Antithetic regulation by β-adrenergic receptors of G_q receptor signaling via phospholipase C underlies the airway β-agonist paradox

β-adrenergic receptors (βARs) relax airway smooth muscle and bronchodilate, but chronic β-agonist treatment in asthma causes increased sensitivity to airway constriction (hyperreactivity) and is associated with exacerbations. This paradox was explored using mice with ablated βAR genes (βAR^–/–) and transgenic mice overexpressing airway smooth muscle β₂AR (β₂AR-OE) representing two extremes: absence and persistent activity of airway βAR. Unexpectedly, βAR^–/– mice, lacking these bronchodilating receptors, had markedly decreased bronchoconstrictive responses to methacholine and other G_q-coupled receptor agonists. In contrast, β₂AR-OE mice had enhanced constrictive responses. Contraction to permeabilization with β-escin was unaltered by gene ablation or overexpression. Inositol phosphate accumulation by G_q-coupled M₃-muscarinic, thromboxane-A₂, and 5-HT₂ receptors was desensitized in airway smooth muscle cells from βAR^–/– mice and sensitized in cells from β₂AR-OE mice. Thus, βAR antithetically regulates constrictive signals, affecting bronchomotor tone/reactivity by additional means other than direct dilatation. Studies of signaling elements in these pathways revealed the nodal point of this cross talk as phospholipase C-β1, whose expression was altered by βAR in a direction and magnitude consistent with the physiologic and cellular responses. These results establish a mechanism of the β-agonist paradox and identify a potential asthma modifier gene (phospholipase C-β1), which may also be a therapeutic target in asthma when chronic β-agonists are required.

Functional regulation of G protein–coupled receptors (GPCRs) is a dynamic process, such that integration of multiple signals leads to an adaptive cellular response and end-organ homeostasis. Regulatory events, however, can also be maladaptive, contributing to disease pathophysiology, and can limit the therapeutic efficiency of agents targeted to these receptors (1–3). One of the most readily observed regulatory phenomena of GPCRs is agonist-promoted desensitization (3, 4), which has been extensively investigated with the β₂-adrenergic receptor (β₂AR). Early events include phosphorylation of the receptor by GPCR kinases and the second messenger-dependent protein kinase A, while prolonged agonist exposure results in a decrease in receptor expression (termed downregulation). The signals evoked by GPCRs and the accompanying regulatory events, however, do not occur in isolation of other cellular processes or physiologic responses. This propensity for one GPCR signal to alter another has been termed cross talk and serves to further modify receptor function and coordinate multiple signaling events leading to global modification of responses within the cell or organ.

In the treatment of asthma, inhalation of β-agonists is the most common and effective approach for acute relief of bronchoconstriction, and chronic use of β-agonists is not infrequent. The primary target for these agonists is the βAR expressed on airway smooth muscle, which acts to relax constricted muscle and thus bronchodilate. A common physiologic consequence of chronic β-agonist use, however, is an increase in bronchoconstrictive responses (5–7). Such responses are assessed in humans by measuring airflow after provocation with graded inhalations of agents such as methacholine or histamine. This enhancement of contractile sensitivity observed with chronic β-agonist use has been referred to as hyperresponsiveness. Similarly, the functional antagonism of β-agonists to constrictive stimuli (termed the bronchoprotective effect) also wanes over time with chronic use. These effects on airway contractility are thought to predispose asthmatic patients using β-agonists on a regular basis, and potentially during abrupt withdrawal, to episodes of acute bronchospasm. Consistent with these physiologic events, frequent β-agonist use is associated with increases in asthma exacerbations and other indices of morbidity and mortality (8–10). Indeed, some investigators have supported the notion of gradual withdrawal of β-agonists as a therapeutic approach in resistant asthma (11). Analogous to the use of βAR antagonists in the treatment of heart failure, the concept of “reverse pharmacology” has been raised as a potentially viable strategy for diseases such as asthma, where chronic agonists are used (12). Airway smooth muscle tone and reactivity is primarily affected by G_αs-coupled receptors (relaxation) and G_αq-coupled receptors (contraction) (13). The former include βARs (primarily the β₂AR subtype in humans), the PGE₂ receptor, and the vasoactive intestinal peptide receptor. Smooth muscle relaxation results from a decrease in intracellular Ca²⁺, a consequence of cAMP-dependent protein kinase A phosphorylation of multiple proteins (14). The contractile G_αq-coupled receptors include muscarinic acetylcholine subtype 3 receptor (M₃R), thromboxane A₂ receptor (TXA₂R), a 5-hydroxytryptamine receptor (likely subtype 2; 5-HT₂R), cysteinyl leukotriene, histamine, platelet-activating factor, and tachykinin receptors. Smooth muscle contraction occurs by receptor-mediated activation of phospholipase C (PLC), leading to inositol 1,4,5 trisphosphate (IP₃) production and release of Ca²⁺ from the sarcoplasmic reticulum (14).

This incongruity in β-agonist therapy has been the source of considerable debate, in part because no cohesive mechanism accounts for the potential deleterious effects of chronic administration, the benefits of minimized usage, or the implied paradoxical regulation by βAR of airway sensitivity to contraction. Recent development of mice lacking βAR, as well as those overexpressing βAR in airway smooth muscle, provides a means to identify heretofore unrecognized βAR signaling events in airway smooth muscle that regulate in vivo bronchial tone and responsiveness. The overexpression of β₂AR results in signaling even in the absence of agonist, since there is an increase in the proportion of receptors in the spontaneously active (termed R*) state, thereby resulting in a continuous level of signaling. The knockout and overexpressing mice thus represent two conditions: a total lack of βAR activity and persistent activity in the airway. In this study we use these mice to define this paradoxical regulation and have identified the molecular basis whereby airway βAR amplify or attenuate G_αq receptor signaling in direct proportion to βAR activity, which we refer to as antithetic regulation.

To determine whether ablation of βAR signal transduction altered intrinsic airway reactivity or constrictive receptor signaling, we initially measured the bronchoconstrictor response to the M₃R agonist methacholine in βAR^–/– mice with whole-body plethysmography (Figure 1, a and b). Strain-matched wild-type mice showed a robust dose-dependent bronchoconstrictor response, with a maximum Penh of 4.9 ± 0.24. In contrast, methacholine-induced bronchoconstriction in the βAR^–/– mice was only 1.40 ± 0.07 (P < 0.001). In addition, the βAR^–/– mouse response was less sensitive than that of wild-type mice, with an ED₂₀₀ for methacholine of approximately 20 mg/ml compared with approximately 5 mg/ml for wild-type mice. This result was somewhat unexpected, because we had anticipated that the loss of a potent bronchoprotective pathway would serve to augment bronchoconstrictor responses. To determine whether this response was specific for methacholine, we exposed mice to the bronchoconstrictor U46619, which is a thromboxane mimetic acting at the TXA₂R. Once again, we found that bronchoconstriction was substantially diminished in the βAR^–/– mice (Figure 1b).

Figure 1

βAR^–/– mice display paradoxic contractile responses as assessed by whole-body plethysmography. Mice inhaled the indicated agents in an unrestrained state in a small-animal body box, and Penh was measured as described in Methods. (a) A representative dose-response study with methacholine (MCh) is shown. (b) Mean results from eight to twelve mice in each group in response to methacholine and the thromboxane analogue U46619 are shown. *P < 0.001 versus wild type.

Although Penh has been reported to correlate with other measurements of airway resistance (17), some have questioned whether Penh accurately reflects the extent of acute bronchoconstriction under certain circumstances (21). Our results described above therefore prompted additional, confirming studies using an anesthetized, intubated mouse model, which provides a direct measure of airway responses in absolute resistance units in the lung (Figure 2). As shown, initial studies showed that the contractile response to methacholine in wild-type mice was ablated by coinhalation of the βAR agonist isoproterenol, confirming that the model detects the expected constrictive as well as dilatory responses in normal mice and that the βAR^–/– mice are indeed physiologic knockouts of βAR responses in the airway. Basal (“resting”) airway resistance, measured after aerosolization of PBS alone, was not found to be different between βAR^–/– and wild-type mice (1.04 ± 0.08 versus 1.36 ± 0.11 cm H₂O/ml/s; P = 0.07). Remarkably, the maximal increase in resistance to methacholine was severely blunted in the βAR^–/– mice (3.0 ± 0.9 versus 24 ± 6.2 cm H₂O/ml/s for wild type), and the response to serotonin was essentially absent (Figure 2).

Figure 2

In vivo airway resistance responses to contractile ligands are decreased in βAR^–/– mice. Intubated mice were administered aerosolized solutions of PBS, methacholine (MCh) (80 mg/ml), serotonin (20 mg/ml), and methacholine with 1.0 mg/ml isoproterenol (MCh + Iso) in PBS, and then airway resistance was measured after each administration. Baseline resistance was not different between wild-type and βAR^–/– mice (see text), but the latter had virtually no contractile responses to methacholine or serotonin (P < 0.001 by ANOVA). Results are from four to six mice of each genotype. *Not different (P > 0.05) from baseline resistance.

We next measured contractile activity in excised tracheal rings so that we could specifically isolate the physiologic response of airway smooth muscle (Figure 3). In wild-type mice, acetylcholine-mediated maximal active isometric force was 13.3 ± 2.1 mN/mm². In contrast, active force attained with βAR^–/– rings was only 5.8 ± 0.6 mN/mm². In addition, the contractile response to U46619 was approximately sevenfold lower in the βAR^–/– mice compared with wild type. Importantly, though, the response following permeabilization with β-escin, which evokes contraction by an influx of extracellular Ca²⁺, was retained in βAR^–/– rings and indeed was slightly greater than wild type (6.6 ± 0.6 versus 4.5 ± 0.6 mN/mm²; P = 0.04). These latter results indicated that the intrinsic ability of airway smooth muscle to generate force (i.e., contract) is not altered by βAR gene ablation.

Figure 3

Airway smooth muscle contractions to G_αq-coupled receptor agonists are impaired in βAR^–/– tracheal rings. The contractile responses to the M₃R agonist acetylcholine (ACh) (10 nM−1.0 mM) and the TXA₂R agonist U46619 (1.0 nM–0.1 mM) were ascertained in tracheal rings from βAR^–/– mice and wild-type littermates. Shown is the maximal absolute isometric force evoked by the indicated agents. Maximal responses to acetylcholine and U46619 were depressed in the βAR^–/– mice, while the contractile responses to the cell-permeabilizing agent β-escin (500 μM) were preserved. Results are from five to seven experiments. *P = 0.005; ⁺P = 0.024.

The results from these three types of physiologic studies were in complete agreement and indicated that βAR gene ablation decreased responsiveness of airway smooth muscle to exogenous contractile agents. Because the agonists used in the above studies each contract airway smooth muscle through activation of receptors that couple to G_αq (M₃R, 5HT₂R, and TXA₂R), we considered that an active cross talk between these signaling pathways and the G_αs-coupled βAR was influenced by the complete loss of βAR signaling in the βAR^–/– mice. To test this hypothesis, we measured agonist stimulation of inositol phosphate production, a downstream consequence of G_αq-coupled receptor activation, in primary cultures of airway smooth muscle cells derived from the two mouse lines. Compared with cells from wild-type littermates, inositol phosphate accumulation was substantially lower in cells from the βAR^–/– mice in response to agonists acting at all three receptors (Figure 4).

Figure 4

Antithetic regulation of G_αq receptor signaling by βAR activity in isolated airway smooth muscle cells. Primary airway smooth muscle cell cultures were established from tracheas from the βAR^–/–, the β₂AR-OE, and their wild-type (WT) littermates. Maximal whole-cell inositol phosphate accumulation in response to carbachol (CCh), serotonin, and U46619 were reduced in βAR^–/– cells and increased in the β₂AR-OE cells (P < 0.01 for all responses versus wild-type littermates). Results are from five experiments performed in triplicate.

Although β-agonists are potent acute bronchodilators, numerous studies have suggested that chronic use of these agents promotes increased airway hyperreactivity or loss of protective effect in asthmatics (see Introduction). This observation, taken together with our finding that βAR signaling modulates the responsiveness of airway smooth muscle to contractile agonists by a mechanism other than direct opposition, suggested the possibility that chronic βAR stimulation could have the opposite physiologic and biochemical effects to those found in the mice without βAR activity. To address this, we used transgenic mice with targeted overexpression of β₂AR (β₂AR-OE) in airway smooth muscle. β₂AR expression in cultured airway smooth muscle cells from these mice was 580 ± 140 fmol/mg (as determined by ¹²⁵I-CYP radioligand binding), which represents approximately tenfold overexpression compared with wild-type cells. This expression provides for persistent activation of this signaling pathway specifically in smooth muscle cells of the airway (16). In cultured airway smooth muscle cells we found that the inositol phosphate responses to carbachol, serotonin, and U46619 were indeed increased in cells from β₂AR-OE mice compared with cells from wild-type littermates (Figure 4). We expected, then, that the physiologic response to contractile agents would be enhanced in the β₂AR-OE mice, as well. To test this, we used the isolated tracheal ring preparations since in this assay the β₂AR-OE mice are not influenced by circulating epinephrine (as would be the case in the intact animal), thereby maximizing sensitivity for detecting potential subtle differences. Using this approach (Figure 5), we found that the concentration-response curves between the tracheal rings from the wild-type and β₂AR-OE mice were clearly different (P < 0.001), and maximal active force generated by β₂AR-OE rings in response to acetylcholine was modestly, but significantly, greater (approximately 33%) than the force generated by the wild-type mice (Figure 5).

Figure 5

β₂AR-OE mice have increased contractile responses in airway smooth muscle. Tracheal rings from the β₂AR-OE mice and wild-type littermates were contracted by the indicated concentrations of acetylcholine (ACh). The overall responses differed (P < 0.001 by ANOVA), and the maximal response was approximately 33% greater for the β₂AR-OE rings (P < 0.001). Results are from six experiments. P values of individual post hoc comparisons: *P = 0.02; **P < 0.001.

Taken collectively, these data in mice that lacked and overexpressed βAR indicated that G_αq-coupled receptor signal transduction was proportionately regulated by βAR activity in airway smooth muscle cells. Since the response to multiple contractile agonists was similarly attenuated, we considered that a signaling component common to these three contractile receptors was a likely nodal point for the tight control by way of such cross talk. We directed our investigation to signaling components distal to the receptor but upstream of Ca²⁺-mediated contraction, particularly the relevant G protein (G_q) and the effector phospholipase Cβ. Using Western blot analysis, we found that G_αq content in βAR^–/– and β₂AR-OE airway smooth muscle cells was not different compared with their respective control (wild-type) cells (Figure 6). PLC-β1 content in the βAR^–/– cells, however, was decreased by over 60% compared with wild-type–derived cells (23,350 ± 3,536 versus 69,640 ± 2,442 RU, respectively; Figure 6a). This decrease is consistent with the decreased agonist-promoted inositol phosphate production and the decreased contractile responses observed in the βAR^–/– mice. Since the signaling and contractile functions were increased in the β₂AR-OE, we expected that PLC-β1 expression would be increased as well, if the evolving paradigm was correct. Indeed, PLC-β1 expression in cells from β₂AR-OE mice was increased twofold over those from wild-type littermates (76,700 ± 8,226 versus 38,280 ± 3,279 RU, respectively; Figure 6b). Neither mouse line had altered levels of G_αs, the cognate G protein for βAR.

Figure 6

Antithetic regulation of PLC-β1 expression by βAR activity in airway smooth muscle cells. (a) PLC-β1 was decreased approximately 60% in βAR^–/– cells (P < 0.001) and (b) increased twofold in β₂AR-OE cells (P = 0.01), compared with their wild-type littermates. In contrast, expression levels of G_αq and G_αs were not different.

The mechanistic basis of enhanced airway responsiveness to constrictive stimuli during chronic β-agonist treatment of asthma has been elusive. This treatment-related phenomena has been considered a physiologically relevant adverse effect, particularly since the increased use of β-agonists has been associated with increased asthma morbidity and mortality (8–10). The enhanced sensitivity to contractile stimuli has been thought to be consistent with a greater propensity for acute flares of bronchospasm, also termed loss of “asthma control,” during chronic β-agonist therapy (11). While β-agonists administered on a chronic basis are efficacious, their use in asthma has been called into question because of the aforementioned deleterious physiologic effects on airway reactivity and these epidemiologic associations. One hypothesized mechanism of this altered hyperresponsiveness, or loss of bronchoprotective effect, has been that desensitization of the β2AR by chronic agonists lessens the opposition to contractile events (7, 22). We show here, however, that cross talk between βAR and G_αq receptor signaling results in enhanced signaling of the contractile pathway. So, rather than simply a loss of relaxation, the program evoked by chronic β-agonists includes a gain of contractile signaling.

In the current work, we approached this issue using mice with an absolute lack of βAR activity or persistent βAR activation in airway smooth muscle. We assumed that these extreme conditions would provide the greatest opportunity to delineate the interplay between βAR and constrictive receptors. The initial findings with the βAR^–/– mice using whole-body plethysmography were somewhat surprising. These mice lacked the bronchodilatory βAR, so the expectation was that they would have enhanced responsiveness to contractile agents. This potentially “unopposed” state paradoxically resulted in a marked decrease in airway responsiveness to the inhaled M₃R agonist methacholine, however. Subsequent studies in an intubated mouse model confirmed that this response is indeed occurring in the airway and also revealed that serotonin-promoted contraction was also markedly reduced in the βAR^–/– mice. Tracheal ring preparations, which provided the most direct approach to isolate airway smooth muscle responses, again confirmed hyporesponsiveness to M₃R activation, as well as to the thromboxane analogue U46619. The contractile responses to cell permeabilization by β-escin, which is due to an influx of Ca2+, were equivalent between wild-type and βAR^–/– mice, indicating that the underlying contractile mechanisms are intact in the knockout mice.

Importantly, the three contractile agonists used in these studies act through receptors (M₃R, TXA₂R, and 5-HT₂R) that couple to G_αq, suggesting a common mechanism. We thus concentrated on shared elements of these signaling pathways, since it seemed unlikely that expression of all three of these receptors would be reduced. Contraction of smooth muscle by G_αq-coupled receptors is due to IP₃ receptor–mediated Ca²⁺ release from the sarcoplasmic reticulum. Since β-escin responses were equivalent, it seemed reasonable that the adaptive event(s) were proximal to the Ca²⁺-dependent step, particularly at the level of G_αq, PLC isoforms, inositol phosphates (or their precursors), or the IP₃ receptor. In cultured airway smooth muscle cells inositol phosphate production in response to all three agonists was significantly reduced in βAR^–/– cells compared with those from wild-type mice, further narrowing the sites of potential interaction. Subsequent studies revealed that expression of PLC-β1 was markedly reduced in the βAR^–/– cells, while levels of G_αq and G_αs were not appreciably changed.

This cross talk between bronchodilating and bronchoprotective pathways suggests that an adaptive program that promotes a defined equilibrium is in play so as to maintain bronchomotor tone or reactivity within a specific range. If this is so, we hypothesized that an antithetic response would be evident with the two extremes of βAR activation, manifested as opposite physiologic, signaling, and protein-expression events. This was indeed found, as indicated by our results from the overexpressing mice. In these β₂AR-OE mice, the sensitivity to methacholine and the maximal contractile response were increased. In addition, inositol phosphate production from activation of M₃R, TXA₂R, and 5-HT₂R were all increased in airway smooth muscle cells from β₂AR-OE mice compared with wild type. Finally, consistent with the physiologic and cell-based signaling and the cross-talk paradigm, expression of PLC-β1 was found to be increased in these mice with persistent β₂AR activation.

The current results with βAR knockout mice and low-level β₂AR-overexpressing mice need to be reconciled with our earlier report with markedly overexpressed β₂AR (approximately 75-fold over endogenous levels) in airway smooth muscle (16). In that study, it was found that the bronchodilating effect of such overexpression resulted in resistance to bronchoconstriction. This scenario is consistent with the notion that airway tone and responsiveness is ultimately set by the net effect of G_q and G_s signaling events. While we have shown in the current report that persistent β₂AR signaling can augment G_q signaling, it is clear from the high overexpressor data that this can be overcome, at some point, by increasing G_s signaling to extraordinary levels. In essence, overexpression to this extent obscures the physiologic sequelae of altered G_q signaling due to such marked, virtually “fixed,” bronchodilatation. Further confirmation of the βAR-G_q receptor cross talk comes from the βAR knockout mice, where overexpression issues are not a concern. In these mice the alterations in G_q signaling at the physiologic and biochemical level and PLC-β1 regulation were the opposite of the β₂AR overexpressors in each case, which indicates a dynamic and directional regulation. In some ways the biology of extreme overexpression has also been experienced by us (23, 24) and others (25) with overexpressing β₂AR in the heart. With very high expression levels, physiologic function (rate and force of contraction) was maximal in the absence of agonist (25). Only with mice expressing β₂AR at a lower level could meaningful physiologic and biochemical correlates be established (23, 24).

Reported here, our results in airway smooth muscle delineate one mechanism by which β-agonists act to increase the potential for airway hyperresponsiveness and support a clinical benefit for minimizing β-agonist use when possible. They also identify a potential asthma-modifier gene, PLC-β, which may also represent a novel target for therapy. Inhibitors of one or more of the PLC isoforms would be predicted to decrease G_αq receptor–mediated bronchoconstriction and minimize the deleterious enhancement of contractile sensitivity induced by β-agonists. Virtually all the endogenous constrictive substances implicated in the bronchospastic component of asthma, including acetylcholine, thromboxane, histamine, leukotrienes, and prostaglandins, act through receptors that couple to G_αq. These are released in neuronal, autocrine, paracrine, or humoral programs instigated by the inflammatory state of the disease. Interestingly, agents that are antagonists at individual G_αq receptors (such as montelukast, ipratropium, and seratrodast) have limited efficacy in treating acute bronchospasm in asthma, which may be due to the large number of constrictive receptors that are activated in the disease. Thus, agents that decrease the activity of a common distal element in the pathway may be more useful. In combination with a β-agonist, both interventions could lead to persistent bronchodilatation without increased airway hyperresponsiveness.

This current study also appears to distinguish between desensitization of airway βAR and airway hyperresponsiveness, both of which occur with chronic β-agonists. While βAR desensitization may contribute to less-effective dilatation, the current data do not support this as the major mechanism of β-agonist–induced airway hyperreactivity for two reasons. First, the ultimate desensitization of βAR function occurs when expression is absent, as is the case with the βAR^–/– mice. These mice, however, do not have enhanced constrictive responses, but indeed have a marked reduction in this parameter. Second, the transgenic expression of β₂AR on airway smooth muscle mimics a persistently activated state due to the receptor overexpression. We have no evidence for desensitization of receptors in such mice (16). Taken together, the results indicate that β₂AR activation initiates and maintains events that lead to enhanced constrictive responses. Of note, some studies suggest that partial β-agonists evoke fewer adverse effects than full agonists (reviewed in ref. 10) and that certain β₂AR polymorphisms with altered downregulation phenotypes influence hyperresponsiveness (26, 27). Based on the current work, these observations reflect how the trigger for the events leading to enhanced contractile responsiveness by way of β₂AR activation can be modified. Furthermore, with the identification of this cross talk event, the genes encoding the PLC isoforms are candidates that should be interrogated for polymorphisms (28), given that there is interindividual variability in β-agonist–promoted bronchial hyperreactivity and bronchoprotection in humans. Concerning regulatory regions within the PLC gene that may be influenced by βAR activity, we are unaware of any reports of potential control motifs or transcription-binding sites. Indeed, the genomic organization of the PLC-β1 gene has been discerned only recently (29).

In conclusion, we have identified a mechanism by which chronic airway βAR activation leads to an increase in airway hyperresponsiveness over time during chronic activation. In contrast to the notion that this pathophysiologic outcome is due to βAR desensitization, we find that βAR activation initiates cross talk with the constrictive G_αq–coupled receptor pathway, leading to enhanced signaling and airway hyperresponsiveness. Such regulation is due to modulation of PLC-β1 expression. Furthermore, this process is antithetic in nature; cessation of βAR activation decreases G_αq receptor signaling and constrictive airway responsiveness. This provides a mechanistic basis for the beneficial clinical effect of limiting chronic β-agonist administration in the management of bronchospastic diseases such as asthma. Whether there is a role for “reverse” or “paradoxic” pharmacology (12) in the treatment of asthma, analogous to the apparent effects of βAR antagonists in heart failure, remains speculative. Since the deleterious cross talk between βARs and G_αs receptors appears to be by altered expression of PLC, however, novel therapeutics targeting this enzyme may have substantial benefit, particularly when chronic β-agonists are required.

The authors thank Fahema Rahman for technical support and Esther Getz for manuscript preparation. This work is supported by NIH grants U01-HL65899 (Pharmacogenetics Research Network, to S.B. Liggett) and HL03986 (to D.W. McGraw).

See the related Commentary beginning on page 495.

Conflict of interest: The authors have declared that no conflict of interest exists.

Nonstandard abbreviations used: G protein–coupled receptors (GPCRs); β-adrenergic receptor (βAR); muscarinic acetylcholine subtype 3 receptor (M₃R); thromboxane A₂ receptor (TXA₂R); 5-hydroxytryptamine subtype 2 receptor (5-HT₂R); phospholipase C (PLC); inositol 1,4,5 trisphosphate (IP₃); enhanced pause (Penh); airway resistance (R_aw); relative units (RUs); ¹²⁵I-cyanopindolol (¹²⁵I-CYP); β₂AR overexpression (β₂AR-OE).

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