[Introduction of dinitrosyl iron complexes with thiol-containing ligands into animal organism by inhalation method].

The possibility of water-soluble dinitrosyl iron complexes (DNIC) with thiol-containing ligands introduction into lungs and other tissues of mice by free inhalation of little drops (2-3 microns diameter) of the solutions of these complexes was investigated. Little drops of 2-20 mM solutions of the complexes were obtained by using an inhalation apparatus (compressor nebulizer). A cloud of these little drops was then inhaled by animals in a closed chamber. A maximal amount of protein-bound DNICs formed in mouse lungs was 0.6 micromoles per kilogram of tissue weight. The amount of DNIC in lungs, liver and blood decreased to the undetected level within 2-3 hours after inhalation. No cytotoxic effect of DNIC formed in lungs on Mycobacterium tuberculosis was found in mice infected with these mycobacteria.

[Dinitrosyl iron complexes with glutathione recover rats with experimental endometriosis].

The effect of binuclear dinitrosyl iron complexes (DNIC) with glutathione on endometrioid tumors in rats with experimental endometriosis has been studied. The latter was induced by an autotransplantation model, where two fragments of endometrium with myometrium (2 x 2 mm) from the left uterine horn was grafted to the inner surface of the anterior abdominal wall. The test animals received intraperitoneal injections of 0.5 ml DNIC-glutathione at the dose of 12.5 micromole per kg daily for 12 days 28 days after operation. The injections resulted in more than a 2-fold decrease in the total volume of both large tumors formed from grafts and small additive tumors formed nearby grafts. The disappearance of the additive tumors was also observed in test animals. The EPR signal with g(av) = 2.03 characteristic of protein bound DNIC with thiol-containing ligands was recorded in livers, graft and additive tumors of test and control animals pointing out intensive generation of nitric oxide in rats with experimental endometriosis. Ribonucleotide reductase activation discovered by doublet the EPR signal at g = 2.0 with 2.3 mT hyperfine structure splitting was found in small tumors. The cytotoxic effect of DNIC-glutathione on endometrioid tumors was suggested to be due to DNIC degradation nearby the tumors induced by iron chelating compounds released from the tumors. The degradation resulted in release of a high amount of nitric oxide molecules and nitrosonium ions from DNICs affecting the tumors by way of the cytotoxic effect.

[Physico-chemical features of dinitrosyl iron complexes with natural thiol-containing ligands underlying biological activities of these complexes].

Current notions and new experimental data of the authors on physico-chemical features of dinitrosyl iron complexes with natural thiol-containing ligands (glutathione or cysteine), underlying the ability of the complexes to act as NO molecule and nitrosonium ion donors, are considered. This ability determines various biological activities of dinitrosyl iron complexes--inducing long-lasting vasodilation and thereby long-lasting hypotension in human and animals, inhibiting pellet aggregation, increasing red blood cell elasticity, thereby stimulating microcirculation, and reducing necrotic zone in animals with myocardial infarction. Moreover, dinitrosyl iron complexes are capable of accelerating skin wound healing, improving the function of penile cavernous tissue, blocking apoptosis development in cell cultures. When decomposed dinitrosyl iron complexes can exert cytotoxic effect that can be used for curing infectious and carcinogenic pathologies.

[The sources of inorganic sulfur in the process of cluster protein Fnr[4Fe-4S]2+ reconstruction in Escherichia coli cells cultivated with NO-donating agents].

Dinitrosyl iron complexes (DNICs) with thiol ligands--binuclear and mononuclear--inhibited aidB gene expression in E. coli cells. This process is due to the nitrosylation of the active center in iron-sulfur protein Fnr [4Fe-4S]2+ by low-molecular DNICs. The next step is transformation of the above DNICs into the DNICs with the thiol groups in the apo-form of Fnr protein. These nitrosylated proteins are characterized by the EPR signal with g perpendicular = 2.04 and g parallel 1 = 2,014. An addition of sulfur containing L-Cys or N-A-L-Cys as well as Na2S to the cells lead to the increasing in the aidB gene expression simultaneously with an appearance of the EPR signal with g perpendicular = 2.04 and g parallel = 2.02 as the characteristics of the DNICs with persulfide (R-S-S-) ligands. We suppose that the recovery of the aidB gene activity was due to the accumulation of inorganic sulfur in the cells and reconstruction of the active center in Fnr[4Fe-4S]2+. It appears that the above process is the function of L-cysteine-desulfurase protein which repaired the active center of Fnr[4Fe-4S]2+ protein using the sulfur from L-Cys or N-A-L-Cys after its deacetylation. On the other side the ions of inorganic sulfur being reacted with SH-groups led to the transformation of DNIC with thiol ligands into the persulfides. Na2S was the most potent activator of the aidB gene expression in our experiments.

[Detection of autowave distribution of the concentration of a dinitrosyl iron complex with glutathione, formed in an aqueous solution of S-nitrosoglutathione after the addition to it of a mixture of glutathione and ferrous iron].

The formation of dark green concentric autowaves of the distribution of the concentration of dinitrosyl iron complex (DNIC) with glutathione in a thin (0.3 mm thick) layer of 0.5 M solution of S-nitrosoglutathione in 15 mM HEPES buffer (pH 7.7) after applying on its surface of a drop of a solution of glutathione (0.5 mM) and ferrous iron (1 mM) in the same buffer of volume 10 microl was detected. At regular intervals, the picture of autowaves changed for 0.4-0.6 s over a period of 3 s after the application of the drop onto the solution. Then the structured picture of the distribution of DNIC dissipated followed by a uniform green coloring of the solution caused by a uniform distribution of DNIC in it. It is assumed that the formation of autowaves is a consequence of the autooscillatory mode of the existence of a chemical system formed in a mixture of NO, low-molecular-weight thiols, and ferrous iron ions. DNIC with thiolate ligands and S-nitrosothiols arising in this system have a capacity for interconversion, and it is this process that may underlie the autooscillatory, autowave mode of functioning of the system. It is not ruled out that the existence of this system in cells and tissues of living organisms may provide the spatial and temporal organization of the regulation of the biological action of NO and its different endogenous compounds and derivatives.

[Effect of exogenous donors of nitric oxide and inhibitors of its enzymatic synthesis on experimental ischemic thrombosis in conjunctive veins of the rabbit eyes].

The beneficial action of dinitrosyl iron complex with glutathione on conjunctive veins of eyes in rabbits with experimental thrombosis of conjunctive veins has been demonstrated. Aqueous solutions of dinitrosyl iron complexes were added subconjunctively at doses of 5.4-8.1 micromole per eye. The average duration of thrombosis by the action of dinitrosyl iron complex decreased from 6.4 days in control animals to 2 days. The addition of dinitrosyl iron complex resulted in blood flow recovery in occlusive vessels and prevented ischemia and necrosis of tissues. The enhancement of hemorrhagic activity induced by dinitrosyl iron complexes was abrogated with combined addition of the nonselective NO synthase inhibitor N-nitro-L-arginine. In contrast, S-nitrosoglutathione affected adversely the veins: the duration of thrombosis in experimental thrombosis of conjunctive veins increased to 7 days. Intensive hemorhage developed in the conjunctive. The formation of protein-bound dinitrosyl iron complexes was observed by the EPR method in eye tissues after the subconjunctive or parabulbar addition of dinitrosyl iron complex with glutathione. This was not the case when the complex was injected intravenously. It was shown that dinitrosyl iron complex with glutathione induces the blockade of pellet aggregation or strengthens the fibrinolytic activity of plasma of patients with eye vessel pathology. The beneficial action of dinitrosyl iron complexes on conjunctive veins was proposed to be due to the capacity of dinitrosyl iron complexes to donate NO primarily to its biological targets. The release of free NO molecules in large amounts is not characteristic for dinitrosyl iron complexes. This process is characteristic of S-nitrosoglutathione, which sharply increases the probability of the accumulation of peroxynitrite, which produces a toxic effect on cells and tissues.

[Quasi-adaptive response to alkylating agents in Escherichia coli and Ada-protein functions].

In 2005 we have described in exponentially growing E. coli cells a new fundamental genetic phenomenon,--quasi-adaptive response to alkylating compounds (quasi-Ada). Phenotypic expression of quasi-Ada is similar to the true Ada response. However, in contrast to the letter, it develops in the course of pretreatment of the cells by a sublethal dose of nonalkylating agent, an NO-containing dinitrosyl iron complex with glutathione (DNICglu). To reveal the mechanisms of quasi-adaptation and its association with the function of the Ada regulatory protein, here we used a unique property of dual gene expression regulation of aidB1 gene, a part of the Ada-regulon, namely its relative independence from Ada protein in anaerobic conditions. Based on the results of aidB1 gene expression analysis an EPR spectra of E. coli MV2176 cells (aidB1::lacZ) in aerobic and anaerobic conditions after the corresponding treatments, we conclude that the function and the spatial structure of meAda and [(Cys-)2Fe+(NO+)2]Ada are identical and thus the nitrosylated protein represents a regulator of the Ada regulon gene expression during quasi-adaptation development.

[Protonation of nitrite is an obligatory stage in the generation of nitric oxide from nitrite in biological systems].

The yield of nitric oxide from 1 mM sodium nitrite differs 200 times when the process was initiated by 10 mM sodium dithionite in the solution of 5 or 150 mM HEPES-buffer (pH 7.4). Dithionite acted both as a strong reductant and an agent that induced a local acidification of solutions without notable change in pH value. The amount of nitric oxide was estimated by the EPR method by measuring the incorporation of nitric oxide to water-soluble complexes of Fe with N-methyl-D-glucamine dithiocarbamate (MGD), which led to the formation of EPR-detectable mononitrosyl iron complexes with MGD (MNIC-MGD). Ten seconds after dithionite addition, the concentration of MNIC - MGD complexes reached 2 microM in 5 mM HEPES-buffer in contrast to 0.01 microM in 150 mM HEPES-buffer. The difference was suggested to be due to a higher life-time of zones with decreased pH values in a weaker weak buffer solution. The life-time was high enough to ensure the protonation of a part of nitrite. The resulting nitrous acid was decomposed to form nitric oxide. The difference in the formation of nitric oxide from nitrite was also observed in weak and strong buffer solutions in the presence of hemoglobin (0.3 mM) or serum albumin (0.5 mM). However, the ratios of nitric oxide yields in weak and strong buffer did not exceed 3-4 times. The increase in the formation of nitric oxide from nitrite was characteristic for the solutions containing both proteins. Large amounts of nitric oxide formed from nitrite was observed in mouse liver preparation subjected to freezing-thawing procedure followed by incubation in 150 mM HEPES-buffer (pH 7.4) and addition of dithionite. The proposition was made that the presence of zones with low pH value in cells and tissues can ensure the predominant operation of the acid mechanism formation of nitric oxide from nitrite. The contribution of the formation of nitric oxide from nitrite catalyzing with heme-containing proteins nitrite reductases can be minor one under these conditions.

[Formation of paramagnetic nitrosyl complexes of non heme iron in animals with participation of nitric oxide from exogenous and endogenous sources]. / Obrazovanie paramagnitnykh nitrozil'nykh kompleksov negemovogo zheleza v organizme zhivotnykh pri uchastii oksida azota iz ékzogennykh i éndogennykh istochnikov.

It was found that thiosulfate has a stabilizing effect on exogenous and endogenous dinitrosyl-iron complexes in mice treated with bacterial lipopolysaccharide. It was assumed that thiosulfate protects dinitrosyl-iron complexes from the destructive influence of superoxide and peroxinitrite whose enhanced synthesis, together with the synthesis of nitric oxide, is initiated in mice by the lipopolysaccharide. For the first time, the formation of dinitrosyl-iron complexes was demonstrated, which occurs with the participation of nitric oxide generated enzymatically via the L-arginine-dependent pathway. The injection of exogenous dinitrosyl-iron complexes with thiosulfate, which, together with diethyldithiocarbamate, provide the formation of exogenous mononitrosyl iron-diethyldithiocarbamate complexes, made it possible to use the ABC method, which markedly enhances the efficiency of scavenging of endogenous nitric oxide in mice treated with lipopolysaccharides.

[Model corasol-induced seizures are followed by increase of nitric oxide generation and are abolished by mexidol and alpha-tocopherol]. / Model'nye korazolovye sudorogi soprovozhdaiutsia usileniem generatsii okisi azota i ustraniaiutsia meksidolom i al'fa-tokoferolom.

The effect of mexidol and alpha-tocopherol on the onset and development of acute epilepsy model was studied in Wistar rats using penthylenetetrazole induced convulsions. The intensity of the nitric oxide (NO) production in the cerebral cortex was determined by a direct method using electron paramagnetic resonance. The rate of lipid peroxidation (LPO) was estimated by measuring the level of secondary products (thiobarbituric acid reactive species). The peak of penthylene-tetrazole convulsions is accompanied by a significant increase in the levels of both NO and LPO products. Mexidol (150 mg/kg) and alpha-tocopherol (100 mg/kg) hindered the development of model convulsions, prevented NO buildup, and inhibited LPO growth. It is suggested that suppression of the excess NO production in the cortex and inhibition of the LPO enhancement can be involved in the mechanism of action of antiepileptic drugs.

[Semax prevents elevation of nitric oxide generation caused by incomplete global ischemia in the rat brain]. / Semaks preduprezhdaet povyshenie generatsii oksida azota v mozge krys, obuslovlennoe nepolnoi global'noi ishemiei.

A twofold increase in the nitric oxide (NO) production and a moderate increase in the content of secondary products of lipid peroxidation was observed in Wistar rats with incomplete global ischemia model induced by the bilateral occlusion of common carotid arteries. A clear correlation was observed between the NO content in the rat brain and the level of neurological disturbance manifestations in the ischemized animals. The synthetic peptide semax (a fragment of ACTH4-7 Pro-Gly-Pro) in a dose of 0.3 mg/kg prevented from the development of both neurological disturbances and excess NO production in the rat brain cortex.

[The effect of anticonvulsants on the nitric oxide content and level of lipid peroxidation in the brain of rats in model seizure states]. / Vliianie antikonvul'santov na soderzhanie oksida azota i uroven' perekisnogo okisleniia lipidov v mozge krys pri model'nykh sudorozhnykh sostoianiiakh.

EPR spectroscopy was performed to study the effect of anticonvulsants lamotrigine and phenobarbital on nitric oxide generation in rat brain on models of a convulsive seizure caused by exposure to maximal electric shock or corasole injection. The intensity of lipid peroxidation (LPO) was studied at the same time. The anticonvulsants under study proved to be capable of suppressing to a various degree intensification of nitric oxide generation and increase of LPO intensity in the rat cerebral cortex induced by convulsions. This widens the existing idea of the mechanism of the effect of antiepileptic agents.

[NO-Dependent mechanisms of adaptation to hypoxia]. / NO-zavisimye mekhanizmy adaptatsii k gipoksii.

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.

[Prevention of nitric oxide hyperproduction by adaptation to stressor effects]. / Preduprezhdenie giperproduktsii oksida azota s pomoshch'iu adaptatsii k stressornym vozdeistviiam.

As was shown earlier, acute hypotensive shock induced by nitrogen oxide (NO) hyperproduction can be prevented by dosed adaptation to environmental factors. In this work we tested the hypothesis that the mechanism of this adaptive effect is based on limiting NO hyperproduction. It was shown that rat adaptation to stress completely prevented arterial depression and sharply increased revival rate of the animals after heat shock. Hypotension induced by heat shock was accompanied by almost 2.5-fold increase in NO production (EPR analysis) as compared to the control. At the background of preadaptation, heat shock did not increase NO production relative to the animals not subjected to heat shock. The data obtained agree with the proposal that the adaptation initiates NO-dependent mechanisms of limiting NO hyperproduction. Such limiting seems to be negatively regulated by NO.

[The seizures induced by N-methyl-D,L-aspartate administration are accompanied by an enhancement of nitric oxide generation and of lipid peroxidation processes in the rat brain]. / Sudorogi, vyzvaemye vvedeniem N-metil-D,L-aspartata, soprovozhdaiutsia usileniem generatsii oksida azota i protsessov perekisnogo okisleniia lipidov v mozge krys.

EPR-spectrometry was performed to study the nitrous oxide (NO) content and the intensity of lipid peroxidation (LPO) in the brain cortex of rats during convulsions induced by intracerebral injection of N-methyl-D,L-aspartate (NMDLA). It was shown that the convulsions were attended with a fourfold increase in the NO content and activation of LPO in the rat brain cortex. Disocilpin injection fully prevented the development of convulsions as well as increase in the NO level and LPO activation caused by NMDLA injection. N-nitro-L-arginine had an anticonvulsive effect and prevented increase in the NO content but did not cause any noticeable effect on LPO intensity in the brain cortex.