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Fig. 2 | Respiratory Research

Fig. 2

From: Soluble adenylyl cyclase mediates hydrogen peroxide-induced changes in epithelial barrier function

Fig. 2

sc19220, an EP1 receptor antagonist, attenuates H2O2-associated changes in ISC and transepithelial resistance and permeability. Panel a. Representative Ussing chamber experiment showing H2O2-induced ISC with current pulses before and after H2O2 exposure. The EP1 receptor antagonist sc19220 (20 μM) not only diminishes H2O2-induced increases in ISC (upward arrow head), but also rescues the decrease in membrane resistance associated with apical H2O2 exposure. Compare the pulse size of the sc19220 pretreated cells (red trace) with the much larger control pulses (black trace) after prolonged H2O2 exposure (downward arrow head). Panel b. Fully differentiated NHBE cells were mounted in Ussing chambers, treated with 10 μM amiloride followed by an EP1 or EP4 receptor antagonist in the apical and basolateral compartments for 20 min. The cells were then stimulated with H2O2 apically in the presence of current pulses to determine the membrane resistance before and after H2O2 exposure. Only the inhibition of the EP1 receptor with sc19220 (20 μM, n = 40 cultures from 26 donors) and not the inhibition of the EP4 receptor with GW627368X (200 nM, n = 6 cultures from 6 donors) or AH23848 (5 μM, n = 4 cultures from 4 donors, * = p < 0.05 compared to control), attenuates the decrease in resistance associated with H2O2 exposure. Panel c. Permeability of cultures treated with sc19220 and H2O2 were not significantly different to untreated control (n = 3 filters from 3 lungs, * = p < 0.05). Panel d. The protective effect of sc19220 on membrane resistance appeared to be an apical phenomenon, since only apical and not basolateral pre-treatment with the EP1 antagonist diminished the effects of H2O2 (n = 5 cultures from 5 donors, p < 0.05). Panel e, NHBE cultures were fixed and then stained with anti-EP1 receptor (green), acetylated α-tubulin (cilia, red) and DAPI (nuclei, blue) and then imaged by confocal microscopy. Confocal sections of combined color channels through the cilia (left), apical membrane (center) and nuclei (right) are shown with indications of the plane in the Z-stack below each panel. The images showed anti-EP1 receptor antibody (green) had an apical localization with a concentration near the apical junction between cells. Bars = 20 μm

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