Bestrophin expression and function in the human pancreatic duct cell line, CFPAC-1

Laura L Marsey, John P Winpenny

Research output: Contribution to journalArticlepeer-review

13 Citations (Scopus)

Abstract

Pancreatic duct epithelial cells (PDECs) have been shown to express calcium activated chloride channels (CaCCs) and there is evidence for their involvement in fluid secretion from these cells. The molecular identity of the CaCC in PDECs remains unknown. Recently, the bestrophin family of proteins have been proposed as a potential molecular candidate for CaCCs. Expression of bestrophins is strongly correlated with the function of CaCCs in a variety of tissues. In the present study, the expression of bestrophins has been investigated in the cystic fibrosis pancreatic duct cell line, CFPAC-1. Iodide efflux analysis was used to characterise native CaCCs in CFPAC-1 cell monolayers. Efflux was induced with the addition of UTP (100 microM, 10.2 +/- 1.5 nmol min(-1)), which was blocked by the chloride channel blockers niflumic acid (81%) and DIDS (90%). The UTP-stimulated iodide efflux was shown to be Ca(2+) dependent and cAMP independent. RT-PCR analysis of RNA isolated from CFPAC-1 cells demonstrated positive identification of all four human bestrophin mRNAs. Western blot of CFPAC-1 cell protein isolates with antibodies specific to human bestrophin 1 (hBest1) showed that hBest1 protein was expressed in this cell line. HBest1 was present on the cell surface, demonstrated using biotinylation and confocal imaging, as well as in the cytoplasm. SiRNA-mediated silencing of hBest1 in CFPAC-1 cells reduced the UTP-stimulated iodide efflux by around 40%. This study provides evidence that the bestrophins are expressed in pancreatic duct cells and, more specifically, that hBest1 plays a role in the CaCCs found in these cells.

Original languageEnglish
Pages (from-to)2211-24
Number of pages14
JournalThe Journal of Physiology
Volume587
Issue numberPt 10
DOIs
Publication statusPublished - 15 May 2009

Keywords

  • Adenosine Triphosphate/pharmacology
  • Bestrophins
  • Cell Line, Tumor
  • Cell Membrane/metabolism
  • Chelating Agents/pharmacology
  • Chloride Channels/antagonists & inhibitors
  • Egtazic Acid/analogs & derivatives
  • Endoplasmic Reticulum/metabolism
  • Epithelial Cells/drug effects
  • Eye Proteins/genetics
  • Gene Expression/physiology
  • Golgi Apparatus/metabolism
  • Humans
  • Iodides/metabolism
  • Ionomycin/pharmacology
  • Lysosomes/metabolism
  • Muscle Proteins/genetics
  • Pancreatic Ducts/cytology
  • RNA, Small Interfering/genetics
  • Uridine Triphosphate/pharmacology

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