title: Chloride transporting capability of Calu-3 epithelia following persistent knockdown of the cystic fibrosis transmembrane conductance regulator, CFTR.
abstract: OBJECTIVE
  Calu-3 cells are derived from serous cells of human lung submucosal glands, a prime target for therapy in cystic fibrosis (CF). Calu-3 cells can be cultured to form epithelia capable of transepithelial transport of chloride. A CF Calu-3 cell is not available.
  
  
  METHODS
  A retroviral vector was used to cause persistent down regulation of CFTR using siRNA methodology, in Calu-3 cells. A Calu-3 cell line with CFTR content less than 5% of the original line has been established. Epithelia grown using the modified cells have been used in comparative studies of transporting capability.
  
  
  RESULTS
  All aspects of cAMP activated chloride secretion were attenuated in the epithelia with reduced CFTR content. However transporting capability was reduced less than the CFTR content. From studies with the CFTR channel inhibitor, GlyH-101, it was concluded that wild type Calu-3 cells have a reserve of CFTR channels not located in the membrane, but available for replacement, while in the modified Calu-3 cell line there was little or no reserve. Lubiprostone, a putative ClC-2 activator, increased transepithelial chloride secretion in both modified and wild type Calu-3 epithelia. Modified Calu-3 epithelia with the residual CFTR currents blocked with GlyH-101 responded equally well to lubiprostone as those without the blocking agent.
  
  
  CONCLUSIONS
  It appears that lubiprostone is capable of stimulating a non-CFTR dependent transepithelial chloride secretion in Calu-3 monolayers, with obvious implications for CF therapy. Cell lines, however, do not always reflect the behaviour of the native tissue with integrity.
authors.affiliation: Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK.
authors.forename: L J
authors.initials: LJ
authors.lastname: MacVinish
authors.forename: G
authors.initials: G
authors.lastname: Cope
authors.forename: A
authors.initials: A
authors.lastname: Ropenga
authors.forename: A W
authors.initials: AW
authors.lastname: Cuthbert
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000527:Alprostadil / Q000031:analogs & derivatives / Q000494:pharmacology; D000090007:CLC-2 Chloride Channels; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D018118:Chloride Channels / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D003550:Cystic Fibrosis / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000172:deficiency / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D005227:Fatty Acids / Q000494:pharmacology; D005822:Genetic Vectors; D006801:Humans; D000068238:Lubiprostone; D008168:Lung / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008564:Membrane Potentials; D018721:Muscarinic Agonists / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D013997:Time Factors
chemical_list: C505032:CFTR protein, human; D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels; D002712:Chlorides; D005227:Fatty Acids; D018721:Muscarinic Agonists; D034741:RNA, Small Interfering; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D000068238:Lubiprostone; D002217:Carbachol; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D000527:Alprostadil