Calcium-stimulated Cl- secretion in Calu-3 human airway cells requires CFTR.
Human airway serous cells secrete antibiotic-rich fluid, but, in cystic fibrosis (CF), Cl(-)-dependent fluid secretion is impaired by defects in CF transmembrane conductance regulator (CFTR) Cl- channels. Typically, CF disrupts adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion but spares Ca(2+)-mediated secretion. However, in CF airway glands, Ca(2+)-mediated secretion is also greatly reduced. To determine the basis of Ca(2+)-mediated Cl- secretion in serous cells, we used thapsigargin to elevate intracellular Ca2+ concentration ([Ca2+]i) in Calu-3 cells, an airway cell line bearing some similarities to serous cells. Cells were cultured using conventional and air interface methods. Short-circuit current (Isc) and transepithelial conductance (Gte) were measured in confluent cell layers. Thapsigargin stimulated large, sustained changes (delta) in Isc and Gte, whereas forskolin stimulated variable and smaller increases. delta Isc was decreased by basolateral bumetanide, quinidine, barium, or diphenylamine-2-carboxylate (DPAC) but was unaffected by high apical concentrations of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), 4,4'-dinitrostilbene-2,2'-disulfonic acid, and calixarene. Isc was measured after permeabilizing the basolateral membrane and establishing transmembrane ion gradients. Unstimulated apical membranes displayed high Cl- conductance (GCl) that was decreased by DPAC but not by DIDS. Apical GCl could be increased by elevating intracellular cAMP concentration but not [Ca2+]i. We conclude that CFTR channels are the exclusive GCl pathway in the apical membrane and display approximately 60% of maximum conductance at rest. Thus elevated [Ca2+]i increases K+ conductance to force Cl- through open CFTR channels. We hypothesize that loss of CFTR channels causes diminution of cholinergically mediated gland secretions in CF.
Moon
S
S
Cystic Fibrosis Research Laboratory, Stanford University, California 94305-2130, USA.
Singh
M
M
Krouse
M E
ME
Wine
J J
JJ
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D002034:Bumetanide / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D002712:Chlorides / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000502:physiology*; D004553:Electric Conductivity; D004847:Epithelial Cells / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008168:Lung; D008954:Models, Biological; D019284:Thapsigargin / Q000494:pharmacology
C505032:CFTR protein, human; D002712:Chlorides; D002034:Bumetanide; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D019284:Thapsigargin; D002217:Carbachol; D007545:Isoproterenol; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium