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Pak 1 (p21-activated kinase) is involved in the regulation of airway smooth muscle (ASM) contraction in vitro. We hypothesized that the genetic disruption of Pak1 would attenuate in vivo airway responsiveness to acetylcholine (ACh) in non-sensitized mice. Male C57B/6 wild type (WT) and Pak1 knockout (KO) mice were anesthetized, tracheostomized, and ventilated with a computer-controlled small-animal mechanical ventilator (flexiVent, SCIREQ, Montreal, Quebec) with tidal volume of 10 mL/kg, PEEP of 4 cmH 2 O, and rate of 150-170 breaths per minute. Resistance was measured with a 2.5 Hz, 1-second sine wave oscillation. Following determination of baseline resistance, measurements were repeated every 30 seconds for 5 minutes following each aerosol of ACh (0.3, 1, 3, 5, 7, 10, 33, and 50 mg/mL) delivered via an in-line nebulizer. Baseline airway resistance was similar for WT and Pak1 KO mice (0.595 vs. 0.594 cmH 2 O.s/mL; pϭ 0.75). Median airway resistance achieved statistical significance at 7 mg/mL (1.52 vs. 0.85 cmH 2 O.s/mL; p ϭ 0.032). The mean maximal resistance following ACh challenge was greater for WT compared to Pak1 KO mice (3.4 vs. 1.6 cmH 2 O.s/mL; p ϭ 0.0005). Isolated tracheas from WT and Pak1 KO mice were then excised, mounted securely to steel cannulas on each end and placed in a tissue bath containing physiologic saline solution (PSS) at 37C (flexiVent IAM, SCIREQ, Montreal, Quebec). The lumen of the airway and tubing connected to a pressure transducer were filled with PSS. A computer controlled syringe was used to raise the initial transmural pressure to 4.5 cm H 2 0. Pressure was recorded during isovolumetric contractions via increasing doses of acetylcholine (10 -8 M -10 -4 M). Mean pressure generation by the isolated tracheas was greater for WT compared to Pak1 KO at the first and second doses (10 -8 M: 8.45 vs. 4.425 cmH 2 O, pϭ 0.0136; 10 -7 M: 8.73 vs. 4.9 cmH 2 O, pϭ 0.031). There was no difference in maximal pressure generation for WT and Pak1 KO tracheas (10 -4 M: 11.01 vs. 11.12 cmH 2 O; pϭ 0.92), indicating a decreased sensitivity to acetylcholine mediated constriction of airway smooth muscle in Pak1 KO mice. We conclude that Pak1 is an important determinant of airway responsiveness in vivo and in vitro. Furthermore, these findings are a direct effect of decreased sensitivity by Pak1 KO mouse airway smooth muscle to acetylcholine. Background: During pregnancy, neurofibromas often enlarge or develop for the first time in females with neurofibromatosis type 1 (NF1). Lysophosphatidic acid (LPA) is a prototypic lysophospholipid that has been implicated in tumor progression. LPA modulates cell migration and survival of Schwann cells (SCs), and interestingly, LPA is made in increasing concentrations throughout pregnancy. SCs are the tumorigenic cells in the development of neurofibromas in NF1. Given the temporal nature of LPA production and neurofibroma formation during pregnancy, we hypothesized that LPA may be a candidate molecule that promotes Schwann cell (SC) migration and survival and pote...