Abstract
Isoprostanes are biologically active molecules, produced when reactive oxygen species mediate the peroxidation of membrane polyunsaturated fatty acids. Previous work has demonstrated that the isoprostane 8-iso-prostaglandin E 2 (PGE2) stimulates cystic fibrosis transmembrane conductance regulator (CFTR)-mediated transepithelial anion secretion across the human airway epithelial cell line, Calu-3. Since isoprostanes predominantly achieve their effects via binding to prostanoid receptors, we hypothesized that this 8-iso-PGE2 stimulation of CFTR activity was the result of the isoprostane binding to a prostanoid receptor. Using RT-PCR, immunoblotting, and immunofluorescence, we here demonstrate that Calu-3 cells express the EP 1-4 and FP receptors, and localize these proteins in polarized cell monolayers. Using iodide efflux as a marker for CFTR-mediated Cl- efflux, we investigate whether prostanoid receptor agonists elicit a functional response from Calu-3 cells. Application of the agonists PGE2, misoprostol (EP2, EP3, and EP4) and PGE 1-OH (EP3 and EP4) stimulate iodide efflux; however, iloprost, butaprost, sulprostone, and fluoprostenol (agonists of the EP1, EP2, EP3, and FP receptors, respectively) have no effect. The iodide efflux seen with 8-iso-PGE2 is abolished by the EP4 receptor antagonist AH23848, the CFTR inhibitor 172, and inhibition of PKA and the PI3K pathway. In conclusion, we demonstrate that although Calu-3 cells possess numerous prostanoid receptors, only the EP 4 subtype appears capable of eliciting a functional iodide efflux response, which is mediated via the EP4 receptor. We propose that 8-iso-PGE2, acting via EP4 receptor, could play an important role in the CFTR-mediated response to oxidant stress, and which would be compromised in the CF airways.
Original language | English |
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Pages (from-to) | 143-152 |
Number of pages | 10 |
Journal | American Journal of Respiratory Cell and Molecular Biology |
Volume | 38 |
Issue number | 2 |
DOIs | |
Publication status | Published - Feb 2008 |
ASJC Scopus Subject Areas
- Molecular Biology
- Pulmonary and Respiratory Medicine
- Clinical Biochemistry
- Cell Biology