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

Andrade YN, Fernandes J, Vázquez E, Fernández-Fernández JM, Arniges M, Sánchez TM, Villalón M, Valverde MA - J. Cell Biol. (2005)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Grup de Fisiologia Cellular i Molecular, Unitat de Senyalització Cellular, Universitat Pompeu Fabra, Barcelona 08003, Spain.

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.

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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.
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fig4: 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.

Mentions: All the functional data shown point to a TRPV4-like channel as the mediator in the increased [Ca2+]i required for the CBF autoregulatory response. Molecular identification of TRPV4 in hamster oviductal ciliated cells was investigated by single-cell RT-PCR and Western blot. Hamster TRPV4 has not yet been cloned, so we used primers directed against a region of the TRPV4 sequence highly conserved across species. Fig. 4 a shows single amplicons of ∼500 bp obtained from two hamster oviductal ciliated cells. Subsequent sequencing of the bands confirmed the expression of TRPV4 in the ciliated cells. Fig. 4 b shows a Western blot obtained with an antibody generated against a COOH-terminal epitope of the human TRPV4 protein. The antibody identified a double band of the expected size in human TRPV4-transfected HEK cells and a single band in hamster oviductal cells.


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

Andrade YN, Fernandes J, Vázquez E, Fernández-Fernández JM, Arniges M, Sánchez TM, Villalón M, Valverde MA - J. Cell Biol. (2005)

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.
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Related In: Results  -  Collection

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fig4: 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.
Mentions: All the functional data shown point to a TRPV4-like channel as the mediator in the increased [Ca2+]i required for the CBF autoregulatory response. Molecular identification of TRPV4 in hamster oviductal ciliated cells was investigated by single-cell RT-PCR and Western blot. Hamster TRPV4 has not yet been cloned, so we used primers directed against a region of the TRPV4 sequence highly conserved across species. Fig. 4 a shows single amplicons of ∼500 bp obtained from two hamster oviductal ciliated cells. Subsequent sequencing of the bands confirmed the expression of TRPV4 in the ciliated cells. Fig. 4 b shows a Western blot obtained with an antibody generated against a COOH-terminal epitope of the human TRPV4 protein. The antibody identified a double band of the expected size in human TRPV4-transfected HEK cells and a single band in hamster oviductal cells.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Grup de Fisiologia Cellular i Molecular, Unitat de Senyalització Cellular, Universitat Pompeu Fabra, Barcelona 08003, Spain.

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.

Show MeSH
Related in: MedlinePlus