Fig 2: Effect of NHE1 with ezrin binding mutation on PASMC function. Bar graphs represent mean ± SEM values for (A) migration (n = 3 each) and (B) proliferation (n = 5 each) in PASMCs infected with AdGFP (control) or NHE1 that is unable to bind ezrin (AdMut).

Fig 3: Effect of hypoxia on phosphorylation of ezrin. (A) Representative confocal images showing enhanced immunofluorescence for phosphorylated ezrin (p-ezrin; Alexa 488) in smooth muscle cells (indicated by positive staining for smooth muscle specific a-actin; SMA; Cy3) in a small pulmonary vessel in mice exposed to normoxia (top) or chronic hypoxia (CH; 10% O2, 3 weeks; bottom). Bar graph represents mean number of vessels (out of 20 randomly selected) staining positive for p-ezrin and SMA. Data from 1 normoxic and 1 CH lung. (B) Immunoblot shows protein levels for p-ezrin in pulmonary arteries (PA) from normoxic and chronically hypoxic rats and in PASMCs from control rats exposed to control (C) or hypoxic (H; 4% O2; 24 h) conditions. Bar graph shows mean ± SEM for fold change (compared to C) in p-ezrin protein levels (normalized to ß-actin) in control and hypoxic PA (n = 3 each) and PASMCs (n = 5 each). (C) Immunoblot demonstrating p-ezrin co-immunoprecipitates with SMA. Similar results were obtained in four separate experiments. (D) Immunoblot shows enhanced NHE1 and SMA immunoprecipitation with p-ezrin in hypoxic (H) compared to control (C) PASMCs. IgG was used as a control. Similar results were obtained in two other experiments. * indicates significant difference (p < 0.05) compared to control.

Fig 4: Evidence for NHE1/actin interactions in hypoxic PASMCs. Rat PASMCs were exposed to control (C; 21% O2) or hypoxic (H; 4% O2) conditions for 24 h. (A) Proteins obtained following immunoprecipitation using anti-NHE1 were probed for expression of smooth muscle specific a—actin (SMA). In the first lane for both precipitate and input (total cell lysate; control), IgG was used in place of anti-NHE1 as a control. A similar increase in SMA/NHE1 interaction with hypoxia was observed in a second experiment. (B) Immunofluorescence images showing NHE1 (green) and SMA (red) expression in control (left) and hypoxic (right) PASMCs. Bar graph shows mean ± SEM for percent of cells exhibiting NHE1 that associated with actin filaments (n = 6 each). (C) Confocal images showing individual SMA (left) and NHElimages (middle) for a control cell prior to photobleaching, and FRET values after acceptor photobleaching (right) for a control (top) and hypoxic (bottom) PASMC. Bar graphs show mean ± SEM FRET efficiency between control and hypoxic PASMCs (n = 10 cells from three separate experiments for each condition). * indicates significant difference (p < 0.05) compared to control.

Fig 5: Increased NHE1 levels and cell function. (A) lmmunoblot showing overexpression of NHE1 protein achieved using an adenoviral construct containing wild-type NHE1 (AdNHE1) tagged with hemagglutinin (HA). Top panel shows expressed protein, while middle panel shows total NHE1 (expressed plus native) in cells infected with AdNHE1 or AdGFP (control). ß-Tubulin (ß-tub) was used as a loading control. Bar graph shows mean ± SEM values for total NHE1 protein (n = 4 each). (B–D) Bar graphs show mean ± SEM for (B) Na+/H+ exchange (NHE) activity (n = 7 each); (C) proliferation (n = 6 each), and (D) migration (n = 5 each) in PASMCs infected with AdGFP and AdNHE1. * indicates significant difference (p < 0.05) compared to AdGFP.