1 Laboratoire de Physiologie des Régulations Cellulaires, Unité Mixte de Recherche 6558, 86022 Poitiers; 2 Laboratoire de Chimie Organique, Faculté de Médecine et de Pharmacie, 86005 Poitiers, France; 3 Laboratorio di Genetica Molecolare, Istituto Giannina Gaslini, 16148 Genova, Italy; and 4 Department of Medical Biochemistry, University of Wales, College of Medicine, Cardiff CF14 4XN, United Kingdom
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ABSTRACT |
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The pharmacological activation of the
cystic fibrosis gene protein cystic fibrosis transmembrane conductance
regulator (CFTR) was studied in human airway epithelial Calu-3 cells,
which express a high level of CFTR protein as assessed by Western blot
and in vitro phosphorylation. Immunolocalization shows that CFTR is
located in the apical membrane. We performed iodide efflux, whole cell patch-clamp, and short-circuit recordings to demonstrate that the novel
synthesized xanthine derivative 3,7-dimethyl-1-isobutylxanthine (X-33)
is an activator of the CFTR channel in Calu-3 cells. Whole cell current
activated by X-33 or IBMX is linear, inhibited by glibenclamide and
diphenylamine-2-carboxylate but not by DIDS or TS-TM calix[4]arene.
Intracellular cAMP was not affected by X-33. An outwardly rectifying
Cl current was recorded in the absence of cAMP and X-33
stimulation, inhibited by DIDS and TS-TM calix[4]arene. With the use
of short-circuit recordings, X-33 and IBMX were able to stimulate a
large concentration-dependent CFTR transport that was blocked by
glibenclamide but not by DIDS. Our results show that manipulating the
chemical structure of xanthine derivatives offers an opportunity to
identify further specific activators of CFTR in airway cells.
cystic fibrosis transmembrane conductance regulator; chloride conductance; pharmacology
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INTRODUCTION |
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CYSTIC FIBROSIS (CF),
the most common lethal autosomal recessive genetic disease, is caused
by mutations of the CF gene, which normally encodes a multifunctional
and integral membrane protein, the CF transmembrane conductance
regulator (CFTR) (35). The native protein is a
Cl channel located in the apical membrane of epithelial
cells, where it mediates Cl
transepithelial transport
(for review see Ref. 25). CFTR mutations lead to an
impaired or absent Cl
conductance (13, 25),
which generates defective Cl
transport across the
epithelium and perturbs the quantity and composition of epithelial
fluids. This results in the manifestations of the disease, which
include airway obstruction and infection, pancreatic failure, male
infertility, and elevated levels of salt in sweat (for review see Ref.
23).
When the CFTR channel binds ATP at nucleotide binding domains and is
phosphorylated at multiple sites within the R domain by cAMP-dependent
kinases, it opens and generates a Cl flux
(25). CFTR, mainly physiologically regulated by processes increasing intracellular cAMP, can also be stimulated by
cAMP-independent pathways. Compounds such as
p-bromotetramisole or levamisole (4, 6),
genistein (29),
5-chloro-2-hydroxyphenyl-1,3-dihydro-2H-benzimidazol-2-one (NS-004) (15, 22), 1-ethyl-2-benzimidazolinone (1-EBIO)
(15), 6-hydroxy-10-chlorobenzo[c]quinolizinium (MPB-07), and
6-hydroxy-7-chlorobenzo[c]quinolizinium (MPB-27)
(5) have been proposed as new CFTR activators. All these
molecules activate CFTR without altering the cell cAMP content. These results suggest other mechanisms of activation, such as the
inhibition of endogenous CFTR-associated phosphatases (3, 4) or perhaps the direct binding to nucleotide binding fold (NBF) 1 or/and 2. Indeed, genistein has been shown to directly bind to
NBF-2 and to compete with ATP binding on that site (34).
One goal of our group is to design pharmacological tools for research
study and therapeutic application in CF. We recently showed that the
use of synthetic xanthines may be important in discovering potent
activators of the wild-type CFTR in Chinese hamster ovary (CHO)
transfected cells by using iodide efflux and cell-attached patch-clamp
experiments (9). Calu-3 cells, which are derived from a
pulmonary adenocarcinoma, express high levels of CFTR mRNA and protein
(21, 38) and secrete a cAMP-dependent Cl
(38) and bicarbonate (16) fluid. The
preponderance of CFTR channels in Calu-3 cells allowed further
examination of channel properties in a well-differentiated epithelial
cell. Moreover, Calu-3 cells resemble serous gland cells (21,
38), which have been identified as critical components in
mucosal defenses and are implicated in CF lung disease (20,
43) and, therefore, are a relevant model to test the efficacy of
CFTR activators.
We first describe in vitro phosphorylation and immunolocalization of CFTR on Calu-3 cells and report experiments using iodide efflux, whole cell recordings, and short-circuit techniques showing the effects of xanthine derivatives, including a novel compound obtained by chemical synthesis, 3,7-dimethyl-1-isobutylxanthine (X-33).
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METHODS |
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Cell culture.
Native CHO cells [CFTR() CHO] or CHO cells stably transfected with
pNUT vector containing wild-type CFTR [CFTR(+) CHO] were provided by
J. R. Riordan and X.-B. Chang (Scottsdale, AZ) (40). Cells cultured at 37°C in 5% CO2 were maintained in
-MEM containing 7% fetal bovine serum, 0.5%
L-glutamine, 0.5% antibiotics (50 IU/ml penicillin and 50 µg/ml streptomycin), and 100 µM methotrexate, as described
previously in detail (4, 40). Calu-3, a cell line of human
pulmonary origin (38), was cultured at 37°C in 5%
CO2 and maintained in DMEM-Ham's F-12 (1:1) nutritive mix
supplemented by 10% FCS and 1% antibiotics (50 IU/ml penicillin and
50 µg/ml streptomycin).
Cell membrane preparation.
Calu-3, CFTR() CHO, and CFTR(+) CHO cells were removed from cell
culture flasks by EDTA treatment and washed several times in
Ca2+- and Mg2+-free PBS. Cell membranes were
obtained as previously described (36) by lysing whole
cells in Tris buffer (20 mM Tris · HCl, pH 7.45, 10 mM NaCl, 1 mM EDTA, 2 mM MgCl2, 1 mM dithiotreitol, 0.2 mg/ml
benzamidine, 1 µg/ml pepstatin A, 0.1 mM phenylmethylsulfonyl fluoride, 20 µg/ml aprotinin, and 20 µg/ml leupeptin), and cell homogenates were centrifuged at 25,000 g for 30 min. The
pellets containing crude membranes were resuspended in Tris buffer and precipitated with 4% TCA (final concentration). After centrifugation, pellets of membrane proteins were solubilized in SDS-PAGE sample buffer
(62 mM Tris · HCl, pH 6.8, 2% SDS, 10% glycerol, 5%
2-mercaptoethanol, and 0.001% bromphenol blue) to obtain a protein
concentration of 2-4 mg/ml.
Antibodies. For immunoblotting, a monoclonal antibody raised against the COOH terminus of CFTR was obtained from Genzyme (Cambridge, MA). For immunoprecipitation and immunolocalization studies, an antibody was raised against a peptide consisting of the 23 COOH-terminal amino acids of CFTR, as previously described for a CFTR antibody directed at the first nucleotide binding domain region (33). Briefly, the peptide was synthesized and coupled to keyhole limpet hemagglutinin (10 mg peptide/8 mg keyhole limpet hemagglutinin; Cambridge Research Biochemicals, Northwich, Cheshire, UK), and antisera were prepared by injection of conjugates (100-200 µg/ml), emulsified in Freund's adjuvant, intradermally into rabbits. The antisera were affinity purified using peptide coupled to CH-Sepharose 4B (Pharmacia), with elution of antibody fractions in 0.1 M glycine-HCl, pH 2.5. The antibody to CD59 (BRIC229) was obtained from the National Blood Service (Bristol, UK).
Electrophoresis and immunoblotting. SDS-PAGE of solubilized membrane proteins (50 µg) was performed by the method of Laemmli (32), with a 7% separating gel. After electrophoresis, proteins were electrotransferred to a polyvinylidene difluoride (PVDF) membrane at 50 V for 90 min at 4°C in a 25 mM Tris · HCl-0.7 M glycine buffer. Unreactive sites on the PVDF membrane were blocked with nonfat dry milk. The PVDF membrane was then incubated for 1 h at room temperature with a primary antibody raised against the COOH terminus of CFTR (Genzyme) at 0.4 µg/ml. After several washes, the membrane was incubated with a secondary antibody, horseradish peroxidase-labeled sheep anti-mouse IgG. Immunoreactive bands were visualized with a commercial enhanced chemiluminescence system (Amersham Pharmacia Biotech, Buckinghamshire, UK).
Immunoprecipitation and phosphorylation of CFTR.
Calu-3, CFTR() CHO, and CFTR(+) CHO cells were lysed in ice-cold RIPA
buffer (50 mM Tris · HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10 mM iodoacetate, 1 mM
phenylmethylsulfonyl fluoride, and 10 µg/ml each of chymostatin, pepstatin A, antipain, aprotinin, and leupeptin) for 30 min on ice.
Nuclear and cell debris were removed by microcentrifugation (13,000 g) for 15 min at 4°C. The supernatant (50 µl, ~4 mg
protein/ml) was incubated with affinity-purified CFTR antibody (80 µg/ml) for 90 min at 4°C, and the antibody-CFTR complex was
precipitated with Pansorbin (10% suspension of Staphylococcus
aureus cells prewashed in RIPA buffer). The precipitate was
washed, resuspended in phosphorylation buffer (50 mM
Tris · HCl, pH 7.5, 10 mM MgCl2, 0.1 mg/ml BSA),
and phosphorylated in vitro (60 min at 37°C) using the catalytic
subunit of protein kinase A (PKA, 75 nM) and 10 µCi of
[
-32P]ATP. Phosphorylation was terminated by addition
of RIPA buffer, and after several washes the immune complex was
dissociated by solubilization in electrophoresis sample buffer (0.125 M
Tris · HCl, pH 6.8, 5% SDS, 25% sucrose, 5%
2-mercaptoethanol) for 15 min at 37°C. Samples were subjected to
SDS-PAGE on 7% separating gel and then Coomassie blue staining. Gels
were dried and autoradiographed overnight using Hyperfilm-MP
(Amersham). Lanes from the autographs were scanned by densitometry
(model GS-670, Bio-Rad) using Molecular Analyst software.
Immunolocalization of CFTR.
Calu-3 cells, grown on coverslips to ~80% confluence, were fixed for
5 min at 20°C in 5% acetic acid in ethanol. All subsequent steps
were carried out in PBS containing 0.1% fish gelatin and 1% BSA at
room temperature. Cells were permeabilized with 0.2% (vol/vol) Triton
X-100 for 20 min, and nonspecific protein binding sites were blocked by
incubation first with 50 mM glycine for 30 min and then with 10%
normal goat serum for 1 h. Cells were incubation with primary
antibody (1:100 dilution) for 18 h at 4°C, washed three times
for 15 min each, and incubated with secondary antibody conjugated with
FITC (anti-rabbit IgG for CFTR; anti-mouse IgG for CD59) for 1 h
and then washed three times. Slides were then mounted in Fluorosave
Reagent (Calbiochem, La Jolla, CA). Fluorescence was detected using
confocal laser scanning microscopy on a Leitz Fluovert FU microscope
(Leica, Germany) fitted with a TCS4D scanner (Leica). Confocal images
were collected at a magnification of ×100 under oil immersion and
displayed as Kalman averages on a 512 × 512-pixel 72-dpi screen.
Images were processed using Scanware software (Leica), and image
manipulation was performed using Corel Photopaint.
cAMP measurement. Calu-3 cells were incubated in the presence or absence of test compounds. After a 5-min incubation period at 37°C, the reaction was stopped by addition of 250 µl of 12% TCA at 4°C. An RIA kit (RIANEN, NEN Life Science Products) was used to determine cAMP levels.
Iodide efflux experiments.
CFTR Cl channel activity was assayed by measuring the
rate of iodide (125I) efflux, as previously described
(5). All experiments were performed at 37°C. Calu-3
cells grown in 12-well plates were washed twice with 2 ml of efflux
buffer containing (in mM) 137 NaCl, 4.4 KCl, 0.3 KH2PO4, 0.3 NaH2PO4,
4.2 NaHCO3, 1.3 CaCl2, 0.5 MgCl2, 0.4 MgSO4, 5.6 glucose, and 10 HEPES, pH 7.5. Cells were
then incubated in efflux medium containing 1 µM KI (1 µCi
Na125I/ml; NEN, Boston, MA) for 1 h at 37°C to
permit the iodide to reach equilibrium. Cells were washed with efflux
medium. After 1 min, the medium was removed to be counted and quickly
replaced by 1 ml of the same medium. This procedure was repeated every 1 min for 11 min. The first three aliquots were used to establish a
stable baseline in efflux buffer alone. Efflux medium containing the
appropriate drug was used for the remaining aliquots. At the end of the
incubation, the medium was recovered and cells were solubilized in 1 ml
of 1 N NaOH. The radioactivity was determined using a gamma counter
(LKB). The fraction of initial intracellular 125I lost
during each time point was also determined, and time-dependent rates of
125I efflux were calculated from
ln(125It1/125It2)/(t1
t2), where
125It is the intracellular
125I at time t, and t1
and t2 are successive time points
(41). Curves were constructed by plotting rates of
125I vs. time. All comparisons were based on maximal values
for the time-dependent rates (peak rates), with exclusion of the points used to establish the baseline. Values are means ± SE of
n separate experiments. Differences were considered
statistically significant using the Student's t-test when
P < 0.05.
Patch-clamp experiments.
Whole cell recordings were performed on Calu-3 cells plated on 35-mm
petri dishes and cultured at 37°C in 5% CO2. Currents were recorded with a List EPC-7 patch-clamp amplifier. The membrane potential was clamped to 40 mV and pulsed from
80 to +80 mV by
20-mV steps. Pipettes with resistance of 2-3 M
were pulled from
borosilicate glass capillary tubing (GL150-T10, Clark Electromedical, Reading, UK) using a two-step vertical puller (Narishige). They were
connected to the head stage of the amplifier through an Ag-AgCl pellet.
Seal resistances of 10-30 G
were obtained. The pipette solution
contained (in mM) 113 L-aspartic acid, 113 CsOH, 27 CsCl, 1 NaCl, 1 MgCl2, 1 EGTA, 3 MgATP, and 10 TES, pH 7.2;
osmolality was 285 mosM. The external solution consisted of (in mM) 145 NaCl, 4 CsCl, 1 MgCl2, 1 CaCl2, 5 glucose, and
10 TES, pH 7.4; osmolality was 315 mosM. Results were analyzed with the
pCLAMP6 package software (pCLAMP, Axon Instruments). All experiments
were performed at room temperature. Cells were stimulated with
xanthines or appropriate compounds at the concentration indicated
(dissolved in DMSO; final DMSO concentration 0.1%). In control
experiments, the currents were not altered by DMSO.
Synthesis of X-33. The starting material for X-33 was theobromine prepared in a solution of dry dimethylformamide (75 ml), which was stirred under nitrogen before the addition of NaH (5.5 mM). The mixture was then brought to 75°C for 30 min before the addition of the isobutyl bromide (5.5 mM). After 4-10 h at 75°C, the reaction mixture was cooled and hydrolyzed. The solvent was evaporated, and the dry residue was extracted with methylene chloride. The solution was dried over Na2SO4 and purified by column chromatography on silica gel (elution with ethyl acetate). The product was further purified by sublimation. 1H-NMR spectra, infrared spectra, and elemental analysis were consistent with the assigned structures: X-33, white powder, 0.75 g (63%), melting point 145°C (9).
Short-circuit current measurements. Calu-3 cells were seeded on Snapwell permeable supports (Corning Costar) at a density of 5 × 105 cells/cm2. The media on the apical (0.5 ml) and basolateral (2 ml) sides were changed daily. Short-circuit current (Isc) was measured after 8 days of culture on Snapwell inserts. The inserts were mounted in a modified Ussing chamber (Corning Costar) filled on both sides with 5 ml of a Krebs bicarbonate solution containing (in mM) 126 NaCl, 0.4 KH2PO4, 2.1 K2HPO4, 1 MgSO4, 1 CaCl2, 24 NaHCO3, 10 glucose, and 0.04 phenol red. During the experiments, this solution was kept at 37°C and continuously bubbled with 5% CO2-95% air. The epithelium was short-circuited with a voltage clamp (model 558-C5, Dept. of Bioengineering, University of Iowa) connected to apical and basolateral chambers with Ag-AgCl electrodes. The potential difference and the fluid resistance between potential sensing electrodes were compensated. The Isc was recorded on a chart recorder (model L6512, Linseis). The Calu-3 cells occasionally showed some amiloride-sensitive current. To remove Na+ transport, all experiments were performed in the presence of 10 µM amiloride in the apical solution.
Chemicals.
All products were obtained from Sigma Chemical (St. Louis, MO), except
-MEM and DMEM-Ham's F-12 nutritive mix, which were acquired from
Fisher and GIBCO BRL. TS-TM calix[4]arene
(5,11,17,23-tetrasulfonato-25,26,27,28-tetramethoxy-calix[4]arene) was a generous gift of Drs. Singh and Bridges (University of Alabama at Birmingham).
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RESULTS |
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Expression and in vitro phosphorylation of CFTR in CHO and Calu-3
cells.
Calu-3 is an airway epithelial cell line that has retained most of the
characteristics of serous gland stem cells (38). CFTR
expression was analyzed using SDS-PAGE and immunoblotting. Results from
Calu-3, CFTR(+) CHO, and CFTR() CHO cells are shown in Fig.
1A. In Calu-3 cells, the major
CFTR form was a 175-kDa protein, as determined by molecular mass
standard, which appreciatively comigrates with permanently expressed
CFTR protein in CHO cells (Fig. 1A, lane 2). The smaller
145-kDa protein expressed in CFTR(+) CHO cells was visually absent in
Calu-3 cells. No immunoreactivity was detected in CFTR(
) CHO cells
(Fig. 1A, lane 3). As previously described
(10), the 175-kDa protein represents the mature, fully glycosylated CFTR and the 145-kDa protein represents the immature or
core-glycosylated CFTR. In addition, the upper band migrating at 175 kDa coincided with the phosphorylated band obtained by CFTR
immunoprecipitation followed by phosphorylation of immunoprecipitates by the catalytic subunit of PKA in Calu-3 and CFTR(+) CHO cells (Fig.
1B, lanes 1 and 2).
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CFTR is in the apical membrane of Calu-3 cells.
CFTR was shown to have a predominantly apical location (Fig.
2A). It showed the same
pattern of localization (Fig. 2B) as the GPI-anchored apical
membrane protein CD59 (14). This was seen in
x-y scans as a patch of immunofluorescence in the first confocal section (<1 µm from the apical surface). The labeling appeared as a ring of fluorescence at 3 µm from the apical surface and was not detectable 6-8 µm into the cell, thus having
characteristics of an apical location. Specificity of CFTR
immunofluorescence was shown by the demonstration that it was abolished
by preabsorption of antibody with COOH-terminal peptide (Fig.
2C). Figure 2, A,d and B,d, show the
side view (x-z scans) of CFTR and CD59 immunofluorescence, which also clearly indicates an apical location for both proteins.
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Stimulation of CFTR-mediated iodide efflux by xanthine derivatives
in Calu-3 cells.
We examined the effects of IBMX and X-33 on Calu-3 cells. IBMX and X-33
were able to generate an iodide efflux significantly different
(P < 0.0001) from the control conditions, i.e., in the absence of agonist (Fig. 3A).
Addition of 250 µM IBMX or 250 µM X-33 increased the peak rate of
iodide efflux from 0.09 ± 0.01 (n = 12) to
0.19 ± 0.01 (n = 24) and 0.15 ± 0.01 (n = 16), respectively (Fig. 3A). Caffeine
(1,3,7-trimethylxanthine) at 250 µM was ineffective in stimulating an
efflux from Calu-3 cells (data not shown).
8-Cyclopentyl-1,3-dipropylxanthine (CPX) was also applied at three
different concentrations (10 nM, 10 µM, and 250 µM), but no
significant iodide efflux was observed (data not shown). Forskolin (5 µM, n = 4) stimulates CFTR, as shown by the increase
of peak rates (Fig. 3B). This forskolin-induced iodide
efflux was strongly inhibited by 100 µM glibenclamide
(n = 4; Fig. 3B) but was not affected by the
outwardly rectifying Cl channel inhibitor
(39) TS-TM calix[4]arene (200 nM, n = 4; Fig. 3B). The average dose-response relationships for IBMX
and X-33 are presented in Fig. 3, C and D,
respectively. These curves were obtained by normalizing the mean peak
rates at various xanthine concentrations to that obtained with the
saturating concentration. The EC50 values for IBMX and X-33
were 45 and 100 µM, respectively.
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cAMP levels. We tested the possibility that activation by X-33 might be due to elevation of cAMP. In resting Calu-3 cells, the cellular cAMP content was 0.55 ± 0.05 pmol cAMP/well (n = 6; Fig. 3E). As expected, forskolin (5 µM) increased the cAMP level measured after 5 min (7.14 ± 0.71 pmol cAMP/well, n = 6; Fig. 3E). In contrast, the corresponding cAMP level determined in the presence of xanthine derivatives X-33 (250 µM, n = 6), CPX (10 nM, n = 3), and caffeine (250 µM, n = 3) was not increased compared with the basal level (Fig. 3E). These results argue against a role of cAMP in mediating the effect of X-33 on CFTR. IBMX (250 µM, n = 6) induced a weak elevation of cAMP content, significantly different from the basal level (P < 0.05; Fig. 3E).
Activation of CFTR current by the xanthine derivative X-33.
It is established that Calu-3 cells are well polarized in culture
(39), and we showed in Fig. 2 that CFTR is apically
located. The iodide efflux data were completed by whole cell recordings to characterize the currents in Calu-3 cells. Figure
4 presents typical whole cell currents
and associated current-voltage (I-V) plots in the presence
or absence of activator in the bath. In control patch-clamp experiments
on Calu-3 cells (Fig. 4, A and C;
n = 20), i.e., in the absence of any activator, no
current was recorded. We first used forskolin to test for the presence of cAMP-dependent Cl current. The addition of 5 µM
forskolin (Fig. 4B) stimulated a time-independent,
nonrectifying conductance in 11 of 17 (65%) Calu-3 cells, indicating
the presence of a linear Cl
-selective current typical of
functional CFTR. The forskolin-activated Cl
conductance
(Fig. 4C) had a current density of 17 ± 3.9 pA/pF (n = 11) when measured at +60 mV. The increase was
statistically significant from the basal current (P < 0.001).
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Effects of Cl current inhibitors on X-33-activated
current.
The experiments described above show that CFTR activated by X-33 (Fig.
5) or forskolin (Fig. 3B) was not sensitive to the outwardly
rectifying Cl
current blocker calixarene. To confirm that
this Cl
current was mediated by the CFTR Cl
channel, we examined the effects of two other Cl
channel
blockers, the sulfonylurea glibenclamide and the arylaminobenzoate diphenylamine-2-carboxylate (DPC), both known as CFTR inhibitors (37). Results of such experiments are illustrated in Fig.
6, which shows representative current
traces, time course of current, and I-V relationships in
Calu-3 cells. The linear and nonrectifying X-33-activated
Cl
current was reduced to the basal level in the presence
of 100 µM glibenclamide [16.2 ± 2.5 pA/pF with X-33
(n = 11) and 2.7 ± 0.4 pA/pF with glibenclamide
(n = 3) at +60 mV; Fig. 6, A and C]. A time course of the activation and inhibition of
current from the same whole cell recording is shown in Fig.
6A,c. Addition of X-33 (250 µM) after 3 min of recording
increased current within 6 min. In the presence of X-33, addition of
glibenclamide (100 µM) at 7 min inhibited the majority of the current
in 3 min. Overall, glibenclamide inhibited ~83% of the current (Fig.
6D). A similar result was obtained in iodide efflux
experiments with glibenclamide (Fig. 3B). We also examined
the effects of DPC. As shown in Fig. 6, B and C,
500 µM DPC induced a rapid and voltage-independent inhibition of
X-33-activated current (0.55 ± 0.15 pA/pF, n = 3, at +60 mV). The time course (Fig. 6B,c) shows that, in the
presence of X-33, DPC completely inhibited the current in 2 min. The
effect of DPC is partially and slowly reversed (Fig. 6B,c).
The maximal inhibition was 97% (Fig. 6D). The fact that DPC
inhibition was voltage-independent is rather surprising but could be
explained by the high concentration (500 µM). Flufenamic acid (FFA,
500 µM, n = 3) was also found to inhibit forskolin-
or X-33-activated Cl
current (not shown).
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Isc measurement.
We have measured the effects of X-33 on transepithelial ion transport
under Isc conditions. Indeed, in this cell
preparation, increases in Isc are an indication
of anion secretion (16). Application of X-33 (10-250
µM) in apical and basolateral solutions determined a fast increase of
Isc. The response consisted in an initial peak
that was followed by a sustained phase (Fig.
7A). At 100 µM, the peak and
the sustained phase (measured 15 min after X-33 application) were
14.7 ± 0.6 and 10.4 ± 1.1 µA/cm2
(n = 4), respectively. The current activated by X-33
was not blocked by DIDS (Fig. 7B). Similar results were
obtained using iodide efflux and whole cell recordings (data not
shown). On the contrary, glibenclamide strongly blocked (Fig.
7A) or prevented (Fig. 7C) the effect of X-33.
The sensitivity to glibenclamide and not to DIDS again suggested that
X-33 activates the cAMP-dependent Cl channel, i.e., CFTR,
as observed using iodide efflux and whole cell patch-clamp techniques.
Consistent with this assumption, X-33 was ineffective when applied
after stimulation with 5 µM forskolin (Fig. 7D), a
concentration at which the cAMP-dependent current is maximally
activated.
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Outwardly rectifying Cl current in Calu-3 cells.
It is now clear that the activity of CFTR in Calu-3 cells is not
affected by DIDS or TS-TM calix[4]arene, as reported here. During
whole cell patch-clamp experiments, we sometimes recorded a DIDS- and
TS-TM calix[4]arene-sensitive Cl
current. Figure
9 shows representative whole cell current
traces and corresponding I-V curves (Fig. 9E)
observed in 10 of 43 (23%) Calu-3 cells. In those cells, for high
positive pulses (i.e., greater than +60 mV), a small outwardly
rectifying Cl
current, which deactivates with time, was
observed in the absence of forskolin (Fig. 9A). Addition of
5 µM forskolin to the bath leads to the activation of a weakly
rectifying linear Cl
current (Fig. 9B), which
was partly inhibited by 100 µM glibenclamide (Fig.
9C). The remaining glibenclamide-insensitive current was outwardly rectifying, deactivates with time, and was abolished in the
presence of external 200 µM DIDS (Fig. 9D). These data indicate that an outwardly rectifying Cl
channel is
expressed in Calu-3 cells, which could be activated by a
cAMP-independent mechanism. Haws et al. (26), indeed,
described at a single-channel level the properties of an outwardly
rectifying Cl
channel in Calu-3 cells. This channel may
be responsible for the outwardly rectifying whole cell Cl
current we recorded here. Because DIDS also inhibits several Cl
transporters, we tested the effect of TS-TM
calix[4]arene, a compound related to DIDS and a more potent blocker
of outwardly rectifying Cl
channels (37,
39). In the experiment described in Fig.
10, an outwardly rectifying
Cl
current was spontaneously activated (Fig.
10A). The addition of 200 nM TS-TM calix[4]arene
completely and rapidly inhibited this outwardly rectifying
Cl
current (Fig. 10B; n = 5).
Then, in the continuous presence of TS-TM calix[4]arene, 5 µM forskolin activated a linear CFTR current (Fig. 10C).
This experiment reveals that TS-TM calix[4]arene inhibited outwardly
rectifying Cl
, but not CFTR, current in Calu-3 cells and
further demonstrates that the cAMP-activated Cl
current
in this cell is mainly, if not exclusively, due to CFTR. In the 20 cells tested for activation by X-33, 11 responded by a linear CFTR
Cl
current but none by an outwardly rectifying
Cl
current, which clearly indicates the specificity of
the xanthine derivative for CFTR.
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DISCUSSION |
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Since the first electrophysiological characterization of the
Cl channel CFTR, the protein product of the CF gene
(35), studying its pharmacology has became possible and
appears to be of high importance not only to understand its regulation
and role in normal and CF epithelia but because our knowledge of the
pharmacology of CFTR (for review see Ref. 37) may serve as
a model for the general pharmacology of Cl
channels.
Calu-3 cells, which are derived from a pulmonary adenocarcinoma,
express a high level of CFTR mRNA and protein (21, 38), show cAMP-dependent Cl (38) and bicarbonate
(16) secretions, and resemble serous gland cells
(26), the major pulmonary cells implicated in CF lung
disease (20). In this study we showed that CFTR is in the apical membrane and is phosphorylated by PKA. Another type of outwardly
rectifying Cl
current was also identified but was
unlikely to contribute to the cAMP-dependent Cl
current
in this cell.
CFTR and non-CFTR Cl currents in Calu-3 cells.
Despite the description of the basic properties of CFTR channels in
this cell line (26, 42), little was known about their whole cell characteristics and pharmacology. At a single-channel level,
Haws et al. (26) characterized two types of
Cl
channels in Calu-3 cells: CFTR and outwardly
rectifying depolarization-induced Cl
channels. In
cell-attached recordings, CFTR has a linear I-V relationship
with a single conductance of 7.1 pS, is voltage independent, and is
activated by cAMP-elevating agents. We showed in this report that the
cAMP-activated iodide efflux and CFTR Cl
current were
inhibited by glibenclamide, DPC, and FFA but not by DIDS and TS-TM
calix[4]arene.
Xanthine as CFTR activator.
The story of xanthine derivatives as modulators of CFTR began in 1991, when Drumm et al. (17) demonstrated that wild-type CFTR,
F508 CFTR, and other mutants expressed in Xenopus oocytes could be activated by IBMX in a concentration-dependent manner, but in
the presence of forskolin (17). Then, in the absence of
forskolin, the methylxanthine drug IBMX by itself was shown to activate
normal and mutated CFTR Cl
channels in epithelia
(3, 25, 26) and transfected cells (4, 37).
IBMX, 1,3-dipropyl-7-methylxanthine, and the synthesized xanthines X-33
and X-32 stimulate CFTR in transfected CHO cells without altering ATP
contents and with little effect on intracellular cAMP level (9; this
study). Nanomolar concentrations of CPX were reported to increase
36Cl efflux in CFPAC-1 pancreatic cells (homozygous for the
F508 allele) (19). Moreover, Arispe et al.
(1) found that CPX could activate wild-type CFTR in planar
lipid bilayer after a modest exposure to PKA and ATP. However, CPX
seems to be more selective for
F508 CFTR than for the wild-type.
Cohen et al. (11) proposed that this agent activates CFTR
by a direct interaction on NBF-1. However, some authors have not
observed any acute activation of CFTR by CPX (9, 27, 31),
whereas others have reported a potentiation of the response to
forskolin on
F508 CFTR-expressing cells consistent with that
obtained with IBMX (27, 28).
Xanthine as CFTR activator in Calu-3 cells.
Having demonstrated, along with others, that CFTR Cl
channel is the main cAMP-dependent apical Cl
pathway in
Calu-3 cells (26, 38; this study), we have investigated the effect of
xanthine derivatives on CFTR. As expected, IBMX elicited a significant
and concentration-dependent (EC50 ~45 µM) iodide efflux
and whole cell current with all the characteristics of CFTR-mediated
Cl
transport. The synthetic xanthine X-33 was found to be
effective in Calu-3 cells by the use of three different techniques
(iodide efflux, whole cell patch-clamp, and short-circuit
measurements). The I-V relationship of the activation of
Cl
transport was linear and time independent in whole
cell recordings, with an EC50 of ~100 µM. The
X-33-activated iodide efflux, whole cell current, and
Isc were remarkably DIDS insensitive and
glibenclamide sensitive. More importantly, the effect of X-33 on Calu-3
cells was insensitive to calix[4]arene. This demonstrates that the
outwardly rectifying Cl
current found in this preparation
is not affected by this agent or by forskolin. The other xanthines
tested, caffeine and CPX, were poorly effective in activating a
Cl
current.
Mechanisms of xanthine-dependent activation of CFTR.
Xanthine derivatives, depending on their structure, may act as
inhibitors of phosphodiesterases (PDEs) (2) or
phosphatases (3, 12) or as antagonists of adenosine
receptors (7). Activation of CFTR channels using xanthine
derivatives was initially attributed to inhibition of PDE activity,
which would elevate cAMP by inhibiting its degradation. However, IBMX
increased the maximal forskolin-dependent F508-CFTR activity without
increasing cellular cAMP, suggesting a cAMP-independent mechanism
(28). A similar conclusion was drawn previously by Chappe
et al. (9). They showed that, among the nonspecific PDE
inhibitors IBMX, theophylline (1,3-dimethylxanthine), caffeine, and
8-cyclopentyltheophylline, only IBMX and theophylline were able to open
CFTR (9). In this study, the xanthine derivative X-33 did
not alter the intracellular cAMP content, as previously shown in
CFTR(+) CHO cells (9). Moreover, the specific PDE
inhibitors rolipram and milrinone failed to open CFTR channels in
CFTR(+) CHO cells (9). For these reasons, it is reasonable
to think that xanthine derivatives activate CFTR through a
cAMP-independent mechanism. In particular, substituted xanthines IBMX
and theophylline inhibit alkaline phosphatase (3, 12).
Becq et al. (4) showed that these two compounds slowed the
rundown of CFTR channel activity in excised membrane patches. Xanthine
derivatives are also known to be antagonists of adenosine receptor A (7). Using pancreatic duct cells expressing the
F508 mutation (CFPAC cells), Eidelman et al. (19)
initially reported the activation of Cl
current by CPX, a
potent A1 adenosine-receptor antagonist (19). No human A1 adenosine-receptor mRNA was later detected in
these cells (30), excluding this receptor as a mediator of
CPX-elicited Cl
efflux. The authors then suggested that
the action of CPX on
F508-CFPAC cells represents a novel site of
action apparently unrelated to a known adenosine receptor. Casavola et
al. (8) reported that CPX decreased intracellular pH,
which would be linked to the inhibition of
Na+/H+ exchange. It is not known, however,
whether the action of CPX on the Cl
efflux may be
correlated with the variation of intracellular pH or with some other
intracellular mechanisms. More recently, Kunzelman et al.
(31) reported that the CPX-induced Cl
efflux
observed in CF cells could not be attributed to a direct activation of
F508 but, on the contrary, may be due to a pH-dependent mechanism.
Finally, a controversy appeared when other groups failed to reproduce
the effect of CPX (27, 31). Some authors showed a biphasic
response to CPX, i.e., a stimulation for low concentrations (10-30 nM), whereas higher concentrations (100 nM-10
µM) caused inhibition of 36Cl efflux (19, 24,
30). In our experience (4, 9; this study), stimulation of CFTR
by CPX has not been observed in CHO cells stably transfected with
wild-type CFTR and in Calu-3 cells, whatever the concentration used (10 nM, 10 µM, and 250 µM).
Structure-function study.
We are presently using a series of alkyl-substituted xanthine
derivatives to study the effects on CFTR of chemical modifications of
the xanthine skeleton (9). Previously, we showed a
correlation between the potency of a series of 1,3,7-trialkylxanthine
derivatives and the opening of the CFTR Cl channel
(9). To understand the substitutions implicated in the
activation of CFTR, caffeine was chosen in this study, because it
differs from X-33 only at N-1 (methyl and isobutyl, respectively). This
modification leads to a potent activator (X-33) and a far less active
one (caffeine). The alkyl substitution at N-1 thus appears to be
important for CFTR activation. However, the same modification
(isobutyl) at N-3 produces a more potent activator (IBMX). So we can
hypothesize that the presence of a long alkyl bond at N-1 generates
agents with high potency on CFTR activation, but a second important
site seems to be located at N-3. Although X-33 is about one-half as
potent as IBMX, X-33 has no effect on the cell cAMP levels, and it is
extremely important to increase the spectrum of compounds capable of
elevating specifically the CFTR activity. Our data suggested that
xanthines substituted at C-8 (CPX) failed to open CFTR in Calu-3 cells,
which suggests that the general mechanism of CFTR opening by xanthines
required alkyl substitution (at N-1 or N-3) and vacancy at C-8 in CHO
and Calu-3 cells. The role of CFTR in the Cl
conductance
of several tissues (e.g., heart, nephron, or exocrine pancreas) may be
clarified by the use of specific activators. For example, the cardiac
CFTR channel has been recently shown to be activated by levamisole
(18), a compound we have identified by its effects in
pancreatic duct cells (3) and in CHO cells (4).
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ACKNOWLEDGEMENTS |
---|
The authors thank Ashvani Singh and Robert Bridges for the generous gift of calixarene.
![]() |
FOOTNOTES |
---|
* L. Bulteau and R. Dérand contributed equally to these results.
This work was supported by postdoctoral fellowships to L. Bulteau and a thesis grant to R. Dérand from the Association Française de Lutte contre la Mucoviscidose, by institutional grants from Centre National de la Recherche Scientifique, and by Association Française de Lutte contre la Mucoviscidose Grant P-97003.
Address for reprint requests and other correspondence: F. Becq, Laboratoire de physiologie des régulations cellulaires, UMR 6558, 40 ave. du recteur Pineau, 86022 Poitiers, France (E-mail: frederic.becq{at}univ-poitiers.fr).
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 January 2000; accepted in final form 8 August 2000.
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