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. 2014 Dec 15;172(1):173–184. doi: 10.1111/bph.12934

Figure 4.

Figure 4

EP4 stimulation of CFTR activates sAC through Epac and increased [Ca2+]i. (A) NHBE ALI cultures in Ussing chambers were stimulated with Cay10598 (50 nM) in the presence or absence of H89 (10 μM), CFTRinh172 (10 μM) or 4,4′-dinitro-stilbene-2,2′-disulphonic acid (DNDS; 100 μM) (n = 4 lung donors for each inhibitor, *P < 0.05). Both the kinase inhibitor H89 and the CFTR inhibitor blocked EP4 receptor-mediated anion secretion, while DNDS has no effect, consistent with CFTR activation. (B) ISC traces of NHBE ALI cultures mounted in Ussing chambers and stimulated with Cay10598 (50 nM) in the presence or absence of KH7 (25 μM) or MDL12,330A (25 μM). (C) Anion secretion was significantly reduced by each inhibitor (n = 5 lung donors, *P < 0.05). (D) NHBE ALI cultures were stimulated with Cay10598 in the presence of different concentrations of KH7 (n = 3–6 lung donors at each concentration, apparent IC50 = 8 μM). (E) NHBE ALI cultures were loaded with fura2-AM, mounted in a perfusion chamber and imaged by epifluorescence microscopy. Addition of Cay10598 (100 nM) to the perfusate increased [Ca2+]i that returned towards baseline after removal of the agonist (representative trace is the mean fura-2 ratio recorded from regions of interest in 16 cells from a single lung donor ± SEM, and is representative of experiments with three individual donors). (F) The Epac inhibitor ESI-09 blocked H2O2-induced changes in ISC with an apparent IC50 = 0.8 μM (n = 1–5 lung donors at each concentration), and ESI-09 (10 μM) attenuated Cay10598-induced anion secretion (G; n = 4 lung donors, *P < 0.05 compared with control no ESI-09). (H) Pretreatment with ESI-09 (10 μM) also attenuated changes in Cay10598-induced fura-2 fluorescence (representative trace of the mean fura-2 ratio recorded from regions of interest in 19 cells from a single donor ± SEM, and is representative of experiments with three individual donors). (I) Cay10598-induced fura-2 fluorescence changes were significantly altered by ESI-09 (10 μM; n = 4 lung donors, P < 0.05).