Bhatnagar V, Srirangam A, Abburi R
Department of Biochemistry, All India Institute of Medical Sciences, New Delhi.
Mol Cell Biochem. 1998 Oct;187(1-2):57-65. doi: 10.1023/a:1006870621424.
B16/F10 murine melanoma cells were grown for 24 and 36 h in Dulbecco's modified Eagle medium in presence of 10-20 mM trisodium citrate. The intracellular melanin concentration and the melanin secreted in the extracellular medium was estimated. It is observed that 20 mM citrate stimulates extracellular melanin secretion in B16/F10 melanoma cells by 200% at 36 h treatment. The intracellular melanin content increased by 90%. This stimulatory effect of citrate was totally abolished when these cells were grown in presence of 1 mM phenyl thiourea, a specific inhibitor of tyrosinase activity. Citrate (0.1-5 mM) had no effect on dopa oxidase activity either at pH 5.0 or at pH 6.8. There was no increase in the tyrosinase specific activity in presence of citrate. The increased melanin synthesis was shown to be due to stimulation of cellular tyrosine hydroxylase activity by citrate. It has been suggested that enhanced melanin synthesis results in an increased production of metabolites that are toxic to the growth of melanoma cells. We have studied the effect of citrate on cellular proliferation. Following 24 and 36 h treatment with citrate, the cells exhibited a dose-dependent decrease in proliferation. In presence of 20 mM citrate the cell number was only up to 50% of the control cultures after 36 h of incubation. The growth retardation was not due to cytotoxicity. Citrate, a natural metabolite, is a unique molecule which may be involved in the regulation of melanin biosynthetic pathway, since it enhances melanogenesis by increasing the hydroxylase activity of tyrosinase which is the regulatory enzyme of this pathway. These observations add further support to the critical role of intramelanosomal pH in regulation of melanogenesis.
将B16/F10小鼠黑色素瘤细胞在含有10 - 20 mM柠檬酸钠的杜氏改良 Eagle培养基中培养24小时和36小时。估计细胞内黑色素浓度以及分泌到细胞外培养基中的黑色素量。观察到在36小时处理时，20 mM柠檬酸盐可使B16/F10黑色素瘤细胞的细胞外黑色素分泌增加200%。细胞内黑色素含量增加了90%。当这些细胞在1 mM苯硫脲（酪氨酸酶活性的特异性抑制剂）存在的情况下生长时，柠檬酸盐的这种刺激作用完全消失。柠檬酸盐（0.1 - 5 mM）在pH 5.0或pH 6.8时对多巴氧化酶活性均无影响。在柠檬酸盐存在的情况下，酪氨酸酶的比活性没有增加。结果表明，黑色素合成增加是由于柠檬酸盐刺激了细胞酪氨酸羟化酶活性。有人提出，黑色素合成增强会导致对黑色素瘤细胞生长有毒性的代谢产物产量增加。我们研究了柠檬酸盐对细胞增殖的影响。用柠檬酸盐处理24小时和36小时后，细胞增殖呈剂量依赖性下降。在20 mM柠檬酸盐存在的情况下，孵育36小时后细胞数量仅为对照培养物的50%。生长迟缓并非由于细胞毒性。柠檬酸盐作为一种天然代谢产物，是一种独特的分子，可能参与黑色素生物合成途径的调节，因为它通过增加酪氨酸酶（该途径的调节酶）的羟化酶活性来增强黑色素生成。这些观察结果进一步支持了黑素小体内pH在调节黑色素生成中的关键作用。
