Mechanisms of Cell Volume Regulation in Liver

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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.