Inhibition of PAK Reduces Airway Responsiveness Figure 2. In vivo airway reactivity of Pak12/2 mice was lower than that of WT mice to aerosolized (A) and intra-venous (B) acetylcholine. Resistance in response to increasing concentrations of aerosolized acetylcholine (ACh) for wild-type (WT; N = 8) and Pak12/2 mice (N = 8) (A); values are means 6 SEM. There was no difference in resistance at baseline. When analyzed by repeated measures ANOVA, resistance increased with increasing ACh dose (p,0.0001), Pak12/2 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 12/2 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 Pak12/2 mice (N = 4) (B); values are means 6 SEM. There was no difference in resistance at baseline. When analyzed by repeated measures ANOVA, resistance for Pak12/2 mice increased less with increasing ACh dose (p,0.0001) compared to WT mice. Post-hoc analysis demonstrated Pak12/2 compared to WT mice had significantly lower resistances at ACh concentrations $0.42 mg (p,0.05). doi:10.1371/journal.pone.0042601.g002 Pharmacologic inhibition of Pak inhibits Responsiveness of Murine Tracheal Segments in vitro The effect of IPA3 on the contractility of murine tracheal segments to ACh was evaluated by concurrently stimulating pairs of tracheas with cumulative concentrations of acetylcholine (ACh) (1029 to 1023 M) or 5-hydroxytryptamine (5-Ht) (1029 to 1024) and then measuring the isometric contractile force at each concentration. The ACh or 5-Ht was then washed out and one trachea from each pair was treated with 100 mM IPA3 to inhibit PAK. The other trachea was treated with vehicle (1% DMSO). Tracheal segments were incubated in IPA3 or vehicle for 2 hours, after which a dose responsive curve to ACh or 5-Ht was repeated. The contractile responses to ACh and 5-Ht were significantly lower in tracheas treated with IPA3 compared to tracheas treated with vehicle (Figure 7). Pharmacologic inhibition of Pak inhibits Responsiveness of Human Bronchial Rings in vitro The effect of IPA3 on the contractility of human bronchial rings to ACh was evaluated by concurrently stimulating pairs of bronchial rings with cumulative concentrations of acetylcholine (ACh) (1029 to 1023 M) as described above. The contractile responses to ACh were significantly lower in bronchi treated with IPA3 compared to bronchi treated with vehicle (Figure 8). The inhibition of PAK activation by IPA3 was confirmed by western blot for PAK T423 phosphorylation (data not shown). Discussion Our results demonstrate that inhibiting the activity of p21- activated protein kinase is an effective approach for reducing airway responsiveness in vivo. Mice with a genetic deletion of Pak1 exhibited significantly lower airway reactivity to acetylcholine in vivo, and the aerosol administration of a synthetic small molecule Pak inhibitor also reduced airway responsiveness in mice in vivo. Furthermore, isolated tracheal segments from Pak12/2 mice exhibited reduced contractility to ACh in vitro, indicating that the decrease in airway reactivity observed in vivo in Pak12/2 mice reflected a decrease in force generation by the airway smooth muscle. As no differences in the structure of the airways and lung parenchyma were detected in Pak12/2 and WT animals, decreased airway responsiveness did not appear to result from other factors that can alter airway responsiveness. Taken in sum, our observations provide strong evidence that the reduction of Pak activity lowers airway responsiveness in vivo, and that this effect is caused by a reduction in the contractility of the airway smooth muscle. The fact that Pak inhibition was also effective in reducing airway smooth muscle responsiveness in human bronchial tissues, which suggests that this approach could be effective in humans in vivo also. We could not detect any differences in the structure or physical properties of the lungs from Pak12/2 mice as compared to WT mice. The quantity of airway smooth muscle within the airway wall was the same in the WT and Pak12/2 mice and airway morphometry was similar. There were also no differences between WT and Pak12/2 mice in lung parenchymal structure or function. These results suggest the lower level of airway reactivity in Pak12/ 2 mice are unlikely to be due to differences in lung structure. Previous studies that have evaluated the functions of other organ systems in Pak12/2 mice have also not detected major develop- mental abnormalities or organ defects [12,17,18]. Taken in sum, our findings in the Pak12/2 mice provide strong evidence that their decreased airway responsiveness in vivo results from the absence of Pak1 in their airway smooth muscle tissue, and that this reflects a critical role for Pak1 in the contractile activation of airway smooth muscle. PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e42601