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Theses and Dissertations

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Browsing Theses and Dissertations by Author "Amberg, Gregory, committee member"

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    A novel approach to real-time monitoring of erythrocyte ATP release as a function of hypoxia
    (Colorado State University. Libraries, 2020) Smith, Meghan E., author; Dinenno, Frank, advisor; Lark, Daniel, committee member; Amberg, Gregory, committee member
    Background: Matching blood flow to tissue oxygen demand is essential for maintaining metabolic homeostasis and sustaining human life. Recent studies suggest that red blood cells (RBCs) play a role in local vasodilatory signaling by releasing ATP in response to hypoxia. RBC ATP release and overall function are impaired with age and disease. Until now, luciferin/luciferase bioluminescence is the only method described to quantify ATP release from RBCs. Here, we describe a novel approach where ATP release is measured as a function of hypoxia continuously and in real time using an Oroboros Oxygraph O2K respirometer. Purpose: This report describes the development and application of this new approach. Methods: We obtained blood samples from 10 healthy, young adults (18-35y) via venipuncture. Washed RBCs were diluted to 5% hematocrit and added to the glass chamber of a calibrated Oxyfluorimeter along with 5μM Mg-G. Nitrogen gas was constantly injected into the chamber at 1 ml/min to decrease PO2. An LED-based fluorescence detection device monitored Mg-G fluorescence, which was used to calculate extracellular [ATP]. Results: When 5% HCT RBCs were exposed to 30 minutes of hypoxia, Mg-G fluorescence (V) continuously increased. During 30 minutes of progressive hypoxia, PO2 in the chamber decreased from 121.9 ± 1.3 to 9.8 ± 0.8 mmHg, and D extracellular [ATP] from normoxia (μM) increases from 0 to 6,985.0 ± 793.6 μM. Extracellular [ATP] accumulates markedly when PO2 in the chamber reaches 50.60 ± 1.52 mmHg. Conclusion: Using this novel method, we identified a PO2 threshold at which extracellular ATP accumulates rapidly, which is consistent with the range of PO2 that elicits Hb desaturation in RBCs. This approach may allow for detailed mechanistic studies into the relationship between hypoxia, Hb desaturation, and RBC ATP release.
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    Mechanisms of impaired red blood cell ATP release in older adults: implications for altered vascular control with age
    (Colorado State University. Libraries, 2018) Racine, Matthew L., author; Dinenno, Frank A., advisor; Amberg, Gregory, committee member; Chicco, Adam, committee member; Gentile, Christopher, committee member
    The following dissertation is comprised of a series of experiments with the overall aim of determining the mechanisms of impaired ATP release from red blood cells (RBCs) of healthy older adults in response to hemoglobin deoxygenation and identifying a potential role of this impairment in the declines in vascular control of peripheral blood flow with advancing age. Advancing age is the primary risk factor for cardiovascular disease (CVD), which is the leading cause of death in societies today and is strongly associated with arterial dysfunction. Furthermore, impairments in vascular control and the subsequent regulation of tissue blood flow and oxygen delivery contribute to vascular pathologies such as atherosclerosis and ischemic disease, as well as the age-associated declines in functional capacity, exercise tolerance, and overall quality of life. Thus, understanding the mechanisms of the age-related impairments in vascular control and identifying potential therapeutic targets holds significant potential for reducing the healthcare burden associated with a rapidly aging population. Accordingly, the ultimate goal of this dissertation is to determine if an in vivo pharmacological approach can be utilized to treat the age-related declines in RBC ATP release, thereby restoring circulating ATP responses and subsequent vascular control during the physiological stimuli of hypoxia and exercise in healthy older adults. The key novel findings of this dissertation are that (i) age-associated declines in RBC deformability are the primary mechanism of impaired deoxygenation-induced ATP release from RBCs of healthy older adults; (ii) primary (healthy) aging is not associated with a global decline in RBC function given that inhibition of cyclic AMP hydrolysis by phosphodiesterase 3 did not improve deoxygenation-induced ATP release from RBCs of older adults and that the cellular responses to Gi protein activation remained intact with age; and (iii) that systemic Rho-kinase inhibition via administration of fasudil improves the age-related impairments in vascular control and circulating ATP during systemic hypoxia and exercise, which may be related to enhanced RBC ATP release and NO bioavailability. These findings are the first to identify a role for Rho-kinase inhibition in improving these physiological responses in healthy older adults and are therefore clinically significant for aging population in which impaired vascular control contributes to elevations in cardiovascular disease risk and declines in exercise tolerance, functional independence and overall quality of life.
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    Role of the endothelium in modulating sympathetic vasoconstriction in contracting skeletal muscle of young and older adults
    (Colorado State University. Libraries, 2016) Hearon, Christopher M., author; Dinenno, Frank A., advisor; Amberg, Gregory, committee member; Chicco, Adam, committee member; Gentile, Christopher, committee member
    Aerobic capacity is a powerful independent predictor of all-cause mortality in healthy and disease populations. Healthy (primary) ageing is associated with a decline in maximal aerobic capacity, exercise intolerance and elevated risk for ischemic cardiovascular disease. Specifically, ageing is characterized by impaired regulation of vascular tone during exercise, due in part to lower vasodilatory signaling and elevated sympathetic vasoconstrictor activity in the peripheral vasculature. Impaired regulation of peripheral vascular tone results in attenuated blood flow and oxygen delivery to contracting skeletal muscle during exercise and is a primary contributor to the age-associated decline in aerobic capacity. The overall aim of this dissertation is to determine the vascular signaling mechanisms responsible regulating sympathetic vasoconstrictor signaling during exercise in young healthy adults and translate these findings to improve vascular function during exercise in older adults. The regulation of blood flow and oxygen delivery during exercise depends on the proper integration of local vasodilatation and neural sympathetic vasoconstriction. In healthy humans, the integration of these competing signals results in attenuation of sympathetic vasoconstriction, or “sympatholysis”, to ensure adequate blood flow to contracting skeletal muscle. The signaling mechanisms responsible for sympatholysis in healthy humans are unknown. To date, the only exogenous vasodilator shown to mimic exercise in its ability to attenuate sympathetic vasoconstriction in humans is adenosine triphosphate (ATP). The first aim of this dissertation is to determine if smooth muscle cell hyperpolarization (via activation of inwardly-rectifying potassium (KIR) channels), the primary vasodilatory pathway of ATP, is responsible for ATP-mediated attenuation of sympathetic vasoconstriction. In contrast to smooth muscle specific signaling, vasodilatory stimuli such as ATP and exercise can act through endothelium-dependent pathways. The second aim of this dissertation tests the hypothesis that endothelium-dependent signaling is capable of attenuating sympathetic vasoconstriction during exercise in young healthy humans. With age, impaired endothelial function and elevated sympathetic vasoconstrictor activity results in impaired functional sympatholysis. The third aim is to determine if augmentation of endothelium-dependent signaling during exercise improves age-associated impairments in functional sympatholysis. The primary findings of this dissertation are that 1) similar to exercise, the ability of ATP to attenuate sympathetic vasoconstriction is independent of smooth muscle cell hyperpolarization via activation of KIR channels, 2) activation of endothelium-dependent signaling during exercise significantly enhances the ability of contracting skeletal muscle to attenuate sympathetic vasoconstriction, and 3) that augmentation of endothelium-dependent signaling during exercise significantly improves functional sympatholysis in older adults. These findings are the first to identify endothelium-dependent modulation of sympathetic vasoconstriction in humans, and identifies vascular signaling pathways capable of improving the regulation of vascular tone during exercise in older adults. These findings are clinically significant for patient populations and disease states characterized by impaired functional sympatholysis including ageing, hypertension, and heart failure.