GROWTH FACTOR CA++ SIGNALING IN ALCOHOLIC LIVER DISEASE

描述了一个研究计划，该计划将针对 生长因子介导的人体内Ca ~（2+）反应的表征 使用肝活检材料的实质肝细胞。 这项研究涉及 关于表皮生长因子的母基金的延期申请 (EGF)和肝细胞生长因子（HGF）对肝脏的作用。 的 匈牙利合作者的医生经验和他的经验 测量生长因子对单个细胞中Ca 2+稳态的影响， 细胞将被结合起来，将先进的技术应用于人类肝细胞。 首先，将集中注意力建立可靠的技术 用于测量负载Fura-2的单个人中的细胞内Ca 2 + 肝细胞表征这些细胞的“正常”Ca 2+稳态， 强调最近描述的HGF的作用。 的 受体酪氨酸激酶参与HGF诱导Ca ~（2+） 将通过酪氨酸激酶抑制剂评价动员。 的量 的非线粒体和线粒体池中可释放的Ca 2 + 将通过微灌注特定的 Ca 2+动员剂，如毒胡萝卜素和线粒体解偶联剂。 此外，纯化的抗体针对不同的成分， 信号通路（G蛋白，磷脂酶C同工酶）将被 显微注射到人肝细胞中。 这种强大的方法具有 有可能对与下列问题有关的问题作出非常具体的回答： 人肝脏中的腺苷诱导的信号转导。 第二，生长因子诱导的信号转导和Ca 2+稳态 将在酒精中毒的整个病理演变过程中进行研究。 肝病（ALD）。 我们假设，细胞的变化， 肝细胞伴有细胞内Ca 2+的特异性改变 在严重程度上对应于ALD阶段的体内平衡。 更高 基础Ca 2+和细胞对Ca 2+作用的反应性降低 动员激素和生长因子预计与一个 降低对钙离子敏感的池螯合Ca 2+的能力。 非线粒体和线粒体Ca 2+池大小的评估 将允许评估细胞内的异常再分布， Ca2+。 转化生长因子β（TGF-β）的抑制特性 以及蛋白激酶C刺激佛波醇的作用 还将测定酯对HGF诱导的Ca 2+反应的影响。 Ca 2+稳态和HGF相关早期生化事件的知识 在整个项目中获得的人实质肝细胞的 与制定新的预防战略直接相关， 酒精性肝病的诊断和治疗

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.