprivation (24 h). An unexpected finding was that hypothalamic resistin mRNA remained unaffected by fasting. The present experiments examined the localization and possible regulation of hypothalamic resistin protein. Using immunohistochemistry we observed a complex network of resistin+ fibres extending rostrally from the arcuate nucleus of the hypothalamus (ARC) to the preoptic area. Labelled cell bodies occurred only in the ARC and in a periventricular region of the dorsal hypothalamus. Hypothalamic resistin immunoreactivity (ir) was unaffected by fasting (48 h) or by a high fat diet, but the periventricular staining was greatly increased in the lactating mouse. Marked reductions in resistin+ fibres were seen in brain tissue from: (a) ob/ob mice, (b) young mice made underweight for their age by raising them in large litters (20 pups per litter) and (c) mice with hypothalamic lesions induced by monosodium glutamate (MSG) or gold thioglucose (GTG). We speculate that the resistin-ir deficit in genetically obese mice, and in severely underweight mice, could be due to low or absent leptin. In contrast, though MSG- and GTG-treated mice have high levels of circulating leptin, in the presence of excessive visceral fat deposits, we hypothesize that damage to the ARC destroys the resistin+ cell bodies. This latter supposition led us to an additional hypothesis, that resistin-ir would be contained in neurons expressing the proopiomelanocortin (POMC) gene. This proved to be correct. Double label immunofluorescence histochemistry revealed that α-MSH-ir, a marker for POMC neurons, was co-localized with resistin-ir. In conclusion, our data reveal a second example of an adipocytokine co-localized with a hypothalamic neuropeptide. We reported previously that leptin was co-localized with oxytocin and vasopressin. RT-PCR analysis confirmed that resistin mRNA is readily detectable in ARC, but further work is required to determine whether the resistin gene is expressed in POMC neurons or if resistin is specifically accumulated by these cells. Nonetheless, our data suggest that the hypothalamus is a target tissue for resistin.

There is evidence that alpha-melanocyte-stimulating hormone (alpha-MSH) has immunomodulatory and anti-inflammatory actions within the brain. In this study, we tested whether these actions are due to inhibition of the synthesis of nitric oxide (NO) and prostaglandins induced by lipopolysaccharide (LPS). Since melanocortin subtype MC4 receptor has been detected in the hypothalamus, we investigated the effect of central administration of alpha-MSH and HS024 (a selective MC4 receptor antagonist) on the gene expression of inducible, neuronal and endothelial NO synthase (iNOS, nNOS and eNOS) and on cyclooxygenase (COX-1 and COX-2) expression in the mediobasal hypothalamus (MBH) of LPS-treated male Wistar rats. Peripheral administration of LPS (250 microg/rat, 3 h) induced iNOS and COX-2 gene expression in the MBH. This stimulatory effect was reduced by alpha-MSH (3 nmol/rat) injected 30 min before LPS. alpha-MSH and HS024 (1 nmol/rat) alone had no effect on iNOS and COX-2 expression. The action of alpha-MSH on LPS-induced iNOS and COX-2 mRNA levels was not observed in the presence of HS024, suggesting that MC4-R may be involved in the modulatory effect of alpha-MSH. None of these treatments produced any modifications in nNOS, eNOS and COX-1 expression in MBH. The increase in serum corticosterone levels induced by LPS was attenuated by alpha-MSH. Both LPS and alpha-MSH decreased serum LH and prolactin levels. HS024 failed to modify the inhibitory effects of LPS and alpha-MSH on prolactin release but reverted the effect of LPS on LH secretion, indicating that MC4-R activation may be involved in the effects of alpha-MSH on LH secretion in male rats. When we examined the in vitro effect of LPS (10 microg/ml) and LPS plus interferon-gamma (IFN-gamma, 100 ng/ml) on iNOS expression in MBH, an increase in iNOS mRNA levels was observed only in the presence of LPS + IFN-gamma. This stimulatory effect was attenuated in the presence of alpha-MSH (5 microM), which by itself had no effect. No changes were found in nNOS, eNOS, COX-1 or COX-2 expression. These results indicate that alpha-MSH reduces the induction of iNOS and COX-2 gene expression at the hypothalamic level during endotoxemia and suggest that endogenous alpha-MSH may exert an inhibitory tone on iNOS and COX-2 transcription via MC4 receptors acting as a local anti-inflammatory agent within the hypothalamus.

The intermediate lobe of rodent pituitaries is involved in the regulation of prolactin (PRL) secretion from the anterior lobe. In a previous study, we demonstrated the stimulatory effect of alpha-melanocyte-stimulating hormone (alpha-MSH) on PRL release and the expression of melanocortin-3 receptors (MC3-Rs) in cultured mouse pituitary cells. The aim of the present study was to clarify whether alpha-MSH directly stimulates PRL release through the MC3-Rs by determining the cell type of MC3-R-expressing cells in the mouse pituitary anterior lobe. Northern blot analysis revealed a 2.7-kb transcript for MC3-R mRNA in the anterior and neurointermediate lobes of pituitary glands of adult male and female mice. Dual cellular localization of MC3-R mRNA and PRL or growth hormone (GH) in the mouse pituitary glands was performed by in situ hybridization analysis of MC3-R mRNA followed by immunocytochemical detection of PRL or GH. MC3-R mRNA was detected in most mammotropes and some somatotropes. alpha-MSH increased PRL release and stimulated DNA replication in mammotropes, and these effects were blocked by SHU9119, an antagonist of MC3-R and MC4-R. These results indicate that alpha-MSH stimulates PRL release and proliferation of mammotropes through MC3-Rs, and suggest that alpha-MSH from intermediate lobes can regulate mammotrope functions in the mouse pituitary.

Mutations in the melanocortin-4 receptor (MC4-R) cause obesity in both mice and humans, and the receptor is presumed to have an important role in the regulation of energy homeostasis. The MC4-R is expressed in discrete sets of neurons in the central nervous system, and thus it has been technically difficult to study the regulation of expression and the signaling mechanisms of this receptor. We report here a neuronal cell line that exhibits endogenous functional expression for the MC4-R. Initially, RT-PCR analysis showed the presence of MC4-R RNA in the hypothalamic GT1-1 and GT1-7 cells. In addition, GT1-7 cells expressed melanocortin-3 receptor while the GT1-1 subclone specifically expressed predominantly the MC4-R RNA. High-affinity binding sites were demonstrated in the GT1-1 and GT1-7 cells for NDP-α melanocyte-stimulating hormone (MSH; Ki = 1.1 × 10–10 and 1.8 × 10–10M) and agouti-related protein (AGRP; Ki = 1.548 × 10–9 and 1.663–9M). α-MSH-stimulated cAMP production in GT1-1 cells with an EC50 of 2.2 × 10–8M, and cAMP production was inhibited in the presence of AGRP, an endogenous antagonist of the MC4-R. Stimulation of gonadotropin-releasing hormone (GnRH) secretion was achieved with 1 nM to 1µM concentrations of NDP-α-MSH while no GnRH secretion was observed when the GT1-1 cells were treated with AGRP. The data presented here show that GT1-1 cells specifically express a functional MC4-R that couples to GnRH release.

The melanotrope population of the frog intermediate lobe consists of two subtypes of cells, referred to as high-(HD) and low-density (LD) melanotrope cells, which differ markedly in their basal morphofunctional features as well as their in vitro response to hypothalamic factors, such as the stimulator thyrotropin-releasing hormone (TRH) and the inhibitor dopamine. In this study, we have investigated whether other major hypothalamic regulators of the release of alpha-melanocyte-stimulating hormone (alpha-MSH), such as gamma-aminobutyric acid (GABA) and neuropeptide Y (NPY), also differentially regulate frog melanotrope subpopulations. Our results show that in LD cells, both factors markedly inhibited proopiomelanocortin (POMC) mRNA accumulation and alpha-MSH secretion. In contrast, the secretory and biosynthetic activity of HD cells was not modified by GABA. NPY inhibited POMC transcript accumulation and tended to reduce alpha-MSH secretion in HD cells, yet these effects were less pronounced than those evoked in LD cells. In addition, GABA and NPY inhibited the KCl-induced rise in cytosolic free calcium levels in both subpopulations. Taken together, these results further indicate that frog melanotrope subpopulations are differentially regulated by the hypothalamus and strongly suggest that the intensity of such regulation is directly related to the activity of the cell subset. Thus, the LD subpopulation represents a highly responsive cell subset which is regulated by multiple neuroendocrine factors (TRH, dopamine, GABA and NPY), whereas the hormone storage HD subpopulation shows a moderate response to single stimulatory (TRH) and inhibitory (NPY) inputs.

The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) occurs in CNS tissue in neurological disorders, infection, and injury. Its excessive production is believed to contribute to local pathology, in which case modulation of TNF-alpha production should promote survival of neural tissue. The neuropeptide alpha-melanocyte stimulating hormone [alpha-MSH (1-13)] inhibits TNF-alpha production in vivo and in vitro, and in this research we tested the capacity of the peptide, and of an anti-inflammatory COOH-terminal tripeptide fragment of it, to inhibit TNF-alpha production induced by bacterial endotoxin in cells of a human glioma line (A-172, anaplastic astrocytoma cells). Both peptides were effective, although the alpha-MSH (1-13) sequence was more potent. Preincubation of the cells with alpha-MSH (1-13) markedly increased its effectiveness. The anticytokine effect of alpha-MSH in glioma cells may be mediated by human melanocortin-1 receptors; mRNA for this receptor subtype was isolated from resting A-172 cells. These results, combined with prior evidence of effectiveness of alpha-MSH molecules in modulating inflammatory processes and of their low toxicity, suggest that the molecules may be useful in the treatment of CNS disorders that have an inflammatory component.

The brain is an immune-privilege site. To understand the mechanism of immune privilege in the brain, human cerebrospinal fluid (CSF) was examined for characteristics associated with fluids derived from other immune-privileged tissues. We first assayed for CSF suppression of effector T cell inflammatory activities. Primed T cells were activated with antigen and antigen-presenting cells in the presence of normal human or rabbit CSF, and T cell proliferation and interferon-gamma production were assayed. Human and rabbit CSF enhanced antigen-stimulated lymph node T cell proliferation and human CSF suppressed IFN-gamma production. T cell proliferation was suppressed by a low molecular weight (5 kDA) fraction of CSF and by transiently acidified unfractionated CSF. Normal CSF, similar to fluids from other immune-privileged sites, has the capacity to suppress production of proinflammatory lymphokines by antigen-stimulated effector T cells. Normal CSF also contains factors that have the potential to suppress effector T cell proliferation. Human CSF was assayed for factors known to mediate immunosuppression in other immune-privileged sites. Human CSF contained the immunosuppressive cytokine-transforming growth factor-beta (1.7 +/- 0.6 ng/ml), and the immunosuppressive neuropeptides alpha-melanocyte stimulating hormone (60 +/- 11 pg/ml), and vasoactive intestinal peptide (42 +/- 3 ng/ml). Much as fluids from other immune-privileged sites, CSF contains immunosuppressive cytokines that prevent activation of inflammatory-mediating (delayed-typed hypersensitivity) T cells. This suggests that, similar to other immune-privileged sites, cytokines and neuropeptides mediate immunosuppression in the brain.

The neuropeptide Α-melanocyte-stimulating hormone (Α-MSH) is known to suppress cytokine-mediated inflammation. In addition, we previously found that Α-MSH suppressed the production of the proinflammatory cytokine interferon (IFN)-Γ by antigen-stimulated primed lymph node T cells. This immunosuppressive activity of Α-MSH on lymph node T cell cultures is similar to that of interleukin (IL)-4. To further examine the potential ‘IL-4 like’ activities of Α-MSH, antigen-stimulated lymph node T cell cultures were treated with Α-MSH in the presence of neutralizing anti-IL-4 antibodies. The enhanced production of IFN-Γ caused by the presence of anti-IL-4 alone in the T cell cultures was squelched by Α-MSH. This demonstrated that in these cultures, Α-MSH regulation of IFN-Γ production operates in a fashion similar to that of endogenous IL-4. Addition of exogenous IL-4 to antigen-stimulated lymph node T cell cultures did not intensify Α-MSH down-regulation of IFN-Γ production, and the addition of Α-MSH to IL-4-treated cultures did not further depress IFN-Γ production. These and the previous results suggest that the mechanism of Α-MSH suppression of IFN-Γ production in the antigen-stimulated T cell cultures is similar to, but independent of, IL-4. When antigen-presenting cells (APCs) were the only cells in the antigen-stimulated T cell cultures treated with a-MSH, there was a significant reduction (60-70%) of APC elicitation of IFN-y production by untreated primed T cells. Moreover, Α-MSH prevented IFN-Γ production by 80-100% when primed T cells were the only cells treated with a-MSH. The mechanism of a-MSH prevention of antigen-stimulated IFN-Γ production in T cell cultures involves both APC and primed T cells. Since Α-MSH is constitutively present in the skin, serum, brain and eye, this neuropeptide may play an important immunomodulatory role linking the nervous system and the rest of the body. In particular, Α-MSH may be critical to the phenomenon of immune privilege in the eye and brain.

The neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH) has potent antipyretic and antiinflammatory properties. When administered systemically, the naturally occurring molecule and its COOH-terminal tripeptide sequence inhibit inflammation induced by peripherally applied irritants and intradermal injections of mediators of inflammation such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF alpha). We recently found that alpha-MSH can act solely within the brain to inhibit inflammation caused by a general irritant applied to the skin. This activity appears to be shared with salicylate drugs and the combined observations suggest the existence of descending neurogenic antiinflammatory signals capable of modulating inflammation in peripheral tissues. To improve our knowledge of the scope of this action of the peptide, alpha-MSH was injected into the cerebral ventricles (i.c.v.) of mice that had received intradermal injections in the ear of mediators of inflammation: IL-1 beta, IL-8, leukotriene B4, and platelet-activating factor. The centrally administered peptide inhibited the actions of all of these proinflammatory agents as determined from comparisons with measures of ear edema over time in control animals; this indicates that the central peptide can alter inflammation induced in the periphery by major mediators of inflammation. In tests confined to IL-1 beta, central administration of alpha-MSH(11-13) was also effective. These findings support the concept of a descending neurogenic antiinflammatory influence promoted by an action of alpha-MSH within the brain, an inhibitory influence that is not restricted to modulation of just one or a limited set of the mediators of inflammation.

alpha-Melanocyte-stimulating hormone (alpha-MSH) modulates inflammatory processes in models of acute inflammation and in models of sepsis/septic shock/adult respiratory-distress syndrome (ARDS). Because this neuropeptide inhibits actions of cytokines and other mediators of imflammation that are also believed to underlie aspects of chronic inflammation, tests were performed to compare the effects of repeated administration of the peptide with those of prednisolone and saline on the development of adjuvant arthritis in rats. alpha-MSH (50 micrograms), injected i.p. twice daily, markedly inhibited the clinical and histological signs of experimental arthritis and moderated the weight loss observed in control animals. Prednisolone (100 mg/kg), given twice per day, prevented development of arthritis but caused marked and progressive weight loss. The results confirm the potent anti-inflammatory influence of alpha-MSH, in this case in a model of chronic inflammation that has immune components.

While dopamine (DA) is known to inhibit pituitary intermediate lobe proopiomelanocortin (POMC) peptide secretion and synthesis in most species, its influence on anterior-lobe (AL) POMC peptide synthesis and secretion is less clear. We, therefore, sought to determine the effects of daily treatment with the DA receptor antagonist, domperidone (DOM), on secretion of the POMC peptides adrenocorticotropic hormone (ACTH) and alpha-melanocyte-stimulating hormone (alpha-MSH) from the dog pituitary, and on concentrations of another pituitary hormone regulated by DA, prolactin (PRL). Dogs treated for 7 days with DOM had significantly higher peak ACTH concentrations in response to corticotropin-releasing hormone (CRH) injection (329 +/- 37 pg/ml, mean +/- SD) than did controls (164 +/- 42 pg/ml). PRL was also significantly (p0.05) increased in samples collected on a daily basis after DOM injections (9.5 +/- 4.6 vs. 4.3 +/- 3.3 ng/ml in controls). However, plasma alpha-MSH concentrations were unaffected by DOM. In a subsequent study, dogs were again treated daily with DOM or vehicle (controls), and additionally were given dexamethasone (DEX) to block AL ACTH release. DEX-treated controls showed low daily and CRH-stimulated ACTH and cortisol concentrations (generally below assay sensitivity). In contrast, DEX + DOM-treated dogs had daily mean ACTH concentrations ranging from 10 +/- 8.1 to 32 +/- 26 pg/ml and mean peak post-CRH ACTH concentrations of 174 +/- 16 pg/ml. Although daily cortisol concentrations were below assay sensitivity, the mean peak post-CRH cortisol concentration was 6.7 +/- 1.8 micrograms/dl, indicating that the immunoreactive ACTH was biologically active.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma ACTH, beta-endorphin and alpha-melanocyte-stimulating hormone levels were evaluated in the inferior petrosal sinuses and in the periphery of 22 patients affected by Cushing's disease, 11 patients with other pituitary diseases subjected to simultaneous and bilateral inferior petrosal sinus sampling and in the peripheral blood of 15 normal subjects. In patients with Cushing's disease ACTH, beta-endorphin and alpha-melanocyte-stimulating hormone levels in the inferior petrosal sinus ipsilateral to the adenoma were significantly higher than in the periphery and in the contralateral inferior petrosal sinus (p < 0.01, p < 0.01 and p < 0.05, respectively). In patients with other pituitary diseases only ACTH and beta-endorphin, but not alpha-melanocyte-stimulating hormone levels in both inferior petrosal sinuses were significantly higher than in the periphery (p < 0.01). Furthermore, no difference was found in the peripheral blood among patients with Cushing's disease, patients with other pituitary diseases and normal subjects for ACTH and beta-endorphin level. By contrast, in patients with Cushing's disease peripheral alpha-melanocyte-stimulating hormone levels were significantly higher than in patients with other pituitary diseases and in normal subjects (p < 0.05). In conclusion, the results of the present study suggest that only in patients with Cushing's disease, but not in patients with other pituitary diseases, alpha-melanocyte-stimulating hormone is released by adenomatous pituitary corticotrophs together with ACTH and beta-endorphin.

The purpose of the present study was to provide neurochemical and endocrinological evidence that dopamine (DA) neurons terminating in the intermediate lobe of the rat pituitary originate in the periventricular nucleus of the hypothalamus. One week following surgical separation of the periventricular nucleus from the mediobasal hypothalamus, DA and 3,4-dihydroxyphenyl-acetic acid (DOPAC) concentrations in the intermediate lobe were reduced by 50%, and this was accompanied by an increase in plasma alpha-melanocyte-stimulating hormone (alpha-MSH) concentrations. In contrast, this procedure had no effect on concentrations of prolactin in the plasma, or DA or DOPAC in the median eminence, the region of the mediobasal hypothalamus containing terminals of tuberoinfundibular DA neurons. Electrical stimulation of the periventricular nucleus increased the ratio of DOPAC/DA in the intermediate lobe and reduced the concentrations of alpha-MSH in the plasma, whereas in these same animals the DOPAC/DA ratio in the median eminence and concentrations of prolactin in the plasma were unaltered. These results indicate that approximately 50% of all the DA neurons terminating in the intermediate lobe of the rat pituitary originate in or project through the periventricular nucleus of the hypothalamus, and that these DA neurons regulate the secretion of alpha-MSH from intermediate lobe melanotrophs.

The effects of ovariectomy and estrogen on prolactin secretion and/or the activity of tuberoinfundibular dopamine (TIDA) neurons were examined by either concurrently measuring concentrations of prolactin in plasma and 3,4-dihydroxyphenylacetic acid (DOPAC) in the median eminence of female rats or by determining the rate of DA synthesis (accumulation of 3,4-dihydroxyphenylalanine (DOPA) after the administration of a decarboxylase inhibitor) in the median eminence. For comparison, concentrations of alpha-melanocyte-stimulating hormone (alpha MSH) in plasma and DOPAC in the intermediate lobe of the pituitary (an index of the activity of tuberohypophysial DA neurons) were also determined. Ovariectomy produced a time-dependent decrease in the accumulation of DOPA and the concentrations of DOPAC in the median eminence and prolactin in plasma with maximal effects occurring by 7 days. Estrogen administration to ovariectomized rats increased plasma prolactin and median eminence DOPAC concentrations to levels comparable to those in diestrous controls. In contrast, neither ovariectomy nor estrogen replacement altered the concentrations of alpha MSH in plasma or DOPAC in the intermediate lobe. Administration of the DA agonist bromocriptine blocked the ability of estrogen to increase plasma prolactin and median eminence DOPAC concentrations. Also, administration of antiserum to rat prolactin blocked the stimulatory action of estrogen on median eminence DOPAC concentrations. Taken together, these results indicate that the stimulatory effect of estrogen on the activity of TIDA neurons is mediated by prolactin.

The effects of cocaine on rat plasma and brain alpha-melanocyte-stimulating hormone (alpha-MSH) levels have been studied by means of a specific radioimmunoassay. The selected brain areas were the hypothalamus, septum-nucleus accumbens and hippocampus. Cocaine given subcutaneously decreased the alpha-MSH levels in the peripheral blood. Pimozide, a dopaminergic antagonist, had an opposite effect: it increased the alpha-MSH levels in the peripheral blood. Combined treatment with cocaine + pimozide resulted in a decrease in the pimozide-induced increase in alpha-MSH levels in the blood. Cocaine and pimozide or the combination of cocaine + pimozide were ineffective on the alpha-MSH levels in the hypothalamus and septum-accumbens brain regions. In the hippocampus, cocaine in the dose applied induced a slight but not significant decrease in the alpha-MSH level. Pimozide caused a significant decrease in the hippocampal alpha-MSH level which disappeared at 60 min. Cocaine prevented the pimozide-induced depletion of alpha-MSH. The data indicate that cocaine may act as a dopaminergic agonist in the mechanism of control of alpha-MSH secretion from the intermediate lobe of the pituitary. The alpha-MSH levels in the brain are controlled by different mechanisms. In some brain areas, the dopaminergic system has no action; in others the mechanisms might be similar to but slightly different from that in the pituitary.

Pro-opiomelanocortin (POMC) is processed to adrenocorticotropic hormone (ACTH) and β-lipotropin in corticotropes of the anterior lobe, and to α-melanocyte-stimulating hormone (α-MSH) and β-endorphin in melanotropes of the intermediate lobe (IL) of the pituitary gland. While ACTH secretion is predominantly under the stimulatory influence of the hypothalamic factors, hormone secretion of the IL is tonically inhibited by neuroendocrine dopamine (NEDA) neurons. Lobe-specific POMC processing is not absolute. For example, D2 type DA receptor (D2R)-deficient mice have elevated plasma ACTH levels, although it is known that corticotropes do not express D2R(s). Moreover, observations that suckling does not influence α-MSH release, while it induces an increase in plasma ACTH is unexplained. The aim of the present study was to investigate the involvement of the NEDA system in the regulation of ACTH secretion and the participation of the IL in ACTH production in lactating rats. Untreated and estradiol (E2)-substituted ovariectomized (OVX) females were also studied. The concentration of ACTH in the IL was higher in lactating rats than in OVX rats, while the opposite change in α-MSH level of the IL was observed. DA levels in the IL and the neural lobe were lower in lactating rats than in OVX rats. Suckling-induced ACTH response was eliminated by pretreatment with the DA receptor agonist, bromocriptine (BRC). Inhibition of DA biosynthesis by α-methyl-p-tyrosine (αMpT) and blockade of D2R by domperidone (DOM) elevated plasma ACTH levels, but did not influence plasma α-MSH levels in lactating rats. The same drugs had opposite effects in OVX and OVX + E2 animals. In lactating mothers, BRC was able to block ACTH responses induced by both αMpT and DOM. Surgical denervation of the IL elevated basal plasma levels of ACTH. Taken together, these data indicate that melanotropes synthesize ACTH during lactation and its release from these cells is regulated by NEDA neurons.

Ovariectomized and adrenalectomized rats kept in a reversed lighting system received either 5 micrograms (once) or 1 microgram estradiol benzoate (EB) daily for 2, 3, or 4 days. These treatments induced sexual receptivity in a proportion of the rats, and alpha-melanocyte-stimulating hormone (alpha-MSH; 20 micrograms/rat s.c.) enhanced this proportion. In rats made receptive by 5 micrograms EB alone, the dopamine (DA) activity in preoptic area and arcuate nucleus was significantly reduced, but the alpha-MSH concentrations were not affected 54-56 h after EB treatment. Addition of alpha-MSH significantly increased the DA activity in ventromedial nucleus (VMN) and zona incerta, but this action is unlikely to account for its stimulation of sexual receptivity, since this effect was not blocked by 0.1 mg/kg haloperidol. Treatment with 100 mg/kg Dopa, which induces a presynaptic DA action, stimulated the receptivity in the EB-primed rats and selectively increased the concentration of alpha-MSH in the VMN, while 50 mg/kg Dopa plus 50 mg/kg benserazide, which induces a postsynaptic DA activity, inhibited the receptivity and reduced the alpha-MSH levels in the VMN. It is suggested that estradiol stimulates the female sexual receptivity by reducing the activity of a dopaminergic system in arcuate nucleus and preoptic area. alpha-MSH does not appear to affect this action, although it enhances the effect of steroids on the sexual behaviour, probably at the level of the VMN. The secretion of alpha-MSH may be under a dopaminergic inhibitory control, and the peptide may autoregulate its own secretion via this dopaminergic system which is sited in zona incerta and VMN.

The involvement of histaminergic neurons in the neuroendocrine regulation of the release of the pro-opiomelanocortin-derived peptides adrenocorticotropin (ACTH; anterior pituitary lobe) and alpha-melanocyte-stimulating hormone (alpha-MSH; intermediate pituitary lobe) was studied in conscious male rats. Pretreatment with the histamine (HA) synthesis inhibitor (S)-alpha-fluoromethylhistidine (100 mumol/kg i.p. at -6 h or 400 mumol/kg i.p. at -20 and -6 h) had no effect on basal ACTH release but decreased basal alpha-MSH release. The two doses of (S)-alpha-fluoromethylhistidine inhibited by 35 and 50% the ACTH response to inhibited the ACTH and alpha-MSH response to ether stress by 50 and 70%, respectively. Intracerebroventricular administration of the HA metabolism inhibitor SKF91488 (400 or 800 nmol at -15 min) stimulated basal secretion of ACTH and alpha-MSH dose-dependently and augmented slightly the restraint- and ether-stress-induced release of ACTH and alpha-MSH. Intracerebroventricular infusion of the H1 receptor antagonist mepyramine (0.37 mumol) or the H2 receptor antagonists cimetidine (0.40 mumol) or ranitidine (0.40 mumol) inhibited or prevented the alpha-MSH response to intracerebroventricular administration of HA (0.27 mumol), restraint or ether stress. Pretreatment with the dopamine receptor agonist bromocriptine or the beta-adrenergic receptor antagonist propranolol inhibited the alpha-MSH response to HA or stress. The results indicate that hypothalamic histaminergic neurons participate in the neuroendocrine regulation of the pro-opiomelanocortin-derived peptides from the anterior (ACTH) and intermediate (alpha-MSH) pituitary lobe of male rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The purpose of the present study was to examine the acute effects of stress on the secretion of alpha-melanocyte-stimulating hormone (alpha MSH) and the activity of tuberohypophysial dopamine (DA) neurons in female and male rats. The activity of tuberohypophysial DA neurons was estimated by measuring the accumulation of 3,4-dihydroxyphenylalanine (DOPA) following administration of the decarboxylase inhibitor NSD 1015, and the concentrations of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the intermediate and neural lobes of the posterior pituitary. The combination of brief (2 min) ether exposure followed by 30 min of supine restraint (immobilization in the supine position) decreased the rate of DOPA accumulation in the intermediate, but not in the neural lobe of both female and male rats. Similarly, brief ether exposure followed by 10, 20 or 30 min of supine restraint increased plasma alpha MSH concentrations and decreased DOPAC concentrations in the intermediate lobe of female and male rats. In the absence of ether, tube restraint (confinement in a cylindrical acrylic tube) increased alpha MSH secretion and decreased intermediate lobe DOPAC concentrations, whereas ether in the absence of physical restraint had no effect. These results suggest that the stress-induced activation of alpha MSH secretion in both female and male rats may be due, in part, to a decrease in the activity of tuberohypophysial DA neurons in the intermediate lobe of the posterior pituitary.

The effects of the kappa-opioid receptor agonist trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzene- acetamide methanesulfonate hydrate (U-50488) were examined on alpha-melanocyte-stimulating hormone (alpha-MSH) secretion and the activity of tuberohypophysial dopamine (DA) neurons in the male rat. Tuberohypophysial DA neuronal activity was estimated by measuring: (1) the rate of DA synthesis [accumulation of 3,4-dihydroxyphenylalanine (DOPA) following inhibition of aromatic L-amino acid decarboxylase], and (2) DA metabolism [concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC)] in the intermediate lobe of the pituitary. U-50488 produced a dose- and time-dependent increase in plasma concentrations of alpha-MSH which was accompanied by a decrease in the accumulation of DOPA and in the intermediate lobe. The effects of U-50488 were blocked by pretreatment with the DA agonist apomorphine but not by the beta-adrenergic antagonist propranolol. The effects of U-50488 on plasma alpha-MSH concentrations and intermediate-lobe DOPA accumulation were blocked by pretreatment with the selective kappa-opioid receptor antagonist nor-binaltorphimine. These results indicate that U-50488, by acting on kappa-opioid receptors, inhibits the activity of intermediate-lobe tuberohypophysial DA neurons, and through this action increases the secretion of alpha-MSH from melanotrophs.

The proopiomelanocortin (POMC) gene and its peptide products are under complex regulation in the pituitary by multiple hormonal, neurohormonal and neurotransmitter factors. Corticotropin-releasing factor (CRF) stimulates the release of POMC-derived peptides in both anterior and intermediate lobes of the pituitary, while having differential long-term effects on levels of POMC mRNA in the two pituitary lobes in vivo. In the present study, we have analyzed the release of POMC-derived peptides, as well as changes in POMC gene transcription, primary transcript, nuclear mRNA and cytoplasmic mRNA levels following acute and chronic in vivo CRF administration in an attempt to elucidate the mechanism whereby this tissue-specific differential regulation occurs. Subcutaneous injection of CRF led to a substantial increase in plasma adrenocorticotropin, but only a minor sustained rise in plasma alpha-melanocyte-stimulating hormone. CRF was shown to induce rapid, time-dependent increases in POMC gene transcription in both anterior and intermediate pituitary lobes, which were reflected by increases in the level of POMC primary transcript in the nucleus. POMC primary transcript remained 2-fold elevated in the anterior lobe for at least 4 h after a single injection of CRF; in contrast, CRF stimulation of POMC primary transcript in the intermediate lobe was short-lived, and had returned to control values by 60 min after injection. After 7 days of repetitive CRF administration, POMC primary transcript, mature nuclear mRNA and cytoplasmic mRNA were 2.5 to 3.0-fold elevated in the anterior pituitary.(ABSTRACT TRUNCATED AT 250 WORDS)

We previously reported a patient with generalized vitiligo improved by oral administration of the drug for menopausal syndrome (sex hormone-thyroid powder mixture). In this study, we reevaluated the efficiency of this drug for vitiligo, and examined its pharmacological action in melanogenesis.

These studies were undertaken to characterize the secretion of the pro-opiomelanocortin (POMC)-peptides adrenocorticotropin (ACTH), immunoreactive (ir)-beta-endorphin (beta-EP), and ir-alpha-melanocyte-stimulating hormone (ir-alpha-MSH) in the conscious sheep. When blood samples were taken at 10-min intervals, a distinct POMC-peptide and cortisol ultradian rhythm was observed, and 4 of the 7 possible POMC-peptide pulse patterns were defined. Three of these patterns included pulsatile ACTH secretion, and 70% were followed by a rise in plasma cortisol. The fourth pulse pattern consisted of concordant ir-beta-EP and ir-alpha-MSH secretion and was not temporally correlated with cortisol secretion. When an intensified rate of venous sampling was used (2-min intervals over 1 h), the POMC-peptide and cortisol pulse frequencies were increased. Although the mean POMC-peptide and cortisol pulse amplitudes remained unaltered, the mean cortisol pulse amplitude was decreased. Five of the 7 possible POMC-peptide pulse patterns were noted, and 3 of these were identical to those observed during the 10-min sampling procedure. Chronic bromocriptine treatment decreased the plasma levels and pulse amplitudes of ir-beta-EP and ir-alpha-MSH, increased the ir-beta-EP pulse interval, and abolished concordant ir-beta-EP and ir-alpha-MSH secretion. Neither the plasma levels nor the ultradian rhythms of ACTH and cortisol were affected by the treatment. Chronic dexamethasone administration abolished the ACTH ultradian rhythm, reduced the plasma levels of ACTH, ir-beta-EP, and ir-alpha-MSH, while plasma cortisol levels became undetectable. Although the ir-alpha-MSH interpulse interval was increased, the ir-beta-EP and ir-alpha-MSH ultradian rhythms were not abolished. We conclude that: (1) an ultradian rhythm characterizes the secretion of ACTH, ir-beta-EP, and ir-alpha-MSH from the ovine pituitary; (2) this rhythm is due to the pulsatile secretion of peptides from the adenohypophysis and intermediate lobe; (3) the adenohypophysis and intermediate lobes appear to secrete POMC-peptides in synchrony and asynchrony, and (4) intensified rates of venous sampling unmask the appearance of high-frequency POMC-peptide and cortisol pulses in vivo in the sheep.

The present study describes and compares the topography of alpha-melanocyte-stimulating hormone (alpha-MSH)-immunoreactive neurons in the forebrain of rats and horses. Two groups of immunoreactive cells were present in both species. These groups were distinguished by their cross-immunoreactivity and topography. The topography of cell bodies with cross-immunoreactivity for peptides derived from pro-opiomelanocortin (POMC) including adrenocorticotropin, 16K, alpha-MSH and beta-endorphin was similar in both species. However, the topography of perikarya which stained only for alpha-MSH, and not for other POMC-derived neuropeptides, was subject to pronounced species differences. In rats, perikarya which stained only for alpha-MSH (alpha 2-system) have a specific distribution in the dorsal and lateral hypothalamic regions from the level of the midoptic chiasm to the premamillary region of the posterior hypothalamus. At anterior levels of the rat brain, the cells are bipolar and oriented horizontally. Posteriorly, all alpha 2-group perikarya are stellate in rats. In the equine species, alpha 2-group cell bodies occupy a shorter anterioposterior field extending from the posterior level of the paraventricular nucleus to the supramamillary nucleus. Stellate cells are mixed with bipolar cells at all levels of the alpha 2-field in the equine hypothalamus. Further, in contrast to the continuous cell group found in rats, the alpha 2-group divides into perifornical and periventricular subpopulations at posterior levels of the equine hypothalamus. The results from this study suggest that the topography of the arcuate opiocortin cell pool, which contains peptides derived from POMC, is similar in phylogenetically diverse mammalian species whereas the topography of the cell pool staining only for alpha-MSH is subject to species differences. In both species, the major terminus of alpha 2-neurons appears to be the cerebral cortex and hippocampus. It has been proposed that alpha-MSH is one of several neuropeptides concerned with memory and behavior. Thus, species differences in the topography of the alpha 2-pool may reflect evolutionary changes in the importance of this peptide to these physiological processes.