Taylor B S, Kim Y M, Wang Q, Shapiro R A, Billiar T R, Geller D A
Department of Surgery, University of Pittsburgh, Pa., USA.
Arch Surg. 1997 Nov;132(11):1177-83. doi: 10.1001/archsurg.1997.01430350027005.
The expression of inducible nitric oxide synthase (iNOS) contributes to the systemic manifestations of sepsis.
To determine whether nitric oxide (NO) can exert negative feedback regulation on iNOS gene expression.
Molecular biology research laboratory of the department of surgery.
Isolated rat hepatocytes were cultured with a cytokine mix consisting of tumor necrosis factor alpha, interleukin 1 beta, and interferon gamma in the presence or absence of the NO donor S-nitroso-N-acetyl-D,L-penicillamine.
Nitrite and nitrate (NO2- and NO3-) levels were assayed. Hepatocyte iNOS messenger RNA and protein levels were assessed. Electromobility shift assays were performed for NF-kappa B DNA binding activity. Finally, iNOS enzyme activity was determined using high-performance liquid chromatography.
Cytokine mix-induced hepatocyte iNOS mRNA and protein production and the addition of the NO donor S-nitroso-N-acetyl-D,L-penicillamine markedly attenuated iNOS mRNA and protein levels. Gel shift assays of the nuclear extracts disclosed that decreased cytokine mix-induced DNA binding activity for NF-kappa B in a concentration-dependent manner. Finally, NO failed to significantly inhibit iNOS enzyme activity.
These data indicate that NO down-regulates iNOS gene transcription, and that the effect is mediated in part by inhibiting NF-kappa B activity. These results identify a novel negative feedback mechanism whereby NO down-regulates iNOS gene expression, possibly to limit overproduction during the septic response.
诱导型一氧化氮合酶（iNOS）的表达参与脓毒症的全身表现。
确定一氧化氮（NO）是否能对iNOS基因表达发挥负反馈调节作用。
外科分子生物学研究实验室。
在有或无NO供体S-亚硝基-N-乙酰-D,L-青霉胺存在的情况下，将分离的大鼠肝细胞与由肿瘤坏死因子α、白细胞介素1β和干扰素γ组成的细胞因子混合物一起培养。
检测亚硝酸盐和硝酸盐（NO2-和NO3-）水平。评估肝细胞iNOS信使核糖核酸和蛋白质水平。进行电泳迁移率变动分析以检测核因子κB（NF-κB）的DNA结合活性。最后，使用高效液相色谱法测定iNOS酶活性。
细胞因子混合物诱导肝细胞iNOS信使核糖核酸和蛋白质生成，而添加NO供体S-亚硝基-N-乙酰-D,L-青霉胺可显著减弱iNOS信使核糖核酸和蛋白质水平。对核提取物进行的凝胶迁移分析显示，细胞因子混合物诱导的NF-κB的DNA结合活性以浓度依赖方式降低。最后，NO未能显著抑制iNOS酶活性。
这些数据表明，NO下调iNOS基因转录，且该效应部分是通过抑制NF-κB活性介导的。这些结果确定了一种新的负反馈机制，即NO下调iNOS基因表达，这可能是为了限制脓毒症反应期间的过度生成。
