Autowave mode of functioning of the system nitric oxide + free iron + thiols may ensure the regulation of biological action of nitric oxide and its endogenic compounds

It has been shown earlier that, in a system NO + Fe2+ + thiols in aqueous solution, an oscillatory mode of changes with time in the concentration of paramagnetic dinitrosyl iron complexes with thiol-containing legends and S-nitrosothiols formed in this system and in the concentration of free iron (not included into dinitrosyl iron complexes) can be realized. It is assumed that, in this system, autowaves can arise, which ensure periodic changes with time and space in the concentration of the system constituents. These changes may underlie the regulation of the physiologic effect of nitric oxide, dinitrosyl iron complexes, and S-nitrosothiols as agents affecting various intracellular and tissue targets.     

Effect of dinitrosyl iron complexes with thiol-containing ligands on the penile cavernosum bodies in rats

A beneficial effect of dinitrosyl iron complexes (DNIC) with thiol-containing ligands on penile cavernus tissue was shown in rats subjected to penile denervation. Histological and histochemical investigations demonstrated that intracavernous injections of dinitrosyl iron complexes (2 times per one week during 6 months) blocked the reinforcement of endothelial cell proliferation in the tissue characteristic of the cavernous tissue when the penile nerve was removed. On the other hand, treatment with dinitrosyl iron complexes led to the preservation of mitotic activity of smooth myocytes and protected against the appearance in these cells of collagenase, an indicator of muscle transformation into fibrous tissue. It was shown that the process of fibrous transformation of myocytes correlates with a decrease in the mitotic activity of fibroblasts in the adventive part of cavernosa. The mitotic activity increased in cavernous tissue in the absence of dinitrosyl iron complexes. The efficiency of long-term action of dinitrosyl iron complexes on the erection in both intact animals and animals subjected to neuroectomy of cavernous tissue nerve was shown. The injection of low-molecular dinitrosyl iron complexes to the cavernous tissue resulted in the formation of protein-bound dinitrosyl iron complexes in the tissue, which were detected by the EPR technique. It is assumed that these dinitrosyl iron complexes function as a depot of nitric oxide, providing long-lasting penis erection.     

A role of iron ions in the SOS DNA repair response induced by nitric oxide in Escherichia coli

An induction of the SOS DNA repair response by physiological nitric oxide donors (dinitrosyl iron complexes (DNIC) with thiols and S-nitrosothiols (RSNO)) was studied in E. coli cells. DNIC with thiols were the most effective SOS-inducers. Being more toxic, RSNO mediated a similar response at 10-100 microM, but they were inactive at concentrations above 0.5 mM. Pretreatment of the cells with chelating agents, o-phenanthroline and picolinic acid, prevented induction of the SOS response by all NO-donors used and led to a decrease in the DNIC-type EPR signal that appeared after incubation of the cells with DNIC or S-nitrosoglutathione (GSNO). Analysis of these effects revealed a dual role of iron ions in reactivity and toxicity of the NO-donating agents. On one hand, they could stabilize GSNO in the form of less toxic DNIC, and, on the other hand, they took part in the formation of the SOS-inducing signal by NO-donating agents.     

Detection of autowave distribution of the concentration of a dinitrosyl iron complex with glutathione formed in an aqueous solution of S-nitrosoglutathione after addition 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 a drop of a solution of glutathione (0.5 mM) and ferrous iron (1 mM) in the same buffer of volume 10 μL was detected. At regular intervals, the picture of autowaves changed in time intervals of 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.     

Interaction between dinitrosyl iron complexes and intermediate products of oxidative stress

The interaction between glutathione-containing dinitrosyl iron complexes and superoxide radicals has been studied under the conditions of superoxide radical generation in mitochondria and in a model system xanthine-xanthine oxidase. It has been shown that both superoxide radical and hydroxyl radical are involved in the destruction of dinitrosyl iron complexes. At the same time, iron contained in dinitrosyl iron complex, apparently, does not catalyze the decomposition of hydrogen peroxide with the formation of hydroxyl radical. It has been found that dinitrosyl iron complexes with different anion ligands inhibit effectively the formation of phenoxyl probucol radical in a hemin-H2O2 a system. In this process, different components of the dinitrosyl iron complexes take part in the antioxidant action of these complexes.     

Effects of reactive species of oxygen and nitrogen on iron ion release from ferritin and synthesis of dinitrosyl iron complexes

Using EPR spectroscopy it was established that Fe ions released from ferritin under the action of glutathione and superoxide took part in the formation of dinitrosyl complexes of iron with glutathione (DNIC). The reaction between O2-. and NO resulted in the formation of peroxynitrite, which oxidized glutathione to the thiyl radical. In these conditions, DNIC did not inhibit the formation of thiyl radicals but effectively slowed down the oxidative destruction of beta-carotene by peroxynitrite and free radicals of lipids. In the presence of glutathione, the inversion of the antioxidant properties of DNIC into prooxidant ones took place. S-nitrosoglutathione prevented this inversion and suppressed the free-radical oxidation of beta-carotene induced by ferritin. It was proposed that the equilibrium between S-nitrosoglutathione, DNIC, "free Fe" ions and ferritin may determine the balance between prooxidant and antioxidant processes in living organisms.     

The mechanisms of S-nitrosothiol decomposition catalyzed by iron.

The mechanisms of S-nitrosothiol transformation into paramagnetic dinitrosyl iron complexes (DNICs) with thiol- or non-thiol ligands or mononitrosyl iron complex (MNICs) with N-methyl-D-glucamine dithiocarbamate catalyzed by iron(II) ions under anaerobic conditions were studied by monitoring EPR or optical features of the complexes and S-nitrosothiols. The kinetic investigations demonstrated the appearance of short-living paramagnetic mononitrosyl-iron complex with L-cysteine prior to the formation of stable dinitrosyl-iron complex with cysteine in the solution of iron(II)-citrate complex (50-100 microM), S-nitrosocysteine (400 microM), and L-cysteine (20 mM) in 100 mM Hepes buffer (pH 7.4). The addition of deoxyhemoglobin (100 microM) did not influence the process, which points to a direct interaction between S-nitrosocysteine and iron(II) ions to yield DNIC. The reaction of DNIC-cysteine formation is first- and second-order in iron and S-nitrosocysteine, respectively. The third-order rate constant is (1.0 +/- 0.2) x 10(5) M(-2) s(-1) (estimated from EPR results) or (2.0 +/- 0.1) x 10(4) M(-2) s(-1) (estimated by optical method). A similar process of DNIC-cysteine formation was observed in a solution of iron(II)-citrate complex, L-cysteine, and NO-proline (200 microM) as a NO* donor. The appearance of a less stable dinitrosyl-iron complex with phosphate was detected when solutions of iron(II)-citrate containing 100 mM phosphate buffer (pH 7.4) were mixed with S-nitrosocysteine or NO-proline. The rapid formation of DNIC with phosphate was followed by its decay. When the concentration of L-cysteine in solutions was reduced from 20 to 1 mM, the life-time of the DNIC-cysteine diminished notably; this was caused by consumption of L-cysteine in the process of DNIC-cysteine formation from S-nitrosocysteine and iron. Thus, L-cysteine is consumed. Formation of DNIC with glutathione was also observed in a solution of glutathione (20 mM), S-nitrosoglutathione (400 microM), and iron(II) complex (800 microM) in 100 mM Hepes buffer (pH 7.4), but the rate of formation was about 10 times slower than the formation of the DNIC-cysteine. The rate of MNIC-MGD formation from iron(II)-MGD complexes and S-nitrosocysteine was first-order in both reactants. The second-order rate constant for this reaction, estimated from EPR measurements, was 30 +/- 5 M(-1) s(-1). Rate constants of MNIC-MGD formation from iron(II)-MGD and the more stable S-nitrosoglutathione and S-nitroso-D,L-penicillamine were equal to 3.0 +/- 0.3 and 0.3 +/- 0.05 M(-1) s(-1), respectively. Thus, the concerted mechanism of DNIC and MNIC formation from S-nitrosothiols and iron(II) ions can be suggested to be predominant.     

Inhibition of platelet aggregation by dinitrosyl iron complexes with low molecular weight ligands

The dinitrosyl iron complexes (DNIC) with thiosulphate, cysteine or phosphate were shown to inhibit in vitro (in citrate plasma) the human platelet aggregation induced by ADP, collagen or adrenaline. This effect cannot be explained by the toxic action of DNIC on the platelet membrane, since DNIC-pretreated platelets are capable of aggregating under the action of 10(-8) M/ml of phorbol ester, which is known to cause direct activation of protein kinase C. The antiaggregatory activity of DNIC exceeds that of Na-nitroprusside and seems to be due to nitric oxide capable to activate guanylate cyclase of platelets. Using the EPR method, it was shown that addition of DNIC to platelet-enriched plasma results in a rapid transfer of Fe(NO)2 groups to the coupled RS(-)-groups proteins of plasma and, apparently, of platelet membrane proteins. These protein DNIC seem to be the source of NO which inhibits human platelet aggregation.     

Formation of a new type of dinitrosyl iron complexes bound to cysteine modified with methylglyoxal

It has been shown that interaction of cysteine dinitrosyl iron complexes with methylglyoxal leads to the formation of a new type of dinitrosyl iron complexes, EPR spectrum of these complexes essentially differs from spectra of dinitrosyl iron complexes containing unmodified thiol. The products of the cysteine reaction with methylglyoxal are hemithioacetals, Schiff bases and thiazolidines, which most likely serve as ligands for the new type of dinitrosyl iron complexes. It has been shown that the new type of dinitrosyl iron complexes as cysteine dinitrosyl iron complexes, which are physiological donors of nitric oxide, exert a vasodilator effect. It has also been found that the oxidative destruction of the new type of dinitrosyl iron complexes occurs at normal oxygen partial pressure, but these dinitrosyl iron complexes remain rather stable under hypoxia modeling. An assumption that the destruction of the new type of dinitrosyl iron complexes is caused by the formation of a bound peroxynitrite-containing intermediate is made.     

The interaction of ferritin and myoglobin as inductors of lipid peroxidation. The role of reactive oxygen and nitrogen species

The effect of iron dinitrosyl complexes, S-nitrosoglutathione, and glutathione on free radical oxidation of rat heart mitochondria induced by tert-butyl hydroperoxide and metmyoglobin or their combination with ferritin was studied. It was shown that iron dinitrosyl complexes or the combination of S-nitrosoglutathione and glutathione inhibited most effectively the peroxidation of mitochondrial membranes. It was found that ferritin stimulated the prooxidant action of metmyoglobin. Using EPR spectroscopy, it was established that, in conditions of O2*- generation, the destruction of iron dinitrosyl complexes took place. Iron dinitrosyl complexes also inhibited the formation of thiyl radicals, which appeared during O2*- generation in the system containing glutathione and S-nitrosoglutathione. It is essential that the formation of iron dinitrosyl complexes in this reaction system took place with the involvement of ferritin. It was proposed that the prooxidant action of ferritin and myoglobin could be inverted to the antioxidant one.     

Nitric oxide initiates iron binding to neocuproine.

It was demonstrated that two species of paramagnetic dinitrosyl iron complex (DNIC) with neocuproine form under the following conditions: in addition of neocuproine to a solution of DNIC with phosphate; in gaseous NO treatment of a mixture of Fe(2+) + neocuproine aqueous solutions at pH 6.5-8; and in addition of Fe(2+)--citrate complex + neocuproine to a S-nitrosocysteine (cys-NO) solution. The first form of DNIC with neocuproine is characterized by an EPR signal with g-factor values of 2.087, 2.055, and 2.025, when it is recorded at 77K. At room temperature, the complex displays a symmetric singlet at g = 2.05. The second form of DNIC with neocuproine gives an EPR signal with g-factor values of 2.042, 2.02, and 2.003, which can be recorded at a low temperature only.The revealed complexes are close to DNIC with cysteine in their stability. The ability of neocuproine to bind Fe(2+) in the presence of NO with formation of paramagnetic DNICs warrants critical reevaluation of the statement that neocuproine is only able to bind Cu(+) ions. It was suggested that the observed affinity of neocuproine to iron was due to transition of Fe(2+) in DNIC with neocuproine to Fe(+). In experiments on cys-NO, it was shown that the stabilizing effect of neocuproine on this compound could be due to neocuproine binding to the iron catalyzing decomposition of cys-NO.     

Autowave mode of the functioning of the nitric oxide + free iron + thiols system as a basis for control of the biological action of nitric oxide and its endogenous compounds

The NO + Fe²⁺ + thiols system in an aqueous solution has been found earlier to exhibit temporal oscillatory changes in the concentration of paramagnetic dinitrosyl iron complexes with thiol-containing ligands and S-nitrosothiols, as well as in the concentration of free iron (not included in the complexes). It is proposed that autowaves can appear in this system characterized by periodic changes in the concentrations of its components in time and space. Such changes may form a basis for the control of the physiological effects of nitric oxide, dinitrosyl iron complexes, and S-nitrosothiols as agents affecting various cellular and tissue targets.     

Dinitrosyl iron complexes with cysteine suppress the development of experimental endometriosis in rats

Administration of dinitrosyl iron complexes (DNIC) with cysteine suppressed the development of experimental (surgically induced) endometriosis in rats: the mean size of endometrioma was 1.85 times smaller if 0.5 mL of a 5 mM aqueous solution of DNIC had been injected daily for 10 days. It is supposed that NO molecules and nitrosonium ions (NO⁺), released from DNIC rapidly decomposed in the organism, prove cytotoxic for endometrioid tissue.     

Coordination of iron ions in the form of histidinyl dinitrosyl complexes does not prevent their genotoxicity

Formation of dinitrosyl iron complexes (DNICs) was observed in a wide spectrum of pathophysiological conditions associated with overproduction of NO. To gain insight into the possible genotoxic effects of DNIC, we examined the interaction of histidinyl dinitrosyl iron complexes (HIS-DNIC) with DNA by means of circular dichroism. Formation of DNIC was monitored by EPR and FT/IR spectroscopy. Vibrational bands for aquated HIS-DNIC are reported. Dichroism results indicate that HIS-DNIC changes the conformation of the DNA in a dose-dependent manner in 10 mM phosphate buffer (pH 6). Increase of the buffer pH or ionic strength decreased the effect. Comparison of HIS-DNIC DNA interaction with the effect of hydrated Fe²⁺ ion revealed many similarities. The importance of iron ions in HIS-DNIC induced genotoxicity is confirmed by plasmid nicking assay. Treatment of pUC19 plasmid with 1 μM HIS-DNIC did not affect the plasmid supercoiling. Higher concentrations of HIS-DNIC induced single strand breaks. The effect was completely abrogated by addition of deferoxamine, a specific strong iron chelator. Our data reveal that formation of HIS-DNIC does not prevent DNA from iron-induced damage and imply that there is no direct interrelationship between iron–NO coordination and their mutual toxicity modulation.     

The cytoprotective effect of nitrite is based on the formation of dinitrosyl iron complexes

Nitrite protects various organs from ischemia–reperfusion injury by ameliorating mitochondrial dysfunction. Here we provide evidence that this protection is due to the inhibition of iron-mediated oxidative reactions caused by the release of iron ions upon hypoxia. We show in a model of isolated rat liver mitochondria that upon hypoxia, mitochondria reduce nitrite to nitric oxide (NO) in amounts sufficient to inactivate redox-active iron ions by formation of inactive dinitrosyl iron complexes (DNIC). The scavenging of iron ions in turn prevents the oxidative modification of the outer mitochondrial membrane and the release of cytochrome c during reoxygenation. This action of nitrite protects mitochondrial function. The formation of DNIC with nitrite-derived NO could also be confirmed in an ischemia–reperfusion model in liver tissue. Our data suggest that the formation of DNIC is a key mechanism of nitrite-mediated cytoprotection.     

The source of the iron binding with nitric oxide in activated macrophages]

No decrease in iron-sulphur centers was found in cultured macrophage cells (J774) after the treatment with nitric oxide (10(-7) M NO/10(7) cells) during 5 min. The center content was controlled by the electron spin resonance (ESR) method. The macrophages pretreated with dithionite + methyl viologen showed the formation of dinitrosyl iron complexes (DNIC) with a characteristic ESR signal at g approximately 2.03. The data suggest that loosely bound nonheme iron (free iron) mostly contributes to the formation of these complexes. Iron from iron-containing proteins does not release from these centers under the direct action of nitric oxide. The iron-sulphur centers can be destroyed by the products of nitric oxide oxidation (NO2, N2O3, etc.) as oxidizing and acid agents.     

Physicochemistry of dinitrosyl iron complexes with thiolate ligands underlying their beneficial effect in endometriosis

Exogenous dinitrosyl iron complexes (DNIC) with thiolate ligands as NO and NO⁺ donors are capable of exerting both regulatory and cytotoxic effects on diverse biological processes similarly to those characteristic of endogenous nitric oxide. Regulatory activity of DNIC (vasodilatory, hypotensive, suppressing thrombosis, increasing erythrocyte elasticity, accelerating skin wound healing, inducing penile erection, etc.) is determined by their capacity of NO and NO⁺ transfer to biological targets of the latter (heme- and thiol-containing proteins, respectively) due to higher affinity of the proteins for NO and NO⁺ than that of DNIC. Cytotoxic activity of DNIC is provided by rapid DNIC decomposition under action of iron-chelating compounds, resulting in appearance of NO and NO⁺ in cells and tissues in high amounts. The latter mechanism is suggested to cause the blocking effect of DNIC as cytotoxic effectors on the development of benign endometrial tumors in rats with experimental endometriosis. It is also proposed that a similar mechanism can operate to cause at least a delay of malignant tumor proliferation under action of DNIC.     

Distribution and pharmacokinetics of dinitrosyl iron complexes in rat organs

Dinitrosyl iron complexes (DNICs) have been traced in rat blood and organs after intravenous infusion of Oxacom. It is shown that the active principle (DNIC with glutathione) is rapidly distributed through the organism and deposited in blood and organs as protein-bound DNICs. The specific levels of DNIC in the main body organs are comparable, whereas its apparent lifetimes relate as blood < heart = lung < liver < kidney. Spin trapping assays indicate that protein-bound DNICs are a major but not the only form of NO deposition; the next largest depot is most probably formed by S-nitrosothiols. The gradual release of NO from such pools ensures the smooth and prolonged hypotensive effect of Oxacom.     

Detection of nitrite and nitrosocompounds in chemical systems and biological liquids by the calorimetric method

The capacity of nitrite, S-nitrosothiols (RS-NO), dinitrosyl iron complexes (DNICs) with thiolcontaining ligands, and nitrosoamines to inhibit catalase has been used for the selective determination of these compounds in purely chemical systems and biological liquids: cow milk and colostrum. The limiting sensitivity of the method is 50 nM. A comparison of the results of the determinations of RS-NO, DNIC, and nitrite by the catalase method and the Griess method conventionally used for nitrite detection showed that, firstly, Griess reagents decompose DNIC and RS-NO to form nitrite. Therefore, the Griess method cannot be used for nitrite determination in solutions of these substances. Secondly, Griess reagents interact with complexes of mercury ions with RS-NO, inducing the release of nitrosonium ions from the complex followed by the hydrolysis of nitrosonium to nitrite. Thus, the proposition about the spontaneous decay of the complexes of mercury ions with RS-NO is incorrect. Keeping in mind a high sensitivity of the method, the use of catalase as an enzyme detector of nitrosocompounds allows one to detect these compounds in neutral medium without prior purification of the object, thereby preventing artificial effects due to noncontrolled modifications of the compounds under study.