The Experts below are selected from a list of 252 Experts worldwide ranked by ideXlab platform
Nenad Blau - One of the best experts on this subject based on the ideXlab platform.
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Tetrahydrobiopterin, its Mode of Action on Phenylalanine Hydroxylase, and Importance of Genotypes for Pharmacological Therapy of Phenylketonuria
Human mutation, 2013Co-Authors: Caroline Heintz, Richard G. H. Cotton, Nenad BlauAbstract:In about 20%-30% of phenylketonuria (PKU) patients (all phenotypes of PAH deficiency), Phe levels may be controlled through phenylalanine hydroxylase cofactor Tetrahydrobiopterin therapy. These patients can be diagnosed by an oral Tetrahydrobiopterin challenge and are characterized by mutations coding for proteins with substantial residual PAH activity. They can be treated with a commercially available synthetic form of Tetrahydrobiopterin, either as a monotherapy or as adjunct to the diet. This review article summarizes molecular and metabolic bases of PKU and the importance of the Tetrahydrobiopterin loading test used for PKU patients. On the basis of in vitro residual PAH activity, more than 1,200 genotypes from patients challenged with Tetrahydrobiopterin were categorized as predictive for Tetrahydrobiopterin responsiveness or non-responsiveness and correlated with the loading test, phenotype, and residual in vitro PAH activity. The coexpression of two distinct PAH mutant alleles revealed possible dominance effects (positive or negative) by one of the mutations on residual activity as result of interallelic complementation. The treatment of the transfected cells with Tetrahydrobiopterin showed an increase in residual PAH activity with several mutations coexpressed.
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6 pyruvoyl tetrahydropterin synthase deficiency with mild hyperphenylalaninemia
Annals of Neurology, 2005Co-Authors: Michelle Demos, Nenad Blau, Keith Hyland, Paula J. Waters, Hilary Vallance, Yolanda Lillquist, Nawal Makhseed, Mary B. ConnollyAbstract:Severe 6-pyruvoyl-Tetrahydrobiopterin synthase deficiency is a Tetrahydrobiopterin deficiency disorder that presents in infancy with developmental delay, seizures, and abnormal movements associated with hyperphenylalaninemia usually detectable by neonatal phenylketonuria screening programs. We describe an 8-year-old girl with delay, seizures, and dystonia with mild hyperphenylalaninemia detected in late childhood. The diagnosis of 6-pyruvoyl-Tetrahydrobiopterin synthase deficiency was made by analysis of pterins in urine, pterins and neurotransmitters in cerebrospinal fluid, and enzyme assay. The patient improved clinically taking oral Tetrahydrobiopterin, levodopa/carbidopa, and 5-hydroxytryptophan. This treatable condition may not always be detected by routine population screening for hyperphenylalaninemia. Ann Neurol 2005;58:164–167
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6‐Pyruvoyl‐tetrahydropterin synthase deficiency with mild hyperphenylalaninemia
Annals of neurology, 2005Co-Authors: Michelle Demos, Nenad Blau, Keith Hyland, Paula J. Waters, Hilary Vallance, Yolanda Lillquist, Nawal Makhseed, Mary B. ConnollyAbstract:Severe 6-pyruvoyl-Tetrahydrobiopterin synthase deficiency is a Tetrahydrobiopterin deficiency disorder that presents in infancy with developmental delay, seizures, and abnormal movements associated with hyperphenylalaninemia usually detectable by neonatal phenylketonuria screening programs. We describe an 8-year-old girl with delay, seizures, and dystonia with mild hyperphenylalaninemia detected in late childhood. The diagnosis of 6-pyruvoyl-Tetrahydrobiopterin synthase deficiency was made by analysis of pterins in urine, pterins and neurotransmitters in cerebrospinal fluid, and enzyme assay. The patient improved clinically taking oral Tetrahydrobiopterin, levodopa/carbidopa, and 5-hydroxytryptophan. This treatable condition may not always be detected by routine population screening for hyperphenylalaninemia. Ann Neurol 2005;58:164–167
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epidermal h2o2 accumulation alters Tetrahydrobiopterin 6bh4 recycling in vitiligo identification of a general mechanism in regulation of all 6bh4 dependent processes
Journal of Investigative Dermatology, 2001Co-Authors: Karin U Schallreuter, Nenad Blau, Jeremy Moore, John M Wood, Wayne D Beazley, Eva M J Peters, Lee K Marles, Stefanie C Behrenswilliams, Reinhard Dummer, Beat ThönyAbstract:It has been shown in vivo that patients with the depigmentation disorder vitiligo accumulate hydrogen peroxide (H2O2) accompanied by low catalase levels and high concentrations of 6- and 7-biopterin in their epidermis. Earlier it was demonstrated that epidermal 4a-OH-Tetrahydrobiopterin dehydratase, an important enzyme in the recycling process of 6(R)-L-erythro 5,6,7,8 Tetrahydrobiopterin (6BH4), has extremely low activities in these patients concomitant with a build-up of the abiogenic 7-isomer (7BH4), leading to competitive inhibition of epidermal phenylalanine hydroxylase. A topical substitution for the impaired epidermal catalase with a pseudocatalase effectively removes epidermal H2O2, yielding a recovery of epidermal 4a-OH-Tetrahydrobiopterin dehydratase activities and physiologic 7BH4 levels in association with successful repigmentation demonstrating recovery of the 6BH4 recycling process. Examination of recombinant enzyme activities, together with 4a-OH-Tetrahydrobiopterin dehydratase expression in the epidermis of untreated patients, identifies H2O2-induced inactivation of this enzyme. These results are in agreement with analysis of genomic DNA from these patients yielding only wild-type sequences for 4a-OH-Tetrahydrobiopterin dehydratase and therefore ruling out the previously suspected involvement of this gene. Furthermore, our data show for the first time direct H2O2 inactivation of the important 6BH4 recycling process. Based on this observation, we suggest that H2O2 derived from various sources could be a general mechanism in the regulation of all 6BH4-dependent processes.
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Tetrahydrobiopterin and inherited hyperphenylalaninemias.
The Turkish journal of pediatrics, 1996Co-Authors: Nenad Blau, Beat Thöny, Marco Spada, Alberto PonzoneAbstract:Tetrahydrobiopterin deficiency, a variant of hyperphenylalaninemia, may be caused by deficiency of one of the following enzymes: guanosine triphosphate cyclohydrolase 1,6-pyruvoyltetrahydropterin synthase, dihydropteridin reductase and pterin-4a-carbinolamine dehydratase. The first two enzymes are involved in the biosynthesis of Tetrahydrobiopterin, the last two in its regeneration. Although these diseases are rare, early detection by selective screening is essential for the treatment and outcome. Tetrahydrobiopterin deficiencies are very heterogenous ranging from mild forms requiring only marginal if any treatment to severe forms which are in some cases very difficult to treat. All variants of Tetrahydrobiopterin deficiency can be differentiated from the classical phenylketonuria (PKU) by measurement of pterin metabolites in patients' urine, Tetrahydrobiopterin loading test, and by dihydropteridine reductase activity in erythrocytes from the Guthrie card.
Ernst R. Werner - One of the best experts on this subject based on the ideXlab platform.
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Tetrahydrobiopterin Compounds Prolong Allograft Survival Independently of Their Effect on Nitric Oxide Synthase Activity
Transplantation, 2006Co-Authors: Gerald Brandacher, Gabriele Werner-felmayer, Manuel Maglione, Peter Obrist, Stefan Schneeberger, Guntram Thoeni, Oliver A. Wrulich, Raimund Margreiter, Ernst R. WernerAbstract:Background. In previous work, the four-amino analogue of Tetrahydrobiopterin, a novel, selective inhibitor of inducible nitric oxide synthase, has been shown to prolong survival of murine cardiac allografts. Methods. To further elucidate the underlying molecular immunosuppressive mechanism, we compared the effect of four-amino Tetrahydrobiopterin with that of the unsubstituted parent compound Tetrahydrobiopterin and of N 6 (iminoethyl)-L-lysine (L-NIL), a nonpterin inhibitor of inducible nitric oxide synthase using a murine cardiac transplant model. We analyzed allograft survival, intragraft gene expression in grafts by microarray and real-time polymerase chain reaction, graft nitrotyrosine staining by immunohistochemistry and plasma nitrite plus nitrate levels by high-performance liquid chromatography. Results. Allograft survival was significantly prolonged by Tetrahydrobiopterin and cyclosporin A, but not by L-NIL althoughdecreasedplasmanitriteplusnitratelevelsconfirmednitricoxidesynthaseinhibitioninvivo.Ascomparedto allogeneic untreated controls, intragraft peroxynitrite formation and hence nitrotyrosine staining was lowered in all groups except in cyclosporine A-treated animals. Gene expression profiles obtained by microarray analysis demonstrated that cyclosporine A was able to counteract the expression changes of more than half of the genes differently expressed in syngeneic grafts versus allografts, whereas Tetrahydrobiopterin compounds and L-NIL showed only smaller influences on gene expression profiles. Conclusions. These results demonstrate that the four-amino substitution, which is essential for inhibition of nitric oxide synthase, is not required for the immunosuppressive effect of Tetrahydrobiopterin compounds. We describe a novel immunosuppressive role of pharmacologically applied Tetrahydrobiopterin.
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Tetrahydrobiopterin and nitric oxide: mechanistic and pharmacological aspects.
Experimental biology and medicine (Maywood N.J.), 2003Co-Authors: Ernst R. Werner, Gabriele Werner-felmayer, Regine Heller, Antonius C. F. Gorren, Bernd MayerAbstract:In previous minireviews in this journal, we discussed work on induction of Tetrahydrobiopterin biosynthesis by cytokines and its significance for nitric oxide (NO) production of intact cells as well as functions of H4-biopterin identified at this time for NO synthases (Proc Soc Exp Biol Med 203: 1-12, 1993; Proc Soc Exp Biol Med 219: 171-182, 1998). Meanwhile, the recognition of the importance of Tetrahydrobiopterin for NO formation has led to new insights into complex biological processes and revealed possible novel pharmacological strategies to intervene in certain pathological conditions. Recent work could also establish that Tetrahydrobiopterin, in addition to its allosteric effects, is redox-active in the NO synthase reaction. In this review, we summarize the current view of how Tetrahydrobiopterin functions in the generation of NO and focus on pharmacological aspects of Tetrahydrobiopterin availability with emphasis on endothelial function.
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Immunosuppressive Effects of the 4-Amino Analogue of Tetrahydrobiopterin
Chemistry and Biology of Pteridines and Folates, 2002Co-Authors: Ernst R. Werner, Gabriele Werner-felmayer, Gerald Brandacher, Soheyl Bahrami, Wolfgang Strohmaier, Raimund MargreiterAbstract:Nitric oxide synthases catalyze a complex reaction converting L-arginine to citrulline and nitric oxide. In addition to heme, FAD, FMN and NADPH, nitric oxide synthases require Tetrahydrobiopterin as cofactor. Tetrahydrobiopterin is known to stabilize the active, dimeric conformation of the enzyme (1), which has a high-spin heme iron (2) and an increased affinity for the substrate L-arginine (3). In addition to these allosteric roles, an electronic contribution of the Tetrahydrobiopterin cofactor to the nitric oxide synthase reaction has been intensely searched for. The most convincing evidence for such an electronic contribution provided the detection of Tetrahydrobiopterin-derived radicals being formed in dependence of the nitric oxide synthase reaction (4, 5, 6).
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l ascorbic acid potentiates endothelial nitric oxide synthesis via a chemical stabilization of Tetrahydrobiopterin
Journal of Biological Chemistry, 2001Co-Authors: Regine Heller, Bernd Mayer, Anett Unbehaun, Berit Schellenberg, Gabriele Wernerfelmayer, Ernst R. WernerAbstract:Ascorbic acid has been shown to stimulate endothelial nitric oxide (NO) synthesis in a time- and concentration-dependent fashion without affecting NO synthase (NOS) expression or l-arginine uptake. The present study investigates if the underlying mechanism is related to the NOS cofactor Tetrahydrobiopterin. Pretreatment of human umbilical vein endothelial cells with ascorbate (1 microm to 1 mm, 24 h) led to an up to 3-fold increase of intracellular Tetrahydrobiopterin levels that was concentration-dependent and saturable at 100 microm. Accordingly, the effect of ascorbic acid on Ca(2+)-dependent formation of citrulline (co-product of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) was abolished when intracellular Tetrahydrobiopterin levels were increased by coincubation of endothelial cells with sepiapterin (0.001-100 microm, 24 h). In contrast, ascorbic acid did not modify the pterin affinity of endothelial NOS, which was measured in assays with purified Tetrahydrobiopterin-free enzyme. The ascorbate-induced increase of endothelial Tetrahydrobiopterin was not due to an enhanced synthesis of the compound. Neither the mRNA expression of the rate-limiting enzyme in Tetrahydrobiopterin biosynthesis, GTP cyclohydrolase I, nor the activities of either GTP cyclohydrolase I or 6-pyruvoyl-tetrahydropterin synthase, the second enzyme in the de novo synthesis pathway, were altered by ascorbate. Our data demonstrate that ascorbic acid leads to a chemical stabilization of Tetrahydrobiopterin. This was evident as an increase in the half-life of Tetrahydrobiopterin in aqueous solution. Furthermore, the increase of Tetrahydrobiopterin levels in intact endothelial cells coincubated with cytokines and ascorbate was associated with a decrease of more oxidized biopterin derivatives (7,8-dihydrobiopterin and biopterin) in cells and cell supernatants. The present study suggests that saturated ascorbic acid levels in endothelial cells are necessary to protect Tetrahydrobiopterin from oxidation and to provide optimal conditions for cellular NO synthesis.
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Tetrahydrobiopterin, cytokines, and nitric oxide synthesis
Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York N.Y.), 1998Co-Authors: Ernst R. Werner, Gabriele Werner-felmayer, Bernd MayerAbstract:Nitric oxide synthases require a surprisingly rich selection of cofactors to perform the conversion of L-arginine to citrulline and nitric oxide (NO): NADPH, FAD, FMN, heme and Tetrahydrobiopterin. In a previous minireview in this journal we summarized work concerning the induction of Tetrahydrobiopterin biosynthesis by cytokines, which yields increased intracellular Tetrahydrobiopterin concentrations supporting NO formation by intact cells (P.S.E.B.M. 203:1-12). The present review updates work on the induction of Tetrahydrobiopterin biosynthesis by cytokines, and summarizes recent advances in research of Tetrahydrobiopterin dependence of the NO synthase reaction. Studies using recombinant NO synthases and site-directed mutations thereof have localized several amino acids critical for Tetrahydrobiopterin binding, which are discussed in reference to the recently published crystal structure of the dimer of the oxygenase domain of murine inducible NO synthase with substrate and pterin. Allosteric actions of Tetrahydrobiopterin on NO synthases are stabilization of dimers, stabilization of a conformation with high-spin heme iron, and support of binding of the substrate L-arginine. Since the 4-amino analog of Tetrahydrobiopterin, which is a dihydropteridine reductase inhibitor, supports these allosteric actions but inhibits the enzyme activity, Tetrahydrobiopterin appears to play a redox-active role in stimulating the NO synthase reaction in addition to its allosteric actions on NO synthases. Amelioration of endothelial dysfunction by Tetrahydrobiopterin in animal models and in humans in vivo has been observed. It remains to be investigated, however, to what extent the role of Tetrahydrobiopterin as cofactor of NO synthases contributes to these in vivo effects of Tetrahydrobiopterin.
Bernd Mayer - One of the best experts on this subject based on the ideXlab platform.
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Tetrahydrobiopterin and nitric oxide: mechanistic and pharmacological aspects.
Experimental biology and medicine (Maywood N.J.), 2003Co-Authors: Ernst R. Werner, Gabriele Werner-felmayer, Regine Heller, Antonius C. F. Gorren, Bernd MayerAbstract:In previous minireviews in this journal, we discussed work on induction of Tetrahydrobiopterin biosynthesis by cytokines and its significance for nitric oxide (NO) production of intact cells as well as functions of H4-biopterin identified at this time for NO synthases (Proc Soc Exp Biol Med 203: 1-12, 1993; Proc Soc Exp Biol Med 219: 171-182, 1998). Meanwhile, the recognition of the importance of Tetrahydrobiopterin for NO formation has led to new insights into complex biological processes and revealed possible novel pharmacological strategies to intervene in certain pathological conditions. Recent work could also establish that Tetrahydrobiopterin, in addition to its allosteric effects, is redox-active in the NO synthase reaction. In this review, we summarize the current view of how Tetrahydrobiopterin functions in the generation of NO and focus on pharmacological aspects of Tetrahydrobiopterin availability with emphasis on endothelial function.
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l ascorbic acid potentiates endothelial nitric oxide synthesis via a chemical stabilization of Tetrahydrobiopterin
Journal of Biological Chemistry, 2001Co-Authors: Regine Heller, Bernd Mayer, Anett Unbehaun, Berit Schellenberg, Gabriele Wernerfelmayer, Ernst R. WernerAbstract:Ascorbic acid has been shown to stimulate endothelial nitric oxide (NO) synthesis in a time- and concentration-dependent fashion without affecting NO synthase (NOS) expression or l-arginine uptake. The present study investigates if the underlying mechanism is related to the NOS cofactor Tetrahydrobiopterin. Pretreatment of human umbilical vein endothelial cells with ascorbate (1 microm to 1 mm, 24 h) led to an up to 3-fold increase of intracellular Tetrahydrobiopterin levels that was concentration-dependent and saturable at 100 microm. Accordingly, the effect of ascorbic acid on Ca(2+)-dependent formation of citrulline (co-product of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) was abolished when intracellular Tetrahydrobiopterin levels were increased by coincubation of endothelial cells with sepiapterin (0.001-100 microm, 24 h). In contrast, ascorbic acid did not modify the pterin affinity of endothelial NOS, which was measured in assays with purified Tetrahydrobiopterin-free enzyme. The ascorbate-induced increase of endothelial Tetrahydrobiopterin was not due to an enhanced synthesis of the compound. Neither the mRNA expression of the rate-limiting enzyme in Tetrahydrobiopterin biosynthesis, GTP cyclohydrolase I, nor the activities of either GTP cyclohydrolase I or 6-pyruvoyl-tetrahydropterin synthase, the second enzyme in the de novo synthesis pathway, were altered by ascorbate. Our data demonstrate that ascorbic acid leads to a chemical stabilization of Tetrahydrobiopterin. This was evident as an increase in the half-life of Tetrahydrobiopterin in aqueous solution. Furthermore, the increase of Tetrahydrobiopterin levels in intact endothelial cells coincubated with cytokines and ascorbate was associated with a decrease of more oxidized biopterin derivatives (7,8-dihydrobiopterin and biopterin) in cells and cell supernatants. The present study suggests that saturated ascorbic acid levels in endothelial cells are necessary to protect Tetrahydrobiopterin from oxidation and to provide optimal conditions for cellular NO synthesis.
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Tetrahydrobiopterin, cytokines, and nitric oxide synthesis
Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York N.Y.), 1998Co-Authors: Ernst R. Werner, Gabriele Werner-felmayer, Bernd MayerAbstract:Nitric oxide synthases require a surprisingly rich selection of cofactors to perform the conversion of L-arginine to citrulline and nitric oxide (NO): NADPH, FAD, FMN, heme and Tetrahydrobiopterin. In a previous minireview in this journal we summarized work concerning the induction of Tetrahydrobiopterin biosynthesis by cytokines, which yields increased intracellular Tetrahydrobiopterin concentrations supporting NO formation by intact cells (P.S.E.B.M. 203:1-12). The present review updates work on the induction of Tetrahydrobiopterin biosynthesis by cytokines, and summarizes recent advances in research of Tetrahydrobiopterin dependence of the NO synthase reaction. Studies using recombinant NO synthases and site-directed mutations thereof have localized several amino acids critical for Tetrahydrobiopterin binding, which are discussed in reference to the recently published crystal structure of the dimer of the oxygenase domain of murine inducible NO synthase with substrate and pterin. Allosteric actions of Tetrahydrobiopterin on NO synthases are stabilization of dimers, stabilization of a conformation with high-spin heme iron, and support of binding of the substrate L-arginine. Since the 4-amino analog of Tetrahydrobiopterin, which is a dihydropteridine reductase inhibitor, supports these allosteric actions but inhibits the enzyme activity, Tetrahydrobiopterin appears to play a redox-active role in stimulating the NO synthase reaction in addition to its allosteric actions on NO synthases. Amelioration of endothelial dysfunction by Tetrahydrobiopterin in animal models and in humans in vivo has been observed. It remains to be investigated, however, to what extent the role of Tetrahydrobiopterin as cofactor of NO synthases contributes to these in vivo effects of Tetrahydrobiopterin.
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structural analysis of porcine brain nitric oxide synthase reveals a role for Tetrahydrobiopterin and l arginine in the formation of an sds resistant dimer
The EMBO Journal, 1995Co-Authors: Peter Klatt, Kurt Schmidt, Dieter Lehner, Otto Glatter, H P Bachinger, Bernd MayerAbstract:Abstract Nitric oxide synthases (NOSs), which catalyze the formation of the ubiquitous biological messenger molecule nitric oxide, represent unique cytochrome P-450s, containing reductase and mono-oxygenase domains within one polypeptide and requiring Tetrahydrobiopterin as cofactor. To investigate whether Tetrahydrobiopterin functions as an allosteric effector of NOS, we have analyzed the effect of the pteridine on the conformation of neuronal NOS purified from porcine brain by means of circular dichroism, velocity sedimentation, dynamic light scattering and SDS-polyacrylamide gel electrophoresis. We report for the first time the secondary structure of NOS, showing that the neuronal isozyme contains 30% alpha-helix, 14% antiparallel beta-sheet, 7% parallel beta-sheet, 19% turns and 31% other structures. The secondary structure of neuronal NOS was neither modulated nor stabilized by Tetrahydrobiopterin, and the pteridine did not affect the quaternary structure of the protein, which appears to be an elongated homodimer with an axial ratio of approximately 20/1 under native conditions. Low temperature SDS-polyacrylamide gel electrophoresis revealed that Tetrahydrobiopterin and L-arginine synergistically convert neuronal NOS into an exceptionally stable, non-covalently linked homodimer surviving in 2% SDS and 5% 2-mercaptoethanol. Ligand-induced formation of an SDS-resistant dimer is unprecedented and suggests a novel role for Tetrahydrobiopterin and L-arginine in the allosteric regulation of protein subunit interactions.
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Kinetics and Mechanism of Tetrahydrobiopterin-induced Oxidation of Nitric Oxide
The Journal of biological chemistry, 1995Co-Authors: Bernd Mayer, Ernst R. Werner, Peter Klatt, Kurt SchmidtAbstract: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.
Gabriele Werner-felmayer - One of the best experts on this subject based on the ideXlab platform.
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Tetrahydrobiopterin Compounds Prolong Allograft Survival Independently of Their Effect on Nitric Oxide Synthase Activity
Transplantation, 2006Co-Authors: Gerald Brandacher, Gabriele Werner-felmayer, Manuel Maglione, Peter Obrist, Stefan Schneeberger, Guntram Thoeni, Oliver A. Wrulich, Raimund Margreiter, Ernst R. WernerAbstract:Background. In previous work, the four-amino analogue of Tetrahydrobiopterin, a novel, selective inhibitor of inducible nitric oxide synthase, has been shown to prolong survival of murine cardiac allografts. Methods. To further elucidate the underlying molecular immunosuppressive mechanism, we compared the effect of four-amino Tetrahydrobiopterin with that of the unsubstituted parent compound Tetrahydrobiopterin and of N 6 (iminoethyl)-L-lysine (L-NIL), a nonpterin inhibitor of inducible nitric oxide synthase using a murine cardiac transplant model. We analyzed allograft survival, intragraft gene expression in grafts by microarray and real-time polymerase chain reaction, graft nitrotyrosine staining by immunohistochemistry and plasma nitrite plus nitrate levels by high-performance liquid chromatography. Results. Allograft survival was significantly prolonged by Tetrahydrobiopterin and cyclosporin A, but not by L-NIL althoughdecreasedplasmanitriteplusnitratelevelsconfirmednitricoxidesynthaseinhibitioninvivo.Ascomparedto allogeneic untreated controls, intragraft peroxynitrite formation and hence nitrotyrosine staining was lowered in all groups except in cyclosporine A-treated animals. Gene expression profiles obtained by microarray analysis demonstrated that cyclosporine A was able to counteract the expression changes of more than half of the genes differently expressed in syngeneic grafts versus allografts, whereas Tetrahydrobiopterin compounds and L-NIL showed only smaller influences on gene expression profiles. Conclusions. These results demonstrate that the four-amino substitution, which is essential for inhibition of nitric oxide synthase, is not required for the immunosuppressive effect of Tetrahydrobiopterin compounds. We describe a novel immunosuppressive role of pharmacologically applied Tetrahydrobiopterin.
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Tetrahydrobiopterin and nitric oxide: mechanistic and pharmacological aspects.
Experimental biology and medicine (Maywood N.J.), 2003Co-Authors: Ernst R. Werner, Gabriele Werner-felmayer, Regine Heller, Antonius C. F. Gorren, Bernd MayerAbstract:In previous minireviews in this journal, we discussed work on induction of Tetrahydrobiopterin biosynthesis by cytokines and its significance for nitric oxide (NO) production of intact cells as well as functions of H4-biopterin identified at this time for NO synthases (Proc Soc Exp Biol Med 203: 1-12, 1993; Proc Soc Exp Biol Med 219: 171-182, 1998). Meanwhile, the recognition of the importance of Tetrahydrobiopterin for NO formation has led to new insights into complex biological processes and revealed possible novel pharmacological strategies to intervene in certain pathological conditions. Recent work could also establish that Tetrahydrobiopterin, in addition to its allosteric effects, is redox-active in the NO synthase reaction. In this review, we summarize the current view of how Tetrahydrobiopterin functions in the generation of NO and focus on pharmacological aspects of Tetrahydrobiopterin availability with emphasis on endothelial function.
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Immunosuppressive Effects of the 4-Amino Analogue of Tetrahydrobiopterin
Chemistry and Biology of Pteridines and Folates, 2002Co-Authors: Ernst R. Werner, Gabriele Werner-felmayer, Gerald Brandacher, Soheyl Bahrami, Wolfgang Strohmaier, Raimund MargreiterAbstract:Nitric oxide synthases catalyze a complex reaction converting L-arginine to citrulline and nitric oxide. In addition to heme, FAD, FMN and NADPH, nitric oxide synthases require Tetrahydrobiopterin as cofactor. Tetrahydrobiopterin is known to stabilize the active, dimeric conformation of the enzyme (1), which has a high-spin heme iron (2) and an increased affinity for the substrate L-arginine (3). In addition to these allosteric roles, an electronic contribution of the Tetrahydrobiopterin cofactor to the nitric oxide synthase reaction has been intensely searched for. The most convincing evidence for such an electronic contribution provided the detection of Tetrahydrobiopterin-derived radicals being formed in dependence of the nitric oxide synthase reaction (4, 5, 6).
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Tetrahydrobiopterin, cytokines, and nitric oxide synthesis
Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York N.Y.), 1998Co-Authors: Ernst R. Werner, Gabriele Werner-felmayer, Bernd MayerAbstract:Nitric oxide synthases require a surprisingly rich selection of cofactors to perform the conversion of L-arginine to citrulline and nitric oxide (NO): NADPH, FAD, FMN, heme and Tetrahydrobiopterin. In a previous minireview in this journal we summarized work concerning the induction of Tetrahydrobiopterin biosynthesis by cytokines, which yields increased intracellular Tetrahydrobiopterin concentrations supporting NO formation by intact cells (P.S.E.B.M. 203:1-12). The present review updates work on the induction of Tetrahydrobiopterin biosynthesis by cytokines, and summarizes recent advances in research of Tetrahydrobiopterin dependence of the NO synthase reaction. Studies using recombinant NO synthases and site-directed mutations thereof have localized several amino acids critical for Tetrahydrobiopterin binding, which are discussed in reference to the recently published crystal structure of the dimer of the oxygenase domain of murine inducible NO synthase with substrate and pterin. Allosteric actions of Tetrahydrobiopterin on NO synthases are stabilization of dimers, stabilization of a conformation with high-spin heme iron, and support of binding of the substrate L-arginine. Since the 4-amino analog of Tetrahydrobiopterin, which is a dihydropteridine reductase inhibitor, supports these allosteric actions but inhibits the enzyme activity, Tetrahydrobiopterin appears to play a redox-active role in stimulating the NO synthase reaction in addition to its allosteric actions on NO synthases. Amelioration of endothelial dysfunction by Tetrahydrobiopterin in animal models and in humans in vivo has been observed. It remains to be investigated, however, to what extent the role of Tetrahydrobiopterin as cofactor of NO synthases contributes to these in vivo effects of Tetrahydrobiopterin.
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Determination of Tetrahydrobiopterin biosynthetic activities by high-performance liquid chromatography with fluorescence detection
Methods in Enzymology, 1997Co-Authors: E R Werner, H Wachter, Gabriele Werner-felmayerAbstract:Publisher Summary Tetrahydrobiopterin serves as cofactor of phenylalanine 4-monooxygenase, tyrosine 3-monooxygenase, glyceryl-ether monooxygenase, and nitric oxide synthase. Because intracellular Tetrahydrobiopterin concentrations affect the amount of metabolites of, for example, nitric oxide synthase formed by intact cells, regulation of the biosynthesis of Tetrahydrobiopterin is of interest. This chapter discusses assays of the three biosynthetic enzymes involved in the formation of Tetrahydrobiopterin from guanosine 5'-triphospate (GTP) based on highperformance liquid chromatography (HPLC) with fluorescence detection. While the assay of GTP cyclohydrolase I, the first enzyme of the pathway that is regulated by cytokines, as well as the assay for sepiapterin reductase, can be run with materials that are commercially available, 6-pyruvoyltetrahydropterin synthase assays are performed with two purified enzymes, GTP cyclohydrolase I and sepiapterin reductase, to synthesize the labile substrate and to help convert the unstable product to a stable metabolite.
Zvonimir S. Katusic - One of the best experts on this subject based on the ideXlab platform.
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long term vitamin c treatment increases vascular Tetrahydrobiopterin levels and nitric oxide synthase activity
Circulation Research, 2003Co-Authors: Livius V Duscio, Sheldon Milstien, Darcy M Richardson, Leslie A Smith, Zvonimir S. KatusicAbstract:In cultured endothelial cells, the antioxidant, L-ascorbic acid (vitamin C), increases nitric oxide synthase (NOS) enzyme activity via chemical stabilization of Tetrahydrobiopterin. Our objective was to determine the effect of vitamin C on NOS function and Tetrahydrobiopterin metabolism in vivo. Twenty-six to twenty-eight weeks of diet supplementation with vitamin C (1%/kg chow) significantly increased circulating levels of vitamin C in wild-type (C57BL/6J) and apolipoprotein E (apoE)--deficient mice. Measurements of NOS enzymatic activity in aortas of apoE-deficient mice indicated a significant increase in total NOS activity. However, this increase was mainly due to high activity of inducible NOS, whereas eNOS activity was reduced. Significantly higher Tetrahydrobiopterin levels were detected in aortas of apoE-deficient mice. Long-term treatment with vitamin C restored endothelial NOS activity in aortas of apoE-deficient mice, but did not affect activity of inducible NOS. In addition, 7,8-dihydrobiopterin levels, an oxidized form of Tetrahydrobiopterin, were decreased and vascular endothelial function of aortas was significantly improved in apoE-deficient mice. Interestingly, vitamin C also increased Tetrahydrobiopterin and NOS activity in aortas of C57BL/6J mice. In contrast, long-term treatment with vitamin E (2000 U/kg chow) did not affect vascular NOS activity or metabolism of Tetrahydrobiopterin. In vivo, beneficial effect of vitamin C on vascular endothelial function appears to be mediated in part by protection of Tetrahydrobiopterin and restoration of eNOS enzymatic activity.
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Tetrahydrobiopterin, nitric oxide and regulation of cerebral arterial tone
Progress in neurobiology, 1997Co-Authors: Hiroyuki Kinoshita, Masato Tsutsui, Sheldon Milstien, Zvonimir S. KatusicAbstract:Abstract Tetrahydrobiopterin is an essential cofactor required for activity nitric oxide synthases. Existing evidence suggests that, during activation of constitutive and inducible isoforms of nitric oxide synthase, Tetrahydrobiopterin is needed for allosteric and redox activation of enzymatic activity. However, precise mechanisms underlying the role of Tetrahydrobiopterin in regulation of nitric oxide formation is not fully understood. In cerebral and peripheral arteries, increased availability of Tetrahydrobiopterin can augment production of nitric oxide. In contrast, in arteries depleted of Tetrahydrobiopterin, production of nitric oxide is impared. Proinflammatory cytokines enhance mRNA expression of the rate-limiting enzyme of Tetrahydrobiopterin biosynthesis, GTP cyclohydrolase I and stimulate production of Tetrahydrobiopterin. The ability of vascular tissues to synthesize Tetrahydrobiopterin plays an important role in regulation of nitric oxide synthase under physiological conditions as well as during inflammation and sepsis. More recent studies concerning expression and function of recombinant nitric oxide in genetically suggest that availability of Tetrahydrobiopterin is important for production of nitric oxide in genetically engineered blood vessels. In this review, mechanisms regulating availability of intracellular Tetrahydrobiopterin and its role in control of vascular tone under physiological and pathological conditions will be discussed.
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Effect of Tetrahydrobiopterin on endothelial function in canine middle cerebral arteries.
Circulation research, 1996Co-Authors: Masato Tsutsui, Sheldon Milstien, Zvonimir S. KatusicAbstract:Tetrahydrobiopterin is an essential cofactor required for activation of NO synthase. However, in intact arteries, the exact role of Tetrahydrobiopterin in the regulation of NO synthase activity is not fully understood. The present study was designed to determine the effect of increasing intracellular Tetrahydrobiopterin levels on endothelial function in isolated canine middle cerebral arteries. The arterial segments were incubated in MEM for 24 hours at 37°C in the presence or absence of a Tetrahydrobiopterin precursor, sepiapterin (10−4 mol/L), and/or superoxide dismutase (150 U/mL). The rings were suspended for isometric tension recording. Tetrahydrobiopterin levels were assayed by high-performance liquid chromatography. Production of cGMP was measured by radioimmunoassay. Incubation with sepiapterin markedly increased intracellular Tetrahydrobiopterin levels. In sepiapterin-treated arteries, endothelium-dependent relaxations to calcium ionophore A23187 and intracellular cGMP levels were significantly r...
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Tetrahydrobiopterin and Dysfunction of Endothelial Nitric Oxide Synthase in Coronary Arteries
Circulation, 1995Co-Authors: F Cosentino, Zvonimir S. KatusicAbstract:Background The l-arginine/nitric oxide pathway plays a key role in the regulation of arterial tone. Biosynthesis of nitric oxide requires activation of nitric oxide synthase in the presence of Tetrahydrobiopterin as a cofactor. Biochemical studies demonstrated that activation of purified nitric oxide synthase at suboptimal concentrations of Tetrahydrobiopterin leads to production of hydrogen peroxide. The present experiments were designed to determine whether in coronary arteries inhibition of Tetrahydrobiopterin synthesis may favor nitric oxide synthase–catalyzed production of hydrogen peroxide. Methods and Results Primary branches of canine left anterior descending artery were incubated for 6 hours in minimum essential medium in the presence or in the absence of the Tetrahydrobiopterin synthesis inhibitor 2,4-diamino-6-hydroxypyrimidine (DAHP; 10−2 mol/L). Arterial rings were suspended for isometric tension recording. Production of cGMP was measured by radioimmunoassay. Experiments were performed in the...
