Arginine-based inhibitors of nitric oxide synthase: therapeutic potential and challenges.

In the past three decades, nitric oxide has been well established as an important bioactive molecule implicated in regulation of cardiovascular, nervous, and immune systems. Therefore, it is not surprising that much effort has been made to find specific inhibitors of nitric oxide synthases (NOS), the enzymes responsible for production of nitric oxide. Among the many NOS inhibitors developed to date, inhibitors based on derivatives and analogues of arginine are of special interest, as this category includes a relatively high number of compounds with good potential for experimental as well as clinical application. Though this group of inhibitors covers early nonspecific compounds, modern drug design strategies such as biochemical screening and computer-aided drug design have provided NOS-isoform-specific inhibitors. With an emphasis on major advances in this field, a comprehensive list of inhibitors based on their structural characteristics is discussed in this paper. We provide a summary of their biochemical properties as well as their observed effects both in vitro and in vivo. Furthermore, we focus in particular on their pharmacology and use in recent clinical studies. The potential of newly designed specific NOS inhibitors developed by means of modern drug development strategies is highlighted.

New role for L-arginine in regulation of inducible nitric-oxide-synthase-derived superoxide anion production in raw 264.7 macrophages.

Dietary supplementation with L-arginine was shown to improve immune responses in various inflammatory models. However, the molecular mechanisms underlying L-arginine effects on immune cells remain unrecognized. Herein, we tested the hypothesis that a limitation of L-arginine could lead to the uncoupled state of murine macrophage inducible nitric oxide synthase and, therefore, increase inducible nitric-oxide-synthase-derived superoxide anion formation. Importantly, we demonstrated that L-arginine dose- and time dependently potentiated superoxide anion production in bacterial endotoxin-stimulated macrophages, although it did not influence NADPH oxidase expression and activity. Detailed analysis of macrophage activation showed the time dependence between LPS-induced iNOS expression and increased O(2)(∙-) formation. Moreover, downregulation of macrophage iNOS expression, as well as the inhibition of iNOS activity by NOS inhibitors, unveiled an important role of this enzyme in controlling O(2)(∙-) and peroxynitrite formation during macrophage stimulation. In conclusion, our data demonstrated that simultaneous induction of NADPH oxidase, together with the iNOS enzyme, can result in the uncoupled state of iNOS resulting in the production of functionally important levels of O(2)(∙-) soon after macrophage activation with LPS. Moreover, we demonstrated, for the first time that increased concentrations of L-arginine further potentiate iNOS-dependent O(2) (∙-) formation in inflammatory macrophages.

Addition of carvedilol to University Wisconsin solution improves rat steatotic and nonsteatotic liver preservation.

Here we examine the effect of adding carvedilol (CVD) to University of Wisconsin (UW) solution on the preservation of steatotic and nonsteatotic livers during cold ischemia and after normothermic reperfusion. We used an isolated perfused rat liver model. The following protocols were evaluated. Protocol 1 concerned the effect of CVD after cold ischemia. Steatotic and nonsteatotic livers were preserved for 24 hours in UW solution alone or with CVD. Livers without cold ischemia were used as controls. Transaminases were evaluated in the flushing effluent. Protocol 2 involved the effect of CVD after reperfusion. Both liver types were preserved for 24 hours in UW solution alone or with CVD and then perfused ex vivo for 2 hours at 37 degrees C. Livers flushed and perfused without ischemia were used as controls. Hepatic injury and functionality [transaminases, bile production, and hepatic clearance of sulfobromophthalein (BSP)] were evaluated after reperfusion. In addition, factors potentially involved in hepatic ischemia-reperfusion injury, including oxidative stress (malondialdehyde and superoxide anion levels), mitochondrial damage (glutamate dehydrogenase activity), microcirculatory disorders (flow rate and vascular resistance), and adenosine triphosphate (ATP) depletion, were evaluated after reperfusion. After cold ischemia, steatotic livers preserved in UW solution showed higher transaminase levels than nonsteatotic livers. After reperfusion, steatotic livers preserved in UW solution showed higher transaminase levels and lower bile production and BSP clearance than nonsteatotic livers. Alterations in the perfusion flow rate and vascular resistance, mitochondrial damage, and reduced ATP content were more evident in steatotic livers preserved in UW solution. The addition of CVD to UW solution reduced hepatic injury, obstructed its mechanisms, and improved hepatic functionality in both liver types. We conclude that CVD is a useful additive for UW solution that improves the preservation of steatotic and nonsteatotic livers subjected to prolonged cold ischemia.

Effect of polyunsaturated fatty acids on the reactive oxygen and nitrogen species production by raw 264.7 macrophages.

BACKGROUND: Polyunsaturated fatty acids (PUFAs) can affect various functions of the immune system including inflammatory responses. An oxidative burst of phagocytes accompanied by reactive oxygen species (ROS) and reactive nitrogen species (RNS) formation is one of the phagocyte functions that could be modulated by PUFAs. AIM OF THE STUDY: To investigate the effects of omega-3 (alpha-linolenic, docosahexaenoic, eicosapentaenoic) and omega-6 (arachidonic, linoleic) PUFAs on lipopolysaccharide (LPS)-stimulated ROS and RNS production by the murine macrophage cell line RAW 264.7. METHODS: Murine peritoneal macrophages RAW 264.7 were stimulated with LPS (0.1 microg/ml) and treated with 0.1-100 microM omega-3 or omega-6 PUFAs for either 8 (ROS production) or 20 h (RNS production). The cytotoxicity of PUFAs was evaluated by an ATP (adenosine triphosphate) test after both 8 and 20 h of treatment with PUFAs. Changes in ROS production by LPS-treated macrophages subsequently activated with phorbol myristate acetate (PMA) or opsonized zymosan particles (OZP) were determined by luminol-enhanced chemiluminescence, whilst the production of RNS was determined as the concentration of nitrites in cell supernatants (Griess reaction). Changes in inducible nitric oxide synthase (iNOS) expression were evaluated by Western blot analysis. The antioxidant properties of PUFAs were tested by TRAP (total peroxyl radical-trapping antioxidant parameter) assay. RESULTS: All PUFAs in 100 microM concentration except eicosapentaenoic acid decreased ROS production. The effect was most significant when docosahexaenoic acid was used. Arachidonic acid decreased PMA-activated ROS production even in 1 and 10 microM concentrations. On the other hand, 10 and 100 microM eicosapentaenoic acid potentiated ROS production. As concerns RNS production, all the fatty acids that were tested in a concentration of 100 microM decreased iNOS expression and nitrite accumulation. Fatty acids had no significant effect on the viability and proliferation of RAW 264.7 cells. The TRAP assay confirmed that none of the tested PUFAs exerted any significant antioxidant properties. CONCLUSION: High concentrations of PUFAs of both omega-3 and omega-6 groups can inhibit ROS and RNS formation by stimulated macrophages. The expression of iNOS can also be inhibited. This effect, together with the absence of antioxidant activity and cytotoxic properties, indicates that PUFAs can participate in the regulation of enzymes responsible for reactive species production.

Molecular targets of the natural antioxidant pterostilbene: effect on protein kinase C, caspase-3 and apoptosis in human neutrophils in vitro.

OBJECTIVE: Pterostilbene, a naturally occurring phenolic derivative, exhibits various pharmacological effects, e.g. anti-cancerous, antioxidant, anti-inflammatory and anti-diabetic. Based on our previous study, we assessed the cellular and molecular effects of pterostilbene on human neutrophils and in cell free systems. Experimental and theoretical molecular descriptors of stilbene derivatives were also determined. METHODS: We assessed the antioxidant properties of pterostilbene using cell free system and computational methods. The effect of pterostilbene on protein kinase C activation/phosphorylation was detected by special anti-phospho protein kinase C antibodies. Membrane associated changes determining the life span of neutrophils and human recombinant caspase-3 assay were examined. RESULTS: Pterostilbene possessed comparable antioxidant properties as resveratrol in cell free system. Computational methods were used to establish the molecular characteristics of stilbene derivatives. The values of electronic parameters suggest a slight enhancement of electron donor properties of pterostilbene compared to resveratrol. Phosphorylation and thus activation of protein kinase C alpha/beta II in activated neutrophils was not decreased by pterostilbene. Pterostilbene in concentrations of 10-100 µM was found to inhibit the activity of human caspase-3 purified enzyme and did not influence cell viability significantly. CONCLUSION: Pterostilbene, an analog of resveratrol, was identified as a good natural antioxidant compound. However, reducing the oxidative burst of human neutrophils during their activation in vitro with pterostilbene does not include protein kinase C phosphorylation pathway. Pterostilbene showed dose dependent activation/inhibition of caspase-3 enzyme activity.

Formation of reactive oxygen and nitrogen species in the presence of pinosylvin - an analogue of resveratrol.

OBJECTIVE: Formation of reactive oxygen species in neutrophils of rats with adjuvant arthritis and generation of nitric oxide in RAW 264.7 macrophages were analysed in the presence of pinosylvin. METHODS AND RESULTS: The method of chemiluminescence was used for the detection of reactive oxygen species in blood of rats with adjuvant arthritis. Pinosylvin (50 mg/kg, daily, p.o.) and methotrexate (0.4 mg/kg, twice a week, p.o.) were applied separately or in a combination over a period of 28 days from the day of immunisation. Adjuvant arthritis was accompanied by a significantly increased number of neutrophils, by elevated concentration of oxidants in blood and by excessive responsiveness of neutrophils to stimulation with PMA. In rats treated with methotrexate, all these changes were significantly reduced and the inhibition became more pronounced when methotrexate was applied in the combination with pinosylvin; the monotherapy with pinosylvin did not induce any detectable changes in the parameters tested. Under in vitro conditions, pinosylvin inhibited formation of nitric oxide (NO) in macrophages, as demonstrated by the decreased concentration of nitrite - the end-product of NO metabolism (assessed by Griess' method), by the reduced expression of inducible NO synthase (detected by Western blot), and by the failure of pinosylvin to scavenge nitric oxide (measured amperometrically in cell-free system). CONCLUSION: The observed ability of pinosylvin to decrease concentration of reactive oxygen and nitrogen species, along with its capacity to enhance the efficacy of methotrexate in arthritis treatment may shed more light into the pharmacological potential of this prospective natural substance.

Different effect of two synthetic coumarin-stilbene hybrid compounds on phagocyte activity.

OBJECTIVE: Activated phagocytes, generating a variety of powerful inflammatory mediators, such as oxygen and nitrogen species, may participate in oxidative stress-mediated inflammation and organ toxicity. At present, great attention is devoted to the important class of phenolic compounds - coumarins - due to their antiinflammatory/antioxidant activities. We compared two synthetic phenylcoumarins: 7-hydroxy-3-(4´-hydroxyphenyl) coumarin (HHC; 0.01-100 µmol/l) and its hydrogenated analogue: 7-hydroxy-3-(4´-hydroxyphenyl)-3,4-dihydrocoumarin (HHDC; 0.01-100 µmol/l) as their ability to inhibit reactive oxygen species (ROS) generation in human neutrophils and nitric oxide (NO) production by RAW 264.7 macrophages in vitro, with respect to some of their physicochemical characteristics. METHODS: ROS production was measured with luminol-enhanced chemiluminescence (CL) in the microplate luminometer Immunotech LM-01T, nitrite formation was determined by the Griess reaction - spectrophotometrically. The radical scavenging assays were employed to assess the antiradical activity values. The relevant physico-chemical parameters of the compounds tested, electronic and hydrophobic, were determined experimentally as well as by suitable computational programmes. RESULTS: Both HHC and HHDC were found to decrease significantly (p<0.01) CL of whole blood stimulated with phorbol myristate acetate (PMA) from the concentration of 1 µmol/l. While HHC significantly inhibited CL stimulated by A23187 and opsonized zymosan (OpZ), HHDC was ineffective. Unlike HHDC, HHC in the concentrations of 10 and 100 µmol/l significantly (p<0.01) reduced NO formation in lipopolysaccharide (LPS) -stimulated murine macrophages RAW 264.7. HHC possessed the higher free radical reducing efficacy in accordance with its more favourable values of electronic parameters in comparison with HHDC. CONCLUSIONS: Our results show the different inhibitory effects of HHC and HHDC on phagocytic activity that might be the result of their diverse free radical scavenging properties and lipophilicity features.

Serotonin and its metabolites reduce oxidative stress in murine RAW264.7 macrophages and prevent inflammation.

In this study, we focused on comparing the effects of serotonin and its metabolites on the functions of RAW264.7 cells (emphasis on oxidative burst and production of nitric oxide and cytokines), thereby expanding the scope of existing knowledge with advent of novel findings in this field. Changes in production of reactive oxygen species (ROS) by RAW264.7 cells after treatment with serotonin, N-acetylserotonin and melatonin were determined using the chemiluminescence (CL) assay. To exclude the direct scavenging effects of the studied compounds on the CL response, the antioxidant properties of all respective compounds were measured using TRAP and amperometrical method. Nitric oxide (NO) production was measured by Griess reagent and inducible NO synthase (iNOS) expression by Western blot. Cytokine production was assessed using the Mouse Cytokine Panel A Array kit and ELISA. We showed that all tested compounds were able to reduce oxidative stress, as well as inhibit production of inflammatory cytokines by macrophages. Of the tested compounds, serotonin and N-acetylserotonin were markedly better antioxidants than melatonin. In comparison, other effects of tested compounds were very similar. It can be concluded that antioxidant capacity of tested compounds is a major advantage in the early stages of inflammation. Since plasma concentrations of N-acetylserotonin and melatonin are lower than serotonin, it can be deduced that serotonin plays a key role in modulation of inflammation and the regulatory functions of immune cells, while also protecting cells against oxidative stress.

Continuous electrochemical monitoring of nitric oxide production in murine macrophage cell line RAW 264.7.

In this study, we realized the continual and long-term electrochemical detection of NO production by stimulated macrophages using modified porphyrinic microsensor. The NO release from RAW 264.7 cells stimulated by lipopolysaccharide started 5 h after the lipopolysaccharide administration. After reaching its maximum at the sixth hour, the stable level of NO production was observed between the seventh and 12th hour of the experiment. This phase was followed by a gradual decline in NO production. A close correlation between the NO signal detected with microelectrode and nitrite accumulation, which had been determined in supernatants removed from stimulated cells, was observed. This finding was utilized for the calibration of the electrochemical experiment. The presence of iNOS enzyme, which constitutes a main requirement for NO production by stimulated macrophages, was confirmed by Western blot analysis of iNOS protein expression at key time points of the corresponding electrochemical experiment. The capability of our microsensor to instantaneously monitor the changes in the NO production by stimulated RAW 264.7 cells was demonstrated by the immediate decrease in the signal due to NO as a response to the addition of iNOS inhibitor into the cell culture medium.

The effect of uric acid on homocysteine-induced endothelial dysfunction in bovine aortic endothelial cells.

OBJECTIVES: Elevated plasma uric acid indicates an increased risk of cardiovascular diseases associated with endothelial dysfunction. However, the role of uric acid in the pathogenesis of endothelial dysfunction is still a matter of debate. It is not clear whether uric acid is a real causative risk factor, an inert marker, or even a protective molecule with respect to its antioxidant properties. We have studied the effect of uric acid on intact endothelial cells as well as cells with homocysteine-induced endothelial dysfunction. DESIGN: Bovine aortic endothelial cells were treated with uric acid (100 - 600 muM) and homocysteine (100 muM) or with uric acid only. After 24 hours, the cells were stimulated with 1 mug/ml of calcium ionophore A23187, and nitric oxide (NO) production was measured electrochemically with the use of a NO-sensitive microelectrode. The expression of endothelial nitric oxide synthase (eNOS) and eNOS phosphorylation at Ser1179 was estimated with the use of Western blotting. Interaction between NO and uric acid was measured with a NO electrode. Superoxide generation was measured with the use of the fluorescence dye MitoSox Red. RESULTS: Homocysteine strongly diminished A23187-induced NO release. 100 muM uric acid slightly restored NO production; higher concentrations were ineffective. Interestingly, a dose-dependent decrease of NO release was observed in the cells treated only with uric acid. Uric acid did not scavenge NO and did not change eNOS protein expression or phosphorylation at Ser1179, but dose-dependently increased superoxide production in A23187-stimulated cells. CONCLUSION: In conclusion, uric acid decreased NO bioavailability and enhanced superoxide generation in A23187-stimulated bovine aortic endothelial cells.

Increased markers of oxidative stress in plasma of patients with chronic pancreatitis.

OBJECTIVES: Chronic pancreatitis (CP) is a heterogeneous disease defined as chronic inflammatory changes of the pancreatic tissue caused by variety of aetiologies. Oxidative stress accompanying the inflammatory processes has been suggested as an important factor contributing to CP development. The aim of this study was to determine levels of lipid peroxidation products malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), together with nitrites and the total antioxidant capacity in the plasma of patients with CP and control subjects. DESIGN: One hundred and five patients with chronic pancreatitis and twenty seven healthy controls were included into this study. Levels of MDA and 4-HNE were analyzed using high-performance liquid chromatography. The total antioxidant capacity of plasma against peroxyl radicals was evaluated using chemiluminescent determination. Nitrites were determined using Griess reaction. Biochemical and haematological parameters were measured by standard methods. RESULTS: The plasma levels of both MDA and 4-HNE, together with the plasma levels of nitrites, were significantly higher in CP patients, compared to healthy controls. The total antioxidant capacity did not differ significantly. Biochemical parameters were in the normal range. The MDA and 4-HNE levels correlated positively with the levels of high-density lipoprotein cholesterol. Nitrite levels correlated positively with C-reactive protein, total white blood cells, and triglycerides. CONCLUSION: The significantly increased plasma levels of MDA, 4-HNE, and nitrites indicate that oxidative stress is present in patients with CP and that it may play a role in initiation and maintenance of inflammation within the pancreatic tissue in CP patients.

Interactions of oxidatively modified calf skin collagen with platelets and phagocytes.

OBJECTIVES: The effects of non-modified and oxidatively modified calf skin collagen type I on platelet aggregation and the oxidative burst of phagocytes were examined in the framework of a general hypothesis that collagen, platelets and phagocytes cooperate to modulate the oxidative burst of phagocytes and the extent of oxidative stress. MATERIALS AND METHODS: Calf skin collagen type I was subjected to oxidative modification by hydrogen peroxide or hydroxyl radical. Thermal denaturation of collagen was performed in a spectrophotometer equipped with a temperature gradient device. The aggregation of isolated human platelets obtained after differential centrifugation was measured using a dual-channel aggregometer. The production of reactive oxygen species by human whole blood phagocytes was evaluated by luminol-enhanced chemiluminescence. RESULTS: Oxidative modification of collagen samples was characterized by a decrease in denaturation transition temperature. Oxidatively modified samples showed a modified SDS-PAGE pattern, evidencing a significant destruction of the collagen. All oxidatively modified collagen samples, independent of the oxidation treatment applied, lost their platelet-aggregating and phagocyte oxidative burst-inducing activity. CONCLUSION: The results suggest that reactive oxygen species were able to modify collagen. On the other hand, oxidatively modified collagen lost its activating properties towards platelets and phagocytes.

H1-antihistamines and oxidative burst of professional phagocytes.

OBJECTIVES: We analysed and compared the effect of five H1-antihistamines on stimulated oxidative burst at extra- and intracellular level of isolated and stimulated human polymorphonuclear leukocytes. DESIGN: Oxidative burst of isolated human neutrophils was studied by means of luminol and isoluminol enhanced chemiluminescence. RESULTS: The following rank order of potency for H1-antihistamines to decrease chemiluminescence was evaluated extracellularly: dithiaden> loratadine> chlorpheniramine> brompheniramine> pheniramine and at intracellular site: loratadine> dithiaden. CONCLUSION: H1-antihistamines differ substantially according to their chemical structure in suppressing oxidative burst both at extra- and intracellular site of isolated stimulated human neutrophils.

Alternation of retinoic acid induced neural differentiation of P19 embryonal carcinoma cells by reduction of reactive oxygen species intracellular production.

OBJECTIVES: Intracellularly generated reactive oxygen species (ROS) are thought to modulate redox sensitive signaling pathways and thus regulate cell physiology including proliferation and differentiation. However, the role of ROS in neuronal differentiation of embryonic pluripotent cells is unknown. For this reason, the modification of retinoic acid (RA) induced neuronal differentiation of mouse embryonal carcinoma cells P19 by selected ROS scavengers and flavoprotein inhibitor was evaluated. METHODS: Intracellular ROS was evaluated by flowcytometry. Cellular redox status was evaluated based on total levels of reduced thiol groups in cells. The activity of the RA responsive element (RARE) was evaluated by luciferase reporter assay. The RA-induced neuronal differentiation was determined based on changes in the expression of protein markers characteristic for undifferentiated (Oct-4) and neuron-like cell differentiated cells (N-cadherin and III-beta tubulin). RESULTS: RA increased the intracellular ROS production that was accompanied by a decrease in thiol groups in cells. The ROS scavengers and flavoprotein inhibitor reduced RA-induced ROS production, RA-induced activity of RARE, and it decreased the RA-induced expression of N-cadherin and III-beta tubulin. CONCLUSIONS: Our data outline a role of ROS as important molecules in the transduction of an intracellular signal during the neuronal differentiation of ES cells.

Inhibition of superoxide generation and myeloperoxidase release by carvedilol after receptor and nonreceptor stimulation of human neutrophils.

OBJECTIVES: To compare three stimuli which activate human neutrophils with different signal transduction mechanisms, in order to better localize the effect of the beta-adrenoceptor antagonist carvedilol (CARV) on superoxide generation (O2*-) and myeloperoxidase release (MPO). The effect of CARV [0.1-100 micromol/l] on O2*- generation and MPO release from isolated human neutrophils was studied after specific receptor activator N-formyl-methionyl-leucyl-phenylalanine (fMLP) and nonreceptor phorbol-12-myristate-13-acetate (PMA) and calcium ionophor (A23187) stimuli. METHODS: O2*- generation was measured as superoxide dismutase inhibitable reduction of cytochrome c and MPO release as the oxidation of o-dianisidine in the presence of hydrogen peroxide in a spectrophotometer Hewlet Packard 8452 A at respective 550 and 463 nm. RESULTS: CARV had no effect on O2*- generation and MPO release in nonstimulated cells. In the concentration 10 and 100 micromol/l, it significantly decreased fMLP and PMA stimulated O2*- generation and MPO release. Incubation of neutrophils with CARV [100 micromol/l] caused significant inhibition of O2*- generation and MPO release induced by A23187. Wortmannin, a specific inhibitor of 1-phosphatidylinositol-3-kinase, inhibited significantly only fMLP stimulated O2*- generation. CARV [100 micromol/l] with wortmannin [50 nmol/l] further decreased O2*- generation after the same stimulus. CONCLUSION: CARV decreased O2*- generation and MPO release from isolated human neutrophils both by membrane-operating stimulus - fMLP and membrane bypassing activators - PMA and A 23187. This fact, together with effect the of wortmannin, indicates that the inhibition may be attributed to the non-specific action of CARV and its interference with phospholipase D signaling pathway, which plays only a minor role in proteinkinase C stimulated O2*- generation.

Effect of carvedilol on the production of reactive oxygen species by HL-60 cells.

OBJECTIVES: The generation of reactive oxygen species (ROS) by phagocytes is one of the irreplaceable microbicidal tools of innate immunity. It has been reported in our previous studies that short-term treatment by carvedilol ex vivo inhibits ROS generation. The purpose of this study was to investigate the long-term effect of carvedilol on phagocytes. METHODS: Human leukemia HL-60 cells differentiated into granulocyte-like cells were used as the model. Final concentrations of carvedilol were 0.1-100 micromol/l. The production of ROS by HL-60 cells was measured using luminol-enhanced chemiluminescence (CL). RESULTS: Carvedilol in concentrations 0.1-10 micromol/l did not exhibit any toxic effect on cells (measured using bioluminescent bacteria Photorhabdus luminescens subsp. thracensis). One hour's treatment with 10 micromol/l carvedilol significantly decreased both spontaneous and activated CL of cells. Conversely, no inhibitory effects on CL were observed in 10 micromol/l carvedilol after 48 h incubation; lower concentrations of carvedilol even slightly increased the CL activity of HL-60 cells. A significant increase in spontaneous CL activity was detected in cells incubated with 10 micromol/l carvedilol in comparison with the control. Powerful antioxidative properties of carvedilol against peroxyl radical (ORAC assay) were proved. No scavenging of nitric oxide (electrochemical method) was observed. CONCLUSIONS: Long-term influence of carvedilol can induce an increase in the generation of phagocyte-derived ROS and potentially also other inflammatory mediators. The increased ROS production is compensated for by antioxidative properties of carvedilol although the increased production of inflammatory mediators could affect the proper function of immune system.

The effect of aldose reductase inhibition by JMC-2004 on hyperglycemia-induced endothelial dysfunction.

OBJECTIVES: An increased glucose utilization by aldose reductase (ALR-2) has been implicated in the pathogenesis of diabetic vascular complications. In this process, several mechanisms are involved, including the depletion of cofactors required for the action of antioxidant enzymes or endothelial NO synthase. In this study, the effect of a novel ALR-2 inhibitor JMC-2004 on hyperglycemia-induced endothelial dysfunction was studied. METHODS: Bovine aortic endothelial cells (BAEC) were treated with glucose (30 mM), JMC-2004 (0.01mM), or glucose and JMC-2004 for 24 h. The cells were then stimulated with calcium ionophore A23187 after which NO production was measured electrochemically using a porphyrine-coated carbon NO electrode. Nitrite concentrations were determined in the cell supernatants. The peroxyl and hydroxyl radical-scavenging activity of JMC-2004 was measured with luminol-enhanced chemiluminescence. The expression of eNOS was determined by Western blotting. JMC-2004 IC50 for ALR-2 was determined colorimetrically with D-glyceraldehyde as a substrate. RESULTS: Incubating the cells with 30 mM glucose strongly diminished A23187- induced NO production. Treatment with JMC-2004 restored NO production by 40% without affecting eNOS expression. This effect was probably antioxidantindependent, since JMC-2004 did not have any antioxidant capacity. JMC-2004 exerted high selectivity towards ALR-2. CONCLUSIONS: ALR-2 inhibition with JMC-2004 was able to abolish hyperglycemia- induced endothelial dysfunction in bovine aortic endothelial cells.

Ultrafiltered pig leukocyte extract (IMUNOR) decreases nitric oxide formation and hematopoiesis-stimulating cytokine production in lipopolysaccharide-stimulated RAW 264.7 macrophages.

A low-molecular-weight (<12 kDa) ultrafiltered pig leukocyte extract, IMUNOR, was tested in experiments in vitro on non-stimulated and lipopolysaccharide (LPS)-stimulated murine RAW 264.7 macrophages in order to assess modulation of nitric oxide (NO) production (measured indirectly as the concentration of nitrites), hematopoiesis-stimulating activity of the supernatant of the macrophage cells (ascertained by counting cell colonies growing from progenitor cells for granulocytes and macrophages (GM-CFC) in vitro), and the release of hematopoiesis-stimulating cytokines. No hematopoiesis-stimulating activity and cytokine or NO production were found in the supernatant of non-stimulated macrophages. It was found that IMUNOR does not influence this status. Supernatant of LPS-stimulated macrophages was characterized by hematopoiesis-stimulating activity, as well as by the presence of nitrites, interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). A key role in the hematopoiesis-stimulating activity of the supernatant of LPS-stimulated macrophages could be ascribed to G-CSF since the formation of the colonies could be abrogated nearly completely by monoclonal antibodies against G-CSF. IMUNOR was found to suppress all the mentioned manifestations of the LPS-activated macrophages. When considering these results together with those from our previous in vivo study revealing stimulatory effects of IMUNOR on radiation-suppressed hematopoiesis, a hypothesis may be formulated which postulates a homeostatic role of IMUNOR, consisting in stimulation of impaired immune and hematopoietic systems but also in cutting back the production of proinflammatory mediators in cases of overstimulation which threats with undesirable consequences.

Nitric oxide sensor based on carbon fiber covered with nickel porphyrin layer deposited using optimized electropolymerization procedure.

Electropolymerization regime of meso-tetrakis(3-methoxy-4-hydroxyphenyl) porphyrin is optimized to yield films possessing both electrocatalytical and permselective properties towards nitric oxide oxidation. The sensor composed of electrochemically oxidized carbon fiber, covered solely with nickel porphyrin derivative layer electropolymerized using our method, is characterized by high selectivity towards nitrite (1:600), ascorbate (1:8000) and dopamine (>1:80), determined by constant potential amperometry at 830 mV (vs. Ag/AgCl). Selectivity for ascorbate and dopamine as well as detection limit for NO (1.5 nM at S/N=3) is 5-10 times better than parameters usually reported for Nafion coated porphyrinic sensors. Nafion coating can further enhance selectivity properties as well as aids to the stability of the sensors' responses.

The effects of berberine on reactive oxygen species production in human neutrophils and in cell-free assays.

The health benefits of berberine have been recognized for years. Even so, its effects on human neutrophils, the first line of immune defense, have not been reported. The purpose of this study was to investigate the effects of berberine on the human neutrophil oxidative burst. Reactive oxygen species production was analyzed by luminol-enhanced chemiluminescence. The analysis was performed in spontaneous and stimulated (phorbol myristate acetate (PMA) or opsonized zymosan particles (OZP)) whole blood and isolated neutrophils in the presence or absence of berberine. The effects of berberine on oxidant production in cell-free assays were evaluated using luminescence (H2O2-peroxidase-luminol) and fluorescence (Oxygen Radical Absorbance Capacity - ORAC) techniques. Berberine decreased the production of reactive oxygen species in human whole blood and isolated neutrophils stimulated with either PMA or OZP with a different efficiency (EC50 was 69 µM and 197 µM for PMA and OZP, respectively). The effect was more pronounced in isolated neutrophils. Cell-free assays showed the antioxidant activity of berberine against peroxyl radicals and hydrogen peroxide. Based on our results, we suggest that the effects of berberine on reactive oxygen species production in human neutrophils are due to its antioxidant activity.