The proposed studies address the mechanisms responsible for regulated transport by cholangiocytes, the epithelial cells that line the lumen of intrahepatic bile ducts and contribute importantly to the volume and composition of bile. Liver diseases associated with impaired bile flow (cholestasis) account for significant morbidity and mortality in both children and adults. While diverse in etiology, all cholestatic disorders converg on a final common pathway characterized by a decrease in bile flow. Bile flow produces shear forces at the apical surface, and based on evidence that cholangiocytes translate this mechanical stimulus into regulated membrane transport, the studies described in this proposal evaluate the role of newly identified mechanosensitive pathways in bile formation. The studies are based on observations by the applicant that increased fluid-flow, or shear, at the apical membrane of cholangiocytes is a potent stimulus for i) ATP release, ii) increases in [Ca ]i, and iii) regulated Cl and Na transport. Collectively these observations support the following working hypothesis: Mechanosensitive signaling serves as a powerful mechanism for autocrine/ paracrine regulation of cholangiocyte transport and bile formation.
The Specific Aims are: 1) To evaluate the role of TRPV4 in the initiation of flow-induced mechanosensitive Ca2+ signaling;and to define the specific roles of shear force, kinase signaling, and the actin cytoskeleton on channel regulation;2) To determine the cellular mechanisms responsible for mechanosensitive ATP release and to define the functional relationships between SNARE proteins, TRPV4, and Ca2+-signaling on ATP vesicle fusion and release;and 3) To evaluate the cellular signals responsible for the coordination and integration of flow-induced mechanosensitive Cl- and Na + transport at the apical membrane with emphasis on the purinergic receptor- and kinase-linked signaling pathways. An integrated approach combining electrophysiology, molecular biology, and fluorescence imaging will be applied to the study of single ion channels, intact cholangiocytes, and novel models of biliary epithelium that retain secretory polarity. The long-term goal of these studies is to define the cellular mechanisms involved in cholangiocyte secretion, and to identify the physiologic factors that contribute to bile formation. Despite substantial effort, today there are no effective and available therapies to modulate the volume and composition of bile. Accordingly, efforts to define the molecular targets involved are a high priority given the prevalence and impact (both economic and personal) of related disorders of bile flow. Thus, these studies are directly relevant to the agency's mission to improve the overall public health and decrease the burden of liver and biliary diseases in the United States.
Chronic liver disease is currently the 12th leading cause of death, accounting for 27,000 deaths and approximately 1.6 billion in economic costs per year in the U.S., cholestatic liver diseases associated with poor bile flow comprise a significant proportion of these disorders. In fact, they comprise the majority of liver diseases in children an are the leading indication for childhood liver transplantation. Consequently, defining the cellular mechanisms responsible for biliary fluid and electrolyte transport will serve as a basis for the development of therapeutic interventions to modulate bile formation for the treatment of cholestatic liver diseases.
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