Results Expression of Pak1 isoform is inhibited in airway tissues from Pak12/2 mice We evaluated the expression of Pak1, Pak2 and Pak3 in WT and Pak12/2 murine tracheas and isolated murine airway smooth muscle by immunoblot. Pak1 was detected in extracts from isolated tracheal smooth muscle tissues and whole tracheas from WT mice, but not in Pak12/2 mice (Figure 1A, B). Pak2 and Pak3 isoforms were also expressed at similar levels in tracheal smooth muscle from both WT and Pak12/2 mice, indicating that the expression of Pak2 and Pak3 were not altered in the Pak1 knockout mice (Figure 1A). Genetic Deletion of Pak1 suppresses Pulmonary Responsiveness in vivo We evaluated the role of Pak1 in the regulation of changes in pulmonary resistance in response to ACh in vivo by comparing responses of WT and Pak12/2 mice. Dose-response curves depicting increases in pulmonary resistance above baseline for increasing concentrations of inhaled ACh are illustrated in Figure 2A. There were no significant differences in the baseline resistances for either group. However, aerosolized ACh elicited significantly smaller increases in pulmonary resistance in Pak12/2 mice compared to WT mice (P,0.05). We also evaluated the effect of the route of agonist delivery upon airway responsiveness by performing dose-response curves in response to intravenous challenge with ACh (Figure 2B). Again, there were no significant differences for baseline resistances in animals challenged with intravenous ACh, and ACh elicited significantly smaller increases in pulmonary resistance in Pak12/2 mice compared to WT mice (P,0.05). The fact that the similar results were obtained using intravenous versus aerosolized ACh indicates the effect of Pak deletion on airway reactivity cannot be Figure 1. Pak1, Pak2 and Pak3 isoforms were detected in WT murine tracheal smooth muscle by immunoblot. No Pak1 was detected in extracts of isolated tracheal smooth muscle (A) or whole tracheas (B) from Pak12/2 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. doi:10.1371/journal.pone.0042601.g001 Inhibition of PAK Reduces Airway Responsiveness attributed to alterations in the epithelial barrier function in Pak12/2 mice. Pharmacologic inhibition of Pak suppresses Pulmonary Responsiveness In Vivo We evaluated the effect of a small molecule synthetic Pak inhibitor (IPA3) on pulmonary reactivity to ACh in WT mice in vivo. IPA3 allosterically inhibits the catalytic activity of Pak1, 2 and 3 isoforms [11,12]. Pak inhibitor IPA3 (20 ml of 5 mM, 1%DMSO) or Vehicle (1% DMSO) was nebulized into the ventilator circuit 60 minutes prior to bronchial challenge with aerosolized ACh. The respiratory response to inhaled ACh of WT mice receiving IPA3 was significantly decreased compared to that of WT mice that received Vehicle alone (Figure 3A). In separate series of experiments, tracheas were isolated from WT animals that had received nebulized IPA3 in vivo to confirm the inhibition of Pak activation by IPA3. Phosphorylation of Thr 423 in the Pak catalytic domain occurs with its activation and is critical for its full catalytic function toward exogenous substrates [15,16]. Following removal of the epithelium from isolated tracheas, immunoblots were performed on tracheal extracts to measure the phosphorylation of Pak on tyrosine 423. ACh elicited a smaller increase in Pak phosphorylation at Thr 423 in tracheal smooth muscle from IPA3-treated animals than in tracheas from WT animals, indicating that the dose of aerosolized IPA3 was effective at inhibiting Pak activation in vivo (Figure 3B). Genetic Deletion of Pak1 Suppresses the Contractility of isolated Tracheal segments in vitro We evaluated the in vitro contractility of tracheal segments isolated from Pak12/2 mice. Tracheal segments from WT and Pak12/2 mice were isolated, the epithelium removed, and the segments suspended in organ chambers for the measurement of isometric contractile force in response to increasing concentrations of ACh. Tracheal segments from Pak12/2 mice developed significantly lower forces in response to ACh compared to tracheas from WT mice (P,0.05) (Figure 4). Genetic Deletion of Pak1 does not affect lung parenchymal structure or function The effect of Pak1 deletion on the elastic properties of the lungs was evaluated by comparing pressure-volume curves obtained in WT and Pak12/2 animals in vivo. Mechanical ventilation was interrupted and the pulmonary system was slowly cycled between the end-expiratory pressure of 2.5 cm H2O and an airway pressure of 30 cm H2O. There were no significant differences in lung volume at 30 cm H2O between WT and Pak12/2 mice. There were also no differences in the pressure volume curves of the WT and Pak12/2 animals (Figures 5A, B). Alveolar size was compared in lungs from WT and Pak12/2 animals by measuring mean linear intercepts from histological sections taken from lungs that had been formalin fixed at 30 cm H2O. There were no significant differences in mean linear intercept for WT and Pak12/2 animals, indicating no differences in alveolar size (Figure 5C). Genetic deletion of Pak1 does not affect airway structure The effects of Pak1 deletion on the structure of the airways were evaluated by morphometric assessments of histologic sections of lungs from WT and Pak12/2 mice. There were no significant differences in the area of the airway wall or the areas of smooth muscle and epithelium (Figure 6). PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e42601