Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an integral membrane protein which probably functions as a cAMP-dependent chloride channel, cause the disease cystic fibrosis (CF). In addition to the failure of cAMP-regulated chloride secretion, the CF phenotype in the lung includes increased net sodium reabsorption, increased sulfation of macromolecules, and increased vulnerability to chronic pseudomonas infection. It is uncertain whether these features of CF are a consequence of the chloride transport defect, a consequence of abnormal processing of mutant CFTR in intracellular compartments, or a consequence of another action of CFTR. By overexpressing a pseudogene for thc R domain of the CFTR molecule in an airway epithelial cell line, we have developed a cell line (pCEP-R 9HTEo-) which lacks cAMP-dependent chloride secretion, but does not express mutant CFTR. This line affords the opportunity to distinguish between the consequences of the mutant protein per se and the ion transport defect. As controls we will use 9HTEo- cells line transfected only with the parent plasmid, pCEP, and a cell line which expresses antisense to CFTR (pCEP-AS). We will assess sodium transport, the composition of macromolecules, and adherence of pseudomonas in these cells. In addition to the 9HTEo- lines, we will develop other sets of cell lines which over express the R domain or antisense, including the normal airway cell line Steph, which forms tight junctions and is thus suitable for testing in the Ussing chamber, an easier system for assessing epithelial electrolyte transport; and the intestinal cell line HT29, a subclone of which has been induced to package in granules and secrete mucin. These lines will be used for detailed studies of sodium transport and macromolecular processing and secretion respectively. Finally, we will develop a transgenic mouse which expresses the R domain in its airway epithelial cells driven by the CC-10 promoter. After verifying that the chloride transport phenotype in the mice is similar to that observed in the human cells, we will test in these animals the impact of failure of regulated chloride secretion on growth, lung histopathology vunerablity to acute and chronic pseudomonas infection, composition of macromolecular secretion, and sodium transport. Using these models in vitro and in vivo, we will obtain a clearer picture of the pathophysiology of CF in the lung.
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