Novel insights into the electrochemical detection of nitric oxide in biological systems.

In recent years, microsensor technologies have made a rapid expansion into different fields of physical sciences, engineering, and biomedicine. For analyses of various biomolecules, novel sensors and detection platforms in the electrochemical field have been reported recently. The most important applications based on microelectromechanical systems dramatically reduce the need of manipulation steps with samples, while improving data quality and quantitative capabilities. This is also the case of a special class of electrochemical sensors that allow direct, real-time and non-invasive measurements of nitric oxide, whose determination is crucial for the purposes of basic research, as well as of preclinical and clinical studies. Therefore, this minireview will focus on the description of recent discoveries in the electrochemical determination of nitric oxide, released in different in vitro systems.

The effects of chloroquine and hydroxychloroquine on nitric oxide production in RAW 264.7 and bone marrow-derived macrophages.

Chloroquine, an antimalarial drug, can also be used in the regulation of the immune system, e.g. it is used in the treatment of autoimmune diseases. In this study we investigated the effects of chloroquine and its hydroxy-derivative on nitric oxide (NO) production in two different cell types: (i) immortalized mouse macrophage cell line RAW 264.7 and (ii) mouse bone marrow-derived macrophages (BMDM). The cells were treated with different concentrations (1-100 µM) of chloroquine or hydroxychloroquine and stimulated with lipopolysaccharide for 24 h to induce NO production. Measurement of nitrites by the Griess reaction was used to evaluate the production of NO. Expression of inducible NO synthase was evaluated with Western blot and ATPcytotoxicity test was used to measure the viability of the cells. Our results showed that both chloroquine and its hydroxy-derivative inhibited NO production in both cell types. However, based on the results of LD50 these inhibitory effects of both derivatives were due to their cytotoxicity. The LD50 values for chloroquine were 24.77 µM (RAW 264.7) and 24.86 µM (BMDM), the LD50 for hydroxychloroquine were 13.28 µM (RAW 264.7) and 13.98 µM (BMDM). In conclusion, hydroxychloroquine was more cytotoxic than its parent molecule. Comparing the two cell types tested, our data suggest that there are no differences in cytotoxicity of chloroquine or hydroxychloroquine for primary cells (BMDM) or immortalized cell line (RAW 264.7).

Modulation of neutrophil oxidative burst via histamine receptors.

Histamine has the ability to influence the activity of immune cells including neutrophils and plays a pivotal role in inflammatory processes, which are a complex network of cellular and humoral events. One of the main functions manifested by activated neutrophils is oxidative burst, which is linked to the production of reactive oxygen species; therefore, the effects of histamine receptor agonists and antagonists on the oxidative burst of neutrophils is reviewed. A role for the well-characterized histamine H1 and H2 receptors in this process is discussed and compared to that of the recently discovered H4 receptor.

Uric acid modulates vascular endothelial function through the down regulation of nitric oxide production.

Endothelial dysfunction characterized by decreased nitric oxide (NO) bioavailability is the first stage of coronary artery disease. It is known that one of the factors associated with an increased risk of coronary artery disease is a high plasma level of uric acid. However, causative associations between hyperuricaemia and cardiovascular risk have not been definitely proved. In this work, we tested the effect of uric acid on endothelial NO bioavailability. Electrochemical measurement of NO production in acetylcholine-stimulated human umbilical endothelial cells (HUVECs) revealed that uric acid markedly decreases NO release. This finding was confirmed by organ bath experiments on mouse aortic segments. Uric acid dose-dependently reduced endothelium-dependent vasorelaxation. To reveal the mechanism of decreasing NO bioavailability we tested the effect of uric acid on reactive oxygen species production by HUVECs, on arginase activity, and on acetylcholine-induced endothelial NO synthase phosphorylation. It was found that uric acid increases arginase activity and reduces endothelial NO synthase phosphorylation. Interestingly, uric acid significantly increased intracellular superoxide formation. In conclusion, uric acid decreases NO bioavailability by means of multiple mechanisms. This finding supports the idea of a causal association between hyperuricaemia and cardiovascular risk.

Differentiating between intra- and extracellular chemiluminescence in diluted whole-blood samples.

INTRODUCTION: The differentiation between extra- and intracellular production of reactive oxygen species (ROS) in whole blood was measured by luminol- and isoluminol-enhanced chemiluminescence (CL). METHODS: Azide (total CL inhibition), azide + horseradish peroxidase (HRP, restoring extracellular CL), superoxide dismutase + catalase (depleting extracellular ROS) and HRP (enhancing extracellular CL) were used to modulate luminol- and isoluminol-enhanced CL (10(-6) -10(-3) m luminophores) of 125× diluted whole blood which was activated by both calcium ionophore A23187 (Ca-I) and opsonized zymosan particles (OZP) separately. RESULTS: Both activators stimulated intra- and extracellular production of ROS. Luminol-enhanced CL of Ca-I-activated samples detected the intracellular ROS, and with the addition of HRP detected the extracellular CL as well. CL enhanced with isoluminol in concentrations of 10(-4) m or less was mostly extracellular. There was a mixture of intra- and extracellular CL in OZP-activated samples, probably because of the ingestion of luminophore molecules. CONCLUSION: Measurement of Ca-I-activated CL enhanced with 10(-4) m luminol is recommended for the detection of intracellular ROS. The addition of HRP leads to the detection of overall ROS production while the OZP-activated system with its addition of HRP can only be used to detect overall ROS production. Ca-I-activated CL enhanced with 10(-4) m isoluminol and with addition of HRP is recommended for the detection of extracellular CL.

Carvedilol and adrenergic agonists suppress the lipopolysaccharide-induced NO production in RAW 264.7 macrophages via the adrenergic receptors.

The interaction of adrenergic agonists and/or antagonists with the adrenergic receptors expressed on immunologically active cells including macrophages plays an important role in regulation of inflammatory responses. Our study investigated the effects of carvedilol, a unique vasodilating beta-adrenergic antagonist, and endogenous adrenergic agonists (adrenalin, noradrenalin, and dopamine) and/or antagonists (prazosin, atenolol) on lipopolysaccharide-stimulated nitric oxide (NO) production from murine macrophage cell line RAW 264.7. The production of NO was determined as the concentration of nitrites in cell supernatants (Griess reaction) and inducible nitric oxide synthase (iNOS) protein expression (Western blot analysis). Scavenging properties against NO were measured electrochemically. Carvedilol in a concentration range of 1, 5, 10 and 25 microM inhibited iNOS protein expression and decreased the nitrite concentration in cell supernatants. Adrenalin, noradrenalin or dopamine also inhibited the iNOS protein expression and the nitrite accumulation. Prazosine and atenolol prevented the effect of both carvedilol and adrenergic agonists on nitrite accumulation and iNOS expression in lipopolysaccharide-stimulated cells. These results, together with the absence of scavenging properties of carvedilol against NO, imply that both carvedilol and adrenergic agonists suppress the lipopolysaccharide-evoked NO production by macrophages through the activation and modulation of signaling pathways connected with adrenergic receptors.

The influence of wine polyphenols on reactive oxygen and nitrogen species production by murine macrophages RAW 264.7.

The aim was to study the antioxidant properties of four wine polyphenols (flavonoids catechin, epicatechin, and quercetin, and hydroxystilbene resveratrol). All three flavonoids exerted significant and dose-dependent scavenging effects against peroxyl radical and nitric oxide in chemical systems. The scavenging effect of resveratrol was significantly lower. All polyphenols decreased production of reactive oxygen species (ROS) by RAW264.7 macrophages. Only quercetin quenched ROS produced by lipopolysaccharide-stimulated RAW264.7 macrophages incubated for 24 h with polyphenols. Quercetin and resveratrol decreased the release of nitric oxide by these cells in a dose-dependent manner which corresponded to a decrease in iNOS expression in the case of quercetin. In conclusion, the higher number of hydroxyl substituents is an important structural feature of flavonoids in respect to their scavenging activity against ROS and nitric oxide, while C-2,3 double bond (present in quercetin and resveratrol) might be important for inhibition of ROS and nitric oxide production by RAW 264.7 macrophages.

Dealcoholized red and white wines decrease oxidative stress associated with inflammation in rats.

In vitro experiments have demonstrated that polyphenols exhibit antioxidant and anti-inflammatory activities. The present study was designed to test whether dealcoholized red (DRW) and white (DWW) wines can decrease the oxidative stress associated with inflammation in vivo. Rats were fed for 15 d either a control diet or one supplemented with DRW or DWW. Finally, a granuloma was induced by subcutaneous administration of carrageenan. Although DRW showed higher antioxidant activity in vitro than DWW, both wines decreased the number of cells recruited into the granuloma pouch. Malondialdehyde decreased in plasma and inflammatory exudate from rats fed with DRW- and DWW-rich diets. Moreover, the concentration of NO increased in exudate, which correlates with the increase in the citrulline:arginine ratio. Polymorphonuclear leucocytes from the inflammatory exudate of rats fed dealcoholized wines showed decreased superoxide anion (O*2) production and increased NO production ex vivo. This change in NO production resulted from increased expression and activity of inducible NO synthase (EC 1.14.13.39). Moreover, the up regulation of cyclo-oxygenase-2 (EC 1.14.99.1) protein expression observed in rats fed the DRW-rich diet was not related to a direct effect of NO. The present results indicate that the non-alcoholic compounds of wines not only improve antioxidant status in an inflammatory situation, but also limit cell infiltration, possibly through a decrease in O*2 and an increase in NO production.

Phagocyte-derived oxidants and plasma antioxidants in haemodialysed patients.

OBJECTIVE: Oxidative stress is one of the important complications occurring in haemodialysis. The aim of the study was to determine haemodialysis-induced changes in oxidative burst of phagocytes and the antioxidative properties of plasma. METHODS: Twenty-seven patients and 50 healthy controls were examined. Oxidative burst of phagocytes and plasma antioxidative potential were measured luminometrically. Concentrations of major plasma antioxidants (vitamin E, bilirubin and uric acid) were also determined. RESULTS: Phagocyte chemiluminescence was higher in patients before haemodialysis compared with that in controls and decreased after haemodialysis compared with predialysis status. A significant increase in plasma antioxidative potential and uric acid was found in patients before haemodialysis. These parameters decreased after haemodialysis compared with both predialysis and control values. CONCLUSIONS: The higher generation of phagocyte-derived oxidants and the decline in plasma antioxidative properties after haemodialysis confirm insufficient antioxidant defence in patients with chronic renal failure.

Blood phagocyte activation during open heart surgery with cardiopulmonary bypass.

Open heart surgery with a cardiopulmonary bypass (CPB) is associated with a systemic inflammatory response which significantly contributes to adverse postoperative complications. The purpose of this study was to characterize the activation of blood phagocytes during open heart surgery with CPB. Blood samples were collected during and up to 24 h after surgery. The production of reactive oxygen species (ROS) in whole blood, the expression of surface molecules by blood phagocytes and complement activity in the plasma were determined. A cDNA microarray analysis of leukocyte RNA profile of genes was performed related to the inflammatory response. Activation of the complement was already observed at the beginning of CPB. This was followed by an increase in the neutrophil number and in both spontaneous and opsonized zymosan-activated ROS production after the onset of reperfusion. The activation of blood phagocytes was affirmed by changes in surface receptors involved in the adhesion and migration of leukocytes (CD11b, CD62L and CD31). Gene arrays also confirmed the activation of leukocytes 4 h after reperfusion. In conclusion, open heart surgery with a cardiopulmonary bypass was found to be associated with a rapid and pronounced activation of blood phagocytes and complement activation which was partly independent at the onset of CPB.

Inhibition of chemiluminescence by carvedilol in the cell-free system, whole human blood and blood cells.

Carvedilol inhibits luminol-enhanced chemiluminescence of reactive oxygen metabolites in vitro. In this study it was found that, in the cell-free system, carvedilol dose-dependently decreased chemiluminescence in the following ranking order of radicals: hydroxyl radical > hydrogen peroxide > superoxide radical. The inhibition of myeloperoxidase was significant with carvedilol concentrations of 10 and 100 micromol/l and manifested in the concentration-dependent shift of chemiluminescence peaks to the right. In whole blood, carvedilol in concentrations of 10 and 100 micromol/l significantly inhibited chemiluminescence induced by both receptor-bypassing stimuli (A23187, PMA) and receptor-operating stimuli (fMLP, OpZ). Carvedilol dose-dependently inhibited chemiluminescence of isolated human polymorphonuclear leucocytes in the ranking order of stimuli: A23187 > OpZ > fMLP. In the presence of blood platelets, carvedilol did not substantially change chemiluminescence induced by fMLP and OpZ, while it was much more effective on chemiluminescence stimulated with calcium ionophore A23187. This could be the result of the supportive effect of serotonin liberated from platelets by A23187.

The effect of intestinal ischemia duration on changes in plasma antioxidant defense status in rats.

The purpose of this study was to follow up the changes in antioxidative adaptive mechanisms induced by various periods of small intestinal ischemia in Wistar rats. The superior mesenteric artery was occluded for 15, 30, 45, 60 and 90 min. After the respective ischemic intervals, a reperfusion was set for 120 min. Samples of the serum and intestinal mucosa were taken at the end of ischemia or at the end of reperfusion. Total radical-trapping antioxidant parameter (TRAP) of the serum and the oxidative burst of neutrophils were evaluated using luminol-enhanced chemiluminescence. Individual antioxidants in the serum and the concentration of thiobarbituric acid reactive substances (TBARs) in both serum and intestinal mucosa were measured spectrophotometrically. Increased activation of circulating neutrophils was found after the reperfusion irrespective of the duration of ischemia. TRAP of the serum was increased at the end of the ischemia lasting from 30 to 90 min. This effect was further enhanced by the subsequent reperfusion period. Ascorbate and urate contributed considerably to the TRAP value especially after reperfusion following 60 and 90 min of ischemia. On the other hand, no significant changes in albumin and bilirubin serum concentrations were observed. Contrary to the mobilized antioxidative mechanisms, increased lipid peroxidation was observed in both serum and mucosa samples.

Silkworm (Bombyx mori) hemocytes do not produce reactive oxygen metabolites as a part of defense mechanisms.

To investigate whether hemocytes of Bombyx mori (Lepidoptera) larvae produce reactive oxygen species (ROS) as part of the oxidative killing of invading pathogens, the production of ROS was measured as a luminol- and lucigenin-enhanced chemiluminescence of unstimulated or stimulated (zymosan particles, phorbol myristate acetate, calcium ionophore, rice starch or Xenorhabdus nematophila) hemolymph. No detectable ROS production was found. The spontaneous and activated ROS production measured with hemocytes, i.e. under the conditions when the antioxidative potential of hemolymph plasma was eliminated, was again undetectable. Likewise, ROS production by isolated hemocytes was observed by spectrophotometric (NBT test, cytochrome c assay) and fluorimetric (using dihydrorhodamine and hydroethidine probes) methods. Hence none of the experimental approaches used indicated the production of ROS by hemocytes of B. mori larvae as part of their immune response.

IL-10 does not affect oxidative burst and expression of selected surface antigen on human blood phagocytes in vitro.

Cytokines play a major role in the control of inflammatory responses, participate in the regulation of blood phagocyte activities and as such are used for immunomodulatory therapy. In the present study, the influence of IL-10 on human blood phagocyte activity in the presence/absence of IL-6, IL-8 and TNF-alpha was tested in vitro. Our research analyzed the effects of cytokines on the production of reactive oxygen species measured by chemiluminescence and flow cytometry, and on the expression of surface molecules (CD11b, CD15, CD62L, CD31) measured by flow cytometry. IL-10 had no inhibitory effect on reactive oxygen species production and the expression of any examined adhesion molecule by resting or stimulated blood phagocytes within 3 h of incubation. Conversely, TNF-alpha, IL-6, and IL-8 increased reactive oxygen species production and the expression of CD11b and CD15 on both neutrophils and monocytes and decreased the expression of CD62L. These priming effects of the tested pro-inflammatory cytokines were not affected by IL-10. The obtained results suggest that IL-10 does not directly control blood phagocyte activation. These results also provide better information about the contribution of IL-6, IL-8 and TNF-alpha to the regulation of blood phagocyte-mediated inflammatory processes.

Comparison of the bioluminescence of Photorhabdus species and subspecies type strains.

Natural bioluminescence of all recently accepted Photorhabdus species and subspecies type strains (bacterial symbionts of entomopathogenic nematodes) was measured using a commercial luminometer; optimum conditions for the measurement were described. Cultures emitted reliably measurable bioluminescence with characteristic level and kinetics for each strain. Bioluminescence of all strains was significantly higher at 37 than at 25 degrees C at the beginning of the measurement, no effect of bacterial concentration on the intensity of bioluminescence was observed. The technique can provide reliable and quick information for the determination of Photorhabdus taxons.

[Determination of the antioxidant activity of potential scavengers of nitric oxide]. / Stanovení antioxidacní aktivity potenciálních scavengeru oxidu dusnatého.

Nitrogen oxide is a relatively stable, highly reactive radical, which develops in the organism by oxidation of the guanidine nitrogen of the amino acid L-arginine by the action of NO-synthase with the development of L-citrulline. It participates in a number of physiological and pathological processes. The difference between the physiological and pathological concentration of nitrogen oxide is very small, and for that reason we search for suitable methods of its determination and the substances influencing the level of nitrogen and thus decreasing its overproduction. Testing of scavenger activity against NO was performed by the method following Griess (spectrophotometric determination of nitrites as the oxidation products of NO), which was compared with the HPLC (high-performance liquid chromatography) method developed by the present authors. The antiradical effect of the compounds being assayed (acetylsalicylic acid, piracetam, paracetamol, serotonin, stobadin dihydrochloride, 5-hydroxy-L-tryptophan, N-acetyl-L-cysteine, L-glutathion, and N-acetyl-DL-penicillamine) against NO was compared with the so-called total antioxidant activity--TRAP (total radical-trapping antioxidant parameter), which corresponds to the capability to trap peroxyl radicals. On the basis of the obtained data, piracetam and acetylsalicylic acid show no antioxidant activity, paracetamol shows probably moderate scavenging action against nitrogen oxide, and other compounds tested (primarily 5-hydroxy-L-tryptophan, serotonin, stobadin) are strong antioxidants against both NO and peroxyl radical.

Time course of leukocyte response and free radical release in an early reperfusion injury of the superior mesenteric artery.

The sequence of changes in circulating immune cells and in free radical production was studied during the small intestine reperfusion. Rat small intestine ischemia/reperfusion was induced by a 45 min superior mesenteric artery occlusion followed by a 4-hour reperfusion. Samples of peripheral blood were collected every 20 min during reperfusion. While the number of polymorphonuclear leukocytes increased significantly both in the sham-operated controls and the experimental group (about 400 per cent at the end of reperfusion), a decrease in lymphocyte counts to 60 per cent was observed in the experimental group only. Although there were no changes in the counts of total T lymphocytes, a significant reduction in B cell counts was observed. Flow-cytometrical measurements showed no changes in the Tc subpopulation, while the Th subpopulation increased in the experimental group only. Free radical generation in blood (luminometric measurements) increased gradually and reached an eight-fold level by the end of reperfusion in both groups. Thus, it has been shown that the increase in free radical production is mainly due to the increased number of polymorphonuclear leukocytes mobilized already at the initial stages of reperfusion. The reduction in B lymphocyte population is probably due to homing mechanisms