Geller D A, Nussler A K, Di Silvio M, Lowenstein C J, Shapiro R A, Wang S C, Simmons R L, Billiar T R
Department of Surgery, University of Pittsburgh, PA 15261.
Proc Natl Acad Sci U S A. 1993 Jan 15;90(2):522-6. doi: 10.1073/pnas.90.2.522.
Nitric oxide (NO.) is a short-lived mediator which can be induced in a variety of cell types and produces many physiologic and metabolic changes in target cells. The inducible or high-output NO. synthase (NOS) pathway was first characterized in macrophages activated by lipopolysaccharide (LPS) and interferon gamma (IFN-gamma). Hepatocytes also express an inducible NOS following exposure to the combination of endotoxin (LPS) and tumor necrosis factor (TNF), interleukin 1 (IL-1), and IFN-gamma. In this study, to identify which of these cytokines, if any, was acting to induce the gene expression for hepatocyte NOS, we measured the levels of rat hepatocyte NOS mRNA by Northern blot analysis after stimulation by various combinations of endotoxin and cytokines in vitro. We found the mRNA for hepatocyte NOS to be a single band at approximately 4.5 kilobases which was maximally up-regulated (approximately 70-fold) by the combination of TNF, IL-1, IFN-gamma, and LPS. Abundance of NOS mRNA peaked 6-8 hr after stimulation and then declined by 25% at 24 hr. Unstimulated hepatocytes in vitro showed only a trace mRNA band after prolonged autoradiographic exposure. As single agents, TNF and IL-1 were the most effective inducers of hepatocyte NOS mRNA. Combinations of two or three stimuli revealed strong synergy between TNF, IL-1, and IFN-gamma. The increased mRNA levels correlated with elevated nitrogen oxide release and cGMP levels in the culture supernatants. Dexamethasone and cycloheximide inhibited induction of mRNA for hepatocyte NOS in a dose-dependent fashion. The addition of NG-monomethyl-L-arginine had no effect on mRNA levels but effectively blocked NO. formation. The inducible hepatocyte NOS mRNA was also detected in rat hepatocytes following chronic hepatic inflammation triggered by Corynebacterium parvum injection in vivo. These data demonstrate that the inducible NOS is functional in rat hepatocytes both in vitro and in vivo and that this pathway is under complex control. Endotoxin and inflammatory cytokines act synergistically to up-regulate gene expression for hepatocyte NOS, whereas glucocorticoids down-regulate the mRNA.
一氧化氮（NO.）是一种半衰期较短的介质，可在多种细胞类型中诱导产生，并在靶细胞中引起许多生理和代谢变化。诱导型或高产量一氧化氮合酶（NOS）途径最初是在脂多糖（LPS）和干扰素γ（IFN-γ）激活的巨噬细胞中得以表征。肝细胞在暴露于内毒素（LPS）、肿瘤坏死因子（TNF）、白细胞介素1（IL-1）和干扰素γ的组合后也会表达诱导型NOS。在本研究中，为了确定这些细胞因子中是否有任何一种在诱导肝细胞NOS的基因表达，我们通过体外对内毒素和细胞因子的各种组合进行刺激后，采用Northern印迹分析来测量大鼠肝细胞NOS mRNA的水平。我们发现肝细胞NOS的mRNA是一条约4.5千碱基的单带，在TNF、IL-1、IFN-γ和LPS的组合作用下被最大程度地上调（约70倍）。NOS mRNA的丰度在刺激后6 - 8小时达到峰值，然后在24小时下降25%。体外未受刺激的肝细胞在长时间放射自显影曝光后仅显示出一条痕量mRNA带。作为单一因子，TNF和IL-1是肝细胞NOS mRNA最有效的诱导剂。两种或三种刺激的组合显示出TNF、IL-1和IFN-γ之间有很强的协同作用。mRNA水平的升高与培养上清液中氮氧化物释放和cGMP水平的升高相关。地塞米松和环己酰亚胺以剂量依赖的方式抑制肝细胞NOS mRNA的诱导。添加NG-单甲基-L-精氨酸对mRNA水平没有影响，但有效地阻断了NO.的形成。在体内注射微小棒状杆菌引发慢性肝脏炎症后，在大鼠肝细胞中也检测到了诱导型肝细胞NOS mRNA。这些数据表明，诱导型NOS在大鼠肝细胞的体外和体内均有功能，并且该途径受到复杂的调控。内毒素和炎症细胞因子协同作用以上调肝细胞NOS的基因表达，而糖皮质激素则下调mRNA。
