BALB/c mice were purchased from Japan SLC, Inc. (Shizuoka, Japan), and all experiments involving the mice followed protocols approved by the Animal Care and Use Committees of Hamamatsu University School of Medicine, Shizuoka, Japan (license number H31-047) and were performed in accordance with the relevant guidelines and regulations.


GLY and TIO were purchased from Sigma-Aldrich (St. Louis, MO, USA) and dissolved in phosphate-buffered saline (PBS). GLY was used at 300µM for the intranasal administration to mice and at 100 nM for the organ culture of murine tracheal tissue and primary NHBE cell culture under air–liquid interface (ALI) conditions. TIO was used at 100 nM for culture conditions. 2-Aminoethoxydiphenylborane (2-APB), an inhibitor of inositol-trisphosphate receptor, was purchased from Tocris Bioscience (Bristol, UK) and used at 40 μM. N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide, dihydrochloride (H89), a protein kinase inhibitor with a high specificity for protein kinase A (PKA), was purchased from Cayman Chemical (Ann Arbor, MI, USA) and used at 10 μM. Telenzepine, M1 receptor antagonist, was purchased from Tocris Bioscience (Bristol, UK) and used at 10 μM. Gallamine, M2 receptor antagonist, was purchased from Sgma-Aldrich and used at 30 μM. 4-DAMP, M3 receptor antagonist, was purchased from Abcam (Cambridge, UK) and used at 10 μM. Thapsigargin were purchased from Sigma-Aldrich and used at 1 μM.

Isolation of murine trachea and organ culture of tracheal tissue

Tracheas were taken from 9 to 11-week-old female BALB/c mice and placed in cold collection medium solution (Dulbecco’s Modified Eagle Medium with sodium pyruvate solution) kept in an ice bath. Excess fat and connective tissue debris were immediately removed from the trachea using forceps. The membranous portion of the trachea was then excised to expose the ciliated epithelium in the trachea. After the dissection, the tracheal tissue was cultured in 2 mL of culture medium in the presence or absence of GLY or TIO in 35-mm culture dishes at 37 °C for 60 min as an organ culture. The tracheal epithelium was then observed and analyzed at room temperature (23–28 °C) using a dedicated microscope.

Daily intranasal administration of glycopyrronium to mice

The 8-week-old female BALB/c mice were anesthetized with a 3% isoflurane oxygen mixture and administered 25 µL of GLY solution (300 µM) or PBS alone intranasally once daily for 7 days. Mice were sacrificed on day 8, after which tracheas were taken. After the organ culture of trachea was prepared as described above, the tracheal epithelium was immediately observed and analyzed at room temperature (23–28 °C) using a dedicated microscope.

Cell culture

Primary NHBE cells were purchased from Lonza (catalog no. CC-2541; Basel, Switzerland). NHBE cells in a submerged condition were cultured on 6.5-mm Transwell (Corning, NT, USA) using commercially available bronchial epithelial growth medium (BEGM; Lonza) and incubated at 37 °C in a humidified atmosphere with 5% carbon dioxide. When the NHBE cells reached full confluency in an immersed culture condition, the cells were transferred to an ALI culture condition using ALI culture medium (HBTEC Air–Liquid Interface Differentiation Medium; Lifeline Cell Technology, Frederick, MD, USA), as previously described with slight modifications [3, 4, 23, 24]. The cells were then cultured for 4 weeks in ALI conditions to facilitate polarization and ciliary differentiation for subsequent experiments [25, 26].

Analysis of cilia-driven flow

Ciliary transport on the surface of the intact murine trachea was analyzed after transient organ culture, as previously described [10]. To visualize the cilia-driven flow, tracheal tissue was placed in a 35-mm culture dish with the luminal face down in 2 mL of culture medium containing 0.2-μm-diameter polystyrene beads (0.2-μm red fluorescent beads: Thermo Fisher Scientific, Waltham, MA, USA). Methylcellulose (M0512, Sigma-Aldrich, St. Louis, MO, USA) was added to the culture medium at a concentration of 0.5% to stabilize the movement of fluorescent beads by increasing the viscosity of the medium. The movement of the beads under the tracheal epithelium was observed from the bottom of the dish using an inverted fluorescence microscope (Eclipse TE2000-U, Nikon, Tokyo, Japan) equipped with a CFI Plan Fluor objective lens (Nikon) and a CCD camera (Hamamatsu Photonics, Hamamatsu, Japan). The optimal configuration of the lens for tracking the movement of the beads, which were 0.5–2 mm from the chamber bottom, was × 20 magnification, an NA of 0.5, and a long working distance (2.1 mm). The velocity of each bead was calculated by dividing the width of the field of view by the time each individual bead took to travel across the field. The fluid movement velocity (cilia-driven flow) was calculated using the beads’ migration distance and travel time by tracking individual fluorescent beads using Aquacosmos image analysis software (Hamamatsu Photonics). Three or more independent tracheal samples in each condition were analyzed. In each tracheal sample, 10 fields of view and 5 beads in each field were evaluated, which meant that cilia-driven flows of 50 beads in one trachea were analyzed. In each experiment, mean data were calculated by determining the average of cilia-driven flows for each trachea, and then averaging data for all tracheae in each condition. The rainbow trace was depicted using a macro for the free software, ImageJ, from Hiratsuka laboratory of JAIST (https://www.jaist.ac.jp/ms/labs/hiratsuka/, in Japanese only).

Analysis of the ciliary beating orientation

The analysis was performed as described previously [8,9,10] with slight modifications. After the murine tracheal tissue was transiently cultured in culture medium, the cilia tips of the ciliated cells were labeled with Indian ink diluted with culture medium (1:100) to analyze the intact cilia. The motility of the ink-labeled cilia tips was recorded using HAS-L1 and HAS-U1 high-speed cameras (DITECT Co. Ltd, Tokyo, Japan) at 300 fps to reflect ciliary motility. The recording was performed at 23–28 °C. CBF was determined by subjecting the original traces to a fast Fourier transform using Microsoft Excel. The ciliary beating amplitude, effective stroke velocity, recovery stroke velocity, and the ratio of effective stroke velocity to recovery stroke velocity were calculated from the movement of the cilia tips. The CBF data are presented as the median (ranges). The ciliary beating amplitude, effective stroke velocity, recovery stroke velocity, and effective stroke velocity-to-recovery stroke velocity ratio are presented as the mean ± SEM. Fully differentiated NHBE cells cultured under ALI conditions for 4 weeks were also used to measure CBF. Three independent tracheal samples and 10 ink-labeled cilia in each sample were analyzed (n = 30 ink-labeled cilia for each condition). Kymographs of ciliary beating were depicted with a macro embedded in ImageJ.

Monitoring of intracellular calcium ion concentrations

Intracellular calcium ion (Ca2+) concentrations were measured using fura-2/AM, a fluorescent Ca2+ indicator. NHBE cells cultured under ALI conditions for 4 weeks were incubated with 2.5 μM fura 2-AM (Dojindo, Kumamoto, Japan) for 30 min at 37 °C, and the cells were treated with GLY or TIO at each concentration. Fluorescent images of fura-2/AM were acquired and quantified every 30 s or 120 s from individual cells with a fluorescence analyzer (Aquacosmos, Hamamatsu Photonics, Hamamatsu, Japan) using an ultra-high sensitivity camera. Changes in the fluorescence ratio (F340/F380) of fura-2 were used to express changes in the intracellular Ca2+ concentrations. Thapsigargin (TG), an inhibitor of the endoplasmic reticulum Ca2+-ATPase pump, which causes a transient increase of intracellular Ca2+ concentrations [27], was used for positive controls.

Analysis of protein kinase A (PKA) activity

NHBE cells cultured under ALI conditions for 4 weeks were treated with GLY for 60 min. The cells were then harvested using RIPA buffer (ATTO, Tokyo, Japan) and PKA activity was determined using nonradioactive PKA activity assay kits (Enzo Life Sciences, Ann Arbor, MI, USA), according to the manufacturer’s instructions. Briefly, the PKA substrate microtiter plates were soaked in kinase assay dilution buffer at room temperature. The cell lysates (1 μg of protein) were subsequently added, followed by the addition of adenosine triphosphate (ATP) to initiate the reaction. After incubation at 30 °C for 90 min, the phosphor-specific substrate antibody was added, and the reaction was incubated at room temperature for 1 h. The HRP-conjugated secondary anti-rabbit IgG was subsequently added to each well, and incubated for an additional 30 min. The TMB substrate solution was added to each well, and further incubated for 30 min. Lastly, the stop solution was added, and the 96-well plate was read at 450 nm in a microplate reader.

Adenosine triphosphate (ATP) measurements

ATP concentrations were measured in culture supernatants using an ATP assay kit based on luminometric techniques (Lucifell 250 plus, Kikkoman Biochemifa, Tokyo, Japan) according to the manufacturer’s protocol. In total, 100 µL of culture medium from murine tracheal tissue with or without GLY were used. Briefly, 100 µL of the ATP extraction reagent was added to each sample, and after 20 s, luciferin-luciferase (100 µL) was added to each sample. The luminescence of each sample was measured using a Lumitester C-100 (Kikkoman Biochemifa).

Statistical analysis

All statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University), which is a graphical user interface for R software (version 2.4–0, The R Foundation for Statistical Computing, Vienna, Austria). A student’s t-test or Tukey’s test were used to compare means among groups. Distribution of CBF data deviated from normal distribution, and were analyzed by the Mann–Whitney U-test or Kruskal–Wallis test based on the number of groups. All data, except for CBF, are presented as means ± SEM. The CBF data are presented as the median (range). Statistical significance was assigned when p values were ≤ 0.05.

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