In allergic asthma Beta 2 adrenergic receptors (β2ARs) are important mediators of bronchorelaxation and, paradoxically, asthma development. This contradiction is likely due to the activation of dual signaling pathways that are downstream of G proteins or β-arrestins. Our group has recently shown that β-arrestin-2 acts in its classical role to desensitize and constrain β2AR-induced relaxation of both human and murine airway smooth muscle. To assess the role of β-arrestins in regulating β2AR function in asthma, we and others have utilized β-arrestin-1 and -2 knockout mice. However, it is unknown if genetic deletion of β-arrestins in these mice influences β2AR expression in the airways. Furthermore, there is lack of data on compensatory expression of βAR subtypes when either of the β-arrestins is genetically deleted, thus necessitating a detailed βAR subtype expression study in these β-arrestin knockout mice. Here we standardized a radioligand binding methodology to characterize and quantitate βAR subtype distribution in the airway smooth muscle of wild-type C57BL/6J and β-arrestin-1 and β-arrestin-2 knockout mice. Using complementary competition and single-point saturation binding assays we found that β2ARs predominate over β1ARs in the whole lung and epithelium-denuded tracheobronchial smooth muscle of C57BL/6J mice. Quantification of βAR subtypes in β-arrestin-1 and β-arrestin-2 knockout mouse lung and epithelium-denuded tracheobronchial tissue showed that, similar to the C57BL/6J mice, both knockouts display a predominance of β2AR expression. These data provide further evidence that β2ARs are expressed in greater abundance than β1ARs in the tracheobronchial smooth muscle and that loss of either β-arrestin does not significantly affect the expression or relative proportions of βAR subtypes. As β-arrestins are known to modulate β2AR function, our analysis of βAR subtype expression in β-arrestin knockout mice airways sets a reference point for future studies exploiting these knockout mice in various disease models including asthma.
Citation: Hegde A, Strachan RT, Walker JKL (2015) Quantification of Beta Adrenergic Receptor Subtypes in Beta-Arrestin Knockout Mouse Airways. PLoS ONE 10(2): e0116458. https://doi.org/10.1371/journal.pone.0116458
Academic Editor: James Porter, University of North Dakota, UNITED STATES
Received: September 3, 2014; Accepted: December 10, 2014; Published: February 6, 2015
Copyright: © 2015 Hegde et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: All relevant data are within the paper.
Funding: This work was supported by the National Institutes of Health (NIH) grants HL084123 and HL093103 to JKLW. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIH. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Bronchoconstriction is one of the salient features of asthma which is reversible by agonist-mediated activation of the β2 adrenergic receptor (β2AR), a prototypical G protein-coupled receptor (GPCR). In addition to bronchodilation, β2ARs also mediate bronchoprotection in asthmatic airways . By virtue of these properties β2AR agonists remain the primary line of therapy to treat asthmatic bronchospasm.
In humans, agonist activation of β2ARs leads to airway smooth muscle (ASM) relaxation through activation of Gαs, cAMP accumulation and activation of protein kinase A (PKA) . The distribution of βAR subtypes in human airways supports the notion that β2ARs mediate bronchorelaxation. Specifically, the distribution of β1AR and β2AR in human lung was reported to be 30:70 ; however, β1ARs were not detected in human bronchus . βARs of human ASM and airway epithelium are known to be entirely of the β2 subtype . βAR distribution (β1AR:β2AR) has also been studied in the airways of other animals such as pig (28:72), guinea pig (15:85), horse (26–20:74–80), dog (23:77) and rat (15:85) [6–13].
Given that mus musculus is one of the most commonly used species for allergic asthma models, a clear understanding of how murine airway βAR subtype expression compares to that of humans is essential to the interpretation of translational studies examining bronchodilation. Similar to that of humans, the distribution of murine βAR subtypes is heterogeneous in various tissues including lung [14, 15]. βAR expression has been studied in mouse tracheal epithelial and ASM cells. Henry et al reported more β2AR than β1AR expression in mouse tracheal epithelium (71% β2AR) but more β1AR than β2AR in ASM (69% β1AR) and that mouse isolated tracheal smooth muscle relaxations were mediated by β1AR [16, 17]. However, as in humans, airways distal to the trachea play a predominant role in determining airway resistance and recent functional data show that bronchial smooth muscle β2ARs play an important role in mediating bronchorelaxation in mice . However, quantitative receptor expression data from murine airways is sparse in the asthma literature.
Because many asthma studies use genetically altered murine strains, interpretation of β-agonist effects on bronchoprotection and bronchorelaxation must also consider the effect of those genetic alterations on β2AR expression levels. Although measurement of total βAR expression is informative, changes in β2AR expression may be counterbalanced by changes in β1AR expression. This is particularly relevant given the recent use of β-arrestin knockout (KO) mice to study asthma. β-arrestins are so named because the β2AR was the first receptor substrate for which they were shown to terminate or “arrest” G protein-dependent cell signaling . β-arrestin KO mice are a valuable tool for asthma research since loss of β-arrestin-1 expression has been shown to reduce airway bronchoconstriction (manuscript in preparation) while loss of β-arrestin-2 expression results in enhanced beta-agonist-mediated bronchorelaxation  and significant protection from development of the asthma phenotype . However, interpretation of airway hyperresponsiveness (AHR) and bronchodilation data in these mice must take into consideration the absence of β-arrestins, not only because β-arrestins modulate airway bronchoconstriction and bronchorelaxation, but also because genetic deletion of β-arrestins may affect the expression of βARs, especially in the airways. Thus, a detailed knowledge of βAR subtype expression in β-arrestin KO mice is required for complete interpretation of AHR data. Here we standardized a radioligand binding methodology to determine if the genetic deletion of β-arrestin proteins has any impact on βAR expression in murine whole lung. Specifically, we used complementary competition and saturation binding assays to quantify βAR subtype distribution in the lung and epithelia-denuded ASM of wild-type C57BL/6J and β-arrestin-1 KO and β-arrestin-2 KO mice.
All animal experiments were approved by the Institutional Animal Care and Use Committee at Duke University Medical Center and were performed in accordance with the standards established by the US Animal Welfare Acts.
Naïve mice (8–16 weeks of age) were euthanized in a CO2 chamber and exsanguinated. The thorax was cut open, trachea, bronchi and whole lung were quickly removed and freed of surrounding tissue. The tracheal and bronchial epithelium was denuded under microscope using Cotton tipped applicators (Puritan Medical Products Co., LLC, Maine, USA). Separated lung and trachea-bronchi were immediately placed in homogenization buffer (25 mM Tris HCl, pH 7.4, 5 mM EDTA, pH 8) with protease inhibitors (1 mM Phenyl methyl sulfonyl fluoride, 5 μg/ml Leupeptin, 10 μg/ml Benzamidine, 0.7 μg/ml Pepstatin and 10 μg/ml Aprotinin) on ice. After homogenization, unwanted cell and tissue debris were removed from the tissue lysate by a slow-speed centrifugation at 200 g for 10 min at 4°C and the supernatant was centrifuged at 38000 g for 20 min at 4°C to get membrane pellets. The pellet obtained was resuspended in ice-cold binding buffer (75 mM Tris HCl, pH 7.4, 2 mM EDTA, pH 8, 12.5 mM MgCl2) with protease inhibitors (5 μg/ml Leupeptin, 10 μg/ml Benzamidine, 0.7 μg/ml Pepstatin and 10 μg/ml Aprotinin) and kept frozen at -80°C until binding assay. Protein concentration was measured using Bradford reagent.
Competition radioligand binding assays
Competition binding assays measured the proportions of βAR subtypes expressed in lung membrane preparations. Similar to previous approaches that quantified receptor subtype proportions based on the differential affinity of a cold competitor for those receptor subtypes [21, 22], we used a serial dilution of the β2AR-selective antagonist ICI-118551 (0.316 pM to 3.16 μM) to displace (125I)-CYP from the β2AR and β1AR with high and low affinity, respectively. In brief, frozen membrane samples were resuspended in ice-cold binding assay buffer (50 mM Tris HCl, pH 7.4, 2 mM EDTA, 12.5 mM MgCl2, 180 μg/ml ascorbic acid) to yield a final membrane amount of 1.5–8 μg (lung) and 11–80 μg (tracheobronchial smooth muscle) in binding reactions containing 60 pM (125I)-CYP and buffer (total binding) or ICI-118551 competitor. Non-specific binding was determined in the presence of 10 μM propranolol. Pilot assays were conducted on each membrane sample to ensure that less than 10% of the total radioligand was bound. Following a 90 min incubation at room temperature, assays were terminated via harvesting onto Whatman GF/B glass fiber filters and washing with 8 mL of cold wash buffer (50 mM Tris HCl, pH 7.4, 2 mM EDTA, 12.5 mM MgCl2). Bound radioactivity was measured using a Packard Cobra gamma counter (Packard). Normalized data were best fit to a two-site competition binding model in GraphPad Prism (GraphPad Inc., La Jolla, CA) to yield the percentage of β1AR and β2AR present in each sample (Fig. 1). Log Ki values were fit directly in Prism by setting (125I)-CYP Kd equal to 0.115 nM and 0.034 nM for the β1AR and β2AR, respectively, as determined from proof-of-concept saturation binding studies (Fig. 2).
A. Competitive displacement of a non-selective antagonist radioligand from a mixed population of receptors (50:50) by a subtype-selective competitor was simulated. Data were generated by fitting affinities of the antagonist ICI-118551 for the β2AR (Log Kd = -9.26) and the β1AR (Log Kd = -6.52)  to a two-site competitive binding model in GraphPad Prism. Due to its >500-fold selectivity for the β2AR, ICI-118551 displaces radioligand from β2ARs at low concentrations and from β1ARs at high concentrations to produce a biphasic inhibition curve. The deconvolution of high and low affinity states quantifies the fraction of each receptor subtype. In the case of ICI-118551, subtype 1 represents the β2AR and subtype 2 represents the β1AR. B-D. Competition binding between (125I)-CYP (60 pM) and ICI-118551 (0.3 pM to 10 μM) detected 36% β1AR and 64% β2AR in WT mouse whole lung (B), 43% β1AR and 57% β2AR in βarr1 KO whole lung (C), and 33% β1AR and 67% β2AR in βarr2 KO whole lung (D). Binding parameters can be found in Table 2, with the data representing the mean ± SEM of 3–6 independent experiments performed in duplicate.
A. Proof-of-concept saturation experiments with β1AR-overexpressing membranes demonstrate that 500 nM CGP-20712A completely displaces (125I)-CYP from all available β1ARs, whereas 100 nM ICI-11855 is sufficiently low to not detect the β1AR. Total β1AR was set to 100% based on the displacement of (125I)-CYP by 10 μM propranolol. B. Proof-of-concept saturation experiments with β2AR-overexpressing membranes demonstrate that 100 nM ICI-118551 completely displaces (125I)-CYP from all available β2ARs, whereas 500 nM CGP-20712A is sufficiently low to not detect the β2AR. Total β2AR was set to 100% based on the displacement of (125I)-CYP by 10 μM propranolol. Plotted data represent the individual means of three experiments performed in duplicate. Data were fit to a one-site saturation model in GraphPad Prism.
Saturation radioligand binding assays
A complementary approach for measuring the proportions of βAR subtypes expressed on the membranes of lung and epithelia-denuded tracheobronchial smooth muscle is the single point saturation assay. In this approach, 500 nM CGP-20712A (β1AR-specific) and 100 nM ICI-118551 (β2AR-specific) were used to displace the nonselective βAR antagonist (125I)-cyanopindolol ((125I)-CYP, Perkin Elmer, MA, USA) from the β1AR and β2AR, respectively. These concentrations were based on their reported affinity for each βAR subtype  and were verified to only detect the desired βAR subtype in saturation experiments on β1AR-overexpressing  and β2AR-overexpressing  cell membranes (Fig. 2). The total βAR pool (set to 100%) was determined using 10 μM propranolol. In brief, frozen membrane samples were resuspended in ice-cold binding assay buffer (50 mM Tris HCl, pH 7.4, 2 mM EDTA, 12.5 mM MgCl2, 180 μg/ml ascorbic acid) to yield a final membrane amount of 1.5–8 μg (lung) and 11–80 μg (tracheobronchial smooth muscle) in binding reactions containing 500 pM (125I)-CYP and buffer (total binding) or competitor (non-specific binding). Pilot assays were conducted on each membrane sample to ensure that less than 10% of the total radioligand was bound. Assays were incubated and terminated as described above. Bound radioactivity was measured using a Packard Cobra gamma counter (Packard). Specific binding was calculated as the difference between total and nonspecific binding and expressed as fmol/mg protein given a specific activity of 4005 cpm/fmol. Control saturation binding assays using 5–750 pM (125I)-CYP and 0.1–0.2 μg βAR overexpressing membranes were fit via a one-site saturation model in GraphPad Prism.
Data were expressed as mean ± SEM. GraphPad Prism software version 5.04 (GraphPad Software, La Jolla, CA, USA) was used for nonlinear curve fitting, regression analysis and statistical calculations. Data derived from the competition experiments were best fit by a two-site binding model as determined by F test (p < 0.05). One way ANOVA was used to determine significant differences between genotypes, with the significance threshold set to p < 0.05.
Analysis of βAR subtype expression in whole lung of wild-type C57BL/6J, β-arrestin-1 KO, and β-arrestin-2 KO mice by ICI-118551 competition binding
Subtype-selective ligands have been previously used to quantify the relative proportions of receptors in various animal tissues [21, 22]. Here we quantified βAR subtypes (Table 1) in whole lung of wild-type C57BL/6J (Fig. 1B) and β-arrestin-deficient mice (Fig. 1C and D) by measuring the competitive displacement of the non-selective βAR antagonist (125I)-CYP by the β2AR-selective antagonist ICI-118551. We chose ICI-118551 based on its >500-fold selectivity for the β2AR over the β1AR , thus providing accurate deconvolution of the two βAR subtypes using a two-site competition binding model .
Consistent with a heterogeneous population of βAR subtypes [14, 15], ICI-118551 competition curves from whole lung membranes of wild-type mice were shallow and best fit by a two-site binding model comprising high affinity for the β2AR (pKi = 10.69 ± 0.06) and low affinity for the β1AR (pKi = 7.21 ± 0.11) (Fig. 1B) (Table 2). Calculating the fraction of receptors in each affinity state revealed that wild-type mouse lung contains 36% β1AR and 64% β2AR (Table 1).
We next quantified β1AR and β2AR expression in whole lung of β-arrestin-1 KO and β-arrestin-2 KO mice to determine if genetic deletion of β-arrestin alters receptor expression. In β-arrestin-1 KO mouse whole lung we found that the relative proportions of β1AR (43%) and β2AR (57%) and ICI-118551 affinities for each subtype were comparable to wild-type mice (Fig. 1C and Tables 1 and 2). Similar proportions of β1AR (33%) and β2AR (67%) and ICI-118551 affinities were detected in whole lung from β-arrestin-2 KO mice (Fig. 1D and Table 1). Small variations in βAR subtypes indicated that individual deletion of β-arrestins in the mouse lung does not alter antagonist binding or the proportion of βAR subtypes.
Competition binding assays were not performed on the tracheobronchial tissue of wild-type and β-arrestin deficient mice given the limitations associated with collecting large amounts of tracheobronchial tissue. Single-point saturation assays were used for this tissue as reported below.
Analysis of βAR subtype expression in whole lung of wild-type C57BL/6J, β-arrestin-1 KO, and β-arrestin-2 KO mice by single-point saturation
We developed a single-point saturation binding assay to quickly and efficiently calculate receptor density (Bmax). Specifically, empirically-determined concentrations (Fig. 2) of CGP-20712A (a β1AR selective antagonist) and ICI-118551 (a β2AR selective antagonist) were used to displace a saturating concentration of the non-selective βAR antagonist (125I)-CYP (500 pM) in a subtype-selective manner from a pool of βARs. A saturating concentration of the non-selective antagonist propranolol (10 μM) was used to determine the total pool of βARs (set to 100%) in each membrane sample. Proof-of-concept experiments in which 10 μM propranolol was used to detect the maximal amount of each βAR subtype in overexpressing cell lines revealed that 500 nM CGP-20712A was sufficiently low to occupy all β1ARs (Fig. 2A) but not detect the β2AR (Fig. 2B). Similarly, 100 nM ICI-118551 was sufficiently low to occupy all β2ARs (Fig. 2A) but not detect the β1AR (Fig. 2B). These concentrations were also consistent with calculations of fractional occupancy for competitive binding between two ligands .
When used to assess the proportion of βAR subtypes in the whole lung of wild-type C57BL/6J mice, single-point saturation experiments yielded 32% β1AR and 67% β2AR (Fig. 3A and Table 1). This was in good agreement with the proportion of βAR determined from ICI-118551 competition experiments (Fig. 1B). The proportions of β1AR and β2AR in whole lung of β-arrestin-1 KO (38% and 63%, respectively) (Fig. 3B) and β-arrestin-2 KO mice (32% and 61%, respectively) (Fig. 3C) were also comparable to the results from competition experiments, showing 1) no effect of genotype on βAR subtype expression and 2) that the two approaches yielded equivalent information. Additionally, as shown in Table 2, the total βAR receptor density in the lung of C57BL/6J mice (598 ± 130 and 292 ± 79 fmol/mg for β2AR and β1AR, respectively) was in good agreement with total βAR expression measured in our prior study (1004 ± 54 fmol/mg) .
The competitive displacement of the non-selective radiolabeled antagonist (125I)-cyanopindalol (CYP) (500 pM) by 500 nM CGP-20712A and 100 nM ICI-118551 quantifies the proportions of β1AR and β2AR, respectively. Propranolol, a nonselective βAR blocker, gives a measure of total βAR present in each tissue. A. WT: β1AR = 32 ± 3%; β2AR = 67 ± 2%; 100% corresponds to 887.2 ± 168 fmol/mg. B. βarr1 KO: β1AR = 38 ± 1%; β2AR = 63 ± 2%; 100% corresponds to 1072 ± 222 fmol/mg. C. βarr2 KO: β1AR = 32 ± 0.5%; β2AR = 61 ± 2%; 100% corresponds to 1221 ± 277 fmol/mg. Data represent the mean ± SEM of 3 independent experiments performed in quadruplicate.
Analysis of βAR subtype expression in epithelia-denuded tracheobronchial smooth muscle of wild-type C57BL/6J, β-arrestin-1 KO, and β-arrestin-2 KO mice by single-point saturation
We next quantified βAR subtypes in the tracheobronchial tissue of mice given that bronchial smooth muscle β2ARs mediate bronchorelaxation in mice . Using single-point saturation analysis we determined for the first time that epithelia-denuded tracheobronchial smooth muscle of wild-type C57BL/6J mice contains 12% β1AR and 64% β2AR (Fig. 4A). Similar levels of expression were observed in β-arrestin-1 KO mice (13% β1AR and 60% β2AR; Fig. 4B) and β-arrestin-2 KO mice (14% β1AR and 65% β2AR; Fig. 4C) (Table 1).
The competitive displacement of the non-selective radiolabeled antagonist (125I)-cyanopindalol (CYP) (500 pM) by 500 nM CGP-20712A and 100 nM ICI-118551 quantifies the proportions of β1AR and β2AR, respectively. Propranolol, a nonselective βAR blocker, gives a measure of total βAR present in each tissue. A. WT: β1AR = 12 ± 5%; β2AR = 64 ± 3%; 100% corresponds to 208.2 ± 28 fmol/mg. B. βarr1 KO: β1AR = 13 ± 4%; β2AR = 60 ± 4%; 100% corresponds to 213 ± 55 fmol/mg. C. βarr2 KO: β1AR = 14 ± 4%; β2AR = 65 ± 2%; 100% corresponds to 255.7 ± 82 fmol/mg. Data represent the mean ± SEM of 3 independent experiments performed in quadruplicate.
Radioligand binding assays are extremely powerful tools to study receptor expression and subtype proportion under normal and disease states and during administration of drug therapies. Binding methods exploit the basic principle of competitive binding between nonselective radioligands and selective cold ligands to quantitate the proportion of receptor subtypes [21, 22, 27]. Here we standardized an approach using complementary competition and saturation binding assays to evaluate the βAR subtype distribution in murine wild-type and β-arrestin KO whole lung. Consistently, we found comparable receptor density results between ICI-118551 competition and single-point saturation assays. Thus, the single-point saturation assay can be used to quantify βAR subtypes, the main advantage being reduction in the amount of tissue sample required for each assay. This permits radioligand binding analysis with fewer animals and improved cost effectiveness. Our careful validation of subtype-specific ligand concentrations and assay conditions, both of which are critical to the success of radioligand binding experiments, is an advance over previous reports in which βAR subtypes were measured. Our work may also provide a partial explanation for the discrepancy in murine airway distribution of βAR subtypes reported in the literature [16, 17] and below we provide further comment on these differences. Furthermore, we denuded the tracheal and bronchial epithelium and used only the smooth muscle for membrane preparation since mouse tracheal epithelium expresses a higher density of β2AR than ASM . The airways distal to the trachea influence the airway resistance during bronchoconstriction  and ASM is an important effector cell type involved in AHR, the cardinal feature of asthma [28, 29]. By carefully denuding the epithelium, we were able to focus our analysis on the receptors expressed in the smooth muscle and thus provide correct reference values of βAR subtypes involved in bronchodilation in these β-arrestin KO mice.
We report that the β2AR subtype was predominantly expressed in whole lung and epithelium-denuded tracheobronchial smooth muscle membranes of C57BL/6J and β-arrestin-1 KO and β-arrestin-2 KO mice. The data provide evidence that β2ARs are expressed in greater abundance than β1ARs in the tracheal smooth muscle. Until recently, the general assumption was that mouse airways use β1ARs to relax and this was primarily based on the receptor expression studies by Henry and colleagues who showed that β1AR expression predominates [16, 17]. Several reasons may account for the discrepant results. First, in our study, the tracheal and bronchial epithelium was separated from the smooth muscle, but this was not the case in the Henry and colleague experiments [16, 17]. By carefully removing epithelial cells we were able to unambiguously quantify the proportion of βAR subtypes in the airway smooth muscle extending to the bronchi. Secondly, we used cell membranes for direct binding studies whereas Henry and colleagues used quantitative autoradiography.
The large excess of β2AR over β1AR expression found in the current study is supportive of results from our prior study which showed that β-agonist-mediated bronchorelaxation does not occur in β2AR-KO mice . Taken together, these results point to an important role for β2ARs, not β1ARs, in mediating murine bronchorelaxation . Collectively, the murine β2AR expression and functional evidence vouches for the suitability of this species as a model to study the effects of β-agonists in human allergic asthma.
Agonist-mediated activation of the β2AR is responsible for the reversal of bronchoconstriction in human and murine asthma. Paradoxically, expression and activation of β2ARs is required for development and pathogenesis of the asthma phenotype in mice and worsening of the asthma phenotype in humans [30–32]. Increasing evidence suggests that separate G protein- and β-arrestin-dependent signaling pathways downstream of β2AR are responsible for these paradoxical effects of β2AR activation in asthma [20, 32, 33]. We previously showed that β-arrestin-2 deletion protects mice against allergen-induced asthma  and enhances β2AR-induced relaxation in murine ASM . Thus, therapeutic strategies that inhibit β-arrestin-2 functions or bias β2AR signaling toward the Gs/cAMP, or away from the β-arrestin-mediated, signaling pathway may be beneficial in asthma [34, 35]. In this context, β-arrestin KO mice represent valuable investigative tools in our lab and elsewhere for the study of asthma; however, measuring βAR subtype expression density in the lung and airways is essential to the full interpretation of asthma phenotypes. Herein, we are the first to show that genetic deletion of either β-arrestin-1 or -2 does not affect the expression of lung or tracheobronchial β1- or β2-ARs in naive mice. Going forward it will be important to understand how various models of allergen exposure may impact the expression of lung and airway βARs and β-arrestins. For example, we previously showed that allergen (OVA) sensitization and chronic allergen challenge leads to a significant reduction in whole lung expression of total βARs (from 1004 ± 54 fmol/mg to 598 ± 88 fmol/mg)  and an elevation in whole lung expression of β-arrestin-2 (under second review Chen et al., Am J Respir Cell Mol Biol—“Genetic deletion of β-arrestin-2 mitigates established airway hyperresponsiveness in a murine asthma model”). Utilization of the methods described herein will facilitate the measurement of βAR subtype expression, especially in tissues that are limited in size, thus providing information needed for correct interpretation of lung mechanics data in a variety of murine models of asthma.
In summary, our study provides the first detailed reference levels of lung and airway βAR subtype densities in β-arrestin-1 KO and β-arrestin-2 KO mice. Our data substantiate the notion that β2ARs mediate murine bronchial smooth muscle bronchorelaxation. Our study also demonstrates that genetic deletion of β-arrestin-1 or -2 does not significantly alter the expression of β1 – or β2-ARs in naïve whole lung or tracheobronchial airway smooth muscle cells.
We thank Prof. Robert J. Lefkowitz for generous support of resources. We thank Barbara Theriot for technical support. Dr. Gianluigi Pironti and Dr. Seungkirl Ahn kindly provided cell membranes for proof-of-concept saturation studies.
Conceived and designed the experiments: AH RTS JKLW. Performed the experiments: AH. Analyzed the data: AH RTS. Contributed reagents/materials/analysis tools: AH RTS JKLW. Wrote the paper: AH RTS JKLW. Acquired, analyzed or interpreted the data: AH RTS JKLW. Drafted and revised the article for important intellectual content: AH RTS JKLW. Approved the final version to be published: AH RTS JKLW. Agreed to be accountable for all aspects of the work: AH RTS JKLW.
- 1. Abisheganaden J, Boushey HA (1998) Long-acting inhaled beta 2-agonists and the loss of "bronchoprotective" efficacy. Am J Med 104: 494–497. pmid:9626035
- 2. Penn RB, Bond RA, Walker JK (2014) GPCRs and arrestins in airways: implications for asthma. Handb Exp Pharmacol 219: 387–403. pmid:24292841
- 3. Engel G (1981) Subclasses of beta-adrenoceptors—a quantitative estimation of beta 1- and beta 2- adrenoceptors in guinea pig and human lung. Postgrad Med J 57 Suppl 1: 77–83. pmid:6272254
- 4. Ikeda T, Anisuzzaman AS, Yoshiki H, Sasaki M, Koshiji T, et al. (2012) Regional quantification of muscarinic acetylcholine receptors and beta-adrenoceptors in human airways. Br J Pharmacol 166: 1804–1814. pmid:22300233
- 5. Carstairs JR, Nimmo AJ, Barnes PJ (1985) Autoradiographic visualization of beta-adrenoceptor subtypes in human lung. Am Rev Respir Dis 132: 541–547. pmid:2864008
- 6. Carswell H, Nahorski SR (1983) Beta-adrenoceptor heterogeneity in guinea-pig airways: comparison of functional and receptor labelling studies. Br J Pharmacol 79: 965–971. pmid:6317123
- 7. Abraham G, Kottke C, Dhein S, Ungemach FR (2003) Pharmacological and biochemical characterization of the beta-adrenergic signal transduction pathway in different segments of the respiratory tract. Biochem Pharmacol 66: 1067–1081. pmid:12963495
- 8. Barnes PJ, Nadel JA, Skoogh BE, Roberts JM (1983) Characterization of beta adrenoceptor subtypes in canine airway smooth muscle by radioligand binding and physiological responses. J Pharmacol Exp Ther 225: 456–461. pmid:6132993
- 9. Finkel MS, Quirion R, Pert C, Patterson RE (1984) Characterization and autoradiographic distribution of the beta-adrenergic receptor in the rat lung. Pharmacology 29: 247–254. pmid:6093158
- 10. Goldie RG, Papadimitriou JM, Paterson JW, Rigby PJ, Self HM, et al. (1986) Influence of the epithelium on responsiveness of guinea-pig isolated trachea to contractile and relaxant agonists. Br J Pharmacol 87: 5–14. pmid:3006855
- 11. Goldie RG, Papadimitriou JM, Paterson JW, Rigby PJ, Spina D (1986) Autoradiographic localization of beta-adrenoceptors in pig lung using [125I]-iodocyanopindolol. Br J Pharmacol 88: 621–628. pmid:3017489
- 12. Goldie RG, Spina D, Rigby PJ, Paterson JW (1986) Autoradiographic localisation of ascorbic acid-dependent binding sites for [125I]iodocyanopindolol in guinea-pig trachea. Eur J Pharmacol 124: 179–182. pmid:3720838
- 13. Minneman KP, Hegstrand LR, Molinoff PB (1979) Simultaneous determination of beta-1 and beta-2-adrenergic receptors in tissues containing both receptor subtypes. Molecular pharmacology 16: 34–46. pmid:39246
- 14. Ota A, Matsui H, Asakura M, Nagatsu T (1993) Distribution of beta 1- and beta 2-adrenoceptor subtypes in various mouse tissues. Neurosci Lett 160: 96–100. pmid:7902545
- 15. Lin R, Degan S, Theriot BS, Fischer BM, Strachan RT, et al. (2012) Chronic treatment in vivo with beta-adrenoceptor agonists induces dysfunction of airway beta(2)-adrenoceptors and exacerbates lung inflammation in mice. Br J Pharmacol 165: 2365–2377. pmid:22013997
- 16. Henry PJ, Goldie RG (1990) Beta 1-adrenoceptors mediate smooth muscle relaxation in mouse isolated trachea. Br J Pharmacol 99: 131–135. pmid:2158831
- 17. Henry PJ, Rigby PJ, Goldie RG (1990) Distribution of beta 1- and beta 2-adrenoceptors in mouse trachea and lung: a quantitative autoradiographic study. Br J Pharmacol 99: 136–144. pmid:1970491
- 18. Lefkowitz RJ, Shenoy SK (2005) Transduction of receptor signals by beta-arrestins. Science 308: 512–517. pmid:15845844
- 19. Deshpande DA, Theriot BS, Penn RB, Walker JK (2008) Beta-arrestins specifically constrain beta2-adrenergic receptor signaling and function in airway smooth muscle. FASEB J 22: 2134–2141. pmid:18337459
- 20. Walker JK, Fong AM, Lawson BL, Savov JD, Patel DD, et al. (2003) Beta-arrestin-2 regulates the development of allergic asthma. J Clin Invest 112: 566–574. pmid:12925697
- 21. De Lean A, Hancock AA, Lefkowitz RJ (1982) Validation and statistical analysis of a computer modeling method for quantitative analysis of radioligand binding data for mixtures of pharmacological receptor subtypes. Mol Pharmacol 21: 5–16. pmid:6982395
- 22. Rohrer DK, Desai KH, Jasper JR, Stevens ME, Regula DP Jr, et al. (1996) Targeted disruption of the mouse beta1-adrenergic receptor gene: developmental and cardiovascular effects. Proc Natl Acad Sci U S A 93: 7375–7380. pmid:8693001
- 23. Baker JG (2005) The selectivity of beta-adrenoceptor antagonists at the human beta1, beta2 and beta3 adrenoceptors. Br J Pharmacol 144: 317–322. pmid:15655528
- 24. Rapacciuolo A, Suvarna S, Barki-Harrington L, Luttrell LM, Cong M, et al. (2003) Protein kinase A and G protein-coupled receptor kinase phosphorylation mediates beta-1 adrenergic receptor endocytosis through different pathways. J Biol Chem 278: 35403–35411. pmid:12821660
- 25. Shenoy SK, McDonald PH, Kohout TA, Lefkowitz RJ (2001) Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin. Science 294: 1307–1313. pmid:11588219
- 26. Kenakin T (2009) A Pharmacology Primer: Theory, Applications, and Methods Third Edition Preface. Pharmacology Primer: Theory, Applications, and Methods, 3rd Edition: Xv–Xv.
- 27. Davenport A, Russell F (1996) Radioligand Binding Assays: Theory and Practice. In: Mather S., editor. Current Directions in Radiopharmaceutical Research and Development. Springer Netherlands. pp. 169–179.
- 28. Black JL, Roth M (2009) Intrinsic asthma: is it intrinsic to the smooth muscle? Clin Exp Allergy 39: 962–965. pmid:19438582
- 29. Zuyderduyn S, Sukkar MB, Fust A, Dhaliwal S, Burgess JK (2008) Treating asthma means treating airway smooth muscle cells. Eur Respir J 32: 265–274. pmid:18669785
- 30. Nguyen LP, Lin R, Parra S, Omoluabi O, Hanania NA, et al. (2009) Beta2-adrenoceptor signaling is required for the development of an asthma phenotype in a murine model. Proc Natl Acad Sci U S A 106: 2435–2440. pmid:19171883
- 31. Thanawala VJ, Forkuo GS, Al-Sawalha N, Azzegagh Z, Nguyen LP, et al. (2013) beta2-Adrenoceptor agonists are required for development of the asthma phenotype in a murine model. Am J Respir Cell Mol Biol 48: 220–229. pmid:23204390
- 32. Walker JK, Penn RB, Hanania NA, Dickey BF, Bond RA (2011) New perspectives regarding beta(2)-adrenoceptor ligands in the treatment of asthma. Br J Pharmacol 163: 18–28. pmid:21175591
- 33. Luttrell LM, Lefkowitz RJ (2002) The role of beta-arrestins in the termination and transduction of G-protein-coupled receptor signals. J Cell Sci 115: 455–465. pmid:11861753
- 34. Deng H, Fang Y (2012) Anti-inflammatory gallic Acid and wedelolactone are G protein-coupled receptor-35 agonists. Pharmacology 89: 211–219. pmid:22488351
- 35. Spina D (2014) Current and novel bronchodilators in respiratory disease. Curr Opin Pulm Med 20: 73–86. pmid:24247039