thiourea and pimagedine

Topics: Animals; Arginine; Disease Models, Animal; Enzyme Induction; Enzyme Inhibitors; Guanidines; Humans; Nitric Oxide Synthase; omega-N-Methylarginine; Rats; Shock, Septic; Thiourea

The present study was conducted to investigate the effect of salubrinal on nitric oxide mediated endoplasmic reticulum stress signaling and neuronal apoptosis. Rotenone treatment to neuro2a cells caused significantly decreased cell viability, increased cytotoxicity, augmented nitrite levels, increased nitrotyrosine level and augmented level of key ER stress markers (GRP-78, GADD153 and caspase-12). These augmented levels of ER stress markers could be attenuated with pretreatment of nitric oxide synthase inhibitor-aminoguanidine as well as with salubrinal. The rotenone treatment to neuro2a cells also triggered the ER stress induced up regulation of various signaling factors of unfolded protein response involving pPERK, ATF4, p-IRE1α, XBP-1 and ATF-6. Pretreatment of salubrinal significantly attenuated the activation of transmembrane kinases (PERK and IRE1) and ATF6 and restored the rotenone induced altered level of other UPR related signaling factors. Rotenone induced dephosphorylation of eIF2α was also inhibited with salubrinal treatment. Biochemically rotenone treatment to neuro2a cells caused the reactive oxygen species generation, depleted mitochondrial membrane potential and increased intra cellular calcium level which was attenuated with salubrinal treatment. Rotenone treatment to neuro2a cells also caused neuronal apoptosis, DNA fragmentation and chromatin condensation which were attenuated with salubrinal treatment. In conclusion, the findings suggested that rotenone causes the augmented level of nitric oxide which contributes in ER stress and could be inhibited by both aminoguanidine and/or salubrinal treatment. Further, salubrinal treatment attenuates the nitric oxide induced ER stress axis PERK:IRE1α:ATF-6 and inhibits the DNA damage and neuronal apoptosis.

Topics: Activating Transcription Factor 6; Animals; Calcium Signaling; Cell Line; Cinnamates; DNA Damage; eIF-2 Kinase; Endoplasmic Reticulum Stress; Endoribonucleases; Enzyme Inhibitors; Guanidines; Membrane Potential, Mitochondrial; Mice; Neurons; Nitric Oxide; Nitric Oxide Synthase; Protein Serine-Threonine Kinases; Rotenone; Signal Transduction; Thiourea; Uncoupling Agents

Previous studies have shown that in situ exposure to arsenic induced increased vascular leakage. However, the underlying mechanism remains unclear. Reactive nitrogen and oxygen species such as nitric oxide (NO) and hydroxyl radical (OH(-)) are known to affect vascular permeability. Therefore, the goal of our present studies is to investigate the functional impact of the generation of NO or OH(-) on arsenic-induced vascular leakage. Vascular permeability changes were evaluated by means of Evans blue (EB) assay. Rats were anesthetized and intravenously injected with EB. Permeability changes were induced in back skin by intradermal injections of sodium arsenite mixed with NOS inhibitor: N(omega)-Nitro-L-arginine methyl ester (L-NAME) or aminoguanidine (AG) and OH(-) scavenger: 1,3 Dimethyl-2 thiourea (DMTU). Experiments were also performed to determine whether DMTU mixed with L-NAME would further inhibit arsenic-induced vascular leakage as compared with attenuation effects by either DMTU or L-NAME. One hour after administration, EB accumulated in the skin was extracted and quantified. Both L-NAME (0.02, 0.1 and 0.5 micromol/site) and DMTU (0.05, 0.2 and 1.2 micromol/site) inhibited the increase in vascular leakage induced by arsenite. However, only high dose (1 micromol/site) of AG significantly attenuated arsenite-induced vascular leakage. In contrast, neither D-NAME (0.02, 0.1 and 0.5 micromol/site) nor AG (0.04 and 0.2 micromol/site) attenuated increased vascular leakage by arsenic. DMTU mixed with L-NAME caused no further inhibition of arsenic-induced vascular leakage by either DMTU or L-NAME. The techniques of India ink and immunostaining were used to demonstrate both vascular labeling and nitrotyrosine staining in tissue treated with arsenic. L-NAME apparently reduced the density of leaky vessels and the levels of peroxynitrite staining induced by arsenite. These results suggest that NO, OH(-) and peroxynitrite play a role in increased vascular permeability induced by arsenic exposure.

Topics: Animals; Arsenites; Blood Vessels; Capillary Permeability; Carbon; Dose-Response Relationship, Drug; Drug Therapy, Combination; Enzyme Inhibitors; Evans Blue; Free Radical Scavengers; Guanidines; Hydroxyl Radical; Injections, Intradermal; Injections, Intravenous; Male; NG-Nitroarginine Methyl Ester; Nitric Oxide; Peroxynitrous Acid; Rats; Rats, Sprague-Dawley; Skin; Sodium Compounds; Thiourea; Tyrosine

Nitric oxide (NO) involvement in intestinal ischemia-reperfusion (I/R) injury has been widely suggested but its protective or detrimental role remains still question of debate. Here, we examine the impact of supplementation or inhibition of NO availability on intestinal dysmotility and inflammation caused by mesenteric I/R in mice. Ischemia 45min and reperfusion 24h were performed by superior mesenteric artery occlusion in female Swiss mice. Saline-treated sham-operated (S) or normal mice without surgery (N) served as controls. Drugs were subcutaneously injected 0, 4, 8, and 18 h after ischemia. Upper gastrointestinal transit (GIT, estimated through black marker gavage), intestinal myeloperoxidase activity (MPO), intestinal malondialdehyde levels (MDA), Evans blue extravasation (EB), intestinal histological damage, and mean arterial pressure (MAP) were considered. In I/R mice, GIT was significantly delayed compared to S and N groups; MPO activity and EB extravasation enhanced, whereas MDA levels did not change. Compared to N and S groups, in I/R mice selective iNOS inhibitor P-BIT significantly prevented motor, MPO and EB changes; putative iNOS inhibitor aminoguanidine significantly counteracted GIT delay but not neutrophil recruitment and the increase in vascular permeability; NOS inhibitor l-NAME and NO precursor l-arginine were scarcely or no effective. Furthermore, in S mice aminoguanidine caused a significant increase of MPO activity reverted by H(1) histamine receptor antagonist pre-treatment. Unlike P-BIT, aminoguanidine and l-NAME injection increased MAP. These findings confirm a detrimental role for iNOS-derived NO overproduction during reperfusion. Aminoguanidine-associated neutrophil recruitment suggests that this drug could act through mechanisms additional to iNOS inhibition involving both eNOS blockade, as indicated by its hemodynamic effects, and indirect activation of H(1) histamine receptors.

Topics: Animals; Blood Pressure; Enzyme Inhibitors; Female; Gastrointestinal Transit; Guanidines; Intestine, Small; Malondialdehyde; Mice; Mice, Inbred Strains; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase Type II; Peroxidase; Reperfusion Injury; Thiourea

The activity of neuronal nitric oxide synthase (nNOS) in homogenates of adult Fasciola hepatica was measured by the direct radiometric assay of the production of L-[3H]citrulline. This is the first radiometric study of the activity of nNOS in a fluke. The effect of arginase was tested. In the presence of L-valine, which is an inhibitor of arginase, the formation of L-[3H]citrulline decreased from 12% to 38%, depending on the time of incubation. This means that the arginase activity in the worm is high, and has to be taken into consideration when measuring the activity of nNOS. When co-factors, such as H4B, and NADPH, were omitted the formation of L-[3H]citrulline decreased significantly (29%). The effects of several nNOS inhibitors were tested. N(omega)-nitro-L-arginine (L-NAME), aminoguanidine and S-methyl-L-thiocitrulline added at a concentration of 1 mM inhibited the L-[3H]citrulline formation by 28%, 15% and 14%, respectively. Chelation of Ca2+ with 1 mM EGTA resulted in a 40% decrease in the formation of L-[3H]citrulline. These results indicate the presence of nNOS activity in homogenates of F. hepatica.

Topics: Animals; Arginase; Cattle; Citrulline; Enzyme Inhibitors; Fasciola hepatica; Guanidines; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Radiometry; Thiourea; Valine

The in vivo role of nitric oxide in inflammatory cell migration, vascular permeability and the development of hyperresponsiveness to methacholine (MCh) was studied in rats 24 h following ovalbumin (OVA) challenge. The NO synthase (NOS) inhibitors N(G)-mono-methyl-L-arginine (L-NMMA; nonselective), aminoguanidine (two-fold inducible NOS-selective), N(omega)-nitro-L-arginine methyl ester (L-NAME; 2000-fold endothelial cell NOS-selective) or S-methyl-L-thiocitrulline (100-fold neuronal NOS-selective) were administered (100 mg x kg(-1) s.c.) to OVA-sensitized Piebald-Virol-Glaxo rats on 3 consecutive days during which they were challenged with allergen (1% OVA). Responses to inhaled MCh were measured in anaesthetized animals 24 h after OVA challenge. Cellular inflammation and vascular permeability were assessed using bronchoalveolar lavage (BAL) fluid collected 30 min after administration of Evans blue (50 mg x kg(-1) i.v.). OVA challenge in sensitized animals induced hyperresponsiveness to MCh, inflammatory cell influx and increased leakage of Evans blue into the BAL fluid (n=9, p<0.001). Aminoguanidine was effective in inhibiting the allergen-induced cellular influx and microvascular leakage (n=9, p<0.001) without altering responses to MCh. This effect was reserved by L-arginine. L-NAME (n=5, p<0.01) and S-methyl-L-thiocitrulline (n=6, p<0.001) further potentiated the allergen-induced hyperresponsiveness without altering cellular inflammation. L-NMMA attenuated both the OVA-induced cellular influx and Evans blue leakage (n=8, p<0.001) as well as further potentiating the hyperresponsiveness to MCh (p<0.05). From these studies, it is suggested that, in allergic Piebald-Virol-Glaxo rats, nitric oxide production by inducible nitric oxide synthase plays a role in the migration of inflammatory cells and increase in vascular permeability following allergen challenge, whereas nitric oxide produced by the constitutively expressed neuronal nitric oxide synthase limits hyperresponsiveness to methacholine.

Topics: Animals; Bronchoalveolar Lavage Fluid; Bronchoconstrictor Agents; Citrulline; Disease Models, Animal; Enzyme Inhibitors; Guanidines; Hypersensitivity; Male; Methacholine Chloride; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; omega-N-Methylarginine; Rats; Thiourea

The inducible isoform of nitric oxide synthase (iNOS) is expressed after systemic administration of lipopolysaccharide (LPS). The importance of expression of iNOS in blood vessels is poorly defined. Because nitric oxide from iNOS may alter vasomotor function, we examined effects of LPS on vasomotor function in carotid arteries from iNOS-deficient mice. We studied contraction of the carotid artery from wild-type and iNOS-deficient mice in vitro 12 h after injection of LPS (20 mg/kg ip). Contractile responses to PGF2alpha (3-30 microM) and thromboxane A2 analog (U-46619; 3-100 nM) were evaluated using vascular rings from mice treated with vehicle or LPS. Maximum force of contraction generated by rings in response to PGF2alpha was 0.39 +/- 0.02 and 0.25 +/- 0.01 (SE) g (n = 14) in vehicle and LPS-treated wild-type mice, respectively (P < 0.001 vs. vehicle). Thus LPS reduced constrictor responses in wild-type mice. Thiocitrulline and aminoguanidine (inhibitors of iNOS) improved contractile responses from LPS-treated wild-type vessels. Indomethacin also improved constrictor responses in arteries from wild-type mice injected with LPS. In contrast, contraction of the carotid arteries in response to PGF2alpha and U-46619 was not impaired in LPS-treated iNOS-deficient mice, and contraction was not altered by inhibitors of iNOS. Expression of iNOS mRNA was confirmed using RT-PCR in carotid arteries from wild-type mice after injection of LPS but not vehicle. PCR products for iNOS were not observed in iNOS-deficient mice. These findings provide the first direct evidence that iNOS mediates impairment of vascular contraction after treatment with LPS.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Carotid Arteries; Citrulline; Dinoprost; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Guanidines; Heterozygote; In Vitro Techniques; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle Contraction; Muscle, Smooth, Vascular; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; RNA, Messenger; Thiourea; Transcription, Genetic; Vasoconstriction

The identification of human inflammatory cells that express inducible nitric oxide synthase and the clarification of the role of inducible nitric oxide synthase in human infectious or inflammatory processes have been elusive. In neutrophil-enriched fractions from urine, we demonstrate a 43-fold increase in nitric oxide synthase activity in patients with urinary tract infections compared with that in neutrophil-enriched fractions from noninfected controls. Partially purified inducible nitric oxide synthase is primarily membrane associated, calcium independent, and inhibited by arginine analogues with a rank order consistent with that of purified human inducible nitric oxide synthase. Molecular, biochemical, and immunocytochemical evidence unequivocally identifies inducible nitric oxide synthase as the major nitric oxide synthase isoform found in neutrophils isolated from urine during urinary tract infections. Elevated inducible nitric oxide synthase activity and elevated nitric oxide synthase protein measured in patients with urinary tract infections and treated with antibiotics does not decrease until 6-10 d of antibiotic treatment. The extended elevation of neutrophil inducible nitric oxide synthase during urinary tract infections may have both antimicrobial and proinflammatory functions.

Topics: Adult; Aged; Anti-Bacterial Agents; Arginine; Bacterial Infections; Blotting, Western; Canavanine; Cell Membrane; Citrulline; Female; Guanidines; Humans; Immunohistochemistry; Isoenzymes; Leukocyte Common Antigens; Male; Middle Aged; Neutrophils; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Nitroarginine; omega-N-Methylarginine; Ornithine; Polymerase Chain Reaction; RNA, Messenger; Sulfonamides; Thiourea; Trifluoperazine; Urinary Tract Infections

The N-methyl-D-aspartate (NMDA) receptor-nitric oxide (NO) pathway has been linked to opiate withdrawal. Pretreatments with four inhibitors of NO synthase, 7-nitro indazole, 3-bromo-7-nitro indazole, S-methyl-L-thiocitrulline and aminoguanidine, which exhibit different isoform selectivity in vitro, were evaluated for their ability to attenuate signs of naloxone-precipitated morphine withdrawal. In separate experiments, effects of NO synthase inhibitors on blood pressure were measured in naive and morphine-dependent rats. 7-Nitro indazole, 3-bromo-7-nitro indazole and S-methyl-L-thiocitrulline, which are specific inhibitors of the constitutive isoforms, produced dose-dependent reductions of several signs of withdrawal. Blood pressure was unaffected by the indazoles, whereas S-methyl-L-thiocitrulline produced a strong vasoconstrictor response. Aminoguanidine, which selectively inhibits inducible NO synthase, reduced fewer signs of opioid withdrawal, had a lower relative potency and exhibited no vasopressor activity. These data suggest that constitutive isoforms, but not the inducible isoform of NO synthase, have a primary role in NO-mediated processes that modulate the opioid withdrawal syndrome in the rat.

Topics: Analysis of Variance; Animals; Blood Pressure; Citrulline; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Induction; Enzyme Inhibitors; Guanidines; Heart Rate; Indazoles; Isoenzymes; Male; Morphine; Naloxone; Narcotic Antagonists; Nitric Oxide Synthase; Rats; Rats, Inbred F344; Substance Withdrawal Syndrome; Thiourea

A series of substituted aminoguanidines and amino-substituted isothioureas have been examined as inhibitors of nitric oxide (NO) synthase (NOS) isoforms. Each of the agents produced a time- and concentration-dependent inactivation of the NO-forming activity of the affinity-purified NOS isoforms. These inactivations required exposure of NOS to the drug under conditions that supported catalysis, consistent with the proposal that they act as alternate substrate, mechanism-based inactivators. Of the aminoguanidines examined, 2-ethylaminoguanidine was the most efficient inactivator, exhibiting vs. iNOS an apparent KI value of 120 microM as measured at 100 microM arginine and a k(inact max) value of 0.48 min(-1) and thus an apparent second-order rate constant for inactivation of 4.0 mM(-1)min(-1). 2-Ethylaminoguanidine displayed a high isoform selectivity for the iNOS compared with the nNOS and eNOS isoforms. 2-Ethylaminoguanidine inactivated NO synthetic activity in cytokine-induced RAW 264.7 cells as measured at 100 microM extracellular arginine with an apparent KI value of 55 microM and a k(inact max) value of 0.09 min(-1). The inactivated RAW 264.7 cell NO synthetic capability was restored over a 3-hr period after drug removal to a value 60% of its pretreatment value. This recovery occurred despite the presence of cycloheximide sufficient to inhibit protein synthesis by >99%. 1-Amino-S-methylisothiourea by contrast with the aminoguanidines was identified as a mechanism-based inactivator selective for the nNOS isoform. In contrast to S-isopropylisothiourea, which was found to be both cell penetrant and reversible, 1-amino-S-methylisothiourea appeared cell impermeable and inhibited NOS enzyme "irreversibly."

Topics: Animals; Cattle; Cytokines; Guanidines; Kinetics; Mice; NADPH Oxidases; Nitric Oxide; Nitric Oxide Synthase; Structure-Activity Relationship; Thiourea

The contractile response of isolated guinea pig trachea to histamine was potentiated in the presence of the histamine N-methyltransferase (HMT) inhibitor SKF 91488, whereas the diamine oxidase inhibitor aminoguanidine was without effect. SKF 91488 shifted in a concentration-dependent fashion the concentration-response curves to histamine to lower concentrations with the maximum by 1 log unit. The trachea contained significant HMT activity (45.4 +/- 5.0 pmol/min/mg protein). In situ hybridization to detect HMT mRNA indicated that HMT mRNA was present in the epithelium and endothelium, being more abundant in the former. Removal of the epithelium shifted the concentration-response curves to histamine to lower concentrations by 0.8 log unit, and SKF 91488 caused only a slight shift of histamine concentration-response curves in tissues denuded of epithelium. These findings suggest that HMT regulates the contractile response of guinea pig trachea to histamine, and epithelial removal-induced bronchial hyperresponsiveness to histamine is largely explained by the loss of HMT in the epithelium.

Topics: Animals; Dimaprit; Dose-Response Relationship, Drug; Guanidines; Guinea Pigs; Histamine; Histamine N-Methyltransferase; Male; Muscle Contraction; Muscle, Smooth; Thiourea; Trachea

1,4-Dithiothreitol (DTT; 1 mM, 30 min preincubation) produced a small, non-specific potentiation of spasmogenic activity in longitudinal muscle strips of guinea-pig small intestine. A direct comparison of contractile responses elicited by histamine and a range of H1- and non-H1-receptor agonists indicated that DTT produced a significantly greater potentiation of H1-receptor responses. This apparently selective increase in tissue sensitivity to histamine H1-receptor agonists did not appear to be a consequence of the inhibition of histamine N-methyl transferase or diamine oxidase activity. Potentiation of the responses to histamine by DTT was still observed in the presence of SKF 91488 (10 microM) and aminoguanidine (1 microM). The potentiation elicited by DTT was readily reversed by the sulphydryl oxidizing agent dithiobis-(2-nitrobenzoic acid) (DTNB). This suggests that the mechanism of action of DTT involves the reduction of disulphide bonds. Exposure of ileal smooth muscle to DTT following desensitization with histamine (100 X EC50 [- DTT]) resulted in a 6.9 +/- 0.7 fold shift of the concentration-response curve to lower agonist concentrations. Conversely, following potentiation of the response to histamine with DTT, exposure of the tissue to desensitizing concentrations of histamine resulted in a dextral shift of the dose-response curve (dose ratio = 39.5 +/- 1.2) to higher agonist concentrations. The results of this study suggest that DTT may be a useful tool with which to investigate histamine H1-receptor mechanisms in ileal smooth muscle.

Topics: Acetylcholine; Animals; Dimaprit; Dithionitrobenzoic Acid; Dithiothreitol; Dose-Response Relationship, Drug; Female; Guanidines; Guinea Pigs; Histamine; Ileum; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth; Pyridines; Pyrilamine; Receptors, Histamine; Receptors, Histamine H1; Thiourea

Spermidine (SPD) cytotoxicity is mediated largely through SPD oxidation by serum amine oxidases at both 37 degrees C and 43 degrees C. Alkaline sucrose gradient data suggest random induction of DNA strand breaks. A dose response with time at 37 degrees C or 43 degrees C (constant SPD) or with SPD concentration (constant time at 43 degrees C) was observed in terms of both DNA strand breaks and cell survival. The generation of peroxide was demonstrated in the absence of cells by the addition of SPD to medium containing 10% fetal bovine serum. Toxicity of SPD was reduced in saline, with catalase-thiourea and aminoguanidine, and enhanced by prior depletion of glutathione. Thermotolerance induced 16 h previously did not protect against SPD toxicity, suggesting that the reactive species from spermidine oxidation and heat damage do not produce related subcellular lesions.

Topics: Animals; Buthionine Sulfoximine; Cell Line; Cell Survival; Cricetinae; Cricetulus; DNA; Dose-Response Relationship, Drug; Female; Glutathione; Guanidines; Hot Temperature; Methionine Sulfoximine; Ovary; Oxidation-Reduction; Spermidine; Thiourea

Intravenous administration of 10-40 micrograms/Kg epinephrine to mice leads to a transient 50% increase in skin histamine, followed by an increase in blood histamine. Delayed inhibition by alpha-fluorometyl histidine (alpha FMH), suggests that these changes follow stimulation of pre-formed tissue histidine decarboxylase.

Topics: Animals; Dimaprit; Epinephrine; Guanidines; Histamine; Histamine Release; Kinetics; Male; Methylhistidines; Mice; Skin; Skin Physiological Phenomena; Thiourea

The Dimaprit analog, S-[4-(N,N-dimethylamino)-butyl]isothiurea (SKF Compound 91488), which is a noncompetitive inhibitor (Ki, 0.9 microM) of histamine-N-methyltransferase in vitro, inhibited the methylation of labeled histamine in mice. When administered in combination with the diamine oxidase inhibitor, aminoguanidine, an almost complete block of histamine metabolism was observed. The inhibition was dependent on dose of drug and was apparent with doses of 50 mg/kg and greater. Since Compound 91488 is reported to have no agonist activity of its own, the drug may help to identify the actions of histamine released from endogenous stores in response to physiological and pathological stimuli.

Topics: Amine Oxidase (Copper-Containing); Animals; Brain; Dimaprit; Guanidines; Histamine; Histamine N-Methyltransferase; Kidney; Male; Methylation; Methyltransferases; Mice; Rats; Thiourea