slides
Chloride transport in normal and cystic fibrosis epithelial cells
- Publication date
- 29 January 1992
- Publisher
- The experimental work described in this thesis was aimed at a better
understanding of the pathogenesis of CF with the emphasis on
electrophysiological studies on Cl" transport. Chloride transport was studied with
Ussing chamber, isotopic ('25I")-efflux and fluorescent (SPQ) measurements as
well as with the patch-damp technique. In search for a suitable cell culturing
model, we studied C!" transport in CF keratinocytes, which are easy to obtain and
would provide us with sufficient cell material (Chapter 3). However, the use of
keratinocytes appeared to be limited to primary cell cultures which can only be
passaged for maximal 10 times, necessatating the use of many different and often
heterogeneous N and CF cell lines necessary. The availibility of a continuously
growing cell line with CF genotype and phenotype would provide a more
homogeneous model for biochemical, electrophysiological and genetic studies. We
have therefore immortalized a CF nasal polyp cell line with a hybrid SV40/ Ad12
virus, and have characterized this cell line biochemically and electrophysiologically
as discussed in Chapter 4. A continuously growing homogeneous cell line selected
for its chloride secreting characteristics would also he of much interest for CF research. The subclone 19A of the human colon carcinoma cell line HI29 is such
a cell line, easy to maintain in culture without the multiple addition;; to the
medium required for culturing keratinocytes and primary nasal polyp cells and
without the use of feeder cells. These colonocytes can be coru;idered as a model
for the study of Cl路 transport in normal cells. In Chapter 5 we describe the patchclamp
experiments with HI29.ci19A cells performed to characterize the cAMPdependent
regulation of the outwardly rectifYing chloride channel which was
thought to be defectively regulated in CF. Apart from cAMP regulation of
chloride channels, we also found that G-proteiru; are involved in the regulation
of a different type of chloride channel in intestinal membranes. Chapter 6
describes the vesicle and patch-damp experiments leading to the identification of
a novel potential signal transduction pathway for chloride transport in intestinal
cells.
The homology between CFIR and different types of pump proteins like MDR led
us to verify the hypothesis that CFIR could function as a pump with substrates
exerting secondary effects on chloride channels. In Chapter 7 MDR-substrates and
-blockers were tested on epithelial chloride traru;port and on the outwardly
rectifYing chloride channel in particular.