TRPV4 channel is involved in the coupling of fluid viscosity changes to epithelial ciliary activity

Abstract

Autoregulation of the ciliary beat frequency (CBF) has been proposed as the mechanism used by epithelial ciliated cells to maintain the CBF and prevent the collapse of mucociliary transport under conditions of varying mucus viscosity. Despite the relevance of this regulatory response to the pathophysiology of airways and reproductive tract, the underlying cellular and molecular aspects remain unknown. Hamster oviductal ciliated cells express the transient receptor potential vanilloid 4 (TRPV4) channel, which is activated by increased viscous load involving a phospholipase A(2)-dependent pathway. TRPV4-transfected HeLa cells also increased their cationic currents in response to high viscous load. This mechanical activation is prevented in native ciliated cells loaded with a TRPV4 antibody. Application of the TRPV4 synthetic ligand 4alpha-phorbol 12,13-didecanoate increased cationic currents, intracellular Ca(2+), and the CBF in the absence of a viscous load. Therefore, TRPV4 emerges as a candidate to participate in the coupling of fluid viscosity changes to the generation of the Ca(2+) signal required for the autoregulation of CBF.

Figure 1.

Effect of viscous loading on CBF. (a) Time course of CBF changes in hamster oviduct ciliated cells exposed to 5% (4.8 cP), 12% (21.5 cP), 20% (73 cP), and 30% (200 cP) dextran solutions. (b) Effect of viscous load on the CBF recorded after 20-min exposure to increased viscosity. CBF recorded at steady-state conditions (15–25 min) after exposure to 4.8 (c) or 73 cP (d) in the absence of extracellular Ca²⁺ or in the presence of 100 μM Gd³⁺. Results are the mean ± SEM of 5–10 separate cultures. Significant differences (P < 0.05) between groups are marked with different letters.

Figure 2.

Dextran-activated calcium entry pathway. (a) Cytosolic Ca²⁺ signal obtained in hamster oviductal ciliated cells exposed to 5 or 20% dextran and the effect of extracellular Ca²⁺ removal and 100 μM Gd³⁺ on the 20% dextran-induced Ca²⁺ signal. Traces are representative of five to six experiments under each condition. (b and c) Whole-cell cationic currents recorded in oviductal ciliated cells dialyzed with CsCl-containing pipette solution under control (1 cP) and 5% dextran (4.8 cP) (b) and 20% dextran solution alone (73 cP) or containing 100 μM Gd³⁺ (c). (d) Average current density measured at −100 mV and +100 mV under the following conditions: control (n = 27), 20% dextran (n = 15); 20% dextran + Gd³⁺ (n = 10); and 5% dextran (n = 11). *, P < 0.05, compared with control. (e) Oscillatory pattern of the cationic current obtained in a single cell exposed to 20% dextran.

Figure 3.

Effect of the TRPV4 activator 4αPDD on cytosolic calcium, cationic currents, and CBF. (a, left) Cytosolic Ca²⁺ response of ciliated oviductal cells to 4αPDD (1 μM). Effect of removal of extracellular Ca²⁺ (middle) and 100 μM Gd³⁺ (right) on the 4αPDD response. Traces are means ± SEM obtained from 6–16 cells per culture (repeated on at least three cultures). (b) Whole-cell cationic currents recorded in an oviductal ciliated cell dialyzed with CsCl-containing pipette solution under control conditions, after application of 1 μM 4αPDD, and after application of 1 μM 4αPDD + 100 μM Gd³⁺. (c) Average current density measured at −100 mV and +100 mV under the following conditions: control (n = 15), 1 μM 4αPDD (n = 6), and 4αPDD + 100 μM Gd³⁺ (n = 3). *, P < 0.05, compared with control. (d) Time course of CBF response to 1 μM 4αPDD (n = 6).

Figure 4.

Molecular identification of dextran-activated cationic currents. (a) Detection of TRPV4 in two oviductal ciliated cells (lanes 2 and 3) by RT-PCR. (lane 1) Negative control where the cDNA was omitted. (b) Western blot showing bands at the predicted molecular mass for TRPV4 (∼100 kD) in hamster oviduct and kidney. Untreated HEK-293 and HEK-293 cells transfected with the human TRPV4 (isoform a) were used as negative and positive controls, respectively. (c) Mean current density recorded in oviduct ciliated cells dialyzed with NMDG-Cl solutions containing either 3.2 μg/ml (dilution 1:500) of TRPV4 antibody preabsorbed with a 10-fold excess of antigen (left; n = 5) or TRPV4 antibody alone (right; n = 6). Cells were superfused with 20% dextran solutions 10 min after the establishment of the whole-cell configuration. (d) Confocal images of HeLa cells transfected with EGFP (top) or EGFP+TRPV4 (bottom). EGFP fluorescence signal (green) and TRPV4 signal (red) are shown. (e) Whole-cell cationic currents recorded in HeLa cells with CsCl-containing pipette solution under control (1 cP), 20% dextran solution (73 cP), and washout. (f) Average current density measured at −100 mV in HeLa cells transfected with EGFP (n = 9) or EGFP+TRPV4 (n = 11). *, P < 0.05, compared with control.

Figure 5.

PLA ₂ -dependent activation of TRPV4 under high viscous conditions. (a) Whole-cell currents recorded in a cell dialyzed with NMDG-Cl solutions and bathed consecutively in control solutions, 100 μM pBPB, 20% dextran + pBPB, and 1 μM 4αPDD. Basal current levels recovered after washout. (b) Average current density measured at −100 mV and +100 mV with NMDG-Cl–containing pipette solutions and Ca²⁺-free bathing solution under the following conditions: control (n = 16), 20% dextran (n = 16), pBPB (n = 12), and pBPB + 20% dextran (n = 12). *, P < 0.05, compared with control. (c) CBF recorded at steady-state conditions (15–25 min) under control (1 cP) and 20% dextran solutions (73 cP) in the absence or presence of 50 μM AACOCF₃. Results are the mean ± SEM of 5–10 separate cultures. Significant differences (P < 0.05) between groups are marked with different letters.