Determination Of Nitric Oxide Production Research Articles

Detection and Quantification of Programmed Cell Death in Chlamydomonas reinhardtii: The Example of S-Nitrosoglutathione.

Chlamydomonas (Chlamydomonas reinhardtii) is a unicellular model alga that has been shown to undergo programmed cell death (PCD) that can be triggered in response to different stresses. We have recently shown that Chlamydomonas is particularly well suited to the study and quantification of PCD. We have shown for the first time that S-nitrosoglutathione (GSNO), a nitric oxide (NO) donor, is able to induce PCD and can be used as a study system in Chlamydomonas. In this article, we provide a simple and robust protocol for quantifying GSNO-induced PCD, which can be adapted to any other treatment. We explain how to detect NO production in the cell following GSNO treatment. We show how PCD can be identified simply by analyzing the degradation profile of genomic DNA. We also provide an easy and reproducible cell death quantification protocol, which makes it possible to follow the course of PCD over time and highlight very fine differences in the number of affected cells between different samples. Key features • Use of S-nitrosoglutathione (GSNO) as a means to study programmed cell death (PCD) in Chlamydomonas. • Discrimination of PCD vs. necrosis. • In vivo determination of NO production in the cell. • A simple, robust protocol for PCD quantification.

2-DEOXYGLUCOSE PROMOTES ANTI-INFLAMMATORY POLARIZATION OF PERITONEAL MACROPHAGES IN MICE WITH LEWIS LUNG CARCINOMA

Summary. Aim: to investigate the effect of 2-deoxyglucose at a wide range of concentrations on the polarization of peritoneal macrophages of intact mice and mice with Lewis lung carcinoma. Object and methods: peritoneal macrophages obtained from intact female C57BL/6 mice and Lewis lung carcinoma-bearing mice. isolation of peritoneal macrophages, determination of nitric oxide production and arginase activity. Results: 2-deoxyglucose does not affect nitric oxide production and arginase activity of peritoneal macrophages of intact mice. 2-deoxyglucose at a concentration of 10 mM significantly (by 17%, p<0.05) increases arginase activity in peritoneal macrophages of mice with Lewis lung carcinoma. Conclusions: It was revealed that peritoneal macrophages in mice with Lewis lung carcinoma have a pro-inflammatory M1 phenotype. The addition of 2-deoxyglucose at a concentration of 10 mM to the incubation medium of peritoneal macrophages obtained from mice with Lewis lung carcinoma promotes a switch in macrophage polarization to the M2 phenotype.

8-O-β-D-Glucopyranosyl-2-methylchromone, a new chromone glycoside from the Tibetan medicine plant of Swertia punicea Hemsl

A new chromone glycoside, 8-O-β-D-Glucopyranosyl-2-methylchromone (1), together with eight known compounds (2–9) were isolated from the Tibetan medicine plant of Swertia punicea. All compounds of this plant were reported for the first time. The structures of these metabolites were elucidated by analysis of their HR–ESI–MS, 1D and 2D NMR spectroscopic data and comparison with data reported in the literature. In vitro test, all compounds were evaluated for their anti-inflammatory activity through the determination of nitric oxide production. Compounds 1–2 were evaluated for cytotoxic activities against three human cancer cell lines (HeLa, MDA-MB-231 and A375) by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. Furthermore, the chemotaxonomic significance of these compounds has also been described.

Construction of a Biocompatible and Antioxidant Multilayer Coating by Layer-by-Layer Assembly of κ-Carrageenan and Quercetin Nanoparticles

The present work aimed at the construction and characterization of a multilayer coating based on κ-carrageenan and quercetin-loaded lecithin/chitosan nanoparticles (Np) by the layer-by-layer technique and the evaluation of its antioxidant capacity and potential cytotoxicity in vitro. The multilayered coating was successfully self-assembled, as confirmed by UV-Vis spectroscopy, contact angle, atomic force microscopy (AFM), and scanning electron microscopy (SEM). Multilayered coatings showed to have antioxidant capacity, with a DPPH• radical scavenging activity of 31.32 ± 3.13% and a result of the FRAP assay of 799.41 ± 95.39 μM of ferrous ion (Fe2+) equivalent. These coatings were also shown to be devoid of cell toxicity, as evaluated by determination of nitric oxide production and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. The alveolar macrophages culture was tested in the presence of the κ-carrageenan/quercetin-Np multilayer coating and showed a cell viability of 91.3 ± 9.6%. These results suggest that this multilayered coating is adequate for surfaces modification in view of biomedical and food industry applications.

Anti-inflammatory activity of nanoemulsions of essential oil from Rosmarinus officinalis L.: in vitro and in zebrafish studies.

The essential oil from Rosmarinus officinalis L. (OERO) has bioactive compounds with anti-inflammatory activity. The objective of this study was to evaluate the anti-inflammatory potency of nanoemulsions containing essential oil of Rosmarinus officinalis L. (NOERO, NECHA, NECULT, and NECOM) in vitro and in vivo. This study was accomplished in a quantitative format through tests with diphenyl picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), cellular antioxidant activity (CCA), determination of nitric oxide production, cellular viability and anti-inflammatory activity in zebrafish. OERO's were submitted to the analysis-coupled gas chromatography-mass spectrometry (GC-MS), which highlighted 1,8-cineol and camphor as major compounds. NOEROs were obtained by a low-energy method and presenting the medium size smaller than 200nm. The efficiency of encapsulation by spectrometry and gas chromatographic analysis was 67.61 and 75.38%, respectively. In the CCA assay, all of the samples presented percentage values of inhibition similar to the quercetin pattern, indicating antioxidant activity. In the test for determination of NO·, all of the samples inhibited the production of NO· when compared to LPS, and NOEROS were more effective than OEROS to 5µg/mL. In the cell viability assay, the cells remained viable after contact with the samples, demonstrating an absence of cytotoxicity. This study showed that all nanoemulsions (NECHA, NECULT, and NECOM) showed no toxicity to macrophages, besides demonstrating antioxidant activity and potentiation of the essential oil effect in the proliferation of viable fibroblasts. Nanoemulsions has also shown the ability to potentiate the anti-inflammatory action of essential oils by exerting immunomodulatory activity by inhibiting the production of the pro-inflammatory mediator nitric oxide. The results obtained with NECHA in zebrafish confirm the hypothesis that prominent terpenic compounds, alpha-pinene, 1,8-cineole, and camphor, became more available at the target sites, inhibiting the inflammatory process in this animal species.

Downregulation of tetrahydrobiopterin inhibits tumor angiogenesis in BALB/c-nu mice with hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a highly vascular tumor, and treatment options for patients of advanced-stage are limited. Nitric oxide (NO), which is derived from endothelial nitric oxide synthase (eNOS), provides crucial signals for angiogenesis in the tumor microenvironment. Tetrahydrobiopterin (BH4) is an essential cofactor eNOS and represents a critical determinant of NO production. To examine whether treatment of 2,4-diamino-6-hydroxypyrimidine (DAHP) inhibits angiogenesis of HCC, BALB/c-nu mice were injected with HepG-2 cells with DAHP. Supplemental DAHP treatment decreased K-ras mRNA transcripts, inhibition of phosphorylation of eNOS and Akt, inhibition of guanosine triphosphate cyclohydrolase (GTPCH), and decreased significantly NO synthesis, and then inhibited angiogenesis, compared with the results observed in the saline group. Histopathology demonstrated angiogenesis and tumor formation were significantly inhibited in HCC. DAHP downregulates GTPCH protein expression, corresponding to decreased levels of BH4 and the contents of NO. In addition, DAHP downregulates eNOS and Akt protein expression, corresponding to decreased eNOS phosphorylation at Ser1177 and Akt phosphorylation, compared with the saline control. We suggest that DAHP, recognized as a specific competitive inhibitor of GTPCH, can decrease tumor BH4 and NO by the inhibition of the wild-type Ras-PI3K/Akt pathway, and then inhibiting angiogenesis, and may provide a novel and promising way to target BH4 synthetic pathways to inhibit angiogenesis and to control potential progression of HCC. Whether DAHP has a therapeutic potential will require more direct testing in humans.

Quantitative determination of nitric oxide production in haemocytes: Nitrite reduction activity as a potential pathway of NO formation in haemolymph of Galleria mellonella larvae

The generation of nitric oxide by Galleria mellonella larvae haemocytes has been investigated. For this purpose, a fluorescent method, specific for detection of NO, has been developed. The method is based on the application of fluorescence probe DAF-FM diacetate and nitronyl nitroxyl radical, NNR, which accelerates the NO-dependent formation of fluorescence product, DAF-FM triazole. The key feature of the method is the registration and analysis of differential kinetics, namely, the difference of kinetics obtained in samples with NNR and without NNR. This approach allows us to exclude any other kinetic processes not related to triazole formation. The differential kinetics were calibrated versus NO generation rate and the resulting low limit of method sensitivity was obtained as about 0.4–0.5nM/min. The generation of nitric oxide by the haemocytes of insects treated with LPS in vivo has been detected at a rate of 0.5–0.7nM/min. However, the production of NO in haemocyte suspensions treated with both the substrate, l-arginine, and the inhibitor, l-NAME, of NOS, has not been detected within method sensitivity. These data provide only the upper level of NO generation by haemocytes but cannot be used to draw definite conclusions about NOS as a source of NO. Meanwhile, it is known, that NO can be formed by NOS independent mechanism. Indeed, we have observed a significant increase in NO generation in the samples of haemocytes intracellularly loaded with nitrite. Moreover, adding nitrite to lysed haemocytes results in the higher NO generation rate. After addition of 500μM nitrite, the rates of NO generation in the samples are determined to be 2 and 20–30nM/min, respectively. The nitrite/nitrate content of haemocytes and lymph were found to be 5 and 50μM, respectively. The detected nitrite reduction activity of haemocytes allowed us to estimate the generation rate of nitric oxide as 0.05–0.5nM/min from endogenous nitrite. It is thus assumed that the observed nitrite reduction activity in haemocytes is dominant in the increased NO production during immune response of the G. mellonella larvae.

Tetrahydrobiopterin protects the kidney from ischemia–reperfusion injury

Tetrahydrobiopterin (BH4) is an essential cofactor for the nitric oxide (NO) synthases and represents a critical determinant of NO production. BH4 depletion during ischemia leads to the uncoupling of the synthases, thus contributing to reperfusion injury due to increased superoxide formation. To examine whether BH4 supplementation attenuates ischemia-reperfusion injury, we clamped the left renal arteries of male Lewis rats immediately following right-side nephrectomy. BH4 tissue levels significantly decreased after 45 min of warm ischemia compared with levels in non-ischemic controls. Histopathology demonstrated significant tubular damage and increased peroxynitrite formation. Intravital fluorescent microscopy found perfusion deficits in the microvasculature and leakage of the capillary mesh. Supplemental BH4 treatment before ischemia significantly reduced ischemia-induced renal dysfunction, and decreased tubular histologic injury scores and peroxynitrite generation. BH4 also significantly improved microcirculatory parameters such as functional capillary density and diameter. These protective effects of BH4 on microvasculature were significantly correlated with its ability to abolish peroxynitrite formation. We suggest that BH4 significantly protects against acute renal failure following ischemia reperfusion. Whether BH4 has a therapeutic potential will require more direct testing in humans.

Nitric oxide production: an easily measurable condition index for vertebrates

Nitric oxide (NO) is a multifunctional signalling molecule, acting as a vasodilator, neurotransmitter, and modulator of inflammatory processes. It also participates in killing parasites, virus-infected cells, and tumor cells by formation of peroxynitrite, one of the most important initiators of the free radical damage. Uncontrolled production of NO can lead to nitrosative stress, causing damages to proteins and DNA and cell injury and death. Determination of NO production in animals is potentially informative for description of individual variation in physiological condition, health state, and work load; however, it’s potential in ecophysiological research has remained almost totally unexplored. Here, we describe application of a simple, precise, and inexpensive spectrophotometric assay for determination of NO production from 5 to 10 μL plasma samples of passerine birds. The method is based on estimation of concentrations of nitrate and nitrite—the stable end products of nitric oxide oxidation. The principle of the assay is reduction of nitrate to nitrite by copper-coated cadmium granules, followed by color development with Griess reagent. NO production in captive greenfinches (Carduelis chloris L.) was significantly repeatable over 6-day period (r = 0.35). Injection of an inflammatory agent phytohemagglutinin into wing web resulted in 21% higher levels of NO production at the third day after treatment as compared to saline-injected birds. These findings, consistently with further evidence from veterinary and biomedical literature, suggest that measuring NO production appears an efficient and robust tool for monitoring individual condition and assessment of the magnitude of innate immune response, pathogenicity of infections, and physical effort. We propose that this assay, which is easily applicable in field studies, has an excellent potential in ecophysiological research, particularly in the rapidly developing fields of immunoecology and conservation physiology.

Fluorescence Determination of Nitric Oxide Production in Stimulated and Activated Platelets

Nitric oxide (NO) is quantitatively determined in platelets prior to, and after, stimulation with adenosine triphosphate (ATP) or activation with adenosine diphosphate (ADP). Platelets obtained from the whole blood of rabbits were loaded with the fluorescence probe diaminodifluorofluorescein diacetate (DAF-FM DA), and the subsequent NO production was measured as a fluorescent benzotriazole. Experiments were performed to determine the effect of probe concentration and probe incubation time in the platelets prior to measurement of the fluorescence. This information, combined with the method of multiple standard additions, was then employed to determine the moles of intracellular NO in the platelets (2.7 +/- 0.3) x 10(-16) mol of NO/platelet and the basal level of extracellular NO in the platelet sample (9.9 +/- 2.2) x 10(-18) mol of NO/platelet. Moreover, this method was used to quantitatively determine the amount of NO released from platelets whose NO production was stimulated with ATP (a nitric oxide synthase stimulus) or ADP, a substance known to result in NO production through platelet aggregation. When stimulated with ATP, the NO released from the platelets was determined to be (2.0 +/- 0.1) x 10(-17) mol of NO/platelet. When activated with ADP, the platelets released (2.8 +/- 0.3) x 10(-17) mol of NO/platelet. The difference between the extracellular basal levels of NO and that after stimulation with either ATP or ADP is in agreement with current estimates of NO release from platelets. Therefore, we conclude that a fluorescence determination of NO using the DAF family of probes, in combination with the method of multiple standard additions, can be employed to quantitatively determine the basal levels of NO in platelets, as well as the amount of NO released from stimulated and/or activated platelets.

Release of Erythrocyte-Derived ATP, a Recognized Stimulus of Nitric Oxide Production, Is Increased upon Incubation of Erythrocytes with C-Peptide.

Red blood cells (RBCs), when traversing microvascular beds, are subjected to mechanical deformation. It is established that RBCs release nanomolar to micromolar amounts of adenosine triphosphate (ATP), a recognized stimulus of nitric oxide (NO) production in vivo. The finding that ATP is released from RBCs in response to mechanical deformation, suggests that the RBC may be an important determinant of NO production as these cells traverse the intact circulation. An example of this potential importance of the RBC exists in diabetic complications. It is well established that patients with Type I diabetes mellitus, when compared to non-diabetics, have RBCs that are less deformable and more susceptible to oxidant stress. In conjunction with these findings, it has recently been reported that RBCs obtained from Type II diabetics release less ATP (91 +/− 10 nM) upon deformation than subjects without the disease (190 +/− 8 nM). These findings suggest that a membrane flexibilizer capable of increasing RBC-derived ATP in response to mechanical deformation may prove beneficial in improving microvascular flow. C-peptide, a 31 amino acid peptide, that connects the A and B chains of insulin, has been thought to have no significant biological role in vivo. Recently, it has been reported that C-peptide increases the deformability of erythrocytes obtained from the whole blood of patients with Type I diabetes mellitus. However, there has been no previous study to determine if C-peptide will actually increase the deformation-induced release of ATP from RBCs. Here, data is presented showing that adding nanomolar concentrations of C-peptide RBCs obtained from rabbits increases the ability of these cells to release ATP over time. When a 7% hematocrit of RBCs was incubated with a solution containing 6.6 nM C-peptide and pumped through tubing having an inside diameter that approximates resistance vessels in vivo (i.e., ~50 μm), the amount of ATP released from the RBCs increased from 0.246 ± 0.033 μM to 0.692 ± 0.140 μM (n = 10 rabbits, p<0.001) over a period of 8 hours. In the absence of the peptide, an aliquot from the same sample resulted in no significant increase in ATP release. Under conditions of non-flow, RBCs incubated with the peptide showed no statistically significant increase in ATP release. Additionally, RBCs that were incubated in the peptide containing 1 mM glybenclamide, a proven inhibitor of RBC-derived ATP, released 50% less ATP than a solution of RBCs in the absence of the inhibitor. Our results suggest that adding C-peptide to rabbit RBCs increases their ability to release ATP. Subsequent studies have shown the activity of C-peptide is depleted within 3–4 days after the solution is prepared. However, spectra obtained from a MALDI-TOF spectrometer clearly indicate that the peptide is still present in the solution exactly as it was on the day it was prepared. This suggests that there may be an activated form of the peptide that is responsible for the increase in RBC deformability and deformation-induced ATP release from these cells. In conclusion, these results suggest that C-peptide should not be considered as a biomarker alone and may have important interactions with RBCs in vivo.

Indirect determination of nitric oxide production by reduction of nitrate with a freeze–thawing-resistant nitrate reductase from Escherichia coli MC1061

Preparation of a nitrate reductase lysate of Escherichia coli MC1061 to measure nitrate and nitrite in biologic fluids is described. To obtain the crude bacterial lysate containing nitrate reductase activity, E. coli MC1061 was subjected to 16–20 freeze–thawing cycles, from −70 to 60 °C, until nitrite reductase activity was ⩽25%. Nitrate reductase activity was detected mainly in the crude preparation. To validate the nitrate reduction procedure, standard nitrate solutions (1.6–100 μM) were incubated with the nitrate reductase preparation for 3 h at 37 °C, and nitrite was estimated by the Griess reaction in a microassay. Nitrate solutions were reduced to nitrite in a range of 60–70%. Importantly, no cofactors were necessary to perform nitrate reduction. The biological samples were first reduced with the nitrate reductase preparation. After centrifugation, samples were deproteinized with either methanol/ether or zinc sulfate and nitrite was quantified. The utility of the nitrate reductase preparation was assessed by nitrate + nitrite determination in serum of animals infected with the protozoan Entamoeba histolytica or the bacteria E. coli and in the supernatant of cultured lipopolysaccharide-stimulated RAW 264.7 mouse macrophages. Our results indicate that the nitrate reductase-containing lysate provides a convenient tool for the reduction of nitrate to determine nitrate + nitrite in biological fluids by spectrophotometric methods.

Novel cardioprotective effects of tetrahydrobiopterin after anoxia and reoxygenation: Identifying cellular targets for pharmacologic manipulation

Contemporary cardioprotective strategies to prevent perioperative ischemia-reperfusion injury have focused on the l-arginine nitric oxide pathway. Tetrahydrobiopterin is an absolute cofactor required for the enzyme nitric oxide synthase and is thus a critical determinant of nitric oxide production. We hypothesized that ischemia-reperfusion results in diminished levels of tetrahydrobiopterin, which might represent a key cellular defect underlying endothelial and myocyte dysfunction after ischemia-reperfusion. To this aim, we examined the effects of tetrahydrobiopterin supplementation in (1) an in vivo experimental model of global ischemia-reperfusion and (2) an in vitro human ventricular heart cell model of simulated ischemia-reperfusion. Measures of endothelial function, oxidant production, cell survival, and cardiac function were used to assess outcome. In study 1 Wistar rats were divided into one of 2 groups (n = 10 per group). One group received tetrahydrobiopterin (25 mg x kg(-1) x d(-1) for 7 days), and the other group served as the control group. Hearts were subjected to 30 minutes of ischemia followed by 30 minutes of reperfusion, and left ventricular developed pressure, left ventricular systolic pressure, and left ventricular end-diastolic pressure were determined by using the modified Langendorff technique. In study 2 we quantitated myocardial malondialdehyde, a marker of lipid peroxidation, in ventricular tissues from both groups of animals using butanol phase extraction and spectrophotometric analysis. In study 3 coronary vascular responses were determined in vascular segments of the left coronary artery in both groups of animals after ischemia-reperfusion. Endothelium-dependent and endothelium-independent vasodilatation to acetylcholine and sodium nitroprusside, respectively, were compared between groups. In study 4, using a human ventricular heart cell model of simulated ischemia-reperfusion, we studied the effects of tetrahydrobiopterin (20 micromol/L) on cellular injury (as assessed by means of trypan blue uptake). After ischemia-reperfusion, myocardial dysfunction was evidenced by a decrease in left ventricular developed pressure and an increase in left ventricular end-diastolic pressure (P =.01 compared with baseline). Hearts from tetrahydrobiopterin-treated rats exhibited protection against ischemia-reperfusion injury (left ventricular developed pressure: 74 +/- 4 vs control 42 +/- 8 mm Hg, P =.01; left ventricular end-diastolic pressure: 12 +/- 3 vs 34 +/- 7 mm Hg, P =.01). Furthermore, tetrahydrobiopterin treatment attenuated the rise in malondialdehyde levels after ischemia-reperfusion (P =.01). After reperfusion, coronary endothelial function to acetylcholine was attenuated (P =.003 vs sham-treated mice), whereas responses to sodium nitroprusside remained unchanged. Tetrahydrobiopterin-treated rats exhibited an improvement in acetylcholine-mediated vasorelaxation (P =.01 vs ischemia-reperfusion group). Cellular injury, as assessed by means of trypan blue uptake, was higher in human ventricular heart cells subjected to simulated ischemia-reperfusion; this effect was prevented with tetrahydrobiopterin treatment (P =.001). Supplemental tetrahydrobiopterin provides a novel cardioprotective effect on left ventricular function, endothelial-vascular reactivity, oxidative damage, and cardiomyocyte injury after ischemia-reperfusion injury and might represent an important cellular target for future operative myocardial protection strategies.

Infection of a canine macrophage cell line with Leishmaniainfantum: determination of nitric oxide production and anti-leishmanial activity

We have previously shown that resistance to Leishmania infantum in dogs is associated with a Th1 type of immune response. In this study, we use a canine macrophage cell line (030-D) that can readily be infected with this protozoan parasite. Our aim is to further characterize the effector mechanisms involved in killing of Leishmania parasite in dogs. We observed that activation of 030-D cells by incubation with a supernatant derived from a Leishmania-specific T cell line containing IFN-γ, TNF-α and interleukin-2 (IL-2) resulted in enhanced nitric oxide (NO) production by these cells. In addition, we observed enhanced anti-leishmanial activity of infected 030-cells after activation. Both, NO production and anti-leishmanial activity were abrogated by addition of L-N G-nitroargininemethyl ester ( L-NAME), an analogue of L-arginine. Thus, NO play an important role in the anti-leishmanial activity of these canine macrophages. We propose the infection of the 030-D cell line as a good in vitro model to further investigate parasite-host cell interactions in dogs, a natural host of Leishmania parasites.