title: Characterization of H+ and HCO3- transporters in CFPAC-1 human pancreatic duct cells.
abstract: OBJECTIVE
  To characterize H+ and HCO3- transporters in polarized CFPAC-1 human pancreatic duct cells, which were derived from a cystic fibrosis patient with the DeltaF508 CFTR mutation.
  
  
  METHODS
  CFPAC-1 cells were seeded at high density onto permeable supports and grown to confluence. The cells were loaded with the pH-sensitive fluorescent dye BCECF, and mounted into a perfusion chamber, which allowed the simultaneous perfusion of the basolateral and apical membranes. Transmembrane base flux was calculated from the changes in intracellular pH and the buffering capacity of the cells.
  
  
  RESULTS
  Our results showed differential permeability to HCO3-/CO2 at the apical and basolateral membranes of CFPAC-1 cells. Na+/ HCO3- co-transporters (NBCs) and Cl-/ HCO3- exchangers (AEs) were present on the basolateral membrane, and Na+/H+ exchangers (NHEs) on both the apical and basolateral membranes of the cells. Basolateral HCO3- uptake was sensitive to variations of extracellular K+ concentration, the membrane permeable carbonic anhydrase (CA) inhibitors acetazolamide (100 micromol/L) and ethoxyzolamide (100 micromol/L), and was partially inhibited by H2-DIDS (600 micromol/L). The membrane-impermeable CA inhibitor 1-N-(4-sulfamoylphenylethyl)-2,4,6-trimethylpyridine perchlorate did not have any effect on HCO3- uptake. The basolateral AE had a much higher activity than that in the apical membrane, whereas there was no such difference with the NHE under resting conditions. Also, 10 micromol/L forskolin did not significantly influence Cl-/ HCO3- exchange on the apical and basolateral membranes. The administration of 250 micromol/L H2-DIDS significantly inhibited the basolateral AE. Amiloride (300 micromol/L) completely inhibited NHEs on both membranes of the cells. RT-PCR revealed the expression of pNBC1, AE2, and NHE1 mRNA.
  
  
  CONCLUSIONS
  These data suggest that apart from the lack of CFTR and apical Cl-/ HCO3- exchanger activity, CFPAC-1 cells express similar H+ and HCO3- transporters to those observed in native animal tissue.
authors.affiliation: Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne, Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom.
authors.forename: Zoltan
authors.initials: Z
authors.lastname: Rakonczay
authors.forename: Amy
authors.initials: A
authors.lastname: Fearn
authors.forename: Péter
authors.initials: P
authors.lastname: Hegyi
authors.forename: Imre
authors.initials: I
authors.lastname: Boros
authors.forename: Michael A
authors.initials: MA
authors.lastname: Gray
authors.forename: Barry E
authors.initials: BE
authors.lastname: Argent
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000086:Acetazolamide / Q000494:pharmacology; D027321:Anion Transport Proteins / Q000378:metabolism*; D001639:Bicarbonates / Q000378:metabolism; D002460:Cell Line; D002463:Cell Membrane Permeability; D005016:Ethoxzolamide / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010183:Pancreatic Ducts / Q000187:drug effects / Q000502:physiology*; D011188:Potassium / Q000378:metabolism
chemical_list: D027321:Anion Transport Proteins; D001639:Bicarbonates; D000086:Acetazolamide; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D011188:Potassium; D005016:Ethoxzolamide