Eumelanin and phaeomelanin contents of human epidermis and cultured melanocytes.

There are two chemically distinct types of melanin: the red-yellow phaeomelanins and the brown-black eumelanins. While both melanins have been detected in human epidermis and cultured melanocytes, it is unknown how the phaeomelanin/eumelanin ratio in human melanocytes maintained in vitro relates to that in the epidermis from which they were isolated. This study uses high-performance liquid chromatography to quantify the eumelanin and phaeomelanin contents of epidermis and/or cultured melanocytes from 12 Europeans with lightly pigmented skin and 9 non-Europeans with more deeply pigmented skin. Epidermis from non-Europeans contained the highest levels of both eumelanin and phaeomelanin and had the lowest phaeomelanin/eumelanin ratios. In contrast, while cultured melanocytes from non-Europeans also had higher levels of eumelanin and phaeomelanin than melanocytes from Europeans, there was no difference in the phaeomelanin/eumelanin ratios in the two groups. However, the phaeomelanin/eumelanin ratios were higher in the cultured melanocytes than in the corresponding epidermis so that while eumelanin was the predominant melanin in these epidermis, phaeomelanin was the major melanin in the cultured melanocytes. These observations may have important implications for the use of cultured human melanocytes in the study of melanogenesis in man.

Nle4DPhe7 alpha-melanocyte-stimulating hormone increases the eumelanin:phaeomelanin ratio in cultured human melanocytes.

In mammals, melanin exists in two chemically distinct forms: the red-yellow phaeomelanin and the brown-black eumelanin. Although administration of the pigmentary hormone alpha-melanocyte-stimulating hormone (alpha MSH) and its synthetic analogue Nle4DPhe7 alpha MSH induces skin darkening in man, the increases in melanogenesis in cultured human melanocytes in response to these peptides are relatively small. However, it is possible that MSH affects the eumelanin:phaeomelanin ratio rather than total cellular melanin. Thus, this study examined the specific effects of Nle4DPhe7 alpha MSH on the two melanins in cultured human melanocytes, quantifying eumelanin and phaeomelanin by high performance liquid chromatography. Nle4DPhe7 alpha MSH induced significant increases in the eumelanin content of these cells while having lesser and varied effects on the levels of phaeomelanin. As a consequence, the eumelanin:phaoemelanin ratio was increased in every culture. These results demonstrate that Nle4DPhe7 alpha MSH affects melanin type in human melanocytes and suggest a possible mechanism by which this peptide induces skin darkening in man.

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.

ACTH stimulates melanogenesis in cultured human melanocytes.

While ACTH is known to induce skin pigmentation in man, its effects on cultured human melanocytes have not been investigated. Using a culture system free of artificial mitogens, we report for the first time that ACTH stimulates melanogenesis in cultured human melanocytes. While ACTH, alpha-MSH and the synthetic alpha-MSH analogue Nle4DPhe7 alpha-MSH all stimulate the activity of tyrosinase, the rate limiting enzyme in melanogenesis, and all produce a 50% increase in the melanin content of the cells at a concentration of 10(-8)-10(-7) mol/l, the shapes of the dose response curves differ: those for the MSH peptides are sigmoidal while those for ACTH are biphasic. In addition, human melanocytes are able to respond to concentrations of ACTH comparable with physiological plasma levels. We suggest that ACTH may be relatively more important than alpha-MSH as a pigmentary hormone in man and could have a physiological role in skin pigmentation.