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Division of Reproductive Biology, Department of Obstetrics and Gynecology, and
* Immunology and Allergy, Department of Pediatrics, Stanford University School of Medicine, Stanford, California, USA
1Correspondence: Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, 300 Pasteur Dr., Room A344, Stanford, CA 94305-5317, USA. E-mail: marco.conti{at}stanford.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-coupled receptor stimulation was increased up to fourfold in the airway of PDE4D-/- mice when compared with wild-type. This increase in cAMP was associated with an increased sensitivity to PGE2-induced relaxation of the PDE4D-/-tracheas. Furthermore, a blockade of prostanoid accumulation in PDE4D-/- tracheas restored the response to muscarinic cholinergic stimulation in vitro and in vivo. These results demonstrate that PDE4D plays a key role in balancing relaxant and contracting cues in airway smooth muscle, suggesting that natural mutations in the PDE4D gene have profound effects on airway tone.—Méhats, C., Jin, S.-L. C., Wahlstrom, J., Law, E., Umetsu, D., Conti, M. PDE4D plays a critical role in the control of airway smooth muscle contraction.
Key Words: cyclic AMP • metabolism • second messengers • trachea
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
A net increase in cAMP concentration may be the consequence of the activation of adenylyl cyclases (AC), resulting in the synthesis of cAMP, or of inhibition of cyclic nucleotide phosphodiesterases (PDEs), which are responsible for degradation of the cyclic nucleotides. Physiological and pharmacological studies have recently underscored the important role of PDEs in the control of airway function (2)
. Among the 11 known mammalian PDE families, the PDE4 family is of particular interest in ASM function. Profiling of the PDE expression in the ASMs of various species suggests an important role of this family in the control of cAMP levels in these cells. Moreover, PDE4 inhibition functionally blunts spontaneous or ligand-induced contractions in smooth muscle preparations (3
4
5)
. The PDE4 family is encoded by four genes (PDE4A-D) that generate multiple isoforms by using multiple promoters or alternative splicing (6)
.
To investigate the physiological role of PDE4, our laboratory generated mice with a targeted disruption of different pde4 genes in an attempt to distinguish the role of PDE4 isotypes within the whole organism and within different cell lineages (7
, 8)
. When the phenotype of the PDE4D null mouse was tested in an asthma model by measuring airway resistance, a complete absence of response to methacholine, a muscarinic cholinergic agonist, was observed in both sensitized and naive PDE4D-/-mice (9)
. The cholinergic parasympathetic nerves are the dominant neural bronchoconstrictors in humans and rodents (10)
. In addition, an increased parasympathetic tone has been associated with clinical asthma or the worsening of asthma symptoms (11)
. Thus, the findings in the PDE4D-/-mice suggested that inactivation of PDE4D alters airway response to parasympathetic stimuli. The current study was undertaken to determine whether smooth muscle contractility is affected in the PDE4D-/- mice and to identify the biochemical mechanisms involved in the inhibitory effect of PDE4D ablation on airway resistance.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 8)
. Wild-type and homozygous null mice used in all experiments were 3–6 months of age and had a mixed genetic background of C57BL/6 and 129/Ola. All experiments involving animals were approved by the Administrative Panel on Laboratory Animal Care at Stanford University.
In vitro contractile studies
Mice were killed by cervical dislocation and the entire trachea was promptly removed and placed in aerated (95% O2/5% CO2) Krebs buffer (glucose 11.1 mM, KCl 4.7 mM, NaCl 118 mM, CaCl2 2.5 mM, MgCl2 0.5 mM, NaH2PO4 1 mM, and NaHCO3 25 mM). The trachea was carefully cleared of adherent connective tissue and suspended for tension recordings in a 10 mL organ bath containing aerated Krebs buffer maintained at 35°C. Tissues were allowed to equilibrate for 60 min under an optimal resting tension of 0.9 g, with the bath medium exchanged every 15 min. At the end of the equilibration period, KCl (60 mM) was added to the bath and the response of the trachea recorded. After washings and return to basal tone, concentration dependency curves were constructed with the cumulative addition, at intervals of 2 min, of either carbachol (10-9 to 10-4 M, final concentration) or arginine vasopressin (AVP, 10-9 to 10-6 M) freshly dissolved in water. When used, propranolol (10-5 M), indomethacin (10-6 M), or RpcAMP (100 µM, BIOLOG Life Science Institute, Bremen, Germany) was added 10 min before construction of the carbachol concentration dependency curve. In one set of experiments, tracheas of wild-type animals were precontracted with carbachol (10-5 M) or AVP (10-6 M), and the response to rolipram was measured after a single addition of the inhibitor (10-5 M). In another set of experiments, the tracheas were precontracted with carbachol (10-5M) in the presence of indomethacin and concentration dependency curves were constructed with the cumulative addition, at intervals of 2 min, of PGE2 (10-10 to 10-5 M). Isometric contractile responses were measured with FT.03 force displacement transducers (Grass Instruments Co., Quincy, MA, USA) and processed by the Maclab/8e Software package (AD Instruments Ltd., Hastings, UK). Areas under the tension curve were measured for a given time. Results were expressed as a percentage of either the resting tone or the carbachol precontraction.
PGE2 production by isolated trachea
At the end of the equilibration period, the bath medium was exchanged; after a 5 min resting time, an aliquot of bath medium was collected. Additional aliquots were collected after 5 min of contractions induced by KCl (60 mM) and 5 min of contractions induced by carbachol (10-5M). The samples were immediately snap-frozen in liquid nitrogen and stored at -80°C until use. At the time of measurement, the samples were thawed and 50 µL aliquots were assayed for PGE2 content by enzyme immunoassay (Cayman Chemical Co, Ann Arbor, MI, USA) according to the manufacturer’s instructions. The assay has a high specificity for PGE2 with minimal cross-reactivity (<0.01%) for other prostanoids such as PGD2 and PGF2. Results are expressed as pg/mL.
cAMP-phosphodiesterase assay
The entire trachea, free of serosa, was homogenized in ice-cold hypotonic buffer (Tris-HCl 20 mM pH 8.0, NaF 50 mM, EDTA 1 mM, EGTA 0.2 mM, Na2PO4 10 mM, ß-mercaptoethanol 5 mM, and a protease inhibitor cocktail: leupeptin 0.5 µg/mL, aprotinin 4 µg/mL, benzamidine 50 mM, pepstatin 0.7 µg/mL, soybean trypsin inhibitor 10 µg/mL, and PMSF 10 µg/mL added freshly before use) using an all-glass homogenizer. Aliquots of the homogenates were assayed for cAMP PDE activity according to the method of Thompson and Appleman (12)
as detailed previously (9)
. PDE activities were measured with 1 µM [3H]-cAMP as a substrate. PDE4 activity was defined as the fraction of cAMP PDE activity inhibited by 10 µM rolipram. Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with BSA as a standard.
In vitro cAMP responses
Lung tissue was cut into slices 
3 x 2 x 1 mm. The tissue slices were incubated in 1 mL of HEPES-buffered MEM at 37°C in the absence or presence of test drugs. The reaction was terminated by the addition of trichloroacetic acid (TCA) to a final concentration of 5%. The tissue slices were homogenized with a Dounce homogenizer before centrifugation at 1500 x g for 30 min. Supernatants were extracted five times with water-saturated ether and cAMP content was then measured by RIA as described by Harper and Brooker (13)
. Protein concentration was measured in the TCA pellet (resuspended in NaOH 1 N) according to the Lowry method.
Antibodies
For immunoprecipitation, monoclonal PDE4D-selective antibodies (M3S1, 1:30), PDE4B-selective polyclonal antibodies (K118, 1:30), and PDE4A-selective antibodies (AC55, 1:30) were used (14)
. For Western blot experiments, monoclonal PDE4D-selective antibodies (61D10E, 1:10,000; a gift from Icos Corp., Seattle, WA, USA) were used. Second-step horseradish peroxidase-conjugated anti-murine antibodies (1:5,000) were purchased from Amersham (Amersham Pharmacia Biotech, Inc., Piscataway, NJ, USA).
Immunoprecipitation
Tracheas were homogenized in ice-cold hypotonic buffer and the protein concentration determined. Aliquots (100 µg protein) were immunoprecipitated using antibodies immobilized on protein G or A-Sepharose (Amersham Pharmacia Biotech, Inc.) for the monoclonal and polyclonal antibodies respectively, following the method of Iona et al. (14)
.
Western blot analysis
Tracheas were homogenized in ice-cold hypotonic buffer and the protein concentration determined. Samples (40 µg protein) were boiled in Laemmli buffer (15)
, subjected to electrophoresis on an 8% SDS-PAGE, and blotted onto Immobilon-P transfer membrane (Millipore Corp., Bedford, MA, USA). Membranes were blocked in TBS-Tween 20 0.1% containing 5% nonfat milk. The PDE4 polypeptides were detected using specific antibodies and visualized by use of the ECL detection reagents (Amersham Pharmacia Biotech, Inc.).
In situ hybridization
Tracheas were dissected from PDE4D+/+ and PDE4D-/-mice, embedded in OCT (Tissue-Tek, Torrance, CA, USA) and cut into 8 µm sections using a cryostat (Leica CM180, Deerfield, IL, USA). The sections were mounted on slides coated with poly-L-lysine, fixed in 4% paraformaldehyde, and stored at -80°C until use. The antisense and sense probes for PDE4D were labeled with [35S]UTP (1000 Ci/mmol; NEN DuPont, Boston, MA, USA). Hybridization and washing were adapted from Tsafriri et al. (16)
. The sections were hybridized under coverslips overnight at 50°C in formamide 50%, NaCl 30 mM, Tris-HCl 10 mM pH 8.0, EDTA 5 mM, 1x Denhardt’s solution, yeast tRNA 1 mg/mL, and 10% dextran sulfate. After RNase (25 µg/mL) treatment, slides were washed to a final stringency of 0.1x SSC at room temperature. After 2–3 wk of exposure to NTB2 emulsion (Eastman Kodak, Rochester, NY, USA), sections were developed, counterstained, and mounted with Permount (Fisher Scientific, Fair Lawn, NJ, USA) for observation with bright- and dark-field illumination and photographed with an AxioCam (Zeiss, Thornwood, NY, USA).
Autoradiographic localization of muscarinic cholinergic receptors
Eight-micrometer cryostat sections from tracheas of PDE4D+/+ and PDE4D-/- mice were incubated for 2 h at room temperature in 1 nM [3H]QNB (30 Ci/mmol; NEN Dupont), followed by two 5 min rinses in ice-cold PBS as described by Wamsley et al. (17)
. Nonspecific binding was determined by 1 µM atropine. The slides were quickly dried by a stream of cold dry air and stored at 4°C in boxes containing Drierite until photographic emulsions were applied. The photographic emulsion exposure and development were executed as described in the in situ hybridization section.
Measurement of airway responsiveness in vivo
Airway responsiveness was assessed by methacholine-induced airflow obstruction from conscious mice placed in a whole-body plethysmograph (model PLY 3211, Buxco Electronics, Troy, NY, USA) as described by Hansen et al. (9)
. Indomethacin (5 mg/kg, i.v.) or vehicle (Na2CO3, 5 mM) was injected 15 min before exposure to methacholine. Measurements of methacholine responsiveness were obtained by exposure for 2 min to NaCl 0.9% (Portable Ultrasonic, 5500D, DeVilbiss Health Care, Somerset, PA, USA) followed by incremental doses of aerosolized methacholine (2.5–80 mg/mL). Pulmonary airflow obstruction was measured by Penh using the following formula: Penh = [(Te/RT) - 1] x (PEF/PIF), where Penh = enhanced pause (dimensionless), Te = expiratory time, RT = relaxation time, PEF = peak expiratory flow (mL/s), and PIF = peak inspiratory flow (mL/s). Results were expressed for each methacholine concentration as the percentage of baseline Penh values after 0.9% NaCl exposure.
Materials
Unless otherwise stated, all drugs and reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Data analysis
Concentration dependency curves were analyzed by nonlinear regression using commercially available software (GraphPad-Prism, GraphPad Software, San Diego, CA, USA) and used to calculate pD2 (-Log[EC50]) and Emax. For concentration dependency curves, significance of the difference was assessed by two-way ANOVA and Bonferroni’s post-test; otherwise one-way ANOVA followed by Student's t test, two tailed for unpaired samples, was applied. The difference was considered significant when P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). Measurement of contractility of tracheas from PDE4D-/-mice demonstrated a large reduction in the response to the muscarinic cholinergic agonist, with a significant decrease in maximal efficacy (Emax: 149.0±6.4 vs. 226.4±10.5, P<0.001), as well as a fivefold decrease in sensitivity (pD2: -5.94±0.22 vs. -6.63±0.22, P<0.01). We observed no difference in resting tone or in the morphological properties between the PDE4D+/+ and PDE4D-/-tracheas (data not shown). Conversely, the responses of the wild-type and PDE4B-/- mouse tracheas to carbachol were not different statistically with respect to Emax or pD2 (Fig. 1B
).
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Normal response of the PDE4D-/- trachea to KCl and AVP
To rule out the possibility that the decreased response of the PDE4D-/-trachea to carbachol is due to an anatomical defect or a generalized loss of contractile response of the smooth muscle, tracheas were challenged with 60 mM KCl to induce membrane depolarization. At this concentration, KCl induced comparable contractile responses in tracheas from PDE4D-/- and wild-type littermates (Fig. 2
A).
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We have previously reported that in vivo serotonin-induced resistance is normal in naive and sensitized PDE4D-/- mice, suggesting a normal ability of the airways to contract. However, as reported in other studies (18
, 19)
, no constriction in response to serotonin was obtained in vitro with either PDE4D+/+ or PDE4D-/- tracheas (data not shown). To monitor contractility induced by GPCR-activated pathways other than the muscarinic cholinergic pathway (18)
, we used arginine vasopressin (AVP) (1 µM), which has been shown to be a potent constrictor of mouse trachea (18)
. AVP induced a sustained contraction of both PDE4D+/+ and PDE4D-/- tracheas with no statistical difference in pD2 or Emax (Fig. 2B
).
Selective inhibition of the carbachol-induced contraction by rolipram in the PDE4D+/+ trachea
To determine whether the PDE4D-/-phenotype could be reproduced by an acute inhibition of PDE4 activity, the PDE4-selective inhibitor rolipram (10-5 M) was added to wild-type tracheas at rest or preconstricted with carbachol (10-5 M), KCl (67 mM), or AVP (10-6 M). Although it decreased the carbachol-induced contraction in a manner similar to the inactivation of the PDE4D gene, rolipram had no effect on KCl- or AVP-induced contractions nor did it affect the resting tone (Fig. 3
). Thus, a preferential disruption of the cholinergic-mediated contraction was observed with acute PDE4 inhibition or ablation of the PDE4D gene.
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Expression of PDE4D in mouse trachea
We have reported previously that PDE4D is the predominant isoform expressed in the mouse lung (9)
. To further define the site of PDE4D expression in the airways, PDE activity was measured in the whole trachea free of serosa. As reported in Fig. 4
A, PDE activity measured in the trachea is comparable to that found in lung tissue (9)
. In the presence of rolipram, 
50% of the activity was inhibited. In tracheas from PDE4D-/- mice, a 45–50% reduction in total PDE activity was detected and the residual PDE activity was minimally inhibited by rolipram. Immunoprecipitation of PDE activity with antibodies specific for PDE4D, PDE4B, and PDE4A subtypes confirmed that PDE4D is the predominant PDE4 expressed in this tissue with a small contribution from PDE4A and PDE4B. Moreover PDE4A and PDE4B activities were not increased in PDE4D-/- compared with wild-type (Fig. 4B
), suggesting that compensation does not occur. Western blot studies were then conducted to identify the PDE4D variants expressed in the airways. One immunoreactive protein with an apparent molecular mass of 93 kDa was present in the tracheas of wild-type and absent in PDE4D-/-mice (Fig. 4C
). Some nonspecific bands of lower apparent molecular mass and corresponding to mouse IgG also were detected in both PDE4D+/+ and PDE4D-/-tissues.
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To determine the exact site of PDE4D expression, in situ hybridization was performed on sections of tracheas from PDE4D+/+ and PDE4D-/- mice using a probe specific for PDE4D. Hybridization signals were detected in the smooth muscle and epithelium of wild-type tracheas (Fig. 5
A). However, no positive signal was observed in the trachea sections from PDE4D-/- mice (Fig. 5B
) or in the cartilage. Localization of the muscarinic cholinergic receptors, as determined by autoradiography using [3H]QNB, revealed high specific binding within the tracheal smooth muscle layer (Fig. 5C
) demonstrating an overlap with the expression of PDE4D. No difference in the density of the muscarinic binding sites was detected between PDE4D+/+ and PDE4D-/- tracheas (data not shown).
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Basal cAMP content and cAMP accumulation in response to GPCR activation
Because PDE4 isoforms catalyze cAMP hydrolysis, we examined the effect of PDE4D ablation on cAMP accumulation in airways. In the absence of exogenous stimuli, cAMP content in the lung tissue was significantly higher in the PDE4D-/- mice compared with the wild-type littermates (PDE4D+/+: 16.13±1.70, PDE4D-/-: 31.09±2.73 pmol/mg protein, P<0.0001) (Fig. 6
A). When stimulated for 15 min with isoproterenol, PGE2, or forskolin, cAMP accumulation was increased fourfold in the PDE4D-/- lung slices compared with the PDE4D+/+-slices (Fig. 6B
). Incubation in the presence of rolipram caused a further increase in cAMP in the wild-type but not in the PDE4D-/- slices. Cyclic AMP accumulation in the PDE4D+/+ lung slices stimulated by isoproterenol in the presence of rolipram was comparable to that in the PDE4D-/- mice (Fig. 6C
).
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Reversal of tracheal hyporesponsiveness to carbachol in the presence of indomethacin
Given the marked increase in cAMP signaling after activation of AC in the PDE4D-/- mice, we surmised that the presence of endogenous activators of AC, such as prostanoids or catecholamines, might counteract the carbachol-induced contraction in PDE4D-/- mice. Therefore, tracheas from the wild-type and PDE4D-/- mice were preincubated for 10 min with either propranolol, a nonselective ß-adrenergic antagonist, or indomethacin, a cyclooxygenase inhibitor. No effect of propranolol on carbachol-induced contractions could be detected in PDE4D+/+ or PDE4D-/- tracheas (Fig. 7
A). Conversely, preincubation with indomethacin caused a complete reversal of the phenotype, with the response of the PDE4D-/- trachea now being similar to that of the wild-type trachea (Fig. 7B
, Emax: 233.50±9.91 vs. 232.50±7.56%; pD2: -6.21±0.19 vs. -6.52±0.16). Moreover, preincubation of the tracheas with the protein kinase A inhibitor RpcAMP restored the response of the PDE4D-/- trachea to carbachol to a level comparable to that in the wild-type (Fig. 7C
, Emax: 207.70±5.52 vs. 205.10±5.09%; pD2: -6.94±0.15 vs. -6.83±0.14).
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PGE2 concentration in the organ bath and relaxation of the trachea in response to exogenous PGE2
On the basis of the cyclooxygenase inhibitor data, one could hypothesize that the defect of the carbachol response in the PDE4D-/- trachea is due to an increased release of prostanoids during experimental manipulations. To test this possibility, the concentration of PGE2 was measured in the organ bath. Under unstimulated conditions, all tracheas produced PGE2, with no difference in the PDE4D+/+ and PDE4D-/- mice (Fig. 8
A). Furthermore, there was no difference in the level of PGE2 between wild-type and PDE4D-/- trachea in the presence of KCl or carbachol. Thus, in our experimental conditions a difference in the PGE2 content does not explain the difference in the response between the PDE4D+/+ and PDE4D-/- tracheas. Cumulative concentration-response curves for exogenous PGE2 were then constructed with PDE4D+/+ and PDE4D-/- tracheas previously constricted with 10 µM carbachol in the presence of indomethacin (Fig. 8B
). PGE2 relaxed the carbachol-precontracted trachea in a concentration-dependent manner. The tracheas reached a complete relaxation at 1 µM PGE2, consistent with the previously published studies (20
, 21)
. Although the maximal effect was identical in the two mouse strains, the concentration curve generated with the PDE4D-/- trachea was significantly shifted to the left compared with the wild-type (pD2= -7.86±0.16 vs. pD2=-7.15±0.13, P<0.01).
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In vivo treatment with indomethacin restores the response to methacholine in PDE4D-/- mice
To determine whether endogenous prostanoids in vivo in naive PDE4D-/- mice prevent bronchoconstriction in response to methacholine, indomethacin was injected 10 min before administration of increasing concentrations of methacholine, and airway hyperactivity was monitored thereafter. As previously reported, untreated PDE4D-/- mice show no sign of response to methacholine, even at the highest dose of 80 mg/mL (Fig. 9
). Conversely, the indomethacin treatment restored a response to methacholine in the PDE4D-/- mice, although the magnitude of the response remained lower than the treatment-matched wild-type littermates (data not shown). These data suggest that increased sensitivity to endogenous prostanoids in vivo is one cause of the loss of muscarinic cholinergic-induced bronchoconstriction.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because in vivo airway resistance is affected by several factors in addition to airway smooth muscle tone (e.g., mucous secretion and upper airway obstruction), we used an ex vivo trachea model to evaluate contractility in response to muscarinic cholinergic agonists. Measurement of the contractile response to carbachol demonstrated a marked decrease in both maximal efficiency and potency of this cholinergic agonist to cause contraction in the PDE4D-/- trachea. This decreased response was observed in all preparations tested and was mimicked by the incubation of tracheas from the wild-type mice with the PDE4-selective inhibitor rolipram. Conversely, inactivation of PDE4B had no effect on the tracheal contraction. In the trachea, PDE4 contributes to 50% of total PDE activity, an amount similar to that reported for human airways (3
, 4)
and nearly all PDE4 expressed belongs to the PDE4D subfamily. PDE4B accounts for a small amount of rolipram-sensitive activity, thus explaining the lack of an effect of the PDE4B ablation on ASM. No compensatory activity from other PDE4 genes or other PDE families was seen in the PDE3D-/- airways, an observation that agrees with our previous reports (7
, 9)
. Furthermore, rolipram reproduces the phenotype of the PDE4D-/- trachea in the wt mice and did not affect cAMP accumulation in response to Gs-coupled receptors in the PDE4D-/- lungs. Collectively, these data demonstrate that the loss of PDE4 activity is indeed responsible for the PDE4D-/- phenotype. Moreover, we provide data that the expression of PDE4D mRNAs overlaps with the expression of binding sites for muscarinic cholinergic agonists within the smooth muscle. The binding pattern of the muscarinic agonist is consistent with earlier reports in the ferret (22)
and suggests a direct effect of PDE4D isoforms in muscarinic cholinergic signaling. Taken together, these data suggest a selective impairment of muscarinic cholinergic signaling in the trachea of PDE4D-/- mice.
The presence of indomethacin in the organ bath completely reversed the PDE4D-/- phenotype, underscoring the existence of an endogenous relaxant prostanoid tone in the isolated trachea. This tone has no effect on contraction in the airways in the wild-type mice. Our conclusion is based on observations that indomethacin has a minimal effect in wild-type tracheas (our results) and that ablation of the PGE2 receptor EP2 does not modify the response of the trachea to carbachol (21)
. Therefore, only after the ablation of PDE4D does the prostaglandin tone become relevant in the in vitro model, and most likely in vivo as well.
The content of PGE2, the predominant prostaglandin accumulating in the upper respiratory tract (23)
, was used as an index of cyclooxygenase activity. PGE2 content was similar in PDE4D-/- and PDE4D+/+ trachea baths, suggesting that an increase in the production of relaxant prostanoids is not the cause of the differences between the two mouse strains. Conversely, an increase in sensitivity to relaxant PGE2 is present in the PDE4D-/- trachea, as demonstrated by a fivefold increase in PGE2 potency to induce relaxation compared with the wild-type. Moreover, blocking the PKA also reversed the phenotype of the PDE4D-/- trachea, and PGE2 induced an exaggerated accumulation of cAMP in the lungs. Thus, airways from the PDE4D-/- mice are in a state of relaxation due to a tonic cAMP/PKA pathway that can be overcome only partially by cholinergic stimulation. A synergism was suggested between prostanoids and PDE4 in isolated human airways (4)
. Moreover, the inhibition of endogenous PGE2 production has been shown to diminish the action of the PDE4 inhibitors in models of chemotaxis and contraction in gel (24)
. Together, these data infer that PGE2-increased sensitivity is a critical factor in the impact of PDE4D ablation on airway contractility.
The in vivo response of the PDE4D-/- mice to methacholine is completely lost, whereas residual contraction to carbachol is present in vitro. Although indomethacin to some extent restores the response in vivo, it is probable that additional activators of AC contribute to the reduction of a cholinergic stimulation. Underwood et al. reported that rolipram in the presence of propranolol failed to inhibit ovalbumin-induced bronchoconstriction in guinea pigs, although no effect of indomethacin on PDE4 inhibition of airway resistance in response to antigen was observed in this model (25)
. In allergic mice, treatment with propranolol inhibits airway hyperresponsiveness (26)
. Moreover, in these models the removal of adrenal abolished the effects of PDE4 inhibitors on bronchoconstriction (25
, 27)
. Thus, it is likely that several ligands that activate GPCRs and cAMP signaling contribute to the total loss of muscarinic cholinergic stimuli observed in vivo. It is also possible that prostaglandin tone in vivo is higher than that measured in vitro.
Of the five muscarinic cholinergic receptors, M3 and M2 have been implicated in airway constriction. M3 receptors are coupled to phosphoinositide turnover and calcium mobilization, whereas M2 receptors are coupled to Gi and decrease cAMP production. We have shown previously that carbachol was unable to decrease cAMP levels in the PDE4D-/- lungs (9)
, suggesting some dysfunction of the M2 receptor response in PDE4D-/- airways. However, carbachol contracts tracheas from the M2 receptor knockout mice with a decrease in potency compared with the wild-type littermates but exhibits no difference in maximal efficacy (28)
. Nonetheless, pharmacological antagonism of the M2 receptors increased the sensitivity of wild-type tracheas to relaxant PGE2 (29)
. Forskolin was shown to be more potent in relaxing tracheas from mice lacking the M2 receptor (30)
. These findings suggest that increased sensitivity to PGE2 in the PDE4D-/- trachea may be due in part to a loss of M2 receptor signaling. In addition, carbachol-induced contractions are decreased by 40% in the M3 receptor knockout trachea, a decrease comparable to that seen in the PDE4D-/- trachea (31)
. Thus, PDE4D inactivation most likely affects both M2 and M3 receptor signaling pathways.
Despite the loss of the cholinergic contractile response, the responses to the depolarizing agent KCl and to AVP were identical in PDE4D+/+ and PDE4D-/- tracheas. This observation is reminiscent of the normal response to serotonin observed in vivo in the PDE4D-/- mice (9)
. Because neither KCl nor AVP signals through Gi in smooth muscle as do the muscarinic agonists (32)
, one can speculate that their actions are not blocked by an altered cAMP degradation. In addition, contraction of the ASM is primarily dependent on both calcium influx, through the opening of Ca2+ channels, and the release of Ca2+ sequestered within the sarcoplasmic reticulum (SR). It has been shown that contraction in response to Ach or carbachol in ASM is solely dependent on Ca2+ movement from the SR, whereas KCl and AVP can both use extra and intracellular Ca2+ (33)
. From these data, it can be surmised that the absence of PDE4D does not affect contractions dependent on Ca2+ influx but blocks responses mediated by Ca2+ mobilization from intracellular stores. The release of Ca2+ is the result of the activation of the IP3 receptors, whereas the reuptake of Ca2+ within the SR is dependent on the opening of the Ca2+/ATPase pump. The SR also expresses the ryanodine receptor, RyR, another type of Ca2+permeable channel. Although the exact role is debatable, it has been proposed that activation of RyR in ASM results in relaxation instead of contraction possibly through depletion of the Ca2+ storage or via the generation of sparks (1)
. These three different components of Ca2+ homeostasis have been shown to be substrates of PKA, and IP3 receptors and RyR exist in macromolecule complexes containing more than one signaling molecule scaffolded by A-kinase anchoring proteins (AKAP) (34
35
36)
. Moreover, PDE4D is present in a signaling complex with PKA through interaction with an AKAP (37
, 38)
and has been described in a complex with RyR (39)
. The involvement of PDE4D in such complexes in ASM remains to be investigated, but would provide a means for cross-talk between Ca2+ and cAMP in contraction regulation.
In conclusion, the present study provides a mechanistic rationale for the absence of cholinergic stimulation in the airways of PDE4D-/- mice. PDE4D is a major determinant of the threshold of action and the balance between contractile and relaxing cues. Moreover, our data infer that a PDE4D inhibitor may induce or enhance the bronchodilatatory response to endogenous prostanoids. This phenotype stresses the pharmacological potential of PDE4D inhibitors in airway diseases associated with smooth muscle contraction and opens the possibility that inherited mutations in the PDE4D gene affect airway constriction.
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ACKNOWLEDGMENTS |
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Received for publication April 7, 2003. Accepted for publication June 12, 2003.
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REFERENCES |
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 A. Hu, G. Nino, J. S. Grunstein, S. Fatma, and M. M. Grunstein Prolonged heterologous {beta}2-adrenoceptor desensitization promotes proasthmatic airway smooth muscle function via PKA/ERK1/2-mediated phosphodiesterase-4 induction Am J Physiol Lung Cell Mol Physiol, June 1, 2008; 294(6): L1055 - L1067. [Abstract] [Full Text] [PDF]
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 C. K. Billington, I. R. Le Jeune, K. W. Young, and I. P. Hall A Major Functional Role for Phosphodiesterase 4D5 in Human Airway Smooth Muscle Cells Am. J. Respir. Cell Mol. Biol., January 1, 2008; 38(1): 1 - 7. [Abstract] [Full Text] [PDF]
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 D. Peter, S. L. C. Jin, M. Conti, A. Hatzelmann, and C. Zitt Differential Expression and Function of Phosphodiesterase 4 (PDE4) Subtypes in Human Primary CD4+ T Cells: Predominant Role of PDE4D J. Immunol., April 15, 2007; 178(8): 4820 - 4831. [Abstract] [Full Text] [PDF]
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 M. D. Houslay, G. S. Baillie, and D. H. Maurice cAMP-Specific Phosphodiesterase-4 Enzymes in the Cardiovascular System: A Molecular Toolbox for Generating Compartmentalized cAMP Signaling Circ. Res., April 13, 2007; 100(7): 950 - 966. [Abstract] [Full Text] [PDF]
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K. Omori and J. Kotera Overview of PDEs and Their Regulation Circ. Res., February 16, 2007; 100(3): 309 - 327. [Abstract] [Full Text] [PDF]
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 J. Cheng and J. P. Grande Cyclic Nucleotide Phosphodiesterase (PDE) Inhibitors: Novel Therapeutic Agents for Progressive Renal Disease Experimental Biology and Medicine, January 1, 2007; 232(1): 38 - 51. [Abstract] [Full Text] [PDF]
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 A. T. Bender and J. A. Beavo Cyclic Nucleotide Phosphodiesterases: Molecular Regulation to Clinical Use Pharmacol. Rev., September 1, 2006; 58(3): 488 - 520. [Abstract] [Full Text] [PDF]
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 J. Cheng, M. M. D. Encarnacion, G. M. Warner, C. E. Gray, K. A. Nath, and J. P. Grande TGF-{beta}1 stimulates monocyte chemoattractant protein-1 expression in mesangial cells through a phosphodiesterase isoenzyme 4-dependent process Am J Physiol Cell Physiol, October 1, 2005; 289(4): C959 - C970. [Abstract] [Full Text] [PDF]
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M. J. Lynch, G. S. Baillie, A. Mohamed, X. Li, C. Maisonneuve, E. Klussmann, G. van Heeke, and M. D. Houslay RNA Silencing Identifies PDE4D5 as the Functionally Relevant cAMP Phosphodiesterase Interacting with {beta}Arrestin to Control the Protein Kinase A/AKAP79-mediated Switching of the {beta}2-Adrenergic Receptor to Activation of ERK in HEK293B2 Cells J. Biol. Chem., September 30, 2005; 280(39): 33178 - 33189. [Abstract] [Full Text] [PDF]
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 S. Liu, A. Veilleux, L. Zhang, A. Young, E. Kwok, F. Laliberte, C. Chung, M. R. Tota, D. Dube, R. W. Friesen, et al. Dynamic Activation of Cystic Fibrosis Transmembrane Conductance Regulator by Type 3 and Type 4D Phosphodiesterase Inhibitors J. Pharmacol. Exp. Ther., August 1, 2005; 314(2): 846 - 854. [Abstract] [Full Text] [PDF]
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 P. J. Barnes and R. A. Stockley COPD: current therapeutic interventions and future approaches Eur. Respir. J., June 1, 2005; 25(6): 1084 - 1106. [Abstract] [Full Text] [PDF]
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 D. A. Wallace, L. A. Johnston, E. Huston, D. MacMaster, T. M. Houslay, Y.-F. Cheung, L. Campbell, J. E. Millen, R. A. Smith, I. Gall, et al. Identification and Characterization of PDE4A11, a Novel, Widely Expressed Long Isoform Encoded by the Human PDE4A cAMP Phosphodiesterase Gene Mol. Pharmacol., June 1, 2005; 67(6): 1920 - 1934. [Abstract] [Full Text] [PDF]
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 H.-F. Tang, Y.-H. Song, J.-C. Chen, J.-Q. Chen, and P. Wang Upregulation of Phosphodiesterase-4 in the Lung of Allergic Rats Am. J. Respir. Crit. Care Med., April 15, 2005; 171(8): 823 - 828. [Abstract] [Full Text] [PDF]
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A. P. Barnes, G. Livera, P. Huang, C. Sun, W. K. O'Neal, M. Conti, M. J. Stutts, and S. L. Milgram Phosphodiesterase 4D Forms a cAMP Diffusion Barrier at the Apical Membrane of the Airway Epithelium J. Biol. Chem., March 4, 2005; 280(9): 7997 - 8003. [Abstract] [Full Text] [PDF]
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 J. Cheng, M. A. Thompson, H. J. Walker, C. E. Gray, M. M. Diaz Encarnacion, G. M. Warner, and J. P. Grande Differential regulation of mesangial cell mitogenesis by cAMP phosphodiesterase isozymes 3 and 4 Am J Physiol Renal Physiol, November 1, 2004; 287(5): F940 - F953. [Abstract] [Full Text] [PDF]
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