The role of ROS on beta-2-adrenergic receptor function in human airway

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PROJECT ABSTRACT Asthma is a chronic inflammatory disease of the lungs that is characterized by airway obstruction and bronchospasm. While there are many factors that contribute to development of asthma and other inflammatory airway diseases, the generation of reactive oxygen species (ROS) within the airway epithelium leads to exacerbation of airway inflammation and contributes greatly to pathophysiology of asthma. The gold-standard for treatment of asthma is inhalation of β2-adrenergic receptor (β2AR) agonists, which relax the airway tissues and reduce bronchospasms, allowing for greater pulmonary function. However, chronic use of β2-agonists leads to loss of bronchodilatory efficacy, a process termed β2-agonist tachyphylaxis, and for which, no mechanistic explanation exists. Recently, our laboratory has demonstrated that agonism of β2AR leads to robust formation of ROS, and that ROS are required for β2AR function, as inhibition of ROS abrogates both β2AR-mediated G-protein and β-arrestin signaling. These results suggest that some level of ROS are required for stabilization of functional β2AR, and indeed, we show that β2AR function is strictly regulated by ROS- dependent transient oxidation of β2AR-cysteine residues to form cysteine-S-Sulfenic acids, an effect that significantly influences receptor function. Importantly, our results also suggest that over-oxidation of β2AR S- Sulfenic acids to higher-order irreversible oxidation states inhibits β2AR function. We hypothesize that the highly oxidative environment of asthma, coupled with the heightened ROS generation upon chronic β2-agonist use, could contribute to β2-agonist tachyphylaxis by causing over-oxidation of β2AR. The overall goal of this project is to examine the mechanisms of ROS generation in normal and asthma-diseased primary airway epithelial cells and to test the hypothesis that over-oxidation of β2AR facilitates clinical tachyphylaxis. We proposed two specific aims, using both primary human airway epithelial cells and animal models of chronic and severe asthma, to reach our goals: [1] To characterize the cellular mechanisms of β2AR-induced ROS generation in normal and asthma-diseased airway epithelial cells [2] To assess the effects of β2AR oxidation on β2-agonist tachyphylaxis. Results of this research will shed important light on the role of ROS as regulators of β2AR function in asthma, and reveal the importance of ROS and β2AR oxidation in facilitating clinical tachyphylaxis.