MECHANISMS OF LIVER CELL INJURY

我们的总体目标是确定关键的细胞机制， 最终导致致命的肝细胞损伤。 我们将测试小说将军 非溶酶体蛋白水解是一种基本机制的假设 在缺氧、氧化应激和胆汁中介导致死细胞损伤 盐细胞毒性。 我们将使用现有的和互补的生化技术 和细胞生物学技术，以确定调节和 致死性肝细胞损伤过程中蛋白水解的机制方面。 的 提案有两个具体目标。首先，我们将确定细胞 致死过程中蛋白酶活性的定位、调节和鉴定 直接测试肝细胞损伤：a）蛋白质水解的假设 在所有肝细胞类型的细胞损伤期间发生， 主要通过非溶酶体蛋白酶，B）假设， 细胞损伤过程中的蛋白水解受细胞ATP、胞浆ATP和胞浆ATP的调节。 pH、囊泡pH和/或胞质游离钙;和c）假设 特定的可识别的蛋白酶种类负责 细胞损伤过程中发生的蛋白水解。 第二，我们将确定 蛋白水解作为致死性肝细胞损伤原因的机制作用 直接测试：a）假设蛋白水解活性先于 细胞死亡的开始;和B）抑制 蛋白水解活性延迟致死细胞损伤的发生。 的 研究将使用分离的大鼠的均质培养物和悬浮液 肝细胞、肝窦内皮细胞和肝内胆汁 导管上皮细胞 在分散的细胞悬液中，总溶酶体 和非溶酶体蛋白水解，细胞杀伤率，ATP消耗， 蛋白酶类特异性蛋白水解将在基础条件下测量。 条件和细胞损伤期间。 使用单个培养细胞， 荧光探针和多参数数字化视频显微镜， 胞浆pH、囊泡pH、胞浆游离钙和蛋白酶类别 特异性蛋白水解将使用可商购的和 新型合成荧光探针。 我们的长期目标是提供 关于蛋白质水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 损伤 干预战略必须建立在理解 导致细胞死亡的细胞机制 成功 完成这项建议可以确定一种蛋白酶负责 致命的肝细胞损伤 结果可能导致识别 治疗方式有效的肝组织的介绍， 缺氧、氧化应激和胆汁淤积伴毒性胆汁潴留 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.