PURINERGIC AXIS OF CARDIAC BLOOD VESSELS

这项研究的目的是解释 细胞外腺嘌呤核苷酸，特别是腺苷5 '- 三磷酸（ATP），在正常心脏内皮细胞（EC）功能 参与血液调节的因子的释放 血流在正常心脏功能和心血管疾病中可能是重要的。 疾病已经显示EC释放ATP和autacoids，例如 内皮依赖性舒张因子和前列环素对 各种刺激，包括ATP本身。此外，我们建议， 可以在细胞外将腺苷5 '-二磷酸（ADP）转化为ATP， 维持ATP介导的冠状动脉舒张的存在， ATP释放位点下游的阻力血管。我们建议 控制ATP在EC外的出现， 转化为腺苷是局部血液的重要成分 正常心脏的血流控制。由于尚不清楚ATP的释放 或由EC将ADP转化为ATP的过程受到调节，具体目标有 设计如下： 1.测试假设，从EC释放ATP和 可以在完整的冠状动脉中测量EC将ADP转化为ATP ATP释放可支持三磷酸核苷 阵此外，为了验证ADP转化的假设， 对ATP的作用可以在原位被抑制，并确定缺氧对 细胞外生成ATP。 2.检验心脏EC释放ATP的假设， 激动剂在腔内和腔外均发生，并测试 缺氧和复氧对这个过程的影响。 3.探索EC释放ATP的方式并检验假设 受刺激的EC产生ATP与 机械地，与EC释放一氧化氮的能力。 4.评估胞外核苷二磷酸激酶和胞外 腺苷酸激酶在内皮细胞胞外产生ATP中的作用 缺氧和复氧对这些酶活性的影响。 我们将从生物化学，药理学， 分子、细胞生物学和显微镜方法， 血管、原代培养物中的细胞和亚细胞级分。

The goal of the proposed research is to explain the role of extracellular adenine nucleotides, particularly adenosine 5'- triphosphate (ATP), in normal cardiac endothelial cell (EC) function where release of factors that participate in the regulation of blood flow may be important in normal cardiac function and in cardiovascular disease. ECs have been shown to release ATP and autacoids such as endothelium-dependent relaxing factor and prostacyclin in response to various stimuli, including ATP itself. In addition, we propose that ECs can convert adenosine 5'-diphosphate (ADP) to ATP extracellularly to maintain the presence of ATP-mediated relaxation of the coronary and resistance vessels down-stream from the site of ATP release. We suggest that control of ATP appearance outside the EC and its eventual conversion to adenosine is an important component of regional blood flow control in the normal heart. As it is not clear how release of ATP or conversion of ADP to ATP by ECs is regulated, specific aims have been designed as follows: 1. Test the hypothesis that the release of ATP from ECs and the conversion of ADP to ATP by ECs can be measured in an intact coronary artery and that ATP release can support nucleoside triphosphate formation. Furthermore, to test the hypothesis that conversion of ADP to ATP can be inhibited in situ and determine the affect of hypoxia on extracellular generation of ATP. 2. Test the hypothesis that cardiac EC release of ATP in response to agonists occurs both luminally and abluminally and test the effect of hypoxia and re-oxygenation on this process. 3. Explore the manner in which ECs release ATP and test the hypothesis that the elaboration of ATP by stimulated ECs is associated, mechanistically, with the ability of ECs to release nitric oxide. 4. Assess the role of ecto-nucleoside diphosphate kinase and ecto- adenylate kinase in the extracellular production of ATP by ECs and test effects of hypoxia and reoxygenation on the activity of these enzymes. We will approach these aims with biochemical, pharmacological, molecular, cell biological and microscopic methods employing intact blood vessels, cells in primary culture and subcellular fractions.