QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.107.135582v1 325/3/1016    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Noël, S. Articles by Becq, F. Search for Related Content PubMed PubMed Citation Articles by Noël, S. Articles by Becq, F.

CELLULAR AND MOLECULAR

Parallel Improvement of Sodium and Chloride Transport Defects by Miglustat (n-Butyldeoxynojyrimicin) in Cystic Fibrosis Epithelial Cells

Institut de Physiologie et Biologie Cellulaires, Université de Poitiers, Centre National de la Recherche Scientifique, Poitiers, France (S.N., F.B.); and Department of Biochemistry, Erasmus University Medical Center, Rotterdam, The Netherlands (M.W., A.G.M.B., H.R.D.J.).

Received December 18, 2007; accepted February 27, 2008.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Cystic fibrosis, an autosomal recessive disease frequently diagnosed in the Caucasian population, is characterized by deficient Cl^- transport due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. A second major hall-mark of the disease is Na⁺ hyperabsorption by the airways, mediated by the epithelial Na⁺ channel (ENaC). In this study, we report that in human airway epithelial CF15 cells treated with the CFTR corrector miglustat (n-butyldeoxynojyrimicin), whole-cell patch-clamp experiments showed reduced amiloride-sensitive ENaC current in parallel with a rescue of defective CFTR Cl^- channel activity activated by forskolin and genistein. Similar results were obtained with cells maintained in culture at 27°C for 24 h before electrophysiology experiments. With monolayers of polarized CF15 cells, short-circuit current (Isc) measurements also show normalization of Na⁺ and Cl^- currents. In excised nasal epithelium of cftr^{F508del/F508del} mice, like with CF15 cells, we found normalization of amiloride-sensitive Isc. Moreover, oral administration of miglustat (6 days) decreased the amiloride-sensitive Isc in cftr^{F508del/F508del} mice but had no effect on cftr^-/- mice. Our results thus show that rescuing the trafficking-deficient F508del-CFTR by miglustat down-regulates Na⁺ absorption. A miglustat-based treatment of CF patients may thus have a beneficial effect both on Cl^- and Na⁺ transports.

It is now well documented that CFTR also regulates many transport proteins and cellular functions due to large and dynamic macromolecular complexes that contain CFTR, signaling molecules and transport proteins (for review, see Kunzelmann et al., 2000; Guggino and Stanton, 2006). In favor of this concept, several studies showed that the functional interaction between CFTR and ENaC regulates both epithelial Cl^- and Na⁺ conductances (Guggino and Stanton, 2006). However, it remains unclear why CF tissues display such a high ENaC activity. In addition, no studies have examined the effect of F508del-CFTR correction on Na⁺ hyperabsorption in native tissues. Finally, other studies argued against a regulation of ENaC by CFTR after coexpression in Xenopus laevis oocytes (Nagel et al., 2005).

We recently showed that miglustat rescues the trafficking-deficient F508del-CFTR to the plasma membrane in human airway epithelial cells but also in the intestine of F508del-CFTR mice (Norez et al., 2006; Antigny et al., 2008). Miglustat is now evaluated in CF patients within a pilot phase 2a clinical trial (http://clinicaltrials.gov/). In this report, we addressed the question whether miglustat, by rescuing F508del-CFTR abnormal trafficking, also down-regulates ENaC-dependent sodium hyperabsorption. To this aim, we have studied endogenous CFTR and ENaC channels in miglustat-corrected human airway epithelial CF15 cells and in cftr^{F508del/F508del} mice. We demonstrate that the rescue of endogenous F508del-CFTR by miglustat or by low temperature in human airway and excised nasal epithelium of cftr^{F508del/F508del} mice is paralleled by a down-regulation of Na⁺ absorption.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Cell Culture. The human nasal epithelial JME/CF15 cell line, derived from a F508del-CFTR homozygous patient (Jefferson et al., 1990), was grown at 37°C in 5% CO₂ under standard culture conditions, in Dulbecco's modified Eagle's medium-Ham's F-12 (3:1) nutritive mix supplemented by 10% fetal bovine serum, 100 IU/ml penicillin and 100 µg/ml streptomycin, 5 µg/ml insulin, 5 µg/ml transferrin, 5.5 µM epinephrine, 180 µM adenine, 2 nM 3,3',5-triiodo-L-thyronine sodium salt, and 1.1 µM hydrocortisone (Cao et al., 2005; Norez et al., 2006). All culture media and antibiotics were from Invitrogen (Carlsbad, CA). Fetal bovine serum was from PerbioScience (Brebières, France). Hormones and growth factors were from Sigma-Aldrich (St. Louis, MO). Cells were seeded in 35-mm plastic dishes for whole-cell patch-clamp recordings and on 12-mm snap wells (diameter, 1.13 cm²; pores, 3 µm; Corning Life Sciences, Acton, MA) for Ussing chamber experiments. Medium was renewed at 2-day interval.

Patch-Clamp Experiments. Perforated whole-cell patch-clamp experiments were performed on CF15 cells at room temperature. Currents were recorded with an RK-400 patch-clamp amplifier (Biologic, Grenoble, France). I-V relationships were built by clamping the membrane potential to -20 mV and by pulses from -140 to +100 mV (20-mV increments). Pipettes with resistance of 3 to 4 M were pulled from borosilicate glass capillary tubing (GC150-TF10; Clarke Electromedical Instruments, Pangbourne, UK) using a two-step vertical puller (Narishige, Tokyo, Japan). They were filled with the following solution: 20 mM NaCl, 100 mM L-aspartic acid, 100 mM CsOH, 1 mM MgCl₂, 20 mM CsCl, 4 mM EGTA, and 10 mM HEPES, pH 7.2. Amphotericin B (100 µg/ml) was dissolved ex temporane. Pipettes were connected to the head of the patch-clamp amplifier through an Ag-AgCl pellet. Seal resistances ranging from 6 to 20 G were obtained. Results were analyzed with the pClamp 6.0.2 package software (pClamp; Molecular Devices, Sunnyvale, CA). The external bath solution contained 150 mM NaCl, 6 mM CsCl, 1 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose, and 10 mM HEPES, pH 7.4. The liquid potential was corrected before seal establishment. Pipette capacitances were electronically compensated in cell-attached mode. To standardize experiments, recordings were performed only when the input resistance had a value 15 M. The mean value of access resistances was 11.7 ± 1.6 M (n = 42). Membrane capacitances were measured in the whole-cell mode by fitting capacitance currents, obtained in response to a hyperpolarization of 6 mV, with a first order exponential and by integrating the surface of the capacitance current. Mean values of membrane capacitance were 30.7 ± 8.2 pF (n = 42). For graphic representations, I-V relationship was normalized to 1 pF to remove variability due to differences in cell sizes.

Animals. Rotterdam homozygous F508del-CFTR mice (cftr^tm1Eur) and their litter mate controls (FVB inbred, 14–17 weeks old, weight between 20 and 30 g) were kept on solid food in a pathogen-free environment. cftr^-/- mice (cftr^tm2Cam) were backcrossed for 12 generations into the FVB background (Scholte et al., 2004). Animals were anesthetized by an i.p. injection of a cocktail containing ketamine (10 mg/ml), xylazine (1.5 mg/ml), and diazepam (0.6 mg/ml). Nasal epithelia were dissected away from mice and mounted in a mini-Ussing chamber (exposed tissue area, 1.13 mm²) (De Jonge et al., 2004).

Short-Circuit Current Measurements. For short-circuit current (Isc) measurements, we seeded CF15 cells on semipermeable membrane snap well inserts (exposed surface area, 1.13 cm²). When cells were forming an impermeable monolayer (transepithelial resistance 500 /cm²), short-circuit current recordings were performed. Snap wells were mounted in a vertical Ussing chamber (Harvard Apparatus Inc., Holliston, MA). Cells were bathed in Meyler buffer (apical and basolateral side) containing the following: 120 mM NaCl, 1.2 mM CaCl₂, 1.2 mM MgCl₂, 0.8 mM K₂HPO₄, 3.3 mM KH₂PO₄, 25 mM NaHCO₃, and 10 mM D-glucose, pH 7.4 (gassed with 95% O₂-5% CO₂ at 37°C). Current magnitude was referred to the apical side of the monolayer. Miglustat was added directly to the culture medium (100 µM, 2 h, 37°C). Ex vivo short-circuit currents across isolated nasal epithelium were recorded first under control conditions using continuous oxygenated (95% CO₂-5% O₂) and temperature-controlled (37°C) Meyler solution, and measurements were repeated on the same tissue after 2-h incubation in Meyler solution containing 100 µM miglustat. For other technical details, see elsewhere (Noel et al., 2006).

Oral Administration of Miglustat to Mice and Isc Measurements. To evaluate the effect of in vivo miglustat treatment on amiloride-sensitive current in nasal epithelium, we administrated 1200 mg/kg/day miglustat by gavage to cftr^{F508del/F508del} or cftr^-/- (FVB) mice. Control groups received vehicle, i.e., PBS solution only. After 6 days, we dissected the nasal epithelium from mice of the two groups and recorded the amiloride-sensitive Isc ex vivo.

Pharmacological Agents. The specific CFTR inhibitor CFTR_inh-172 (Ma et al., 2002) was from Calbiochem (San Diego, CA). Forskolin was from LC Laboratories (Woburn, MA). Miglustat was purchased from Toronto Research Chemicals (Toronto, ON, Canada). All other chemicals were obtained from Sigma-Aldrich. All chemicals were dissolved in DMSO (final concentration in DMSO < 0.1%) except miglustat, which was dissolved in water for all in vitro and ex vivo experiments and in PBS for oral administration. The currents were not altered by DMSO alone.

Data Analysis. All the data are presented as mean value ± S.E.M., where n refers to the number of experiments and N to the number of animals. The unpaired Student's t test was used to compare sets of data. All graphs are plotted with GraphPad Prism 4.0 for Windows (GraphPad Software Inc., San Diego, CA). Values of P < 0.05 were considered as statistically significant: *, P < 0.05; **, P < 0.01; and ***, P < 0.001. N.S. difference was P > 0.05.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Miglustat Reduces Amiloride-Sensitive Na⁺ Current. Perforated whole-cell patch-clamp experiments were performed to measure the impact of miglustat treatment on ENaC and CFTR currents in human airway epithelial CF cells (cells maintained 2 h at 37°C in a culture medium containing 100 µM miglustat; Fig. 1). JME/CF15 cells derived from the nasal airway epithelium of a CF patient (homozygous F508del-CFTR; Jefferson et al., 1990) were not responsive to forskolin (fsk)/genistein (gst) stimulation but displayed a significant amiloride-sensitive Na⁺ current (Tong et al., 2004; Cao et al., 2005). In the first series of experiments, we identified and characterized ENaC currents in several bath conditions (as indicated on the top of each panel) for control CF15 cells cultured at 37°C (Fig. 1, left traces), for temperature (27°C)-corrected cells (Fig. 1, middle traces) and for miglustat-corrected cells (Fig. 1, right traces). For each cell for which a perforated whole-cell experiment was possible, we first recorded basal currents (Fig. 1A) and then added 100 µM amiloride in the bath solution (Fig. 1B). By subtracting the residual current in the presence of amiloride from the basal current, we obtained the mean value for amiloride-sensitive current normalized to the cell capacitance and calculated the mean current densities (pA/pF; Fig. 2). At +100 mV, we obtained mean values of 7.8 ± 2.3 pA/pF for control cells (n = 12), 0.52 ± 0.35 pA/pF for low temperature-corrected cells (n = 7, P < 0.05), and 1.7 ± 0.8 pA/pF for miglustat-corrected cells (n = 10, P < 0.05). Then, on the same cell, we added the cocktail containing 10 µM forskolin plus 30 µM genistein to activate CFTR currents (Fig. 1C). Under this condition, we only observed activation of linear currents with miglustat-corrected (n = 10) and low temperature-corrected (n = 7; Figs. 1C and 2) cells. In the presence of fsk/gst in the bath, the corresponding experimental reversal potential E_rev was -33.6 ± 3.4 mV for miglustat and -32.6 ± 2.9 mV for low temperature-corrected cells, both values being near the theoretical Nernst potential for the Cl^- ion (E_Cl- =-33 mV) showing stimulation of chloride-selective currents. We determined the current density, after adding amiloride, for the Cl^- currents recorded with miglustat-treated cells (current density at +100 mV, 11.6 ± 3.2 pA/pF, n = 10) and with temperature-corrected CF15 cells (current density at +100 mV, 16.5 ± 4.9 pA/pF, n = 7). No activation of Cl^- current was recorded in untreated CF15 cells (current density of 1.75 ± 0.27 pA/pF, n = 12). To prove that the Cl^- current activated after amiloride in the presence of fsk/gst was due to F508del-CFTR, we perfused 10 µM of the specific CFTR inhibitor CFTR_inh-172 (Ma et al., 2002). This maneuver fully inhibited Cl^- currents in miglustat- and temperature-corrected cells (Fig. 1D).

View larger version (33K): [in this window] [in a new window]   Fig. 1. Perforated whole-cell patch-clamp experiments on homozygous F508del/F508del airway epithelial CF15 cells. Currents were recorded in uncorrected cells (left, noted 37°C), temperature-corrected cells (middle, 27°C), or miglustat-corrected cells (right). For each condition, the example given is the complete sequence of experiments shown for the same cell. The current was recorded in the control condition (A), after addition of amiloride (B), in the presence of amiloride + cocktail composed of forskolin plus genistein (C) and finally after adding CFTRinh-172 (D). Concentrations used are as follows: amiloride, 100 µM; forskolin, 10 µM; genistein, 30 µM; CFTRinh-172, 10 µM; and miglustat, 100 µM.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Mean voltage-current density relationships of the ENaC Na+ and CFTR Cl- currents on F508del/F508del airway epithelial CF15 cells. The residual current in the presence of amiloride was subtracted from the basal current. The CFTR Cl- current corresponds to fsk/gst-activated current. The number of experiments is n = 12 for control 37°C, n = 10 for miglustat, and n = 7 for low temperature. Concentrations used are as follows: forskolin, 10 µM; genistein, 30 µM; and miglustat, 100 µM.

Activation of CFTR Does Not Influence ENaC Currents in CF15 Cells. In a second series of experiments, we wished to evaluate the effect of a preactivation of CFTR by fsk/gst on the amiloride-sensitive current on CF15 cells in the same experimental conditions as in Fig. 1. To that end, we reversed the protocol for channel activation, i.e., we activated CFTR first and then measured amiloride-sensitive ENaC currents. Figure 3A shows spontaneous control currents in resting CF15 cells. Adding fsk/gst in the experimental chamber activated a linear Cl^- current only in miglustat-corrected cells (Fig. 3B, right traces, current density at +100 mV of 13.1 ± 3.1 pA/pF, n = 4, data not shown). As expected, no Cl^- current was activated in untreated cells (Fig. 3B, left traces). The Cl^- current density at +100 mV was 6.8 ± 0.9 pA/pF in the control condition and 5.8 ± 0.8 pA/pF in the presence of fsk/gst in the bath (N.S.; data not shown). After adding amiloride to the bath to block the activity of ENaC, the residual current in miglustat-corrected cells (Fig. 3C, right traces) was inhibited by CFTR_inh-172 (Fig. 3D, right traces) indicating that F508del-CFTR channels were active. We measured the corresponding amiloride-sensitive ENaC current and calculated the amiloride-sensitive current density at +100 mV (Fig. 4). For miglustat-corrected cells, we found no significant difference between the amiloride-sensitive current with fsk/gst in the bath (1.76 ± 0.3 pA/pF at +100 mV, n = 4, N.S.) or without (1.7 ± 0.8 pA/pF at +100 mV, n = 10). Likewise, no significant effect of fsk/gst was noted on the magnitude of the amiloride-sensitive current in untreated CF15 cells (7.8 ± 2.3 pA/pF in the control condition, n = 12; 6.7 ± 1.7 pA/pF in the presence of fsk/gst, n = 9, N.S.) (Fig. 4). In addition, we performed several additional experiments to determine whether miglustat could by itself activate membrane conductances in CF cells. When perfusing miglustat in the experimental chamber bathing CF15 cells (cultured at 37°C), we were not able to record any conductances (data not shown). Moreover, using iodide efflux methods, we did not detect any stimulation of efflux in the presence of this agent. Finally, with CF15 cells treated for 2 h by this corrector, no modulation of either volume- or calcium-dependent-iodide efflux was noted (data not shown; see also Norez et al., 2006). Taken together, these experiments show that the iminosugar miglustat is not a channel activator but rather a F508del-CFTR corrector.

View larger version (25K): [in this window] [in a new window]   Fig. 3. Effect of forskolin/genistein on ENaC currents in CF15 cells. Shown are representative current recordings for a single control CF15 cell (37°C, left traces) and incubated for 2 h with miglustat (right traces) under control conditions (A), after addition of the cocktail forskolin/genistein (B), in the presence of amiloride + forskolin/genistein (C), and after addition of CFTRinh-172 in the presence of amiloride + forskolin/genistein (D). Concentrations used are as follows: amiloride, 100 µM; forskolin, 10 µM; genistein, 30 µM; CFTRinh-172, 10 µM; and miglustat, 100 µM.

View larger version (11K): [in this window] [in a new window]   Fig. 4. Effect of forskolin/genistein on the mean voltage-current density relationships of the ENaC current at +100 mV. The residual current in the presence of amiloride was subtracted from the basal current (black bars) or from the fsk/gst-activated current (open bars). The number of experiments is indicated on each bar graph. Concentrations used are as follows: forskolin, 10 µM; genistein, 30 µM; and miglustat, 100 µM. N.S. difference was P > 0.05.

Miglustat Reduces Amiloride-Sensitive Isc in Polarized CF15 Cells. We performed Ussing chamber experiments on CF15 cells that we seeded on semipermeable snap well membrane in control conditions and after 2-h incubation in culture medium containing miglustat (Fig. 5). The mean value of transepithelial resistance was 531 ± 26 /cm² (n = 4) for untreated cells monolayers and increased to 658 ± 39 /cm² (n = 4, P < 0.05) for miglustat-corrected cells monolayers. Adding amiloride to the apical compartment induced a change of Isc, i.e., inhibition of apical Na⁺ absorption (Fig. 5A). We found Isc of -2.05 ± 1.1 µA/cm² for miglustat-corrected CF15 cells monolayers (n = 4) and -7.43 ± 0.7 µA/cm² (n = 4) for control monolayers (P < 0.01; Fig. 5B). Then, addition of 10 µM fsk (basolateral side) and 30 µM gst (both sides) stimulated a glibenclamide-sensitive Isc, i.e., activation of apical F508del-CFTR-dependent Cl^- secretion, for miglustat-corrected monolayers (Fig. 5B) but not for control monolayers (Fig. 5A). The Isc was 8.3 ± 0.6 µA/cm² for miglustat-corrected monolayers (n = 4) and 0.18 ± 0.09 µA/cm² for control monolayers (n = 4, P < 0.001) (Fig. 5C).

View larger version (9K): [in this window] [in a new window]   Fig. 5. Effect of miglustat on Isc recorded in CF15 cells monolayers. CF15 cells were cultured at 37°C on semipermeable membranes in the absence (A) or in the presence (B) of miglustat (100 µM during 2 h in the culture medium). The effect of apical application of 100 µM amiloride, basolateral application of 10 µM forskolin and bilateral 30 µM genistein, then bilateral 500 µM glibenclamide was recorded. C, mean values of amiloride-sensitive Isc (left) and fsk/gst-sensitive Isc (right) from control CF15 cells monolayers (open bars) and miglustat-treated monolayers (black bars) (n = 4 for each condition). **, P < 0.01; ***, P < 0.001.

View larger version (8K): [in this window] [in a new window]   Fig. 6. Ex vivo effect of miglustat on amiloride-sensitive Isc recorded in nasal epithelium from F508del-CFTR or wild-type mice. Shown are mean values of amiloride-sensitive Isc recorded ex vivo in CF tissue under control conditions (open bars) and after incubation during 2 h in Meyler solution containing 100 µM miglustat (filled bars). For details, see Materials and Methods (n = 7 for each mice type). *, P < 0.05; **, P < 0.01.

Effect of Oral Administration of Miglustat on Ex Vivo Bioelectrics of cftr^{F508del/F508del} and cftr^-/- Mice. We administered 1200 mg/kg/day miglustat to cftr^{F508del/F508del} and cftr^-/- mice by gavage for 6 days. This concentration has been applied previously to demonstrate therapeutic benefits of miglustat in a mouse model of Sandhoff disease (Andersson et al., 2004). The control group received PBS only. On day 6 (i.e., after 12 applications), we dissected nasal epithelium from the different mice groups and recorded the amiloride-sensitive Isc. We found significantly reduced amiloride-sensitive Isc for nasal epithelium of cftr^{F508del/F508del} mice (Isc = -12.3 ± 6.2 µA/cm², n = 10 mice) who received miglustat, compared with the PBS group (Isc = -28.2 ± 3.5 µA/cm², n = 12 mice) (P < 0.05; Fig. 7). To learn whether the inhibition of ENaC-mediated Na⁺ absorption in nasal epithelium is a consequence of the F508del-cftr rescue by miglustat in this tissue or is due to a cftr-independent effect, we have repeated the in vivo study with cftr^-/- mice. However, no effect was noted between the two groups of cftr^-/- mice (PBS, Isc = -27.3 ± 17.0 µA/cm², n = 5 mice; miglustat, Isc = -24.0 ± 4.3 µA/cm², n = 5 mice; N.S.; Fig. 7). Because there is no effect on KO mice, the effect of miglustat is due to F508del-cftr rescue; therefore, this corrector has no direct effect on ENaC activity.

View larger version (10K): [in this window] [in a new window]   Fig. 7. Effect of oral administration of miglustat (6 days) on amiloride-sensitive Isc in mice nasal epithelium mounted in Ussing chambers. Bars graphs, amiloride-sensitive Isc both in cftrF508del/F508del (left) and cftr-/- (right) nasal epithelium dissected away from mice who received oral administration of miglustat (filled bars) or PBS vehicle only (open bars) during 6 days. For cftrF508del/F508del mice, N(PBS) = 12 mice and N(Miglustat) = 10 mice; for cftr-/- mice, N(PBS) = 5 mice and N(Miglustat) = 5 mice. *, P < 0.05, N.S. difference was P > 0.05.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Nowadays, the majority of clinical treatments of CF targets the secondary manifestations of the pulmonary disease (inhaled antibiotics and recombinant human DNase). However, CFTR-directed treatments will probably arise in the near future due to our expanded knowledge of transepithelial ion transport pharmacology and molecular biology. One approach for correcting the basic defect in CF (also called protein therapy) aims at creating conditions to restore cAMP-dependent chloride transport and, hence, rehydration, of the airway surface in priority. However, it remains uncertain whether such a therapy is able to restore all pleiotropic functions of CFTR (Vankeerberghen et al., 2002). In the present study, we addressed one particular aspect of this question by analyzing the consequence of CFTR correction for the activity of ENaC of treating airway epithelial cells with miglustat, an agent that we showed able to restore functional and mature F508del-CFTR in epithelial cells (Norez et al., 2006; Antigny et al., 2008) and that is now evaluated in a phase 2a clinical trial for homozygous F508del patients (http://clinicaltrials.gov/). The major findings of the present study are summarized hereafter. In CF15 human airway epithelial cells incubated for 2 h at 37°C in the presence of 100 µM miglustat, a cAMP-dependent and CFTR_inh-172-sensitive F508del-CFTR current was restored in parallel to the reduction in amplitude of the amiloride-sensitive ENaC Na⁺ current. In miglustat-treated cells, the magnitude of the amiloride-sensitive current was similar in the presence or absence of forskolin/genistein (to open CFTR channels), arguing that the activation of F508del-CFTR by forskolin/genistein is not required for down-regulation of ENaC. Finally, a CFTR-dependent normalization of amiloride-sensitive Na⁺ absorption in response to miglustat was observed both in human airway epithelial cells and in nasal epithelia of cftr^{F508del/F508del} mice.

CFTR is a pleiotropic ion channel, i.e., apart from its ability to transport chloride ions as an ionic channel, it also regulates many other transport proteins and cellular functions due to large and dynamic macromolecular complexes that contain CFTR, signaling molecules, and transport proteins (Vankeerberghen et al., 2002; Guggino and Stanton, 2006). Abnormal Na⁺ transport in CF-affected airway epithelia has been suggested by many in vivo and in vitro observations in humans and mice, showing increased amiloride-sensitive transepithelial potentials in CF (Knowles et al., 1981, 1983; Boucher et al., 1986; Grubb and Boucher, 1997; Mall et al., 1998). Although not completely solved, it becomes apparent that interaction between CFTR and ENaC may involve PDZ-domain proteins and kinases (Guggino and Stanton, 2006). It is particularly important that the functional and reciprocal interaction between CFTR and ENaC regulates both epithelial Cl^- and Na⁺ conductances (Stutts et al., 1995, 1997). However, it remains unclear why CF airway epithelia display such a high ENaC activity. Despite numerous studies, the molecular mechanism (direct or indirect) is still unknown. A potential direction of investigation to clear these points will have to address protein-protein interactions between CFTR and ENaC, which have been studied only in few reports. Berdiev et al. (2007) recently demonstrated a direct physical interaction between CFTR and the three ENaC subunits by fluorescence resonance energy transfer analysis. In the same study, these experiments were confirmed by coimmunoprecipitation. Nevertheless, these results argue that CFTR and - and β-rENaC interact in a complex. However, to our knowledge, no results concerning physical interactions between ENaC subunits and CFTR mutant proteins, and in particular the F508del mutant, have yet been shown.

We also found that the CFTR activators forskolin/genistein on whole-cell current, examined under control condition and in the presence of amiloride, had no influence on amiloride-sensitive ENaC currents in CF15 cells. This outcome is at variance with previous studies showing that the CFTR regulation of ENaC in human intestinal (Mall et al., 1998) and colonic epithelial cells (Ecke et al., 1996) required active CFTR channels. The mechanism of reciprocal regulation of CFTR and ENaC is currently under investigation. It was initially observed in MDCK cells expressing both ENaC and CFTR and was subsequently demonstrated in X. laevis (Stutts et al., 1995; Mall et al., 1996). ENaC inhibition by CFTR was demonstrated when , β, and ENaC subunits were coexpressed in oocytes of X. laevis with wild-type CFTR but not with F508del-CFTR (Stutts et al., 1995; Mall et al., 1996). In these studies, ENaC was inhibited during stimulation by agonist raising intracellular cAMP. A few studies showed that ENaC inhibition by CFTR also takes place in cells expressing both proteins endogenously (Ecke et al., 1996; Letz and Korbmacher, 1997) and was operational in normal human airways but not in CF patient tissues (Mall et al., 1998). It is now admitted that these findings may explain the typical enhanced amiloride-sensitive Na⁺ conductance and increased reabsorption of electrolytes observed in CF airways, two phenomena that are leading to highly viscous mucus and reduced mucociliary clearance (Zhang et al., 1996).

Note that it has been recently shown that overexpression of βENaC in mice led to a CF-like phenotype even in the presence of functional CFTR channels (Mall et al., 2004). Thus, inhibition of ENaC activity alone might already be of therapeutic value in CF. However, despite the fact that F508del-CFTR-mediated chloride secretion can be restored by a number of physical or pharmacological maneuvers in vitro and for some, in vivo (reviewed in Becq, 2006; MacDonald et al., 2007), a parallel change in amiloride-sensitive Na⁺ transport has not been frequently reported. The protein repair agent 4-phenylbutyrate (Buphenyl), which has been clinically evaluated in F508del-homozygous CF patients, partially restored CFTR function but had no effect on nasal amiloride-sensitive potential (Rubenstein and Zeitlin, 1998). In contrast, curcumin not only corrected CFTR functions but also affected the amiloride-sensitive response in cftr^{F508del/F508del} mice (Egan et al., 2004). Thus, these results suggest that a single pharmacological agent should be, in principle, capable of sufficient correction of the defects in CF cells to produce clinical benefits. However, a preliminary phase 1 clinical trial with oral curcumin was rather disappointing and did not show correction of CFTR (http://www.cff.org). Oral administration of miglustat in cftr^{F508del/F508del} mice resulted in reduction of amiloride-sensitive Isc. Therefore, our study demonstrates potential normalization of cAMP-dependent Cl^- secretion and amiloride-sensitive Na⁺ absorption by a treatment with a single agent, the CFTR corrector miglustat. Moreover, the persistence of ENaC inhibition in cftr^{F508del/F508del} animals receiving miglustat, together with a lack of effect on cftr^-/- mice, offer direct proof that the rescue of F508del-cftr and ENaC down-regulation are linked.

How does miglustat affect both CFTR and ENaC transports in CF cells? Earlier, we provided evidence that miglustat prevents, at least in part, the interaction of the mutant channel with the ER-resident molecular chaperone calnexin (Norez et al., 2006). Preventing the calnexin interaction with the mutant protein in the ER has been regarded as one of the major mechanism of rescue (Egan et al., 2004; Norez et al., 2006). During an extensive study to better understand the mechanism of action of miglustat on epithelial CF cells, we also observed that this agent has no direct effect either on the Cl^- channel activity of CFTR or on the activity of other non-CFTR Cl^- channels. Finally, as far as the literature showed, in epithelial CF cells, the three ENaC subunits are all expressed and located at the apical plasma membrane. However, because F508del-CFTR is retained in the ER, the equilibrium between Cl^- secretion and Na⁺ absorption is affected due to the absence of negative control of ENaC by CFTR as proposed by several authors (Stutts et al., 1995; Mall et al., 1996, 1998; Letz and Korbmacher, 1997; Kunzelmann et al., 2000; Berdiev et al., 2007). Moreover, the ENaC conductance is not inhibited by F508del-CFTR (Mall et al., 1996, 1998), and overexpression of the β-subunit of ENaC produces CF-like lung disease in a mouse model (Mall et al., 2004). Therefore, we propose that miglustat indirectly affects the transport of Na⁺ in CF cells through its effect as an 1,2-glucosidase inhibitor to perturb the F508del-CFTR/calnexin molecular interaction in the ER. Further studies will be needed to understand how rescuing F508del-CFTR from its intracellular retention re-establish the control of ENaC activity and thus normalize the transport of Na⁺ in CF cells.

In summary, in this report, we demonstrate that the rescue of endogenous F508del-CFTR by miglustat (or by low temperature) in human airway and nasal epithelial cells of cftr^{F508del/F508del} mice is accompanied by a down-regulation of Na⁺ transport. Because the balance between CFTR-dependent Cl^- secretion and ENaC-dependent Na⁺ reabsorption regulates the net amount of salt and water in airway periciliary fluid and thus the ability to clear bacteria and other noxious agents from the lungs, our findings predict that miglustat may not only ameliorate chloride transport but also sodium hyperabsorption.

	Acknowledgements

 
We thank Nathalie Bizard for cell culture maintenance and James Habrioux for excellent assistance.

	Footnotes

 
This study was supported by specific grants from Vaincre La Mucoviscidose and MucoVie66. S.N. was supported by a studentship from MucoVie66.

Part of this work were previously presented as an abstract as follows: Noel S, Wilke M, De Lange HR, and Becq F (2006) Rescue of F508del-CFTR processing defect by miglustat down-regulates sodium absorption in homozygous F508del-CFTR mice and human nasal cell (Abstract number 114). Pediatr Pulmonol Suppl. 29:247.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.135582.

ABBREVIATIONS: CF, cystic fibrosis; CFTR, CF transmembrane conductance regulator; F508del, deletion of phenylalanine at 508 position of CFTR protein; ER, endoplasmic reticulum; ENaC, epithelial sodium channel; miglustat, n-butyldeoxynojyrimicin; Isc, short-circuit current; PBS, phosphate-buffered saline; CFTR_inh-172, 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone; DMSO, dimethylsulfoxide; fsk, forskolin; Gst, genistein.

Address correspondence to: Dr. Frédéric Becq, Institut de Physiologie et Biologie Cellulaires, Centre National de la Recherche Scientifique Unité Mixte de Recherche 6187, Université de Poitiers, 40 Avenue du Recteur Pineau, 86022 Poitiers, France. E-mail: frederic.becq{at}univ-poitiers.fr

	References

Top Abstract Materials and Methods Results Discussion References

 

Andersson U, Smith D, Jeyakumar M, Butters TD, Borja MC, Dwek RA, and Platt FM (2004) Improved outcome of N-butyldeoxygalactonojirimycin-mediated substrate reduction therapy in a mouse model of Sandhoff disease. Neurobiol Dis 16: 506-515.[CrossRef][Medline]

Antigny F, Norez C, Becq F, and Vandebrouck C (2008) Calcium homeostasis is abnormal in cystic fibrosis airway epithelial cells but is normalized after rescue of F508del-CFTR. Cell Calcium 43: 175-183.[CrossRef][Medline]

Barbry P and Ladzunski M (1996) Structure and regulation of the amiloride-sensitive epithelial sodium channel. Ions Channels 4: 115-167.

Becq F (2006) On the discovery and development of CFTR chloride channel activators. Curr Pharm Des 12: 471-484.[CrossRef][Medline]

Berdiev BK, Cormet-Boyaka E, Tousson A, Qadri YJ, Oosterveld-Hut HM, Hong JS, Gonzales PA, Fuller CM, Sorscher EJ, Lukacs GL, et al. (2007) Molecular proximity of cystic fibrosis transmembrane conductance regulator and epithelial sodium channel assessed by fluorescence resonance energy transfer. J Biol Chem 282: 36481-36488.[Abstract/Free Full Text]

Boucher RC (2007) Cystic fibrosis: a disease of vulnerability to airway surface dehydration. Trends Mol Med 13: 231-240.[CrossRef][Medline]

Boucher RC, Stutts MJ, Knowles MR, Cantley L, and Gatzy JT (1986) Na⁺ transport in cystic fibrosis respiratory epithelia: abnormal basal rate and response to adenylate cyclase activation. J Clin Invest 78: 1245-1252.[Medline]

Canessa CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger JD, and Rossier BC (1994) Amiloride-sensitive epithelial Na⁺ channel is made of three homologous subunits. Nature 367: 463-467.[CrossRef][Medline]

Cao L, Owsianik G, Becq F, and Nilius B (2005) Chronic exposure to EGF affects trafficking and function of ENaC channel in cystic fibrosis cells. Biochem Biophys Res Commun 331: 503-511.[CrossRef][Medline]

Cheng SH, Gregory RJ, Marshall J, Paul S, Souza DW, White GA, O'Riordan CR, and Smith AE (1990) Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell 63: 827-834.[CrossRef][Medline]

De Jonge HR, Ballmann M, Veeze H, Bronsveld I, Stanke F, Tummler B, and Sinaasappel M (2004) Ex vivo CF diagnosis by intestinal current measurements (ICM) in small aperture, circulating Ussing chambers. J Cyst Fibros 3: 159-163.[Medline]

Ecke D, Bleich M, and Greger R (1996) The amiloride inhibitable Na⁺ conductance of rat colonic crypt cells is suppressed by forskoline. Pflugers Arch 431: 984-986.[Medline]

Egan ME, Pearson M, Weiner SA, Rajendran V, Rubin D, Glöckner-Pagel J, Canny S, Du K, Lukacs GL, and Caplan MJ (2004) Curcumin, a major constituent of turmeric, corrects cystic fibrosis defects. Science 304: 600-602.[Abstract/Free Full Text]

Grubb BR and Boucher RC (1997) Enhanced colonic Na⁺ absorption in cystic fibrosis mice versus normal mice. Am J Physiol Gastrointest Liver Physiol 272: G393-G400.[Abstract/Free Full Text]

Guggino WB and Stanton BA (2006) New insights into cystic fibrosis: molecular switches that regulate CFTR. Nat Rev Mol Cell Biol 7: 426-436.[CrossRef][Medline]

Jefferson DM, Valentich JD, Marini FC, Grubman SA, Iannuzzi MC, Dorkin HL, Li M, Klinger KW, and Welsh MJ (1990) Expression of normal and cystic fibrosis phenotypes by continuous airway epithelial cell lines. Am J Physiol Lung Cell Mol Physiol259: L496-L505.[Abstract/Free Full Text]

Kartner N, Hanrahan JW, Jensen TJ, Naismith AL, Sun SZ, Ackerley CA, Reyes EF, Tsui LC, Rommens JM, Bear CE, et al. (1991) Expression of the cystic fibrosis gene in non-epithelial invertebrate cells produces a regulated anion conductance. Cell 64: 681-691.[CrossRef][Medline]

Knowles M, Gatzy J, and Boucher R (1981) Increased bioelectric potential difference across respiratory epithelia in cystic fibrosis. N Engl J Med 305: 1489-1495.[Abstract]

Knowles MR, Stutts MJ, Spock A, Fischer N, Gatzy JT, and Boucher RC (1983) Abnormal ion permeation through cystic fibrosis respiratory epithelium. Science 221: 1067-1070.[Abstract/Free Full Text]

Kunzelmann K, Schreiber R, Nitschke R, and Mall M (2000) Control of epithelial Na⁺ conductance by the cystic fibrosis transmembrane conductance regulator. Pflugers Arch 440: 193-201.[Medline]

Letz B and Korbmacher C (1997) cAMP stimulates CFTR-like Cl^- channels and inhibits amiloride-sensitive Na⁺ channels in mouse CCD cells. Am J Physiol Cell Physiol272: C657-C666.[Abstract/Free Full Text]

Ma T, Thiagarajah JR, Yang H, Sonawane ND, Folli C, Galietta LJ, and Verkman AS (2002) Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion. J Clin Invest 110: 1651-1658.[CrossRef][Medline]

MacDonald KD, McKenzie KR, and Zeitlin PL (2007) Cystic fibrosis transmembrane regulator protein mutations: "class" opportunity for novel drug innovation. Paediatr Drugs 9: 1-10.[Medline]

Mall M, Hipper A, Greger R, and Kunzelmann K (1996) Wild type but not deltaF508 CFTR inhibits Na⁺ conductance when coexpressed in Xenopus oocytes. FEBS Lett 381: 47-52.[CrossRef][Medline]

Mall M, Bleich M, Greger R, Schreiber R, and Kunzelmann K (1998) The amiloride-inhibitable Na⁺ conductance is reduced by the cystic fibrosis transmembrane conductance regulator in normal but not in cystic fibrosis airways. J Clin Invest 102: 15-21.[Medline]

Mall M, Grubb BR, Harkema JR, O'Neal WK, and Boucher RC (2004) Increased airway epithelial Na⁺ absorption produces cystic fibrosis-like lung disease in mice. Nat Med 10: 487-493.[CrossRef][Medline]

Nagel G, Barbry P, Chabot H, Brochiero E, Hartung K, and Grygorczyk R (2005) CFTR fails to inhibit the epithelial sodium channel ENaC expressed in Xenopus laevis oocytes. J Physiol 564: 671-682.[Abstract/Free Full Text]

Noel S, Faveau C, Norez C, Rogier C, Mettey Y, and Becq B (2006) Discovery of pyrrolo[2,3-b]pyrazines derivatives as submicromolar affinity activators of wild type, G551D, and F508del cystic fibrosis transmembrane conductance regulator chloride channels. J Pharmacol Exp Ther 319: 349-359.[Abstract/Free Full Text]

Norez C, Noel S, Wilke M, Bijvelds M, Jorna H, Melin P, DeJonge H, and Becq F (2006) Rescue of functional delF508-CFTR channels in cystic fibrosis epithelial cells by the alpha-glucosidase inhibitor miglustat. FEBS Lett 580: 2081-2086.[CrossRef][Medline]

Pind S, Riordan JR, and Williams DB (1994) Participation of the endoplasmic reticulum chaperone calnexin (p88, IP90) in the biogenesis of the cystic fibrosis transmembrane conductance regulator. J Biol Chem 269: 12784-12788.[Abstract/Free Full Text]

Riordan JR (1993) The cystic fibrosis transmembrane conductance regulator. Annu Rev Physiol 55: 609-630.[CrossRef][Medline]

Rubenstein RC and Zeitlin PL (1998) A pilot clinical trial of oral sodium 4-phenylbutyrate (Buphenyl) in deltaF508-homozygous cystic fibrosis patients: partial restoration of nasal epithelial CFTR function. Am J Respir Crit Care Med 157: 484-490.[Medline]

Scholte BJ, Davidson DJ, Wilke M, and De Jonge HR (2004) Animal models of cystic fibrosis. J Cyst Fibros 3: 183-190.[CrossRef][Medline]

Stutts MJ, Canessa CM, Olsen JC, Hamrick M, Cohn JA, Rossier BC, and Boucher RC (1995) CFTR as a cAMP-dependent regulator of sodium channels. Science 269: 847-850.[Abstract/Free Full Text]

Stutts MJ, Rossier BC, and Boucher RC (1997) Cystic fibrosis transmembrane conductance regulator inverts protein kinase A-mediated regulation of epithelial sodium channel single channel kinetics. J Biol Chem 272: 14037-14040.[Abstract/Free Full Text]

Tong Z, Illek B, Bhagwandin VJ, Verghese GM, and Caughey GH (2004) Prostasin, a membrane-anchored serine peptidase, regulates sodium currents in JME/CF15 cells, a cystic fibrosis airway epithelial cell line. Am J Physiol Lung Cell Mol Physiol287: L928-L935.[Abstract/Free Full Text]

Vankeerberghen A, Cuppens H, and Cassiman JJ (2002) The cystic fibrosis transmembrane conductance regulator: an intriguing protein with pleiotropic functions. J Cyst Fibros 1: 13-29.[CrossRef][Medline]

Welsh MJ and Smith AE (1993) Molecular mechanisms of CFTR chloride channel dysfunction in cystic fibrosis. Cell 73: 1251-1254.[CrossRef][Medline]

Zhang Y, Yankaskas J, Wilson J, and Engelhardt JF (1996) In vivo analysis of fluid transport in cystic fibrosis airway epithelia of bronchial xenografts. Am J Physiol Cell Physiol 270: C1326-C1335.[Abstract/Free Full Text]

This article has been cited by other articles:

				B. Lubamba, J. Lebacq, P. Lebecque, R. Vanbever, A. Leonard, P. Wallemacq, and T. Leal Airway Delivery of Low-Dose Miglustat Normalizes Nasal Potential Difference in F508del Cystic Fibrosis Mice Am. J. Respir. Crit. Care Med., June 1, 2009; 179(11): 1022 - 1028. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.107.135582v1 325/3/1016    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Noël, S. Articles by Becq, F. Search for Related Content PubMed PubMed Citation Articles by Noël, S. Articles by Becq, F.