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Basolateral chloride loading by the anion exchanger type 2: role in fluid secretion by the human airway epithelial cell line Calu‐3
Author(s) -
Huang Junwei,
Shan Jiajie,
Kim Dusik,
Liao Jie,
Evagelidis Alexandra,
Alper Seth L.,
Hanrahan John W.
Publication year - 2012
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2012.236919
Subject(s) - secretion , chemistry , bumetanide , ion transporter , microbiology and biotechnology , epithelial polarity , forskolin , cell culture , epithelium , sodium–hydrogen antiporter , biophysics , biochemistry , sodium , biology , cell , membrane , in vitro , organic chemistry , genetics
Key points• The companion paper provided evidence for basolateral anion exchange during cAMP stimulation of chloride and fluid secretion by Calu‐3 monolayers; however, the molecular basis of this transport was not identified. • To test the role of AE2, an anion exchanger expressed at the basolateral membrane of Calu‐3 and many other epithelial cells, we used lentivirus‐mediated RNA interference to generate a stable Calu‐3 AE2 knock‐down cell line and characterized its fluid and anion transport properties. • AE2 knock‐down suppressed fluid secretion and increased the fraction of cAMP‐stimulated anion secretion that was sensitive to bumetanide inhibition. • Basolateral Cl − /HCO 3 − exchange was nearly abolished in AE2 knock‐down cells. • We conclude that AE2 is active during forskolin‐stimulated fluid secretion and mediates chloride uptake and bicarbonate recycling at the basolateral membrane.Abstract Anion exchanger type 2 (AE2 or SLC4A2) is an electroneutral Cl − /HCO 3 − exchanger expressed at the basolateral membrane of many epithelia. It is thought to participate in fluid secretion by airway epithelia. However, the role of AE2 in fluid secretion remains uncertain, due to the lack of specific pharmacological inhibitors, and because it is electrically silent and therefore does not contribute directly to short‐circuit current ( I sc ). We have studied the role of AE2 in Cl − and fluid secretion by the airway epithelial cell line Calu‐3. After confirming expression of its mRNA and protein, a knock‐down cell line called AE2‐KD was generated by lentivirus‐mediated RNA interference in which AE2 mRNA and protein levels were reduced ≥90%. Suppressing AE2 increased the expression of the cystic fibrosis transmembrane conductance regulator (CFTR) by ∼70% without affecting the levels of NKCC1 (Na + –K + –2Cl − cotransporter) or NBCe1 (Na + –nHCO 3 − cotransporter). cAMP agonists stimulated fluid secretion by parental Calu‐3 and scrambled shRNA cells >6.5‐fold. In AE2‐KD cells this response was reduced by ∼70%, and the secreted fluid exhibited elevated pH and [HCO 3 − ] as compared with the control lines. Unstimulated equivalent short‐circuit current ( I eq ) was elevated in AE2‐KD cells, but the incremental response to forskolin was unaffected. The modest bumetanide‐induced reductions in both I eq and fluid secretion were more pronounced in AE2‐KD cells. Basolateral Cl − /HCO 3 − exchange measured by basolateral pH‐stat in cells with permeabilized apical membranes was abolished in AE2‐KD monolayers, and the intracellular alkalinization resulting from basolateral Cl − removal was reduced by ∼80% in AE2‐KD cells. These results identify AE2 as a major pathway for basolateral Cl − loading during cAMP‐stimulated secretion of Cl − and fluid by Calu‐3 cells, and help explain the large bumetanide‐insensitive component of fluid secretion reported previously in airway submucosal glands and some other epithelia.