The mucociliary clearance (MCC) process that removes foreign particles and pathogens is the primary innate defense mechanism in the airways. Major components of MCC, i.e., ion transport, mucin secretion, and ciliary beat frequency, are regulated by extracellular ATP and adenosine, acting on cell surface purinergic receptors. Given the physiological importance of purinergic regulation of MCC activities, the objective of this dissertation was to elucidate signaling elements and pathways relevant for ATP release from airway epithelial cells. The protease activated receptor (PAR) agonist thrombin elicited a rapid Ca2+-dependent release of ATP. In contrast, the P2Y2 receptor agonist UTP caused negligible ATP release, despite promoting a robust Ca2+ response. Thrombin-elicited ATP release was associated with Rho activation, was accompanied by enhanced cellular uptake of the hemichannel fluorescence probe propidium iodide in a Ca2+ - and Rho kinase-dependent manner, and was inhibited by connexin/pannexin hemichannel blockers. These studies suggested that thrombin promotes ATP release from airway epithelial cells via Rho- and Ca2+-dependent activation of connexin/pannexin hemichannels. Similarly to thrombin, hypotonic challenge triggered ATP release, which was accompanied by RhoA activation, MLC phosphorylation, and dye uptake. ATP release and dye uptake in hypotonic challenge-stimulated cells were inhibited by transfecting cells with a dominant negative mutant of RhoA, and by inhibiting or knocking-down pannexin 1. Transient receptor potential 4 (TRPV4) inhibitors reduced RhoA activation, dye uptake, and ATP release. Thus, hypotonic stress-induced ATP release occurs via Rho-dependent pannexin 1 hemichannel opening, and TRPV4 likely transduces osmotic stress into Rho-mediated ATP release. In goblet cells, PAR agonists stimulated the concomitant release of mucins and ATP, which was dependent on intracellular Ca2+-mobilization and cytoskeletal reorganization. Mucin granules contained ATP, but levels of ADP and AMP within granules exceeded those of ATP. Direct release of ADP/AMP from mucin granules likely represents an important source of ASL adenosine, promoting A2b receptor-dependent ion/water secretion necessary for mucin hydration. In sum, this dissertation suggests a major mechanism for ATP release from non-mucous cells, i.e., Rho-dependent pannexin 1 opening. These studies also reveal that PARs promote Ca2+-regulated secretion of ATP/ADP/AMP-rich mucin granules from goblet cells
