MECHANISMS OF LIVER CELL INJURY

我们的总体目标是定义关键的细胞机制 最终导致致命的肝细胞损伤。我们将测试小说《将军》 非溶酶体蛋白降解是一种基本机制的假说 介导缺氧、氧化应激和胆汁中的致死细胞损伤 盐的细胞毒性。我们将采用现代和互补的生化 和细胞生物学技术，以确定调控和 致死性肝细胞损伤过程中蛋白质分解的机制。这个 提案有两个具体目标。首先，我们将确定细胞 致死过程中蛋白水解酶活性的定位、调控和鉴定 直接测试对肝细胞的损伤：a)蛋白分解的假说 在所有类型的肝细胞中发生在细胞损伤期间，并发生 主要由非溶酶体蛋白水解酶，b)假设 细胞损伤过程中的蛋白分解受细胞内、胞浆三磷酸腺苷的调节 PH、囊泡pH和/或胞浆游离钙；以及c)假设 可识别的特定类别的蛋白水解酶负责 在细胞损伤过程中发生的蛋白质分解。第二，我们将确定 蛋白分解在致死性肝细胞损伤中的机制作用 直接检验：a)蛋白质分解活性先于 细胞死亡的开始；以及b)假设抑制 蛋白分解活性可延缓致死性细胞损伤的发生。这个 研究将使用同种培养和分离的大鼠悬液 肝细胞、肝窦内皮细胞和肝内胆汁 导管上皮细胞。在分散的细胞悬浮液中，完全的，溶酶体 和非溶酶体蛋白分解、细胞杀伤率、ATP耗竭和 在碱性条件下，将测量特定的蛋白水解酶 在条件和细胞损伤期间。利用单个培养的细胞， 荧光探头和多参数数字化视频显微镜， 胞浆pH、囊泡pH、胞浆游离钙和蛋白水解酶 特定的蛋白质分解将使用商业上可用的和 新型合成荧光探针。我们的长期目标是提供 关于蛋白质分解作为一种机制的新的基本信息 临床相关细胞模型中肝细胞的致死性损伤 受伤。干预策略必须以理解为前提 导致细胞死亡的细胞机制。成功 完成这项提议可能会确定一种负责 致命性肝细胞损伤。这一结果可能导致对 治疗方法对肝组织的临床表现有效 缺氧、氧化应激和胆汁淤积伴有毒胆汁滞留 盐。

Our OVERALL OBJECTIVE is to define the critical cellular mechanisms culminating in lethal liver cell injury. We will test the novel GENERAL HYPOTHESIS that NON-LYSOSOMAL PROTEOLYSIS IS AN ESSENTIAL MECHANISM MEDIATING LETHAL CELL INJURY DURING ANOXIA, OXIDATIVE STRESS AND BILE SALT CYTOTOXICITY. We will employ current and complementary biochemical and cell biological techniques to ascertain the regulatory and mechanistic aspects of proteolysis during lethal liver cell injury. The proposal has two SPECIFIC AIMS. FIRST, we will determine the cellular localization, regulation and identity of protease activity during lethal liver cell injury by directly testing: a) the HYPOTHESIS that proteolysis occurs during cell injury in all liver cell types, and occurs predominantly by nonlysosomal proteases, b) the HYPOTHESIS that proteolysis during cell injury is regulated by cellular ATP, cytosolic pH, vesicular pH and/or cytosolic free calcium; and c) the HYPOTHESIS that specific identifiable classes of proteases are responsible for the proteolysis occurring during cell injury. SECOND, we will determine the mechanistic role of proteolysis as a cause of lethal liver cell injury by directly testing: a) the HYPOTHESIS that proteolytic activity precedes the onset of cell death; and b) the HYPOTHESIS that inhibition of proteolytic activity delays the onset of lethal cell injury. The studies will employ homogeneous cultures and suspensions of isolated rat liver hepatocytes, sinusoidal endothelial cells and intrahepatic bile duct epithelial cells. In dispersed cell suspensions, total, lysosomal and non-lysosomal proteolysis, rates of cell killing, ATP depletion, and protease class specific proteolysis will be measured under basal conditions and during cell injury. Employing single cultured cells, fluorescent probes and multiparameter digitized video microscopy, cytosolic pH, vesicular pH, cytosolic free calcium, and protease class specific proteolysis will be quantitated using commercially available and novel synthetic fluorescent probes. Our LONG-TERM GOALS are to provide new fundamental information regarding proteolysis as a mechanism of lethal injury of liver cells during clinically relevant models of cell injury. Interventional strategies must be predicated on an understanding of the cellular mechanisms which lead to cell death. Successful completion of this proposal could identify a protease responsible for lethal liver cell injury. The results may lead to the identification of treatment modalities effective in the presentation of liver tissue during anoxia, oxidative stress, and cholestasis with retention of toxic bile salts.