OXYGEN SENSING AND CELL SIGNALING IN HYPOXIA

描述(改编自申请者的摘要)：尽管是手机 在常氧状态下，呼吸是由代谢需要决定的，有证据表明 细胞可以通过减少能量需求来适应低氧，从而降低 对ATP的使用和对氧气的需求。在复氧时，代谢正常 过程恢复，细胞活动恢复。一种减少 低氧时的能量需求，同时保持高能磷酸盐水平 (低氧适应)在严重缺氧时可能通过保存 用于基本过程的ATP。低氧适应需要细胞内的氧气 能够检测PO2的传感器。尽管可能存在多个传感器， 数据表明，细胞色素氧化酶在低氧时起着感应器的作用。 适应。特殊目标1将测试细胞色素氧化酶功能 作为氧气感受器在低氧期间通过降低其表观Vmax。这将是 在测试过程中使用能降低酶的Vmax的抑制剂 常氧，确定它们是否激活了低氧适应 回应。氧气传感器在缺氧过程中的激活必须与 随后激活细胞内信号级联，这是 最终抑制三磷酸腺苷的利用。《特定目标2》将测试 认为活性氧(ROS)是第二信使的假说 在这个信号通路中。完整细胞中依赖PO2的ROS的产生 将被研究并与该酶的功能相关联。其他 研究将确认线粒体是否是ROS的来源，并将 将这些信号与低氧适应反应的激活联系起来。 对分离的线粒体的研究将确定其位置和机制 低氧时PO2依赖的ROS的产生。总的来说，这些研究 将阐明线粒体在缺氧时产生ROS的机制 并将这些信号与细胞色素氧化酶和缺氧的功能联系起来 适应反应。假说是下游的信号元件 ROS的抑制导致对ATP依赖的酶系统的抑制。具体目标3 将开始测试蛋白激酶的功能假设 低氧反应中的下游信号元件。参与其中的 在先前研究的基础上，将测试该途径中的蛋白激酶C 证明其被ROS或低氧激活。的长期目标是 该项目是为了确定氧气传感机制和下游 参与低氧适应的信号序列。这些研究将 确定一种新的细胞氧检测途径，并可能有助于澄清 了解细胞如何适应低氧条件。

DESCRIPTION (Adapted from the applicant's abstract): Although cellular respiration is set by metabolic need during normoxia, evidence suggests that cells can adapt to hypoxia by reducing their energy demand, thereby lowering the use of ATP and the need for O2. At reoxygenation, normal metabolic processes are restored and cellular activity recovers. An ability to reduce energy demands in hypoxia while preserving high energy phosphate levels (hypoxic adaptation) may be protective during severe hypoxia by conserving ATP for essential processes. Hypoxic adaptation requires a cellular O2 sensor capable of detecting PO2. Although multiple sensors likely exist, data suggest that cytochrome oxidase acts as the sensor during hypoxic adaptation. Specific Aim 1 will test whether cytochrome oxidase functions as the O2 sensor during hypoxia by decreasing its apparent Vmax. This will be tested using inhibitors that reduce the Vmax of the oxidase during normoxia, determining whether these activate the hypoxic adaptation response. Activation of the O2 sensor during hypoxia must be coupled to subsequent activation of an intracellular signaling cascade, which ultimately inhibits ATP utilization. Specific Aim 2 will test the hypothesis that reactive oxygen species (ROS) function as a second messenger in this signaling pathway. The PO2-dependent ROS generation in intact cells will be studied and correlated with the function of the oxidase. Other studies will confirm whether mitochondria are the source of the ROS and will link these signals to the activation of the hypoxic adaptation response. Studies with isolated mitochondria will identify the sites and mechanisms of PO2-dependent ROS generation during hypoxia. Collectively, these studies will clarify the mechanisms of mitochondrial ROS generation during hypoxia and link these signals to the function of cytochrome oxidase and the hypoxic adaptation response. The hypothesis is that signaling elements downstream of ROS lead to inhibition of ATP-dependent enzyme systems. Specific Aim 3 will begin to test the hypothesis that protein kinases function as downstream signaling elements in the hypoxic response. The involvement of protein kinase C in this pathway will be tested, based on previous studies demonstrating its activation by ROS or by hypoxia. The long term goal of this project is to identify O2 sensing mechanisms and the downstream signaling sequence involved in hypoxic adaptation. These studies will identify a novel pathway of cellular O2 detection, and may help clarify understanding of how cells adapt to lowered O2 conditions.