[Nitric oxide and electrogenic metals (Ca, Na, K) in epidermal cells].

Using atomic emission spectrometry and EPR analysis metal-ligand homeostasis (MLH) has been studied in epidermal cells of 954 liquidators of the Chernobyl accident and 947 healthy individuals. A possible association of the redox status with the quantitative changes in the MLH, which could be used as discriminators of oxidative/nitrosative stress, attracts special interest. Characteristic features of oxidative stress mainly related to electrogenic metals (Ca, K, Na), were found not only among the liquidators examined, but also in some healthy individuals (18.1%); this suggests the presence of oxidative/nitrosative stress of non-radiation origin. Correlation between intracellular production of nitric oxide (NO) with quantitative changes in the electrogenic metals may indicate the possible involvement of NO in the generation of an electric potential of the cell.

Diagnosis of nitrosative stress by quantitative EPR-spectroscopy of epidermal cells.

Quantitative assay of nitric oxide (NO) and iron (Fe) was carried out by the electron paramagnetic resonance (EPR) method in epidermal derivative (hair) of 45 liquidators of the Chernobyl accident, 30 employees of radiological institutions having contact with radiation, and 50 students of Vladimir State University, most of which were diagnosed with iron deficiency, namely iron deficiency anemia (4 persons) and latent iron deficiency (34 persons). Measurements were performed on a Radiopan EPR-spectrometer. It was found that the method of quantitative EPR-spectroscopy using diethyldithiocarbamate as a trap in vitro could be successfully used to measure NO-radical activity in such a biological substrate as hair. It was found that the intensity of NO-radical signal in the spectrogram depends on the Fe level (not only in the analyzed substrate, but also in the whole organism).

Chronic hepatitis C: quantitative EPR analysis of nitrogen oxide and copper in patients' blood.

Measurements of nitrogen oxide and copper in the blood of 57 patients with chronic viral hepatitis C was carried out before antiviral therapy by electron paramagnetic resonance on a Radiopan EPR spectrometer. The results indicate elevated levels of nitrogen oxide and copper in the blood of these patients in comparison with normal subjects. Comparison of these findings with the results of a previous analysis of redox status of patients with chronic viral hepatitis C indicate that this disease is characterized by a significant pro-oxidant shift in the realization of redox processes and disorders in the metal ligand homeostasis (at least as regards copper).

Paradoxical effects of two oximes on nitric oxide production by purified NO synthases, in cell culture and in animals.

We have studied the impact of two novel compounds TO-85 (2,6-di-(alpha-aziridino-alpha-hydroxyiminomethyl)pyridine and TO-133 (bis-(diaziridinoglyoximato)copper), designed as NO donors, on nitrite production by cell cultures, NO production in rat tissues and their ability to inhibit purified NO synthases (NOS). Both substances induced considerable increase of nitrite production in cell cultures. When NO production was assayed in rat organs by means of ESR using Fe(DETC) as a spin trap the anticipated NO-increasing activity of TO-85 was observed only in kidneys; the NO level increasing almost 10-fold. Treatment of rats with TO-133, decreased the NO concentration in brain cortex, cerebellum and liver. When the drugs were administered to animals with high level of iNOS expression induced by LPS, TO-85 did not significantly modify the LPS-induced NO production; administration of TO-133 caused a significant decrease of NO production in blood, brain cortex and cerebellum. Only high concentrations of TO-85 were capable of inhibiting iNOS (IC50=7 mM), the substance inhibited eNOS at lower concentrations (IC50=250 microM). Inhibitory activities of TO-85 on nNOS were dependent on BH4 concentrations, suggesting eventual competition of TO-85 with BH4 when the substance interacts with nNOS. TO-133 reduced eNOS activity with IC50=200 microM, nNOS activity with IC50=200 microM, iNOS activity was not much affected by this substance. Thus, the two tested compounds manifest opposite effects on NO production by purified enzymes and in cell culture. The pattern of the NO synthesis modification in a living animal appears to be even more complex. Our results stress the importance of direct measurements of NO in the tissues using the ESR method.

EPR investigation of in vivo inhibitory effect of guanidine compounds on nitric oxide production in rat tissues.

The aim of the present study was to evaluate in vivo effects on NO production of pharmacologically widely used, commercially available NOS inhibitors, structurally related to guanidine. We compared the NO inhibitory potency and selectivity of L-NAME, aminoguanidine and guanabenz in tissues of normal and LPS-stimulated rats using ex vivo EPR measurements of the NO radical in its complex with dithiocarbamate-Fe(II). The tissues studied were the brain cortex, kidney, liver, heart and testis. Differential inhibitory effects were seen for L-NAME, aminoguanidine and guanabenz when applied during basal or LPS-stimulated conditions. Aminoguanidine exerted inhibition of NO only after stimulation with LPS. Guanabenz had little effect on NO in liver, kidney, testis and heart under normal conditions, while it reduced the basal NO in brain cortex. After stimulation with LPS guanabenz afforded a partial inhibition of the NO formation in all tissues studied. L-NAME was a potent inhibitor of NO synthesis in all tested tissues, both during basal and LPS stimulated conditions. Our results show that compounds containing a guanidine moiety might possess different NOS inhibitory profiles in vivo.

Increased synthesis of nitric oxide in rat brain cortex due to halogenated volatile anesthetics confirmed by EPR spectroscopy.

BACKGROUND: Halogenated volatile anesthetics (HVAs) are considered to be inhibitors of nitric oxide synthase (NOS). On other hand, NO mediates the vasodilation produced by HVAs. Thus, both increase and decrease of NO concentration in brain tissues are possible during anesthesia. Previously, we have observed an increase of NO content in rat brain cortex under halothane anesthesia. The goal of this study was to determine whether the observed phenomenon was general for this anesthetic group, if it was specific for brain cortex, and if the NO increase was due changes in NOS activity. METHODS: NO scavengers were injected to adult rats 30 min prior to anesthesia. Rats were anesthetized by inhalation of an O2 mixture with volatile anesthetics (1.5% for halothane; 1% for isoflurane, 2% for sevoflurane). After 30 min of anesthesia, rats were decapitated and brain cortex, cerebellum, liver, heart, kidneys and testes were dissected, frozen in liquid nitrogen and subjected to EPR spectroscopy. Nitric oxide content was determined quantitatively based on the intensity of the NO-Fe-DETC complex spectrum and its comparison with the calibration curve. RESULTS: In rats anesthetized with HVAs, we observed a greater than twofold increase of NO content in brain cortex as compared to the nonanesthetized animals. No significant changes were detected in other organs. The NOS inhibitor N(omega)-nitro-L-arginine abolished the increase of NO content in brain produced by volatile anesthetics. CONCLUSION: The action of volatile anesthetics is coupled with an increase of NO content in the cortex dependent on NOS activity.

[Role of nitric oxide (NO) in the mechanism of anesthetic action]. / Role oksida azota (NO) v mekhanizme deistviia sredstv dlia narkoza.

Published reports on modifications of nitric oxide (NO) synthase (NOS) activity and NO production in the brain during development of anesthesia induced by the most common inhalational (halothane, isoflurane, sevoflurane, enflurane) and intravenous (ketamine, barbiturates, propofol, etomidate) anesthetics are reviewed. According to a popular universally acknowledged hypothesis, inhibition of NOS activity and blockade of NO neurotransmitter function are important steps in the mechanism of action of anesthetics. There are data which confirm the validity of this hypothesis for all above-listed drugs, but there are also data which disagree with it. Some scientists find that anesthesia has no effect on NOS activity and NO production, others found that the enzyme activity and NOS gene expression increased under the effect of anesthesia. Published reports and authors' data on a drastic increase of NO content in the cerebral cortex in halothane anesthesia are discussed. The effects of narcotics on NO-mediated changes in vascular tone are analyzed.

Improved method for EPR detection of DEPMPO-superoxide radicals by liquid nitrogen freezing.

5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) is frequently used as a spin trap for the measurement of superoxide by EPR spectrometry. However, its half life is fairly short in room temperature. We here show that superoxide radicals trapped by DEPMPO can be successfully recorded at -196 degrees C. Moreover, we show that the signal intensity remains unaltered for up to 7 days, when the samples are stored in liquid nitrogen. Our new approach for measurement of superoxide should greatly simplify the studies of this important radical in biological systems.

N-Hydroxyguanidine compound 1-(3,4-dimethoxy- 2-chlorobenzylideneamino)-3-hydroxyguanidine inhibits the xanthine oxidase mediated generation of superoxide radical.

We here show that the novel N-hydroxyguanidine derivative PR5 (1-(3, 4-dimethoxy-2-chlorobenzylideneamino)-3-hydroxyguanidine) is acting as an alternative electron acceptor in xanthine oxidase catalyzed oxidation of xanthine. The reduction product is the corresponding guanidine derivative 1-(3, 4-dimethoxy-2-chlorobenzylideneamino)guanidine (PR9). The reaction occurs under both anaerobic and aerobic conditions. Moreover, EPR measurements show that the action of PR5 is associated with the inhibition of superoxide radical formation seen under aerobic conditions. PR5 also supports xanthine oxidase catalyzed anaerobic oxidation of NADH. Kinetic studies indicate that increasing xanthine concentration significantly increases the apparent K(m) of PR5, but it remains unaltered by changing NADH concentration. Moreover, the molybdenum center inhibitor allopurinol inhibits the PR5-sustained oxidation of xanthine and NADH equally well, whereas the flavin adenine dinucleotide site inhibitor diphenyliodonium (DPI) markedly inhibits only the PR5-sustained oxidation of NADH. We suggest that PR5 binds and becomes reduced at the molybdenum center of the xanthine oxidase. We also found that both PR5 and its reduction product PR9 can inhibit the oxygen-sustained xanthine oxidase reaction. The properties of PR5 suggest that it is a member of a novel class of compounds which we have termed xanthine oxidase electron acceptor-inhibitor drugs. The potential use of xanthine oxidase electron acceptor-inhibitors in the prevention of free radical mediated tissue damage in organ ischemia-reperfusion diseases is discussed.

Drastic increase in nitric oxide content in rat brain under halothane anesthesia revealed by EPR method.

A drastic increase in nitric oxide (NO) content was revealed by the EPR method in rat brain cortex and cerebellum under halothane anesthesia. The NO scavenger diethyldithiocarbamate sodium salt (DETC) and ferrous citrate were injected into adult rats 30-60 min before anesthesia. Rats were anesthetized by inhalation of a halothane-oxygen mixture (1%, 1.5%, 2%, or 4%). After different times of anesthesia, rats were decapitated, and brain cortex and cerebellum were dissected, frozen in liquid nitrogen, and subjected to EPR spectroscopy. The concentration of NO was determined from the NO-Fe-DETC radical spectrum. In control animals, NO content in the cerebellum was only 68% of that in the cortex. We observed a time-dependent increase in NO content in the cortex and cerebellum of rats anesthetized with 1.5% halothane. In brain cortex, the NO level increased to six times that of waking animals after 30 min and remained at this level up to 60 min of anesthesia. In cerebellum the changes were less drastic, the NO level showing only a 2-fold increase. The same effect was produced by 1% and 2% halothane. Ketamine, chloral hydrate, and pentobarbital were used as reference drugs. None of these anesthetics produced effects similar to those of halothane. In ketamine-anesthetized rat brain, the NO content slightly decreased. Pentobarbital and chloral hydrate produced an insignificant increase in NO. Data are discussed in the context of possible interference of halothane in the regulation of nitric oxide synthase activity.