Figure 5. Bronchoconstriction induces the release of biologically active TGF-β leading to contractile protein expression.

Human MRC-5 fibroblasts were stimulated for 1 hour with TGF-β₁ (2 ng/mL), methacholine (MCh; 10 µM) or medium (basal), or with conditioned media obtained from lung slice cultures treated for 2 days with and without 10 µM methacholine. MRC-5 cell lysates were analysed for phosphorylated (ser 423/425) and total Smad-3. Representative blots and quantified data of Smad-3 phosphorylation in response to conditioned media are shown in (A). Data shown are the means ± SE of 4 independent experiments. ^*: p<0.05, compared to basal (paired Student's t-test with two-tailed distribution). Lung slices were pre-treated with latrunculin A (0.3 µM), SB431542 (0.3 µM), or medium (basal) for 30 min, followed by 2 days of treatment with methacholine (MCh; 10 µM), TGF-β₁ (2 ng/mL), or medium (basal) (B). Lung slice lysates were analysed for the presence of sm-myosin, using ß-actin as a loading control. Blots shown are representative of 3 experiments. Human MRC-5 fibroblasts were stimulated for 1 hour with conditioned media obtained from lung slice cultures after treatment with methacholine (MCh; 10 µM), TGF-β (2 ng/mL) or medium (basal), in the absence and presence of latrunculin A (0.3 µM) or SB431542 (0.3 µM) (C). MRC-5 cell lysates were analysed for phosphorylated (ser 423/425) and total Smad-3. Blots shown are representative of 3 experiments. Lung slices were pre-incubated with latrunculin A (0.3 µM), SB431542 (0.3 µM), or medium (basal) for 30 min, followed by 2 days stimulation with methacholine (MCh; 10 µM), TGF-β₁ (2 ng/mL), histamine (His, 1 µM), KCl (K⁺, 60 mM) or medium (basal) (D). Lung slice lysates were analysed for sm-myosin, using β-actin as a loading control. Blots shown are representative of 3 experiments.