1.0 mg. Measurements of resistance were obtained every 10 sec- onds for a minute and then the next Ach dose was administered. Evaluation of PAK inhibitor in vivo (IPA3) The effects of the small molecule Pak inhibitor IPA3 [13] on in vivo pulmonary responsiveness was evaluated in WT animals. IPA3 (5 mM) was dissolved in 1% DMSO and 20 ml was nebulized into the ventilator circuit 60 minutes prior to perform- ing the Ach dose response curves. Control animals received nebulized 1% DMSO prior to bronchial challenge. Pressure-volume curves of the lungs The pulmonary elastic properties were evaluated from quasi- static pressure-volume curves obtained in vivo. Mechanical ventilation of the anesthetized animals was interrupted and the pulmonary system was slowly cycled between the end-expiratory pressure of 2.5 cm H2O and a maximal airway pressure of 30 cmH2O. Measurement of Contractility of Isolated Murine Tracheal Segments and Human Bronchial Rings In Vitro Pak12/2 and WT mice were euthanized with pentobarbital (12 mg/100 g intra-peritoneal) and intact tracheal segments were removed and dissected free of the epithelium. The isolated tracheal segments were mounted on wires and attached to Grass force transducers in tissue baths for the measurement of isometric force in response to increasing doses of ACh. In some experiments, tracheas were isolated from WT mice to evaluate the effect of Pak inhibition on the contractile responses of airway tissues. Dose-response curves to ACh (1029 to 1023 M) or 5-hydroxytryptamine (5-Ht) (1029 to 1024 M) were performed after 30 min incubation in physiologic saline. After washing the agonists from the tissues, one of each pair of the tracheal segments was treated with 100 mM IPA3 dissolved in 1% DMSO for 2 hours and the other segment was treated with 1% DMSO alone. A dose-response curve to ACh or 5-HT was then repeated on each pair of tissues concurrently. Human bronchial rings (5 mm diameter, 2 mm wide) were dissected from lung specimens and cleaned of connective tissue. Rings were attached to force transducers as described above and equilibrated in a tissue bath for 30–60 min. Two rings were obtained from each lung specimen and studied concurrently. Dose-response curves to ACh (1029 to 1023 M) were performed after 30 min incubation in physiologic saline. After washing ACh References 1. Dechert MA, Holder JM, Gerthoffer WT (2001) p21-activated kinase 1 participates in tracheal smooth muscle cell migration by signaling to p38 Mapk. American Journal of Physiology - Cell Physiology 281: C123–C132. 2. Knaus UG, Bokoch GM (1998) The p21Rac/Cdc42-activated kinases (PAKs). International Journal of Biochemistry & Cell Biology 30: 857–862. 3. Pacheco A, Chernoff J (2010) Group I p21-activated kinases: emerging roles in immune function and viral pathogenesis. International Journal of Biochemistry & Cell Biology 42: 13–16. 4. Sells MA, Knaus UG, Bagrodia S, Ambrose DM, Bokoch GM, et al. (1997) Human p21-activated kinase (Pak1) regulates actin organization in mammalian cells. Current Biology7(3):202–10. 5. Zhao Z-s, Manser E (2005) PAK and other Rho-associated kinases–effectors with surprisingly diverse mechanisms of regulation. Biochemical Journal 386: 201–214. 6. McFawn PK, Shen L, Vincent SG, Mak A, Van Eyk JE, et al. (2003) Calcium- independent contraction and sensitization of airway smooth muscle by p21- activated protein kinase. American Journal of Physiology - Lung Cellular & Molecular Physiology 284: L863–L870. 7. Van Eyk JE, Arrell DK, Foster DB, Strauss JD, Heinonen TY, et al. (1998) Different molecular mechanisms for Rho family GTPase-dependent, Ca2+- independent contraction of smooth muscle. Journal of Biological Chemistry 273: 23433–23439. Inhibition of PAK Reduces Airway Responsiveness from the tissue, one of the bronchial rings was treated with 100 mM IPA3 dissolved in 1% DMSO for 2 hours and the other ring was treated with 1% DMSO alone. A dose-response curve to ACh was then repeated on each tissue concurrently. Tissues from each group were quick frozen in liquid nitrogen for biochemical analysis to confirm the inhibition of Pak activation by IPA3. Analysis of protein expression and phosphorylation Proteins were extracted from pulverized murine tracheal smooth muscle, whole murine tracheal segments or human bronchial rings. Tissue extracts were then boiled in sample buffer (1.5% dithiothreitol, 2% SDS, 80 mM Tris-HCl (pH 6.8), 10% glycerol and 0.01% Bromophenol Blue) for 5 min, separated using SDS polyacrylamide gel electrophoresis (SDS-PAGE), and trans- ferred to nitrocellulose. Membranes were probed with antibodies followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (Ig) (Jackson, Immunoresearch Co.). Proteins were visualized by enhanced chemiluminescence (ECL). Antibodies against Pak1, Pak2, Pak3 and Pak Thr423 were obtained from Cell Signaling. Lung and Airway Histology Lung were excised and fixed with 10% formalin at a distending pressure of 20 cm H2O, sectioned into blocks, embedded in paraffin, and a 5-mm-thick section were stained with Masson’s Trichrome. Tissues were visualized by light microscopy and images captured with a digital camera (SPOT, Diagnostic Instruments, Inc, MI), and morphometric measurements from the fixed tissues were obtained from the digital images using imaging software (Metamorph, version 5.01r, Universal Imaging). For airways, the internal perimeters were calculated from the luminal border of the airway epithelium. The cross-sectional areas of the airway wall, airway smooth muscle and epithelium were determined by point counting, as previously described [21]. For the parenchyma, alveolar size was quantified by measuring the mean linear intercept, which is the number of alveolar walls intersecting a line of known length [22,23]. Author Contributions Conceived and designed the experiments: WCH JC DWC SJG RST. Performed the experiments: WCH WZ ZX HG. Analyzed the data: WCH WZ ZX HG SJG RST. Contributed reagents/materials/analysis tools: JC DWC. Wrote the paper: WCH SJG RST. 8. Wang R, Li QF, Anfinogenova Y, Tang DD (2007) Dissociation of Crk- associated substrate from the vimentin network is regulated by p21-activated kinase on ACh activation of airway smooth muscle. American Journal of Physiology - Lung Cellular & Molecular Physiology292(1):L240–8. 9. Bokoch GM (2003) Biology of the p21-activated kinases. Annual Review of Biochemistry 72: 743–781. 10. Molli PR, Li DQ, Murray BW, Rayala SK, Kumar R (2009) PAK signaling in oncogenesis. Oncogene 28: 2545–2555. 11. Wirth A, Schroeter M, Kock-Hauser C, Manser E, Chalovich JM, et al. (2003) Inhibition of contraction and myosin light chain phosphorylation in guinea-pig smooth muscle by p21-activated kinase 1. Journal of Physiology 549: 2–500. 12. Allen JD, Jaffer ZM, Park S-J, Burgin S, Hofmann C, et al. (2009) p21-activated kinase regulates mast cell degranulation via effects on calcium mobilization and cytoskeletal dynamics. Blood 113: 2695–2705. 13. Deacon SW, Beeser A, Fukui JA, Rennefahrt UEE, Myers C, et al. (2008) An isoform-selective, small-molecule inhibitor targets the autoregulatory mechanism of p21-activated kinase. Chemistry & Biology 15: 322–331. 14. Yi C, Maksimoska J, Marmorstein R, Kissil JL (2010) Development of small- molecule inhibitors of the group I p21-activated kinases, emerging therapeutic targets in cancer. Biochemical Pharmacology 80: 683–689. PLOS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e42601