7-8-dihydrobiopterin and sepiapterin

Nitric oxide, generated by the nitric oxide synthase (NOS) enzymes, plays pivotal roles in cardiovascular homeostasis and in the pathogenesis of cardiovascular disease. The NOS cofactor, tetrahydrobiopterin (BH4), is an important regulator of NOS function, since BH4 is required to maintain enzymatic coupling of L-arginine oxidation, to produce NO. Loss or oxidation of BH4 to 7,8-dihydrobiopterin (BH2) is associated with NOS uncoupling, resulting in the production of superoxide rather than NO. In addition to key roles in folate metabolism, dihydrofolate reductase (DHFR) can 'recycle' BH2, and thus regenerate BH4. It is therefore likely that net BH4 cellular bioavailability reflects the balance between de novo BH4 synthesis, loss of BH4 by oxidation to BH2, and the regeneration of BH4 by DHFR. Recent studies have implicated BH4 recycling in the direct regulation of eNOS uncoupling, showing that inhibition of BH4 recycling using DHFR-specific siRNA and methotrexate treatment leads to eNOS uncoupling in endothelial cells and the hph-1 mouse model of BH4 deficiency, even in the absence of oxidative stress. These studies indicate that not only BH4 level, but the recycling pathways regulating BH4 bioavailability represent potential therapeutic targets and will be discussed in this review.

Topics: Alcohol Oxidoreductases; Animals; Biological Transport; Biopterins; Dihydropteridine Reductase; Endothelial Cells; GTP Cyclohydrolase; Humans; Methotrexate; Mice; Nitric Oxide; Nitric Oxide Synthase; Oxidation-Reduction; Pterins; Tetrahydrofolate Dehydrogenase; Vascular Diseases

Cells in target organs such as liver do not generally incorporate tetrahydrobiopterin (BH4) in its fully reduced form. Instead, they transiently take up BH4 from the extracellular fluid, instantaneously oxidize it and then expel virtually all of it. However, a small but stable accumulation of BH4 was observed after BH4 administration to the cell cultures. This accumulation was inhibited by methotrexate, an inhibitor of dihydrofolate reductase, a phenomenon that was first suggested based on results of in vitro studies which used established cell lines such as RBL2H3 and PC12. These cells also take up dihydrobiopterin (BH2) and reduce it to enzymically active BH4. Their ability to accumulate usable BH4 upon BH4 administration was attributed to the incorporation of BH2, which in typical experiments was produced by the cells as well as by auto-oxidation of BH4. Most cells of the various cell lines so far examined behaved similarly in culture. Our in vivo work with individual mice demonstrated that administration of sepiapterin, BH2, and BH4 was comparably effective in raising BH4 levels in target organs. BH4 accumulation in various tissues after supplementation with BH4, BH2 or sepiapterin was also inhibited by methotrexate, as in the case of our cell culture system. It was concluded that the elevation in BH4 by supplementation was mainly through a "salvage pathway" that included BH2 as the key intermediate in the production of BH4 through the action of dihydrofolate reductase.

Topics: Animals; Biopterins; Cells, Cultured; Methotrexate; Mice; Pterins; Tetrahydrofolate Dehydrogenase

Sepiapterin reductase deficiency is a rare, under-recognized, autosomal recessively inherited disorder of neurotransmitter metabolism.. Five new patients from 3 unrelated Saudi consanguineous families are reported. Symptoms began at 6 months, with delay to diagnosis averaging 8 years. All 5 patients presented with severe symptoms including axial hypotonia, dystonia, and cognitive impairment, associated with hyper-reflexia (4 patients), spasticity (4 patients), bulbar dysfunction (4 patients), and oculogyric crisis (2 patients) with diurnal fluctuation and sleep benefit. Cerebrospinal fluid neurotransmitters analysis showed a typical pattern with increased sepiapterin and increased 7,8-dihydrobiopterin. Analysis of the SPR gene identified 3 novel mutations: c.1A > G, c.370T > C, and c.527C > T. Patient one, with early diagnosis, is currently developing within the normal range. The 4 other patients showed significant improvement in their motor function, but only mild improvement in their cognitive dysfunction.. Our cases illustrate the difficulties in the diagnosis of sepiapterin reductase deficiency in infancy, and the importance of early recognition and management.

Topics: Adolescent; Alcohol Oxidoreductases; Biopterins; Child; Delayed Diagnosis; Dystonia; Female; Humans; Infant; Male; Metabolism, Inborn Errors; Mutation; Psychomotor Disorders; Pterins

Here, evidence suggests that nitric oxide synthases (NOS) of tumor cells, in contrast with normal tissues, synthesize predominantly superoxide and peroxynitrite. Based on high-performance liquid chromatography analysis, the underlying mechanism for this uncoupling is a reduced tetrahydrobiopterin:dihydrobiopterin ratio (BH4:BH2) found in breast, colorectal, epidermoid, and head and neck tumors compared with normal tissues. Increasing BH4:BH2 and reconstitution of coupled NOS activity in breast cancer cells with the BH4 salvage pathway precursor, sepiapterin, causes significant shifts in downstream signaling, including increased cGMP-dependent protein kinase (PKG) activity, decreased β-catenin expression, and TCF4 promoter activity, and reduced NF-κB promoter activity. Sepiapterin inhibited breast tumor cell growth in vitro and in vivo as measured by a clonogenic assay, Ki67 staining, and 2[18F]fluoro-2-deoxy-D-glucose-deoxyglucose positron emission tomography (FDG-PET). In summary, using diverse tumor types, it is demonstrated that the BH4:BH2 ratio is lower in tumor tissues and, as a consequence, NOS activity generates more peroxynitrite and superoxide anion than nitric oxide, resulting in important tumor growth-promoting and antiapoptotic signaling properties.. The synthetic BH4, Kuvan, is used to elevate BH4:BH2 in some phenylketonuria patients and to treat diseases associated with endothelial dysfunction, suggesting a novel, testable approach for correcting an abnormality of tumor metabolism to control tumor growth.

Topics: Animals; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; beta Catenin; Biopterins; Cell Line, Tumor; Cyclic GMP-Dependent Protein Kinases; Disease Progression; Heterografts; Humans; Mice, Nude; Neoplasms; NF-kappa B; Nitric Oxide Synthase; Peroxynitrous Acid; Pterins; Superoxides; Transcription Factor 4; Transcription Factors

Diabetic nephropathy (DN) is the most common cause of end-stage renal disease worldwide. The accumulation of advanced glycation end products (AGE) is a key mediator of renal tubular hypertrophy in DN. Elimination of tetrahydrobiopterin (BH(4)) and nitric oxide (NO) bioavailability may contribute to the aggravation of DN. The present study aims to explore any possible beneficial effect of exogenous BH(4) in alleviating the AGE-induced renal tubular hypertrophy in DN. Thus, renal tubular cells were treated with BH(4), BH(2), sepiapterin, or DAHP in the presence of AGE. We found that AGE (but not non-glycated BSA) markedly reduced NO production and increased hypertrophy index in these cells. Exogenous BH(4)/BH(2) and sepiapterin treatments attenuated AGE-inhibited the iNOS/NO/GTPCH I protein synthesis. Moreover, BH(4) and BH(2) significantly reversed AGE-enhanced the JAK2-STAT1/STAT3 activation. The abilities of BH(4) and BH(2) to inhibit AGE-induced renal cellular hypertrophy were verified by the observation that BH(4) and BH(2) inhibited hypertrophic growth and the protein synthesis of p27(Kip1) and α-SMA. These findings indicate for the first time that exogenous BH(4) and BH(2) attenuate AGE-induced hypertrophic effect at least partly by increasing the iNOS/GTPCH I synthesis and NO generation in renal tubular cells.

Topics: Actins; Biopterins; Cell Enlargement; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p27; Diabetic Nephropathies; Gene Expression Regulation; Glycation End Products, Advanced; GTP Cyclohydrolase; Humans; Janus Kinase 2; Kidney Tubules; Nitric Oxide; Nitric Oxide Synthase Type II; Pterins; Signal Transduction; STAT1 Transcription Factor; STAT3 Transcription Factor; Sugar Acids

Tetrahydrobiopterin (BH4) is a major endogenous vasoprotective agent that improves endothelial function by increasing nitric oxide (NO) synthesis and scavenging of superoxide and peroxynitrite. Therefore, administration of BH4 is considered a promising therapy for cardiovascular diseases associated with endothelial dysfunction and oxidative stress. Here we report on a novel function of BH4 that might contribute to the beneficial vascular effects of the pteridine. Treatment of cultured porcine aortic endothelial cells with nitroglycerin (GTN) or 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) resulted in heme oxidation of soluble guanylate cyclase (sGC), as evident from diminished NO-induced cGMP accumulation that was paralleled by increased cGMP response to a heme- and NO-independent activator of soluble guanylate cyclase [4-([(4-carboxybutyl)[2-(5-fluoro-2-([4'-(trifluoromethyl)biphenyl-4-yl]methoxy)phenyl)ethyl]amino]methyl)benzoic acid (BAY 60-2770)]. Whereas scavenging of superoxide and/or peroxynitrite with superoxide dismutase, tiron, Mn(III)tetrakis(4-benzoic acid)porphyrin, and urate had no protective effects, supplementation of the cells with BH4, either by application of BH4 directly or of its precursors dihydrobiopterin or sepiapterin, completely prevented the inhibition of NO-induced cGMP accumulation by GTN and ODQ. Tetrahydroneopterin had the same effect, and virtually identical results were obtained with RFL-6 fibroblasts, suggesting that our observation reflects a general feature of tetrahydropteridines that is unrelated to NO synthase function and not limited to endothelial cells. Protection of sGC against oxidative inactivation may contribute to the known beneficial effects of BH4 in cardiovascular disorders associated with oxidative stress.

Topics: Animals; Aorta; Biopterins; Cardiovascular Diseases; Cells, Cultured; Cyclic GMP; Endothelial Cells; Fibroblasts; Guanylate Cyclase; Heme; Nitric Oxide; Nitric Oxide Synthase; Nitroglycerin; Oxidation-Reduction; Oxidative Stress; Peroxynitrous Acid; Pterins; Receptors, Cytoplasmic and Nuclear; Soluble Guanylyl Cyclase; Superoxides; Swine

Tetrahydrobiopterin (BH(4)) is synthesized de novo in particular cells, but in the case of a systemic or local BH(4) deficiency, BH(4) supplementation therapy is applied. BH(4)-responsive PKU has also been effectively treated with BH(4) supplementation. However, the rapid clearance of the supplemented BH(4) has prevented the therapy from being widely accepted. Deposition of BH(4) after supplementation involves oxidation of BH(4) to dihydrobiopterin (BH(2)) and subsequent conversion to BH(4) by the salvage pathway. This pathway is known to be almost ubiquitous in the body. However, the mechanism for the redistribution and exclusion of BH(4) across the plasma membrane remains unclear. The aim of this work was to search for the key transporter of the uptake precursor of the salvage pathway. Based on the observed sensitivity of pterin transport to nitrobenzylthioinosine (NBMPR), we examined the ability of ENT1 and ENT2, representative equilibrative nucleoside transporters, to transport sepiapterin (SP), BH(2) or BH(4) using HeLa cell and Xenopus oocyte expression systems. hENT2 was capable of transporting the pterins with an efficiency of SP>BH(2)>BH(4). hENT1 could also transport the pterins but less efficiently. Non-transfected HeLa cells and rat aortic endothelial cells were able to incorporate the pterins and accumulate BH(4) via uptake that is likely mediated by ENT2 (SP>BH(2)>BH(4)). When exogenous BH(2) was given to mice, it was efficiently converted to BH(4) and its tissue deposition was similar to that of sepiapterin as reported (Sawabe et al., 2004). BH(4) deposition after BH(2) administration was influenced by prior treatment with NBMPR, suggesting that the distribution of the administered BH(2) was largely mediated by ENT2, although urinary excretion appeared to be managed by other mechanisms. The molecular basis of the transport of SP, BH(2), and BH(4) across the plasma membrane has now been described for the first time: ENT2 is a transporter of these pterins and is a plausible gateway to the salvage pathway of BH(4) biosynthesis, at least under conditions of exogenous pterin supplementation. The significance of the gateway was discussed in terms of BH(2) uptake for BH(4) accumulation and the release for modifying the intracellular BH(2)/BH(4) ratio.

Topics: Animals; Biopterins; Endothelial Cells; Endothelium, Vascular; Equilibrative Nucleoside Transport Proteins; Female; HeLa Cells; Humans; In Vitro Techniques; Mice; Mice, Inbred C57BL; Oocytes; Pterins; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Thioinosine; Xenopus laevis

Pterins belong to a class of heterocyclic compounds present in a wide range of living systems and accumulate in the skin of patients affected by vitiligo, a depigmentation disorder. The study of the emission of 7,8-dihydropterins is difficult because these compounds are more or less unstable in the presence of O(2) and their solutions are contaminated with oxidized pterins which have much higher fluorescence quantum yields (Φ(F)). In this work, the emission properties of six compounds of the dihydropterin family (6-formyl-7,8-dihydropterin (H(2)Fop), sepiapterin (Sep), 7,8-dihydrobiopterin (H(2)Bip), 7,8-dihydroneopterin (H(2)Nep), 6-hydroxymethyl-7,8-dihydropterin (H(2)Hmp), and 6-methyl-7,8-dihydropterin (H(2)Mep)) have been studied in aqueous solution. The fluorescence characteristics (spectra, Φ(F), lifetimes (τ(F))) of the neutral form of these compounds have been investigated using the single-photon-counting technique. Φ(F) and τ(F) values obtained lie in the ranges 3-9 × 10(-3) and 0.18-0.34 ns, respectively. The results are compared to those previously reported for oxidized pterins.

Topics: Biopterins; Neopterin; Oxidation-Reduction; Oxygen; Pterins; Quantum Theory; Solutions; Spectrometry, Fluorescence; Water

1. Uncoupling of nitric oxide synthase (NOS) has been implicated in the pathogenesis of left ventricular (LV) dysfunction in diabetes mellitus. In the present study, we investigated the role of NOS uncoupling in oxidative/nitrosative stress and LV dysfunction in the diabetic mouse heart. 2. Diabetes was induced in wild-type (WT), endothelial (e) NOS knockout (eNOS(-/-)), inducible (i) NOS knockout (iNOS(-/-)) and neuronal (n) NOS knockout (nNOS(-/-)) mice by streptozotocin (STZ) treatment. 3. In the diabetic heart, iNOS, but not eNOS or nNOS, expression was increased. Levels of malondialdehyde (MDA), 4-hydroxy-noneal (HNE) and nitrotyrosine (NT), as markers of oxidative/nitrosative stress, were increased in the diabetic mouse heart, but the increase in oxidative/nitrosative stress was significantly repressed in the iNOS(-/-) diabetic mouse heart. Levels of nitrite and nitrate (NO(x)), as an index of nitric oxide, bioavailability were significantly decreased in the iNOS(-/-) diabetic mouse heart. 4. Oral administration of sepiapterin (10 mg/kg per day), a precursor of tetrahydrobiopterin (BH(4)), significantly increased BH(4) and the BH(4)/BH(2) ratio in diabetic mouse heart. Similarly, sepiapterin inhibited the formation of HNE, MDA and NT in diabetic hearts from all three genotypes, but the increase in NO(x) following sepiapterin treatment was significantly attenuated in the iNOS(-/-) diabetic mouse heart. Percentage fractional shortening (FS), evaluated by echocardiography, decreased significantly in all genotypes of diabetic mice. Sepiapterin significantly increased percentage FS in diabetic mice, except in iNOS(-/-) mice. 5. These results suggest that sepiapterin inhibits uncoupling of NOS and improves LV function presumably by increasing iNOS-derived nitric oxide in the diabetic heart.

Topics: Animals; Biopterins; Cardiotonic Agents; Coenzymes; Diabetes Mellitus, Experimental; Diabetic Cardiomyopathies; Enzyme Inhibitors; Lipid Peroxidation; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide; Nitric Oxide Synthase; Pterins; Tyrosine; Ventricular Dysfunction, Left; Ventricular Function, Left

An elevation of oxidized forms of tetrahydrobiopterin (BH(4)), especially dihydrobiopterin (BH(2)), has been reported in the setting of oxidative stress, such as arteriosclerotic/atherosclerotic disorders, where endothelial nitric oxide synthase (eNOS) is dysfunctional, but the role of BH(2) in the regulation of eNOS activity in vivo remains to be evaluated. This study was designed to clarify whether increasing BH(2) concentration causes endothelial dysfunction in rats. To increase vascular BH(2) levels, the BH(2) precursor sepiapterin (SEP) was intravenously given after the administration of the specific dihydrofolate reductase inhibitor methotrexate (MTX) to block intracellular conversion of BH(2) to BH(4). MTX/SEP treatment did not significantly affect aortic BH(4) levels compared with control treatment. However, MTX/SEP treatment markedly augmented aortic BH(2) levels (291.1 ± 29.2 vs. 33.4 ± 6.4 pmol/g, P < 0.01) in association with moderate hypertension. Treatment with MTX alone did not significantly alter blood pressure or BH(4) levels but decreased the BH(4)-to-BH(2) ratio. Treatment with MTX/SEP, but not with MTX alone, impaired ACh-induced vasodilator and depressor responses compared with the control treatment (both P < 0.05) and also aggravated ACh-induced endothelium-dependent relaxations (P < 0.05) of isolated aortas without affecting sodium nitroprusside-induced endothelium-independent relaxations. Importantly, MTX/SEP treatment significantly enhanced aortic superoxide production, which was diminished by NOS inhibitor treatment, and the impaired ACh-induced relaxations were reversed with SOD (P < 0.05), suggesting the involvement of eNOS uncoupling. These results indicate, for the first time, that increasing BH(2) causes eNOS dysfunction in vivo even in the absence of BH(4) deficiency, demonstrating a novel insight into the regulation of endothelial function.

Topics: Acetylcholine; Analysis of Variance; Animals; Biopterins; Blood Pressure; Dose-Response Relationship, Drug; Endothelium, Vascular; Folic Acid Antagonists; Male; Methotrexate; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase Type III; Nitroprusside; Oxidation-Reduction; Phosphorylation; Protein Multimerization; Pterins; Rats; Rats, Wistar; Superoxide Dismutase; Superoxides; Tetrahydrofolate Dehydrogenase; Up-Regulation; Vasodilation; Vasodilator Agents

Rat aortic endothelial cells were cultured on a porous membrane to form a monolayer sheet. They efficiently accumulated tetrahydrobiopterin (BH(4)) by uptake of sepiapterin but did so only moderately by uptake of dihydrobiopterin. The endothelial cell sheet preferentially took up the pterins from the apical side. Accordingly, a dense accumulation of ENT2-like immunoreactivity was visualized on the apical surface of the cell sheet. The findings suggest that vascular endothelial cells receive BH(4) precursors directly from the blood stream rather than from ablumenal tissues.

Topics: Animals; Aorta; Biopterins; Endothelial Cells; Nitric Oxide; Pterins; Rats

Previously we showed that Brown Norway (BN/Mcw) rats are more resistant to myocardial ischemia-reperfusion (I/R) injury than Dahl S (SS/Mcw) rats due to increased nitric oxide (x NO) generation secondary to increased heat shock protein 90 (HSP90) association with endothelial nitric oxide synthase (NOS3). Here we determined whether increased resistance to I/R injury in BN/Mcw hearts is also related to tetrahydrobiopterin (BH(4)) and GTP cyclohydrolase I (GCH-1), the rate-limiting enzyme for BH(4) synthesis. We observed that BH(4) supplementation via sepiapterin (SP) and inhibition of GCH-1 via 2,4-diamino-6-hydroxypyrimidine (DAHP) differentially modulate cardioprotection and that SP alters the association of HSP90 with NOS3. BH(4) levels were significantly higher and 7,8-dihydrobiopterin (BH(2)) levels were significantly lower in BN/Mcw than in SS/Mcw hearts. The BH(4)-to-BH(2) ratio in BN/Mcw was more than two times that in SS/Mcw hearts. After I/R, BH(4) decreased and BH(2) increased in hearts from both strains compared with their preischemia levels. However, the increase in BH(2) in SS/Mcw hearts was significantly higher than in BN/Mcw hearts. Real-time PCR revealed that BN/Mcw hearts contained more GCH-1 transcripts than SS/Mcw hearts. SP increased recovery of left ventricular developed pressure (rLVDP) following I/R as well as decreased superoxide (O(2)(x-)) and increased x NO in SS/Mcw hearts but not in BN/Mcw hearts. DAHP decreased rLVDP as well as increased O(2)(x-) and decreased x NO in BN/Mcw hearts compared with controls but not in SS/Mcw hearts. SP increased the association of HSP90 with NOS3. These data indicate that BH(4) mediates resistance to I/R by acting as a cofactor and enhancing HSP90-NOS3 association.

Topics: Animals; Biopterins; Disease Models, Animal; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; GTP Cyclohydrolase; HSP90 Heat-Shock Proteins; Hypoxanthines; Myocardial Ischemia; Myocardial Reperfusion Injury; Myocytes, Cardiac; Nitric Oxide; Nitric Oxide Synthase Type III; Pterins; Rats; Rats, Inbred BN; Rats, Inbred Dahl; RNA, Messenger; Species Specificity; Superoxides; Ventricular Function, Left; Ventricular Pressure

Adult rat cardiac myocytes typically display a phenotypic response to cytokines manifested by low or no increases in nitric oxide (NO) production via inducible NO synthase (iNOS) that distinguishes them from other cell types. To better characterize this response, we examined the expression of tetrahydrobiopterin (BH4)-synthesizing and arginine-utilizing genes in cytokine-stimulated adult cardiac myocytes. Intracellular BH4 and 7,8-dihydrobiopterin (BH2) and NO production were quantified. Cytokines induced GTP cyclohydrolase and its feedback regulatory protein but with deficient levels of BH4 synthesis. Despite the induction of iNOS protein, cytokine-stimulated adult cardiac myocytes produced little or no increase in NO versus unstimulated cells. Western blot analysis under nonreducing conditions revealed the presence of iNOS monomers. Supplementation with sepiapterin (a precursor of BH4) increased BH4 as well as BH2, but this did not enhance NO levels or eliminate iNOS monomers. Similar findings were confirmed in vivo after treatment of rat cardiac allograft recipients with sepiapterin. It was found that expression of dihydrofolate reductase, required for full activity of the salvage pathway, was not detected in adult cardiac myocytes. Thus, adult cardiac myocytes have a limited capacity to synthesize BH4 after cytokine stimulation. The mechanisms involve posttranslational factors impairing de novo and salvage pathways. These conditions are unable to support active iNOS protein dimers necessary for NO production. These findings raise significant new questions about the prevailing understanding of how cytokines, via iNOS, cause cardiac dysfunction and injury in vivo during cardiac inflammatory disease states since cardiac myocytes are not a major source of high NO production.

Topics: Alcohol Oxidoreductases; Animals; Arginase; Biopterins; Cells, Cultured; Cytokines; GTP Cyclohydrolase; Heart Transplantation; Intracellular Signaling Peptides and Proteins; Male; Myocytes, Cardiac; Nitric Oxide; Nitric Oxide Synthase Type II; Phosphorus-Oxygen Lyases; Proteins; Pterins; Rats; Rats, Inbred Lew; Rats, Inbred WF; Rats, Sprague-Dawley; RNA, Messenger; Tetrahydrofolate Dehydrogenase; Time Factors

Hyperhomocysteinemia is a risk factor for cardiovascular diseases that induces endothelial dysfunction. Here, we examine the participation of endothelial NO synthase (eNOS) in the homocysteine-induced alterations of NO/O(2)(-) balance in endothelial cells from human umbilical cord vein. When cells were treated for 24 h, homocysteine dose-dependently inhibited thrombin-activated NO release without altering eNOS phosphorylation and independently of the endogenous NOS inhibitor, asymmetric dimethylarginine. The inhibitory effect of homocysteine on NO release was associated with increased production of reactive nitrogen and oxygen species (RNS/ROS) independent of extracellular superoxide anion (O(2)(-)) and was suppressed by the NOS inhibitor L-NAME. In unstimulated cells, L-NAME markedly decreased RNS/ROS formation and the ethidium red fluorescence induced by homocysteine. This eNOS-dependent O(2)(-) synthesis was associated with reduced intracellular levels of both total biopterins (-45%) and tetrahydrobiopterin (-80%) and increased release of 7,8-dihydrobiopterin and biopterin in the extracellular medium (+40%). In addition, homocysteine suppressed the activating effect of sepiapterin on NO release, but not that of ascorbate. The results show that the oxidative stress and inhibition of NO release induced by homocysteine depend on eNOS uncoupling due to reduction of intracellular tetrahydrobiopterin availability.

Topics: Antioxidants; Arginine; Ascorbic Acid; Biopterins; Blotting, Western; Cells, Cultured; Dose-Response Relationship, Drug; Endothelium, Vascular; Ethidium; Fluorescent Dyes; Homocysteine; Humans; L-Lactate Dehydrogenase; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type III; Oxidative Stress; Phosphorylation; Pterins; Reactive Nitrogen Species; Superoxides; Thrombin; Time Factors

We previously reported that acute incubation with tetrahydrobiopterin (BH4) or sepiapterin, a cofactor for endothelial nitric oxide synthase and a stable precursor of BH4, respectively, enhanced the acetylcholine (Ach)-induced relaxation of isolated small mesenteric arteries (SMA) from diabetic (db/db) mice. In this study, we investigated the effect of chronic oral supplementation of sepiapterin (10 mg x kg-1 x day-1) to db/db mice on endothelium function, biopterin levels and lipid peroxidation in SMA. Oral dietary supplementation with sepiapterin had no effect on glucose, triglyceride, cholesterol levels and body weight. SMA from db/db mice showed enhanced vascular reactivity to phenylephrine, which was corrected with sepiapterin supplementation. Furthermore, Ach, but not sodium nitroprusside-induced relaxation, was improved with sepiapterin supplementation in db/db mice. BH4 levels and guanosine triphosphate cyclohydrolase I activity in SMA were similar in db/+ and db/db mice. Sepiapterin treatment had no effects on BH4 or guanosine triphosphate cyclohydrolase I activity. However, the level of dihydrobiopterin+biopterin was higher in SMA from db/db mice, which was corrected following sepiapterin treatment. Thiobarbituric acid reactive substance, malondialdehyde, a marker of lipid peroxidation, was higher in SMA from db/db mice, and was normalized by sepiapterin treatment. These results indicate that sepiapterin improves endothelial dysfunction in SMA from db/db mice by reducing oxidative stress. Furthermore, these results suggest that decreased biosynthesis of BH4 may not be the basis for endothelial dysfunction in SMA from db/db mice.

Topics: Acetylcholine; Administration, Oral; Animals; Biopterins; Diabetes Mellitus; Disease Models, Animal; Drug Administration Schedule; Drug Therapy, Combination; Endothelium, Vascular; GTP Cyclohydrolase; Lipid Peroxidation; Male; Malondialdehyde; Mesenteric Artery, Inferior; Mice; Mice, Inbred C57BL; Neopterin; Oxidative Stress; Phenylephrine; Pterins; Vasoconstriction; Vasodilation

Topics: Biopterins; Female; Humans; Hydro-Lyases; Infant, Newborn; Kinetics; Phenylalanine; Pteridines; Pterins

The ability of the enzyme dihydrofolate reductase to catalyze the formation of tetrahydrobiopterin from dihydrobiopterin was used to develop a method for measuring the activity of this enzyme in vivo. This method can be used to determine the activity of the enzyme in tissues as well as the extent and duration of inhibition of the enzyme by antifolates. Sepiapterin, which is converted to dihydrobiopterin by the enzyme sepiapterin reductase, was as effective a precursor as dihydrobiopterin and has been used in these studies because of its greater stability relative to dihydrobiopterin. Assay conditions must be established for each tissue and enzyme activity can be determined either by measuring the rate of disappearance of dihydrobiopterin or the rate of formation of tetrahydrobiopterin.

Topics: Animals; Biopterins; Brain; Folic Acid Antagonists; Injections, Intraventricular; Liver; Male; Methotrexate; Mice; Pteridines; Pterins; Pyrimidines; Rats; Tetrahydrofolate Dehydrogenase

Pterins inhibit rat liver GTP cyclohydrolase I activity noncompetitively. Reduced pterins, such as 7,8-dihydro-D-neopterin, (6R,S)-5,6,7,8-tetrahydro-D-neopterin, 7,8-dihydro-L-biopterin, (6R)-5,6,7,8-tetrahydro-L-biopterin, L-sepiapterin, and DL-6-methyl-5,6,7,8-tetrahydropterin are approximately 12-times more potent as inhibitors than are oxidized pterins, such as D-neopterin, L-biopterin, and isoxanthopterin. They are also 12-times more potent than folates, such as folic acid, dihydrofolic acid, (+/-)-L-tetrahydrofolic acid, and aminopterin. The Ki values for 7,8-dihydro-D-neopterin, 7,8-dihydro-L-biopterin, and (6R)-5,6,7,8-tetrahydro-L-biopterin are 12.7 microM, 14.4 microM, and 15.7 microM, respectively. These results suggest that mammalian GTP cyclohydrolase I may be regulated by its metabolic end products.

Topics: Aminohydrolases; Animals; Biopterins; Folic Acid; GTP Cyclohydrolase; Kinetics; Liver; Neopterin; Oxidation-Reduction; Pteridines; Pterins; Rats; Xanthopterin

We have found a new ability of sepiapterin reductase, which has been known to show a strict substrate specificity for the 6-lactyl sidechain of sepiapterin to produce 6-dihydroxypropyl sidechain of dihydrobiopterin in the biosynthesis of tetrahydrobiopterin, to reduce many dicarbonyl compounds with NADPH as effectively utilized substrates. By analysis of diacetyl reduction by purified sepiapterin reductase, it was observed that both of the carbonyl groups of the compound are finally sequentially reduced by the enzyme with NADPH to hydroxyl groups. And we expect that this enzyme may reduce "Compound X", which is an intermediate of tetrahydrobiopterin synthesis and would be a dicarbonyl derivative of pteridine (Tanaka et. al., 1980), to dihydrobiopterin via sepiapterin.

Topics: Acetoin; Alcohol Oxidoreductases; Animals; Biopterins; Diacetyl; Erythrocytes; Gramicidin; Ketones; Kinetics; NADP; Oxidation-Reduction; Pteridines; Pterins; Rats; Spectrophotometry; Substrate Specificity

Topics: Animals; Biopterins; Humans; Kidney; Liver; Male; Pteridines; Pterins; Rats

A patient with atypical phenylketonuria and normal liver dihydropteridine reductase and phenylalanine-4-hydroxylase activities excreted neopterin but not biopterin or dihydrobiopterin in urine. The oral administration of L-sepiapterin (1 mg/kg body weight) lowered serum-henylalanine from 17.1 to 1.1 mg/dl within 6 h. Comparable responses were observed after oral administration of L-erythro-7, 8-dihydrobiopterin or L-erythro-5, 6, 7, 8-tetrahydrobiopterin (each given in a dose of 2.5 mg/kg body weight). The results indicate a 7, 8-dihydrobiopterin synthetase deficiency in the patient.

Topics: Alcohol Oxidoreductases; Biopterins; Child, Preschool; Female; Guanosine Triphosphate; Humans; Phenylalanine; Phenylketonurias; Pteridines; Pterins