Effects of CFTR gene silencing by siRNA or the luminal application of a CFTR activator on fluid secretion from guinea-pig pancreatic duct cells
Effects of CFTR gene silencing by siRNA or the luminal application of a CFTR activator on fluid secretion from guinea-pig pancreatic duct cells
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DOI：
10.1016/j.bbrc.2011.06.093
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发表时间：
2011-07-15
影响因子：
3.1
通讯作者：
Goto, Hidemi
中科院分区：
文献类型：
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作者：
Ko, Shigeru B. H.;Yamamoto, Akiko;Goto, Hidemi
目标：囊性纤维化跨膜传导调节因子（CFTR）是在胰管细胞的顶端质膜中表达的环AMP调节的氯离子通道，其中其在液体分泌中起重要作用。本研究的目的是阐明CFTR氯离子通道对豚鼠胰腺离子和液体分泌的作用，方法是在分离培养的胰管中，通过RNA干扰或通过腔应用CFTR选择性激活剂MPB 91来操纵CFTR的表达。使用从豚鼠小肠中分离的cDNA，通过聚合酶链反应产生CFTR基因片段并直接测序。根据获得的序列设计了两种不同的小干扰RNA（siRNA）的RNA双链体。使用视频显微镜测量来自分离的豚鼠胰管的液体分泌。结果：豚鼠CFTR由1481个氨基酸残基组成，在胰管中表达的CFTR氯通道和水通道分别用CFTR和水通道AQP1的抗体进行免疫印迹。发现在人CFTR的氨基酸残基403和404之间插入另外的谷氨酰胺残基。与MPB91不存在下的液体分泌相比，在MPB91存在下，毛喉素刺激的完整胰管的液体分泌显著更高。与来自用乱序阴性对照dsRNA转染的导管的液体分泌相比，用CFTR特异性siRNA转染的胰管中的基础和毛喉素刺激的液体分泌减少了类似于50%。CFTR和AQP1蛋白的量分别减少到对照的34%和45%，respectively：CFTR氯离子通道的活性或CFTR蛋白的表达量决定了离体豚鼠胰管的液体分泌速率。(C)2011 Elsevier Inc. All rights reserved.
Aims: The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP regulated chloride channel expressed in the apical plasma membrane of pancreatic duct cells where it plays an important role in fluid secretion. The purpose of this study was to elucidate the role of the CFTR chloride channel on ion and fluid secretion from the guinea-pig pancreas by manipulating the expression of CFTR by RNA interference or by luminal application of a CFTR selective activator, MPB91, in isolated cultured pancreatic ducts.Materials and methods: Using cDNA isolated from the guinea-pig small intestine, fragments of the CFTR gene were generated by polymerase chain reaction and directly sequenced. Two different RNA duplexes for small interference RNA (siRNA) were designed from the sequence obtained. Fluid secretion from the isolated guinea-pig pancreatic ducts was measured using video-microscopy. The amount of CFTR chloride channel or AQP1 water channel expressed in pancreatic ducts was examined by immunoblotting with antibodies against CFTR or AQP1, respectively.Results: Guinea-pig CFTR consists of 1481 amino acid residues. An additional glutamine residue was found to be inserted between amino acid residues 403 and 404 of human CFTR. Forskolin-stimulated fluid secretion from intact pancreatic ducts was significantly higher in the presence of MPB91 compared to fluid secretion in the absence of MPB91. Both basal and forskolin-stimulated fluid secretion in pancreatic ducts transfected with CFTR specific siRNAs were reduced by similar to 50% compared to fluid secretion from ducts transfected with scrambled negative control dsRNAs. The amount of CFTR and AQP1 proteins was reduced to 34% and 45% of control, respectively.Conclusions: The activity of the CFTR chloride channel or the amount of CFTR protein expressed determines the rate of fluid secretion from the isolated guinea-pig pancreatic ducts. (C) 2011 Elsevier Inc. All rights reserved.