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8-iso-PGE₂ Stimulates Anion Efflux from Airway Epithelial Cells via the EP₄ Prostanoid Receptor

¹ Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada

Correspondence and requests for reprints should be addressed to Dr. Elizabeth A. Cowley, Department of Physiology and Biophysics, Dalhousie University, Halifax, NS, B3H 4H7 Canada. E-mail: elizabeth.cowley{at}dal.ca

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 E₂ (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 PGE₁-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 EP₄ 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.

Key Words: oxidant stress • Calu-3 cells • iodide efflux • isoprostane

CLINICAL RELEVANCE This article contains important new information that will be of considerable interest to researchers in the cystic fibrosis community, and to scientists concerned with airway physiology and inflammatory lung disease.