8-iso-PGE2 stimulates anion efflux from airway epithelial cells via the EP4 prostanoid receptor.
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.
Joy
Andrew P
AP
Department of Physiology and Biophysics, Dalhousie University, Halifax, NS, B3H 4H7 Canada.
Cowley
Elizabeth A
EA
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
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