Peter I. Mordvintcev

On-line detection of nitric oxide formation in liquid aqueous phase by electron paramagnetic resonance spectroscopy.

A method for the detection of the nitric oxide radical (NO) in oxygen-containing aqueous solution by means of electron paramagnetic resonance spectroscopy (EPR) is described. NO evolving from the spontaneous decomposition of 3-morpholinosydnonimine (SIN-1) was trapped by Fe(2+)-diethyldithiocarbamate (DETC) complex dissolved in yeast cell membranes. The resulting mononitrosyl-Fe(2+)-(DETC)2 complex was stable and exhibited a characteristic EPR signal at g perpendicular = 2.04 and g parallel = 2.02 with an unresolved triplet hyperfine structure at g perpendicular in frozen solution and an isotropic triplet signal at gav = 2.03 at 37 degrees C. The amount of NO trapped was calculated from the amplitude of one of the triplet lines calibrated by means of a dinitrosyl-Fe(2+)-thiosulfate standard. The lower detection limit of NO was 0.5 nmol/(ml x h) due to a low background NO signal. The upper detection limit was about 10 nmol NO/40 mg traps (DETC-loaded yeast cells), because of saturation of traps. The trapping efficiency approached 60% under anaerobic conditions and with low concentrations of SIN-1, but decreased progressively with higher concentrations and in the presence of oxygen. Nitrite (up to 0.1 mM) did not increase the background NO level. The sensitivity was sufficient to follow the rate of NO release from SIN-1 on-line at 37 degrees C in a flat quartz cuvette. The time course of NO release detected by EPR spectrometry correlated with the time course of nitrite accumulation measured by diazotation. In conclusion, this method will permit the on-line detection of NO formation from endogenous and pharmacological sources in oxygen-containing aqueous media.

The potent vasodilating and guanylyl cyclase activating dinitrosyl‐iron(II) complex is stored in a protein‐bound form in vascular tissue and is released by thiols

We studied the biological activity, stability and interaction of dinitrosyl‐iron(II)‐L‐cysteine with vascular tissue. Dinitrosyl‐iron((II)‐L‐cysteine was a potent activator of purified soluble guanylyl cyclase (EC50 (nM with and 100 nM without superoxide dismutase) and relaxed noradrenaline‐precontracted segments of endothelium‐denuded rabbit femoral artery (EC50 10 nM superoxide dismutase). Pre‐incubation (5 min; 310 K) of endothelium‐denuded rabbit aortic segments with dinitrosyl‐iron(II)‐L‐cysteine (0.036–3.6 mM) resulted in a concentration‐dependent formation of a dinitrosyl‐iron(II complex with protein thiol groups, as detected by ESR spectroscopy. While the complex with proteins was stable for 2 h at 310 K, dinitrosyl‐iron(II)‐L‐cysteine in aqueous solution (30–360 μM) decomposed completely within 15 min, as indicated by disappearance of its isotropic ESR signal at g av = 2.03 (293 K). Aortic segments pre‐incubated with dinitrosyl‐iron(II)‐L‐cysteine released a labile vasodilating and guanylyl cyclase activating factor. Perfusion of these segments with N‐acetyl‐L‐cysteine resulted in the generation of a low molecular weight dinitrosyl‐iron(II)‐dithiolate from the dinitrosyl‐iron(II) complex with proteins, as revealed by the shape change of the ESR signal at 293 K. The low molecular weight dinitrosyl‐iron(II)‐dithiolate accounted to an enhanced guanylyl cyclase activation and vasodilation induced by the aortic effluent. We conclude that nitric oxide (NO) produced by, or acting on vascular cells can be stabilized and stored as a dinitrosyl‐iron(II) complex with protein thiols, and can be released from cells in the form of a low molecular weight dinitrosyl‐iron(II)‐dithiolate by intra‐ and extracellular thiols.

Nitric oxide promotes seizure activity in kainate-treated rats.

L-Arginine-derived nitrogen monoxide (NO) formation was determined in different regions of the rat brain during kainate-induced seizures. NO was trapped in vivo as a paramagnetic mononitrosyl-iron diethyldithiocarbamate complex, the concentration of which was determined ex vivo by cryogenic electron spin resonance spectroscopy. Basal NO formation (0.3-0.8 nmol g-1 tissue 30 min-1) was detected in the brain of control rats. In kainate-injected rats NO formation was increased six-fold within 30-60 min in the amygdala/temporal cortex region, and up to 12-fold, though more slowly, in the remaining cortex. The kainate-elicited convulsions and NO formation were attenuated in animals pretreated with either 7-nitroindazole, a specific inhibitor of neuronal NO synthase, or diazepam. These findings identify NO as a proconvulsant mediator in kainate-evoked seizures.

Diethyldithiocarbamate inhibits induction of macrophage NO synthase

We investigated whether sodium diethyldithiocarbamate (DETC), an inhibitor of the nuclear transcription factor kappa B (NFkappaB), modulates induction of NO synthase (NOS) in murine bone marrow‐derived macrophages. A short exposure (between 1 and 16 h) of L929‐cell medium‐preconditioned macrophages to E. coli lipopolysaccharide (LPS) significantly increased the level of NOS mRNA, and elicited NO formation as detected by electron spin resonance spectroscopy and by the release of nitrite. DETC (0.1–1 mM) present during stimulation with LPS prevented the increase in NOS mRNA and the expression of NOS activity. These findings suggest that NFkappaB is involved in the signal transduction pathway linking stimulation of macrophages by LPS with transcription of, the gene encoding inducible NOS.

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.

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.

In Vivo Spin Trapping of Glyceryl Trinitrate–Derived Nitric Oxide in Rabbit Blood Vessels and Organs

BACKGROUND The objectives of this study were (1) to assess glyceryl trinitrate (GTN)-derived nitric oxide (NO) formation in vascular tissues and organs of anesthetized rabbits in vivo, (2) to establish a correlation between tissue NO levels and a biological response, and (3) to verify biotransformation of GTN to NO by cytochrome P-450. METHODS AND RESULTS NO was trapped in tissues in vivo as a stable paramagnetic mononitrosyl-iron-diethyldithiocarbamate complex [NOFe(DETC)2]. After removal of the tissues, NO was determined by cryogenic electron spin resonance spectroscopy. NO formation in vitro was assessed by spin trapping and by activation of soluble guanylyl cyclase. The GTN-elicited decrease in coronary perfusion pressure was monitored in isolated, constant-flow perfused rabbit hearts. NO was not detected in control tissues. In GTN-treated rabbits, NO formation was higher in organs than in vascular tissues and higher in venous than in arterial vessels. In isolated hearts, ventricular NO levels and decreases in coronary perfusion pressure achieved by GTN were closely correlated. Purified cytochrome P-450 catalyzed NO formation from GTN in a P-450-NADPH reductase- and NADPH-dependent fashion. CONCLUSIONS Since GTN-derived NO formation in myocardial tissue correlates to the GTN-elicited vasodilator response, we conclude that GTN-derived NO detected in vivo correlates with the systemic effects of GTN. Therefore, the higher rate of NO formation detected in veins compared with arteries explains the preferential venodilator activity of GTN. High NO formation in cytochrome P-450-rich organs in vivo and efficient NO formation from GTN by cytochrome P-450 in vitro highlights the importance of this pathway for NO formation from GTN in the intact organism.

Quantification of nitric oxide in biological samples by electron spin resonance spectroscopy

Abstract We describe a method for the detection of the nitric oxide radical (NO) in aqueous media, cells, and tissues in the presence of oxygen that is based on the trapping of NO by the ferrous iron-diethyldithiocarbamate complex (Fe(DETC) 3 ). Yeast, cells, and tissues provide the ferrous iron to accumulate this complex in the hydrophobic membrane compartment upon incubation with DETC. Therefore, trapping NO generated in cells and tissues requires only preincubation with DETC, whereas in cell-free media DETC-loaded yeast must be added. Fe(DETC) 3 avidly and nearly quantitatively binds NO, yielding a stable paramagnetic mononitrosyl complex (NOFe(DETC) 2 ) exhibiting a characteristic electron spin resonance (ESR) signal with g τ = 2.035 and g | = 2.02 in the frozen state. The amount of NO trapped is calculated by calibration with a standard, and the intracellular free ferrous iron content can be determined by titration with exogenously added NO. The detection limit is 0.05 nmol NO in sample volumes of 0.7 ml, depending on the quality of the ESR instrument. The method is also suitable for on-line recording of NO formation proceeding in aqueous incubates exposed to the magnetic field and for measurement of NO formation in living organisms.

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.