Inhibition of PAK Reduces Airway Responsiveness Figure 3. Aerosolized IPA3 inhibited airway contractility in vivo and suppressed Pak activation. 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 in vivo 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). Results 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. doi:10.1371/journal.pone.0042601.g003 PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e42601 Figure 4. Tracheas isolated from Pak12/2 mice showed reduced contractility to ACh in vitro. Isometric force generation (% of maximal force to ACh stimulation in WT mice) was significantly lower in tracheas isolated from Pak12/2 (grey squares) mice compared to WT (black diamonds) mice (N = 10 or 11 in each group, p,0.01). doi:10.1371/journal.pone.0042601.g004 We showed that the synthetic Pak inhibitor, IPA3, was effective at inhibiting ACh-induced Pak activation in airway tissues both in vivo and in vitro. We also showed that IPA3 inhibits the contractile response of airway tissues to 5-Ht in vitro, indicating that the effects of IPA3 are not agonist-specific. IPA3 is an allosteric Pak inhibitor that binds outside the ATP-binding pocket and specifically inhibits the auto-activation of Pak isoforms 1, 2 and 3 [13,14]. Treatment of WT mice with IPA3 inhibited airway responsiveness in vivo and in vitro similarly to that observed in the Pak12/2 mice. These results provide further support for our conclusion that the reduced airway responsiveness in Pak12/2 mice results from the loss of Pak1 activation in airway smooth muscle in response to agonist stimulation, rather than from an unidentified developmental defect caused by the absence of Pak1. IPA3 also inhibited the contractility of isolated human bronchial tissues to ACh, confirming that this inhibitor is also effective in human airway smooth muscle tissue. This is the first study to demonstrate a role for Pak1 in the regulation of airway responsiveness in vivo. Previous studies that have evaluated the role of Pak in the regulation of smooth muscle contractile proteins have yielded inconsistent findings. Some of these studies have shown an inhibitory effect of Pak on contractile protein activation, suggesting that it would inhibit smooth muscle