Pharmacological study on NO and biopterin synthesis in osteoblastic cells

il -1 β、tnf - α和ifn - γ结合的促炎细胞因子引起四氢生物terin （BH_4）生物合成限速酶GTP环水解酶I （GTP- ch）和诱导型一氧化氮合酶（iNOS） mRNA的表达。我们首次在成骨细胞中发现GTP-CH基因的表达。GTP-CH和iNOS mrna的表达分别在3和9小时达到峰值。这两个基因的表达均引起BH_4和NO水平升高。利用GTP-CH活性抑制剂2,4 -二氨基-6-羟基嘧啶（DAHP）的药理研究表明，BH_4参与iNOS的活性，但不参与iNOS mRNA的诱导。使用核因子(NF)-kappaB抑制剂和活化蛋白-1 （AP-1）激活的结果表明，通过NF-kappaB和AP-1的激活，这两个基因对细胞因子的反应发生了共诱导。细胞因子和更多的s -亚硝基-n -乙酰- d， l-青霉胺（SNAP），一种NO产生物，降低细胞活力；而在细胞内转化为BH_4的sepapterin则使其增加。通过乳酸脱氢酶（LDH）释放和流式细胞术评估的凋亡细胞死亡来检测细胞毒性，表明细胞因子诱导的细胞活力降低可能是基于细胞凋亡导致的细胞死亡，而不是像坏死那样的溶解性死亡。在sepapterin的存在下，细胞因子治疗导致DNA片段量的统计学显著减少。另外，NO清除剂2-（4-羧基苯基）- 4,4,5,5 -四甲基咪唑-1-氧基3-氧化物（carboxy-PTIO）可以阻断DNA的断裂。这些结果表明，细胞因子诱导的凋亡细胞死亡是由NO引起的，并受到BH_4的保护，成骨细胞对促炎细胞因子的反应既激活了NO的产生，又抑制了BH_4的产生。细胞因子诱导的凋亡细胞死亡可能是刺激过程优于抑制过程的结果。少

Proinflammatory cytokines, a combination of IL-1beta, TNF-alpha, and IFN-gamma, caused mRNA expression of GTP cyclohydrolase I (GTP-CH), the rate-limiting enzyme in tetrahydrobiopterin (BH_4) biosynthesis, as well as that of inducible nitric oxide synthase (iNOS) in a well-characterized osteoblastic clone MC3T3-E1 cell line. We found the expression of GTP-CH gene in osteoblasts for the first time. The expression of GTP-CH and iNOS mRNAs was found to be maximal at 3 and 9 hr, respectively. The expression of both genes elicited increases in BH_4 and NO levels. Pharmacological studies using 2, 4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of GTP-CH activity showed that BH_4 is involved in the activity of iNOS, but not in the induction of iNOS mRNA.The results using an inhibitor of nuclear factor (NF)-kappaB and activating protein-1 (AP-1) activation suggested that coinduction of the two genes in response to cytokines occurred via activation of NF-kappaB and AP-1.Cytokines as well as … More S-nitroso-N-acetyl-D, L-penicillamine (SNAP), an NO generator, decreased cell viability ; but sepiapterin, which was converted intracellularly to BH_4, increased it. The examination of cytotoxicity measured in terms of lactate dehydrogenase (LDH) release and apoptotic cell death assessed by flow cytometric analysis showed that cytokine-induced reduction of cell viability may be based upon cell death by apoptosis, but not lytic death as in necrosis. In the presence of sepiapterin, cytokine treatment resulted in a statistically pronounced reduction in the amount of DNA fragmentation. Furthermore, the DNA fragmentation could be blocked by 2-(4-carboxy-phenyl)-4, 4, 5, 5-tetramethylimidazole-1-oxyl 3-oxide (carboxy-PTIO), a NO scavenger. These results suggest that cytokine-induced apoptotic cell death is attributed to NO and is protected by BH_4, and that osteoblastic cells in response to proinflammatory cytokines operate both a stimulatory process resulting in NO production and an inhibitory one resulting in BH_4 production for apoptotic cell death. Cytokine-induced apoptotic cell death may be a consequence of the predominance of the stimlatory process over the inhibitory process. Less