Mac E. Hadley

alpha-Melanocyte-stimulating hormone (alpha-MSH) reversibly darkens frog skins by stimulating melanosome movement (dispersion) within melanophores. Heat-alkali treatment of alpha-MSH results in prolonged biological activity of the hormone. Quantitative gas chromatographic analysis of the hydrolyzed heat-alkali-treated peptide revealed partial racemization particularly at the 4(methionine) and 7(phenylalanine) positions. [Nle4]-alpha-MSH, a synthetic analogue of alpha-MSH, reversibly darkens frog skins and also exhibits prolonged activity after heat-alkali treatment. Synthesis of [Nle4, D-Phe7]-alpha-MSH provided an analogue with prolonged biological activity identical to that observed with heat-alkali-treated alpha-MSH or [Nle4]-alpha-MSH. [Nle4, D-Phe7]-alpha-MSH was resistant to enzymatic degradation by serum enzymes. In addition, this peptide exhibited dramatically increased biological activity as determined by frog skin bioassay, activation of mouse melanoma adenylate cyclase, and stimulation of mouse melanoma cell tyrosinase activity. This Nle4, D-Phe7 synthetic analogue of alpha-MSH is a very porent melanotropin, 26 times as potent as alpha-MSH in the adenylate cyclase assay. The resistance of the peptide to enzymatic degradation and its extraordinarily potent and prolonged biological activity should make this analogue of alpha-MSH an important molecular probe for studying the melanotropin receptors of both normal and abnormal (melanoma) melanocytes.

The cloning of the melanocyte-stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) receptors (MC1-R and MC2-R, respectively) recently has led to the identification of three additional melanocortin receptors, MC3-R, MC4-R, and MC5-R. The MC2 receptor primarily recognizes only ACTH peptides, but the other four receptors all recognize alpha-melanocyte-stimulating hormone (alpha-MSH) and potent alpha-MSH agonists such as [Nle4,D-Phe7]alpha-MSH-NH2 and Ac-Nle4-c[Asp5,D-Phe7,Lys10]alpha-MSH-(4-10)-NH2 as well as ACTH. The absence of any known physiological role for these new receptors, expressed both in the brain (MC3-R and MC4-R) and throughout a number of peripheral tissues (MC5-R), has necessitated as search for potent and receptor selective agonists and antagonists. We report here that analogues of the superpotent cyclic agonist analogue Ac-Nle4-c[Asp5,D-Phe7, Lys10]alpha-MSH-(4-10)-NH2, in which a bulky aromatic amino acid is substituted in the 7-position, can produce potent and selective antagonists for melanocortin receptors. Thus, the D-p-iodophenylalanine7-containing analogue Ac-Nle4-c[Asp5,D-Phe(pI)7,Lys10]alpha-MSH-(4-10)-NH2 is a potent antagonist (pA2 = 10.3) in the classical frog skin (Rana pipiens) assay (MC1-R), as is the D-2'-naphthylalanine7 (D-Nal(2)7)-containing analogue Ac-Nle4-c[Asp5,D-Nal(2)7,Lys10]alpha-MSH-(4-10)-NH2 (pA2 > 10.3). Interestingly, the D-p-chloro- and D-p-fluorophenylalanine7-containing analogues lacked antagonist activities at all melanotropin receptors, and both exhibited full agonist potency in the frog skin assay. The activity of these analogues also was examined at four mammalian melanocortin receptors. Interestingly, Ac-Nle4-c[Asp5,(D-Nal(2)7,Lys10] alpha-MSH-(4-10)-NH2 was found to be a potent antagonist of the MC4-R (pA2 = 9.3) with minimal agonist activity, a less potent antagonist of the MC3-R (pA2 = 8.3) with minimal agonist activity, and a full agonist of the MC1 and MC5 receptors. Surprisingly, Nle4-c[Asp5,D-Phe(pI)7,Lys10]alpha-MSH was found to be a potent agonist at the cloned human MC1-R (EC50 = 0.055 nM) and mouse MC1-R (EC50 = 0.19 nM) but had potent antagonist activities at the human MC4-R (pA2 = 9.7) and human MC3-R (pA2 = 8.3) with significant partial agonist activities (EC50 = 0.57 and 0.68 nM, respectively) as well. Thus, highly potent and receptor selective antagonist analogues can arise from substitution of the D-Phe7 residue with a bulky aromatic amino acid. These analogues can be used to help determine the functional roles of these receptors.

Rapid color changes of amphibians are mediated by three types of dermal chromatophores, xanthophores, iridophores, and melanophores, which comprise a morphologically and physiologically distinct structure, the dermal chromatophore unit. Xanthophores, the outermost element, are located immediately below the basal lamella. Iridophores, containing light-reflecting organelles, are found just beneath the xanthophores. Under each iridophore is found a melanophore from which processes extend upward around the iridophore. Finger-like structures project from these processes and occupy fixed spaces between the xanthophores and iridophores. When a frog darkens, melanosomes move upward from the body of the melanophore to fill the fingers which then obscure the overlying iridophore. Rapid blanching is accomplished by the evacuation of melanosomes from these fingers. Pale coloration ranging from tan to green is provided by the overlying xanthophores and iridophores. Details of chromatophore structure are presented, and the nature of the intimate contact between the chromatophore types is discussed.