Effects of CFTR gene silencing by siRNA or the luminal application of a CFTR activator on fluid secretion from guinea-pig pancreatic duct cells
| Autor: | Hidemi Goto, Shigeru B. H. Ko, Michael A. Gray, Sakiko Azuma, Hiroshi Ishiguro, Akiko Yamamoto, Frédéric Becq, Kaori Kamimura, Miyuki Nakakuki, Haizhen Song |
|---|---|
| Rok vydání: | 2011 |
| Předmět: |
congenital
hereditary and neonatal diseases and abnormalities Small interfering RNA Guinea Pigs Biophysics Cystic Fibrosis Transmembrane Conductance Regulator In Vitro Techniques Biology Biochemistry Sequence Analysis Protein medicine Animals Humans Secretion Gene Silencing RNA Small Interfering Molecular Biology Pancreatic duct Aquaporin 1 Activator (genetics) Pancreatic Ducts Sequence Analysis DNA Cell Biology respiratory system Molecular biology Cystic fibrosis transmembrane conductance regulator Small intestine Body Fluids medicine.anatomical_structure Gene Knockdown Techniques biology.protein Chloride channel Female Pancreas Quinolizines |
| Zdroj: | Biochemical and Biophysical Research Communications. 410:904-909 |
| ISSN: | 0006-291X |
| DOI: | 10.1016/j.bbrc.2011.06.093 |
| Popis: | 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 ∼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. |
| Databáze: | OpenAIRE |
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