Endothelial nitric oxide synthase (eNOS) modification by hypochlorous acid and the development of a hypochlorous acid oxidation assay

Oxidative stress is an important contributor in the pathogenesis and progression of vascular complications, diabetes, hypercholesterolemia and atherosclerosis. A manifestation of vascular oxidative stress is endothelial dysfunction, characterized by a decline in nitric oxide (NO) levels, due to the inactivation of the enzyme endothelial nitric oxide synthase (eNOS). One oxidant with a major role during inflammation is hypochlorous acid (HOCl), a potent 2-electron oxidant produced by the macrophage enzyme myeloperoxidase (MPO). HOCl has numerous biological targets and has been repeatedly reported to modify proteins in atherosclerotic lesions and endothelial cells. This thesis focuses on (1) the implications of HOC1 on the function and structure of eNOS in cultured endothelial cells and (2) the development of an in vitro assay to test for HOCl oxidation that can be applied to screening HOCl scavengers.;The effects of HOCl on eNOS dimer stability, phosphorylation status and interactions with HSP90 were determined. The results demonstrate that HOCl at high concentrations (100muM) destabilizes eNOS, an effect reversible with sepiapterin. At moderate concentrations (50muM) of HOCl, increased eNOS Ser-1179 phosphorylation and HSP90 association were observed. Moreover, HOCl modulated the MAPK signaling pathway in endothelial cells by upregulating ERK and p38 phosphorylation. Mass spectrometry analysis of HOCl treated eNOS revealed signs of protein oxidation and unfolding.;A straightforward and quantifiable assay in a 96-well microtiter plate format was developed to assess HOCl-induced oxidation. The method uses 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) that allows a lower limit of detection and higher sensitivity than the standard BSA protein carbonyl formation assay. HOCl from 1 to 250muM induced a robust and dramatic linear increase in DCFH-DA fluorescence. Thiol-containing amino acids known to inhibit HOCl-induced oxidation were also effective at inhibiting DCFH-DA fluorescence and HOCl mediated cell death. Analysis with UV/VIS spectroscopy indicated that DCFH-DA is specific for HOCl over H 2O2 and NO. Finally, mass spectrometry confirmed that HOCl resulted in chemical modifications to DCFH-DA with the production of novel products. The described DCF assay can be applied for screening small molecules with HOCl inhibitory effects in the treatment of inflammatory diseases.