Activity of endothelial nitric oxide synthase: substrates, modulators and products

In this thesis, the activity of endothelial nitric oxide synthase (eNOS) and the effects of its substrates, modulators and products are described. eNOS produces nitric oxide (NO), which is involved in vascular biology. L-arginine and BH4 are both essential factors for adequate eNOS function. Reduced levels of the substrate or cofactor lead to the formation of superoxide by eNOS, a process referred to as eNOS uncoupling. In all studies described here, a microvascular endothelial cell line (bEnd.3) is used. These cells express high levels of eNOS but lack neuronal or inducible NOS. In addition, bEnd.3 cells produce relatively large amounts of NO, which facilitates the detection of subtle differences in NO production as a consequence of treatment with agonists or antagonists. Despite the high concentrations of NO produced by bEnd.3 cells, determining the exact NO concentration is a chalenge. In chapter 3, we describe that electron paramagnetic resonance (EPR) is an excellent tool to quantify the amount of NO produced by stimulated and unstimulated bEnd.3 cells. In chapter 4 it is shown that localisation of eNOS at cell-cell contact site proofs to be essential for eNOS activity. In contrast to earlier reports, we show in chapter 5 that addition of L-arginine leads to an increase in eNOS uncoupling. Addition of BH4 prevents eNOS uncoupling. In chapter 6 and 7 the effects of elevated levels of free fatty acids and hypoalbuminaemia, respectively, on eNOS activity, NO production and down-stream effectors are discussed. In the presence of high concentrations of free fatty acids, eNOS activity is decreased and mitochondrial ROS production is increased. Mitochondrial-derived ROS can induce BH4 oxidation, resulting in a shortage of the cofactor and subsequent eNOS uncoupling. Hypoalbuminemia, which is associated with increased risk of cardiovascular disease, results in increased eNOS activity and NO production in vitro. Endothelium-independent relaxation was markedly blunted in analbuminemic rats, while endothelium-dependent dilatation was slightly, but significantly, increased. This implies that in vivo hypoalbuminemia reduces vascular NO sensitivity. We show that low albumin as such seems to enhance, rather than diminish, eNOS-mediated endothelial NO production. In chapter 8, we show a marked increase in NO production under anoxic conditions, even though the normal arginine pathway of NO formation is blocked due to absence of oxygen. The anoxic release of NO is mediated by eNOS. This phenomenon is attributed to anoxic reduction of intracellular nitrite by eNOS. Its magnitude and duration suggests that nitrite reductase activity of eNOS is relevant for fast NO delivery in hypoxic vascular tissues. In chapter 9, the results presented in the previous chapters are discussed in a broader context. The importance of eNOS and its possible therapeutic benefits in the struggle against endothelial dysfunction and cardiovascular disease are discussed

Activity of endothelial nitric oxide synthase: substrates, modulators and products

In this thesis, the activity of endothelial nitric oxide synthase (eNOS) and the effects of its substrates, modulators and products are described. eNOS produces nitric oxide (NO), which is involved in vascular biology. L-arginine and BH4 are both essential factors for adequate eNOS function. Reduced levels of the substrate or cofactor lead to the formation of superoxide by eNOS, a process referred to as eNOS uncoupling. In all studies described here, a microvascular endothelial cell line (bEnd.3) is used. These cells express high levels of eNOS but lack neuronal or inducible NOS. In addition, bEnd.3 cells produce relatively large amounts of NO, which facilitates the detection of subtle differences in NO production as a consequence of treatment with agonists or antagonists. Despite the high concentrations of NO produced by bEnd.3 cells, determining the exact NO concentration is a chalenge. In chapter 3, we describe that electron paramagnetic resonance (EPR) is an excellent tool to quantify the amount of NO produced by stimulated and unstimulated bEnd.3 cells. In chapter 4 it is shown that localisation of eNOS at cell-cell contact site proofs to be essential for eNOS activity. In contrast to earlier reports, we show in chapter 5 that addition of L-arginine leads to an increase in eNOS uncoupling. Addition of BH4 prevents eNOS uncoupling. In chapter 6 and 7 the effects of elevated levels of free fatty acids and hypoalbuminaemia, respectively, on eNOS activity, NO production and down-stream effectors are discussed. In the presence of high concentrations of free fatty acids, eNOS activity is decreased and mitochondrial ROS production is increased. Mitochondrial-derived ROS can induce BH4 oxidation, resulting in a shortage of the cofactor and subsequent eNOS uncoupling. Hypoalbuminemia, which is associated with increased risk of cardiovascular disease, results in increased eNOS activity and NO production in vitro. Endothelium-independent relaxation was markedly blunted in analbuminemic rats, while endothelium-dependent dilatation was slightly, but significantly, increased. This implies that in vivo hypoalbuminemia reduces vascular NO sensitivity. We show that low albumin as such seems to enhance, rather than diminish, eNOS-mediated endothelial NO production. In chapter 8, we show a marked increase in NO production under anoxic conditions, even though the normal arginine pathway of NO formation is blocked due to absence of oxygen. The anoxic release of NO is mediated by eNOS. This phenomenon is attributed to anoxic reduction of intracellular nitrite by eNOS. Its magnitude and duration suggests that nitrite reductase activity of eNOS is relevant for fast NO delivery in hypoxic vascular tissues. In chapter 9, the results presented in the previous chapters are discussed in a broader context. The importance of eNOS and its possible therapeutic benefits in the struggle against endothelial dysfunction and cardiovascular disease are discussed

Nitric oxide synthase reduces nitrite to NO under anoxia.

Item does not contain fulltextCultured bEND.3 endothelial cells show a marked increase in NO production when subjected to anoxia, even though the normal arginine pathway of NO formation is blocked due to absence of oxygen. The rate of anoxic NO production exceeds basal unstimulated NO synthesis in normoxic cells. The anoxic release of NO is mediated by endothelial nitric oxide synthase (eNOS), can be abolished by inhibitors of NOS and is accompanied by consumption of intracellular nitrite. The anoxic NO release is unaffected by the xanthine oxidase inhibitor oxypurinol. The phenomenon is attributed to anoxic reduction of intracellular nitrite by eNOS, and its magnitude and duration suggests that the nitrite reductase activity of eNOS is relevant for fast NO delivery in hypoxic vascular tissues

Reduction enhances yields of nitric oxide trapping by iron-diethyldithiocarbamate complex in biological systems.

Item does not contain fulltextThe mechanism of NO trapping by iron-diethylthiocarbamate complexes was investigated in cultured cells and animal and plant tissues. Contrary to common belief, the NO radicals are trapped by iron-diethylthiocarbamates not only in ferrous but in ferric state also in the biosystems. When DETC was excess over endogenous iron ligands like citrate, ferric DETC complexes were directly observed with EPR spectroscopy at g=4.3. This was the case when isolated spinach leaves, endothelial cultured cells were incubated in the medium with 2.5mM DETC or mouse liver was perfused with 100mM DETC solution. After trapping NO, the nitrosylated Fe-DETC adducts are mostly in diamagnetic ferric state, with only a minor fraction having been reduced to paramagnetic ferrous state by endogenous biological reductants. In actual in vivo trapping experiments with mice, the condition of excess DETC was not met. The substantial quantities of iron in animal tissues were bound to ligands other than DETC, in particular citrate. These non-DETC complexes appear as roughly equal mixtures of ferric and ferrous iron. The presence of NO favors the replacement of non-DETC ligands by DETC. In all biological systems considered here, the nitrosylated Fe-DETC adducts appear as mixture of diamagnetic and paramagnetic states. The diamagnetic ferric nitrosyl complexes may be reduced ex vivo to paramagnetic form by exogenous reductants like dithionite. The trapping yields are significantly enhanced upon exogenous reduction, as proven by NO trapping experiments in plants, cell cultures and mice

Low albumin levels increase endothelial NO production and decrease vascular NO sensitivity

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