Am J Physiol Cell Physiol
279: C1925–C1937, 2000.
Properties of CFTR activated by the xanthine
derivative X-33 in human airway Calu-3 cells
Received 20 January 2000; accepted in final form 8 August 2000
Bulteau, Laurence, Renaud Dérand, Yvette Mettey,
Thierry Métayé, M. Rachel Morris, Ceinwen M. McNeilly, Chiara Folli, Luis J. V. Galietta, Olga ZegarraMoran, Malcolm M. C. Pereira, Chantal Jougla, Robert
L. Dormer, Jean-Michel Vierfond, Michel Joffre, and
Frédéric Becq. Properties of CFTR activated by the xanthine derivative X-33 in human airway Calu-3 cells. Am J
Physiol Cell Physiol 279: C1925–C1937, 2000.—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
(CF), the most common lethal autosomal
recessive genetic disease, is caused by mutations of the
CF gene, which normally encodes a multifunctional
CYSTIC FIBROSIS
* L. Bulteau and R. Dérand contributed equally to these results.
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
@univ-poitiers.fr).
http://www.ajpcell.org
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 cAMPindependent pathways. Compounds such as p-bromotetramisole or levamisole (4, 6), genistein (29),
5-chloro-2-hydroxyphenyl-1,3-dihydro-2H-benzimidazol2-one (NS-004) (15, 22), 1-ethyl-2-benzimidazolinone (1EBIO) (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
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.
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society
C1925
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LAURENCE BULTEAU*,1 RENAUD DÉRAND*,1 YVETTE METTEY,2 THIERRY MÉTAYÉ,1
M. RACHEL MORRIS,4 CEINWEN M. MCNEILLY,4 CHIARA FOLLI,3
LUIS J. V. GALIETTA,3 OLGA ZEGARRA-MORAN,3 MALCOLM M. C. PEREIRA,4
CHANTAL JOUGLA,1 ROBERT L. DORMER,4 JEAN-MICHEL VIERFOND,2
MICHEL JOFFRE,1 AND FRÉDÉRIC BECQ1
1
Laboratoire de Physiologie des Régulations Cellulaires, Unité Mixte de Recherche 6558,
86022 Poitiers; 2Laboratoire de Chimie Organique, Faculté de Médecine et de Pharmacie,
86005 Poitiers, France; 3Laboratorio di Genetica Molecolare, Istituto Giannina Gaslini,
16148 Genova, Italy; and 4Department of Medical Biochemistry, University of Wales,
College of Medicine, Cardiff CF14 4XN, United Kingdom
C1926
ACTIVATION OF CFTR IN CALU-3 CELLS BY X-33
METHODS
Cell culture. Native CHO cells [CFTR(⫺) CHO] or CHO
cells stably transfected with pNUT vector containing wildtype 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 ␮g/ml streptomycin), and 100 ␮M 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 ␮g/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 䡠 HCl, pH 7.45, 10 mM NaCl, 1 mM EDTA, 2 mM MgCl2,
1 mM dithiotreitol, 0.2 mg/ml benzamidine, 1 ␮g/ml pepstatin A, 0.1 mM phenylmethylsulfonyl fluoride, 20 ␮g/ml aprotinin, and 20 ␮g/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 䡠 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 ␮g/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 ␮g) 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 䡠 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 ␮g/ml. After
several washes, the membrane was incubated with a secondary antibody, horseradish peroxidase-labeled sheep antimouse 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 䡠 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 ␮g/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 ␮l, ⬃4 mg protein/ml) was
incubated with affinity-purified CFTR antibody (80 ␮g/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 䡠 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 ␮Ci 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 䡠 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
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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).
ACTIVATION OF CFTR IN CALU-3 CELLS BY X-33
matography 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. Shortcircuit 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 shortcircuited 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 ␮M 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-tetrasulfonato25,26,27,28-tetramethoxy-calix[4]arene) was a generous gift
of Drs. Singh and Bridges (University of Alabama at Birmingham).
RESULTS
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).
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
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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 ␮l 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 ␮M KI (1
␮Ci 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 chro-
C1927
C1928
ACTIVATION OF CFTR IN CALU-3 CELLS BY X-33
patch of immunofluorescence in the first confocal section (⬍1 ␮m from the apical surface). The labeling
appeared as a ring of fluorescence at 3 ␮m from the
apical surface and was not detectable 6–8 ␮m 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.
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 ␮M IBMX or 250 ␮M 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 ␮M
was ineffective in stimulating an efflux from Calu-3
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Fig. 1. Identification of the cystic fibrosis transmembrane conductance regulator (CFTR) protein in Calu-3 cells. A: Western blot
analysis of protein extracts from Calu-3 (lane 1), CFTR(⫹) Chinese
hamster ovary (CHO, lane 2), and CFTR(⫺) CHO (lane 3) cells. B:
immunoprecipitation and phosphorylation of CFTR from Calu-3
(lane 1), CFTR(⫹) CHO (lane 2), and CFTR(⫺) CHO (lane 3) cells.
The immunoprecipitates were phosphorylated in the presence of the
catalytic subunit of protein kinase A and [␥-32P]ATP. Samples were
subjected to SDS-PAGE and analyzed by autoradiography. Positions
of molecular weight markers are shown on right. Arrows, 175- and
145-kDa CFTR proteins.
cells (data not shown). 8-Cyclopentyl-1,3-dipropylxanthine (CPX) was also applied at three different concentrations (10 nM, 10 ␮M, and 250 ␮M), but no significant iodide efflux was observed (data not shown).
Forskolin (5 ␮M, n ⫽ 4) stimulates CFTR, as shown by
the increase of peak rates (Fig. 3B). This forskolininduced iodide efflux was strongly inhibited by 100 ␮M
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 ␮M, respectively.
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 ␮M) 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 ␮M, n ⫽
6), CPX (10 nM, n ⫽ 3), and caffeine (250 ␮M, 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 ␮M,
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 ␮M 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).
To further prove that the forskolin-activated current
was due to CFTR activity, we performed ion-substitution experiments. Replacing all except 8 mM of the Cl⫺
in the bath solution with an equimolar amount of
iodide led to a shift of the reversal potential toward
positive potentials, from ⫺37 ⫾ 0.3 to ⫹28 ⫾ 2 mV (n ⫽
3) in the presence or absence of forskolin, indicating a
larger permeability for Cl⫺, i.e., a selectivity of Cl⫺ ⬎⬎
I⫺, consistent with a CFTR channel (26, 40).
ACTIVATION OF CFTR IN CALU-3 CELLS BY X-33
C1929
We then tested the effects of the two xanthine derivatives IBMX and X-33 (Fig. 4, B and C). Addition of 250
␮M IBMX to the bath caused a statistically significant
increase in CFTR current amplitude compared with
basal current [when measured at ⫹60 mV, basal ⫽
2.4 ⫾ 0.3 pA/pF (n ⫽ 20) and IBMX ⫽ 20.4 ⫾ 4.2 pA/pF
(n ⫽ 4); Fig. 4C]. In CFTR(⫺) CHO cells, forskolin,
IBMX, or X-33 failed to activate any current (n ⫽ 5 for
each, data not shown). Addition to the bath of 250 ␮M
X-33 caused the activation of a time-independent, nonrectifying conductance in 11 of 20 (55%) Calu-3 cells
(Fig. 4, B and C). These increases were significantly
different from basal current [16.2 ⫾ 2.5 pA/pF (n ⫽ 11)
in the presence of X-33 and 2.4 ⫾ 0.3 pA/pF (n ⫽ 20) in
control conditions at ⫹60 mV]. Overall, X-33 activates
a Cl⫺ conductance with CFTR-like kinetics (time- and
voltage-independent, linear I-V relationship) in Calu-3
cells similar to that obtained for CFTR(⫹) CHO cells
(not shown).
We then quantified the relationship between the
X-33 concentration and the activated current in whole
cell membranes from Calu-3 cells. The effects of increasing concentrations of X-33 were observed in the
presence of 200 nM TS-TM calix[4]arene, a specific
blocker of outwardly rectifying Cl⫺ current (39). A time
course of the X-33 concentration-dependent activation
of current in a Calu-3 cell is shown in Fig. 5A. Figure
5B shows averaged I-V curves obtained for different
concentrations of X-33 applied to the bath. A complete
dose-response relationship (Fig. 5C) was obtained by
normalizing mean current amplitudes at various X-33
concentrations to that obtained with 250 ␮M X-33 (n ⫽
4 for each concentration). This concentration was chosen because it is likely to maximally activate the X-33dependent current. Elevating the X-33 concentration
from 50 to 100 ␮M increased significantly the CFTR
current. A concentration ⬎250 ␮M generated a saturating X-33-dependent current. The EC50 was consistent with the iodide efflux data.
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 ␮M 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.
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Fig. 2. Confocal images of CFTR and CD59 in Calu-3 cells. Calu-3 cells grown on glass coverslips were fixed,
permeabilized, and labeled for immunofluorescence using antibody to CFTR raised against COOH-terminal
peptide (A), CD59 (B), and CFTR preabsorbed with COOH-terminal peptide (10 ␮g/ml for 18 h at 4°C; C). a–c: x-y
scans at ⬍1 ␮m (a), 3 ␮m (b), and 6 ␮m (c) from the apical surface of the cell. Note fluorescence signal in the apical
sections, the dark area inside the ring of fluorescence at 3 ␮m, and lack of fluorescence toward the basolateral end
of the cell (6–8 ␮m). d: x-z scan with strong apical fluorescence of CFTR and CD59. Total height of the x-z scan was
50 ␮m, indicating the height of cells to be ⬃10 ␮m.
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ACTIVATION OF CFTR IN CALU-3 CELLS BY X-33
6A,c. Addition of X-33 (250 ␮M) after 3 min of recording
increased current within 6 min. In the presence of
X-33, addition of glibenclamide (100 ␮M) 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 ␮M
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
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Fig. 3. A: activation of CFTR-mediated iodide efflux by xanthine derivatives in Calu-3 cells [vehicle (䊐, n ⫽ 12), 250
␮M IBMX (F, n ⫽ 24), and 250 ␮M X-33 (, n ⫽ 16), as indicated by arrow]. B: averaged curves of iodide efflux
comparing the strong inhibition of the forskolin (Fsk)-induced efflux by 100 ␮M glibenclamide (■, n ⫽ 4) with the
lack of inhibitory effect of 200 nM TS-TM calix[4]arene (}, n ⫽ 4). 䊐, Basal conditions (n ⫽ 4); Œ, 5 ␮M forskolin (n ⫽
4). Rates of iodide efflux are plotted as a function of time. Stimulatory agents, added at 3 min, were present during
remainder of efflux period. Errors bars, SE. Arrow indicates the time the drug was added. C and D: dose-response
curves for xanthine-activated 125I efflux in Calu-3 cells. Cells were treated with IBMX (C, n ⫽ 4 for each
concentration, means ⫾ SE) or X-33 (D, n ⫽ 2 for each concentration, means ⫾ SE) at 10–500 ␮M. E: effect of
forskolin and xanthine derivatives on cAMP in Calu-3 cells. Forskolin was tested at 5 ␮M, and xanthine derivatives
were tested at 250 ␮M. CPX, 8-cyclopentyl-1,3-dipropylxanthine.
ACTIVATION OF CFTR IN CALU-3 CELLS BY X-33
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Fig. 4. Activation of CFTR current in Calu-3 cells. Representative current traces for CFTR are shown in 3
representative Calu-3 cells before (A) and after (B) application of activator: 5 ␮M forskolin, 250 ␮M IBMX, or 250
␮M X-33. Step protocol consisted of 300-ms voltage steps from ⫺80 to ⫹80 mV from a holding potential of ⫺40 mV.
Dashed lines, zero-current level. Capacitances for the 3 cells are 47, 44, and 55 pF, respectively. C: current-voltage
(I-V) curves (means ⫾ SE) for CFTR current before (n ⫽ 20) and after application of forskolin (n ⫽ 11), IBMX (n ⫽
4), and X-33 (n ⫽ 11).
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ACTIVATION OF CFTR IN CALU-3 CELLS BY X-33
X-33 (10–250 ␮M) 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 ␮M, the peak and the
sustained phase (measured 15 min after X-33 application) were 14.7 ⫾ 0.6 and 10.4 ⫾ 1.1 ␮A/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
explained by the high concentration (500 ␮M). Flufenamic acid (FFA, 500 ␮M, n ⫽ 3) was also found to
inhibit forskolin- or X-33-activated Cl⫺ current (not
shown).
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
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Fig. 5. Concentration-response relationship for the activation of
CFTR current by X-33. A: time course of X-33-activated CFTR
current. Whole cell current at ⫹40 mV is plotted as a function of time
in the presence of 200 nM TS-TM calix[4]arene (calix), increasing
concentrations of X-33, and 100 ␮M glibenclamide (glib). B: I-V
curves for CFTR current with different concentrations of X-33 (n ⫽ 4
for each concentration). Cells were held at ⫺40 mV and clamped with
300-ms voltage pulses from ⫺80 to ⫹80 mV. C: percentage of the
maximal response expressed as current density (means ⫾ SE) for a
depolarizing step to ⫹40 mV normalized to the maximum value
obtained in the presence of 250 ␮M X-33. *P ⬍ 0.05; ns, not significant (i.e., P ⬎ 0.05).
Fig. 6. Effect of Cl⫺ channel blockers on X-33-activated CFTR currents. A and B: representative current traces and time course of
current in 2 Calu-3 cells. Capacitances are 66 pF (A) and 46 pF (B).
a and b: Membrane currents elicited by stepping from the holding
potential of ⫺40 mV to a series of test potentials from ⫺80 to ⫹80 mV
in 20-mV increments. X-33-activated currents were measured before
(a) and after (b) exposure to glibenclamide (100 ␮M) or diphenylamine-2-carboxylate (DPC, 500 ␮M). c: Time course of activation
and inhibition of CFTR current at ⫹40 mV. X-33 (250 ␮M), glibenclamide, and DPC were applied (horizontal bars). Dashed lines, zero
current level. C: I-V curves for X-33-activated CFTR current before
(n ⫽ 11) and after application of inhibitors (n ⫽ 3 for each inhibitor).
D: percentage of current inhibition at ⫹80 mV. Numbers in parentheses indicate the number of cells tested for each inhibitor.
ACTIVATION OF CFTR IN CALU-3 CELLS BY X-33
C1933
Fig. 7. Effect of X-33 on short-circuit current (Isc) in Calu-3 cells.
Time course of Isc in 4 representative experiments (A–D) is shown.
Time of application of X-33 (100 ␮M, apical and basolateral), glibenclamide (500 ␮M, apical and basolateral), DIDS (500 ␮M, apical),
and forskolin (5 ␮M, apical and basolateral) is indicated by arrows.
using iodide efflux and whole cell patch-clamp techniques. Consistent with this assumption, X-33 was
ineffective when applied after stimulation with 5 ␮M
forskolin (Fig. 7D), a concentration at which the cAMPdependent current is maximally activated.
We have tested three other xanthines, IBMX, CPX,
and caffeine, to compare their effectiveness with that of
X-33. On a qualitative basis, the biphasic time course
of the Isc activated by IBMX or caffeine was very
similar to that of X-33 and forskolin (Fig. 8A). However, IBMX was more potent, since at 10 ␮M, a concentration at which X-33 and caffeine had a small
effect, this xanthine elicited almost a maximal current
increase (Fig. 8B). On the contrary, caffeine was less
Fig. 8. Xanthine-dependent Isc increase in Calu-3 epithelia. A: representative effects of IBMX and caffeine at 100 ␮M. B: summary of
xanthine effects at different concentrations. Values are means ⫾ SE
of the current measured at the peak from ⱖ4 experiments.
Fig. 9. A DIDS-sensitive Cl⫺ current is present in Calu-3 cells.
Representative current traces in a Calu-3 cell in control conditions
(A) and in the presence of 5 ␮M forskolin with (C and D) or without
(B) inhibitors are shown. E: corresponding I-V curves of instantaneous current (arrows on current traces; n ⫽ 10 for basal and n ⫽ 3
for each other condition). Capacitance is 34 pF. Glibenclamide, 100
␮M; DIDS, 200 ␮M.
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effective, since only a weak effect was obtained at 250
␮M. CPX was also found to have no effect (10 nM and
10 ␮M) or a weak effect (250 ␮M), as observed in iodide
efflux (data not shown).
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 ␮M forskolin to the bath leads to the
activation of a weakly rectifying linear Cl⫺ current
(Fig. 9B), which was partly inhibited by 100 ␮M glibenclamide (Fig. 9C). The remaining glibenclamide-
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ACTIVATION OF CFTR IN CALU-3 CELLS BY X-33
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insensitive current was outwardly rectifying, deactivates with time, and was abolished in the presence of
external 200 ␮M DIDS (Fig. 9D). These data indicate
that an outwardly rectifying Cl⫺ channel is expressed
in Calu-3 cells, which could be activated by a cAMPindependent 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 ␮M 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.
DISCUSSION
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-
Fig. 10. Effect of TS-TM calix[4]arene on outwardly rectifying Cl⫺
currents in Calu-3 cells. Representative current traces in the absence
(A) or presence (B and C) of 200 nM TS-TM calix[4]arene in the bath
are shown. C: subsequent activation of a linear current by 5 ␮M
forskolin in the presence of TS-TM calix[4]arene. Capacitance is
54 pF.
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.
Non-CFTR channels were observed in 17% of unstimulated cell-attached patches and 8% of stimulated
ACTIVATION OF CFTR IN CALU-3 CELLS BY X-33
tionship of the activation of Cl⫺ transport was linear
and time independent in whole cell recordings, with an
EC50 of ⬃100 ␮M. 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 ⌬F508CFTR 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 CPXelicited 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 CPXinduced 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
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excised patches in Calu-3 cells (26). An outwardly
rectifying depolarization-induced Cl⫺ channel was observed in unstimulated patches of Calu-3 cells but not
in cell-attached patches (26, 42). In 13% of excised
patches exposed to ATP plus PKA, Haws et al. (26)
recorded outwardly rectifying depolarization-induced
plus CFTR channels. In good agreement with these
data, we recorded outwardly rectifying Cl⫺ current in
only 23% of the unstimulated Calu-3 cells but in 0% of
stimulated cells studied here. Since this current is
activated mainly in the absence of cAMP, this suggests
that the outwardly rectifying Cl⫺ current is not directly under the control of a cAMP pathway. The pharmacology of outwardly rectifying Cl⫺ current is based
on the inhibitory effect of stilbene derivatives such as
DIDS, SITS, or 4,4⬘-dinitrosostilbene-2,2⬘-disulfonate
(39). Recently, TS-TM calix[4]arene, a compound related to DIDS and a more potent blocker of outwardly
rectifying Cl⫺ channels, was developed (39). We have
also shown here that DIDS or TS-TM calix[4]arene
inhibited the outwardly rectifying Cl⫺ current.
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 wildtype 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 concentrationdependent (EC50 ⬃45 ␮M) 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 patchclamp, and short-circuit measurements). The I-V rela-
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ACTIVATION OF CFTR IN CALU-3 CELLS BY X-33
The authors thank Ashvani Singh and Robert Bridges for the
generous gift of calixarene.
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.
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stimulation for low concentrations (10–30 nM), whereas
higher concentrations (100 nM–10 ␮M) 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 wildtype CFTR and in Calu-3 cells, whatever the concentration used (10 nM, 10 ␮M, and 250 ␮M).
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 1,3,7trialkylxanthine 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).
In conclusion, the pharmacology of CFTR is at the
point where numerous families of chemicals of interest
have been discovered, which opens a very exciting
avenue toward the development of agents of high importance for the knowledge of the physiology of epithelia and other tissues, including the heart, and of the
pathophysiology, including CF. Solving the structureactivity relationships of these chemicals will help build
further steps toward those goals in the future.
ACTIVATION OF CFTR IN CALU-3 CELLS BY X-33
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