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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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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).
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fig3: 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).

Mentions: Functional TRPV4 channels in oviductal ciliated cells were also evaluated using a synthetic activator of TRPV4, 4α-phorbol 12,13-didecanoate (4αPDD; Watanabe et al., 2002), in the absence of viscous load (1 cP). Intracellular Ca2+ in ciliated cells increased in response to 4αPDD (Fig. 3 a), and, occasionally, showed an oscillatory pattern (not depicted). This response was prevented in the absence of extracellular Ca2+ (Fig. 3 a, middle) or in the presence of Gd3+ (Fig. 3 a, right), a blocker of nonselective cation channels. A whole-cell current with characteristics similar to that obtained with 20% dextran solutions was recorded when ciliated cells were exposed to 1 μM 4αPDD (Fig. 3, b and c). Analysis of the reversal potential of the currents activated by 20% dextran solutions (−4.7 ± 2.4 mV; n = 15) and 4αPDD (−5.5 ± 2.4 mV; n = 6) are not statistically different but showed a right shift compared with control currents (−12 ± 1.3 mV; n = 21) as previously described (Watanabe et al., 2002). Addition of 4αPDD also resulted in the increase of the CBF (Fig. 3 d) in the absence of mechanical stimuli (1 cP), thus adding support to the hypothesis linking the activity of TRPV4 channels to the control of CBF at high viscous loads. Changes in flow superfusion (1–15 ml/min) in the absence of dextran did not activate cationic currents, whereas exposure to 20% dextran solutions activated them either in the absence of fluid flow or under continuous perfusion at 1–3 ml/min. Therefore, it appears that high viscous load by itself is the main trigger of the response, although we cannot completely discard a shear stress component in the response elicited by high dextran solutions.


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)

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).
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fig3: 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).
Mentions: Functional TRPV4 channels in oviductal ciliated cells were also evaluated using a synthetic activator of TRPV4, 4α-phorbol 12,13-didecanoate (4αPDD; Watanabe et al., 2002), in the absence of viscous load (1 cP). Intracellular Ca2+ in ciliated cells increased in response to 4αPDD (Fig. 3 a), and, occasionally, showed an oscillatory pattern (not depicted). This response was prevented in the absence of extracellular Ca2+ (Fig. 3 a, middle) or in the presence of Gd3+ (Fig. 3 a, right), a blocker of nonselective cation channels. A whole-cell current with characteristics similar to that obtained with 20% dextran solutions was recorded when ciliated cells were exposed to 1 μM 4αPDD (Fig. 3, b and c). Analysis of the reversal potential of the currents activated by 20% dextran solutions (−4.7 ± 2.4 mV; n = 15) and 4αPDD (−5.5 ± 2.4 mV; n = 6) are not statistically different but showed a right shift compared with control currents (−12 ± 1.3 mV; n = 21) as previously described (Watanabe et al., 2002). Addition of 4αPDD also resulted in the increase of the CBF (Fig. 3 d) in the absence of mechanical stimuli (1 cP), thus adding support to the hypothesis linking the activity of TRPV4 channels to the control of CBF at high viscous loads. Changes in flow superfusion (1–15 ml/min) in the absence of dextran did not activate cationic currents, whereas exposure to 20% dextran solutions activated them either in the absence of fluid flow or under continuous perfusion at 1–3 ml/min. Therefore, it appears that high viscous load by itself is the main trigger of the response, although we cannot completely discard a shear stress component in the response elicited by high dextran solutions.

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