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J Biol Chem, Vol. 274, Issue 45, 32241-32247, November 5, 1999

Transgenic Overexpression of ₂-Adrenergic Receptors in Airway Smooth Muscle Alters Myocyte Function and Ablates Bronchial Hyperreactivity^*

Dennis W. McGraw, Susan L. Forbes, Lisa A. Kramer, David P. Witte^§, Christopher N. Fortner^¶, Richard J. Paul^¶, and Stephen B. Liggett^**

From the Departments of  Medicine,  Molecular Genetics, and ^¶ Physiology, University of Cincinnati College of Medicine, Cincinnati Ohio 45267 and ^§ Children's Hospital Medical Center, Cincinnati, Ohio 45229

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

₂-Adrenergic receptors (₂AR) act to relax airway smooth muscle and can serve to counteract hyperresponsiveness, although the effect may not be ablative even in the presence of exogenous agonist. Within this signaling cascade that ultimately transduces smooth muscle relaxation, a significant "spare receptor" pool has been hypothesized to be present in the airway. In order to modify the relationship between ₂AR and downstream effectors, transgenic mice (TG) were created overexpressing ₂AR ~75-fold in airway smooth muscle using a mouse smooth muscle -actin promoter. While >90% of these receptors were expressed on the smooth muscle cell surface, the percentage of receptors able to form the agonist-promoted high affinity complex was less than that found with nontransgenic (NTG) cells (R_H = 18 versus 36%). Nevertheless, ₂AR signaling was found to be enhanced. Intact airway smooth muscle cells from TG had basal cAMP levels that were greater than NTG cells. A marked increase in agonist-stimulated cAMP levels was found in the TG (~200% stimulation over basal) compared with NTG (~50% over basal) cells. Adenylyl cyclase studies gave similar results and also showed a 10-fold lower EC₅₀ for TG cells. Tracheal rings from TG mice that were precontracted with acetylcholine had an enhanced responsiveness (relaxation) to -agonist, with a 60-fold decrease in the ED₅₀, indicating that the enhanced signaling imposed by overexpression results in an increase in the coordinated function of the intact airway cells. In vivo studies showed a significantly blunted airway resistance response to the inhaled bronchoconstrictor methacholine in the TG mice. Indeed, with -agonist pretreatment, the TG mice displayed no response whatsoever to methacholine. These results are consistent with ₂AR being the limiting factor in the transduction system. Increases in the initial component of this transduction system (the ₂AR) are sufficient to markedly alter signaling and airway smooth muscle function to the extent that bronchial hyperresponsiveness is ablated, consistent with an anti-asthma phenotype.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Asthma is a chronic inflammatory disorder of the airways in which airflow obstruction occurs due to an active constriction of airway smooth muscle of the bronchi and airway mucous accumulation. Bronchial smooth muscle cells express numerous G protein-coupled receptors that modulate contractility including ₂-adrenergic receptors (₂AR),¹ which act to relax, and muscarinic receptors, which act to contract, the muscle (1). Agonists to the former and antagonists to the latter receptors are utilized clinically for reversal of bronchoconstriction. The propensity for airway smooth muscle to constrict in asthma has been termed airway hyperresponsiveness (2). Thus, a hallmark physiological finding in patients with asthma is hyperresponsiveness of the bronchi to inhalation of constrictive agents such as the muscarinic agonist methacholine. This constrictive response in asthmatics is thought to be ultimately due to sensitization of pathways, such as the cholinergic system, that culminate in airway smooth muscle contraction. Typically, nonasthmatics have no detectable airway response to inhalation of methacholine, and thus airway hyperreactivity has become a defining physiologic parameter.

₂AR are cell surface receptors that couple to the stimulatory guanine nucleotide-binding protein (G_s), activating adenylyl cyclase. Increased intracellular cAMP mediates relaxation of airway smooth muscle by activation of protein kinase A. Protein kinase A acts to phosphorylate myosin light chain kinase, cell surface K⁺ channels, a Na⁺/K⁺ ATPase, phospholamban, and one or more pumps that lead to sarcoplasmic reticulum uptake of Ca²⁺ and acts to inhibit the production of inositol phosphates (3, 4). The net effect is a decrease in intracellular Ca²⁺ and phosphorylation of contractile proteins leading to relaxation. The ₂AR subtype is the predominant AR expressed on bronchial smooth muscle in humans (5), although there are some conflicting data regarding the role of the ₁AR subtype in bronchodilation in various other species (6-9). Within the airway, luminal epithelial cells also express ₂AR, and some evidence suggests that activation of these receptors contributes to smooth muscle relaxation via an unknown mediator (10).

The hierarchy of signaling pathways that establishes physiologic bronchomotor tone is not well established. The principle endogenous agonist for airway ₂AR is epinephrine. Thus, in the absence of significant elevations in circulating epinephrine, adrenergic control of smooth muscle tone may be primarily due to "basal" coupling of receptor to its effector. Chronic exposure to exogenous agonists in the treatment of bronchospasm can result in desensitization of the ₂AR response (tachyphylaxis) (11). Furthermore, the asthmatic inflammatory milieu itself appears to promote a desensitized ₂AR (12). These issues have promoted the concept that maintenance or augmentation of nonagonist (basal), and agonist-promoted, ₂AR function in the airway could favorably alter myocyte signaling and airway physiology to effectively block hyperresponsiveness. Due to the dynamic nature of ₂AR regulation, such attempts by pharmacologic or genetic means may lead to feedback regulation of the receptor, G_s, or other downstream components of the transduction pathway, thus minimizing the impact at the cellular and physiologic level. Such overexpression has also been shown to cause "promiscuous" coupling of receptors to G proteins that are not natively activated in cells with physiologic levels of receptor (13). Extensive functional coupling of airway smooth muscle ₂AR to G_i could serve to inhibit adenylyl cyclase and possibly promote mitogen-activated protein kinase activation (14), adversely affecting cell growth and possibly airway contractility itself (3). Promiscuous G_q coupling would mimic M3 muscarinic receptor activity and act to bronchoconstrict. Finally, data from some studies suggest that there are significant "spare ₂AR" expressed on airway smooth muscle (15-17), such that increasing the number or function of these receptors would have no discernible effect on signaling or physiologic function. Similarly, it may be that other components of the pathway leading to relaxation are limiting factors, such that augmentation of AR-promoted relaxation by increased receptor expression may not be possible.

The current study was undertaken to further understand the relationship between airway smooth muscle ₂AR expression and signaling, smooth muscle function, and airway responses within the context of bronchial hyperreactivity and to specifically address the multiple issues regarding augmentation of ₂AR function as stated above. We thus created transgenic mice overexpressing the ₂AR on smooth muscle using a smooth muscle -actin promoter. This was carried out in FVB/N mice, a strain that displays a moderate degree of bronchial hyperresponsiveness in the absence of antigenic challenge. Such overexpression had profound effects on cellular signaling, smooth muscle function, and bronchial hyperresponsiveness.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Transgenic Mice-- Smooth muscle-specific expression of the human ₂AR in transgenic mice was achieved by using the mouse smooth muscle -actin promoter (18) (a gift from Dr. A. Strauch). The construct was prepared by cloning the 1.5-kb HindIII/PshAI fragment encoding the human ₂AR (1.2 kb of ORF and 0.3 kb of 3'-untranslated region) upstream of the SV40 polyadenylation sequence in the plasmid pNNO3. The 3.6-kb smooth muscle -actin promoter fragment, termed SMP8 (18), was then subcloned into a BamHI site 5' to the ₂AR ORF. Orientation of each fragment was confirmed by sequence analysis and restriction enzyme digestion. The transgenic construct (~5.9 kb) was excised from the plasmid by NotI digestion and microinjected into male pronuclei of fertilized zygotes from superovulated FVB/N mice. Surviving zygotes were implanted into pseudopregnant foster mothers who gave birth to founders. Transgene-positive founder mice were identified by Southern blot analysis of genomic DNA derived from tail clips. Founders expressing the transgene were mated with nontransgenic FVB/N mice. Subsequent screening for the hemizygous transgene-positive progeny was by polymerase chain reaction analysis of the genomic DNA using a forward primer in the ₂AR ORF (5'-GGAGCAGAGTGGATATCACG-3') and a reverse primer in the SV40 polyadenylation region (5'-GTCACACCACAGAAGTAAGG-3'). Hemizygous mice from generations 2-4 between the ages of 10 and 14 weeks were used for all studies.

RNA Studies-- Ribonuclease protection assays were used to quantitate the amount of transgene mRNA in total RNA prepared from whole lung homogenates. A ³²P-labeled antisense riboprobe corresponding to the distal 500 base pairs of the human ₂AR ORF was prepared. This portion of the human transcript has only ~75% homology with the mouse ₂AR mRNA. A radiolabeled antisense riboprobe for -actin was also utilized to account for any potential differences in gel loading. Ribonuclease protection assays were carried out as previously reported (19) by hybridizing 20 µg of total cellular RNA with both the ₂AR and actin riboprobes. To localize transgene expression within the lung, in situ hybridization was performed on lung sections as described previously (20). Briefly, lungs were rapidly dissected, fixed in 4% paraformaldehyde, cryoprotected with 30% sucrose in PBS, and frozen in OCT. Cryostat sections (7 µm) were then mounted on saline-coated slides. An antisense cRNA probe for the human ₂AR was prepared as described above for the ribonuclease protection assay studies except that the probe was labeled with [³⁵S]UTP using a commercially available kit (Stratagene). A sense cRNA probe was generated from the same plasmid template using SP6 polymerase for use as a negative control. Hybridization was performed with 0.5-1.0 × 10⁶ cpm of labeled probe in a final volume of 30 µl/slide. Following overnight incubation at 42 °C, the sections were treated with 50 µg/ml RNase A and 100 units/ml RNase T1 for 30 min at 37 °C and washed to a final stringency of 0.1× standard citrate saline at 50 °C. Slides were dipped in NTB2 emulsion (Eastman Kodak Co.) diluted 1:1 with 0.6 M ammonium acetate, and exposed for 2 weeks, after which they were developed with D19 developer (Kodak) and counterstained with hematoxylin and eosin.

Airway Smooth Muscle Cell Cultures-- Tracheal smooth muscle cells were cultured from explants of excised tracheas using a modification of previously described methods (21). The entire trachea between the larynx and main stem bronchi was removed and placed in a sterile Petri dish containing room temperature Hanks' balanced saline solution supplemented with a 2× concentration of antibiotic-antimycotic solution (Life Technologies, Inc.). After additional surrounding tissue was removed with the aid of a dissecting microscope, the tracheal segment was split longitudinally and dissected into 2-3-mm squares. All of the segments from a single trachea were then placed intima side down in a sterile 60-mm dish. After allowing the explants to adhere, 2.5 ml of Dulbecco's modified Eagle's medium supplemented with 20% FCS and 2× antibiotic-antimycotic was added to cover the explants. The explants were incubated at 37 °C in a humidified environment of 95% air, 5% CO₂. After the first 3 days of cell growth, the concentration of FCS was reduced to 10%, and the antibiotic-antimycotic was reduced to 1×. Explanted trachea was removed when the outgrowing cells became locally confluent. Once the 60-mm dish became confluent, the cells were harvested by trypsinization and passed into a single 75-cm² flask. Tracheal smooth muscle cells were subsequently passaged at a 1:4 ratio. Greater than 90% of these cells from each donor mouse were smooth muscle cells, as determined by immunohistochemistry performed with an antibody raised against smooth muscle -actin (see below). All experiments were performed on confluent cells at matched passage numbers 3-6.

Radioligand Binding and cAMP Studies-- To prepare membranes from lung tissue, lungs from an individual mouse were homogenized with a Polytron (Brinkman) in 10 ml of hypotonic lysis buffer (5 mM Tris, pH 7.4, 2 mM EDTA) containing the protease inhibitors leupeptin, aprotinin, benzamidine, and soybean trypsin inhibitor (10 µg/ml each). Detached smooth muscle cells from primary cultures were processed similarly. Homogenates were centrifuged at 40,000 × g for 10 min at 4 °C. The pellets were washed and centrifuged two additional times, after which the pellets were suspended in assay buffer (75 mM Tris, pH 7.4, 12.5 mM MgCl₂, 2 mM EDTA). For determination of receptor expression, radioligand binding was carried out with ¹²⁵I-CYP as described (22). The fraction of receptors in the high affinity binding state was determined by competition experiments performed in the absence of GTP using 40 pM ¹²⁵I-CYP and 16 concentrations of isoproterenol ranging from 10⁴ to 10¹⁰ M as described previously (23). Competition data were fit to a two-site model when this fit was statistically (p < 0.05) better than a one-site fit by F-test, using Prizm software (GraphPad, San Diego, CA). Receptor density on the cell surface was assessed using methods previously described (24). Briefly, cells grown in monolayers were detached with 0.25% trypsin for 5 min at 37 °C. After trypsin activity was neutralized by the addition of fetal calf serum, the cells were washed with PBS and resuspended in Dulbecco's modified Eagle's medium. The resuspended cells were incubated in a volume of 500 µl with 400 pM ¹²⁵I-CYP at 37 °C for 60 min in the absence or presence of the hydrophobic antagonist propranolol (1 µM) or the hydrophilic antagonist CGP 12177 (10 µM). Binding to receptors localized throughout the cell was defined as that displaced by propranolol, while cell surface binding was defined as that displaced by CGP12177 (24). Bound radioactivity was separated by filtration and washing over GF/C glass fiber filters. Cyclic AMP content of attached mouse smooth muscle cells in culture exposed to 10 µM isoproterenol or carrier for 10 min was measured by an acetylated radioimmunoassay method as described (19). Adenylyl cyclase activity was measured in membranes prepared as above using column chromatography as described previously (25).

Ex Vivo Smooth Muscle Studies-- Studies of mouse tracheal contractility have been reported in detail elsewhere (26). Briefly, tracheas were excised and dissected free of surrounding tissues and cut into rings of approximately 5 mm in length. The trachea rings were mounted on stainless steel wires connected to isometric force transducers. The rings were then immersed in a physiologic saline solution (118 mM NaCl, 4.73 mM KCl, 1.2 mM MgCl₂, 0.026 mM EDTA, 1.2 mM KH₂PO₄, 2.5 mM CaCl₂, 25 mM NaH₂CO₃, and 11 mM glucose) maintained at 37 °C and bubbled with 95% O₂, 5% CO₂ to maintain a pH of 7.4. Each tracheal ring was stretched to a tension of 5 millinewtons, an optimal passive tension for maximizing active force (27). After a 20-min equilibration period, contraction/relaxation cycles with 10 µM acetylcholine were performed until consistent forces were observed. Cumulative concentration-isometric force curves were then generated to acetylcholine (1 nM to 30 µM). After rinsing, tracheas were contracted with 10 µM acetylcholine (approximately the ED₈₀), and cumulative concentration-relaxation curves were generated to isoproterenol (30 pM to 10 µM). Concentration-response relations were fitted to a logistic equation (26).

In Vivo Airway Physiology-- Airway responsiveness to methacholine was measured noninvasively in conscious, unrestrained mice using a whole body plethysmograph (Buxco Electronics, Troy, NY) (28). Using this system, the volume changes that occur during a normal respiratory cycle are recorded as the pressure difference between the animal-containing chamber and a reference chamber. The resulting signal is used to calculate respiratory frequency, minute volume, tidal volume, and enhanced pause (Penh). Penh is a unitless value that is a function of the peak inspiratory (PIP) and peak expiratory (PEP) pressures and the timing of expiration and is calculated as follows,

(Eq. 1)

where Pause is the ratio of time spent in the last third of expiration relative to early expiration. Penh has been shown to closely correlate with invasive measurements of airway resistance (28) and was used as the measure of airway responsiveness in this study. Mice were placed in the chamber and allowed to adjust to their surroundings for 10 min. They were then exposed to aerosolized PBS (to establish baseline), followed by increasing concentrations of methacholine (2.5-80 mg/ml). Aerosolization was for 3 min, and respiratory measurements were recorded and averaged for the subsequent 5 min after each dose. The degree of bronchoconstriction was expressed as the percentage change in Penh relative to the PBS base line. On a separate day, the mice were submitted to the same protocol except that they were first treated with aerosolized albuterol (1.0 mg/ml) for 20 min. The concentration-response data for each individual mouse were fit to a sigmoid curve by an iterative least squares technique, and the dose of methacholine required to double baseline Penh (ED₂₀₀) was derived.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

From a total of 40 mice screened, three SMP8-₂AR founder mice were identified. Subsequent matings with nontransgenic mice showed that one of the three founders was mosaic. In lines established from the other two founder mice (denoted as lines 79 and 95), the transgene was inherited in ~50% of the offspring with equal distribution between male and female mice. Hemizygous mice from these two lines were further characterized with respect to transgene copy number, mRNA expression, receptor density, and histologic analysis. Southern blot analysis of genomic DNA prepared from tail clips showed that the transgene copy number was about 1 copy for line 95 and about two copies for line 79 (data not shown). To confirm that the SMP8-₂AR transgene was being expressed in the lungs of the transgenic positive mice, total cellular RNA prepared from whole lung homogenates of transgenic and nontransgenic animals was subjected to ribonuclease protection assays. For these experiments, we used an antisense RNA probe corresponding to the distal 500 base pairs of the human ₂AR ORF. Because this region has <75% homology with the mouse sequence, only the human sequence results in a full-length protected fragment. As shown in Fig. 1, SMP8-₂AR mRNA was present in lungs from mice that screened positive for the transgene, but was absent in nontransgenic littermates. Quantitation of band density showed that there was no significant difference in transgene expression between lines 79 and 95. 

View larger version (74K): [in this window] [in a new window]   Fig. 1.   Ribonuclease protection assays of SMP8-2AR transgene expression in the lung. Total cellular RNA (20 µg) was prepared from whole lung homogenates and simultaneously hybridized with a human 2AR and a mouse -actin riboprobe. Markers and full-length, undigested probes are shown on the left. The -actin band was present in all samples. A protected fragment corresponding to the human 2AR mRNA was present in transgenic mice but absent in NTG littermates. When band density of the 2AR fragment was normalized for -actin expression, transgene expression for the two transgenic lines was not significantly different.

In situ hybridization was performed to verify that expression of the transgene was directed to airway smooth muscle. Previous studies have shown that ₂AR mRNA is heavily expressed in airway smooth muscle (5, 7). We therefore used the same species-specific cRNA probe described above for the ribonuclease protection assay analysis so that we could limit detection to that of the transgene only. As shown in Fig. 2, specific hybridization (appearing as white dots) was observed in the SMP8-₂AR mice (A and B) but was absent in nontransgenic mice (C and D). These studies clearly show that expression of the SMP8-₂AR transcript was confined to airway smooth muscle, and to a lesser extent in pulmonary vascular smooth muscle, with no signal observed in the bronchial epithelium or alveolar lining cells (Fig. 2, A and B). Additional studies with in situ hybridization or radioligand binding showed increased expression in smooth muscle of stomach, colon, and uterus (data not shown).

View larger version (104K): [in this window] [in a new window]   Fig. 2.   In situ hybridization of SMP8-2AR mice and nontransgenic controls. Cryosections of lungs and tracheas were hybridized with a species-specific human 2AR antisense riboprobe to detect transgene expression. Dark field photomicrographs are shown. An arrow denotes areas of smooth muscle. A, longitudinal section of trachea from an SMP8-2AR transgenic mouse shows that the hybridization signals (white dots) are localized exclusively to smooth muscle. B, a cross-section of lung parenchyma from a SMP8-2AR transgenic mouse shows that the transgene was expressed in the smooth muscle of the airways but was absent in the bronchial epithelium and alveoli. C and D, no specific hybridization signals were present in either the trachea or peripheral lung of nontransgenic mice.

₂AR expression was further quantitated by radioligand binding assays with ¹²⁵I-CYP. Initial studies with membrane preparations from whole lung homogenates showed no differences in receptor density between transgenic and nontransgenic mice. However, previous studies have shown that >90% of ₂ARs in the lung are localized to cells (type I and type II pneumocytes and capillary endothelium) that line the alveoli (5, 7). Indeed, based on the densities of AR as assessed by autoradiography (5, 7) and the extensive surface area of alveoli (29), the ratio of total airway smooth muscle versus alveolar AR is ~1:1000 or more. Thus, we felt it was unlikely that using a whole lung preparation would detect enhanced smooth muscle expression in the transgenic mice. Since the in situ hybridization experiments demonstrated transgene expression in airway smooth muscle, we measured ¹²⁵I-CYP binding in smooth muscle cells cultured from tracheal explants. As indicated in Fig. 3, immunohistochemistry studies showed that >90% of these cells had the morphologic characteristics of smooth muscle cells and stained positive for smooth muscle -actin. Saturation radioligand binding studies showed that the ₂AR was significantly overexpressed in tracheal smooth muscle cells from transgenic mice when compared with the level of expression in smooth muscle cells derived from nontransgenic mice (Fig. 4). AR density in airway smooth muscle cells from transgenic lines 79 and 95 were both ~75 times greater than that of cells from nontransgenic mice (2510 ± 229 and 2218 ± 167 versus 33 ± 6 fmol/mg protein, respectively, n = 4, p < 0.001). Given that the levels of transgene mRNA and receptor protein were equivalent in the two transgenic lines, the majority of the remaining pharmacological and physiological studies were carried out with mice of line 95. 

View larger version (59K): [in this window] [in a new window]   Fig. 3.   Isolation and culture of tracheal smooth muscle cells. Primary cultures of tracheal smooth muscle cells were derived from tracheal explants as described under "Experimental Procedures." To confirm that the cells were of smooth muscle origin, immunohistochemistry was performed using an anti-smooth muscle -actin antibody and an FITC-conjugated secondary antibody. A, phase contrast microscopy of isolated cells. B, immunofluorescence of the same cells stained with the anti-smooth muscle -actin antibody. As shown in this representative experiment, >90% of the cells stain positively for smooth muscle -actin.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   2AR expression in primary cultures of tracheal smooth muscle cells. Primary cultures of tracheal smooth muscle cells were derived from tracheal explants as described under "Experimental Procedures." AR density was determined in membranes prepared from NTG and SMP8-2AR transgenic smooth muscle cells by radioligand binding with 125I-CYP. 10 µM propranolol was used to define nonspecific binding. Results represent the mean ± S.E. of four independent experiments for each group.

Radioligand binding with ¹²⁵I-CYP was also carried out in whole cells, using the competitors propranolol (hydrophobic) and CGP12177 (hydrophilic) to identify total cellular versus cell surface ₂AR, respectively. These studies showed that the vast majority of ₂AR (95 ± 1.5%, n = 4) of transgenic smooth muscle cells were expressed on the cell surface. This distribution was similar to what was found in cells from nontransgenic mice, although quantitation was difficult due to the low expression of receptors in these cells. Having determined that the transgenic ₂AR have a normal cellular distribution, we next assessed whether they had the capacity to form the high affinity agonist/receptor/G_s complex. In cells derived from nontransgenic and transgenic mice, agonist (isoproterenol) competition data in studies carried out in the absence of guanine nucleotide were best fit to a two-site model, while in the presence of 100 µM GTP the data were best fit to a single site model. However, in the absence of GTP, the proportion of receptors in the high affinity state (%R_H) was lower in membranes from transgenic cells compared with those of nontransgenic cells (%R_H = 18 ± 4.2 versus 36 ± 6.7). Taken together, the radioligand binding studies indicate that expression of ₂AR is significantly greater in transgenic smooth muscle cells compared with nontransgenic and that such expression is localized primarily to the cell surface but that a smaller fraction of these receptors is capable of physically coupling to G_s.

To assess whether receptor-mediated adenylyl cyclase stimulation was enhanced in SMP8-₂AR mice, we measured both cAMP content (Fig. 5A) in intact tracheal smooth muscle cells and adenylyl cyclase activities in smooth muscle cell membranes (Fig. 5B). We found that basal cAMP contents in airway smooth muscle cells from transgenic mice were ~2.8-fold greater than those of cells from nontransgenic mice (3.70 ± 0.41 versus 1.34 ± 0.05 pmol/ml, respectively, n = 5, p < 0.001). Similarly, isoproterenol-stimulated cAMP content in transgenic airway smooth muscle cells was also significantly greater than that of cells from the nontransgenic controls (11.78 ± 1.62 versus 2.05 ± 0.10 pmol/ml, respectively, n = 5, p < 0.001). When assessed as percentage of stimulation over basal levels, the isoproterenol-stimulated cAMP levels of cells from the transgenic mice (~200%) was markedly greater than what was observed in nontransgenic cells, which amounted to only ~50% over basal. Studies of adenylyl cyclase activity in cell membranes gave similar results. Basal activities were greater in transgenic as compared with nontransgenic membranes (273 ± 39.3 versus 172 ± 7.8, pmol/min/mg, n = 4, p < 0.05), as were maximal isoproterenol-stimulated activities (609 ± 74.7 versus 251 ± 7.3, pmol/min/mg, n = 4, p < 0.005). The percentage of isoproterenol stimulation over basal level was ~125 versus ~46%, respectively. The dose-response curve for isoproterenol stimulation of adenylyl cyclase from SMP8-₂AR membranes was left shifted compared with nontransgenic, with an EC₅₀ of 21 ± 1.4 versus 213 ± 107 nM (n = 4, p < 0.05). Of note, a less than doubling of basal adenylyl cyclase or cAMP levels by isoproterenol in cultured airway smooth muscle cells from other species has been previously reported (30), although higher levels have also been noted (21). This variability is probably due to assay conditions or species variation. However, in our work, smooth muscle cells from the two lines were studied under identical conditions in a paired manner, so it is the difference observed between transgenic and nontransgenic cells that is the critical finding.

View larger version (8K): [in this window] [in a new window]   Fig. 5.   cAMP production and adenylyl cyclase activity in isolated tracheal smooth muscle cells. A, cAMP levels were measured in intact smooth muscle cells that were grown in 24-well plates and treated with either vehicle or various concentrations of isoproterenol for 10 min at 37 °C. Reactions were stopped by the addition of HCl, and cAMP was measured by radioimmunoassay. Results are from four independent experiments. B, adenylyl cyclase activity was measured in membranes prepared from tracheal smooth muscle cells of transgenic and nontransgenic mice. Reactions were carried out in the presence of the indicated concentrations of isoproterenol for 10 min at 37 °C. Detection of [32P]cAMP produced was determined by column chromatography. Shown is a representative dose-response curve. Data for basal and maximal (10 µM isoproterenol) agonist-stimulated activity in pmol/min/mg are summarized in the bar graph (n = 4 experiments).

These studies thus clearly demonstrate that overexpression of ₂AR in airway smooth muscle results in enhanced adenylyl cyclase activity at baseline and in response to agonist. The data are consistent with the multistate model of G protein-coupled receptors, where in the non-agonist-bound state a small proportion of receptors spontaneously achieve the active conformation (R*). With overexpression, the number of receptors in this state at any one time is increased sufficiently to alter "basal" coupling, as shown by the increased cAMP levels and adenylyl cyclase activities of airway smooth muscle cells in the absence of agonist. Maximal constitutive activation of G_s-adenylyl cyclase was not observed, however, since isoproterenol resulted in yet further stimulation of cAMP in the transgenic derived cells. The percentage stimulation by agonist over base line was significantly greater as compared with nontransgenic littermates. Taken together, the data are consistent with the ₂AR being a limiting factor in the receptor-G_s-adenylyl cyclase cascade in native airway smooth muscle cells. This is in direct contrast to other studies (15-17), which have concluded that there is a substantial receptor reserve on airway smooth muscle. Although ₂AR expression was increased ~75-fold in the current study, we did not observe commensurate increases in basal or agonist-stimulated cAMP responses. A very similar finding has been reported with transgenic ₂AR overexpression in the heart. In one such study, we obtained a ~45-fold overexpression of wild-type human ₂AR in the hearts of transgenic mice, yet basal and isoproterenol-stimulated adenylyl cyclase activities were increased only 3-4-fold over nontransgenic activities (31). Milano et al. (32) had a 200-fold increase in cardiac ₂AR expression, with 2-fold increases in basal and isoproterenol-stimulated activities. These findings have been interpreted as being consistent with other elements of the transduction cascade (G_s, adenylyl cyclase) becoming limiting factors when AR expression is markedly increased, such that proportional increases in signaling in relation to receptor expression are not observed. Indeed, in the current study a smaller percentage of ₂AR in transgenic smooth muscle cells can form the high affinity receptor-G_s complex as compared with nontransgenic cells, probably indicating insufficient G_s to accommodate all of the overexpressed receptors.

To determine whether this enhanced signaling observed in cells results in regulation of coordinated smooth muscle function of the airway, we measured responses to agonist ex vivo using tracheal ring preparations. For these studies, tracheal rings dissected from SMP8-₂AR and nontransgenic mice were mounted in the same organ bath, and relaxation in response to isoproterenol was measured. Initial studies showed that the constriction response to acetylcholine was equivalent between rings derived from transgenic and nontransgenic mice (Fig. 6A). For isoproterenol dose-response experiments, rings were preconstricted by incubation with 10 µM acetylcholine. The sensitivity to isoproterenol was found to be markedly enhanced in the tracheal rings from SMP8-₂AR mice (Fig. 6B). As shown, response curves for isoproterenol for these mice were shifted to the left (60-fold decrease in ED₅₀) compared with nontransgenic tracheal rings (ED₅₀ = 0.64 ± 0.07 versus 40.0 ± 7.1 nM, respectively, n = 4, p < 0.001). However, the maximal extent of relaxation was equivalent. This enhancement of isoproterenol-induced relaxation in SMP8-₂AR mice was blocked by pretreatment with the selective ₂AR antagonist ICI 118,551 (0.1 µM) (Fig. 6C), indicating that the response observed in these mice was directly the result of ₂AR activation from transgenic overexpression of the receptor rather than a change in some other factor caused by insertion of the transgene. While these differences in smooth muscle relaxation ex vivo are quite significant, the fact that the maximal degree of agonist-mediated relaxation in the transgenic rings was not greater than that of the nontransgenics suggests that at the level of this physiologic response there may be other factors that limit further relaxation regardless of the number of ₂AR expressed. Since in intact smooth muscle cells we do observe a leftward shift in the response curve as well as an increase in the maximal response, the limiting factor(s) may be in elements that are necessary after the early signal transduction events.

View larger version (9K): [in this window] [in a new window]   Fig. 6.   Ex vivo assessment of airway function in transgenic and nontransgenic mice. Tracheal contractility was measured using ring preparations that were mounted on wires connected to pressure transducers and immersed in organ baths. The absolute contractile force evoked by acetylcholine is shown in A. In B and C, the relaxation response to isoproterenol is shown. Rings were precontracted to the same extent with 10 µM acetylcholine for these studies. The dose response for the SMP8-2AR rings was shifted to the left (ED50 ~60-fold less) compared to nontransgenic rings. See "Results and Discussion" for mean results. Pretreatment with ICI 118,551 abolished the enhanced isoproterenol sensitivity in SMP8-2AR mice, indicating that the effect was specifically due to overexpression of the 2AR (C). Curves shown represent the mean ± S.E. of data generated from four different mice in each group.

The above results indicated that transgenic mice overexpressing the ₂AR in airway smooth muscle have enhanced signaling in isolated cells and tracheal rings. To determine whether this resulted in altered physiologic function of the airways in vivo, studies were carried out in intact mice using a rodent whole body plethysmography system. We hypothesized that this persistent ₂AR signaling would result in a state of relative hyporesponsiveness to bronchoconstriction by methacholine. Thus, the responses to inhaled methacholine alone and methacholine after inhalation of the -agonist albuterol were measured (Fig. 7). These results show that the maximal level of bronchoconstriction induced by methacholine in the SMP-₂AR mice was significantly less than that of nontransgenic mice (Penh = 250 ± 26 versus 558 ± 42% of base line, respectively, p < 0.001). In addition, the sensitivity to methacholine was altered in the SMP-₂AR mice, with the ED₂₀₀ = 35.7 ± 10.6 mg/ml as compared with 12.1 ± 1.90 mg/ml in the nontransgenic species (p < 0.02). Even more striking were the responses to methacholine after exposure to the -agonist albuterol. As shown, when SMP8-₂AR mice were pretreated with albuterol, methacholine caused no increase in Penh, even at the highest dose used in this study (80 mg/ml) (Fig. 7). In contrast, in nontransgenic mice methacholine responsiveness after albuterol was present, with the maximal bronchoconstriction being 373 ± 33% of base line. It is interesting to note that the extent of bronchoconstriction in nontransgenic mice treated with albuterol was greater than that observed in the transgenic mice in the absence of agonist (Penh = 373 ± 33 versus 250 ± 26% of base line, p = 0.01). These results are entirely consistent with the intact smooth muscle cell studies, where maximal cAMP content after agonist exposure in the nontransgenic cells was similar to non-agonist-exposed levels in the transgenic cells. The in vivo studies are also consistent with the ex vivo organ bath results. Here, trachea overexpressing ₂AR maximally relaxed in the presence of low concentrations of agonist, while nontransgenic rings showed no demonstrable response at these concentrations (Fig. 6B). It should be noted, however, that while cellular cAMP/adenylyl cyclase, tracheal ring, and whole body plethysmography studies are complementary, each has constraints that limit interpretation in isolation. In cells, our studies are confined to measurement of effector (adenylyl cyclase) activity or its product (cAMP). While these experiments and radioligand binding studies can assess coupling of the receptor to this pathway, they do not provide information regarding post-cAMP events relevant to smooth muscle function (i.e. relaxation). The tracheal ring studies require preconstriction with acetylcholine in order to derive a signal and may not directly relate relaxation to airflow in vivo, the latter being influenced by additional physiologic variables. The whole animal plethysmography studies are limited by the doses of the drugs that are tolerated, could additionally be influenced by endogenous catecholamines, and are not easily amenable to measurements in response to multiple doses of agonist. Given the whole cell cAMP and the in vivo plethysmography results, it would not be unexpected for the tracheal ring studies to show a decreased basal force (tension) and a leftward shift in the acetylcholine dose response for the transgenic rings. However, tracheal rings must be stretched to some extent in order to obtain a transducer signal. Thus, basal as well as acetylcholine-induced constriction are under conditions in such preparations that are not analogous to our whole cell or in vivo studies.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   In vivo measurement of airway hyperreactivity in conscious, unrestrained mice. Airway responsiveness to methacholine was assessed using a rodent whole body plethysmography system to measure Penh (see "Experimental Procedures"). The mice were first treated with aerosolized PBS to establish base-line function, which was then followed by increasing doses of aerosolized methacholine. On a separate day, the same mice were first pretreated with aerosolized albuterol (1.0 mg/ml) for 20 min. Penh values are reported as percentage of the PBS (base-line) value. Bronchoconstriction was greatest in the untreated, NTG control mice (n = 12) with a maximal increase in Penh of 558 ± 42%. Pretreatment with albuterol lowered the maximal increase in Penh to 373 ± 33% (p < 0.02). The maximum Penh achieved in the transgenic mice (250 ± 26%, n = 9) was less than that of both the untreated (p < 0.001) and the pretreated NTG mice (p < 0.02). When SMP8-2AR mice were pretreated with albuterol, no methacholine-induced bronchoconstriction was observed (maximum Penh was 138 ± 11%, p > 0.05 compared with base line).

Our studies constitute the first transgenic overexpression of ₂AR in a tissue where the receptor acts to inhibit a physiologic response. Previous reports with transgenic overexpression of the receptor in the heart, where the receptor stimulates contraction, have shown increased cardiac inotropy and chronotropy (31, 32). These studies suggested that ₂AR overexpression might be useful therapeutically to overcome the depressed chronotropic state in congestive heart failure. Subsequent studies with very high ₂AR overexpression in genetic models of cardiac hypertrophy/heart failure have revealed an increased mortality (33, 34). In our current work, we have overexpressed the receptor in a cell type where the physiologic response of ₂AR is a decrease, rather than an increase, in contraction. In mice up to 14 months old, we have not found structural remodeling of the airway, alterations in smooth muscle morphology, or pathologic consequences in the lung of ₂AR overexpression in airway smooth muscle. Our results clearly indicate that the ₂AR-mediated relaxation response in airway smooth muscle can accommodate increased receptor levels with an increase in base-line and agonist-promoted function. The notion that there are sufficient spare receptors on native airway smooth muscle to limit the effectiveness of enhanced expression or function is thus not supported, at least in mouse lung.

The potential for transgenic overexpression of ₂AR in smooth muscle to decrease cellular levels of G_s has been suggested in studies with transfection of ₂AR into NG108 cells (35). Such a decrement might have consequences for ₂AR and other G_s-coupled receptors endogenously expressed on the airway and could limit the effectiveness of the transgene. Using the isolated airway smooth muscle cells, G_s content as assessed by Western blots was not different in cells derived from transgenic mice compared with nontransgenic littermates (data not shown); nor were the levels of the G_i isoform G_i2 different. We also examined the potential for agonist-promoted desensitization of physiologic ₂AR responses in this setting of transgenic overexpression of the receptor. Transgenic mice were implanted with osmotic minipumps administering isoproterenol for 3 continuous days and then studied by plethysmography. The methacholine concentration-response curve in transgenic mice pretreated with isoproterenol remained essentially flat after acute albuterol, with a maximal Penh of 128 ± 1% of base line (compared with 146 ± 12% in the absence of isoproterenol pretreatment, p > 0.05). So agonist-promoted desensitization of ₂AR function at the physiologic level is not observed in the SMP8-₂AR transgenic mice, probably due to the extensive overexpression to a point such that some fraction of spare receptors are in fact present.

In conclusion, we have shown that the ₂AR of airway smooth muscle represent a limiting element of the signal transduction pathway. This constraint is alleviated by increased expression, which enhances basal and isoproterenol-stimulated levels of intracellular cAMP. Such an increase has a significant impact on airway smooth muscle function, ultimately decreasing bronchial hyperresponsiveness. Given the central importance of bronchial hyperresponsiveness to the asthmatic phenotype, these mice can be considered to be in an anti-asthmatic state. As such, overexpression of ₂AR in airway smooth muscle may be a potential genetic therapy for asthma.

	ACKNOWLEDGEMENTS

We thank Craig Weber and Cheryl Theiss for technical assistance and Mary Ann Rosensweet for manuscript preparation.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This work was supported by National Institutes of Health Grants HL45967, HL41496, and HL54829.

^** To whom correspondence should be addressed: University of Cincinnati College of Medicine, 231 Bethesda Ave., Cincinnati, OH 45267-0564. Tel.: 513-558-4831; Fax: 513-558-0835; E-mail: Stephen. Liggett{at}UC.edu.

	ABBREVIATIONS

The abbreviations used are: ₂AR, ₂-adrenergic receptor(s); G_s, stimulatory guanine nucleotide-binding protein; Penh, enhanced pause; NTG, nontransgenic; kb, kilobase; ORF, open reading frame; PBS, phosphate-buffered saline; CYP, cyanopindolol.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

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