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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Merck Frosst Centre for Therapeutic Research, Pointe Claire–Dorval, Quebec, Canada
Received January 11, 2005; accepted April 29, 2005.
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Abstract |
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). Most CFTR mutations lead to either a reduced expression or proteins with a compromised chloride conductance in response to physiological stimuli. The major disease mutation 
F508-CFTR with a decreased epithelial expression in CF patients (Kalin et al., 1999) remains partially functional with a reduced open probability and sensitivity to stimulation by cAMP agonists (Wang et al., 2000a). The functional defects of CFTR mutants result in multiple organ dysfunctions, including a severely impaired airway mucociliary clearance that leads to chronic secondary bacteria infections and respiratory failure in CF patients.
It is being increasingly recognized that there is an elevated inflammation in CF patients, which plays a major role in the pathogenesis of CF lung disease (Chmiel et al., 2002). Early pulmonary inflammation with neutrophilia can be detected in CF infants before the onset of bacterial colonization (Khan et al., 1995). Leukotriene B4 is elevated in the epithelial lining fluid of CF patients (Konstan et al., 1993). CF tissues have abnormally high levels of proinflammatory arachidonic acid with values of the heterozygous parents halfway between CF patients and healthy controls (Freedman et al., 2004). These data suggest the possible presence of an excessive eicosanoid-based inflammatory response in CF patients and CF carriers. Diseases associated with CFTR mutations continue to widen. In addition to the classic CF, approximately 3.3% of the U.S. population are CF carriers who are predisposed to a number of related airway diseases, including chronic bronchitis and rhinosinusitis (Kostuch et al., 2000; Wang et al., 2000b).
The key regulatory event for CFTR activation is the PKA-mediated serine phosphorylations of its R-domain, which facilitates its ATP binding, with the sequential ATP hydrolysis triggering the channel opening and closing. CFTR is dynamically regulated by the activities of adenylyl cyclase (AC) and cAMP-phosphodiesterase (PDE) through the activation of PKA. Multiple signaling elements, including receptors, G proteins, AC, PKA, CFTR, and protein phosphatase 2C cluster at the apical membrane of epithelial cells to ensure the specificity of the cAMP-mediated CFTR activation (Zhu et al., 1999; Sun et al., 2000; Huang et al., 2001a). cAMP hydrolysis in airway epithelial cells is mainly regulated through PDE4s and PDE3s (Wright et al., 1998). There are four PDE4 (4A to 4D) and two PDE3 (3A and 3B) genes, each producing multiple spliced variants with specific tissue distribution and subcellular localization. PDE4s are abundantly expressed in proinflammatory cells and airway epithelial cells. PDE4 inhibition attenuates the overproduction of many proinflammatory mediators and cytokines, including arachidonic acid, leukotrienes, reactive oxygen species, and tumor necrosis factor-
, and suppresses the infiltration of neutrophils and eosinophils in inflamed airways (Torphy, 1998). PDE4D, the major cAMP-PDE species in bronchial epithelia, plays a dominant role in controlling airway smooth muscle contraction (Mehats et al., 2003). A number of second-generation PDE4 inhibitors, exemplified by roflumilast [3-(cyclopropyl-methoxy)-N-(3,5-dichloropyridin-4-yl)-4-di-fluoromethoxy benzamide], are presently in advanced clinical development and may emerge as new therapeutics for asthma and COPD (Huang et al., 2001b).
Restoring CFTR activity and simultaneously reducing the excessive airway inflammatory response may represent a promising strategy in CF treatment. In addition, enhancing the mucociliary clearance via CFTR activation may reduce the respiratory disorders among CF carriers and improve the lung function of COPD patients. This partly stems from the observation that 1) levels of the residual CFTR activity seem to be predictive of the CF disease severity; and 2) cAMP-elevating agents, either through receptor stimulation, AC activation, or nonselective PDE or PDE3 inhibition, activated the wild type-CFTR and partially restored the defective chloride conductance of many CFTR mutants, including F508-CFTR from CF mice and patients (Haws et al., 1996; Kelley et al., 1997; Al Nakkash and Hwang, 1999). Effects of PDE4 inhibitors on CFTR activity had been controversial. Rolipram [4-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-pyrolidinone] and RP-73401 were either inactive or weakly modulated the chloride conductance of Calu3 and T84 cells under conditions where PDE3 inhibition potently increased the short-circuit current measurement (Kelley et al., 1995; O'Grady et al., 2002; Cobb et al., 2003), whereas RS25344 significantly augmented the adenosine-induced apical anion conductance in Calu3 cells (Barnes et al., 2005). By directly monitoring the chloride secretion using 125I as tracer in T84 monolayer, we report here that PDE4 inhibitors, particularly the PDE4D-selective inhibitors, potently augmented CFTR-mediated iodide secretion with their efficacy coupled to the AC activation state.
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Materials and Methods |
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; Reid, 2002; Claveau et al., 2004). Forskolin, H89, and trequinsin [9,10-dimethoxy-2-mesitylimino-3-methyl-2,3,6,7-tetrahydro-(6,1-a)-isoquinolin-4-one, HCl] were from Calbiochem (San Diego, CA). Sodium iodide (125I) with a typical specific activity of 2000 Ci/mmol was from Draximage Inc. (Kirkland, QC, Canada). Cell Culture. T84 cells (American Type Culture Collection, Manassas, VA) were grown as a monolayer in a 1:1 mixture of Dulbecco's modified Eagle's medium/F-12 supplemented with 5% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. The epithelial cells (1 x 106 cells/well) were plated onto the 9.5-cm2 culture dishes and incubated at 37°C in a humidified atmosphere of 5% CO2 in air. Culture medium was changed every 48 h. It becomes confluent in 4 to 5 days. Only cell viability exceeded 90% by trypan blue exclusion was used for further experiments.
125I Efflux Assay. Confluent T84 monolayer in six-well dishes with a density of 4 x 106 cells/well was labeled with 5 µCi/ml Na125I in 1 ml of HPBR buffer (135 mM NaCl, 5 mM KCl, 3.33 mM NaH2PO4, 0.83 mM Na2HPO4, 1 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 5 mM HEPES, pH 7.2) for 30 min at 37°C. Labeled cells were washed with 3 x 5 ml of HPBR buffer within 30 s to completely remove extracellular radioactivity. Efflux of the intracellular 125I was sampled by replacing 80% (0.8 ml) of the incubation buffer with fresh buffer every 30 s up to 9 min. Activators were added via the assay buffer immediately after the third buffer replacement at 1.5 min. Temperature was maintained at 37°C. NaOH (1 ml, 0.1 N) was added after 9 min to lyse the cells by sonication (2 min, Branson model 2510 bath sonicator). The radioactivity in each time point and the final cell lysate were determined on a Microbeta liquid scintillation counter in 24-well format. The maximal forskolin-induced efflux rate over the DMSO control typically ranged from 0.35 to 0.45 min-1 for cells up to 10 passages. Studies investigating multiple conditions were paired using cells of same passage.
cAMP Measurement. Confluent T84 cells in 96-well plate were incubated with drug in 200 µl of efflux assay buffer at RT. After quenched with 20 µl of 1 N HCl, cells were lysed by sonication for 2 min. The acidic lysate (20 µl) was neutralized with 80 µl of 250 mM Tris-HCl, pH 7.5, and its cAMP levels were quantified using the cAMP-Biotrak SPA screening assay kit (Amersham Biosciences, Inc.) per manufacturer's directions.
PDE Activity Assay. Confluent T84 cells (
100 x 106) were harvested by trypsinization and washed twice using ice-cold phosphate-buffered saline by centrifugation. Cells were suspended in 1 ml of a buffer containing 50 mM HEPES, pH 7.5, 5 mM EDTA, 0.05 mM dithiothreitol, 1 mM N--benzoyl-L-arginine ethyl ester, 1 mM benzamide, 10 µg/ml pepstatin A, and Complete EDTA-free proteases inhibitors tablet. After sonication (5 x 10-s pulses; power setting 3; 4°C), the lysate was collected after a 10,000g spin (30 min at 4°C). The lysate cAMP-PDE activity was determined by monitoring the hydrolysis of 0.1 µM [3H]cAMP in 10 mM MgCl2 and 50 mM HEPES, pH 7.2, using the SPA-PDE assay kit from Amersham Biosciences, Inc. (Laliberte et al., 2000). The potencies of inhibitors against multiple PDEs in Table 1 were determined using 0.1 µM [3H]cAMP for PDE1, PDE4s, PDE7A, and PDE8B and 0.01 µM [3H]cAMP for PDE3A and PDE3B under the same assay conditions as detailed previously (Claveau et al., 2004). Under the conditions, they were close to the apparent Ki. PDE1 was purified from dog heart, and other PDEs were human recombinant enzymes.
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Phosphor-CREB Induction and Western Blot Analysis. Confluent T84 monolayer in six-well plate at 37°C was incubated with compounds or DMSO in efflux buffer for specified time. After the removal of incubation buffer, 300 µl of ice-cold lysis buffer [50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na4P2O7, 0.1 mM vanadate, 250 µM H2O2 plus protease inhibitor (EDTA-free) cocktail tablet] was added. Cells were lysed in the bath sonicator (2 min/4°C). Supernatant was collected after a 10,000g centrifugation (10 min/4°C) and concentrated by Speed-Vac. Approximately 100 µg of protein was loaded per lane on SDS-polyacrylamide gel electrophoresis under denaturing condition. After electrophoresis, proteins were transferred to polyvinylidene difluoride membranes, and blocked with 5% skim milk in TTBS [20 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 0.1% (v/v) Tween 20] for >60 min. After briefly washing in TTBS, it was incubated with 1:1000 anti-phosphor-CREB (pCREB) (Ser-133, polyclonal; Cell Signaling Technology Inc., Beverly, MA) in 5% bovine serum albumin/TTBS overnight at 4°C. After 3 x 15 min washes with TTBS, the membranes were incubated with 1:10,000 anti-rabbit IgG horseradish peroxidase (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in 5% skim milk/TTBS for 1 h at RT. After 3 x 15 min washes with Tris-buffered saline containing 0.3% Tween 20, it was developed with the ECL kit (Amersham Biosciences, Inc.) and digitized with LAS-1000 Plus Image Reader (Fuji Photo Film Co. Ltd., Tokyo, Japan). For detecting CREB, the membrane was stripped using Restore stripping buffer (Pierce Chemical, Rockford, IL) at RT for 15 min and washed 3 x 15 min with TTBS. After blocking with 5% skim milk/TTBS for 1 h, the membrane was incubated with 1:1000 polyclonal anti-CREB (Cell Signaling Technology Inc.) and detected using the same secondary antibody as described above.
Data Analysis. 125I efflux rate was calculated following [In(R2)–In(R1)]/(t2 - t1), where Rx is the percentage of radioactivity remaining in the monolayer at time tx as described previously (Rogers et al., 1990). Data are expressed as mean ± S.E. of three or more independent experiments unless otherwise specified. Dose-response curves and IC50 values were analyzed by a nonlinear iterative regression routine with Grafit (Erithacus Software, Horley, Surrey, UK).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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; Sun et al., 2000; O'Grady et al., 2002). Previous studies also established that its cAMP-mediated chloride secretion, sensitive to the overexpression of a mutant PKA, occurred primarily through CFTR channel (Rogers et al., 1990; Bell and Quinton, 1992). In addition, T84 monolayer possesses several key characteristics that are similar to native secretory epithelial cells, including the formation of tight junctions and the maintenance of a vectorial chloride transport in the confluent stage. Thus, it provides the advantage of a nonrecombinant system to study CFTR regulation in response to AC activation and PDE inhibition (Halm et al., 1988).
To dissect the expression of cAMP-PDEs in T84 cells, the hydrolysis of cAMP by T84 lysate was titrated with the PDE4 inhibitor L-826,141 (Cpd-A), roflumilast, and the PDE3 inhibitor trequinsin. As summarized by their intrinsic potencies against the multiple recombinant cAMP-PDEs in Table 1, Cpd-A and roflumilast are potent PDE4 inhibitors, with IC50 values ranging from 0.2 to 2.4 nM and from 0.1 to 0.6 nM for the inhibition of PDE4A, 4B, 4C, and 4D, respectively. Cpd-A and roflumilast are >400- and 10,000-fold more potent than their weaker PDE3 inhibition, respectively (Claveau et al., 2004). Trequinsin, with IC50 value of 0.05 nM against PDE3A and 3B, is >6000-fold more potent compared with its weaker PDE4 inhibition. The inhibition of the cAMP-PDE activity of T84 lysate by Cpd-A and roflumilast were biphasic (Fig. 1A). Each inhibited 55% of the total activity during the first titration phase, with inflection points at 0.4 and 0.1 nM, respectively. These values were consistent with their intrinsic potencies against the recombinant PDE4s listed in Table 1. Approximately 30% of the total activity was further inhibited by higher concentrations of Cpd-A with a second inflection point near 3000 nM, which is likely the consequence of its weaker PDE3 inhibition. This is supported by the biphasic titration of trequinsin from its potent PDE3 inhibition and weaker PDE4 inhibition in Fig. 1B. Its first and second titration phases each eliminated 30 and 55% of the total activity with the corresponding inflection points at 0.1 and 400 nM, respectively. Thus, PDE4 and PDE3 accounted for approximately 55 and 30% of the cAMP-PDE activity, respectively, in T84 lysate under the assay conditions. Most of the remaining activity (10 to 15% of the total) was inhibited by 10 µM 3-isobutyl-1-methylxanthine, a nonselective PDE inhibitor. This was thought to be contributed by PDE1 from its sensitivity toward 8-me-thoxymethyl-3-isobutyl-1-methylxanthine (O'Grady et al., 2002). The abundant PDE4 and PDE3 expression in T84 cells compared well with that in airway epithelial cells.
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Forskolin-Stimulated Dynamic 125I Efflux in T84 Cells. To monitor chloride secretion in T84 cells, we selected the well established 125I- efflux assay for its sensitivity and robustness. Previous head to head studies comparing 125I- and 36Cl- have established that 125I- is an ideal alternative for the highly penetrating 36Cl- in monitoring chloride secretion from T84 cells (Venglarik et al., 1990). After removing extracellular 125I- through extensive buffer washing, the efflux of intracellular 125I- into the incubation media was continuously monitored every 30 s by replacing only 80% of the incubation buffer with fresh buffer over a 9-min period. The partial buffer replacement minimized physical perturbation to the monolayer from repeated buffer exchange, thus significantly improved data reproducibility over that of previous procedures. Activators were added via the efflux buffer at 1.5 min, with their concentrations maintained throughout the remaining time course. More than 85% of the intracellular 125I- was released into the culture media over the 9-min duration in response to a saturating concentration (10 µM) of forskolin stimulation (Fig. 2A, 
). More than 90% of the released radioactivity comigrated with authentic 125I- by high-performance liquid chromatography analysis, indicating a negligible biotransformation of the tracer. Compared with the spontaneous efflux (Fig. 2B, (
), significantly elevated 125I- efflux slowly emerged after >5.5 min incubation with 0.1 µM forskolin (Fig. 2B, Fsk, ). Higher forskolin concentrations elicited a faster and transient increase in 125I efflux in a dose-dependent manner that remained elevated at approximately 2 to 3 times that of the spontaneous level at the end of the 9-min period. The peak efflux rate extrapolated from the efflux curves saturated in response to increasing forskolin concentrations with an EC50 of 0.55 µM. In addition, the increased forskolin concentration progressively reduced the latency to reach the maximum efflux response (response time) as marked in Fig. 2B. These results are consistent with the presence of a tightly coupled AC-cAMP-CFTR signaling in T84 cells, reflecting a progressively activated CFTR from increased AC activation by forskolin. The iodide secretion peaked within 60 s after 30 µM forskolin stimulation, illustrating the rapid responsiveness of the system. The efflux rate peaked and then fell rapidly above 1 µM forskolin. The later phase of the biphasic response that causes the efflux rate to decline could be contributed either by tracer depletion, local ATP depletion, or desensitization. The forskolin-stimulated iodide efflux and its responsiveness in T84 cells echoed that in CFTR-overexpressing cells (Haws et al., 1996).
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Synergistic CFTR Activation from Dual PDE3 and PDE4 Blockade under the Basal AC State. In the absence of forskolin, the iodide efflux was insensitive to the presence of up to 10 µM Cpd-A or 1 µM trequinsin, with their efflux curves overlapped with the spontaneous efflux of the cell (Fig. 3, 
). Since these concentrations were >1000-fold above their intrinsic PDE4 and PDE3 potencies, respectively, a complete suppression of PDE4 and PDE3 activity would be expected under the conditions, even after considering the potentially reduced potency from increased protein binding in the whole cell environment. Yet, the ablation of PDE3 or PDE4 activity alone, under the basal AC state, was insufficient to activate the iodide secretion. On the other hand, dual PDE4 and PDE3 blockade (10 µM Cpd-A plus 1 µM trequinsin; Treq) induced a significantly elevated iodide secretion (Fig. 3, 
), yielding an efflux curve that has a comparable peak efflux rate but with a prolonged duration to that elicited by 0.3 µM forskolin alone (Fig. 3, 
). These data suggest that dual suppression of PDE3 and PDE4 activity is synergistic, instead of additive, in stimulating the cAMP-mediated CFTR activation under the basal AC state with a limited cAMP turnover.
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Increased Efficacy of PDE3 and PDE4 Inhibitors upon Adenylyl Cyclase Activation. In contrast to the lack of efficacy under the basal AC state, PDE3 or PDE4 inhibition each potently augmented iodide secretion after forskolin stimulation. The presence of 0.1 µM forskolin, which only slightly increased the efflux after >5.5 min incubation (Fig. 2B, ), significantly increased the efficacy of 1 µM Cpd-A by elevating the efflux rate to 0.055 min-1 over the vehicle control, with the enhanced efflux emerged after 3 min instead. Under the same AC state, 0.1 and 1 µM trequinsin augmented the peak efflux to 0.09 and 0.16 min-1, respectively. Further AC activation by increasing forskolin to 0.3 µM amplified the responsiveness of the iodide secretion toward PDE4 and PDE3 inhibition, as represented by the efflux curves of Cpd-A (Fig. 4A) and trequinsin (Fig. 4B). Cpd-A (3 nM) (Fig. 4A, ) or 1 nM trequinsin (Fig. 4B, ) each significantly prolonged the iodide efflux induced by 0.3 µM forskolin (Fig. 4, A and B, ), and 3 nM roflumilast elicited a similar response to that of Cpd-A (data not shown). Higher inhibitor concentrations augmented the peak efflux rate in a dose-dependent and biphasic manner, with the response of Cpd-A nearly superimposed onto that of roflumilast (Fig. 4C). The first phases of Cpd-A and roflumilast's biphasic responses elevated the peak efflux rate by 0.06 min-1 over the forskolin response, with inflection points between 2 to 5 nM. Their second response phases started above 1000 nM, increasing the peak efflux rate by another 0.09 min-1. In comparison, trequinsin augmented the peak efflux rate by 0.09 min-1 over the forskolin response with an initial inflection point between 1 to 3 nM. Its second response phase occurred above 100 nM, elevating the peak efflux rate by another 0.05 min-1, which is comparable with that derived from PDE4 inhibition by Cpd-A or roflumilast. The potentiating effects through PDE3 or PDE4 inhibition start to diminish at higher AC activation states, with a minimal augmentation detected at or above 10 µM forskolin (data not shown).
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) which was comparable with that from a full AC activation by 10 µM forskolin (Fig. 5, 
). The combination of higher PDE3 and PDE4 inhibitor concentrations did not augment the peak efflux rate nor shorten the response time further, supporting that PDE3 and PDE4 are mainly responsible for degrading the cAMP pool involved in CFTR activation in T84 cells. Compared with forskolin stimulation, there remains a delayed response in activating the chloride secretion from blocking cAMP degradation through PDE3 plus PDE4 inhibition. The exact timing difference remains to be quantified with a faster sampling protocol.
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CFTR Activation, Paralleled with Phosphor-CREB Formation, Is Blocked by H89. pCREB induction is a surrogate for PKA activation from cAMP elevation. The CFTR activation through PDE4 inhibition was compared with increased pCREB formation under the same condition. Treatment of T84 cells with 1 µM Cpd-A alone caused no pCREB induction, echoing its negligible stimulation of iodide secretion. In contrast to the robust activation of iodide secretion after 2 min, 0.3 µM forskolin nonsignificantly elevated the pCREB level over the DMSO control after 10 min (1.6-fold; p < 0.2; Fig. 6). Further cAMP elevation by combining 0.3 µM forskolin and 1 µM Cpd-A resulted in a statistically significant pCREB induction over the DMSO control (1.8-fold at 5 min, p < 0.02; 3-fold at 10 min, p < 0.001; Fig. 6). Thus, pCREB induction is a less sensitive marker for PKA activation in comparison with the active iodide secretion.
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66% reduced pCREB formation (Fig. 7B).
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). The (S)-isomer stereoselectively inhibited PDE4D with an IC50 of 1.4 nM under our assay conditions (Table 1). It is >140-fold more potent against PDE4D in comparison with its inhibition of PDE4A, 4B, and 4C. In addition, the (S)-isomer is an 28-fold more potent PDE4D inhibitor than the less potent and less selective (R)-isomer. As shown in Fig. 8, significantly augmented iodide secretion over the forskolin-control was detected at 1 nM (p < 0.05; n = 4) and 5 nM (p < 0.001; n = 4) of the (S)-isomer, which are comparable with that elicited by the nonselective Cpd-A or roflumilast within a similar concentration range. The efficacy of the (R)-isomer decreased in parallel by approximately 10- to 20-fold at augmenting the peak efflux rate. Thus, the efficacy difference of the two stereoisomers closely matched their PDE4D potency difference, supporting that PDE4D is the predominant PDE4 component in regulating the active iodide secretion in T84 cells.
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CFTR Activation Dissociated from Global Intracellular cAMP Elevation. The cAMP content of T84 cells in response to forskolin stimulation and PDE4 or PDE3 inhibition was analyzed as detailed under Materials and Methods. Significantly elevated cAMP was only detected after 10-min treatment with >1 µM forskolin (Fig. 9). In the absence of forskolin, treatment with 1 µM Cpd-A, roflumilast, and trequinsin separately or their combination elicited a negligible global cAMP elevation. In the presence of 0.3 µM forskolin, elevated cAMP was only detected after treatment with above 5 µM trequinsin [FSK*/Treq(5)] or roflumilast [FSK*/Rof (5)], which are >1000-fold higher than the minimal dose capable of prolonging the efflux duration. Therefore, the global cAMP elevation from either forskolin stimulation or in combination with PDE3 or PDE4 inhibition all dissociated from their more potent activation of iodide secretion. It is also less sensitive than the pCREB induction index.
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Discussion |
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The localized cAMP-CFTR signaling relies on the formation of a supermolecular assembly for specificity and efficiency. Previous studies have elegantly demonstrated the presence of a cAMP microdomain near its production site, with restricted diffusion access of cAMP to the bulky cytosol. PDE inhibitors reduced the compartmentalized response to various degrees (Rich et al., 2001; Jurevicius et al., 2003). A macromolecular complex including 
2-adrenoceptor, PKA, and CFTR through the interaction of protein kinase A anchoring proteins has been identified after receptor stimulation (Sun et al., 2000; Naren et al., 2003). Augmentation of the adenosine-induced apical anion conductance by RS25344 supports the PDE4 proximity to the signaling complex (Barnes et al., 2005). Stimulation of the 2-adrenoceptor also rapidly recruits -arrestins with bound PDE4s to the plasma membrane within minutes as part of the desensitization machinery to limit the spread of the cAMP pool (Baillie et al., 2003). Some PDE4s are associated with PKA via protein kinase A anchoring proteins and/or activated by PKA-mediated phosphorylations, which provide additional controls to ensure a localized cAMP signaling through PDE4 regulation (Laliberte et al., 2002; Conti et al., 2003). Despite its lower abundance in T84 cells, PDE3 inhibition by trequinsin seems to be a more effective activator of iodide efflux with a quicker response, compared with that from PDE4 inhibition by Cpd-A under an identical AC state. It is unclear whether the enhanced PDE3/CFTR coupling is due to their potential proximity or from the approximately 10-fold enhanced cAMP affinity of PDE3. Previous subfractionation study indicated a similarly abundant PDE4 over PDE3 distribution on the T84 membrane (O'Grady et al., 2002). Whether PDE3, PDE4, or PDE4D is physically associated with the CFTR regulatory complex remains to be clarified.
Airway disease from mucus accumulation, recurring bacterial colonization, and chronic inflammation is the major cause of morbidity and mortality in CF. Asthma, COPD, and chronic bronchitis are also characterized by chronic airway inflammation, mucus-congested airways, and hyperplasia of goblet cells. The shifted balance from water secretion to mucus secretion around the periciliary layer of the disease airways may compromise the mucociliary clearance process, with the resulting accumulation of mucus, bacterial, and host-response products from infiltrating neutrophils and eosinophils contributing significantly to the pathogenesis of disease states (O'Byrne and Postma, 1999). In contrast to CF, most asthma, COPD, and chronic bronchitis patients have functional CFTR channel. Agents such as PDE4 and PDE4D inhibitors that can reduce the exuberant inflammation response and simultaneously enhance mucociliary clearance through CFTR activation in airway may provide additional benefits over antiinflammatory treatment alone in their management. Cpd-A and roflumilast belong to the second generation nonselective PDE4 inhibitors, effectively suppressing the overproduction of leukotrienes and a variety of proinflammatory cytokines in vitro and in animal models (Hatzelmann and Schudt, 2001; Claveau et al., 2004). Treatment with roflumilast at 0.5 mg once daily has significantly improved airway function in asthmatic and COPD patients, with its plasma concentration reached a Cmax of 3.8 ng/ml (9.5 nM) and the active N-oxide metabolite being several-fold higher (Reid, 2002). Since the combined exposure is higher above the minimal dose required for CFTR activation in T84 cells, it is possible that an enhanced airway mucociliary clearance from CFTR activation may have contributed to its improved airway function in clinic. Slightly increased diarrhea incidence has been noted as a treatment-related adverse event in roflumilast trials with its mechanism(s) unresolved. Whether the enhanced secretary response signified a possible CFTR activation in the gastrointestinal track remains to be clarified. The present data clearly demonstrated that the cellular efficacy of PDE inhibitors is coupled to the AC activation state. If the flux-mediated sensitivity phenomenon is transferred in vivo, it would support the notion that PDE4 inhibitors are more effective at targeting disorders and potentiating pathways undergoing higher cAMP turnover. In view of the high flux sensitivity of PDE4 inhibitors in activating CFTR identified here, one might expect that the combination of an oral PDE4 inhibitor with an inhaled -agonist may augment their efficacy and improve the therapeutic index further from the airway-specific activation of cAMP signaling.
In summary, the cAMP-mediated CFTR activation in T84 cells is almost exclusively under PDE3 and PDE4 (mainly PDE4D) regulation. The similarly abundant PDE4 and PDE3 expression in human airway epithelia supports an analogous CFTR regulation there. In view of the increased cardiotonic risk from the chronic administration of PDE3 inhibitors, restoring or enhancing the airway mucociliary clearance and suppressing the inflammatory responses with the emerging PDE4 inhibitors may provide exciting new opportunities to combat cystic fibrosis and other airway diseases, including asthma, COPD, and chronic bronchitis in near future.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: CFTR, cystic fibrosis transmembrane conductance regulator; CF, cystic fibrosis; PKA, protein kinase A; AC, adenylyl cyclase; PDE, 3',5'-cyclic nucleotide phosphodiesterase; COPD, chronic obstructive pulmonary disease; RP-73401, 3-(cyclopentyloxyl)-N-(3,5-dichloro-pyridin-4-yl)-4-methoxybenzamidine; Cpd-A (L-826,141), active enantiomer of 4-[2-(3,4-bis-difluoromethoxyphenyl)-2-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-ethyl]-3-methylpyridine-1-oxide; Cpd-B, 2-(4-fluorophenoxy)-N-[(1S)-1-(4-methoxyphenyl)ethyl]nicotinamide; Cpd-C, 2-(4-fluorophenoxy)-N-[(1R)-1-(4-methoxyphenyl)ethyl]nicotinamide; H89, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide; DMSO, dimethyl sulfoxide; RS25344, 8-aza-1-(3-nitrophenyl)-3-(4-pyridylmethyl)-2,4-quinazolinedione; RT, room temperature; TTBS, 20 mM Tris-HCl, pH 7.5, 50 mM NaCl, and 0.1% (v/v) Tween 20; pCREB, phosphor-cAMP response element-binding protein; CREB, cAMP response element-binding protein.
Address correspondence to: Dr. Zheng Huang, Merck Frosst Center for Therapeutic Research, P.O. Box 1005, Pointe Claire–Dorval, QC H9R 4P8, Canada. E-mail: zheng_huang{at}merck.com
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3. The potency of roflumilast and Cpd-A have recently been disclosed (Claveau et al., 2004
