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Published ahead of print on March 23, 2004, doi:10.1165/rcmb.2004-0012OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 140-146, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2004-0012OC

Activation of Airway Cl– Secretion in Human Subjects by Adenosine

Karen Hentchel-Franks*, David Lozano*, Valerie Eubanks-Tarn, Bryan Cobb, Lijuan Fan, Robert Oster, Eric Sorscher and J. P. Clancy

Departments of Pediatrics and Medicine, the Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama; and Department of Human Genetics, Georgetown University, Washington, DC

Address correspondence to: J. P. Clancy, M.D., Department of Pediatrics, 620 ACC, 1600 7th Avenue South, Birmingham, AL 35233. E-mail: jclancy{at}peds.uab.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated cystic fibrosis (CF) transmembrane conductance regulator (CFTR) regulation by A2 adenosine (Ado) receptors and ß2 adrenergic receptors in CFTR-corrected CFBE41o- airway cells and human subjects. CFBE41o- cells stimulated with Ado (10 µM), isoproterenol (Iso, 10 µM), or Ado + Iso (10 µM each) elevated cyclic AMP (cAMP) above control conditions (P < 0.001), with the Iso conditions increasing cAMP ~ 10-fold above that produced by Ado alone (P < 0.001). All agonist conditions had similar effects on short circuit current at 10 and 25 µM, with no further currents produced by subsequent stimulation with forskolin (20 µM). CFTR dependence was demonstrated by glybenclamide block of agonist-stimulated currents. Nasal potential difference studies in normal (n = 50) subjects demonstrated that Ado (10 µM) and Ado + Iso (10 µM each) produced more polarization compared with Iso (10 µM Ado increase = 44%, 10 µM Ado + Iso increase = 52%, P < 0.05 for each condition compared with Iso alone). Studies completed in patients with CF (n = 10, "severe" genotypes) confirmed that Ado-stimulated polarization was CFTR-dependent. Together, these results indicate that Ado is a potent Cl– secretagogue in vivo, with relatively small effects on cAMP levels despite strong effects on CFTR-dependent short circuit current and nasal Cl– transport. These findings support growing evidence indicating a role for Ado regulation of CFTR-dependent Cl– secretion in vivo.

Abbreviations: ß2 adrenergic receptor, ß2 AR • A2B adenosine receptor, A2B AR • African American, AA • adenosine, Ado • A kinase anchoring proteins, AKAPs • cyclic AMP, cAMP • cystic fibrosis, CF • CF transmembrane conductance regulator, CFTR • G protein coupled receptor, GPCR • short circuit current, Isc • isoproterenol, Iso • potential difference, P.D. • protein kinase, PK


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cystic fibrosis (CF) is a common, lethal genetic disorder due to abnormal function of the cystic fibrosis transmembrane conductance regulator protein (CFTR). Defects in CFTR are manifest by abnormal ion transport in the epithelia where it is expressed, including absent cyclic AMP (cAMP)-stimulated anion secretion (1, 2). CFTR normally resides in the apical membrane of airway epithelia, and is found in both surface and glandular cells of the large and small airways (3). It is a member of the traffic ATPase family with inherent Cl– channel activity (4–7). CFTR also positively regulates other Cl– transport pathways (8) and downregulates Na+ transport (9). Activation of CFTR is complex, and is dependent upon ATP binding and hydrolysis at nucleotide binding domains 1 and 2, coupled with protein kinase (PK)A- and possibly PKC-dependent phosphorylation of its regulatory (R) domain (6, 10–12).

Regulation of CFTR in vivo is believed to be accomplished through activation of surface G protein–coupled receptors (GPCRs) that release Gs, stimulate adenyl cyclase, and raise cAMP, which in turns activates PKA. Two distinct GPCRs are expressed in airway epithelial cells and activate CFTR through this mechanism, including A2B adenosine receptors (A2B ARs), and ß2 adrenergic receptors (ß2 ARs). Both have been shown to couple to CFTR through spatial compartmentalization, but in different and incompletely understood ways. For example, recent studies by Huang and colleagues demonstrated that extracellular adenosine (Ado) could activate CFTR by stimulation of A2B ARs within a single, inside-out membrane patch (13). This activation was mediated through AKAP interactions, and indicated that all of the components necessary to activate CFTR through this receptor were found within the same patch of membrane. In addition to raising cAMP, Ado stimulates additional second messenger systems in Calu-3 and other CFTR-expressing epithelial cells that may also contribute to Cl– transport (14, 15). ß2 ARs, on the other hand, contain a c-terminal PDZ-binding domain similar to that found in CFTR that provides a plausible mechanism for ß2 AR activation of CFTR through domain interactions mediated by ezrin-binding protein 50 (16, 17).

Previously we found that Ado was a potent agonist of CFTR-dependent Cl– transport in the nasal epithelium of mice, comparing favorably with ß receptor agonists (isoproterenol [Iso] and albuterol), in addition to stimulation with forskolin (15, 18). In the current study, we investigated the capacity of Ado to activate Cl– transport in human surface airway cells expressing CFTR in vitro, and in the airway epithelium of human subjects. Ion transport was measured in vivo by the nasal potential difference assay in subjects with and without CF. We compared this with Cl– secretion produced by Iso, a ß receptor agonist that is commonly used in human nasal potential difference (P.D.) assays to detect CFTR activity (19). Our studies provide evidence that Ado is a potent agonist of CFTR-dependent Cl– secretion in vivo, and that addition of Ado to nasal P.D. protocols may improve our ability to detect CFTR activity in human subjects.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
CFBE41o- Cell Culture, CFTR Expression, and Ussing Chamber Studies
CFBE41o- cells ({Delta}F508/{Delta}F508) were the generous gift of Dr. D. Gruenert (University of San Francisco, San Francisco, CA) (20). Cells were grown in Dulbecco's modified Eagle's medium/F12 media + 10% fetal bovine serum and 1% penicillin + streptomycin on fibronectin-coated tissue culture flasks until ~ 90% confluent. The cells were then seeded onto 6.5-mm diameter fibronectin-coated Costar Transwell filters at a density of ~ 5 x 105 cells/filter. Media was changed every other day on the basolateral surface until filters were confluent (~ 5 d post seeding) with a resistance > 300 {Omega} · cm, and then transfected with Ad5-CFTR (CFTR under regulatory control of the CMV promoter, contained in a replication deficient adenovirus vector with deletion of the E1A early genes; generous gift of Dr. J. Zabner, University of Iowa, Iowa City, IA) at an MOI of 50 for 6 h. The transfecting media was then removed, and confluent cells were kept at an air–liquid interface until 48 h after transfection (resistance of 300–500 {Omega} · cm). Inserts were mounted in an Ussing chamber, and short circuit current (Isc) was measured under voltage clamp conditions similar to previously described studies (15, 21). Briefly, cells grown 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 Ringers solutions containing (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. Isc measurements were obtained by using 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 using 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 Na gluconate, all other components as above, + 100 µM amiloride. Increasing concentrations of Ado or Iso were added to the mucosal bathing solutions as indicated (10 min of observation at each concentration), followed by 20 µM forskolin (mucosal and serosal). A quantity of 200 µM glybenclamide was added to the mucosal bathing solution, effectively blocking the stimulated Isc.

Detection of A2B ARs
To detect the A2B adenosine receptor, CFBE41o- cells were lysed with RIPA buffer (150 mM NaCl, 1% NP-40, 0.05% Na-deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM tris-Cl, 10 mg/ml PMSF, at pH = 8.0). Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis using pre-cast 16% gels (Novex gels; Invitrogen Corporation, Carlsbad, CA), and electrophoretically blotted onto PVDF membranes. The membranes were then blocked with 1% bovine serum albumin in phosphate-buffered saline for 30 min, washed three times with phosphate-buffered saline + 0.1% tween, and then probed using 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 the brain human A2B AR cDNA (1:500 dilution) for 2 h. The membranes were washed three times, and incubated with secondary antibody (1:1,000 dilution, goat anti-rabbit antibody conjugated to alkaline phosphatase; Southern Biotechnology Associates, Birmingham, AL) for 2 h. Membranes were washed three times, and then developed with 5-bromo-4-chlor-3-indolyl-phosphate (BCIP) and 4-nitro blue tetrazolium chloride in carbonate buffer (pH = 9.8). Control conditions were probed with nonimmune rabbit IgG during primary antibody incubation.

Detection of cAMP
Celluar cAMP was measured using an ELISA-based detection kit as previously described (15) (Cayman Chemicals, Ann Arbor, MI). Briefly, cells grown on 35-mm dishes (~ 7 x 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, resuspended in phosphate buffer, and the cAMP levels were quantified per manufacturer's directions. For all experiments, papaverine (nonspecific nonxanthine phosphodiesterase inhibitor, 100 µM) was included to improve cAMP detection. Xanthine-based inhibitors were avoided, as drugs of this class commonly interact with Ado receptors (22).

Human Nasal P.D. Studies
Human studies were approved by the University of Alabama at Birmingham Institutional Review Board and the UAB General Clinical Research Center Scientific Advisory Committee. Signed informed consent (adults) and assent of minors (< 19 yr) was obtained prior to nasal P.D. measurements. All studies were completed through the UAB General Clinical Research Center. All subjects were in stable condition at the time of the study. Inclusion criteria included age > 12 yr, and for patients with CF a diagnosis based on two positive sweat [Cl–] results, CF lung and pancreatic disease, and two identified class I, II, or III CFTR mutations. Exclusion criteria included active nasal/sinus symptoms, symptomatic asthma, recent change in nasal medications (within 2 wk), and inability to undergo three nasal P.D. measurements. Patients on nasal medications were requested to remain on them until completion of the three measurements. All measurements were completed over a 2-wk period in an attempt to minimize variation. For our studies, we used a nasal P.D. protocol previously validated in human subjects and standardized to the protocol developed by Knowles and colleagues (19). Briefly, a series of stopcocks was configured to allow perfusion of the following solutions through the port at the tip of the exploring catheter: Ringer's solution containing 10–4 M amiloride (Solution A); a low Cl– solution (in mM, 2.4 K2HPO4; 0.4 KH2PO4; 115 NaGluconate; 25 NaHCO3; 1.24 CaGluconate2) containing 10–4 M amiloride (Solution B); Solution B with added Ado, Iso, or Ado + Iso (10–5 M; Solution C); and Solution B with added Ado, Iso, or Ado + Iso (2.5 x 10–5 M; Solution D). All test solutions were perfused at a rate of 5.0 cc/min. In each nostril, the largest P.D. readings in Ringer's at 1, 2, and 3 cm (lumen negative) were averaged and taken as the average baseline P.D. The catheter tip was then placed at the most negative P.D. site (generally the same distance and site for each nostril for each of the three measurements) and maintained for superperfusion measurements with a disposable face shield (Splash Shield, Inc., Woburn, MA) adapted to hold the catheter for the duration of the protocol.

For each perfusion condition, a steady-state recording was obtained. The recording lasted at least 1 min (for Solutions A and B) before proceeding to the next solution within the sequence. Because the 3-min time point following the change to low [Cl–]/Iso is taken as a highly sensitive and specific measure of wild-type CFTR activity, all patients and control subjects were monitored for at least 3 min during perfusion with Solutions C and D. To ensure standardization of the assay, measurements in this study were performed by the same three investigators using the same nasal PD apparatus. Several readings were obtained for data analysis, including: (i) "baseline P.D." as described above; (ii) "change due to amiloride" (the difference between the starting lactated Ringer's P.D. and the stable P.D. after perfusion with solution A); (iii) "change low [Cl–]/10 µM agonist" (difference between the lowest stable P.D. following addition of amiloride before perfusion with solution C, and the P.D. after 3 min of perfusion with solution C); and (iv) "change low [Cl–]/25 µM agonist" (difference between the completion of perfusion with solution C, and the P.D. after 3 min of perfusion with solution D). Tracings in patients and control subjects were not included in data analysis if catheter movement occurred. The final analysis included ~ 95% of all tracings.

Each subject (50 normal control subjects, 10 patients with CF) was studied in all three conditions on separate occasions over a 2-wk period. The first and second P.D.s were performed with either Ado or Iso in an alternating fashion, and the third P.D. in the series was the combination of Ado and Iso. All nasal P.D. tracings were then coded by an investigator not involved in the study, such that all data were stripped of identifiers (including diagnosis or condition). Each nasal P.D was then scored independently by two investigators. The values for the above measurements were entered into a database, and mean values of both investigator scores for each nostril were tabulated and used for subsequent analysis.

Statistical Analysis
For Isc and cAMP measurements, descriptive statistics (mean and SEM) and paired and unpaired t tests were performed using SigmaStat software (Jandel, CA). For nasal P.D. data, comparisons among the three agonist conditions (Ado, Iso, Ado + Iso) and between the two study groups (normal subjects, patients with CF) were performed simultaneously; in addition, comparisons among the three agonist conditions were performed separately for each study group due to highly significant differences between the study groups and to the fact that the sample size in the group of patients with CF was considerably smaller than that in the group of control subjects. These comparisons were performed using mixed-models repeated measures ANOVA, assuming an unstructured covariance matrix. Tukey's multiple comparisons test was then used to determine which specific pairs of means were significantly different. Analyses of nasal P.D. data were performed with the use of SAS software (version 9.0; SAS Institute Inc., Cary, NC). All statistical tests were two-sided and were performed at a 5% significance level (i.e., {alpha} = 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Stimulation of CFBE41o- Cells with Ado and Iso Raises cAMP and Activates Isc
To compare the relationship between Ado and Iso stimulation of cAMP and Cl– secretion in vitro, CFBE41o- cells were exposed to increasing concentrations of Ado, Iso, or the combination of Ado + Iso as described in MATERIALS AND METHODS. Figure 1A shows the effect of the agonists (10 µM) on cellular cAMP. Although all agonist conditions increased cAMP levels above control levels (compared with papaverine alone, P < 0.001) the Ado-stimulated cAMP was significantly less than the levels produced by Iso (P < 0.001). The combination of Ado and Iso failed to raise cAMP levels above that of Iso alone, indicating that maximal cAMP stimulation had been achieved. Complimentary studies performed in Calu-3 cells (airway serous cell phenotype) had similar results (Figure 1B). Figure 1C compares the effects of the agonist conditions on Isc in CFBE41o- cells expressing CFTR (48 h after transduction with Ad-CFTR as described in MATERIALS AND METHODS). In all three conditions, mucosal stimulation with 10 µM Ado, Iso, or Ado + Iso activated Isc. Higher concentrations of agonists (25 µM, mucosal) and subsequent stimulation with the potent cAMP-elevating agonist forskolin (20 µM, mucosal and serosal) failed to further increase Isc. Control conditions (no CFTR expression) failed to activate Isc by Ado, Iso, or forskolin. In previously reported studies performed in Calu-3 cell monolayers, we found similar CFTR activation by A2B AR and ß2 AR agonists (15, 17). Complimentary experiments evaluating the efficiency of Ad-GFP transduction indicated that ~ 50% of CFBE41o- cells grown on permeable supports exhibited GFP expression under the same conditions as the Isc studies (Figure 1D). Figure 1E depicts a Western blot of CFBE41o- cell lysates, confirming A2B AR expression (right panel). Controls included identification of the receptor in a second airway epithelial cell line previously demonstrated to express this membrane protein (Calu-3 cells) (13, 15), and failure to detect A2B AR in HL60 cells (promyelocyte cell line; Figure 1E, left panel). Figure 1F shows an example of forskolin-activated Isc in CFBE41o- cells after Ad-CFTR transfection. Brisk activation is seen, and all of the stimulated current is sensitive to glybenclamide block. Together, these studies indicate that both Ado and Iso raise cAMP and activate CFTR-dependent Isc in CFTR-expressing human airway cells grown on permeable supports, and provide a dosing rationale for studies in human subjects. Similar patterns of activation of Isc were seen after stimulation with all three agonist conditions at 10 µM, despite large differences in cAMP production by Ado compared with Iso (Figures 1A and 1B).








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  		Figure 1. (A) Comparison of receptor-stimulated cAMP production in CFBE41o- cells. cAMP levels were increased for Ado (shaded bar), Iso (striped bar), and Ado + Iso (open bar) compared with control (papaverine alone, black bar; *P < 0.001, 10 µM agonist exposure for 10 min to Ado, Iso, Ado + Iso). cAMP levels were increased for Iso and Ado + Iso compared with Ado alone ({dagger}P < 0.001) (n = 4 dishes per condition). (B) Comparison of receptor-stimulated cAMP production in Calu-3 cells. cAMP levels were increased for Ado (shaded bar), Iso (striped bar), and Ado + Iso (open bar) compared with control (papaverine alone, black bar; *P < 0.001, 10 µM agonist exposure for 10 min to Ado, Iso, Ado + Iso). cAMP levels were increased for Iso and Ado + Iso compared with Ado alone ({dagger}P < 0.001) (n = 4 dishes per condition). (C) Comparison of Isc stimulated by Ado, Iso, and forskolin in CFTR-corrected CFBE41o- cells. AdCFTR was expressed in CFBE41o- cells as described in MATERIALS AND METHODS. A low Cl– gradient (LoCl–, mucosal, 6 mM) was established at the first arrow, and amiloride (A, 100 µM, mucosal) was added as shown (second arrow). Both receptor agonists (mucosal addition) had similar effects on Isc at 10 and 25 µM. Total currents were not significantly different between the three conditions. Subsequent stimulation with forskolin (F, 20 µM, mucosal and serosal) did not stimulate further Isc. All agonist-stimulated currents were sensitive to blockade with glybenclamide (G, 200 µM, mucosal). Cells without CFTR introduced (black circles) showed no stimulated currents following all maneuvers (n = 8–12 filters per condition). Diamonds, Ado + Iso; squares, Ado; triangles, Iso. (D) Expression of Ad-GFP in CFBE41o- cell cultures. En face image of CFBE41o- cells grown on filters, 48 h after infection with AdCFTR (left panel, MOI = 50) or AdGFP (right panel, MOI = 50). Approximately 50% of cells display GFP expression (visual inspection). (E) Western blot of CFBE41o- cells identifying A2B Ado receptors. Left panel: a characteristic ~ 40-kD band is identified in CFBE41o- and Calu-3 cells but not HL60 cell lysates. Right panel: negative control (nonimmune rabbit IgG replaced the primary antibody). (F) Forskolin-stimulated Isc in CFBE41o- cells after AdCFTR expression. Representative tracing of AdCFTR-expressing CFBE41o- cells showing effects of mucosal low (Cl–) gradient (LoCl), amiloride (A, 100 µM, mucosal), forskolin (F, 20 µM, mucosal and serosal), and glybenclamide (G, 200 µM, mucosal) on Isc.

 
Ado Activates CFTR-Dependent Cl– Transport in Human Subjects
Based on the results of our in vitro studies and our previous experience in murine nasal P.D. measurements, we investigated the effects of Ado on ion transport in human subjects using the nasal P.D. assay. This bioelectric measurement of airway ion transport is frequently used to detect CFTR function, and to discriminate between a normal and a CF phenotype. For these studies, we used a protocol similar to that shown in our in vitro experiments, comparing Ado, Iso, and the combination of Ado and Iso at two concentrations (10 and 25 µM). We chose to investigate relatively low doses of Ado based on our findings in CFTR-corrected CFBE41o- cells, and because high concentrations of Ado have previously been suggested to elicit mast cell degranulation in human nasal studies (23).

Table 1 summarizes demographic information in the control subjects and in subjects with CF. For the group without CF, there was a slightly higher incidence of female participants (70% in control subjects versus 60% in subjects with CF) and higher mean age than the CF group. The subjects with CF all carried the diagnosis of CF based on positive sweat Cl– values, disease involving at last two organ systems, and the identification of two class I, II, or III CF-causing mutations. Nine out of ten subjects with CF were homozygous for the {Delta}F508 mutation.


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  		TABLE 1. Demographic information in normal subjects and in subjects with CF

 
Table 2 summarizes nasal P.D. results in our normal subjects for the three agonist conditions. Data shown are the mean (± SD) from individual nostrils (two measurements/subject). Similar results were obtained when data were analyzed as mean P.D. change for both nostrils per subject and "best response" per subject (data not shown). As expected, the baseline, amiloride-sensitive, and low [Cl–] + amiloride components of the nasal P.D. were similar across the 3 d of study for each condition. No statistically significant differences were found for any of these parameters for the different days of study. A quantity of 10 µM Ado and Ado + Iso produced greater polarization than 10 µM Iso alone, with a 44% (Ado) and a 52% (Ado + Iso) increase in stimulated P.D. (P < 0.05, also see Figure 2 for comparisons of stimulated Cl– secretion within the normal group). Further stimulation with 25 µM agonist (3-min perfusion) produced minimal further polarization in any of the three conditions (NS). When comparing the change in P.D. after perfusion with amiloride + low [Cl-] + 10 µM agonist, the mean stimulated P.D. for both the Ado and the Ado + Iso conditions were mildly increased over the Iso condition alone (P > 0.05). The additional 3-min perfusion with 25 µM agonist increased the total measured Cl– secretion slightly in all three conditions.


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  		TABLE 2. Nasal P.D. values in subjects without CF

 


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  		Figure 2. Mean change in P.D. (± SD) in normal subjects after perfusion with Iso, Ado, and Ado + Iso (10 µM [open bars], and 10 µM + 25 µM [shaded bars]). All changes in P.D. are after switch from amiloride (100 µM) and low [Cl-] perfusion. The dotted line is the change in P.D. produced by 10 µM Iso (gold standard). *P < 0.05 compared with 10 µM Iso alone condition.

 
Table 3 summarizes nasal P.D. results for our subjects with CF. Data shown are the mean (± SD) from individual nostrils (two measurements/subject). Similar results were obtained when data were analyzed as mean P.D. change for both nostrils per subject and "best response" per subject (data not shown). As expected, differences between the group with CF and the control group were seen for all measured parameters for each agonist condition (P < 0.001). For comparisons of conditions within the group with CF, no differences were seen in terms of baseline, amiloride-sensitive P.D., and the change in P.D. after perfusion with amiloride + low [Cl–] for the different days of study. For comparisons of the change in P.D. after agonist perfusion, the Ado and Ado + Iso conditions displayed less depolarization than the Iso condition alone. There was a trend toward significance for the change in P.D. after perfusion with low [Cl–] solution + amiloride + Ado (10 µM), and the change in P.D. after perfusion with low [Cl–] + amiloride + [Ado + Iso (10 µM)] compared with low [Cl–] + amiloride + Iso alone (10 µM, 0.05 < P < 0.10, see Table 3). Figure 3 shows mean and individual responses (scatter points) for the group with CF after perfusion with solutions B and C (Low [Cl–]/amiloride/10 µM agonist, right panel).


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  		TABLE 3. Nasal P.D. values in subjects with CF

 


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  		Figure 3. Mean change in P.D. (bar, ± SD) in subjects with CF after perfusion with low [Cl–] solution containing amiloride (100 µM), and subsequent perfusion with Iso, Ado, and Ado+Iso (10 µM). Individual responses for each nostril for each subject in the three conditions are shown in scatter points. 0.05 < {dagger}P < 0.10 (Ado, and Ado+Iso compared with Iso alone).

 
Figure 4 shows the sequential nasal P.D. measures of Cl– secretion (Solutions B and C, change in P.D. after perfusion with low [Cl–] + amiloride + 10 µM agonist) in our control subjects (1–50), whereas Figure 5 shows similar data for our subjects with CF (1–10). Each value is the mean (± SD) of the six separate nasal P.D. readings (two nostrils, three agonist conditions) obtained for each subject. There was no consistent trend seen in the magnitude of Cl– secretory responses in the normal group obtained over the course of the study (~ 24 mo) except for poor detection of Cl– secretion during an isolated period in the normal group (subjects 14–21), suggesting that time or accumulated experience was not a contributing factor to the results. On review of our records, the failure to detect Cl– secretion in normal subjects 14–21 was believed to be due to erroneous storage of agar bridges for this period in a low electrolyte solution. Because we could not definitely exclude other etiologies for the reduced responses, data from these subjects was included in the final analysis. Similar Cl– secretory results were obtained in the normal group when the data from these individuals were excluded (change P.D. [mV ± S.D.] 10 µM drug [*Ado = –8.09 {7.92}, {dagger}Ado + Iso = –7.48 {7.97}, Iso = –5.17 {6.46}] *P = 0.001 compared with Iso, {dagger}P = 0.077; change low [Cl–] + 10 µM drug [Ado = –19.82 {15.01}, Ado + Iso = –19.17 {14.21}, Iso = –19.17 {14.21}]). Data variability also remained relatively stable throughout the course of the study. Similar measurements for the group with CF (Figure 5) showed no consistent trend in this value over the course of the study.



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  		Figure 4. Comparison of mean stimulated Cl– secretion produced by perfusion with amiloride (100 µM) + Low [Cl–] + 10 µM agonists (mean of all agonist conditions, ± SD) for each normal subject (n = 50).

 


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  		Figure 5. Comparison of mean stimulated Cl– secretion produced by perfusion with amiloride (100 µM) + Low [Cl–] + 10 µM agonists (mean of all agonist conditions, ± SD) for each subject with CF (n = 10).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results of our study establish that Ado is a potent Cl– secretagogue in vivo, activating CFTR-dependent Cl– transport across the nasal epithelium of normal subjects. The change in potential difference produced by 10 µM Ado was greater than that seen with Iso alone, and similar to that seen with Ado + Iso (10 µM, P < 0.05 for Ado and Ado+ Iso compared with Iso alone). This difference was lost with 25 µM agonists, suggesting that all conditions achieved maximal activation of CFTR at the higher concentration studied. When combined with the change in P.D. after perfusion with amiloride + low [Cl–] solution, similar trends were seen for all three experimental conditions after 3 min of perfusion with 10 µM agonists, and after subsequent perfusion for 3 min with 25 µM agonists.

Using an in vitro human airway cell model of Cl- transport, both Ado and Iso potently stimulated CFTR-dependent Cl– secretion. Interestingly, similar patterns of Cl– transport were seen in all three agonist conditions, despite large (~ 10-fold) differences in cAMP produced by Ado compared with Iso and Ado + Iso. The comparable effects of the agonists on Isc were also somewhat surprising, given the known differences in ligand affinities of Ado for A2 receptors (low-affinity Ado receptor, with Kd reported as ~1–20 µM) relative to that of isoproterenol for ß2 ARs (< 1 µM) (22, 24). These results are consistent with the recent findings of Huang and colleagues in Calu-3 cells, in which A2B Ado receptor activation of CFTR was found to be tightly compartmentalized and efficiently transduced through AKAP-based protein:protein interactions (13). These results are also consistent with previous findings from our laboratory identifying differences in signaling pathways activated by A2B AR and ß2 AR agonists in epithelial cells (15, 25). Although both Ado and ß2 AR agonists raise cAMP in a variety of airway cell lines (Calu-3, IB3, CFBE41o-), Ado also raises cell Ca++ in Calu-3 cells in contrast to albuterol (ß2 AR) or forskolin, which have no effect. Furthermore, Ado has been shown to induce arachidonic acid release from airway cells and other epithelial cells that express A2 Ado receptors, suggesting that this agonist can mobilize cellular PLA2 that in turn contributes to Ado-stimulated Cl– secretion (14, 15). Recent studies published by Naren and colleagues identified protein:protein interactions that exist between CFTR, ezrin-binding protein 50, and the ß2 AR (17). These PDZ-based interactions were shown to be an important component of ß2 AR:CFTR signaling that were disrupted following CFTR phosphorylation. We speculate that some of the differences between the levels of cAMP and the activation of CFTR may therefore be independent of the recently described physical links between CFTR and these distinct GPCRs, possibly including stimulation of additional (cAMP-independent) signaling pathways that contribute to CFTR activity and/or Cl– transport.

Recent studies by Donaldson and colleagues highlight purinergic levels and regulation of Cl– secretion and CFTR in airway cells (13, 26, 27). Some of these data suggest that Ado, A2B Ado receptors, and CFTR may play a pivotal role in setting the airway surface liquid depth in primary human airway cell monolayer cultures, and in this way serve as a natural regulator of CFTR activity in vivo. A2B AR regulation of CFTR has also been described in Calu-3 cells, a serous glandular cell line that expresses high levels of CFTR (13, 15, 21). Glandular function has been a topic of considerable interest in CF, and work by Joo and colleagues (28) and Ballard and coworkers (29) have recently addressed the importance of airway gland function in CF pathogenesis.

Phosphorylated nucleosides such as ATP and UTP have been shown to activate CFTR-independent Cl– secretion in normal and CF airway epithelial cell cultures and in human subjects through stimulation of P2Y2 purinergic receptors (30, 31). We recently reported that phosphorylated Ado nucleosides, including ATP, ADP, and AMP, all contribute to the regulation of anion secretion in Calu-3 cells through A2 receptors, presumably through dephosphorylation by surface ectonucleotidases (21). P2 purinergic receptors, in contrast, appear to play a relatively limited role in anion secretion in these cells. Together, these studies highlight the significance of Ado as a signaling molecule to regulate CFTR activity, and the importance of understanding the role of this regulatory pathway in both glandular secretion and CF pathogenesis.

The nasal P.D. results observed in subjects with CF (Table 3 and Figure 3) suggest that Ado may have very small Cl– stimulatory effects in some subjects beyond that seen with Iso. The nature of this effect is unknown, but previous studies from our laboratory have shown that Ado stimulation of A2B ARs can activate surface localized mutant CFTR molecules, including {Delta}F508 CFTR after growth at permissive temperatures, G551D CFTR, and R117H CFTR (15, 32). As the majority of our study patients were homozygous for the {Delta}F508 CFTR mutation, it seems unlikely that this mechanism explains the small effects seen, unless small amounts of {Delta}F508 CFTR are available for partial activation at the nasal epithelial cell membrane (33). Alternatively, Ado has been shown to regulate other channels such as those mediating K+ transport, which may in turn increase the driving force for Cl– secretion across the nasal mucosa (34). A1 Ado receptors have also been implicated in stimulating Cl– transport in normal and CF airway cells; however, this effect has been demonstrated after treatment with receptor antagonists (35, 36). Finally, Ado is weak agonist for P2Y2 receptors, and stimulation of this receptor class could theoretically contribute to the small effects seen in some members of the CF group. The differences between Ado and Iso-stimulated P.D. were more pronounced at lower (10 µM) concentrations compared with 25 µM stimulation in both the normal and the CF groups, arguing against this interpretation. Furthermore, the additive effects of these agonists were minimal in the normal group (and absent at the higher concentration), suggesting stimulation of a common Cl– secretion pathway by both receptor agonists.

Ado is a ubiquitous signaling molecule, contributing to regulation of a variety of organ processes, ranging from ion transport and inflammation to neural function, vascular tone, and cardiac activity (36). Ado has been shown to signal mast cell degranulation, and after metabolism from AMP may serve as a bronchoprovocative agent to detect allergic-based asthma in human subjects (37). AMP has been found to be quite specific in this regard, as studies in normal subjects and a limited number of control patients with airway disease did not induce bronchoconstriction. Based on Ado's extremely short half life (seconds) and its possible role in provoking bronchoconstriction, it appears unlikely that the parent molecule (adenosine) would serve a therapeutic, lower airway role in the treatment of CF. A better understanding of Ado signaling in airway cells, however, may identify additional physiologic regulators of Cl– secretion and CFTR.

Finally, performance of the nasal potential difference in human subjects has the potential for multiple technique-related inaccuracies, which may have been responsible for the unusually low Cl– secretory responses seen with all agonists for normal subjects 14–21 (Figure 4). Subsequent to this study, Standard Operating Procedures for performance of the nasal P.D. have been developed by the Cystic Fibrosis Therapeutic Development Network. As researchers continue to refine this endpoint for studies of airway ion transport in vivo, standardized procedures coupled with meticulous execution and data review need to be employed in an ongoing fashion to ensure that results from protocols (particularly multi-center studies) continue to provide useful, reproducible, and meaningful data.

In summary, our results indicate that Ado is a potent activator of CFTR-dependent Cl– transport in human subjects. It compares favorably with the current gold standard ß2 AR agonist (Iso), increasing mean stimulated Cl– secretion at lower concentrations. Future nasal P.D. studies of Ado compared with Iso in patients with CF carrying partial function CFTR mutations, or with retained pulmonary function, may provide useful information regarding the relative contribution of these two pathways to airway ion transport in subjects with various disease manifestations.


   Acknowledgments
 
Research was supported by the National Heart, Lung and Blood Institute (HL67088) the Cystic Fibrosis Foundation (CFF R464), the National Institute of Diabetes and Digestive and Kidney Diseases (P30 DK54781, P50 DK53090), and the National Center for Research Resources (M01 RR00032). The authors thank Cassie Woodley for her expert help in preparing this manuscript.


   Footnotes
 
Conflict of Interest Statement: K.H.-F. has no declared conflict of interest; D.L. has no declared conflict of interest; V.E.-T has no declared conflict of interest; B.C. has no declared conflict of interest; L.F. has no declared conflict of interest; R.O. has no declared conflict of interest; E.S. has no declared conflict of interest; and J.P.C. has no declared conflict of interest.

* Both authors contributed equally to the work included in this manuscript. Back

Received in original form January 12, 2004

Received in final form March 8, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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