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.

Myeloperoxidase induces the priming of platelets.

The release of myeloperoxidase (MPO) from polymorphonuclear neutrophils is a hallmark of vascular inflammation and contributes to the pathogenesis of vascular inflammatory processes. However, the effects of MPO on platelets as a contributory mechanism in vascular inflammatory diseases remain unknown. Thus, MPO interaction with platelets and its effects on platelet function were examined. First, dose-dependent binding of MPO (between 1.7 and 13.8nM) to both human and mouse platelets was observed. This was in direct contrast to the absence of MPO in megakaryocytes. MPO was localized both on the surface of and inside platelets. Cytoskeleton inhibition did not prevent MPO localization inside the three-dimensional platelet structure. MPO peroxidase activity was preserved upon the MPO binding to platelets. MPO sequestered in platelets catabolized NO, documented by the decreased production of NO (on average, an approximately 2-fold decrease). MPO treatment did not affect the viability of platelets during short incubations; however, it decreased platelet viability after long-term storage for 7 days (an approximately 2-fold decrease). The activation of platelets by MPO was documented by an MPO-mediated increase in the expression of surface platelet receptors P-selectin and PECAM-1 (of about 5 to 20%) and the increased formation of reactive oxygen species (of about 15 to 200%). However, the activation was only partial, as MPO did not induce the aggregation of platelets nor potentiate platelet response to classical activators. Nor did MPO induce a significant release of the content of granules. The activation of platelets by MPO was connected with increased MPO-treated platelet interaction with polymorphonuclear leukocytes (an approximately 1.2-fold increase) in vitro. In conclusion, it can be suggested that MPO can interact with and activate platelets, which can induce priming of platelets, rather than the classical robust activation of platelets. This can contribute to the development of chronic inflammatory processes in vessels.

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.

The expression of NADPH oxidases and production of reactive oxygen species by human lung adenocarcinoma epithelial cell line A549.

Controlled production of reactive oxygen species (ROS) by NADPH oxidases in non-phagocytic cells has recently been suggested to participate in the regulation of cellular functions. Due to the role of ROS in control of cellular functions, precise and accurate detection of ROS is of essential importance. However, various methodological approaches currently used for ROS determination vary in sensitivity, specificity, as well as in requirements for specialized equipment. In this study, human lung epithelial cell line A549 was screened for expression of NADPH oxidases NOX1, NOX2, NOX4, NOX5, DUOX1 and DUOX2 by quantitative RT-PCR. Fluorometric, colorimetric, and chemiluminometric methods were applied to determine ROS production. A549 cells were found to significantly express NOX1, NOX2, DUOX1 and DUOX2. ROS production by A549 cells was detected with fluorometric probes 2',7'-dichlorofluorescein- diacetate, dihydroethidium, and amplex red or colorimetric probe nitrobluetetrazolium. The production of ROS detected by these probes was partially reduced by NADPH oxidase inhibitor diphenyleneiodonium. The inhibitory effect of diphenyleneiodonium was the most significant regarding amplex red detection of phorbol myristate acetateactivated ROS production. In contrast to other probes, neither cytochrome c colorimetric determination nor luminol- and L-012-amplified chemiluminescence, regardless of the addition of horseradish peroxidase, exerted sufficient sensitivity to detect ROS production by A549. The results revealed differences among methods used for ROS formation measurement by human lung epithelial cell line A549 and highlighted the sensitivity of fluorometric determination for this purpose.

New luminescence-based approach to measurement of luciferase gene expression reporter activity and adenosine triphosphate-based determination of cell viability.

The assay employing firefly luciferase as the end-point reporter is one of the most popular gene reporter systems. However, the physiological conditions of cells may affect the reporter gene expression, which makes an assessment of cell viability desirable. Estimates of cell viability may be based on different principles. We tested for correlations between various cell viability assessments, including luminescent determination of adenosine triphosphate in whole-cell lysate, and the reporter luciferase activity in pluripotent embryonic and colon adenocarcinoma cells. Luciferase activity in cell lysate from both cell lines cultured under different conditions correlated with the amount of viable cells assessed by all of the methods employed. Importantly, it was also possible to carry out adenosine triphosphate determination in cell lysates prepared in the buffer originally designed for determining luciferase activity; it correlated significantly with adenosine triphosphate determination in cells lysed in the buffer originally designed for adenosine triphosphate determination. The results suggest that the assessment of live cells by determining adenosine triphosphate can be multiplexed with a luciferase reporter gene assay, which allows independent monitoring of both reporter expression and cell viability.

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.

Neural differentiation potentiated by the leukaemia inhibitory factor through STAT3 signalling in mouse embryonal carcinoma cells.

LIF is a cytokine playing a key role in the regulation of self-renewal and maintenance of undifferentiated state in mouse ES cells. The response of pluripotent cells to LIF is mediated mainly by the STAT3 and ERK signalling pathways. Recently, we have shown that LIF potentiated retinoic acid-induced neural differentiation of pluripotent mouse embryonal carcinoma P19 cells. Here we demonstrate that pro-neural effects of LIF and partially also of retinoic acid are abolished by inhibition of the JAK2->STAT3 signalling pathway. In contrast, inhibition of the MEK1->ERK signalling pathway does not exhibit any effect. These results suggest that in neurogenic regions, cooperative action of LIF and other neuro-differentiation-inducing factors, such as retinoic acid, may be mediated by the STAT3 signalling pathway.

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.

Neural differentiation of pluripotent mouse embryonal carcinoma cells by retinoic acid: inhibitory effect of serum.

In both embryonal carcinoma (EC) and embryonic stem (ES) cells, the differentiation pathway entered after treatment with retinoic acid (RA) varies as it is based upon different conditions of culture. This study employs mouse EC cells P19 to investigate the effects of serum on RA-induced neural differentiation occurring in a simplified monolayer culture. Cell morphology and expression of lineage-specific molecular markers document that, while non-neural cell types arise after treatment with RA under serum-containing conditions, in chemically defined serum-free media RA induces massive neural differentiation in concentrations of 10(-9) M and higher. Moreover, not only neural (Mash-1) and neuroectodermal (Pax-6), but also endodermal (GATA-4, alpha-fetoprotein) genes are expressed at early stages of differentiation driven by RA under serum-free conditions. Furthermore, as determined by the luciferase reporter assay, the presence or absence of the serum does not affect the activity of the retinoic acid response element (RARE). Thus, mouse EC cells are able to produce neural cells upon exposure to RA even without culture in three-dimensional embryoid bodies (EBs). However, in contrast to standard EBs-involving protocol(s), neural differentiation in monolayer only takes place when complex signaling from serum factors is avoided. This simple and efficient strategy is proposed to serve as a basis for neurodifferentiation studies in vitro.

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.

[Antioxidative action of pyridoindoles and N-(alkoxyphenyl)-2-(2-oxo-1-aza-1-cycloalkyl) acetamides in biological, enzymatic and chemical systems]. / Antioxidacní aktivita pyridoindolu a N-(alkoxyfenyl)-2-(2-oxo-1-aza-1-cykloalkyl) acetaminu v biologickém, enzymatickém a chemickém systému.

The luminol-enhanced chemiluminiscence method was used to investigate the antioxidative activity of N-(alkoxyphenyl)-2-(2-oxo-1-aza-1-cykloalkyl) acetamides studied as potential cognitive enhancers and stobadine acylderivatives which form prodrugs with increased lipophilicity. The effect on the production of reactive oxygen metabolites by activated leukocytes was studied in vitro. Furthermore, the total radical-trapping antioxidant parameter was evaluated as the peroxyl radical-trapping capacity and the scavenging effect on the superoxide anion radical (generated by the enzymatic system hypoxanthine/xanthine oxidase) and on the hydroxyl radical (produced in Fenton reaction) were studied. The antioxidative properties of the tested substances were compared with that of stobadine dihydrochloride. Only stobadine and its butyrylderivative have been demonstrated to possess free radical scavenging activity in all systems. Cinnamoylstobadine inhibited only the leukocyte chemiluminiscence activity. The potential cognitive enhancers did not show any antioxidant activity.

Differential effects of v-Jun and c-Jun proteins on v-myb-transformed monoblasts.

The v-myb oncogene of avian myeloblastosis virus transforms myelomonocytic cells in vitro. The line of v-myb-transformed chicken monoblasts BM2 can be induced to terminal differentiation using phorbol esters. The fact that Jun proteins are up-regulated in the phorbol ester-treated BM2 cells prompted us to investigate the role of the Jun proteins in regulation of myeloid differentiation. We ectopically expressed v-jun and c-jun in BM2 cells and evaluated their effects on differentiation and proliferation. c-Jun up-regulated the transactivation activity of v-Myb and induced a proliferation block and differentiation of BM2 cells. In contrast, v-Jun down-regulated v-Myb transactivation causing no dramatic effects on BM2 cells. This confirms that there is no strong correlation between transcriptional activation and strength of oncogenic transformation by v-Myb. Both c-Jun and v-Jun proteins affected sensitivity of BM2 cells to retinoic acid and phorbol ester. Sensitivity of BM2 cells to retinoic acid was enhanced by both Jun proteins, while sensitivity to phorbol 12-myristate 13-acetate was reduced by v-Jun. These data suggest thate Jun plays a major role in macrophage differentiation.

Influence of polysulfone and hemophan hemodialysis membranes on phagocytes.

The aim of the study was to compare the effect of hemophane and polysulfone membranes on the phagocyte-derived production of reactive oxygen species (ROS) as well as on neutrophil CD11b and CD62L expression in patients undergoing regular hemodialysis. The effects of hemodialysis membranes were also studied in in vitro conditions after coincubating them with differentiated HL-60 cells. ROS production was measured using chemiluminometric and flow cytometric methods. Expression of CD11b, CD62L and mitochondrial membrane potential were detected by monoclonal antibodies and by the JC-1 fluorescent probe, respectively. Depressed ROS production was observed in patients already before dialysis. Further decrease in ROS production and an increase in CD11b expression were observed especially in patients after hemophan hemodialysis. Decreased ROS production and increased CD11b expression were observed also after incubation of HL-60 cells with hemophan membranes. Mitochondrial membrane potential dropped only after incubating cells with hemophan membranes proving its more serious adverse effects in comparison with the polysulfone membrane. In conclusion, deleterious effects of hemodialysis on the metabolic activity of phagocytes were proved. Combining chemiluminescent and flow cytometric methods for the detection of ROS production and determining mitochondrial membrane potential can be useful tools for the analysis of material biocompatibility.

Peri- and post-operative course of cytokines and the metabolic activity of neutrophils in human liver transplantation.

Peri- and post-operative (up to day 7 after surgery) neutrophil chemiluminescence and the plasma concentrations of interleukin 6 (IL-6), IL-8, IL-10 and tumour necrosis factor alpha (TNF-alpha) were evaluated in the blood of patients undergoing liver transplantation. IL-6, IL-8 and IL-10 levels increased during early reperfusion and then returned to normal mostly within the first post-operative day. TNF-alpha was increased during the whole period observed. Spontaneous as well as activated neutrophil chemiluminescence was depressed in early reperfusion and remained low during the whole period followed. Samples collected during early reperfusion provided positive correlation for IL-6 vs IL-8 as well as for IL-6 and IL-8 vs chemiluminescence. The data were also evaluated with respect to the outcome of transplantation. Since IL-8, IL-10 and TNF-alpha levels increased significantly during the first post-operative week, mainly in a group of patients who developed serious complications within the first month after surgery, we proved a connection between peri- and early post-operative induction of cytokine release and the outcome of liver allograft transplantation.