Glutathione regulates nitric oxide synthase in cultured hepatocytes
Glutathione regulates nitric oxide synthase in cultured hepatocytes
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DOI：
10.1097/00000658-199701000-00009
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发表时间：
1997-01-01
影响因子：
9
通讯作者：
Billiar, TR
中科院分区：
文献类型：
--
作者：
Harbrecht, BG;DiSilvio, M;Billiar, TR
目的 作者确定了培养肝细胞中谷胱甘肽和一氧化氮 (NO) 合成之间的关系。背景数据摘要 谷胱甘肽是许多酶的辅助因子，它的存在对于诱导型巨噬细胞一氧化氮合酶 (iNOS) 的最大酶活性至关重要，该酶可产生活性一氧化氮自由基。肝细胞含有大量的谷胱甘肽，这种重要的三肽在受到缺血/再灌注或内毒素血症应激的肝细胞中会减少。内毒素血症还诱导炎症细胞因子的合成，导致 iNOS 从肝细胞产生一氧化氮，这表明当细胞内谷胱甘肽减少时，肝细胞可能试图合成一氧化氮。 1,3-双(氯乙基)-1-亚硝基脲 (BCNU) 抑制谷胱甘肽。暴露于细胞因子后，通过上清液亚硝酸盐水平、胞浆 INOS 酶活性和 iNOS mRNA 水平评估 NO 合成。结果 用丁硫氨酸亚磺酰亚胺抑制谷胱甘肽合成或用 BCNU 抑制谷胱甘肽还原酶活性可抑制亚硝酸盐合成。通过 Northern 印迹分析检测，丁硫氨酸亚磺酰亚胺和 BCNU 均抑制 iNOS mRNA 的诱导。外源性谷胱甘肽增加了细胞因子刺激的 INOS 诱导，克服了 BCNU 的抑制作用，并增加了完整肝细胞、诱导肝细胞胞浆和部分纯化的肝细胞 iNOS 的亚硝酸盐产量。结论在培养的肝细胞中，最佳一氧化氮合成需要足够的谷胱甘肽水平。这一发现主要是由于对 INOS mRNA 水平的影响，尽管谷胱甘肽也参与 iNOS 酶活性的调节。
Objective The authors determine the relationship between glutathione and nitric oxide (NO) synthesis in cultured hepatocytes.Summary Background Data Glutathione is a cofactor for a number of enzymes, and its presence is essential for maximal enzyme activity by the inducible macrophage nitric oxide synthase (iNOS), which produces the reactive nitric oxide radical. Hepatocytes contain substantial quantities of glutathione, and this important tripeptide is decreased in hepatocytes stressed by ischemia/reperfusion or endotoxemia. Endotoxemia also induces the synthesis of inflammatory cytokines that result in the production of nitric oxide from hepatocytes by iNOS, suggesting that hepatocytes may be attempting to synthesize nitric oxide at times when intracellular glutathione is reduced.Methods Hepatocytes were cultured with buthionine sulfoximine and 1,3-bis(chloroethyl)-1-nitrosourea (BCNU) to inhibit glutathione. After exposure to cytokines, NO synthesis was assessed by supernatant nitrite levels, cytosolic INOS enzyme activity, and iNOS mRNA levels.Results Inhibition of glutathione synthesis with buthionine sulfoximine or inhibition of glutathione reductase activity with BCNU inhibited nitrite synthesis. Both buthionine sulfoximine and BCNU inhibited the induction of iNOS mRNA, as detected by Northern blot analysis. Exogenous glutathione increased cytokine-stimulated INOS induction, overcame the inhibitory effects of BCNU, and increased nitrite production by intact hepatocytes, induced hepatocyte cytosol, and partially purified hepatocyte iNOS.Conclusions In cultured hepatocytes, adequate glutathione levels are required for optimal nitric oxide synthesis. This finding is predominantly due to an effect on INOS mRNA levels, although glutathione also participates in the regulation of iNOS enzyme activity.