The goal of this K08 application is to facilitate development of essential skills that will allow the PI to become a successful academician and achieve independent scientific investigator status. The respiratory epithelium serves as a barrier, a regulator of the content of airway surface liquid, and a source of cytokines and other products that regulate airway physiology. In addition to cyclic extensions and contraction, airway epithelial cells are exposed to lumenal shear stress, defined as the frictional force per unit surface area, generated by airflow. The shear stress sensed by airway epithelia is continuously changing under both physiologic and pathologic conditions. We have exciting novel data suggesting that shear stress in airway epithelial cells leads to changes in airway epithelial permeability. Therefore, dynamic modulation of the airway epithelial barrier could be an important mechanism by which epithelial cells transduce lumenal information into subepithelial responses. Our preliminary studies indicate that shear stress induces TRPV4 activation with subsequent L-type voltage gated channel activation with subsequent increases in intracellular calcium. The increase in intracellular calcium in airway epithelial cells leads to concerted changes in epithelial paracellular permeability by modulating actin rearrangement and NO production, as well as in transmembrane permeability by modulating AQP5 abundance. We propose to study the role of shear stress in altering both paracellular and transmembrane permeability as well as dissect the underlying mechanisms regulating these effects. In SA#1we will investigate the effects of shear stress on airway epithelial permeability using primary cultured human airway epithelial cells (NHBE) and.ex vivo isolated mouse trachea. In SA#2 we will define the role for and the mechanisms (TRPV4, L-type channel) of shear-induced calcium flux in modulating epithelial permeability using pharmacologic inhibition and genetic manipulation. In SA#3 we will study the role od shear-induced increases in calcium on NO production and its effect on permeability. Through these complimentary techniques and coursework, the PI will develop new skills and generate novel data regarding the effects of mechanical shear stress on airway epithelial cell regulation and function, a little-examined area likely to be of immense importance to normal lung function, as well as conditions of altered shear including exercise and asthma.
