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 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.

Iron catalyzes both decomposition and synthesis of S-nitrosothiols: optical and electron paramagnetic resonance studies.

Formation of S-nitrosothiols was demonstrated in 1-50 mM aqueous solutions of cysteine or glutathione (cys-NO or GS-NO, respectively) upon contact of thiols with gaseous nitric oxide under a pressure of 50-600 mm Hg and anaerobic conditions. The yield of S-nitrosothiols was increased by mixing with NO plus air at a molar ratio [NO]/[O2 from air] of no less than 40. In this instance, the S-nitrosothiol formation was optimum at a NO pressure of 100-150 mm Hg. The addition of 0.25 mM o-phenanthroline, a selective Fe2+ chelator, to thiol solutions prior to the treatment with NO or NO + air completely blocked the formation of S-nitrosothiols. On the other hand, this process was potentiated by the addition of Fe2+ but not Cu2+ ions. These data indicated a crucial influence of Fe2+ on the process. The contact of o-phenanthroline with S-nitrosothiols synthesized by a routine method (treatment of thiol solutions with the NO + NO2 mixture at pH <1) did not induce their degradation at pH 3-10. Moreover, o-phenanthroline strikingly enhanced the cys-NO stability at neutral pH. Cysteine, glutathione, and desferal, a selective Fe3+ chelator, exerted a similar effect on cys-NO. The stabilizing effect of thiols on cys-NO was accompanied by the formation of dinitrosyl-iron complexes with thiol-containing ligands containing admixed (intrinsic) iron (1-2 microM). The addition of Fe2+ at a concentration higher than 10 microM abolished the stabilizing effect of thiols on cys-NO. Therefore iron can induce both degradation and synthesis of S-nitrosothiols. According to the proposed mechanisms such opposite effects of iron on S-nitrosothiols are determined by the ratio between S-nitrosothiols, thiols, iron, and NO in the reaction system.

The influence of antioxidants and cycloheximide on the level of nitric oxide in the livers of mice in vivo.

When injected into mice prior to the NO generation increase induced with lipopolysaccharide (LPS) from Escherichia coli, exogenous antioxidants diethyldithiocarbamate (DETC) or phenazan (sodium 3.5-di-tert-butyl-4-oxiphenylpropionate) as well as the inhibitor of protein biosynthesis, cycloheximide (CHI) attenuated the NO production in mouse liver in vivo. These data demonstrated the key role of free radicals, which were likely, active oxygen species, in the synthesis of inducible NO-synthase (iNOS) responsible for the NO production in this organ. Similar effects of phenazan and CHI were observed in livers of mice treated with gamma-irradiation or LPS + Fe(2+)-citrate, which suggested that these treatments also induced 1NOS synthesis through initiating the action of active oxygen species. The rate of NO synthesis was estimated by accumulation of paramagnetic mononitrosyl iron complexes with DETC (MNIC-DETC) detected using the EPR method. The formation of MNIC-DETC complexes was found in the brain of mice pre-treated with LPS + Fe(2+)-citrate which seemed to be due to iNOS synthesis stimulated by this treatment.

[S-nitrosothiols and dinitrosothiol iron complexes as a source of nitric oxide in animals]. / S-nitrozotioly i dinitrozotiol'nye kompleksy zheleza kak istochniki oksida azota v organizmakh zhivotnykh.

It was established by EPR method, that S-nitrosocysteine or S-nitrosohomocysteine rapid destructured with releasing nitric oxide (NO) in blood, forming with hemoglobin paramagnetic hemoglobin nitrosyl complexes (Hb-NO). In the presence of exogenous iron in blood dinitrosyl non-heme iron complexes (DNIC) with thiol-containing ligands, i.e. 2,03 complexes arose in blood. These complexes occurred in large quantity if it was introduced to animals low molecular DNIC with cysteine or homocysteine in relation iron/thiol in complexes and solution 1:20 or 1:2. 2,03 complexes in organisms was stable. So, the system of DNIC S-nitrosothiols prevailed the S-nitrosothiols conversation into DNIC.

[Exogenous iron and gamma-radiation induces synthesis of NO-synthetase in mouse liver]. / Ekzogennoe zhelezo i gamma-obluchenie indutsiruiut sintez NO-sintetazy v pecheni myshei.

The inhibitor of protein biosynthesis, cycloheximide (CHI) and the exogenous antioxidant, phenazan, attenuated the synthesis of nitric acid oxide (NO) in mouse liver in vivo induced by gamma-irradiation, bacterial lipopolysaccharide (LPS) or LPS+Fe(2+)-citrate treatment of experimental animals. The rate of NO synthesis was followed by accumulation of paramagnetic mononitrosyl iron complexes with the exogenous ligand--diethyldithiocarbamate (MNIC-DETC). The latter were formed as a result of NO binding to selective NO traps (DETC complexes with exogenous or endogenous Fe2+ ions) and measured by the EPR method. A conclusion is drawn that the activation of NO biosynthesis under the action of gamma-irradiation, LPS or LPS+Fe(2+)-citrate treatment was due to the induction of NO synthase synthesis inhibited by CHI. This process is initiated by active oxygen species, presumably due to the activation of the transcription factor, NFkB protein. The accumulation of active oxygen species was inhibited by the antioxidant, phenazan.

[Dinitrosyl complexes of iron with thiol-containing ligands and their reverse conversion into nitrosothiols]. / Dinitrozil'nye kompleksy zheleza s tiolsoderzhashchimi ligandami i ikh obratimoe prevrashchenie v nitrozotioly.

Bovine serum albumin (BSA) is capable of forming dinitrosyl complexes with iron (DNIC) containing one thiol and one nonthiol ligand and yielding an EPR signal at the following values of the g-factor: g1 = 2.046; g2 = 2.03; g3 = 2.012. During interaction with L-cysteine or N-acetyl-L-cysteine the complex symmetry increased due to the substitution within DNIC of the nonthiol ligand of BSA for cysteine; such DNIC was characterized by an EPR signal with an axial-symmetrical tensor of the g-factor. At high cysteine concentrations all of the Fe(NO)-groups appeared to be transferred from BSA to cysteine to form DNIC with cysteine yielding an EPR signal with a g-factor of g perpendicular = 2.037; g parallel = 2.012. The lifetime of DNIC-BSA was about 24 hrs, whereas that of DNIC-cysteine was less than 1 min due to cysteine oxidation in the air. In 0.5 M HCl DNIC-BSA and DNIC-cysteine were reversibly converted into appropriate nitrosothiols characterized by intensive adsorption at 340-360 nm. Upon subsequent neutralization of the solution and addition of the substituent (cysteine or dithionite) these nitrosothiols were converted into DNIC. In the absence of iron cysteine and dithionite caused reductive destruction of protein and low molecular weight nitrosothiols to liberate nitrogen oxide. This property of nitrosothiols makes them distinct from those DNIC, in which cysteine acts exclusively as a scavenger of Fe(NO)-groups by shifting the equilibrium between the protein and low molecular weight DNIC towards the latter.

EPR evidence for nitric oxide production from guanidino nitrogens of L-arginine in animal tissues in vivo.

Administration of Fe(2+)-citrate complex (50 mg/kg of FeSO4 or FeCl2 plus 250 mg/kg of sodium citrate) subcutaneously in the thigh or Escherichia coli lipopolysaccharide (LPS, 1 mg/kg) intraperitoneally, (i.p.) to mice induced NO formation in the livers in vivo at the rate of 0.2-0.3 micrograms/g wet tissue per 0.5 h. The NO synthesized was specifically trapped with Fe(2+)-diethyldithiocarbamate complex (FeDETC2), formed from endogenous iron and diethyldithiocarbamate (DETC) administered i.p. 0.5 h before decapitation of the animals. NO bound with this trap resulted in the formation of a paramagnetic mononitrosyl iron complex with DETC (NO-FeDETC2), characterized by an EPR signal at g perpendicular = 2.035, g parallel = 2.02 with triplet hyperfine structure (HFS) at g perpendicular. This allowed quantification of the amount of NO formed in the livers. An inhibitor of enzymatic NO synthesis from L-arginine, NG-nitro-L-arginine (NNLA, 50 mg/kg) attenuated the NO synthesis in vivo. L-Arginine (500 mg/kg) reversed this effect. Injection of L-[guanidineimino-15N2]arginine combined with Fe(2+)-citrate or LPS led to the formation of the EPR signal of NO-FeDETC2 characterized by a doublet HFS at g perpendicular, demonstrating that the NO originates from the guanidino nitrogens of L-arginine in vivo.

Effect of diethyldithiocarbamate on the activity of nitric oxide-releasing vasodilators.

The effect of diethyldithiocarbamate (DETC, 10(-3) M) on the vasorelaxant activity of acetylcholine, nitric oxide (NO), nitrite, glyceryl trinitrate or dinitrosyl iron cysteine complexes was studied in isolated rat aortic rings contracted with norepinephrine. Pretreatment of these segments with DETC attenuated the vasorelaxation induced by vasodilators and prevented the subsequent restoration of vessel tone, whereas DETC added after the vasodilators induced a rapid restoration of tone. The inhibitory effect of DETC was due to the trapping of NO by a complex of DETC with Fe2+ formed in the tissue.

Similarity between the vasorelaxing activity of dinitrosyl iron cysteine complexes and endothelium-derived relaxing factor.

Dinitrosyl iron complexes with cysteine (DNIC) induced a concentration-dependent relaxation of pre-contracted (norepinephrine, 10(-7) M) de-endothelialized ring segments of rat aorta. The vasodilator response was more similar to acetylcholine (ACh)-induced relaxation in intact aortic rings than to nitric oxide (NO)-induced relaxation. The time course of tone recovery after maximal concentrations (10(-5) M) of DNIC was similar to the time course of tone recovery after endothelium-dependent relaxation induced by ACh, whereas the restoration of tone after NO was much faster. Vessel tone was restored by oxyhemoglobin (10(-5) M) in all cases. The results suggest that DNIC with cysteine may function as endothelium-derived relaxing factor in the vessels.

[L-arginine--an endogenous source of nitric oxide in animal tissues in vivo]. / L-arginin--éndogennyi istochnik oksida azota v tkaniakh zhivotnykh in vivo.

According to EPR data, NG-mononitro-L-arginine (MNA) being intraperitoneally injected to inbred albino mice in the dose of 70-700 mg/kg strongly decreases the formation of mononitrosyl iron complexes (MNIC) with the exogenous ligand, diethyldithiocarbamate (DETC) in liver cells. Simultaneous injections of experimental mice with MNA (70 mg/kg) and L-arginine (700 mg/kg) are unaccompanied by the formation of MNIC-DETC complexes. It is concluded that nitric oxide (NO) which is produced in mouse liver in vivo and which provides for the formation of MNIC complexes with DETC is generated by L-arginine via an enzymatic reaction which is competitively inhibited by MNA. Besides, MNA causes reversible inhibition and augmented synthesis of NO formed in mouse liver after the injection of the exogenous lipopolysaccharide of E. coli.

[Supercarcinogens and supermutagens--nitrosoalkoxyalkylamines--effective producers of nitrogen oxide]. / Superkantserogeny i supermutageny--nitrozoalkoksialkilaminy--éffektivnye produtsenty okisi azota.

Nitrosoalcoxyalkylamines are more efficient producers of nitrogen oxide in vivo than nitrosoalkylamines. Production of nitrogen oxide was monitored by EPR. The patterns of denitrosylation have been studied. The ability of the studied compounds to produce nitrogen oxide in vivo may be related to their carcinogenic and mutagenic action.

[ATP synthesis in the membrane fragments of Staphylococcus aureus cells induced by a sudden increase in pH]. / Sintez ATF v membranno-stenochnykh fragmentakh kletok Staphylococcus aureus, indutsirovannyi skachkoobraznym povysheniem pH.

Membrane fragments of St. aureus are able to synthesize up to 7 nmol/mg protein of ATP after a jump-like increase of pH value. The presence of respiratory substrates is obligatory. The effect is realized only during the jump, if pH passes of 7.4 value. ADP phosphorylation induced by pH-jump is completely inhibited by DCCD.

[The effect of respiration substrates on the ATPase activity of uncoupled mitochondria]. / Vliianie substratov dykhaniia na ATFaznuiu aktivnost' razobshchennykh mitokhondrii.

Mg-dependent ATPase activity in aging uncoupled mitochondria is 30% reduced by 2 mM of succinate. The results show that redox state of mitochondria electron-transport chain affects the activity and apparently modifies the structure of the enzyme performing ATP synthesis and hydrolysis.

[The effect of external factors on ATP synthesis during sudden pH increase in mitochondria]. / Vliianie vneshnikh faktorov na sintez ATF pri skachkoobraznom povyshenii pH v mitokhondriiakh.

Fast alkaline jump in aging uncoupled mitochondria results in the ATP synthesis. This process is completely inhibited with oligomycin and by 60-70% with FCCP. The NADH experiments have shown that mitochondrial membranes do not change their orientation during aging.

PH-jump-induced ADP phosphorylation in mitochondria.

Mitochondria, uncoupled by aging or by freeze-thaw treatment, are able to synthesize ATP from ADP and Pi after a fast increase (but not decrease) in the external pH. The maximal ATP yield (approx. 2.5 ATP molecules/electron-transport chain per pH jump) can be obtained under the following conditions: (1) the pH change during the jump must exceed 0.7 pH units; (2) in the course of this change, the pH of the mitochondrial suspension must cross the pH 8.1-8.3 value. This pH-jump-induced ATP synthesis is completely inhibited by oligomycin.