title: 8-iso-PGE2 stimulates anion efflux from airway epithelial cells via the EP4 prostanoid receptor.
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) (PGE(2)) 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-PGE(2) 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 PGE(2), misoprostol (EP(2), EP(3), and EP(4)) and PGE(1)-OH (EP(3) and EP(4)) stimulate iodide efflux; however, iloprost, butaprost, sulprostone, and fluoprostenol (agonists of the EP(1), EP(2), EP(3), and FP receptors, respectively) have no effect. The iodide efflux seen with 8-iso-PGE(2) is abolished by the EP(4) 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 EP(4) receptor. We propose that 8-iso-PGE(2), acting via EP(4) receptor, could play an important role in the CFTR-mediated response to oxidant stress, and which would be compromised in the CF airways.
authors: Department of Physiology and Biophysics, Dalhousie University, Halifax, NS, B3H 4H7 Canada.
authors: Andrew P
authors: AP
authors: Joy
authors: Elizabeth A
authors: EA
authors: Cowley
mesh_terms: D000838:Anions; D015153:Blotting, Western; D002460:Cell Line; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000502:physiology; D015232:Dinoprostone / Q000031:analogs & derivatives* / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006801:Humans; D007454:Iodides / Q000378:metabolism*; D017136:Ion Transport; D028421:Isoprostanes / Q000494:pharmacology*; D018078:Receptors, Prostaglandin E / Q000502:physiology*; D058309:Receptors, Prostaglandin E, EP4 Subtype; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014132:Trachea / Q000187:drug effects* / Q000378:metabolism
chemical_list: D000838:Anions; D007454:Iodides; D028421:Isoprostanes; C545650:PTGER4 protein, human; D018078:Receptors, Prostaglandin E; D058309:Receptors, Prostaglandin E, EP4 Subtype; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D015232:Dinoprostone; C011987:8-isoprostaglandin E2