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E R Werner

Publications and source records attributed to E R Werner.

At least 73 records · Page 4Linked to original sources
PubMed1996

Differential regulation of inducible nitric oxide synthase production in bovine and caprine macrophages.

Inducible nitric oxide synthase (iNOS) regulation in human and murine macrophages in vitro differs considerably. In this study, expression of macrophage iNOS in ruminants was addressed. Nitric oxide (NO) output by cattle and goat macrophages was as different as that by human and mouse macrophages. Bovine macrophages activated by heated Salmonella dublin or lipopolysaccharide (LPS) expressed high levels of iNOS mRNA, protein, and enzyme activity. Analogously cultured caprine macrophages did not respond to these and other activators by NO generation and iNOS expression. The lack of response was not due to general unresponsiveness to stimuli. Caprine iNOS mRNA was induced by stimulation of caprine macrophages with LPS, as shown by reverse transcription polymerase chain reaction. The level of mRNA expression in activated goat macrophages was lower than in resting bovine macrophages. A caprine 372-bp iNOS mRNA fragment that was sequenced closely resembled the bovine counterpart. This points to species-specific iNOS gene regulation.

Animals↗
PubMed1996

Pteridine and nitrite/nitrate formation in experimental septic and traumatic shock.

Bacterial lipopolysaccharides (LPS) induce the activity of guanosine triphosphate (GTP)-cyclohydrolase I (GTP-CHI), the first enzyme in the biosynthesis of tetrahydrobiopterin (H4bip) from GTP in endothelial cells and macrophages. In these and other cells, LPS also acts costimulatory with cytokines, i.e., mainly tumor necrosis factor-alpha (TNF-alpha). H4bip is the cofactor for nitric oxide synthase (NOS). We were interested in comparing the pteridine and nitrate levels in two baboon models: a hyperdynamic sepsis model and a hemorrhagic traumatic shock model. Our results show a similar response of pteridines (H4bip, neopterin) and nitrite/nitrate levels to an immune stimulus. LPS, which peaks rapidly, induces a sustained increase in pteridine levels in septic animals. Since hemorrhagic animals show very little response in terms of cytokine production, it was not possible to measure the induction of neopterin and nitrite/nitrate. This information could aid our understanding of the regulatory mechanisms in various forms of experimental shock.

Animals↗
PubMed1995

Nitric oxide formation as predictive parameter for acute graft-versus-host disease after human allogeneic bone marrow transplantation.

Due to the accumulation of evidence concerning a putative role of nitric oxide (NO) in graft-versus-host disease (GVHD), we performed follow-up measurements of the stable end-products of NO, nitrite/nitrate (NO2-/NO3-) in plasma of patients undergoing allogeneic (n = 16) and autologous (n = 6, as a control) bone marrow transplantation. NO2-/NO3- concentrations were set in relation to the clinical course and to serum levels of soluble tumor necrosis factor receptor 75 (sT-NFrec 75) and neopterin, both of which are known to be sensitive indicators of cellular immune activation phenomena involving macrophages in vivo, and endogenous interleukin (IL)-10, a major T helper cell type 2 (TH-2)-derived cytokine and potent inhibitor of macrophage activation and NO formation. A significant rise of NO2-/NO3- levels was observed in patients with GVHD and preceded the onset of clinical symptoms by up to 3 days. In contrast to indicators of macrophage activation, i.e., neopterin and sT-NFrec75, NO2-/NO3- concentrations were not significantly altered from baseline levels during infectious complications, as NO2-/NO3- concentrations did not fluctuate in patients after autologous engraftment. During episodes of acute GVHD, NO2-/NO3- concentrations showed a strong positive correlation with levels of plasma neopterin and sTNFrec 75, but were also significantly related to IL-10. In non-GVHD patients, a negative correlation between IL-10 and NO2-/NO3- concentrations was evident. Therefore, NO2-/NO3- determination may be a valuable early indicator of the initiation of human GVHD. Our results provide some further insights concerning cytokine-related metabolic changes in the course of human GVHD in vivo which may prove useful in the development of new therapeutic approaches for this disease.

Acute Disease↗
PubMed1995

Kinetics and mechanism of tetrahydrobiopterin-induced oxidation of nitric oxide.

A Clark-type nitric oxide-sensitive electrode was used for electrochemical determination of NO oxidation kinetics. Reaction with molecular oxygen followed second-order rate law with respect to NO with an overall rate constant of 9.2 +/- 0.33 x 10(6) M-2 s-1. Tetrahydrobiopterin, an essential cofactor of NO synthases, was found to induce rapid oxidation of NO in a 1:1 stoichiometry. The reaction required the presence of oxygen, was zero order with respect to NO and first order with respect to tetrahydrobiopterin, completely blocked by 5,000 units/ml superoxide dismutase, and mimicked by a superoxide-generating system. Purified brain NO synthase produced no detectable NO unless high amounts of superoxide dismutase were present. NO synthase-catalyzed citrulline formation was inhibited by superoxide dismutase (5,000 units/ml) in an oxyhemoglobin-sensitive manner, indicating that NO induces feedback inhibition of NO synthase. NO-stimulated soluble guanylyl cyclase was inhibited by tetrahydrobiopterin at half-maximally active concentrations of 2 microM. The present data suggest that NO is inactivated to peroxynitrite by superoxide generated in the course of tetrahydrobiopterin autoxidation.

Amino Acid Oxidoreductases↗
PubMed1995

In search of a function for tetrahydrobiopterin in the biosynthesis of nitric oxide.

(6R)-5,6,7,8-Tetrahydro-L-biopterin(H4biopterin) is well known as a cofactor of enzymes that hydroxylate aromatic amino acids. More recent work has revealed an essential role of H4biopterin in the biosynthesis of nitric oxide (NO), an intercellular messenger molecule synthesized from L-arginine by different NO synthase isozymes in many species and tissues. While the function of H4biopterin in aromatic amino acid hydroxylation is well established, the role of this pteridine in NO synthesis is, as yet, elusive. Current experimental evidence hints at a dual mode of action of H4biopterin, involving both an allosteric effect on the NO synthase protein and participation as a reactant in L-arginine oxidation. As discussed in detail in the present article, the latter effect of this pteridine may be related to the protection of NO synthase from feedback inhibition by NO.

Amino Acid Oxidoreductases↗
PubMed1995

Brain biopterin metabolism in chronic experimental hepatic encephalopathy.

Chronic hepatic encephalopathy (HE) is a neuropsychiatric syndrome associated with a substantial increase in the brain L-tryptophan (L-TRP) level. Moreover, a supranormal L-TRP hydroxylating activity in the brain suggests an induced enzymatic process in chronic HE. GTP-cyclohydrolase I (GTPCHI) and tetrahydrobiopterin (BH4) are two major factors besides L-TRP that are involved in regulating the brain L-TRP hydroxylating activity. We therefore determined the GTPCHI activity, the total biopterin and the BH4 concentrations in the neocortex and mesencephalon-pons of portacaval shunted (PCS) rats. The encephalopathic component in PCS rats was accounted for by studying open field behaviour. The acute effects of a single parenteral L-TRP challenge were also evaluated. The basal GTPCHI activities in PCS rats were decreased bu 50% (p < 0.05) compared to controls in both investigated brain regions. No significant alterations in brain total biopterin or BH4 levels were present. The PCS rats exhibited a clearly reduced spontaneous locomotor activity. After the exogenous L-TRP load only a lower GTPCHI activity in the neocortex of PCS rats was recorded. We conclude that a perturbation in the brain biopterin metabolism is concomitantly present with behavioural abnormalities in the chronic PCS condition and that the acute effects of a superimposed L-TRP load do not aggravate these disturbances.

Animals↗
PubMed1995

Serum nitrite plus nitrate in infection with human immunodeficiency virus type-1.

To get a measure of the extent of induction of nitric oxide synthase in infection with human immunodeficiency virus type-1 (HIV-1) in vivo, we estimated serum nitrite plus nitrate concentrations in 110 HIV-1 infected individuals compared to 76 blood donors. To monitor cytokine action and to measure induction of pteridine synthesis, we determined in parallel neopterin, biopterin, soluble tumor necrosis factor-alpha receptor 55 and 75, and beta 2-microglobulin. Serum nitrite plus nitrate concentrations were elevated in patients as compared to blood donor controls. In sera of patients, nitrite plus nitrate levels correlated significantly with neopterin, soluble tumor necrosis factor receptor 55 and 75, and beta 2-microglobulin. Nitrite plus nitrate levels were higher and correlations were stronger in groups of patients with lower CD4+ cell count. These results suggest that cytokine-mediated nitric oxide synthesis occurs in individuals with HIV-1 infection.

Adult↗
PubMed1995

Detection of bacterial pyrogens on the basis of their effects on gamma interferon-mediated formation of neopterin or nitrite in cultured monocyte cell lines.

In a number of mammalian cell types, pteridine biosynthesis from guanosine 5'-triphosphate and formation of nitric oxide from L-arginine are induced by gamma interferon (IFN-gamma) and bacterial lipopolysaccharide (LPS). We assessed the possibility of using such metabolic alterations for the in vitro detection of pyrogens. Products from gram-negative and gram-positive bacteria and related synthetic compounds were tested for their potential to induce either of these pathways. Stimulation of pteridine biosynthesis was monitored as the formation of neopterin in the human myelomonocytic cell line THP-1. The formation of nitric oxide was determined as nitrite in murine J774A.1 macrophage cultures. The substances tested included toxic and detoxified parts of LPS and lipid A from Escherichia coli, Salmonella typhimurium, Salmonella minnesota, and Klebsiella pneumoniae as well as lipoteichoic acid and toxic shock syndrome toxin 1 from Staphylococcus aureus. Furthermore, two cell wall compounds from Mycobacterium tuberculosis, trehalose 6,6'-dimycolate and N-acetylmuramyl-L-alanyl-D-isoglutamine, which are active components of Freund's adjuvant, were used. When applied as a single stimulus, only the whole LPS molecule potently stimulated neopterin or nitrite formation. Lipid A and products from gram-positive bacteria were weakly active. For neopterin formation, lipid A required the presence of fetal calf serum. Besides detoxified LPS and independently from the presence of serum, all bacterial compounds tested strongly increased the effects mediated by IFN-gamma. Our results show that bacterial pyrogens can be detected by monitoring the formation of neopterin or nitrite. This may provide a basis for the development of an in vitro assay for the detection of pyrogenic contamination with the aim of replacing the currently used animal test.

Animals↗
PubMed1994

Pteridine biosynthesis and nitric oxide synthase in Physarum polycephalum.

Physarum polycephalum, an acellular slime mould, serves as a model system to study cell-cycle-dependent events since nuclear division is naturally synchronous. This organism was shown to release isoxanthopterin which is structurally related to tetrahydrobiopterin, a cofactor of aromatic amino acid hydroxylases and of nitric oxide synthases (NOSs) (EC 1.14.13.39). Here, we studied Physarum pteridine biosynthesis in more detail and found that high amounts of tetrahydrobiopterin are produced and NOS activity is expressed. Physarum pteridine biosynthesis is peculiar in as much as 7,8-dihydroneopterin aldolase (EC 4.1.2.25), an enzyme of folic acid biosynthesis usually not found in organisms producing tetrahydrobiopterin, is detected in parallel. NOS purified from Physarum depends on NADPH, tetrahydrobiopterin and flavins. Enzyme activity is independent of exogenous Ca2+ and is inhibited by arginine analogues. The purified enzyme (with a molecular mass of 130 kDa) contains tightly bound tetrahydrobiopterin and flavins. During the synchronous cell cycle of Physarum, pteridine biosynthesis increases during S-phase whereas NOS activity peaks during mitosis, drops at telophase and peaks again during early S-phase. Our results characterize Physarum pteridine biosynthesis and NOS and suggest a possible link between NOS activity and mitosis.

Amino Acid Oxidoreductases↗
PubMed1994

Synthesis and characterization of 3H-labelled tetrahydrobiopterin.

We synthesized [3'-3H]-5,6,7,8-tetrahydrobiopterin from [8,5'-3H]guanosine 5'-triphosphate ([8,5'-3H]GTP) using GTP cyclohydrolase (EC 3.5.4.16), 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase (EC 1.1.1.153). After purification by cation-exchange h.p.l.c. a solution of radiochemically pure (> 95%) [3'-3H]-5,6,7,8-tetrahydrobiopterin with a specific activity of 9.2 Ci/mmol was obtained. The product proved well suited for studying the binding of tetrahydrobiopterin to nitric-oxide synthase.

Amino Acid Oxidoreductases↗
PubMed1994

Iron regulates nitric oxide synthase activity by controlling nuclear transcription.

Recently, it was reported that nitric oxide (NO) directly controls intracellular iron metabolism by activating iron regulatory protein (IRP), a cytoplasmic protein that regulates ferritin translation. To determine whether intracellular iron levels themselves affect NO synthase (NOS), we studied the effect of iron on cytokine-inducible NOS activity and mRNA expression in the murine macrophage cell line J774A.1. We show here that NOS activity is decreased by about 50% in homogenates obtained from cells treated with interferon gamma plus lipopolysaccharide (IFN-gamma/LPS) in the presence of 50 microM ferric iron [Fe(3+)] as compared with extracts from cells treated with IFN-gamma/LPS alone. Conversely, addition of the iron chelator desferrioxamine (100 microM) at the time of stimulation with IFN-gamma/LPS increases NOS activity up to 2.5-fold in J774 cells. These effects of changing the cellular iron state cannot be attributed to a general alteration of the IFN-gamma/LPS signal, since IFN-gamma/LPS-mediated major histocompatibility complex class II antigen expression is unaffected. Furthermore, neither was the intracellular availability of the NOS cofactor tetrahydrobiopterin altered by treatment with Fe(3+) or desferrioxamine, nor do these compounds interfere with the activity of the hemoprotein NOS in vitro. We demonstrate that the mRNA levels for NOS are profoundly increased by treatment with desferrioxamine and reduced by Fe(3+). The half-life of NOS mRNA appeared not to be significantly altered by administration of ferric ion, and NOS mRNA stability was only slightly prolonged by desferrioxamine treatment. Nuclear run-off experiments demonstrate that nuclear transcription of cytokine-inducible NOS mRNA is strongly increased by desferrioxamine whereas it is decreased by Fe(3+). Thus, this transcriptional response appears to account quantitatively for the changes in enzyme activity. Our results suggest the existence of a regulatory loop between iron metabolism and the NO/NOS pathway.

Amino Acid Oxidoreductases↗
PubMed1994

Identification of imidazole as L-arginine-competitive inhibitor of porcine brain nitric oxide synthase.

Imidazole acts as a heme-site inhibitor of nitric oxide synthase (NOS). We used this compound to investigate whether the substrate L-arginine binds directly to the heme or to a separate domain of brain NOS. Enzyme kinetic experiments showed that imidazole enhanced the apparent Km for L-arginine without affecting maximal enzyme activity, and binding studies revealed that the inhibitor displaced the radioligand NG-nitro-L-[3H]arginine in a concentration-dependent fashion. These results demonstrate that imidazole exerts its effects on NOS in an L-arginine-competitive manner and that the substrate site of the enzyme may be identical with the prosthetic heme group.

Amino Acid Oxidoreductases↗
PubMed1994

The pteridine binding site of brain nitric oxide synthase. Tetrahydrobiopterin binding kinetics, specificity, and allosteric interaction with the substrate domain.

Nitric oxide (NO) synthases contain FAD, FMN, heme, and (6R)-5,6,7,8-tetrahydro-L-biopterin as prosthetic groups. We have characterized the pteridine-binding site of purified brain NO synthase, using 3H-labeled (6R)-5,6,7,8-tetrahydro-L-biopterin as radioligand. Association of [3H]tetrahydrobiopterin followed second-order kinetics (kon = 1.3 x 10(6) M-1 min-1), the dissociation reaction was reversible and first-order (koff = 3.2 x 10(-1) min-1), yielding a kinetic KD of 0.25 microM. Binding of the radioligand was competitively antagonized by several pteridine derivatives with the following order of potency (KI): 7,8-dihydro-L-biopterin (2.2 microM), (6S)-5,6,7,8-tetrahydro-L-biopterin (19 microM), (6R,S)-6-methyl-5,6,7,8-tetrahydropterin (240 microM), and 6,7-dimethyl-5,6,7,8-tetrahydropterin (> 1 mM). The affinity of NO synthase for tetrahydrobiopterin was increased 6-fold in the presence of 0.1 mM L-arginine (KD = 37 nM), and, conversely, tetrahydrobiopterin enhanced the affinity of the enzyme for 3H-labeled NG-nitro-L-arginine about 2-fold. 7-Nitroindazole, which presumably binds to the heme group of NO synthase, competitively inhibited binding of [3H]tetrahydrobiopterin and [3H]NG-nitro-L-arginine with similar Ki values (0.1 microM). Functional as well as binding studies revealed that 7-nitroindazole was competitive with both L-arginine and tetrahydrobiopterin. Our data indicate that brain NO synthase exhibits a highly specific binding site for (6R)-5,6,7,8-tetrahydro-L-biopterin, which allosterically interacts with the substrate domain and may be located proximal to the prosthetic heme group of NO synthase.

Allosteric Regulation↗
PubMed1994

Enhancement of hydrogen peroxide-induced luminol-dependent chemiluminescence by neopterin depends on the presence of iron chelator complexes.

We have previously shown that neopterin, 6-D-erythro-trihydroxypropyl-pteridine, synthesized by human monocytes/macrophages upon stimulation by interferon-gamma, enhances toxicity of reactive oxygen at neutral or slightly alkaline pH (7.5), but not at acidic pH (below 6.5). In the present study, we explored in more detail the necessary requirements for neopterin to modulate the effects of hydrogen peroxide in a luminol-dependent chemiluminescence assay. We demonstrate that neopterin enhances hydrogen peroxide effects only in the presence of iron chelator complexes like iron-(III)- or iron-(II)-EDTA or iron-(III)-DTPA. Thus, iron chelator complexes together with neopterin may play an important role in macrophage-mediated effector mechanisms.

Biopterins↗
PubMed1994

Molecular mechanisms of inhibition of porcine brain nitric oxide synthase by the antinociceptive drug 7-nitro-indazole.

7-Nitro-indazole (7-NI) has been described as novel nitric oxide synthase (NOS) inhibitor with in vivo selectivity for the neuronal isozyme [Moore et al. Br. J. Phaarmac. 110, 219-224 (1993)]. In the present study we have used purified porcine brain NOS to investigate the molecular mechanisms of enzyme inhibition by 7-NI. The drug was competitive with L-arginine, exhibited a kinetic KI of 2.8 microM, and additionally induced a slight reduction in Vmax. As a cytochrome P-450, NOS catalyzes a heme-mediated reduction of molecular oxygen, resulting in the formation of H2O2 in the absence of L-arginine. 7-NI turned out as a potent inhibitor of H2O2 formation (IC50 = 0.28 +/- 0.096 microM) but did not affect flavin-mediated electron transfer. Thus, 7-NI resembled imidazole, a known heme-site inhibitor of NOS. We found that imidazole was a purely competitive inhibitor of L-citrulline formation (KI = 263 microM) and blocked H2O2 formation at similar concentrations (IC50 = 280 +/- 38 microM). In accordance with their L-arginine-competitive effects in the citrulline assay, both drugs antagonized binding of radiolabeled NG-nitro-L-arginine (L-NNA), a high affinity probe for reversible labelling of the substrate site of NOS [Klatt et al., J. Biol. Chem. 269, 14781-14787 (1994)]. The calculated KI values for 7-NI and imidazole were 0.09 +/- 0.024 microM and 200 +/- 63 microM, respectively. Finally, binding of radiolabelled tetrahydrobiopterin, a NOS cofactor with unknown function, was also antagonized by 7-NI with a KI of 0.12 +/- 0.023 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗