recently described small molecule inhibitor of Gbc signaling [27] (Acros Organics). Measurement of in vivo bronchoconstrictor responsiveness At 24 hr following OVA challenge, under initial general anesthesia with intramuscular injections of xylazine (10 mg/Kg) and ketamine (50 mg/Kg), rabbits were tracheotomized, para- lyzed with pancuronium bromide (0.05 mg/Kg), and mechani- cally ventilated via an intra-tracheal cannula. The cannula was connected to a pneumotachograph to monitor airflow and a pressure transducer to record transthoracic pressure. As previously described [69], the flow and pressure signals were digitized and analyzed to determine breath-to-breath changes in respiratory system resistance (Rrs) and dynamic compliance (Cdyn) elicited by i.v. bolus injections of cumulatively increasing doses of MCh (0.001–0.15 mg/Kg). Lung histology and BALF analysis of inflammation Following assessment of MCh responsiveness, the animals were sacrificed with an overdose of sodium pentobarbital (100 mg/Kg) and, similar to the approach previously described in OVA- senstized rabbits [68] the lungs were lavaged in situ with a total of 25 ml of normal saline, delivered by slowly injecting and aspirating five 5-ml aliquots via a pliable plastic catheter inserted into the intra-tracheal tube. The lungs were then excised and fixed in 10% formalin under a constant pressure of 20 cmH20. The BALF return averaged between 67 and 79% of the total instilled volume. Total and differential cell counts in the BALF samples were assessed in Wright-stained cytospin preparations, with at least 500 cells counted per cytospin preparation. The levels of IL-13 and IFN-c in the BALF supernatants were determined using ELISA kits (R&D Systems). Paraffin-embedded 4 mM sections of lung tissue obtained from the lower lobes of both formalin-fixed lungs were stained with hematoxylin and eosin (H&E) and examined in a blinded manner to assess inflammation. Preparation and ex vivo sensitization of rabbit ASM tissues In separate experiments, na¨ıve NZW rabbits were sacrificed with sodium pentobarbital (100 mg/Kg) and the tracheae were excised, cleaned of loose connective tissue, and the epithelium was removed by gently scraping with a cotton-tipped applicator, as previously described [7,24]. The isolated tracheae were divided into equal ring segments, and each alternate segment was incubated overnight either with vehicle alone or serum isolated from control (non-sensitized) or OVA-sensitized rabbits at 24 hr following OVA inhalation challenge, or with IL-13 (50 ng/ml), both in the absence and presence of pretreatment with the anti- Gbc blocking peptide (20 mM), MPS peptide alone (20 mM), gallein (10 mM), the ERK1/2 inhibitor, U0126 (5 mM), or the PDE4 inhibitor rolipram (10 mM). The tissues were exposed to each of these treatments for ,3 hr prior to incubation with the sera or IL-13 preparations, and the treatments were maintained throughout the ensuing overnight period (,18 hr) leading to the following pharmacodynamic studies. Pharmacodynamic studies of ASM tissue responsiveness Following incubation under the different treatment conditions, the ASM tissue segments were placed in organ baths containing the same concentrations of their respective pharmacological treatments in modified Krebs-Ringer solution aerated with 5% CO2 in O2 and attached to force transducers to monitor isometric G Protein bc-Subunit Signaling in Asthma tension. As previously described [7–10], cholinergic contractility to cumulatively administered acetylcholine (ACh; 1029 to 1023 mol/ L) was assessed and, after rinsing with fresh buffer, relaxation dose- response curves to isoproterenol (1029 to 1024 mol/L) were generated following initial half-maximal contraction of the tissues with ACh. The constrictor and relaxation dose-response curves were analyzed with respect to each tissue’s maximal isometric contractile force (Tmax) to acetylcholine and maximal relaxation response (Rmax) to isoproterenol. Assay of cAMP PDE activity Levels of total cAMP PDE activity were determined, as previously described [10,11], using a colorimetric, non-radioactive enzymatic assay (Biomol), in control and passively sensitized ASM tissues, and in lung tissue sections isolated from the control and OVA-sensitized rabbits under the different treatment conditions. Following the above treatments, ASM and lung tissues sections were immediately frozen and then stored at 280uC. At the subsequent time of analysis, the tissue samples were thawed, then finely minced and homogenized in ice-cold 30 mM N-2-hydro- xyethylpiperazine-N9-ethane sulfonic acid (pH 7.4) containing 0.1% Trion X-100, and PDE activity was standardized to protein content in the tissue samples [10,11]. Culture and treatment of ASM cells HASM cells were grown in smooth muscle basal medium (SmBm) supplemented with 10% FBS (BioWhittaker) and maintained throughout in a humidified incubator containing 5% CO2 in air at 37uC. The experimental protocols involved growing the cells to ,95% confluence in the above medium. Thereafter, in separate experiments, the cells were starved in unsupplemented Ham’s F12 media for 24 hr, treated with different concentrations and for varying durations with IL-13, and then examined for induced changes in c-Src and ERK1/2 phosphorylation and PDE4D mRNA expression in the absence and presence of specific inhibitors, as described. Transfection of ASM cells with adeno-bARK-ct Adenovirus (adeno)-bARK-ct, an adenovirus vector encoding the bARK1 carboxyl-terminal domain which blocks Gbc signaling [14,15], and adeno-b-gal, an adenovirus vector expressing lacZ as a negative control, were constructed using the AdenoX adenovirus construction kit (BD-Clontech). Recombinant plaques were isolated and propagated in HEK293 cells (Invitrogen), with viral purification using the cesium chloride gradient method, and viral titer detected by plaque assay. HASM cells were transfected with either of the adenoviral vectors at a MOI of 100, and experiments were conducted at 24 hr following the adenoviral transfections. Immunoblot analysis of c-Src and ERK1/2 phosphorylation Levels of c-Src and ERK1/2 proteins, as well as phosphorylated c-Src at residue Tyr416 and ERK1/2 proteins, were detected by Western blot analysis of lysates isolated from HASM cells before and at various times after treatment with IL-13 in the absence and presence of specific inhibitors, as described. Following protein extraction and the addition of gel loading buffer, the extracts were loaded on a 10% SDS-PAGE gel for immunoblotting after transfer to a PVDF membrane. The membranes were then incubated overnight with monoclonal mouse anti-human primary antibodies directed against c-Src, phospho-c-SrcTyr416, ERK and phospho- ERK1/2 (Cell Signaling Technology), and levels were detected by ECL after a 1-hr incubation with a 1:2,000 dilution of HRP- PLoS ONE | www.plosone.org 13 February 2012 | Volume 7 | Issue 2 | e32078