Figure 5.

Inhibition of the V-type ATPase increases the ASL pH and decreases ASL viscosity in porcine non-CF Sm airways. ASL pH was measured using a ratiometric fluorescent dye (SNARF–dextran), which showed that the pH increased with bafilomycin treatment under both (A) bicarbonate-containing conditions (N = 11) and (B) bicarbonate-free conditions (N = 7). Lines plus connected dots indicate experimental pairs. *P < 0.05 by paired t test. (C) ASL viscosity was assayed using fluorescence recovery after photobleaching, showing that viscosity in Sm airways is controlled by pH, which is set by V-type ATPase. Lines plus connected dots indicate experimental pairs (N = 6). (D) siRNA knockdown of V0 d2 in cultured Sm airway epithelial cells increases the ASL pH. (E and F) Sm airway cells immunostained for V0D2, cells treated with Si-CTL versus Si-ATP6V0D2. Green indicates positive staining for V0D2, whereas red indicates F-actin staining. Scale bars, 120 μm. *P < 0.05 by paired t test. Si-ATP6V0D2 = siRNA 1 targeting V0D2 transcripts; Si-CTL = scrambled siRNA CTL.