Cilostazol and rolipram were purchased from Biomol (Plymouth Meeting, PA). Forskolin was obtained from Tocris (Ellisville, MO). Dithiobissuccinimidyl propionate (DSP) was obtained from Pierce (Rockford, IL). Adenosine, IBMX, cpt-cAMP, latrunculin B (Lat B), carbachol, and indomethacin were purchased from Sigma-Aldrich (St. Louis, MO).
Cell Culture and Transfections
HEK293 cells were cultured in DMEM-F12 media (Invitrogen, Carlsbad, CA) containing 10% serum and 1% penicillin/streptomycin and maintained in 5% CO2 incubator at 37°C. Calu-3 cell line was purchased from ATCC (Manassas, VA) and cultured in MEM media (Invitrogen) containing 15% serum, 1% penicillin/streptomycin, 1 mM sodium pyruvate, and 1× nonessential amino acids. Lipofectamine 2000 (Invitrogen) was used to express plasmids containing CFTR or PDE3A in both HEK293 and Calu-3 cell lines according to manufacturer's instructions. Stable cell lines were generated by selection using 0.4 mg/ml G418 (geneticin).
Full-length PDE3A was cloned into pcDNA3.1(−) containing yellow fluorescent protein (YFP) or cyan fluorescent protein (CFP), thereby generating pcDNA3.1(−)-CFP-PDE3A and pcDNA3.1(−)-YFP-PDE3A. Toward this, primers were designed with XhoI and Asp7181 sites at the 5′ and 3′ end of full-length PDE3A. A Flag or hemagglutinin (HA) tag was inserted in the putative first outer loop of full-length PDE3A between amino acids 104 and 105 using site-directed mutagenesis. Site-specific primers were designed with Flag or HA sequence in the middle of the primer and the double-stranded plasmid containing full-length PDE3A was mutated using PCR with QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA). Primer design and reaction conditions were according to the protocol recommended by the manufacturer. Human PDE3A constructs spanning the whole protein (1-255, 255-750, 751-1035, and 1035-1141 aa) were PCR-amplified. The PCR-cleaned DNA was further used for ligation-independent cloning (LIC) in pTriEx-4 and pET-41 vectors using Ek/LIC cloning kit from Novagen (EMD Chemicals, Gibbstown, NJ).
Submucosal Gland Secretion
Submucosal gland secretion was monitored as described by Wine's lab (Joo et al., 2001
). Freshly collected pig trachea was placed in cold Krebs-Ringer bicarbonate buffer (120 mM NaCl, 25 mM NaHCO3
, 3.3 mM KH2
, 0.8 mM K2
, 1.2 mM MgCl2
, 1.2 mM CaCl2
, 10 mM d
-glucose, and 1 μM indomethacin). The submucosal layer was carefully dissected from the cartilage, and a 1-cm piece was mounted in a chamber with the mucosal side up. The mucosal side was wiped and quickly air-dried with 95% O2
and 5% CO2
gas. A thin layer of water-saturated mineral oil was applied to the mucosal side. The tissue was constantly maintained at 37°C and gassed with 95% O2
and 5% CO2
after mounting. To establish a baseline, Krebs-Ringer bicarbonate buffer was added to the serosal side. The PDE3 inhibitor cilostazol (100 μM), CFTRinh-172 (50 μM), Lat B (10 μM), or cilostazol (100 μM)/Lat B (10 μM) were added to the serosal side after monitoring basal secretion. Carbachol (10 μM) was added at the end of the experiment to check for the viability of submucosal glands. Images were collected at 1-min time intervals with a digital camera (Motic Images 2.0 ML software, Richmond, BC, Canada) attached to a stereoscopic microscope (National Optical, San Antonio, TX) and analyzed using ImageJ software (NIH; http://rsb.info.nih.gov/ij/
). A 1 × 1-mm grid was placed on the tissue in the last image for area measurements. Volume was calculated from area using the formula v = Πr3
, and the secretion rate was calculated as slope of volume-versus-time plot by fitting at least four points using linear regression.
Immunohistochemistry and Immunofluorescence Microscopy
Pig trachea were paraffin-embedded and sectioned. The slides with sections were treated with protease to retrieve the antigen. The antigens sections were blocked with PBS (containing 4% bovine serum albumin [BSA] and 0.2% Triton X-100) inside a humidifying chamber for 2 h. The slides were treated with rabbit polyclonal α-PDE3A (Santa Cruz Biotechnology, Santa Cruz, CA) at 1:50 dilution overnight. Normal rabbit IgG was used for negative control. The slides were then treated for 1 h with secondary antibody α-rabbit AlexaFluor 488 (Invitrogen) at 1:500 dilution and 1:1000 dilution of propidium iodide. Images were taken on a Carl Zeiss confocal microscope (Thornwood, NY).
Calu-3 cells were grown on glass-bottom dishes and fixed with 3.7% paraformaldehyde. The fixed cells were blocked, treated with antibodies, and imaged as described above.
Short-circuit Current Measurements
Polarized lung serosal cells (Calu-3) monolayers were grown on Costar Transwell permeable supports (Cambridge, MA; filter area 0.33 cm2
) until they reached a resistance of ~1500 Ù and then mounted in an Ussing chamber. Short-circuit currents (Isc
) were measured as described previously (Li et al., 2005
). Epithelia were bathed in Ringer solution (in mM; Basolateral: 140 NaCl, 5 KCl, 0.36 K2
, 0.44 KH2
, 1.3 CaCl2
, 0.5 MgCl2
, 4.2 NaHCO3
, 10 HEPES, 10 glucose, pH 7.2, [Cl−
] = 149), and low Cl−
Ringer solution (in mM; Apical: 133.3 Na-gluconate, 5 K-gluconate, 2.5 NaCl, 0.36 K2
, 0.44 KH2
, 5.7 CaCl2
, 0.5 MgCl2
, 4.2 NaHCO3
, 10 HEPES, 10 mannitol, pH 7.2, [Cl−
] = 14.8) at 37°C, and gassed with 95% O2
and 5% CO2
. The PDE3-specific inhibitor cilostazol (10–100 μM), the PDE4 inhibitor rolipram (2–10 μM), or a combination of both inhibitors was added to the apical and basolateral sides. Adenosine was added to the apical and basolateral sides. Forskolin (20 μM) was added to both sides for maximal response. CFTRinh-172 (20 μM) was added to the apical side. For Isc
measurements with actin cytoskeleton disruption of the cells, Lat B (1 μM) was added to both apical and basolateral sides after pretreating the cells with Lat B (1 μM) for 30 min.
Iodide Efflux Assay
Human embryonic kidney (HEK) 293 cells expressing wild-type CFTR were grown on 60-mm dishes. Forty-eight hours later, iodide efflux was monitored as previously described (Naren et al., 2003
). Briefly, cells were loaded for 60 min at room temperature with loading buffer (136 mM NaI, 137 mM NaCl, 4.5 mM KH2
, 1 mM CaCl2
, 1 mM MgCl2
, 10 mM glucose, 5 mM HEPES, pH 7.2). Extracellular NaI was washed away thoroughly (seven times) with efflux buffer (136 mM NaNO3
replacing 136 mM NaI in the loading buffer), and cells were equilibrated for 1 min in a final 1-ml aliquot. The first four aliquots were used to establish a stable baseline in efflux buffer alone. Agonist (1 μM adenosine with or without 100 μM cilostazol) was added to the efflux buffer, and samples were collected every minute for 6 min in the continued presence of agonists (i.e., the efflux buffer used for subsequent replacements also contained agonists at the same concentration). The iodide concentration of each aliquot was determined using an iodide-sensitive electrode (Thermo Scientific, Waltham, MA) and converted to iodide content (nanomoles/minute). HEK293 parental cells in the presence of forskolin were used as a negative control.
Fluorescence Resonance Energy Transfer Microscopy and Data Analysis
For ratiometric fluorescence resonance energy transfer (FRET), Calu-3 cells or HEK293 cells expressing CFP-EPAC-YFP were grown on glass-bottom dishes (MatTek, Ashland, MA), washed twice with Hanks' balanced salt solution (HBSS), and mounted on an inverted Olympus microscope (IX51, U-Plan Fluorite 60× 1.25 NA oil-immersion objective, Melville, NY). Cells were maintained in HBSS in the dark at room temperature. After establishing the baseline, PDE inhibitors were added as indicated. Ratiometric FRET imaging was performed as described previously (Li et al., 2007
). Briefly, images were collected using cooled electron microscope (EM)-CCD camera (Hamamatsu, Bridgewater, NJ) controlled by Slidebook 4.2 software (Intelligent Imaging Innovations, Denver, CO). Light source used was 300-W xenon lamp with a neutral density filter. JP4 CFP/YFP filter set was used for image capture (Chroma, Brattleboro, VT), which includes a 430/25-nm excitation filter, a double dichroic beam splitter and two emission filters (470/30 nm for CFP and 535/30 nm for FRET emission) alternated by filter-change controller Lambda 10-3 (Sutter Instruments, Novato, CA). Time-lapse images were captured with 100–300-ms exposure time and 1-min time intervals. After background subtraction, multiple regions of interest (10–20) were selected (three to five cells) for data analysis using ratio module. The emission ratio (CFP/FRET) was obtained from CFP and FRET emission of background subtracted cells.
For direct sensitized emission FRET, HEK293 cells were transiently transfected with pcDNA3.1(−)-CFP-PDE3A, or pcDNA3.1(−)-YFP-CFTR or both using Lipofectamine 2000 (Invitrogen). Single transfected cells were used to acquire CFP- or YFP-only images for bleed-through calculations. Double-transfected cells were used for data collection using CFP/YFP filter sets. After acquiring images without PKA agonists as the 0 time point images, PKA-activating cocktail (in μM; forskolin 10, IBMX 100, cpt-cAMP 200) was added, and images were acquired at 2-, 4-, 6-, 8-, and 10-min intervals. FRET calculations were performed as described (Galperin and Sorkin, 2003
). Corrected FRET (FRETc) was calculated on a pixel-by-pixel basis for the entire image by using the equation: FRETc = FRET − (0.5 × CFP) − (0.06 × YFP), where FRET, CFP, and YFP correspond to background-subtracted images of cells coexpressing CFP-PDE3A and YFP-CFTR acquired through the FRET, CFP, and YFP channels, respectively. The 0.5 and 0.06 are the fractions of bleed-through of CFP and YFP fluorescence through the FRET filter channel, respectively. FRETc was normalized with donor CFP intensity (FRETc/CFP) to give the normalized corrected FRET (N-FRETc). The intensity of FRETc images was presented in monochrome mode, stretched between the low and high renormalization values according to a temperature-based lookup table, with black indicating low values and white indicating high values. All calculations were performed using FRET module of the SlideBook 4.2 software (Intelligent Imaging).
Coimmunoprecipitation and Immunoblotting
For detection of PDE3A and PDE3B expression in Calu-3 cells using Western blotting, the cells were lysed in lysis buffer (1× PBS, containing 0.2% Triton X-100 and protease inhibitors 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, 1 μg/ml leupeptin, and 1 μg/ml aprotinin). The lysate was centrifuged at 16,000 × g for 10 min at 4°C, and the clear supernatant was mixed with 5× Laemmli sample buffer (containing 2.5% β-mercaptoethanol), denatured, subjected to SDS-PAGE on 4–15% gel (Bio-Rad, Hercules, CA), transferred to PVDF membrane, and immunoblotted for PDE3A using a α-PDE3A pAb (Santa Cruz) or for PDE3B using α-PDE3B pAb (Santa Cruz). Calu-3 cells overexpressing HA-PDE3A were lysed, and the clear supernatant was used to probe for HA-PDE3A using α-HA (Sigma) using the method described above. To knockdown PDE3A expression, we used PDE3A shRNA (Santa Cruz) in Calu-3 cells, lysed the cells and immunoblotted for PDE3A using a α-PDE3A pAb (Santa Cruz).
For detecting PDE3A localization in Calu-3 cells and HEK293 cells using Western blotting, the cells were washed with 1× PBS, harvested and pelleted at 600 × g for 5 min at 4°C. The pellet was resuspended in hypotonic lysis buffer (10 mM HEPES, 1 mM EDTA, protease inhibitor cocktail, pH 7.2) by incubation for 10 min. The suspension was homogenized at 4°C by 10 strokes in Dounce homogenizer, followed by 15 strokes in the presence of equal volume of sucrose buffer (500 mM sucrose, 1 mM EDTA, 10 mM HEPES, pH 7.2). The lysate was spun at 6000 × g for 20 min at 4°C to obtain postmitochondrial supernatant. Crude membrane was collected by centrifuging the supernatant at 100,000 × g for 45 min at 4°C. The pellet was resuspended in isotonic buffer (250 mM sucrose, 1 mM EDTA, 10 mM HEPES, pH 7.2) and immunoblotted for PDE3A using a PDE3A pAb (Santa Cruz) using the method described above.
For coimmunoprecipitation of CFTR and PDE3A, HEK293 cells transfected with Flag-CFTR and HA-PDE3A or only HA-PDE3A were lysed and centrifuged (16,000 × g for 10 min at 4°C) using the method described above. The clear supernatant was subjected to immunoprecipitation using α-Flag beads (Sigma). The immunoprecipitated beads were washed three times with lysis buffer, and the proteins were eluted from the beads using 5× Laemmli sample buffer (containing 2.5% β-mercaptoethanol). The eluates were immunoblotted for PDE3A using PDE3A mAb (Novus Biologicals, Littleton, CO). For immunoprecipitation of PDE3A and CFTR in Calu-3 cells, the cell lysate was immunoprecipited with α-PDE3A and blotted for CFTR. For immunoprecipitation of PDE3A and PDE4D in Calu-3 cells, the lysate was immunoprecipited with α-PDE4D pAb (Abcam, Cambridge, MA) and blotted for PDE3A. All these coimmunoprecipitation experiments were performed using the same protocol.
For detecting the minimum domain of PDE3A responsible for interacting with CFTR, we overexpressed HIS-S–tagged constructs of PDE3A spanning the whole protein and full-length Flag-CFTR in HEK293 cells. The total protein from the cell lysate was immunoprecipitated with α-Flag beads (Sigma) and probed with α-S-HRP (horse radish peroxidase; EMD Chemicals) using the method described above.
For immunoprecipitating cross-linked complex, cells expressing Flag-CFTR and HA-PDE3A or only Flag-CFTR were washed twice with 1× PBS (containing 0.1 mM calcium chloride and 1 mM magnesium chloride) and incubated with or without Lat B (1 μM) for 30 min at 37°C. The cells were then treated with thiol-cleavable, amine-reactive, homobifunctional cross-linker DSP (1 mM) for 5 min. DSP was removed and RIPA buffer (140 mM NaCl, 1% Nonidet P40, 0.5% Na-deoxycholate, 0.1% Na-dodecyl sulfate, and 50 mM Tris-HCl, pH 8.0) containing protease inhibitor cocktail was added to quench the reaction and lyse the cells. The lysate was spun at 20,000 rpm for 10 min at 4°C. After taking 100 μl of total protein for input, the rest of the protein was immunoprecipitated using α-HA beads (Sigma) overnight. The beads were washed twice with RIPA buffer and the cross-linked complex was eluted with 100 mM glycine (pH 2.2) and quickly neutralized with 150 mM Tris (pH 8.8). For cleaving the disulphide bond of DSP and separating the proteins, 2.5% β-mercaptoethanol was added to the sample Laemmli buffer. The proteins were immunoblotted for CFTR using M3A7 CFTR mAb (Millipore, Billerica, MA).
For all the immunoblotting experiments, we loaded samples of equal amounts of total protein.
Calu-3 cells expressing Flag- or HA-PDE3A were grown on 35-mm dishes, fixed with 3.7% formaldehyde for 10 min, blocked with 1% BSA for 30 min, and treated with α-Flag or α-HA HRP (0.2 μg/ml) for 90 min. The HRP substrate 1-step Ultra TMB (Pierce) was then added to the dishes for ~20 min, and the reaction was stopped by adding equal amount of 2 M H2SO4. The absorbance was read at 450 nm.
To detect the effects of PKA-phosphorylation on surface expression levels of PDE3A, Calu-3 cells expressing Flag-PDE3A were pretreated with forskolin (20 μM) for 10 min, fixed, surface-labeled, and then assayed as described above.
To detect the effects of Lat B on PDE3A surface expression levels, Calu-3 cells expressing HA-PDE3A were pretreated with Lat B (1 μM) for 30 min and then surface-labeled as described above.
Surface Biotinylation and Immunoblotting
HEK293 cells expressing HA-PDE3A were surface biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (Pierce) for 1 h at 4°C, lysed, and immunoprecipitated using α-HA agarose beads (Sigma). The biotinylated purified HA-PDE3A was pulled down using streptavidin beads (Pierce) for 1 h at room temperature. The beads were spun down to collect unbound fraction, and the beads contained the bound fraction. The proteins were mixed with 5× Laemmli sample buffer (containing 2.5% β-mercaptoethanol), denatured, subjected to SDS-PAGE on 4–15% gel (Bio-Rad), transferred to PVDF membrane, and immunoblotted for PDE3A using PDE3A mAb (Novus Biologicals).
Calu-3 cells stably expressing HA-PDE3A were grown on 35-mm glass-bottom dishes (MatTek). Cells were washed twice with PBS containing 6 mM glucose and 1 mM sodium pyruvate (PBS/Glu/NaPyr) and blocked with PBS/Glu/NaPyr containing 4% BSA for 10 min. Cells were then incubated with biotin α-HA antibody (1 μg/ml, Sigma) for 15 min, washed five times followed by a second incubation with streptavidin-conjugated Qdot-655 (0.1 nM, Invitrogen) for 2 min, washed extensively eight times, and immediately mounted on an Olympus inverted microscope (IX51). The images were captured with Hamamatsu EM-CCD camera at 1–3 frames per second for 1–3 min with 50-ms exposure time, 100× oil-immersion objective (NA 1.40), xenon (300-W lamp) light source, and SlideBook 4.2 software. Qdot 655-A BrightLine high brightness and contrast single band filter set (Semrock, Rochester, NY) was used for collecting data. Single-particle tracking (SPT) was done using the particle-tracking module of SlideBook 4.2 software, which generates trajectories and calculates the mean squared displacement (MSD). The diffusion coefficient (D) was calculated by linear squares fitting using points 1–5 on the MSD curve. Five to 10 cells were used for plotting histograms of the diffusion coefficient.
To monitor changes in lateral diffusion of PDE3A with cytoskeletal disruption, cells were pretreated with Lat B (1 μM, 30 min), and Lat B was also added to the buffer during the course of the experiment.
AlphaScreen Assay for PDE3A–CFTR Interaction
AlphaScreen FLAG (M2) detection kit (Perkin Elmer, Waltham, MA) was used to detect the interaction between purified full-length biotin-(HA)-PDE3A and Flag-wt-CFTR. In brief, starting from a 100 nM final concentration, biotin-(HA)-PDE3A was serially diluted (in 1/2 log dilution series) in assay buffer (1× PBS, 0.1% BSA, 0.05% Tween 20 [vol/vol], pH 7.2) containing Flag-wt-CFTR (100 nM final concentration). The resulting solutions were incubated at room temperature for 30 min. Each sample solution (15 μl) was transferred to a white opaque 384-well microplate (OptiPlate-384, Perkin Elmer) in triplicates and into which anti-FLAG (M2) acceptor beads (5 μl, 20 μg/ml final concentration) were added and incubated for 30 min at room temperature. Streptavidin donor beads (5 μl, 20 μg/ml final concentration) were then added and incubated for 2 h at room temperature. The plate was read on an EnVision 2103 Multilabel Reader (Perkin Elmer).
Cell-attached Single-Channel Recording
Single-channel recordings were obtained from Calu-3 cells as described previously (Li et al., 2007
). The pipette solution contained either forskolin or cilostazol (10–20 μM) to activate CFTR channels. Both bath and pipette solution contained (in mM) 140 N
-glucamine, 140 HCl, 2 CaCl2
, 2 MgCl2
, and 10 HEPES, pH 7.4. Single-channel currents were recorded at a test potential of +80 mV (reference to the cell interior) delivered from the recording electrode, filtered at 100 Hz, and sampled at 2 kHz.
Statistical analyses were done using Student's t test (two-tailed) or ANOVA (single-factor), and p < 0.01 or p < 0.05 was considered significant. All the results are represented as mean ± SEM, with n equaling the number of experiments.