Cardiovascular disease remains the number one killer of both men and women in the United States. To date, research efforts have been directed at understanding the development of coronary artery disease and its role in myocardial ischemia, injury, and infarction, which are due to alterations in coronary blood flow. The investigators will attempt to characterize the mechanism that results in the regulation of coronary artery blood flow in normal physiological states and may eventually provide the potential for increasing coronary blood flow during compromised cardiovascular states. Specifically, they will investigate the extracellular mediators that may be involved in this regulation of coronary artery blood flow. The mediators to be examined are the adenyl purines (i.e., ATP, ADP, and adenosine). It is hypothesized that the cardiac endothelial cells can enzymatically generate extracellular ATP to act in the blood vessel as an autocrine or paracrine hormone. The proposed research will: 1) determine whether human coronary artery endothelial cells can enzymatically generate extracellular ATP; 2) characterize the ecto-kinase(s) associated with the plasma membrane of human coronary artery endothelial cells; and 3) inhibit the function of the ecto-kinase(s) associated with the plasma membrane of human coronary artery endothelial cells. The investigators will use human coronary artery endothelial cells obtained from the Duke Comprehensive Cancer Center Cell Culture Facility, which obtain their cells from Clonetics or ATCC. The generation of ATP will be measured by HPLC radioactive determination and chemiluminesecence. Protein determination and identification will be accompanied with SDS-PAGE and Western blot technique. Antisense oligonucleotides will be utilized to inhibit the function of the ecto-kinase(s). The data obtained will be analyzed using the Student's t-test and repeated measures ANOVA. The results will form the basis for future in vivo studies and intervention research.
