Form Of Nitric Oxide Synthase Research Articles

Kininase 1 As a Preclinical Therapeutic Target for Kinin B1 Receptor in Insulin Resistance.

Kinin B1 receptor (B1R) contributes to insulin resistance, an early event in type 2 diabetes, through the upregulation and activation of the inducible form of nitric oxide synthase (iNOS), pro-inflammatory cytokines and the oxidative stress. This study addresses the hypothesis that inhibition of kininase 1 (carboxypeptidase M, CPM), the key enzyme involved in the biosynthesis of B1R agonists, could exert the same beneficial effects to B1R antagonism in insulin resistance. Male Sprague-Dawley rats were made insulin resistant with a drinking solution containing 10% D-glucose for a period of 9 weeks. Control rats received tap water. During the last week, kininase 1 was blocked with Mergetpa (1 mg kg−1 twice daily, s.c.) and the impact was determined on insulin resistance (HOMA index), metabolic hormone levels, oxidative stress and the expression of several markers of inflammation by western blot and qRT-PCR. Glucose-fed rats displayed hyperglycemia, hyperinsulinemia, hyperleptinemia, insulin resistance, hypertension, positive body weight gain, and enhanced expression of B1R, CPM, iNOS, and IL-1β in renal cortex, aorta and liver. Markers of oxidative stress (superoxide anion and nitrotyrosine expression) were also enhanced in aorta and renal cortex. Mergetpa reversed and normalized most of those alterations, but failed to affect leptin levels and hypertension. Pharmacological blockade of kininase 1 (CPM) exerted similar beneficial effects to a 1-week treatment with a B1R antagonist (SSR240612) or an iNOS inhibitor (1,400 W). These data reinforce the detrimental role of B1R in insulin resistance and recommend CPM as a new therapeutic target.

A Novel Capacitive Biosensor for the Detection of Small Molecule S-Nitrosothiols

Small molecule S-nitrosothiols are a class of endogenous chemicals which are produced by various forms of nitric oxide synthase. The regulation of these molecules has been shown to play a role in control of a variety of bodily processes and disease models including breathing, blood pressure, pulmonary hypertension, and asthma. However, these molecules are extremely labile, making in vivo detection extremely challenging as most small molecule S-nitrosothiols exists at very low concentrations in the body. To overcome this challenge, we have developed a capacitive biosensor which employs an organic semiconductor that readily covalently crosslinks to all free amines, free thiols, and S-nitrosylated thiols in solution. Samples were treated with formaldehyde to block all free amines and free thiols, leaving only S-nitrosylated thiols. S- nitrosothiol bonding to the semiconducting surface of the sensing electrode changes its capacitance, allowing for extremely sensitive detection of S-nitrosothiols in biological samples. We will present evidence of attomolar detection of S-nitrosocysteine which can be abolished by the addition of Mercury to the fixing buffer, or by exposing the sample to UV light during fixing, both methods of degrading S-nitrosothiols. We will also present evidence of the presence of small molecule S-nitrosothiols in blood, saliva, and other biological samples.

Role of α-1 Anti Trypsin, A Constitutive Form of Nitric Oxide Synthase in Cyclic AMP Mediated Inhibition of Platelet Aggregation

Role of α-1 Anti Trypsin, A Constitutive Form of Nitric Oxide Synthase in Cyclic AMP Mediated Inhibition of Platelet Aggregation

NITRIC OXIDE SYNTHASE ACTIVITY AND ITS CONCENTRATION IN THE TISSUES OF HUMAN THYROID CARCINOMAS.

The study of the activity of the constitutive form of nitric oxide synthase (cNOS) revealed that in the papillary thyroid carcinomas it corresponded to that detected in unchanged extratumoral tissue, while the enzyme activity in follicular carcinoma was half lesser. At the same time, the activity of inducible nitric oxide synthase (іNOS) was higher in the papillary and follicular carcinomas. Such changes in the enzyme activity were associated with an increase in its level in papillary carcinomas, and with minor changes in follicular carcinomas. In medullary carcinomas the parameters under study corresponded to those in unchanged tissue, and in the papillary carcinoma metastases without changes in enzyme activity of nitric oxide formation, the level of the latter was much higher. Elevated levels of nitric oxide and іNOS activity in papillary thyroid carcinomas did not depend significantly on the aggression characteristics of the latter, being however absent in tumors of T4 category on a background of reduced cNOS activity and less expressed in tumors surrounded by the tissue in the presence of a chronic thyroiditis. Furthermore, in the papillary carcinomas of papillary or follicular structure nitric oxide level did not differ from the normal range, being slightly higher in tumors of solid or heterogeneous structure with presence of solid areas, whereas in carcinomas of papillary-follicular structure it was twice, and in tissue of solidinsular structure three times higher. іNOS hyperactivity was observed in the carcinomas of different structure, except for tumors of solid structure, in the tumor of which enzyme activity was within the normal range, and in tumor of solid-insular structure where it was significantly higher (as well as cNOS activity) compared with tumors of other structure. Nitric oxide generating system is involved in the transformation of thyroid cells and progression of tumor growth, including through apoptosis regulation, as shown by the results of an analysis of data obtained both in the present study and previously. The nature of such involvement in papillary thyroid carcinomas with different histological structure is different. Key words: nitric oxide; constitutive and inducible nitric oxide synthase; thyroid carcinoma; apoptosis.

Participation of oleic acid in the formation of the aortic aneurysm in Marfan syndrome patients

Marfan syndrome (MFS) is associated with progressive aortic dilatation and endothelial dysfunction that lead to early acute dissection and rupture of the aorta and sudden death. Alteration in fatty acid (FA) metabolism can stimulate nitric oxide (NO) overproduction which increases the activity of the inducible form of NO synthase (iNOS) that is involved in endothelial dysfunction. We evaluated the participation of FA in the formation of thoracic aneurysms in MFS and its relation to the iNOS. Oleic acid (OA), iNOS, citrulline, nitrates and nitrites, TGF-β1, TNF-α, monounsaturated FA and NO synthase activity were significantly increased (p<0.05) in tissue from the aortas of MFS. Saturated FA, eNOS and HDL were significantly decreased (p<0.05). Arachidonic acid, delta-9 desaturase tended to increase and histological examination showed an increase in cystic necrosis, elastic fibers and collagen in MFS. The increase in OA contributes to the altered pathway of iNOS, which favors endothelial dysfunction and formation of the aortic aneurysms in MFS.

The Emerging Role of Arginase in Endothelial Dysfunction in Diabetes.

Diabetes mellitus is a major risk factor for the development of cardiovascular disease due to increased vascular inflammatory and oxidative stress favouring atherogenesis. Endothelial dysfunction has received increasing attention as a potential contributor to the pathogenesis of vascular disease in diabetes mellitus. Although the underlying cause of endothelial dysfunction is multifactorial, a key factor is impairment of the bioavailability of nitric oxide (NO). Emerging evidence suggest that upregulation of arginase is of central importance for reduced NO bioavailability due to competition for the substrate L-arginine between arginase and the endothelial form of NO synthase. Arginase is also associated with increased oxidative stress, further impairing NO bioavailability. Upregulation of arginase has been suggested to be a key factor driving endothelial dysfunction in diabetes. The present review describes the regulation of arginase in relation to diabetes and arginase as a potential therapeutic target to improve endothelial function in experimental models and the clinical setting of diabetes mellitus.

Alcoholic Liver Disease: A Mouse Model Reveals Protection by Lactobacillus fermentum

Objectives:Alcoholism is one of the most devastating diseases with high incidence, but knowledge of its pathology and treatment is still plagued with gaps mostly because of the inherent limitations of research with patients. We developed an animal model for studying liver histopathology, Hsp (heat-shock protein)-chaperones involvement, and response to treatment.Methods:The system was standardized using mice to which ethanol was orally administered alone or in combination with Lactobacillus fermentum following a precise schedule over time and applying, at predetermined intervals, a battery of techniques (histology, immunohistochemistry, western blotting, real-time PCR, immunoprecipitation, 3-nitrotyrosine labeling) to assess liver pathology (e.g., steatosis, fibrosis), and Hsp60 and iNOS (inducible form of nitric oxide synthase) gene expression and protein levels, and post-translational modifications.Results:Typical ethanol-induced liver pathology occurred and the effect of the probiotic could be reliably monitored. Steatosis score, iNOS levels, and nitrated proteins (e.g., Hsp60) decreased after probiotic intake.Conclusions:We describe a mouse model useful for studying liver disease induced by chronic ethanol intake and for testing pertinent therapeutic agents, e.g., probiotics. We tested L. fermentum, which reduced considerably ethanol-induced tissue damage and deleterious post-translational modifications of the chaperone Hsp60. The model is available to test other agents and probiotics with therapeutic potential in alcoholic liver disease.

Comparative Study of Catecholaminergic and Nitroxidergic Neurons in the Vasomotor Nuclei of the Caudal Part of the Brainstem in Rats

Immunohistochemical studies in 12 Wistar rats detected tyrosine hydroxylase (TH) and the neuronal form of nitric oxide synthase (nNOS) to address the distribution of catecholaminergic and nitroxidergic neurons, respectively, in the vasomotor nuclei of the medulla oblongata and pons. The highest frequency of TH expression was seen in neurons located in these nuclei and some reticular nuclei (the gigantocellular, paragigantocellular, and caudal nuclei of the pons), though the proportions of immunoreactive neurons in these structures were no greater than 8–14%. In other nuclei (the reticular parvocellular and oral nucleus of the pons, the spinal nucleus of the trigeminal nerve), this proportion ranged from 1 to 3%. Neurons of this type were consistently not detected in a large group of nuclei with confirmed vasomotor functions. Structures with many catecholaminergic neurons generally contained fewer nNOS-positive cells than nuclei with limited numbers of TH-positive neurons.

Farnesol quells oxidative stress, reactive gliosis and inflammation during acrylamide-induced neurotoxicity: Behavioral and biochemical evidence

Acrylamide (ACR) is an industrial pollutant, to which humans are exposed through chemicals associated with day to day human life and contributes to neurological disorders. The role of reactive gliosis upon toxic insults remains paradoxical, and the immunomodulatory events during ACR intoxication remain obscure. In view of this, the present study investigated ACR-induced (20mg/kgb.wt for 4weeks) neurodegeneration in the context of oxidative stress and associated inflammatory events and the ability of farnesol, a sesquiterpene, to mitigate reactive gliosis in the brain of Swiss albino mice. Farnesol supplementation (100mg/kgb.wt.) showed a marked improvement in gait performance, neuromuscular function and fine motor coordination and attenuated ACR-induced diminution in glutathione (GSH) with parallel reduction in lipid peroxidation (LPO), protein carbonyls, hydroxide, hydroperoxide and nitrite levels. Farnesol treatment significantly ameliorated ACR-mediated histological aberrations and reactive gliosis by downregulating Glial fibrillary acidic protein (GFAP) and Ionizsed calcium-binding adapter molecule-1 ​(Iba-1) in the cortex, hippocampus and striatum. Further, ACR stimulated increase in levels of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β) and inducible form of nitric oxide synthase (iNOS) were considerably decreased by farnesol. In conclusion, our findings indicate that farnesol exerts neuroprotective efficacy during ACR-induced neuropathology by suppressing reactive gliosis and associated inflammatory events.

The Influence of Low Doses of Zearalenone on Distribution of Selected Active Substances in Nerve Fibers Within the Circular Muscle Layer of Porcine Ileum.

The aim of this study was to investigate, whether low doses (25 % of no observable adverse effect levels values) of zearalenone (ZEN) can affect the expression of active substances in nerve fibers in the muscular layer of porcine ileum. The study was performed on ten immature pigs divided into two groups: experimental group (n = 5), where zearalenone (10 μg/kg body weight) was given for 42 days, and control animals (n = 5), where placebo was administered. Fragments of ileum of all animals were processed for single-labelling immunofluorescence technique using the antibodies against vasoactive intestinal peptide, neuronal form of nitric oxide synthase, cocaine and amphetamine regulatory peptide, galanin, pituitary adenylate cyclase-activating peptide-27 and substance P. The number of nerve fibers immunoreactive to particular substances was evaluated by the counting of nerves per observation field (0.1 mm2). Low doses of zearalenone caused the clear changes in the expression of substances studied. The number of nerve fibers immunoreactive to the majority of substances increased in experimental animals. The exception was only galanin, the expression of which was less after administration of zearalenone. The obtained results for the first time show that even low doses of zearalenone can affect the nerve fibers in the digestive tract.

Benfotiamine attenuates inflammatory response in LPS stimulated BV-2 microglia.

Microglial cells are resident immune cells of the central nervous system (CNS), recognized as key elements in the regulation of neural homeostasis and the response to injury and repair. As excessive activation of microglia may lead to neurodegeneration, therapeutic strategies targeting its inhibition were shown to improve treatment of most neurodegenerative diseases. Benfotiamine is a synthetic vitamin B1 (thiamine) derivate exerting potentially anti-inflammatory effects. Despite the encouraging results regarding benfotiamine potential to alleviate diabetic microangiopathy, neuropathy and other oxidative stress-induced pathological conditions, its activities and cellular mechanisms during microglial activation have yet to be elucidated. In the present study, the anti-inflammatory effects of benfotiamine were investigated in lipopolysaccharide (LPS)-stimulated murine BV-2 microglia. We determined that benfotiamine remodels activated microglia to acquire the shape that is characteristic of non-stimulated BV-2 cells. In addition, benfotiamine significantly decreased production of pro-inflammatory mediators such as inducible form of nitric oxide synthase (iNOS) and NO; cyclooxygenase-2 (COX-2), heat-shock protein 70 (Hsp70), tumor necrosis factor alpha α (TNF-α), interleukin-6 (IL-6), whereas it increased anti-inflammatory interleukin-10 (IL-10) production in LPS stimulated BV-2 microglia. Moreover, benfotiamine suppressed the phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and protein kinase B Akt/PKB. Treatment with specific inhibitors revealed that benfotiamine-mediated suppression of NO production was via JNK1/2 and Akt pathway, while the cytokine suppression includes ERK1/2, JNK1/2 and Akt pathways. Finally, the potentially protective effect is mediated by the suppression of translocation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the nucleus. Therefore, benfotiamine may have therapeutic potential for neurodegenerative diseases by inhibiting inflammatory mediators and enhancing anti-inflammatory factor production in activated microglia.

The effect of a selective neuronal nitric oxide synthase inhibitor 3-bromo 7-nitroindazole on spatial learning and memory in rats

Since the discovery of nitric oxide (NO) as a neuronal messenger, its way to modulate learning and memory functions is subject of intense research. NO is an intercellular messenger in the central nervous system and is formed on demand through the conversion of L-arginine to L-citrulline via the enzyme nitric oxide synthase (NOS). Neuronal form of nitric oxide synthase may play an important role in a wide range of physiological and pathological conditions. Therefore the aim of this study was to investigate the effects of chronic 3-bromo 7-nitroindazole (3-Br 7-NI), specific neuronal nitric oxide synthase (nNOS) inhibitor, administration on spatial learning and memory performance in rats using the Morris water maze (MWM) paradigm. Male rats received either 3-Br 7-NI (20mg/kg/day) or saline via intraperitoneal injection for 5days. Daily administration of the specific neuronal nitric oxide synthase (nNOS) inhibitor, 3-Br 7-NI impaired the acquisition of the MWM task. 3-Br 7-NI also impaired the probe trial. The MWM training was associated with a significant increase in the brain-derived neurotrophic factor (BDNF) mRNA expression in the hippocampus. BDNF mRNA expression in the hippocampus did not change after 3-Br 7-NI treatment. L-arginine significantly reversed behavioural parameters, and the effect of 3-Br 7-NI was found to be NO-dependent. There were no differences in locomotor activity and blood pressure in 3-Br 7-NI treated rats. Our results may suggest that nNOS plays a key role in spatial memory formation in rats.

Exercise and NO production: relevance and implications in the cardiopulmonary system.

This article reviews the existing knowledge about the effects of physical exercise on nitric oxide (NO) production in the cardiopulmonary system. The authors review the sources of NO in the cardiopulmonary system; involvement of three forms of NO synthases (eNOS, nNOS, and iNOS) in exercise physiology; exercise-induced modulation of NO and/or NOS in physiological and pathophysiological conditions in human subjects and animal models in the absence and presence of pharmacological modulators; and significance of exercise-induced NO production in health and disease. The authors suggest that physical activity significantly improves functioning of the cardiovascular system through an increase in NO bioavailability, potentiation of antioxidant defense, and decrease in the expression of reactive oxygen species-forming enzymes. Regular physical exercises are considered a useful approach to treat cardiovascular diseases. Future studies should focus on detailed identification of (i) the exercise-mediated mechanisms of NO exchange; (ii) optimal exercise approaches to improve cardiovascular function in health and disease; and (iii) physical effort thresholds.

Low-dose ribavirin treatments attenuate neuroinflammatory activation of BV-2 Cells by interfering with inducible nitric oxide synthase.

Microglia play a key role in defending central nervous system from various internal and external threats. However, their excessive and/or chronic activation is associated with deleterious effects in a variety of neurodegenerative diseases. Previously, we have shown that ribavirin when applied in clinically relevant dosage (10 μM) modulates activated microglia in complex fashion inducing both anti- and proinflammatory effects, simultaneously causing cytotoxicity. Here, we examined potential of low-dose ribavirin (0.1 and 1 μM) to modulate activated BV-2 microglia. Morphological and functional activation of BV-2 cells was achieved with lipopolysaccharide (LPS) stimulation. Our results demonstrated that low-dose ribavirin did not induce cell death, while 10 μM ribavirin promoted LPS induced apoptosis. We determined that 1 μM ribavirin was equally efficient in deactivation of LPS induced morphological changes as 10 μM ribavirin treatment. Ribavirin showed halfway success in reducing markers of functional activation of microglia. Namely, none of the doses had effect on LPS triggered production of proinflammatory cytokine tumor necrosis factor alpha. On the other hand, low-dose ribavirin proved its effectiveness in reduction of another inflammatory mediator, nitric oxide, by inhibiting inducible form of nitric oxide synthase. Our results imply that low-dose ribavirin may alleviate nitrosative stress during neuroinflammation.

Nitric oxide in liver fibrosis: The role of inducible nitric oxide synthase.

The inducible form of nitric oxide synthase (iNOS) is expressed in hepatic cells in pathological conditions. Its induction is involved in the development of liver fibrosis, and thus iNOS could be a therapeutic target for liver fibrosis. This review summarizes the role of iNOS in liver fibrosis, focusing on 1) iNOS biology, 2) iNOS-expressing liver cells, 3) iNOS-related therapeutic strategies, and 4) future directions.

Distribution of NADPH-Diaphorase and Neuronal NO Synthase in the Nuclei of the Medulla Oblongata in Rats

The distribution of nitroxidergic neurons in the medulla oblongata nuclei in Wistar rats (n = 8) was studied using histochemical (NADPH-diaphorase) and immunohistochemical methods with antisera to the neuronal form of nitric oxide synthase (nNOS). NADPH-diaphorase was found in large and small neurons in the sensory, autonomic, and motor nuclei. The latter were found to contain particularly large numbers of neurons expression this enzyme activity. In contrast to NADPH-diaphorase, nNOS in the corresponding nuclei was always present in smaller quantities of mainly smaller neurons. The sensory nuclei (solitary tract nucleus, reticular parvocellular and lateral nuclei, spinal nucleus of the trigeminal nerve) showed 1.5‐3 times more nNOS neurons than the motor nuclei. In some nuclei (the nucleus ambiguus, the nucleus of the hypoglossal nerve) containing large numbers of NADPH-diaphorase-positive neurons, immunoreactive cells were particularly rare.

Microglia P2Y₆ receptors mediate nitric oxide release and astrocyte apoptosis.

BackgroundDuring cerebral inflammation uracil nucleotides leak to the extracellular medium and activate glial pyrimidine receptors contributing to the development of a reactive phenotype. Chronically activated microglia acquire an anti-inflammatory phenotype that favors neuronal differentiation, but the impact of these microglia on astrogliosis is unknown. We investigated the contribution of pyrimidine receptors to microglia-astrocyte signaling in a chronic model of inflammation and its impact on astrogliosis.MethodsCo-cultures of astrocytes and microglia were chronically treated with lipopolysaccharide (LPS) and incubated with uracil nucleotides for 48 h. The effect of nucleotides was evaluated in methyl-[3H]-thymidine incorporation. Western blot and immunofluorescence was performed to detect the expression of P2Y6 receptors and the inducible form of nitric oxide synthase (iNOS). Nitric oxide (NO) release was quantified through Griess reaction. Cell death was also investigated by the LDH assay and by the TUNEL assay or Hoechst 33258 staining.ResultsUTP, UDP (0.001 to 1 mM) or PSB 0474 (0.01 to 10 μM) inhibited cell proliferation up to 43 ± 2% (n = 10, P <0.05), an effect prevented by the selective P2Y6 receptor antagonist MRS 2578 (1 μM). UTP was rapidly metabolized into UDP, which had a longer half-life. The inhibitory effect of UDP (1 mM) was abolished by phospholipase C (PLC), protein kinase C (PKC) and nitric oxide synthase (NOS) inhibitors. Both UDP (1 mM) and PSB 0474 (10 μM) increased NO release up to 199 ± 20% (n = 4, P <0.05), an effect dependent on P2Y6 receptors-PLC-PKC pathway activation, indicating that this pathway mediates NO release. Western blot and immunocytochemistry analysis indicated that P2Y6 receptors were expressed in the cultures being mainly localized in microglia. Moreover, the expression of iNOS was mainly observed in microglia and was upregulated by UDP (1 mM) or PSB 0474 (10 μM). UDP-mediated NO release induced apoptosis in astrocytes, but not in microglia.ConclusionsIn LPS treated co-cultures of astrocytes and microglia, UTP is rapidly converted into UDP, which activates P2Y6 receptors inducing the release of NO by microglia that causes astrocyte apoptosis, thus controlling their rate of proliferation and preventing an excessive astrogliosis.

Relationship between macrophages in mouse uteri and angiogenesis in endometrium during the peri-implantation period

The objective of this study is to examine the change in macrophage numbers, inducible form of NO synthase (iNOS), and vascular endothelial growth factor (VEGF) expression both before and after embryo implantation in the uterine tissue of mice. In order to explore the mechanism of macrophages in endometrial angiogenesis, 8-week-old female mice were divided into three groups: pregnant group, pseudopregnant group (mated to male mice that had been vasectomized), and estrous group (unmated). Individuals from these three groups were sacrificed at time intervals D1.5 to D6.5. Formalin-fixed paraffin-embedded tissue was used for immunocytochemical localization of Mφ, iNOS, and VEGF utilizing standard methodology. The proportion of macrophages in the peripheral blood was determined by flow cytometry, and the relationship between macrophage, iNOS, and VEGF expression was analyzed. The proportion of peripheral blood macrophages in the pregnancy group was significantly higher than that in the other groups. The results of immunohistochemistry determined that the macrophages exhibited changes in both numbers and distribution. The number of macrophages in the endometrium of the pregnancy and pseudopregnancy groups was significantly higher than that in the control (estrous) group. In the pregnancy group, macrophage numbers dramatically decreased and gradually transferred to the perimetrium on D4.5. Immunostaining revealed strong staining in the pregnancy group and weaker staining in the pseudopregnant and control groups for both iNOS and VEGF. There was strong, dense immunostaining at the implantation site for both iNOS and VEGF, whereas light immunostaining was seen in interimplantation tissues on D5.5 to D6.5. In the pregnant group, peripheral blood and uterine macrophage proportions were negatively correlated, whereas the amount of macrophages, iNOS, and VEGF expression in the endometrium were positively correlated. The expression of iNOS and VEGF in the endometrium also displayed a strong positive correlation. In conclusion, during embryo implantation, macrophages levels decreased in the uterus, whereas the number of peripheral macrophages increased, suggesting that macrophages may migrate into the peripheral blood and uterus to adapt for pregnancy. Additionally, an increase in the expression of iNOS and VEGF was observed during the implantation window, implying that iNOS and VEGF may play an important role in promoting embryo implantation. The positive correlation between macrophages, iNOS, and VEGF in the implanting uterus implied that macrophages might regulate iNOS and VEGF during the implantation process.

Characterization of anti-inflammatory responses of norepinephrine in hepatitis induced by LPS: Effects on expression of IL-6, TNF-α and iNOS in liver of mice

Under septic conditions, Kupffer cells produce pro-inflammatory mediators which contribute to hepatic dysfunction, and whose levels can be modulated by endogenous factors including neurotransmitters such as norepinephrine (NE). We investigated the ability of NE to suppress Kupffer cell activation, in particular its effects on induction and activity of the inducible form of nitric oxide synthase (NOSII), interleukin 6 (IL-6) and tumor necrosis factor-alpha (TNF-α). In the present study used twenty one male NMRI mice (25 ± 5 g) that they divided into tree groups (n = 7). Negative control (normal saline), positive control (LPS), NE (∼100 nM) is injected through the mouse tail vein 30 minutes before inducts inflammation by LPS (5 mg/kg for intraperitoneal). Changes in the levels of expression of TNF-α, IL-6 and iNOS genes in the liver induced by LPS injection for two hours studied by a semi quantitative RT-PCR method. The results showed that administration of LPS increased hepatic TNF-α, IL-6 and iNOS mRNAs. NE (∼100 nM) reduces TNF-α, IL-6 and iNOS mRNA levels two hours after the injection. The most important have remarked effect norepinephrine in reducing iNOS; on the other hand, NE has a better influence on iNOS in liver tissue. These results indicate that high concentration of NE effectively suppress LPS-induced TNF-α, IL-6 and iNOS expression from liver tissue, suggesting that the down regulatory effect of NE on pro-inflammatory cytokine may represent a mechanism responsible for their beneficial effects in preventing inflammatory responses and tissue damage in sepsis.

Cardiopathogenic mediators generated by GATA4 signaling upon co-activation with endothelin-1 and Trypanosoma cruzi infection

Trypanosoma cruzi (Tc), the etiological agent of Chagas disease, triggers multiple responses in the myocardium, a central organ of infection and pathology in the host. Parasite-driven induction of diverse regulators of cardiovascular function, including the vasoconstrictor endothelin-1 (ET-1), the inducible form of nitric oxide synthase (iNOS) and the B-type natriuretic peptide (BNP), has been linked to the development of severe chagasic cardiomyopathy. Our current goal was to analyze the participation of the zinc finger transcription factor GATA4, critically implicated in pathological cardiac hypertrophic response, in the generation of key mediators involved in the pathogenesis of Tc-elicited heart dysfunction. In this study, we found that the combined effects of Tc and ET-1 on atrial myocytes promoted the protein expression, phosphorylation and DNA-binding activity of GATA4, leading to augmented protein levels of iNOS and increased nitric oxide release. Moreover, Tc- and ET-1-co-activation of cardiomyocytes resulted in enhanced GATA4-dependent secretion of BNP. Accordingly, mice with chronic chagasic cardiomyopathy showed increased expression of GATA4, iNOS and BNP at inflammatory lesions in cardiac muscle. Our findings support a role for the GATA4 signaling pathway in the myocardial production of pathogenic mediators associated with Chagas heart disease, and may help define novel therapeutic targets.