Mechanisms of Cell Volume Regulation in Liver

描述(由申请人提供)：调节体积的能力对细胞的生存至关重要。这在肝脏中尤其如此，由于其在营养加工、代谢和胆汁形成中的作用，它受到渗透负荷的动态变化。失去容量控制会导致不可逆的细胞肿胀，这是急性和慢性肝损伤的一个标志。鉴于这个问题的重要性，这个项目的目标是了解在渗透胁迫期间肝细胞体积是如何维持的，进而器官功能是如何维持的。众所周知，这种应激导致细胞结构发生显著变化，而质膜离子通道在肝细胞肿胀后的体积恢复中起着关键作用。该实验室最近的工作表明，酪氨酸激酶Src在协调这些活动中扮演了一个新的角色。这一提议将检验这一假设，即Src作为一个分子开关，将来自体积敏感传感器的信号传递到下游效应器，后者重组细胞骨架，并通过钾和氯通道激活液体和电解质运动，以将细胞体积维持在生理状态。其具体目的是：(1)建立体积敏感的Src激活的动力学和细胞定位；(2)确定Src在体积敏感的细胞骨架重组中的作用；(3)阐明Src如何调节体积敏感的离子通道的激活。目标1中的研究将确定Src如何影响整合素、粘着斑激酶(FAK)以及Src效应器Vav和磷脂酶C(PLC)伽马的体积敏感细胞定位和调节功能。目标1的研究还将测试在缺血再灌注损伤的动物模型中，Src的表达是否影响结果，缺血再灌注损伤是一种以病理性肝细胞肿胀为表现的紊乱。Aim 2的研究将确定抑制Src效应器是否改变肿胀诱导的肌动蛋白动力学，以及抑制这些动力学是否减弱体积恢复。AIM 3的研究将测试在肿胀时，依赖于Src的细胞外信号受体(ERK)激酶是否受PLC Gamma和/或Vav的调节，以及ERK效应因子磷脂酶A2是否调节容量敏感的钾和氯通道。建议的研究将采用多种方法，包括活细胞成像、膜片钳记录、体内和体外腺病毒基因传递，以实现这些目标。总之，这些研究将提供在生理条件下对肝细胞体积调节起关键作用的信号通路的综合图景，并将为发现在肝损伤过程中发挥细胞保护作用的体积敏感信号蛋白提供基础。肝细胞在处理营养物质的过程中会膨胀，为了保持完好，它们会释放盐和水，以收缩回正常大小。该项目将研究肝细胞如何保持其大小和形状，这对于保护肝脏免受毒素或低血流造成的损伤至关重要。通过识别防止肝细胞失控肿胀的基本过程，这项研究将为开发有效的治疗肝损伤的药物提供重要信息。

DESCRIPTION (provided by applicant): The ability to regulate volume is critical for the survival of cells. This is particularly true in the liver, which is subjected to dynamic changes in osmotic load as a result of its role in nutrient processing, metabolism, and bile formation. Loss of volume control leads to irreversible cell swelling that is a hallmark of acute and chronic liver injury. Given the importance of this problem, the goals of this project are to understand how hepatocyte volume, and by extension organ function, are maintained during osmotic stress. It is known that such stress imposes dramatic changes in cell architecture, and that plasma membrane ion channels are crucial to volume restoration after hepatocellular swelling. Recent work from this laboratory has suggested a novel role for the tyrosine kinase Src in coordinating these actions. This proposal will test the hypothesis that Src serves as a molecular switch that relays signals from volume-sensitive sensors to downstream effectors that reorganize the cytoskeleton and activate fluid and electrolyte movement through potassium and chloride channels, to maintain cell volume within a physiological state. The Specific Aims are: (1) to establish the kinetics and cellular localization of volume-sensitive Src activation, (2) to determine the role of Src in volume-sensitive cytoskeletal reorganization, and (3) to elucidate how Src regulates the activation of volume-sensitive ion channels. Studies in Aim 1 will determine how Src influences the volume-sensitive cellular localization and regulatory function of integrins, focal adhesion kinase (FAK), and the Src effectors Vav and phospholipase C (PLC) gamma. Studies in Aim 1 will also test whether the expression of Src influences outcome in an animal model of ischemia-reperfusion injury, a disorder manifested by pathological hepatocyte swelling. Studies in Aim 2 will determine whether inhibition of Src effectors modifies swelling-induced actin dynamics, and whether inhibition of these dynamics attenuates volume recovery. Studies in Aim 3 will test whether Src- dependent extracellular signal receptor (ERK) kinases are regulated upon swelling by PLC gamma and/or Vav, and whether the ERK effector phospholipase A2 regulates volume-sensitive potassium and chloride channels. The proposed studies will employ a variety of approaches, including live cell imaging, patch clamp recording, and in vivo and in vitro adenovirus gene delivery, to accomplish these aims. Collectively, these studies will provide an integrated picture of signaling pathways that are key to liver cell volume regulation under physiological conditions, and they will provide a basis for discovery of volume sensitive signaling proteins that serve cytoprotective roles during liver injury. Liver cells swell during the processing of nutrients, and to remain intact, they release salt and water to shrink back to their normal size. This project will examine how liver cells maintain their size and shape, which is essential for protection against liver injury produced by toxins or low blood flow. By identifying basic processes that prevent uncontrolled liver cell swelling, this research will provide important information for the development of effective drugs for treatment of liver injury.