Melanocortin-5 receptor and sebogenesis

The melanocortins (α-MSH, β-MSH, γ-MSH, and ACTH) bind to the melanocortin receptors and signal through increases in cyclic adenosine monophosphate to induce biological effects. The melanocortin MC₅ and MC₁ receptors are expressed in human sebaceous glands, which produce sebum, a lipid mixture of squalene, wax esters, triglycerides, cholesterol esters, and free fatty acids that is secreted onto the skin. Excessive sebum production is one of the major factors in the pathogenesis of acne. The expression of melanocortin MC₅ receptor has been associated with sebocyte differentiation and sebum production. Sebaceous lipids are down-regulated in melanocortin MC₅ receptor-deficient mice, consistent with the observation that α-MSH acts as a sebotropic hormone in rodents. These findings, which suggest that melanocortins stimulate sebaceous lipid production through the MC₅ receptor, led to our search for MC₅ receptor antagonists as potential sebum-suppressive agents. As predicted, an antagonist was shown to inhibit sebocyte differentiation in vitro, and to reduce sebum production in human skin transplanted onto immunodeficient mice. The melanocortin MC₅ receptor antagonists may prove to be clinically useful for the treatment of sebaceous disorders with excessive sebum production, such as acne.     

Fish melanocortin system

Melanocortin signalling is mediated by binding to a family of G protein-coupled receptors that positively couple to adenylyl cyclase. Tetrapod species have five melanocortin (MC₁–MC₅) receptors. The number of receptors varies in fish, zebrafish, for example, having six melanocortin receptors, with two copies of the melanocortin MC₅ receptor, while pufferfish have 4 receptors with no melanocortin MC₃ receptor and one copy of melanocortin MC₅ receptor. Fish genomes also exhibit orthologue genes for agouti-signalling protein (ASP) and -related protein (AGRP). AGRP expression is confined to a small area in the hypothalamus but ASP is expressed in the skin. Fish melanocortin MC₂ receptor is specific for ACTH and requires the cooperation of accessory proteins (MRAP) to reach functional expression. The four other melanocortin MC receptors distinctively bind MSHs. The interaction of α-MSH and melanocortin MC₁ receptor plays a key point in the control of the pigmentation and mutations of melanocortin MC₁ receptor are responsible for reduced melanization. Both melanocortin MC₄ and MC₅ receptor are expressed in the hypothalamus, and central melanocortin MC₄ receptor expression is thought to regulate the energy balance through the modulation of feeding behaviour. In addition, the peripheral melanocortin system also regulates lipid metabolism by acting at hepatic melanocortin MC₂ and MC₅ receptors. Both sea bass melanocortin MC₁ and MC₄ receptors are constitutively expressed in vitro and both ASP and AGRP work as inverse agonists but only after inhibition of the phosphodiesterase system. Accordingly, the overexpression of AGRP and ASP transgenes promotes obesity and reduces melanization in zebrafish, respectively.     

Use of chimeric melanocortin-2 and -4 receptors to identify regions responsible for ligand specificity and dependence on melanocortin 2 receptor accessory protein

The melanocortin 2 (MC₂) receptor differs from other melanocortin family members in its pharmacological profile and reliance on an accessory protein, MC₂ receptor accessory protein (MRAP), for surface expression and signal transduction. To identify features of the MC₂ receptor responsible for these characteristics, we created chimeras between MC₂ and MC₄ receptors and expressed these in CHO cells, where MRAP is essential for trafficking and signaling by MC₂ but not MC₄ receptors. Replacing the first transmembrane segment of the MC₂ receptor with the corresponding region from the MC₄ receptor allowed some surface expression in the absence of an accessory protein, while ACTH-induced cAMP production remained entirely MRAP-dependent. On the other hand, replacing the last two transmembrane domains, third extracellular loop and C-terminal tail of the MC₄ receptor with the corresponding regions from the MC₂ receptor resulted in MRAP-dependent signaling. Surprisingly, replacing the second and third transmembrane domains and the intervening first extracellular loop of MC₂ receptors with MC₄ sequences generated a chimera (2C2) that responded to both adrenocorticotropic hormone (ACTH) and to the potent MSH analog 4-norleucine-7-d-phenylalanine-α-melanocyte stimulating hormone (NDP-α-MSH), which does not activate native MC₂ receptors. The 2C2 chimeric receptor was able to respond to NDP-α-MSH without MRAP, but MRAP shifted the EC50 value for NDP-α-MSH to the left and caused constitutive activity. These results identify the first transmembrane domain as important for surface expression and regions from the second to third transmembrane segments of the MC₂ receptor as important for MRAP dependent-signal transduction and ligand specificity.     

Chondroprotective and anti-inflammatory role of melanocortin peptides in TNF-α activated human C-20/A4 chondrocytes

BACKGROUND AND PURPOSE

Melanocortin MC₁ and MC₃ receptors, mediate the anti-inflammatory effects of melanocortin peptides. Targeting these receptors could therefore lead to development of novel anti-inflammatory therapeutic agents. We investigated the expression of MC₁ and MC₃ receptors on chondrocytes and the role of α-melanocyte-stimulating hormone (α-MSH) and the selective MC₃ receptor agonist, [DTRP⁸]-γ-MSH, in modulating production of inflammatory cytokines, tissue-destructive proteins and induction of apoptotic pathway(s) in the human chondrocytic C-20/A4 cells.

EXPERIMENTAL APPROACH

Effects of α-MSH, [DTRP⁸]-γ-MSH alone or in the presence of the MC_3/4 receptor antagonist, SHU9119, on TNF-α induced release of pro-inflammatory cytokines, MMPs, apoptotic pathway(s) and cell death in C-20/A4 chondrocytes were investigated, along with their effect on the release of the anti-inflammatory cytokine IL-10.

KEY RESULTS

C-20/A4 chondrocytes expressed functionally active MC_1,3 receptors. α-MSH and [DTRP⁸]-γ-MSH treatment, for 30 min before TNF-α stimulation, provided a time-and-bell-shaped concentration-dependent decrease in pro-inflammatory cytokines (IL-1β, IL-6 and IL-8) release and increased release of the chondroprotective and anti-inflammatory cytokine, IL-10, whilst decreasing expression of MMP1, MMP3, MMP13 genes.α-MSH and [DTRP⁸]-γ-MSH treatment also inhibited TNF-α-induced caspase-3/7 activation and chondrocyte death. The effects of [DTRP⁸]-γ-MSH, but not α-MSH, were abolished by the MC_3/4 receptor antagonist, SHU9119.

CONCLUSION AND IMPLICATIONS

Activation of MC₁/MC₃ receptors in C-20/A4 chondrocytes down-regulated production of pro-inflammatory cytokines and cartilage-destroying proteinases, inhibited initiation of apoptotic pathways and promoted release of chondroprotective and anti-inflammatory cytokines. Developing small molecule agonists to MC₁/MC₃ receptors could be a viable approach for developing chondroprotective and anti-inflammatory therapies in rheumatoid and osteoarthritis.     

Development of a high throughput screen for allosteric modulators of melanocortin-4 receptor signaling using a real time cAMP assay

The melanocortin MC₄ receptor is a potential target for the development of drugs for both obesity and cachexia. Melanocortin MC₄ receptor ligands known thus far are orthosteric agonists or antagonists, however the agonists, in particular, have generally exhibited unwanted side effects. For some receptors, allosteric modulators are expected to reduce side-effect profiles. To identify allosteric modulators of the melanocortin MC₄ receptor, we created HEK293 cell lines coexpressing the human melanocortin MC₄ receptor and a modified luciferase-based cAMP sensor. Monitoring luminescence as a readout of real-time intracellular cAMP concentration, we demonstrate that this cell line is able to report melanocortin agonist responses, as well as inverse agonist response to the physiological AgRP peptide. Based on the MC4R-GLO cell line, we developed an assay that was shown to meet HTS standards (Z′ = 0.50). A pilot screen run on the Microsource Spectrum compound library (n = 2000) successfully identified 62 positive modulators. This screen identified predicted families of compounds: β₂AR agonists – the β₂AR being endogenously expressed in HEK293 cells, an adenylyl cyclase activator and finally a distribution of phosphodiesterase (PDE) inhibitors well characterized or recently identified. In this last category, we identified a structural family of coumarin-derived compounds (imperatorin, osthol and prenyletin), along with deracoxib, a drug in veterinary use for its COX2 inhibitory properties. This latter finding unveiled a new off-target mechanism of action for deracoxib as a PDE inhibitor. Overall, these data are the first report of a HTS for allosteric modulators for a Gs protein coupled receptor.     

Discovery of novel melanocortin4 receptor selective MSH analogues

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]α-MSH(4–11) displayed specific MC₄ receptor selectivity. The preference order of the different MC receptor subtypes for the novel [Cys⁴D-Nal⁷Cys¹¹]α-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]α-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 (K_i 3.2 nM, which is ∼300 fold higher affinity than for α-MSH), was also demonstrated to antagonize α-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]α-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.

     

Melanocortin MC4 receptor expression sites and local function

The melanocortin MC₄ receptor plays an important role in energy metabolism, but also affects blood pressure, heart rate and erectile function. Localization of the receptors that fulfill these distinct roles is only partially known. Mapping of the melanocortin MC₄ receptor has been stymied by the absence of a functional antibody. Several groups have examined mRNA expression of the melanocortin MC₄ receptor in the rodent brain and transgenic approaches have also been utilized to visualize melanocortin MC₄ receptor expression sites within the brain. Ligand expression and binding studies have provided additional information on the areas of the brain where this elusive receptor is functionally expressed. Finally, microinjection of melanocortin MC₄ receptor ligands in specific nuclei has further served to elucidate the function of melanocortin MC₄ receptors in these nuclei. These combined approaches have helped link the anatomy and function of this receptor, such as the role of paraventricular hypothalamic nucleus melanocortin MC₄ receptor in the regulation of food intake. Intriguingly, however, numerous expression-sites have been identified that have not been linked to a specific receptor function such as those along the optic tract and olfactory tubercle. Further research is needed to clarify the function of the melanocortin MC₄ receptor at these sites.     

Effects of melanocortins on adrenal gland physiology

The melanocortin-2-receptor (MC₂ receptor), also known as the ACTH receptor, is a critical component of the hypothalamic–pituitary–adrenal axis. The importance of MC₂ receptor in adrenal physiology is exemplified by the condition familial glucocorticoid deficiency, a potentially fatal disease characterised by isolated cortisol deficiency. MC₂receptor mutations cause ~ 25% of cases. The discovery of a MC₂ receptor accessory protein MRAP, mutations of which account for ~ 15%–20% of familial glucocorticoid deficiency, has provided insight into MC₂ receptor trafficking and signalling. MRAP is essential for the functional expression of MC₂ receptor. MRAP2, a novel homolog of MRAP, can also facilitate MC₂ receptor cell surface expression and function. Like MRAP, MRAP2 is a small transmembrane domain glycoprotein capable of homodimerising. In addition, MRAP/MRAP2 can heterodimerise. The presence of MRAP2 adrenal expression suggests a possible role for MRAP2 in adrenal physiology, which has yet to be elucidated. Importantly, new data shows that the MRAPs can interact with all the other melanocortin receptors (MC_1,3,4,5 receptor). In contrast to MC₂ receptor, this interaction results in reduced melanocortin receptor surface expression and signalling. MRAP2 is predominantly expressed in brain. Hypothalamic expression has been demonstrated for both MRAP and MRAP2. The ability of MRAPs to modulate different members of the melanocortin receptor family in a bidirectional manner is intriguing. Furthermore, central nervous system expression of MRAPs points to a role beyond MC₂ receptor mediated adrenal steroidogenesis.     

Melanocortin MC1 receptor in human genetics and model systems

The melanocortin MC₁ receptor is a G-protein coupled receptor expressed in the melanocytes of the skin and hair and is known for its key role in the regulation of human pigmentation. Melanocortin MC₁ receptor activation after ultraviolet radiation exposure results in a switch from the red/yellow pheomelanin to the brown/black eumelanin pigment synthesis within cutaneous melanocytes; this pigment is then transferred to the surrounding keratinocytes of the skin. The increase in melanin maturation and uptake results in tanning of the skin, providing a physical protection of skin cells from ultraviolet radiation induced DNA damage. Melanocortin MC₁ receptor polymorphism is widespread within the Caucasian population and some variant alleles are associated with red hair colour, fair skin, poor tanning and increased risk of skin cancer. Here we will discuss the use of mouse coat colour models, human genetic association studies, and in vitro cell culture studies to determine the complex functions of the melanocortin MC₁ receptor and the molecular mechanisms underlying the association between melanocortin MC₁ receptor variant alleles and the red hair colour phenotype. Recent research indicates that melanocortin MC₁ receptor has many non-pigmentary functions, and that the increased risk of skin cancer conferred by melanocortin MC₁ receptor variant alleles is to some extent independent of pigmentation phenotypes. The use of new transgenic mouse models, the study of novel melanocortin MC₁ receptor response genes and the use of more advanced human skin models such as 3D skin reconstruction may provide key elements in understanding the pharmacogenetics of human melanocortin MC₁ receptor polymorphism.     

Pharmacological properties of adenosine receptors and adenosine binding proteins

Two major subtypes of adenosine receptors occur in different tissues which have been distinguished by pharmacological and biochemical criteria. The A₁ adenosine receptor has a high-affinity for adenosine and mediates inhibition of adenylyl cyclase, whereas the A₂ adenosine receptor usually has a lower affinity and mediates stimulation of the enzyme. Furthermore, evidence has been obtained that A₁ receptors increase the conductance of receptor-regulated potassium channels, induce inactivation of calcium channels, and modulate the breakdown of phosphoinositides by phospholipase C. Selective agonists and antagonists have been developed for both receptor subtypes. In addition, both adenosine receptors have extensively been characterized by radioligand binding studies. Suitable radioligands for the A₁ receptor are the agonist [³H]2-chloro-N⁶-cyclopentyladenoisine (CCPA) and the antagonist [³H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX)and for the A_2a receptor [³H]2-[p-(carboxyethyl)phenethylamino]- 5′-N-carboxamidoadenosine (CGS 21860). Furthermore, photoaffinity ligands were developed from adenosine derivatives, which can be covalently incorporated into the binding unit of both receptor subtypes. With this approach, it has been shown that the A₁ receptor has an apparent molecular weight of approximately 36 kDa and the A_2a receptor of 45 kDa. A second approach to elucidate the structure of adenosine receptors involves the purification of receptor protein by affinity chromatography. With this procedure, cerebral A₁ receptors have been purified to apparent homogeneity. More recently, the structure of receptor subtypes has been elucidated by cloning the receptors from a cDNA library. Furthermore, a novel adenosine binding with [³H] 5′ -N-ethylcarboxamidoadenosine ([³H]NECA). The pharmacological profile of this NECA-binding protein has been determined in competition experiments with adenosine receptor ligands. It can be distinguished from that of A_2a adenosine receptors and other adenosine binding proteins. We propose the name A_x for this unique adenosine binding protein. © 1993 Wiley-Liss, Inc.     

The role of melanocortins in body weight regulation: opportunities for the treatment of obesity

Five G-protein-coupled melanocortin receptors (MC₁–MC₅) are expressed in mammalian tissues. The melanocortin receptors support diverse physiological functions, including the regulation of hair color, adrenal function, energy homeostasis, feed efficiency, sebaceous gland lipid production and immune and sexual function. The melanocortins (adrenocorticotropic hormone (ACTH), -melanocyte-stimulating hormone (-MSH), β-MSH and γ-MSH) are agonist peptide ligands for the melanocortin receptors and these peptides are processed from the pre-prohormone proopiomelanocortin (POMC). Peptide antagonists for the melanocortin MC₁, MC₃ and MC₄ receptors include agouti-related protein (AgRP) and agouti. Diverse lines of evidence, including genetic and pharmacological data obtained in rodents and humans, support a role for the melanocortin MC₃ and MC₄ receptors in the regulation of energy homeostasis. Recent advances in the development of potent and selective peptide and non-peptide melanocortin receptor ligands are anticipated to help unravel the roles for the melanocortin receptors in humans and to accelerate the clinical use of small molecule melanocortin mimetics.     

Physiological roles of the melanocortin MC3 receptor

The melanocortin MC₃ receptor remains the most enigmatic of the melanocortin receptors with regard to its physiological functions. The receptor is expressed both in the CNS and in multiple tissues in the periphery. It appears to be an inhibitory autoreceptor on proopiomelanocortin neurons, yet global deletion of the receptor causes an obesity syndrome. Knockout of the receptor increases adipose mass without a readily measurable increase in food intake or decrease in energy expenditure. And finally, no melanocortin MC₃ receptor null humans have been identified and associations between variant alleles of the melanocortin MC₃ receptor and diseases remain controversial, so the physiological role of the receptor in humans remains to be determined.     

The early origin of melanocortin receptors, agouti-related peptide, agouti signalling peptide, and melanocortin receptor-accessory proteins, with emphasis on pufferfishes, elephant shark, lampreys, and amphioxus

There are conflicting theories about the evolution of melanocortin MC receptors while only few studies have addressed the evolution of agouti-related peptide (AgRP) and agouti signalling peptide (ASIP), which are antagonists at the melanocortin receptors (MCRs), or the melanocortin MC₂ receptor accessory proteins (MRAP1 and MRAP2). Previously we have cloned melanocortin MC receptors (MC_a and MC_b) genes in river lamprey and here we identify orthologues to these melanocortin MC receptor sequences in the sea lamprey. We investigate the putative presence of the melanocortin MC receptor genes in lancelet (amphioxus; Branchiostoma floridae) but we find it unlikely that such gene exists, due to a sharp drop in sequence similarity beyond sequence clusters of known receptors. We show the presence of AgRP and ASIP in elephant shark, a cartilaginous fish belonging to the subclass of Elasmobranchii. However, we do not find any of these genes in lamprey or lancelet after detailed analysis of both targeted and whole proteome regular expression scans. We found MRAP2, but not MRAP1, to be present in elephant shark and sea lamprey while Fugu (T. rubripes) has both genes. This study shows that the most ancient presence of these melanocortin-related sequences is found in elephant shark and lampreys considering the current available sequence data.     

Recent advances in the development of melanocortin-4 receptor agonists

The melanocortin receptors are an area of intense current research, both in academia and in the pharmaceutical industry. The large body of evidence to support a critical role for the melanocortin-4 receptor (MC₄R) in energy homeostasis (as well as indications of involvement in other interesting physiological processes) has prompted research efforts to investigate its pharmacology. This review will focus on recent advances toward the identification of potential therapeutic agents working via activation of MC₄R, supplementing the more general review on ‘Ligands to the melanocortins’ published in this journal in 2001 [1]. Several patent applications have been filed in the last year on compounds with MC₄R agonist activity for a variety of indications, most notably the treatment of feeding and body weight disorders and sexual dysfunction.     

Implication of the melanocortin-3 receptor in the regulation of food intake

The melanocortin system is well recognized to be involved in the regulation of food intake, body weight, and energy homeostasis. To probe the role of the MC₃ in the regulation of food intake, JRH322-18 a mixed MC₃ partial agonist/antagonist and MC₄ agonist tetrapeptide was examined in wild type (WT) and melanocortin 4 receptor (MC₄) knockout mice and shown to reduce food intake in both models. In the wild type mice, 2.0 nmol of JRH322-18 statistically reduced food intake 4 h post icv treatment into satiated nocturnally feeding wild type mice. The same dose in the MC₄KO mice significantly reduced cumulative food intake 24 h post treatment. Conditioned taste aversion as well as activity studies supports that the decreased food intake was not due to visceral illness. Since these studies resulted in loss-of-function results, the SHU9119 and agouti-related protein (AGRP) melanocortin receptor antagonists were administered to wild type as well as the MC₃ and MC₄ knockout mice in anticipation of gain-of-function results. The SHU9119 ligand produced an increase in food intake in the wild type mice as anticipated, however no effect was observed in the MC₃ and MC₄ knockout mice as compared to the saline control. The AGRP ligand however, produced a significant increase in food intake in the wild type as well as the MC₃ and MC₄ knockout mice and it had a prolonged affect for several days. These data support the hypothesis that the MC₃ plays a subtle role in the regulation of food intake, however the mechanism by which this is occurring remains to be determined.     

Synthesis and biological evaluation on hMC3, hMC4 and hMC5 receptors of γ‐MSH analogs substituted with l‐alanine

Abstract: To elucidate the molecular basis of the interaction of the native dodecapeptide γ‐MSH with the melanocortin receptors, we performed a structure?activity study in which we systematically replaced l ‐Ala in each position of this peptide. Here we report the binding affinity and agonist potency on human MC3R, MC4R and MC5R. Intracellular cAMP concentration was measured on CHO cells, and binding assays were carried out using membranes prepared from these cell lines which stably express hMC3R, hMC4R and hMC5R. Our results indicate that the last four amino acids in the C‐terminal region of γ‐MSH are not important determinants of biological activity and selectivity at human melanocortin receptors. Interesting results were obtained when l ‐Ala was substituted for His⁶, Phe⁷, Arg⁸ and Trp⁹. For these peptides, the affinity and activity at all three human receptors (MC3R, MC4R and MC5R) decreased significantly, demonstrating that the His‐Phe‐Arg‐Trp sequence in γ‐MSH is important for activity at these three melanocortin receptors. Similar results were obtained when Met³ was replaced with l ‐Ala, suggesting the importance of this position in the interaction with all three receptors. This study highlights the role played by the His‐Phe‐Arg‐Trp sequence in receptor binding and in agonist activity of γ‐MSH.     

Comparative pharmacology of human adenosine receptor subtypes – characterization of stably transfected receptors in CHO cells

Four adenosine receptor subtypes of the family of G protein-coupled receptors, designated A₁, A_2A, A_2B and A₃ are currently known. In this study all human subtypes were stably transfected into Chinese hamster ovary (CHO) cells in order to be able to study their pharmacological profile in an identical cellular background utilizing radioligand binding studies (A₁, A_2A, A₃) or adenylyl cyclase activity assays (A_2B). The A₁ subtype showed the typical pharmacological profile with 2-chloro-N⁶-cyclopentyladenosine (CCPA) as the agonist with the highest affinity and a marked stereoselectivity for the N⁶-phenylisopropyladenosine (PIA) diastereomers. In competition with antagonist radioligand biphasic curves were observed for agonists. In the presence of GTP all receptors were converted to a single low affinity state indicating functional coupling to endogenous G proteins. For A_2A adenosine receptors CGS 21680 (2-[p-(2-carboxyethyl)phenylethylamino]-5′-N-ethylcarboxamidoadenosine) and N-ethylcarboxamidoadenosine (NECA) were found to be the most potent agonists followed by R- and S-PIA with minor stereoselectivity. The relative potencies of agonists for the A_2B adenosine receptor could only be tested by measurement of receptor-stimulated adenylyl cyclase activity. NECA was the most potent agonist with an EC₅₀-value of 2.3 μM whereas all other compounds tested were active at concentrations in the high micromolar range. Inhibition of NECA-stimulated adenylyl cyclase identified xanthine amino congener (XAC; 8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]oxy]phenyl]-1,3-dipropylxanthine) as the most potent antagonist at this receptor subtype. The A₃ receptor was characterized utilizing the nonselective agonist [³H]NECA. The N⁶-benzyl substituted derivatives of adenosine-5′-N-methyluronamide (MECA) turned out to be the most potent agonists. The notion of xanthine-insensitivity of the A₃ receptor should be dropped at least for the human receptor as xanthines with submicromolar affinity were found. Overall, the pharmacological characteristics of the human receptors are similar to other species with some species-specific characteristics. In this study we present for the first time the comparative pharmacology of all known human adenosine receptor subtypes. The CHO cells with stably transfected adenosine receptors provide an identical cellular background for such a pharmacological characterization. These cells are valuable systems for further characterization of specific receptor subtypes and for the development of new ligands. Received: 14 July 1997 / Accepted: 26 September 1997     

Melanocortins protect against multiple organ dysfunction syndrome in mice

BACKGROUND AND PURPOSE

Melanocortins reverse circulatory shock and improve survival by counteracting the systemic inflammatory response, and through the activation of the vagus nerve-mediated cholinergic anti-inflammatory pathway. To gain insight into the potential therapeutic value of melanocortins against multiple organ damage following systemic inflammatory response, here we investigated the effects of the melanocortin analogue [Nle⁴, D-Phe⁷]α-MSH (NDP-α-MSH) in a widely used murine model of multiple organ dysfunction syndrome (MODS).

EXPERIMENTAL APPROACH

MODS was induced in mice by a single intraperitoneal injection of lipopolysaccharide followed, 6 days later (= day 0), by zymosan. After MODS or sham MODS induction, animals were randomized to receive intraperitoneally NDP-α-MSH (340 µg·kg⁻¹ day) or saline for up to 16 days. Additional groups of MODS mice were concomitantly treated with the melanocortin MC₄ receptor antagonist HS024, or the nicotinic acetylcholine receptor antagonist chlorisondamine, and NDP-α-MSH.

KEY RESULTS

At day 7, in the liver and lung NDP-α-MSH, significantly reduced mRNA expression of tumour necrosis factor-α (TNF-α), increased mRNA expression of interleukin-10 and improved the histological picture, as well as reduced TNF-α plasma levels; furthermore, NDP-α-MSH dose-dependently increased survival rate, as assessed throughout the 16 day observation period. HS024 and chlorisondamine prevented all the beneficial effects of NDP-α-MSH in MODS mice.

CONCLUSIONS AND IMPLICATIONS

These data indicate that NDP-α-MSH protects against experimental MODS by counteracting the systemic inflammatory response, probably through brain MC₄ receptor-triggered activation of the cholinergic anti-inflammatory pathway. These findings reveal previously undescribed effects of melanocortins and could have clinical relevance in the MODS setting.     

Adenosine receptor agonists

Adenosine, a naturally-occurring nucleoside, modulates a variety of physiological and pathophysiological processes. The effects of adenosine are mediated via a family of cell surface G-protein-coupled receptors designated into four subtypes, A₁, A_2A, A_2B and A₃. The adenosine receptors have widespread tissue distribution and are often co-expressed in the same cell type. Research on adenosine receptors over the past few decades has resulted in the molecular cloning of the four subtypes from multiple species, significant progress in identifying selective agonists and antagonists and an increased understanding of the particular roles adenosine receptor subtypes play in physiological processes. This knowledge has continued to fuel considerable interest in pursuing adenosine receptors as therapeutic targets. For example, adenosine receptor agonists have been proposed for the treatment of heart arrhythmias, inflammatory diseases and in diagnosing coronary artery disease. In general, adenosine receptor agonists are derivatives of the physiological agonist, adenosine. The development of adenosine receptor agonists has been limited by an essential requirement for retention of the ribose moiety for agonist activity. Despite this restriction, significant progress has been made in the identification of potent and selective adenosine receptor agonists, some of which have entered clinical trials.