Vasak D. Mikoyan

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.

Nitric oxide donor induces HSP70 accumulation in the heart and in cultured cells

As our group has shown, the NO‐synthase inhibitor L‐NNA decreased 2–3 times heat shock‐induced synthesis of the heat shock protein HSP70 (FEBS Lett. 370 (1995) 159–162). It was suggested that NO is involved in such induction. In the present study, it was found that (1) injection of the NO donor dinitrosyl iron complex (DNIC) into rats results in accumulation of HSP70 in the heart; (2) heat shock is accompanied by increased generation of NO (EPR assay) and HSP70 accumulation in cultured cells; (3) DNIC induces HSP70 accumulation in cultured cells not exposed to heat shock.

Dinitrosyl iron complexes with glutathione suppress experimental endometriosis in rats

Dinitrosyl iron complexes (DNIC) with glutathione exert a cytotoxic effect on endometrioid tumours in rats with surgically induced experimental endometriosis. Intraperitoneal treatment of rats (Group 1) with DNIC (12.5μmoles/kg, daily, for 12 days), beginning with day 4 after the surgical operation (implantation of two 2mm-thick uterine fragments onto the abdominal wall) followed by 14-day keeping of animals on a standard feeding schedule (without medication) resulted in complete inhibition of the growth of endometrioid implants (EMI) in the majority of experimental animals. The ratio of mean EMI volumes in control and experimental rats of Group 1 was 14:1. In Group 2 rats, the use of a similar treatment protocol 4 weeks after surgery changed this ratio to 1.4:1. Noteworthy, the decrease of this ratio was irrelevant to deceleration of EMI growth at later periods after surgery. The histopathological analysis of EMI samples from experimental rats of Group 2 demonstrated complete disappearance of endometrial cysts suggesting a cytotoxic effect of DNIC on the tumours. The data obtained demonstrate that DNIC with glutathione and, probably, with other thiol-containing ligands hold considerable promise in the design of drugs for treating endometriosis in female patients.

Redox conversions of dinitrosyl iron complexes with natural thiol-containing ligands

Using the electron paramagnetic resonance (EPR) and optical spectrophotometric methods, it has been established that biologically active, water-soluble dinitrosyl iron complexes (DNIC) with glutathione are predominantly represented by the diamagnetic binuclear form (B-DNIC) even in the presence of a 10-fold excess of glutathione non-incorporated into DNIC at neutral pH. With the increase in рН to 10-11, B-DNIC are fully converted into the paramagnetic mononuclear form (М-DNIC) with a characteristic EPR signal at g⊥=2.04, g‖=2.014 and gaver.=2.03. After treatment with a strong reducing agent sodium dithionite, both М- and B-DNIC are converted into the paramagnetic form with a characteristic EPR signal at g⊥=2.01, g‖=1.97 and gaver.=2.0. Both forms display similar absorption spectra with absorption bands at 960 and 640nm and a bend at 450nm. After oxidation by atmospheric oxygen, this situation is reversed, which manifests itself in the disappearance of the EPR signal at gaver.=2.0 and complete regeneration of initial absorption spectra of М- or B-DNIC with characteristic absorption bands at 390 or 360 and 310nm, respectively. Treatment of bovine serum albumin (BSA) solutions with gaseous NO in the presence of Fe(2+) and cysteine yields BSA-bound М-DNIC (М-DNIC-BSA). After treatment with sodium dithionite, the latter undergo transformations similar to those established for low-molecular М-DNIC with glutathione. Based on the complete coincidence of the optical and the EPR characteristics of sodium dithionite-treated М- and B-DNIC and other findings, it is suggested that sodium dithionite-reduced B-DNIC are subject to reversible decomposition into М-DNIC. The reduction and subsequent oxidation of М- and B-DNIC are interpreted in the paradigm of the current concepts of the initial electronic configurations of М- and B-DNIC (d(7) ({Fe(NO)2}(7)) and d(7)-d(7) ({Fe(NO)2}(7)-{Fe(NO)2}(7)), respectively).

The effect of dinitrosyl iron complexes with glutathione and S-nitrosoglutathione on the development of experimental endometriosis in rats: a comparative studies.

It has been established that intraperitoneal bolus administration of S-nitrosoglutathione (GS-NO) (12.5μmoles/kg; 10 injections in 10 days), beginning with day 4 after transplantation of two 2-mm autologous fragments of endometrial tissue onto the inner surface of the abdominal wall of rats with surgically induced (experimenta) endometriosis failed to prevent further growth of endometrioid (EMT) and additive tumors, while treatment of animals with dinitrosyl iron complexes (DNIC) with glutathione (12.5μmoles/kg, 10 injections in 10 days) suppressed tumor growth virtually completely. The histological analysis of EMT samples of GS-NO-treated rats revealed pathological changes characteristic of control (non-treated with GS-NO or DNIC) rats with experimental endometriosis. EPR studies established the presence of the active form of ribonucleotide reductase, a specific marker for rapidly proliferating tumors, in EMT samples of both control and GS-NO-treated animals. Noteworthy, in small-size EMT and adjacent tissues of DNIC-treated rats the active form of ribonucleotide reductase and pathological changes were not found.

Redox activities of mono- and binuclear forms of low-molecular and protein-bound dinitrosyl iron complexes with thiol-containing ligands

EPR, optical, electrochemical and stopped-flow methods were used to demonstrate that Fe(NO)2 fragments in paramagnetic mononuclear and diamagnetic binuclear forms of dinitrosyl iron complexes with glutathione are reversibly reduced by a two-electron mechanism to be further transformed from the initial state with d(7) configuration into states with the d(8) and d(9) electronic configurations of the iron atom. Under these conditions, both forms of DNIC display identical optical and EPR characteristics in state d(9) suggesting that reduction of the binuclear form of DNIC initiates their reversible decomposition into two mononuclear dinitrosyl iron fragments, one of which is EPR-silent (d(8)) and the other one is EPR-active (d(9)). Both forms of DNIC produce EPR signals with the following values of the g-factor: g⊥=2.01, g||=1.97, gaver.=2.0. M-DNIC with glutathione manifest an ability to pass into state d(9), however, only in solutions with a low content of free glutathione. Similar transitions were established for protein-bound М- and B-DNIC with thiol-containing ligands.

Nitrite protonation as a necessary stage in the generation of nitric oxide from nitrite in biological systems

The yields of nitric oxide from 1 mM and 10 mM sodium dithionite in 5 or 150 mM solutions of HEPES buffer (pH 7.4) differed by a factor of 200. Dithionite acted as both a strong reducing agent and an agent responsible for local acidification of the solutions without significant changes in pH. The concentration of nitric oxide was estimated by electron paramagnetic resonance (EPR) by monitoring its incorporation into water-soluble complexes of Fe with N-methyl-D-glucamine dithiocarbamate (MGD), which resulted in the formation of EPR-detectable mononitrosyl complexes of iron. Ten seconds after dithionite addition, the concentration of mononitrosyl iron complexes reached 2 μM, whereas it did not become greater than 0.01 μM in 5 mM HEPES buffer. It has been suggested that this difference results from a longer lifetime of a localized decrease in pH in a weaker buffer solution. This time could be long enough for the protonation of some nitrite molecules. Nitrous acid thus formed decomposed to nitric oxide. A difference in nitric oxide formation from nitrite in weak and strong buffer solutions was also observed in the presence of hemoglobin (0.3 mM) or serum albumin (0.5 mM). However, in the weak buffer the nitric oxide yield was only three-four times greater than in the strong buffer. An increase in the nitric oxide yield from nitrite was observed in solutions containing both proteins. A significant amount of nitric oxide from nitrite was formed in mouse liver preparation subjected to freezing and thawing procedure followed by slurrying in 150 mM HEPES buffer (pH 7.4) and dithionite addition (10 mM). We suggest that the presence of zones with lowered pH values in cells and tissues may be responsible for the predominance of the acidic mechanism of nitric oxide formation from nitrite. The contribution of nitric oxide formation from nitrite catalyzed by heme-containing proteins as nitrite reductases may be minor under these conditions.

The binuclear form of dinitrosyl iron complexes with thiol-containing ligands in animal tissues.

It has been established that treatment of mice with sodium nitrite, S-nitrosoglutathione and the water-soluble nitroglycerine derivative isosorbide dinitrate (ISDN) as NO donors initiates inxa0vivo synthesis of significant amounts of EPR-silent binuclear dinitrosyl iron complexes (B-DNIC) with thiol-containing ligands in the liver and other tissues of experimental mice. This effect is especially apparent if NO donors are administered to mice simultaneously with the Fe2+-citrate complex. Similar results were obtained in experiments on isolated liver and other mouse tissues treated with gaseous NО inxa0vitro and during stimulation of endogenous NO synthesis in the presence of inducible NO synthase. B-DNIC appeared in mouse tissues after inxa0vitro treatment of tissue samples with an aqueous solution of diethyldithiocarbamate (DETC), which resulted in the transfer of iron-mononitrosyl fragments from B-DNIC to the thiocarbonyl group of DETC and the formation of EPR-detectable mononitrosyl iron complexes (MNIC) with DETC. EPR-Active MNIC with N-methyl-d-glucamine dithiocarbamate (MGD) were synthesized in a similar way. MNIC-MGD were also formed in the reaction of water-soluble MGD-Fe2+ complexes with sodium nitrite, S-nitrosoglutathione and ISDN.

Dinitrosyl iron complexes with natural thiol-containing ligands in aqueous solutions: Synthesis and some physico-chemical characteristics (A methodological review)

Two approaches to the synthesis of dinitrosyl iron complexes (DNIC) with glutathione and l-cysteine in aqueous solutions based on the use of gaseous NO and appropriate S-nitrosothiols, viz., S-nitrosoglutathione (GS-NO) or S-nitrosocysteine (Cys-NO), respectively, are considered. A schematic representation of a vacuum unit for generation and accumulation of gaseous NO purified from the NO2 admixture and its application for obtaining aqueous solutions of DNIC in a Thunberg apparatus is given. To achieve this, a solution of bivalent iron in distilled water is loaded into the upper chamber of the Thunberg apparatus, while the thiol solution in an appropriate buffer (рН 7.4) is loaded into its lower chamber. Further steps, which include degassing, addition of gaseous NO, shaking of both solutions and formation of the Fe2+-thiol mixture, culminate in the synthesis of DNIC. The second approach consists in a stepwise addition of Fe2+ salts and nitrite to aqueous solutions of glutathione or cysteine. In the presence of Fe2+ and after the increase in рН to the physiological level, GS-NO or Cys-NO generated at acid media (pHxa0<xa04) are converted into DNIC with glutathione or cysteine. Noteworthy, irrespective of the procedure used for their synthesis DNIC with glutathione manifest much higher stability than DNIC with cysteine. The pattern of spin density distribution in iron-dinitrosyl fragments of DNIC characterized by the d7 electronic configuration of the iron atom and described by the formula Fe+(NO+)2 is unique in that it provides a plausible explanation for the ability of DNIC to generate NO and nitrosonium ions (NO+) and the peculiar characteristics of the EPR signal of their mononuclear form (M-DNIC).

Decomposition of water-soluble mononitrosyl iron complexes with dithiocarbamates and of dinitrosyl iron complexes with thiol ligands in animal organisms

EPR studies have shown that water-soluble mononitrosyl iron complexes with N-methyl-d-glucamine dithiocarbamate (MNIC-MGD) (3 micromol) injected to intact mice were decomposed virtually completely within 1h. The total content of MNIC-MGD in animal urine did not exceed 30 nmol/ml. In the liver, a small amount of MNIC-MGD were converted into dinitrosyl iron complexes (30 nmol/g of liver tissue). The same was observed in intact rabbits in which MNIC-MGD formation was induced by endogenous or exogenous NO binding to NO traps, viz., iron complexes with MGD. In mice, the content of MNIC-MGD in urine samples did not change after bacterial lipopolysaccharide-induced expression of iNOS. It was supposed that MNIC-MGD decomposition in intact animals was largely due to the release of NO from the complexes and its further transfer to other specific acceptors. In mice with iNOS expression, the main contribution to MNIC-MGD decomposition was made by superoxide ions whose destructive effect is mediated by an oxidative mechanism. This effect could fully compensate the augmented synthesis of MNIC-MGD involving endogenous NO whose production was supported by iNOS. Water-soluble dinitrosyl iron complexes (DNIC) with various thiol-containing ligands and thiosulfate injected to intact mice were also decomposed; however, in this case the effect was less pronounced than in the case of MNIC-MGD. It was concluded that DNIC decomposition was largely due to the oxidative effect of superoxide ions on these complexes.