Abstract
Oxidative stress is implicated in the pathogenesis of many inflammatory pulmonary diseases, including cystic fibrosis (CF). Delineating how oxidative stress stimulates CF transmembrane conductance regulator (CFTR) in airway epithelial cells is useful, both to increase the understanding of airways host defense and suggest therapeutic approaches to reduce the oxidant stress burden in the CF lung. Using the airway epithelial cell line Calu-3, we investigated the hypothesis that hydrogen peroxide (H2O2), which stimulates anion efflux through CFTR, does so via the production of prostaglandin E2 (PGE2). Using iodide efflux as a biochemical marker of CFTR activity and short circuit current (Isc) recordings, we found that the H2O2-stimulated efflux was abolished by cyclooxygenase-1 inhibition and potentially also involves microsomal prostaglandin E synthase-1 activity, implicating a role for PGE2 production. Furthermore, H2O2 application resulted in a rapid release of PGE2 from Calu-3 cells. We additionally hypothesized that the PGE2 subtype 4 (EP4) receptor was involved in mediating this response. In the presence of (4Z)-7-[(rel-1S,2S,5R)-5-((1,1′-biphenyl-4-yl)methoxy)-2-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid (AH23848) (which blocks the EP4 receptor), the H2O2-stimulated response was abolished. To investigate this finding in a polarized system, we measured the increase in Isc induced by H2O2 addition in the presence and absence of AH23848. H2O2 induced a robust increase in Isc, which was significantly attenuated in the presence of AH23848, suggesting some role for the EP4 receptor. In conclusion, with H2O2 as a model oxidant stress, stimulation of CFTR seems to involve PGE2 production and likely EP4 receptor activation in Calu-3 airway epithelial cells. This mechanism would be compromised in the CF airways.
Footnotes
This work was supported by Cystic Fibrosis Canada.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS:
- CF
- cystic fibrosis
- CFTR
- CF transmembrane conductance regulator
- PG
- prostaglandin
- PGE2
- prostaglandin E2
- EP4
- PGE2 subtype 4
- ROS
- reactive oxygen species
- H2O2
- hydrogen peroxide
- COX
- cyclooxygenase
- Isc
- short circuit current
- mPGES-1
- microsomal prostaglandin E synthase-1
- HPRT
- hypoxanthine guanine phosphoribosyltransferase
- PCR
- polymerase chain reaction
- qPCR
- quantitative PCR
- RT-PCR
- reverse transcription-PCR
- ELISA
- enzyme-linked immunosorbent assay
- MAFP
- methyl arachidonyl fluorophosphate
- ATK
- arachidonil-trifluoromethyl-ketone
- ANOVA
- analysis of variance
- TP
- thromboxane
- PI3K
- phosphotidylinositol-3-kinase
- PLA2
- phospholipase A2
- cPLA2
- cytosolic PLA2
- IL
- interleukin
- INDO
- indomethacin
- SC560
- 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole
- Cay10404
- 3-(4-methylsulphonylphenyl)-4-phenyl-5-trifluoromethylisoxazole
- Cay10589
- 2-[[4-[([1,1′-biphenyl]-4-ylmethyl)amino]-6-chloro-2-pyrimidinyl]thio]-octanoic acid
- Cay10526
- 4-(benzo[b]thiophen-2-yl)-3-bromo-5-hydroxydihydrofuran-2(3H)-one
- AH23848
- (4Z)-7-[(rel-1S,2S,5R)-5-((1,1′-biphenyl-4-yl)methoxy)-2-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid
- GlyH101
- N-(naphthalen-2-ylamino)-acetic acid(3,5-dibromo-2,4,-dihydroxy-benzylidene)-hydrazide
- SQ29,548
- [1S-[1α,2α(Z),3α,4α]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid
- LY-294002
- 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one.
- Received August 19, 2011.
- Accepted February 13, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics
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