Signal transduction system in osteoclasts through tetrahydrobiopterin-mediated production of NO and cGMP.

一氧化氮（NO）抑制破骨细胞活性，但其信号转导机制尚不清楚。cGMP和cGMP依赖性蛋白激酶信号通路介导NO的多种生理作用，可能包括骨吸收。NO合成酶（NOS）最近被证明需要四氢生物蛋白（BH4）作为其活性的天然辅因子。因此了解破骨细胞中BH4含量通过激活NOS并生成NO和cGMP对骨吸收的调节机制具有重要意义。然而，对于破骨细胞中BH4的合成能力尚不清楚。因此，我们研究了BH4合成酶在破骨细胞中的出现情况和BH4的含量，利用RT-PCR法扩增了BH4生物合成途径最后一步酶sepapterin reductase的cDNA，并用大肠杆菌进行了表达。获得了显示酶活性的蛋白，并纯化到均质性。制备了针对该蛋白的多克隆抗体。这有助于检测破骨细胞中bh4合成的水平和定位。从兔大腿骨的骨髓中分离出破骨细胞含量大于95%的细胞团，但由于其含量不足以测定细胞中BH4或NO的含量，分离难度很大。每10^5个细胞中检测到的BH4和NO含量相当显著。由此我们发现破骨细胞具有合成BH4的能力，我们可以建立一个试剂盒来分析BH4、NO、cGMP的形成与破骨之间的关系。

Nitric oxide (NO) inhibits osteoclast activity, but the signal transduction mechanism is not understood. cGMP and cGMP-dependent protein kinase signling pathway mediate several physiologic effects of NO possiblly including bone resorption. NO synthase (NOS) was recently shown to require tetrahydrobiopter in (BH4) as an natural cofactor in its activity. Thus it is of great interest to know the regulation mechanism of bone resorption by BH4 content in the osteoclast through activation of NOS and the resulted formations of NO and cGMP.However, nothing is known about the ability of BH4 synthesis in osteoclasts. Therefore, we studied the occurence of BH4-synthesyzing enzymes and BH4 content in the osteoclaset, cDNA of sepiapterin reductase, an enzyme involved in the last step of the biosynthetic pathway of BH4, was amplified by RT-PCR method and expressed by E.coli. The protein which shows the enzyme activity has been obtained and purified to homogeneity. And the polyclonal antibody was prepared against the protein. These are usefull for detecting the level and localization of BH4-synthesis in the osteoclasts. We could isolated cell mass containing more than 95% osteoclasts from bonemarrows of rabbit thigh bones with great difficulty as the enough amount to assay BH4 or NO content in the cell. And contents of BH4 and NO detected per 10^5cells were quite significant. Thus we have found that the osteoclast has the ability for BH4 synthesis and we could established a kit for analysis the relation between the formation of BH4, NO,and cGMP,and osteoclasis.