PMC

Effect of ionomycin on the [Ca²⁺]_i and CBF of ciliated cells in lung slices. (A) A fluorescence and (C) phase–contrast image showing the basal fluorescence of Fluo-3AM in the epithelial cells, and the position of a selected ROI in a ciliated cell. (B) The increase in fluorescence in the epithelial cells after addition of 5 μM ionomycin. The cross-hair sights indicates the position of the ROI used in this figure. (D) Ionomycin (5 μM) increased the fluorescence ratio (F/F₀) in the selected ROI. (E) Representative data showing no change in CBF in a lung slice after addition of 5 μM ionomycin (n = 112 cells, 4 mice).

Effect of temperature changes on CBF in ciliated cells of small airways. (A) A representative experiment of a lung slice perfused with sHBSS maintained at RT (∼ 20°C) and with a basal CBF of 12.17 Hz. After 25 s of recording, “warm” sHBSS (∼ 31°C) was perfused which increased the CBF to a maximum value of 26.5 Hz before declining and stabilizing after ∼ 120 s at 23.1 Hz. (B) Summary of the increase in CBF from experiments performed as described in (A). CBF is significantly higher at 28–34°C (21.88 ± 0.66 Hz) than at RT (12.45 ± 0.88 Hz; *P < 0.05; n = 45 cells, 3 mice). (Values are mean ± SEM).

Measurement of CBF in small airways of mice. (A) A low-power phase–contrast image showing a small airway obtained with the lung slice preparation. The lumen is devoid of agarose, but filled with sHBSS. (B) A medium-power image of epithelial cells and SMCs of the small airways and agarose-filled alveoli obtained with DIC microscopy. (C) A high-power DIC image showing two ciliated cells. The cross-hair sight indicates the position of the region of interest used to measure CBF. (D) The waveform of the variation in gray intensity extracted from a 250-ms sequence of digital images (60 frames) recorded at 240 fps. Waveform shows the three phases of the beat cycle: recovery phase, effective phase and rest phase. Each beat cycle period (P; bidirectional arrow) was used to calculate CBF (1/period).

A comparison of the basal CBF and the effects of ATP on CBF in noncultured and cultured lung slices or tracheal rings. (A) The basal CBF of tracheal rings (20.19 ± 0.84 Hz; n = 102 cells, 5 mice) and lung slices (21.65 ± 0.47 Hz; n = 112 cells, 5 mice) are similar, whereas both are significantly higher than the basal CBF of cultured outgrowths from tracheal rings (*13.23 ± 0.72 Hz; n = 56 cells, 6 mice; P < 0.05) or lung slices (**15.73 ± 0.70 Hz; n = 112 cells, 7 mice; P < 0.05). CBF measured at RT in lung slices is significantly lower (***13.52 ± 0.70 Hz; n = 188 cells, 5 mice; P < 0.05) than CBF measured at ∼ 30°C. (B) ATP (5 μM) induced a similar increase in CBF in both tracheal rings (45 ± 3%; n = 64 cells, 5 mice) and tracheal cultures (51 ± 5%; n = 22 cells, 6 mice). However, CBF decreased gradually in tracheal rings to reach the prestimulatory basal rate after ∼ 2 min, while CBF was sustained in tracheal cultures. (Values are mean ± SEM.)

Effect of ATP on CBF in the small airways of mice. (A–C) Representative measurements of CBF during 100 s in 3 cells from different lung slices. Application of ATP (A) 1 μM, (B) 5 μM, and (C) 10 μM did not change CBF (temperature 29.6, 31.2, and 30.6°C, respectively). (D) Summary of CBF measurements in response to ATP 1 μM (19.35 ± 0.77 Hz; n = 44 cells, 3 mice), 5 μM (21.04 ± 0.67 Hz; n = 72 cells, 3 mice), 10 μM (21.02 ± 0.82 Hz; n = 112 cells, 4 mice), 100 μM (20.29 ± 1.08 Hz; n = 48 cells, 3 mice), and 500 μM (19.76 ± 0.71 Hz; n = 48 cells, 3 mice) with respect to the control CBF (1 μM ATP 19.30 ± 1.25 Hz; ATP 5 μM, 21.48 ± 0.84 Hz; ATP 10 μM, 21.05 ± 0.97 Hz; ATP 100 μM, 19.33 ± 0.87 Hz) obtained from the first 10 s of recording in the absence of ATP. The temperature during the experiments was between 29.3 and 34°C. (Values are mean ± SEM.)

Effect of ATP on Ca²⁺ signaling in epithelial cells of lung slices. (A and B) Fluorescence images obtained with confocal microscopy and Fluo-3AM. The basal fluorescence was low before addition of ATP (A) and increased with addition of 5 μM ATP (B), indicating an increase in [Ca²⁺]_i. The cross-hair sight indicates the position of the ROI used in this figure. (C) A phase–contrast image showing the localization of ROI on the slice. (D) In the ciliated cell, 5 or 10 μM ATP induced a large and transient increase of fluorescence. Slices were exposed to only one concentration of ATP. The arrow indicates the peak used to compare the maximum levels of fluorescence reached between different experiments. (E) Summary of the increase of the fluorescence ratio (F/F₀, peak) after addition of 1, 5, 10, or 100 μM ATP. No significant differences were found between the different concentrations tested. Each concentration was tested at least three times with three different mice.