A novel approach to study mechanisms of age-related dysfunction in hypoxia-induced erythrocyte ATP release

项目概要： 使血流和氧气输送与组织氧气需求相匹配是最重要的基础之一， 生理过程。最近的研究表明，红细胞（RBC）感觉缺氧，并作出反应， 释放ATP。RBC衍生的ATP引起血管舒张，改善局部血流和氧气输送 通过与内皮嘌呤能（P2）受体结合。我们的实验室和其他实验室已经证明， ATP释放在健康的老年人以及II型糖尿病和肺动脉高压患者中受损。 高血压目前研究缺氧诱导的RBC ATP释放的方法仅限于静态测量 ATP在氧合（PO 2）的离散水平，因此理解缺氧诱导的关键障碍 RBC ATP释放是指无法同时实时测量PO 2和ATP释放。我们的初步 数据表明，用镁绿色（Mg-G）的荧光血氧测定法可以同时测量ATP的释放， 实时PO 2，允许精确量化RBC功能的变量，包括总ATP释放， 启动ATP释放所需的PO 2、ATP释放的峰值速率等。虽然它是公认的 缺氧时ATP释放调节的最终途径是通过泛连接蛋白-1通道， 刺激红细胞ATP释放的机制对缺氧的反应仍不清楚。红细胞变形性 与缺氧诱导的ATP释放有关，我们已经证明，改善变形能力， 来自老年人的RBC恢复ATP释放。最近的数据表明， 通道Piezo 1在剪切介导的红细胞ATP释放中的作用，然而Piezo 1在缺氧诱导的红细胞ATP释放中的作用 释放未知。因此，这一探索性研究建议的总体目标是建立我们的小说 同时实时监测红细胞ATP释放和PO 2的方法，并探讨Piezo 1的作用 在年轻人和老年人缺氧时刺激ATP释放。在具体目标1.1和1.2中，我们将使用 连续，同时测量PO 2和ATP，以确定RBC ATP释放参数， 进行性缺氧我们将通过证明缺氧时ATP释放被消除来验证我们的方法 通过泛连接蛋白-1通道阻断。在具体目标2.1和2.2中，我们将确定是否刺激 Piezo 1通道是年轻人缺氧诱导的红细胞ATP释放所必需的， Piezo 1通道的刺激解释了老年人RBC ATP释放的损伤。我们还将 确定机械敏感性Piezo 1通道的药理学刺激是否逆转了年龄相关的 RBC ATP释放受损。拟议研究的结果将建立一种新的方法， 研究红细胞在缺氧过程中的生理学，并将提供有关红细胞在缺氧过程中的机械作用的第一个数据。 Piezo 1在年轻人和老年人缺氧诱导的红细胞ATP释放中的作用我们的研究结果可能会推动 未来的研究旨在改善老年人和各种患者人群的循环ATP， 由于血流和氧气输送的局部调节受损而引起的运动不耐受或组织缺血。

PROJECT SUMMARY: Matching blood flow and oxygen delivery to tissue oxygen demand is one of the most essential fundamental physiological processes. Recent studies show that red blood cells (RBCs) sense hypoxia and respond by releasing ATP. RBC-derived ATP causes vasodilation that improves local blood flow and oxygen delivery via binding to endothelial purinergic (P2) receptors. Our laboratory and others have demonstrated that RBC ATP release is impaired in healthy older adults, as well as patients with type II diabetes and pulmonary hypertension. Current methodology to study hypoxia-induced RBC ATP release is limited to static measures of ATP at discrete levels of oxygenation (PO2), and thus the critical barrier to understanding hypoxia-induced RBC ATP release is the inability to simultaneously measure PO2 and ATP release in real-time. Our preliminary data indicates that fluo-oximetry with magnesium green (Mg-G) can simultaneously measure ATP release and PO2 in real-time, allowing for precise quantification of variables of RBC function that include total ATP release, the PO2 required to initiate ATP release, peak rate of ATP release, and others. Although it is well established that the final conduit for regulated ATP release during hypoxia occurs via pannexin-1 channels, the mechanisms stimulating RBC ATP release in response to hypoxia remain unclear. RBC deformability has been linked with hypoxia-induced ATP release, and we have demonstrated that improving deformability of RBCs from older adults restores ATP release. Recent data implicate the mechanically activated cation channel Piezo1 in shear-mediated RBC ATP release, however the role of Piezo1 in hypoxia-induced RBC ATP release is unknown. Therefore, the overall goal of this exploratory research proposal is to establish our novel approach for monitoring real-time RBC ATP release and PO2 simultaneously, and to explore the role of Piezo1 in stimulating ATP release during hypoxia in young and older adults. In Specific Aims 1.1 and 1.2, we will use continuous, simultaneous measurement of PO2 and ATP to define parameters of RBC ATP release during progressive hypoxia. We will validate our approach by demonstrating ATP release during hypoxia is abolished via pannexin-1 channel blockade. In Specific Aims 2.1 and 2.2, we will determine whether stimulation of Piezo1 channels is requisite for hypoxia-induced RBC ATP release in young adults, and whether reduced stimulation of Piezo1 channels explains the impairment in RBC ATP release in older adults. We will also determine whether pharmacological stimulation of mechanosensitive Piezo1 channels reverses the age-related impairment in RBC ATP release. The findings from the proposed studies will establish a novel approach for studying RBC physiology during hypoxia, and will provide the first data regarding the mechanistic role of Piezo1 in hypoxia-induced RBC ATP release in young and older adults. Our results could be the impetus for future studies designed to improve circulating ATP in older adults and various patient populations suffering from exercise intolerance or tissue ischemia due to impaired local regulation of blood flow and oxygen delivery.