2-2--(hydroxynitrosohydrazono)bis-ethanamine and Hypoxia

Soluble guanylyl cyclase (sGC) is a heterodimer. The dimerization of the enzyme is obligatory for its function in mediating actions caused by agents that elevate cyclic guanosine monophosphate (cGMP). The present study aimed to determine whether sGC dimerization is modulated by thiol-reducing agents and whether its dimerization influences relaxations in response to nitric oxide (NO).. The dimers and monomers of sGC and cGMP-dependent protein kinase (PKG) were analysed by western blotting. The intracellular cGMP content was measured by enzyme-linked immunosorbent assay. Changes in isometric tension were determined in organ chambers. In isolated porcine coronary arteries, the protein levels of sGC dimer were decreased by the thiol reductants dithiothreitol, l-cysteine, reduced l-glutathione and tris(2-carboxyethyl) phosphine. The effect was associated with reduced cGMP elevation and attenuated relaxations in response to nitric oxide donors. The dimerization of sGC and activation of the enzyme were also decreased by dihydrolipoic acid, an endogenous thiol antioxidant. Dithiothreitol at concentrations markedly affecting the dimerization of sGC had no significant effect on the dimerization of PKG or relaxation in response to 8-Br-cGMP. Relaxation of the coronary artery in response to a NO donor was potentiated by hypoxia when sGC was partly inhibited, coincident with an increase in sGC dimer and enhanced cGMP production. These effects were prevented by dithiothreitol and tris(2-carboxyethyl) phosphine.. These results demonstrate that the dimerization of sGC is exquisitely sensitive to thiol reductants compared with that of PKG, which may provide a novel mechanism for thiol-dependent modulation of NO-mediated vasodilatation in conditions such as hypoxia.

Topics: Animals; Coronary Vessels; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Dimerization; Dithiothreitol; Female; Guanylate Cyclase; Hypoxia; In Vitro Techniques; Male; Nitric Oxide; Nitroso Compounds; Protein Structure, Quaternary; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase; Sulfhydryl Reagents; Sus scrofa; Vasodilation

Hypoxia/reoxygenation (H/R) in vitro induced cerebral endothelial dysfunction is mediated by superoxide production. However, the intracellular pathways involved remain unclear. The present study was designed to investigate the involvement of Rho-kinase and its interaction with nitric oxide (NO) in cerebral endothelial dysfunction after H/R. Arterial diameter and intraluminal pressure were simultaneously measured in vitro on rat posterior cerebral arteries. Vascular NO production was determined by measuring stable NO metabolites nitrate/nitrite. H/R selectively inhibited cerebral vasodilation to the endothelium-dependent agonist acetylcholine (ACh, 0.01 to 10 micromol/L) and caused NO deficiency. H/R-impaired vasodilation to ACh was reversed by Y27632 (1 micromol/L), a specific inhibitor of Rho-kinase, but not by chelerythrine (1 micromol/L), a selective inhibitor of protein kinase C. Y27632 had no protective effect in the presence of N-nitro-L-arginine methyl ester (L-NAME; 100 micromol/L), a specific endothelial NO synthase inhibitor. L-NAME (100 micromol/L) alone failed to modulate H/R-impaired vasodilation, so did L-arginine (3 mmol/L), a substrate for NO synthase. However, a stable NO donor diethylenetetra amine-NONOate (5 micromol/L) normalized H/R-impaired dilation to ACh. In conclusion, H/R-induced endothelial dysfunction is associated with activation of Rho-kinase-dependent pathway and NO deficiency. Pretreatment with either Y27632 or the stable NO donor profoundly prevented H/R-mediated cerebral endothelial dysfunction.

Topics: Acetylcholine; Alkaloids; Amides; Animals; Benzophenanthridines; Cerebral Arteries; Endothelium, Vascular; Enzyme Inhibitors; Hypoxia; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Male; Muscle, Smooth, Vascular; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Oxygen; Phenanthridines; Protein Serine-Threonine Kinases; Pyridines; Rats; Rats, Sprague-Dawley; rho-Associated Kinases; Superoxides; Triazenes; Vasodilation; Vasodilator Agents

The role of nitric oxide (NO) and reactive oxygen species (ROS) in regulating capillary perfusion was studied in the hamster cheek pouch model during normoxia and after 20 min of exposure to 10% O2-90% N2. We measured PO2 by using phosphorescence quenching microscopy and ROS production in systemic blood. Identical experiments were performed after treatment with the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA) and after the reinfusion of the NO donor 2,2'-(hydroxynitrosohydrazono)bis-etanamine (DETA/NO) after treatment with L-NMMA. Hypoxia caused a significant decrease in the systemic PO2. During normoxia, arteriolar intravascular PO2 decreased progressively from 47.0 +/- 3.5 mmHg in the larger arterioles to 28.0 +/- 2.5 mmHg in the terminal arterioles; conversely, intravascular PO2 was 7-14 mmHg and approximately uniform in all arterioles. Tissue PO2 was 85% of baseline. Hypoxia significantly dilated arterioles, reduced blood flow, and increased capillary perfusion (15%) and ROS (72%) relative to baseline. Administration of L-NMMA during hypoxia further reduced capillary perfusion to 47% of baseline and increased ROS to 34% of baseline, both changes being significant. Tissue PO2 was reduced by 33% versus the hypoxic group. Administration of DETA/NO after L-NMMA caused vasodilation, normalized ROS, and increased capillary perfusion and tissue PO2. These results indicate that during normoxia, oxygen is supplied to the tissue mostly by the arterioles, whereas in hypoxia, oxygen is supplied to tissue by capillaries by a NO concentration-dependent mechanism that controls capillary perfusion and tissue PO2, involving capillary endothelial cell responses to the decrease in lipid peroxide formation controlled by NO availability during low PO2 conditions.

Topics: Animals; Capillaries; Cricetinae; Enzyme Inhibitors; Hypoxia; Male; Mesocricetus; Mouth Mucosa; Nitric Oxide; Nitric Oxide Donors; omega-N-Methylarginine; Oxygen; Reactive Oxygen Species; Triazenes

The activity of hypoxia-inducible factor 1 (HIF-1) is primarily determined by stability regulation of its alpha subunit, which is stabilized under hypoxia but degraded during normoxia. Hydroxylation of HIF-1alpha by prolyl hydroxylases (PHDs) recruits the von Hippel-Lindau (pVHL) E3 ubiquitin ligase complex to initiate proteolytic destruction of the alpha subunit. Hypoxic stabilization of HIF-1alpha has been reported to be antagonized by nitric oxide (NO). By using a HIF-1alpha-pVHL binding assay, we show that NO released from DETA-NO restored prolyl hydroxylase activity under hypoxia. Destabilization of HIF-1alpha by DETA-NO was reversed by free radical scavengers such as NAC and Tiron, thus pointing to the involvement of reactive oxygen species (ROS). Therefore, we examined the effects of ROS on HIF-1alpha stabilization. Treatment of cells under hypoxia with low concentrations of the superoxide generator 2,3-dimethoxy-1,4-naphthoquinone lowered HIF-1alpha protein stabilization. In vitro HIF-1alpha-pVHL interaction assays demonstrated that low-level ROS formation increased prolyl hydroxylase activity, an effect antagonized by ROS scavengers. While determining intracellular ROS formation we noticed that reduced ROS production under hypoxia was restored by the addition of DETA-NO. We propose that an increase in ROS formation contributes to HIF-1alpha destabilization by NO donors under hypoxia via modulation of PHD activity.

Topics: 1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; Acetylcysteine; Carcinoma, Hepatocellular; Cell Line; Humans; Hydroxylation; Hypoxia; Naphthoquinones; Nitric Oxide; Nitric Oxide Donors; Procollagen-Proline Dioxygenase; Reactive Oxygen Species; Triazenes; Tumor Cells, Cultured; Von Hippel-Lindau Tumor Suppressor Protein

Excitation-contraction coupling is modulated by nitric oxide (NO) which otherwise has either beneficial or detrimental effects on myocardial function during hypoxia-reoxygenation. This work aimed at characterizing the variations of electromechanical delay (EMD) induced by anoxia-reoxygenation within the developing heart and determining whether atrial and ventricular EMD are modulated by NO to the same extent.. Hearts of 4 or 4.5-day-old chick embryos were excised and submitted in vitro to normoxia (45 min), anoxia (30 min) and reoxygenation (60 min). Electrocardiogram and atrial and ventricular contractions were simultaneously recorded throughout experiment. Anoxia-reoxygenation-induced chrono-, dromo-and inotropic disturbances and changes in EMD in atrium (EMDa) and ventricle (EMDv) were investigated in control hearts and in hearts exposed to 0.1, 1, 10, 50 and 100 microM of DETA-NONOate (a NO donating agent) or to 50 microM of L-NAME (a NOS inhibitor).. Under normoxia, heart rate, PR interval, ventricular shortening velocity, EMDa and EMDv were similar in control, L-NAME-treated and DETA-NONOate-treated hearts. Under anoxia, cardiac activity became markedly erratic within less than 10 min in all groups. At the onset of reoxygenation, EMDv was increased by about 300% with respect to the preanoxic value while EMDa did not vary significatively. Compared to control conditions, L-NAME or DETA-NONOate had no influence on the negative chrono-, dromo- and inotropic effects induced by anoxia-reoxygenation. However, L-NAME prolonged EMDv during anoxia and delayed EMDv recovery during reoxygenation while 100 microM DETA-NONOate had the opposite effects. EMDa was neither affected by NOS inhibitor nor NO donor. At the end of reoxygenation, all the investigated parameters returned to their basal values.. This work provides evidence that a NO-dependent pathway is involved in regulation of the ventricular excitation-contraction coupling in the anoxic-reoxygenated developing heart.

Topics: Animals; Chick Embryo; Chickens; Electrocardiography; Electrophysiology; Heart; Heart Atria; Heart Ventricles; Hypoxia; In Vitro Techniques; Myocardial Contraction; Myocardium; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitroso Compounds; Oxygen; Reperfusion Injury; Signal Transduction; Time Factors

Nitric oxide (NO) shows cytotoxicity, and its reaction products with reactive oxygen species, such as peroxynitrite, are potentially more toxic. To examine the role of O2 in the NO toxicity, we have examined the proliferation of cultured human umbilical vein endothelial cells in the presence or absence of NO donor, ((Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-++ +ium-1,2-diolate) (DETA-NONOate) (100-500 microM), under normoxia (air), hypoxia (< 0.04% O2) or hyperoxia (88-94% O2). It was found that the dose dependency on NONOate was little affected by the ambient O2 concentration, showing no apparent synergism between the two treatments. We have also examined the effects of exogenous NO under normoxia and hyperoxia on the cellular activities of antioxidant enzymes involved in the H2O2 elimination, since many of them are known to be inhibited by NO or peroxynitrite in vitro. Under normoxia DETA-NONOate (500 microM) caused 25% decrease in catalase activity and 30% increases in glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities in 24h. Under hyperoxia NO caused about 25% decreases in activities of catalase, glutathione reductase and glucose-6-phosphate dehydrogenase. The H2O2 removal rate by NO-treated cells was computed on the mathematical model for the enzyme system. It was concluded that the cellular antioxidant function is little affected by NO under normoxia but that it is partially impaired when the cells are exposed to NO under hyperoxia.

Topics: Catalase; Cell Division; Cells, Cultured; Endothelium, Vascular; Glucosephosphate Dehydrogenase; Glutathione Peroxidase; Glutathione Reductase; Humans; Hyperoxia; Hypoxia; Nitric Oxide Donors; Nitroso Compounds; Oxidoreductases; Oxygen; Phosphogluconate Dehydrogenase