Fig. 5. Loss of β2AR in response to RSV infection.
(A) Cultured HASMCs were treated with sterile control medium or were infected with rrRSV at an MOI of 1. Twenty-four hours later, isolated plasma membranes were subjected to [125]I-CYP (cyanopindolol, a β2AR agonist) β2AR binding at a saturation concentration of 250 pM. A Wilcoxon matched-pair signed-rank test was performed, with P values calculated by two-tailed test. *P < 0.05; N = 3 experiments. CPM, counts per minute. (B) pβ2AR-reactive protein expression was analyzed by Western blotting in HASMCs pretreated with vehicle, 100 nM MG-132, or 2.5 μM batimastat (Bat) before being treated with sterile medium or being infected with rrRSV at an MOI of 1 for 24 hours. Top: The cells were then analyzed by Western blotting with antibody against pβ2AR. Actin was used as a loading control. Bottom: The bar graph shows the intensity of the band corresponding to the 35-kDa anti-pβ2AR–reactive band normalized to that of the actin band. Results from five individual blots are presented as box-and-whisker plots. Data are means ± SEM and were analyzed by Kruskal-Wallis test with Dunn’s correction for multiple comparisons. P values were calculated by two-tailed test. **P < 0.01 and ****P < 0.0001 compared to the RSV only treatment. (C and D) Cultured HASMCs were pretreated with the indicated concentrations of propranolol with or without albuterol (Alb), as indicated, for 1 hour before being incubated with sterile control medium (C), UVrrRSV, or rrRSV (MOI of 1) for 24 hours. Top: The cells were lysed and analyzed by Western blotting with antibodies against pβ2AR (C) and RSV-G protein (D). Actin was used as a loading control. Bottom: The graphs show the relative band intensities, normalized to that of actin, for pβ2AR (C) and RSV-G protein (D). Results from three individual blots are presented as box-and-whisker plots (N = 3). Data are means ± SEM and were analyzed by Kruskal-Wallis test with Dunn’s correction for multiple comparisons. P values were calculated by two-tailed test. *P < 0.05 and **P < 0.01 compared to the RSV only treatment. (E) HASMCs embedded in a 1.5% collagen matrix were incubated with sterile medium or rrRSV for 48 hours before being subjected to a 30-min treatment with vehicle or 200 μM albuterol followed by 200 μM methacholine. Images of the collagen gels were taken at 10-min intervals for 30 min. Data are from three independent experiments and are expressed as the percentage change in gel contraction from time zero. Data are means ± SEM and were analyzed by one-way analysis of variance (ANOVA) using Bonferroni’s multiple comparison test. *P < 0.05 and ***P < 0.001 compared to the methacholine treatment only. The black horizontal bars in the Western blots indicate that the blots are not contiguous.