Swelling-activated Ca2+ Entry via TRPV4 Channel Is Defective in Cystic Fibrosis Airway Epithelia
2004; Elsevier BV; Volume: 279; Issue: 52 Linguagem: Inglês
10.1074/jbc.m409708200
ISSN1083-351X
AutoresMaite Arniges, Esther Vázquez, José M. Fernández‐Fernández, Miguel A. Valverde,
Tópico(s)Ion channel regulation and function
ResumoThe vertebrate transient receptor potential cationic channel TRPV4 has been proposed as an osmo- and mechanosensor channel. Studies using knock-out animal models have further emphasized the relevance of the TRPV4 channel in the maintenance of the internal osmotic equilibrium and mechanosensation. However, at the cellular level, there is still one important question to answer: does the TRPV4 channel generate the Ca2+ signal in those cells undergoing a Ca2+-dependent regulatory volume decrease (RVD) response? RVD in human airway epithelia requires the generation of a Ca2+ signal to activate Ca2+-dependent K+ channels. The RVD response is lost in airway epithelia affected with cystic fibrosis (CF), a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator channel. We have previously shown that the defective RVD in CF epithelia is linked to the lack of swelling-dependent activation of Ca2+-dependent K+ channels. In the present study, we show the expression of TRPV4 in normal human airway epithelia, where it functions as the Ca2+ entry pathway that triggers the RVD response after hypotonic stress, as demonstrated by TRPV4 antisense experiments. However, cell swelling failed to trigger Ca2+ entry via TRPV4 channels in CF airway epithelia, although the channel's response to a specific synthetic activator, 4α-phorbol 12,13-didecanoate, was maintained. Furthermore, RVD was recovered in CF airway epithelia treated with 4α-phorbol 12,13-didecanoate. Together, these results suggest that defective RVD in CF airway epithelia might be caused by the absence of a TRPV4-mediated Ca2+ signal and the subsequent activation of Ca2+-dependent K+ channels. The vertebrate transient receptor potential cationic channel TRPV4 has been proposed as an osmo- and mechanosensor channel. Studies using knock-out animal models have further emphasized the relevance of the TRPV4 channel in the maintenance of the internal osmotic equilibrium and mechanosensation. However, at the cellular level, there is still one important question to answer: does the TRPV4 channel generate the Ca2+ signal in those cells undergoing a Ca2+-dependent regulatory volume decrease (RVD) response? RVD in human airway epithelia requires the generation of a Ca2+ signal to activate Ca2+-dependent K+ channels. The RVD response is lost in airway epithelia affected with cystic fibrosis (CF), a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator channel. We have previously shown that the defective RVD in CF epithelia is linked to the lack of swelling-dependent activation of Ca2+-dependent K+ channels. In the present study, we show the expression of TRPV4 in normal human airway epithelia, where it functions as the Ca2+ entry pathway that triggers the RVD response after hypotonic stress, as demonstrated by TRPV4 antisense experiments. However, cell swelling failed to trigger Ca2+ entry via TRPV4 channels in CF airway epithelia, although the channel's response to a specific synthetic activator, 4α-phorbol 12,13-didecanoate, was maintained. Furthermore, RVD was recovered in CF airway epithelia treated with 4α-phorbol 12,13-didecanoate. Together, these results suggest that defective RVD in CF airway epithelia might be caused by the absence of a TRPV4-mediated Ca2+ signal and the subsequent activation of Ca2+-dependent K+ channels. The TRPV4 channel, a member of the transient receptor potential (TRP) 1The abbreviations used are: TRP, transient receptor potential; RVD, regulatory volume decrease; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; CFBE, cystic fibrosis bronchial epithelium; HEK, human embryonic kidney; RT, reverse transcription; 4α-PDD, 4α-phorbol 12,13-didecanoate; CFT1, cystic fibrosis tracheal epithelium; hIK, intermediate conductance; AA, arachidonic acid; PLA2, phospholipase A2. family of channels, was first identified as a nonselective cation channel rapidly activated under hypotonic conditions when expressed heterologously (1Strotmann R. Harteneck C. Nunnenmacher K. Schultz G. Plant T.D. Nat. Cell Biol. 2000; 2: 695-702Crossref PubMed Scopus (809) Google Scholar, 2Liedtke W. Choe Y. Marti-Renom M.A. Bell A.M. Denis C.S. Sali A. Hudspeth A.J. Friedman J.M. Heller S. Cell. 2000; 103: 525-535Abstract Full Text Full Text PDF PubMed Scopus (1100) Google Scholar, 3Wissenbach U. Bodding M. Freichel M. Flockerzi V. FEBS Lett. 2000; 485: 127-134Crossref PubMed Scopus (257) Google Scholar). Subsequent studies have shown that TRPV4 channels possess multiple activation and regulatory sites that allow them to integrate distinct physical and chemical stimuli from the environment, offering a wide range of possible physiological roles (4Nilius B. Vriens J. Prenen J. Droogmans G. Voets T. Am. J. Physiol. 2004; 286: C195-C205Crossref PubMed Scopus (376) Google Scholar). TRPV channels can also respond to mechanical stress (5Gao X. Wu L. O'Neil R.G. J. Biol. Chem. 2003; 278: 27129-27137Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 6Suzuki M. Mizuno A. Kodaira K. Imai M. J. Biol. Chem. 2003; 278: 22664-22668Abstract Full Text Full Text PDF PubMed Scopus (600) Google Scholar, 7Liedtke W. Tobin D.M. Bargmann C.I. Friedman J.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14531-14536Crossref PubMed Scopus (287) Google Scholar), heat (8Guler A.D. Lee H. Iida T. Shimizu I. Tominaga M. Caterina M. J. Neurosci. 2002; 22: 6408-6414Crossref PubMed Google Scholar, 9Watanabe H. Vriens J. Suh S.H. Benham C.D. Droogmans G. Nilius B. J. Biol. Chem. 2002; 277: 47044-47051Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar), acidic pH (6Suzuki M. Mizuno A. Kodaira K. Imai M. J. Biol. Chem. 2003; 278: 22664-22668Abstract Full Text Full Text PDF PubMed Scopus (600) Google Scholar), endogenous ligands (10Watanabe H. Vriens J. Prenen J. Droogmans G. Voets T. Nilius B. Nature. 2003; 424: 434-438Crossref PubMed Scopus (814) Google Scholar), and synthetic agonists such as 4α-phorbol 12,13-didecanoate (4Nilius B. Vriens J. Prenen J. Droogmans G. Voets T. Am. J. Physiol. 2004; 286: C195-C205Crossref PubMed Scopus (376) Google Scholarα-PDD) (11Watanabe H. Davis J.B. Smart D. Jerman J.C. Smith G.D. Hayes P. Vriens J. Cairns W. Wissenbach U. Prenen J. et al.J. Biol. Chem. 2002; 277: 13569-13577Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar). Although many studies have implied the involvement of TRPV4 channel in osmosensation (7Liedtke W. Tobin D.M. Bargmann C.I. Friedman J.M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14531-14536Crossref PubMed Scopus (287) Google Scholar, 12Mizuno A. Matsumoto N. Imai M. Suzuki M. Am. J. Physiol. 2003; 285: C96-C101Crossref PubMed Scopus (299) Google Scholar), the role of this channel in the regulatory volume decrease (RVD) response triggered in many cells under hypotonic conditions has not been evaluated. Exposure of cells to hypotonic solutions results in cell swelling followed, in most cases, by an RVD response that returns the cells to their original size. This process involves the activation of ionic pathways that permit the passive loss of electrolytes (typically via K+ and Cl– channels) and osmotically obliged water (13Hoffmann E.K. Dunham P.B. Int. Rev. Cytol. 1995; 161: 173-262Crossref PubMed Scopus (443) Google Scholar). In several cell types, entry of extracellular Ca2+ and subsequent activation of Ca2+-dependent K+ channels is required for an effective RVD (14McCarty N.A. O'Neil R.G. Physiol. Rev. 1992; 72: 1037-1061Crossref PubMed Scopus (331) Google Scholar, 15Pasantes-Morales H. Morales-Mulia S. Nephron. 2000; 86: 414-427Crossref PubMed Scopus (80) Google Scholar). The recent molecular and functional characterization of the TRPV4 channel has suggested its participation in the generation of this Ca2+ signal and the triggering of RVD, although this hypothesis needs to be tested. Human airway epithelia show a typical Ca2+-dependent RVD under hypotonic conditions (16Fernandez-Fernandez J.M. Nobles M. Currid A. Vazquez E. Valverde M.A. Am. J. Physiol. 2002; 283: C1705-C1714Crossref PubMed Scopus (86) Google Scholar), but this RVD response is lost in cystic fibrosis (CF) airways (17Vazquez E. Nobles M. Valverde M.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5329-5334Crossref PubMed Scopus (55) Google Scholar). The cellular defect associated with the impaired RVD in CF epithelia is mainly linked to the dysfunction of volume-sensitive K+ channels (17Vazquez E. Nobles M. Valverde M.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5329-5334Crossref PubMed Scopus (55) Google Scholar, 18Valverde M.A. O'Brien J.A. Sepulveda F.V. Ratcliff R.A. Evans M.J. College W.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9038-9041Crossref PubMed Scopus (56) Google Scholar, 19Valverde M.A. Vazquez E. Munoz F.J. Nobles M. Delaney S.J. Wainwright B.J. Colledge W.H. Sheppard D.N. Cell Physiol. Biochem. 2000; 10: 321-328Crossref PubMed Scopus (26) Google Scholar, 20Belfodil R. Barriere H. Rubera I. Tauc M. Poujeol C. Bidet M. Poujeol P. Am. J. Physiol. 2003; 284: F812-F828Google Scholar), although defective swelling-activated Cl– channels have also been reported (21Barriere H. Belfodil R. Rubera I. Tauc M. Poujeol C. Bidet M. Poujeol P. Am. J. Physiol. 2003; 284: F796-F811Crossref PubMed Scopus (38) Google Scholar). It has been proposed that the dysfunction of K+ channels required for the RVD response might be related to the lack of a Ca2+ signal in CF epithelia, at least in airway and small intestine epithelia (17Vazquez E. Nobles M. Valverde M.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5329-5334Crossref PubMed Scopus (55) Google Scholar, 18Valverde M.A. O'Brien J.A. Sepulveda F.V. Ratcliff R.A. Evans M.J. College W.H. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9038-9041Crossref PubMed Scopus (56) Google Scholar, 19Valverde M.A. Vazquez E. Munoz F.J. Nobles M. Delaney S.J. Wainwright B.J. Colledge W.H. Sheppard D.N. Cell Physiol. Biochem. 2000; 10: 321-328Crossref PubMed Scopus (26) Google Scholar). In this study, we have evaluated the contribution of the TRPV4 channel to the RVD response in normal and CF airway epithelia; in doing so, we have achieved two specific goals. First, we have demonstrated that the TRPV4 channel is the only pathway mediating the swelling-activated Ca2+ entry required to achieve a full RVD in human tracheal epithelial cells. Second, we have shown that the impaired RVD response in CF airway epithelia is caused by a misregulation of TRPV4, suggesting that hypotonic activation of TRPV4 channels is CFTR-dependent. Cells—The cystic fibrosis tracheal epithelial cell line CFT1 was obtained from a CF ΔPhe508/ΔPhe508 patient, and the reverted non-CF cell line CFT1-LCFSN was generated by stable transfection of CFT1 cells with wild-type CFTR (22Yankaskas J.R. Conrad M. Kovai D. Lazarowsky E. Paradiso A.M. Rinehart C.A. Sarkadi B. Schlegel R. Boucher R.C. Am. J. Physiol. 1993; 264: C1219-C1230Crossref PubMed Google Scholar). Both cell lines were grown in Ham's F12 medium (Invitrogen) as described previously (17Vazquez E. Nobles M. Valverde M.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5329-5334Crossref PubMed Scopus (55) Google Scholar). The cystic fibrosis bronchial epithelial cell line CFBE was also obtained from a CF ΔPhe508/ΔPhe508 patient. CFBE cells were grown in modified Eagle's medium with Earle's salts (Invitrogen), 10% fetal bovine serum (Invitrogen), and 1% gentamycin (Sigma). The three cell lines were cultured in fibronectin-coated flasks (16Fernandez-Fernandez J.M. Nobles M. Currid A. Vazquez E. Valverde M.A. Am. J. Physiol. 2002; 283: C1705-C1714Crossref PubMed Scopus (86) Google Scholar). Cells were seeded onto plastic dishes or glass coverslips and used for experiments within 2–3 days. Cytosolic Ca2+ Measurements—Cells were incubated in isotonic bath solution containing 2 μm fura-2 AM (Molecular Probes, Leiden, The Netherlands) for 30 min at room temperature. The cells were then washed thoroughly before initiating the experiment. Video microscopic measurements of [Ca2+]i were obtained using an Olympus IX70 inverted microscope (Hamburg, Germany) with a 40× oil-immersion objective (Olympus). A Polychrome IV monochromator (Till Photonics, Martinsried, Germany) supplied the excitation light (340 and 380 nm), which was directed toward the cells in the field of view by a 505DR dichromatic mirror (Omega Optical, Brattleboro, VT). Fluorescence images were collected by a digital charge-coupled device camera (Hamamatsu Photonics, Hamamatsu City, Japan), after their passage through a 535DF emission filter (Omega Optical), using the AquaCosmos software program (Hamamatsu Photonics). 340/380 nm ratio images were computed every 5 s. The isotonic bathing solution contained 2.5 mm KCl, 140 mm NaCl, 1.2 mm CaCl2, 0.5 mm MgCl2, 5 mm glucose, and 10 mm HEPES (310 milliosmolal, pH 7.35). The hypotonic bathing solution (220 mosm, pH 7.35) was prepared by omitting 50 mm NaCl from the isotonic solution and adjusting the osmolality with d-mannitol where necessary. Ca2+-free extracellular solutions were obtained by replacing CaCl2 with MgCl2. All chemicals were purchased from Sigma-Aldrich except iberiotoxin (Alomone Laboratories, Jerusalem, Israel). Volume Measurements: Morphometric Analysis—Cell volume experiments were carried out at room temperature. Cells were seeded on glass coverslips and were placed in a recording chamber after 3–4 h. The cells were subsequently washed with isotonic solution. Well defined single round cells were selected and digital images acquired using Aquacosmos software (Hamamatsu). The individual cell volume was calculated as described previously (23Bond T.D. Ambikapathy S. Mohammad S. Valverde M.A. J. Physiol. 1998; 511: 45-54Crossref PubMed Scopus (61) Google Scholar, 24Lock H. Valverde M.A. J. Biol. Chem. 2000; 275: 34849-34852Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) and presented as the ratio of volume at time t divided by that measured at time t = 0. Electrophysiological Recordings—Ionic currents were measured at room temperature using the whole-cell recording mode of the patch-clamp technique (25Hamill O.P. Marty A. Neher E. Sakmann B. Sigworth J. Pfluegers Arch. Eur. J. Physiol. 1981; 391: 85-100Crossref PubMed Scopus (15187) Google Scholar) through a D-6100 amplifier (List Medical, Darm-stadt, Germany). The pClamp8 software (Axon Instruments, Foster City, CA) was used for pulse generation, data acquisition, and subsequent analysis. Whole-cell cationic currents were recorded using boro-silicate glass electrodes (2–4 MΩ) filled with a solution containing 120 mm N-methyl-d-glucamine chloride, 1 mm MgCl2, 1 mm EGTA, 30 mm HEPES, 4 mm ATP, and 0.1 mm GTP (290 milliosmolal, pH 7.25). Bath solution contained 125 mm NaCl, 1.5 mm MgCl2, 1 mm EGTA, and 10 mm HEPES (pH 7.36, 305 milliosmolal (adjusted with d-mannitol)). Cells were held at 0 mV and ramps from –140 mV to +100 mV with a duration of 200 ms were applied at a frequency of 0.2 Hz. Ramp data were acquired at 10 KHz and low-pass filtered at 1 kHz. Whole-cell K+ (Maxi K+ and hIK) currents were measured as described previously (16Fernandez-Fernandez J.M. Nobles M. Currid A. Vazquez E. Valverde M.A. Am. J. Physiol. 2002; 283: C1705-C1714Crossref PubMed Scopus (86) Google Scholar, 17Vazquez E. Nobles M. Valverde M.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5329-5334Crossref PubMed Scopus (55) Google Scholar). Pipette solution contained 140 mm KCl, 2 mm MgCl2, 0.15 mm CaCl2, 0.5 mm EGTA, and 10 mm HEPES (290 milliosmolal, pH 7.25; the intracellular free Ca2+ concentration was 50 nm as calculated using EqCal from Biosoft (Cambridge, UK)). The isotonic bathing solution contained 100 mm NaCl, 5 mm KCl, 1.2 mm CaCl2, 0.5 mm MgCl2, 5 mm glucose, 10 mm HEPES, and 90 mm d-mannitol (305 milliosmolal; pH 7.35). The hypotonic bathing solution (215 milliosmolal) was prepared by omitting d-mannitol from the isotonic solution. Ca2+-free bath solutions, containing 0 Ca2+, 1.5 mm MgCl2, and 1 mm EGTA, were also used in several experiments. Cells were clamped at –80 mV and pulsed for 400 ms from –100 mV to +100 mV in 20-mV steps. RNA Extraction and RT-PCR—RNA extraction and RT-PCR were performed as described previously (16Fernandez-Fernandez J.M. Nobles M. Currid A. Vazquez E. Valverde M.A. Am. J. Physiol. 2002; 283: C1705-C1714Crossref PubMed Scopus (86) Google Scholar). In brief, RNA was isolated from CFT1-LCFSN, CFT1, and CFBE cells using the Nucleospin RNA II kit (Macherey-Nagel, Germany), according to the manufacturer's instructions. Total RNA (1–2 μg) was reverse-transcribed to cDNA. GADPH was used as a positive control, and negative controls (absence of the oligodT primer or MMLV-RT) were also performed. We used the following primer pair for PCR amplification of TRPV4: forward, 5′-CCTCTTCCCCGACAGCAAC-3′, and reverse, 5′-CCCCAGTGAAGAGCGTAATG-3′, as described previously (16Fernandez-Fernandez J.M. Nobles M. Currid A. Vazquez E. Valverde M.A. Am. J. Physiol. 2002; 283: C1705-C1714Crossref PubMed Scopus (86) Google Scholar), which gives a 500 bp product (nucleotides 1154–1653; GenBank™ accession number NM_021625, isoform a) and a 319 product in case of isoform b (nucleotides 1154–1473; GenBank™ accession number NM_147204). The amplicons were confirmed by direct sequencing. Cloning of Human TRPV4 (hTRPV4) and Transient Expression in HEK-293 Cells—Total RNA was extracted from human tracheal epithelial CFT1-LCFSN cells using the Nucleospin RNA II Kit (Macherey-Nagel, Düven, Germany). The full-length TRPV4 cDNA was amplified by an RT-PCR-based protocol (OneStep RT-PCR kit; Qiagen) using the following profile: 1 h at 50 °C, 15 min at 90 °C, 45 cycles of 1 min at 94 °C, 1 min at 55 °C and 3.5 min plus 1 s/cycle at 68 °C ending with 10 min at 68 °C. The primers used were: forward, 5′-AAGCATGGCGGATTCCAGCGAAG-3′ (nucleotides 86–108); and reverse, 5′-CTAGAGCGGGGCGTCATCAGTC-3′ (nucleotides 2684–2705). The fully sequenced TRPV4 cDNA corresponded to the GenBank™ accession number NM_021625 (isoform a) containing a synonymous single nucleotide polymorphism in position 761 (dbSNP reference number rs3825394). The cDNA was inserted into the eukaryotic pcDNA-3 expression vector and transfected into HEK-293 cells, grown as described previously (26Vriens J. Watanabe H. Janssens A. Droogmans G. Voets T. Nilius B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 396-401Crossref PubMed Scopus (496) Google Scholar), using a linear polyethylenimine derivative (polycation ExGen500; Fermentas MBI). Cells were seeded in 35-mm dishes at 80% confluence and transfected with 3 μg of the construct, together with the reporter pEGFPN1 at a 10:1 ratio or with pEGFPN1 alone (control). Antisense Oligonucleotides—CFT1-LCFSN cells were seeded in 12-mm wells at 80% confluence and were exposed to 1 μm of TRPV4 antisense morpholino oligonucleotide with a sequence 5′-GCCTTCGCTGGAATCCGCCAT-3′ which hybridizes to the first 21 nucleotides (90–110 bp) of both isoform a (GenBank NM_021625) and isoform b (GenBank NM_147204) of the human TRPV4 or to 1 μm β-globin antisense morpholino oligonucleotide with the sequence 5′-CCTCTTACCTCAgTTACAATTTATA-3′ as negative control (Gene Tools, Corvallis, OR) plus 100 ng of pEGFP plasmid (BD Biosciences Clontech) diluted into 25 μl of serum-free medium. Cells were transfected by a Lipofectamine Plus (Invitrogen) procedure following the manufacturer's instructions. 24 h after transfection, cells were trypsinized and seeded for performing experiments within 1–2 days. Western Blot—88 μg of total protein was obtained from CFT1-LCFSN cells harvested 96 h after transfection with TRPV4/β-globin antisense (6 μm). The protein was separated on a polyacrylamide gel (8%) and electrotransferred to nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). Total protein from HEK-293 cells transfected with TRPV4 was used as a control. The nitrocellulose membrane was incubated in blocking buffer (5% nonfat dry milk in TTBS (100 mm Tris-HCl, pH 7.5, 150 mm NaCl, and 0.1% Tween 20)) for 1 h at room temperature, followed by incubation overnight at 4 °C in blocking buffer containing TRPV4 antibody (1:500 dilution). This antibody was raised against a synthetic peptide representing the C-terminal sequence of human TRPV4 (CDGHQQGYPRKWRTDDAPL) and then subjected to affinity purification with the peptide antigen. The membranes were washed with blocking buffer to remove the nonspecific binding and incubated with horseradish peroxidase-conjugated donkey antirabbit IgG (Amersham Biosciences) at a dilution of 1:2000 in blocking buffer for 1 h at room temperature. Nitrocellulose membranes were incubated with an enhanced chemiluminescence reagent (SuperSignal West Femto, Chemiluminescent Substrate; Pierce) and films (Amersham Biosciences) were exposed to the membrane. Statistics—Results are expressed as means ± S.E of n observations. Student's t test was performed to examine statistical significance. p ± 0.05 was considered significant. Swelling-activated Ca2+ Entry in Normal and CF Human Tracheal Epithelial Cells—CFT1 tracheal epithelial cells, derived from a ΔPhe508/ΔPhe508 patient, show a typical CF ion transport phenotype, including impaired RVD, whereas the CFT1-LCFSN cells, obtained by stable transfection of CFT1 cells with wild-type CFTR, show both cAMP-dependent ion secretion and RVD (17Vazquez E. Nobles M. Valverde M.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5329-5334Crossref PubMed Scopus (55) Google Scholar). The RVD response in the recovered tracheal cells (CFT1-LCFSN), as well as in normal human bronchial cells, relies on the activation of Ca2+-dependent K+ channels after the influx of Ca2+ (16Fernandez-Fernandez J.M. Nobles M. Currid A. Vazquez E. Valverde M.A. Am. J. Physiol. 2002; 283: C1705-C1714Crossref PubMed Scopus (86) Google Scholar, 17Vazquez E. Nobles M. Valverde M.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5329-5334Crossref PubMed Scopus (55) Google Scholar). Therefore, we evaluated whether CF airway epithelial cells might posses a defect in the activation of swelling-dependent Ca2+ entry. Using the Ca2+-sensitive fura-2 dye, we observed that CFT1-LCFSN cells triggered a significant Ca2+ signal in response to a 30% hypotonic shock (Fig. 1A), whereas CFT1 cells showed negligibly low changes (Fig. 1B). Removal of extracellular Ca2+ or addition of Gd3+ abolished the Ca2+ response of CFT1-LCFSN cells to hypotonic stress (results not shown). The molecular nature of the Ca2+ entry pathway was subsequently investigated. Functional Expression of TRPV4 Channel in Airway Tracheal Epithelial Cells—A possible candidate to mediate Ca2+ entry in response to cell swelling is the TRPV4 channel (4Nilius B. Vriens J. Prenen J. Droogmans G. Voets T. Am. J. Physiol. 2004; 286: C195-C205Crossref PubMed Scopus (376) Google Scholar). Therefore, we evaluated the expression and function of TRPV4 channels and their contribution to RVD in normal and CF airway epithelial cells. Expression of TRPV4 channel in human normal (CFT1-LCFSN cells) and CF tracheal epithelial cells (CFT1) was analyzed by RT-PCR. Fig. 1C shows the amplification of two specific bands corresponding to TRPV4 isoforms a (500 bp) and b (319 bp) in CFT1-LCSFN and CFT1 cells. The activity of the TRPV4 channel was evaluated using a synthetic activator, 4α-phorbol 12,13-didecanoate (4α-PDD), specific for the TRPV4 channel (11Watanabe H. Davis J.B. Smart D. Jerman J.C. Smith G.D. Hayes P. Vriens J. Cairns W. Wissenbach U. Prenen J. et al.J. Biol. Chem. 2002; 277: 13569-13577Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar). The intracellular Ca2+ signals induced by 1 μm 4α-PDD in the non cystic fibrosis tracheal cell line CFT1-LCSFN (Fig. 2A; mean peak 340/380 ratio increase: 0.16 ± 0.02; n = 13) and the cystic fibrosis CFT1 cell line (Fig. 2B; mean increase 0.10 ± 0.06; n = 30) were not statistically significant. The response to 4α-PDD in both cell lines was abolished in the absence of extracellular Ca2+ (Fig. 2, C–D) or in the presence of 100 μm Gd3+ (Fig. 2, E–F), a blocker of TRPV4 channels (1Strotmann R. Harteneck C. Nunnenmacher K. Schultz G. Plant T.D. Nat. Cell Biol. 2000; 2: 695-702Crossref PubMed Scopus (809) Google Scholar). The activity of TRPV4 was also evaluated electrophysiologically. Application of 1 μm 4α-PDD induced typical TRPV4 cationic currents that rectified inwardly when pipettes were loaded with impermeant cation-containing solutions (Fig. 2, G–H). Taken together, our data support the presence of TRPV4 in both human normal and CF tracheal epithelial cells, although its activation by cell swelling appears only in non-CF cells. Defective activation of TRPV4 in CF cells under hypotonic conditions might explain the impaired Ca2+-dependent RVD observed in different CF epithelia (17Vazquez E. Nobles M. Valverde M.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5329-5334Crossref PubMed Scopus (55) Google Scholar, 19Valverde M.A. Vazquez E. Munoz F.J. Nobles M. Delaney S.J. Wainwright B.J. Colledge W.H. Sheppard D.N. Cell Physiol. Biochem. 2000; 10: 321-328Crossref PubMed Scopus (26) Google Scholar). This hypothesis relies on the assumption that TRPV4 channels are essential in providing the Ca2+ entry pathway to trigger the RVD response in airway epithelia. Therefore, we next investigated the role of TRPV4 in the RVD response in CFT1-LCFSN. TRPV4 Channel Activity Is Required for RVD in Tracheal Epithelial Cells—Many reports have demonstrated the activation of TRPV4 channels after cell swelling under hypotonic conditions (for review, see Ref. 4Nilius B. Vriens J. Prenen J. Droogmans G. Voets T. Am. J. Physiol. 2004; 286: C195-C205Crossref PubMed Scopus (376) Google Scholar). However, none have shown that the activation of TRPV4 channels provides the actual Ca2+ signal needed to trigger the RVD response. To test this hypothesis, we incubated CFT1-LCSFN cells with TRPV4 antisense oligonucleotides. We also used a control antisense oligonucleotide directed against an unrelated protein, β-globin. As illustrated in Fig. 3E, TRPV4 antisense specifically reduced TRPV4 protein expression levels compared with control β-globin antisense (α-tubulin expression was unaltered; results not shown). Next, the hypotonic-induced activity of Ca2+-dependent KCNN4 potassium channels, needed for the RVD response (17Vazquez E. Nobles M. Valverde M.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5329-5334Crossref PubMed Scopus (55) Google Scholar), and the RVD itself were evaluated. Swelling-dependent activation of KCNN4 currents, which occurs only in the presence of extracellular Ca2+ (results not shown), was prevented in TRPV4 antisense-treated cells (Fig. 3A) but preserved in CFT1-LCFSN cells treated with control antisense (Fig. 3B). Morphometric cell volume analysis showed that anti-TRPV4 oligonucleotides inhibited the RVD response (Fig. 3C) in CFT1-LCFSN cells. Cells incubated with control oligonucleotides maintained the RVD response, recovering to nearly 100% of their original volume after 25 min (Fig. 3D). The fact that both activation of Ca2+-dependent K+ channels and RVD are prevented in those cells incubated with TRPV4 antisense oligonucleotide provides the first direct piece of evidence relating the activity of TRPV4 with the RVD response. Human CF Bronchial Epithelial Cells Also Show Defective Ca2+-dependent RVD—Human bronchial epithelial cells also show an RVD that depends on the entry of Ca2+ and the activation of Maxi K+ channels (KCNMA1) (16Fernandez-Fernandez J.M. Nobles M. Currid A. Vazquez E. Valverde M.A. Am. J. Physiol. 2002; 283: C1705-C1714Crossref PubMed Scopus (86) Google Scholar). However, no data exist on the RVD response in human CF bronchial epithelial cells. The presence of the two TRPV4 isoforms (a and b) in a human CF bronchial epithelial cell line (CFBE) obtained from a patient homozygous for the ΔPhe508 mutation was determined by RT-PCR (Fig. 4A). CFBE cells exposed to 30% hypotonicity, like human CF tracheal cells (CFT1) (17Vazquez E. Nobles M. Valverde M.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5329-5334Crossref PubMed Scopus (55) Google Scholar), were unable to regulate their volume and remained swollen during the 20-min exposure to hypotonic solution (Fig. 4B). Similar to CF tracheal cells (CFT1), CFBE cells did not respond with a significant increase in intracellular Ca2+ when exposed to hypotonic solutions (Fig. 4C) but did respond to 1 μm 4α-PDD (Fig. 4D). The response to 4α-PDD was absent when cells were incubated in Ca2+-free solutions or in the presence of 100 μm Gd3+ (results not shown). As mentioned above, RVD in normal human bronchial epithelial cells requires the activation of Maxi K+ channels (16Fernandez-Fernandez J.M. Nobles M. Currid A. Vazquez E. Valverde M.A. Am. J. Physiol. 2002; 283: C1705-C1714Crossref PubMed Scopus (86) Google Scholar). Maxi K+ currents in CFBE, identified by their inhibition by 100 nm iberiotoxin (results not shown), failed to respond to hypotonic conditions (Fig. 4E). Current density at –100 mV was 37.7 ± 4.19 pA/pF (n = 13) under isotonic conditions and 39.3 ± 4.9 pA/pF (n = 8) under hypotonic conditions. Together, these results suggest that human CF tracheal and bronchial cells lack an effective RVD because of a deficient Ca2+ influx via the TRPV4 channel and the subsequent activation of Ca2+-dependent K+ channels. Activation of TRPV4 by 4α-PDD Restores RVD in CF Airway Cells—The possibility to revert the defective RVD in human CF airway cells using the TRPV4 activator 4α-PDD was evaluated next. CFT1 and CFBE cells underwent a significant RVD when exposed to hypotonic conditions in the presence of 1 μm 4α-PDD (Fig. 5, A and D). Whereas CF tracheal cells required the presence of 4α-PDD throughout the whole hypotonic stimulus for an effective RVD, CF bronchial cells rapidly down-regulate their volume immediately after the addition of 4α-PDD. The diffe
Referência(s)