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The effect of N-acetylcysteine on chloride efflux from airway epithelial cells
Örebro University, School of Health and Medical Sciences.
Örebro University, School of Health and Medical Sciences.
Örebro University, School of Health and Medical Sciences.
CF-center, Department of Women's and Children's Health, Academic Hospital, SE-75185 Uppsala, Sweden.
2010 (English)In: Cell Biology International, ISSN 1065-6995, E-ISSN 1095-8355, Vol. 34, no 3, p. 245-252Article in journal (Refereed) Published
Abstract [en]

Defective chloride transport in epithelial cells increases mucus viscosity and leads to recurrent infections with high oxidative stress in patients with CF (cystic fibrosis). NAC (N-acetylcysteine) is a well known mucolytic and antioxidant drug, and an indirect precursor of glutathione. Since GSNO (S-nitrosoglutathione) previously has been shown to be able to promote Cl efflux from CF airway epithelial cells, it was investigated whether NAC also could stimulate Cl efflux from CF and non-CF epithelial cells and through which mechanisms. CFBE (CF bronchial epithelial cells) and normal bronchial epithelial cells (16HBE) were treated with 1 mM, 5 mM, 10 mM or 15 mM NAC for 4 h at 37°C. The effect of NAC on Cl transport was measured by Cl efflux measurements and by X-ray microanalysis. Cl efflux from CFBE cells was stimulated by NAC in a dose-dependent manner, with 10 mM NAC causing a significant increase in Cl efflux with nearly 80% in CFBE cells. The intracellular Cl concentration in CFBE cells was significantly decreased up to 60% after 4 h treatment with 10 mM NAC. Moreover immunocytochemistry and Western blot experiments revealed expression of CFTR channel on CFBE cells after treatment with 10 mM NAC. The stimulation of Cl efflux by NAC in CF airway epithelial cells may improve hydration of the mucus and thereby be beneficial for CF patients.

Place, publisher, year, edition, pages
London: Portland Press Ltd , 2010. Vol. 34, no 3, p. 245-252
Keyword [en]
airway epithelium, chloride transport, cystic fibrosis, N-acetylcysteine, Medical cell biology, Morphology, cell biology, pathology
National Category
Cell and Molecular Biology Medical and Health Sciences Cell and Molecular Biology
Research subject
Medicine
Identifiers
URN: urn:nbn:se:oru:diva-12451DOI: 10.1042/CBI20090007OAI: oai:DiVA.org:oru-12451DiVA: diva2:369841
Note
Georgia Varelogianni, Igor Oliynyk, Godfried M Roomans are also affiliated w. Department of Medical Cell Biology, Uppsala University, Box 571, SE-75123 Uppsala, SwedenAvailable from: 2010-11-12 Created: 2010-11-12 Last updated: 2018-01-12Bibliographically approved
In thesis
1. Advances in Pharmacological Treatment of Cystic Fibrosis
Open this publication in new window or tab >>Advances in Pharmacological Treatment of Cystic Fibrosis
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cystic fibrosis (CF) is an inborn, hereditary disease, due to mutations in the gene for a cAMP-activated chloride (Cl-) channel, the cystic fibrosis transmembrane conductance regulator (CFTR). As a result of impaired ion and water transport,the airway mucus is abnormally viscous, which leads to bacterial colonization.Recurrent infections and inflammation result in obstructive pulmonary disease.Similar changes in the pancreas lead to pancreatic insufficiency.Several compounds have been tested to improve transepithelial ion transport in CF patients, either via activation of the mutant CFTR, or via stimulation of alternative chloride channels. The main purpose of this thesis was to find substances that might correct the defective ion transport in epithelial cells in CFand could be useful for the pharmacological treatment of CF patients. Long-term treatment with the macrolide antibiotic azithromycin (AZM)improved clinical parameters and lung function in CF patients and increased Cl- transport in CF bronchial epithelial cells (CFBE) (Paper I); although mRNA expression of the CFTR gene remained unchanged.In contrast, pre-exposure to the mucolytic antioxidant N-acetylcysteine (NAC) increased CFTR protein expression and was associated with increased Cl- efflux from CFBE cells (Paper II). Clinical trials of this substance might be warranted. Duramycin has been the subject of clinical trials that finished in June2009. Up till now, no results from this study are available. The effect of this substance on Cl- efflux from three CF and three non-CF cell lines (Paper III) was disappointing. An effect was found only in CFBE cells, the effect was minimal, occurred in a narrow concentration range, and was not associated with an increase in the intracellular calcium concentration [Ca2+]i. The fact that NO-donors stimulated Cl- efflux from CFBE cells (but did notchange [Ca2+]i) after several hours of preincubation suggests that these substances may be a potentially interesting group of compounds for the treatment of CF (Paper IV). A model for the effect of NO-donors on Cl- efflux is presented.

Abstract [sv]

Cystisk fibros (CF) är en medfödd, ärftlig, sjukdom, som förorsakas av en mutation i en gen som innehåller koden för en kloridkanal som aktiveras av cykliskt AMP (cystic fibrosis transmembrane conductance regulator, CFTR). Som en följd av otillräcklig transport av joner och vatten är slemmet i luftvägarna onormalt segt, vilket leder till att det koloniseras av bakterier. Upprepade infektioneroch inflammation av luftvägarna leder slutligen till obstruktiv lungsjukdom.Liknande förändringar i bukspottkörteln leder till att också detta organ inte fungerar. Flera kemiska ämnen har testats för sin förmåga att förbättra jontransporten över epitelet hos CF-patienter. Detta skulle kunna göras antingen genom aktivering av det muterade CFTR-proteinet, eller genom stimulering av alternativa kloridkanaler. Huvudsyftet med den forskning som beskrivs i denna avhandling var att hitta kemiska substanser som skulle kunna korrigera den defekta jontransporten i epitelceller hos CF-patienter, och därför vara nyttiga för behandlingen av patienterna. Behandling under längre tid med azithromycin (AZM), ett makrolidantibiotikum,förbättrade CF-patienternas kliniska status och lungfunktion,samt ökade kloridutflödet från CF bronkialepitelceller (CFBE-celler) (Arbete I).Däremot ändrades inte uttrycket av mRNA för CFTR-genen. I kontrast till detta ökade uttrycket av CFTR-proteinet om CFBE-cellerna utsattes för den slemlösande anti-oxidanten N-acetylcystein (NAC), vilket ledde till ökat kloridutflöde från denna cellinje (Arbete II). Det vore rimligt att utföra kliniska prövningar av detta ämne. Duramycin har testats i kliniska prov som slutade i juni 2009, men några resultatfrån dessa prov har inte offentliggjorts än. Effekten av detta ämne på kloridutflödet från tre CF-cellinjer och tre icke-CF cellinjer (Arbete III) var en besvikelse. Duramycin hade endast effekt på CFBE-celler, effekten var mycket liten, förekom endast i ett litet koncentrationsområde av duramycin, och var inte kopplad till en ökning av den intracellulära kalciumkoncentrationen [Ca2+]i. Att ämnen som avger kväveoxid (NO) stimulerade kloridutflödet från CFceller (men inte påverkade [Ca2+]i) efter några timmar, visar att denna grupp av ämnen kan vara potentiellt intressant för behandlingen av CF (arbete IV). En modell för effekten av NO på kloridtransporten i CF-celler presenteras.

Place, publisher, year, edition, pages
Örebro: Örebro university, 2010. p. 88
Series
Örebro Studies in Medicine, ISSN 1652-4063 ; 50
Keyword
Cystic fibrosis, CFTR, chloride transport, N-acetylcysteine, NO-donors, duramycin, intracellular calcium, azithromycin
National Category
Medical and Health Sciences
Research subject
Medicine
Identifiers
urn:nbn:se:oru:diva-12424 (URN)978-91-7668-773-4 (ISBN)
Public defence
2010-12-17, Konferensrum Hjälmaren, USÖ, M-huset, S. Grev Rosengatan, Örebro, 09:15
Opponent
Supervisors
Available from: 2010-11-12 Created: 2010-11-08 Last updated: 2017-10-17Bibliographically approved
2. Chloride transport and inflammation in cystic fibrosis airways
Open this publication in new window or tab >>Chloride transport and inflammation in cystic fibrosis airways
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cystic fibrosis (CF) is one of the most common lethal, autosomal recessive inherited diseases among Caucasians. It is caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel activated by cyclic AMP. The disturbed electrolyte transport caused by the impaired CFTR channel leads to defective mucociliary clearance, airway obstruction and chronic airway infections.

Three different substances (N-acetylcysteine, duramycin and ambroxol) were tested in order to investigate if they could improve Cl- transport across epithelia and if so, through which mechanisms.

N-acetylcysteine (NAC), a well-known mucolytic and anti-oxidant drug increased significantly Cl-efflux from cystic fibrosis bronchial epithelial (CFBE) cells. It was shown that NAC can (partially) correct the Cl- transport deficiency in CF airway epithelial cells withF508-CFTR through a direct effect on CFTR (Paper I).

Duramycin increased Cl- efflux from CF airway epithelial cell in a very narrow concentration range. That effect is not associated with an increase in the intracellular calcium concentration (Paper II). The fact that results of a clinical study of duramycin, carried out several years ago, have not been published suggests that duramycin is not only ineffective in vitro, but also in vivo.

Ambroxol has been widely used as supplementary therapy for the treatment of many respiratory diseases. It was shown that 8h treatment with ambroxol caused a nearly six-fold increase of Cl- efflux from CF airway epithelial cells, which brings the Cl- efflux up to the level of cells with wild-type CFTR (Paper III).

Moreover, we studied the role of the innate immunity system in CF airway epithelial cells. High levels of Nod1 were found both in CF and non-CF airway epithelial cells, but a decreased expression of Nod2, and decreased levels of the pro-inflammatory cytokines IL-6 and IL-8 were found in CF airway epithelial cells compared to non-CF cells. The decreased levels of Nod2 suggest that the CF-cells are less able to recognize pathogens such as Pseudomonas aeruginosa, which might lead to a higher uptake of the pathogen (Paper IV).

Place, publisher, year, edition, pages
Örebro: Örebro universitet, 2011. p. 65
Series
Örebro Studies in Medicine, ISSN 1652-4063 ; 62
Keyword
Cystic fibrosis, CFTR, chloride efflux, N-acetylcysteine, ambroxol, duramycin, Nod-like receptors, cytokines
National Category
Medical and Health Sciences
Research subject
Medicine
Identifiers
urn:nbn:se:oru:diva-19126 (URN)978-91-7668-830-4 (ISBN)
Public defence
2011-11-25, Seminarierum Berguven (F1), Universitetssjukhuset, Örebro, 13:00 (English)
Opponent
Supervisors
Available from: 2011-10-04 Created: 2011-10-04 Last updated: 2017-10-17Bibliographically approved

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Publisher's full texthttp://www.cellbiolint.org/cbi/034/cbi0340245.htm

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Varelogianni, GeorgiaOliynyk, IgorRoomans, Godfried M.Johannesson, Marie

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