Anatoly F. Vanin

Nitrite as Regulator of Hypoxic Signaling in Mammalian Physiology

In this review we consider the effects of endogenous and pharmacological levels of nitrite under conditions of hypoxia. In humans, the nitrite anion has long been considered as metastable intermediate in the oxidation of nitric oxide radicals to the stable metabolite nitrate. This oxidation cascade was thought to be irreversible under physiological conditions. However, a growing body of experimental observations attests that the presence of endogenous nitrite regulates a number of signaling events along the physiological and pathophysiological oxygen gradient. Hypoxic signaling events include vasodilation, modulation of mitochondrial respiration, and cytoprotection following ischemic insult. These phenomena are attributed to the reduction of nitrite anions to nitric oxide if local oxygen levels in tissues decrease. Recent research identified a growing list of enzymatic and nonenzymatic pathways for this endogenous reduction of nitrite. Additional direct signaling events not involving free nitric oxide are proposed. We here discuss the mechanisms and properties of these various pathways and the role played by the local concentration of free oxygen in the affected tissue.

Dinitrosyl iron complexes with thiolate ligands: physico-chemistry, biochemistry and physiology.

Some present-day concepts on the origin and functional activities of dinitrosyl iron complexes (DNIC) with thiolate ligands are considered. Nitric oxide (NO) including to DNIC increases its stability and ensures effective targeting of NO to organs and tissues. DNIC have a square-planar structure; unpaired electron is localized on the d(z2) orbital of the d(7) iron atom. The formula of DNIC appears as [(RS(-))(2)Fe(+)(NO(+))(2)....((-)SR)(2)](-); electron spin is S=1/2. Conversion of an originally diamagnetic group, Fe(2+)(NO)(2) with electron configuration d(8), into a paramagnetic Fe(+)(NO(+))(2) group is a result of disproportionation of NO ligands and substitution of newly generated NO(-) for NO. The nitrosonium ions present in DNIC impart to them high nitrosylating activity, e.g., ability to induce S-nitrosylation of thiols. The ability of S-nitrosothiols to form DNIC in a direct reaction with bivalent iron is a prerequisite to effective mutual conversions of DNIC and S-nitrosothiols. In this work, I consider some mechanisms of destructive effects of low-molecular DNIC on active centers of iron-sulfur proteins, ability of DNIC to express certain genes, to activate guanylate cyclase, to exert hypotensive, vasodilator effects, to inhibit platelet aggregation, to accelerate wound healing and to produce potent erective action. Recently a stabilized powder-like polymeric composition based on dimeric glutathione DNIC the water-soluble polymer in which was used as a filling agent was designed. The advantages of this stable DNIC-glutathione preparation include their ability to retain their physico-chemical and functional activities within at least one year. At present, the preparation undergo testing as a base for the design of a wide variety of broad-spectrum drugs.

The potent vasodilating and guanylyl cyclase activating dinitrosyl‐iron(II) complex is stored in a protein‐bound form in vascular tissue and is released by thiols

We studied the biological activity, stability and interaction of dinitrosyl‐iron(II)‐L‐cysteine with vascular tissue. Dinitrosyl‐iron((II)‐L‐cysteine was a potent activator of purified soluble guanylyl cyclase (EC50 (nM with and 100 nM without superoxide dismutase) and relaxed noradrenaline‐precontracted segments of endothelium‐denuded rabbit femoral artery (EC50 10 nM superoxide dismutase). Pre‐incubation (5 min; 310 K) of endothelium‐denuded rabbit aortic segments with dinitrosyl‐iron(II)‐L‐cysteine (0.036–3.6 mM) resulted in a concentration‐dependent formation of a dinitrosyl‐iron(II complex with protein thiol groups, as detected by ESR spectroscopy. While the complex with proteins was stable for 2 h at 310 K, dinitrosyl‐iron(II)‐L‐cysteine in aqueous solution (30–360 μM) decomposed completely within 15 min, as indicated by disappearance of its isotropic ESR signal at g av = 2.03 (293 K). Aortic segments pre‐incubated with dinitrosyl‐iron(II)‐L‐cysteine released a labile vasodilating and guanylyl cyclase activating factor. Perfusion of these segments with N‐acetyl‐L‐cysteine resulted in the generation of a low molecular weight dinitrosyl‐iron(II)‐dithiolate from the dinitrosyl‐iron(II) complex with proteins, as revealed by the shape change of the ESR signal at 293 K. The low molecular weight dinitrosyl‐iron(II)‐dithiolate accounted to an enhanced guanylyl cyclase activation and vasodilation induced by the aortic effluent. We conclude that nitric oxide (NO) produced by, or acting on vascular cells can be stabilized and stored as a dinitrosyl‐iron(II) complex with protein thiols, and can be released from cells in the form of a low molecular weight dinitrosyl‐iron(II)‐dithiolate by intra‐ and extracellular thiols.

Nitric oxide synthase reduces nitrite to NO under anoxia.

Abstract.Cultured 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.

Nitric oxide promotes seizure activity in kainate-treated rats.

L-Arginine-derived nitrogen monoxide (NO) formation was determined in different regions of the rat brain during kainate-induced seizures. NO was trapped in vivo as a paramagnetic mononitrosyl-iron diethyldithiocarbamate complex, the concentration of which was determined ex vivo by cryogenic electron spin resonance spectroscopy. Basal NO formation (0.3-0.8 nmol g-1 tissue 30 min-1) was detected in the brain of control rats. In kainate-injected rats NO formation was increased six-fold within 30-60 min in the amygdala/temporal cortex region, and up to 12-fold, though more slowly, in the remaining cortex. The kainate-elicited convulsions and NO formation were attenuated in animals pretreated with either 7-nitroindazole, a specific inhibitor of neuronal NO synthase, or diazepam. These findings identify NO as a proconvulsant mediator in kainate-evoked seizures.

Endothelium-derived relaxing factor is a nitrosyl iron complex with thiol ligands

A hypothesis is put forward on the nature of the endothelium‐derived relaxing factor (EDRF) which is released from vascular endothelial cells by acetylcholine, bradykinin and other agonists. It is suggested that EDRF is a nitrosyl iron complex with low‐molecular thiol ligands, most probably with cysteine. Its active principle is nitric oxide (NO). This free radical is stabilized by inclusion into the iron complex, which promotes NO transfer within the cell and between cells. Subsequent release of NO from these complexes results from thiol group oxidation.

EPR evidence for nitric oxide production from guanidino nitrogens of L-arginine in animal tissues in vivo.

Administration of Fe(2+)-citrate complex (50 mg/kg of FeSO4 or FeCl2 plus 250 mg/kg of sodium citrate) subcutaneously in the thigh or Escherichia coli lipopolysaccharide (LPS, 1 mg/kg) intraperitoneally, (i.p.) to mice induced NO formation in the livers in vivo at the rate of 0.2-0.3 micrograms/g wet tissue per 0.5 h. The NO synthesized was specifically trapped with Fe(2+)-diethyldithiocarbamate complex (FeDETC2), formed from endogenous iron and diethyldithiocarbamate (DETC) administered i.p. 0.5 h before decapitation of the animals. NO bound with this trap resulted in the formation of a paramagnetic mononitrosyl iron complex with DETC (NO-FeDETC2), characterized by an EPR signal at g perpendicular = 2.035, g parallel = 2.02 with triplet hyperfine structure (HFS) at g perpendicular. This allowed quantification of the amount of NO formed in the livers. An inhibitor of enzymatic NO synthesis from L-arginine, NG-nitro-L-arginine (NNLA, 50 mg/kg) attenuated the NO synthesis in vivo. L-Arginine (500 mg/kg) reversed this effect. Injection of L-[guanidineimino-15N2]arginine combined with Fe(2+)-citrate or LPS led to the formation of the EPR signal of NO-FeDETC2 characterized by a doublet HFS at g perpendicular, demonstrating that the NO originates from the guanidino nitrogens of L-arginine in vivo.

Similarity between the vasorelaxing activity of dinitrosyl iron cysteine complexes and endothelium-derived relaxing factor

Dinitrosyl iron complexes with cysteine (DNIC) induced a concentration-dependent relaxation of pre-contracted (norepinephrine, 10(-7) M) de-endothelialized ring segments of rat aorta. The vasodilator response was more similar to acetylcholine (ACh)-induced relaxation in intact aortic rings than to nitric oxide (NO)-induced relaxation. The time course of tone recovery after maximal concentrations (10(-5) M) of DNIC was similar to the time course of tone recovery after endothelium-dependent relaxation induced by ACh, whereas the restoration of tone after NO was much faster. Vessel tone was restored by oxyhemoglobin (10(-5) M) in all cases. The results suggest that DNIC with cysteine may function as endothelium-derived relaxing factor in the vessels.

Nitric oxide is involved in heat-induced HSP70 accumulation

Heat shock potentiated the nitric oxide production (EPR assay) in the liver, kidney, heart, spleen, intestine, and brain. The heat shock‐induced sharp transient increase in the rate of nitric oxide production preceded the accumulation of heat shock proteins (HSP70) (Western blot analysis) as measured in the heart and liver. In all organs the nitric oxide formation was completely blocked by the NO‐synthase inhibitor Nω‐nitro‐L‐arginine (L‐NNA). L‐NNA also markedly attenuated the heat shock‐induced accumulation of HSP70. The results suggests that nitric oxide is involved in the heat shock‐induced activation of HSP70 synthesis.

Complexes of Fe2+ with diethyldithiocarbamate or N-methyl-d-glucamine dithiocarbamate as traps of nitric oxide in animal tissues: comparative investigations

In EPR experiments on mice it was demonstrated that a hydrophobic complex Fe2+ with diethyldithiocarbamate (DETC) is a more efficient selective NO trap than a hydrophilic complex Fe2+ with N-methyl-D-glutamine dithiocarbamate (MGD). This difference can be due to the higher stability of paramagnetic nitrosyl iron complex with DETC (MNIC-DETC) formed by NO binding to Fe2+-DETC in animal tissues in vivo. The complex analogue MNIC-MGD is reversibly oxidized in animal blood to transform into the diamagnetic EPR-silent form. The latter is detectable also in urine of animals, especially of those treated with bacterial lipopolysaccharide which initiates the enhanced NO production in the organism. We suggest that NO2 or peroxynitrite formed from endogenous NO can serve as an agent reversibly oxidizing MNIC-MGD in these animals.