Ischemia and reperfusion trigger the rapid release of endothelial granules containing mediators of vascular[unreadable] inflammation and thrombosis, including P-selectin, interleukin-8, and von Willebrand factor. P-selectin is[unreadable] translocated from the interior of the endothelial cell to the exterior surface, where it mediates leukocyte[unreadable] adherence to the endothelial wall. Interleukin-8 is released into the blood, where it can activate leukocytes,[unreadable] increasing the interactions between leukocytes and endothelial cells. Endothelial exocytosis is thus an early step[unreadable] in leukocyte trafficking into the ischemic myocardium.[unreadable] The over-all goal of this project is to characterize the mechanisms by which exocytosis of endothelial granules[unreadable] leads to injury in the post-ischemic myocardium. We have previously identified components of the exocytic[unreadable] machinery of endothelial cells. We subsequently discovered that nitric oxide inhibits vascular inflammation by[unreadable] inhibiting specific proteins that mediate exocytosis. We then developed a novel polypeptide that interferes with[unreadable] the exocytic machinery, inhibits exocytosis, and decreases vascular inflammation. We now propose to extend[unreadable] these studies to explore the mechanisms of exocytosis in post-ischemic myocardium.[unreadable] We first plan to study how hypoxia activates exocytosis. We will focus on the role of signaling[unreadable] intermediates such as calcineurin which regulate exocytosis by post-translational modifications of the exocytic[unreadable] machinery. We will then characterize the role of one particular component of the exocytic machinery in postischemic[unreadable] inflammation. We will finally define a novel mechanism which regulates a major stress response[unreadable] pathway in hypoxic endothelial cells. Each of these aims includes a wide range of studies, from recombinant[unreadable] proteins to transduced cells to animal experiments. These studies will increase our understanding of the[unreadable] molecular mechanisms regulating endothelial exocytosis, and will lead to novel therapies that inhibit[unreadable] inflammation and injury to the post-ischemic myocardium.