The discovery that mammalian cells have the ability to synthesize the free radical nitric oxide (NO) has stimulated an extraordinary impetus for scientific research in all the fields of biology and medicine. Since its early description as an endothelial-derived relaxing factor, NO has emerged as a fundamental signaling device regulating virtually every critical cellular function, as well as a potent mediator of cellular damage in a wide range of conditions. Recent evidence indicates that most of the cytotoxicity attributed to NO is rather due to peroxynitrite, produced from the diffusion-controlled reaction between NO and another free radical, the superoxide anion. Peroxynitrite interacts with lipids, DNA, and proteins via direct oxidative reactions or via indirect, radical-mediated mechanisms. These reactions trigger cellular responses ranging from subtle modulations of cell signaling to overwhelming oxidative injury, committing cells to necrosis or apoptosis. In vivo, peroxynitrite generation represents a crucial pathogenic mechanism in conditions such as stroke, myocardial infarction, chronic heart failure, diabetes, circulatory shock, chronic inflammatory diseases, cancer, and neurodegenerative disorders. Hence, novel pharmacological strategies aimed at removing peroxynitrite might represent powerful therapeutic tools in the future. Evidence supporting these novel roles of NO and peroxynitrite is presented in detail in this review.

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Nitric Oxide and Peroxynitrite in Health and Disease

Vascular endothelial growth factor (VEGF) is a highly specific mitogen for vascular endothelial cells. Five VEGF isoforms are generated as a result of alternative splicing from a single VEGF gene. These isoforms differ in their molecular mass and in biological properties such as their ability to bind to cell-surface heparan-sulfate proteoglycans. The expression of VEGF is potentiated in response to hypoxia, by activated oncogenes, and by a variety of cytokines. VEGF induces endothelial cell proliferation, promotes cell migration, and inhibits apoptosis. In vivo VEGF induces angiogenesis as well as permeabilization of blood vessels, and plays a central role in the regulation of vasculogenesis. Deregulated VEGF expression contributes to the development of solid tumors by promoting tumor angiogenesis and to the etiology of several additional diseases that are characterized by abnormal angiogenesis. Consequently, inhibition of VEGF signaling abrogates the development of a wide variety of tumors. The various VEGF forms bind to two tyrosine-kinase receptors, VEGFR-1 (flt-1) and VEGFR-2 (KDR/flk-1), which are expressed almost exclusively in endothelial cells. Endothelial cells express in addition the neuropilin-1 and neuropilin-2 coreceptors, which bind selectively to the 165 amino acid form of VEGF (VEGF165). This review focuses on recent developments that have widened considerably our understanding of the mechanisms that control VEGF production and VEGF signal transduction and on recent studies that have shed light on the mechanisms by which VEGF regulates angiogenesis.

Vascular endothelial growth factor (VEGF) and its receptors

Resveratrol, a constituent of red wine, has long been suspected to have cardioprotective effects. Interest in this compound has been renewed in recent years, first from its identification as a chemopreventive agent for skin cancer, and subsequently from reports that it activates sirtuin deacetylases and extends the lifespans of lower organisms. Despite scepticism concerning its bioavailability, a growing body of in vivo evidence indicates that resveratrol has protective effects in rodent models of stress and disease. Here, we provide a comprehensive and critical review of the in vivo data on resveratrol, and consider its potential as a therapeutic for humans.

Therapeutic potential of resveratrol: the in vivo evidence.

The incretin system: glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes

The objective of this study was to determine whether endogenous nitric oxide (NO) inhibits leukocyte adhesion to vascular endothelium. This was accomplished by superfusing a cat mesenteric preparation with inhibitors of NO production, NG-monomethyl-L-arginine (L-NMMA) or NG-nitro-L-arginine methyl ester (L-NAME), and observing single (30-microns diameter) venules by intravital video microscopy. Thirty minutes into the superfusion period the number of adherent and emigrated leukocytes, the erythrocyte velocity, and the venular diameter were measured; venular blood flow and shear rate were calculated from the measured parameters. The contribution of the leukocyte adhesion glycoprotein CD11/CD18 was determined using the CD18-specific monoclonal antibody IB4. Both inhibitors of NO production increased leukocyte adherence more than 15-fold. Leukocyte emigration was also enhanced, whereas venular shear rate was reduced by nearly half. Antibody IB4 abolished the leukocyte adhesion induced by L-NMMA and L-NAME. Incubation of isolated cat neutrophils with L-NMMA, but not L-NAME, resulted in direct upregulation of CD11/CD18 as assessed by flow cytometry. Decrements in venular shear rate induced by partial occlusion of the superior mesenteric artery in untreated animals revealed that only a minor component of L-NAME-induced leukocyte adhesion was shear rate-dependent. The L-NAME-induced adhesion was inhibited by L-arginine but not D-arginine. These data suggest that endothelium-derived NO may be an important endogenous modulator of leukocyte adherence and that impairment of NO production results in a pattern of leukocyte adhesion and emigration that is characteristic of acute inflammation.

https://www.pnas.org/content/pnas/88/11/4651.full.pdf

Nitric oxide: an endogenous modulator of leukocyte adhesion.

Citrulline is a product of arginine degradation by nitric oxide synthase and is a precursor for arginine synthesis in animal cells. After arginine is incorporated into proteins, it may undergo methylation to form N(G)-monomethylarginine, which may be converted to asymmetric dimethylarginine and symmetric dimethylarginine. The degradation of these methylated proteins produces free methylarginines. This chapter focuses on the analysis of these amino acids in biological samples (including plasma/serum, urine, cell culture medium, and tissues) using high-performance liquid chromatography that involves precolumn derivatization with o-phthaldialdehyde. Fluorescence is monitored at excitation and emission wavelengths of 340 and 455 nm, respectively. Detection limits are 5 nM for amino acids. The assays are linear between 1 and 100 microM for citrulline and arginine and between 0.1 and 10 microM for methylarginines. These chromatographic methods are highly sensitive, specific, accurate, and easily automated and provide a useful tool to study the regulation of the arginine-nitric oxide pathway.

Analysis of Citrulline, Arginine, and Methylarginines Using High‐Performance Liquid Chromatography

Polyamines are essential for cell proliferation; therefore, we hypothesized that arginase I or arginase II activities, via production of ornithine for polyamine synthesis, may be limiting for proli...

Activities of arginase I and II are limiting for endothelial cell proliferation

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.

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

Nitric oxide (NO) is a signaling molecule synthesized from L-arginine by NO synthase in animals. Increasing evidence shows that NO regulates the mammalian metabolism of energy substrates and that these effects of NO critically depend on its concentrations at the reaction site and the period of exposure. High concentrations of NO (in the micromolar range) irreversibly inhibit complexes I, II, III, IV, and V in the mitochondrial respiratory chain, whereas physiological levels of NO (in the nanomolar range) reversibly reduce cytochomrome oxidase. Thus, NO reduces oxygen consumption by isolated mitochondria to various extents. In intact cells, through cGMP and AMP-activated protein kinase signaling, physiological levels of NO acutely stimulate uptake and oxidation of glucose and fatty acids by skeletal muscle, heart, liver, and adipose tissue, while inhibiting the synthesis of glucose, glycogen and fat in the insulin-sensitive tissues, and enhancing lipolysis in white adipocytes. Chronic effects of physiological levels of NO in vivo include stimulation of angiogenesis, blood flow, mitochondrial biogenesis, and brown adipocyte development. Modulation of NO-mediated pathways through dietary supplementation with L-arginine or its precursor L-citrulline may provide an effective, practical strategy to prevent and treat metabolic syndrome, including obesity, diabetes, and dyslipidemia in mammals, including humans. © 2013 BioFactors, 39(4):383–391, 2013

Nitric oxide and energy metabolism in mammals

Mast cells accumulate within solid tumors and can release many angiogenic factors, suggesting that they may modulate vascularization of tumors. Stem cell factor (SCF) stimulates mast cell migration, proliferation, and degranulation and therefore may influence mast cell behavior within tumors. We investigated the contribution of SCF to tumor angiogenesis by manipulating its level in mammary tumors. Sense or antisense cDNA fragments of rat SCF were ligated into an episomal expression vector. Ethylnitrosourea-induced rat mammary tumor cell lines were transfected with vector containing either control (no insert, C-P), sense (S-P), or antisense (AS-P) SCF DNA. The functional nature of the transfectants was confirmed by measuring SCF in cell lysates and conditioned media. Immunohistochemical analysis of the tumors induced in Berlin-Druckrey rats by these transfected cells demonstrated that mast cell number and microvascular density were significantly higher in S-P tumors and significantly lower in AS-P tumors, compared with C-P tumors. The expression of von Willebrand factor, an endothelial cell marker, showed a similar pattern. AS-P tumors were significantly smaller than either C-P or S-P tumors. These data suggest that SCF modulates tumor growth and angiogenesis via the involvement of mast cells.

https://cancerres.aacrjournals.org/content/canres/60/23/6757.full.pdf

Cynthia J. Meininger

Papers

Analysis of Citrulline, Arginine, and Methylarginines Using High‐Performance Liquid Chromatography

Activities of arginase I and II are limiting for endothelial cell proliferation

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

Nitric oxide and energy metabolism in mammals

Modulation of Tumor Angiogenesis by Stem Cell Factor