Nitric oxide (NO) is an unusual messenger molecule with diverse roles. NO is made by NO synthase (NOS) which converts arginine and oxygen into citrulline and NO. Several isoforms of NOS which converts arginine and oxygen into citrulline and NO. Several isoforms of NOS exist, including neuronal NOS, endothelial NOS, and macrophage NOS. NO serves many functions: neurons secrete it as a neurotransmitter, endothelial cells release it as a vasodilator, and macrophages produce it during inflammation as an effector molecule which can kill pathogens and tumor cells. NO may have an important role in post-ischemic myocardial inflammation. NO reduced leukocyte adherence to endothelial cells and inhibits superoxide production by activated leukocytes; and exogenous NO reduces infarct size. However, NO can damage cells. This proposal explores the roles of NO during cardiac ischemia, reperfusion, and post-ischemic inflammation, focusing on the regulation of the enzyme that makes NO. To explore the effects of oxygen upon NOS, first the enzymology of purified NOS isoforms will be measured by a NOS catalytic assay as oxygen partial pressures are varied. To determine its transcriptional regulation, NOS mRNA, and NOS protein will be assayed by Northern and Western blots in endothelial cells or macrophages grown in vitro under hypoxic and reoxygenation conditions. Post-translational modification of NOS by phosphorylation and subcellular localization will be assessed in vitro as well by phosphopeptide mapping. To show which cells in the myocardium can produce NO, sections from in vivo ischemic or reperfused hearts will be analyzed by immunohistochemistry. These results will show how NOS is regulated normally in endothelial cells, and how it might be induced in inflammatory cells during inflammatory cells during inflammation following ischemia. To further characterize the regulation of NOS in coronary arteries, the regulatory region of the endothelial cell NOS gene will be isolated. The transcriptional regulation of the endothelial NOS gene will be analyzed by transfection of deletions of regulatory region during hypoxic conditions. Footprinting and gel-shift assays will define the precise DNA sequences responsible for inducing endothelial NOS transcription. If the transcription of endothelial NOS is regulated by the oxygen partial pressure, then the regulatory sites where oxygen-sensing transcription factors bind will be isolated. These experiments will test the hypothesis that NOS activity and expression change in the heart during ischemia and post-ischemic inflammation. These changes, it is postulated, are mediated by the oxygen partial pressure, NOS co-factor levels, calcium concentrations, phosphorylation, and by oxygen- sensing transcription factors. The NOS regulatory region will be isolated to determine its transcriptional regulation. These studies will define the different levels of regulation of NOS.