Effects of long-acting muscarinic antagonists on promoting ciliary function in airway epithelium

Abstract

Background: Mucociliary clearance (MCC) is an essential defense mechanism in airway epithelia for removing pathogens from the respiratory tract. Impaired ciliary functions and MCC have been demonstrated in asthma and chronic obstructive pulmonary disease (COPD). Long-acting muscarinic antagonists (LAMAs) are a major class of inhaled bronchodilators, which are used for treating asthma and COPD; however, the effects of LAMAs on ciliary function remain unclear. This study aimed to identify the effects of LAMAs on airway ciliary functions.

Methods: Wild-type BALB/c mice were treated with daily intranasal administrations of glycopyrronium for 7 days, and tracheal samples were collected. Cilia-driven flow and ciliary activity, including ciliary beat frequency (CBF), ciliary beating amplitude, effective stroke velocity, recovery stroke velocity and the ratio of effective stroke velocity to recovery stroke velocity, were analyzed by imaging techniques. Using in vitro murine models, tracheal tissues were transiently cultured in media with/without LAMAs, glycopyrronium or tiotropium, for 60 min. Cilia-driven flow and ciliary activity were then analyzed. Well-differentiated normal human bronchial epithelial (NHBE) cells were treated with glycopyrronium, tiotropium, or vehicle for 60 min, and CBF was evaluated. Several mechanistic analyses were performed.

Results: Intranasal glycopyrronium administration for 7 days significantly increased cilia-driven flow and ciliary activity in murine airway epithelium. In the murine tracheal organ culture models, treatment with glycopyrronium or tiotropium for 60 min significantly increased cilia-driven flow and ciliary activity in airway epithelium. Further, we confirmed that 60-min treatment with glycopyrronium or tiotropium directly increased CBF in well-differentiated NHBE cells. In the mechanistic analyses, neither treatment with glycopyrronium nor tiotropium affected intracellular calcium ion concentrations in well-differentiated NHBE cells. Glycopyrronium did not increase protein kinase A activity in well-differentiated NHBE cells. Moreover, glycopyrronium had no effect on extracellular adenosine triphosphate concentration.

Conclusions: LAMAs exert a direct effect on airway epithelium to enhance ciliary function, which may improve impaired MCC in asthma and COPD. Further investigations are warranted to elucidate the underlying mechanisms of the effects of LAMAs on the promotion of airway ciliary function.

Keywords: Airway epithelium; Asthma; COPD; Ciliary beat frequency; Long-acting muscarinic antagonists; Mucociliary clearance.

Fig. 1

The effects of the intranasal administration of glycopyrronium on cilia-driven flow and ciliary motility Wild-type (WT) mice were treated with glycopyrronium (GLY) or phosphate-buffered saline (PBS) intranasally for 7 days, The trachea was then removed. Cilia-driven flow and ciliary motility were evaluated. A. Representative bead trajectories of cilia-driven flow (rainbow trace for 4.4 s). B. the histogram of the cilia-driven flows (150 beads from 3 tracheas) in each condition. C. the bar chart of the cilia-driven flow demonstrated that 7-day GLY administration significantly increased cilia-driven flow (PBS, 7.30 ± 0.15 μm/s; GLY, 9.04 ± 0.08 μm/s; n = 3 tracheas in each condition). D. Kymographs of the ciliary beating. E. GLY significantly increased ciliary beating frequency (CBF; PBS, 15.23 [8.20–22.27] Hz; GLY, 18.17 [10.55–23.44] Hz; n = 30 cilia in each condition). F. The ciliary beating amplitude did not differ between the conditions (PBS, 4.18 ± 0.14 μm; GLY, 4.23 ± 0.15 μm). G and H. The effective stroke velocity (G) (PBS, 503.7 ± 21.1 μm/s; GLY, 785.7 ± 45.1 μm/s) and recovery stroke velocity (H) (PBS, 396.8 ± 15.3 μm/s; GLY, 675.3 ± 43.3 μm/s) were significantly increased by GLY administration compared with the control. I. The ratio of effective stroke velocity to recovery stroke velocity (E/R ratio) did not differ between the two conditions (PBS, 1.29 ± 0.05; GLY, 1.20 ± 0.05)

Fig. 2

The short-term effects of glycopyrronium on cilia-driven flow and ciliary motility Murine tracheal epithelia were incubated for 1 h with/without glycopyrronium (GLY) in the culture medium. The cilia-driven flow and ciliary motility were then evaluated. A. Representative bead trajectories of cilia-driven flow (rainbow trace for 4.4 s). B. the histogram of the cilia-driven flows (200 beads from 4 tracheas) in each condition. C. the bar chart of the cilia-driven flow demonstrated that GLY significantly increased cilia-driven flow (control, 8.63 ± 0.39 μm/s; GLY, 11.85 ± 1.21 μm/s; n = 4 tracheas in each condition). D. Kymographs of ciliary beating. E. GLY significantly increased ciliary beat frequency (CBF; control, 13.48 [10.55–19.92] Hz; GLY, 18.75 [15.23–24.61] Hz; n = 30 cilia in each condition). F. The ciliary beating amplitude did not differ between the conditions (control, 4.31 ± 0.16 μm; GLY, 4.29 ± 0.15 μm). G and H. The effective stroke velocity (G) (control, 504.4 ± 20.7 μm/s; GLY, 734.9 ± 40.3 μm/s) and recovery stroke velocity (H) (control, 393.9 ± 15.1 μm/s; GLY, 602.2 ± 35.5 μm/s) were significantly increased by GLY treatment compared with the control. I. The ratio of effective stroke velocity to recovery stroke velocity (E/R ratio) did not differ between the two conditions (control, 1.30 ± 0.05; GLY, 1.25 ± 0.05). Ctrl, control

Fig. 3

The short-term effects of tiotropium on cilia-driven flow and ciliary motility Murine tracheal epithelia were incubated for 1 h with/without tiotropium (TIO) in the culture medium. Cilia-driven flow and ciliary motility were then evaluated. A. Representative bead trajectories of cilia-driven flow (rainbow trace for 4.4 s). B. the histogram of the cilia-driven flows (200 beads from 4 tracheas) in each condition. C. the bar chart of the cilia-driven flow demonstrated that TIO significantly increased cilia-driven flow (control, 8.26 ± 0.11 μm/s; GLY, 10.59 ± 0.72 μm/s; n = 4 tracheas in each condition). D. Kymographs of ciliary beating. E. TIO significantly increased ciliary beat frequency (CBF; control, 17.58 [10.55–23.44] Hz; TIO, 19.92 [14.06–24.61] Hz; n = 30 cilia in each condition). F. The ciliary beating amplitude did not differ between the conditions (control, 4.56 ± 0.13 μm; TIO, 4.56 ± 0.10 μm). G and H. The effective stroke velocity (G) (control, 606.9 ± 33.3 μm/s; TIO, 794.0 ± 27.9 μm/s) and recovery stroke velocity (H) (control, 494.0 ± 30.7 μm/s; TIO, 683.1 ± 29.7 μm/s) were significantly increased by TIO treatment compared with the control. I. The ratio of effective stroke velocity to recovery stroke velocity (E/R ratio) did not differ between the two conditions (control, 1.29 ± 0.07; TIO, 1.20 ± 0.04). Ctrl, control

Fig. 4

The effects of glycopyrronium or tiotropium on ciliary motility in normal human bronchial epithelial cells Well-differentiated normal human bronchial epithelial (NHBE) cells cultured under air–liquid interface (ALI) conditions for 4 weeks were treated for 1 h with/without glycopyrronium (GLY) or tiotropium (TIO) in the culture medium. Ciliary beat frequency (CBF) was then evaluated. A. Kymographs of ciliary beating. B. GLY significantly increased CBF (CBF; control, 11.72 [9.38–14.06] Hz; GLY, 12.89 [9.38–17.58] Hz; n = 30 cilia in each condition). C. Kymographs of ciliary beating. D. TIO significantly increased CBF (control, 9.38 [4.69–11.72] Hz; TIO, 10.55 [5.86–15.23] Hz; n = 30 cilia in each condition). Ctrl, control

Fig. 5

Involvement of intracellular calcium ion in the glycopyrronium or tiotropium-mediated promotion of ciliary function The murine tracheal tissues were incubated with glycopyrronium (GLY) or tiotropium (TIO) with/without 2-Aminoethoxydiphenylborane (2-APB) for 1 h. Cilia-driven flow and ciliary beat frequency (CBF) were then evaluated. A and B. 2-APB had few effects on GLY-mediated increases of cilia-driven flow (A) (control, 8.73 ± 0.15 μm/s; GLY, 11.66 ± 0.64 μm/s; GLY + 2-APB, 11.04 ± 0.84 μm/s; n = 4 tracheas in each condition) and CBF (B) (control, 14.06 [7.03–19.92] Hz; GLY, 18.17 [12.89–22.27] Hz; GLY + 2-APB, 16.40 [10.55–19.92] Hz; n = 30 cilia in each condition). C and D. 2-APB had few effects on TIO-mediated increases of cilia-driven flow (C) (control, 8.46 ± 0.34 μm/s; TIO, 10.37 ± 0.48 μm/s; TIO + 2-APB, 9.54 ± 0.38 μm/s; n = 3 tracheas in each condition) and CBF (D) (control, 14.06 [10.55–19.92] Hz; TIO, 17.58 [11.72–21.09] Hz; TIO + 2-APB, 16.40 [12.89–19.92] Hz; n = 30 cilia in each condition). E and F. Intracellular Ca²⁺ concentrations were measured using Fura-2/AM in normal human bronchial epithelial cells. Neither treatment with GLY (E) nor TIO (F) changed the fluorescence ratio (F340/F380) of Fura-2 until 50 min (n = 20 cells). Ctrl, control

Fig. 6

Examination of involvement of protein kinase A in the glycopyrronium-mediated increase of ciliary beat frequency Well-differentiated normal human bronchial epithelial (NHBE) cells were incubated with glycopyrronium (GLY) with or without H89, a protein kinase inhibitor with a high specificity for protein kinase A (PKA), for 1 h. Ciliary beat frequency (CBF) and PKA activity was then evaluated. A. H89 had few effects on GLY-mediated increases in CBF (control, 7.62 [5.86–10.55] Hz; GLY, 9.38 [7.03–12.89] Hz; GLY + H89, 8.79 [7.03–12.89] Hz; n = 30 cilia in each condition). B. GLY treatment had few effects on PKA activity (control, 100 ± 14.95%; GLY, 94.53 ± 18.14%; n = 3). The results are presented as percent of control. Ctrl, control