The general goal of the proposed research is to improve our understanding of the complex interaction between nitric oxide (NO), hemoglobin, oxygen, carbon dioxide, and thiols in the blood and tissue In recent years, many hypotheses have been suggested regarding the production and transport of NO in the blood and various factors that may affect these processes However, disagreement still exists over the true mechanisms of action and transport of NO. Several specific questions remain (1) How do the complex interactions of intracellular calcium, oxygen, shear stress, and thiol levels affect the amount of NO produced by the endothelium and delivered to tissue? (2) Do nitrosothiols or nitrosylhemoglobin (SNO-I-Ib) act as a facilitated carrier mechanism by storing NO and transporting it to areas where it is needed? (3) Can mathematical modeling assist in determining the most probable transport mechanisms for NO? The proposed research seeks to answer these questions using a combination of in vitro and in vivo experimental studies and mathematical modeling Experimental studies have been designed to provide vital information for the mathematical modeling and will be used to test model validity and evaluate hypothesized mechanisms of NO transport In vitro studies will be conducted in a parallel-plate flow chamber using rat endothelial cells. Nitric oxide release will be stimulated using neurohumoral mediators and measured under basal conditions. Additional conditions, including altered oxygen and carbon dioxide levels, various levels of shear stress and addition of thiols will be imposed. In vivo studies will be conducted in the arterioles and venules of the rat mesentery. NO levels will be measured under normal physiological conditions, during hypoxia and hypercapnia and under altered thiol, shear stress and hematocrit levels. The effects of these interventions on NO production, transport, and distribution will be measured, incorporated into the mathematical model, and used to test its validity under various ranges of conditions. The mathematical model will simulate the production, mass transport, feedback regulation, and biochemical mechanisms of action of NO in the microcirculation and tissue. Quantitative data obtained from the validated model will be used to predict parameters that cannot be measured in vivo, analyze the hypotheses and further the understanding of NO production and transport mechanisms, and used to shape future experimental studies. Understanding NO transport mechanisms is important clinically since altered NO regulation has been implicated in pathophysiological conditions
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