title: Cholangiocytes exhibit dynamic, actin-dependent apical membrane turnover.
abstract: The present studies of cholangiocytes used complementary histological, biochemical, and electrophysiological methods to identify a dense population of subapical vesicles, quantify the rates of vesicular trafficking, and assess the contribution of the actin cytoskeleton to membrane trafficking. FM 1-43 fluorescence measured significant basal rates of total exocytosis (1.33 +/- 0.16% plasma membrane/min) in isolated cholangiocytes and apical exocytosis in cholangiocyte monolayers. Cell surface area remained unchanged, indicating that there was a concurrent, equivalent rate of endocytosis. FM 1-43 washout studies showed that 36% of the endocytosed membrane was recycled to the plasma membrane. 8-(4-Chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP; cAMP analog) increased exocytosis by 71 +/- 31%, whereas the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS; protein kinase A inhibitor) diminished basal exocytosis by 53 +/- 11%. A dense population of 140-nm subapical vesicles arose, in part, from apical membrane endocytosis. Phalloidin staining showed that a subpopulation of the endocytosed vesicles was encapsulated by F-actin. Furthermore, membrane trafficking was inhibited by disrupting the actin cytoskeleton with cytochalasin D (51 +/- 13% of control) or jasplakinolide (58 +/- 9% of control). These studies indicate that there is a high rate of vesicular trafficking at the apical membrane of cholangiocytes and suggest that both cAMP and the actin cytoskeleton contribute importantly to these events.
authors.affiliation: Division of Gastroenterology/Hepatology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA. brian.doctor@uchsc.edu
authors.forename: R Brian
authors.initials: RB
authors.lastname: Doctor
authors.forename: Rolf
authors.initials: R
authors.lastname: Dahl
authors.forename: Laura
authors.initials: L
authors.lastname: Fouassier
authors.forename: Gordan
authors.initials: G
authors.lastname: Kilic
authors.forename: J Gregory
authors.initials: JG
authors.lastname: Fitz
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001652:Bile Ducts / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D001692:Biological Transport / Q000502:physiology*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D016764:Cell Polarity; D000242:Cyclic AMP / Q000378:metabolism; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism; D047630:Depsipeptides; D004705:Endocytosis / Q000502:physiology; D005089:Exocytosis / Q000502:physiology; D005456:Fluorescent Dyes / Q000378:metabolism; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010456:Peptides, Cyclic / Q000494:pharmacology; D051381:Rats; D022161:Transport Vesicles / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D000199:Actins; D000970:Antineoplastic Agents; D047630:Depsipeptides; D005456:Fluorescent Dyes; D019384:Nucleic Acid Synthesis Inhibitors; D010456:Peptides, Cyclic; C057531:jasplakinolide; D015638:Cytochalasin D; D000242:Cyclic AMP