Detection of nitric oxide in tissue by spin trapping EPR spectroscopy and triacetylglycerol extraction

A modified method based on EPR spin trapping and triacetylglycerol extraction was used for tissue nitric oxide (NO) detection at room temperature. NO signal intensity was stable for about 1.5 h and the detection limit of this method was less than 200 pmol g^–1 tissue. Using this method, we report evidence that NO production in vivo can be inhibited by adriamycin in mice livers.     

In vivo detection of nitric oxide distribution in mice

This paper discusses in vivo detection of nitric oxide (NO) distribution in endotoxin-treated mice using L-band (1.1 GHz) electron paramagnetic resonance spectroscopy (EPR) in combination with the hydrophilic NO trapping complex: N-methyl-D-glucamine dithiocarbamate and iron (MGD-Fe). MGD-Fe-NO complex is found in the upper abdomen (liver region), lower abdomen (kidney and urinary bladder) and head region of ICR mice. Experiments with nitric oxide synthase (NOS) inhibition and ¹⁵N-labeled L-arginine as NOS substrate verify the origin of trapped NO from L-arginine. However, contribution from a 'nonenzymatic' NO generation pathway can not be ruled out. This paper further examines potential artifacts, which may arise in experiments using dithiocarbamate-iron complexes as NO trapping agents.     

Alternative pathways of nitric oxide formation in Lactobacilli: Evidence for nitric oxide synthase activity by EPR

The study of the ability of Lactobacillus plantarum 8P-A3 to synthesize nitric oxide (NO) showed that this strain lacks nitrite reductase. However, analysis by the EPR method revealed the presence of nitric oxide synthase activity in this strain. Like mammalian nitric oxide synthase, lactobacillar NO synthase is involved in the formation of nitric oxide from L-arginine. L. plantarum 8P-A3 does not produce NO in the denitrification process. The regulatory role of NO in symbiotic bacteria is emphasixed.     

Changes in nitric oxide in heart of intact and sympathectomized rats of different age

Nitric oxide production in heart tissues of rats of different age in the norm and after pharmacological sympathectomy was studied by electron spin resonance spin-trapping. Rats at the age of 14, 21, 70, and 100 days were used in the experiment. The concentration of nitric oxide produced in rat heart tissues proved to considerably decrease during ontogeny. Pharmacological sympathectomy notably decreased nitric oxide production in the heart in 14-and 21-day-old rats: the nitric oxide concentration in the spin trap as well as the level of R and T conformers of hemoglobin nitrosyl complexes decreased. In 70-day-old rats, pharmacological sympathectomy had no notable effect on the level of nitric oxide-containing paramagnetic complexes.     

Simultaneous detection of NOS-3 protein expression and nitric oxide production using a flow cytometer

Nitric oxide (NO) is involved in the regulation of SMC proliferation during intimal hyperplasia as has been shown by the inhibitory effect on intimal hyperplasia of adenovirus-mediated ceNOS overexpression in injured arteries in pig. Good assays to quantify the NO-producing enzymes, i.e., NO synthases (NOS), are essential to analyze the mechanism of action of NO in this process. We have developed novel flow cytometric assays for the simultaneous detection of NOS-3 protein, using NOS-3 specific antibodies, and NO production using 4,5-diaminofluorescein-diacetate (DAF-2/DA). The presence of NOS-3 protein and NO production is demonstrated on human A549 and HepG2 cells infected with a NOS-3 adenovirus (Ad.NOS-3). A comparative study showed that the flow cytometric assays are equally sensitive as Western blot analysis, the citrulline assay, or the Sievers assay. On human endothelial and SMC, NOS-3 protein and NO production were simultaneously detected with the assays, both under basal conditions and after Ad.NOS-3transduction. Simultaneous analysis of NOS-3 protein and NO production, made possible by the here-described novel flow cytometric assays, is of significant value to those investigating NOS-3 and NO.     

Spin Trapping Isotopically-Labelled Nitric Oxide Produced from [15n]L-Arginine and [17ojdioxygen by Activated Macrophages Using a Water Soluble Fe++-Dithiocarbamate Spin Trap

The unique capabilities of EPR spin trapping of nitric oxide based on a ferrous-dithiocarbamate spin trap have been demonstrated in a study verifying the source of the nitrogen and oxygen atoms in nitric oxide produced from activated macrophages. Spin trapping experiments were performed during nitric oxide generation from activated mouse peritoneal macrophages using the ferrous complex of N-methyl D-glucam-ine dithiocarbamate as a spin trap. When ¹⁵N-substituted arginine was given to the activated macrophages in the presence of the spin trap, a characteristic EPR spectrum of the nitric oxide spin adduct was obtained, which indicates the presence of the ^l5N atom in the nitric oxide molecule. The hyperfine splitting (hfs) constant of the ^l5N nucleus was 17.6 gauss. When ^l7O-containing dioxygen (55%) was supplied to the medium, an EPR spectrum consistent with the “O-substituted nitric oxide spin adduct was observed in the composite spectrum. The hfs of “O was estimated to be 2.5 gauss. The ^l4NO spin adduct observed after prolonged incubation in the medium which contains [^l5N]L-arginine as the only extracellular source of arginine demonstrates that arginine is recycled through its metabolite in activated macrophages.     

Beneficial effect of gaseous nitric oxide on the healing of skin wounds

Intermittent daily exposures (60 s) to NO-containing gas flow (NO dose of 500 ppm) generated by air-plasma unit "Plason" improves healing of skin wounds in rats. The gas flow treatment shortened the recovery time of both aseptic and purulent wounds (300 mm2 area) by nearly a third. The treatment allows to achieve a marked improvement in the histological, histochemical, and electron-microscopic characteristics of the affected tissue. The mechanism of this phenomenon was studied by spin trapping method. The NO status of the wound tissue was investigated with EPR by following the formation of paramagnetic mononitrosyl complexes with iron-diethyldithiocarbamate, or with the heme groups in hemoglobin or myoglobin. For the first 5 min after a gas treatment with the exposure of 60s, detectable NO levels in the affected tissue were slightly lowered with respect to untreated controls. At subsequent times, treated tissues showed the formation of large quantities of nitroso-iron complexes: At 30-40 min after gas exposure, their levels were nearly two orders of magnitude higher than soon after (15 s-5 min) the exposure. The data demonstrate that the accumulation of nitrosyl-iron complexes reflects a sharp rise in endogenous NO production inside the affected tissue. Paradoxically, the beneficial effect of gaseous NO treatment can be mediated by the formation of limited quantities of peroxynitrite due to the reaction between exogenous NO and superoxide anions generated in high amount in wound tissue. This peroxynitrite has a strong prooxidant effect and can activate various antioxidant systems which diminish the amount of superoxide anions in wound tissue. The reduced superoxide levels allow to increase the contents of endogenous NO in gas-treated tissues. Therefore, the beneficial action of the treatment is attributed to enhanced NO bioavailability.     

昆虫一氧化氮及其合酶的研究进展

一氧化氮作为一种重要的信息分子 ,参与调节昆虫嗅觉、视觉、机械感受、发育、机体防御及学习行为。该文从生理、生化、形态定位以及信号转导几方面综述了有关昆虫一氧化氮及其合酶的最新研究进展。     

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.     

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.     

Plasma nitric oxide and iron concentrations in exercised rats are negatively correlated

The aim of this study was to investigate the effect of strenuous exercise on plasma nitric oxide and iron (PI) concentrations in rats. The rats were divided into six groups: 3, 6 and 12 months of the exercise (swimming) groups and their corresponding controls. At the end of experimental periods, blood samples were collected to measure plasma NOx (nitrate and nitrite) and iron concentrations and other hematological indices. The correlative analysis of plasma NOx with PI in the exercised and the control rats was performed. The results showed that plasma NOx concentration was significantly greater and PI lower in the 3, 6, and 12 months of the exercise groups compared to their sedentary controls (p < 0.01). However, the duration of strenuous exercise had no significant effect on plasma NOx or PI contents. A negative correlation between plasma NOx and PI levels was found in all three exercise groups (r = -0.750, -0.578, and -0.808 and p < 0.01, 0.05, 0.01 respectively), but not in the sedentary control groups. These results imply that strenuous exercise may lead to an increase in plasma NOx concentration as well as a low iron level. They also suggest the possibility that the increased NO production might be associated with the development of the lower iron status in exercise.     

Studies on nitrosyl hemes in Ni(II)–Fe(II) hybrid hemoglobins

Subunit heterogeneity within a particular subunit in hemoglobin A have been explored with electron paramagnetic resonance spectroscopy using the nitrosyl hemes in Ni-Fe hybrid Hb under various solution conditions. Our previous studies on the crystal structure of NiHb demonstrated the presence of subunit heterogeneity within alpha-subunit. To further cross check this hypothesis, we made a hybrid Hb in which either the alpha- or beta-subunit contains iron, which alone can bind to NO. By this way dynamic exchange between penta- and hexa-coordinated forms within a subunit was confirmed. Upon the addition of inositol hexa phosphate (IHP) to these hybrids, R to T state transition is observed for [alpha(2)(Fe-NO)beta(2)(Ni)] but such a direct transformation is less marked in [alpha(2)(Ni)beta(2)(Fe-NO)]. Hence the bond between N(epsilon) and Fe is fundamental to the structure-function relation in Hb, as the motion of this nitrogen triggers the vast transformation, which occurs in the whole molecule on attachment of NO.     

Measurement of acetylcholine-induced endothelium-derived nitric oxide in aorta using a newly developed catheter-type nitric oxide sensor

Intra-aortic measurement of nitric oxide (NO) would provide valuable insights into NO bioavailability in systemic circulation and vascular endothelial function. In the present study, we thus developed a catheter-type NO sensor to measure intra-aortic NO concentration in vivo. An NO sensor was encased and fixed in a 4-Fr catheter. The sensor was then located in the thoracic aorta via the femoral artery through a 7-Fr catheter to measure intra-aortic plasma NO concentration in vivo in anesthetized dogs. Infusion of acetylcholine (10 microg/kg) increased base-to-peak plasma NO level in the aorta by 2.4+/-0.4 nM (n=7). After 20-min infusion of N(G)-methyl-L-arginine (NO synthase inhibitor), changes in plasma NO concentration in response to acetylcholine were attenuated significantly (1.8+/-0.4 nM, P<0.003, n=7). In conclusion, the newly developed catheter-type NO sensor successfully measured acetylcholine-induced changes in intra-aortic plasma concentration of endothelium-derived NO in vivo and demonstrated applicability to direct evaluation of intravascular NO bioavailability.     

甲醛炎性痛增强大鼠海马NOS表达和NO生成

目的：观察甲醛炎性痛过程中大鼠痛行为、海马一氧化氮合酶（NOS）活性及一氧化氮（NO）含量的变化以及变化的时程及区域特征。方法：采用辐射热甩尾法测定大鼠痛阈变化;采用NADPH—d组织化学法和硝酸还原酶法分别测定大鼠海马NOS表达和No含量。结果：皮下注射甲醛溶液后,大鼠出现伤害性感受反应及痛阈降低。注射甲醛后6h,海马CA1、CA2～3区及DG区NOS阳性细胞数目、阳性细胞染色深度均显著增加。海马NO含量亦显著增加;注射甲醛后12h时这些改变最为显著,48h时恢复至对照组水平。结论：甲醛炎性痛可诱导海马NOS活性增强及NO生成增多．这种改变可发生在海马各区．并具有一定的时程特征。     

一氧化氮在铁诱导的大鼠心肌损伤中的作用

采用Langendorff灌流大鼠心脏和酶解分离的心肌细胞为实验模型,研究铁负荷下心肌损伤情况以及一氧化氮（NO）在铁诱导的心肌损伤中的地位。结果显示:（1）心肌铁负荷（Fe-HQ）可使分离心肌细胞舒张期细胞长度缩短、收缩幅度和速度降低,离体灌流心脏左室发展压（LVDP）、±dp/dtmax、冠脉流量呈现双相变化;冠脉流出液中乳酸脱氢酶（LDH）、肌酸激酶（CK）释放量和心肌丙二醛（MDA）增高。（2）NO的前体L-精氨酸（L-argi-nine,L-Arg）引起心肌细胞舒张期细胞长度缩短、收缩幅度降低。离体灌流心脏LVDP、冠脉流量、和±dp/dtmax增高,用K-H液复灌后可恢复正常。（3）L-Arg预处理,再行Fe-HQ灌流,与单纯的L-Arg或Fe-HQ组相比,心肌细胞舒张期细胞长度、收缩幅度和速度减小;离体灌流心脏LVDP、±dp/dtmax、心率和冠脉流量明显下降,冠脉流出液中LDH、CK增加。（4）Nω-硝基-L-精氨酸甲酯（L-NAME）和Fe-HQ合并灌流后,与单纯Fe-HQ组相比,心肌细胞舒张期细胞长度、收缩幅度和速度增加。L-NAME可阻断Fe-HQ引起的LVDP、左室舒张末压（LVEDP）和±dp/dtmax降低,冠脉流出液中LDH、CK增高。（5）用Triton X-100短暂处理以去除冠脉内皮后,与保留冠脉内皮的心肌相比,Fe-HQ引起的LVDP和±dp/dtmax的一过性增高现象被抑制,但     

Postanoxic functional recovery of the developing heart is slightly altered by endogenous or exogenous nitric oxide

Nitric oxide synthase (NOS) is strongly and transiently expressed in the developing heart but its function is not well documented. This work examined the role, either protective or detrimental, that endogenous and exogenous NO could play in the functioning of the embryonic heart submitted to hypoxia and reoxygenation. Spontaneously beating hearts isolated from 4-day-old chick embryos were either homogenized to determine basal inducible NOS (iNOS) expression and activity or submitted to 30 min anoxia followed by 100 min reoxygenation. The chrono-, dromo- and inotropic responses to anoxia/reoxygenation were determined in the presence of NOS substrate (L-arginine 10 mM), NOS inhibitor L-NIO (1–5 mM), or NO donor (DETA NONOate 10–100 M). Myocardial iNOS was detectable by immunoblotting and its activity was specifically decreased by 53% in the presence of 5 mM L-NIO. L-Arginine, L-NIO and DETA NONOate at 10 M had no significant effect on the investigated functional parameters during anoxia/reoxygenation. However, irrespective of anoxia/reoxygenation, DETA NONOate at 100 M decreased ventricular shortening velocity by about 70%, and reduced atrio-ventricular propagation by 23%. None of the used drugs affected atrial activity and hearts of all experimental groups fully recovered at the end of reoxygenation. These findings indicate that (1) by contrast with adult heart, endogenously released NO plays a minor role in the early response of the embryonic heart to reoxygenation, (2) exogenous NO has to be provided at high concentration to delay postanoxic functional recovery, and (3) sinoatrial pacemaker cells are the less responsive to NO.     

Medium-throughput ESR detection of superoxide production in undetached adherent cells using cyclic nitrone spin traps

Abstract

Spin trapping with cyclic nitrones coupled to electron spin resonance (ESR) is recognized as a specific method of detection of oxygen free radicals in biological systems, especially in culture cells. In this case, the detection is usually performed on cell suspensions, which is however unsuitable when adhesion influences free radical production. Here, we performed ESR detection of superoxide with four spin traps (5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DEPMPO; 5-diisopropoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DIPPMPO; (4R*, 5R*)-5-(diisopropyloxyphosphoryl)-5-methyl-4-[({[2-(triphenylphosphonio)ethyl]carbamoyl}oxy)methyl]pyrroline N-oxide bromide, Mito-DIPPMPO; and 6-monodeoxy-6-mono-4-[(5-diisopropoxyphosphoryl-5-methyl-1-pyrroline-N-oxide)-ethylenecarbamoyl-(2,3-di-O-methyl) hexakis (2,3,6-tri-O-methyl)]-β-cyclodextrin, CD-DIPPMPO) directly on RAW 264.7 macrophages cultured on microscope coverslip glasses after phorbol 12-myristate 13-acetate (PMA) stimulation. Distinct ESR spectra were obtained with each spin trap using this method. CD-DIPPMPO, a recently published phosphorylated cyclic nitrone bearing a permethylated β-cyclodextrin moiety, was confirmed as the most specific spin trap of the superoxide radical, with exclusive detection of the superoxide adduct. ESR detection performed on cells attached to coverslips represents significant advances over other methods in terms of simplicity, speed, and measurement under near-physiological conditions. It thus opens the way for numerous applications, such as medium-throughput screening of antioxidants and reactive oxygen species (ROS)-modulating agents.     

Enhancement of nitric oxide production by association of nitric oxide synthase with N-methyl-D-aspartate receptors via postsynaptic density 95 in genetically engineered Chinese hamster ovary cells: real-time fluorescence imaging using nitric oxide sensitive dye

The current quantitative study demonstrates that the recruitment of neuronal nitric oxide synthase (nNOS) beneath N-methyl-D-aspartate (NMDA) receptors, via postsynaptic density 95 (PSD-95) proteins significantly enhances nitric oxide (NO) production. Real-time single-cell fluorescence imaging was applied to measure both NO production and Ca(2+) influx in Chinese hamster ovary (CHO) cells expressing recombinant NMDA receptors (NMDA-R), nNOS, and PSD-95. We examined the relationship between the rate of NO production and Ca(2+) influx via NMDA receptors using the NO-reactive fluorescent dye, diaminofluorescein-FM (DAF-FM) and the Ca(2+)-sensitive yellow cameleon 3.1 (YC3.1), conjugated with PSD-95 (PSD-95-YC3.1). The presence of PSD-95 enhanced the rate of NO production by 2.3-fold upon stimulation with 100 microm NMDA in CHO1(+) cells (expressing NMDA-R, nNOS and PSD-95) when compared with CHO1(-) cells (expressing NMDA-R and nNOS lacking PSD-95). The presence of nNOS inhibitor or NMDA-R blocker almost completely suppressed this NMDA-stimulated NO production. The Ca(2+) concentration beneath the NMDA-R, [Ca(2+)](NR), was determined to be 5.4 microm by stimulating CHO2 cells (expressing NMDA-R and PSD-95-YC3.1) with 100 microm NMDA. By completely permealizing CHO1 cells with ionomycin, a general relationship curve of the rate of NO production versus the Ca(2+) concentration around nNOS, [Ca(2+)](NOS), was obtained over the wide range of [Ca(2+)](NOS). This sigmoidal curve had an EC(50) of approximately 1.2 microm of [Ca(2+)](NOS), implying that [Ca(2+)](NR) = 5.4 microm can activate nNOS effectively.     

Interaction of caveolin-1, nitric oxide, and nitric oxide synthases in hypoxic human SK-N-MC neuroblastoma cells

Neuroblastoma cells are capable of hypoxic adaptation, but the mechanisms involved are not fully understood. We hypothesized that caveolin-1 (cav-1), a plasma membrane signal molecule, might play a role in protecting neuroblastoma cells from oxidative injury by modulating nitric oxide (NO) production. We investigated the alterations of cav-1, cav-2, nitric oxide synthases (NOS), and NO levels in human SK-N-MC neuroblastoma cells exposed to hypoxia with 2% [O2]. The major discoveries include: (i) cav-1 but not cav-2 was up-regulated in the cells exposed to 15 h of hypoxia; (ii) NO donor 1-[N, N-di-(2-aminoethyl) amino] diazen-1-ium-1, 2-diolate up-regulated the expression of cav-1, whereas the non-selective NOS inhibitor N(G)-nitro-L-arginine methyl ester and inducible NOS (iNOS) inhibitor 1400W each abolished the increase in cav-1 expression in the hypoxic SK-N-MC cells. These results suggest that iNOS-induced NO production contributes to the up-regulation of cav-1 in the hypoxic SK-N-MC cells. Furthermore, we studied the roles played by cav-1 in regulating NO, NOS, and apoptotic cell death in the SK-N-MC cells subjected to 15 h of hypoxic treatment. Both cav-1 transfection and cav-1 scaffolding domain peptide abolished the induction of iNOS, reduced the production of NO, and reduced the rates of apoptotic cell death in the hypoxic SK-N-MC cells. These results suggest that increased expression of cav-1 in response to hypoxic stimulation could prevent oxidative injury induced by reactive oxygen species. The interactions of cav-1, NO, and NOS could be an important signal pathway in protecting the neuroblastoma cells from oxidative injury, contributing to the hypoxic tolerance of neuroblastoma cells.     

Detection of Free Radicals by Microdialysis/Spin Trapping Epr Following Focal Cerebral Ischemia-Reperfusion and a Cautionary Note on the Stability of 5,5-Dimethyl-1-Pyrroline N-Oxide (DMPO)

We have examined free radical production in a rat model of focal cerebral ischemia using microdialysis coupled with EPR analysis. A microdialysis probe was inserted 2 mm into the cerebral cortex, supplied by the right middle cerebral artery (MCA), and after a 2-hour washout period with artificial cerebral spinal fluid (ACSF), the perfusate solution was changed to ACSF containing the spin trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO). No free radicals were detected by DMPO during the pre-ischemia period. Both common carotid arteries and the right MCA were then ligated for 90 minutes. Microdialysate collected every 15 min during the ischemic period demonstrated predominantly superoxide or peroxyl radical production. After release of the occlusive sutures, hydroxyl radical became apparent initially, then thiyl and carbon centered radicals appeared later in samples collected every 15 min for two hours following cortical reperfusion. Careful studies on the purification and stability of DMPO solution were performed to circumvent artifacts and spurious signals.