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. Cholangiocytes are also the cellular site of injury in many cholestatic liver diseases associated with abnormal bile flow such as cystic fibrosis (CF). Cl- channels in the apical membrane of cholangiocytes provide the driving force for secretion and we have previously identified and characterized TMEM16A as a Ca2+-activated Cl- which is activated by ATP binding purinergic (P2) receptors. In preliminary studies described in this proposal, we characterize an apical membrane Ca2+ channel, TRPV4, which translates stimuli at the apical membrane of cholangiocytes to increases in [Ca ]i, ATP release, P2 receptor binding, and 2+ TMEM16A activation. Together these studies suggest that TRPV4, P2 receptors, and TMEM16A together represent a functional signaling complex in the apical cholangiocyte membrane which contributes to biliary secretion and bile formation. Accordingly, the Specific Aims are designed to address the following working hypothesis: We propose that an apical signaling complex coordinates and regulates cholangiocyte secretion in response to luminal stimuli including fluid-flow, bile acids, and ATP; represents an essential and critical determinant of ductular bile formation; and provides potential therapeutic targets to improve bile flow in the treatment of cholestatic liver diseases.
The Specific Aims are: 1) to assess the functional roles of the apical signaling complex comprised of the Ca2+ channel, TRPV4, the Cl- channel, TMEM16A, and the purinergic receptor, P2Y2, in the integration and coordination of cholangiocyte secretion and bile formation; 2) to critically evaluate the cellular signals responsible for the regulation of th TRPV4 -P2R- TMEM16A signaling complex and to define the specific roles of i) exocytosis, ii) intracellular Ca2+ and Ca2+-binding proteins, and iii) kinase signaling pathways in channel localization and open probability; and 3) to determine the effects of cholestasis, utilizing cystic fibrosis as a model, on apical-directed signaling; and conversely to determine if targeting specific members of the signaling complex can serve as a therapeutic strategy for cholestatic conditions. An integrated approach combining electrophysiology, molecular biology, and fluorescence imaging will be applied to study of single cholangiocytes, intact and polarized cholangiocyte monolayers, and a novel live bile duct-cannulated murine model which we have recently developed. 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 bil formation. Understanding the physiologic stimuli and molecular basis of cholangiocyte membrane ion permeability and secretion will provide novel insights into bile formation in health and disease; and provide a foundation for development of novel choleretics targeting cholangiocyte plasma membrane receptors and channels. 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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