Mechanisms of Cell Volume Regulation in Liver

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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. Disordered 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 hepatocellular 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. This maintains 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. It will also be tested whether the expression of Src or its effectors is altered in human liver diseases manifested by hepatocyte ballooning. Studies in Aim 2 will test whether inhibition of Src effectors modifies swelling-induced actin dynamics, and whether inhibition of these dynamics attenuates volume recovery. Studies in Aim 3 will determine 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 are expected to provide an integrated picture of mechanisms by which cell volume is regulated in the liver. Since cell volume control is essential to the maintenance of organ level function, an understanding of these mechanisms will provide new ways to prevent organ dysfunction in the setting of liver disease.