title: Early to Late Endosome Trafficking Controls Secretion and Zymogen Activation in Rodent and Human Pancreatic Acinar Cells.
abstract: OBJECTIVE
  Pancreatic acinar cells have an expanded apical endosomal system, the physiological and pathophysiological significance of which is still emerging. Phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2) is an essential phospholipid generated by PIKfyve, which phosphorylates phosphatidylinositol-3-phosphate (PI(3)P). PI(3,5)P2is necessary for maturation of early endosomes (EE) to late endosomes (LE). Inhibition of EE to LE trafficking enhances anterograde endosomal trafficking and secretion at the plasma membrane by default through a recycling endosome (RE) intermediate. We assessed the effects of modulating PIKfyve activity on apical trafficking and pancreatitis responses in pancreatic acinar cells.
  
  
  METHODS
  Inhibition of EE to LE trafficking was achieved using pharmacological inhibitors of PIKfyve, expression of dominant negative PIKfyve K1877E, or constitutively active Rab5-GTP Q79L. Anterograde endosomal trafficking was manipulated by expression of constitutively active and dominant negative Rab11a mutants. The effects of these agents on secretion, endolysosomal exocytosis of lysosome associated membrane protein (LAMP1), and trypsinogen activation in response to high-dose CCK-8, bile acids and cigarette toxin was determined.
  
  
  RESULTS
  PIKfyve inhibition increased basal and stimulated secretion. Adenoviral overexpression of PIKfyve decreased secretion leading to cellular death. Expression of Rab5-GTP Q79L or Rab11a-GTP Q70L enhanced secretion. Conversely, dominant-negative Rab11a-GDP S25N reduced secretion. High-dose CCK inhibited endolysosomal exocytosis that was reversed by PIKfyve inhibition. PIKfyve inhibition blocked intracellular trypsin accumulation and cellular damage responses to high CCK-8, tobacco toxin, and bile salts in both rodent and human acini.
  
  
  CONCLUSIONS
  These data demonstrate that EE-LE trafficking acutely controls acinar secretion and the intracellular activation of zymogens leading to the pathogenicity of acute pancreatitis.
authors.affiliation: Department of Nutritional Sciences, University of Wisconsin, Madison, WI, USA 53706.
authors.forename: Scott W
authors.initials: SW
authors.lastname: Messenger
authors.affiliation: Department of Nutritional Sciences, University of Wisconsin, Madison, WI, USA 53706.
authors.forename: Diana Dh
authors.initials: DD
authors.lastname: Thomas
authors.affiliation: Department of Nutritional Sciences, University of Wisconsin, Madison, WI, USA 53706.
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Cooley
authors.affiliation: Department of Nutritional Sciences, University of Wisconsin, Madison, WI, USA 53706.
authors.forename: Elaina K
authors.initials: EK
authors.lastname: Jones
authors.affiliation: Department of Nutritional Sciences, University of Wisconsin, Madison, WI, USA 53706.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Falkowski
authors.affiliation: Department of Nutritional Sciences, University of Wisconsin, Madison, WI, USA 53706.
authors.forename: Benjamin K
authors.initials: BK
authors.lastname: August
authors.affiliation: Department of Surgery, University of Wisconsin, Madison, WI, USA 53706.
authors.forename: Luis A
authors.initials: LA
authors.lastname: Fernandez
authors.affiliation: Department of Internal Medicine, Yale University School of Medicine, New Haven Connecticut, USA ; Department of Cell Biology, Yale University School of Medicine, New Haven Connecticut, USA ; Veterans Administration Connecticut Healthcare, West Haven, Connecticut, USA.
authors.forename: Fred S
authors.initials: FS
authors.lastname: Gorelick
authors.affiliation: Department of Nutritional Sciences, University of Wisconsin, Madison, WI, USA 53706.
authors.forename: Guy E
authors.initials: GE
authors.lastname: Groblewski
keywords: PIKfyve; endosome; pancreatitis; trypsin