Figure 7. Pak inhibitor, IPA3, reduced the contractility of isolated murine tracheal segments to ACh and 5-Ht in vitro. Curves show responses of tracheal segments normalized to maximal response to ACh or 5-Ht prior to incubation with vehicle or IPA3. IPA3 treatment (open circles) significantly inhibited contractile responses to (A) ACh (n = 4) and (B) 5-Ht (n = 4) compared to responses of vehicle- treated tissues (black squares). Values are means 6 SEM, significantly different, * p,0.05. doi:10.1371/journal.pone.0042601.g007 contractility [9,17], whereas other studies indicate that Pak may contribute to the activation of contractile proteins and active tension development [8,12,13,19]. Most of these studies were performed using Triton skinned muscle tissues and exogenous recombinant Pak [5–7,16] or by expressing recombinant Pak in cultured cells [20]. The observations obtained using these preparations may not reflect the function of endogenous Pak in intact smooth muscle tissues under physiologic conditions. The results of our current studies evaluating airway responsiveness under physiologic conditions in vivo and in vitro clearly demonstrate that Pak is a critical intermediate in the contractile activation of airway smooth muscle and that its inhibition reduces airway responsiveness. In conclusion, our findings demonstrate that Pak is an effective molecular target for inhibiting airway smooth muscle contraction and suppressing airway reactivity in vivo. This suggests that Pak inhibition could provide a novel strategy for reducing airway hyperresponsiveness in diseases such as asthma. Materials and Methods Ethics Statement The protocols for these studies were approved by the Indiana University Institutional Animal Care and Use Committee Inhibition of PAK Reduces Airway Responsiveness Figure 8. Pak inhibitor, IPA3, reduced the contractility of isolated human bronchial segments to ACh in vitro. Curves show responses of bronchial rings normalized to maximal ACh response obtained prior to incubation with vehicle or IPA3. IPA3 treatment (open circles) significantly inhibited contractile responses to ACh compared to responses of vehicle-treated bronchi (black squares). Values are means 6 SEM, (n = 3 pairs of bronchi), * p,0.05. doi:10.1371/journal.pone.0042601.g008 (IACUC) and Indiana University Institutional Review Board, which waived the need for written consent for the use of surgical waste material for our research study and the tissue was, and remained de-identified. Animals Pak12/2 mice on a C57BL/6 background were backcrossed 6 generations as previously described [10,15]. Pak12/2 mice and Wild type (WT) C57BL/6 mice were studied at 8 weeks of age. Human Airway Tissues Fresh non-identified human lung tissue from surgical waste was collected within 1–2 hours of removal from the lungs and preserved in physiologic saline solution. Tissues were studied in vitro immediately after they were obtained. In Vivo Pulmonary Measurements WT and Pak12/2 mice were anesthetized with pentobarbital (6 mg/100 gm intra-peritoneal). Following tracheotomy, an 18- gauge cannula was inserted and securely tied with 4.0 braided silk. The animals were then mechanically ventilated with a computer- controlled small-animal ventilator (FlexiVent, SCIREQ, Mon- treal, Quebec) using a tidal volume of 8 mL/kg at a rate of 180 breaths/min and PEEP of 3.5 cm H20. Pulmonary resistance was measured by the computer controlled ventilator by interrupting ventilation and imposing a 2.5 Hz sinusoidal signal and then resuming ventilation. Airway Challenge Following measurement of baseline resistance, animals under- went airway challenge with normal saline and then increasing concentrations of acetylcholine (ACh) or methacholine (MCh) by either aerosol challenge or intravenous challenge. For aerosol challenge, 20 mL of each solution of ACh ranging from 0.3 to 50 mg/ml was nebulized in line with the ventilation circuit over 10 seconds. Resistance was then measured every 30 seconds for the next 4 minutes to record a maximal response for each ACh dose. For intravenous Challenge, increasing volumes (3–100 mL) of acetylcholine (10 mg/ml) were infused through a jugular vein catheter for 20 seconds to deliver Ach doses ranging from 0.03– PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e42601