We have previously observed that melanocytes produce nitric oxide in response to ultraviolet radiation and lipopolysaccharide and in this study have examined how these responses are affected by α-melanocyte-stimulating hormone. Nitric oxide production by cultured cells was measured electrochemically in real time using an ISO-nitric oxide sensor probe. B16 mouse melanoma cells released nitric oxide in response to lipopolysaccharide and the effects were enhanced in cells that had been grown in the presence of 1011-109 M α-melanocyte-stimulating hormone prior to stimulation. At concentrations in excess of 109 M α-melanocyte-stimulating hormone decreased nitric oxide production. Preincubation with lipopolysaccharide, a well-known inducer of inducible nitric oxide synthase, also increased nitric oxide production but this response was reduced by α-melanocyte-stimulating hormone. α-Melanocyte-stimulating hormone also increased the levels of nitric oxide produced in response to ultraviolet radiation (20–100 mJ per cm2) in B16 cells. The same effect was seen in human melanocytes and as this was inhibited by aminoguanidine would appear to involve an induction of inducible nitric oxide synthase. Reverse transcription–polymerase chain reaction showed that melanocytic cells express inducible nitric oxide synthase mRNA. Western blotting analysis and immunocytochemistry confirmed the presence of inducible nitric oxide synthase protein in B16 cells and FM55 human melanoma cells and that the levels were increased in response to α-melanocyte-stimulating hormone. α-Melanocyte-stimulating hormone, however, decreased inducible nitric oxide synthase protein expression, which occurred in response to lipopolysaccharide. These results suggest that α-melanocyte-stimulating hormone regulates nitric oxide production in melanocytic cells by modulating the induction of inducible nitric oxide synthase. Additional experiments showed that nitric oxide increased melanin production by B16 cells and human melanocytes. This is in keeping with a melanogenic role for nitric oxide but whether its production by melanocytes in response to α-melanocyte-stimulating hormone is associated with such a role or whether it has some other significance relating to melanocyte differentiation or in mediating immunomodulatory actions of α-melanocyte-stimulating hormone remains to be seen.
Keywords
melanogenesis
nitric oxide synthase
ultraviolet radiation
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1Presented in part at the XVIth International Pigment Cell Conference, Anaheim, October 1996 and the European Society for Pigment Cell Research Meeting, Bordeaux, October 1997.
D. Szatkowski was a Socrates exchange student from the University of Gdansk, Gdansk, Poland. P. Manning is now at Department of Neurology, Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne, NE2 4HH, U.K.
Valverde P, Manning P, Graham A., McNeil CJ, Thody AJ: Melanocytes produce superoxide anion and nitric oxide in response to low doses of ultraviolet radiation. J Invest Dermatol 107:509 1996 (abstr.)
