RELEASE AND METABOLISM OF EXTRACELLULAR ATP IN THE HEART

本项目的总体假设是，在基础条件下，心肌细胞在细胞外心脏间质空间组成性地释放和代谢腺嘌呤核苷酸，这种核苷酸的释放和代谢在缺血和缺氧应激下显著增加。研究表明，释放的腺嘌呤核苷酸在心脏调节中起着两种重要作用：1)作为肌细胞P2核苷酸受体的自分泌/旁分泌激动剂；2)作为定位生成腺苷的底物，用于自分泌/旁分泌激活A1和A3受体。通过诱导信号通路抵消缺氧和缺血的影响，这些肌细胞受体的激活将提供对代谢应激的快速反馈反应。这一假设的一个推论是，心肌细胞本身是外链ATP酶和其他外链核苷酸酶活性的主要位点，这些活性既清除释放的ATP，又在心肌细胞表面产生腺苷。这些研究在生理学上具有重要意义，因为它们将为肌细胞表面核苷酸和核苷水平的高度局部变化提供新的见解，这些变化有助于信号通路的自分泌调节，这些信号通路调节心脏基础收缩力和对缺氧和缺血应激的生物能量适应。这些研究是新颖的，因为分析心肌细胞表面的核苷酸和核苷水平有助于信号通路的自分泌调节，这些信号通路调节心脏的基础收缩力和对缺氧和缺血应激的生物能量适应。这些研究是新颖的，因为核苷酸释放和细胞外代谢的分析将利用新开发的方法，提供定量灵敏度，时间分辨率和空间定位，这在以前的心肌细胞或其他细胞研究中是不可能的。这些研究是及时的，因为最近的研究已经发现了许多编码不同外核苷酸酶的新基因，但这些基因在心肌细胞中的表达和功能尚未被探索。最后，拟议的项目与该项目的总体主题高度相关，因为它试图定义腺嘌呤核苷酸通量（细胞内心脏生物能量学的一个核心方面）如何与调节对生物能量应激的适应的细胞外信号级联相耦合。

The overall hypothesis for this project is that cardiac myocytes constitutively release and metabolize adenine nucleotides in the extracellular cardiac interstitial space under basal conditions, and that this nucleotide release and metabolism is significantly increased in response to ischemic and hypoxic stress. It is proposed that released adenine nucleotides play two important roles in cardiac regulation: 1) as autocrine/paracrine agonists of myocyte P2 nucleotide receptors; and 2) as substrates for the localized generation of adenosine used in autocrine/paracrine activation of A1 and A3 receptors. By inducing signaling pathways that counteract the effects of hypoxia and ischemia, activation of these myocyte receptors will provide a rapid feedback response to metabolic stress. A corollary of this hypothesis is that cardiac myocytes per se are major sites of th ecto-ATPase and other ectonucleotidase activities that both scavenge released ATP and generate adenosine at the myocyte cell surface. The proposed studies are physiologically significant because they will provide new insights regarding now highly localized changes in nucleotide and nucleoside levels at the myocyte cell surface contribute to the autocrine regulation of signaling pathways that modulate basal cardiac contractility and bioenergetic adaptation to hypoxic and ischemic stress. The studies are novel because the analysis of nucleotide and nucleoside levels at the myocyte cell surface contribute to the autocrine regulation of signaling pathways that modulate basal cardiac contractility and bioenergetic adaptation to hypoxic and ischemic stress. The studies are novel because the analysis of nucleotide release and extracellular metabolism will utilize newly developed methods that provide a quantitative sensitivity, temporal resolution, and spatial localization not possible in previous studies with cardiac myocytes or other cells. The studies are timely because recent research has identified many new genes encoding distinct ecto-nucleotidases, but the expression and function of these genes in cardiac myocytes has not been explored. Finally, the proposed project is highly relevant to the overall theme of the Program Project because it seeks to define how adenine nucleotide flux, a central aspect of intracellular cardiac bioenergetics, may be coupled to extracellular signaling cascades that regulate adaptation to bioenergetic stress.