Low molecular mass dinitrosyl nonheme-iron complexes up-regulate noradrenaline release in the rat tail artery

BACKGROUND: Dinitrosyl nonheme-iron complexes can appear in cells and tissues overproducing nitric oxide. It is believed that due to their chemical nature these species may be implicated in certain pathophysiological events. We studied the possible role of low molecular mass dinitrosyl iron complexes in the control of noradrenaline release in electrically stimulated rat tail artery. RESULTS: A model complex, dinitrosyl-iron-thiosulfate (at 1–10 μM) produced a concentration-dependent enhancement of electrical field stimulated [(3)H]noradrenaline release (up to 2 fold). At the same time, dinitrosyl-iron-thiosulfate inhibited neurogenic vasoconstriction, consistent with its nitric oxide donor properties. A specific inhibitor of cyclic GMP dependent protein kinase, Rp-8pCPT-cGMPS, partially inhibited the effect of dinitrosyl-iron-thiosulfate on neurogenic vasoconstriction, but not on [(3)H]noradrenaline release. Another model complex, dinitrosyl-iron-cysteine (at 3 μM) elicited similar responses as dinitrosyl-iron-thiosulfate. Conventional NO and NO+ donors such as sodium nitroprusside, S-nitroso-L-cysteine or S-nitroso-glutathione (at 10 μM) had no effect on [(3)H]noradrenaline release, though they potently decreased electrically-induced vasoconstriction. The "false complex", iron(II)-thiosulfate showed no activity. CONCLUSIONS: Low molecular mass iron dinitrosyl complexes can up-regulate the stimulation-evoked release of vascular [(3)H]noradrenaline, apparently independently of their NO donor properties. This finding may have important implications in inflammatory tissues

Trans Fatty Acids Induce Vascular Inflammation and Reduce Vascular Nitric Oxide Production in Endothelial Cells

Intake of trans fatty acids (TFA), which are consumed by eating foods made from partially hydrogenated vegetable oils, is associated with a higher risk of cardiovascular disease. This relation can be explained by many factors including TFA's negative effect on endothelial function and reduced nitric oxide (NO) bioavailability. In this study we investigated the effects of three different TFA (2 common isomers of C18 found in partially hydrogenated vegetable oil and a C18 isomer found from ruminant-derived—dairy products and meat) on endothelial NF-κB activation and nitric oxide (NO) production. Human endothelial cells were treated with increasing concentrations of Elaidic (trans-C18:1 (9 trans)), Linoelaidic (trans-C18:2 (9 trans, 12 trans)), and Transvaccenic (trans-C18:1 (11 trans)) for 3 h. Both Elaidic and Linoelaidic acids were associated with increasing NF-κB activation as measured by IL-6 levels and phosphorylation of IκBα, and impairment of endothelial insulin signaling and NO production, whereas Transvaccenic acid was not associated with these responses. We also measured superoxide production, which has been hypothesized to be necessary in fatty acid-dependent activation of NF-κB. Both Elaidic acid and Linoelaidic acid are associated with increased superoxide production, whereas Transvaccenic acid (which did not induce inflammatory responses) did not increase superoxide production. We observed differential activation of endothelial superoxide production, NF-κB activation, and reduction in NO production by different C18 isomers suggesting that the location and number of trans double bonds effect endothelial NF-κB activation

AMP-activated protein kinase activation and NADPH oxidase inhibition by inorganic nitrate and nitrite prevent liver steatosis

Advanced age and unhealthy dietary habits contribute to the increasing incidence of obesity and type 2 diabetes. These metabolic disorders, which are often accompanied by oxidative stress and compromised nitric oxide (NO) signaling, increase the risk of adverse cardiovascular complications and development of fatty liver disease. Here, we investigated the therapeutic effects of dietary nitrate, which is found in high levels in green leafy vegetables, on liver steatosis associated with metabolic syndrome. Dietary nitrate fuels a nitrate–nitrite–NO signaling pathway, which prevented many features of metabolic syndrome and liver steatosis that developed in mice fed a high-fat diet, with or without combination with an inhibitor of NOS (L-NAME). These favorable effects of nitrate were absent in germ-free mice, demonstrating the central importance of host microbiota in bioactivation of nitrate. In a human liver cell line (HepG2) and in a validated hepatic 3D model with primary human hepatocyte spheroids, nitrite treatment reduced the degree of metabolically induced steatosis (i.e., high glucose, insulin, and free fatty acids), as well as drug-induced steatosis (i.e., amiodarone). Mechanistically, the salutary metabolic effects of nitrate and nitrite can be ascribed to nitrite-derived formation of NO species and activation of soluble guanylyl cyclase, where xanthine oxidoreductase is proposed to mediate the reduction of nitrite. Boosting this nitrate–nitrite–NO pathway results in attenuation of NADPH oxidase-derived oxidative stress and stimulation of AMP-activated protein kinase and downstream signaling pathways regulating lipogenesis, fatty acid oxidation, and glucose homeostasis. These findings may have implications for novel nutrition-based preventive and therapeutic strategies against liver steatosis associated with metabolic dysfunction.</p

eNOS Protects from Atherosclerosis Despite Relevant Superoxide Production by the Enzyme in apoE−/− Mice

All three nitric oxide synthase (NOS) isoforms are expressed in atherosclerotic plaques. NOS enzymes in general catalyse NO production. However, under conditions of substrate and cofactor deficiency, the enzyme directly catalyse superoxide formation. Considering this alternative chemistry, the effects of NOS on key events in spontaneous hyperlipidemia driven atherosclerosis have not been investigated yet. Here, we evaluate how endothelial nitric oxide synthase (eNOS) modulates leukocyte/endothelial- (L/E) and platelet/endothelial- (P/E) interactions in atherosclerosis and the production of nitric oxide (NO) and superoxide by the enzyme. Intravital microscopy (IVM) of carotid arteries revealed significantly increased L/E-interactions in apolipoproteinE/eNOS double knockout mice (apoE(-/-)/eNOS(-/-)), while P/E-interactions did not differ, compared to apoE(-/-). eNOS deficiency increased macrophage infiltration in carotid arteries and vascular cell adhesion molecule-1 (VCAM-1) expression, both in endothelial and smooth muscle cells. Despite the expression of other NOS isoforms (inducible NOS, iNOS and neuronal NOS, nNOS) in plaques, Electron Spin Resonance (ESR) measurements of NO showed significant contribution of eNOS to total circulating and vascular wall NO production. Pharmacological inhibition and genetic deletion of eNOS reduced vascular superoxide production, indicating uncoupling of the enzyme in apoE(-/-) vessels. Overt plaque formation, increased vascular inflammation and L/E- interactions are associated with significant reduction of superoxide production in apoE(-/-)/eNOS(-/-) vessels. Therefore, lack of eNOS does not cause an automatic increase in oxidative stress. Uncoupling of eNOS occurs in apoE(-/-) atherosclerosis but does not negate the enzyme's strong protective effects

A multi-level analytical approach for detection and visualization of intracellular NO production and nitrosation events using diaminofluoresceins

Diaminofluoresceins are widely used probes for detection and intracellular localization of NO formation in cultured/isolated cells and intact tissues. The fluorinated derivative, 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM), has gained increasing popularity in recent years due to its improved NO-sensitivity, pH-stability, and resistance to photo-bleaching compared to the first-generation compound, DAF-2. Detection of NO production by either reagent relies on conversion of the parent compound into a fluorescent triazole, DAF-FM-T and DAF-2-T, respectively. While this reaction is specific for NO and/or reactive nitrosating species, it is also affected by the presence of oxidants/antioxidants. Moreover, the reaction with other molecules can lead to the formation of fluorescent products other than the expected triazole. Thus additional controls and structural confirmation of the reaction products are essential. Using human red blood cells as an exemplary cellular system we here describe robust protocols for the analysis of intracellular DAF-FM-T formation using an array of fluorescence-based methods (laser-scanning fluorescence microscopy, flow cytometry and fluorimetry) and analytical separation techniques (reversed-phase HPLC and LC-MS/MS). When used in combination, these assays afford unequivocal identification of the fluorescent signal as being derived from NO and are applicable to most other cellular systems without or with only minor modifications

Novel activation of non-selective cationic channels by dinitrosyl iron–thiosulfate in PC12 cells

Low molecular mass dinitrosyl iron complexes (DNICs) are nitrosating agents and it is known that the dinitrosyl iron moiety can be transferred to proteins. The aim of the present study was to determine if the formation of protein-bound dinitrosyl iron can modulate ionic channel activity.In PC12 cells, dinitrosyl iron-thiosulfate (50 μM) caused irreversible activation of a depolarizing inward current (IDNIC). IDNIC was partially inhibited by the metal chelator diethyldithiocarbamate (DETC, 1 mM), but not by the reducing/denitrosylating agent dithiothreitol (DTT, 5 mM).The activation of IDNIC was not reproduced by application of nitric oxide (NO·, 100 μM), S-nitrocysteine (200 μM) or ferrous iron-thiosulfate (50 μM), and was not prevented by the irreversible guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 1 μM). Similarly, intracellular perfusion of dinitrosyl iron-thiosulfate (100 μM) did not result in activation of IDNIC.Ion replacement experiments show that the DETC-sensitive component of IDNIC is a non-selective cationic current. In accordance, IDNIC was blocked by antagonists of receptor-operated calcium entry, gadolinium (25 μM) and SK&F 96365 (25 μM).Single-channel measurements from outside-out patches reveal that the DETC-sensitive component of IDNIC is an inward current carried by a cationic channel having a conductance of 50 pS.The present observations suggest that the formation of ion channel-bound dinitrosyl iron represents another mechanism of regulation of ion channel activity by NO·-related species, which may be particularly important in pathophysiological processes where NO· is overproduced