This proposal focuses on defining the role of Nox oxidase-derived hydrogen peroxide, and cytosolic NADPH and NADH redox systems in processes that control signaling mechanisms regulating vascular smooth muscle force responses to physiological changes in PO2.Studies in this application investigate the importance of our new evidence that cytosolic NADPH and possibly NADH levels control a baseline Nox oxidase-derived hydrogen peroxide-mediated relaxation of bovine pulmdnary arteries which is removed by hypoxia, resulting in contraction. On the other hand, bovine coronary arteries maintain a lower level of NADPH and the NADPH generating pentose phosphate pathway (PPP) enzyme glucose-6-phosphate dehydrogenase. We hypothesize that differences in the function of the PPP in coronary arteries results in a hypoxia-elicited oxidation of cytosolic NADPH, which activates relaxation through a mechanism we have shown to be controlled by the PPP.Studies in Aim 1 will investigate how PO2 regulates the expression of vascular responses involving Nox-derived hydrogen peroxide.
Aim 2 focuses on understanding how PO2 regulates cytosolic NADH and NADPH redox-linked signaling mechanisms that contribute to the control of force generation. Studies in Aim 3 are to define the origins of differences in the control of Nox oxidase activity and cytosolic NAD(P)H redox that contribute to hypoxia-elicited contractile and relaxing responses observed in pulmonary and coronary arteries. Isolated endothelium-removed coronary and pulmonary conduit and resistance arteries will be studied with perturbations that alter Nox activity and expression, and cytosolic NADH and NADPH redox to define how signaling systems regulated by these processes may function in PO2-elicited responses. The control of cytosolic NAD(P)H redox and oxidant production by PO2 will be examined with a combination of metabolic measurements, tissue fluorescence and chemiluminescent techniques, and cellular fluorescence imaging approaches which are designed to characterize the ROS and redox aspects of the signaling mechanisms that are involved. Rat arteries will also be examined to identify similarities and differences in mechanisms of PO2-elicited responses controlled by Nox and cytosolic NAD(P)H redox. These studies should help define the origins of differences in physiological responses of pulmonary and coronary arteries to changes in O2 tension.
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