NITRIC OXIDE AND HEPATIC FUNCTION IN SEPSIS AND TRAUMA

脓毒症和创伤中的一氧化氮与肝功能

• 批准号: 6179662
• 负责人: TIMOTHY R BILLIAR
• 金额: $ 23.14万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6179662
• 关键词: biological signal transduction; blood toxicology; cGMP dependent protein kinase; cell death; cysteine endopeptidases; enzyme activity; enzyme inhibitors; gel mobility shift assay; gene expression; glutathione; iron; laboratory mouse; laboratory rat; liver cells; liver function; liver infection; nitric oxide; nitric oxide synthase; nitroso compounds; northern blottings; polymerase chain reaction; tissue /cell culture; trauma; tumor necrosis factor alpha; western blottings

The inducible nitric oxide synthase (iNOS) is upregulated diffusely during sepsis and clearly contributes to hemodynamic instability and organ injury during septic shock. However, in stark contrast to the damaging effects of iNOS in many tissues, in the liver upregulation of iNOS has a protective function. Sustained, high-level iNOS expression has no toxic effects on hepatocytes, and even low-level expression is protective. We now hypothesize that upregulation of iNOS in the liver during acute inflammatory states (i.e., sepsis) is part of a protective response that limits the toxicity of pro-inflammatory mediators, including TNFalpha. We have shown that NO can directly inhibit TNFalpha-induced signaling pathways leading to hepatocyte death. By activating soluble guanylate cyclase, NO inhibits the activation of the caspase cascade by TNFalpha, and via S-nitrosylation NO directly inhibits caspase protease activity. We have also shown that NO can regulate gene expression in hepatocytes to promote cell survival. We will now pursue the mechanistic basis of these observations in three aims. AIM I: To determine how NO/cGMP/G-kinase inhibits TNFalpha signaling in hepatocytes. Experiments under Aim I will define the level at which cGMP and the cGMP-dependent kinase inhibit TNFalpha signaling in hepatocytes. As part of this objective, we will identify the substrates for G-kinase that mediate the protective actions. AIM II: To determine the pathways leading to efficient S-nitrosylation of caspase in hepatocytes. Under Aim II, we will identify the factors that lead to efficient S-nitrosylation of caspases in hepatocytes. Factors that are likely to be important and that will be tested include the levels of glutathione and intracellular iron as well as the ratio of NO to O2-. AIM III: To identify iNOS-induced protective genes in hepatocytes. Under Aim III, we will complete our analysis of NO-regulated genes in hepatocytes using differential display. We have already identified 11 candidate genes, and we will characterize genes that contribute to the hepatoprotective actions of iNOS. Upon completion of the three aims, it is expected that we will have an understanding of the factors that render hepatocytes less susceptible to injury and dysfunction during sepsis. By defining the protective mechanisms in hepatocytes, we will gain insights into the molecular mechanisms that lead to cellular toxicity in sepsis in susceptible organs.

在败血症期间，诱导型一氧化氮合酶（INOS）扩散地上调，并且在败血性休克期间显然有助于血液动力学不稳定性和器官损伤。 但是，与iNOS在许多组织中的破坏作用形成鲜明对比，在iNOS的肝上调中具有保护功能。持续的高级iNOS表达对肝细胞没有毒性作用，甚至低水平的表达也是保护性的。 现在，我们假设在急性炎症状态下（即败血症）在肝脏中的iNOS上调是保护性反应的一部分，该反应限制了包括TNFALPHA在内的促炎性介质的毒性。 我们已经表明，不能直接抑制TNFalpha诱导的信号传导途径，导致肝细胞死亡。通过激活可溶性鸟苷酸环化酶，NO抑制tnfalpha抑制caspase cascad的激活，并通过s-硝基化抑制NO直接抑制caspase蛋白酶活性。我们还表明，不能调节肝细胞中的基因表达以促进细胞存活。 现在，我们将以三个目标来追求这些观察的机械基础。 目的I：确定/cGMP/g-激酶如何抑制肝细胞中的tnfalpha信号传导。 AIM I下的实验将定义CGMP和CGMP依赖性激酶在肝细胞中抑制TNFALPHA信号传导的水平。 作为此目标的一部分，我们将确定介导保护行为的G-激酶的底物。 AIM II：确定导致肝细胞中caspase有效的S-硝基化的途径。 在AIM II下，我们将确定导致肝细胞中胱天蛋白酶有效的S-硝基化的因素。 可能很重要并将测试的因素包括谷胱甘肽和细胞内铁的水平以及NO与O2-的比率。 AIM III：鉴定肝细胞中iNOS诱导的保护基因。 在AIM III下，我们将使用差分显示来完成对肝细胞中无调基因的分析。 我们已经确定了11个候选基因，我们将表征有助于iNOS的肝保护作用的基因。 三个目标完成后，预计我们将了解败血症期间肝细胞不易受伤和功能障碍的因素。 通过定义肝细胞中的保护机制，我们将了解导致易感器官败血症细胞毒性的分子机制。