Basolateral chloride loading by the anion exchanger type 2: role in fluid secretion by the human airway epithelial cell line Calu‐3
Bibliographic record
Abstract
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.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".