<i>In vivo</i> airway reactivity of Pak1<i>^−/−</i> mice was lower than that of WT mice to aerosolized (A) and intra-venous (B) acetylcholine.

<p>Resistance in response to increasing concentrations of aerosolized acetylcholine (ACh) for wild-type (WT; N = 8) and Pak1<i>^−/−</i> mice (N = 8) (A); values are means ± SEM. There was no difference in resistance at baseline. When analyzed by repeated measures ANOVA, resistance increased with increasing ACh dose (p<0.0001), Pak1<i>^−/−</i> mice had a significantly smaller slope of the dose response curve (p<0.03), and a significantly smaller increase in resistance compared to WT mice (p<0.03). Post-hoc analysis demonstrated Pak 1<i>^−/−</i> compared to WT mice had significantly smaller resistances at all ACh concentrations ≥7.5 mg/ml (p<0.05). Resistance in response to increasing concentrations of intravenous acetylcholine (ACh) for wild-type (WT; N = 4) and Pak1<i>^−/−</i> mice (N = 4) (B); values are means ± SEM. There was no difference in resistance at baseline. When analyzed by repeated measures ANOVA, resistance for Pak1<i>^−/−</i> mice increased less with increasing ACh dose (p<0.0001) compared to WT mice. Post-hoc analysis demonstrated Pak1<i>^−/−</i> compared to WT mice had significantly lower resistances at ACh concentrations ≥0.42 mg (p<0.05).</p

Comparison of the Lung Parenchyma (N = 3–5 in each group) for WT (black) and Pak1^−/− (grey) mice.

<p>There were no significant differences for lung volumes at 30 cmH₂O (p>0.37) (A); pressure volume curves normalized to volume at 30 cmH₂O (B); or Alveolar Mean Linear Intercepts (MLI) (p>0.40) (C).</p

Comparison of Lung Histology for WT and <i>Pak1</i>^−/− mice.

<p>Airway wall area (A), airway smooth muscle area (B), and airway epithelium area (C) were not significantly different between the WT (black squares) and <i>Pak1</i>^−/− (grey circles) mice when assessed by ANOVA adjusting for airway perimeter (p>0.15). (N = 5 mice in each group).</p

Aerosolized IPA3 inhibited airway contractility <i>in vivo</i> and suppressed Pak activation.

<p>When assessed by repeated ANOVA, resistance increased with increasing ACh dose (p<0.0001), and IPA3 dissolved in 1% DMSO (N = 3) and aerosolized 1-hour prior to bronchial challenge of WT mice significantly reduced the slope of the increase in resistance (p<0.0001), as well as the magnitude of the increase in resistance compared to control vehicle (1%DMSO; N = 5) (p<0.001) (A). Post-hoc analysis indicated that IPA3 treatment resulted in lower resistances at MCh doses ≥33 mg/ml (p<0.05). Tracheal smooth muscle isolated from WT mice treated <i>in vivo</i> with IPA3 demonstrated significantly lower Pak activation as assessed by Pak T423 phosphorylation following stimulation with ACh compared to airway smooth muscle isolated from WT mice (B). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042601#s2" target="_blank">Results</a> represent 2 samples of tracheal smooth muscle from each group. Each sample consisted of pooled tracheal muscle tissues from 3 separate mice with the same treatment.</p

Pak1, Pak2 and Pak3 isoforms were detected in WT murine tracheal smooth muscle by immunoblot.

<p>No Pak1 was detected in extracts of isolated tracheal smooth muscle (A) or whole tracheas (B) from <i>Pak1</i>^−/− mice. Immunoblots of tracheal smooth muscle were obtained from tracheal smooth muscle extracts pooled from 3 mice of each type. Whole trachea extracts were each from a single mouse.</p

Tracheas isolated from <i>Pak1</i>^−/− mice showed reduced contractility to ACh in vitro.

<p>Isometric force generation (% of maximal force to ACh stimulation in WT mice) was significantly lower in tracheas isolated from <i>Pak1</i>^−/− (grey squares) mice compared to WT (black diamonds) mice (N = 10 or 11 in each group, p<0.01).</p