Pharmacological study on NO and biopterin synthesis in osteoblastic cells

成骨细胞合成NO和生物蝶呤的药理学研究

基本信息

批准号：
09671912
负责人：
TOGARI Akifumi
金额：
$ 1.98万
依托单位：
Aichi-Gakuin University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09671912/
关键词：
Osteoblast Nitric Oxide Biopterin Apoptosis Cytokine mRNA NF-χB RT-PCR

项目摘要

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

促炎细胞因子（IL-1β、TNF-α 和 IFN-γ 的组合）引起 GTP 环水解酶 I (GTP-CH)（四氢生物蝶呤 (BH_4) 生物合成中的限速酶）的 mRNA 表达，以及诱导型细胞因子的表达。我们在特征明确的成骨细胞克隆 MC3T3-E1 细胞系中发现了一氧化氮合酶 (iNOS) 的表达。首次在成骨细胞中发现 GTP-CH 和 iNOS mRNA 的表达分别在 3 小时和 9 小时达到最大。使用药理学研究，这两种基因的表达均引起 BH_4 和 NO 水平的增加。 2、4-二氨基-6-羟基嘧啶(DAHP)，一种GTP-CH活性抑制剂，表明BH_4参与iNOS的活性，但不参与iNOS的活性。 iNOS mRNA 的诱导。使用核因子 (NF)-kappaB 抑制剂和激活蛋白 1 (AP-1) 激活的结果表明，这两个基因响应细胞因子的共同诱导是通过 NF-kappaB 和 AP- 的激活发生的。 1.细胞因子以及 NO 生成剂 S-亚硝基-N-乙酰基-D、L-青霉胺 (SNAP) 会降低细胞活力；但墨蝶呤会被转化；通过流式细胞术分析评估乳酸脱氢酶（LDH）释放和细胞凋亡来测量细胞毒性，结果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，但并非如此。在存在墨蝶呤的情况下，细胞因子处理导致DNA断裂量明显减少。此外，DNA断裂可以被阻断。 2-(4-羧基-苯基)-4, 4, 5, 5-四甲基咪唑-1-氧基 3-氧化物 (羧基-PTIO)，一种 NO 清除剂这些结果表明细胞因子诱导的细胞凋亡归因于 NO。并受到 BH_4 的保护，并且成骨细胞对促炎细胞因子的反应既会产生 NO 产生的刺激过程，也会产生 BH_4 的抑制过程细胞因子诱导的细胞凋亡可能是刺激过程优于抑制过程的结果。