Liver disorders associated with impaired bile flow (cholestasis) are a leading cause of liver disease in children and adults. Lack of precise knowledge regarding the molecular basis of bile formation is an obstacle for the development of successful treatments. Based on evidence that intrahepatic bile duct epithelial cells (cholangiocytes) contribute importantly to the volume and composition of bile, the studies described in this proposal evaluate the role of the small molecule ATP as an extracellular autocrine/paracrine factor that modulates cholangiocyte secretion, and thereby the volume and composition of bile. The studies are based on observations by the applicant and others that indicate that i) ATP is released into bile by cholangiocytes, ii) purinergic (P2) receptors are expressed on the plasma membranes of these cells, and iii) exogenous ATP stimulates an increase in biliary secretion. Collectively these observations support the working hypothesis that regulated ATP release into bile serves as a powerful mechanism for regulating cholangiocyte secretory functions and bile formation.
The specific aims are: 1) to evaluate the role of exocytosis of ATP-enriched vesicles in regulated ATP release;2) to characterize the roles of integrin-, actin-, and kinase-linked mechanotransduction pathways in translating flow/shear stress and cell volume changes to ATP release;and 3) to determine the role of ATP-binding cassette (ABC) proteins Familial Intrahepatic Cholestasis-1 (FIC-1) and Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) on ATP release and assess whether mutations in these proteins, which result in cholestatic liver disease, are associated with altered nucleotide signaling. An integrated approach combining electrophysiology, molecular biology, and fluorescence imaging will be applied to study of single ion channels, intact cholangiocytes, and novel models of biliary epithelium that retain secretory polarity. Additionally, a novel method combining chemiluminescence with total internal reflection fluorescence (TIRF) microscopy for the visualization of ATP-vesicle trafficking and exocytosis in living cells is described. The long-term goal of these studies is to define the cellular mechanisms involved in cholangiocyte secretion in order to develop new and effective strategies for the treatment of cholestatic liver disorders. 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. (1;2). 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 and are the leading indication for childhood liver transplantation (3). Unfortunately, few therapeutic options exist and progression to end stage liver disease is either fatal or requires liver transplantation in the majority of these disorders. 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. This proposal is in response to the Action Plan for Liver Disease Research report by the Liver Disease Subcommittee of the Digestive Diseases Interagency Coordinating Committee of the National Institutes of Health, to ?understand the process, pathway, and molecules that underlie normal liver functioning, which can then be applied to understanding cellular and molecular disease processes (2).? The long-term goal of these studies therefore is to define the cellular mechanisms involved in cholangiocyte secretion, and to identify the physiologic factors that contribute to bile formation. Understanding the physiologic stimuli and molecular basis of cholangiocyte ATP release and the factors regulating P2 receptor-linked cholangiocyte secretion will provide novel insights into bile formation in health and disease and serve as the basis for new and innovative choleretics based on nucleotide receptor stimulation.
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