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Journal Article 2007; 28(1): 39-44 PubMed PMID: 17277728 Citation  Keywords:  Acetylcholine:pharmacology, Animals, Cholinergic Agents:pharmacology, Enzyme Inhibitors:pharmacology, Female, Ganglia, Sympathetic:enzymology, Guanidines:pharmacology, NG-Nitroarginine Methyl Ester:pharmacology, Nitric Oxide:physiology, Nitric Oxide Synth.   

: Both peripheral innervation and nitric oxide (NO) participate in ovarian steroidogenesis. Considering the existence of the nitric oxide/ nitric oxide synthase system in the peripheral neural system and in the ovary, the aim of this work was to analyze if the liberation of NO in the ovarian compartment of prepubertal rats is of ovarian and/or ganglionic origin. The analysis is carried out from a physiological point of view using the experimental coeliac ganglion--Superior Ovarian Nerve--ovary model with and without ganglionic cholinergic stimulus Acetylcholine (Ach) 10(-6) M. Non selective and selective inhibitors of the synthase nitric oxide enzyme were added to the ovarian and ganglionic compartment, and the liberation of nitrites (soluble metabolite of the nitric oxide) in the ovarian incubation liquid was measured. We found that the non-selective inhibitor L-nitro-arginina methyl ester (L-NAME) in the ovarian compartment decreased the liberation of nitrites, and that Aminoguanidine (AG) in two concentrations in a non-dose dependent form provoked the same effect. The addition of Ach in ganglion magnified the effect of the inhibitors of the NOS enzyme. The most relevant results after the addition of inhibitors in ganglion were obtained with AG 400 and 800 microM. The inhibition was made evident with and without the joint action of Ach in ganglion. These data suggest that the greatest production of NO in the ovarian compartment comes from the ovary, mainly the iNOS isoform, though the coeliac ganglion also contributes through the superior ovarian nerve but with less quantity....

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Journal Article 2006; 27(Suppl 2): 160-163 PubMed PMID: 17159805 Citation  Keywords:  Animals, Carbazoles:pharmacology, Cells, Cultured, Immunity, Cellular:drug effects, Immunity, Innate:drug effects, Macrophages:drug effects, Mice, N-Formylmethionine Leucyl-Phenylalanine:pharmacology, Nitric Oxide Synthase Type II:metabolism, Peroxidase:m.   

OBJECTIVES: Neutrophils and macrophages are critical components of our innate host defenses. They are a potential source of nitric oxide (NO) and superoxide, known to play an important role in many physiological processes including defense, immune and inflammatory responses. Myeloperoxidase (MPO), a major NO scavenger and a marker of oxidative stress, as well as increased inducible nitric oxide synthase (iNOS) expression, may affect the NO-superoxide balance, critical for cellular redox balance in the cardiovascular system at physiologic conditions. The effect of carvedilol was studied on stimulated superoxide generation, MPO release and iNOS expression in phagocytes by using receptor operating stimuli [N-formyl-Met-Leu-Phe (FMLP), lipopolysaccharide (LPS) and specific inhibitors (wortmannin, propranolol).

METHODS: Superoxide generation was measured as superoxide dismutase inhibitable reduction of cytochrome c (550 nm), MPO activity as the oxidation of o-dianisidine in the presence of hydrogen peroxide in a spectrophotometer Hewlet Packard 8452 A (463 nm). Expression of iNOS (Western-blot analysis) in RAW 264.7 cell line (murine macrophages) was stimulated by lipopolysaccharide (LPS).

RESULTS: Carvedilol dose-dependently decreased superoxide generation and MPO release from intact neutrophils stimulated by FMLP. In the highest concentration tested, carvedilol pronounced the effect of wortmannin [inhibitor of phospholipase D (PLD) regulatory pathway] and significantly decreased LPS stimulated iNOS expression in RAW 264.7 cell line.

CONCLUSION: The conceivable cumulative non-specific membrane effect of carvedilol and its effect on PLD signalling pathway contribute to the decrease of both superoxide generation and MPO release, thus supporting the restoration of NO-superoxide balance....

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Comparative Study 2006; 27(Suppl 2): 31-34 PubMed PMID: 17159774 Citation  Keywords:  Hyaluronic Acid:chemistry, Hydrogen Peroxide:pharmacology, Manganese Compounds:pharmacology, Models, Biological, Molecular Weight, Oxides:pharmacology, Peroxynitrous Acid:chemical synthesis, Viscosity,.   

OBJECTIVES: This contribution presents the results of the kinetics of HA degradation by peroxynitrite, which represents one of the main reactive oxygen species degrading various biomacromolecules under inflammatory conditions.

METHODS: Two simple procedures have been adapted to prepare sodium peroxynitrite: the first containing an excess of H(2)O(2), and the second in which the H(2)O(2) excess had been decomposed by MnO(2) treatment. The kinetics of hyaluronan degradation by action of peroxynitrite was monitored by rotational viscometry.

RESULTS: High-molecular-weight hyaluronan was degraded by peroxynitrite. The degradation was increased in the presence of ONOO(-) previously treated by MnO(2) in order to remove residual hydrogen peroxide. One of the reasons of this finding could be that by the action of the residual metal the pathway of ONOO(-) decomposition starts to be manifested immediately on mixing traces of metals originally present in the HA sample with the ions of manganese.

CONCLUSIONS: Trace amounts of transition metal(s) should be taken into consideration on evaluating the experimental results. Purchase of the marketed peroxynitrite product appears to be the appropriate approach to simplify and standardize the quality of ONOONa....

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Comparative Study 2006; 27(5): 675-678 PubMed PMID: 17159822 Citation  Keywords:  5-alpha-Dihydroprogesterone:pharmacology, Animals, Cell Line, Cell Survival:drug effects, Dose-Response Relationship, Drug, Lipopolysaccharides:pharmacology, Mice, Microglia:cytology, Nitric Oxide:secretion, Pregnanolone:pharmacology, Progesterone:metabol.   

OBJECTIVE: Microglial cells, important immunosurveillance cells in the nervous system, respond to pathogens with an increase in inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) release. Because excessive release of NO may be neurotoxic, tight regulation of nitrosative stress is required.

DESIGN AND SETTING: We cultured the murine microglial cell-line, BV-2, with bacterial lipopolysaccharide (LPS) to induce NO synthesis and quantified the impact of progesterone and its metabolites, 5alpha-dihydroprogesterone (DHP) and 5alpha-3alpha-tetrahydroprogesterone (THP), on NO release.

RESULTS: Our in vitro experiments showed that in BV-2 cells LPS-induced NO release is suppressed by progesterone and THP. Both neurosteroids decreased NO release by about 40% when used at a concentration of 10 microM. NO release was less sensitive to DHP. This neurosteroid decreased NO release only by 20% when used at a concentration of 10 microM. NO release was sensitive to N(G)-methyl-L-arginine (L-NMMA), a completive inhibitor of NOS, indicating that LPS-induced NO release is mediated by NOS activity. Trypan blue exclusion experiments showed that the ratio of viable to dead cells did not vary using different concentrations of progesterone. Furthermore, progesterone did not increase apoptosis or necrosis when estimated by the distribution of DAPI-labelled condensed chromatin.

CONCLUSION: These experiments indicate that the decline in NO release is mainly due to modulation of NOS activity or expression. Because progesterone, DHP, and THP are synthesized in astrocytes and oligodendrocytes, these neurosteroids may locally suppress an immune response....

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Comparative Study 2006; 27(5): 615-621 PubMed PMID: 17159817 Citation  Keywords:  Adult, Epitopes:immunology, Fatigue Syndrome, Chronic:blood, Fatty Acids:immunology, Female, Humans, Immunoglobulin M:blood, Male, Middle Aged, Oxidative Stress:immunology, Proteins:immunology, Reactive Nitrogen Species:immunology, Reactive Oxygen Species.   

: There is now some evidence that chronic fatigue syndrome (CFS) is accompanied by signs of oxidative stress and by a decreased antioxidant status. The aim of the present study was to examine whether CFS is accompanied by an immune response to neoepitopes of a variety of modified lipids and proteins indicating damage caused by oxidative and nitrosative stress. Toward this end we examined serum antibodies to fatty acids (oleic, palmitic and myristic acid), by-products of lipid peroxidation, i.e. azelaic acid and malondialdehyde (MDA), acetylcholine, S-farnesyl-L-cysteine, and N-oxide modified amino-acids in 14 patients with CFS, 14 subjects with partial CFS and 11 normal controls. We found that the prevalences and mean values for the serum IgM levels directed against oleic, palmitic and myristic acid, MDA, azelaic acid, S-farnesyl-L-cysteine, and the N-oxide derivates, nitro-tyrosine, nitro-phenylalanine, nitro-arginine, nitro-tryptophan, and nitro-cysteinyl were significantly greater in CFS patients than in normal controls, whereas patients with partial CFS took up an intermediate position. There were significant and positive correlations between the serum IgM levels directed against fatty acids, MDA and azelaic acid and the above N-oxide-derivates and the severity of illness (as measured by the FibroFatigue scale) and symptoms, such as aches and pain, muscular tension and fatigue. The results show that CFS is characterized by an IgM-related immune response directed against disrupted lipid membrane components, by-products of lipid peroxidation, S-farnesyl-L-cysteine, and NO-modified amino-acids, which are normally not detected by the immune system but due to oxidative and nitrosative damage have become immunogenic....

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Journal Article 2005; 26(6): 799-805 PubMed PMID: 16380691 Citation  Keywords:  Chromatography, High Pressure Liquid, Dihydropyridines:analysis, Histamine:chemistry, Malondialdehyde:chemistry, Neurotoxins:chemistry, Nitrogen Compounds:analysis, Protein Carbonylation,.   

OBJECTIVES: To explore the mechanism of carbonyl stress-related toxification on neurotransmitter histamine and the potential de-carbonylation function of histamine.

METHODS: The reaction mixture of malondialdehyde (MDA) and histamine (HA) at pH7.4, 37 degrees C was assayed by high performance liquid chromatography (HPLC), spectrophotometry and spectrofluorometry. The reaction products were assayed by LC/MS.

RESULTS: In physiological condition, the reaction of MDA and HA yielded two products: a nonfluorescent enaminal derivatives and a fluorescent 1,4-dihydropyridine adducts. The fluorescence maxima of the fluorescent products (Ex. 393 nm/Em. 464 nm) were similar to those of lipofuscin pigment. The fluorescence intensity of reaction mixture was in direct proportion to the MDA concentration.

CONCLUSIONS: HA can react with MDA to form stable products, a non-fluorescent enamine (product 1) and a fluorescent 1,4-dihydropyridine (product 2) which are ceroid/lipofuscin-related adducts. The reaction of HA with MDA may reveal toxic effect of unsaturated carbonyls in the brain and may reflect a novel de-carbonylation function of histamine under various pathological conditions....

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Journal Article 2005; 26(4): 373-376 PubMed PMID: 16136006 Citation  Keywords:  Adenoma:blood supply, Apoptosis, Humans, Hyperplasia, Immunoenzyme Techniques, Neovascularization, Pathologic:metabolism, Nitric Oxide Synthase Type II:metabolism, Nitric Oxide Synthase Type III:metabolism, Parathyroid Glands:blood supply, Parathyroid Neo.   

OBJECTIVE: The aim of the presented study was to perform the immunohistochemical detection of endothelial (eNOS) and inducible (iNOS) isoform of nitric oxide synthase in the adenomatous and hyperplastic parathyroid gland in relation to the apoptotic process.

DESIGN AND SETTING: Tissue samples from 12 patients with parathyroid gland adenoma (PGA) and 10 patients with secondary parathyroid gland hyperplasia (PGH) were collected during surgery at the Department of Otorhinolaryngology and Head and Neck Surgery of The First Faculty of Medicine in Prague.

METHODS: Three-step immunoperoxidase reaction on acetone-fixed cryostat sections was performed using both polyclonal and monoclonal antibodies against eNOS and iNOS. The detection of apoptotic cells was done using antibody against cleaved caspase-3 as an apoptotic marker.

RESULTS: The immunoreactivity to eNOS antibody was observed in the endothelial lining of vessels in PGA, PGH and in the rim of normal parathyroid gland adjacent to PGA sample. Variable expression of eNOS was confirmed in arteries, arterioles, capillaries and veins in the glandular parenchyma as well as in the surrounding connective tissue. There was no iNOS immunoreactive cell detected in any examined sample. No apoptotic cells were detected.

MAIN RESULT: Our findings confirm that eNOS is regularly expressed in the vasculature of PGA and PGH.

CONCLUSION: eNOS observed in the vasculature of the enlarged parathyroid glands can serve as a factor that contributes to the viability of hypertrophic pathologic tissue. The lack of stimulating signals may be a reason for negative iNOS detection and negligible apoptotic rate....

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Journal Article 2005; 26(4): 407-412 PubMed PMID: 16136000 Citation  Keywords:  Animals, Blood Pressure:drug effects, Cyclooxygenase Inhibitors:pharmacology, Disease Models, Animal, Hypotension:blood, Ibuprofen:pharmacology, Nitrates:blood, Nitrites:blood, Rabbits, Shock, Septic:blood,.   

OBJECTIVES: The purpose of this study was to investigate the effects of ibuprofen on plasma nitrite/nitrate levels, as indirect indicators of nitric oxide, in correlation with blood pressure in a rabbit model of endotoxin-induced shock.

METHODS: A total of 28 rabbits were randomly divided into four groups. Control group received physiological saline, while endotoxin (ETX, E. Coli, 055:B5, 2 mg/kg, i.v.) was administered to the rabbits in the other groups: group II receiving only ETX, in addition to ETX group III received ibuprofen (30 mg/kg) 30 minutes after ETX administration, whilst the group IV received ibuprofen (30 mg/kg) 30 minutes before ETX. Arterial blood pressure and plasma levels of nitrite/nitrate were determined immediately before (time 0) and 30, 60, 90, 120, 180, 240, and 300 minutes after ETX administration.

RESULTS: ETX administered groups had significantly higher plasma levels of nitrite/nitrate than the control group, at all consecutive measurements except at time O. Treatment with ibuprofen, either before or after ETX, partly restored the elevated levels of nitrite/nitrate. ETX also caused a significant decrease in blood pressure which was prevented in ibuprofen treated groups.

CONCLUSION: Results from this study indicate that administration of ibuprofen prevents sudden reductions in blood pressure by inhibiting excessive production of nitric oxide in rabbit model of endotoxin-induced shock and this may be of importance for providing crucial time for therapeutic intervention and survival in septic shock....

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