Bereta M, Bereta J, Georgoff I, Coffman F D, Cohen S, Cohen M C
Department of Pathology, Hahnemann University, Philadelphia, Pennsylvania 19102.
Exp Cell Res. 1994 Jun;212(2):230-42. doi: 10.1006/excr.1994.1139.
In response to exposure to the inflammatory cytokines tumor necrosis factor-alpha (TNF) and interferon-gamma (IFN-gamma), murine brain microvascular endothelial cells (MME) synthesize the cell surface molecule, vascular cell adhesion molecule-1 (VCAM-1), and the intracellular enzyme, inducible nitric oxide synthase (iNOS). However, iNOS synthesis requires the presence of both TNF and IFN-gamma, while VCAM-1 can be induced by either cytokine alone. We examined the induction of VCAM-1 and iNOS under a variety of conditions to better define the regulation of TNF and IFN-gamma signal transduction pathways in MME. We utilized the analysis of steady-state levels of iNOS mRNA as well as the measurement of MME-released NO-EDRF (nitric oxide as an endothelium-derived relaxing factor) activity and accumulation of nitrite in the culture medium to define iNOS expression and activity. VCAM-1 expression was determined by flow cytometric analysis. Our data indicate that low density lipoproteins inhibited cytokine-induced iNOS activity by affecting the steady-state levels of iNOS mRNA. Methylxanthines (caffeine and theophylline) as well as several calcium-mobilizing agents inhibited the expression/activity of both iNOS and VCAM-1 in MME. The effectiveness of these agents was dependent upon the degree of disruption in cell calcium homeostasis during cytokine treatment. Cells which had been pretreated with calcium-modulating drugs and then washed and allowed to return to normal calcium homeostasis showed little to no effect from these agents. In addition, our results suggest that NO produced by iNOS acts as a metabolic switch during inflammation by inhibiting oxidative phosphorylation and forcing vascular endothelial cells to temporarily utilize anaerobic energy metabolism.
为响应炎性细胞因子肿瘤坏死因子-α（TNF）和干扰素-γ（IFN-γ）的刺激，小鼠脑微血管内皮细胞（MME）合成细胞表面分子血管细胞黏附分子-1（VCAM-1）和细胞内酶诱导型一氧化氮合酶（iNOS）。然而，iNOS的合成需要TNF和IFN-γ同时存在，而VCAM-1可由单独一种细胞因子诱导产生。我们在多种条件下检测了VCAM-1和iNOS的诱导情况，以更好地界定MME中TNF和IFN-γ信号转导途径的调控机制。我们利用对iNOS mRNA稳态水平的分析以及对MME释放的NO-EDRF（作为内皮源性舒张因子的一氧化氮）活性的测定和培养基中亚硝酸盐的积累情况来界定iNOS的表达和活性。通过流式细胞术分析确定VCAM-1的表达。我们的数据表明，低密度脂蛋白通过影响iNOS mRNA的稳态水平来抑制细胞因子诱导的iNOS活性。甲基黄嘌呤（咖啡因和茶碱）以及几种钙动员剂抑制MME中iNOS和VCAM-1的表达/活性。这些药物的有效性取决于细胞因子处理期间细胞钙稳态的破坏程度。预先用钙调节药物处理然后洗涤并使其恢复正常钙稳态的细胞对这些药物几乎没有影响。此外，我们的结果表明，iNOS产生的NO在炎症过程中通过抑制氧化磷酸化并迫使血管内皮细胞暂时利用无氧能量代谢而起到代谢开关的作用。
