Changes in beta 2-adrenoceptor and other signaling proteins produced by chronic administration of 'beta-blockers' in a murine asthma model

Abstract

Background: We have previously reported that chronic treatment with certain 'beta-blockers' reduces airway hyperresponsiveness (AHR) to methacholine in a murine model of asthma.

Methods: Airway resistance was measured using the forced oscillation technique in ovalbulmin-sensitized and ovalbulmin-challenged mice treated with several beta-adrenoceptor (beta-AR) ligands. We used the selective beta 2-AR ligand ICI 118,551 and the preferential beta 1-AR ligand metoprolol to investigate the receptor subtype mediating the beneficial effect. Expression of beta-ARs was evaluated using immunofluorescence. We evaluated several signaling proteins by western blot using lung homogenates, and measured the relaxation of the isolated trachea produced by EP2 and IP receptor agonists.

Results: Four findings were associated with the decreased AHR after chronic beta-blocker treatment: (1) the highly selective beta 2-AR antagonist/inverse agonist, ICI 118,551 produced the bronchoprotective effect; (2) beta 2-AR up-regulation resulted from chronic 'beta-blocker' treatment; (3) reduced expression of certain proteins involved in regulating bronchial tone, namely, Gi, phosphodiesterase 4D and phospholipase C-beta 1; and (4) an enhanced bronchodilatory response to prostanoid agonists for the IP and EP2 receptors.

Conclusions: These data suggest that in the murine model of asthma, several compensatory changes associated with either increased bronchodilator signaling or decreased bronchoconstrictive signaling, result from the chronic administration of certain 'beta-blockers'.

Fig. 1

Effects of treatment with subtype specific β-AR ligands and nadolol on airway hyperresponsiveness to methacholine in murine model of asthma. Cumulative concentration–response curves to methacholine-induced increases in airway resisitance (R_aw) in NS/NC and S/C mice treated with or without 50% DMSO (A), S/C mice treated with or without ICI118,551 (ICI) in 50% DMSO (B), and S/C mice treated with or without metoprolol 10 mg/kg (C), metoprolol 20 mg/kg (D) for 7 days, and nadolol 250 ppm (E) for 28 days. For the β-AR subtype experiments, treatment started from day 21 to day 28. For nadolol, treatment was from days 1 to 28. Values represent mean and vertical lines show S.E.M. (n = 7–10). #, P < 0.001 compared with NS/NC; *, P < 0.05 compared with DMSO; $, P < 0.01, @, P < 0.001 compared with S/C.

Fig. 2

Immunofluoroscence staining for specific β-AR subtypes. Tissue sections of the lung obtained from NS/NC (n = 6), S/C (n = 5) ICI 118,551 (ICI; n = 6), metoprolol 10 mg/kg/d (Met 10; n = 3), and metoprolol 20 mg/kg/d (Met 20; n = 3), were immunostained with β₁-AR (A) and β₂-AR (B) specific antibodies. The fluorescence intensity of the images was localized predominately to the airway epithelium. Tissues incubated only with secondary antibodies (C) were used as negative controls. Original magnification is 20 ×.

Fig. 3

Morphometric analysis to quantify the expression of β-AR subtypes. Measured fluorescence intensity from the airway epithelium of NS/NC and S/C mice, and S/C mice treated with ICI 118,551 (ICI), metoprolol 10 mg/kg/d (Met 10), and metoprolol 20 mg/kg/d (Met 20) (A); tissue sections immunostained with β₁-AR and β₂-AR specific antibodies from the left cardiac ventricle of NS/NC (n = 6) mice (B). Tissues incubated only with secondary antibodies were used as negative controls. Original magnification is 20 ×. Data represents the mean ± S.E.M. (n = 3–6) mice. *, P < 0.05 compared with S/C; #, P < 0.05 compared with NS/NC.

Fig. 4

Immunoblotting of PDE4D, Gα_i3 and PLC-β1. Western blotting of PDE4D in cytosolic fraction (A), Gα_i3 in membranous fraction of lung homogenates (B) and PLC-β1 in trachea and large bronchi homogenate (C). Expression of β-actin was also measured as a loading control. Representative band images are shown in the upper panels. Densitometric analysis of protein of interest/β-actin ratios are shown in the lower panels as mean ± S.E.M. (n = 6–13). Nad Chr (nadolol chronic). *, P < 0.05 compared with S/C; #, P < 0.05 compared with NS/NC.

Fig. 5

Effects of nadolol chronic treatment on isolated hyperresponsive mouse trachea. Cumulative concentration–response curves to cicaprost (A) and CAY 10399 (B) in the mouse isolated trachea after methacholine precontraction. Each point represents mean and vertical lines show S.E.M. (n = 5–6). The methacholine contraction was obtained by the addition of the approximate EC₈₀ methacholine, 3 μM for (A) and 10 μM for (B) methacholine and produced a contraction of 2.436 ± 0.362 g and 2.646 ± 0.451 g, respectively. Nad Chr (nadolol chronic). *, P < 0.05 compared with S/C.