Bicarbonate-dependent chloride transport drives fluid secretion by the human airway epithelial cell line Calu-3.
Anion and fluid secretion are both defective in cystic fibrosis (CF); however, the transport mechanisms are not well understood. In this study, Cl(-) and HCO(3)(-) secretion was measured using genetically matched CF transmembrane conductance regulator (CFTR)-deficient and CFTR-expressing cell lines derived from the human airway epithelial cell line Calu-3. Forskolin stimulated the short-circuit current (I(sc)) across voltage-clamped monolayers, and also increased the equivalent short-circuit current (I(eq)) calculated under open-circuit conditions. I(sc) was equivalent to the HCO(3)(-) net flux measured using the pH-stat technique, whereas I(eq) was the sum of the Cl(-) and HCO(3)(-) net fluxes. I(eq) and HCO(3)(-) fluxes were increased by bafilomycin and ZnCl(2), suggesting that some secreted HCO(3)(-) is neutralized by parallel electrogenic H(+) secretion. I(eq) and fluid secretion were dependent on the presence of both Na(+) and HCO(3)(-). The carbonic anhydrase inhibitor acetazolamide abolished forskolin stimulation of I(eq) and HCO(3)(-) secretion, suggesting that HCO(3)(-) transport under these conditions requires catalysed synthesis of carbonic acid. Cl(-) was the predominant anion in secretions under all conditions studied and thus drives most of the fluid transport. Nevertheless, 50-70% of Cl(-) and fluid transport was bumetanide-insensitive, suggesting basolateral Cl(-) loading by a sodium-potassium-chloride cotransporter 1 (NKCC1)-independent mechanism. Imposing a transepithelial HCO(3)(-) gradient across basolaterally permeabilized Calu-3 cells sustained a forskolin-stimulated current, which was sensitive to CFTR inhibitors and drastically reduced in CFTR-deficient cells. Net HCO(3)(-) secretion was increased by bilateral Cl(-) removal and therefore did not require apical Cl(-)/HCO(3)(-) exchange. The results suggest a model in which most HCO(3)(-) is recycled basolaterally by exchange with Cl(-), and the resulting HCO(3)(-)-dependent Cl(-) transport provides an osmotic driving force for fluid secretion.
Department of Physiology, McGill University, Montr´eal, QC H3G 1Y6, Canada.
Jiajie
J
Shan
Jie
J
Liao
Junwei
J
Huang
Renaud
R
Robert
Melissa L
ML
Palmer
Scott C
SC
Fahrenkrug
Scott M
SM
O'Grady
John W
JW
Hanrahan
D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D002034:Bumetanide / Q000494:pharmacology; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism*; D017136:Ion Transport; D034741:RNA, Small Interfering / Q000235:genetics; D012137:Respiratory System / Q000166:cytology / Q000378:metabolism; D049994:Sodium Potassium Chloride Symporter Inhibitors / Q000494:pharmacology
D001639:Bicarbonates; C505032:CFTR protein, human; D002712:Chlorides; D034741:RNA, Small Interfering; D049994:Sodium Potassium Chloride Symporter Inhibitors; D002034:Bumetanide; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D000242:Cyclic AMP