Woollhead Alison M, Baines Deborah L
Division of Basic Medical Sciences, Ion Channels and Cell Signaling Centre, St. Georges' University of London, Cranmer Terrace, Tooting, London SW17 0RE, United Kingdom.
J Biol Chem. 2006 Feb 24;281(8):5158-68. doi: 10.1074/jbc.M509947200. Epub 2005 Dec 22.
Elevation of intracellular cAMP increases fluid re-absorption in the lung by raising amiloride-sensitive Na+ transport through the apically localized epithelial, amiloride-sensitive Na+ channel (ENaC). However, the signaling pathways mediating this response are still not fully understood. We show that inhibition of protein-tyrosine kinase (PTK) with Genistein and protein kinase A (PKA) with KT5720, decreased forskolin-stimulated amiloride-sensitive short circuit current (I(sc)) across H441 adult human lung epithelial cell monolayers. KT5720 also decreased basal I(sc). Stable expression of green fluorescent protein (GFP)-labeled human alphaENaC in H441 cells was used to investigate dynamic changes in the cellular localization of this protein in response to forskolin. Reverse transcription-PCR and immunoblotting analysis revealed two clones expressing a truncated (alphaC3-5) and full-length (alphaC3-3) EGFP-halphaENaC protein. Only the alphaC3-3 clone displayed dome formation and exhibited a 50% increase in basal and forskolin-stimulated amiloride-sensitive I(sc) indicating that the full-length protein was required for functional activity. Apical surface biotinylation and real-time confocal microscopy demonstrated that EGFP-halphaENaC (alphaC3-3) translocated to the apical membrane in response to forskolin in a Brefeldin A-sensitive manner. This effect was completely inhibited by Genistein but only partially inhibited by KT5720. Forskolin also induced a reduction in the height of cells within alphaC3-3 monolayers, indicative of cell shrinkage. This effect was inhibited by KT5720 but not by Genistein or Brefeldin A. These data show that forskolin activates PKA-sensitive cell shrinkage in adult human H441 lung epithelial cell monolayers, which induces a PTK-sensitive translocation of EGFP-halphaENaC subunits to the apical membrane and increases amiloride-sensitive Na+ transport.
细胞内cAMP水平的升高通过增强顶端定位的上皮性、阿米洛利敏感性钠通道（ENaC）介导的阿米洛利敏感性钠转运，增加肺内液体重吸收。然而，介导这种反应的信号通路仍未完全明确。我们发现，用金雀异黄素抑制蛋白酪氨酸激酶（PTK）以及用KT5720抑制蛋白激酶A（PKA），可降低毛喉素刺激的跨H441成人肺上皮细胞单层的阿米洛利敏感性短路电流（I(sc)）。KT5720还降低了基础I(sc)。利用在H441细胞中稳定表达绿色荧光蛋白（GFP）标记的人αENaC，研究该蛋白在细胞内定位的动态变化对毛喉素的反应。逆转录聚合酶链反应（RT-PCR）和免疫印迹分析显示，有两个克隆表达截短型（αC3-5）和全长型（αC3-3）的EGFP-hαENaC蛋白。只有αC3-3克隆形成穹顶，并在基础和毛喉素刺激的阿米洛利敏感性I(sc)方面表现出50%的增加，表明全长蛋白是功能活性所必需的。顶端表面生物素化和实时共聚焦显微镜显示，EGFP-hαENaC（αC3-3）以对布雷菲德菌素A敏感的方式响应毛喉素转运至顶端膜。金雀异黄素完全抑制了这种效应，但KT5720仅部分抑制。毛喉素还导致αC3-3单层内细胞高度降低，表明细胞收缩。这种效应被KT5720抑制，但未被金雀异黄素或布雷菲德菌素A抑制。这些数据表明，毛喉素激活成人H441肺上皮细胞单层中PKA敏感的细胞收缩，这诱导了PTK敏感的EGFP-hαENaC亚基向顶端膜的转运，并增加了阿米洛利敏感性钠转运。
