Tsatmali M, Graham A, Szatkowski D, Ancans J, Manning P, McNeil C J, Graham A M, Thody A J
Department of Biomedical Sciences, University of Bradford, Bradford, UK.
J Invest Dermatol. 2000 Mar;114(3):520-6. doi: 10.1046/j.1523-1747.2000.00879.x.
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 alpha-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 10-11-10-9 M alpha-melanocyte-stimulating hormone prior to stimulation. At concentrations in excess of 10-9 M alpha-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 alpha-melanocyte-stimulating hormone. alpha-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 alpha-melanocyte-stimulating hormone. alpha-Melanocyte-stimulating hormone, however, decreased inducible nitric oxide synthase protein expression, which occurred in response to lipopolysaccharide. These results suggest that alpha-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 alpha-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 alpha-melanocyte-stimulating hormone remains to be seen.
我们之前观察到黑素细胞会响应紫外线辐射和脂多糖产生一氧化氮，在本研究中，我们研究了α-黑素细胞刺激素如何影响这些反应。使用ISO-一氧化氮传感器探头通过电化学方法实时测量培养细胞产生的一氧化氮。B16小鼠黑素瘤细胞会响应脂多糖释放一氧化氮，并且在刺激前于10-11-10-9 Mα-黑素细胞刺激素存在下生长的细胞中，这种效应增强。在α-黑素细胞刺激素浓度超过10-9 M时，一氧化氮产生减少。用脂多糖（一种众所周知的诱导型一氧化氮合酶诱导剂）预孵育也会增加一氧化氮产生，但这种反应会被α-黑素细胞刺激素减弱。α-黑素细胞刺激素还增加了B16细胞中响应紫外线辐射（20-100 mJ每平方厘米）产生的一氧化氮水平。在人黑素细胞中也观察到了相同的效应，并且由于这种效应被氨基胍抑制，似乎涉及诱导型一氧化氮合酶的诱导。逆转录聚合酶链反应表明黑素细胞表达诱导型一氧化氮合酶mRNA。蛋白质印迹分析和免疫细胞化学证实了B16细胞和FM55人黑素瘤细胞中存在诱导型一氧化氮合酶蛋白，并表明其水平会响应α-黑素细胞刺激素而增加。然而，α-黑素细胞刺激素会降低响应脂多糖而出现的诱导型一氧化氮合酶蛋白表达。这些结果表明α-黑素细胞刺激素通过调节诱导型一氧化氮合酶的诱导来调节黑素细胞中一氧化氮的产生。额外的实验表明一氧化氮增加了B16细胞和人黑素细胞中黑色素生成量。这与一氧化氮在黑色素生成中的作用一致，但黑素细胞响应α-黑素细胞刺激素产生一氧化氮是否与这种作用相关，或者它是否与黑素细胞分化或介导α-黑素细胞刺激素的免疫调节作用有其他某种意义，仍有待观察。
