Bicarbonate-dependent chloride transport drives fluid secretion by the human airway epithelial cell line Calu-3

Jiajie Shan, Jie Liao, Junwei Huang, Renaud Robert, Melissa L Palmer, Scott C. Fahrenkrug, Scott M O'Grady, John W. Hanrahan

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

Anion and fluid secretion are both defective in cystic fibrosis (CF); however, the transport mechanisms are not well understood. In this study, Cl- and HCO3- 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 (Isc) across voltage-clamped monolayers, and also increased the equivalent short-circuit current (Ieq) calculated under open-circuit conditions. Isc was equivalent to the HCO3- net flux measured using the pH-stat technique, whereas Ieq was the sum of the Cl- and HCO3- net fluxes. Ieq and HCO3- fluxes were increased by bafilomycin and ZnCl2, suggesting that some secreted HCO3- is neutralized by parallel electrogenic H+ secretion. Ieq and fluid secretion were dependent on the presence of both Na+ and HCO3-. The carbonic anhydrase inhibitor acetazolamide abolished forskolin stimulation of Ieq and HCO3- secretion, suggesting that HCO3- 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 HCO3- 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 HCO3- secretion was increased by bilateral Cl- removal and therefore did not require apical Cl-/HCO3- exchange. The results suggest a model in which most HCO3- is recycled basolaterally by exchange with Cl-, and the resulting HCO3--dependent Cl- transport provides an osmotic driving force for fluid secretion.

Original languageEnglish (US)
Pages (from-to)5273-5297
Number of pages25
JournalJournal of Physiology
Volume590
Issue number21
DOIs
StatePublished - Nov 1 2012

Fingerprint

Fluids and Secretions
Colforsin
Bicarbonates
Chlorides
Epithelial Cells
Cell Line
Anions
Sodium-Potassium-Chloride Symporters
Carbonic Acid
Carbonic Anhydrase Inhibitors
Bumetanide
Cystic Fibrosis Transmembrane Conductance Regulator
Acetazolamide
Cystic Fibrosis

Cite this

Bicarbonate-dependent chloride transport drives fluid secretion by the human airway epithelial cell line Calu-3. / Shan, Jiajie; Liao, Jie; Huang, Junwei; Robert, Renaud; Palmer, Melissa L; Fahrenkrug, Scott C.; O'Grady, Scott M; Hanrahan, John W.

In: Journal of Physiology, Vol. 590, No. 21, 01.11.2012, p. 5273-5297.

Research output: Contribution to journalArticle

Shan, Jiajie ; Liao, Jie ; Huang, Junwei ; Robert, Renaud ; Palmer, Melissa L ; Fahrenkrug, Scott C. ; O'Grady, Scott M ; Hanrahan, John W. / Bicarbonate-dependent chloride transport drives fluid secretion by the human airway epithelial cell line Calu-3. In: Journal of Physiology. 2012 ; Vol. 590, No. 21. pp. 5273-5297.
@article{6c0ad6523c5c4bed9db292ad628c35ec,
title = "Bicarbonate-dependent chloride transport drives fluid secretion by the human airway epithelial cell line Calu-3",
abstract = "Anion and fluid secretion are both defective in cystic fibrosis (CF); however, the transport mechanisms are not well understood. In this study, Cl- and HCO3- 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 (Isc) across voltage-clamped monolayers, and also increased the equivalent short-circuit current (Ieq) calculated under open-circuit conditions. Isc was equivalent to the HCO3- net flux measured using the pH-stat technique, whereas Ieq was the sum of the Cl- and HCO3- net fluxes. Ieq and HCO3- fluxes were increased by bafilomycin and ZnCl2, suggesting that some secreted HCO3- is neutralized by parallel electrogenic H+ secretion. Ieq and fluid secretion were dependent on the presence of both Na+ and HCO3-. The carbonic anhydrase inhibitor acetazolamide abolished forskolin stimulation of Ieq and HCO3- secretion, suggesting that HCO3- 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 HCO3- 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 HCO3- secretion was increased by bilateral Cl- removal and therefore did not require apical Cl-/HCO3- exchange. The results suggest a model in which most HCO3- is recycled basolaterally by exchange with Cl-, and the resulting HCO3--dependent Cl- transport provides an osmotic driving force for fluid secretion.",
author = "Jiajie Shan and Jie Liao and Junwei Huang and Renaud Robert and Palmer, {Melissa L} and Fahrenkrug, {Scott C.} and O'Grady, {Scott M} and Hanrahan, {John W.}",
year = "2012",
month = "11",
day = "1",
doi = "10.1113/jphysiol.2012.236893",
language = "English (US)",
volume = "590",
pages = "5273--5297",
journal = "Journal of Physiology",
issn = "0022-3751",
publisher = "Wiley-Blackwell",
number = "21",

}

TY - JOUR

T1 - Bicarbonate-dependent chloride transport drives fluid secretion by the human airway epithelial cell line Calu-3

AU - Shan, Jiajie

AU - Liao, Jie

AU - Huang, Junwei

AU - Robert, Renaud

AU - Palmer, Melissa L

AU - Fahrenkrug, Scott C.

AU - O'Grady, Scott M

AU - Hanrahan, John W.

PY - 2012/11/1

Y1 - 2012/11/1

N2 - Anion and fluid secretion are both defective in cystic fibrosis (CF); however, the transport mechanisms are not well understood. In this study, Cl- and HCO3- 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 (Isc) across voltage-clamped monolayers, and also increased the equivalent short-circuit current (Ieq) calculated under open-circuit conditions. Isc was equivalent to the HCO3- net flux measured using the pH-stat technique, whereas Ieq was the sum of the Cl- and HCO3- net fluxes. Ieq and HCO3- fluxes were increased by bafilomycin and ZnCl2, suggesting that some secreted HCO3- is neutralized by parallel electrogenic H+ secretion. Ieq and fluid secretion were dependent on the presence of both Na+ and HCO3-. The carbonic anhydrase inhibitor acetazolamide abolished forskolin stimulation of Ieq and HCO3- secretion, suggesting that HCO3- 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 HCO3- 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 HCO3- secretion was increased by bilateral Cl- removal and therefore did not require apical Cl-/HCO3- exchange. The results suggest a model in which most HCO3- is recycled basolaterally by exchange with Cl-, and the resulting HCO3--dependent Cl- transport provides an osmotic driving force for fluid secretion.

AB - Anion and fluid secretion are both defective in cystic fibrosis (CF); however, the transport mechanisms are not well understood. In this study, Cl- and HCO3- 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 (Isc) across voltage-clamped monolayers, and also increased the equivalent short-circuit current (Ieq) calculated under open-circuit conditions. Isc was equivalent to the HCO3- net flux measured using the pH-stat technique, whereas Ieq was the sum of the Cl- and HCO3- net fluxes. Ieq and HCO3- fluxes were increased by bafilomycin and ZnCl2, suggesting that some secreted HCO3- is neutralized by parallel electrogenic H+ secretion. Ieq and fluid secretion were dependent on the presence of both Na+ and HCO3-. The carbonic anhydrase inhibitor acetazolamide abolished forskolin stimulation of Ieq and HCO3- secretion, suggesting that HCO3- 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 HCO3- 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 HCO3- secretion was increased by bilateral Cl- removal and therefore did not require apical Cl-/HCO3- exchange. The results suggest a model in which most HCO3- is recycled basolaterally by exchange with Cl-, and the resulting HCO3--dependent Cl- transport provides an osmotic driving force for fluid secretion.

UR - http://www.scopus.com/inward/record.url?scp=84868096271&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84868096271&partnerID=8YFLogxK

U2 - 10.1113/jphysiol.2012.236893

DO - 10.1113/jphysiol.2012.236893

M3 - Article

VL - 590

SP - 5273

EP - 5297

JO - Journal of Physiology

JF - Journal of Physiology

SN - 0022-3751

IS - 21

ER -