a, XZ confocal images of PCL at 0, 6, and 48 h after mucosal addition of 20 μl of PBS containing Texas-red dextran to NL and CF bronchial epithelial cultures. b, mean PCL height with time taken from NL (squares, n = 9) and CF (circles, n = 8) cultures. Note that the blue-shaded region denotes normal height of outstretched cilia (i.e. normal PCL height). c, electron micrographs of bronchial epithelial culture surfaces fixed with OsO₄/PFC 48 h after PBS addition. d, bar graphs depicting changes in transepithelial electric potential difference (V_t) in response to bumetanide (10⁻⁴ M, basolateral) and amiloride (3 × 10⁻⁴ M, apical) in NL (open bars, n = 6) and CF (closed bars, n = 7) cultures at 0 and 48 h. All scale bars are 7 μm. Data shown are the mean ± S.E. *, significantly different from t = 0. †, significantly different between NL and CF cultures.

a, airflow induced in vivo shear stress as a function of airway generation at airway walls (see the supplemental material). The trachea is denoted by generation 0. b, xz confocal images of PCL immediately (0) and 48 h after mucosal PBS addition to NL and CF epithelia cultured under phasic motion. c, mean PCL heights after 48 h of phasic motion culture for NL (open bars, n = 7) and CF (closed bars, n = 8). d, bumetanide (bumet.) and amiloride (amil.)-sensitive V_t in NL (open bars, n = 7) and CF (closed bars, n = 4) cultures before (0) and after 48 h of phasic motion. e, rotational mucus transport rates in static CF cultures (closed bars, n = 12) and phasic motion cultures (gray bars, n = 14) 48 h after volume addition. Data are shown as mean ± S.E. *, data significantly different between NL and CF cultures. †, data significantly different from t = 0. ‡, significantly different between static and phasic motion.

Mean PCL [ATP] (a) and mean serosal bath [ATP] (b) obtained from CF cultures 1 h after 50 μl of PBS addition under variable phasic motion (both, n = 4). NL PCL [ATP] was not significantly different from CF PCL ATP (1.9 ± 0.6, 12 ± 4, 95 ± 13, and 131 ± 41 nM at 0, 0.006, 0.6, and 6 dynes/cm², respectively; all n = 4 and p < 0.05). c, mean PCL height after 48 h of phasic motion in the presence of mucosal apyrase (apyr., 5 units/ml) and/or 8-SPT (10 μM) in NL (open bars; n = 6 – 8) and CF (closed bars; n = 5–7). d, bumetanide (bumet.) and amiloride (amil.)-sensitive changes in V_t in NL (open bars, n = 8) and CF (closed bars, n = 4) cultures before (0) and 48 h after PBS addition/phasic motion in the presence of 5 units/ml mucosal apyrase. Note that amiloride and bumetanide values for CF cultures at 48 h are significantly different from the values without apyrase at 48 h (Fig. 3d). e, simultaneous measurements of V_t and intracellular calcium (Ca²⁺_i) in CF cultures perfused bilaterally with Ringer solution. Left, changes in V_t/Ca²⁺_i induced by stopping then restarting mucosal perfusion (denoted by arrows). Right, altered perfusion rates in the presence of mucosal apyrase (5 units/ml). The mean changes in V_t and Ca²⁺_i responses to phasic perfusion with KBR were −7.8 ± 0.3 mV and 198 ± 12 nM, respectively (p < 0.05; n = 7); the changes in each parameter were significantly reduced in the presence of apyrase; ΔV_t = −0.2 ± 0.1 mV, ΔCa²⁺ = 3 ± 2 nM, respectively (p < 0.05; n = 6). Data shown as the mean ± S.E. *, data significantly different between NL and CF cultures. †, data significantly different from t = 0. ‡, data significantly different from apyrase. §, data significantly different from 8-SPT.

a, NL airway epithelia under static conditions coordinately regulate the rates of Na⁺ absorption and Cl⁻ secretion to adjust PCL volume from an “excessive” PCL height (25 μm) to the physiologic PCL height with time. The blue color depicts PCL height as referenced to extended cilia. b, in CF epithelia, the higher basal rate of Na⁺ absorption, the failure to inhibit Na⁺ transport rates, and the failure to initiate Cl⁻ secretion under static conditions lead to PCL depletion (note “flattened” cilia). c, NL airway epithelia under phasic motion respond to increased nucleotide/nucleoside release into PCL by shifting the balance toward Cl⁻ secretion via CFTR (and CaCC; not shown) and a higher PCL. d, CF cultures under phasic motion conditions release sufficient ATP into the PCL to inhibit Na⁺ absorption and initiate CaCC-mediated Cl⁻ secretion to restore PCL to a physiologically adequate height.

a, xz confocal images of PCL immediately (0) and 6 and 48 h after mucosal addition of PBS with 5 units/ml apyrase to NL and CF bronchial epithelia. b, mean PCL height over 48 h for NL cultures exposed to apyrase (5 units/ml; closed squares, n = 9) or apyrase and 8-SPT (10⁻⁵ M; triangles, n = 5); CF cultures (n = 7) exposed to apyrase are denoted by red circles. c, change in V_t in response to bumetanide (bumet., 10⁻⁴ M, basolateral) and amiloride (amil., 3 × 10⁻⁴ M, apical) in NL (open bars, n = 8) and CF (closed bars, n = 4) cultures before (0 h) and 48 h after apyrase-exposure. d, mean PCL height after mucosal the addition of 8-SPT (10⁻⁵ M) alone at t = 0 to NL (squares, n = 7) and CF (circles, n = 6) cultures. Data shown as the mean ± S.E. †, data significantly different from t = 0. ‡, data significantly different between NL and CF cultures.

Mean PCL height after 3 h of phasic motion in the presence of a CFTR antagonist (CFTR_inh172; 10 μM) or CFTR_inh172 and a CaCC antagonist (DIDS; 100 μM). NLs (open bars; n = 6) and CFs (closed bars; n = 6). Data are shown as the mean ± S.E. *, data significantly different between NL and CF cultures. †, data significantly different from control. ‡, data significantly different from CFTR_inh172.

a, XZ confocal images of PCL (red) covering control or RSV-gfp-infected ciliated cells (green) before (0) and 48 h after mucosal PBS addition to CF cultures under phasic motion conditions. b, PCL height 48 h after the addition of PBS for control CF cultures (closed bars; n = 10) and CF cultures infected with RSV-gfp (gray bars, n = 10). Also shown are mucosal ATP (c) and Mucosal Ap₄A (d) hydrolysis rates in CF epithelia in control (closed bars) and RSV-infected cultures (gray bars). Both, n = 3. e, PCL height 48 h after the addition of PBS for CF cultures infected with RSV-gfp (gray bars, n = 5), and for cultures exposed to PCL and serosal media, but not virus, from RSV-infected cultures for 48 h under phasic motion conditions (CF transfer, open bars, n = 5). Data are shown as the mean ± S.E. *, difference (p < 0.05) between control and RSV-infected CF epithelia. †, difference (p < 0.05) between RSV-infected and CF-RSV media-exposed epithelia.