Melanocyte-stimulating Hormone Peptides Research Articles

A Genomic Profile of Local Immunity in the Melanoma Microenvironment Following Treatment with α Particle-Emitting Ultrasmall Silica Nanoparticles.

An α particle-emitting nanodrug that is a potent and specific antitumor agent and also prompts significant remodeling of local immunity in the tumor microenvironment (TME) has been developed and may impact the treatment of melanoma. Biocompatible ultrasmall fluorescent core–shell silica nanoparticles (C′ dots, diameter ∼6.0 nm) have been engineered to target the melanocortin-1 receptor expressed on melanoma through α melanocyte-stimulating hormone peptides attached to the C′ dot surface. Actinium-225 is also bound to the nanoparticle to deliver a densely ionizing dose of high-energy α particles to cancer. Nanodrug pharmacokinetic properties are optimal for targeted radionuclide therapy as they exhibit rapid blood clearance, tumor-specific accumulation, minimal off-target localization, and renal elimination. Potent and specific tumor control, arising from the α particles, was observed in a syngeneic animal model of melanoma. Surprisingly, the C′ dot component of this drug initiates a favorable pseudopathogenic response in the TME generating distinct changes in the fractions of naive and activated CD8 T cells, Th1 and regulatory T cells, immature dendritic cells, monocytes, MΦ and M1 macrophages, and activated natural killer cells. Concomitant upregulation of the inflammatory cytokine genome and adaptive immune pathways each describes a macrophage-initiated pseudoresponse to a viral-shaped pathogen. This study suggests that therapeutic α-particle irradiation of melanoma using ultrasmall functionalized core–shell silica nanoparticles potently kills tumor cells, and at the same time initiates a distinct immune response in the TME.

A Novel Targeting Approach for Cancer Treatment Based on Photodynamic Therapy

Over the last decades, the development of effective and selective therapies suitable for the treatment of tumors has been one of the major ambitions of scientific research. Among this multiplicity, photodynamic therapy (PDT) represents a non-invasive form of cancer treatment. Its molecular mechanism relies on the combined action of three non-toxic components: a photosensitizer (PS), light with the appropriate wavelength in the visible range and oxygen naturally dissolved in cells. The selective anticancer effect is achieved by irradiating the PS with light. The process generates cytotoxic reactive oxygen species, mainly singlet oxygen, damaging the area of PS distribution. The introduction of targeting capabilities while designing an effective photoactive compound is critical for the development of a PS-delivery system, endowed with selectivity towards cancer cells only. Furthermore, it is essential to know the location at which the damage occurred, localizing the PS molecule inside the cell environment before triggering the cytotoxic reaction. Hence, we present a protein-based nanocarrier with multifunctional capabilities for the targeting and treatment of melanoma cancer cells (A375). The system is built starting from the streptavidin-biotin complex, whose non-covalent interaction is one of the strongest known in nature (dissociation constant ≈10−14 mol/L). We functionalized streptavidin by attaching it to eosin 5-isothiocyanate, a molecule with strong photosensitizing effects, and performed FCS measurements to assess the effectiveness of the binding. The targeting ability will be introduced with the addition of a biotinylated melanocyte-stimulating hormone peptide (MSH) that binds to a receptor over-expressed on melanoma cells. Subsequently, we will administer such complex to melanoma cells and monitor the fluorescence signal of eosin on the plasma membranes, in order to follow the uptake and induce the photosensitizing effects.

The regulation of melanocyte-stimulating hormone on the pigment granule dispersion in the xanthophores and melanophores of the large yellow croaker (Larimichthys crocea)

The large yellow croaker (Larimichthys crocea) is one of the most valuable economic mariculture fish in China, and its yellowness in skin is considered as the most important traits that determine the market value. The fish skin colors present naturally silvery white during the daytime and golden yellow during the night-time. In the present study, we found that the xanthosomes of xanthophores aggregated during the daytime and dispersed during the night-time, while the melanosomes of melanophores always dispersed all day long in vivo. We further cloned three proopiomelanocortin (POMC) genes of the large yellow croaker. All these POMC genes were expressed in the pituitary, but only POMC-C mRNA was expressed in the skin. The results of single-cell RT-PCR showed that the POMC-C mRNA was expressed in isolated xanthophores but not in melanophores. The xanthophores, but not melanophores, were found in the ventral skin, where only one subtype of melanocortin receptor (MCR), i.e. MC5R, was expressed. MC1R, MC4R and MC5R mRNAs were detected in the dorsal skin, where there existed both the xanthophores and melanophores. The results of single-cell RT-PCR showed that only MC5R mRNA was expressed in the xanthophores, while only MC1R mRNA was detected in the melanophores. Thirty min after in vivo and in vitro treatment with melanocyte-stimulating hormone (MSH) peptides (α-MSH, Des-Ac-α-MSH, α-MSH-C and β-MSH-C), dispersion degrees of the xanthosomes and melanosomes significantly increased, even under light exposure. Interestingly, when we immediately observed the dorsal scales that have been maintained under in vitro complete dark condition without MSH peptides treatment for at least 30 min, the melanosomes aggregated as expected, but the xanthosomes dispersed. Both α-MSH and α-MSH-C exhibited similar, but stronger effects than Des-Ac-α-MSH on dispersion of both the xanthosomes and melanosomes. Although the two MSH peptides (α-MSH-C and β-MSH-C) are generated from POMC-C, the former showed stronger effect on xanthosome dispersion than the latter. Forskolin, an Adenylyl cyclase activator, dispersed pigment granules in both the xanthophores and melanophores. H 89 2HCl, a specific inhibitor of protein kinase A blocked α-MSH-induced pigment granule dispersion of xanthophores and melanophores. Taken together, our results indicated that the dispersion of melanosomes is stimulated by MSH, possibly secreted from pituitary, through endocrine pathway. The yellowness of the large yellow croaker skin was mainly due to the dispersion of xanthosomes. The dispersion of xanthosomes was regulated by MSH that is possibly released both from the pituitary through endocrine pathway and from the xanthophores through autocrine pathway.

Lentivirus‐Mediated α‐Melanocyte‐Stimulating Hormone Overexpression in the Hypothalamus Decreases Diet Induced Obesity in Mice

Melanocyte stimulating hormone (MSH) derived from the pro-hormone pro-opiomelanocortin (POMC) has potent effects on metabolism and feeding that lead to reduced body weight in the long-term. To determine the individual roles of POMC derived peptides and their sites of action, we created a method for the delivery of single MSH peptides using lentiviral vectors and studied the long-term anti-obesity effects of hypothalamic α-MSH overexpression in mice. An α-MSH lentivirus (LVi-α-MSH-EGFP) vector carrying the N'-terminal part of POMC and the α-MSH sequence was generated and shown to produce bioactive peptide in an in vitro melanin synthesis assay. Stereotaxis was used to deliver the LVi-α-MSH-EGFP or control LVi-EGFP vector to the arcuate nucleus (ARC) of the hypothalamus of male C57Bl/6N mice fed on a high-fat diet. The effects of 6-week-treatment on body weight, food intake, glucose tolerance and organ weights were determined. Additionally, a 14-day pairfeeding study was conducted to assess whether the weight decreasing effect of the LVi-α-MSH-EGFP treatment is dependent on decreased food intake. The 6-week LVi-α-MSH-EGFP treatment reduced weight gain (8.4 ± 0.4 g versus 12.3 ± 0.6 g; P < 0.05), which was statistically significant starting from 1 week after the injections. The weight of mesenteric fat was decreased and glucose tolerance was improved compared to LVi-EGFP treated mice. Food intake was decreased during the first week in the LVi-α-MSH-EGFP treated mice but subsequently increased to the level of LVi-EGFP treated mice. The LVi-EGFP injected control mice gained more weight even when pairfed to the level of food intake by LVi-α-MSH-EGFP treated mice. We demonstrate that gene transfer of α-MSH, a single peptide product of POMC, into the ARC of the hypothalamus, reduces obesity and improves glucose tolerance, and that factors other than decreased food intake also influence the weight decreasing effects of α-MSH overexpression in the ARC. Furthermore, viral MSH vectors delivered stereotaxically provide a novel tool for further exploration of chronic site-specific effects of POMC peptides.

Neuroendocrine activity of the melanocyte

More than 15 years ago, we have proposed that melanocytes are sensory and regulatory cells with computing capability, which transform external and/or internal signals/energy into organized regulatory network(s) for the maintenance of the cutaneous homeostasis. This concept is substantiated by accumulating evidence that melanocytes produce classical stress neurotransmitters, neuropeptides and hormones, express corresponding receptors and these processes are modified and/or regulated by ultraviolet radiation, biological factors or stress. Examples of the above are catecholamines, serotonin, N-acetyl-serotonin, melatonin, proopiomelanocortin-derived adrenocorticotropic hormone, beta-endorphin or melanocyte-stimulating hormone peptides, corticotropin releasing factor, related urocortins and corticosteroids including cortisol and corticosterone as well as their precursors. Furthermore, their production is not random, but hierarchical and follows the structures of classical neuroendocrine organizations such as hypothalamic-pituitary-adrenal axis, serotoninergic, melatoninergic and catecholaminergic systems. An example of an intrinsic but overlooked neuroendocrine activity is production and secretion of melanogenesis intermediates including l-DOPA or its derivatives that could enter circulation and act on distant sites. Such capabilities have defined melanocytes as neuroendocrine cells that not only coordinate cutaneous but also can affect a global homeostasis.

Regions of Melanocortin 2 (MC2) Receptor Accessory Protein Necessary for Dual Topology and MC2 Receptor Trafficking and Signaling

MRAP, melanocortin 2 (MC2) receptor accessory protein, is required for trafficking by the MC2 (ACTH) receptor. MRAP is a single transmembrane protein that forms highly unusual antiparallel homodimers. We used molecular complementation to ask where MRAP achieves dual topology. Fragments of yellow fluorescent protein (YFP) were fused to the NH2 or COOH terminus of MRAP such that YFP fluorescence could occur only in antiparallel homodimers; fluorescence was present in the endoplasmic reticulum. MRAP retained dual topology after deletion of most of the amino terminus. In contrast, deletion of residues 31-37, just NH2-terminal to the transmembrane domain, forced MRAP into a single Nexo/Ccyt orientation and blocked its ability to promote MC2 receptor trafficking and homodimerize. When the transmembrane domain of MRAP was replaced with the corresponding region from RAMP3, dual topology was retained but MRAP was inactive. Insertion of MRAP residues 29-37 conferred dual topology to RAMP3, normally in an Nexo/Ccyt orientation. When expressed with MRAPDelta1-30, MRAPDelta10-20, or MRAPDelta21-30, MC2 receptor was localized on the plasma membrane but unable to respond to ACTH. Residues 18-21 of MRAP were critical; MC2 receptor expressed with MRAP(18-21A) localized to the plasma membrane but did not bind 125I-ACTH or increase cAMP in response to ACTH. A newly identified MRAP homolog, MRAP2, lacks amino acids 18LDYI21 of MRAP and, like MRAP(18-21A), allows MC2 receptor trafficking but not signaling. MRAP2 with an LDYI insertion functions like MRAP. These results demonstrate that MRAP not only facilitates MC2 receptor trafficking but also allows properly localized receptor to bind ACTH and consequently signal.

Novel selective melanocortin 4 receptor antagonist induces food intake after peripheral administration

We synthesized a new series of small cyclic melanocyte-stimulating hormone (MSH) analogues and screened them for binding affinity at the four MSH binding melanocortin (MC) receptors. We identified a novel substance HS131, with about 20-fold higher affinity for the MC4 receptor than the MC3 receptor. This substance proved to be antagonist for all the four MC receptors in a cAMP assay. HS131 is a six amino acid long peptide, has a molecular weight below 1000, and has only two amino acids in common with the natural MSH peptides. HS131 potently and dose dependently increased food intake after i.c.v. administration. Moreover, s.c. administration of HS131 (1.0 mg/kg) increased food intake, suggesting that HS131 may be able to pass the blood brain barrier. This cyclic low molecular weight peptidomimetic will enable studies of the functional role of the MC4 receptors by peripheral administration and it may be used as a template for further development of low molecular weight substances for the MC receptors.

Presence of melanocortin (MC4) receptor in spiny dogfish suggests an ancient vertebrate origin of central melanocortin system.

We report the cloning, expression, pharmacological characterization and tissue distribution of a melanocortin (MC) receptor gene in a shark, the spiny dogfish (Squalus acanthias) (Sac). Phylogenetic analysis showed that this receptor is an ortholog of the MC4 subtype, sharing 71% overall amino acid identity with the human (Hsa) MC4 receptor. When expressed and characterized by radioligand binding assay for the natural MSH (melanocyte-stimulating hormone) peptides alpha-, beta-, and gamma-MSH, the SacMC4 receptor showed pharmacological properties very similar to the HsaMC4 receptor. Stimulation of SacMC4 receptor transfected cells with alpha-MSH caused a dose-dependent increase in intracellular cAMP levels. The SacMC4 receptor has Ala in position 59 where all other cloned MC receptors have Glu. We confirmed that this was not due to individual polymorphism and subsequently mutated the residue 'back' to Glu but the mutation did not affect the pharmacological properties of the receptor. SacMC4 receptor mRNA was detected by RT-PCR in the optic tectum, hypothalamus, brain stem, telencephalon and olfactory bulb but not in cerebellum or in peripheral tissues. This study describes the first characterization of an MC receptor in a cartilaginous fish, the most distant MC receptor gene cloned to date. Conservation of gene structure, pharmacological properties and tissue distribution suggests that this receptor may have similar roles in sharks as in mammals and that these were established more than 450 million years ago.

Alpha-melanocyte-stimulating hormone peptide analogs labeled with technetium-99m and indium-111 for malignant melanoma targeting.

Previous studies have shown that the compact structure of a rhenium-cyclized alpha--melanocyte-stimulating hormone peptide analog, [Cys3410,D-Phe7]alpha-MSH(3--13), or Re-CCMSH, significantly enhanced its in vivo tumor uptake and retention. In this study, the metal chelate 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was coupled to the N-terminus of Re-CCMSH in order to develop a melanoma-targeting peptide that could be labeled with a wider variety of imaging and therapeutic radionuclides. Biodistribution properties of indium-111 ((111)In)--labeled DOTA-Re-CCMSH were compared with the non-DOTA-containing technetium-99m ((99m)Tc)--CCMSH in murine melanoma--bearing C57 mice to determine the effects of DOTA on tumor uptake and whole-body clearance. The tumor targeting capacity and clearance kinetics of (111)In-DOTA-Re-CCMSH were also compared with other related cyclic and linear (111)In-labeled DOTA-alpha-MSH complexes. The in vivo distribution data showed that the conjugation of DOTA to Re-CCMSH did not reduce its initial tumor uptake kinetics but did enhance its tumor retention and renal clearance properties. The tumor uptake of (111)In-DOTA-Re-CCMSH was significantly higher than the other (111)In-DOTA--coupled cyclic or linear alpha-MSH analogs used in this study. Moreover, (111)In-DOTA-Re-CCMSH displayed lower radioactivity accumulation in normal tissues of interest than its non-Re-cyclized counterpart, (111)In-DOTA-CCMSH; the disulfide bond--cyclized (111)In-DOTA-CMSH; or the linear (111)In-DOTA-NDP. Peptide cyclization via rhenium coordination significantly enhanced the tumor targeting and renal clearance properties of DOTA-Re-CCMSH, making it an excellent candidate for melanoma radiodetection and radiotherapy.

Chimeric melanocortin MC1 and MC3 receptors: identification of domains participating in binding of melanocyte-stimulating hormone peptides.

The melanocortin receptors MC1 and MC3 are G protein-coupled receptors that have substantial structural similarities and bind melanocyte peptides but with different affinity profiles. We constructed a series of chimeric MC1/MC3 receptors to identify the epitopes that determine their selectivities for natural melanocyte peptides and synthetic analogues. The chimeric constructs were made by a polymerase chain reaction that used identical regions in or just outside transmembranes (TM) 1, 4, and 6 and divided the receptors into four segments. Saturation and competition studies on the expressed chimeric proteins indicate that TM1, TM2, TM3, and TM7 are involved in the subtype-specific binding of melanocyte peptides to these receptors. The results support the hypothesis that TM4 and TM5 may not contribute to the ligand-binding specificity of the MC receptors. This is the first report to describe the subtype-specific hormone-binding domains of the melanocortin receptor family.

Evidence indicating that the extracellular loops of the mouse MC5 receptor do not participate in ligand binding

The mMC5 receptor was cloned from a genomic library, mutated in the extracellular loops (EL's), expressed and tested for binding to melanocyte stimulating hormone (MSH) peptides. The EL's show low amino acid homology within the MC receptor family. Two mutants of the mMC5 receptor were created in order to investigate the participation of these regions in ligand binding. The EL1 and EL3 were separately altered by multiple mutagenesis so that their amino acid sequences became identical with the hMC1 receptor. The mutants were expressed in COS cells and found to bind peptide ligands in the same fashion as the wild type mMC5 receptor clone. The results indicate that the amino acids that were mutated in the mMC5 receptor do not participate in binding of MSH peptides. Comparison of the wild type mMC5 receptor with the hMC5 receptor showed that it has the same potency order for the MSH peptides but considerably higher affinity than the hMC5 receptor.

Desensitization of melanotropin receptors in COS-7 cells

Non-transfected COS-7 cells have been found to possess functional melanotropin receptors on their cell surface. These receptors, and the properties of the melanocyte stimulating hormone (MSH) peptides can be characterized by measuring melanotropin stimulation of cAMP accumulation in the cells. In these cells we studied the ultra-long lasting super agonist [Nle 4-D-Phe 7]-α-MSH (NDP-α-MSH), and compared it with the endogenous MSH peptides with respect to potency, maximal activity, duration of action, and rate of desensitization. Surprisingly, NDP- α-MSH did not act as a full agonist in COS-7 cells. In multiple experiments, it could stimulate cAMP accumulation to approximately 50% of the level of α-MSH, β-MSH and adrenocorticotropic hormone (ACTH). The MSH receptor mediating this activity is unknown. The time course of cAMP accumulation, and the duration of receptor activation was also investigated. In contrast to other systems, NDP-α-MSH did not induce prolonged activity, with respect to cAMP accumulation, in COS-7 cells. The MSH receptors present in COS-7 were found to desensitize rapidly subsequent to pretreatment by any of the MSH peptides. As expected for a partial agonist, the activity of NDP-α-MSH desensitized more rapidly than any of the full agonists. Surprisingly, desensitization induced by pretreatment with NDP-α-MSH also occurred more rapidly than desensitization induced by the other MSH analogs.

Ultraviolet B and Melanocyte-Stimulating Hormone (MSH) Stimulate mRNA Production for ∝MSH Receptors and Proopiomelanocortin-Derived Peptides in Mouse Melanoma Cells and Transformed Keratinocytes

Cell lines of cutaneous origin, namely melanocytes and keratinocytes, were previously demonstrated to exhibit functional melanocyte-stimulating hormone (MSH) receptors that are up-regulated by ultraviolet (UV) radiation and by MSH itself. In this study, it is demonstrated that UVB irradiation, exposure to MSH, or exposure to N6,O2-dibutyryl cyclic adenosine monophosphate stimulates production of mRNAs for both alpha MSH receptors and proopiomelanocortin in cultured mouse Cloudman S91 melanoma cells, and that UVB stimulates production and release of MSH and adrenocorticotropin peptides in both melanoma cells and transformed PAM 212 mouse keratinocytes. The results add support to the hypothesis that the effects of UVB on cutaneous melanogenesis are mediated through a series of coordinated events in which MSH receptors and proopiomelanocortin-derived peptides play a central role.

Melanocyte-Stimulating Hormone: A Regulator of Human Melanocyte Physiology

Although the melanocyte-stimulating hormone (MSH) is well-recognised as a pigmentary hormone in animals, its role in human pigmentation is still a matter for conjecture, not least because cultured human melanocytes have proved to be relatively refractory to the peptide. However, recent work has shown that human melanocytes can respond to MSH peptides and the related adrenocorticotropic hormone. While the pigmentary responses are the most studied, they are by no means the only effects of these peptides on human melanocytes. This article reviews recent work on the responses of human melanocytes to MSH peptides and demonstrates that these peptides may be key regulators of human melanocyte physiology.

Cultured human melanocytes respond to MSH peptides and ACTH.

Although the administration of melanocyte-stimulating hormone (MSH) peptides results in skin darkening in man, cultured human melanocytes have been reported to be unresponsive to these peptides. This may be a consequence of the conditions under which the cells were maintained in vitro, particularly the use of phorbol esters and cholera toxin as melanocyte mitogens. By culturing the cells in the absence of these additives, we demonstrate that alpha-MSH and its synthetic analogue Nle4DPhe7 alpha-MSH (NDP-MSH) induce dose-related increases in melanin content and tyrosinase activity and affect cell morphology in the majority of human melanocyte cultures. In addition, NDP-MSH induces increases in tyrosinase mRNA and tyrosinase-related protein-1 (TRP-1) mRNA. The dose-response curves for the MSH peptides are sigmoidal and the two peptides are equipotent in their effects on human melanocytes. Adrenocorticotropic hormone (ACTH) also affects morphology and stimulates melanogenesis and tyrosinase activity in human melanocytes. However, the dose-response curves for ACTH are biphasic, and the melanocytes respond to lower concentrations of ACTH than MSH peptides, similar to those normally present in human plasma. These findings may be important in understanding the role of these pro-opiomelanocortin peptides in human skin pigmentation.

Alpha-melanocyte stimulating hormone and its analogue Nle4DPhe7 alpha-MSH affect morphology, tyrosinase activity and melanogenesis in cultured human melanocytes.

Although melanocyte stimulating hormone (MSH) peptides are known to stimulate pigmentation in man, previous reports suggest that human melanocytes are relatively unresponsive to these peptides in vitro. This may be related to the conditions under which the melanocytes were cultured. Thus, we have re-investigated the in vitro effects of MSH peptides using human melanocytes cultured in the absence of artificial mitogens. Human melanocytes were incubated with alpha-MSH or its potent analogue Nle4Dphe7 alpha-MSH for 3 days. After 18 hours, melanocyte morphology had evolved from mainly bipolar to dendritic in approximately 66% of cultures. Nle4DPhe7 alpha-MSH produced dose-related increases in both tyrosinase activity and melanin content although the degree of response was variable and tyrosinase activity was the relatively more responsive to the peptide. Similar results were obtained with alpha-MSH, but, although the effect on melanin content was similar to that of Nle4DPhe7 alpha-MSH, the effect on tyrosinase activity was less marked. The preliminary EC50 values for the actions of the MSH peptides suggest that they may be equipotent in their actions on human melanocytes. In addition, we have demonstrated that the common melanocyte mitogens 12-O-tetradecanoyl phorbol-13-acetate (TPA) and cholera toxin affect basal melanogenesis and modulate the effects of the MSH peptides. However, not all melanocyte cultures showed melanogenic responses to the MSH peptides. Ability to respond was unrelated to basal levels of tyrosinase activity or melanin content. In at least some cultures, morphological and melanogenic responses appear to be independent of one another.(ABSTRACT TRUNCATED AT 250 WORDS)

Melanotropic activity of gamma MSH peptides in melanoma cells

We studied the pigmentary activity of the peptides gamma 1, gamma 2 and gamma 3 melanocyte stimulating hormone (MSH), which differ in the structure of their Ctermini, using hamster and mouse melanoma cell lines responsive to betaMSH by increasing tyrosinase activity. Gamma 1MSH alone or in combination with betaMSH had no effect on either cell line. Gamma 2MSH alone was biologically inactive but potentiated betaMSH stimulation of tyrosinase activity. Gamma 3MSH at high concentration (10 μM) induced tyrosinase activity and dendrite formation in the hamster melanoma line. When added together with betaMSH, gamma 3MSH partially inhibited the tyrosinase activity response to betaMSH. Thus, gammaMSH peptides have low intrisic melanotropic activity in mammalian melanoma cells; the specific pigmentary responses appear to be affected by the structure of the Cterminal portion.

In situ melanin assay for MSH using mouse B16 melanoma cells in culture

A sensitive in situ melanin assay using cultured mouse B16 melanoma cells is described for structure-activity studies with melanocyte-stimulating hormone (MSH) peptides. B16 Cells were seeded at a density of 2500 cells per well in 96-well microtest tissue culture plates; after 24 h the cells were incubated in the presence of serial dilutions of MSH peptides for 3 to 5 days. The melanin released into the medium of each well was then determined spectrophotometrically at a wavelength of 405 nm using an automatic microplate reader calibrated against synthetic melanin. Studies with α-MSH, [Nle 4, d-Phe 7]-α-MSH, [3′-iodo-Tyr 2]-α-MSH, adrenocorticotropin (ACTH)(1–24), and ACTH(1–39) showed that the peptides had identical intrinsic activities and that the relative potencies were similar to those obtained with a tyrosinase assay. The EC 50 of α-MSH was 27 p m, i.e., about five- to sevenfold lower than that in the assays for tyrosinase intracellular melanin. Thus, the new assay represents the most sensitive melanoma cell assay for MSH available to date.

Structural studies of alpha-melanocyte-stimulating hormone and a novel beta-melanocyte-stimulating hormone from the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias.

A melanocyte-stimulating hormone (MSH) has been isolated from extracts of the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias by gel-filtration and ion-exchange chromatography. It had approximately 1% of the potency of mammalian alpha-MSH on bioassays in vitro on frog skin and dogfish skin. Sequence analysis revealed it to be a hexadecapeptide with the following primary structure: Asp-Gly-Asp-Asp-Tyr-Lys-Phe-Gly-His-Phe-Arg-Trp-Ser-Val-Pro-Leu. It appears to be related to the beta-MSH species of mammalian species but has only the sequence -His-Phe-Arg-Trp- in common with the heptapeptide core -Met-Glu-His-Phe-Arg-Trp-Gly- which is characteristic not only of the MSH peptides but also of the adrenocorticotrophins and lipotrophins studied so far. An alpha-MSH was also isolated, 50% of which was amidated at the C-terminus group. Sequence data from this study taken in conjunction with those from a previous study (Lowry & Chadwick, 1970b) revealed it to be a tridecapeptide which is identical with the N-terminal sequence of dogfish adrenocorticotrophin.

Melanocyte-stimulating hormone activity of synthetic MSH and ACTH peptides, in vivo and in vitro.

SIMULTANEOUSLY performed in vivo and in vitro determinations of the melanocyte-stimulating hormone (MSH) activity of a large number of synthetic MSH and ACTH peptides have not been reported previously. The work recorded here compares by both methods the MSH activity of 10 stereoisomeric pentapeptide fragments of MSH and 8 synthetic peptides related to ACTH. The MSH activity of most of the stereoisomers appeared greater in vivo than in vitro. When, these compounds were assayed at various time intervals, some of the pentapeptides revealed differences which may help partially to explain this discrepancy in activity between the two methods. These differences may also help in understanding the alterations of α-MSH induced by sodium hydroxide1,2. In addition, preliminary studies were performed on the inhibitory properties of the stereoisomers.