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A modified technique for the impregnation of lanthanum tracer to study the integrity of tight junctions on cells grown on a permeable substrate
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.
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.
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2006 (English)In: Microscopy research and technique (Print), ISSN 1059-910X, E-ISSN 1097-0029, Vol. 69, no 10, 776-783 p.Article in journal (Refereed) Published
Abstract [en]

Ionic lanthanum is commonly used to trace permeability pathways across epithelia and endothelia in biological electron microscopy. A method for obtaining a uniformly dense precipitate of lanthanum is described. The method, which is a modification of the technique described by Shaklai and Tavassoli (1977) was suitable for fixation of cell cultures grown on permeable filter inserts and was successfully applied to study opening of tight junctions by hypertonic solutions in the airway epithelial cell line 16HBE14o(-). The preparation method formed the basis for a semi-quantitative morphological determination in which the tight junctions were subdivided as "intact," "weakened," and "open." By using this modified technique, it could be demonstrated that opening of tight junctions in airway epithelial cells increased, with increasing osmolarity with electrolytes having a stronger effect than nonelectrolytes. A significant linear relationship was found between the osmolarity of the medium and the open state of the tight junctions (as determined by the semi-quantitative morphological technique) or the transepithelial electrical resistance.

Place, publisher, year, edition, pages
2006. Vol. 69, no 10, 776-783 p.
Keyword [en]
lanthanum impregnation, airway epithelial cells, permeable substrate, tight junctions, hypertonic conditions, transepithelial resistance, permeability
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-24744DOI: 10.1002/jemt.20347ISI: 000241261400002PubMedID: 16865714OAI: oai:DiVA.org:uu-24744DiVA: diva2:52518
Available from: 2007-02-07 Created: 2007-02-07 Last updated: 2017-12-07Bibliographically 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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Nilsson, HarrietDragomir, AncaAhlander, AndersLjungkvist, MarianneRoomans, Godfried M.

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