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Determination of chloride efflux by X-ray microanalysis versus MQAE-fluorescence
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
2002 (English)In: Microscopy research and technique (Print), ISSN 1059-910X, E-ISSN 1097-0029, Vol. 59, no 6, 531-355 p.Article in journal (Refereed) Published
Abstract [en]

The importance of chloride channels for the cell is demonstrated by a number of serious human diseases that are due to mutations in chloride channels. The most well-known of these diseases is cystic fibrosis. Investigations into the mechanisms of the disease and possible treatments require the study of chloride fluxes at the level of individual cells. The present study compares two methods for studies of chloride transport: X-ray microanalysis and MQAE fluorescence with image analysis. As an experimental system, the cAMP-activated chloride channel in cultured respiratory epithelial cells was chosen. Both methods showed that stimulation with the cAMP-elevating agents forskolin and IBMX decreased the chloride content of the cells by about 20-27%. Inducing a driving force for chloride by replacing extracellular chloride by nitrate resulted in a chloride efflux that was significantly increased in the presence of forskolin and IBMX. This study shows that X-ray microanalysis and MQAE fluorescence are adequate and comparable methods for measuring cAMP-dependent chloride transport in individual cells.

Place, publisher, year, edition, pages
2002. Vol. 59, no 6, 531-355 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-89969DOI: 10.1002/jemt.10234PubMedID: 12467030OAI: oai:DiVA.org:uu-89969DiVA: diva2:161957
Available from: 2002-10-11 Created: 2002-10-11 Last updated: 2013-03-22Bibliographically approved
In thesis
1. Towards Pharmacological Treatment of Cystic Fibrosis
Open this publication in new window or tab >>Towards Pharmacological Treatment of Cystic Fibrosis
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

S-nitrosogluthatione is an endogenous substance, present at decreased levels in the lungs of CF patients and was recently found to induce mature CFTR in airway epithelial CF cell lines. We show that S-nitrosoglutathione in physiological concentrations increases the presence of ΔF508 CFTR in the cell membrane and induces cAMP dependent chloride transport in cystic fibrosis airway epithelial cells. The properties of S-nitrosoglutathione include other potential benefits for the CF patient and make this agent an interesting candidate for pharmacological treatment of CF that needs to be further evaluated.

Genistein was found to increase the chloride efflux in both normal and ΔF508 cells without stimulation of cAMP elevating agents and without prior treatment with phenylbutyrate. Genistein, in concentrations close to those that can be detected in plasma after a high soy diet, could induce chloride efflux in cells with the ΔF508 CFTR mutation and its possible use in the treatment of CF should therefore be further investigated.

Studies on nasal epithelial cells from CF patients showed cAMP dependent chloride efflux in some of the patients with severe genotypes. This may complicate in vitro evaluation of clinical treatment of these patients. The presence of cAMP dependent chloride transport did not necessarily lead to a milder phenotype. Other factors than CFTR may influence the clinical development of the disease.

Cystic fibrosis (CF) is the most common monogenetic disease among Caucasians. A defective cAMP regulated chloride channel (cystic fibrosis transmembrane conductance regulator, CFTR) in epithelial cells leads to viscous mucus, bacterial infections, inflammation and tissue damage in the lungs that cause death in 95% of the cystic fibrosis patients. There is no cure for the disease although existing treatment has dramatically prolonged the life expectancy. The aim of this thesis was to study pharmacological agents for their ability to restore the cellular deficiency in CF airway epithelial cells. X-ray microanalysis, MQAE fluorescence and immunocytochemistry were used to evaluate the effects.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2002. 53 p.
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 0282-7476 ; 1185
Cell biology, cystic fibrosis, airway epithelium, genotype, CFTR, chloride transport, genistein, phenotype, phenylbutyrate, S-nitrosoglutathione, Cellbiologi
National Category
Cell and Molecular Biology
Research subject
urn:nbn:se:uu:diva-2634 (URN)91-554-5409-7 (ISBN)
Public defence
2002-11-01, B21, BMC, Uppsala, 09:15
Available from: 2002-10-11 Created: 2002-10-11Bibliographically approved

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