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

项目总结： 使血流和氧气输送与组织需氧量相匹配是最基本的基础之一 生理过程。最近的研究表明，红细胞(RBC)感知缺氧并通过 释放三磷酸腺苷。红细胞衍生的三磷酸腺苷引起血管扩张，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人已经证明了红细胞 健康老年人以及II型糖尿病和肺心病患者的ATP释放受损 高血压。目前研究低氧诱导的红细胞ATP释放的方法仅限于静态测量 不连续氧合水平(PO2)的ATP，因此是理解低氧诱导的关键障碍 红细胞ATP释放是指不能同时实时测量PO2和ATP的释放。我们的预赛 数据表明，镁绿(mg-G)荧光血氧法可以同时测量ATP的释放和 实时PO2，允许对包括总ATP释放在内的RBC功能变量进行精确量化， 启动ATP释放所需的PO2、ATP释放的峰值速率等。尽管它已经建立得很好了 低氧过程中调节ATP释放的最终通道是通过pAnnin-1通道， 低氧刺激红细胞释放三磷酸腺苷的机制尚不清楚。红细胞变形性具有 与缺氧诱导的ATP释放有关，我们已经证明，改善血管的变形性 来自老年人的红细胞可以恢复ATP的释放。最近的数据表明，机械活化的阳离子 Piezo1通道在剪切介导的红细胞ATP释放中的作用，但Piezo1在缺氧诱导的红细胞ATP中的作用 释放情况尚不清楚。因此，这一探索性研究方案的总体目标是建立我们的小说 实时监测RBC-ATP释放和PO2的方法及Piezo1的作用 在年轻人和老年人缺氧时刺激ATP释放的作用。在具体的目标1.1和1.2中，我们将使用 连续、同时测定氧分压和三磷酸腺苷以确定红细胞三磷酸腺苷释放的参数 进行性缺氧。我们将通过证明在缺氧过程中ATP释放被取消来验证我们的方法 通过pAnnexin-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 PIEZO1通道是缺氧诱导的年轻人红细胞ATP释放所必需的，以及是否减少 刺激Piezo1通道可以解释老年人红细胞ATP释放的障碍。我们还将 确定药物刺激机械敏感的Piezo1通道是否逆转与年龄相关的 红细胞三磷酸腺苷释放受损。拟议研究的结果将建立一种新的方法 研究缺氧时的红细胞生理学，将提供有关低氧的机制作用的第一批数据。 PIEZO1在缺氧诱导的年轻人和老年人红细胞ATP释放中的作用。我们的结果可能是推动 未来的研究旨在改善老年人和不同患者群体的循环ATP 由于血流和氧气输送的局部调节受损而导致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.