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CFTR and tight junctions in cultured bronchial epithelial cells
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. (Godfried Roomans)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology. (Godfried Roomans)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. (Per Artursson)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Family Medicine and Clinical Epidemiology.
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2010 (English)In: Experimental and molecular pathology (Print), ISSN 0014-4800, E-ISSN 1096-0945, Vol. 88, no 1, 118-127 p.Article in journal (Refereed) Published
Abstract [en]

Airway epithelial salt and water transport takes place through paracellular and transcellular pathways. This transport depends critically on the epithelial sodium channel (ENaC) and the cystic fibrosis transmembrane conductance regulator (CFTR), operating in concert with the paracellular pathway through the tight junctions (TJs). Normal (16HBE14o-), cystic fibrosis (CFBE41o-), and corrected CFBE41o- (CFBE41o pCep4) airway epithelial cell lines were cultured under isotonic conditions. Transepithelial electrical resistance (TEER) was measured as indicator of the tightness of the cultures. Morphology was investigated by immunofluorescence and paracellular permeability by lanthanum nitrate or [14C] mannitol as permeability markers. CFBE41o pCep4 cells developed lower TEER than CFBE41o- cells. Addition of a specific inhibitor of CFTR (CFTRinh-172) to 16HBE14o- and CFBE pCep4 cells resulted in a time-dependent increase in TEER whereas stimulation of CFTR by IBMX and forskolin caused a decrease. Permeability to lanthanum and [14C] mannitol was lower in 16HBE14o- cells exposed to CFTRinh-172 and in CFBE41o- cells compared to untreated 16HBE14o- and CFBE41o pCep4 cells, respectively. 16HBE14o- cells exposed to IBMX and forskolin showed higher permeability to lanthanum but lower permeability to [14C] mannitol compared to control. Immunofluorescence revealed a disorganisation of F-actin and a-tubulin in 16HBE14o- cells exposed to CFTRinh-172, which was not seen in untreated cultures. A higher degree of disorganised F-actin and a-tubulin was also seen in CFBE41o- cells compared to CFBE41o- pCep4 cells. Changes in F-actin and a-tubulin in 16HBE14o- cells exposed to IBMX and forskolin were also seen, although these were less apparent. These results suggest the possibility of an interaction between the activity of CFTR and the TJ protein complex, probably via the cytoskeleton.

Place, publisher, year, edition, pages
2010. Vol. 88, no 1, 118-127 p.
Keyword [en]
CFTR, transepithelial electrical resistance, tight junctions, cytoskeleton
National Category
Medical and Health Sciences
Research subject
Biology with specialization in Molecular Cell Biology; Molecular Medicine
Identifiers
URN: urn:nbn:se:uu:diva-99427DOI: 10.1016/j.yexmp.2009.09.018ISI: 000274500100017PubMedID: 19818767OAI: oai:DiVA.org:uu-99427DiVA: diva2:208571
Available from: 2009-03-18 Created: 2009-03-13 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Studies of Tight Junctions and Airway Surface Liquid in Airway Epithelium with Relevance to Cystic Fibrosis
Open this publication in new window or tab >>Studies of Tight Junctions and Airway Surface Liquid in Airway Epithelium with Relevance to Cystic Fibrosis
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cystic fibrosis (CF) is a multi-organ autosomal recessive disease of fluid-transporting epithelia, due to a mutation in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. CFTR is a cAMP-regulated Cl-channel involved in various regulatory processes. Salt and water transport depend on CFTR and the epithelial sodium channel (ENaC), operating in concert with the paracellular pathway through the tight junctions (TJ). The ionic composition of the ASL has been assumed to be altered in CF, resulting in a fatal accumulation of viscous mucus in the airways.

ASL samples were collected from tracheal and nasal fluid in normal and transgenic CF mice and from the fluid covering the apical surface of normal bronchial cells (16HBE14o-) and a CF human bronchial cell line (CFBE41o-). Analysis of the elemental content of the ASL was performed by X-ray microanalysis. The ASL contained more Na and Cl in CFTR-deficient or DF508-CFTR-containing cells than in control cells with wild- type CFTR.

The relation between osmolarity and TJ permeability was examined by the addition of salt or sugar (295-700 mOsm) to 16HBE14o- cells, where the integrity of TJ was evaluated by transepithelial electrical resistance (TEER) measurements. Studies of interaction between the activity of CFTR, TJ and cytoskeleton were performed in CFBE41o-, plasmid corrected CFBE41o- (CFBE41o pCep4), and 16HBE14o- cells exposed to an inhibitor of CFTR (CFTRinh-172). The TJ were investigated by determining the paracellular permeability to lanthanum ions or with [14C] mannitol. Cytoskeletal changes were evaluated by immunofluorescence.

Hyperosmotic stress resulted in opening of TJ. Inhalation of hypertonic salt or sugar solutions may open TJ, leading to enhanced paracellular water transport and increased ASL volume, diluted mucus and enhanced mucociliary clearance. This may explain the beneficial effect of this treatment for CF-patients.

In healthy airway epithelial cells, inhibition of CFTR by CFTRinh-172 resulted in an increased TEER, whereas stimulation of CFTR by IBMX/forskolin caused a decrease. The paracellular permeability was inversely proportional to TEER. Immunofluorescence revealed a disorganization of cytoskeletal proteins in CF-cells. These results point toward a possible interaction between the activity of CFTR and TJ protein complex, presumably via the cytoskeleton.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 91 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 435
Identifiers
urn:nbn:se:uu:diva-99411 (URN)978-91-554-7464-5 (ISBN)
Public defence
2009-04-27, B:7:101a, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2009-04-06 Created: 2009-03-13 Last updated: 2010-01-13Bibliographically approved

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