Department of Physiology, McGill University, Montr´eal, QC, Canada.
J Physiol. 2012 Nov 1;590(21):5299-316. doi: 10.1113/jphysiol.2012.236919. Epub 2012 Jul 16.
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.
阴离子交换体 2 型（AE2 或 SLC4A2）是一种电中性的 Cl(-)/HCO(3)(-)交换体，表达于许多上皮细胞的基底外侧膜。它被认为参与了气道上皮的液体分泌。然而，由于缺乏特异性的药理学抑制剂，以及由于它是电沉默的，因此不会直接导致短路电流（I(sc)），AE2 在液体分泌中的作用仍然不确定。我们研究了 AE2 在气道上皮细胞系 Calu-3 的 Cl(-)和液体分泌中的作用。在确认其 mRNA 和蛋白表达后，通过慢病毒介导的 RNA 干扰生成了一个敲低细胞系 AE2-KD，其中 AE2 mRNA 和蛋白水平降低了 90%。抑制 AE2 增加了囊性纤维化跨膜电导调节因子（CFTR）的表达，增加了约 70%，而不影响 NKCC1（Na(+)-K(+)-2Cl(-)共转运体）或 NBCe1（Na(+)-nHCO(3)(-)共转运体）的水平。cAMP 激动剂刺激亲本 Calu-3 和 scrambled shRNA 细胞的液体分泌超过 6.5 倍。在 AE2-KD 细胞中，这种反应减少了约 70%，并且分泌的液体的 pH 和[HCO(3)(-)]比对照系升高。未刺激的等效短路电流（I(eq)）在 AE2-KD 细胞中升高，但对 forskolin的递增反应不受影响。在 AE2-KD 细胞中，适度的布美他尼诱导的 I(eq)和液体分泌的减少更为明显。用透化顶端膜的细胞进行基底外侧 pH -stat 测量的基底外侧 Cl(-)/HCO(3)(-)交换在 AE2-KD 单层中被废除，并且由于基底外侧 Cl(-)去除而导致的细胞内碱化减少了约 80%在 AE2-KD 细胞中。这些结果表明，AE2 是 Calu-3 细胞 cAMP 刺激 Cl(-)和液体分泌时基底外侧 Cl(-)加载的主要途径，并有助于解释先前在气道粘膜下腺和其他一些上皮细胞中报道的大剂量布美他尼不敏感的液体分泌成分。
