Low-density lipoprotein (LDL) and its oxidized derivatives are hypothesized to impair vascular function by increasing superoxide anion (O.). To investigate mechanisms in situ, isolated carotid arteries were incubated with native LDL (nLDL) or minimally oxidized LDL (mmLDL). With the use of en face fluorescent confocal microscopy and hydroethidine, an oxidant-sensitive fluorescent probe, we found that nLDL increased O. in vascular endothelium greater than fourfold by an N(omega)-nitro-L-arginine methyl ester (L-NAME)-inhibitable mechanism. In contrast, mmLDL increased O. in vascular endothelium greater than eightfold by mechanisms that were partially inhibited by L-NAME and allopurinol and essentially ablated by diphenyleneiodium. These data indicate that both nLDL and mmLDL uncouple endothelial nitric oxide synthase (eNOS) activity and that mmLDL also activates xanthine oxidase and NADPH oxidoreductase to induce greater increases in O. generation than nLDL. Western analysis revealed that both lipoproteins inhibited A-23187-stimulated association of heat shock protein 90 (HSP90) with eNOS without inhibiting phosphorylation of eNOS at serine-1179 (phospho-eNOS), an immunological index of electron flow through the enzyme. As HSP90 mediates the balance of.NO and O. generation by eNOS, these data provide new insight into the mechanisms by which oxidative stress, induced by nLDL and mmLDL, uncouple eNOS activity to increase endothelial O. generation.
Flavin-containing monooxygenases (FMOs) are important oxidative drug metabolizing enzymes. FMO3 is the primary human adult liver FMO enzyme, but is developmentally regulated. FMO3 promoter characterization using in vitro DNA binding assays with HepG2 cell and fetal and adult liver nuclear protein, as well as FMO3/reporter construct transient expression in HepG2 cells, provided evidence for specific mechanisms contributing to both developmental and constitutive adult regulation. NFY, USF1, an unidentified GC box binding protein, and YY1 appear to play major roles regulating constitutive FMO3 transcription, while Pbx(2) as a heterodimer with an unidentified Hox isoform also may contribute to FMO3 developmental expression.
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