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Functional Aspects of Epithelia in Cystic Fibrosis and Asthma
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP activated chloride channel in the apical membrane of epithelial cells, is defective in patients with cystic fibrosis (CF). Research efforts are focused on chloride channel function in order to find a cure for the disease.

Genistein increased chloride transport in normal and delF508-CFTR cultured airway epithelial cells without cAMP stimulation. Prior pretreatment with phenylbutyrate did not affect the rate of the genistein-stimulated chloride efflux in these cells.

S-nitrosoglutathione is an endogenous bronchodilator, present in decreased amounts in the lungs of CF patients. We studied the effect of GSNO on chloride (Cl-) transport in primary nasal epithelial cells from CF patients homozygous for the delF508-CFTR mutation, as well as in two CF cell lines, using a fluorescent Cl- indicator and X-ray microanalysis. GSNO increased chloride efflux in the CF cell lines and in primary nasal epithelial cells from CF patients. This effect was partly mediated by CFTR. If the cells were exposed to GSNO in the presence of L-cysteine, Cl- transport was enhanced after 5 min, but not after 4 h. GSNO may be a candidate for pharmacological treatment of CF patients.

Chloride transport properties of cultured NCL-SG3 sweat gland cells were investigated. The CFTR protein was neither functional nor expressed in these cells. Ca2+-activated chloride conductance was confirmed and the putative Ca2+-activated chloride channel (CaCC) was further characterized in term of its pharmacological sensitivity.

Corticosteroids, the primary treatment for asthma, cause necrosis/apoptosis of airway epithelial cells. It was investigated whether a newer generation of drugs used in asthma, leukotriene receptor antagonists, had similar effects. Both montelukast and dexamethasone, but not beclomethasone or budesonide induced apoptosis/necrosis in superficial airway epithelial cells. Montelukast and corticosteroids also caused decreased expression of intercellular adhesion molecule -1 (ICAM-1) in epithelial but not endothelial cells.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2008. , p. 91
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 361
Keywords [en]
Cell biology, cystic fibrosis, CFTR, chloride transport, airway epithelium, sweat gland, asthma, corticosteroids, montelukast
Keywords [sv]
Cellbiologi
Identifiers
URN: urn:nbn:se:uu:diva-8905ISBN: 978-91-554-7224-5 (print)OAI: oai:DiVA.org:uu-8905DiVA, id: diva2:172206
Public defence
2008-06-05, B7:113a, BMC, Husargatan 3, Uppsala, 13:15
Opponent
Supervisors
Available from: 2008-05-15 Created: 2008-05-15 Last updated: 2013-09-20Bibliographically approved
List of papers
1. Activation of CFTR by genistein in human airway epithelial cell lines
Open this publication in new window or tab >>Activation of CFTR by genistein in human airway epithelial cell lines
2003 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 308, no 3, p. 518-522Article in journal (Refereed) Published
Abstract [en]

Cystic fibrosis (CF) is caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel expressed in epithelial cells. The effects of genistein and 4-phenylbutyrate (PBA) on CFTR were studied in three human airway epithelial cell lines expressing wild-type or DeltaF508 CFTR: Calu-3, CFSMEo-, and CFBE41o- cells. The cells were loaded with the fluorescent dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) and chloride efflux was studied. Forskolin and 3-isobutyl-1-methylxanthine (IBMX) induced chloride efflux in Calu-3 cells but not in CF lines. Genistein (2.5-50 microM) alone was able to induce chloride efflux in all cell lines. Genistein did not enhance the effect of forskolin and IBMX. PBA had little or no effect on genistein-induced chloride efflux. The effect of genistein seen at low concentrations makes genistein interesting for possible pharmacological treatment of CF, since it is known that similar concentrations can be obtained in plasma by a soy-rich diet.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-207884 (URN)10.1016/S0006-291X(03)01436-0 (DOI)12914781 (PubMedID)
Available from: 2013-09-20 Created: 2013-09-20 Last updated: 2017-12-06Bibliographically approved
2. Activation of chloride transport in CF airway epithelial cell lines and primary CF nasal epithelial cells by S-nitrosoglutathione
Open this publication in new window or tab >>Activation of chloride transport in CF airway epithelial cell lines and primary CF nasal epithelial cells by S-nitrosoglutathione
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2006 (English)In: Respiratory research (Online), ISSN 1465-9921, E-ISSN 1465-993X, Vol. 7, p. 124-Article in journal (Refereed) Published
Abstract [en]

Background: It has been suggested that low mu M concentrations of S-nitrosoglutathione (GSNO), an endogenous bronchodilator, may promote maturation of the defective cystic fibrosis (CF) transmembrane conductance regulator ( CFTR). Because nitric oxide ( NO) and GSNO levels appear to be low in the CF airway, there is an interest in the possibility that GSNO replacement could be of therapeutic benefit in CF.

Methods: The effect of GSNO on chloride (Cl-) transport was investigated in primary nasal epithelial cells obtained from CF patients homozygous for the delF508 mutation, as well as in two CF cell lines (CFBE and CFSME), using both a fluorescent Cl- indicator and X-ray microanalysis. Maturation of delF508 CFTR was determined by immunoblotting.

Results: Treatment with 60 mu M GSNO for 4 hours increased cAMP-induced chloride efflux in nasal epithelial cells from 18 out of 21 CF patients, but did not significantly affect Cl- efflux in cells from healthy controls. This Cl- efflux was confirmed by measurements with a fluorescent Cl- indicator in the CFBE and CFSME cell lines. The effect of GSNO on Cl- efflux in CFBE cells could be inhibited both by a specific thiazolidinone CFTR inhibitor (CFTRinh-172) and by 4,4'-diisothiocyanatodihydrostilbene- 2,2'-disulfonic acid (H2DIDS). X-ray microanalysis showed that, following 4 hours incubation with 60 mu M GSNO, cAMP agonists caused a decrease in the cellular Cl- concentration in CFBE cells, corresponding to Cl- efflux. GSNO exposure resulted in an increase in the protein expression and maturation, as shown by immunoblot analysis. GSNO did not increase the cytosolic Ca2+ concentration in cultured airway epithelial cells.

Conclusion: Previous studies have suggested that treatment with GSNO promotes maturation of delF508-CFTR, consistent with our results in this study. Here we show that GSNO increases chloride efflux, both in the two CF cell lines and in primary nasal epithelial cells from delF508-CF patients. This effect is at least in part mediated by CFTR. GSNO may be a candidate for pharmacological treatment of the defective chloride transport in CF epithelial cells.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-24748 (URN)10.1186/1465-9921-7-124 (DOI)000241188400001 ()17022806 (PubMedID)
Available from: 2007-02-07 Created: 2007-02-07 Last updated: 2017-12-07Bibliographically approved
3. The effect of S-nitrosoglutathione and L-cysteine on chloride efflux from cystic fibrosis airway epithelial cells
Open this publication in new window or tab >>The effect of S-nitrosoglutathione and L-cysteine on chloride efflux from cystic fibrosis airway epithelial cells
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2011 (English)In: Experimental and molecular pathology (Print), ISSN 0014-4800, E-ISSN 1096-0945, Vol. 90, no 1, p. 79-83Article in journal (Refereed) Published
Abstract [en]

The endogenous bronchodilator, S-nitrosoglutathione (GSNO), has been proposed as a possible pharmacological remedy that reverses the Delta F508-CFTR (cystic fibrosis transmembrane conductance regulator) maturation defect and increases CFTR-mediated chloride efflux in cultured cystic fibrosis airway epithelial cells (CFBE41o(-)). It has also been reported that L-cysteine enhanced S-nitrosothiol uptake and increased the intracellular S-nitrosothiol levels, likely through transnitrosation chemistry. The present study investigated whether L-cysteine augmented the effect of GSNO on chloride efflux from CF airway epithelial cells. Treatment with 10 mu M GSNO combined with 20 mu M L-cysteine resulted in increased chloride efflux from CFBE41o(-) cells after 5 minutes exposure compared to the control efflux rate and to the efflux rate in the presence of L-cysteine alone as measured using the fluorescent dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE). Chloride efflux rates from these cells after 4 h exposure to GSNO and L-cysteine were not different from control. Treatment with 10 mu M GSNO alone increased chloride efflux from CFBE41o(-) cells after 4 h but not at shorter incubation times. GSNO with or without L-cysteine did not alter epithelial tight junction integrity. In conclusion, a combination of GSNO with L-cysteine led to significant increase in chloride efflux in CFBE41o(-) cells but the effect was transient and not sustained beyond minutes.

Keywords
Cystic fibrosis, S-nitrosoglutathione, L-cysteine, Chloride efflux, Airway epithelial cells
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-148133 (URN)10.1016/j.yexmp.2010.10.005 (DOI)000286349300013 ()20965165 (PubMedID)
Available from: 2011-03-02 Created: 2011-03-02 Last updated: 2017-12-11Bibliographically approved
4. Chloride transport in NCL-SG3 sweat gland cells: channels involved
Open this publication in new window or tab >>Chloride transport in NCL-SG3 sweat gland cells: channels involved
2007 (English)In: Experimental and molecular pathology (Print), ISSN 0014-4800, E-ISSN 1096-0945, Vol. 83, no 1, p. 47-53Article in journal (Refereed) Published
Abstract [en]

The aim of the study was to assess whether NCL-SG3, the only immortalized sweat gland cell line available, can be used as an in vitro model to study chloride ion transport in cultured sweat gland cells. Cl efflux was measured using the MQAE dye fluorescence technique after stimulating the cells with different agonists. A significant stimulation of chloride efflux was achieved with the calcium ionophore A23187 resulting in an efflux rate of 0.9 mM/s. Both ATP and UTP activated chloride efflux in these cells, with the ATP response being larger. IBMX and forskolin stimulation did not induce a rate of chloride efflux above the basal level. Immunocytochemistry showed no detectable CFTR in NCL-SG3 cells. This finding was confirmed with flow cytometry analysis. Niflumic acid (20 and 100 μM NFA) and 4,4′-diisothiocyanatodihydrostilbene-2,2′-disulfonic acid (H2DIDS) (100 ìM) decreased the rate of ATP-stimulated chloride efflux significantly (0.40 and 0.31 mM/s with NFA, 0.37 mM/s with H2DIDS). Gadolinium (20 ìM) had no effect on the chloride transport rate. In conclusion, the NCL-SG3 cells retain some of the aspects of human sweat gland epithelium, such as the ability to form cell–cell contacts. The CFTR protein is neither functional nor expressed in cultured NCL-SG3 sweat gland cells. Ca2+-activated chloride conductance is confirmed and the putative Ca2+-activated chloride channel (CaCC) is further characterized in term of its pharmacological sensitivity. The NCL-SG3 sweat gland cell line can be used to investigate the characteristics of the CaCC and to identify the channel.

Keywords
Biological Markers, Calcium/metabolism, Cell Line, Chloride Channels/*metabolism, Chlorides/*metabolism, Epithelial Cells/metabolism, Humans, Immunohistochemistry, Ion Channel Gating, Ion Transport, Microscopy; Electron; Transmission, Sweat Glands/*metabolism/ultrastructure
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-17161 (URN)10.1016/j.yexmp.2007.02.003 (DOI)000247716800008 ()17383636 (PubMedID)
Available from: 2008-06-17 Created: 2008-06-17 Last updated: 2017-12-08Bibliographically approved
5. Corticosteroids and Montelukast: Effects on Airway Epithelial and Human Umbilical Vein Endothelial Cells
Open this publication in new window or tab >>Corticosteroids and Montelukast: Effects on Airway Epithelial and Human Umbilical Vein Endothelial Cells
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2010 (English)In: Lung, ISSN 0341-2040, E-ISSN 1432-1750, Vol. 188, no 3, p. 209-216Article in journal (Refereed) Published
Abstract [en]

Our primary objective was to investigate the possible contribution of controller medications to asthmatic airway remodeling, by (1) comparing the apoptotic and necrotic effects of several corticosteroids and montelukast on cultured airway human bronchial surface epithelial (16HBE) and submucosal (Calu3) cells; (2) measuring epithelial shedding potential and desmosome length in response to a cytokine challenge, with or without co-administered corticosteroids; and (3) studying corticosteroids and montelukast effects on inter-cellular adhesion molecule (ICAM) expression in both 16HBE and human umbilical vein endothelial cells (HUVEC). For this purpose, apoptosis, necrosis, and ICAM expression were quantified by flow cytometry, with 16HBE cells sensitive to both the apoptotic and necrotic effects of dexamethasone and montelukast; Calu3 cells sensitive only to budesonide. Transmission electron microscopy revealed decreased desmosome length in the presence of cytokines (TNF-alpha and INF-gamma), with corticosteroids counteracting this reduction. Dexamethasone, beclomethasone, and montelukast decreased versus increased ICAM-1 expression in airway epithelial cells and HUVEC, respectively. For conclusions, bronchial surface epithelial and submucosal cells exhibit a different sensitivity profile toward dexamethasone, budesonide, and montelukast, with corticosteroids preventing cytokineinduced desmosomal damage in 16HBE cells. The studied drugs led to increased ICAM-1 expression in endothelium, potentially facilitating inflammatory cell migration into lung tissue.

Keywords
Corticosteroids, Leukotriene receptor antagonist, Montelukast, Airway epithelium, Apoptosis, Desmosomes, ICAM
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-136340 (URN)10.1007/s00408-010-9227-6 (DOI)000276910400004 ()
Available from: 2010-12-13 Created: 2010-12-11 Last updated: 2017-12-11Bibliographically approved

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