[Mono- and Binuclear Dinitrosyl Iron Complexes with Thiol-containing Ligands in Various Biosystems].

It has been shown that dinitrosyl iron complexes with thiol-containing ligands, bound with modified bovine serum albumin with high amount of thiol groups, appeared in baker yeast or in animal tissues in the presence of exogenous or endogenous nitric oxide, respectively, are represented predominantly by EPR-silent binuclear form. This form can be transformed into EPR-active mononuclear form of dinitrosyl iron complexes with an increase in pH to basic values, into EPR-active form of mononuclear iron nitrosyl complexes in case of bielectronic recovery of the binuclear form of dinitrosyl iron complexes or under the action of dithiocarbamate derivatives. The latter induced the transformation of dinitrosyl iron complexes into EPR-active mononitrosyl iron complexes with dithiocarbamates. A significant amount of binuclear dinitrosyl iron complexes with thiol-containing ligands in living systems and identical biological activity of these complexes and endogenous nitric oxide systems allow of considering endogenous binuclear dinitrosyl iron complexes as a "working form" of endogenous nitric oxide recognized now as a universal regulator of biological processes.

[Anti-nitrosative system as a factor of malignant tumor resistance to cytotoxic effect of nitrogen monooxide].

The inhibitory action of binuclear dinitrosyl iron complexes with glutathione on the growth of implanted solid tumor in BDF1 mice bearing Lewis lung carcinoma cells was found. The effect was induced by intraperitoneal injection of the binuclear dinitrosyl iron complexes to mice at a dose of 200 µM/kg daily on days 1-5 and 7-11. At the binuclear dinitrosyl iron complexes: free glutathione ratios of 1:1; and 1:10 in solutions, the inhibitory effect of the DNICs reached the level of 70% and 85%, respectively. When B-DNICs were not further infused, intensive tumor growth, a more rapid rate of tumor growth than control, was observed. The selective accumulation of DNICs as well as iron nitrosyl complexes of heme-containing proteins in tumors were detected by EPR method. The latter were found also in the tumors in control animals. Tumor growth delay in course of B-DNIC administration to the mice is supposed to be due to the elaboration of anti-nitrosative defense in tumor tissue in response to the action of NO released from B-DNIC.

[Anti-Tumour Activity of Dinitrosyl Iron Complex with Glutathione and S-Nitrosoglutathione Preparations: Comparative Studies].

The anti-tumor activity of the binuclear form of dinitrosyl iron complexes with glutathione against Lewis lung carcinoma, found earlier upon intraperitoneal administration of the complexes, was also observed when this preparation was injected subcutaneously. A 100 µM/kg subcutaneous dose of the complex being used daily (as calculated per one iron atom in binuclear dinitrosyl iron complexes) for 10 or 15 days, inhibited the tumor growth by 43%. The effect was observed during the first two weeks after tumor transplantation. After that, the tumors began to grow at the rate equal to or even higher than that one for control animals. The mean survival time for treated mice exceeded the control values by 30%. Binuclear dinitrosyl iron complexes administered intraperitoneally was also effective against Ca-755 adenocarcinoma. However, in this case the mean survival time for treated animals increased only by 7%. The anti-tumor activity of S-nitrosoglutathione against Lewis lung carcinoma growth inhibition by 70% and Ca-755 adenocarcinoma growth inhibition by 90% was also shown. However, unlike binuclear dinitrosyl iron complexes the anti-tumor effect of S-nitrosoglutathione decreased when a daily dose of the compound increased (from 200 to 400 µM/kg) The initial anti-tumor effect of binuclear dinitrosyl iron complexes and S-nitrosoglutathione is suggested to be due to NO released from both compounds. A subsequent suppression of the effect is determined by the development of anti-nitrosative and anti-oxidant defense systems in tumors.

[Anti-tumour activity of dinitrosyl iron complexes with glutathione].

The anti-tumour dose-dependent effect of binuclear dinitrosyl iron complexes with glutathione as NO donors on solid tumour in the mouse Lewis lung carcinome was detected. The complexes being injected at the doses of 21, 42, 105 mg/kg daily during 10 days blocked completely the development of the tumour for the first week after tumour cell implantation into animals. After that, the part of tumour cells which remained in intact alive state began to grow at the rate equal to that for control animals. The effect was proposed to be caused via the formation of the anti-nitrosative defense system in the cells as a response to NO attack to cells. It was also hypothesized that this system can be inactivated by higher doses of dinitrosyl iron complexes. The data were obtained which were in line with the hypothesis.

[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.

[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.

[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.

Detection and evaluation of NO stores in awake rats.

We demonstrated and substantiated the possibility of detection and evaluation of NO stores in freely moving awake rats. NO stores were created by administering NO donor or by heat shock and were then detected by hypotensive reaction to diethyldithiocarbamate (blood pressure monitoring) under conditions of NO synthase inhibition. Electron paramagnetic resonance revealed NO release from its stores by incorporation into paramagnetic mononitrosyl-iron complexes with diethyldithiocarbamate. Five hours after administration of NO donor or heat shock diethyldithiocarbamate induced a blood pressure drop and the appearance of electron paramagnetic resonance signals from the mononitrosyl-iron-diethyldithiocarbamate complex in rat heart, liver, kidneys, and brain. The hypotensive reaction to diethyldithiocarbamate and electron paramagnetic resonance signals were absent in control rats.

Nitric oxide increases gene expression of Ca(2+)-ATPase in myocardial and skeletal muscle sarcoplasmic reticulum: physiological implications.

The aim of the study was to verify the hypothesis that NO-dependent regulation of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) gene expression can play an important role in prevention of calcium overload under the influence of detrimental factors. It was shown that 2 hours after the administration of the NO donor dinitrosyl iron complex (DNIC), the gene expression of myocardial SERCA was increased by 20% as compared to the control. In skeletal muscles, the maximum increase in SERCA expression was observed in 6 hours and amounted to 156% as compared with the initial value. Simultaneously DNIC enhanced the resistance of isolated heart and the organism as a whole to damaging effects of intracellular calcium overload induced by post-ischemic reperfusion or vigorous exercise, respectively. The results obtained confirm the existence of NO-dependent activation of SERCA expression and the important role of this mechanism in restriction of calcium overload.

NO-dependent mechanisms of adaptation to hypoxia.

In studying NO-dependent mechanisms of resistance to hypoxia, it was shown that (1) acute hypoxia induces NO overproduction in brain and leaves unaffected NO production in liver of rats; (2) adaptation to hypoxia decreases NO production in liver and brain; and (3) adaptation to hypoxia prevents NO overproduction in brain and potentiates NO synthesis in liver in acute hypoxia. Dinitrosyl iron complex (DNIC, 200 microg/kg, single dose, iv), a NO donor, decreases the resistance of animals to acute hypoxia by 30%. Nomega-nitro-L-arginine (L-NNA, 50 mg/kg, single dose, ip), a NO synthase inhibitor, and diethyl dithiocarbamate (DETC, 200 mg/kg, single dose, iv), a NO trap, increases this parameter 1.3 and 2 times, respectively. Adaptation to hypoxia developed against a background of accumulation of heat shock protein HSP70 in liver and brain. A course of DNIC reproduced the antihypoxic effect of adaptation. A course of L-NNA during adaptation hampered both accumulation of HSP70 and development of the antihypoxic effect. Therefore, NO and the NO-dependent activation of HSP70 synthesis play important roles in adaptation to hypoxia.

[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.

[Selective inhibition of inducible NO-synthase by nonselective inhibitor]. / Otsenka vozmozhnosti izbiratel'nogo ingibirovaniia indutsibel'noi NO-sintazy s pomoshch'iu neselektivnogo ingibitora.

The study has shown that Nw-nitro-L-arginine, a nonselective nitric oxide (NO) inhibitor, in low non-vasoactive doses (10 mg/kg) exerted a protective effect in heat shock as demonstrated by a decrease in the mortality rate and prevention of acute hypotension in rats. The L-NNA in the same dose inhibited the basal NO production but left unaffected a carbachol-activated NO production. The findings suggest a possibility in principle of preferential inhibition of inducible NO-synthase in pathological conditions related to the NO overproduction using non-vasoactive doses of L-NNA the nonselective NO-synthase inhibitor.