The objective of this research is to develop and characterize long- term cultures of the cells from the reabsorptive duct of human eccrine sweat glands, from CF patients and controls, with documentation that these cell lines express the CF and control differentiated phenotypes regarding ion transport properties. The capability of prolonged propagation of large numbers of this primarily affected cell type will greatly facilitate the efforts of molecular geneticists to identify the specific CF genetic abnormality and of physiologists/biochemists to delineate how it is expressed. The proposal describes a consortium of investigators who are already working with primary outgrowth cultures of sweat duct cells. Two concurrent approaches are being taken to attain long- term, expanded cultures, viz. (1) optimization of culture conditions using several growth promoters including feeder cells, and (2) immortalization of cell lines through viral transformation which will be attempted with several agents including SV40 constructs and Epstein-Barr virus. Culture conditions are being developed which induce differentiated transepithelial function -- defined as high, amiloride-sensitive short-circuit current and high (control) or low (CF) Cl- conductance. Cell lines expressing the expected ion conductance properties will be subjected to extensive functional, morphological, and biochemical characterization especially with regard to transepithelial ion transport and patch- clamp studies of ion channels and their regulation. These parameters will serve to establish the functional cell type and its physiological state of differentiation. Based on our current knowledge of the CF ion transport phenotype, several approaches to the development of a screening assay to detect the CF phenotype will be pursued including radioactive tracer (e.g.36C1-) uptake and fluorescent monitoring of cytosolic pH, membrane potential, and perhaps cytosolic (Cl-).
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