Reactive Oxygen Species and Vascular O2 Sensing

描述(由申请人提供)：本提案侧重于确定NOx氧化酶衍生的过氧化氢，以及胞质NADPH和NADH氧化还原系统在控制信号机制中的作用，这些信号机制调节血管平滑肌力对PO2生理变化的反应。在这一应用中的研究探讨了我们的新证据的重要性，即细胞内NADPH和可能的NADH水平控制基线的NOx氧化酶衍生的过氧化氢介导的牛肺动脉的松弛，该松弛被低氧去除，导致收缩。另一方面，牛冠状动脉维持较低水平的NADPH和NADPH产生磷酸戊糖途径(PPP)酶葡萄糖-6-磷酸脱氢酶。我们假设，冠状动脉中PPP功能的不同导致了缺氧引起的胞内NADPH的氧化，这通过我们已经证明受PPP控制的机制激活了松弛。目标1的研究将调查PO2如何调节涉及NOx衍生的过氧化氢的血管反应的表达。目的2重点了解PO2如何调节胞内NADH和NADPH氧化还原相关的信号机制，从而有助于控制力的产生。目标3中的研究是为了确定在控制NOx氧化酶活性和胞浆NAD(P)H氧化还原反应方面存在差异的根源，这些差异有助于在肺和冠状动脉观察到低氧引起的收缩和松弛反应。我们将对去内皮的冠状动脉、肺导管和阻力动脉进行研究，以改变NOx的活性和表达，以及胞浆中NADH和NADPH的氧化还原，以确定受这些过程调控的信号系统在PO2诱导的反应中如何发挥作用。PO2对胞质NAD(P)H氧化还原和氧化剂产生的控制将结合代谢测量、组织荧光和化学发光技术以及细胞荧光成像方法进行研究，这些方法旨在表征所涉及的信号机制的ROS和氧化还原方面。还将检查大鼠动脉，以确定由NOx和胞内NAD(P)H氧化还原控制的PO2引发的反应机制的异同。这些研究应该有助于确定肺和冠状动脉对氧分压变化的生理反应差异的根源。

DESCRIPTION (provided by applicant): 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 pulmonary 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.