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Published ahead of print on April 24, 2003, doi:10.1165/rcmb.2002-0247OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 410-418, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0247OC

Adenosine Receptors and Phosphodiesterase Inhibitors Stimulate Cl- Secretion in Calu-3 Cells

Bryan R. Cobb, Lijuan Fan, Timea E. Kovacs, Eric J. Sorscher and John P. Clancy

Departments of Human Genetics, Medicine, and Pediatrics, and Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama

Address correspondence to: Dr. J. P. Clancy, M.D., Associate Professor, Department of Pediatrics, Associate Scientist, Gregory Fleming James Cystic Fibrosis Research Center, 1600 7th Avenue South, Suite 620 ACC, Birmingham, AL 35233. E-mail: jclancy{at}peds.uab.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We investigated cystic fibrosis transmembrane conductance regulator (CFTR) activation by clinically used phosphodiesterase inhibitors (PDEis) in Calu-3 cell monolayers alone and in combination with A2B adenosine receptor stimulation. This receptor pathway has previously been shown to activate wild-type and mutant CFTR molecules. Several PDEis, including milrinone, cilostazol (Pletal), papaverine, rolipram, and sildenafil (Viagra), produced a short circuit current (Isc) that was glibenclamide-sensitive, achieving 20–85% of forskolin-stimulated Isc. Papaverine, cilostazol, and rolipram also augmented both the magnitude and the duration of Isc following low dose stimulation of adenosine receptors with Ado (0.1–1.0 µM, P < 0.01). Subsequent studies demonstrated that very low concentrations of cilostazol or papaverine (~ 1/2 peak serum concentrations) were sufficient to activate Isc, and both agents markedly augmented Ado-stimulated Isc (1 µM, P < 0.01). Our results provide evidence that select PDEis, at concentrations achieved as part of systemic therapies, can activate CFTR-dependent Isc in Calu-3 cell monolayers. These studies also indicate that PDEis have the capacity to augment an endogenous CFTR-activating pathway in an "in vivo"–like model system, and supports future investigations of these agents relevant to cystic fibrosis.

Abbreviations: adenosine deaminase, ADA • cystic fibrosis, CF • cystic fibrosis transmembrane conductance regulator, CFTR • cilostazol, Cil • 8-Cyclopentyl-1,3-dipropylxanthine, DPCPX • hexokinase, HEXO • short circuit current, Isc • milrinone, Mil • papaverine, Pap • phosphodiesterase, PDE • PDE inhibitor, PDEi • rolipram, Rol • theophylline, Theo


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that is activated through PKA-dependent phosphorylation of its regulatory domain, coupled with ATP binding and hydrolysis at nucleotide binding domains 1 and 2 (1–4). Activation in vivo is believed to be primarily through stimulation of surface receptors that couple to adenylate cyclase and raise cellular cAMP. A2B adenosine receptors have recently been shown to be very important in this regard, as they spatially compartmentalize with CFTR through scaffolding protein interactions with A-kinase anchoring proteins, which in turn modulate CFTR activation by adenylate cyclase and PKAII (5). CFTR is also regulated by phosphodiesterases, which catalyze the conversion of cAMP to 5' AMP (6–11). Phosphodiesterases are attractive therapeutic targets in cystic fibrosis (CF), and inhibitors could theoretically potentiate low level activity of mutant CFTR molecules that spontaneously localize to the cell surface (e.g., R117H CFTR, G551D CFTR) or can be localized to the cell surface at low levels by corrective molecules (e.g., butyrate compounds for rescue of {Delta}F508 CFTR, aminogylcosides to suppress premature stop mutations) (12–15). Previous studies have indicated that certain phosphodiesterase inhibitors (PDEis) can activate CFTR-dependent Cl- transport in nonpolarized cells and CF mice, although demonstration of Cl- secretion in human subjects has been variable (6–11). No studies, however, have comprehensively evaluated the dose–response relationships between PDEis and CFTR activation, quantitatively examined these aspects using "in vivo"–like polarized airway cell monolayer systems, tested the spectrum of clinically approved agents at therapeutic drug levels for the ability to activate CFTR, or measured their possible synergy with A2B adenosine receptors. Studies that systemically evaluate available compounds that activate CFTR are also of particular interest, as these agents could have effects on CFTR function in airway cell types (such as glandular cells) that are not exposed to the cell surface. CFTR expression is high within submucosal glands, and recent studies highlight the importance of glandular cell function and Cl-/anion transport in airway mucociliary clearance (16–20).

In this article, we investigated the ability of several clinically approved PDEis to stimulate anion transport in Calu-3 cells, a cell line of serous glandular origin that expresses high levels of CFTR and A2B adenosine receptors. Our results provide new functional evidence that PDEs contribute to tonic regulation of CFTR in polarized Calu-3 cells, and that PDE inhibition can both activate vectorial Cl- transport and augment adenosine receptor activation of Cl- secretion. Collectively, these studies provide a rationale to further evaluate the effects of PDEis on CFTR and Cl- transport in animal models and humans.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
All cell lines were purchased from American Type Culture Collection (ATCC, Rockville, MD). Calu-3 cells were grown in MEM media (ATCC) + 10% FBS and 1% penicillin + streptomycin. 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 Ussing chamber experiments; Fisher Scientific, Pittsburgh, PA) were coated with human placental collagen extracellular matrix (Becton Dickinson, Franklin Lakes, NJ) at a concentration of 5 µg/cm2 overnight, and then seeded at ~ 1 x 106 cells/cm2. Once the filters were confluent, the media was removed from the apical surface and cells were fed only on the basolateral surface. Resistance was monitored, and the cells were studied when resistance was 300–500 {Omega} · cm2 (generally between 72 and 96 h after establishing an air–liquid interface, or 8–10 d post seeding).

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 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 95% O2 and 5% CO2. Solutions and chambers were maintained at 37°C. Short circuit current (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. For some measurements of stimulated Isc (gradient conditions) the mucosal bathing solution was changed to a low Cl- solution containing (in mM) 1.2 NaCl, 115 Na gluconate, and all other components as above. For studies of anion secretion under nongradient conditions, the mucosal and serosal compartments were continuously bathed with Ringer's solution. For all experiments, amiloride (100 µM) was added to the mucosal surface before stimulation. Amiloride generally had very small effects on Isc (< 1 µA), consistent with previous reports indicating that ENaC is expressed in Calu-3 cells at low levels (21). Increasing concentrations of PDEis were then added to the mucosal and serosal solutions as noted in the text. At the end of most experiments, 200 µM glybenclamide was added to the mucosal bathing solution, effectively blocking the majority of stimulated Isc (~ 90%). Studies investigating multiple conditions were carefully paired and completed on cells of similar age and passage [10–20]. Baseline Isc and Isc following the switch to low [Cl-] mucosal conditions was relatively low (examples shown in Figure 1B). Resting currents in Calu-3 cells have been reported to range from ~ 15–35 µA/cm2 (21–28). The reasons for these differences are uncertain, but may relate to differences in culture conditions, filters used, age of monolayers, buffers used in voltage clamp experiments, cell passage, endogenous cAMP levels, or other unidentified factors. Finally, although Cl- was the predominate anion used in our studies, it is important to note that other anions (such as HCO3-) may contribute to the Isc seen following PDEi or adenosine nucleotide stimulation in these cells (21, 29, 30).



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  		Figure 1. PDEis activate Cl- secretion in Calu-3 cell monolayers. Cells were grown at an air–liquid interface and studied in Ussing chambers under gradient conditions as described in MATERIALS AND METHODS. (A) All PDEis, except for theophylline, activated Isc in a dose-dependent manner ranging from 10–85% of the forskolin response (20 µM). Values represent the percent of forskolin (20 µM) response + SE for each condition (n = 4–8 filters/condition). All agonists were added to the mucosal and serosal compartments at 2 µg/ml (open bars), 20 µg/ml (gray bars), or 200 µg/ml (black bars). Papaverine was the most potent agent to activate Isc at low concentrations (2 µg/ml, *P < 0.02). Because data concerning therapeutic serum concentrations for rolipram were not available, this agent was studied at 1, 10, 100 µM{dagger}. (B) Examples of basal currents, and PDEi-activated Isc (200 µg/ml – arrows). Basal currents before and after the switch to low [Cl-] (mucosal) conditions were small (top left panel, LC) and minimally sensitive to amiloride (Am, 100 µM –mucosal) and glybenclamide blockade. Each pulse = 100 s. Inverted triangles, addition of glybenclamide (200 µM, mucosal). Mil, milrinone; Cil, cilostazol; Rol, rolipram; Sil, sildenafil; Pap, papaverine; Theo, theophylline. Horizontal lines in each PDEi-stimulated panel are basal currents after switch to low [Cl-] (mucosal) conditions.

 
cAMP Measurements
Cellular cAMP was measured using an enzyme-linked immunosorbent assay–based detection kit as previously described (Cayman Chemicals, Ann Arbor, MI [12]). Briefly, cells grown on 60-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.

Statistics
Descriptive statistics (mean and SEM) and tests of statistical significance were performed using SigmaStat software (Jandel, CA). Paired and unpaired t tests were used for studies with continuous, parametric data (Isc, Figures 1, 3, 4, 6, and 7) and the Mann–Whitney U test was used for studies with continuous nonparametric data (Figure 5).



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  		Figure 5. Additive effects of PDE inhibition and Ado-stimulated Isc. Calu-3 cell monolayers under gradient conditions were exposed to PDEis (2 µg/ml, mucosal and serosal) followed by Ado stimulation (1µM, mucosal, n = 4 for each PDEi). (A) Black bars: change in Isc after 5 min exposure to PDEi. Gray bars: change in Isc after 5 min exposure to Ado. Values are means ± SE of stimulated Isc. P values represent Ado stimulation alone compared with Ado with PDEi pretreatment (*P < 0.05, {dagger}P < 0.005). (B) Examples of PDEi- and Ado-stimulated Isc tracings. Each pulse = 100 s.

 
Materials
Adenosine hemi-sodium salt, forskolin, dipyridamole, AMP, adenosine deaminase (ADA), hexokinase (HEXO), and rolipram were purchased from Calbiochem (San Diego, CA). Theophylline, ATP, ADP, 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX), alloxazine, and milrinone were purchased from Sigma Chemicals (St. Louis, MO); papaverine HCl was purchased from Research Biochemicals International (Natick, MA). Cilostazol and sildenafil were obtained from the University of Alabama at Birmingham pharmacy.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PDEis Activate Isc in Calu-3 Cell Monolayers
In our first series of experiments, we screened several isotype-specific and nonspecific PDEis to activate Isc across Calu-3 cell monolayers grown at an air–liquid interface. These screening studies were completed in the presence of a serosal to mucosal [Cl-] gradient, thereby reducing the requirement of the PDEis to stimulate additional ion transport pathways (such as K+ channels) to produce vectorial anion transport. Calu-3 cells were studied because they have an airway serous gland phenotype, polarize, and express high levels of CFTR as well as receptors that couple to CFTR, including A2B adenosine receptors (5, 21–23, 25–34). An example of basal Isc, Isc following the switch to low [Cl-] (mucosal) conditions, and glybenclamide block (200 µM, mucosal) of the basal, unstimulated current is shown in Figure 1B (top left panel). Resting currents were small, suggesting that under the conditions of our studies, CFTR activity before agonist stimulation was low. PDEis were tested at 2, 20, and 200 µg/ml (n = 4–8 filters studied in each condition). The corresponding molar concentrations, therapeutic serum concentrations, and IC50 values are provided in Table 1. This strategy was adopted because serum concentrations (rather than molarity) are used most commonly to correlate drug dose with clinical effects in vivo. The lowest concentrations tested were well above the IC50 of the isotype-specific PDEis for their respective PDEs (10 to ~ 1,000-fold), and the concentration range was inclusive of the IC50 for the nonspecific PDEis. The concentrations used also encompassed therapeutic serum levels for the well-described PDEi theophylline, which has been used for decades to treat a variety of pulmonary disorders (therapeutic serum concentrations of 2–20 µg/ml). Theophylline (nonspecific PDEi), cilostazol (PDE3-specific), and sildenafil (PDE5-specific) are all orally bioavailable and routinely used in clinical medicine to treat non-CF pulmonary diseases and other disorders. We also studied two vasoactive agents that are dosed intravenously (papaverine, a nonspecific PDEi; and milrinone, a PDE3-specific inhibitor), and rolipram (PDE4-specific PDEi). Although PDE4-specific PDEis are not currently approved for human use in the United States, rolipram has been used as part of treatment for depression in Europe and Japan (35). Stimulated Isc was normalized to the percentage of the response produced by forskolin (20 µM), which directly stimulates adenylate cyclase, produces a global increase in cellular cAMP, and potently activates CFTR. At the end of experiments, glybenclamide (a CFTR channel blocker, 200 µM) was added to the apical surface of polarized monolayers (examples shown in Figure 1B), effectively blocking the stimulated Isc produced by the majority of PDEis.


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  		TABLE 1 Characteristics of isotype specific and nonspecific PDEis

 
Monolayers were exposed to increasing concentrations of agonists, and the Isc 10 min after stimulation were compared. In the presence of a serosal to mucosal [Cl-] gradient, papaverine, sildenafil, cilostazol, rolipram, and milrinone (but not theophylline) were able to activate Cl- secretion at 200 µg/ml ranging from ~ 20–85% of the forskolin response (Figure 1A). Nonspecific PDE inhibition with papaverine (2 µg/mL) stimulated Isc most efficiently, producing ~ 35% of the forskolin response at the lowest concentration tested (Figure 1, P < 0.02 compared with all other agonists). Theophylline, a xanthine-based nonspecific PDEi, failed to activate Cl- secretion at all doses studied. In general, all isotype-specific PDEis produced a dose-dependent increase in Cl- secretion, with milrinone achieving > 50% of the forskolin response at 20 µg/ml and close to 85% at the highest dose tested. These concentrations, however, were well above those tolerated clinically (100–300 ng/ml). Cilostazol (PDE3-specific) displayed a clear dose-dependent effect on Isc, achieving 14.92% (± 1.61 SE), 27.35% (± 5.91), and 39.07% (± 5.25) of the forskolin response at 2, 20, and 200 µg/mL, respectively. Dipyridamole, a PDEi with activity against PDE7, PDE8, and PDE11, failed to activate Isc at all concentrations tested (2, 20, and 200 µg/ml; data not shown). Although the majority of stimulated Isc produced by the different PDEis was blocked by glybenclamide (Figure 1B), Cl- secretion produced by high dose milrinone was only partially glybenclamide-sensitive (~ 50%), suggesting that it may have effects on Cl- secretion in Calu-3 cells that are independent of CFTR. The remainder of our studies concentrated on the PDEis that produced only glybenclamide-sensitive currents.

PDEi Effects on cAMP in Calu-3 Cells
To correlate PDEi-stimulated Isc with cAMP production, we measured cell cAMP in Calu-3 cells following PDEi exposure for 10 min at concentrations of 2, 20, and 200 µg/ml (Figure 2). cAMP production was again normalized to forskolin-stimulated cAMP (20 µM). All PDEis produced dose-dependent increases in cAMP concentration. All PDEis tested (except for sildenafil) increased cAMP concentration at 200 µg/mL, ranging from ~ 8–15% of that produced by forskolin. Interestingly, the levels of activated cAMP did not predict the Isc response in all cases. The differences between stimulated Isc and cAMP production were most pronounced at lower agonist concentrations. For example, low dose papaverine (2 µg/ml) produced less than 5% of the cAMP produced by forskolin, yet stimulated Isc to ~ 40% of the forskolin response (Figure 1). Milrinone and rolipram produced comparatively similar increases in cAMP at medium and high concentrations, but had very different effects on Isc. Cilostazol (20 µg/ml) activated Isc to a lesser extent than milrinone (Figure 1), but this was accomplished at cAMP concentrations that were below the sensitivity of our assay. Sildenafil had negligible effects on cAMP, but was nonetheless able to activate Isc at 20 and 200 µg/ml.



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  		Figure 2. PDEis modestly stimulate cAMP production in Calu-3 cells. cAMP levels were measured by ELISA as described (see MATERIALS AND METHODS). Cells were stimulated at 2 µg/ml (open bars), 20 µg/ml (gray bars), or 200 µg/ml (black bars) for 10 min before extraction (n = 4 dishes/condition). Values represent the percent of forskolin (20 µM) response ± SE for each condition.

 
Adenosine Nucleosides Activate Isc in Calu-3 Cell Monolayers through A2B Adenosine Receptors
Due to the interest in developing ways to augment residual or partial CFTR activity, coupled with recent evidence that Ado can serve this function, we were interested in determining whether PDE inhibition might be synergistic with Ado-activation of Cl- secretion (31). We began by characterizing Ado and Ado nucleoside activation of Isc in Calu-3 cell monolayers. The subsequent series of studies was designed to test whether PDE inhibition could augment receptor activation of Isc. We focused our studies on purinergic receptors, because stimulation of receptors within this broad class by adenosine nucleosides represent important paracrine signaling mechanisms used by cells to activate Cl- secretion (36–39). P1 purinoreceptors show a rank order of potency for adenosine nucleosides of Ado > AMP > ADP > ATP, and frequently stimulate (A2A and A2B subtypes) or inhibit (A1 and A3 subtypes) adenylate cyclase and cell cAMP. P2 receptors exhibit the reverse rank order of potency (40, 41), and members of this class have been shown to produce Cl- secretion through elevation of cell calcium (42, 43). One member of the P1 purinergic receptor family (the A2B adenosine receptor) has been linked to CFTR regulation in Calu-3 cells (5, 31, 37). To investigate the predominate purinoreceptor class used by adenosine nucleosides to activate Isc in Calu-3 cells, monolayers were stimulated with ATP, ADP, AMP, and Ado (all agonists were studied at 100 µM; Figure 3, left arrow), followed by treatment with HEXO and ADA. These studies (Figure 3), and those described for Figure 4 were performed under nongradient conditions (i.e., symmetric Ringer's buffer in the mucosal and serosal compartments). HEXO catalyzes the conversion of ATP to ADP (in the presence of glucose), whereas ADA catalyzes the conversion of adenosine to the inactive nucleoside inosine. Figure 3 shows that Cl- secretion produced by all four nucleosides was unaffected by HEXO, but markedly inhibited by ADA treatment (2 U/ml, stimulated Isc produced by the adenosine nucleosides inhibited 38–71%; the remaining currents likely represent the activity of ADP and AMP on purinergic receptors). Currents produced by all of the Ado nucleosides were also glybenclamide sensitive (200 µM – mucosal, data not shown), consistent with CFTR dependence. Subsequent studies investigated the effects of the A2B adenosine receptor antagonist alloxazine on adenosine nucleoside–stimulated Isc (44, 45) (Figure 4, arrows). Alloxazine also had similar inhibitory effects on Isc produced by all of the adenosine nucleosides (50 µM, stimulated Isc produced by the adenosine nucleosides inhibited 35–78%). Subsequent treatment with the A1 adenosine receptor antagonist DPCPX (1 µM) had minimal effects on the remaining Isc. These results, coupled with previous reports identifying A2B adenosine receptors in Calu-3 cells (31), suggest that a predominant mechanism used by adenosine nucleosides to activate Cl- secretion in these cells is through stimulation of A2B adenosine receptors. These effects could occur either after conversion to Ado by surface ectonucleotidases, or by direct adenosine receptor stimulation with higher concentrations of phosphorylated adenosine nucleosides. Ado-stimulated Isc likely represents A2B receptor-coupled activation of CFTR (5, 31), and may also include effects of Ado on other transport pathways (e.g., K+ channels, Na+-K+-2Cl- cotransporters) that in turn promote anion secretion.



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  		Figure 3. Adenosine deaminase inhibits Isc produced by adenosine nucleosides. Calu-3 cell monolayers were studied under nongradient conditions, and stimulated with adenosine nucleosides at the first arrow (closed circles, Ado; open squares, AMP; closed triangles, ADP; open diamonds, ATP; 100 µM each, mucosal and serosal). Isc at various time points is shown. Hexokinase (2 U/ml, mucosal and serosal) and adenosine deaminase (ADA, 2 U/ml, mucosal and serosal) were added at the arrows as indicated. ADA reduced adenosine nucleoside-stimulated Isc produced by all agonists (P <= 0.003, Isc before ADA compared with Isc after ADA for each nucleoside). Values represent the mean ± SE, n = 6 filters in each condition.

 


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  		Figure 4. A2B adenosine receptor blockade with alloxazine inhibits Isc produced by adenosine nucleosides. Calu-3 cell monolayers were studied under nongradient conditions, and stimulated with adenosine nucleosides at the first arrow (closed circles, Ado; open squares, AMP; closed triangles, ADP; open diamonds, ATP; 50 µM each, mucosal and serosal). Isc at various time points is shown. Alloxazine (50 µM, mucosal and serosal) and DPCPX (1 µM, mucosal and serosal) were added at the arrows as indicated. Alloxazine reduced adenosine nucleoside-stimulated Isc produced by all agonists (P <= 0.04, Isc before alloxazine compared with Isc after alloxazine for each nucleoside). Values represent the mean ± SE, n = 5–6 filters in each condition.

 
The observations above provided a rationale to investigate the additive effects of PDE inhibition on Ado-activated Cl- transport. For these studies we stimulated Calu-3 cell monolayers with each PDEi at a low dose (2 µg/ml, mucosal and serosal) for 5 min before stimulation with Ado at concentrations that modestly activate Isc (1 µM mucosal, Figures 5A and 5B). Stimulated Isc 5 min after PDEi addition (black bars) and 5 min after Ado addition (hatched bars) are shown (Figure 5A). Papaverine had a strong additive effect on Ado-stimulated Isc (P < 0.005 compared with Ado only), whereas cilostazol, and to a lesser extent rolipram, produced smaller additive effects with Ado (P < 0.05 compared with Ado only). These three PDEis alone were also able to acutely simulate Isc at 2 µg/ml (whereas sildenafil and theophylline did not) with minimal effects on cAMP production (Figures 2 and 5B).

Papaverine and Cilostazol Stimulate Isc and Augment Ado-Activated Isc
The results from the above studies indicate that several PDEis can activate Cl- secretion in Calu-3 cell monolayers when provided a favorable Cl- secretory gradient (Figures 1 and 5), and that at least two, including cilostazol and papaverine, accomplish this near clinically achievable serum levels. We next investigated the ability of these PDEis to activate Isc in Calu-3 cells under nongradient conditions. The PDEis were studied at 500 ng/ml, a concentration that is ~ 50% of the reported peak active serum level for both cilostazol and papaverine (46). Figure 6 summarizes the time course of Isc produced by both agents compared with Ado (50 µM, mucosal and serosal), which produces maximal Cl- secretion under these conditions that is comparable to forskolin stimulation (data not shown). Both PDEis at low concentrations produced sustained Isc that was > 60% of that produced by Ado at 45 min. To characterize the ability of these PDEis to augment Cl- secretion produced by Ado, monolayers were stimulated with the PDEis for 30 min, and then stimulated with submaximal Ado (Figure 7A, 1 µM Ado, mucosal and serosal) or maximal Ado (Figure 7B, 50 µM Ado, mucosal and serosal). As an additional control PDEi condition, monolayers were also stimulated with sildenafil at half-maximal serum concentration (250 ng/ml, mucosal and serosal). Figure 7A shows that both cilostazol and papaverine augmented Isc stimulated by submaximal Ado, doubling both the peak (P <= 0.05) and the sustained response (P <= 0.001). Sildenafil, in contrast, had no stimulatory effect when combined with Ado. Pretreatment with the combination of cilostazol (500 ng/ml) and sildenafil (250 ng/ml) together failed to augment Ado-stimulated Isc above that produced by cilostazol + Ado (data not shown). cAMP measurements (pMol/1 x 106 cells ± [SE]) following 30 min of costimulation with PDEi and 1 µM Ado correlated with the findings by Isc: (blank = -8.19 [5.13]; Ado = 7.58 [17.79]; Ado + cilostazol = 49.42 [15.98]; Ado + papaverine = 59.79 [9.41]; Ado + sildenafil = -24.05 [19.96]; n = 4 dishes/condition). In the presence of maximal stimulation with Ado (50 µM), the augmenting effect produced by the PDEis was not observed (Figure 7B), suggesting a common Cl- transport pathway used by the two classes of agonists. Because Ado normally binds to A2 adenosine receptors in the low micromolar range (0.5–20 µM), these results suggest that PDEis may work together with A2 adenosine receptor signaling to activate CFTR as part of normal cellular regulation of CFTR (40).



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  		Figure 6. Isc measurements in Calu-3 cell monolayers stimulated with PDEis (open squares, cilostazol; open triangles, papaverine; both were studied at a concentration of 500 ng/ml) compared with Ado, closed diamonds. (50 µM). Experiments were conducted here and in Figure 7 under nongradient conditions. All agonists were added to both the mucosal and serosal surface as indicated. Time course of stimulated Isc produced by cilostazol (Cil) and papaverine (Pap) above basal currents (hatched line) compared with Ado. Both PDEis produced a prolonged (> 45 min) response compared with 50 µM Ado alone (n = 6 filters for each condition, values are mean ± SE).

 


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  		Figure 7. Isc measurements in Calu-3 cell monolayers stimulated with PDEis (cilostazol and papaverine, 500 ng/ml; sildenafil, 250 ng/ml) and Ado at 1.0 µM (A) and 50 µM (B). Closed diamonds represent Ado treatment alone, open squares include Cil and Ado, open diamonds Pap and Ado, and closed circles are Sil and Ado stimulation. All agonists were added to both the mucosal and serosal surface as indicated. (A) Time course of Ado-stimulated Isc alone compared with Ado-stimulated Isc following prestimulation with PDEis (Cil, Pap, Sil). Pretreatment with cilostazol and papaverine augmented the Isc response to Ado stimulation (*P <= 0.05, {dagger}P <= 0.001) compared with Ado alone (n = 6 filters for each condition, values are mean ± SE). (B) Stimulation with maximal Ado (50 µM) overcomes additive effects of the PDEis (n = 6 filters for each condition, values are mean ± SE).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this article, we compared the ability of several clinically relevant PDEis to activate Isc in a polarized, CFTR-expressing airway epithelia. Using Calu-3 cells grown as monolayers, we demonstrated that several PDEis (including a nonspecific PDEi and several isotype-specific PDEis) could activate CFTR-dependent Cl- transport in a dose-dependent manner. PDEi-stimulated Isc did not correlate closely with changes in total cellular cAMP. We also found that two PDEis had the capacity to activate Cl- secretion alone, and produced additive effects on the magnitude and duration of Ado-stimulated Isc under nongradient conditions (cilostazol and papaverine, Figures 6 and 7). These effects were demonstrated at concentrations well within therapeutic serum drug levels (Table 1). Together, these results help delineate the physiologic role of PDEs as regulators of CFTR in polarized Calu-3 cells. They indicate that inhibition of PDE activity is sufficient to activate CFTR and vectorial Cl- transport, and that these enzymes also play an important role in the pathway that governs Cl- secretion produced by P1 purinergic receptor stimulation. Our results also extend our previous studies of PDE inhibition in nonpolarized cell systems (12, 31), and provide a rationale for future studies using available agents in polarized epithelia and in vivo.

The studies described in this article were designed to investigate the effects of several PDEis on Isc using a systematic approach based on active serum levels and increasing stringency (i.e., gradient and nongradient conditions). The results from Figure 1 show that several PDEis can activate CFTR-dependent Isc in polarized airway cell monolayers. The stimulated currents (except for Isc produced by milrinone) were sensitive to glybenclamide blockade (200 µM, mucosal), providing evidence that CFTR activity was responsible for the observed Cl- transport. The dose–response profile of some of these agents, however (such as milrinone and sildenafil, 2 µg/ml), suggest that they would be unlikely to have effects on CFTR activity at clinically tolerated concentrations (see Table 1). Many PDE subtypes, including members of the PDE2, PDE3, PDE4, PDE5, PDE6, PDE7, PDE8, PDE9, and PDE10 families, have been shown to be expressed in mammalian airways and/or lungs by either Northern blot analysis or RT-PCR (47–58). Of these isotypes, PDE3 has most consistently been demonstrated to regulate CFTR activity based on functional studies. Although our studies do not exclude additional PDEs contributing to CFTR regulation, the results seen with the PDE3 inhibitor cilostazol are consistent with previous observations implicating this isotype in PDE CFTR regulation (6, 7, 11, 59).

A primary goal of our investigations was to determine whether clinically available PDEis could influence CFTR activity and Cl- secretion in airway cell monolayers at concentrations observed as part of systemic PDEi therapy. Cilostazol produced moderate and prolonged activation of Isc at concentrations well below peak serum levels that are achieved as part of oral treatment of peripheral vascular disease (500 ng/ml; Figure 6 and Table 1). Furthermore, after confirmation of a primary role for P1 purinergic receptor signaling in adenosine nucleoside activated Isc (Figures 3 and 4), cilostazol augmented submaximal Ado-activated Isc in Calu-3 cells in terms of both magnitude and duration (Figure 7A). These effects on Ado-stimulated Isc were demonstrated at relatively low Ado concentrations (1 µM), suggesting that cilostazol may have the capacity to influence Cl- secretion that occurs as part of normal in vivo signaling. Papaverine had similar effects on Isc below peak serum levels, and augmented Isc produced by submaximal Ado stimulation (Figures 5, 6, and 7). This PDEi, however, requires intravenous administration for clinical effect, and active levels represent local and not systemic concentrations (60). It is therefore less likely that this agent as currently used would be able to modulate CFTR activity in human subjects. To date, 26 PDE genes within 11 isotype families have been identified. Our results with papaverine were somewhat unexpected, and suggest that in contrast to previous reports implicating PDE3 regulation of CFTR (7, 59), additional PDEs may play a role in regulating CFTR in airway serous glandular cells. In our model system, papaverine was capable of activating sustained Isc almost 10-fold above a PDE4-specific PDEi, (Figure 5), and this agent showed the highest potency of all PDEis tested under gradient conditions (Figure 1).

The results from Figures 1 and 2 demonstrate that many PDEis can activate Cl- secretion in the setting of very low (and often undetectable) cAMP levels. Although this could suggest that cAMP-independent mechanisms contribute to PDEi-activated Isc, we favor the interpretation that the different PDEis regulate cAMP pools near CFTR. First, several PDEis alone and at low concentrations (5–10 µM, Table 1) were able to acutely activate Cl- secretion in Calu-3 cell monolayers (Figure 5B). These results suggest that PDEs provide tonic negative regulation to CFTR through reduction of local cAMP, and that a threshold, localized elevation of cAMP (through PDE inhibition) is adequate to activate CFTR. This same pattern was observed with most of the PDEis tested and over a variable range of concentrations. Activation was seen despite structural differences between the compounds, arguing against idiosyncratic affects that produce CFTR activation independent of PDEi activity. Moreover, a number of recent studies in other model systems point toward PDEs participating in compartmentalized protein regulation. For example, PDE3A and 3B have six predicted transmembrane helices, a requirement for binding to the membrane fraction of the endoplasmic reticulum (61). Additionally, certain PDE4 subtypes contain proline-rich SRC homology 3 (SH3)-binding motifs in their amino terminal tails, which could allow interactions with anchoring proteins possessing SH3 domains (62, 63). Finally, PDE6A, B, and C (expressed predominantly in the retina), like CFTR, have a putative PDZ binding domain that may be important for targeting to specific intercellular regions (64). Future studies that investigate the nature of PDE:CFTR compartmentalization and regulation may, therefore, be of particular interest. Our results suggest that removal of tonic negative regulation imparted by PDEs can acutely increase resting CFTR activity in the absence of specific, cAMP-elevating stimuli.

In summary, our data demonstrate that PDEis alone can activate CFTR-dependent Isc across airway cell monolayers. Inhibition of PDE activity can also have additive effects on Isc when combined with A2 adenosine receptor stimulation, increasing the duration and magnitude of receptor-based activation of Cl- secretion. For some PDEis, this was accomplished at concentrations used currently to treat non-CF disorders. These studies support a strategy using endogenous cell signaling pathways to augment surface localized CFTR activity and Cl- secretion, and provide a rationale for further studies of novel PDEis in CF mice and humans.


   Acknowledgments
 
The authors thank Dr. Cathy Fuller and Dr. Kevin Kirk for helpful discussions about this manuscript. This work was supported by NIH-NHLBI RO1 HL67088-02, NIH-NIDDK P50 DK53090, NIH-NIDDK P30 DK54781, and CFF R464. J.P.C. is a Leroy Matthews Award Recipient (CFF).

Received in original form November 12, 2002

Received in final form April 16, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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E. R. Lazarowski, R. Tarran, B. R. Grubb, C. A. van Heusden, S. Okada, and R. C. Boucher
Nucleotide Release Provides a Mechanism for Airway Surface Liquid Homeostasis
J. Biol. Chem., August 27, 2004; 279(35): 36855 - 36864.
[Abstract] [Full Text] [PDF]