A long-acting β2-adrenergic agonist increases the expression of muscarine cholinergic subtype-3 receptors by activating the β2-adrenoceptor cyclic adenosine monophosphate signaling pathway in airway smooth muscle cells

The persistent administration of β2-adrenergic (β2AR) agonists has been demonstrated to increase the risk of severe asthma, partly due to the induction of tolerance to bronchoprotection via undefined mechanisms. The present study investigated the potential effect of the long-acting β2-adrenergic agonist, formoterol, on the expression of muscarinic M3 receptor (M3R) in rat airway smooth muscle cells (ASMCs). Primary rat ASMCs were isolated and characterized following immunostaining with anti-α-smooth muscle actin antibodies. The protein expression levels of M3R and phospholipase C-β1 (PLCβ1) were characterized by western blot analysis and the production of inositol 1,4,5-trisphosphate (IP3) was determined using an enzyme-linked immunosorbent assay. Formoterol increased the protein expression of M3R in rat ASMCs in a time- and dose-dependent manner, which was significantly inhibited by the β2AR antagonist, ICI118,551 and the cyclic adenosine monophosphate (cAMP) inhibitor, SQ22,536. The increased protein expression of M3R was positively correlated with increased production of PLCβ1 and IP3. Furthermore, treatment with the glucocorticoid, budesonide, and the PLC inhibitor, U73,122, significantly suppressed the formoterol-induced upregulated protein expression levels of M3R and PLCβ1 and production of IP3. The present study demonstrated that formoterol mediated the upregulation of M3R in the rat ASMCs by activating the β2AR-cAMP signaling pathway, resulting in increased expression levels of PLCβ1 and IP3, which are key to inducing bronchoprotection tolerance. Administration of glucocorticoids or a PLC antagonist prevented formoterol-induced bronchoprotection tolerance by suppressing the protein expression of M3R.


Introduction
Asthma is a chronic airway inflammatory disease with increasing prevalence worldwide (1,2). The airways of patients with asthma are hyper-responsive to exercise, allergens and contractile agents, including histamine and acetylcholine (ACh) (3,4). Due to bronchodilatation and bronchoprotection, which prevent bronchoconstriction, long-acting β 2 -adrenergic agonists (LABAs), including salmeterol and formoterol, have been widely combined with inhaled corticosteroids to treat patients with asthma that respond poorly to corticosteroid-only based therapies (5). However, LABAs alone increase the risk of asthma-associated mortality (6), possibly due to increased bronchial hyper-responsiveness (7), severe exacerbation of asthmatic symptoms (8) or tolerance to bronchodilation and bronchoprotection (9)(10)(11).
Previous studies have demonstrated that adenylyl cyclase stimulation results in the subsequent activation of cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) associated with LABA-induced rapid bronchodilatation (12,13). By contrast, contractile agonists, including ACh, were revealed to initiate bronchoconstriction as a result of G-protein-coupled muscarinic M3 receptors (M 3 R) binding to airway smooth muscle cells, resulting in the subsequent activation of phospholipase C (PLC) and the production of inositol 1,4,5-trisphosphate (IP 3 ) (12,14). Chilvers et al reported that pretreatment with salmeterol significantly inhibits histamine-stimulated accumulation of IP 3 (15) and McGraw et al demonstrated that transgenic mice overexpressing airway smooth muscle β 2 -adrenoceptor (β 2 AR) agonists significantly increase the expression of PLC-β 1 compared with that of wild-type mice (14), suggesting that the sustained activation of β 2 AR induces the PLCβ 1 -IP 3 signaling pathway via mechanisms that remain to be elucidated.
The present study investigated the effects of formoterol on the expression of M 3 R and the downstream signaling events leading to bronchoprotection tolerance in rat airway smooth muscle cells (ASMCs).
Primary rat ASMC cultures. Male Wistar rats (8 weeks old; 150±50g) were provided by the Animal Center of West China Hospital, (Sichuan University, Chengdu, China). The rats were housed under specific-pathogen-free conditions at 25˚C and maintained on a 12-h light/dark cycle, with access to food and sterile water ad libitum. A total of 52 rats were injected (i.p.) with 10% chloral hydrate to anesthetize them, and then they were sacrificed by cervical vertebra dislocation. Primary rat ASMC cultures were prepared, as previously described (16). Briefly, the trachea of each rat was excised, minced and the cells were allowed to adhere to the culture flasks for 3 h. Fresh culture medium (DMEM+FBS) was subsequently added and the cells were grown to confluency (density, 80 cells at x200 high-power lens) in an incubator at 37˚C with 5% CO 2 . The cultured cells were passaged following trypsinization (0.05%). ASMCs passaged three times were immunostained with anti-α-smooth muscle actin antibodies. Cells between passages four and six, which were >80% confluent, were used for subsequent experiments. The present study was approved by the Biomedical Research Ethics Committee at West China Hospital (Sichuan University, Chengdu, China).
To observe the effect of formoterol on bronchoconstriction prevention (bronchoprotection), the cells were first stimulated with the contractile agonist ACh (10 -4 mmol/l) for 15 min, followed by the above mentioned treatments and analyzed by western blot analysis to determine the protein expression levels of M 3 R and PLCβ 1 , in addition to determining the expression of IP 3 by ELISA.
Western blot analysis. The protein expression levels of M 3 R and PLCβ 1 were measured by western blot analysis. The total cellular protein was extracted using radioimmunoprecipitation assay lysis buffer (1% Triton-X, 0.5% sodium deoxychlate, 0.1% SDS; Sangon Biotech, Shanghai, China), quantified using a bicinchoninic acid assay (Boster, Wuhan, China) and a Model 680 spectrophotometer (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and the total protein concentration was adjusted to 0.8 µg/µl. Equal quantities of protein were subjected to 5% sodium dodecyl sulphate polyacrylamide gel electrophoresis (12.6% separation gel for M 3 R, β 2 AR and β-actin; 10% separation gel for PLCβ1; Sigma-Aldrich) and subsequently transferred onto polyvinylidene fluoride membranes (Merck Millipore). The membranes were blocked for 1 h with Tris-buffered saline containing 0.05% Tween-20 (TBST; Boster) and 5% goat serum (Boster), for M 3 R blots or with 5% (w/v) non-fat milk for the PLCβ 1 and β-actin blots. The membranes were subsequently incubated with primary antibodies against anti-M 3 R (1:500), anti-PLCβ 1 (1:1,000) or anti-β-actin (1:2,000) at 4˚C overnight. Following incubation, the membranes were washed three times with TBST for 10 min and incubated with anti-rabbit (1:20,000) or anti-mouse (1:20,000) secondary antibodies for 1 h at room temperature. The membranes were subsequently washed and the blots were visualized using a Bio-Rad Gel Doc™ XR+ Imaging system and the band densities were quantified using Quantity One software (Bio-Rad Laboratories, Inc.).
ELISA. The levels of IP 3 were determined using an IP3 ELISA kit (Cusabio Biotech Co., Ltd, Wuhan, China), according to the manufacturer's instructions. The ASMC culture medium was removed and the cells were incubated with 0.1 mmol/l HClO 4 for 20 min. The cells were centrifuged at 170 x g for 15 min at room temperature and the supernatant was collected for analysis. An anti-IP 3 detection antibody was added and incubated at 37˚C for 60 min, followed by the addition of substrate solution for 15 min at 37˚C. The reaction was terminated following the addition of stop solution and the plates were read at an absorbance of 450 nm using a Model 680 spectrophotometer (Bio-Rad Laboratories, Inc.). The effect of formoterol on the expression of IP 3 was determined using the following formula: Inhibition of ACh-induced IP 3 accumulation (%) = (IP 3 levels in the control group -IP 3 levels in the treatment group) / IP 3 levels in the control group x 100%.
Statistical analysis. Data are expressed as the mean ± standard deviation and the differences between groups were analyzed using analysis of variance or non-paired Student's t-test if the continuous variables were not normally distributed. The associations between M 3 R and IP 3 or PLCβ 1 were determined using a linear regression model. All statistical analyses were performed using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

Results
Characterization of rat ASMCs. The confluent rat ASMCs were relatively homogeneous, with a hill-and-valley pattern (Fig. 1A). Anti-α-smooth muscle actin (a SMC-specific marker) was diffusely distributed within the cytoplasm and the purification of ASMCs between passages four and six was confirmed to be >95% (Fig. 1B).

A B
The clinical concentration of plasma formoterol is significantly lower than 10 -4 mmol/l (17), therefore, 10 -5 mmol/l formoterol was selected for the subsequent experiments. The immunocytochemical analysis demonstrated that the expression of M 3 R was significantly increased and predominantly located in the cellular membrane (Fig. 4). These results suggested that formoterol upregulated the protein expression of M 3 R in rat ASMCs.
Formoterol regulates the expression of M 3 R through the β 2 AR-cAMP signaling pathway. Pre-treatment with the ICI118,551 β 2 AR antagonist or the SQ22,536 cAMP inhibitor   Rat airway smooth muscle cells were randomly divided into seven groups. The cells were treated with formoterol (10 -5 mmol/l) for 1 h or 24 h. The cells stimulated with cAMP were treated with 10 -5 mmol/l forskolin for 24 h. Inhibition of the β2AR-cAMP-protein kinase A was performed by pretreating the cells with 10 -5 mmol/l ICI118,551, 10 -4 mmol/l SQ22,536 or 10 -5 mmol/l H89 for 24 h. These treatment groups and the control group were subsequently treated with 10 -5 mmol/l formoterol for 2 h. The protein expression of M3R in the rat airway smooth muscle cells was determined by western blot analysis following acetylcholine stimulation for 15 min. (B) Expression of M3R was normalized to the β-actin control. The data are expressed as the mean ± standard deviation from three independent experiments ( * P<0.01, compared with the 1 h incubation group; ∆ P<0.05, compared with the 24 h formoterol treatment group). F1h, 1 h formoterol treatment; F24h, 24 h formoterol treatment; FK, forskolin; ICI+F, formoterol+ICI118,551; SQ+F, formoterol+SQ22,536; H89+F, formoterol+H89; Con, control; M 3 R, muscarinic M 3 receptor.  Fig. 5). However, the H89 PKA inhibitor had no effect on the formoterol-regulated expression of M 3 R (P>0.05). As expected, the forskolin cAMP stimulator caused similar effects as formoterol with respect to the protein expression of M 3 R. The present study demonstrated that 24 h incubation with forskolin significantly increased the protein expression of M 3 R (P<0.01), compared with the control and compared with levels 24 h after treatment with formoterol. These results suggested that formoterol induced the expression of M 3 R through the β 2 AR-cAMP signaling pathway. 3 . The present study demonstrated that formoterol increased the expression of PLCβ 1 in ACh-stimulated rat ASMCs (Fig. 6A and B). Inhibition of the β 2 AR-cAMP signaling pathway using the ICI118,551 or SQ22,536 antagonists inhibited the formoterol-induced upregulation of PLCβ 1 (P<0.05). By contrast, no significant difference was observed in the expression of PLCβ 1 following exposure to the H89 PKA inhibitor in the presence of formoterol (P>0.05). Forskolin had a similar effect on the formoterol-induced expression of PLCβ 1 . In addition, treatment with formoterol for 1 h suppressed the ACh-induced production of IP 3 by ~72.89±2.29%, compared with the 26.58±2.37% inhibition observed following formoterol exposure for 24 h (Fig. 6E). Similarly, ICI118,551 and SQ22,536 also reduced the expression of IP 3 . Positive correlations were observed between M 3 R and PLCβ 1 (R 2 = 0.872; P<0.01) and between M 3 R and IP 3 (R 2 =0.877, P<0.01), as shown in Fig. 6D and E.

Effects of a glucocorticoid and a PLC inhibitor on the formoterol-induced upregulation of M 3 R.
The combined treatment of budesonide and formoterol significantly reduced the expression levels of M 3 R, PLCβ 1 and IP 3 compared with the expression levels observed following treatment with formoterol alone (P<0.05; Fig. 7). In addition, the U73,122 PLC inhibitor significantly decreased the formoterol-induced upregulation

Discussion
Emerging evidence has demonstrated that prolonged adm inist ration of LA BAs increases the r isk of asthma-associated mortality (6) or can seriously exacerbate asthmatic symptoms (8), possibly due to increased bronchial hyper-responsiveness (7) and bronchodilator and bronchoprotection tolerance (9-11) The β 2 AR, fenoterol, induces the upregulation of G-protein-coupled neurokinin receptors and H1 histamine receptors in ASMCs (18,19) This suggests that β 2 AR may lead to increased bronchial responsiveness and bronchodilator tolerance by upregulating the expression of G-protein-coupled receptors. However, bronchoprotection gradually decreases in the presence of sustained administration of LABAs via mechanisms, which remain to be elucidated. M 3 R is a G-protein-coupled receptor predominantly distributed on the membrane surface of ASMCs. In the present study the effects of formoterol, a widely used LABA, on the expression of M 3 R was investigated in rat ASMCs. Formoterol upregulated the expression of M 3 R for at least 48 h, however, the long-term effects of formoterol were not evaluated due to the rapid proliferation of ASMCs in vitro. It has been suggested that stimulation of β 2 AR activates intracellular adenyl cyclase, which catalyzes the conversion of ATP to cAMP, which in turn increases the activity of PKA associated with altered intracellular Ca 2+ homeostasis and results in bronchodilation (12). Treatment with the ICI118,551 β 2 AR antagonist, SQ22,536 cAMP antagonist or H89 PKA antagonist demonstrated that the β 2 AR-cAMP signaling pathway contributed to the formoterol-mediated upregulation of M 3 R via a PKA-independent mechanism. Consistent with these results, it was previously demonstrated that prolonged exposure to the cAMP-responding element-binding (CREB) protein and c-Ets1 (LABA) contribute to mucous cell hypersecretion associated with common respiratory disorders (20), suggesting a role for the β 2 AR-cAMP-CREBs signaling pathway in this process. In addition, β 2 AR agonists increased the cAMP-mediated activation of cGMP-dependent protein kinases leading to smooth muscle relaxation (12,21). cAMP can bind to exchange proteins, which are directly activated by cAMP (Epac) independent of PKA, resulting in the induction of Rap-1-dependent responses in the airway smooth muscles, epithelium and pro-inflammatory immune cells (12,22). However, a previous study revealed that β 2 AR agonists selectively inhibit ASMC migration by interfering with the β 2 AR/PKA signaling pathway and that prolonged treatment with albuterol eliminated the inhibitory effect of β-agonists on ASMC migration (13). This suggested that multiple signaling pathways, including PKA, may be involved in β 2 AR agonist functions. Whether the overexpression of M 3 R from prolonged treatment with formoterol is mediated by a cAMP-responding element-binding protein, through the β 2 AR-cAMP signaling pathway, requires further investigation. In addition, further experiments are required to determine whether downstream signaling proteins, in addition to cAMP, are important in the formoterol-induced overexpression of M 3 R.
McGraw et al demonstrated that the expression of PLCβ 1 is significantly increased in transgenic mice overexpressing airway smooth muscle β 2 AR (14) and Sayers et al reported that a β 2 AR agonist upregulated the protein expression of PLCβ 1 in human ASMCs (23). The present study supported these observations and demonstrated that formoterol exposure increased the protein expression of PLCβ 1 and production of IP 3 in the rat ASMCs. In addition, changes to the expression levels of PLCβ 1 and IP 3 were positively correlated with the expression of M 3 R. Contractile agonists bind to G-protein-coupled M 3 R and trigger the activation of PLC, resulting in the production of IP 3, leading to Ca 2+ release and subsequent airway smooth muscle contraction (12,14). A previous study revealed that salbutamol and salmeterol (short-and LABR) inhibit the histamine-stimulated accumulation of IP 3 in airway smooth muscle cells (15). The data from the present study demonstrated that 24 h pre-treatment with formoterol significantly reduced the ACh-stimulated production of IP 3 . This inhibitory effect on the accumulation of IP 3 , however, was reduced following pre-treatment with formoterol for 24 h (26.58±2.37%) compared with 1 h (72.89±2.29%). These results demonstrated that short-term pre-exposure of ASMCs to formoterol antagonized the accumulation of IP 3 induced by ACh and that this effect was attenuated significantly if the pre-exposure duration was extended, suggesting that this may be a mechanism contributing to bronchoprotection tolerance.
The present study also demonstrated that inhibiting the β 2 AR-cAMP signaling pathway significantly downregulated the formoterol-induced expression of M 3 R and inhibited the production of IP 3 . The expression of M 3 R was negatively correlated to the rate at which production of IP 3 was inhibited, suggesting that M 3 R may be important in bronchoprotection tolerance and that cholinergic antagonists may be used in the potential treatment of patients that respond poorly to LABAs. Furthermore, inhibiting PLCβ 1 significantly reduced the expression of M 3 R and increased the inhibitory effect of formoterol on the production of IP 3 . These results suggested that the inhibition of PLCβ 1 may provide a novel strategy for preventing bronchoprotection tolerance. However, other mechanisms, including the functional desensitization of β 2 AR in mast cells, may also have contributed to bronchoprotection tolerance (24,25). In addition, β 2 AR agonists may result in membrane hyperpolarization by activating K + channels (26).
Combined treatment with LABAs and inhaled corticosteroids is a common for patients with poorly controlled asthma, which is associated with improved pulmonary function and asthma control (27,28). The data presented in the present study revealed that glucocorticoids suppressed the formoterol-induced upregulation of M 3 R, reduced the expression of PLCβ 1 and partially facilitated the formoterol-mediated inhibition of IP 3 production. These observations suggested that the inhibition of the expression of M 3 R may be important in combination therapies designed to prevent bronchoprotection tolerance. However, these studies were performed in vitro, therefore the results require confirmation in experimental animal asthma models and in patients.
In conclusion, the present study demonstrated that formoterol upregulated the protein expression of M 3 R in rat ASMCs following activation of the β 2 AR-cAMP signaling pathway, resulting in an increased expression of PLCβ 1 and IP 3 , which are critical for mediating bronchoprotection tolerance. Administration of a glucocorticoid or PLC antagonist prevented formoterol-induced bronchoprotection tolerance by suppressing the protein expression of M 3 R.