X-band and S-band EPR detection of nitric oxide in murine endotoxaemia using spin trapping by ferro-di(N-(dithiocarboxy)sarcosine)

Ammonium salt of N-(dithiocarboxy)sarcosine (DTCS) chelated to ferrous salt was tested as an NO-metric spin trap at room temperature for ex vivo measurement of (.)NO production in murine endotoxaemia. In a chemically defined in vitro model system EPR triplet signals of NO-Fe(DTCS)(2) were observed for as long as 3 hours, only if samples were reduced with sodium dithionite. This procedure was not necessary for the ex vivo detection of (.)NO in endotoxaemic liver homogenates at X-band or in the whole intact organs at S-band, whereas only a weak signal was observed in endotoxaemic lung. These results suggest that in endotoxaemia not only high level of (.)NO, but also the redox properties of liver and lung might determine the formation of complexes of (.)NO with a spin trap. Nevertheless, both S- and X-band EPR spectroscopy is suitable for (.)NO-metry at room temperature using Fe(DTCS)(2) as the spin trapping agent. In particular, S-band EPR spectroscopy enables the detection of (.)NO production in a whole organ, such as murine liver.     

Spin trapping of vascular nitric oxide using colloid Fe(II)-diethyldithiocarbamate

Currently available EPR spin-trapping techniques are not sensitive enough for quantification of basal vascular nitric oxide (NO) production from isolated vessels. Here we demonstrate that this goal can be achieved by the use of colloid Fe(DETC)(2). Rabbit aortic or venous strips incubated with 250 microM colloid Fe(DETC)(2) exhibited a linear increase in tissue-associated NO-Fe(DETC)(2) EPR signal during 1 h. Removal of endothelium or addition of 3 mM N(G)-nitro-l-arginine methyl ester (L-NAME) inhibited the signal. The basal NO production was estimated as 5.9 +/- 0.5 and 8.3 +/- 2.1 pmol/min/cm(2) in thoracic aorta and vena cava, respectively. Adding sodium nitrite (10 microM) or xanthine/xanthine oxidase in the incubation medium did not modify the intensity of the basal NO-Fe(DETC)(2) EPR signal. Reducing agents were not required with this method and superoxide dismutase activity was unchanged by the Fe(DETC)(2) complex. We conclude that colloid Fe(DETC)(2) may be a useful tool for direct detection of low amounts of NO in vascular tissue.     

NAD(P)H oxidase-derived hydrogen peroxide mediates endothelial nitric oxide production in response to angiotensin II

Recently, it has been shown that the exogenous addition of hydrogen peroxide (H(2)O(2)) increases endothelial nitric oxide (NO(.)) production. The current study is designed to determine whether endogenous levels of H(2)O(2) are ever sufficient to stimulate NO(.) production in intact endothelial cells. NO(.) production was detected by a NO(.)-specific microelectrode or by an electron spin resonance spectroscopy using Fe(2+)-(DETC)(2) as a NO(.)-specific spin trap. The addition of H(2)O(2) to bovine aortic endothelial cells caused a potent and dose-dependent increase in NO(.) release. Incubation with angiotensin II (10(-7) mol) elevated intracellular H(2)O(2) levels, which were attenuated with PEG-catalase. Angiotensin II increased NO(.) production by 2-fold, and this was prevented by Losartan and by PEG-catalase, suggesting a critical role of AT1 receptor and H(2)O(2) in this response(.) In contrast, NO(.) production evoked by either bradykinin or calcium ionophore was unaffected by PEG-catalase. As in bovine aortic endothelial cells, angiotensin II doubled NO(.) production in aortic endothelial cells from C57BL/6 mice but had no effect on NO(.) production in endothelial cells from p47(phox-/-) mice. In contrast, stimulated NO(.) production to a similar extent in endothelial cells from wild-type and p47(phox-/-) mice. In summary, the present study provides direct evidence that endogenous H(2)O(2), derived from the NAD(P)H oxidase, mediates endothelial NO(.) production in response to angiotensin II. Under disease conditions associated with elevated levels of angiotensin II, this response may represent a compensatory mechanism. Because angiotensin II also stimulates O(2)() production from the NAD(P)H oxidase, the H(2)O(2) stimulation of NO(.) may facilitate peroxynitrite formation in response to this octapeptide.     

EPR spectroscopy of common nitric oxide - spin trap complexes

Two commonly used hydrophobic and hydrophilic spin traps for NO, namely Fe2+(DETC)(2)and Fe2+(MGD)(2), respectively, were analyzed via EPR spectroscopy. EPR spectra of trapped NO, together with field position standards, were recorded both in the frozen state and at room temperature. We present a detailed characterization of the EPR spectra of the above paramagnetic NO complexes, concerning g-value, hyperfine splitting and linewidths. This study also provides spectroscopic data required to develop a quantitative and sensitive detection system for nitric oxide both in hydrophobic and hydrophilic aqueous media.     

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.     

In vivo spin trapping of nitric oxide from animal tumors.

Spin trapping/electron paramagnetic resonance (EPR) spectroscopy allows specific detection of nitric oxide (NO) generation, in vivo. However, in order to detect an EPR signal in living organism, usually a stimulation of immune system with LPS is used to achieve higher than physiological NO levels. Here, we report non-invasive spin trapping of NO in tumors of non-treated, living animals. EPR spectroscopy was performed at S-band to detect NO in Cloudman S91 melanoma tumors growing in the tail of living, syngeneic hosts-DBA/2 mice. Iron (II) N-(dithiocarboxy)sarcosine Fe2+(DTCS)(2) was used as the spin trap. The results were confirmed by X-band ex vivo study. A characteristic three-line spectrum of NO-Fe(DTCS)(2) (A(N)=13 G) was observed (n=4, out of total n=6) in non-treated tumors and in tumors of animals treated with l-arginine. Substrate availability did not limit the detection of NO by spin trapping. Half-life time of the NO-Fe(DTCS)(2) in tumor tissue was about 60 min. The feasibility of non-invasive spin trapping/EPR spectroscopic detection of NO generated in tumor tissue in living animals, without additional activation of the immune system, was demonstrated for the first time.     

Electron paramagnetic resonance imaging of nitric oxide organ distribution in lipopolysuccaride treated mice

The recent development of electron paramagnetic resonance (EPR) permits its application for in vivo studies of nitric oxide (NO). In this study, we tried to obtain 3D EPR images of endogenous NO in the abdominal organs of lipopolysuccaride (LPS) treated mice. Male ICR mice, each weighing about 30 g, received 10 mg/kg of LPS intraperitoneally. Six hours later, a spin trapping reagent comprised of iron and an N-dithiocarboxy sarcosine complex (Fe(DTCS)₂, Fe 200 mM, DTCS/Fe = 3) were injected subcutaneously. Two hours after this treatment, the mice were fixed in a plastic holder and set in the EPR system, equipped with a loop-gap resonator and a 1 GHz microwave. NO was detected as an NO-Fe(DTCS)₂ complex, which had a characteristic 3-line EPR spectrum. NO-Fe(DTCS)₂ complexes in organ homogenates were also measured using a conventional X-band EPR system. NO-Fe(DTCS)₂ spectra were obtained in the upper abdominal area of LPS treated mice at 8 h after the LPS injection. 3D EPR tiled and stereoscopic images of the NO distribution in the hepatic and renal areas were obtained at the same time. The NO-Fe(DTCS)₂ distribution in abdominal organs was confirmed in each organ homogenate using conventional X-band EPR. This is the first known EPR image of NO in live mice kidneys.     

Spin trapping agent, phenyl N-tert-butylnitrone, reduces nitric oxide production in the rat brain during experimental meningitis

Phenyl N-tert-butylnitrone (PBN) is a spin trapping agent previously shown to exert a neuroprotective effect in infant rat brain during bacterial meningitis. In the present study, we investigated the effect of systemic PBN administration on nitric oxide (NO) production in a rat model of experimental meningitis induced by lipopolysaccharide (LPS). We assessed the NO concentration in rat brain tissues with an electron paramagnetic resonance (EPR) NO trapping technique. In this model, rats receiving intracisternal LPS administration showed symptoms of meningitis and cerebrospinal fluid (CSF) pleocytosis. The time course study indicated that the concentration of NO in the brain reached the maximum level 8.5h after injection of LPS, and returned to the control level 24 h after the injection. When various doses of PBN (125-400 mg/kg) were injected intraperitoneally 30 min prior to LPS, NO production in the brain was reduced with increasing PBN dose (250 mg/kg suppressed 80% at 8.5h after LPS injection), and white blood cells (WBC) in CSF were significantly decreased. We concluded that reduction of NO generation during bacterial meningitis contributes to the neuroprotective effect of PBN in addition to its possible direct scavenging of reactive oxygen intermediate (ROI).     

Spin trapping agent,phenyl N-tert-butylnitrone,reduces nitric oxide production in the rat brain during experimental meningitis

Phenyl N-tert-butylnitrone (PBN) is a spin trapping agent previously shown to exert a neuroprotective effect in infant rat brain during bacterial meningitis. In the present study, we investigated the effect of systemic PBN administration on nitric oxide (NO) production in a rat model of experimental meningitis induced by lipopolysaccharide (LPS). We assessed the NO concentration in rat brain tissues with an electron paramagnetic resonance (EPR) NO trapping technique. In this model, rats receiving intracisternal LPS administration showed symptoms of meningitis and cerebrospinal fluid (CSF) pleocytosis. The time course study indicated that the concentration of NO in the brain reached the maximum level 8.5h after injection of LPS, and returned to the control level 24 h after the injection. When various doses of PBN (125–400 mg/kg) were injected intraperitoneally 30 min prior to LPS, NO production in the brain was reduced with increasing PBN dose (250 mg/kg suppressed 80% at 8.5h after LPS injection), and white blood cells (WBC) in CSF were significantly decreased. We concluded that reduction of NO generation during bacterial meningitis contributes to the neuroprotective effect of PBN in addition to its possible direct scavenging of reactive oxygen intermediate (ROI).     

EPR quantification of vascular nitric oxide production in genetically modified mouse models.

With increasing use of genetically modified mice to study endothelial nitric oxide (NO) biology, methods for reliable quantification of vascular NO production by mouse tissues are crucial. We describe a technique based on electron paramagnetic resonance (EPR) spectroscopy, using colloid iron (II) diethyldithiocarbamate [Fe(DETC)2], to trap NO. A signal was seen from C57BL/6 mice aortas incubated with Fe(DETC)2, that increased 4.7-fold on stimulation with calcium ionophore A23187 [3.45+/-0.13 vs 0.73+/-0.13au (arbitrary units)]. The signal increased linearly with incubation time (r(2) = 0.93), but was abolished by addition of N(G)-nitro-l-arginine methyl ester (L-NAME) or endothelial removal. Stimulated aortas from eNOS knockout mice had virtually undetectable signals (0.14+/-0.06 vs 3.17+/-0.21 au in littermate controls). However, the signal was doubled from mice with transgenic eNOS overexpression (7.17+/-0.76 vs 3.37+/-0.43 au in littermate controls). We conclude that EPR is a useful tool for direct NO quantification in mouse vessels.     

Simultaneous measurement of NO(*) and PO(2) from tissue by in vivo EPR.

We describe a technique that utilizes electron paramagnetic resonance (EPR) to measure NO(*) and pO(2) directly, and non-invasively, from tissue in vivo. Diethyldithiocarbamate (DETC) was injected with iron so as to complex with NO(*) in the tissue. Gloxy (an oxygen-sensitive, paramagnetic material) was also implanted into the tissue of interest (brain or liver). Because the signals arising from gloxy and NO-Fe-(DETC)(2) did not overlap, they could be monitored and measured simultaneously in vivo. The gloxy was not responsive to NO(*) and/or DETC. As model systems we either injected SNP (an NO(*) donor) into animals and monitored NO(*) and pO(2) simultaneously from brain, or endotoxin (lipopolysaccharide; LPS) was injected in order to induce a septic episode and NO(*) and pO(2) measured from liver. We found a close correlation between levels of SNP-derived NO(*) and brain pO(2) in vivo. During sepsis, liver pO(2) decreased dramatically at 300-360 min after endotoxin injection, and this coincided with decreases in mean arterial blood pressure and increased tissue NO(*) detected. These studies demonstrate the potential usefulness of this technique for making direct in vivo measurements of NO(*) and pO(2) simultaneously from tissue.     

Evidence against the generation of free hydroxyl radicals from the interaction of copper,zinc-superoxide dismutase and hydrogen peroxide

Prior spin trapping studies reported that H(2)O(2) is metabolized by copper,zinc-superoxide dismutase (SOD) to form (.)OH that is released from the enzyme, serving as a source of oxidative injury. Although this mechanism has been invoked in a number of diseases, controversy remains regarding whether the hydroxylation of spin traps by SOD is truly derived from free (.)OH or (.)OH scavenged off the Cu(2+) catalytic site. To distinguish whether (.)OH is released from the enzyme, a comprehensive EPR investigation of radical production and the kinetics of spin trapping was performed in the presence of a series of structurally different (.)OH scavengers including ethanol, formate, and azide. Although each of these have similar potency in scavenging (.)OH as the spin trap 5, 5-dimethyl-1-pyrroline-N-oxide and form secondary radical adducts, each exhibited very different potency in scavenging (.)OH from SOD. Ethanol was 1400-fold less potent than would be expected for reaction with free (.)OH. The anionic scavenger formate, which readily accesses the active site, was still 10-fold less effective than would be predicted for free (.)OH, whereas azide was almost 2-fold more potent than would be predicted. Analysis of initial rates of adduct formation indicated that these reactions did not involve free (.)OH. EPR studies of the copper center demonstrated that while high H(2)O(2) concentrations induce release of Cu(2+), the magnitude of spin adducts produced by free Cu(2+) was negligible compared with that from intact SOD. Further studies with a series of peroxidase substrates demonstrated that characteristic radicals formed by peroxidases were also efficiently generated by H(2)O(2) and SOD. Thus, SOD and H(2)O(2) oxidize and hydroxylate substrates and spin traps through a peroxidase reaction with bound (.)OH not release of (.)OH from the enzyme.     

Epr detection of endogenous nitric oxide in postischemic heart using lipid and aqueous-soluble dithiocarbamate-iron complexes

Spin-trapping techniques combined with electron paramagnetic resonance (EPR) spectroscopy to measure nitric oxide (·NO) production were compared in the ischemic-reperfused myocardium for the first time, using both aqueous-soluble and lipophilic complexes of reduced iron (Fe) with dithiocarbamate derivatives. The aqueous-soluble complex of Fe and N-methyl-D-glucamine dithiocarbamate (MGD) formed MGD2-Fe-NO complex with a characteristic triplet EPR signal (a_N12.5 G and g_iso = 2.04) at room temperature, in native isolated rat hearts following 40 min global ischemia and 15 min reperfusion. Diethyldithiocarbamate (DETC) and Fe formed in ischemic-reperfused myocardium the lipophilic DETC2-Fe-NO complex exhibiting an EPR signal (g = 2.04 and g = 2.02 at 77K) with a triplet hyperfine structure at g. Dithiocarbamate-Fe-NO complexes detected by both trapping agents were abolished by the ·NO synthase inhibitor, N^G-nitro-L-arginine methyl ester. Quantitatively, both trapping procedures provi ded similar values for tissue ·NO production, which were observed primarily during ischemia. Postischemic hemodynamic recovery of the heart was not affected by the trapping procedure. (Mol Cell Biochem 175: 91–97, 1997)     

The registration of nitric oxide production in mammals using a water-soluble complex of N-methyl-D,L-glucamine dithiocarbamate with Fe3+

The level of nitric oxide production in the intact rabbit organism was studied using the water-soluble complex of Fe3+ with MGD as a selective spin trap for nitric oxide. The Fe(3+)-MGD3 complex was injected intravenously. It was shown by the EPR method that this injection resulted in the formation of paramagnetic complexes in the urine, as Cu(2+)-MGD2, and nitric oxide spin adducts: nitric oxide-Fe(2+)-MGD2 and nitric oxide-Fe(3+)-MGD2. The level of nitric oxide production was estimated by the ratio of the total amount of these adducts to the nitric oxide-Fe(2+)-MGD2 level, formed after the addition of excessive S-nitrosoglutathione. This value for intact animals was 1.33 +/- 0.13%.     

Role of nitric oxide in radiation-induced initiation of mammary tumorigenesis in rats.

Nitric oxide (NO) and its reaction products have been shown to cause DNA damage and to be mutagenic. To elucidate whether NO produced by irradiation participates in the initiation of mammary tumorigenesis, we performed experiments using the nitric oxide-specific scavenger Fe(2+)-diethyldithiocarbamate complex (Fe(DETC)(2)) or a selective inhibitor for inducible nitric oxide synthase (iNOS), S,S(')-(4-phenylene-bis(1,2-ethanedinyl))bis-isothiourea (1,4-PB-ITU). Mother rats at day 21 of lactation were injected simultaneously with diethyldithiocarbamate intraperitoneally and Fe(2+)-citrate subcutaneously to form Fe(DETC)(2), in vivo, and then irradiated with 1.5Gy gamma-rays immediately after the injection. An additional injection of chemicals followed twice at 8 and 24h after the irradiation in the same manner. Both control and treated rats were then implanted with diethylstilbestrol pellets as a tumor promoter. The mammary tumor incidence in the experimental group was significantly reduced to one-fourth of that in the irradiated-alone group as the control. On the other hand, when mother rats took drinking water containing 0.005% 1,4-PB-ITU for 6 days from 3 days prior to irradiation at day 21 of lactation, a low tumor incidence in the iNOS inhibitor-treated groups was observed in the 1-year period. This report is the first to show that the NO derived from iNOS is an important radical for radiation-induced initiation of tumorigenesis of mammary glands in rats.     

RhoA/Rho-Kinase and Nitric Oxide in Vascular Reactivity in Rats with Endotoxaemia

RhoA/Rho-kinase (RhoA/ROK) pathway promotes vasoconstriction by calcium sensitivity mechanism. LPS causes nitric oxide (NO) overproduction to induce vascular hyporeactivity. Thus, we tried to examine the role of RhoA/ROK and NO in the regulation of vascular reactivity in different time-point of endotoxaemia. Male Wistar rats were intravenously infused for 10 min with saline or E. coli endotoxin (lipopolysaccharide, LPS, 10 mg/kg) and divided to five groups (n = 8 in each group): (i) Control, sacrificed at 6 h after saline infusion; (ii) LPS1h, sacrificed at 1 h after LPS infusion; (iii) LPS2h, sacrificed at 2 h after LPS infusion; (iv) LPS4h, sacrificed at 4 h after LPS infusion; and (v) LPS6h, sacrificed at 6 h after LPS infusion. LPS1h and LPS2h were regarded as early endotoxaemia, whereas LPS4h and LPS6h were regarded as late endotoxaemia. Indeed, our results showed that LPS reproduced a biphasic hypotension and sustained vascular hyporeactivity to noradrenaline (NA) in vivo. Interestingly, this hyporeactivity did not occur in ex vivo during early endotoxaemia. This could be due to increases of aortic RhoA activity (n = 5, P<0.05) and myosin phosphatase targeting subunit 1 phosphorylation (n = 3, P<0.05). In addition, pressor response to NA and vascular reactivity in early endotoxaemia were inhibited by ROK inhibitor, Y27632. Furthermore, plasma bradykinin was increased at 10 min (24.6±13.7 ng/mL, n = 5, P<0.05) and aortic endothelial NO synthase expression was increased at 1 h (+200%. n = 3, P<0.05) after LPS. In late endotoxaemia, the vascular hyporeactivity was associated with aortic inducible NO synthase expression (n = 3, P<0.05) and an increased serum NO level (n = 8, P<0.05). Thus, an increased RhoA activity could compensate vascular hyporeactivity in early endotoxaemia, and the large NO production inhibiting RhoA activity would lead to vascular hyporeactivity eventually.     

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.     

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.     

Phospholipids modulate superoxide and nitric oxide production by lipopolysaccharide and phorbol 12-myristate-13-acetate-activated microglia

Microglial activation and inflammatory processes have been implicated in the pathogenesis of a number of neurodegenerative disorders. Recently, peroxynitrite (ONOO(-)), the reaction product of superoxide (O(2)(-)) and nitric oxide (NO) both of which can be generated by activated microglia, has been demonstrated to act as a major mediator in the neurotoxicity induced by activated microglia. On the other hand, phospholipids such as phosphatidylserine (PS) and phosphatidylcholine (PC) have been reported to modulate the immune function of phagocytes. We therefore evaluated the effects of liposomes which comprise both PS and PC (PS/PC liposomes) or PC only (PC liposomes) regarding the production of both O(2)(-) and NO by lipopolysaccharide (LPS)/phorbol 12-myristate-13-acetate (PMA)-activated microglia using electron spin resonance (ESR) spin trap technique with a DEPMPO and Griess reaction, respectively. Pretreatment with PS/PC liposomes or PC liposomes considerably inhibited the signal intensity of O(2)(-) adduct associated with LPS/PMA-activated microglia in a dose-dependent manner. In addition, pretreatment with PS/PC liposomes also significantly reduced LPS/PMA-induced microglial NO production. In contrast, pretreatment with PC liposomes had no effect on the NO production. These results indicate that PS/PC liposomes can inhibit the microglial production of both NO and O(2)(-), and thus presumably prevent a subsequent formation of ONOO(-). Therefore, PS/PC liposomes appear to have both neuroprotective and anti-oxidative properties through the inhibition of microglial activation.     

Thiolate coordination to Fe(II)-porphyrin NO centers

The interaction of the Fe(II)-porphyrin NO model complex [Fe(TPP)(NO)] (1, TPP=tetraphenylporphyrin) with thiophenolate ligands and tetrahydrothiophene is explored both computationally and experimentally. Complex 1 is reacted with substituted thiophenolates and the obtained six-coordinate adducts of type [Fe(TPP)(SR)(NO)](-) are investigated in solution using electron paramagnetic resonance (EPR) spectroscopy. From the obtained g values and (14)N hyperfine pattern of the NO ligand it is concluded that the interaction of the thiophenolates with the Fe(II) center is weak in comparison to the corresponding 1-methylimidazole adduct. The strength of the Fe-S bond is increased when alkylthiolates are used as evidenced by comparison with the published EPR spectra of ferrous NO adducts in cytochromes P450 and P450nor, which have an axial cysteinate ligand. These results are further evaluated by density functional (DFT) calculations. The six-coordinate model complex [Fe(P)(SMe)(NO)](-) (1-SMe; P=porphine ligand used for the calculations) has an interesting electronic structure where NO acts as a medium strong sigma donor and pi acceptor ligand. Compared to the N-donor adducts with 1-methylimidazole (1-MeIm), etc., donation from the pi(h)( *) orbital of NO to Fe(II) is reduced due to the stronger trans effect of the alkylthiolate ligand. This is reflected by the predicted longer Fe-NO bond length and smaller Fe-NO force constant for 1-SMe compared to the 1-MeIm adduct. Therefore, the Fe(II)-porphyrin NO adducts with trans alkylthiolate coordination have to be described as Fe(II)-NO(radical) systems. The N-O stretching frequency of these complexes is predicted below 1600cm(-1) in agreement with the available experimental data. In addition, 1-SMe has a unique spin density distribution where Fe has a negative spin density of -0.26 from the calculations. The implications of this unusual electronic structure for the reactivity of the Fe(II)-NO alkylthiolate adducts as they occur in cytochrome P450nor are discussed.