The overall goal of this thesis was to understand the mechanisms underlying beta-adrenergic receptor (beta-AR) regulation of anion secretion and epithelial cell migration on mucociliary clearance and epithelial repair in human airways. The first chapter of the thesis presents an overview of the literature with the purpose of providing background information of mucociliary clearance, airway epithelial cell restitution and specific inflammatory disease conditions where these processes contribute to the innate defense of the airways. Chapters 2 and 3 describe the methods, results and a discussion of experiments that were performed to address the overall goal of the thesis. The final chapter presents a general discussion of the major findings of these studies in the context of what is presently known about the role of beta-AR regulation of epithelial function in airways. Initial experiments presented in Chapter 2 so looked at the effects of growth conditions on beta-AR localization in a human airway epithelial cell model system (Calu-3 cells). The results from these experiments showed that under air-liquid interface (ALI) conditions basolateral stimulation with 1uM epinephrine produced a significant increase in CFTR dependent anion secretion, whereas cells that were grown under liquid liquid interface (LLI) had a much smaller increase secretion when stimulated with the same concentration of epinephrine. These results indicated that basolateral expression of beta-ARs is increased when cells were grown under ALI. Furthermore Calu-3 cells that were apically stimulated with the beta2-AR selective agonist salbutamol produced an increase in anion secretion similar to what was observed with 8cpt-cAMP, a non-metabolizable analog of cyclic AMP. Additionally, when cells were treated with carvedilol, an inverse agonist acting at beta2-ARs, an initial decrease in basal Isc occurred and carvedilol treatment after stimulation with 8cpt-cAMP inhibited anion secretion. Cells pretreated with nocotazole, an agent that disrupts microtubule assembly, blocked the inhibitory effects of carvedilol on anion secretion, suggesting that endocytosis was necessary in order to observe the inhibitory effects of carvedilol. Finally, western blot analysis of apical membrane proteins showed that when cells were treated with carvedilol there was reduced expression of CFTR in the apical membrane, which was not observed following stimulation with epinephrine.Experiments in chapter 3 were designed to investigate the effects of beta-AR agonists on epithelial cell migration. Stimulation of Normal Human Bronchial Epithelial (NHBE) cells and Calu-3 cells), with a beta2-AR agonist produced a significant increase in time to wound closure compared to untreated control cells. Moreover, agonist stimulated cells were rescued when pretreated with beta-AR antagonists propranolol or ICI-118551. The addition of beta-AR agonists epinephrine or salbutamol to CFTR silenced cells (shCFTR) or cells where CFTR was inhibited with 20uM CFTRinh-172 showed no further decrease in migration rate suggesting that inhibition due to a change in CFTR expression or activity. Furthermore beta-AR agonists reduced lamellipodia protrusion similar to what was observed after CFTR inhibition. Overall these results suggest that treatment of airway epithelial cells with beta-AR agonists causes a decrease in migration rate leading to a significant reduction in the time required for complete wound closure. Additionally these results suggest that stimulating cells with carvedilol causes inhibition of cAMP-stimulated anion secretion by promoting retrieval and internalization of CFTR from the apical membrane. These results signify that the chronic use of beta-AR agonists can lead to increased risk for infection in individuals with obstructive airway disease and the use of bias ligands that promote Gs signaling while blocking beta-arrestin signaling may be the key to effectively treating these patients. These drugs would potentially promote wound repair and mucociliary clearance while limiting the risk of increased exacerbations and infection.
University of Minnesota Ph.D. dissertation. December 2014. Major: Animal sciences. Advisor: Scott M. O’Grady. 1 computer file (PDF); xii, 126 pages.
Peitzman, Elizabeth Ruth.
Adrenergic regulation of CFTR-dependent anion secretion and cell migration in airway epithelial cells.
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