GROWTH FACTOR CA++ SIGNALING IN ALCOHOLIC LIVER DISEASE

酒精性肝病中的生长因子 CA 信号传导

• 批准号: 2291729
• 负责人: JOHN R. WILLIAMSON
• 金额: $ 2.15万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2291729
• 关键词: alcoholic liver cirrhosis; biological signal transduction; biopsy; calcium; calcium indicator; growth factor; hormone regulation /control mechanism; human subject; liver cells; transforming growth factors

A research program is described which will be directed towards the characterization of growth factor-medicated Ca2+ responses in human parenchymal liver cells using liver biopsy material. This study relates to an extension of the parent grant concerning epidermal growth factor (EGF) and hepatocyte growth factor (HGF) actions on the liver. The physician experience of the Hungarian collaborator and his experience with measurement of growth factor effects on Ca2+ homeostasis in single cells will be combined to apply advanced techniques to human liver cells. First, attention will be focussed on establishing a reliable technique for measurement of intracellular Ca2+ in fura-2-loaded single human hepatocytes to characterize "normal" Ca2+ homeostasis of these cells with an emphasis on the effects of the recently described HGF. The involvement of a receptor tyrosine kinase in HGF-induced Ca2+ mobilization will be evaluated by tyrosine kinase inhibitors. The amount of releasable Ca2+ from non-mitochondrial and mitochondrial pools of human hepatocytes will be assessed by microperfusion of the specific Ca2+-mobilizing agents such as thapsigargin and mitochondrial uncouplers. Furthermore, purified antibodies raised against different components of the signaling pathway (G-proteins, phospholipase C isozymes) will be microinjected into human hepatocytes. This powerful approach has the potential for giving very specific answers to questions related to hormone-induced signal transduction in human liver. Second, growth factor-induced signal transduction and Ca2+ homeostasis will be investigated throughout the pathological evolution of alcoholic liver disease (ALD). We hypothesize that cellular changes in the hepatocytes are accompanied by specific alterations in cellular Ca2+ homeostasis corresponding in severity to the stage of ALD. A higher basal Ca2+ and a reduced cellular responsiveness to the effect of Ca2+ mobilizing hormones and growth factors is expected in relation to a decrease in the capacity of hormone-sensitive pools to sequester Ca2+. Assessment of the size of non-mitochondrial and mitochondrial Ca2+ pools will allow an evaluation of abnormal redistribution in the intracellular Ca2+. The inhibitory properties of transforming growth factor beta (TGF- beta) as well as the effect of protein kinase C-stimulating phorbol esters on HGF-induced Ca2+ response will be also determined. Knowledge of Ca2+ homeostasis and HGF-related early biochemical events of human parenchymal liver cells obtained throughout this project is of direct relevance to the development of new strategies for the prevention, prognostication, and treatment of alcoholic liver disease.

描述了一项研究计划，该计划将针对 人类生长因子介导的 Ca2+ 反应的表征 使用肝活检材料观察实质肝细胞。 这项研究涉及 延长有关表皮生长因子的家长补助金 (EGF) 和肝细胞生长因子 (HGF) 对肝脏的作用。 这 匈牙利合作者的医生经历和他的经历 测量生长因子对单个细胞中 Ca2+ 稳态的影响 细胞将被组合起来，将先进技术应用于人类肝细胞。 首先，注意力将集中在建立可靠的技术上 用于测量负载 fura-2 的单个人的细胞内 Ca2+ 肝细胞来表征这些细胞的“正常”Ca2+稳态 强调最近描述的 HGF 的影响。 这 受体酪氨酸激酶参与 HGF 诱导的 Ca2+ 动员将通过酪氨酸激酶抑制剂进行评估。 量 从非线粒体和线粒体池中释放的 Ca2+ 人类肝细胞将通过特定的微灌注进行评估 Ca2+ 动员剂，例如毒胡萝卜素和线粒体解偶联剂。 此外，针对不同成分的纯化抗体 信号通路（G 蛋白、磷脂酶 C 同工酶）将是 显微注射到人肝细胞中。 这种强大的方法具有 能够对相关问题给出非常具体的答案 人类肝脏中激素诱导的信号转导。 二、生长因子诱导的信号转导和Ca2+稳态 将在酒精的整个病理演变过程中进行研究 肝病（ALD）。 我们假设细胞发生变化 肝细胞伴随着细胞 Ca2+ 的特异性改变 体内平衡的严重程度与 ALD 阶段相对应。 更高的 基础 Ca2+ 和细胞对 Ca2+ 作用的反应性降低 预计与调动激素和生长因子有关 激素敏感库吸收 Ca2+ 的能力下降。 非线粒体和线粒体 Ca2+ 库大小的评估 将允许评估细胞内的异常重新分布 钙2+。 转化生长因子β（TGF-β）的抑制特性 beta) 以及蛋白激酶 C 刺激佛波醇的作用 酯类对 HGF 诱导的 Ca2+ 反应的影响也将被确定。 了解 Ca2+ 稳态和 HGF 相关的早期生化事件 在该项目中获得的人类实质肝细胞的数量为 与制定新的预防战略直接相关， 酒精性肝病的预测和治疗。