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Physiol. Genomics 27: 171-177, 2006. First published July 18, 2006; doi:10.1152/physiolgenomics.00044.2006
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Received 22 March 2006; accepted in final form 6 July 2006.
Physiological Genomics 27:171-177 (2006)
1094-8341/06 $8.00 © 2006 American Physiological Society

Transcriptional response to persistent ß2-adrenergic receptor signaling reveals regulation of phospholamban, which alters airway contractility

Dennis W. McGraw 1, Kevin M. Fogel 1, Sue Kong 2, Augusto A. Litonjua 3, Evangelia G. Kranias 4, Bruce J. Aronow 2 and Stephen B. Liggett 5

1 Pulmonary and Critical Care Medicine, University of Cincinnati College of Medicine, Cincinnati
2 Division of Biomedical Informatics, Cincinnati Children's Hospital Research Foundation, University of Cincinnati College of Medicine, Cincinnati, Ohio
3 Channing Laboratory, Brigham and Women's Hospital, Boston, Massachusetts
4 Departments of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio
5 Cardiopulmonary Genomics Program, University of Maryland School of Medicine, Baltimore, Maryland


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
ß2-Adrenergic receptors (ß2AR) are expressed on airway smooth muscle cells and act to relax the airway on activation by ß-agonists. These agents are utilized for treating asthma but are associated with adverse outcomes. To ascertain the effects of persistent ß2AR activation on gene expression, cultured airway smooth muscle cells derived from wild-type (WT) and transgenic mice overexpressing ß2AR were subjected to DNA microarray analysis; 319 genes were increased and 164 were decreased. Differential expression was observed in genes from 22 Gene Ontology Slim categories, including those associated with ion transport and calcium ion binding. A 60% decrease (P = 0.008) in phospholamban (PLN), an intracellular Ca2+ concentration ([Ca2+]i)-handling protein that is at a signaling nodal point in cardiomyocytes, was observed in ß2AR-overexpressing cells and confirmed at the protein level. To isolate the physiological effect of decreased PLN in airway smooth muscle, airway contraction and relaxation responses were studied in WT and PLN–/– mice. PLN–/– mice had a markedly reduced constrictive response to methacholine. In contrast, the bronchodilatory effect of ß-agonist was not different between WT and PLN–/– mice. These results revealed an unanticipated therapeutic effect of ß-agonists, PLN downregulation, which acts to decrease airway hyperreactivity. Thus agents that inhibit PLN may act synergistically with the bronchodilating action of ß-agonists. A number of other genes related to [Ca2+]i are also differentially regulated by ß2AR activity, some of which may act to oppose, or augment, the efficacy of chronic ß-agonists. These genes or pathways may also represent additional targets in the treatment of asthma and related obstructive lung diseases.

G protein-coupled receptors; asthma; ß-agonist


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
ß2-ADRENERGIC RECEPTOR (ß2AR) agonists represent the most commonly employed therapy for asthma. These agents bind to ß2AR on airway smooth muscle, which evokes relaxation and counteracts the constrictive signals derived from airway inflammation. ß2AR couple to the stimulatory guanine nucleotide Gs, which activates adenylyl cyclase, catalyzing the conversion of ATP to cAMP. Subsequent activation of protein kinase A (PKA) phosphorylates a number of proteins, leading to relaxation (11). G protein-coupled bronchoconstrictive stimuli are typically mediated by Gq-coupled receptors, such as the M3-muscarinic and cysteinyl leukotriene receptors (7). Of particular concern over the last few decades have been the observed deleterious effects associated with chronic ß-agonist treatment in asthma, which include enhanced sensitivity to constrictive agents (13), decreased bronchodilatation (28), and increased asthma exacerbations and mortality (2, 21, 25). The molecular basis for these events has remained elusive, but recent studies have indicated cross talk between bronchodilatory Gs pathways and constrictive Gq pathways in airway smooth muscle. For example, we recently found antithetic regulation of phospholipase Cß (PLCß) expression by chronic ß2AR activity, which had a substantial impact on airway function (18). Enhanced ß2AR receptor activity caused increased PLCß expression, imparting increased sensitivity of the airways to constrict to agonists that activate Gq-coupled receptors. These types of unexpected findings have prompted us to consider other regulatory events that may occur in airway smooth muscle during continuous activation of ß2AR, particularly "deep-pathway" elements, which are fundamental to smooth muscle contraction and relaxation. Central to receptor Gq-mediated contraction is the mobilization of Ca2+ stores from the endoplasmic reticulum to the cytosol, where intracellular Ca2+ ([Ca2+]i) acts via multiple pathways to induce contraction. This includes the well-described myosin light chain kinase and smooth muscle myosin light chain phosphatase signals, which act to regulate the phosphorylation of myosin and thus smooth muscle contractile status (10). Critical components of [Ca2+]i reuptake are the sarco(endo)plasmic reticulum Ca2+-ATPase-2 (SERCA2), which pumps [Ca2+]i from the cytosol to the endoplasmic reticulum (ER), and phospholamban (PLN), which decreases the Ca2+ affinity of, and thus partially inhibits, SERCA2 under steady-state conditions (17). PKA-mediated phosphorylation of PLN relieves its inhibitory effects on SERCA2, which is one of several proposed mechanisms by which ß2AR mediate smooth muscle relaxation (11).

To investigate the extent and nature of physiologically relevant changes in these fundamental contraction/relaxation pathways evoked by chronic ß2AR activation, we utilized airway smooth muscle cells derived from transgenic mice with smooth muscle-specific overexpression of the receptor. DNA microarrays revealed that ß2AR activation leads to alterations in multiple transcripts of proteins involved with [Ca2+]i-mediated contraction/relaxation. Of particular interest was that PLN transcripts and protein expression in ß2AR-overexpressing airway smooth muscle cells were markedly reduced compared with cells derived from wild-type (WT) mice. A number of other [Ca2+]i-related proteins also exhibited altered expression profiles. To ascertain physiological relevance of the ß2AR-PLN cross talk, airway resistance measurements were carried out in intact WT and PLN–/– mice in response to receptor-mediated contraction and relaxation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Genetically altered mice.
The generation of transgenic mice overexpressing human ß2AR (ß2AR-OE) on airway smooth muscle using the smooth muscle actin promoter has been previously described (19). The airway smooth muscle cells derived from these mice overexpress the ß2AR ~40-fold over endogenous levels as determined by [125I]cyanopindolol radioligand binding (19). Similarly, the generation of PLN–/– mice has been detailed elsewhere (16). Both sets of mice were in the FVB/N background. The protocol for these studies was approved by the University of Cincinnati Institutional Animal Care and Use Committee.

Cell culture.
Primary cultures of murine airway smooth muscle cells were established from tracheal explants of nontransgenic and ß2AR-OE mice as previously reported (19). Briefly, the trachea between the larynx and main stem bronchi was removed and placed in a sterile petri dish containing Hanks' balanced saline solution supplemented with a 2x concentration of antibiotic-antimycotic solution (Life Technologies). After additional surrounding tissue was removed with the aid of a dissecting microscope, the tracheal segment was split longitudinally and dissected into 2- to 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, we added 2.5 ml of Dulbecco's modified Eagle's medium supplemented with 20% FCS and 2x antibiotic-antimycotic to cover the explants. Explanted tracheas were subsequently removed when there was local confluency. Once the initial seed dish became confluent, cells were harvested by trypsinization and passed into 75-cm2 flasks. As previously described (19), nearly all of these cells are smooth muscle cells, as determined by immunohistochemistry performed with an antibody raised against smooth muscle {alpha}-actin. At passages 8–10, cells maintained in the above media at 95% confluence were washed twice with PBS, and total RNA was prepared using TRI Reagent (Molecular Research Center, Cincinnati, OH), with final resuspension in diethyl pyrocarbonate-treated water. Each sample was visually inspected for degradation in agarose gels stained with ethidium bromide, and their quality was further checked for RNase degradation using an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA). Six total RNA samples (20 µg) from WT and six from ß2AR-OE cell lines were utilized for cDNA labeling and microarray hybridization.

DNA microarrays.
Differential gene expression of 8,734 cDNAs was assessed by microarray slides fabricated by the Genomic and Microarray Laboratory, Center for Environmental Genetics, University of Cincinnati (http://microarray.uc.edu/). Briefly, clones from the Incyte Genomics mouse GEM1 Library (Incyte Pharmaceuticals, Palo Alto, CA) were amplified by PCR and printed onto glass slides (Omnigrid microarrayer; GeneMachines, San Carlos, CA). A complete list of the genes represented on the GEM1 microarray can be found at http://microarray.uc.edu/DataBases/Incyte_Mouse_GEM1.xls. cDNA prepared from the RNA samples was labeled with the fluorescent dyes Cy5 and Cy3, using random primers and RT and competitively hybridized to the microarray chip exactly as described previously (1). Fluorescence intensity analyses and background subtraction were performed using an Axon Instruments scanner and GenePix software.

DNA microarray data analyses.
Data normalization and analyses were carried out with GeneSpring GX 7.3 (Agilent Technologies) software. To account for dye swap, the signal channel and control channel measurements for two of the six chips were reversed. Relative expression intensity was calculated as the ratio of the signal from the transgenic mouse sample against the control signal from the labeled nontransgenic mouse reference cDNA for each gene on each array. Then Lowess normalization method was used to eliminate dye-related artifacts caused by nonlinear rates of dye incorporation as well as inconsistencies in the relative fluorescence intensity between some red and green dyes; 20.0% of the data was used to calculate the Lowess fit (29) at each point and was used to adjust the control value for each measurement. A cutoff value of 10 was used if the intensity measurement of the control channel was lower than 10. Per-chip normalization was then applied, in which each measurement was divided by the median intensity value of all measurements on that chip.

The signals for each gene from the six replicates were averaged, and a two-tailed Student's t-test was calculated for each gene to test whether the mean normalized expression value for the gene is statistically different from 1. From all cDNA clones on the chip, differentially expressed genes were identified using t-test P value with Benjamini and Hochberg false discovery rate <0.05. The relative expression patterning of potentially differentially expressed cDNAs was determined using the hierarchical tree clustering algorithm as implemented in the GeneSpring program using Pearson correlation applied to the log ratio of gene expression values.

We used Gene Ontology (GO) Slim implemented in GeneSpring for functional classification of differentially expressed genes and to identify GO terms associated with them. Statistically significant functional groups were selected by comparing gene lists resulted from our analysis with each of the Biological Process and Molecular Function categories in GO Slim using GeneSpring scripts (3). A significance value (P) from hypergeometric Fisher’s exact t-test was calculated for the number of identified genes in a particular category, based on the total number of genes in that category on the array. This value indicates whether there are higher numbers of genes identified in a particular category than one would expect by chance. A P value cutoff of 0.02 was used, and the minimum number of genes in a category to be included in further analysis was set to five.

Western blots.
Primary airway smooth muscle cells derived from WT and ß2AR-OE were lysed and solubilized in 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors (10 µg/ml benzamidine, 10 µg/ml soybean trypsin inhibitor, 10 mg/ml aprotinin, and 5 µg/ml leupeptin) in PBS. After centrifugation at 10,000 g, supernatant protein was quantitated, and equal amounts (40 µg) were fractionated on polyacrylamide gels (6–10%) and transferred to polyvinylidene difluoride membranes. Membranes were washed with 0.1% Tween in Tris-buffered saline and then blocked by incubation in the same buffer also containing 5% nonfat milk for 20 min. Membranes were incubated overnight with anti-phospholamban antibody. (Upstate, Charlottesville, VA) at a titer of 1:500. After further washing and a blocking incubation as above, membranes were incubated with a goat anti-rabbit secondary antibody (diluted 1:6,500). Bands were visualized by enhanced chemiluminescence and quantitated with Scan Analysis software (Biosoft, Cambridge, UK), with the data reported in relative units (RU) of pixel density. To assess the consistency of protein transfer, membranes were stripped, and immunoblots were carried out as above using a GAPDH antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA).

Airway physiology.
Invasive assessment of respiratory mechanics was carried out using an intact, intubated, anesthetized, mouse model similar to that previously reported (18). Briefly, mice were anesthetized with ~60 mg/kg pentobarbital, after which the trachea was cannulated with an 18-gauge metal needle. Mice were then mechanically ventilated using a computer-controlled rodent ventilator (flexiVent; SciReq, Montreal, Canada) to deliver a tidal volume of 10 ml/kg (~250 µl/breath) at a rate of 150 breaths/min, with positive end-expiratory pressure of 2.5 cm H2O. Dynamic lung resistance (R) was determined by fitting a linear first-order single-compartment model of airway mechanics to measurements of airway pressure, volume, and air flow made during application of single sinusoidal perturbation with an amplitude of 150 µl at 2.5 Hz for ~1.2 s using software provided by the manufacturer. Two measurements of R made before administration of methacholine were averaged to establish the baseline. Increasing concentrations of methacholine were subsequently delivered to the airway by transiently diverting the inspiratory limb of the ventilator through the reservoir of an ultrasonic nebulizer for 30 s. R was measured at 30-s intervals for 5 min after each dose, and the maximum R value after each dose was used to establish the dose-response curves. In studies to assess the relaxation effects of inhaled ß-agonist, isoproterenol (1.0 mg/ml) was delivered by aerosol, and then the constrictive response to varying doses of methacholine was determined as described above. The dose of methacholine required to increase R by 200% (ED200) was calculated from nonlinear curves fit to the data by using the Prism software package (GraphPad, San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
As previously shown by our group (6, 19, 27) and others (20), overexpression of the ß2AR in cells results in increased signaling, which mimics persistent activation by agonist. Airway smooth muscle cells from the ß2AR-OE mice thus provide a useful model to ascertain long-term regulatory events that occur in a cell type relevant to chronic treatment of asthma with ß-agonist. Our initial goal was to identify, and broadly classify by pathway analysis, transcripts with altered expression in these cells compared with WT cells. A specific concentration on genes involved in [Ca2+]i flux was pursued. From these data, a significant decrease in a key [Ca2+]i-handling protein was noted and verified, and the specific role of this alteration was explored at the physiological level in genetically modified mice.

DNA microarray analysis of ß2AR-OE airway smooth muscle cells.
The raw data from the arrays are deposited in the Gene Expression Omnibus (GEO) with accession number GSE4499. A hierarchical analysis based on expression patterns of the ß2AR-OE vs. WT smooth muscle cells is shown in Fig. 1. We identified a set of genes whose expression was consistently changed the most by referencing gene expression levels to the WT, applying a P value cutoff using Student's parametric t-test. Transcript levels are shown relative to the distribution of expression for all the genes, with overexpressed genes in red and underexpressed genes in blue. There were 483 transcripts that were increased or decreased significantly at the 95% confidence level compared with WT (see Supplemental Material S1 for this list; the online version of this article contains supplemental data); 319 were increased and 164 were decreased. Using the GO term assignments (from GO Slim), the differentially expressed genes (P = 0.02) were classified as shown in Fig. 2. Among these 22 GO Slim terms, there were 19 categories that displayed only up- or downregulation: 6 were uniquely upregulated and 13 uniquely downregulated. Persistent ß2AR activity uniquely upregulated genes associated with lipid and carbohydrate metabolism, protein modification, transferase activity, and protein kinase activity. Downregulated genes included those associated with cell growth and differentiation, consistent with the generally accepted anti-proliferative effects of ß2AR in airway smooth muscle (26). Similarly, ß2AR have been considered anti-apoptotic (30), which was observed here with downregulation of apoptosis regulator activity genes. Three gene categories were enriched significantly with both up- and downregulated genes: receptor activity, calcium ion binding, and structural molecule activity (dashed lines, Fig. 2). Given the central role of [Ca2+]i regulation in cardiac and smooth muscle contraction and relaxation, we further explored gene regulation in a group of genes consisting of calcium ion binding (GO:0005509) and ion transport (GO:0006811). Shown in Table 1 are those genes within this list that were ≥30% up- or downregulated; 37 were upregulated and 22 were downregulated in ß2AR-OE airway smooth muscle cells compared with WT cells. The expression of PLN, a "nodal point" in cardiac contraction (8), was one of the most downregulated genes, being decreased by ~60% (P = 0.008) in the ß2AR-OE cells. Importantly, the expression of SERCA2a (Atp2a2) was not significantly downregulated; rather, if anything, it trended toward being higher in the ß2AR-OE cells. Thus the PLN-to-SERCA2 ratio was substantially decreased in these airway smooth muscle cells. Calsequestrin 2 was reduced to approximately the same extent as PLN, consistent with its role as a low-affinity [Ca2+]i binding protein localized to the luminal surface of the ER. Western blots of WT and ß2AR-OE airway smooth muscle cells confirmed decreased expression of PLN protein, amounting to ~60% (Fig. 3).


Figure 1
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  		Fig. 1. Hierarchical clustering of differentially expressed genes in ß2-adrenergic receptor (ß2AR)-overexpressing (ß2AR-OE) compared with wild-type (WT) airway smooth muscle cells. From all cDNA clones on the chip, differentially expressed genes were identified using a t-test P value with Benjamini and Hochberg false discovery rate < 0.05. The relative expression patterning of the 483 differentially expressed cDNAs was determined by a hierarchical tree clustering algorithm, using Euclidian distances as implemented in GeneSpring, using Pearson correlation applied to the log ratio of gene expression values. The extent of regulation is indicated by the scale, with the most upregulated being red and the most downregulated blue.

 

Figure 2
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  		Fig. 2. Distribution of differentially expressed genes in ß2AR-overexpressing compared with WT airway smooth muscle cells. Genes from the list of 483 that were increased or decreased at the 95% confidence level, between ß2AR-OE and WT cells, were categorized by Gene Ontology (GO) Slim terms and stratified by up- or downregulation. Categories shown here had ≥5 genes that were differentially regulated and, compared with all genes in the category, had hypergeometric Fishers exact t-test P values ≤ 0.02. Dashed lines indicate the 3 categories with genes that were both up- and downregulated.

 

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  		Table 1. Differentially expressed calcium ion binding or ion transport genes in ß2AR-overexpressing vs. WT airway smooth muscle cells

 

Figure 3
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  		Fig. 3. ß2AR-OE airway smooth muscle cells have downregulated phospholamban (PLN) protein expression. Shown is a representative Western blot with 2 replicates from whole cell lysates derived from WT and ß2AR-OE cells using antibodies to PLN (top) and GAPDH (bottom). PLN expression was decreased ~60%, whereas GAPDH was unchanged.

 
Consequences of decreased PLN on airway smooth muscle responsiveness.
These data indicated that the Ca2+-handling protein PLN is downregulated as a consequence of chronic ß2AR activation. To isolate the potential role of decreased PLN in contraction/relaxation airway responses, PLN–/– mice were utilized in the intact, intubated mouse model. For these studies, airway-constrictive responses were ascertained with varying doses of the aerosolized muscarinic agonist methacholine. As shown, baseline airway resistance was not different between WT and PLN–/– mice; however, PLN–/– mice displayed a marked decrease in the maximal constrictive response (P = 0.005, Fig. 4A). The dose-response curve was also right-shifted in the PLN–/– mice (ED200 = 54 ± 3.3 mg/ml) compared with WT mice (ED200 = 22 ± 3.9 mg/ml). These data indicate that depressed PLN levels result in decreased sensitivity and maximal responsiveness to airway constriction by the Gq-coupled M3-muscarinic receptor. To assess Gs-coupled relaxation, mice were pretreated with the ß-agonist isoproterenol, followed by methacholine dose-response studies (Fig. 4B). As shown for WT mice, acute activation of airway ß2AR results in a rightward shift in the response curve and a ~25% decrease in the maximal contractile response. Given that the contractile response to methacholine in the PLN–/– mice without ß-agonist pretreatment is right-shifted and depressed, it is difficult to quantitate the extent of the relaxation effect of ß-agonist under these conditions. Nevertheless, the maximal response to methacholine was substantially decreased in PLN–/– mice pretreated with isoproterenol (Fig. 4B) compared with control PLN–/– mice; indeed, the response is essentially flat compared with an approximate threefold increase in PLN–/– mice not pretreated with isoproterenol (Fig. 4A). And under the isoproterenol pretreatment condition, the contractions at the two highest concentrations of methacholine were not different between WT and PLN–/– mice (P > 0.1). We conclude, then, that ß-agonist-mediated relaxation is not substantially impaired in PLN–/– mice.


Figure 4
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  		Fig. 4. Phenotype of decreased airway smooth muscle PLN expression. A: mechanically ventilated WT and PLN–/– mice were administered the indicated doses of inhaled methacholine, and airway resistance was measured as described in MATERIALS AND METHODS. PLN mice had a decreased maximal constrictive response and a rightward shift in the dose-response curve compared with WT mice. *P = 0.008 vs. WT. B: mice were pretreated with the ß-agonist bronchodilator isoproterenol, and then methacholine constrictive dose responses were obtained. This pretreatment antagonized constriction with WT and PLN–/– mice (decrease in maximum and rightward shift of the curve), consistent with intact ß2AR-mediated bronchodilatation. #P < 0.05 vs. WT control. +P < 0.05 vs. PLN–/– control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the current work, we explored the effects of chronic ß2AR activation on transcript expression in airway smooth muscle. The basis of the study is centered on the controversy surrounding chronic administration of ß-agonists in the treatment of asthma. As introduced earlier, multiple human studies have shown varying degrees of deleterious effects on certain asthmatic phenotypes with chronic ß-agonist treatment (2, 21, 25, 28). On the other hand, chronic ß-agonist therapy, particularly with long-acting agonists that can provide persistent activation, is associated with group mean improvements in airway function, exacerbation rates, and symptoms (4, 14). So it is not unexpected, then, that some effects of ß-agonist therapy on contraction/relaxation gene expression may be beneficial; i.e., these represent part of the therapeutic response. We found that a number of genes involved in [Ca2+]i-mediated airway smooth muscle contraction/relaxation were altered by chronic ß2AR activity. This is provided as a resource for investigators, and additional studies will be needed to ascertain which changes are significant in terms of airway function. In the current work, ß2AR overexpression was utilized as a model for chronic ß2AR activation by agonists. We recognize, however, that this overexpression strategy may not necessarily mimic chronic ß-agonist exposure. This may be particularly relevant to the effects evoked by full vs. partial ß-agonists, or those with unique binding sites such as salmeterol (15).

Of specific interest was the ~60% reduction of PLN transcript and protein observed with persistent ß2AR activation, due to receptor overexpression in airway smooth muscle cells. We further pursued PLN because of its role in cardiac muscle. The 52-amino acid protein binds to SERCA2 of the sarcoplasmic reticulum (SR) and regulates the affinity of the pump in moving [Ca2+]i from the cytosol to the SR. The sequestration of [Ca2+]i elicits relaxation, and the [Ca2+]i necessary for the next contraction is made available from the replenished SR store. PKA-mediated phosphorylation of PLN decreases its capacity to inhibit SERCA2, thereby enhancing [Ca2+]i reuptake, resulting in enhanced cardiac relaxation and subsequent contraction. In heart failure, where cardiac muscle has depressed basal and ß-agonist-mediated contraction, the PLN-to-SERCA2 ratio is typically increased. Indeed, this alteration is observed in cardiac hypertrophy as well, before chamber dilatation and decompensated failure. Efforts to decrease this ratio by PLN gene ablation have prevented development of certain heart failure phenotypes in various mouse cardiomyopathy models (5, 23).

This central role of PLN prompted us to study the effects of PLN reduction using the PLN–/– mouse, so as to isolate a potential physiological effect from this one perturbation. As shown, ablation of PLN results in a decrease in the maximal airway contraction and a decrease in sensitivity, to the agonist methacholine. These physiological data are similar to those found in mice with ß2AR overexpression on airway smooth muscle (19). In these mice, we found a resistance to bronchoconstriction by methacholine in vivo, which we assumed was entirely due to a direct effect of ß2AR signaling to relaxation. However, it now appears that chronic ß2AR activation has effects on the expression of multiple genes potentially involved in contraction/relaxation, and that one beneficial effect of ß2AR activation is a decrease in PLN, which decreases bronchoconstriction from activation of M3-muscarinic receptors. The M3-muscarininc receptor acts to constrict via coupling to Gq and is one of several highly spasmogenic pathways that are active in asthma. The fact that the physiology of the ß2AR-overexpressing mouse is not identical to that of the PLN–/– mouse (the former has enhanced ß-agonist-mediated relaxation) is consistent with multiple downstream effects evoked by chronic ß2AR activation. The mechanism of the marked decrease in contraction of the PLN–/– mice is not altogether clear, particularly since paradigms established for cardiac muscle, which has a rapid contraction/relaxation cycle, are not readily applicable to tonically contracted smooth muscle. And, of course, ß2AR/PKA act to enhance the rhythmic contraction of cardiac muscle but serve to tonically relax smooth muscle. With decreased PLN, ER [Ca2+]i loading, at least initially, would be expected to be increased and primed for an enhanced contractile response. However, under chronic steady-state conditions, enhanced ER/SR [Ca2+]i influx can be alleviated by vectoral transport to plasma membrane Ca2+ pumps and exchangers (22). This may ultimately leave the ER/SR stores depleted and thus a depressed Gq receptor-mediated contractile response. Alternatively, increased ER/SR reuptake from enhanced SERCA2 activity in the absence of PLN may serve to rapidly "quench" the receptor-mediated rise in free [Ca2+]i and thus depress contraction. Finally, an increased ER/SR [Ca2+]i load has been reported to lead to hyperpolarization of the cell membrane potential because of large-conductance [Ca2+]i-sensitive K+ channel activation, which would be predicted to decrease sensitivity to contraction (12).

We find here that chronic ß2AR activation on airway smooth muscle decreases PLN expression and that this event, in isolation, contributes to the therapeutic effect by decreasing the sensitivity to contract. Thus, in asthma, where inflammation evokes local accumulation of Gq-coupled receptor agonists that act to constrict the airways, chronic ß-agonists bronchodilate and initiate programs that can attenuate bronchial hyperresponsiveness or dampen the effects of other events that increase hyperresponsiveness. It is interesting to note that, despite the fact that ß2AR activate PKA, which phosphorylates PLN and decreases its affinity for SERCA, PLN–/– mice had no apparent alteration in ß-agonist-promoted relaxation. This is consistent with multiple other ß2AR pathways that act to alter membrane polarization and decrease cytosolic [Ca2+]i, leading to relaxation (11). However, the link between persistent ß2AR activity and PKA activation suggests that PKA-mediated phosphorylation of PLN may initiate the feedback mechanism resulting in decreased expression of PLN. Nevertheless, the current data reveal that an unexpected therapeutic benefit of chronic ß-agonists in treating asthma is a decrease in PLN, leading to decreased airway constrictive responses. Interestingly, there have been efforts to develop PLN inhibitors for the treatment of heart failure. On the basis of our results, such agents may also be useful in treating bronchospasm and may be synergistic with ß-agonists.

Finally, this therapeutic effect of ß-agonists on airway contractility via PLN downregulation could be modified by polymorphisms within the pathway. No common nonsynonymous polymorphisms of PLN have been reported (but rare mutations are associated with cardiomyopathy) (9, 24). However, resequencing results [Single Nucleotide Polymorphism database (dbSNP) build 125] indicate common polymorphisms in genes encoding proteins intimately involved in the signal transduction and [Ca2+]i handling of PLN, such as PLCß isoforms, the ryanodine and inositol trisphosphate receptors, SERCA2, and calsequestrin. Thus these polymorphisms may represent novel pharmacogenetic loci for ß-agonist responsiveness in the treatment of obstructive lung disease.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This research was supported by National Heart, Lung, and Blood Institute Grants HL-045967, HL-065899, HL-072068, HL-071609, and HL-26057 and the State of Ohio Computational Medicine Center.


   ACKNOWLEDGMENTS
 
We thank Mary Rose Schwarb and Clare Glinka for technical assistance and Esther Moses for manuscript preparation.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: S. B. Liggett, 20 Penn St., HSF-II, Rm. S-114, Baltimore, MD 21201-1075 (e-mail: sligg001{at}umaryland.edu).


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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