Children’s Hospital Oakland Research Institute, Oakland, California, USA.
Am J Physiol Lung Cell Mol Physiol. 2010 Oct;299(4):L585-94. doi: 10.1152/ajplung.00421.2009. Epub 2010 Jul 30.
Using cell culture models, we have investigated the relative importance of cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated chloride channels (CaCC) in Cl secretion by mucous and serous cells of human airway glands. In transepithelial recordings in Ussing chambers, the CFTR inhibitor CFTR(inh)-172 abolished 60% of baseline Cl secretion in serous cells and 70% in mucous. Flufenamic acid (FFA), an inhibitor of CaCC, reduced baseline Cl secretion by ∼20% in both cell types. Methacholine and ATP stimulated Cl secretion in both cell types, which was largely blocked by treatment with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and partially by mucosal FFA or CFTR(inh)-172 with the exception of methacholine responses in mucous cells, which were not blocked by FFA and partially (∼60%) by CFTR(inh)-172. The effects of ionomycin on short-circuit current (I(sc)) were less than those of ATP or methacholine. Forskolin stimulated Cl secretion only if Cl in the mucosal medium was replaced by gluconate. In whole cell patch-clamp studies of single isolated cells, cAMP-induced Cl currents were ∼3-fold greater in serous than mucous cells. Ionomycin-induced Cl currents were 13 times (serous) or 26 times (mucous) greater than those generated by cAMP and were blocked by FFA. In serous cells, mRNA for transmembrane protein 16A (TMEM16A) was ∼10 times more abundant than mRNA for CFTR. In mucous cells it was ∼100 times more abundant. We conclude: 1) serous and mucous cells both make significant contributions to gland fluid secretion; 2) baseline Cl secretion in both cell types is mediated predominantly by CFTR, but CaCC becomes increasingly important after mediator-induced elevations of intracellular Ca; and 3) the high CaCC currents seen in patch-clamp studies and the high TMEM16A expression in intact polarized cells sheets are not reflected in transepithelial current recordings.
我们使用细胞培养模型，研究了囊性纤维化跨膜电导调节因子 (CFTR) 和钙激活氯离子通道 (CaCC) 在人呼吸道腺黏液和浆液细胞氯离子分泌中的相对重要性。在 Ussing 室的跨上皮记录中，CFTR 抑制剂 CFTR(inh)-172 消除了浆液细胞基础氯离子分泌的 60%，黏液细胞则消除了 70%。氟芬那酸 (FFA)，一种 CaCC 抑制剂，使两种细胞类型的基础氯离子分泌减少了约 20%。乙酰甲胆碱和 ATP 刺激两种细胞类型的氯离子分泌，用 1,2-双(2-氨基苯氧基)乙烷-N,N,N',N'-四乙酸 (BAPTA) 处理可使这种分泌大部分被阻断，用黏膜 FFA 或 CFTR(inh)-172 处理则部分被阻断，但黏液细胞的乙酰甲胆碱反应不受 FFA 阻断，部分被 CFTR(inh)-172 阻断（约 60%）。离子霉素对短路电流 (I(sc)) 的影响小于 ATP 或乙酰甲胆碱。forskolin 仅在黏膜介质中的氯离子被葡萄糖酸盐取代时才能刺激氯离子分泌。在单个分离细胞的全细胞膜片钳研究中，cAMP 诱导的氯离子电流在浆液细胞中比在黏液细胞中约大 3 倍。离子霉素诱导的氯离子电流是 cAMP 诱导的氯离子电流的 13 倍（浆液细胞）或 26 倍（黏液细胞），并被 FFA 阻断。在浆液细胞中，跨膜蛋白 16A (TMEM16A) 的 mRNA 比 CFTR 的 mRNA 丰富约 10 倍。在黏液细胞中，它的丰富度约为 CFTR 的 100 倍。我们的结论是：1) 浆液细胞和黏液细胞均对腺液分泌有重要贡献；2) 两种细胞类型的基础氯离子分泌主要由 CFTR 介导，但在介质诱导细胞内 Ca 升高后，CaCC 变得越来越重要；3) 全细胞膜片钳研究中观察到的高 CaCC 电流和完整极化细胞薄片中高 TMEM16A 表达不能反映在跨上皮电流记录中。
