Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom;
Department of Oral Biology, Semmelweis University, Budapest, Hungary; and.
Am J Physiol Cell Physiol. 2014 Jul 15;307(2):C208-19. doi: 10.1152/ajpcell.00083.2014. Epub 2014 Jun 4.
Despite the importance of airway surface liquid pH in the lung's defenses against infection, the mechanism of airway HCO3- secretion remains unclear. Our aim was to assess the contribution of apical and basolateral Cl-/HCO3- exchangers to Cl- and HCO3- transport in the Calu-3 cell line, derived from human airway submucosal glands. Changes in intracellular pH (pHi) were measured following substitution of Cl- with gluconate. Apical Cl- substitution led to an alkalinization in forskolin-stimulated cells, indicative of Cl-/HCO3- exchange. This was unaffected by the anion exchange inhibitor DIDS but inhibited by the CFTR blocker CFTRinh-172, suggesting that the HCO3- influx might occur via CFTR, rather than a solute carrier family 26 (SLC26) exchanger, as recently proposed. The anion selectivity of the recovery process more closely resembled that of CFTR than an SLC26 exchanger, and quantitative RT-PCR showed only low levels of SLC26 exchanger transcripts relative to CFTR and anion exchanger 2 (AE2). For pHi to rise to observed values (∼7.8) through HCO3- entry via CFTR, the apical membrane potential must reverse to at least +20 mV following Cl- substitution; this was confirmed by perforated-patch recordings. Substitution of basolateral Cl- evoked a DIDS-sensitive alkalinization, attributed to Cl-/HCO3- exchange via AE2. This appeared to be abolished in forskolin-stimulated cells but was unmasked by blocking apical efflux of HCO3- via CFTR. We conclude that Calu-3 cells secrete HCO3- predominantly via CFTR, and, contrary to previous reports, the basolateral anion exchanger AE2 remains active during stimulation, providing an important pathway for basolateral Cl- uptake.
尽管气道表面液体 pH 值在肺部抗感染防御中具有重要意义，但气道 HCO3-分泌的机制仍不清楚。我们的目的是评估顶端和基底外侧 Cl--HCO3-交换器对 Calu-3 细胞系（源自人呼吸道黏膜下腺）中 Cl-和 HCO3-转运的贡献。通过用葡萄糖酸盐替代 Cl-来测量细胞内 pH 值（pHi）的变化。在福司可林刺激的细胞中，顶端 Cl-替代导致碱化，表明 Cl--HCO3-交换。这不受阴离子交换抑制剂 DIDS 的影响，但被 CFTR 阻断剂 CFTRinh-172 抑制，表明 HCO3-内流可能通过 CFTR 发生，而不是最近提出的溶质载体家族 26（SLC26）交换器。恢复过程的阴离子选择性更类似于 CFTR，而不是 SLC26 交换器，定量 RT-PCR 显示相对于 CFTR 和阴离子交换器 2（AE2），SLC26 交换器转录本水平较低。为了使 pHi 通过 CFTR 进入 HCO3-而升高到观察到的值（约 7.8），顶端膜电位必须在 Cl-替代后至少反转至+20 mV；这通过穿孔贴片记录得到证实。基底外侧 Cl-的替代引起 DIDS 敏感的碱化，归因于通过 AE2 的 Cl--HCO3-交换。在福司可林刺激的细胞中，这似乎被消除，但通过阻断 CFTR 顶端 HCO3-的外排而被揭示。我们得出结论，Calu-3 细胞主要通过 CFTR 分泌 HCO3-，与之前的报道相反，基底外侧阴离子交换器 AE2 在刺激期间仍然活跃，为基底外侧 Cl-摄取提供了重要途径。
