Analysis of the Tetrahydrobiopterin Biosynthesizing System in Cultured Neuron-Derived Cells

6R-四氢生物蝶呤(BH4)是一种天然存在的芳香氨基酸羟基酶和一氧化氮合酶(NOS)的必需辅因子。脂多糖是革兰氏阴性杆菌外膜的一种成分，它能增加小鼠神经母细胞瘤细胞株N1E-115的BH4含量，并促进BH4向培养基中的释放。BH4产量的这种变化最初是由于编码GTP环水解酶I(GTPCH)的RNA表达水平提高所致，GTPCH是BH4从头合成途径中的限速酶。此外，位于BH4生物合成途径第二步和最后一步的6-丙酮酰四氢蝶呤合成酶和海风蝶呤还原酶的mRNAs的表达水平也较高。然而，我们在N1E-115细胞中没有发现编码CD14的mRNA表达的证据。CD14是一种膜锚定蛋白，已被认为是巨噬细胞上的内毒素受体。因此，我们在N1E-115细胞中寻找了除CD14外的内毒素受体候选基因。在一系列实验中，我们用逆转录聚合酶链式反应的方法，发现巨噬细胞上编码CD18和Toll样受体4型(T1r-4)的mRNA在巨噬细胞上的表达。肿瘤坏死因子α(肿瘤坏死因子α)也能促进N1E-115细胞产生BH4。我的实验室目前正在研究肿瘤坏死因子-α诱导的BH_4产生增加是否是由于激活了脂多糖刺激共同的途径而引起的。在N1E-115细胞中，我们既没有检测到NO产生的增加，也没有检测到编码神经元型和诱导型NOS的mRNAs表达水平的增加。据推测，这些细胞系中的NOS酶被BH4饱和。

6R-Tetrahydrobiopterin (BH4) is a naturally occurring essential cofactor for aromatic amino acid hydroxylases and nitric oxide (NO) synthase (NOS). Lipopolysaccharide(LPS), a component of the outer membrane of gram-negative bacilli, has been shown to enhance the BH4 content of the murine neuroblastoma cell line N1E-115 as well as the release of BH4 into culture medium. Such alterations in BH4 production were at first caused by the enhancement of the expression level of RNA encoding GTP cyclohydrolase "I"(GTPCH), a rate-limiting enzyme in the BH4 de novo biosynthetic pathway. Moreover, the expression levels of mRNAs encoding 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase which place at the 2nd and the final step in BH4 biosynthesic pathway, respectively. However, we found no evidence in N1E-115 cells showing the expression of mRNA encoding CD14. CD14 is an membrane-anchoring protein and it is already recognized as the LPS receptor on macrophages. Therefore we searched the candidates for LPS receptor except for CD14 in N1E-115 cells. In a series of experiments, we found the expressions of mRNA encoding CD18, another subtype of LPS receptor on macrophages, and type 4 Toll-like receptor (T1r-4) in this cell line by way of reverse transcriptase-polymerase chain reaction. This should be the first evidence that neuron-derived cell line expresses T"I"r-4..Tumor necrotizing factor α(TNF-α) was also capable of enhancing BH4 production in N1E-115 cells. It is currently under investigation in my laboratory whether the increase in BH4 production induced by TNF-α was caused by the activation of the pathway common to the LPS stimulation.In spite of the increase in BH4 production by LPS stimulation. we could detect neither the increase in NO production nor the increase in the expression levels of mRNAs encoding neuronal and inducible NOS in N1E-115 cells. NOS enzymes in these cell lines were speculated to be saturated by BH4.