Ovalbumin (OVA) treatment increases cholinergic airways responsiveness in BALB/c mice. A: time course of OVA sensitization and challenge. On day 0, mice were injected intraperitoneally (IP) with 1 mg of a sterile, endotoxin-free solution of OVA emulsified in 25% (wt/vol, final) alum suspension or an equivalent amount of alum alone for the PBS control group. On day 11, mice were given a similar IP injection as well as an intranasal (IN) inoculation with 1 mg of OVA dissolved in 50 μl of PBS or PBS alone. Mice were allowed to recover and inoculated again with IN PBS or OVA on days 18, 21, 22, and 23. Mice were studied on day 25. B: PBS-treated (▪) or OVA-treated (○) mice were examined for airways resistance using a positive displacement respirometer and increasing doses of nebulized methacholine in PBS (n = 3, means ± SE; *P < 0.05, 2-way ANOVA).

OVA treatment increases phospho-Ser⁹ GSK-3β (pGSK) immunostaining in the airway epithelium and ASM. A and B: H&E-stained lung sections from PBS- and OVA-treated mice. C–F: lung sections were immunostained with anti-phospho-GSK-3β (green, C and D) or anti-α-actin Cy3 conjugate (red, E and F). Nuclei were stained with Hoechst 33342 (blue, C–H). Images were merged (G and H) to show colocalization of phospho-GSK-3β and α-actin. Colocalization varies in intensity from orange (relatively low pGSK) to yellow (higher pGSK). OVA-treated mice (B, D, F, and H) showed increased airway inflammation (B), phospho-GSK-3β (D), α-actin (F), and colocalization of phospho-GSK-3β and α-actin (H). Magnification is ×320, black bars represent 50 μm, and all panels are to the same scale.

OVA treatment increases whole lung pGSK content and decreases whole lung GSK-3β kinase activity. A: lungs from PBS or OVA-sensitized mice were homogenized, and 20 μg of protein was assessed by immunoblotting for α-actin, β-actin, phospho-Ser⁹, and total GSK-3β. For MHC, 50 μg of protein was processed. In addition, GSK-3β was immunoprecipitated from 1 mg of soluble lung protein, and GSK-3β activity was assessed using recombinant tau as a substrate. Changes in phosphorylation were not due to a difference in the GSK-3β content of immunoprecipitates. B: group mean data for pixel densitometry scans using NIH ImageJ software (n = 3; *P < 0.05, ANOVA).

Glycogen synthase kinase-3β (GSK-3β) inhibition increases cell size and contractile protein synthesis in cultured murine airway smooth muscle (ASM) cells. A: mouse ASM cells from control animals were serum-deprived and treated with 10 ng/ml transforming growth factor (TGF)-β or the GSK-3β inhibitors 10 mM LiCl or 50 nM SB-216763. After 3 days of treatment, samples were processed for Western blots using antibodies to α-actin, myosin heavy chain (MHC), phospho-Ser⁹ GSK-3β (pS9 GSK-3β), total GSK-3β, and β-actin. B: group mean data for immunoblots as quantified by densitometry. Data are represented as fold increase over control. Data from 3 individual experiments are shown (*P < 0.05). C: mouse ASM cells were untreated (black line) or treated with 10 ng/ml TGF-β (red line), 50 nM SB-216763 (blue line), or 10 mM LiCl (green line). Cells were trypsinized, immunostained with anti-α-actin-FITC, and processed for flow cytometry. TGF-β, SB-216763, and LiCl each significantly increased cell size (as measured by forward scatter) and α-actin immunostaining (*P < 0.05, 1-way ANOVA; n = 3). D: fluorescence confocal microscopy. Cells were treated with vehicle, TGF-β, LiCl, or SB-216763 immunostained with rabbit anti-pGSK (green channel, top 3 frames) or anti-NFATc3 (green channel, bottom 3 frames) and anti-α-actin-Cy3 (red channel). Nuclei were counterstained with Hoechst 33342 (blue channel). Merged images are shown. In the cytoplasm, colocalization of pGSK or NFATc3 with α-actin is orange to yellow. Localization of pGSK or NFATc3 in the nucleus is pale blue (without α-actin) or white (with α-actin). Both TGF-β and GSK-3β inhibitors increased cell size, pGSK content, nuclear localization of NFAT, and α-actin expression. The white bar is 100 μm, and all images are to the same scale. sm, Smooth muscle.

OVA treatment increases airway inflammation and ASM α-actin immunostaining. A and B: hematoxylin and eosin (H&E)-stained lung sections from PBS- and OVA-treated mice. Inset shows eosinophilic inflammation. C–F: lung sections were immunostained with anti-α-actin (C and D) or mouse IgG (E and F) and peroxidase-conjugated anti-mouse IgG and subsequently developed with diaminobenzidine and NiCl₂. Sections were counterstained with nuclear fast red. G and H: immunofluorescence staining of PBS- and OVA-treated mouse lungs using an anti-α-actin Cy3 conjugate (red) and rabbit anti-pGSK with goat anti-rabbit IgG-Alexa 488 secondary (green channel) antibodies. Nuclei were stained with Hoechst 33342 (blue). OVA-treated mice (B, D, F, and H) showed increased airway inflammation and α-actin immunostaining (D). Magnification is ×200, and black bars represent 100 μm.

OVA treatment increases the number of pGSK (+) ASM. Nuclear counts of α-actin- and pGSK-staining cells were assessed by stereological analyses using an optical disector probe. Nuclei were visualized with Hoechst 33342 (blue), pGSK immunoreactivity (green), and α-actin (red). Colocalization of pGSK and α-actin appears yellow-orange. Using NIH ImageJ, a grid was overlaid on an entire 30-μm-thick z-series stack, as in this figure, and nuclei from α-actin-positive cells were counted as they came into focus through the stack using the optical disector counting rules. Nuclei touching the bright red corners of the indicated squares were not counted, whereas nuclei touching the green borders were allowed. Consistent with stereological counting rules, no immediately adjacent squares were counted. Magnification, ×400.