Melanocortin Receptors: Identification and Characterization by Melanotropic Peptide Agonists and Antagonists

Hormones are chemical messengers released from cells to act on and control the activity of other cells. Hormonal ligands initiate their actions by interacting with receptive substances (Langley, 1906) of the target cells. These receptors are proteins that are either integral components of the cell membrane or are localized cytoplasmically within cells. Ligand-receptor interaction results in either the stimulation or inhibition of cellular activity. Since most hormones bind rather specifically to receptors possessed by their target cells, labeling of hormonal ligands can be utilized to identify and localize cells within an animal. In this report we discuss what is presently known about melanocortin receptors (MCRs) as studied by the use of labeled melanotropic peptide ligands.     

The Administration of an α-MSH Analogue Reduces the Serum Release of IL-1α and TNFα Induced by the Injection of a Sublethal Dose of Lipopolysaccharides in the BALB/c Mouse

The injection of α-MSH or of one of its analogues ([Nle₄-D.Phe₇] α-MSH_4–10) reduced, in vivo, the release of two cytokines (IL-1α and TNFα) involved in inflammation. The inflammatory state was induced in BALB/c mice by intraperitoneal injection of a sublethal dose of lipopolysaccharides (LPS). The assay of these cytokines by ELISA showed a reduction of 20% with α-MSH and between 30 and 60% with the α-MSH analogue. The α-MSH or the analogue was administered in one of two ways: intravenously or subcutaneously. The most efficient method seemed to be the subcutaneous one because it improved the activity 10,000 times more than the intravenous method. Moreover, the analogue induced a regression of mortality in the animals treated by the intravenous method. Our results show that α-MSH and one of its analogues inhibit IL-1α and TNFα, and can be used as anti-inflammatory molecules.     

Linear and Cyclic α-Melanotropin [4–10]-Fragment Analogues That Exhibit Superpotency and Residual Activity

Two analogues of α-MSH (Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂), Ac-[Nle⁴, Asp⁵, D-Phe⁷, Lys¹⁰]α-MSH_4–10NH₂ and Ac-[Nle⁴, Asp⁵, D-Phe⁷, Lys¹⁰] α-MSH_4–10-NH₂, were synthesized, and the melanotropic activities of the peptides were compared in several bioassays. Potencies were determined in the in vitro frog and lizard skin bioassays and in the S91 melanoma cell tyrosinase assay. Both analogues were equipotent or more potent than α-MSH in all bioassays, and the activities of the analogues were prolonged compared to α-MSH. The two analogues were very resistant to inactivation by purified proteolytic enzymes (α-chymotrypsin, trypsin, and pepsin). The two peptides could be topically applied and transdermally delivered across the skin of mice in vivo, resulting in a shift from pheomelanogenesis to eumelanogenesis within follicular melanocytes. The cyclic analogue exhibited greater potency, prolonged activity, and stability against enzyme inactivation than did the linear peptide. The significance of the findings for the further design of melanotropin analogues is discussed, as in the possible relevance of these melanotropin analogues for use in biomedical studies.     

Secretion of melanophore-stimulating hormone (MSH) in long-term cultures of pituitary neurointermediate lobes

Summary Neurointermediate lobes from pituitaries of the frog, Rana berlandieri forreri (Rana pipiens, sensu lato), were maintained in organ culture in media with and without serum for up to six months. The cultured tissues were examined periodically by light microscopy and transmission electron microscopy and by bioassay of the melanophore-stimulating hormone (MSH) secreted and present in the culture media. Light-microscopic observations revealed a high degree of preservation of the pars intermedia at four weeks with isolated areas of some glands maintaining histological integrity for the entire six months. Similarly, at the ultrastructural level the cells appeared morphologically intact and to be actively synthesizing and secreting hormone. Bioassays showed the glands to be continuously secreting MSH; however, larger yields of hormone were obtained in media lacking serum. No significant ultrastructural differences between cells grown in the presence or absence of serum were detected. The difference in concentration of MSH between the two groups therefore apparently results from enzymatic degradation of the hormone by the serum. Organ culture of the vertebrate neurointermediate lobe may provide a unique method for the production of large quantities of MSH and for the study of other melanotropic and opiate peptides as they may be synthesized and secreted by the pars intermedia.     

Corticotropin Peptides and Melanotropins Elevate the Level of Adenosine 3'': 5''-Cyclic Monophosphate in Cultured Murine Brain Cells

Cell cultures derived from mouse and rat brain and consisting mainly of astroblasts are known to respond to several hormones by increasing or decreasing their intracellular concentration of cyclic AMP. In the present study these cultures were analyzed for their susceptibility to various additional hormonal and other neuroactive compounds. Only the peptides of the corticotropin (ACTH)/melanotropin (MSH) family were found active. Their potency for elevating the intracellular level of cyclic AMP decreases in the sequence (values for the half-maximally stimulating concentrations, EC50, in parentheses) ACTH-(1-24) (10 m) greater than alpha-,beta-MSH (30 nm) greater than ACTH (greater than or equal to 100 nm) gamma-MSH, ACTH-(1-10), -(4-10), -(4-11) (greater than or equal to 0.5 microM). The lack of additivity of the maximal effects of the peptides suggests that they all act at the same receptor. The stimulation exerted by these peptides is partially suppressed by hormones known to inhibit cyclic AMP formation in that culture, i.e., noradrenaline (acting via an alpha-adrenergic receptor), adenosine (acting via an A1 receptor), and somatostatin. It is concluded that the receptors for the ACTH/MSH peptides and the inhibitory hormones are located on the same cells, presumably the astroblasts. The maximal response to ACTH and alpha- and beta-MSH depends strongly on the age of culture. The results are discussed in view of the facts that (1) peptides of the ACTH/MSH family affect behavior and learning in animals, and (2) ACTH and alpha-MSH occur in brain.     

Human Epidermal Melanocyte and Keratinocyte Melanocortin Receptors: Visualization by Melanotropic Peptide Conjugated Microspheres (Latex Beads)

The objectives of this research were to determine whether melanocortin receptors are characteristic (constant) membrane markers of human epidermal melanocytes. Methodologies were developed to visualize melanotropin receptors by scanning electron microscopy (SEM). Multiple copies (up to a hundred) of [Nle⁴,D-Phe⁷]α-MSH, a superpotent analog of α-melanocyte stimulating hormone (α-MSH), were conjugated to a macromo-lecular carrier (latex beads: microspheres). Incubation in the presence of the melanotropin-conjugated microspheres resulted in binding of human normal epidermal melanocytes to the beads. Almost every (possibly all) melanocyte possesses melanocortin receptors as visualized by SEM. Specificity of binding of the macromolecular conjugate was demonstrated by several studies: 1) Binding of melanocytes to the microspheres was specific since it could be blocked by prior incubation of the cells in the presence of the unconjugated hormone analog; 2) microspheres lacking bound ligand did not bind to the melanocytes; 3) micro-spheres that were first treated with reducing agents (e.g., dithiothreitol) did not subsequently bind to melanocytes; 4) another peptide hormone ligand (e.g., a substance-P analog) attached to the latex beads failed to bind to the cells; 5) B16/F10 mouse melanoma cells known to express melanocortin receptors bound to the microspheres; and 6) cells of nonmelanocyte origin (e.g., mammary cancer cells, small-cell lung cancer cells, fibroblasts) did not bind to the macromolecular conjugate. One exception was that human epidermal keratinocytes also expressed melanocortin receptors as determined by all the criteria established above for epidermal melanocytes. Thus, cell specific melanocortin receptors appear to be characteristic cell surface markers of epidermal melanocytes and keratinocytes.     

Characterization of Vitiligo Antigens

Patients with vitiligo have circulating antibodies directed in part to pigment cell antigens with MWs of approximately 90, 75, and 40-45 kDs. These antigens are denominated VIT 90, VIT 75, and VIT 40, respectively. To further characterize these “vitiligo” antigens, we examined their relation to antigens defined by a panel of 25 monoclonal antibodies (moab) to pigment cell antigens. We found by immunoprecipitation and SDS-PAGE analysis of ¹²⁵I labelled, detergent soluble, human melanocyte macromolecules, that 24 (83%) of 29 patients with vitiligo had antibodies to one or more vitiligo antigens vs. 2 (7%) of 28 control individuals. Seventeen of the 25 moabs did not react with any labelled antigen in the same lysate. Of the remaining eight moabs, only four precipitated an antigen that co-migrated with one of the vitiligo antigens. Moab TA99, HMSA-5, and TMH-1 (all directed to the 75 kD tyrosinase-related protein [TRP1]) co-migrated with VIT 75. Moab W6/32 (directed to class I HLA antigen) co-migrated with VIT 40. Immunodepletion studies with vitiligo antibodies selectively depleted the antigen defined by W6/32 but not the antigen defined by TA99 and HMSA-5, indicating that VIT 75 was not the 75 kD tyrosinase-related protein. The vitiligo antigens were easily labelled by the lactoperoxidase technique but poorly labelled with ³⁵S-methionine, suggesting they are expressed on the cell surface. These studies indicate that VIT 90 and VIT 75 differ from antigens defined by currently available moabs to pigment cell antigens. VIT 40 appears to share a cross-reactive epitope, or be tightly bound to, class I HLA antigen.     

Linker modification reduced the renal uptake of technetium-99m-labeled Arg-Ala-Asp-conjugated alpha-melanocyte stimulating hormone peptide

The purpose of this study was to examine the biodistribution of ^99mTc-RAD-Arg-(Arg¹¹)CCMSH in B16/F1 melanoma-bearing C57 mice to determine whether the replacement of the Lys linker with an Arg linker could decrease the renal uptake of ^99mTc-RAD-Arg-(Arg¹¹)CCMSH. ^99mTc-RAD-Arg-(Arg¹¹)CCMSH exhibited rapid and high tumor uptake (17.98 ± 4.96% ID/g at 2 h post-injection) in B16/F1 melanoma-bearing C57 mice. As compared to ^99mTc-RAD-Lys-(Arg¹¹)CCMSH, the replacement of the Lys linker with an Arg linker dramatically decreased the renal uptake of ^99mTc-RAD-Arg-(Arg¹¹)CCMSH by 68%, 62%, 73% and 64% at 0.5, 2, 4 and 24 h post-injection, respectively. Flank B16/F1 melanoma lesions were clearly imaged at 2 h post-injection using ^99mTc-RAD-Arg-(Arg¹¹)CCMSH as an imaging probe.     

Activation of Tyrosinase Reduces the Cytotoxic Effects of the Superoxide Anion in B16 Mouse Melanoma Cells

Tyrosinase may protect against oxidative stress by using the superoxide anion (O^?₂) in the production of melanin. We have examined this by comparing its cytotoxic effects in B16/F10 and B16/F10-differential deficient (-DD) mouse melanoma cells that express high and low levels of tyrosinase activity respectively. Xanthine oxidase (XO) was used to generate O^?₂ and cytotoxicity assessed by measuring cell survival. XO increased O^?₂ concentrations and 3 h later dose related decreases in cell survival were seen. F10 cells were more resistant to these cytotoxic effects than the F10-DD cells. [Nle⁴,DPhe⁷]MSH increased tyrosinase activity and melanin content, reduced O^?₂ concentration and increased the resistance of F10 cells to the cytotoxic effects of O^?₂. No such effects were seen in F10-DD cells. The effect of [Nle⁴,DPhe⁷]MSH on the resistance of the F10 cells was time-dependent and noticeable when tyrosinase activity but not melanin was increased. This suggests that it was the activation of tyrosinase rather than the increase in the melanin that provided the protection against O^?₂. In support of this, inhibition of tyrosinase with phenylthiocarbamide reduced the increased resistance induced by [Nle⁴,DPhe⁷]MSH. Moreover, although melanin was capable of scavenging O^?₂ it had little effect at concentrations comparable to those in the activated F10 cells. XO also increased the melanin content of F10 but not F10-DD cells. We conclude that tyrosinase is able to utilise O^?₂ to produce melanin and this provides pigment cells with a unique anti-oxidant mechanism.     

Effects of Ultraviolet Light on Melanocyte Differentiation: Studies with Mouse Neural Crest Cells and Neural Crest‐derived Cell Lines

To evaluate the etiologic role of ultraviolet (UV) radiation in acquired dermal melanocytosis (ADM), we investigated the effects of UVA and UVB irradiation on the development and differentiation of melanocytes in primary cultures of mouse neural crest cells (NCC) by counting the numbers of cells positive for KIT (the receptor for stem cell factor) and for the L ‐3,4‐dihydroxyphenylalanine (DOPA) oxidase reaction. No significant differences were found in the number of KIT‐ or DOPA‐positive cells between the UV‐irradiated cultures and the non‐irradiated cultures. We then examined the effects of UV light on KIT‐positive cell lines derived from mouse NCC cultures. Irradiation with UVA but not with UVB inhibited the tyrosinase activity in a tyrosinase‐positive cell line (NCCmelan5). Tyrosinase activity in the cells was markedly enhanced by treatment with α‐melanocyte‐stimulating hormone (α‐MSH), but that stimulation was inhibited by UVA or by UVB irradiation. Irradiation with UVA or UVB did not induce tyrosinase activity in a tyrosinase‐negative cell line (NCCmelb4). Levels of KIT expression in NCCmelan5 cells and in NCCmelb4 cells were significantly decreased after UV irradiation. Phosphorylation levels of extracellular signal‐regulated kinase 1/2 in cells stimulated with stem cell factor were also diminished after UV irradiation. These results suggest that UV irradiation does not stimulate but rather suppresses mouse NCC. Thus if UV irradiation is a causative factor for ADM lesions, it would not act directly on dermal melanocytes but may act in indirect manners, for instance, via the overproduction of melanogenic cytokines such as α‐MSH and/or endothelin‐1.     

HOMOLOGOUS REGULATION OF MELANOCORTIN-1 RECEPTOR (MC1R) EXPRESSION IN MELANOMA TUMOR CELLS IN VIVO

ABSTRACT

As G protein-coupled receptors (GPCRs) are the target of numerous signaling molecules, including about half of the therapeutic drugs currently used, it is important to understand the consequences of homologous (ligand-induced) receptor regulation. Continuous exposure of GPCRs to agonist in vitro most frequently results in receptor down-regulation, but receptor up-regulation may occur as well. These phenomena are expected to play a role in the physiological adaptation to endogenous ligands and also in the response to repetitive administration of drugs in the clinic. However, there is little information on homologous regulation of GPCRs in vivo. Here, we report on the regulation of melanocortin-1 receptor (MC1R) expression in melanoma cells implanted into mice. Two melanoma cell lines were investigated, D10 and B16F1, which in vitro had previously been shown to undergo homologous receptor up- and down-regulation, respectively. After implantation into mice and exposure to the natural MC1R agonist α-melanocyte-stimulating hormone (α-MSH), cell-surface MC1R expression was evaluated by competition binding experiments in tumor membrane preparations. In B16F1 cells, a single injection of 50 to 500?µg α-MSH induced a rapid but moderate dose-dependent MC1R down-regulation which could be totally reverted within 16–24?h. By continuous administration of α-MSH via osmotic minipumps, MC1R down-regulation was considerably amplified and reached the level observed in vitro, demonstrating that prolonged receptor interaction was necessary to induce a maximal effect in vivo. Similar results were obtained in vitro, which demonstrates that homologous MC1R regulation in B16F1 cells is essentially independent of the physiological environment. In D10 cells, however, up-regulation could not be reproduced in vivo, suggesting that MC1R up-regulation is more dependent on the physiological environment. These results demonstrate the importance of in vivo receptor regulation studies, in particular in view of the potential use of MC1R as a target for melanoma therapy.