A comparison of structure-activity relationships within alpha-MSH on melanophores of Anolis carolinensis and Rana pipiens

We have compared the structure-function relationship of the tridecapeptide alpha-melanocyte stimulating hormone (alpha-MSH) on the melanophores of the lizard Anolis carolinensis and the frog Rana pipiens by determining the melanosome-dispersing potency of 15 shorter peptide sequences and 8 substituted alpha-MSH analogues. Major differences were found between the lizard and the frog in their response to alpha-MSH peptide fragments and analogues. In Anolis, the sequence Ser-Tyr-Ser- is not as important for the pigmentary response as in Rana since alpha-MSH-(4-13) was nearly as potent (89%) as alpha-MSH-(1-13) (100%), whereas in Rana alpha-MSH-(4-13) potency was reduced to 7.5%. In addition, loss of potency due to removal of residues Pro and Val was more marked in Rana (alpha-MSH-(1-11) = 0.1%) than in Anolis (alpha-MSH-(1-11) = 1%), suggesting that this C-terminal sequence is necessary for pigmentary activity in the frog melanophore. These results together with those of other peptide fragments and analogues have led us to define the minimal pigmentary sequence of alpha-MSH as alpha-MSH-(4-12) in Anolis in contrast to alpha-MSH-(1-13) in Rana. This suggests that Anolis and Rana alpha-MSH receptors recognise different message amino acids of the alpha-MSH peptide sequence even though the final response (melanosome dispersion) is the same.     

Nucleotide sequence of cloned cDNA for pro-opiomelanocortin in the amphibian Xenopus laevis

The function of alpha-melanocyte-stimulating hormone (alpha-MSH) is not known in mammals. It is well-established in the amphibian Xenopus laevis in which alpha-MSH mediates the process of adaptation to a dark background. The amino acid sequence of this hormone is, however, not known in amphibians. In order to determine the primary structure of the precursor protein for alpha-MSH, which in mammals has been called pro-opiomelancortin (POMC), we constructed a cDNA library from Xenopus pituitary mRNA. A pool of synthetic oligodeoxyribonucleotide tetradecamers corresponding to part of the mammalian alpha-MSH sequence was used to screen the library. The nucleotide sequence of a 1050-base pair hybridization-positive cDNA clone was determined and the deduced amino acid sequence of Xenopus POMC revealed the sequences of Xenopus gamma-MSH, alpha-MSH, corticotropin-like intermediate lobe peptide, beta-MSH, and beta-endorphin. Interestingly, the N-terminal amino acid of Xenopus alpha-MSH, which is N alpha-acetylated in the biologically active form of the hormone, is different from that of mammalian alpha-MSH. The distribution of the bioactive domains within Xenopus POMC is remarkably similar to that in other known POMC molecules and as in mammals the domains in the amphibian prohormone are flanked on both sides by pairs of basic amino acids.     

Biological activity, binding, and metabolic fate of Ac-[Nle4, D-Phe7]alpha-MSH4-11NH2 with the F1 variant of B16 melanoma cells

The alpha-MSH (alpha-melanocyte-stimulating hormone) agonist, Ac-[Nle4, D-Phe7]alpha-MSH4-11NH2 (hereafter called ND4-11 alpha-MSH), is at least 10-fold more potent than alpha-MSH as a stimulus of tyrosinase activity in F1 variant cells of B16 melanoma. The binding to these cells during an incubation with 5 nM (3H)ND4-11 alpha-MSH at 37 degrees C is maximal at 0-30 min, 22 fmol/10(6) cells, but declines to 40% of this value at 4 hr. in the presence of 5 nM (3H)ND4-11 alpha-MSH at 37 degrees C, the acid soluble (cell surface) radioactivity decreased rapidly from 11.4 fmol/10(6) cells at 5 min to 4.6 fmol/10(6) cells at 4 hr. Chromatographic analysis of media and cellular samples revealed that there was no evidence of degradation of (3H)ND4-11 alpha-MSH in the medium but there was evidence of intracellular degradation of (3H)ND4-11 alpha-MSH. Ammonium chloride (10mM) resulted in an increase in acid resistant radioactivity (internalized hormone) at 4 hr. The binding to F1 variant cells during an incubation with 0.155 nM or 5 nM (3H)ND4-11 alpha-MSH at 4 degrees C was constant from 4 hr to 24 hr. Under these conditions, there was no time-dependent change in the acid soluble radioactivity from 4 to 24 hr. Scatchard analysis of (3H)ND4-11 alpha-MSH binding to F1 variant cells at 4 degrees C demonstrated that there were approximately 4500 receptors per cell and an association constant of 17.1 nM-1. These results are consistent with a process of (3H)ND4-11 alpha-MSH binding to its receptor followed by internalization of the receptor-hormone complex and then intracellular degradation of the hormone.     

Structure and expression of Xenopus prohormone convertase PC2.

The multifunctional prohormone, proopiomelanocortin (POMC), is processed in the melanotrope cells of the pituitary pars intermedia at pairs of basic amino acid residues to give a number of peptides, including alpha-melanophore-stimulating hormone (alpha-MSH). This hormone causes skin darkening in amphibians during background adaptation. Here we report the complete structure of Xenopus laevis prohormone convertase PC2, the enzyme thought to be responsible for processing of POMC to alpha-MSH. A comparative structural analysis revealed an overall amino acid sequence identity of 85-87% between Xenopus PC2 and its mammalian counterparts, with the lowest degree of identity in the signal peptide sequence (28-36%) and the region amino-terminal to the catalytic domain (59-60%). The occurrence of a second, structurally different PC2 protein reflects the expression of two Xenopus PC2 genes. The expression pattern of PC2 in the Xenopus pituitary gland of black- and white-adapted animals was found to be similar to that of POMC, namely high expression in active melanotrope cells of black animals. This observation is in line with a physiological role for PC2 in processing POMC to alpha-MSH.     

Use of an alpha-melanocyte-stimulating hormone analogue to improve alpha-melanocyte-stimulating hormone receptor binding assay in human melanoma

In order to optimize the detection and measurement of alpha-melanocyte-stimulating hormone (alpha-MSH) receptivity in human melanoma cells, and the authors replaced the natural hormone by [Nle4,D-Phe7]-alpha-MSH, a more stable and potent analogue in the receptor binding assay commonly performed with alpha-MSH. The following parameters were investigated: temperature, incubation time, number of cells, and ratio of labelled to unlabelled hormone. Optimal conditions for each assay were determined. The results demonstrate that the analogue has identical binding sites to alpha-MSH, as similar reciprocal displacements of each labelled (125I) hormone by serial dilutions of unlabelled alpha-MSH or [Nle4,D-Phe7]-alpha-MSH (10(-12) M to 10(-6) M) were obtained. To further compare the two hormones, we performed a screening of various human cell lines: ten melanomas and five nonmelanomas. The assay with [Nle4,D-Phe7]-alpha-MSH yielded more receptor expression on six of ten melanoma lines against only four of ten with the natural hormone. In conclusion, the use of radiolabelled [Nle4,D-Phe7]-alpha-MSH analogue instead of labelled alpha-MSH improved both sensitivity and reproducibility in this receptor binding assay on human melanoma lines.     

Recent developments in our understanding of the avian melanocortin system: its involvement in the regulation of pigmentation and energy homeostasis

The mammalian melanocortin system has been established as a crucial regulatory component in an extraordinarily diverse number of physiological functions. In contrast, comparatively little is known about the avian melanocortin system: interest in the physiological role of alpha-MSH in birds has been limited by the fact that birds lack the intermediate lobe of the pituitary, the main source of circulating alpha-MSH in most vertebrates. Recently, however, the main avian melanocortin system genes, including POMC, AGRP, and all the melanocortin receptors, have been cloned and their physiological roles are the beginning to be elucidated. This review outlines our improved understanding of the avian melanocortin system, particularly in relation to two of the most widely studied physiological functions of the melanocortin system in mammals, the regulation of pigmentation and energy homeostasis. The data reviewed here indicate that the melanocortin system has been strongly conserved during vertebrate evolution and that alpha-MSH is produced locally in birds to act as an autocrine/paracrine hormone.     

Melanin concentrating hormone. V. Isolation and characterization of alpha-melanocyte-stimulating hormone from frog pituitary glands

The structure of alpha-melanocyte-stimulating hormone (alpha-MSH) has been determined in the pars intermedia of the frog Rana ridibunda. Pulse-chase labeling of frog neurointermediate lobes with selective amino acids revealed that the composition of frog alpha-MSH is similar to that of alpha-MSH from all mammalian species yet studied. Tryptic mapping of nexly synthetized alpha-MSH generated two fragments with the following amino acid composition: (T1) Trp, Pro, Lys, Gly, Val and (T2) Tyr, Arg, Phe, His, Ser, Glu. Concurrently, alpha-MSH was purified from 100 neurointermediate lobes to apparent homogeneity by reverse-phase HPLC. The sequence of the peptide determined by automated Edman degradation was Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val. The structure of frog alpha-MSH is thus identical to mammalian des-N alpha-acetyl alpha-MSH and differs from the sequence of toad (Xenopus laevis) alpha-MSH only by the first residue (Ser instead of Ala). These results confirm that the sequence of alpha-MSH has been highly preserved during evolution.     

Antipyretic properties of centrally administered α-MSH fragments in the rabbit

alpha-Melanocyte stimulating hormone (alpha-MSH 1-13) has marked antipyretic effects when administered centrally or peripherally in small doses. A C-terminal fragment, alpha-MSH (11-13), contains an antipyretic message sequence of alpha-MSH; however, the lesser potency of this fragment relative to that of the entire molecule suggests that other amino acids of the alpha-MSH sequence are essential for the full antipyretic effect. Graded doses of alpha-MSH (11-13) (Ac LysProVal NH2), alpha-MSH (10-13) (Ac GlyLysProVal NH2), and alpha-MSH (8-13) (Ac ArgTrpGlyLysProVal NH2), were injected into the cerebral ventricles of rabbits made febrile by IV administration of crude interleukin-1. All three fragments reduced fever in a dose-related manner. The (8-13) sequence was much more effective than the other two fragments, and the (10-13) portion was less effective than the (11-13) tripeptide. None of the fragments was as potent as alpha-MSH (1-13). The results confirm that an antipyretic message resides within alpha-MSH (11-13) and sequential addition of amino acids to alpha-MSH (11-13) can both enhance and reduce the potency of the fragment.     

Antiinflammatory activity of a COOH-terminal fragment of the neuropeptide alpha-MSH

The endogenous neuropeptide alpha-melanocyte stimulating hormone (alpha-MSH 1-13), previously found to have marked antipyretic activity, inhibits histamine-induced increases in vasopermeability. The primary antipyretic amino acid message sequence is believed to be the COOH-terminal trieptide, lysine-proline-valine. In recent preliminary research this tripeptide inhibited increases in vasopermeability, raising the possibility that this portion of the alpha-MSH molecule has general antiinflammatory activity. To test this idea, the effects of graded doses of alpha-MSH [11-13] on ear swelling induced by picryl chloride in mice were compared with the effects of saline and a large dose of corticosteroid. Alpha-MSH [11-13] inhibited swelling in a dose-related fashion. This result, together with previous findings, suggests that endogenous circulating alpha-MSH and its COOH-terminal fragments may contribute to modulation of physiological responses in host defense. If this is true, it may be possible to develop new peptide drugs or mimetics based on the tripeptide that are useful in treating inflammation.     

Cyclic lactam analogues of Ac-[Nle4]alpha-MSH4-11-NH2

Two side-chain cyclic lactam analogues of the 4-11 fragment of alpha-melanocyte-stimulating hormone (alpha-MSH), Ac-[Nle4,D-Orn5,Glu8]alpha-MSH4-11-NH2 and Ac-[Nle4,D-Orn5,D-Phe7,Glu8]alpha-MSH4-11-NH2, were prepared on p-methylbenzhydrylamine resin by using a combination of N alpha-Boc and N alpha-Fmoc synthetic strategies with diphenyl phosphorazidate mediated cyclization. The melanotropin activities of these two analogues were examined and compared relative to those of alpha-MSH, Ac-[Nle4]alpha-MSH4-11-NH2, and Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. In the frog (Rana pipiens) skin bioassay, the L-Phe7 17-membered ring cyclic analogue was slightly more potent than the linear Ac-[Nle4]alpha-MSH4-11-NH2 and exhibited prolonged melanotropic bioactivity (greater than or equal to 4 h). In this same assay, the D-Phe7 cyclic analogue was more than 100-fold less potent than the L-Phe cyclic analogue and was 10,000 times less potent than linear Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. In the lizard skin (Anolis carolinensis) bioassay, the L-Phe7 cyclic analogue was 100-fold less potent than Ac-[Nle4]alpha-MSH4-11-NH2, while the D-Phe7 cyclic analogue was 10,000-fold less potent than both Ac-[Nle4]alpha-MSH4-11-NH2 and the D-Phe7 linear derivative Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. The solution conformation of these two cyclic analogues in dimethyl sulfoxide-d6 was examined by 1D and 2D 500-MHz 1H NMR spectroscopy. Our analysis suggests an H bond stabilized C10 (or C13) turn for the D-Phe7 cyclic structure while the L-Phe7 analogue is more conformationally flexible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunocytochemical demonstration of proopiomelanocortin- and other opioid-related substances and a CRF-like peptide in the gut of the american cockroach, Periplaneta americana L

Using the peroxidase-antiperoxidase technique, we showed the presence of peptides which are immunologically resembling mammalian corticotropin releasing hormone (CRF)-, adrenocorticotropic hormone (ACTH)-, beta-endorphin (beta-END)-, alpha-melanocyte stimulating hormone (alpha-MSH)-, methionine-enkephalin (met-ENK)- and leucine enkephalin (leu-ENK)- like immunoreactivity in hundreds to thousands of endocrine cells and nerve fibers in the midgut of the American cockroach Periplaneta americana. In the cockroach hindgut no immunoreactive cell bodies could be observed, although nerve fibers were clearly noticed to be recognized by antisera to CRF, ACTH1-24, ACTH11-24 and beta-END. Nothing is exactly known as to the function(s) of the demonstrated materials, but one can speculate that these numerous immunoreactive cells, might have important paracrine and/or endocrine functions in the insect physiology.     

Alpha-MSH peptides inhibit acute inflammation and contact sensitivity

Alpha-melanocyte stimulating hormone [alpha-MSH(1-13)] occurs within the CNS, skin, circulation and in other body sites. This tridecapeptide and its COOH-terminal tripeptide, alpha-MSH (11-13), have antipyretic and anti-inflammatory actions. Studies of the anti-inflammatory effects of these molecules have been confined mainly to tests of inhibition of histamine and endogenous pyrogen-induced increases in capillary permeability in rabbits and acute inflammation of ear tissue in mice. The aim in the present experiments was to learn if alpha-MSH peptides also antagonize inflammation in two additional models: acute edema induced in the mouse paw and contact sensitivity. Significant anti-inflammatory effects were observed with MSH peptides in both models. These findings converge with previous results to indicate that alpha-MSH peptides modulate inflammation. Because circulating alpha-MSH increases after treatment of animals with endogenous pyrogen or endotoxin, administration of the peptides may simply mimic a naturally occurring modulation of host defense reactions.     

Alpha-MSH peptides inhibit acute inflammation induced in mice by rIL-1 beta, rIL-6, rTNF-alpha and endogenous pyrogen but not that caused by LTB4, PAF and rIL-8.

The neuropeptide alpha-melanocyte stimulating hormone [alpha-MSH(1-13)] occurs in the pituitary, brain, skin and other tissues and receptors for this molecule are likewise widespread. In previous research, this tridecapeptide, which shares its amino acid sequence with ACTH(1-13), was shown to have both potent antipyretic activity and a role in the endogenous control of the febrile response. alpha-MSH(1-13) and its COOH-terminal tripeptide were subsequently found to inhibit inflammation induced by general stimuli such as topical application of an irritant. The aim in the present experiments was to determine if these peptides can inhibit acute inflammatory responses induced in mice by injection of individual cytokines, endogenous pyrogen (EP), a natural cytokine mixture, and other mediators of inflammation. Inflammation induced in the mouse ear by rIL-1 beta, rIL-6 or rTNF-alpha was inhibited by alpha-MSH and a D-valine-substituted analog of alpha-MSH(11-13) whereas substantial doses of alpha-MSH(1-13) did not alter inflammation induced by LTB4, PAF and IL-8. Both peptides inhibited edema caused in the mouse paw by local injection of EP. The results indicate that alpha-MSH molecules antagonize the actions of certain cytokine mediators of inflammation, consistent with previous observations of anti-cytokine activity of these peptides. Failure to inhibit edema caused by LTB4, PAF and IL-8 suggests that, in inflammation induced by general stimuli, such as EP, the peptides act prior to the release of these mediators of the inflammatory response. Because of the anticytokine/anti-inflammatory actions of the alpha-MSH molecules they may be useful in understanding the cytokine network and for treatment of inflammatory diseases.     

Melanin concentrating hormone analogues: contraction of the cyclic structure. II. Antagonist activity

Asp-Thr-Met-Arg-Cys-Met-Val-Gly-Arg-Val-Tyr-Arg-Pro-Cys-Trp-Glu-Val, melanin concentrating hormone (MCH), is a cyclic hormone possessing both MCH-like (melanin granule aggregating effect) and melanocyte stimulating hormone (MSH)-like (melanin granule dispersing effect) activities. Nine ring-contracted analogues were synthesized and characterized for their melanotropic activity on the fish (Synbranchus marmoratus) and frog (Rana pipiens) bioassays. In most cases, these analogues were totally devoid of MCH-like agonist activity, demonstrating the essential role of the disulfide bridge between residues 5 and 14 of the hormone. [Ala5, Cys10]MCH, for example, was totally devoid of MCH-like activity. This analogue, like alpha-MSH, however, antagonized the melanosome aggregating actions of MCH on fish melanocytes. The antagonistic activity of the analogue, like that of alpha-MSH, was Ca2+-dependent. Evidence suggested that this antagonism of MCH activity was related to the intrinsic MSH-like activity of the analogue. These results suggest that MCH and alpha-MSH may be structurally and, therefore, evolutionarily related.     

Alpha-melanocyte-stimulating hormone immunoreactivity in human melanoma metastases extracts

Seven human melanoma metastases were extracted in order to check the possible presence of any alpha-melanocyte stimulating hormone (MSH) immunoreactivity. The aim of that study was to provide some explanation for, mainly, two observations that we have already made and reported: 1) increased plasma alpha-MSH levels in melanoma of tumour-bearing patients as compared with tumour-free patients; 2) the presence of specific alpha-MSH receptors on human melanoma cells in culture. We could measure large amounts of immunoreactive alpha-MSH in all tumours ranging from 0.31 to 4.27 pmoles per g of wet tissue. Further identification of the extracted material by high-performance liquid chromatography revealed compounds of higher molecular weight and more hydrophobic than synthetic alpha-MSH. In addition, purified extracts could also displace 125I-labelled alpha-MSH from its cellular binding sites in an alpha-MSH specific radio-receptor binding assay. Our findings would suggest a possible presence of some hormone precursor(s) inside the melanoma tumours.     

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.     

ACTH(4-12) is the minimal message sequence required to induce the differentiation of mouse epidermal melanocytes in serum-free primary culture

It is well known that alpha-melanocyte stimulating hormone (MSH) induces the differentiation of mouse epidermal melanocytes in vivo and in vitro. Although adrenocorticotropic hormone (ACTH) possesses the same amino acid sequence as MSH does, it is not clear whether the peptide and its fragments induce the differentiation of mouse epidermal melanocytes. In this study, the differentiation-inducing potencies of human ACTH and its fragments were investigated by adding them into a culture medium (0.001-1,000 nM) from the initiation of primary culture of epidermal cell suspensions. Their potencies were compared with the potency of alpha-MSH. After 2-4 days of primary cultures with ACTH(1-13), ACTH(1-17), ACTH(1-24), ACTH(1-39), ACTH(4-12), ACTH(4-13), and alpha-MSH, pigment granules appeared in the cytoplasms and dendrites of melanoblasts that were in contact with the adjacent keratinocyte colonies. By 14 days, cultures contained mostly pigmented melanocytes. The order of potencies of ACTH fragments and alpha-MSH shown by the ED(50) value was as follows: alpha-MSH = ACTH(1-13) = ACTH(1-17) = ACTH(4-12) = ACTH(4-13) > ACTH(1-24) > ACTH(1-39). The length of their peptide chains was inversely proportional to the potency. On the contrary, ACTH(1-4), ACTH(11-24), and ACTH(18-39) failed to induce the differentiation of melanocytes. In contrast, ACTH(1-10), ACTH(4-10), ACTH(4-11), and ACTH(5-12) possessed a weak potency at high doses only (100 and 1,000 nM). These results suggest that ACTH(4-12) is the minimal message sequence required to induce the differentiation of mouse epidermal melanocytes in culture completely. The amino acids of Met(4) and Pro(12) are suggested to be important for its potency.     

Alpha-melanocyte-stimulating hormone exhibits target cell selectivity in its capacity to affect interleukin 1-inducible responses in vivo and in vitro

The ability of i.v.-administered recombinant human interleukin 1 (IL 1 beta) to increase core body temperature, stimulate an increased production of serum amyloid P substance, and augment blood levels of circulating neutrophils in mice was inhibited in a dosage-dependent manner by administration of the neuropeptide alpha-melanocyte-stimulating hormone (alpha-MSH). alpha-MSH administration was also capable of inhibiting the capacity of i.v.-administered IL 1 beta to enhance plasma levels of corticosterone and to depress the generation and/or elicitation of contact hypersensitivity responses to skin-reactive chemicals. An analog of alpha-MSH (Nle4, D-Phe7 alpha-MSH), known to be more potent than native alpha-MSH in a number of melanotropin-sensitive systems, was determined to be more active than alpha-MSH in the modification of these same in vivo responses. Neither alpha-MSH nor its analog were capable of altering the capacity of IL 1 to stimulate increased plasma levels in prostaglandin E2 (PGE2). In vitro, neither alpha-MSH nor its analog were capable of reducing the capacity of IL 1 to stimulate fibroblast production of PGE2 or to augment the proliferation of murine thymocytes exposed to phytohemagglutinin. The apparent selectivity associated with the regulatory influences of alpha-MSH on IL 1-induced responses in vivo suggests that this neuropeptide may function as an endogenous inhibitor of certain immunomodulatory and inflammatory activities of the cytokine IL 1.     

Evidence for Alpha-MSH Binding Sites on Human Scalp Hair Follicles: Preliminary Results

Alpha-MSH, considered an important pigmentation hormone, binds to melanocytes and is thought to stimulate melanogenesis through a cyclic-AMP-dependent mechanism. The binding of alpha-MSH to follicular melanocytes has been investigated in human hair of different colors, ranging from black to blond and senile white. Hairs were plucked, the follicles were cut off, and an alpha-MSH binding assay, using a radiolabeled alpha-MSH analogue, was performed on these bulbs. As controls of each assay, fragments of hairs of the same person were used. The results show a dose-response relationship and the assay seems to be specific for alpha-MSH, because other peptides such as ACTH, beta-LPH and beta-endorphins do not compete for binding sites as alpha-MSH does. These binding sites seem to be present only on melanin synthesizing melanocytes, since the controls and follicles of senile white hair, which do not contain active melanocytes, show negative results. All the assays were performed on raw material, i.e., whole plucked hair follicles. This is the first time that binding sites for alpha-MSH have been demonstrated on human scalp hair follicles. In addition, their presence was found to be associated with active melanin production; their absence was demonstrated on senile white hair follicles.