Effect of melanin concentrating hormone on pigment and adrenal cells in vitro

Highly purified synthetic salmonid melanin concentrating hormone (MCH) and some analogs were investigated for their ability to concentrate the pigment in scale melanophores of the Chinese grass carp, Ctenopharyngodon idellus, to produce melanin dispersion in frog or lizard melanophores and to inhibit alpha-MSH in its action on mouse melanoma and rat adrenal glomerulosa cells in vitro. In the grass carp, MCH produced half-maximal pigment aggregation at 6 X 10(-11) M and its oxidized form at 7 X 10(-11) M. Replacement of the two methionines at position 3 and 6 with norvaline lowered the potency by a factor of 2.7 and with propargylglycine by a factor of about 7. Linear, Cys5,14-Acm-protected MCH was a full agonist of MCH but with a 345-fold lower potency. Iodinated MCH showed similar, low activity. In tetrapods, salmonid MCH and its analogs displayed only marginal pigment dispersion at concentrations greater than 10(-5) M. Alkali-treatment of MCH increased the pigment-dispersing potency by a factor of about 30 whereas the activity for pigment aggregation in the grass carp was destroyed. At high concentrations (10(-6), 10(-5) M) MCH also stimulated tyrosinase activity in B-16 mouse melanoma cells but did not modify the effects of alpha-MSH in this system. By contrast, when tested on rat adrenal glomerulosa cells, salmonid MCH had no effect alone but at a concentration of greater than 10(-10) M it slightly reduced corticosterone production by an alpha-MSH concentration of 10(-7) M. Aldosterone production was not affected and MCH did not influence the response to ACTH.     

Behavioral effects of [4-norleucine, 7-D-phenylalanine]-alpha-melanocyte-stimulating hormone

The behavioral effects of alpha-melanocyte stimulating hormone (alpha-MSH) were compared to an alpha-MSH analogue that had a norleucine substituted for methionine in the four position and a D-phenylalanine substituted for L-phenylalanine in the seven position. [Nle4, D-Phe7]-alpha-MSH has previously been shown to be a superpotent agonist on melanocytes [17]. The present experiments indicate that [Nle4, D-Phe7]-alpha-MSH is equipotent to alpha-MSH in inducing grooming when administered intraventricularly. In contrast, the analogue has the opposite effect of alpha-MSH on performance of a visual discrimination task. alpha-MSH improves visual performance whereas [Nle4, D-Phe7]-alpha-MSH attenuates such performance. The contrasting activities of [Nle4, D-Phe7]-alpha-MSH on the physiological processes described suggest that this analogue may interact with three distinct melanotropin receptors in different ways. On melanocyte receptors the melanotropin analogue is a superagonist, on CNS melanotropin receptors involved in grooming it is equipotent to alpha-MSH, and on CNS receptors involved in attention, learning and memory [Nle4, D-Phe7]-alpha-MSH may be an antagonist of endogenous melanotropin.     

Biological Activities of Melanotropic Peptide Fatty Acid Conjugates

Four fatty acids (FA, palmitic, myristic, decanoic, hexanoic) were individually conjugated to the N-terminus of the α-MSH fragment analog, H-Asp⁵-His⁶-D-Phe⁷-Arg⁸-Trp⁹-Lys¹⁰-NH₂. This resulted in enhanced potency of the conjugates (compared to the unconjugated melanotropin analog) as determined in the lizard skin bioassay and in the mouse melanoma cell tyrosinase bioassay. The shorter conjugates of hexanoic and decanoic acid were at least equipotent to α-MSH in the lizard skin bioassay, whereas the longer myristoyl and palmitoyl analogs were 100 times less active. The myristoyl and palmitoyl conjugates exhibited a “creeping” potency in the lizard skin bioassay—that is, potency of the peptides increased with time in contact with the skins. These observations may be related to the more lipid nature of these FA-conjugates. In the tyrosinase assay, the conjugates were 10–100 times more active than α-MSH or the unconjugated analog. Each of the FA-melanotropic peptide conjugates exhibited prolonged (residual) melanotropic activity in both the lizard skin and melanoma cell bioassays. In other words, after removal of the melanotropin conjugates from contact with the skins or cells, responses were still manifested for hours or days thereafter. As little as 1 hr of contact with melanoma cells resulted in enhanced enzyme activity as measured 48 hr later. Since the conjugates, but not H-[Ast⁵,D-Phe⁷,Lys¹⁰]α-MSH₅-₁₀-NH₂, exhibited prolonged activity, the conversion of reversible agonists to irreversible agonists was demonstrated.     

Chimeric NDP-MSH and MTII melanocortin peptides with agouti-related protein (AGRP) Arg-Phe-Phe amino acids possess agonist melanocortin receptor activity

Agouti-related protein (AGRP) is one of only two known endogenous antagonists of G-protein coupled receptors (GPCRs). Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis, regulation of feeding behavior, and obesity. -Melanocyte stimulating hormone (-MSH) is one of the known endogenous agonists for these receptors. It has been hypothesized that the Arg-Phe-Phe (111–113) human AGRP amino acids may be mimicking the melanocortin agonist Phe-Arg-Trp (7–9) residue interactions with the melanocortin receptors that are important for both receptor molecular recognition and stimulation. To test this hypothesis, we generated thirteen chimeric peptide ligands based upon the melanocortin agonist peptides NDP-MSH (Ac-Ser-Tyr-Ser-Nle⁴-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH₂) and MTII (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH₂). In these chimeric ligands, the agonist DPhe-Arg-Trp amino acids were replaced by the AGRP Arg-Phe-Phe residues, and resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3–5Rs), supporting the hypothesis that the AGRP antagonist ligand Arg-Phe-Phe residues mimic the agonist Phe-Arg-Trp amino acids. Interestingly, the Ac-Ser-Tyr-Ser-Nle⁴-Glu-His-Arg-DPhe-Phe-Gly-Lys-Pro-Val-NH₂ peptide possessed 7 nM mMC1R agonist potency, and is 850-fold selective for the mMC1R versus the mMC3R, 2300-fold selective for the mMC1R versus the mMC4R, and 60-fold selective for the MC1R versus the mMC5R, resulting in the discovery of a new peptide template for the design of melanocortin receptor selective ligands.     

The effect of N-terminal substitutions on the biological activity of MSH fragments

In order to study the role of N-terminal substitutions of peptide sequences related to the active site of α-melanotropin, [Glp⁵]α-MSH(5–10), [Glp⁵, -Phe⁷]α-MSH(5–10), [Sar⁵, -Phe⁷]α-MSH(5–10), [Nle⁴, -Phe⁷]α-MSH(4–10), [N-carbamoyl]α-MSH(5–10), and formyl and acetyl derivatives of α-MSH(5–10), [Gly⁵]α-MSH(5–10) and [Gly⁵, -Phe⁷]α-MSH(5–10), were synthesized in solution. The N-terminal acylations enhance by 2 to 10 times the melanin-dispersing activity of the unsubstituted sequences. Alkylation of the N-terminus does not change the biological activity of the parent peptide, suggesting the necessity of a carbonyl group for increasing the hormonal effect.     

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.     

Regulation of biosynthesis and release of pars intermedia peptides in Rana ridibunda: dopamine affects both acetylation and release of alpha-MSH

Dopamine is likely an important physiological melanotropin release-inhibiting factor in amphibians. This study concerns the effects of dopamine on the biosynthesis and release of peptides from the pars intermedia of the frog Rana ridibunda. Our results show that this secretagogue has no immediate effects on either the rate of biosynthesis of pro-opiomelanocortin nor on the direction of processing of this prohormone. Pulse-chase experiments revealed that dopamine inhibited the release of all newly synthesized POMC-related peptides in a dose-dependent manner. For each dopamine concentration tested, the degree of inhibition exerted on the release of the various newly synthesized peptides by a given concentration of secretagogue was approximately the same, with the exception of that found for the alpha-MSH related peptides. Analysis of the release of these melanotropins was complex because dopamine not only inhibited their release but also, either directly or indirectly, inhibited the acetylation reaction which converts des-N alpha-acetyl alpha-MSH to alpha-MSH. Dopamine was shown to be less potent in inhibiting the release of des-N alpha-acetyl alpha-MSH than inhibiting release of the acetylated form of the peptide. In amphibians a preferential release of the less-bioactive non-acetylated form of MSH under inhibitory conditions induced by dopamine may be of physiological importance.     

Isolation and characterization of A new β-melanotropin from horse pituitary glands

A new β-melanotropin was isolated from horse pituitaries and its primary structure has been determined. The amino acid sequence of the new peptide differed from that of β-melanotropin in the deletion of the NH₂-terminal aspartic acid. Thus, it is designated as [Des-Asp¹]-β-melanotropin. It possessed higher lipolytic activity when compared with β-melanotropin.     

The effect of N-terminal substitutions on the biological activity of MSH fragments.

In order to study the role of N-terminal substitutions of peptide sequences related to the active site of -melanotropin, [Glp⁵]-MSH(5–10), [Glp⁵, -Phe⁷]-MSH(5–10), [Sar⁵, -Phe⁷]-MSH(5–10), [Nle⁴, -Phe⁷]-MSH(4–10), [N-carbamoyl]-MSH(5–10), and formyl and acetyl derivatives of -MSH(5–10), [Gly⁵]-MSH(5–10) and [Gly⁵, -Phe⁷]-MSH(5–10), were synthesized in solution. The N-terminal acylations enhance by 2 to 10 times the melanin-dispersing activity of the unsubstituted sequences. Alkylation of the N-terminus does not change the biological activity of the parent peptide, suggesting the necessity of a carbonyl group for increasing the hormonal effect.