Airway smooth muscle (ASM) contraction is a critical component in the pathophysiology of airway disease. Parasympathetic efferent nerves, mast cells, eosinophils and epithelial cells all release or synthesize substances that modify ASM tone. Although a rise in the intracellular calcium concentration is the normal trigger for smooth muscle contraction, agonist activation renders the myofilaments more sensitive to calcium (Ca2+) and potentiates Ca2+-induced contraction. Agonist activation, moreover, dynamically reorganizes the cytoskeleton. Preliminary studies for this proposal indicate that small G proteins (rho and ras) are important regulators of agonist-activated potentiation of Ca2+-induced contraction and cytoskeletal reorganization in ASM. However, the signaling pathways which link agonist activation via the heterotrimeric and small G proteins to cytoskeletal reorganization and potentiation of Ca2+-induced contraction in ASM are not well understood. Hence our overall goal is to determine how the signalling pathways transduced by the heterotrimeric G proteins, Gq and Gi, regulate the small G proteins, rho and ras, in ASM. We hypothesize that in ASM, activation of Gi is linked to rho, whereas activation of Gq is linked to ras. Using physiological, biochemical, and molecular techniques, we propose to: a) evaluate the signalling pathways by which activation of Gi and Gq induce rho- and ras-mediated potentiation of Ca2+-induced contraction in skinned porcine ASM, b) to identify the pathways by which Gq- and Gi-coupled receptor activation of rho increases formation of actin filaments and phosphorylation of paxillin in intact porcine ASM and cultured human ASM cells, and c) to examine the intermediate steps in the pathways linking Gi- and Gq-coupled receptors to ras and rho in cultured ASM. An understanding of signaling pathways in ASM regulating these novel Ca2+ independent processes that increase force production promises new targets and improved methods for the treatment of airway disease.
