TRPV4 channel participates in receptor-operated calcium entry and ciliary beat frequency regulation in mouse airway epithelial cells

Expression and Localization of TRPV4 in Mouse Ciliated Tracheal Cells.

Fig. 1 shows ciliated tracheal cells from wild-type (TRPV4^+/+) (Fig. 1A) and KO (TRPV4^−/−) mice (Fig. 1B). TRPV4 immunofluorescence (green) was clearly identified in the cilia of TRPV4^+/+ but not in the cilia of TRPV4^−/− cells. Double staining with anti-tubulin antibody (red) to mark the cilia axoneme confirmed the ciliary localization of TRPV4. Additional immunofluorescence images are shown in supporting information (SI) Fig. S1. Cytoplasmic immunoreactivity spots [more pronounced than in hamster oviductal ciliated cells (21)] were detected in both TRPV4^+/+ and TRPV4^−/− cells, a clear indication of their nonspecificity. Molecular identification of TRPV4 was also investigated by Western blot. Fig. 1C shows only a double band of the expected size in TRPV4^+/+ trachea, which also suggests that the cytoplasmic immunoreactivity spots shown in Fig. 1B were TRPV4 nonspecific. Similar nonspecific immunoreactivity has been reported in kidney sections of TRPV4^+/+ and TRPV4^−/− mice probed with an antibody raised against the same C-terminal epitope of rat TRPV4 (29).

Intracellular Ca²⁺ measurements were carried out to test functional expression of TRPV4 in primary cultures of tracheal explants exposed to the relatively specific TRPV4 agonist 4αPDD (10 μM). Monitoring intracellular Ca²⁺ concentration in fura-2 loaded ciliated tracheal cells showed significant increases that commenced at different times in many TRPV4^+/+ cells (Fig. 1D) but were completely absent in TRPV4^−/− ciliated cells (Fig. 1E). A quantitative analysis of the Ca²⁺ signal measured after 10 min in 4αPDD is shown in Fig. 1F. To check whether the TRPV4-mediated Ca²⁺ signals can also be associated with the activation of CBF, we measured CBF in TRPV4^+/+ and TRPV4^−/− tracheal cells by using high-speed digital video microscopy. Basal CBF of ciliated tracheal cells did not differ between TRPV4^+/+ (10.7 ± 0.4 Hz; n = 37) and TRPV4^−/− (10.3 ± 0.3 Hz; n = 37, P > 0.05 vs. TRPV4^+/+, measured at room temperature) and was not affected by removal of extracellular Ca²⁺ (Fig. 2A), in accordance with previous studies suggesting that basal Ca²⁺ CBF is not directly under the influence of Ca²⁺ (3). However, TRPV4^+/+ tracheal cells, unlike their TRPV4^−/− counterparts, had increased CBF in response to 10 μM 4αPDD. Fig. 2B shows the time course of the relative changes in CBF of a TRPV4^+/+ and TRPV4^−/− cells exposed to 4αPDD. Mean increases in CBF are shown in Fig. 2C.

Response of Ciliated Tracheal Cells to Physical Stimuli Activating TRPV4.

We tested the effect of warm temperature and high viscous solutions on the generation of Ca²⁺ signals and modulation of CBF in TRPV4^+/+ and TRPV4^−/− ciliated tracheal cells. Switching the temperature of the bathing solution from 24°C to 38°C triggered a Ca²⁺ response characterized by a peak followed by a slow decline toward the baseline (Fig. 3A), whereas TRPV4^−/− cells showed a reduction in both the Ca²⁺ peak and the more sustained component without significant modification of the time constant of the signal relaxation (0.95 ± 0.12 min for TRPV4^+/+ and 1.12 ± 0.07 min for TRPV4^−/−, P > 0.05). Accordingly, TRPV4^−/− cells exposed to warm temperatures responded with a smaller increase in CBF (Fig. 3B).

Ciliated tracheal cells from TRPV4^+/+ mice (Fig. 3C) responded to high viscous solutions (20% dextran solutions) with an oscillatory Ca²⁺ signal (3.9 ± 0.6 peaks/cell per 10 min) in 19 of 71 cells (26%). However, TRPV4^−/− ciliated cells (Fig. 3D) typically presented an initial transient peak (18 of 85 cells, 21%) with sporadic Ca²⁺ oscillations (1.4 ± 0.1 peaks/cell per 10 min; P < 0.001 vs. TRPV4^+/+). We also tested the impact of TRPV4 disruption on CBF response to high viscous loads. After the addition of solutions containing 5% or 20% dextran, CBF declined to a new stable value within 5 min (Fig. 3E); however, despite the apparent difference in the Ca²⁺ signal pattern between the two genotypes (Fig. 3 C and D), no differences were detected in the maintenance of a steady-state CBF in response to either 5% (4.8 cP) or 20% (73 cP) dextran solutions (Fig. 3E).

Participation of TRPV4 in the ATP-Induced Ca²⁺ Signal.

We have recently reported a cross-talk between the ATP-PLC-inositol trisphosphate receptor (IP₃R) pathway and TRPV4 to initiate and maintain the oscillatory Ca²⁺ signal triggered by mechanical and osmotic stimuli (21). In the present study, the role of TRPV4 in the generation of ATP-mediated Ca²⁺ signals and regulation of CBF was addressed. The Ca²⁺ signal obtained after the addition of 20 μM ATP showed clear differences between the two genotypes. Although ciliated TRPV4^+/+ and TRPV4^−/− cells showed no statistical difference in peak increases in [Ca²⁺], the sustained component was significantly reduced (by ≈30%) in the latter (Fig. 4 A and B) without significant differences in the time constant of the signal relaxation (1.45 ± 0.2 and 0.9 6 ± 0.09 min for TRPV4^+/+ and TRPV4^−/−, P > 0.05). TRPV4^−/− cells also showed a diminished increase in the CBF when exposed to 20 μM ATP (Fig. 4C). Altogether, the data suggested the contribution of TRPV4 to the sustained component of the Ca²⁺ signal and its coupling to the acceleration of CBF induced by 20 μM ATP. As with many other G protein-coupled receptor agonists, the Ca²⁺ signal generated by low ATP concentrations (200 nM) usually presents a more oscillatory pattern instead of a large transient peak followed by sustained elevation (Fig. 4D). Neither the pattern (Fig. 4D) nor the amplitude of the Ca²⁺ signal (Fig. 4E) generated by 200 nM ATP was significantly different in ciliated tracheal cells from TRPV4^+/+ and TRPV4^−/− mice. Accordingly, no differences in the CBF response to 200 nM ATP were detected between the two genotypes (Fig. 4F).

The sustained component of the Ca²⁺ signal recorded with 20 μM ATP can also be evaluated with a Ca²⁺-free protocol. Fig. 5 shows representative Ca²⁺ traces obtained from TRPV4^+/+ (Fig. 5A) and TRPV4^−/− (Fig. 5B) ciliated tracheal cells exposed to 20 μM ATP in the absence of extracellular Ca²⁺ followed by replacement of external Ca²⁺. The Ca²⁺ entry component after Ca²⁺ replacement in the presence of ATP was clearly reduced in TRPV4^−/− cells (Fig. 5C). To distinguish whether TRPV4 participates in the ROCE or SOCE mechanism, we use the sarcoplasmic (endoplasmic) reticulum Ca²⁺ pump inhibitor thapsigargin (TG). The ability of TG to empty the intracellular stores and the subsequent activation of SOCE (without the involvement of receptor activation) provides a well known protocol to functionally differentiate SOCE from ROCE, which is defined to require seven transmembrane receptors, G proteins, and PLC activation. The Ca²⁺ entry component after Ca²⁺ replacement after TG (1 μM) stimulation (SOCE) was not different between TRPV4^+/+ and TRPV4^−/− cells (Fig. 5 D–F). Also, the pattern and amplitude of the peak Ca²⁺ signals generated by ATP and TG in Ca²⁺-free media (reflecting intracellular Ca²⁺ release) were not significantly different between TRPV4^+/+ and TRPV4^−/− cells (2 ± 0.13 and 1.98 ± 0.09 for ATP; 1.83 ± 0.2 and 1.8 ± 0.2 for TG, respectively; P > 0.05).

Chemicals and Solutions.

All chemicals were purchased from Sigma–Aldrich except dextran T-500 (500,000 Daltons; Amersham Pharmacia), fura2-AM (Molecular Probes), Hank's balanced salt solution (HBSS; Gibco), and collagen type I from rat tail (Upstate). Isotonic bathing solutions used for imaging experiments contained (in mM): 140 NaCl; 5 KCl; 1.2 CaCl₂; 0.5 MgCl₂; 5 glucose; 10 Hepes, pH 7.4; and 305 mosmol/liter. Ca²⁺-free extracellular solutions were obtained by replacing CaCl₂ with MgCl₂ and adding 0.5 mM EGTA. CBF measurements were carried out in phenol red-free HBSS supplemented with 25 mM Hepes (pH 7.4). ATP and 4αPDD were dissolved in milli-Q distilled water and ethanol, respectively. The viscosity of the isotonic solution was increased by adding 5% or 20% dextran T-500, which does not change the osmolality (305 mosmol/liter) of the solution.

Primary Cultures of Mouse Tracheal Cells.

Adult (10–14 weeks old) TRPV4 wild-type mice (TRPV4^+/+) and null (TRPV4^−/−) mice generated in a C57Bl6/J background (46) were used for these studies. Primary cultures of airway tracheal epithelial cells were prepared as described in ref. 47. Briefly, the trachea was opened and cut into rings, placed onto 1 mg/ml collagen-coated coverslips, and cultured in DMEM containing 1 mg/liter glucose and supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in 5% CO₂ for 3–4 days. All experiments were carried out with beating ciliated cells. Animals were maintained and experiments were performed according to the guidelines issued by both the Institutional Ethics Committees of the Institut Municipal d'Investigació Mèdica (Universitat Pompeu Fabra) and the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School.

Immunodetection.

Epithelial cell isolation, immunofluorescence, and laser confocal immunolocalization were performed as described in ref. 21. Mouse tracheas were fixed with 4% paraformaldehyde in a 3.7% (wt/vol) sucrose solution before cell isolation using 0.005% Protease XIV dissolved in Ca²⁺-free isotonic solution. Isolated cells were attached to 1.5% gelatin-coated coverslips by spinning at 500 rpm for 3 min using a cytospin (Shandon; Thermo Fisher Scientific) and fixation procedure for 10 min more at room temperature. Single cells were permeabilized with Tween 20 (0.05%) in PBS (15 min at room temperature), and nonspecific interactions were blocked with PBS containing 1.5% BSA, 5% FBS, and 0.05% Tween 20. Isolated epithelial cells were incubated overnight at 4 °C with the primary antibodies diluted in the same blocking solution.

The anti-TRPV4 polyclonal antibody (21, 27) was used at 6.4 μg/ml. A commercial anti-α-tubulin (Sigma-Aldrich) was diluted to 1:500. For immunodetection, we used a goat anti-rabbit IgG Alexa Fluor 488 (Molecular Probes) and a goat anti-mouse IgG Alexa Fluor 555 (Molecular Probes) diluted 1:750 in the same solution used with the primary antibodies. Images were taken at room temperature with an inverted Leica SP2 confocal microscope using a Leica HCX Pl APO 63 × 1.32 NA Oil Ph3 CS objective.

Proteins from TRPV4^+/+ and TRPV4^−/− tracheas were also detected by Western blot technique using the anti-TRPV4 antibody (1:100) as described in refs. 21, 22, and 24.

Measurement of Intracellular [Ca²⁺].

Cytosolic Ca²⁺ signals were determined at room temperature (≈24°C unless otherwise indicated) in ciliated cells loaded with 4.5 μM fura-2·AM (45 min) as described in detail in refs. 24 and 25. Cytosolic [Ca²⁺] increases are presented as the ratio of emitted fluorescence (510 nm) after excitation at 340 nm and 380 nm relative to the ratio measured before cell stimulation (ratio 340/380).

Measurement of CBF.

CBF of cultured ciliated cells was detected and quantified with a high-speed digital imaging system as described in ref. 4. In general, phase-contrast images (512 × 512 pixels) were collected at 120–135 frames s⁻¹ (fps) with a high speed CCD camera using a frame grabber and recording software from Video Savant (IO Industries). CBF was determined from the variation in the light intensity of the image that resulted from the repetitive motion of cilia. Video recordings of beating cilia lasting 1-2 s were analyzed, and the frequency of each ciliary beat cycle was determined from the period of each cycle of the gray-intensity waveform.

Statistics.

All data were expressed as means± SEM. Statistical analysis was performed with the Student's paired or unpaired tests or one-way ANOVA using SigmaPlot or OriginPro software. Bonferroni's test was used for post hoc comparison of means. The criterion for a significant difference was a final value of P < 0.05.