1 Genzyme Corporation, Framingham 01701-9322; and 2 Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215
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ABSTRACT |
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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
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INTRODUCTION |
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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 (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.
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MATERIALS AND METHODS |
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Synthesis of GL-172.
5-Cholenicacid-23,24-bisnor-3-ol (487 mg, 1.41 mmol) was suspended
in 5
-pregnane-3
,11
,17
,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 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).
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.
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 M. 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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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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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 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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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
· 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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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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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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