Brézillon S, Zahm J M, Pierrot D, Gaillard D, Hinnrasky J, Millart H, Klossek J M, Tümmler B, Puchelle E
INSERM Unité 314, IFR 53, Université de Reims, 51092 Reims cedex, France.
J Biol Chem. 1997 Oct 31;272(44):27830-8. doi: 10.1074/jbc.272.44.27830.
To mimic the effect of ischemia on the integrity of airway epithelium and expression of cystic fibrosis transmembrane conductance regulator (CFTR), we induced an ATP depletion of the respiratory epithelium from upper airway cells (nasal tissue) and human bronchial epithelial 16HBE14o- cell line. Histological analysis showed that 2 h of ATP depletion led to a loss of the epithelium integrity at the interface between basal cells and columnar cells. The expression of connexin 43 (Cx43, subunit of the gap junctions) and desmoplakins 1 and 2 (DPs 1 and 2, major components of the desmosomes) proteins was inhibited. After 90 min of ATP depletion, a significant decrease of the transepithelial resistance (25%) was observed but was reversible. Similar results were obtained with the 16HBE14o- human bronchial epithelial cell line. ATP depletion led to actin filaments depolymerization. The expression of the mature CFTR (170 kDa) and fodrin proteins at the apical domain of the ciliated cells was down-regulated. The steady-state levels of CFTR, Cx43, DPs 1 and 2 mRNAs, semiquantified by RT-polymerase chain reaction kinetics, remained constant throughout ATP depletion in nasal tissue as in the homogeneous cell population of 16HBE14o- human bronchial epithelial cell line. This suggests that the down-regulation of these proteins might be posttranscriptional. The intercellular diffusion through gap junctions of Lucifer dye was completely inhibited after 90 min of ATP depletion but was reversible. The volume-dependent and the cAMP-dependent chloride secretion were inhibited in a nonreversible way. Taken together, these results suggest that an ATP depletion in human airway epithelium, mimicking ischemia, may induce a marked alteration in the junctional complexes and cytoskeleton structure concomitantly with a loss of apical CFTR expression and chloride secretion function.
为模拟缺血对气道上皮完整性及囊性纤维化跨膜传导调节因子（CFTR）表达的影响，我们诱导上呼吸道细胞（鼻组织）和人支气管上皮16HBE14o-细胞系的呼吸上皮细胞发生ATP耗竭。组织学分析显示，ATP耗竭2小时导致基底细胞与柱状细胞界面处的上皮完整性丧失。连接蛋白43（Cx43，缝隙连接的亚基）以及桥粒斑蛋白1和2（DPs 1和2，桥粒的主要成分）蛋白的表达受到抑制。ATP耗竭90分钟后，观察到跨上皮电阻显著降低（25%），但该降低是可逆的。在16HBE14o-人支气管上皮细胞系中也获得了类似结果。ATP耗竭导致肌动蛋白丝解聚。纤毛细胞顶端区域成熟CFTR（170 kDa）和血影蛋白的表达下调。通过RT-聚合酶链反应动力学进行半定量分析，在鼻组织以及16HBE14o-人支气管上皮细胞系的均匀细胞群体中，整个ATP耗竭过程中CFTR、Cx43、DPs 1和2的mRNA稳态水平保持恒定。这表明这些蛋白的下调可能发生在转录后水平。ATP耗竭90分钟后，荧光素染料通过缝隙连接的细胞间扩散被完全抑制，但该抑制是可逆的。体积依赖性和cAMP依赖性氯分泌被不可逆地抑制。综上所述，这些结果表明，模拟缺血的人气道上皮细胞ATP耗竭可能诱导连接复合体和细胞骨架结构发生显著改变，同时伴有顶端CFTR表达丧失和氯分泌功能丧失。
