The studies described in this proposal address the cellular mechanisms responsible for secretion by cholangiocytes, the epithelial cells that line the lumen of intrahepatic bile ducts and account for ~40% of bile volume in man. Previous studies indicate that the secretory capacity of cholangiocytes undergoes dynamic changes in response to varying physiologic demands;and have identified multiple pools of intracellular vesicles that can be mobilized rapidly and are sufficient in size to replace up to 40% of the entire cholangiocyte plasma membrane within minutes. Further, the best evidence supports a model wherein one of these vesicular pools is enriched in ATP, and exocytosis leads to a local increase in extracellular nucleotides and initiation of a purinergic signaling cascade. These different vesicular pools are functionally linked to cellular ATP release, activation of purinergic signaling and selective opening of separateC/" channels in response to cAMP, cytosolic Ca2+, and cell volume. Accordingly, the Specific Aims are designed to further address the working hypothesis, which is unchanged, that trafficking (endocytosis and exocytosis) of membrane vesicles containing ion channels provides an early and essential mechanism for modifying the composition and conductance of the cholangiocyte plasma membrane and is essential for the secretory responses to cAMP, Ca2+, and cell volume.
The Specific Aims continue to focus on 1) characterization of the cellular signals responsible for regulation of endocytosis and exocytosis;2) assessment of the functional roles of small conductance K+ (SK2) channels in regulation of cholangiocyte volume and ductular secretion;and 3) definition of the cellular strategies for maintaining the multiprotein signaling complex responsible for apical ATP release, P2 receptor distribution and K+ and CI"secretion. To that end, we have developed combined imaging and biophysical approaches to measurement membrane dynamics and ion transport in real time;and to detection of cellular ATP release in both macroscopic and individual cell models. The long term goal is to define the cellular mechanisms involved in ductular secretion, and to identify the physiologic factors which contribute to bile formation through effects on duct cells. Thus, the findings continue to be directly relevant to diagnosis and management of a broad range of cholestatic disorders characterized by impaired cholangiocyte function;and to development of pharmacologic approaches to modify the volume and composition of bile through effects on duct cells

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (NSS)
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Sherker, Averell H
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University of Texas Sw Medical Center Dallas
Internal Medicine/Medicine
Schools of Medicine
United States
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Feranchak, Andrew P; Lewis, Matthew A; Kresge, Charles et al. (2010) Initiation of purinergic signaling by exocytosis of ATP-containing vesicles in liver epithelium. J Biol Chem 285:8138-47
Dolovcak, Svjetlana; Waldrop, Shar L; Fitz, J Gregory et al. (2010) Copper inhibits P2Y(2)-dependent Ca(2+) signaling through the effects on thapsigargin-sensitive Ca(2+) stores in HTC hepatoma cells. Biochem Biophys Res Commun 397:493-8
Dolovcak, Svjetlana; Waldrop, Shar L; Fitz, J Gregory et al. (2009) 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) stimulates cellular ATP release through exocytosis of ATP-enriched vesicles. J Biol Chem 284:33894-903
Emmett, Daniel S; Feranchak, Andrew; Kilic, Gordan et al. (2008) Characterization of ionotrophic purinergic receptors in hepatocytes. Hepatology 47:698-705
Doctor, R Brian; Johnson, Sylene; Brodsky, Kelley S et al. (2007) Regulated ion transport in mouse liver cyst epithelial cells. Biochim Biophys Acta 1772:345-54
Puljak, Livia; Kilic, Gordan (2006) Emerging roles of chloride channels in human diseases. Biochim Biophys Acta 1762:404-13
Puljak, Livia; Pagliassotti, Michael J; Wei, Yuren et al. (2005) Inhibition of cellular responses to insulin in a rat liver cell line. A role for PKC in insulin resistance. J Physiol 563:471-82
Doctor, R Brian; Matzakos, Thomas; McWilliams, Ryan et al. (2005) Purinergic regulation of cholangiocyte secretion: identification of a novel role for P2X receptors. Am J Physiol Gastrointest Liver Physiol 288:G779-86
Gatof, David; Kilic, Gordan; Fitz, J Gregory (2004) Vesicular exocytosis contributes to volume-sensitive ATP release in biliary cells. Am J Physiol Gastrointest Liver Physiol 286:G538-46
Feranchak, Andrew P; Doctor, R Brian; Troetsch, Marlyn et al. (2004) Calcium-dependent regulation of secretion in biliary epithelial cells: the role of apamin-sensitive SK channels. Gastroenterology 127:903-13

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