ATP released from air passage epithelial cells promotes purinergic receptor-regulated mucociliary clearance activities necessary for innate lung defense. tracheas and dye uptake in main tracheal epithelial cells were impaired in pannexin 1 knockout mice. Hypotonicity-promoted ATP release and dye uptake in main well differentiated human bronchial epithelial cells was accompanied by RhoA activation and myosin light chain phosphorylation and was reduced by the RhoA dominating unfavorable mutant RhoA(T19N) and Rho and myosin light chain kinase inhibitors. ATP release and Rho activation were reduced by highly selective inhibitors of transient receptor potential vanilloid 4 (TRPV4). Lastly, knocking down TRPV4 impaired hypotonicity-evoked air passage epithelial ATP release. Our data suggest that TRPV4 and Rho transduce cell membrane stretch/strain into pannexin 1-mediated ATP release in air passage epithelia. (30) reported L-Glutamine that ATP release from hypotonically swollen main cultures of human bronchial epithelial (HBE) cells was nearly 60% inhibited by pannexin channel blockers or by knocking down pannexin 1 via shRNA. Thus, pannexin 1 is usually a candidate ATP release pathway in hypotonically swollen HBE cells. However, regulatory signaling elements transducing hypotonic/mechanical stress into ATP release have not been recognized. Moreover, the contribution of pannexin 1 to the physiological release of ATP from native airways is usually not known. We recently discovered that activation of lung epithelial cell G protein-coupled protease-activated receptors (PAR) resulted in enhanced release of ATP and uptake of propidium iodide in a Rho-dependent manner (31), suggesting a link between Rho activation and the opening of a propidium iodide-permeable plasma membrane channel. In the present study, we tested the hypothesis that ATP release from hypotonically stimulated air passage epithelial cells entails Rho-regulated opening of pannexin 1 channels and used a pannexin 1 knockout mouse model to assess the contribution of pannexin 1 to the release of ATP from a physiologically relevant air passage tissue, excised tracheas. EXPERIMENTAL PROCEDURES Reagents 2-Phenyl-1,2-benzisoselenazol-3(2H)-one (ebselen), ,-methylene ATP, carbenoxolone, flufenamic acid, propidium iodide, luciferase from 1 by attachment of a strong splice acceptor site between exons 1 and 2 (axis of WD-HBE and MTE cell cultures confirmed that nuclei labeled with propidium iodide were localized in the most lumenal cell layer of the cultures. In main (multilayered) cultures, total nuclei were quantified from the differential interference contrast images. In A549 cells (monolayers), total nuclei were assessed either from the differential interference contrast images or by quantifying propidium iodide staining after permeabilizing the cells with 0.05% Triton X-100. Both methods yield comparable results. RT-PCR Analysis Total RNA was prepared using the RNeasy Mini Kit (Qiagen, Inc., Valencia, CA) and reverse-transcribed using SuperScript III reverse transcriptase (Invitrogen). Standard RT-PCR was performed as explained (31), except that 45 thermocycles (rather TIMP2 than 35) were used L-Glutamine to amplify Panx2 and Panx3 in WD-HBE cells. Amplified products were sequenced at the UNC Genome Analysis. Semi-quantitative PCR was performed in a LightCycler PCR machineR thermal cycler, as explained previously (31). Pannexin primers are explained in supplemental Table 1. Connexin primers were as follows: forward, 5-GGGTTAAGGGAAAGAGCGACC-3 and reverse, 5-CCCCATTCGATTTTGTTCTGC-3. siRNA Oligonucleotides targeting human pannexin 1 (siRNA-70) and its scrambled control (supplemental Table 2) were purchased from Dharmacon, Inc. A549 cells were transfected with 1 g of oligonucleotide using the Amaxa Nucleofector Devicetm and Cell Collection Nucleofector? Kit T (Amaxa Biosystems, Gaithersburg, MD), following the manufacturer’s instructions. Transfected cells were cultured in serum-supplemented DMEM for 48 h prior to assays. shRNA Lentiviral vector manifestation clones (pLKO1/puromycin) made up of shRNAs (supplemental Table 2) were obtained from the Lenti-shRNA core facility of the UNC. Cells were infected with the desired lentivirus (106cfu/35-mm dish) and subsequently selected with 0.5 g/ml puromycin. Cells were used within five passages post-infection. Overexpression of RhoA(T19N) A549 cells were transfected with vacant pcDNA3.1 vector or vector containing RhoA(T19N) insert using the Amaxa L-Glutamine Nucleofector Devicetm and used 48 h post-transfection, as described (31). RhoA Pull-down Assay and MLC Phosphorylation Measurements of GTP-bound RhoA were performed using the Rho activation assay biochem kit (Rhoketing pull-down assay), as explained previously (31). Duplicated membranes were separately blotted with anti-phospho-MLC(Ser-19) antibody or anti-MLC antibodies (Cell Signaling Technology, Inc., Danvers, MA), and immunoblots were revealed and quantified as explained (31). To minimize autocrine opinions via ATP release, hypotonic stress-promoted Rho activation and MLC phosphorylation were assessed in the presence of 5 models/ml apyrase (31). Cell Volume Rules Changes in cell height were assessed to estimate cell volume changes, as explained previously (21). In brief, WD-HBE cells were loaded with 5 m calcein-acetoxymethyl ester (Was) (Molecular Probes, Eugene, OR) for 30 min at 37 C. The apical surface of cultures was equilibrated for 10 min with HBSS+, and the.