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.
