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. 2017 Jan 4:7:39905.
doi: 10.1038/srep39905.

β2-adrenoreceptor Inverse Agonist Down-regulates Muscarine Cholinergic Subtype-3 Receptor and Its Downstream Signal Pathways in Airway Smooth Muscle Cells in vitro

Jian Luo  1 , Yuan-Hua Liu  2 , Wei Luo  3 , Zhu Luo  1 , Chun-Tao Liu  1
Affiliations

β2-adrenoreceptor Inverse Agonist Down-regulates Muscarine Cholinergic Subtype-3 Receptor and Its Downstream Signal Pathways in Airway Smooth Muscle Cells in vitro

Jian Luo et al. Sci Rep. .

Retraction in

Abstract

Mechanisms underlying β2-adrenoreceptor (β2AR) inverse agonist mediated bronchoprotectiveness remain unknown. We incubated ICI118,551, formoterol, budesonide, and formoterol plus budesonide, as well as ICI118,551 or pindolol plus formoterol, ICI118,551 plus forskolin, SQ22,536 or H89 plus formoterol in ASMCs to detect expressions of M3R, PLCβ1 and IP3. The level of M3R in the presence of 10-5 mmol/L ICI118,551 were significantly decreased at 12 h, 24 h and 48 h (P < 0.05), and at 24 h were significantly reduced in ICI118,551 with concentration of 10-5 mmol/L, 10-6 mmol/L, 10-7 mmol/L, and 10-8 mmol/L (P < 0.05). The level of IP3 in 10-5 mmol/L ICI118,551 was significantly diminished at 24 h (P < 0.01), except for that at 1 h, neither was in the level of PLCβ1. A concentration of 10-5 mmol/L ICI118,551 at 24 h showed a significant reduction of M3R level compared to formoterol (P < 0.01), budesonide (P < 0.01), and formoterol + budesonide (P < 0.05), but significant reduction of PLCβ1 and IP3 was only found between 10-5 mmol/L ICI118,551 and formoterol at 24 h, but not in the comparison of budesonide or formoterol + budesonide. Pindolol and H89 could not inhibit the formoterol-induced expression of M3R (P > 0.05), but SQ22,536 significantly antagonized the formoterol-induced M3R expression (P < 0.05). In conclusions, β2AR inverse agonist, ICI118,551, exerts similar bronchoprotective effects to corticosteroids via decreasing the expression of M3R and inhibiting the production of IP3.

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Figures

Figure 1
Figure 1. Primary culture of rat ASMCs.
The primary culture of rat ASMCs were prepared. (A) Confluent cultured ASMCs were visualized under phase-contrast microscopy. (Magnification: ×200) (B) ASMCs were identified by immunocytochemistry staining with an anti-α-SMA antibody. Nuclear were double stained with DAPI. (Magnification: ×200). ASMCs, airway smooth muscle cells; DAPI, 4′,6-diamidino-2-phenylindole; SMA, smooth muscle actin.
Figure 2
Figure 2. ICI118,551 down-regulated the protein expression of M3R.
(A) Cropped gel of M3R protein levels in rat ASMCs determined by Western blotting. The protein extract was obtained from ASMCs treated with 10−5 mM ICI118,551 at indicated time points. (B) The densitometry results of M3R protein levels were normalized to β-actin control. Data were presented as means ± SEM from three independent experiments. DMEM + FBS served as control. *Significant difference as compared to the control group (P < 0.05). ASMCs, airway smooth muscle cells; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; M3R, muscarine cholinergic subtype-3 receptor; SEM, standard error of mean.
Figure 3
Figure 3. ICI118,551 dose-dependently down-regulated the protein expression of M3R.
(A) Cropped gel of M3R protein levels in rat ASMCs determined by Western blotting. The protein extract was isolated from ASMCs treated with increasing doses of ICI118,551 for 24 h. (B) The densitometry results of M3R protein levels were normalized to a β-actin control. Data were presented as means ± SEM from three independent experiments. DMEM + FBS served as control. *Significant difference as compared to the control group (P < 0.05). ASMCs, airway smooth muscle cells; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; M3R, muscarine cholinergic subtype-3 receptor; SEM, standard error of mean.
Figure 4
Figure 4. ICI118,551 had no effect on the expression of PLCβ1.
(A) Cropped gel of PLCβ1 protein levels in rat ASMCs determined by Western blotting. Rat ASMCs were randomly divided into control group, ICI118,551 1 h and 24 h groups, and received different treatments as described previously. (B) The densitometry results of PLCβ1 protein normalized to a β-actin control. Data were presented as means ± SEM from three independent experiments. DMEM + FBS served as control. There were no significant differences among the three group (P > 0.05). ASMCs, airway smooth muscle cells; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; PLCβ1, phospholipase Cβ1; SEM, standard error of mean.
Figure 5
Figure 5. ICI118,551 suppressed the release of IP3 by Ach stimulation.
The levels of IP3 were assayed by ELISA. DMEM + FBS served as control. *Significant difference as compared with control group (P < 0.01). Ach, acetylcholine; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; IP3, 1,4,5-trisphosphate; ELISA, enzyme-linked immunosorbent assay.
Figure 6
Figure 6. Comparison of the effects of ICI118,551, formoterol, budesonide/formoterol on M3R, PLCβ1 and IP3 expression.
Rat ASMCs were randomly divided into five groups. Cells were incubated with 10−5 mM ICI118,551, 10−5 mM formoterol, 10−4 mM budesonide, 10−4 mM budesonide + 10−5 mM formoterol and control group for 24 h. The M3R, PLCβ1 and IP3 levels were determined after 15 min of Ach (10−4 mM) stimulation. Cropped gel of M3R (A) and PLCβ1 (C) protein levels in rat ASMCs were determined by Western blotting. The densitometry results of M3R (B) or PLCβ1 (D) were normalized to β-actin control. The expressions of IP3 were evaluated by ELISA (E). Data were presented as means ± SEM from three independent experiments. DMEM + FBS served as control. *Significant difference as compared with 24 h of ICI118,551 treatment alone (P < 0.05). Ach, acetylcholine; ASMCs, airway smooth muscle cells; B, budesonide; F, formoterol; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; ICI, ICI118,551; IP3, 1,4,5-trisphosphate; M3R, muscarine cholinergic subtype-3 receptor; PLCβ1, phospholipase Cβ1; SEM, standard error of mean.
Figure 7
Figure 7. Comparison of the effects of ICI118,551/pindolol + formoterol, ICI118,551 + forskolin, SQ22,536/H89 + formoterol, and formoterol on M3R expression.
Rat ASMCs were randomly divided into seven groups. Cells were incubated with 10−5 mM ICI118,551 + 10−5 mM formoterol, 10−5 mM pindolol + 10−5 mM formoterol, 10−5 mM ICI118,551 + 10−5 mM forskolin, 10−4 mM SQ22,536 + 10−5 mM formoterol, 10−5 mM H89 + 10−5 mM formoterol, 10−5 mM formoterol, and control group for 24 h. The M3R levels were determined after 15 min of Ach (10−4 mM) stimulation. (A) Cropped gel of M3R protein levels in rat ASMCs determined by Western blotting. (B) The densitometry results of M3R protein levels were normalized to β-actin control. Data were presented as means ± SEM from three independent experiments. DMEM + FBS + formoterol served as control. *Significant difference as compared with 24 h of formoterol treatment alone (P < 0.05). Ach, acetylcholine; ASMCs, airway smooth muscle cells; DMEM, Dulbecco’s modified Eagle’s medium; F, formoterol; FBS, fetal bovine serum; FK, forskolin; ICI, ICI118,551; M3R, muscarine cholinergic subtype-3 receptor; P, pindolol; SEM, standard error of mean; SQ, SQ22,536.
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