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Figure 5 Functional analysis of δ802 ENaC activity in mouse tracheal epithelial (MTE) and alveolar type 2 (mAT2) monolayers. (A) Short-circuit current traces (Isc) in MTE monolayers mounted on an Ussing chamber setup for uninduced δ802Tg (Control, black line) and induced δ802Tg/Cre (δ-ENaC, red line). Primary MTE cells were cultured at the air-liquid interface for 13-15 days. n=3. Arrows indicate the time to add designed concentrations of amiloride. (B) Dose-response relationship of amiloride for δ-ENaC expressing MTE monolayers and controls. Raw data were fitted with the logistic function to compute ki values for amiloride. The resulting ki values were 0.68 ± 0.00 μM and 3.04 ± 0.26 μM, respectively, for control and δ-ENaC group. Data collected when cells were bathed with Na+-free solution were included but not used for fitting curves. n=5. (C) Representative Isc traces for control and δ-ENaC mAT2 monolayer cells. Accumulating amiloride from 1 to 1,000 μM were applied as indicated by arrows. (D) Dose-response curve for amiloride in mAT2 cells. (E) Transepithelial resistance measured with an EVOM meter. Resistance was read with a chopstick meter when the culture medium was replaced every other day. n=7, *** P<0.001 vs controls. (F) Representative Isc traces for α-13 peptide inhibition. α-13 inhibitory peptide (300 μM) was added to the apical counterpart followed by amiloride and finally by Na+-free bath solution as indicated. (G) Average Isc levels in the presence and absence of α-13 inhibitory peptide (300 μM), amiloride (100 μM), and Na+ ions (150 mM). n=6. * P<0.05 vs control. (H) Isc fractions associated with αβγ-ENaC and δ802βγ-ENaC subpopulations as computed as α-13 inhibitable (α-13 peptide sensitive) and the component inhibited by amiloride and removal of bath Na+ ions (α-13 peptide resistant). n=6. * P<0.05 vs controls. (I) α-13 and amiloride-sensitive alveolar fluid clearance in human lungs ex vivo. n=5 per group. * P<0.5 vs control. Data were presented as mean ± s.e.m., mean difference between two groups was computed by student t-test in (E-H).

Image published in: Zhao R et al. (2019)

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