Departments of 1 Human Genetics, 4 Medicine, and 5 Pediatrics, University of Alabama at Birmingham, Birmingham 35233; 2 Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294; and 3 Department of Pediatrics, University of Mississippi, Jackson, Mississippi 39216
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ABSTRACT |
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We investigated adenosine (Ado) activation
of the cystic fibrosis transmembrane conductance regulator (CFTR) in
vitro and in vivo. A2B Ado receptors were identified in
Calu-3, IB-3-1, COS-7, and primary human airway cells. Ado elevated
cAMP in Calu-3, IB-3-1, and COS-7 cells and activated protein kinase
A-dependent halide efflux in Calu-3 cells. Ado promoted arachidonic
acid release from Calu-3 cells, and phospholipase A2
(PLA2) inhibition blocked Ado-activated halide efflux in
Calu-3 and COS-7 cells expressing CFTR. Forskolin- and
2-adrenergic receptor-stimulated efflux were not
affected by the same treatment. Cytoplasmic PLA2
(cPLA2) was identified in Calu-3, IB-3-1, and COS-7 cells,
but cPLA2 inhibition did not affect Ado-stimulated cAMP
concentrations. In cftr(+) and cftr(
/
) mice,
Ado stimulated nasal Cl
secretion that was CFTR dependent
and sensitive to A2 receptor and PLA2 blockade.
In COS-7 cells transiently expressing
F508 CFTR, Ado activated
halide efflux. Ado also activated G551D CFTR-dependent halide efflux
when combined with arachidonic acid and phosphodiesterase inhibition.
In conclusion, PLA2 and protein kinase A both contribute to
A2 receptor activation of CFTR, and components of this
signaling pathway can augment wild-type and mutant CFTR activity.
cystic fibrosis transmembrane conductance regulator; airway epithelia; Calu-3 cells; chloride secretion; nasal potential difference; protein kinase A; phospholipase A2
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INTRODUCTION |
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THE CYSTIC
FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) is a
cAMP-regulated Cl channel and is responsible for
regulating ion transport in many tissues where it is expressed,
including the airways and airway submucosal glands, sweat glands,
gastrointestinal tract, pancreatic and bile ducts, and the reproductive
tract (58, 74). Defective CFTR function disrupts ion
transport, which is believed to contribute to the clinical sequelae
characteristic of cystic fibrosis (CF) (49, 58, 67, 74).
CFTR regulation in vivo is accomplished through ATP binding and
hydrolysis at nucleotide binding domain-1 and -2 and regulation of the
phosphorylation status of the regulatory domain (10, 11,
36). This process is complex and depends on cAMP production
through surface receptors that couple to adenylyl cyclase, cAMP
degradation by phosphodiesterase (PDE) activity (40, 41),
domain-domain interactions within the CFTR (55), protein-protein interactions between the CFTR and associated regulatory proteins (31, 32, 54, 64), and direct phosphorylation and
dephosphorylation of the regulatory domain by protein kinase A (PKA)
and phosphatases (6, 11), respectively. Receptor-based pathways that signal through cAMP are therefore relatively accessible candidates for promoting activation of the CFTR in vivo. This has been
established for
2-adrenergic receptors, which are
commonly stimulated in assays to detect CFTR-dependent Cl
secretion as measured by changes in the nasal potential difference (PD)
in mice and in humans (44).
Adenosine (Ado) is a Cl secretagogue that signals through
P1 purinergic receptors, and recent studies have
demonstrated that A2B Ado receptors (ARs) can tightly
couple to CFTR through PKA. In Calu-3 cells, part of this coupling is
due to compartmentalized signaling of CFTR with adenylyl cyclase and
PKAII through A kinase anchoring protein interactions (34,
71). T84 cells also express A2B-ARs, where they have
been shown to mediate neutrophil-stimulated Cl
secretion
(48, 70). Studies (2-4) have also
suggested a role for phospholipase A2 (PLA2) in
Ado-activated Cl
secretion in T84 cells. The nature of
this role and whether similar signaling contributes to CFTR activation
in other CFTR-expressing cells and tissues, however, are unknown.
In this report, we describe CFTR regulation by A2-ARs in vitro and in vivo and evaluate the contribution of cytoplasmic PLA2 (cPLA2) to A2 receptor signaling in airway cells and the murine airway. Our findings suggest that A2 receptors maximize CFTR activation by signaling through both adenylyl cyclase-PKA and cPLA2 and that components of these receptor signaling pathways can activate common disease-causing CFTR mutations.
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METHODS |
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Cell culture.
All cell lines were purchased from the American Type Culture Collection
(Manassas, VA). COS-7 cells were grown in DMEM plus 10% fetal bovine
serum (FBS) and 1% penicillin plus streptomycin. IB-3-1 cells were
grown in LHC medium plus 3% FBS and 1% penicillin plus streptomycin.
Calu-3 cells were grown in MEM plus 10% FBS and 1% penicillin plus
streptomycin supplemented with nonessential amino acids. To study
polarized Calu-3 cells at an air-liquid interface, polyester
Transwell-Clear Costar filters [0.4-µm pore diameter; 6-mm insert
diameter for arachidonic acid (AA) release and Ussing chamber
experiments and 24-mm insert diameter for immunocytochemistry experiments; Fisher Scientific, Pittsburgh, PA] were coated with human
placental collagen matrix (Becton Dickinson, Franklin Lakes, NJ) at a
concentration of 5 µg/cm2 overnight and then seeded at
~1 × 106 cells/cm2. Once the filters
were confluent (~1 wk), the medium was removed from the apical
surface and the cells were fed only on the basolateral surface. After
48 h, resistance was checked (~1,000-2,000 · cm
for the 6-mm filters and ~400-600
· cm for the 24-mm
filters), and at 72-96 h the cells were studied as described in
Transepithelial short-circuit currents. The primary
human nasal cells were isolated and grown as explant cultures on
Vectabond-treated glass coverslips as previously described
(15) and were studied <1 wk after being seeded.
Transient CFTR expression.
CFTR was transiently expressed in COS-7 cells with a
vaccinia-based expression system as previously described (16,
17). Cells grown on Vectabond-treated glass coverslips were
infected with vaccinia containing the T7 polymerase (generous gift of
Dr. B. Moss, National Institutes of Health, Bethesda, MD) at a
multiplicity of infection of 10 for 30 min. Wild-type (WT),
F508, or G551D CFTR under control of the T7 promoter in the pTM-1
vector was then introduced into the cells in complex with
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium (DOTAP)-propane-1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE; 20 µg DOTAP-DOPE and 5 µg pTM-1 CFTR/5 × 105 cells) for 4 h. These CFTR plasmids in the pTM-1
vector were the generous gift of Dr. S. Cheng (Genzyme, Cambridge, MA).
Cells were then washed in PBS, returned to DMEM plus 10% FBS, and
studied 18-24 h postinfection (for WT CFTR and G551D CFTR) or
after being grown at 29°C for 48 h (
F508 CFTR).
Fluorescence-based halide efflux measurements.
To study CFTR activation, we measured halide efflux in COS-7 cells
transiently expressing WT, F508, or G551D CFTR or in Calu-3 cells
with the halide-quenched dye
6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ; Molecular
Probes, Eugene, OR) as previously described (16, 17).
Briefly, COS-7 cells were seeded on Vectabond-treated glass coverslips,
and Calu-3 cells were seeded on Vectabond-treated coverslips coated
with 5 µg/cm2 of human placental extracellular matrix.
Cells were grown until ~80% confluent. Immediately before study, the
cells were hypotonically loaded with 10 mM SPQ for 10 min and placed in
a quenching NaI buffer. The cells were then placed in a specially
designed perfusion chamber and studied at 23°C. The fluorescence of
individual cells was measured with a Zeiss inverted microscope
(excitation 340 nm, emission >410 nm), a PTI imaging system, and a
Hamamatsu camera. Baseline fluorescence was measured in isotonic NaI
buffer (16, 17), and cells were then perfused with
isotonic dequench buffer (NaNO3 replaced NaI) at the
indicated time point (generally 200 s). The perfusate was then
switched to dequench buffer plus agonist and requenched at the end of
the experiment. Fluorescence was normalized for each cell to its
baseline value, and increases are shown as percent increase in
fluorescence above basal (quenched) fluorescence. Unless otherwise
specified, the means ± SE of all cells were included in the
curves shown.
Protein detection. To detect the A2B-AR, cells were lysed with radioimmunoprecipitation assay buffer (150 mM NaCl, 1% Nonidet P-40, 0.05% sodium deoxycholate, 0.1% SDS, 50 mM Tris-Cl, and 10 mg/ml of phenylmethylsulfonyl fluoride, pH 8.0). For the COS-7 cells, lysates were immunoprecipitated with an isoform-specific polyclonal rabbit anti-A2B-AR-antibody (Alpha Diagnostics, San Antonio, TX) raised against a 16-amino acid sequence corresponding to the third extracellular domain of human brain A2B-AR cDNA as previously described (17). Briefly, anti-A2B-AR antibody was linked to protein A agarose beads and incubated with cell lysates for 2 h. Precleared cell lysates (beads without primary antibody) were used as the negative control. The beads were then washed three times with PBS plus 0.1% Tween, and the immunoprecipitates were released with sample buffer incubated at 37°C for 10 min. For the Calu-3 and IB-3-1 cells, lysates were Western blotted without immunoprecipitation. Proteins were separated by SDS-PAGE with precast 12% gels (Novex gels; Invitrogen, Carlsbad, CA) and electrophoretically blotted onto polyvinylidene difluoride membranes. The membranes were then blocked with 1% BSA in PBS for 30 min, washed three times with PBS plus 0.1% Tween, and probed with anti-A2B-AR antibody (1:1,000 dilution) for 2 h. Negative control membranes were blotted with a fivefold excess (by weight) of A2B-AR-specific neutralizing peptide that was added with the primary antibody (Alpha Diagnostics). The membranes were washed three times and incubated with secondary antibody (1:1,000 dilution of goat anti-rabbit antibody conjugated to alkaline phosphatase; Southern Biotechnology Associates, Birmingham, AL) for 2 h. Membranes were washed three times and developed with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and 4-nitro blue tetrazolium chloride (NBT) in carbonate buffer (pH 9.8). Similar techniques were used to identify cPLA2 in cell lysates using an affinity-purified mouse monoclonal IgG2b antibody raised against the amino-terminal domain of the human cPLA2 cDNA (Santa Cruz Biotechnology, Santa Cruz, CA). This antibody was used to identify cPLA2 protein in cell lysate immunoprecipitates (Calu-3 and IB-3-1 cells; anti-cPLA2 linked to precleared protein G beads; Roche Diagnostics, Indianapolis, IN) or cell lysates alone (COS-7 cells). Proteins were separated by SDS-PAGE with 8% precast gels, and after being blotted onto polyvinylidene difluoride membranes, the membranes were blocked and washed as above and probed with primary antibody (1:1,000). The secondary antibody was a goat anti-mouse IgG alkaline phosphatase conjugate (1:1,000 dilution; Southern Biotechnology Associates), and blots were developed with NBT-BCIP as above.
Immunofluorescence studies. Nasal explant cultures from human surgical specimens were grown on Vectabond-treated coverslips coated with 5 µg/cm2 of human placental extracellular matrix as previously described (15). At 5 days of age, cells were fixed in 4% formaldehyde in PBS (20 min; pH 7.4), washed twice with PBS, and then permeabilized with 0.05% surfactant Triton X-100 (Pierce Endogen, Rockford, IL). The slides were then pretreated with 1% BSA in PBS to block nonspecific protein-binding sites. A2B-AR antigen was detected with the polyclonal rabbit anti-A2B-AR antibody (1:25 dilution) in 1% BSA for 1 h. Cells were then washed three times with PBS over 15 min and incubated with secondary antibody (goat anti-rabbit FITC conjugate, 1:100 dilution in 1% BSA) for 1 h. The cells were mounted with 4',6-diamidino-2-phenylindole Vectashield mounting fluid (Vector Laboratories). For the studies of A2B-AR localization in polarized Calu-3 cells, the A2B-AR antigen was identified with the filter fold technique as previously described (7). Briefly, high-resistance monolayers were fixed with 3% transmission electron microscopy-grade formaldehyde (Touismis, Rockville, MD) for 45 min at room temperature and stained without detergent permeabilization. The fixed monolayers were washed three times with PBS over 15 min, blocked with nonimmune goat serum for 30 min (1:25 dilution; Vector Laboratories), and incubated with primary rabbit anti-A2B-AR antibody (1:100 dilution in nonimmune goat serum) for 1 h at room temperature. Neutralizing peptide at five times (by weight) the concentration of primary antibody was used for negative control filters. Goat anti-rabbit Alexa fluorochromes (1:80 dilution; Molecular Probes) were used to identify the primary antibody. Nuclei were identified with Hoechst 33258 staining (20 µg/ml for 4 min). The filters were folded with the apical side exposed and mounted in 0.1% p-phenylenediamine (Sigma, St. Louis, MO) in PBS-glycerol (1:9 dilution). Digital confocal images were captured and analyzed with an Olympus IX70 inverted reflective fluorescent light microscope at 623 nm excitation with UplanAPO ×100 or U-APO/340 ×40 objectives, a Photometric Sensys digital camera, and IPLab Spectrum software supplemented with Power Microtome extension software (Signalytics, Fairfax, VA).
Transepithelial short-circuit currents.
Calu-3 cells grown as monolayers at an air-liquid interface were
mounted in modified Ussing chambers (Jim's Instruments, Iowa City, IA)
and initially bathed on both sides with identical Ringer solutions
containing (in mM) 115 NaCl, 25 NaHCO3, 2.4 KH2PO4, 1.24 K2HPO4,
1.2 CaCl2, 1.2 MgCl2, and 10 D-glucose (pH 7.4). Bath solutions were vigorously stirred
and gassed with 5% CO2. Solutions and chambers were
maintained at 37°C. Short-circuit current
(Isc) measurements were obtained with an
epithelial voltage clamp (University of Iowa Bioengineering, Iowa City,
IA). A 3-mV pulse of 1 s duration was imposed every 100 s to
monitor resistance, which was calculated with Ohm's law. To measure
stimulated Isc, the mucosal bathing solution was
changed to a low Cl solution containing (in mM) 1.2 NaCl,
115 sodium gluconate, and all other components as above plus 100 µM
amiloride. Increasing concentrations of Ado were added to the mucosal
or serosal bathing solutions (8 min of observation at each Ado
concentration). After cells were stimulated with 100 µM Ado, 200 µM
glibenclamide was added to the mucosal bathing solution, effectively
blocking the stimulated Isc (>90%).
Murine nasal PD measurements.
Cftr(+) and cftr(/
) mice (CFTRunc
mice, C57BL6J genetic background) were studied by a conventional nasal
PD protocol (29, 69). The cftr(
/
) mice
carried two copies of the human cftr cDNA, which contains a
stop codon at position 489 (S489X). Cftr(+) mice included
cftr(+/+) and cftr(+/
) mice, which have been
shown to have similar nasal ion transport characteristics
(42). Genotypes were verified by PCR and dental enamel
characteristics. Both male and female mice ~16-40 wk of age were
studied. Mice were anesthetized with a cocktail consisting of ketamine
(100 mg/ml, 82.5 µl), acepromazine (10 mg/ml, 30 µl), and xylazine
(100 mg/ml, 15 µl) administered by intraperitoneal injection (0.1 ml/g body wt). The mouse tail was gently abraded, placed in lactated
Ringer solution, and connected through a calomel cell to a
high-impedance voltage follower (VF-1; World Precision Instruments,
Sarasota, FL). An exploring bridge was established by connecting a
Ag-AgCl electrode (wire) bridge to a syringe that pumped solutions at a
rate of 180 µl/h. After ~5 min, mice were appropriately somnolent
to permit cannulation of the nostril with a PE-10 cannula pulled to a
tip diameter of ~0.15 mm. The solutions perfused included Ringer
lactate plus amiloride (100 µM; solution 1); a low
Cl
concentration ([Cl
]) solution
containing 2.4 mM K2HPO4, 0.4 mM
KH2PO4, 115 mM sodium gluconate, 25 mM
NaHCO3, 1.24 mM calcium gluconate2, and 100 µM amiloride (solution 2); and solution 3 (solution 2 plus agonist as described in text). Each
superperfused condition was studied for 6 min (total of ~18 min per
protocol per mouse).
AA release. Calu-3 cells, grown to confluence on 35-mm culture dishes coated with 5 µg/ml of human placental collagen or as high-resistance monolayers at an air-liquid interface were washed in PBS and loaded with 1 µCi/ml of [3H]AA overnight (Moravek Biochemical, La Brea, CA). The plates and filters were then washed five times with PBS and placed in MEM plus 10% FBS with and without 100 µM Ado (750-µl volume for the 35-mm plates; 150-µl apical volume and 300-µl basolateral volume for the cells on filters). AA release was quantified for 20 min (cells on plates) and for 20 and 40 min (cells on filters). The cells were lysed (1 N NaOH), and the effluxed AA from each plate was quantified by scintillation counting and normalized to the percent of total number of counts in a manner similar to that previously described (13).
cAMP measurements. Cellular cAMP was measured with an ELISA-based detection kit as previously described (17) (Cayman Chemical, Ann Arbor, MI). Briefly, cells grown on 35-mm dishes (~7 × 106 cells/dish) were stimulated with agonist for 10 min, and the cellular cAMP was extracted with ice-cold ethanol. The supernatants were vacuum dried and resuspended in phosphate buffer, and the cAMP levels were quantified per the manufacturer's directions. For all experiments, papaverine (100 µM; nonspecific nonxanthine PDE inhibitor) was included to improve cAMP detection. Xanthine-based inhibitors were avoided because these commonly interact with Ado receptors (39).
Materials. Ado hemisodium salt, chlorpromazine (CPZ), H-89 Cl, forskolin, zaprinast, and AA were purchased from Calbiochem (San Diego, CA); albuterol (Alb; salbutamol), theophylline, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), 8-phenyltheophylline, rolipram, and milrinone were purchased from Sigma; papaverine HCl was purchased from Research Biochemicals International (Natick, MA).
Statistics.
Descriptive statistics (means ± SE) and tests of statistical
significance were performed with SigmaStat software (Jandel, CA).
Paired and unpaired t-tests were used for samples with
continuous data (cAMP levels, AA levels, stimulated PD measurements,
and Isc), and 2 analysis was used
to compare the number of
F508 CFTR-expressing cells responding to
different agonists (Ado, forskolin, DPCPX, and control). An
-level
of 0.05 was considered statistically significant.
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RESULTS |
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Human airway cells express A2B-ARs.
With a polyclonal anti-A2B-AR antibody, Calu-3 and IB-3-1
cell lysates were probed for A2B receptor expression
through Western blotting. As a positive control, COS-7 cells, which our
laboratory previously demonstrated to endogenously express
A2B-ARs (17), were also evaluated.
Figure 1 shows that in all three cell
types, a specific ~40-kDa band was identified. Using the same
anti-A2B-AR antibody, we investigated
A2B receptor localization in primary human nasal airway
cells (Fig. 2A) and polarized
Calu-3 cells (an airway cell line with a serous phenotype and high
levels of CFTR expression; Fig. 2B) (37, 53).
In both cell types, the antibody detected plasma membrane-localized
A2B-AR. Staining was predominantly along the apical surface
in Calu-3 cells. Functional studies in Ussing chambers demonstrated
that Ado added to either the apical or basolateral surface briskly
activated Isc (Fig. 2C,
left). Ado was a more potent stimulus when added to the
apical membrane than to the basolateral membrane, and
Isc stimulated from either membrane was
sensitive to apical glibenclamide blockade (~90% inhibition of
stimulated Isc produced by 100 µM Ado; P
< 0.05). Although the range of concentrations capable of
activating Isc was most consistent with
A2 receptor stimulation, these experiments do not exclude
the possibility that additional AR subtypes may contribute to
Ado-stimulated Isc. Because
HCO
transport, may contribute to Isc in Calu-3
monolayers (22, 66), further functional studies of CFTR
activity were performed with an SPQ-based halide efflux assay
(15-17).
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Ado stimulates PLA2, adenylyl cyclase, and CFTR
activation.
In previous studies of CFTR regulation by A2B receptors in
COS-7 cells, our laboratory (17) showed that
A2B receptor signaling was a potent stimulus, accomplishing
strong activation of CFTR despite only modest effects on cellular
[cAMP] compared with forskolin. These results were similar to those
reported by Barrett and colleagues (3, 4) in T84 cells. In
subsequent studies by Barrett and Bigby (2), Ado-activated
Isc in T84 monolayers was found to be associated
with AA mobilization and sensitivity to PLA2 inhibition. To
determine whether PLA2 signaling might contribute to
A2 receptor activation of CFTR in airway and COS-7 cells,
we evaluated cells for cPLA2 expression and
cPLA2 activity after Ado stimulation. Figure
3 shows that in Calu-3, IB-3-1, and COS-7
cell lysates, a monoclonal anti-cPLA2 antibody detected an
~110-kDa protein consistent with cPLA2.
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Ado activation of CFTR-dependent Cl transport
in vivo.
The results given in Ado stimulates PLA2,
adenylyl cyclase, and CFTR activation provide a framework in
which to investigate Ado-activated Cl
transport in vivo.
For these studies, we investigated ion transport across the murine
nasal mucosa using the nasal PD, a well-established bioelectric assay
of CFTR activity in vivo. Figure 7 shows
examples of nasal PD tracings from a cftr(+) (Fig.
7A) and a cftr(
/
) (Fig. 7B) mouse.
In the cftr(+) mouse, Ado stimulated further hyperpolarization (Fig. 7A, arrow #3), consistent with
Cl
conductance. In the cftr(
/
) mouse,
depolarization continued during perfusion with a low
[Cl
] solution and a low [Cl
] solution
plus Ado (500 µM). Figure 8A
summarizes a comparison of Ado (100 µM)-, Alb (100 µM)-,
isoproterenol (Iso; 100 µM)-, and forskolin (10 µM)-stimulated
Cl
secretion in cftr(+) mice. Ado was a potent
Cl
secretagogue, producing further polarization in 10 of
12 mice studied (P < 0.005 compared with the
no-agonist or Iso control mice). Alb was also a strong agonist (similar
to forskolin), producing further polarization in six of eight mice
studied (P < 0.05 for Alb and forskolin compared with
Iso and control animals). In contrast, Iso was less predictable,
producing further hyperpolarization in only 5 of 14 mice. Both AR
stimulation with Ado (500 µM) and
2-receptor
stimulation with the more specific
2-receptor agonist Alb (500 µM) failed to activate Cl
conductance in
cftr(
/
) mice (Fig. 8B). These studies confirm that both receptors stimulate CFTR-dependent Cl
transport. Ado-stimulated Cl
conductance in
cftr(+) mice was sensitive to A2 receptor
blockade with 8-phenyltheophylline (P < 0.02),
indicating that Ado activates Cl
conductance
through A2 receptor signaling (Fig. 8C).
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A2B-ARs activate F508 CFTR and G551D CFTR.
The results discussed in Ado activation of CFTR-dependent
Cl
transport in vivo show that Ado and
A2 receptor signaling potently activate CFTR-dependent
Cl
transport in vitro and in vivo. To determine whether
A2B receptor signaling could be used to improve the
activity of common disease-causing CFTR mutations, we transiently
expressed
F508 CFTR (class II mutation) and G551D CFTR
(class III mutation) in COS-7 cells. Figure
10 shows that after localization to the
cell membrane (growth at 29°C for 48 h),
F508 CFTR was
activated by A2B receptor stimulation (10 µM Ado) in a
fashion similar to direct adenylyl cyclase activation with forskolin
(20 µM). Halide efflux was stronger than that produced by stimulation
with DPCPX, an agent that can acutely stimulate
F508 CFTR-dependent
halide efflux in
F508 CFTR-expressing cells [and is currently in
clinical trials as an activator of
F508 CFTR in CF patients
(24, 30); P < 0.001 comparing the
proportion of responding cells stimulated by Ado with DPCPX]. We did
not test the ability of A2B receptor stimulation to
activate
F508 CFTR after prolonged treatment with DPCPX. Activation
of
F508 CFTR was accomplished despite only modest effects of Ado on
[cAMP] compared with forskolin (17).
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DISCUSSION |
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In this report, we investigated the cellular pathways utilized by
Ado to activate CFTR-dependent ion transport. Using a series of
experimental systems, including single cells, polarized cell monolayers, and in vivo measurements in cftr(+) and
cftr(/
) mice, we demonstrated that Ado, through
A2-ARs, activates CFTR-dependent halide transport. These
receptors are expressed in human airway cells and signal in part
through cAMP and PKA. We also demonstrated the dependence of this
signaling pathway on PLA2 activity in vitro and in vivo.
Finally, we showed that the two most common disease-associated CFTR
mutations can be activated by A2B receptors alone (
F508 CFTR) or by using messenger pathways stimulated by A2B
receptors (G551D CFTR). These results help to establish the
physiological role of Ado-stimulated Cl
secretion in vivo
and may identify new targets for improving the function of mutant CFTR molecules.
Ado is a ubiquitous signaling molecule, serving protean functions
ranging from regulation of neurotransmission to cardiovascular tone and
inflammation in many organ systems. Our results, together with those of
other investigators, indicate an important role for Ado as a regulator
of CFTR. Ado and A2 receptors are potent activators of
Cl secretion in primary human airway cell monolayers, in
addition to canine and gerbil airway cells and T84 intestinal cells
(28, 46, 59, 70). In each of these systems,
Cl
secretion has been accomplished in the setting of low
cAMP levels. Part of this high efficiency is due to
compartmentalization of A2B receptors, transduction
proteins, and CFTR (34, 71). Our results suggest that in
addition to spatial compartmentalization, efficient transduction
between A2 receptors and CFTR involves cPLA2
activation and AA signaling. A2 receptors, including
A2A and A2B subtypes, classically signal
through stimulation of adenylyl cyclase, cAMP, and, ultimately, PKA
(56). Although A2A receptors appear to hold
more strictly to this observation, A2B receptors have been
shown to stimulate additional signaling pathways such as phospholipase
C, direct regulation of calcium channels, and, in our experience,
PLA2 (26). These observations are of
particular interest because recent studies (27) have
demonstrated abnormal lipid profiles, including elevated AA
levels, in murine CF tissues. Our findings demonstrate that lipid
signaling may be important to the regulation of CFTR activity by Ado in
vitro and in vivo. The present results therefore set the stage for
future studies designed to test the relationship between membrane lipid
composition, A2 receptors, and CFTR activity.
There are two general classes of PLA2 enzymes:
cPLA2 (three subtypes) and secretory PLA2
(seven subtypes) (33). Both are expressed in many tissues
including the lung and leukocytes. cPLA2 translocates to
cell membranes after activation and primarily releases AA from membrane
lipids. cPLA2 has been shown to play an important
immunologic role in human airway epithelial cells, governing AA release
from epithelia after immunostimulation (76). Studies
(50) have also observed dysregulation of PLA2
in the CF airway, with increased AA release seen after bradykinin
exposure in F508 CFTR-expressing airway cells compared with normal
control cells. AA is the parent molecule of two important inflammatory signaling cascades including 1) the cyclooxygenase (COX)
pathway, which leads to the production of prostanoids and thromboxanes and 2) the lipoxygenase pathway in which AA is metabolized
to 5-hydroperoxyeicosatetraenoic acid, 5-hydroxyeicosatetraenoic acid,
and the leukotrienes A4-E4. Clinical
studies suggest that products of AA metabolism, including
proinflammatory species, have direct relevance to CF-related
inflammation. The COX-1 inhibitor ibuprofen is routinely used in the
pediatric CF population to reduce lung inflammation and slow the
progression of CF lung disease (45). Glucocorticoids,
which suppress COX-2 expression, have also been studied in CF clinical
trials, attenuating the decline in pulmonary function in pediatric CF
patients (25). The relevance of AA signaling pathways to
CFTR activity and ion transport, however, has not been previously
demonstrated. Our results suggest that a buildup in AA may have
beneficial effects on CFTR activity in the context of its activation by
surface receptors.
The mechanism by which A2 receptors activate CFTR-dependent
Cl transport is complex. CFTR activation by
A2 receptors could be blocked by either PKA or
cPLA2 inhibition (Fig. 5). Production of cAMP was not
reduced by PLA2 inhibition in any of the three cell lines
(Fig. 6). Although 10 min of high-dose (100 µM) AA exposure could
stimulate some cAMP production in the two airway cell lines (primarily
Calu-3 cells, likely through the actions of distal metabolites such as
PGI, PGE2, PGF2
or PGD) (73), it failed to augment cAMP production by Ado or acutely activate CFTR
when used alone (COS-7 or Calu-3 cells; Fig. 5). These results suggest
that the additional effects of AA on CFTR-dependent halide transport
were not through influences on total cellular cAMP. Rather,
A2 receptors appear to activate both adenylyl cyclase and
cPLA2 in parallel, with each signaling pathway contributing to the maximization of CFTR activity. PLA2 signaling was
not necessary to activate CFTR through receptors, however, because
2-adrenergic stimulation activated CFTR-dependent
Cl
transport in vitro and in vivo despite
cPLA2 inhibition (Figs. 5 and 9).
Figure 12 is a summary model that
provides three possible mechanisms by which cPLA2 and AA
could contribute to A2 receptor activation of CFTR. It is
possible that Ado and/or AA exerts its effects in part by activating
K+ channels and increasing the driving force for halide
efflux. AA has been shown to have both stimulatory and inhibitory
effects on epithelial K+ channels, and modulation of
basolateral K+ channel activity can strongly influence
transepithelial Cl transport (20, 21). In
related experiments, Ado-activated halide efflux in CFTR-expressing
COS-7 cells was not reduced by two K+ channel blockers,
including barium and tetraethylammonium (10 mM each) (18,
19). These results suggest that the stimulatory effects of Ado
on CFTR seen in our studies were independent of K+ channel
activation. The observation that AA augmented the stimulation of CFTR
by Ado and forskolin (Figs. 5 and 11), but failed to stimulate CFTR
alone in vitro (Fig. 5), suggests that AA may exert a permissive effect
on CFTR activation. Membrane-derived fatty acids, including AA and its
metabolites, have been demonstrated to interact with ion channels and
influence their activation by other signaling molecules, including
nucleotide sensitivity of ATP-activated K+ channels
(5, 65), light-sensitive transient receptor potential, and
transient receptor potential-like channels (14),
small Cl
channels in rabbit parietal cells
(61), the nicotinic acetylcholine receptor
(9), and calcium channels as part of oscillating calcium currents (51). AA does interact with CFTR and alter its
Cl
channel activity. Cytoplasmic AA produces a flickery
block of CFTR in inside-out membrane patches after heterologous
expression (1, 35, 47). Membrane lipids (including AA) can
have differential effects on the function of other channels,
stimulating channel activity on one surface and inhibiting channel
activity on the other (52). We speculate that AA or one of
its metabolites may interact with an external portion of the channel or
with an unidentified regulatory factor that secondarily influences CFTR
Cl
channel function. Alternatively, products of AA
metabolism could accelerate local cAMP production and, in this way,
increase CFTR channel activity. This process would be difficult to
detect with total cellular assays to quantitate [cAMP]. The effect
would need to be quite dramatic, however, because G551D CFTR, which has
previously been shown to be poorly responsive to powerful
phosphorylating stimuli, appears to be activated by costimulation with
AA (Fig. 11B, Refs. 38, 58, and 77). This
occurs despite no detectable change in [cAMP] when combined with Ado
(Fig. 6A) but is most pronounced when combined with PDE
inhibition. Single-channel-based studies will be necessary to help
clarify the mechanism by which cPLA2 and AA promote CFTR
activity. Finally, our results do not exclude the possibility that
additional processes may be involved because CPZ has been shown to have
effects on other cell signaling pathways in addition to
PLA2 (8, 12, 23, 43, 57).
|
The results shown in Figs. 7-9 indicate that Ado is a potent
Cl secretagogue in vivo, activating CFTR-dependent
Cl
transport across the murine nasal mucosa more
predictably than Iso (when studied at 100 µM). This is of relevance
to human nasal PD protocols, which typically use
2-receptor stimulation with Iso to aid in the detection
of CFTR-dependent Cl
transport. Our results suggest that
comparisons of these two receptor pathways in human subjects may
improve the sensitivity of the nasal PD, which could have implications
for clinical studies that use this assay to measure low-level CFTR
function. The importance of PLA2 signaling to Ado
activation of Cl
transport was also demonstrated in vivo
because Ado- but not Alb-stimulated Cl
conductance was
sensitive to PLA2 inhibition.
A2 receptor signaling may also be useful in improving the
activity of disease-related CFTR mutations. Using an in vitro system, we have demonstrated that CFTR mutations from three subclasses (classes
II, III, and IV) can be activated by A2B receptors
(17) (Figs. 10 and 11). Although the expression levels of
the mutant CFTRs were far higher than those found in vivo, the results
shown in Figs. 10 and 11 support the notion that mutant CFTRs, when
localized to the cell membrane, can be activated by endogenous
receptor-based signaling pathways. Current approaches to increase
mutant CFTR activity, including improvement of CFTR biosynthesis
[e.g., suppression of premature stop mutations with aminoglycoside
antibiotics (7, 15, 75)], increase in trafficking of
F508 CFTR to the cell membrane [e.g., treatment with butyrate
compounds (60)], or treatment of surface mutant CFTRs
with activating compounds [e.g., genistein or PDE inhibitors
(38, 68)] may be complemented by the use of
A2 receptor signaling pathways. Our findings also suggest
that common mutant CFTRs should be at least partially responsive to
this signaling pathway when available at the cell membrane.
In summary, our studies demonstrate that A2B-ARs are
expressed in CF and normal airway cells, localizing predominantly to the apical membrane of polarized Calu-3 cells. A2 receptors
mediate activation of CFTR-dependent Cl transport in
vitro and in vivo, utilizing cPLA2 and AA in addition to
cAMP and PKA. Ado and A2-ARs compare favorably with other
agents as activators of CFTR-dependent Cl
conductance,
stimulating Cl
secretion better than the
2-adrenergic receptor agonist Iso in mice and activating
F508 CFTR similar to forskolin in vitro. Our studies therefore
provide a rationale for the investigation of the effects of Ado and
A2 receptor signaling on measured Cl
secretion in humans.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank the University of Alabama at Birmingham (UAB) Adult and Pediatric Ear, Nose, and Throat Departments for providing remnant human tissue for immunocytochemical studies; Dr. Zsuzsa Bebok (Department of Internal Medicine and UAB Cystic Fibrosis Research Center) and Albert Tousson (UAB Imaging Facilities Core) for providing guidance with the immunocytochemistry experiments; and Dr. Kevin Kirk for helpful discussions regarding the manuscript.
![]() |
FOOTNOTES |
---|
This work was supported by Cystic Fibrosis Foundation Grants CLANCY96LO and R464 and National Institutes of Health Grants R01-HL-67088-01, P50-DK-53090, and P30-DK-54781.
J. P. Clancy is a Cystic Fibrosis Foundation Leroy Matthews Award recipient.
Address for reprint requests and other correspondence: J. P. Clancy, Dept. of Pediatrics and Gregory Fleming James Cystic Fibrosis Research Center, 1600 7th Ave. South, Ste. 620 ACC, Birmingham, AL 35233 (E-mail: jclancy{at}peds.uab.edu).
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 10 May 2001; accepted in final form 12 September 2001.
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