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Effect of NO-donors on chloride efflux and intracellular Ca2+ concentration in cystic fibrosis airway epithelial cells
Örebro University, School of Health and Medical Sciences.
Örebro University, School of Health and Medical Sciences.
Department of Pediatrics, University of Wellington School of Medicine, Wellington, New Zealand.
Department of Pediatric Respiratory Medicine, University of Virginia School of Medicine, Charlottesville, VA, USA.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Previous studies have shown that the endogenous bronchodilator, S-nitrosglutathione (GSNO), causes a 4–fold increase in CFTR-mediated chloride (Cl) efflux and improves the trafficking of CFTR to the plasma membrane. The NO-donor GEA3162 had a similar, but smaller, effect on Cl efflux. In order to investigate whether the NO-donor properties of GSNO were relevant for its effect on Cl efflux from airway epithelial cells, the effect of a number of other NO-donors (SNP, SNAP, DETA-NO, and DEA-NONOate) on Cl efflux from CFBE (DF508/DF508) airway epithelial cells was tested. Cl efflux was determined using the fluorescent MQAE-technique. Possible changes in the intracellular Ca2+ concentration were tested by the fluorescent fluo-4 method in a confocal microscope system. Like GSNO, SNP had no immediate effect on Cl efflux, but after 4h incubation with the NO-donor, an increased Cl efflux was found (in the order SNAP > DETA-NO > DEA- NONOate > SNP). The effect of DEA-NONOate on Cl efflux was not significant, and the compound may have (unspecific) deleterious effects on the CFBE cells. None of the compounds that caused significant Cl efflux caused significant changes in the intracellular Ca2+ concentration. It is concluded that the effect of GSNO is, at least in part, due to its properties as an NO-donor, and that the effect is likely to be mediated by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), not on Ca2+-activated Cl channels.

Keywords [en]
Nitric oxide donors, cystic fibrosis, airway epithelium, chloride channels, medical cell biology
National Category
Medical and Health Sciences
Research subject
Medicine
Identifiers
URN: urn:nbn:se:oru:diva-12446OAI: oai:DiVA.org:oru-12446DiVA, id: diva2:369725
Available from: 2010-11-11 Created: 2010-11-11 Last updated: 2017-10-17Bibliographically 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
Keywords
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

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