Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC63
The Prostaglandin E2 type 4 receptor mediates the response to oxidant stress in human airway epithelial cells
E. Cowley1, C. L. Jones1, T. Li1
1. Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada.
Oxidative stress is implicated in the pathogenesis of many inflammatory pulmonary diseases, including cystic fibrosis (CF). CF arises from mutations in the CFTR anion channel, which can be directly activated by oxidant stress. Delineating how oxidative stress stimulates CFTR in airway epithelial cells is useful, both to increase understanding of airways host defence, and also to 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 H2O2, which stimulates anion efflux through CFTR, does so via production of prostaglandin (PG)E2. Using iodide efflux as a biochemical marker of CFTR activity, we found that the H2O2-stimulated iodide efflux was abolished by the cyclooxygenase (COX) inhibitor indomethacin. The response was also abolished by the selective COX-1 inhibitor SC560, while the COX-2 inhibitor Cay10404 had no effect. Since PGE2 is also produced via the activity of microsomal prostaglandin E synthase-1 (mPGES-1), we also investigated the effects of inhibiting this enzyme. Both Cay10589, an mPGES-1 inhibitor, and Cay10526, which decreases mPGES-1 expression, inhibited the H2O2-mediated anion efflux. Therefore, we conclude that the H2O2-stimulated efflux involves PGE2 production via both COX-1 and mPGES-1 activity, while COX-2 is not involved. We next wished to investigate how much of the H2O2-stimulated efflux was mediated via the PGE2 subtype 4 receptor (EP4), since this is the only PGE2 receptor subtype capable of mediating a CFTR-mediated anion efflux in these cells. Here we utilised AH23848, a selective EP4 receptor antagonist. In the presence of AH23848, the H2O2-stimulated response was significantly inhibited, effectively to basal levels. In order to investigate EP4 receptor inhibition in a more physiological, polarized system, we also measured the increase in short circuit current (Isc) induced by application of 1 mM H2O2 to the apical face of Calu-3 cells in the presence and absence of AH23848. In these experiments, H2O2 induced a robust increase in Isc, which was significantly attenuated in the presence of AH 23848 (22.6 ± 3.0 µA/cm-2 n = 9 vs 65.3 ± 10.9 µA/cm-2 for H2O2 alone, n = 12). These results implicate a significant role for the EP4 receptor in mediated the response to anion secretory H2O2, although they do suggest that the complete abolition of the response seen in the non-polarized iodide efflux system may over-estimate its role in the response to oxidant stress. In conclusion, using H2O2 as a model oxidant stress, stimulation of CFTR appears critically dependent both on PGE2 production (via COX-1 and mPGES-1) and the EP4 receptor in Calu-3 airway epithelial cells, a mechanism which would be compromised in the CF airways.
Where applicable, experiments conform with Society ethical requirements