NITRIC OXIDE AND PULMONARY ARTERY ENDOTHELIAL CELL FUNCTION

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Pulmonary vascular endothelial cells are exposed to nitric oxide (NO.) from both exogenous environmental sources and from endogenous cellular sources. Numerous cellular responses to NO. are possible ranging from signalling events related to elevation of cGMP to cytostasis and cytotoxicity. These responses depend on both the concentration of NO. and the length of exposure. In general, transient activation of NO. synthase (NOS) leads to signalling responses while prolonged NO. production following induction of NOS (inflammation) or prolonged exposure to NO. in the environment is associated with dramatic alterations in cellular function and potentially cell death. NO. is a redox active molecule which under aerobic conditions can react with and modify protein sulfhydryls, enzyme iron/sulfur complexes, protein heme iron, and other reactive groups. Recent evidence in the literature and our own preliminary data suggests that NO modifies protein sulfhydryls, particularly those with low pKa's which are associated with the active site of many proteins including dehydrogenases. In this project, we will determine the mechanisms through which NO. modifies active site sulfhydryls in dehydrogenases with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the prototypical enzyme. GAPDH is particularly important in this regard because this enzyme is key for cell viability in endothelial cells since these cells are depend almost exclusively on glycolysis for ATP generation. In other cells types, oxidative inhibition of GAPDH by mediators other than NO. is associated with cell death and involves oxidation of the active site sulfhydryl. Recent work by others using isolated enzyme preparations has established GAPDH as a potential target for NO. modification presumably at the same sulfhydryl. The exact nature of the modification or whether modifications can occur in intact cells is not known. Moreover, the role of cellular antioxidant systems such as the glutathione redox cycle, glutaredoxin, and thioredoxin, in regulating the extent of NO.-induce injury is not known. Our hypothesis is that NO. inhibits GAPDH activity in intact cells via mechanisms similar to those that occur in isolated enzyme preparations and that the extent of inhibition is regulated by the redox status of the cell. Our approach will be to determine mechanisms by which NO. inhibits GAPDH, investigate the reversibility of inhibition and investigate the role of cellular redox mechanisms in regulating these events. Experiments will be carried out using isolated enzyme systems and intact pulmonary artery endothelial cells.