Date of Award
Thomas C. Rich, Silas J. Leavesley
Current asthma therapies include corticosteroids, long acting β2-adrenergic receptor (β2-AR) agonists paired with corticosteroids, or short-acting β2-AR agonists used in rescue inhalers. Chronic use of β-agonists results in diminished effectiveness, and use of long-acting β-agonists can even exacerbate asthma symptoms; this is partly because anti-inflammatory therapy does not directly address the exaggerated airway narrowing due to excessive shortening of airway smooth muscle (ASM), which is the hallmark of asthma. These observations led our collaborators to investigate other potential asthma therapies – bitter taste agonists. 2 Bitter taste agonists, such as chloroquine, activate a subset of Gq-coupled receptors known as bitter taste 2 receptors (TAS2Rs) in airway smooth muscle cells. Activation of these receptors triggers relaxation of human airway smooth muscle cells (HASMCs). This response is markedly different than the response to activation of other Gq-coupled receptors, such as muscarinic receptors, which when activated trigger contraction of HASMCs. The goal of this thesis project is to understand how different Gq-coupled receptor agonists trigger distinct responses in HASMCs. The working hypothesis is that differences in the spatial distributions and/or kinetics (duration) of intracellular calcium (Ca++) signals are responsible for the distinct responses. To test this hypothesis, xv HASMCs were treated with different concentrations of carbachol (1 – 50 µM), a m3 muscarinic acetylcholine receptor (m3 mAChR) agonist; chloroquine (10 – 200 µM), a TAS2R agonist; or histamine (0.5 – 5 µM), a histamine 1 receptor (H1R) agonist, to target different subsets of Gq-mediated receptors. HASMCs were loaded with Cal-520® AM, a green-fluorescent dye that, upon binding to intracellular Ca++, increases its fluorescence intensity. Change in intracellular Ca++ following agonist addition were visualized and measured over time using an Andor WD spinning disk confocal microscope. Distinct spatial differences in Ca++ signals in response the agonist addition were quantified by measuring two parameters – Ca++ signal maximum intensity in the identified signal areas over time and Ca++ signal area changes over time. Distinct kinetic differences in Ca++ signals were quantified by measuring two more parameters: Ca++ signal duration and the number of identified Ca++ responses induced by each agonist per experiment. Furthermore, the distinct changes in peak intensity, signal area, duration, and number of induced Ca++ signals were analyzed for possible concentration dependencies, which could provide future insight into the unique airway responses induced by each agonist.
Bobinger, Hanna Viktoria, "Gq-Mediated Ca** Signals in Human Airway Smooth Muscle Cells" (2021). Undergraduate Theses . 23.