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Two precursors to Chinook salmon (Oncorhynchus tshawytscha) melanin-concentrating hormone, an important factor in teleosts involved in the control of skin pigmentation and stress responsiveness, have been identified from DNA sequence analysis. Both precursors encode proteins of 132 amino acids and they share 107/132 amino acid identities. The biologically active 17-residue peptide is located at the C terminus of both precursors and can be liberated by proteolytic cleavage following two adjacent arginine residues. Additional putative proteolytic processing sites are located within the two precursors. Northern analysis demonstrated an intense hybridization signal of 750 nucleotides in the hypothalamus. Immunocytochemical studies as well as in situ hybridization analyses identify intensely staining cell bodies in the hypothalamus in the area of the lateral tuberal nucleus.

Melanin-concentrating hormone (MCH) is a cyclic heptadecapeptide (H-Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val-O H) that induces aggregation of melanin granules within the melanophores of teleost fishes. Chemical and enzymatic modifications of MCH were conducted in order to deduce the structure-activity relationship using an in vitro bioassay with fish scales, and a radioimmunoassay using a specific antiserum to synthetic MCH. Micro-modification of MCH was employed with the natural peptide, and the modified form was purified by reverse-phase HPLC. MCH1-14 and NPS-Trp15-MCH were equipotent to MCH. Reduction and carboxamidomethylation of MCH caused complete loss of biological activity. Modification of the Tyr residue with tetranitromethane and Arg residues with 1,2-cyclohexadione significantly reduced activity, while oxidation with hydrogen peroxide caused only partial loss (10%) of activity. These results suggest that the configuration of the S-S loop is essential for activity, and Arg and Tyr may play an important role in the biological activity. In the radioimmunoassay, MCH1-14, MCH5-14 and CAM-Cys5,14-MCH showed no cross-reactivity, whereas MCH5-17 and other derivatives gave inhibition slopes parallel to the MCH standard, suggesting that the antigenic determinant of the antiserum is located in the carboxy-terminal.

Inhibition of oxidative phosphorylation by 2,4-dinitrophenol or sodium cyanide promotes complete melanosome aggregation in teleost melanophores. This aggregation is not promoted via the alpha-sympathetic receptor sites because it occurs in the presence of tolazoline hydrochloride, an alpha-receptor blocking agent. Interpretation of these results suggests metabolic energy release is required in the centrifugal direction only.

alpha-MSH and ACTH-like peptides are known to play an important role in the adaptation of many vertebrates to a new environment. These peptides induce pigment dispersion in amphibian melanophores through a receptor-mediated mechanism. In this study we compared the structural requirements of these peptides for melanotropic activity on Xenopus laevis melanophores with those for inducing excessive grooming in the rat. With the exception of ACTH1-24 there is a close resemblance in structure-activity relationships of the fragments and analogs tested in the two bioassays. [Nle4,-D-Phe7]-alpha-MSH is extremely active in both assays. Weak agonists such as [Leu9]-alpha-MSH did not possess antagonistic properties either in the melanophore assay or in the excessive grooming test. The data suggest that the mechanism of action of alpha-MSH-like peptides in rat brain is receptor-mediated like their action on melanophores.

Skin explants of the neotenic form of Ambystoma tigrinum (Axolotl) have been kept in culture during several days. When alpha-MSH or prolactin are added to the medium, a dispersion of melanin in melanosomes is observed. The reaction is reversible in the case of prolactin when the explants are transferred in the medium without hormones. Furthermore with prolactin, expansion of melanin in dermal melanophores is a more permanent and highly reproducible phenomenon, compared with the action of alpha-MSH in the same conditions. The type of action of prolactin could be different of the one of alpha-MSH.

Melanophores of wild-type and periodic albino mutants of Xenopus laevis were successfully cultured in vitro. They proliferated in the presence of alpha-melanocyte-stimulating hormone (alpha-MSH or cyclic adenosine monophosphate (cAMP) at a doubling time of 8-10 days. These proliferating melanophores retained their phenotypes, ability to synthesize melanin, and melanin-dispersing response to MSH stimulation. Neither depigmentation nor selective cell death of periodic albino melanophores was observed for at least 4 months during the cultivation.

alpha-MSH secretion from the amphibian pars intermedia is under inhibitory hypothalamic control, and the catecholamine dopamine is thought to be the physiological MSH release-inhibiting factor. In the present study we evaluated the possible role of the neurotransmitter gamma-aminobutyric acid (GABA) in the regulation of the pars intermedia of Xenopus laevis. Immunocytochemical staining with antibodies to glutamic acid decarboxylase showed the presence of a rich GABAergic network in the intermediate lobe of the pituitary gland. Administration of GABA to superfused neurointermediate lobes caused a rapid and dose-dependent inhibition of basal release of MSH and immunoreactive endorphin. Pulse-chase experiments revealed that GABA gave a coordinate inhibition of the release of all peptides derived from proopiomelanocortin. In vivo administration of GABA resulted in almost complete pigment aggregation in dermal melanophores of both adults and larvae. Altogether, our results indicate that GABA is a physiologically important factor for regulation of the pars intermedia in Xenopus laevis.

Adrenaline was effective in aggregating the melanosomes both in innervated as well as denervated melanophores. Isotonic KCl could induce pigment aggregation only in innervated melanophores. Adrenaline- and K+-induced pigment aggregation response in these melanophores was blocked by phentolamine: propranolol failed to do so. It is suggested that the chromatic nerves in the fish, Labeo rohita are adrenergic and via post-synaptic alpha-adrenoceptors, control the melanosome aggregation.

Functioning of the dermal melanophores was studied in the isolated skin of the Rana temporaria and R. esculenta tadpoles at stages 17-21 and 20-24 (after Kopsch). At all stages we studied melanophores exhibited reaction to light. From stage 18 on repeated alternation of pigment dispersion and aggregation was obtained using melanotropins and melatonin. When observing transition of the melanophores from interphase to mitosis, it was found that dividing dermal melanophores could be distinguished due to changes in their appearance shortly before the end of prophase.

Studies were conducted to investigate the molecular basis for bidirectional pigment granule transport in digitonin-lysed melanophores. Pigment granule dispersion, but not aggregation, required cAMP and resulted in the phosphorylation of a 57 kd polypeptide. cAMP-dependent protein kinase inhibitor prevented this phosphorylation as well as pigment dispersal. In contrast, both pigment aggregation and the concomitant dephosphorylation of the 57 kd polypeptide were blocked by phosphatase inhibitors. These data support a model in which pigment dispersion and aggregation require protein phosphorylation and dephosphorylation, respectively. Furthermore, studies using the ATP analog, ATP gamma S, suggest either that protein phosphorylation alone is sufficient for dispersion or that transport is mediated by a unique force-generating ATPase that can use ATP gamma S for hydrolyzable energy.