Partial correction of defective Clminus secretion in cystic fibrosis epithelial cells by an analog of squalamine

Canwen Jiang1, Edward R. Lee1, Mathieu B. Lane1, Yong-Fu Xiao2, David J. Harris1, and Seng H. Cheng1

1 Genzyme Corporation, Framingham 01701-9322; and 2 Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Defective cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-mediated Cl- transport across the apical membrane of airway epithelial cells is implicated in the pathophysiology of CF lungs. A strategy to compensate for this loss is to augment Cl- transport through alternative pathways. We report here that partial correction of this defect could be attained through the incorporation of artificial anion channels into the CF cells. Introduction of GL-172, a synthetic analog of squalamine, into CFT1 cells increased cell membrane halide permeability. Furthermore, when a Cl- gradient was generated across polarized monolayers of primary human airway or Fischer rat thyroid cells in an Ussing chamber, addition of GL-172 caused an increase in the equivalent short-circuit current. The magnitude of this change in short-circuit current was ~30% of that attained when CFTR was maximally stimulated with cAMP agonists. Patch-clamp studies showed that addition of GL-172 to CFT1 cells also increased whole cell Cl- currents. These currents displayed a linear current-voltage relationship and no time dependence. Additionally, administration of GL-172 to the nasal epithelium of transgenic CF mice induced a hyperpolarization response to perfusion with a low-Cl- solution, indicating restoration of Cl- secretion. Together, these results demonstrate that in CF airway epithelial cells, administration of GL-172 is capable of partially correcting the defective Cl- secretion.

cystic fibrosis transmembrane conductance regulator; nasal potential difference; Ussing chamber; whole cell patch clamp; chloride ion


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE CYSTIC FIBROSIS (CF) transmembrane conductance regulator (CFTR) is a Cl- channel located at the apical membrane of epithelial cells, the activity of which is regulated by phosphorylation and intracellular nucleotides (38, 39). In airway epithelial cells, the CFTR is the major Cl- transport pathway and regulates active ion transport-mediated fluid transport (16, 17, 23, 32, 33, 38). Additionally, CFTR purportedly also regulates other transmembrane proteins such as the amiloride-sensitive Na+ channel (4, 35) and the outwardly rectifying Cl- channel (27). In CF, mutations in the gene encoding CFTR cause defective transepithelial Cl- and fluid transport, with resultant impairment of airway mucociliary clearance (4, 16, 17, 21, 23, 32, 33) and reduction of the bactericidal activity of salt-sensitive defensins (10, 31). These deficits, it is proposed, are responsible for the recurrent infections and subsequent destruction of the lungs in CF patients.

Several therapeutic approaches are being developed concurrently for the treatment of CF. These include 1) use of agents that improve the bactericidal activity and viscosity of the mucous fluid lining the airways (10, 31), 2) use of agents that activate alternative Cl- channels to compensate for the CFTR Cl- channel defect (7, 16, 21, 24, 30), 3) protein and gene augmentation therapy (39), and 4) use of pharmacological agents that rescue the intracellular trafficking defect associated with the most common mutant form of CFTR (5, 6, 15, 25) or that suppress premature stop mutations (3, 44).

Yet another approach to modulating Cl- secretion in CF epithelia involves the generation of synthetic Cl- channels. Examples of such artificial Cl- channels include those generated with the peptide C-K4-M2GlyR (36, 37). Another antibiotic, a synthetic mimetic of squalamine, is also reportedly capable of increasing halide permeability in lipid bilayers (8, 22). These ionophores are useful as antibiotics, presumably because in addition to transporting ions, the ionophores disrupt bacterial cell membranes, leading to leakage of vital cellular constituents and subsequent cell destruction. Because these ionophores will likely be delivered locally to the CF lung with an aerosol, any cytotoxicity that may be associated with these compounds will be limited.

We report here that GL-172, a synthetic mimetic of squalamine (22), is capable of increasing halide permeability in immortalized airway epithelial cells from a CF (Delta F508) patient. In the nasal epithelium of transgenic CF mice, administration of GL-172 induced a significant increase in transepithelial potential difference (PD) in response to perfusion with a low-Cl- solution, indicating a partial restoration of Cl- secretion. These results suggest that in airway epithelia in vitro and in vivo, GL-172 is capable of partially correcting the defective Cl- secretion.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Synthesis of GL-172. 5-Cholenicacid-23,24-bisnor-3beta -ol (487 mg, 1.41 mmol) was suspended in 5beta -pregnane-3alpha ,11beta ,17alpha ,21-tetrol-20-one (THF; 50 ml), and N-hydroxysuccinimide (178 mg, 1.55 mol) and dicyclohexocarbodiimide (437 mg, 2.12 mmol) were added. The reaction mixture was stirred for 3 h in a 50°C oil bath. The reaction mixture was filtered, and a basic (sodium bicarbonate) workup was performed. The resulting crude product (1.09 g) was dry loaded (10 g of silica) onto a silica gel column (95 g) and was eluted with 50% ethyl acetate-hexanes. The eluate was isolated and characterized by 1H-NMR as the hydroxybisnorcholenic acid-NHS-ester (571 mg, 92%). The hydroxybisnorcholenic acid-NHS-ester (460 mg, 1.04 mmol) was dissolved in chloroform (50 ml), and sulfur trioxide-pyridine complex (499 mg, 3.14 mmol) was added. The reaction mixture was stirred for 4 h, and an aqueous workup was performed. The crude sulfonate (570 mg) was used without further purification. A suspension of the sulfonate (50 mg, 0.583 mmol) in dimethylformamide was added to a solution of spermine (189 mg, 0.934 mmol) in dimethylformamide (17.5 ml), and the reaction mixture was stirred for 1.5 h. The solvent was removed, and the resulting crude product was purified by flash column chromatography (50 g of silica gel) eluted with chloroform-methanol-concentrated ammonium chloride step gradients of 40:25:2, 40:25:5, and 40:25:10. The final product was isolated and characterized by 1H-NMR as hydroxybisnorcholenic-spermine-sulfonate (GL-172; 140 mg, 38%).

Cell culture. The immortalized tracheobronchial epithelial cell line CFT1, isolated from a Delta F508 CF patient, was cultured essentially as described previously (40). Briefly, CFT1 cells were seeded on 12-well cell culture plates at a density of 50,000 cells/cm2 and cultured with Ham's F-12 medium supplemented with 2% fetal bovine serum, 5 µg/ml of insulin, 3.7 µg/ml of endothelial cell growth supplement, 25 ng/ml of epidermal growth factor, 30 nM triiodothyronine, 1 µM hydrocortisone, 5 µg/ml of transferrin, and 10 ng/ml of cholera toxin (Life Technologies, Gaithersburg, MD). Primary (normal) human tracheobronchial epithelial (NHBE) cells were purchased from Clonetics (San Diego, CA). NHBE cells were passaged once and then seeded on collagen-coated semipermeable inserts (Millicell-PCF, 0.4-mm pore size, 0.6-cm2 growth area) at a density of 5 × 105 cells/cm2 and grown under air-liquid interface conditions (16, 19) with a 1:1 (vol/vol) mixture of DMEM and bronchial epithelial growth medium supplemented with growth factors and antimicrobials (Clonetics). Transepithelial resistance was monitored every other day, starting on day 3, with an ohmmeter. Fischer rat thyroid (FRT) epithelial cells (29, 47) were cultured the same as NHBE cells except that the culture medium was DMEM supplemented with 5% fetal bovine serum (Sigma, St. Louis, MO).

In studies designed to compare the relative ability of GL-172 and CFTR to facilitate Cl- efflux, CFT1 cells were infected with a recombinant adenovirus vector encoding wild-type human CFTR (Ad2/CFTR-5). The details of the construction and properties of this recombinant adenovirus vector have been described previously (2, 14, 18).

Assessment of Cl- channel activity with fluorescence digital imaging microscopy. Cl- channel activity was assessed with the halide-sensitive fluorophore 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ), as reported previously (15, 20). CFT1 cells growing on coverslips were loaded with SPQ by hypotonic shock for 4 min at room temperature. SPQ fluorescence was initially quenched by incubating the cells for up to 30 min in a NaI buffer of the following composition (in mM): 135 NaI, 2.4 K2HPO4, 0.6 KH2PO4, 1 MgSO4, 1 CaSO4, 10 dextrose, and 10 HEPES, pH 7.4. After baseline fluorescence was measured for 2 min, the NaI solution was replaced with a solution in which NaI was replaced with NaNO3, and fluorescence was measured for another 16 min. GL-172 was added 5 min after anion substitution, and the change in fluorescence was measured. In some control studies, a cocktail of 20 µM forskolin and 100 µM IBMX was added to stimulate the CFTR Cl- channel activity.

An increase in halide permeability is reflected by a rapid increase in SPQ fluorescence. It is the rate of change rather than the absolute change in signal that is the important variable in evaluating anion permeability with this technique. Differences in absolute levels reflect quantitative differences between groups in SPQ loading, size of cells, or number of cells studied. The data are presented as means ± SE of fluorescence at time t minus the baseline fluorescence (average fluorescence measured in the presence of I- for 2 min before ion substitution). For each experiment, 50-100 cells were examined on a given day, and studies under each condition were repeated on at least 2 days. For each experiment, responses were compared with those obtained with control or untreated cells. Cells were scored as positive if they exhibited a rate of change in fluorescence that was greater than that observed in the control cells. There was a broad spectrum of rates of change in SPQ fluorescence observed with responsive cells. Normally, cells were scored as responsive if the slope of the response curve, which is indicative of the rate of increase in SPQ fluorescence, was >= 0.364 after addition of the compound. This value was based on an analysis of >2,000 cells from 20 independent SPQ assays in CFT1 cells, which showed that values < 0.364 represented background noise. The entire field of cells was evaluated, but for clarity of presentation, only the top 10% of responders are illustrated in Fig. 2.

Measurement of transepithelial electrolyte transport. Polarized airway epithelial cells were mounted in modified Ussing chambers (Jim's Instruments, Iowa City, IA) interfaced with electrodes and bathed bilaterally in Krebs-Ringer solution (135 mM NaCl, 2.4 mM K2HPO4, 0.6 mM KH2PO4, 1.2 mM CaCl2, 1.2 mM MgCl2, 25 mM NaHCO3, and 10 mM glucose, pH 7.4) bubbled with 95% O2 and 5% CO2 (18, 41). On the mucosal side, NaCl was replaced with 135 mM sodium gluconate to create a transepithelial Cl- concentration gradient. Transepithelial voltage was measured for 5 min, after which it was clamped to 0 mV and changes in the equivalent short-circuit current (Ieq) were determined. After a stable baseline was achieved, the cells were treated sequentially with 1) 100 µM amiloride (to estimate the activity of the amiloride-sensitive Na+ channel); 2) in the continuous presence of amiloride, a cocktail containing 10 µM forskolin and 100 µM IBMX (to stimulate transepithelial Cl- current through the CFTR Cl- channels); and 3) in the continuous presence of amiloride, forskolin, IBMX, and 10-100 µM 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB; a Cl- channel blocker that inhibits CFTR Cl- channels) (13). Amiloride and NPPB were added to the mucosal solutions, and the forskolin and IBMX mixture was added to the submucosal solutions. To evaluate the activity of GL-172 (in DMSO), increasing amounts of the compound were added to the submucosal solution in the presence of amiloride.

Whole cell patch-clamp recording. Whole cell patch-clamp recordings were performed essentially as described previously (1, 9, 12, 15). Briefly, cells on coverslips were placed in a chamber mounted on a Nikon Diaphot inverted microscope. Patch pipettes had resistances of 2-4 MOmega . Whole cell configuration was achieved with an additional pulse suction to rupture the gigaseal. The pipette (intracellular) solution contained (in mM) 130 CsCl, 20 tetraethylammonium chloride, 10 HEPES, 10 EGTA, 10 Mg-ATP, and 0.1 Li-GTP, pH 7.4. The bath (extracellular) solution contained (in mM) 140 N-methyl-D-glucamine chloride, 2 CaCl2, 1 MgCl2, 0.1 CdCl2, 10 HEPES, 4 CsCl, and 10 glucose, pH 7.4. These solutions were designed to study only Cl- currents because Cl- was the only significant permeant ion in the solutions. Aspartate was used as the replacement anion in experiments in which extracellular Cl- concentration was changed. GL-172 (1, 10, and 100 µM dissolved in DMSO) or an equivalent concentration of DMSO (0.5-1% vol/vol) was added to the bath solutions as indicated. Current recordings were made from the same cells before, during, and after exposure to the solutions containing the different concentrations of GL-172 or DMSO. All experiments were performed at room temperature (22°C). Currents were filtered at 2 kHz. Data acquisition and analysis were performed with pCLAMP 5.5.1 software (Axon Instruments, Foster City, CA).

Nasal PD measurements in transgenic CF mice. GL-172 was administered to the nasal mucosae of FABP-CFTR bitransgenic mice (46) that were obtained from Jackson Laboratory (Bar Harbor, ME). The PD across the nasal epithelia of the CF mice was measured as described previously (11, 14, 19, 43). Briefly, a 23-gauge subcutaneous needle filled with Ringer solution (135 mM NaCl, 2.4 mM K2HPO4, 0.6 mM KH2PO4, 1.2 mM CaCl2, 1.2 mM MgCl2, and 10 mM HEPES, pH 7.4) was used as a reference electrode. The exploring electrode (pulled from PE-20 tubing and filled with Ringer solution) was inserted ~5 mm into the nasal cavity. The electrodes were electrically coupled by agar bridges (3% agar, 1 M KCl) that were inserted into the fluid stream of the flowing bridges and connected by calomel electrodes to a digital voltmeter (ISO-millivoltmeter; World Precision Instruments). Signals were recorded with a strip chart recorder (Servocorder model 6221). After placement of the electrodes, the nasal passage was perfused with Ringer solution through a separate catheter at 5-20 µl/min for 3-5 min with a micropump (model 55-3206; Harvard Apparatus). Once a baseline was achieved, the perfusing solution was switched to Ringer solution containing 100 µM amiloride, and perfusion continued until a new steady state was reached. The perfusing solution was then replaced with a low-Cl- Ringer solution (NaCl was replaced with sodium gluconate) containing GL-172 or DMSO in the presence of amiloride.

Statistical analysis. Data are expressed as means ± SE; n is the number of animals examined or individual experiments performed. Statistical analysis was performed with ANOVA followed by Student-Newman-Keuls tests. In experiments involving only two groups, an unpaired Student's t-test was used to compare the means. P values < 0.05 were considered significant.


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INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Synthesis of GL-172, a synthetic mimetic of squalamine. The structures of squalamine and its synthetic analog GL-172 are shown in Fig. 1. Squalamine is a sterol-spermidine conjugate that was initially isolated from dogfish sharks. This steroid, which is an adduct between spermidine and an anionic bile salt intermediate, has potent antibacterial activity and ionophoric activity that is membrane and ion selective (8). We were intrigued with the possibility that GL-172 might prove useful in restoring the defective Cl- transport shown to be associated with CF cells. To address this possibility, we assessed the relative potency of GL-172 at facilitating net Cl- efflux in vitro and in vivo in airway epithelial cells.


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Fig. 1.   Schematic illustration of the chemical structures of squalamine and GL-172.

Effect of GL-172 on halide permeability as assessed with the SPQ fluorescence assay. To assess the ability of GL-172 to facilitate anion transport in a CF cell line, CFT1 cells (an immortalized human CF airway epithelial cell line with the Delta F508 mutation) were treated with the compound, and Cl- transport was then assayed with the Cl--sensitive fluorescent indicator SPQ. In this assay, an increase in SPQ fluorescence is indicative of Cl- transport. Because CFT1 cells lack CFTR Cl- channel activity, addition of the cAMP agonists forskolin (20 µM) and IBMX (100 µM) caused only a very small increase in SPQ fluorescence in <1% of the cells (19). Addition of 100 µM GL-172 induced a significant increase in SPQ fluorescence in >10% of the CFT1 cells, indicating an increased anion permeability (Fig. 2). This change in anion permeability was unrelated to the addition of DMSO, the solvent used to reconstitute GL-172 (Fig. 2). These results suggest that GL-172 is capable of partially restoring Cl- efflux in CF cells. Although the response to the addition of GL-172 was significant, it was much less than that observed in CFT1 cells that had been infected with Ad2/CFTR-5. In contrast to cells that were treated with GL-172, addition of cAMP agonists to the Ad2/CFTR-infected cells generated significant halide efflux in >90% of the cells (Fig. 2). The rate and extent of change in SPQ fluorescence elicited by the addition of GL-172 were also less than those attained with virus-infected cells, suggesting that GL-172-mediated halide efflux was much lower than that mediated by CFTR channels.


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Fig. 2.   GL-172-induced increase in anion permeability as determined with fluorescence digital imaging microscopy. Cystic fibrosis (CF) airway epithelial cells (CFT1) were loaded with the fluorophore 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). CFTR, CF transmembrane conductance regulator. NO<UP><SUB>3</SUB><SUP>−</SUP></UP> was substituted for I- in the bathing solution at 0 min. GL-172 (dissolved in DMSO) or equal volume of DMSO was added to the bathing solution 5 min after ion substitution (arrow). In CFT1 cells infected with a recombinant adenovirus vector encoding wild-type human CFTR (Ad2/CFTR-5) at a multiplicity of infection of 100, a cocktail of forskolin and IBMX was added instead of GL-172. Data are means ± SE of fluorescence at time t (Ft) minus the baseline fluorescence (F0; average fluorescence measured for 2 min before ion substitution).

Effect of GL-172 on Ieq measurements. To assess the activity of GL-172 in a more physiologically relevant model, Ussing chamber assays were performed with polarized NHBE cells. These cells exhibit functional CFTR Cl- channel activity at the apical membranes. Polarized NHBE cells that developed a transepithelial resistance of >= 1,000 Omega  · cm2 were mounted between two halves of a modified Ussing chamber for Ieq measurements. Figure 3A shows that addition of amiloride (100 µM) to the apical side caused a decrease in Ieq, indicating the presence of an amiloride-sensitive Na+ conductance. In the continuous presence of amiloride, a cocktail of forskolin (10 µM) and IBMX (100 µM) induced a significant increase in Ieq. This increase in Ieq represents the maximum Cl- conductance through cAMP-mediated channels because this cocktail of cAMP agonists stimulates the maximum increase in intracellular cAMP. Data from six independent experiments showed that an average increase in Ieq of 11.3 ± 1.6 µA/cm2 was generated after the addition of these agonists. To ascertain that the increase in Ieq was mediated by cAMP-mediated CFTR Cl- channels, addition of 100 µM NPPB (an inhibitor of CFTR channel activity) inhibited the response (Fig. 3A).


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Fig. 3.   Representative tracings of GL-172-induced changes in short-circuit current (Delta Ieq) in normal human tracheobronchial epithelial (NHBE) cells. Polarized NHBE cells were clamped between 2 halves of an Ussing chamber for Ieq measurements. After addition of 10 µM amiloride (amil), a cocktail of 10 µM forskolin and 100 µM IBMX (F + I; A) or GL-172 (B) at different cumulative concentrations was added to the apical side of the polarized epithelium. Equal concentrations of DMSO, the solvent used to solubilize GL-172, did not cause a significant change in Ieq (data not shown). NPPB, 5-nitro-2-(3-phenylpropylamino)benzoate, an inhibitor of CFTR.

The increase in Ieq in response to the addition of GL-172 is shown in Fig. 3B. GL-172 was dissolved in DMSO and added to the apical side in a cumulative fashion. In the presence of amiloride (100 µM), GL-172 at 1 or 10 µM stimulated an increase in Ieq in ~30% of the cultures examined. Consistent responses were not observed until the concentration of GL-172 in the apical bath was raised to 50 or 100 µM. In cases where an increase in Ieq was observed at 50 or 100 µM, further increases in GL-172 concentration only modestly increased the magnitude of the subsequent responses. The responses were rapid and decayed very slowly, similar to those observed with forskolin and IBMX. Addition of DMSO alone (up to two times the concentration used to dissolve GL-172) did not cause any increase in Ieq.

The data from five independent experiments are summarized in Fig. 4. Statistical analysis suggested that the increase in Ieq induced by GL-172 was significant (P < 0.01) above 1 µM GL-172. The increases in Ieq induced by GL-172 at concentrations of 10 and 100 µM were ~20 and 35%, respectively, of the maximum response obtained with forskolin and IBMX in these cells. This observation of a lower signal with GL-172 is consistent with data obtained with the SPQ assay described in Effect of GL-172 on halide permeability as assessed with the SPQ fluorescence assay. Ussing chamber assays were also performed with FRT cells, which do not exhibit a cAMP-mediated change in Ieq (29). Addition of GL-172 to FRT cells caused a similar increase in Ieq (data not shown). These results suggest that the increase in Ieq induced by GL-172 in NHBE cells was independent of CFTR or other cAMP-mediated Cl- channels. As such, they imply that the GL-172-induced changes in Ieq may have resulted from the insertion of an additional anion conductance pathway in the apical membrane.


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Fig. 4.   Summary of GL-172-induced Delta Ieq in airway epithelial cells. Delta Ieq in response to cumulative concentrations of GL-172 was measured after addition of 100 µM amiloride. Data are means ± SE; n = 5 experiments. **P < 0.01 compared with untreated cells.

Whole cell patch-clamp analysis of CFT1 cells treated with GL-172. To confirm that the signals observed in the above assays were Cl- currents, whole cell patch-clamp experiments were performed on the CFT1 cells. Figure 5 shows representative current tracings from one such experiment. In these studies, the holding potential was 0 mV (which inactivates the voltage-gated Na+ and Ca2+ channels), and the voltage was stepped to potentials ranging from -100 mV to +80 mV in 20-mV increments to activate whole cell currents. Intracellular and extracellular solutions were designed to study only Cl- current because Cl- was the only significant permeant ion in the solutions. Currents from Ca2+ and K+ channels were minimized by omitting K+ from both intra- and extracellular solutions and by inclusion of 100 µM Cd2+ in the extracellular solution and 20 mM tetraethylammonium and 10 mM EGTA in the intracellular solution. The basal currents observed under these conditions are shown in Fig. 5A. Addition of 1% DMSO alone failed to activate whole cell currents (Fig. 5B). Addition of GL-172 at a concentration of 1 or 10 µM also did not cause any increase in whole cell currents in all cells examined (n = 10). In contrast, exposure of CFT1 cells to a higher concentration (30 µM) of GL-172 induced a significant increase in whole cell currents in ~30% of the cells examined (Fig. 5, C and D). At an even higher concentration of 100 µM, GL-172 caused a large increase in whole cell currents in all the cells tested (data not shown). The currents were sustained for 40 min (longest time tested) and were persistent even after washout with control buffer for 45 min. The current-voltage relationships under basal conditions and after addition of GL-172 are summarized in Fig. 5D. The whole cell currents in the cells treated with GL-172 displayed a linear current-voltage relationship and were time independent. Interestingly, these properties are qualitatively similar to those observed with wild-type CFTR Cl- channels (1, 38).


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Fig. 5.   Whole cell patch-clamp analysis of CFT-1 cells after addition of GL-172. Currents shown are in response to voltage steps from a holding potential of 0 mV to potentials ranging from -100 mV to +80 mV in 20-mV increments. Representative whole cell currents from CFT1 cells under basal (untreated; A) and treated (DMSO; B) conditions are shown. C: recording from same CFT1 cells after treatment with GL-172 (30 µM, dissolved in DMSO). D: current-voltage relationships obtained under basal conditions () and after addition of 10 (triangle ) and 30 (open circle ) µM GL-172. Current-voltage relationship was linear and time independent. Data are means ± SE; n = 3-6 experiments.

Effect of GL-172 on the nasal PD of CF mice. To examine the potential utility of GL-172 at restoring Cl- transport in vivo, we also applied this squalamine analog to the nasal epithelium of a mouse model of CF. The nasal mucosae of CF mice have proven to be an invaluable model for evaluating the ability of gene delivery vectors to restore epithelial Na+ and Cl- transport defects (11, 14, 19, 41). Because the utility of CF-null [(-/-)] mice can be limited by intestinal complications (34), we used FABP-CFTR(-/-) bitransgenic mice (46). The nasal epithelium of the FABP-CFTR(-/-) bitransgenic mouse displays the electrophysiological abnormalities observed in CF(-/-) animals and humans with CF (19). Figure 6 shows representative tracings from wild-type and CF mice of the basal PD, changes in PD induced by amiloride, and changes in PD in response to the subsequent substitution of NaCl with sodium gluconate in the presence of amiloride. As our laboratory has reported previously (14), substitution of NaCl with sodium gluconate caused a small depolarization in the CF bitransgenic animals (Fig. 6B) but a significant hyperpolarization in normal mice (Fig. 6A). Addition of GL-172 in the low-Cl- Ringer solution induced a hyperpolarization response in the CF mice, indicating increased anion permeability (Fig. 6C). In three of four mice, GL-172 (100 µM) caused a significant hyperpolarization response (change in PD of 2.5, 3, and 6.5 mV). At a reduced concentration (20 µM) of GL-172, a hyperpolarization (4.2 mV) was observed in only one of three animals examined. Statistical analysis (Fig. 7) indicated that the hyperpolarization response induced by GL-172 (100 µM) was significant (P < 0.05). The magnitude of the response obtained with 100 µM GL-172 was ~30% of that observed in normal mice after perfusion with a low-Cl- solution (Fig. 7). Taken together with the results obtained in CFT1 and FRT cells, these results in the CF mouse model indicate that GL-172 is capable of partially restoring Cl- efflux in CF cells both in vitro and in vivo.


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Fig. 6.   GL-172-induced hyperpolarization in the nasal epithelia of CF mice. Potential difference across nasal epithelia was measured in wild-type [(+/+)] (A) and homozygous FABP-CFTR bitransgenic [(-/-)] CF (B) mice under basal conditions after administration of amil and after Cl- substitution (low Cl-) in the presence of amil. C: tracing from a homozygous [CF(-/-)] mouse that showed a hyperpolarization in response to perfusion with the low-Cl- solution containing GL-172 (100 µM).



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Fig. 7.   Summary of GL-172-induced hyperpolarization in the nasal epithelia of wild-type and CF mice. Changes in potential difference (Delta PD) across the nasal epithelia in response to low-Cl- solution and in the absence and presence of GL-172 (20 and 100 µM, respectively) were measured in the presence of amil (100 µM). Data are means ± SE (n >=  4 mice). *P < 0.05 compared with untreated group.


    DISCUSSION
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ABSTRACT
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Ionophores are small hydrophobic molecules that dissolve in lipid bilayers and increase their permeability to specific inorganic ions. They purportedly operate by shielding the charge of the transported ion so that it can penetrate the hydrophobic interior of the lipid bilayer. There is significant interest in the continued design and synthesis of ionophores and similar membrane-active compounds in the search for novel antibiotic agents. The antibiotic squalamine is a sterol-spermidine conjugate that has recently been isolated from tissues of the dogfish, Squalus acanthias (22). The synthetic sterol-spermine conjugate GL-172, which has structural similarities to squalamine, also exhibits antibiotic properties (8, 26). Additionally, it has also been demonstrated to exhibit unique ionophoric activity with membrane and ion selectivity (8). Specifically, it favors the transport of anions such as Cl- across synthetic lipid bilayers over cations such as Na+. Because CF is characterized by a loss of CFTR Cl- channel activity, we sought to examine the ability of GL-172 to compensate for this specific function in CF airway epithelial cells.

Biochemical and biophysical studies suggest that on lipid membrane surfaces, there can be two discrete forms of the ionophore GL-172: a monomer-active form and a monomer plus aggregate-active form (8). When a critical micelle concentration is reached on the membrane surface, the transition of the monomeric to the "aggregate" form begins to occur with the appearance of channel activity (8). In CF epithelial cells, we observed that an increase in halide permeability or whole cell currents occurred only above a threshold concentration of GL-172. No clear-cut dose-dependent response was observed before this threshold concentration of GL-172 was attained. Although this threshold concentration varied in different cell types and culture conditions, in most cases, it was shown to be ~30 µM. These observed variations may be attributable to differences in the drug-to-cholesterol ratio at the cell membrane in the different cells and culture conditions. Thus we would argue that in CF airway epithelial cells, the characteristics of the activity of GL-172 would favor an ion channel rather than a carrier model.

Maintenance of mucociliary clearance in the airway epithelium requires the coordinate regulation of ciliary motion, airway surface liquid (ASL) depth, and mucin content. The quantity and composition of ASL are controlled by both the surface epithelium and submucosal glands. CFTR is a major Cl- transport pathway in airway cells (23, 38). Although it is not precisely known how mutations in the gene encoding CFTR lead to CF lung disease, which is characterized by recurrent infection, inflammation, and lung destruction, decreased Cl- secretion and increased Na+ absorption (23) are well-documented defects. These changes in ion transport produce alterations in fluid transport across surface and gland epithelia (16, 17, 39, 45). As a consequence, these resultant alterations in water and salt content of ASL purportedly diminish the activity of bactericidal peptides secreted from the epithelial cells (10, 31) and/or impair mucociliary clearance (21).

In cultured CF airway epithelial cells in vitro, we demonstrated that GL-172 increased cell membrane halide permeability as determined by a fluorescence assay performed with the Cl- indicator SPQ and caused an increase in whole cell Cl- currents as measured with patch-clamp techniques. Administration of GL-172 also resulted in Cl- secretion in fully confluent and polarized epithelia in response to a Cl- gradient. The magnitude of the current was ~30% of that generated when CFTR was stimulated maximally with cAMP agonists. Patch-clamp experiments demonstrated that GL-172 increased whole cell Cl- currents. The whole cell Cl- currents exhibited linear and voltage-independent properties. Together, these in vitro results demonstrate that GL-172 is capable of increasing Cl- permeability across the epithelial cell membrane in an ion-selective manner. More importantly, in the nasal epithelia of transgenic CF mice, GL-172 caused a significant PD increase in response to perfusion with a low-Cl- solution, demonstrating its ability to partially restore Cl- secretion. These studies provide evidence that in CF airway epithelial cells in vitro and in vivo, GL-172 is capable of partially correcting defective Cl- secretion. In light of these observations, this compound may have therapeutic potential for the treatment of CF lung disease or for any other disease that might benefit from increased Cl- transport.

It should be noted that in contrast to CFTR Cl- channel activity, which is tightly regulated by physiological pathways, GL-172 exhibits Cl- transport activity when subjected to electrical and chemical gradients. It is unclear whether the introduction of such an unregulated Cl- transport activity in affected airway cells would be of clinical benefit. Furthermore, CFTR has ascribed to it roles in other cellular functions, including the regulation of other transmembrane proteins, intracellular pH, and binding and internalization of bacteria. It would seem unlikely that the mere restoration of Cl- transport would affect all these activities. In this regard, it may be more relevant to contemplate the use of GL-172 as an adjunct for treating patients with CF. It therefore remains to be determined whether restoring Cl- permeability alone is sufficient to correct the clinical phenotype of CF patients. Yet another consideration is the fact that altered active ion and fluid transport in submucosal glands contributes to the pathophysiology of CF lung disease. It is unknown whether GL-172, when administered to the apical aspect of the airway epithelium, will gain access to the submucosal glands.

Another aspect associated with the use of GL-172 pertains to its antibiotic activity. Although it has been reported that GL-172 has no measurable lytic activity in preformed phospholipid vesicles, it nonetheless possesses significant antimicrobial activity (28). If it is effective against gram-positive organisms such as Staphylococcus aureus, an additional benefit might be obtained through its use for CF because these patients invariably become chronically infected with this organism. However, the dose of GL-172 required for its ionophoric activity may not necessarily be similar to that required for its antimicrobial activity.

In summary, we have characterized the properties of the ionophore GL-172 and showed that it is able to facilitate Cl- transport in CF cells. As such, we argue that it may have therapeutic applications for patients with CF. This, however, assumes that the Cl- channel activity of CFTR that is defective in CF is primarily responsible for the pathophysiology. Although there is evidence to support this notion, the relationship between the loss of this activity per se and lung disease has not been unequivocally established.


    ACKNOWLEDGEMENTS

We thank members of the Cystic Fibrosis Research Group for comments and formative discussions throughout the project and S. Fang, S. O'Connor, and members of the Laboratory Animals Research Department for technical assistance. We also thank R. Scheule for constructive comments on the manuscript.


    FOOTNOTES

Address for reprint requests and other correspondence: S. H. Cheng, Genzyme Corp., 31 New York Ave., Framingham, MA 01701-9322 (E-mail: seng.cheng{at}genzyme.com).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 20 March 2001; accepted in final form 13 June 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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