A2 adenosine receptors regulate CFTR through PKA and PLA2

B. R. Cobb1,2, F. Ruiz3, C. M. King2, J. Fortenberry2, H. Greer2, T. Kovacs2, E. J. Sorscher2,4, and J. P. Clancy2,5

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta 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 Delta 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta 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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Omega  · cm for the 6-mm filters and ~400-600 Omega  · 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), Delta 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 (Delta F508 CFTR).

Fluorescence-based halide efflux measurements. To study CFTR activation, we measured halide efflux in COS-7 cells transiently expressing WT, Delta 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 chi 2 analysis was used to compare the number of Delta F508 CFTR-expressing cells responding to different agonists (Ado, forskolin, DPCPX, and control). An alpha -level of 0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP> transport, in addition to Cl- 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).


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 1.   Identification of A2B adenosine (Ado) receptors (ARs). Characteristic 40-kDa band was identified (+) in 3 cell types. MW, molecular mass. Negative controls (-) were precleared beads with cell lysates (COS-7 cell immunoprecipitates, no primary antibody conjugated to beads) or with addition of neutralizing peptide during Western blotting (Calu-3 and IB-3-1 cells, 5-fold excess of neutralizing peptide added during incubation with primary antibody).



View larger version (39K):
[in this window]
[in a new window]
 
Fig. 2.   Immunofluorescence of primary human bronchial epithelial cells and Calu-3 cell monolayers studied with immunofluorescence (A and B, respectively) and in Ussing chambers (C). Cells were prepared and studied as described in METHODS. A, left: A2B-AR antigen is identified with a membrane-staining pattern (green). A, right: negative control cultures (5-fold excess of neutralizing peptide during incubation with primary antibody) eliminated membrane staining. B, top: A2B-AR antigen in Calu-3 monolayers (green) shows predominantly apical membrane staining. B, bottom: addition of neutralizing peptide (as in A) to negative control cultures eliminated membrane staining. C: effects of apical and basolateral addition of Ado on short-circuit current (Isc) in Calu-3 cells. Cells were grown at an air-liquid interface and studied in Ussing chambers. C, left: representative Isc tracings. Top: Calu-3 cells were initially cultured in lactated Ringer solution followed by (from left to right) 1) mucosal low-Cl- concentration ([Cl-]) buffer + amiloride (100 µM), 2) addition of mucosal 1 µM Ado, 3) addition of basolateral 1 µM Ado, and 4) blockade with mucosal 200 µM glibenclamide. Bottom: same experiment as above, except basolateral Ado (1 µM; 2) was added before mucosal Ado (1 µM; 3). Right: Ado was a strong stimulus when added to either membrane. Apical: P = 0.002 for 0.1 µM compared with 1.0 µM and 1.0 µM compared with 10 µM. Basolateral: P < 0.03 for 0.10 µM compared with 1.0 µM; P < 0.001 for 1.0 µM compared with 10 µM; and P < 0.001 for 10 µM compared with 100 µM. Apical-stimulated Isc was greater than basolateral-stimulated Isc for 0.1 and 10 µM (*P < 0.02) and 1.0 µM (dagger P < 0.001). Approximately 90% of the stimulated Isc (100 µM, either membrane) was blocked by glibenclamide (200 µM added to the mucosal compartment; data not shown).

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.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 3.   Identification of cytoplasmic phospholipase A2 (cPLA2) in 3 cell types. Cell lysates were immunoprecipitated with anti-cPLA2 antibody (Calu-3 and IB-3-1 cells) and separated by SDS-PAGE. cPLA2 antigen (~110 kDa) was detected with monoclonal anti-cPLA2 antibody (arrow). Control conditions were precleared beads from cell lysates without primary antibody (Calu-3 and IB-3-1 cells). COS-7 cell lysates were separated by SDS-PAGE and Western blotted with anti-cPLA2 antibody. Controls received no primary antibody during blotting.

Activated PLA2 releases AA from the sn-2 position of membrane phospholipids. To determine whether Ado activated PLA2 and stimulated the release of AA in airway cells that express both A2B receptors and CFTR, Calu-3 cells were grown on plastic and on permeable supports at an air-liquid interface, loaded with [3H]AA, and exposed to Ado (100 µM; Fig. 4). Ado stimulated AA release from Calu-3 cells grown in either condition. AA release is expressed as percent of counts released (basolateral or apical) over total counts; P <=  0.025 (Fig. 4, A and B). AA was released preferentially from the apical compartment, but a detectable amount (approximately [1/10] of apical release) was released from the basolateral surface (Fig. 4C). Studies of the effects of cPLA2 inhibition (with CPZ) or PKA inhibition (with H-89 Cl) on Ado-stimulated AA release were complicated by a mild (approximately twofold) increase in nonspecific AA release from Calu-3 cells after treatment with either inhibitor. Cell viability after treatment with either compound was preserved, however, based on cytotoxicity assay studies (data not shown), SPQ retention and response (Fig. 5), and cAMP production (Fig. 6).


View larger version (9K):
[in this window]
[in a new window]
 
Fig. 4.   Ado stimulates arachidonic acid (AA) release from Calu-3 cells. Cells were loaded with [3H]AA overnight, washed 5 times, and then studied. Values are means ± SE. A: AA release from cells grown on 35-mm plastic dishes stimulated with Ado (100 µM) for 20 min compared with cells in unstimulated dishes (n = 10 dishes/condition). *P < 0.025. B: apical release of AA from Calu-3 cells grown on permeable supports at an air-liquid interface and stimulated with 100 µM Ado compared with cells on unstimulated filters (n = 6 filters/condition). Cells were stimulated for two 20-min time points. Release over the second 20 min is shown. For the entire 40-min period, Ado stimulated AA release by ~20% over the unstimulated condition (P = 0.06). *P = 0.025. C: basolateral release of AA for same 20-min time point. Total no. of counts (released to medium and retained in cells) for the stimulated and unstimulated conditions was not different for cells grown on dishes (A) or on filters (B and C). P = 0.08 for Ado-stimulated compared with control cells. P = 0.06 for entire 40-min time period.



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 5.   Ado activation of halide efflux in cystic fibrosis transmembrane conductance regulator (CFTR)-expressing COS-7 cells (A and B) and Calu-3 cells (C-E). Values are means ± SE in %increase in fluorescence over basal (NaI-quenched fluorescence); n > 40 cells/condition. A: COS-7 cells expressing CFTR were stimulated with Ado (arrows), with activation seen at the higher concentration. Same experiment was performed in the presence of 25 µM AA, from 200 s, with shift of CFTR activation to 0.2 µM Ado. Treatment of cells with chlorpromazine (CPZ; 50 µM) from 200 s blocked CFTR activation by 2.0 µM Ado. AA at 25 and 100 µM (arrows) failed to activate halide efflux. B: COS-7 cells expressing CFTR were stimulated with forskolin (Forsk) alone (20 µM; arrow) or with addition of 25 µM AA (200 s) to forskolin stimulation of CFTR (20 µM). Treatment with CPZ (50 µM) had no effect on forskolin activation. T7 controls (no CFTR) stimulated with AA (25 µM at 200 s) and forskolin (20 µM, arrow) failed to activate halide efflux. C: Calu-3 cells stimulated with Ado (2 µM). Treatment with CPZ (50 µM from 200 s) or H-89 (5 µM for 4 h) blocked Ado-activated halide efflux. AA alone (100 µM; arrow) failed to activate halide efflux. D: Calu-3 cells stimulated with albuterol (Alb; 0.2 µM) activated halide efflux. Treatment with CPZ (50 µM from 200 s) had no effect on Alb activation, whereas treatment with H-89 Cl (5 µM for 4 h) blocked activation of halide efflux. E: Calu-3 cells treated with CPZ (50 µM) from 200 s. High-concentration Ado (100 µM) partially overcame CPZ blockade.



View larger version (40K):
[in this window]
[in a new window]
 
Fig. 6.   Ado stimulates cAMP production in COS-7, IB-3-1, and Calu-3 cells. cAMP levels were measured by ELISA as described (see METHODS). Cells were stimulated for 10 min before extraction, n = 4-12 cultures/condition. Concentrations were 10 µM Ado, 50 µM CPZ, and 100 µM AA. Papaverine (100 µM) was included in all conditions to enhance cAMP detection as previously described (17). Values are means ± SE for each condition. A: COS-7 cells. *P < 0.001 compared with control (100 µM papaverine). B: IB-3-1 cells. *P < 0.02 compared with control. C: Calu-3 cells. *P < 0.001 compared with control. dagger P < 0.02 compared with AA alone. D: Calu-3 cells cAMP dose-response curve. Ado (0.1, 1.0, and 10 µM) stimulation of cAMP production was not inhibited by treatment with 100 µM CPZ. *P < 0.02 for Ado alone compared with papaverine control (100 µM). dagger P < 0.01 for Ado + CPZ compared with papaverine control.

In addition to investigating CFTR activity with Isc measurements, studies were performed with SPQ, an established assay of CFTR activity and a specific assay of halide transport. SPQ-based studies also allowed comparison of A2 receptor-stimulated halide transport in Calu-3 cells with that in COS-7 cells (which fail to polarize). These cells represent a simple cell type used to study A2B receptor regulation of WT and mutant CFTRs because they express A2B receptors but not other purinergic receptors commonly found in airway cells and other complex epithelial cells (17).

Ado-stimulated halide efflux in COS-7 cells expressing CFTR is shown in Fig. 5, A and B. Ado alone activated halide efflux at 2 µM but not at 0.2 µM, consistent with signaling through lower-affinity A2 receptors rather than A1 receptors (Fig. 5A). Treatment of cells with the PLA2 inhibitor CPZ (50 µM) blocked Ado-activated efflux. In contrast, addition of the PLA2 product AA (25 µM) augmented the Ado stimulus, shifting activation of halide efflux to 0.2 µM. AA alone (25 and 100 µM), however, failed to activate halide efflux in CFTR-expressing COS-7 cells. These results suggest that cPLA2 signaling is part of the pathway by which A2 receptors activate CFTR in this model system. The cPLA2 product AA when used alone, however, was insufficient and unable to substitute for Ado. The results therefore suggest that for A2 receptors to activate CFTR, additional signaling pathways need to be stimulated. In control experiments (Fig. 5B), forskolin-activated halide efflux in COS-7 cells (+ CFTR) was not inhibited by CPZ.

To test whether these observations applied to airway cells that express both CFTR and A2B receptors, halide efflux experiments were performed in Calu-3 cells. Ado-activated halide efflux was blocked by CPZ (50 µM), whereas AA alone failed to activate efflux. High concentrations of Ado (100 µM) were able to partially overcome the CPZ blockade (Fig. 5E). The Ado response was also blocked by the PKA inhibitor H-89 Cl (5 µM), indicating that Ado also utilizes adenylyl cyclase and cAMP to activate halide efflux. In control experiments, beta 2-receptor activation of halide efflux with Alb was sensitive to H-89 Cl but not to cPLA2 inhibition with CPZ (Fig. 5D). These results are consistent with those in the COS-7 cells and indicate that distinct differences exist in the mechanisms by which A2 receptors and beta 2-receptors activate CFTR and halide transport in these in vitro systems.

A2-ARs traditionally couple to adenylyl cyclase through Gs and elevate cellular cAMP, and previously, our laboratory (17) has shown that A2B receptor stimulation in COS-7 cells increases cAMP in a dose-dependent manner (17). Whether A2 receptor stimulation of cAMP involves PLA2 signaling, however, is not known. Figure 6 shows that in COS-7, IB-3-1, and Calu-3 cells, Ado (10 µM) increased cAMP. Treatment of cells with the PLA2 inhibitor CPZ (50 µM) had no effect on Ado-stimulated cAMP production in all three cell lines, yet completely abolished Ado-activated halide efflux in both COS-7 and Calu-3 cells (Fig. 5). Production of cAMP by lower doses of Ado (0.1 and 1 µM) was also not inhibited by CPZ treatment (Calu-3 cells; Fig. 6D). These results confirm that A2 receptors do couple to adenylyl cyclase and elevate cAMP but also show that the inhibitory effect that CPZ exerts on Ado stimulation of CFTR is independent of cAMP. Rather, the results shown in Figs. 4-6 suggest that Ado stimulation leads to both cPLA2 and PKA activation, with each pathway required for the stimulation of CFTR.

AA alone (100 µM) had variable effects on [cAMP], stimulating some cAMP production in airway cells (P < 0.001 for the Calu-3 cells compared with control conditions) but not in COS-7 cells. The cAMP produced by high-dose AA appeared to contribute little to CFTR activation. AA alone was insufficient to acutely activate halide efflux in either Calu-3 cells or COS-7 cells expressing CFTR (Fig. 5), and AA had no additive effect on [cAMP] when combined with Ado stimulation in any of the three cell types. This was in contrast to the functional results, such as those shown in Fig. 5A, in which the addition of AA to Ado stimulation appeared to augment CFTR-dependent halide efflux.

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 beta 2-receptor stimulation with the more specific beta 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).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 7.   Examples of nasal potential differences (PDs) in cftr(+) and cftr(-/-) mice. Mice underwent a standard nasal PD protocol as described in METHODS. Solution 1, lactated Ringer + 100 µM amiloride; solution 2, low [Cl-] solution + 100 µM amiloride; solution 3, solution 2 + agonist. Upward deflections are hyperpolarization (lumen negative), conventionally taken to represent Cl- secretion. A: cftr(+) mouse with 100 µM Ado included in solution 3. B: cftr(-/-) mouse with 500 µM Ado included in solution 3. A depolarizing phenotype throughout the entire protocol is seen.



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 8.   Stimulated Cl- transport in cftr(+) (A and C) and cftr(-/-) (B) mice. Change in PD after the switch to solution 3 is shown. A: cftr(+) mice stimulated with Ado (100 µM; n = 12), Alb (100 µM; n = 8), Forsk (10 µM; n = 10), isoproterenol (Iso; 100 µM; n = 14), and low [Cl-] control (no agonist; n = 18) included in solution 3 are shown. *P < 0.005 for Ado compared with ISO or control. dagger P < 0.025 for FORSK compared with ISO or control; Dagger P < 0.05 for Alb compared with ISO or control. B: cftr(-/-) mice stimulated with Ado (500 µM; n = 10) or Alb (500 µM; n = 10). A depolarizing phenotype is seen with both agonists that is similar to that of cftr(-/-) control mice (low-[Cl-] alone in solution 3; n = 14). C: cftr(+) mice stimulated with 50 µM Ado in solution 3 (n = 8) and 50 µM Ado (solution 3) + 100 µM 8-phenyltheophylline (8-PT) in solutions 2 and 3 (n = 10). *P < 0.02 for change in PD after stimulation with solution 3 (+ Ado) compared with solution 2 (no agonist).

We next investigated the contribution of PLA2 to Cl- secretion across the murine nasal mucosa. Figure 9 shows the effect of the PLA2 inhibitor CPZ (100 µM) on Ado- and Alb-stimulated Cl- conductance. Ado (500 µM) strongly stimulated Cl- secretion that was blocked by CPZ treatment (P < 0.02). In contrast, Alb (500 µM)-stimulated Cl- secretion was not affected by CPZ. Costimulation with Ado and Alb (500 µM each) failed to produce additive effects on Cl- conductance, indicating that the two signaling pathways converge on CFTR-dependent and not other (CFTR-independent) Cl- transport pathways (data not shown). These results also confirm our in vitro observations, which demonstrated that A2 receptor activation of CFTR in vivo depends on PLA2 activity.


View larger version (11K):
[in this window]
[in a new window]
 
Fig. 9.   cPLA2 inhibition blocks Ado-stimulated Cl- transport. Nasal PDs were performed in cftr(+) mice as described in Fig. 7 (n = 10-12 mice/condition). Mice were stimulated with Ado (500 µM) or Alb (500 µM) in solution 3 in the presence and absence of CPZ (100 µM, included in solutions 2 and 3). *P < 0.02 for Ado + CPZ compared with Ado alone, Alb alone, or Alb + CPZ.

A2B-ARs activate Delta 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 Delta 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), Delta 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 Delta F508 CFTR-dependent halide efflux in Delta F508 CFTR-expressing cells [and is currently in clinical trials as an activator of Delta 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 Delta F508 CFTR after prolonged treatment with DPCPX. Activation of Delta F508 CFTR was accomplished despite only modest effects of Ado on [cAMP] compared with forskolin (17).


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 10.   A2B receptor activation of halide efflux in Delta F508 CFTR-expressing cells. COS-7 cells expressing Delta F508 CFTR were studied after growth at 29°C for 48 h. Cells were perfused with NaI quenching buffer from 0 to 200 s, NO3 dequenching buffer from 200 to 500 s, and NO3 buffer + agonist from 500 s (arrow). Nos. in parentheses, total no. of responding cells/total no. of screened cells per condition (as described in METHODS). No. of responding cells in each condition was compared by the chi 2 test. Values are means ± SE of responding cells in each stimulated condition for Forsk (20 µM) and Ado (10 µM) or means ± SE of nonresponding cells for the 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 30 nM)-stimulated cells and NO3 control cells. Concentration of DPCPX used has previously been shown to maximally induce acute 36Cl- release from Delta F508 CFTR-expressing cells (24, 30). *P < 0.001 for Ado or Forsk compared with DPCPX or NO3 conditions.

In Fig. 11, COS-7 cells expressing G551D CFTR were stimulated with forskolin, Ado, or AA. A series of PDE inhibitors was also evaluated because previous reports (62, 63, 77) suggested that surface-localized mutant CFTRs might be partially responsive to stimulation with certain PDE inhibitors. Isotype-specific inhibitors (rolipram, PDE-4 specific; milrinone, PDE-3 specific; and zaprinast, PDE-5 and -6 specific) were used at concentrations ~20-fold above the inhibitor constant of their respective PDEs (72). Two nonspecific PDE inhibitors (papaverine and theophylline) were studied at 200 µM. PDE inhibitors such as milrinone have been evaluated in clinical trials for their ability to activate Cl- conductance in normal subjects and in CF patients carrying the G551D CFTR mutation (68). At the concentrations used, none of the PDE inhibitors alone consistently elevated cAMP in COS-7 cells (10-min exposure; data not shown), and none of the stimuli (including Ado) activated G551D CFTR-specific halide efflux (Fig. 11A). WT CFTR-expressing cells stimulated with forskolin are shown for comparison. In Fig. 11B, G551D CFTR-expressing COS-7 cells were exposed to combinations of Ado (200 µM), AA (100 µM), and PDE inhibitors. In contrast to the results in Fig. 11A, A2B receptor stimulation, when combined with AA and PDE inhibition, strongly activated halide efflux in a fashion similar to that produced in WT CFTR-expressing cells. Together, the results shown in Figs. 10 and 11 indicate that the activity of the two most common disease-associated CFTR mutations can be increased dramatically with A2B-AR signaling.


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 11.   Activation of halide efflux in G551D CFTR-expressing cells. A: G551D CFTR-expressing cells compared with wild-type (WT) CFTR-expressing cells. COS-7 cells transiently expressing WT or G551D CFTR were studied with 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) as described in METHODS; n 40 cells/condition. G551D CFTR-expressing cells failed to activate halide efflux when stimulated with Ado (200 µM) + papaverine (PAP; 200 µM), Ado + rolipram (ROL; 20 µM) in combination, or Forsk (20 µM; arrow). WT CFTR-expressing cells stimulated with Forsk (CFTR + Forsk; 20 µM) are shown for comparison. B: G551D CFTR-expressing COS-7 cells stimulated with Ado (200 µM) and AA (100 µM) combined with a series of phosphodiesterase inhibitors (PDEis). Cells were perfused with NO3 buffer + AA from 200 s and Ado + PDEi was added (arrows). Isotype-specific PDEis were studied at concentrations ~20-fold over the inhibition constant of their respective PDEs. Cells were also studied with Ado + AA alone (i.e., no PDEi). Values are means ± SE of all cells studied in each condition (n > 40). Concentrations of PDEis were (in µM) 20 milrinone (MIL), 20 ROL, 200 PAP, 5 ZAP, and 200 theophylline (THEO).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (Delta 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 Delta 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, PGF2alpha 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 beta 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).


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 12.   Three potential mechanisms by which A2 receptors signal CFTR activation. Ado stimulates A2 adenosine receptors (A2-AR), which activate cPLA2 and adenylyl cyclase. AA or products of AA metabolism, when combined with cAMP, helped maximize CFTR transport of Cl-. The mechanism could involve effects on the electrochemical driving force for ion transport through CFTR (i.e., activation of K+ channels) through direct effects of lipid species with CFTR or associated factors (direct effects) or possibly by increasing the rate of cAMP production in the vicinity of CFTR (accelerated cAMP production). COX, cyclooxygenase; LO, lipoxygenase.

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 beta 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 Delta 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 beta 2-adrenergic receptor agonist Iso in mice and activating Delta 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Anderson, MP, and Welsh MJ. Fatty acids inhibit apical membrane chloride channels in airway epithelia. Proc Natl Acad Sci USA 87: 7334-7338, 1990[Abstract].

2.   Barrett, KE, and Bigby TD. Involvement of arachidonic acid in the chloride secretory response of intestinal epithelial cells. Am J Physiol Cell Physiol 264: C446-C452, 1993[Abstract/Free Full Text].

3.   Barrett, KE, Cohn JA, Huott PA, Wasserman SI, and Dharmsathaphorn K. Immune-related intestinal chloride secretion. II. Effect of adenosine on T84 cell line. Am J Physiol Cell Physiol 258: C902-C912, 1990[Abstract/Free Full Text].

4.   Barrett, KE, Huott PA, Shah SS, Dharmsathaphorn K, and Wasserman SI. Differing effects of apical and basolateral adenosine on colonic epithelial cell line T84. Am J Physiol Cell Physiol 256: C197-C203, 1989[Abstract/Free Full Text].

5.   Baukrowitz, T, Schulte U, Oliver D, Herlitze S, Krauter T, Tucker SJ, Ruppersberg JP, and Fakler B. PIP2 and PIP as determinants for ATP inhibition of KATP channels. Science 282: 1141-1144, 1998[Abstract/Free Full Text].

6.   Becq, F, Jensen TJ, Chang XB, Savoia A, Rommens JM, Tsui LC, Buchwald M, Riordan JR, and Hanrahan JW. Phosphatase inhibitors activate normal and defective CFTR chloride channels. Proc Natl Acad Sci USA 91: 9160-9164, 1994[Abstract/Free Full Text].

7.   Bedwell, DM, Kaenjak A, Benos DJ, Bebok Z, Bubien JK, Hong J, Tousson A, Clancy JP, and Sorscher EJ. Suppression of a CFTR premature stop mutation in a bronchial epithelial cell line. Nat Med 3: 1280-1284, 1997[ISI][Medline].

8.   Bhattacharyya, D, and Sen PC. The effect of binding of chlorpromazine and chloroquine to ion transporting ATPases. Mol Cell Biochem 198: 179-185, 1999[ISI][Medline].

9.   Bouzat, CB, and Barrantes FJ. Effects of long-chain fatty acids on the channel activity of the nicotinic acetylcholine receptor. Receptors Channels 1: 251-258, 1993[ISI][Medline].

10.   Carson, MR, Travis SM, and Welsh MJ. The two nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator (CFTR) have distinct functions in controlling channel activity. J Biol Chem 270: 1711-1717, 1995[Abstract/Free Full Text].

11.   Cheng, SH, Rich DP, Marshall J, Gregory RJ, Welsh MJ, and Smith AE. Phosphorylation of the R domain by cAMP-dependent protein kinase regulates the CFTR chloride channel. Cell 66: 1027-1036, 1991[ISI][Medline].

12.   Choi, SY, Kim YH, Lee YK, and Kim KT. Chlorpromazine inhibits store-operated calcium entry and subsequent noradrenaline secretion in PC12 cells. Br J Pharmacol 132: 411-418, 2001[Abstract/Free Full Text].

13.   Choukroun, GJ, Marshansky V, Gustafson CE, McKee M, Hajjar RJ, Rosenzweig A, Brown D, and Bonventre JV. Cytosolic phospholipase A2 regulates Golgi structure and modulates intracellular trafficking of membrane proteins. J Clin Invest 106: 983-993, 2000[Abstract/Free Full Text].

14.   Chyb, S, Raghu P, and Hardie RC. Polyunsaturated fatty acids activate the Drosophila light-sensitive channels TRP and TRPL. Nature 397: 255-259, 1999[ISI][Medline].

15.   Clancy, JP, Bebok Z, Ruiz FE, King C, Jones J, Walker L, Greer H, Hong JS, Wing L, Macaluso M, Lyrene R, and Sorscher EJ. Evidence that systemic gentamicin suppresses premature stop mutations in patients with cystic fibrosis. Am J Respir Crit Care Med 1163: 1682-1692, 2001.

16.   Clancy, JP, Hong JS, Bebok Z, King SA, Demolombe S, Bedwell DM, and Sorscher EJ. Cystic fibrosis transmembrane conductance regulator (CFTR) nucleotide-binding domain 1 (NBD-1) and CFTR truncated within NBD-1 target to the epithelial plasma membrane and increase anion permeability. Biochemistry 37: 15222-15230, 1998[ISI][Medline].

17.   Clancy, JP, Ruiz FE, and Sorscher EJ. Adenosine and its nucleotides activate wild-type and R117H CFTR through an A2B receptor-coupled pathway. Am J Physiol Cell Physiol 276: C361-C369, 1999[Abstract/Free Full Text].

18.   Cobb, BR, Bebok Z, Hong J, King C, Sorscher EJ, and Clancy JP. Wildtype and G551D CFTR activation by A2B adenosine receptors and phosphodiesterase (PDE) inhibitors: [cAMP] and CFTR activity (Abstract). Pediatr Pulmonol Suppl 19: 171, 1999.

19.   Cobb, BR, Hong J, and Clancy JP. PLA2 signaling governs A2B adenosine receptor activation of CFTR (Abstract). Pediatr Pulmonol Suppl 20: 191, 2000.

20.   Devor, DC, and Frizzell RA. Modulation of K+ channels by arachidonic acid in T84 cells. I. Inhibition of the Ca2+-dependent K+ channel. Am J Physiol Cell Physiol 274: C138-C148, 1998[Abstract/Free Full Text].

21.   Devor, DC, and Frizzell RA. Modulation of K+ channels by arachidonic acid in T84 cells. II. Activation of a Ca2+-independent K+ channel. Am J Physiol Cell Physiol 274: C149-C160, 1998[Abstract/Free Full Text].

22.   Devor, DC, Singh AK, Lambert LC, DeLuca A, Frizzell RA, and Bridges RJ. Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells. J Gen Physiol 113: 743-760, 1999[Abstract/Free Full Text].

23.   Dwivedi, Y, and Pandey GN. Effects of treatment with haloperidol, chlorpromazine, and clozapine on protein kinase C (PKC) and phosphoinositide-specific phospholipase C (PI-PLC) activity and on mRNA and protein expression of PKC and PLC isozymes in rat brain. J Pharmacol Exp Ther 291: 688-704, 1999[Abstract/Free Full Text].

24.   Eidelman, O, Guay-Broder C, van Galen PJ, Jacobson KA, Fox C, Turner RJ, Cabantchik ZI, and Pollard HB. A1 adenosine receptor antagonists activate chloride efflux from cystic fibrosis cells. Proc Natl Acad Sci USA 89: 5562-5566, 1992[Abstract].

25.   Eigen, H, Rosenstein BJ, FitzSimmons S, and Schidlow DV. A multicenter study of alternate-day prednisone therapy in patients with cystic fibrosis. Cystic Fibrosis Foundation Prednisone Trial Group. J Pediatr 126: 515-523, 1995[ISI][Medline].

26.   Feoktistov, I, Polosa R, Holgate ST, and Biaggioni I. Adenosine A2B receptors: a novel therapeutic target in asthma? Trends Pharmacol Sci 19: 148-152, 1998[ISI][Medline].

27.   Freedman, SD, Katz MH, Parker EM, Laposata M, Urman MY, and Alvarez JG. A membrane lipid imbalance plays a role in the phenotypic expression of cystic fibrosis in cftr(-/-) mice. Proc Natl Acad Sci USA 96: 13995-14000, 1999[Abstract/Free Full Text].

28.   Furukawa, M, Ikeda K, Oshima T, Suzuki H, Yamaya M, Sasaki H, and Takasaka T. A2 adenosine receptors in Mongolian gerbil middle ear epithelium and their regulation of Cl- secretion. Acta Physiol Scand 163: 103-112, 1998[ISI][Medline].

29.   Grubb, BR, Vick RN, and Boucher RC. Hyperabsorption of Na+ and raised Ca2+-mediated Cl- secretion in nasal epithelia of CF mice. Am J Physiol Cell Physiol 266: C1478-C1483, 1994[Abstract/Free Full Text].

30.   Guay-Broder, C, Jacobson KA, Barnoy S, Cabantchik ZI, Guggino WB, Zeitlin PL, Turner RJ, Vergara L, Eidelman O, and Pollard HB. A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine selectively activates chloride efflux from human epithelial and mouse fibroblast cell lines expressing the cystic fibrosis transmembrane regulator delta F508 mutation. Biochemistry 34: 9079-9087, 1995[ISI][Medline].

31.   Hall, RA, Ostedgaard LS, Premont RT, Blitzer JT, Rahman N, Welsh MJ, and Lefkowitz RJ. A C-terminal motif found in the beta 2-adrenergic receptor, P2Y1 receptor and cystic fibrosis transmembrane conductance regulator determines binding to the Na+/H+ exchanger regulatory factor family of PDZ proteins. Proc Natl Acad Sci USA 95: 8496-8501, 1998[Abstract/Free Full Text].

32.   Hallows, KR, Raghuram V, Kemp BE, Witters LA, and Foskett JK. Inhibition of cystic fibrosis transmembrane conductance regulator by novel interaction with the metabolic sensor AMP-activated protein kinase. J Clin Invest 105: 1711-1721, 2000[Abstract/Free Full Text].

33.   Heller, A, Koch T, Schmeck J, and van Ackern K. Lipid mediators in inflammatory disorders. Drugs 55: 487-496, 1998[ISI][Medline].

34.   Huang, P, Trotter K, Boucher RC, Milgram SL, and Stutts MJ. PKA holoenzyme is functionally coupled to CFTR by AKAPs. Am J Physiol Cell Physiol 278: C417-C422, 2000[Abstract/Free Full Text].

35.   Hwang, TC, Guggino SE, and Guggino WB. Direct modulation of secretory chloride channels by arachidonic and other cis unsaturated fatty acids. Proc Natl Acad Sci USA 87: 5706-5709, 1990[Abstract].

36.   Hwang, TC, Nagel G, Nairn AC, and Gadsby DC. Regulation of the gating of cystic fibrosis transmembrane conductance regulator Cl channels by phosphorylation and ATP hydrolysis. Proc Natl Acad Sci USA 91: 4698-4702, 1994[Abstract].

37.   Illek, B, Tam AW, Fischer H, and Machen TE. Anion selectivity of apical membrane conductance of Calu 3 human airway epithelium. Pflügers Arch 437: 812-822, 1999[ISI][Medline].

38.   Illek, B, Zhang L, Lewis NC, Moss RB, Dong JY, and Fischer H. Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein. Am J Physiol Cell Physiol 277: C833-C839, 1999[Abstract/Free Full Text].

39.   Jacobson, KA, and Galen PJM Adenosine receptors: pharmacology, structure-activity relationships, and therapeutic potential. J Med Chem 35: 407-422, 1992[ISI][Medline].

40.   Kelley, TJ, Al-Nakkash L, Cotton CU, and Drumm ML. Activation of endogenous Delta F508 cystic fibrosis transmembrane conductance regulator by phosphodiesterase inhibition. J Clin Invest 98: 513-520, 1996[Abstract/Free Full Text].

41.   Kelley, TJ, al-Nakkash L, and Drumm ML. CFTR-mediated chloride permeability is regulated by type III phosphodiesterases in airway epithelial cells. Am J Respir Cell Mol Biol 13: 657-664, 1995[Abstract].

42.   Kelley, TJ, Thomas K, Milgram LJ, and Drumm ML. In vivo activation of the cystic fibrosis transmembrane conductance regulator mutant deltaF508 in murine nasal epithelium. Proc Natl Acad Sci USA 94: 2604-2608, 1997[Abstract/Free Full Text].

43.   Khan, SZ, Dyer JL, and Michelangeli F. Inhibition of the type 1 inositol 1,4,5-trisphosphate-sensitive Ca2+ channel by calmodulin antagonists. Cell Signal 13: 57-63, 2001[ISI][Medline].

44.   Knowles, MR, Paradiso AM, and Boucher RC. In vivo nasal potential difference: techniques and protocols for assessing efficacy of gene transfer in cystic fibrosis. Hum Gene Ther 6: 445-455, 1995[ISI][Medline].

45.   Konstan, MW, Byard PJ, Hoppel CL, and Davis PB. Effect of high-dose ibuprofen in patients with cystic fibrosis. N Engl J Med 332: 848-854, 1995[Abstract/Free Full Text].

46.   Lazarowski, ER, Mason SJ, Clarke L, Harden TK, and Boucher RC. Adenosine receptors on human airway epithelia and their relationship to chloride secretion. Br J Pharmacol 106: 774-782, 1992[Abstract].

47.   Linsdell, P. Inhibition of cystic fibrosis transmembrane conductance regulator chloride channel currents by arachidonic acid. Can J Physiol Pharmacol 78: 490-499, 2000[ISI][Medline].

48.   Madara, JL, Patapoff TW, Gillece-Castro B, Colgan SP, Parkos CA, Delp C, and Mrsny RJ. 5'-Adenosine monophosphate is the neutrophil-derived paracrine factor that elicits chloride secretion from T84 intestinal epithelial cell monolayers. J Clin Invest 91: 2320-2325, 1993[ISI][Medline].

49.   Matsui, H, Grubb BR, Tarran R, Randell SH, Gatzy JT, Davis CW, and Boucher RC. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 95: 1005-1015, 1998[ISI][Medline].

50.   Miele, L, Cordella-Miele E, Xing M, Frizzell R, and Mukherjee AB. Cystic fibrosis gene mutation (Delta F508) is associated with an intrinsic abnormality in Ca2+-induced arachidonic acid release by epithelial cells. DNA Cell Biol 16: 749-759, 1997[ISI][Medline].

51.   Mignen, O, and Shuttleworth TJ. I(ARC), a novel arachidonate-regulated, noncapacitative Ca2+ entry channel. J Biol Chem 275: 9114-9119, 2000[Abstract/Free Full Text].

52.   Mochizuki-Oda, N, Negishi M, Mori K, and Ito S. Arachidonic acid activates cation channels in bovine adrenal chromaffin cells. J Neurochem 61: 1882-1890, 1993[ISI][Medline].

53.   Moon, S, Singh M, Krouse ME, and Wine JJ. Calcium-stimulated Cl- secretion in Calu-3 human airway cells requires CFTR. Am J Physiol Lung Cell Mol Physiol 273: L1208-L1219, 1997[Abstract/Free Full Text].

54.   Moyer, BD, Denton J, Karlson KH, Reynolds D, Wang S, Mickle JE, Milewski M, Cutting GR, Guggino WB, Li M, and Stanton BA. A PDZ-interacting domain in CFTR is an apical membrane polarization signal. J Clin Invest 104: 1353-1361, 1999[Abstract/Free Full Text].

55.   Naren, AP, Cormet-Boyaka E, Fu J, Villain M, Blalock JE, Quick MW, and Kirk KL. CFTR chloride channel regulation by an interdomain interaction. Science 286: 544-548, 1999[Abstract/Free Full Text].

56.   Olah, ME, and Stiles GL. Adenosine receptor subtypes: characterization and therapeutic regulation. Annu Rev Pharmacol Toxicol 35: 581-606, 1995[ISI][Medline].

57.   Park, T, Bae S, Choi S, Kang B, and Kim K. Inhibition of nicotinic acetylcholine receptors and calcium channels by clozapine in bovine adrenal chromaffin cells. Biochem Pharmacol 61: 1011-1019, 2001[ISI][Medline].

58.   Pilewski, J, and Frizzell RA. Role of CFTR in airway disease. Physiol Rev 79, Suppl 1: S215-S255, 1999[Medline].

59.   Pratt, AD, Clancy G, and Welsh MJ. Mucosal adenosine stimulates chloride secretion in canine tracheal epithelium. Am J Physiol Cell Physiol 251: C167-C174, 1986[Abstract/Free Full Text].

60.   Rubenstein, RC, and Zeitlin PL. A pilot clinical trial of oral sodium 4-phenylbutyrate (Buphenyl) in Delta F508-homozygous cystic fibrosis patients: partial restoration of nasal epithelial CFTR function. Am J Respir Crit Care Med 157: 484-490, 1998[Abstract/Free Full Text].

61.   Sakai, H, Okada Y, Morii M, and Takeguchi N. Arachidonic acid and prostaglandin E2 activate small-conductance Cl- channels in the basolateral membrane of rabbit parietal cells. J Physiol (Lond) 448: 293-306, 1992[Abstract].

62.   Schultz, BD, Frizzell RA, and Bridges RJ. Rescue of dysfunctional Delta F508-CFTR chloride channel activity by IBMX. J Membr Biol 170: 51-66, 1999[ISI][Medline].

63.   Schultz, BD, Singh AK, Devor DC, and Bridges RJ. Pharmacology of CFTR chloride channel activity. Physiol Rev 79: S109-S144, 1999[Medline].

64.   Short, DB, Trotter KW, Reczek D, Kreda SM, Bretscher A, Boucher RC, Stutts MJ, and Milgram SL. An apical PDZ protein anchors the cystic fibrosis transmembrane conductance regulator to the cytoskeleton. J Biol Chem 273: 19797-19801, 1998[Abstract/Free Full Text].

65.   Shyng, SL, and Nichols CG. Membrane phospholipid control of nucleotide sensitivity of KATP channels. Science 282: 1138-1141, 1998[Abstract/Free Full Text].

66.   Singh, M, Krouse M, Moon S, and Wine JJ. Most basal Isc in Calu-3 human airway cells is bicarbonate-dependent Cl- secretion. Am J Physiol Lung Cell Mol Physiol 272: L690-L698, 1997[Abstract/Free Full Text].

67.   Smith, JJ, Travis SM, Greenberg EP, and Welsh MJ. Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid. Cell 85: 229-236, 1996[ISI][Medline]. [Corrigenda. Cell 87: October 1996, p. 355.]

68.   Smith, SN, Middleton PG, Chadwick S, Jaffe A, Bush KA, Rolleston S, Farley R, Delaney SJ, Wainwright B, Geddes DM, and Alton EW. The in vivo effects of milrinone on the airways of cystic fibrosis mice and human subjects. Am J Respir Cell Mol Biol 20: 129-134, 1999[Abstract/Free Full Text].

69.   Snouwaert, JN, Brigman KK, Latour AM, Malouf NN, Boucher RC, Smithies O, and Koller BH. An animal model for cystic fibrosis made by gene targeting. Science 257: 1083-1088, 1992[ISI][Medline].

70.   Strohmeier, GR, Reppert SM, Lencer WI, and Madara JL. The A2B adenosine receptor mediates cAMP responses to adenosine receptor agonists in human intestinal epithelia. J Biol Chem 270: 2387-2394, 1995[Abstract/Free Full Text].

71.   Sun, F, Hug MJ, Bradbury NA, and Frizzell RA. Protein kinase A associates with cystic fibrosis transmembrane conductance regulator via an interaction with ezrin. J Biol Chem 275: 14360-14366, 2000[Abstract/Free Full Text].

72.   Torphy, TJ. Phosphodiesterase isozymes: molecular targets for novel antiasthma agents. Am J Respir Crit Care Med 157: 351-370, 1998[Free Full Text].

73.   Versteeg, HH, van Bergen en Henegouwen PM, van Deventer SJ, and Peppelenbosch MP. Cyclooxygenase-dependent signaling: molecular events and consequences. FEBS Lett 445: 1-5, 1999[ISI][Medline].

74.   Welsh, MJ, Boat TF, Tsui L-C, and Beaudet AL. Cystic fibrosis. In: The Metabolic Basis of Inherited Disease, edited by Scriver CR, Beaudet AL, Sly WS, and Valle D.. New York: McGraw-Hill, 1995, p. 3799-3876.

75.   Wilschanski, M, Famini C, Blau H, Rivlin J, Augarten A, Avital A, Kerem B, and Kerem E. A pilot study of the effect of gentamicin on nasal potential difference measurements in cystic fibrosis patients carrying stop mutations. Am J Respir Crit Care Med 161: 860-865, 2000[Abstract/Free Full Text].

76.   Wu, T, Levine SJ, Cowan M, Logun C, Angus CW, and Shelhamer JH. Antisense inhibition of 85-kDa cPLA2 blocks arachidonic acid release from airway epithelial cells. Am J Physiol Lung Cell Mol Physiol 273: L331-L338, 1997[Abstract/Free Full Text].

77.   Yang, Y, Devor DC, Engelhardt JF, Ernst SA, Strong TV, Collins FS, Cohn JA, Frizzell RA, and Wilson JM. Molecular basis of defective anion transport in L cells expressing recombinant forms of CFTR. Hum Mol Genet 2: 1253-1261, 1993[Abstract].


Am J Physiol Lung Cell Mol Physiol 282(1):L12-L25
1040-0605/02 $5.00 Copyright © 2002 the American Physiological Society