Lyudmila N. Kubrina

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.

A simple protocol for the synthesis of dinitrosyl iron complexes with glutathione: EPR, optical, chromatographic and biological characterization of reaction products.

The diamagnetic binuclear form of dinitrosyl iron complexes (B-DNIC) with glutathione can be easily synthesized in the air at ambient temperature. The synthetic protocol includes consecutive addition to distilled water of glutathione, which decreases the pH of the test solution to 4.0, a bivalent iron salt (e.g., ferrous sulphate) and sodium nitrite at the molar ratio of 2:1:1, with a subsequent increase in pH to neutral values. Under these conditions, the amount of B-DNIC formed is limited by initial nitrite concentration. In the novel procedure, 20mM glutathione, 10mM ferrous sulfate and 10mM sodium nitrite give 2.5mM B-DNIC with glutathione, while 5mM glutathione remains in the solution. Bivalent iron (5mM) is precipitated in the form of hydroxide complexes, which can be removed from the solution by passage through a paper filter. After the increase in рН to 11 and addition of thiols at concentrations exceeding that of DNIC tenfold, B-DNIC are converted into a mononuclear EPR-active form of DNIC (M-DNIC) with glutathione. B-DNIC preparations synthesized by using new method contain negligible amount of nitrite or S-nitrosoglutathione as a contaminations. All the steps of DNIC synthesis were characterized by using optical, EPR and HPLC methods. A long-lasting hypotensive action of DNIC formed was demonstrated.

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).

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.

Low Rate of Induced Nitric Oxide Synthesis in Immunocompetent Organs Is Characteristic of Hereditary Immune Deficiency in Mice

Nitric oxide is involved in the immune response of the body, which is an extremely important biological function of this compound. At the initial stage of the immune response, activated macrophages produce highly toxic nitric oxide, which suppresses the resistance of bacteria, viruses, and other invasive microorgansims. The cytotoxic and cytostatic effects of nitric oxide are accounted for by its ability to inhibit the respiratory-chain key enzymes and the DNA synthesis in the target cells [1, 2]. In animals and plants, nitric oxide is synthesized by the constitutive and inducible forms of NO synthases (NOSs) from the amino acid L-arginine. Numerous experimental evidence on the location of both constitutive and inducible NOSs, their specific regulation, activity, and functions has been obtained mostly at the cellular level [3]. However, to our knowledge, it had never been shown that the immune status of animals from isogenic lines differing in the activity of only one gene was directly associated with the level of nitric oxide synthesized in immunocompetent organs in pathology. In this study, the content of nitric oxide in the liver and intestine of mice with identified genotypes were compared under the conditions of a model inflammatory process caused by injection of lipopolysaccharide from the E. coli cell wall. Mice of the inbred line C57BL/10SnY (B 10) (control) and those of two mutant lines, C57BL/10-hr rhy (Rhino) and NZB/Orly (NZB), were used in this study. The hairless ( hr ) gene mutation is characterized by pleiotropic effects, including immune disorders. The incidence of leukemia in the HRS mice homozygous for the hr mutation is higher than in their normal sibs. HRS mice exhibit a decreased cellular immune response against sheep red blood cells [4]. In 1984, a new hr gene mutation referred to as rhino-Y was detected in B10.R109 mice in the Laboratory of Experimental Biological Models, Russian Academy of Medical Sciences. A lack of hairs and a coarse skin rugosity increasing with age are characteristic traits of the mutant phenotype. The mutant females are completely infertile, and the males lose their fertility beginning from the age of four to five months [5]. In these animals, an immune pathology condition develops that is accompanied by deposition of immune complexes in the skin, muscles, kidney, and thymus. Visible morphological changes were the following: increased lymph nodes and a reduced thymus, a complete involution of which was sometimes observed by the age of six to eight months. The nature of the rhino-Y mutation is now known and the mutation has been shown to cause similar pathological disorders in mice and humans [6, 7]. The NZB mouse line is generally used in experimental medicine as a biological model. These animals are characterized by autoimmune hemolytic anemia developing with age and by the lups-like nephritis [8].

No-dependent mechanisms of adaptation to hypoxia

Studies of nitrogen oxide (NO)-dependent mechanisms of organism resistance to hypoxia demonstrate that (1) acute hypoxia induces NO hyperproduction in the brain and does not affect NO production in the liver; (2) adaptation to hypoxia decreases NO production in the liver and brain; and (3) adaptation to hypoxia prevents NO hyperproduction in the brain and enhances NO synthesis in the lever during acute hypoxia. An NO donor--dinytrosyl iron complexes (DCI, 200 micrograms/kg, single intravenous (i.v.) introduction)--decreases animal resistance to acute hypoxia by 30%, while introduction of an NO synthase inhibitor--N- nitro-L-arginine (NNA, 50 micrograms/kg, single intraperitoneal (i.p.) introduction)--and an NO trap--diethyldithiocarbamate (DETC, 200 mg/kg, single i.p. introduction)--increases the resistance 1.3 and 2 times, respectively. Adaptation to hypoxia is realized against a background of accumulation of heat shock proteins HSP70 in the liver and brain. Course treatment with DCI reproduces the antihypoxic effect of adaptation to hypoxia. Course treatment with NNA during adaptation to hypoxia prevents both accumulation of HSP70 and development of the antihypoxic effect. Hence, No and NO-dependent activation of HSP70 synthesis play an important role in adaptation to hypoxia.