Polarized distribution of HCO3- transport in human normal and cystic fibrosis nasal epithelia.
The polarized distribution of HCO3- transport was investigated in human nasal epithelial cells from normal and cystic fibrosis (CF) tissues. To test for HCO3- transport via conductive versus electroneutral Cl-/HCO3- exchange (anion exchange, AE) pathways, nasal cells were loaded with the pH probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and mounted in a bilateral perfusion chamber. In normal, but not CF, epithelia, replacing mucosal Cl- with gluconate caused intracellular pH (pHi) to increase, and the initial rates (Delta pH min-1) of this increase were modestly augmented (approximately 26 %) when normal cells were pretreated with forskolin (10 microM). Recovery from this alkaline shift was dependent on mucosal Cl-, was insensitive to the AE inhibitor 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H2DIDS; 1.5 mM), but was sensitive to the cystic fibrosis transmembrane conductance regulator (CFTR) channel inhibitor diphenylamine-2-carboxylate (DPC; 100 microM). In contrast, removal of serosal Cl- caused pHi to alkalinize in both normal and CF epithelia. Recovery from this alkaline challenge was dependent on serosal Cl- and blocked by H2DIDS. Additional studies showed that serosally applied Ba2+ (5.0 mM) in normal, but not CF, cells induced influx of HCO3- across the apical membrane that was reversibly blocked by mucosal DPC. In a final series of studies, normal and CF cells acutely alkaline loaded by replacing bilateral Krebs bicarbonate Ringer (KBR) with Hepes-buffered Ringer solution exhibited basolateral, but not apical, recovery from an alkaline challenge that was dependent on Cl-, independent of Na+ and blocked by H2DIDS. We conclude that: (1) normal, but not CF, nasal epithelia have a constitutively active DPC-sensitive HCO3- influx/efflux pathway across the apical membrane of cells, consistent with the movement of HCO3- via CFTR; and (2) both normal and CF nasal epithelia have Na+-independent, H2DIDS-sensitive AE at their basolateral domain.
Paradiso
Anthony M
AM
Cystic Fibrosis/Pulmonary Research and Treatment Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7248, USA. paradiso@med.unc.edu
Coakley
Raymond D
RD
Boucher
Richard C
RC
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000328:Adult; D000584:Amiloride / Q000494:pharmacology; D001464:Barium / Q000494:pharmacology; D001639:Bicarbonates / Q000378:metabolism*; D001693:Biological Transport, Active / Q000187:drug effects; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D003550:Cystic Fibrosis / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism; D015194:Diffusion Chambers, Culture; D004232:Diuretics / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005452:Fluoresceins; D005470:Fluorometry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D009297:Nasal Mucosa / Q000187:drug effects / Q000378:metabolism*; D062367:ortho-Aminobenzoates / Q000494:pharmacology
D001639:Bicarbonates; C505032:CFTR protein, human; D002712:Chlorides; D004232:Diuretics; D005452:Fluoresceins; D062367:ortho-Aminobenzoates; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D001464:Barium; D000584:Amiloride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; C001209:fenamic acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid