Inhibition Of NO Generation Research Articles

Glutamine metabolism to glucosamine is necessary for glutamine inhibition of endothelial nitric oxide synthesis.

L-Glutamine is a physiological inhibitor of endothelial NO synthesis. The present study was conducted to test the hypothesis that metabolism of glutamine to glucosamine is necessary for glutamine inhibition of endothelial NO generation. Bovine venular endothelial cells were cultured for 24 h in the presence of 0, 0.1, 0.5 or 2 mM D-glucosamine, or of 0.2 or 2 mM L-glutamine with or without 20 microM 6-diazo-5-oxo-L-norleucine (DON) or with 100 microM azaserine. Both DON and azaserine are inhibitors of L-glutamine:D-fructose-6-phosphate transaminase (isomerizing) (EC 2.6.1.16), the first and rate controlling enzyme in glucosamine synthesis. Glucosamine at 0.1, 0.5 and 2 mM decreased NO production by 34, 45 and 56% respectively compared with controls where glucosamine was lacking. DON (20 microM) and azaserine (100 microM) blocked glucosamine synthesis and prevented the inhibition of NO generation by glutamine. Neither glutamine nor glucosamine had an effect on NO synthase (NOS) activity, arginine transport or cellular tetrahydrobiopterin and Ca(2+) levels. However, both glutamine and glucosamine inhibited pentose cycle activity and decreased cellular NADPH concentrations; these effects of glutamine were abolished by DON or azaserine. Restoration of cellular NADPH levels by the addition of 1 mM citrate also prevented the inhibiting effect of glutamine or glucosamine on NO synthesis. A further increase in cellular NADPH levels by the addition of 5 mM citrate resulted in greater production of NO. Collectively, our results demonstrate that the metabolism of glutamine to glucosamine is necessary for the inhibition of endothelial NO generation by glutamine. Glucosamine reduces the cellular availability of NADPH (an essential cofactor for NOS) by inhibiting pentose cycle activity, and this may be a metabolic basis for the inhibition of endothelial NO synthesis by glucosamine.

O-87 - ET-1 augments the inhibition of NO generation with L-NMMA in rabbit arterial strips by increasing L-NMMA uptake

O-87 - ET-1 augments the inhibition of NO generation with L-NMMA in rabbit arterial strips by increasing L-NMMA uptake

Nitrotyrosine formation and its role in various pathological conditions

The formation of peroxynitrite and nitrotyrosine was examined in a variety of in vitro and in vivo animal models and its relation to cell or tissue damage was examined. In polymorphonuclear leukocyte (PMN)-induced injury to cardiac myocytes or endothelial cells, activated PMN produced peroxynitrite. Peroxynitrite appears to be responsible for the injury but it was not a major mediator of endothelial cell injury. In the experiment of ischemia-reperfusion injury of the rat brain nitrotyrosine was formed in the peri-infarct and core-of infarct regions. The degradation curve of nitrotyrosine revealed that its t1/2 was about 2.2 hours. In the radiation-induced lung injury of rats, nitrotyrosine was also formed but it was not the sole mechanism for the injury. Levels of nitrotyrosine correlated with the severity of myocardial dysfunction in the canine model of cytokine-induced cardiac injury. Inhibition of NO generation abolished the formation of peroxynitrite and nitrotyrosine in all experiments. In conclusion; although nitrotyrosine is formed in a variety of pathological conditions where the generation of NO is increased, its presence does not always correlate with the severity of injury.

Role of L-arginine--endogenous NOS inhibitors--endothelin-1 pathway for the vascular remodelling

Mechanisms of vascular remodelling process are complex and poorly understood. We describe herein the role of L-arginine-endogenous NOS inhibitors-endothelin-1 pathway for the vascular remodelling after endothelial denudation of the rabbit carotid artery. It is reportedly known that NO is a vasodilating substance, an inhibitor of platelet aggregation and adhesion, and an inhibitor of vascular smooth muscle cell proliferation and that endothelin-1, of which production is inhibited by NO, is a potent vasoconstrictor and a potent mitogen. An accumulation of endogenous inhibitors (L-NMMA and ADMA) in regenerated endothelial cells after the endothelial denudation was accompanied by the decreased NO generation, the increased endothelin-1 content within the vessel wall and the occurrence of intimal hyperplasia. Endothelin-1 content within the vessel wall was significantly increased after the exogenous L-NMMA administration for 2 weeks, suggesting that accumulated L-NMMA results in the decreased NO generation and, in turn, increases endothelin-1 content. Endothelin-1 facilitated the [3H]-L-NMMA uptake by endothelial cell and brought about the potentiation of L-NMMA-mediated inhibition of NO generation. These results strongly suggest that the L-arginine--endogenous NOS inhibitors--endothelin-1 pathway plays an active role for vascular remodelling.

Prostaglandin D2 inhibits inducible nitric oxide synthase expression in rat vascular smooth muscle cells.

Vascular smooth muscle cells (VSMCs) as well as macrophages have been shown to generate a substantial amount of NO in inflammatory vascular lesions. Prostaglandin (PG) D2 (PGD2) is produced by inflammatory cells, including mast cells and macrophages. We investigated whether PGD2 modulates NO metabolism in rat VSMCs. PGD2 at a concentration of 10(-7) mol/L or greater dose-dependently inhibited nitrite accumulation in the medium of cultured VSMCs stimulated with interleukin 1beta (IL-1beta). In a dose-response analysis of IL-1beta and nitrite accumulation, PGD2 was seen to decrease the maximal ability of VSMCs to generate NO, arguing against competition by PGD2 at cytokine receptors. Northern analysis showed that PGD2 suppresses induction of inducible NO synthase (iNOS) mRNA in IL-1beta-stimulated VSMCs, with consequent inhibition of iNOS protein expression in Western analysis. A thromboxane A2 (TXA2) analogue, U46619 (10(-5) mol/L), produced less inhibition of NO generation than did PGD2. Neither the PGI2 analog carbaprostacyclin nor PGE1 showed any inhibition. PGD2 dose-dependently inhibited NO generation despite the addition of the TXA2 antagonist SQ29548. PGJ2, delta12-PGJ2, and 15-deoxy-delta12,14-PGJ2, all metabolites of PGD2, were as potent as or slightly stronger than PGD2 in the inhibition of NO generation. These data suggest that PGD2 suppresses NO generation in VSMCs by inhibiting iNOS mRNA expression, most likely through the cascade of the PGJ2 series rather than through the TX receptor or cAMP upregulation. Such action makes it likely that PGD2 regulates NO metabolism in vascular lesions.

Role of nitric oxide in tumour progression: lessons from human tumours.

Varied cellular expression and localisation of nitric oxide synthase (NOS) isoforms has been shown in human cancers, including tumours of the breast, ovary, stomach, cervix and central nervous system. Mapping of NOS expression within tumour tissue from breast and gastric cancers shows inducible NOS (iNOS) is expressed predominantly in stromal (macrophage and endothelial) cells, although the level of NOS activity is at least 1-2 orders of magnitude lower than the enzyme activity associated with cytotoxicity and apoptosis. There is evidence that the intratumoural environment may provide chemoattractant signals for monocyte-macrophage recruitment and their subsequent activation via expression of interleukin-4, IgE, and CD23. Such signals lead to induction of iNOS in human macrophages in vitro. The correlation between NOS activity and grade for breast cancer suggests that NO may provide a positive growth signal within the tumour microenvironment. In vivo studies showing increased growth rate, vascular density and invasiveness of a human tumour cell line transfected to constitutively express iNOS support this. Furthermore, in vivo administration of a highly selective inhibitor of iNOS limited invasion and growth rate of iNOS transfected tumours and other murine tumours expressing this isoform. Inhibition of NO generation in the intratumoural microenvironment may prove a useful cancer therapy by preventing angiogenesis, invasion and metastasis.

CD69-Induced Monocyte Apoptosis Involves Multiple Nonredundant Signaling Pathways

Simultaneous stimulation of human monocytes/macrophages or THP1 cells with LPS and an antibody specific for the activation marker CD69 induces apoptosis. Here we demonstrate the involvement of multiple independent signals that are necessary for apoptosis induction. Thus, inhibitors of phospholipase A2and lipoxygenase prevent apoptosis induction. Similarly, the ADP-ribosylating G-protein-reactive pertussis toxin (PTX) but not a mutant toxin lacking the ADP-ribosylating moiety (mPTX) prevents apoptosis induction. Furthermore, inhibition of NO generation abrogates completely the induction of apoptosis by LPS/CD69 ligation. These three pathways can be dissociated from each other in the sense that interventions on the arachidonic acid metabolism or G proteins do not inhibit the generation of NO and that exogenous NO cannot reverse the inhibition of cell death by inhibitors of phospholipase A2or PTX. In addition, both PTX and mPTX affect arachidonic acid mobilization only partially, indicating that the apoptosis-inhibitory effect of PTX (which is not shared by mPTX) cannot be explained by its effect on phospholipase A2activation. Both LPS and anti-CD69 are sufficient on their own to activate cells, as determined by TNF production, NO generation, or arachidonic acid metabolism, but neither LPS nor anti-CD69 can induce apoptosis on their own. Thus, apoptosis induction in this system involves at least three independent signal transduction systems—(i) arachidonic acid metabolism, (ii) NO, and (iii) PTX-sensitive events—each of which is necessary but insufficient to induce monocyte/macrophage apoptosis. These findings underline the complex control of activation-induced apoptosis in cells of the myelomonocytic lineage.

Intestinal reperfusion up-regulates inducible nitric oxide synthase activity within the lung

This study examines the hypothesis that pulmonary inducible nitric oxide synthase (iNOS) activity is up-regulated during intestinal reperfusion and that inhibition of NO generation exacerbates pulmonary microvascular dysfunction. Sprague-Dawley rats underwent intestinal ischemia and reperfusion (IIR) or sham operation (SHAM). Pulmonary iNOS activity was measured by quantitating the conversion of L-arginine (L-Arg) to L-citrulline. Another set of animals undergoing IIR or SHAM received an inhibitor of NOS (NG-nitro-L-arginine methylester; L-NAME; 20 mg/kg intravenously), substrate for NO generation (L-Arg; 300 mg/kg intravenously), or vehicle (normal saline solution; 3 ml). Pulmonary microvascular dysfunction was then quantitated by measuring the extravasation of Evans blue dye (EBD) into the lung. Inducible NOS activity was six times greater in the lungs of animals sustaining IIR when compared with SHAM (p = 0.0005). The concentration of EBD within the lungs of animals sustaining IIR was 30% greater than SHAM (p < 0.05). Inhibiting NOS with L-NAME significantly increased pulmonary EBD concentration of both IIR and SHAM groups when compared with normal saline solution-treated animals (p < 0.0001). Treatment with L-Arg prevented this IIR-induced increase in pulmonary dye extravasation. These data suggest that pulmonary iNOS activity is up-regulated in animals sustaining IIR and that this may serve as a compensatory protective response to remote organ injury.

Acetaminophen blocks spinal hyperalgesia induced by NMDA and substance P

The hypothesis tested was that inhibition of the l-arginine-nitric oxide (NO) pathway may represent a potential central mechanism of action for acetaminophen (paracetamol). Spinal administration of N- methyl- d- aspartate (NMDA, 0.5 nmol), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA, 0.1 nmol) or substance P (SP, 0.5 nmol) to the rat provoked a specific behaviour characterized by biting, scratching and licking (BSD. This behaviour was antagonized by pretreatment with acetaminophen for NMDA and SP but not for AMPA. Further, the antinociceptive effect of acetaminophen was readily reversed by administration of the natural substrate for nitric oxide synthase (NOS), l-arginine, but not by d-arginine. This suggests that the analgesic effect of acetaminophen is related to inhibition of NO generation. Potential mechanisms for this may involve NMDA and SP. Our data suggest that a significant portion of the analgesic effect of acetaminophen, when used clinically, may be related to an interaction with the central nervous system l-arginine-NO pathway.