1-1-diethyl-2-hydroxy-2-nitrosohydrazine and Disease-Models--Animal

Hemolysis and consequent release of cell-free hemoglobin (CFHb) impair vascular nitric oxide (NO) bioavailability and cause oxidative and inflammatory processes. Hydroxyurea (HU), a common therapy for sickle cell disease (SCD), induces fetal Hb production and can act as an NO donor. We evaluated the acute inflammatory effects of intravenous water-induced hemolysis in C57BL/6 mice and determined the abilities of an NO donor, diethylamine NONOate (DEANO), and a single dose of HU to modulate this inflammation. Intravenous water induced acute hemolysis in C57BL/6 mice, attaining plasma Hb levels comparable to those observed in chimeric SCD mice. This hemolysis resulted in significant and rapid systemic inflammation and vascular leukocyte recruitment within 15 minutes, accompanied by NO metabolite generation. Administration of another potent NO scavenger (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) to C57BL/6 mice induced similar alterations in leukocyte recruitment, whereas hemin-induced inflammation occurred over a longer time frame. Importantly, the acute inflammatory effects of water-induced hemolysis were abolished by the simultaneous administration of DEANO or HU, without altering CFHb, in an NO pathway-mediated manner. In vitro, HU partially reversed the Hb-mediated induction of endothelial proinflammatory cytokine secretion and adhesion molecule expression. In summary, pathophysiological levels of hemolysis trigger an immediate inflammatory response, possibly mediated by vascular NO consumption. HU presents beneficial anti-inflammatory effects by inhibiting rapid-onset hemolytic inflammation via an NO-dependent mechanism, independently of fetal Hb elevation. Data provide novel insights into mechanisms of hemolytic inflammation and further support perspectives for the use of HU as an acute treatment for SCD and other hemolytic disorders.

Topics: Anemia, Sickle Cell; Animals; Cell Movement; Cyclic N-Oxides; Disease Models, Animal; Free Radical Scavengers; Hemoglobins; Hemolysis; Humans; Hydrazines; Hydroxyurea; Imidazoles; Inflammation; Leukocytes; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Nitric Oxide; Nitric Oxide Donors; Primary Cell Culture; Tumor Necrosis Factor-alpha; Viscosity; Water

Nitrite has become a topic of interest in the field of medical research because of its potential therapeutic role as an alternative source of nitric oxide (NO). While the bioconversion of nitrite to NO occurs via either nonenzymatic or enzymatic reduction under acidic or hypoxic conditions, little is known about its conversion to NO under normoxic conditions. Because of a recent report of aldehyde dehydrogenase 2 (ALDH2)-catalyzed glyceryl trinitrate (GTN) vasorelaxation by denitration of GTN to 1,2-glyceryl dinitrate (1,2-GDN) and nitrite, we therefore investigated a catalytic activity of ALDH2 for nitrite reduction and subsequent effect on N(ω)-nitro-l-arginine methyl ester (l-NAME)-induced hypertension in normoxic rat. Male Sprague-Dawley rats treated with l-NAME in drinking water for 3 weeks developed hypertension with significantly reduced plasma levels of nitrite and nitrate. The intravenous injection of sodium nitrite lowered the arterial pressure in a dose-dependent manner (17, 50 and 150 μmol/kg). Pretreatment with ALDH2 inhibitors (cyanamide and chloral hydrate) partially inhibited the hypotensive responses to sodium nitrite. In addition, cyanamide significantly delayed the nitrite clearance from plasma and most of the organs examined during the experimental period. These results suggest that ALDH2 may be at least in part involved in nitrite-mediated hypotensive effects and nitrite catalysis in many organs of normoxic rats.

Topics: Aldehyde Dehydrogenase; Aldehyde Dehydrogenase, Mitochondrial; Animals; Antihypertensive Agents; Blood Pressure; Disease Models, Animal; Dose-Response Relationship, Drug; Hydrazines; Hypertension; Male; Mitochondrial Proteins; NG-Nitroarginine Methyl Ester; Nitrates; Nitric Oxide; Nitroglycerin; Rats; Rats, Sprague-Dawley; Sodium Nitrite; Time Factors

Glucocorticoids (GC) remain the first choice of treatment in asthma, but GC therapy is not always effective and is associated with side effects. In a porcine study in our laboratory, simultaneous administration of GC and nitric oxide (NO) attenuated the endotoxin-induced inflammatory response and made GC treatment more effective than inhaled NO or steroids alone. In the present study, we aimed to further investigate the interactions between NO and GC treatment in two murine models of asthma. Inflammation was induced by endotoxin, ovalbumin, or a combination of both. With an animal ventilator and a forced oscillation method (FlexiVent), lung mechanics and airway reactivity to methacholine in response to various treatments were assessed. We also describe histology and glucocorticoid receptor (GR) protein expression in response to inhaled NO treatment [40 ppm NO gas or NO donors sodium nitroprusside (SNP) or diethylamine NONOate (DEA/NO)]. SNP and GC provided protection against bronchoconstriction to a similar degree in the model of severe asthma. When GC-treated mice were given SNP, maximum airway reactivity was further reduced. Similar effects were seen after DEA/NO delivery to GC-treated animals. Using 1-H-[1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (ODQ), a soluble guanylate cyclase inhibitor, we found this effect of NO donors to be mediated through a cGMP-independent mechanism. In the severe model, prolonged NO treatment restored or even increased the nuclear levels of GR. In conclusion, in our murine model of severe asthma GC treatment provided protection to only a limited degree against bronchoconstriction, while concomitant treatment with a NO donor was markedly more potent than the use of either NO or GC alone.

Topics: Administration, Inhalation; Animals; Anti-Asthmatic Agents; Asthma; Bronchial Provocation Tests; Bronchoconstriction; Bronchoconstrictor Agents; Cyclic GMP; Disease Models, Animal; Drug Therapy, Combination; Enzyme Inhibitors; Female; Glucocorticoids; Guanylate Cyclase; Hydrazines; Hydrocortisone; Injections, Intraperitoneal; Lipopolysaccharides; Methacholine Chloride; Mice; Mice, Inbred BALB C; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Ovalbumin; Oxadiazoles; Pneumonia; Quinoxalines; Receptors, Cytoplasmic and Nuclear; Receptors, Glucocorticoid; Respiration, Artificial; Respiratory Mechanics; Soluble Guanylyl Cyclase

Recent studies show that pulmonary vasodilator responses to nitrite are enhanced by hypoxia. However, the mechanism by which nitrite is converted to vasoactive nitric oxide (NO) is uncertain. In the present study, intravenous injections of sodium nitrite decreased pulmonary and systemic arterial pressures and increased cardiac output. The decreases in pulmonary arterial pressure were enhanced when tone in the pulmonary vascular bed was increased with U-46619. Under elevated tone conditions, decreases in pulmonary and systemic arterial pressures in response to nitrite were attenuated by allopurinol in a dose that did not alter responses to the NO donors, sodium nitroprusside and diethylamine/NO, suggesting that xanthine oxidoreductase is the major enzyme-reducing nitrite to NO. Ventilation with a 10% O(2) gas mixture increased pulmonary arterial pressure, and the response to hypoxia was enhanced by N(G)-nitro-l-arginine methyl ester and not altered by allopurinol. This suggests that NO formed by the endothelium and not from the reduction of plasma nitrite modulates the hypoxic pulmonary vasoconstrictor response. Although intravenous injections of sodium nitrite reversed pulmonary hypertensive responses to U-46619, hypoxia, and N(G)-nitro-l-arginine methyl ester, the pulmonary vasodilator response to nitrite was not altered by ventilation with 10% O(2) when baseline pulmonary arterial pressure was increased to similar values in animals breathing room air or the hypoxic gas. These data provide evidence that xanthine oxidoreductase is the major enzyme-reducing nitrite to vasoactive NO, and that this mechanism is not modified by hypoxia.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Allopurinol; Animals; Blood Pressure; Cardiac Output; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme Inhibitors; Hydrazines; Hypoxia; Injections, Intravenous; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroprusside; Oxypurinol; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Sodium Nitrite; Time Factors; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents; Xanthine Oxidase

Vascular tolerance to nitroglycerin (GTN) may be caused by impaired GTN bioactivation due to inactivation of mitochondrial aldehyde dehydrogenase (ALDH2). As relaxation to GTN is reduced but still sensitive to ALDH2 inhibitors in ascorbate deficiency, we compared the contribution of ALDH2 inactivation to GTN hyposensitivity in ascorbate deficiency and classical in vivo nitrate tolerance.. Guinea pigs were fed standard or ascorbate-free diet for 2 weeks. Reversibility was tested by feeding ascorbate-deficient animals standard diet for 1 week. Nitrate tolerance was induced by subcutaneous injection of 50 mg x kg(-1) GTN 4 times daily for 3 days. Ascorbate levels were determined in plasma, blood vessels, heart and liver. GTN-induced relaxation was measured as isometric tension of aortic rings; vascular GTN biotransformation was assayed as formation of 1,2- and 1,3-glyceryl dinitrate (GDN).. Two weeks of ascorbate deprivation had no effect on relaxation to nitric oxide but reduced the potency of GTN approximately 10-fold in a fully reversible manner. GTN-induced relaxation was similarly reduced in nitrate tolerance but not further attenuated by ALDH inhibitors. Nitrate tolerance reduced ascorbate plasma levels without affecting ascorbate in blood vessels, liver and heart. GTN denitration was significantly diminished in nitrate-tolerant and ascorbate-deficient rings. However, while the approximately 10-fold preferential 1,2-GDN formation, indicative for active ALDH2, had been retained in ascorbate deficiency, selectivity was largely lost in nitrate tolerance.. These results indicate that nitrate tolerance is associated with ALDH2 inactivation, whereas ascorbate deficiency possibly results in down-regulation of ALDH2 expression.

Topics: Aldehyde Dehydrogenase; Animals; Ascorbic Acid; Ascorbic Acid Deficiency; Biotransformation; Chloral Hydrate; Disease Models, Animal; Dose-Response Relationship, Drug; Down-Regulation; Drug Tolerance; Enzyme Activation; Enzyme Inhibitors; Female; Guinea Pigs; Hydrazines; Injections, Subcutaneous; Isoflavones; Male; Nitric Oxide; Nitric Oxide Donors; Nitroglycerin; Time Factors; Vasodilation; Vasodilator Agents

Nitric oxide (NO) synthesis is up-regulated in inflammatory bowel disease. However, its role in the pathophysiology of this condition is controversial. The aims of this study were to assess whether nitric oxide administration ameliorates experimental colitis and to determine the possible mechanisms underlying its effects on intestinal inflammation. For this purpose, the NO donor diethylamine NONOate (DETA/NO; 0.01, 0.1, 1, 5, or 10 mg/kg/day), or the DETA moiety, was administered daily to mice with dextran sulfate sodium-induced colitis. Daily body weight and colonic pathologic alterations at Day 10 were determined. Leukocyte endothelial cell interactions in colonic venules were assessed with intravital microscopy, and expression of endothelial cell adhesion molecules was determined using radiolabeled antibodies. IL-12 and IFN-gamma production were measured in intestinal tissue. Colitis induced a significant loss of body weight, reduction of colon length, and increase in colon weight and myeloperoxidase activity. Administration of 1 mg/kg/day DETA/NO significantly attenuated these pathologic changes. The marked increase in leukocyte rolling and adhesion in colonic venules of colitic mice were significantly reduced by administration of 1 mg/kg/day DETA/NO. Development of colitis was associated with a marked increase in endothelial expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and P-selectin. Supplementation with NO significantly attenuated the up-regulation of endothelial intercellular adhesion molecule-1 and P-selectin, but not vascular cell adhesion molecule-1, in colonic tissue. NO abrogated the increase in IL-12 and IFN-gamma mRNA expression in the colon of colitic mice. The DETA moiety alone did not have any effect on any of the parameters studied. In conclusion, exogenous NO supplementation significantly ameliorates dextran sulfate sodium-induced colitis. This effect is related to a reduction in leukocyte recruitment and proinflammatory cytokine production.

Topics: Animals; Body Weight; Cell Adhesion; Cell Adhesion Molecules; Colitis; Colon; Dextran Sulfate; Disease Models, Animal; Dose-Response Relationship, Drug; Epithelium; Hydrazines; Interferon-gamma; Interleukin-12; Leukocytes; Male; Mice; Mice, Inbred Strains; Nitric Oxide Donors; Nitrogen Oxides; Peroxidase; RNA, Messenger; Venules