Fig. 3.
Cyclic compressive stress (CCS) increases apical Ca2+ flux and nitrogen oxide formation in human airway epithelial cells at air-liquid interface (ALI). A: Ca2+ influx into the apex of these cells (n = 16; ALI 5) with each pulse of CCS (blue arrows). This experiment was repeated 3 times. B: sensor response from the system shown in Fig. 1D in apical medium measured after CCS from cystic fibrosis (CF), ciliated cells at ALI untreated (n = 3) or treated with CCS as shown in 1C (n = 6; ALI 3). The S-nitrosothiol signal was higher after CCS. *P < 0.05, CCS vs. buffer; ***P < 0.05, CCS-treated vs. control cells. C: after CCS, F508Del homozygous cells were snap frozen in liquid nitrogen. Blinded reduction-chemiluminescence assay showed S-nitrosothiol content/protein after CCS (n = 9) was greater than control (n = 8; *P = 0.002; ALI 1 and 2). D: assays were also performed on a fresh set of cells using a modified sensor as described in Ref. 53. Left dots represent the individual signals from each set of cells (using the log scale with which the measurements were made), while the right dots represent the geometric mean of the results at left (red: control with standard error; green: cells received CCS). Because this sensor acts as an on/off toggle for the presence or absence of S-nitrosocysteine, we performed serial dilutions of the cell lysate and medium over 4 logs (0.01–100 nM). All cells had S-nitrosothiols [36 ± 12 nM; greater than medium alone (0 ± 0 nM), n = 5 samples, 3 replicates each; P < 0.001]. CCS increased S-nitrosothiol levels to the point that the sensor was saturated (≥100 nM in all samples, n = 5 samples, 3 replicates each, P < 0.001 compared with untreated; ALI 8). E: normal subjects were studied after an overnight fast for fraction of exhaled NO (FENO) analysis before and after airway clearance vest treatment: 10 Hz, 10 cm water, 10 min; n = 8). FENO consistently increased in these subjects with in vivo CCS (*P = 0.006 by paired t-test). ULD, upper limit of detection; LLD, lower limit of detection.