Pharmacological properties of whale shark (Rhincodon typus) melanocortin-2 receptor and melancortin-5 receptor: Interaction with MRAP1 and MRAP2.

In the current study, the whale shark (ws; Rhincodon typus) melanocortin-2 receptor (MC2R) co-expressed with wsMRAP1 in Chinese Hamster Ovary (CHO) Cells could be stimulated in a dose dependent manner by ACTH(1-24) with an EC50 of 2.6 × 10-10 M ± 9.7 × 10-11. When the receptor was expressed alone, stimulation was only observed at [10-6 M]. A comparable increase in sensitivity to stimulation by srDes-Ac-αMSH was also observed when the receptor was co-expressed with wsMRAP1. Furthermore, co-expression with wsMRAP1 significantly increased the trafficking of wsMC2R to the plasma membrane of CHO cells. Surprisingly, co-expression with wsMRAP2 also increased sensitivity to stimulation by ACTH(1-24) and srDes-Ac-αMSH, and increased trafficking of the receptor to the plasma membrane. These observations are in sharp contrast to the response of MC2R orthologs of bony vertebrates which have an obligate requirement for co-expression with MRAP1 for both trafficking to the plasma membrane and activation, whereas, co-expression with MRAP2 increases trafficking, but has minimal effects on activation. In addition, when comparing the activation features of wsMC2R with those of the elephant shark MC2R and red stingray MC2R orthologs, both similarities and differences are observed. The spectrum of features for cartilaginous fish MC2R orthologs will be discussed. A second objective of this study was to determine whether wsMC5R has features in common with wsMC2R in terms of ligand selectivity and interaction with wsMRAP paralogs. While wsMC5R can be activated by either srACTH(1-24) or srDes-Ac-αMSH, and co-expression with wsMRAP1 enhances this activation, wsMRAP1 had no effect on the trafficking of wsMC5R. In addition, co-expression with wsMRAP2 had no positive or negative effect on either ligand sensitivity or trafficking of wsMC5R.

Pharmacological properties of whale shark (Rhincodon typus) melanocortin-2 receptor and melancortin-5 receptor: Interaction with MRAP1 and MRAP2.

In the current study, the whale shark (ws; Rhincodon typus) melanocortin-2 receptor (MC2R) co-expressed with wsMRAP1 in Chinese Hamster Ovary (CHO) Cells could be stimulated in a dose dependent manner by ACTH(1-24) with an EC50 of 2.6 × 10-10 M ± 9.7 × 10-11. When the receptor was expressed alone, stimulation was only observed at [10-6 M]. A comparable increase in sensitivity to stimulation by srDes-Ac-αMSH was also observed when the receptor was co-expressed with wsMRAP1. In addition, co-expression with wsMRAP1 significantly increased the trafficking of wsMC2R to the plasma membrane of CHO cells. Surprisingly, co-expression with wsMRAP2 also increased sensitivity to stimulation by ACTH(1-24) and srDes-Ac-αMSH, and increased trafficking of the receptor to the plasma membrane. These observations are in sharp contrast to the response of MC2R orthologs of bony vertebrates which have an obligate requirement for co-expression with MRAP1 for both trafficking to the plasma membrane and activation, and while co-expression with MRAP2 increases trafficking, it has minimal effects on activation. In addition, when comparing the activation features of wsMC2R with those of the elephant shark MC2R and red stingray MC2R orthologs, both similarities and differences are observed. The spectrum of features for cartilaginous fish MC2R orthologs will be discussed. A second objective of this study was to determine whether wsMC5R has features in common with wsMC2R in terms of ligand selectivity and interaction with wsMRAP paralogs. While wsMC5R can be activated by either srACTH(1-24) or srDes-Ac-αMSH, and co-expression with wsMRAP1 enhances this activation, wsMRAP1 had no effect on the trafficking of wsMC5R. Co-expression with wsMRAP2 had no positive or negative effect on either ligand sensitivity or trafficking of wsMC5R.

Structural Complexity and Plasticity of Signaling Regulation at the Melanocortin-4 Receptor.

The melanocortin-4 receptor (MC4R) is a class A G protein-coupled receptor (GPCR), essential for regulation of appetite and metabolism. Pathogenic inactivating MC4R mutations are the most frequent cause of monogenic obesity, a growing medical and socioeconomic problem worldwide. The MC4R mediates either ligand-independent or ligand-dependent signaling. Agonists such as α-melanocyte-stimulating hormone (α-MSH) induce anorexigenic effects, in contrast to the endogenous inverse agonist agouti-related peptide (AgRP), which causes orexigenic effects by suppressing high basal signaling activity. Agonist action triggers the binding of different subtypes of G proteins and arrestins, leading to concomitant induction of diverse intracellular signaling cascades. An increasing number of experimental studies have unraveled molecular properties and mechanisms of MC4R signal transduction related to physiological and pathophysiological aspects. In addition, the MC4R crystal structure was recently determined at 2.75 Å resolution in an inactive state bound with a peptide antagonist. Underpinned by structural homology models of MC4R complexes simulating a presumably active-state conformation compared to the structure of the inactive state, we here briefly summarize the current understanding and key players involved in the MC4R switching process between different activity states. Finally, these perspectives highlight the complexity and plasticity in MC4R signaling regulation and identify gaps in our current knowledge.

[Melanocortin peptides : Fundamentals, translational research, clinical dermatology, and future perspectives]. / Melanokortinpeptide : Grundlagen, translationale Forschung, klinische Dermatologie und Zukunftsperspektiven.

Melanocortins are peptides that share a common central pharmacophor. Melanin pigmentation of interfollicular epidermis and hair via MC1R remains the key physiologic function of the naturally occurring melanocortin peptides in skin. Moreover, the melanocortins are crucially involved in the ultraviolet light-induced tanning response. Under pathophysiologic conditions, melanocortin peptides induce cutaneous hyperpigmentation, likewise via the MC1R axis, e.g. in patients with Addison's disease, ectopic precursor pro-opiomelanocortin (POMC) syndrome and in those with abnormally elevated melanocortin blood levels. Translational research on α­MSH (melanocyte-stimulating hormones) and their antagonists has further revealed a variety of other biological activities beyond pigmentation. They include cytoprotection, antioxidative effects, regulation of collagen metabolism and fibrosis, sebum production, and cutaneous wound healing. These findings have also promoted the development of novel therapies in clinical dermatology including the exploitation of afamelanotide. In 2015, this agent became the first in-class synthetic α­MSH analogue to be approved in dermatology for the treatment of erythropoetic protoporphyria. In addition to afamelanotide, setmelanotide has recently emerged as a highly selective MC4R agonist useful for the treatment of distinct forms of genetically determined obesity, e.g., POMC deficiency. Future perspectives in dermatology reside in treatment of other difficult-to-treat skin diseases with α­MSH analogues, either with topical or systemic formulations. Moreover, synthetic melanocortin peptide derivatives lacking the central pharmacophor but with maintained anti-inflammatory effects could become a promising strategy for the design of new therapies in dermatology.

Melanocortin-1 Receptor Positively Regulates Human Artery Endothelial Cell Migration.

BACKGROUND/AIMS: Melanocortin receptors (MCRs) belong to a hormonal signalling pathway with multiple homeostatic and protective actions. Microvascular and umbilical vein endothelial cells (ECs) express components of the melanocortin system, including the type 1 receptor (MC1R), playing a role in modulating inflammation and vascular tone. Since ECs exhibit a remarkable heterogeneity, we investigated whether human artery ECs express any functional MCR and whether its activation affects cell migration. METHODS: We used reverse transcription real-time PCR to examine the expression of melanocortin system components in primary human artery ECs. We assessed MC1R protein expression and activity by western blot, immunohistochemistry, cAMP production, and intracellular Ca²âº mobilization assays. We performed gap closure and scratch tests to examine cell migration after stimulation with alpha-melanocyte-stimulating hormone (α-MSH), the receptor highest-affinity natural ligand. We assessed differential time-dependent transcriptional changes in migrating cells by microarray analysis. RESULTS: We showed that human aortic ECs (HAoECs) express a functionally active MC1R. Unlike microvascular ECs, arterial cells did not express the α-MSH precursor proopiomelanocortin, nor produced the hormone. MC1R engagement with a single pulse of α-MSH accelerated HAoEC migration both in the directional migration assay and in the scratch wound healing test. This was associated with an enhancement in Ca²âº signalling and inhibition of cAMP elevation. Time-course genome-wide expression analysis in HAoECs undergoing directional migration allowed identifying dynamic co-regulation of genes involved in extracellular matrix-receptor interaction, vesicle-mediated trafficking, and metal sensing - which have all well-established influences on EC motility -, without affecting the balance between pro- and anticoagulant genes. CONCLUSION: Our work broadens the knowledge on peripherally expressed MC1R. These results indicate that the receptor is constitutively expressed by arterial ECs and provide evidence of a novel homeostatic function for MC1R, whose activation may participate in preventing/healing endothelial dysfunction or denudation in macrovascular arteries.

Analyzing the signaling properties of gar (Lepisosteus oculatus) melanocortin receptors: Evaluating interactions with MRAP1 and MRAP2.

RT-PCR analysis of gar pituitary and brain indicated that different combinations of gar melanocortin receptor mRNAs are present in the same tissues with mRNAs for gar mrap1 and gar mrap2. Against this background, an objective of this study was to determine whether the ligand sensitivity for either ACTH or α-MSH was affected when gar (g) melanocortin receptors (Mcrs) were co-expressed with either of the accessory proteins gMrap1 or gMrap2 in Chinese Hamster Ovary cells. The results indicated that gMc2r has an obligatory requirement for co-expression with gMrap1 in order for the receptor to be activated by hACTH(1-24). In addition, activation of gMc2r did not occur when the receptor was expressed alone or co-expressed with gMrap2. Furthermore, co-expression of gMc2r with gMrap1 followed by stimulation with NDP-MSH resulted in a low level of activation (only at 10-7 M and 10-6 M). However, gMc1r, gMc3r, gMc4r, and gMc5r responded to stimulation by NDP-MSH in a more robust manner. Co-expression of gMc1r, gMc3r, gMc4r, and gMc5r with gMRAP1 had no effect on sensitivity to stimulation by NDP-MSH or hACTH(1-24). Co-expression with gMRAP2 had no negative or positive effect on ligand sensitivity for gMc1r, gMc3r, and gMc5r, however this treatment did increase the activation of CHO cells transfected with gMc4r following stimulation with both hACTH(1-24) (p < 0.001), and NDP-MSH (p < 0.001). Co-expression of gMC5R with either gMRAP1 or gMRAP2 increased trafficking of gMC5R to the plasma membrane. These pharmacological observations are compared to the response of melanocortin receptors from other neopterygian fishes, cartilaginous fishes, and tetrapods to stimulation by ACTH(1-24) and forms of α-MSH.

Elephant shark melanocortin receptors: Novel interactions with MRAP1 and implication for the HPI axis.

The presence of Mrap1 and Mrap2 orthologs in the genome of the elephant shark (es), a cartilaginous fish, presented an opportunity to evaluate the potential interactions between these accessory proteins and melanocortin receptors of a cartilaginous fish. RT-PCR analysis indicated that Mrap1 mRNA was present in interrenal, brain, and pituitary tissue with mRNA for Mc2R, Mc3R, Mc4R, and Mc5r. Co-expression of esMrap1 cDNA with esMc2r cDNA or esMc5r cDNA in CHO cells increased sensitivity to stimulation with ACTH(1-24) 10 fold and 100 fold, respectfully, but had no effect on sensitivity to stimulation with DesAc-αMSH [i.e., ACTH(1-13)NH2] for either receptor, and had no effect on the ligand sensitivity of either esMc3r or esMc4r. Fluorescence image analysis indicated co-localization of esMrap1/esMc2r, and esMrap1/esMc5r on the plasma membrane; however, cell surface ELISA analysis indicated that co-expression with esMrap1 had no effect, positive or negative, on the trafficking of either esMc2r or esMc5r to the plasma membrane. RT-PCR analysis also indicated that Mrap2 mRNA, as well as, mRNAs for Mc2r, Mc3r, Mc4r, and Mc5r could be detected in brain tissue, however no Mrap2 mRNA was detected in interrenal tissue. Co-expression of esMrap2 in CHO cells with, respectively, esMc2r, esMc4r, or esMc5r had no effect on ligand sensitivity. However, co-expression of esMrap2 with esMc3r did lower sensitivity to stimulation by DesAc-αMSH 10 fold. These observations are discussed in the context of the parallel evolution of melanocortin receptors and their accessory proteins, and the hypothalamus/pituitary/adrenal axis and the hypothalamus/pituitary/interrenal axis in bony vertebrates and cartilaginous fishes.

Melanocortin receptor subtypes are expressed on cells in the oligodendroglial lineage and signal ACTH protection.

ACTH, a melanocortin peptide used to treat multiple sclerosis (MS) relapses, acts by stimulating adrenal corticosteroid (CS) production via melanocortin receptor 2 (MC2R), but it may also exert a therapeutic effect independent of CS by stimulating other melanocortin receptors (MCR) distributed in many tissues, including the brain. We reported that oligodendroglia (OL) and oligodendroglial precursor cells (OPC) express MC4R, and that ACTH 1-39 protects OL and OPC in vitro from cell death induced by mechanisms likely involved in white matter damage in MS. This study investigates expression of MC1R, MC2R, MC3R and MC5R in OL and MC4R in OPC using immunocytochemistry with MCR subtype specific antibodies. OL express surface MC1R, MC3R and MC5R, in addition to MC4R. To investigate whether these receptors are functional, we asked if signaling through MCR is involved in ACTH protection of cultured rat OL from apoptosis (staurosporine), or cell death induced by excitotoxicity (glutamate), reactive oxygen species (ROS), or an inflammatory mediator (quinolinic acid). Like ACTH 1-39, MCR subtype specific agonists for MC1R, MC3R, MC4R and MC5R all protected OL from these insults. Conversely, antagonists for MC3R and MC4R blocked ACTH protection of OL. We then investigated the role of MC4R, as a prototype MCR, in protection and proliferation of OPC; MC4R agonists protected OPC and increased their proliferation, while antagonists blocked these effects. Our results demonstrate that MCR on OL and OPC are functional and activate signaling pathways that protect against mechanisms involved in OL damage in MS, suggesting potential beneficial effects in neurologic diseases.

Analyzing the effects of co-expression of chick (Gallus gallus) melanocortin receptors with either chick MRAP1 or MRAP2 in CHO cells on sensitivity to ACTH(1-24) or ACTH(1-13)NH2: Implications for the avian HPA axis and avian melanocortin circuits in the hypothalamus.

In order to better understand the roles that melanocortin receptors (cMCRs) and melanocortin-2 receptor accessory proteins (cMRAP1 and cMRAP2) play in the HPA axis and hypothalamus, adrenal gland and hypothalamus mRNA from 1day-old white leghorn chicks (Gallus gallus), were analyzed by real-time PCR. mRNA was also made for kidney, ovary, and liver. Mrap1 mRNA could be detected in adrenal tissue, but not in any of the other tissues, and mrap2 mRNA was also detected in the adrenal gland. Finally, all five melanocortin receptors mRNAs could be detected in the adrenal gland; mc2r and mc5r mRNAs were the most abundant. To evaluate any potential interactions between MRAP1 and the MCRs that may occur in adrenal cells, individual chick mcr cDNA constructs were transiently expressed in CHO cells either in the presence or absence of a chick mrap1 cDNA, and the transfected cells were stimulated with hACTH(1-24) at concentrations ranging from 10-13M to 10-6M. As expected, MC2R required co-expression with MRAP1 for functional expression; whereas, co-expression of cMC3R with cMRAP1 had no statistically significant effect on sensitivity to hACTH(1-24). However, co-expression of MC4R and MC5R with MRAP1, increased sensitivity for ACTH(1-24) by approximately 35 fold and 365 fold, respectively. However, co-expressing of cMRAP2 with these melanocortin receptors had no effect on sensitivity to hACTH(1-24). Since the real-time PCR analysis detected mrap2 mRNA and mc4r mRNA in the hypothalamus, the interaction between cMC4R and cMRAP2 with respect to sensitivity to ACTH(1-13)NH2 stimulation was also evaluated. However, no effect, either positive or negative, was observed. Finally, the highest levels of mc5r mRNA were detected in liver cells. This observation raises the possibility that in one-day old chicks, activation of the HPA axis may also involve a physiological response from liver cells.

Evaluating the interactions between red stingray (Dasyatis akajei) melanocortin receptors and elephant shark (Callorhinchus milii) MRAP1 and MRAP2 following stimulation with either stingray ACTH(1-24) or stingray Des-Acetyl-αMSH: A pharmacological study in Chinese Hamster Ovary cells.

Previous studies on bony vertebrate MC2R orthologs (i.e., ray finned fishes, amphibians, reptiles, birds, and mammals) have shown that these MC2R orthologs have an obligatory requirement for interaction with bony vertebrate MRAP1 orthologs to a) allow for the trafficking of the MC2R ortholog to the plasma membrane; and b) to allow activation by ACTH, but not by any MSH-sized ligand. In addition, previous studies have found that co-expression of teleost and mammalian MC4R orthologs with corresponding MRAP2 has positive effects on sensitivity to stimulation by αMSH or ACTH. MRAP1 and MRAP2 paralogs have been detected in the genome of a cartilaginous fish (elephant shark), yet two cartilaginous fish MC2R orthologs (elephant shark and red stingray) do not apparently require MRAP1 for trafficking to the plasma membrane when expressed in Chinese Hamster Ovary (CHO) cells, and both orthologs can be activated by either ACTH or MSH-sized ligands. This study was done to determine whether sensitivity to stimulation by ACTH(1-24) or Des-Acetyl-αMSH is affected when stingray (sr) MC1R, MC2R, MC3R, MC4R or MC5R were co-expressed in CHO cells with either elephant shark (es) MRAP1 or esMRAP2. The results indicated that co-expression with heterologous MRAP1 increased the sensitivity of all five stingray melanocortin receptors for srACTH(1-24), but had not statistically significant effect on stimulation by srDes-Acetyl-αMSH for any of the stingray melanocortin receptors. Conversely, co-expression with esMRAP2 only enhanced sensitivity for srDes-Acetyl-αMSH for srMC4R, but had no effect on the other stingray orthologs, and there was no increase in sensitivity for srACTH(1-24) for any of the stingray melanocortin receptors. It appears then that some stingray melanocortin receptors have retained the ability to interact with a cartilaginous MRAP1 paralog. These results are discussed with reference to radiation of MRAP-related accessory proteins in cartilaginous fishes.

α-Melanocyte-Stimulating Hormone Attenuates Neovascularization by Inducing Nitric Oxide Deficiency via MC-Rs/PKA/NF-κB Signaling.

α-melanocyte-stimulating hormone (α-MSH) has been characterized as a novel angiogenesis inhibitor. The homeostasis of nitric oxide (NO) plays an important role in neovascularization. However, it remains unclear whether α-MSH mitigates angiogenesis through modulation of NO and its signaling pathway. The present study elucidated the function and mechanism of NO signaling in α-MSH-induced angiogenesis inhibition using cultured human umbilical vein endothelial cells (HUVECs), rat aorta rings, and transgenic zebrafish. By Griess reagent assay, it was found α-MSH dose-dependently reduced the NO release in HUVECs. Immunoblotting and immunofluorescence analysis revealed α-MSH potently suppressed endothelial and inducible nitric oxide synthase (eNOS/iNOS) expression, which was accompanied with inhibition of nuclear factor kappa B (NF-κB) activities. Excessive supply of NO donor l-arginine reversed the α-MSH-induced angiogenesis inhibition in vitro and in vivo. By using antibody neutralization and RNA interference, it was delineated that melanocortin-1 receptor (MC1-R) and melanocortin-2 receptor (MC2-R) participated in α-MSH-induced inhibition of NO production and NF-κB/eNOS/iNOS signaling. This was supported by pharmaceutical inhibition of protein kinase A (PKA), the downstream effector of MC-Rs signaling, using H89 abolished the α-MSH-mediated suppression of NO release and eNOS/iNOS protein level. Therefore, α-MSH exerts anti-angiogenic function by perturbing NO bioavailability and eNOS/iNOS expression in endothelial cells.

Molecular signatures of human melanocortin receptors for ligand binding and signaling.

Human melanocortin receptors (hMCRs) belong to the seven-transmembrane (TM) domain proteins. There are five hMCR subtypes and each of these receptor subtypes has different patterns of tissue expression and physiological function. The endogenous agonists for hMCRs are α-, ß-, and γ-MSH and ACTH and endogenous antagonists are Agouti and AGRP which are the only known naturally occurring antagonists for the receptors. These peptides have their own profiles regarding the relative potency for specific hMCR subtype. Extensive studies have been performed to examine the molecular basis of the hMCRs for different ligand binding affinity and potency. Studies indicate that natural ligand α-MSH utilizes conserved amino acid residues for MCR specific binding (orthosteric binding) while synthetic ligands utilize non-conserved amino acid residues for receptor subtype specific binding (allosteric binding). ACTH is the only endogenous agonist for hMC2R and more amino acid residues at hMC2R are required for ACTH binding and signaling. HMCR computer modeling provides the detailed information of ligand and MCR interaction. This review provides the latest understanding of the molecular basis of the hMCRs for ligand binding and signaling. This article is part of a Special Issue entitled: Melanocortin Receptors - edited by Ya-Xiong Tao.

The melanocortin system regulates body pigmentation and social behaviour in a colour polymorphic cichlid fish.

The melanocortin system is a neuroendocrine system that regulates a range of physiological and behavioural processes. We examined the extent to which the melanocortin system simultaneously regulates colour and behaviour in the cichlid fish Astatotilapia burtoni We found that yellow males are more aggressive than blue males, in line with previous studies. We then found that exogenous α-melanocyte-stimulating hormone (α-MSH) increases yellowness of the body and dispersal of xanthophore pigments in both morphs. However, α-MSH had a morph-specific effect on aggression, with only blue males showing an increase in the rate of aggression. Exogenous agouti signalling peptide (ASIP), a melanocortin antagonist, did not affect coloration but reduced the rate of aggression in both colour morphs. Blue males had higher cortisol levels than yellow males. Neural gene expression of melanocortin receptors (mcr) and ligands was not differentially regulated between colour morphs. In the skin, however, mc1r and pro-opiomelanocortin (pomc) ß were upregulated in blue males, while asip 1 was upregulated in yellow males. The effects of α-MSH on behaviour and body coloration, combined with morph-specific regulation of the stress response and the melanocortin system, suggest that the melanocortin system contributes to the polymorphism in behaviour and coloration in A. burtoni.

Association of estradiol on expression of melanocortin receptors and their accessory proteins in the liver of chicken (Gallus gallus).

The melanocortin receptor accessory proteins (MRAP and MRAP2) are small single-pass transmembrane proteins that regulate the biological functions of the melanocortin receptor (MCR) family. MCRs comprise five receptors (MC1R-MC5R) with diverse physiological roles in mammals. Five MCR members and two MRAPs were also predicted in the chicken (Gallus gallus) genome. However, little is known about their expression, regulation and biological functions. In this study, we cloned the MRAP and MRAP2 genes. Sequencing analysis revealed that the functional domains of MRAP and MRAP2 were conserved among species, suggesting that the physiological roles of chicken MRAP and MRAP2 could be similar to their mammalian counterparts. Tissue expression analysis demonstrated that MRAP was expressed in the adrenal gland, liver, spleen, glandular stomach and lungs, while MRAP2 is predominantly expressed in the adrenal gland. All five MCRs were present in the adrenal gland, but showed different expression patterns in other tissues. The MC5R was the only MCR member that was expressed in the chicken liver. The expression levels of MRAP in chicken liver were significantly increased at sexual maturity stage, and were significantly up-regulated (P<0.05) when chickens and chicken primary hepatocytes were treated with 17ß-estradiol in vivo and in vitro, respectively; however, expression levels of PPARγ were down-regulated, and no effect on MC5R was observed. Our results suggested that estrogen could stimulate the expression of MRAP in the liver of chicken through inhibiting the expression of transcription regulation factor PPARγ, and MRAP might play its biological role in a different way rather than forming an MRAP/MC2R complex in chicken liver during the egg-laying period.

Stimulation of the hypothalamic arcuate nucleus increases brown adipose tissue nerve activity via hypothalamic paraventricular and dorsomedial nuclei.

Hypothalamic arcuate nucleus (ARCN) stimulation elicited increases in sympathetic nerve activity (IBATSNA) and temperature (TBAT) of interscapular brown adipose tissue (IBAT). The role of hypothalamic dorsomedial (DMN) and paraventricular (PVN) nuclei in mediating these responses was studied in urethane-anesthetized, artificially ventilated, male Wistar rats. In different groups of rats, inhibition of neurons in the DMN and PVN by microinjections of muscimol attenuated the increases in IBATSNA and TBAT elicited by microinjections of N-methyl-d-aspartic acid into the ipsilateral ARCN. In other groups of rats, blockade of ionotropic glutamate receptors by combined microinjections of D(-)-2-amino-7-phosphono-heptanoic acid (D-AP7) and NBQX into the DMN and PVN attenuated increases in IBATSNA and TBAT elicited by ARCN stimulation. Blockade of melanocortin 3/4 receptors in the DMN and PVN in other groups of rats resulted in attenuation of increases in IBATSNA and TBAT elicited by ipsilateral ARCN stimulation. Microinjections of Fluoro-Gold into the DMN resulted in retrograde labeling of cells in the ipsilateral ARCN, and some of these cells contained proopiomelanocortin (POMC), α-melanocyte-stimulating hormone (α-MSH), or vesicular glutamate transporter-3. Since similar projections from ARCN to the PVN have been reported by us and others, these results indicate that neurons containing POMC, α-MSH, and glutamate project from the ARCN to the DMN and PVN. Stimulation of ARCN results in the release of α-MSH and glutamate in the DMN and PVN which, in turn, cause increases in IBATSNA and TBAT.

Melanocortin-4 receptor agonists alleviate intestinal dysfunction in secondary intra-abdominal hypertension rat model.

BACKGROUND: Intra-abdominal hypertension (IAH) is a potentially life-threatening disease. Melanocortin-4 (MC4) receptor activation exhibits life-saving properties. The aim of the present study was to examine whether treatment with the MC4 receptor agonist RO27-3225 ameliorates intestinal injury in IAH rats. METHODS: A total of 72 male Sprague-Dawley rats were randomized into six groups. Group 1 was the sham group. Group 2, the sham + RO group, received RO27-3225 (180 µg/kg, intraperitoneally). IAH was induced in group 3, the IAH group, by blood draw (mean arterial pressure = 30 mm Hg for 90 min) followed by shed blood and/or Ringer solution reinfusion. Intra-abdominal pressure was increased to 20 mm Hg by injecting air into the peritoneal cavity. Group 4, the RO group, was administered RO27-3225 at 5 min after blood draw. Groups 5 and 6 were the chlorisondamine (Chl) and HS024 groups, in which the rats were pretreated with the nicotinic acetylcholine receptor antagonist Chl or selective MC4 receptor antagonist (HS024), respectively, at 2 min before RO27-3225 was administered. RESULTS: RO27-3225 restored mean arterial pressure, reduced tumor necrosis factor-α, and interleukin-1ß messenger RNA expression increased by IAH, alleviated histologic damage, and improved superoxide dismutase activity in the intestine. Compared with the IAH group, the levels of intestinal fatty acid-binding protein, intestinal edema and intestinal permeability were lower in the RO group. Furthermore, the RO27-3225 treatment increased the expression of Rho-associated coiled-coil-containing protein kinase 1 and phosphorylated myosin light chain. Chl and HS024 abrogated the protective effects of RO27-3225. CONCLUSIONS: These data indicate that the MC4 receptor agonist counteracts the intestinal inflammatory response, ameliorating intestinal injury in experimental secondary IAH by MC4 receptor-triggered activation of the cholinergic anti-inflammatory pathway. It may represent a promising strategy for the treatment of IAH in the future.

A fragment of the Escherichia coli ClpB heat-shock protein is a micromolar melanocortin 1 receptor agonist.

The melanocortin system consists of five receptor subtypes (MC1-5R), endogenous agonists derived from the proopiomelanocortin gene transcript, and the antagonists agouti and agouti-related protein. The Escherichia coli heat shock protein ClpB has previously been described as an antigen mimetic to the endogenous melanocortin agonist α-MSH. Herein, we investigated if a fragment of the ClpB protein could directly signal through the melanocortin receptors. We synthesized a complementary fragment of the ClpB protein that partially aligned with α-MSH. Pharmacological assessment of this fragment resulted in no antagonist activity at the MC3R or the MC4R and no agonist activity at the MC4R. Partial receptor activation was observed for the MC3R and MC5R at 100 µM concentrations. This fragment was shown to be a full micromolar MC1R agonist and may serve as a template for future research into selective MC1R ligands.

Adrenocorticotropin hormone 1-39 promotes proliferation and differentiation of oligodendroglial progenitor cells and protects from excitotoxic and inflammation-related damage.

Oligodendroglia (OL) are highly susceptible to damage and, like neurons, are terminally differentiated. It is important to protect OL precursors (OPC) because they are reservoirs of differentiating cells capable of myelination following perinatal insult and remyelination in white matter diseases, including multiple sclerosis (MS). Patients with relapsing-remitting MS are commonly treated with high-dose corticosteroids (CS) when experiencing an exacerbation. Adrenocorticotropin hormone (ACTH), a primary component of another approved MS exacerbation treatment, is a melanocortin peptide that stimulates production of CS by the adrenals. Melanocortin receptors are also found in the central nervous system (CNS) and on immune cells. ACTH is produced within the CNS and may have CS-independent effects on glia. We found that ACTH 1-39 stimulated proliferation of OPC, and to a lesser extent astroglia (AS) and microglia (MG), in rat glial cultures. ACTH accelerated differentiation of PDGFRα(+) OPC to a later stage marked by galactolipid expression and caused greater expansion of OL myelin-like sheets compared with untreated cells. Protective effects of ACTH on OPC were assessed by treating cultures with selected toxic agents, with or without ACTH. At 200 nM, ACTH protected OPC from death induced by staurosporine, glutamate, NMDA, AMPA, kainate, quinolinic acid, H2 O2 , and slow NO release, but not against kynurenic acid or rapid NO release. These agents and ACTH were not toxic to AS or MG. Our findings indicate that ACTH 1-39 provides benefits by increasing the number of OPC, accelerating their development into mature OL, and reducing OPC death from toxic insults.

Synthesis and evaluation of bivalent ligands for binding to the human melanocortin-4 receptor.

Membrane proteins, especially G-protein coupled receptors (GPCRs), are interesting and important theragnostic targets since many of them serve in intracellular signaling critical for all aspects of health and disease. The potential utility of designed bivalent ligands as targeting agents for cancer diagnosis and/or therapy can be evaluated by determining their binding to the corresponding receptors. As proof of concept, GPCR cell surface proteins are shown to be targeted specifically using multivalent ligands. We designed, synthesized, and tested a series of bivalent ligands targeting the over-expressed human melanocortin 4 receptor (hMC4R) in human embryonic kidney (HEK) 293 cells. Based on our data suggesting an optimal linker length of 25±10Å inferred from the bivalent melanocyte stimulating hormone (MSH) agonist, the truncated heptapeptide, referred to as MSH(7): Ac-Ser-Nle-Glu-His-D-Phe-Arg-Trp-NH2 was used to construct a set of bivalent ligands incorporating a hMC4R antagonist, SHU9119: Ac-Nle-c[Asp-His-2'-D-Nal-Arg-Trp-Lys]-NH2 and another set of bivalent ligands containing the SHU9119 antagonist pharmacophore on both side of the optimized linkers. These two binding motifs within the bivalent constructs were conjoined by semi-rigid (Pro-Gly)3 units with or without the flexible poly(ethylene glycol) (PEGO) moieties. Lanthanide-based competitive binding assays showed bivalent ligands binds to the hMC4R with up to 240-fold higher affinity than the corresponding linked monovalent ligands.

Ocular pharmacological and biochemical profiles of 6-thioguanine: a drug repurposing study.

Introduction: The purine analog 6-thioguanine (6TG), an old drug approved in the 60s to treat acute myeloid leukemia (AML), was tested in the diabetic retinopathy (DR) experimental in vivo setting along with a molecular modeling approach. Methods: A computational analysis was performed to investigate the interaction of 6TG with MC1R and MC5R. This was confirmed in human umbilical vein endothelial cells (HUVECs) exposed to high glucose (25 mM) for 24 h. Cell viability in HUVECs exposed to high glucose and treated with 6TG (0.05-0.5-5 µM) was performed. To assess tube formation, HUVECs were treated for 24 h with 6TG 5 µM and AGRP (0.5-1-5 µM) or PG20N (0.5-1-5-10 µM), which are MC1R and MC5R antagonists, respectively. For the in vivo DR setting, diabetes was induced in C57BL/6J mice through a single streptozotocin (STZ) injection. After 2, 6, and 10 weeks, diabetic and control mice received 6TG intravitreally (0.5-1-2.5 mg/kg) alone or in combination with AGRP or PG20N. Fluorescein angiography (FA) was performed after 4 and 14 weeks after the onset of diabetes. After 14 weeks, mice were euthanized, and immunohistochemical analysis was performed to assess retinal levels of CD34, a marker of endothelial progenitor cell formation during neo-angiogenesis. Results: The computational analysis evidenced a more stable binding of 6TG binding at MC5R than MC1R. This was confirmed by the tube formation assay in HUVECs exposed to high glucose. Indeed, the anti-angiogenic activity of 6TG was eradicated by a higher dose of the MC5R antagonist PG20N (10 µM) compared to the MC1R antagonist AGRP (5 µM). The retinal anti-angiogenic effect of 6TG was evident also in diabetic mice, showing a reduction in retinal vascular alterations by FA analysis. This effect was not observed in diabetic mice receiving 6TG in combination with AGRP or PG20N. Accordingly, retinal CD34 staining was reduced in diabetic mice treated with 6TG. Conversely, it was not decreased in diabetic mice receiving 6TG combined with AGRP or PG20N. Conclusion: 6TG evidenced a marked anti-angiogenic activity in HUVECs exposed to high glucose and in mice with DR. This seems to be mediated by MC1R and MC5R retinal receptors.