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PubMed1992

Tetrahydrobiopterin synthesis. An absolute requirement for cytokine-induced nitric oxide generation by vascular smooth muscle.

Nitric oxide (NO) synthesis is induced in vascular smooth muscle cells by lipopolysaccharide (LPS) where it appears to mediate a variety of vascular dysfunctions. In some cell types tetrahydrobiopterin (BH4) synthesis has also been found to be induced by cytokines. Because BH4 is a cofactor for NO synthase, we investigated whether BH4 synthesis is required for LPS-induced NO production in rat aortic smooth muscle cells (RASMC). The total biopterin content (BH4 and more oxidized states) of untreated RASMC was below our limit of detection. However, treatment with LPS caused a significant rise in biopterin levels and an induction of NO synthesis; both effects of LPS were markedly potentiated by interferon-gamma. 2,4-Diamino-6-hydroxypyrimidine (DAHP), a selective inhibitor of GTP cyclohydrolase I, the rate-limiting enzyme for de novo BH4 synthesis, completely abolished the elevated biopterin levels induced by LPS. DAHP also caused a concentration-dependent inhibition of LPS-induced NO synthesis. Inhibition of NO synthesis by DAHP was reversed by sepiapterin, an agent which circumvents the inhibition of biopterin synthesis by DAHP by serving as a substrate for BH4 synthesis via the pterin salvage pathway. The reversal by sepiapterin was overcome by methotrexate, an inhibitor of the pterin salvage pathway. Sepiapterin, and to a lesser extent BH4, dose-dependently enhanced LPS-induced NO synthesis, indicating that BH4 concentration limits the rate of NO production by LPS-activated RASMC. Sepiapterin also caused LPS-induced NO synthesis to appear with an abbreviated lag period phase, suggesting that BH4 availability also limits the onset of NO synthesis. In contrast to the stimulation of LPS-induced NO synthesis, observed when sepiapterin was given alone, sepiapterin became a potent inhibitor of NO synthesis in the presence of methotrexate. This is attributable to a direct inhibitory action of sepiapterin on GTP cyclohydrolase I, an activity which is only revealed after blocking the metabolism of sepiapterin to BH4. Further studies with sepiapterin, methotrexate, and N-acetylserotonin (an inhibitor of the BH4 synthetic enzyme, sepiapterin reductase) indicated that the BH4 is synthesized in RASMC predominantly from GTP; however, a lesser amount may derive from pterin salvage. We demonstrate that BH4 synthesis is an absolute requirement for induction of NO synthesis by LPS in vascular smooth muscle. Our findings also suggest that pterin synthesis inhibitors may be useful for the therapy of endotoxin- and cytokine-induced shock.

Amino Acid Oxidoreductases↗
PubMed1979

Biosynthesis of "drosopterins" by an enzyme system from Drosophila melanogaster.

The red eye pigment of Drosophila melanogaster consists of six complex pteridines known as neodrosopterin, drosopterin, isodrosopterin, fraction e, and aurodrosopterins (2); these pigments are greatly reduced in the purple mutant. Conditions for biosynthesis of these "drosopterins" are described and compared with those for the synthesis of sepiapterin. The enzymes are contained in a soluble, pteridine-free extract obtained between 40 and 60% saturated ammonium sulfate. The results indicate that sepiapterin synthase consists of two enzymes, the first of which provides a precursor for "drosopterin" biosynthesis. The evidence is (1) the purple mutant, low in accumulated sepiapterin and "drosopterins", is known to have approximately 10% of the sepiapterin synthase activity of wild type; (2) unlabeled sepiapterin does not cause isotope dilution of "drosopterin" synthesis; (3) the 600g pellet prepared from a wild-type head homogenate contains "drosopterin" synthesizing activity and no sepiapterin synthase, yet a heat-labile factor in this fraction stimulates sepiapterin synthesis in the 100000g supernatant of wild-type or pr flies; (4) sepiapterin and "drosopterin" syntheses require Mg2+; (5) sepiapterin synthesis is stimulated by NADPH; "drosopterin" synthesis responds to either NADPH or NADH. Although "drosopterins" are complex pteridine-type pigments, we have demonstrated their biosynthesis by soluble enzymes. This allows us to consider investigation into the mechanism by which the amounts of these pigments are regulated.

Animals↗
PubMed1979

Partial purification and some properties of biopterin synthase and dihydropterin oxidase from Drosophila melanogaster.

An enzyme which has been named "biopterin synthase" has been discovered in Drosophila melanogaster. This enzyme, which has been purified 200-fold from extracts of Drosophila, catalyzes the conversion of sepiapterin to dihydrobiopterin, or oxidized sepiapterin to biopterin. The Km values for the two substrates are 63 microM for sepiapterin and 10 microM for oxidized sepiapterin. NADPH is required in this enzymatic reaction. An analysis of enzyme activity during development in Drosophila indicates a correlation between enzyme activity and biopterin content at various development stages. Another enzyme, called "dihyropterin oxidase," was also discovered and partially purified. This enzyme catalyzes the oxidation of dihydropterin compounds to the corresponding pterin compounds. For example, sepiapterin (a dihydroterin) is oxidized to oxidized sepiapterin in the presence of this enzyme. The only dihydropterin that has been tested that is not a substrate for this enzyme is dihydroneopterin triphosphate, the compound thought to be a precursor for all naturally occurring pterins and dihydropterins. Since the action of dihydropterin oxidase is reduced significantly when the concentration of oxygen is very low, it is likely that this enzyme uses molecular oxygen as the oxidizing agent during the oxidation of dihydropterins. Neither NAD+ or NADP+ is required. In the presence of the two enzymes dihydropterin oxidase and biopterin synthase, sepiapterin is converted to biopterin. However, in the presence of biopterin synthase alone, sepiapterin is converted to dihydrobiopterin.

Alcohol Oxidoreductases↗
PubMed1998

Reduction by tetrahydrobiopterin of H2O2-induced endothelial cell injury.

The purpose of this study was to examine the effect of tetrahydrobiopterin, a co-factor of nitric oxide synthase, on H2O2-induced endothelial cell injury. Pretreatment with sepiapterin, a precursor of tetrahydrobiopterin biosynthesis, increased tetrahydrobiopterin content of endothelial cells, and reduced H2O2-induced endothelial cell injury, which was measured by leakage of lactate dehydrogenase. Both the increase in tetrahydrobiopterin content and the protective effect of sepiapterin were prevented by co-pretreatment with N-acetylserotonin, an inhibitor of sepiapterin reductase. Although Ca2+ ionophore ionomycin-induced nitric oxide synthesis was increased by pretreatment with sepiapterin, the protective effect of sepiapterin was not affected by an inhibitor of nitric oxide synthesis. On the other hand, pretreatment with sepiapterin also reduced H2O2-induced rat foetal lung fibroblast cell injury via an increase in tetrahydrobiopterin content, despite rat foetal lung fibroblast cells lacking nitric oxide synthase. Moreover, increase in tetrahydrobiopterin strongly reduced H2O2-induced intracellular oxidative stress. These findings indicate that sepiapterin reduces H2O2-induced endothelial cell injury via an increase in tetrahydrobiopterin content. Although increase in endothelial tetrahydrobiopterin content stimulated nitric oxide production, the protective effect of tetrahydrobiopterin against H2O2-induced endothelial cell injury is unlikely to be related to the stimulation of nitric oxide release from nitric oxide synthase. The protective effect of tetrahydrobiopterin may involve reactive oxygen species-scavenging activity.

Animals↗
PubMed1996

Effects of depletion of intracellular tetrahydrobiopterin in murine erythroleukemia cells.

The biosynthesis of 6(R)-5,6,7,8-tetrahydrobiopterin (BH4) in murine erythroleukemia (MEL) cells is almost completely inhibited by 10 mM, 2,4-diamino-6-hydroxypyrimidine (DAHP), which targets GTP cyclohydrolase. The inhibition results in dephosphorylation of the retinoblastoma gene product, prolongation of the G1-phase in the cell cycle, and subsequent commitment to terminal differentiation of MEL cells. Reversal of the processes by repletion of cellular BH4 with biopterin-related compounds including BH4, 7,8-dihydrobiopterin (7,8-BH2), sepiapterin, and 7,8-dihydroneopterin has generated complicated results. Low micromolar exogenous pterin compounds had little or no effect. At 300 microM or higher, the synthesis of hemoglobin by DAHP-induced MEL cells is significantly inhibited by 7,8-dihydrobiopterin and sepiapterin. However, further cell cycle analysis shows that the inhibition of cell differentiation by 7,8-BH2 and sepiapterin may not be due to the reversal of cell proliferation. Inhibition of BH4 biosynthesis in MEL cells by inhibitors of sepiapterin reductase has also been studied. None of the inhibitors that were tested, including N-chloroacetyl-dopamine and N-acetylserotonin, which are specific for sepiapterin reductase, can block MEL cells in G1-phase or induce the cells to commit to terminal differentiation. Furthermore, inhibitors of sepiapterin reductase are found to reduce or to abolish hemoglobin synthesis in differentiating MEL cells induced by hexamethylene bisacetamide. The mechanism for this is not clear. Not all of the effects caused by the depletion of BH4 synthesis can be rescued by repletion of BH4. These results suggest that BH4 may not regulate proliferation or differentiation of MEL cells as previously thought. Its function in MEL cells is still not clear.

Acetamides↗
PubMed2002

Altered tetrahydrobiopterin metabolism in atherosclerosis: implications for use of oxidized tetrahydrobiopterin analogues and thiol antioxidants.

OBJECTIVE: Tetrahydrobiopterin (BH4) is of fundamental importance for the normal function of endothelial NO synthase. The purpose of this study was to investigate the effects of hyperlipidemia on vascular BH4 levels and the effect of supplementation with sepiapterin in the presence and absence of N-acetylcysteine (NAC). METHODS AND RESULTS: New Zealand White rabbits were fed normal chow (normocholesterolemic [NC] group) or hyperlipidemic chow (hyperlipidemic [HL] group) for 8 to 10 weeks. Mean cholesterol levels were 1465+/-333 and 53+/-17 mg/dL in the HL and NC group, respectively. Markedly diminished BH4 levels were found in the HL group compared with the NC group, but these levels could be restored after 6 hours of incubation with sepiapterin. Peak relaxations to acetylcholine and A23187 were impaired in the HL group. Supplementation with sepiapterin resulted in a further diminution of relaxation in the HL but not NC group. Incubation with NAC for 6 hours failed to raise BH4 levels, whereas NAC in conjunction with sepiapterin raised BH4 levels approximately 221-fold. However, this increase did not improve relaxations to A23187 and acetylcholine. CONCLUSIONS: Prolonged exposure to sepiapterin impairs vasorelaxation in hyperlipidemia despite repletion of endogenous BH4. Antioxidant thiols do not correct this impairment. These studies have implications for the use of sepiapterin in the correction of vasomotor tone in atherosclerosis.

Acetylcholine↗
PubMed1998

Modulation of inducible nitric oxide synthase mRNA stability by tetrahydrobiopterin in vascular smooth muscle cells.

Tetrahydrobiopterin (BH4) regulates inducible nitric oxide synthase (iNOS) as cofactor and allosteric effector. The present paper describes a novel function of BH4 in vascular smooth muscle cells (SMC). By varying BH4 levels with dicumarol (an inhibitor of BH4 synthesis) and sepiapterin (an exogenous source of co-factor), we investigated iNOS expression in activated rat aortic SMC. In sepiapterin-supplemented cells, iNOS protein levels were increased while in dicumarol-treated cells, iNOS levels were diminished. Time-kinetic experiments revealed that inhibition or supplementation of BH4 synthesis had no effects on iNOS induction or transcription rate. However, iNOS mRNA was present over a prolonged time in sepiapterin-supplemented SMC. Analysis of iNOS mRNA levels showed stable iNOS mRNA in sepiapterin-treated cells 8 hours after transcription inhibition, while in dicumarol-treated cells iNOS mRNA disappeared. The decrease of iNOS mRNA by dicumarol was abolished by sepiapterin. These data indicate that BH4 post-transcriptionally stabilizes iNOS mRNA in SMC. By this way BH4 modulates iNOS expression in the vascular system.

Animals↗
PubMed1982

Purification and biosynthesis of quench spot, a drosopterin precursor in Drosophila melanogaster.

Pteridine biosynthesis has been examined in extracts of the heads of Drosophila melanogaster by measuring the conversion of dihydroneopterin triphosphate to sepiapterin and the "drosopterins" (six eye pigments that are dipterin derivatives). These two products share a common first step in the production of an intermediate that is a branch point from which both products are formed. This first step can be catalyzed by sepiapterin synthase or by an enzyme found in particles that sediment at 600g. A substance named "quench spot" was found earlier to be at low levels in the purple mutants that were defective in drosopterin synthesis and to be restored to normal when a suppressor mutant, su(s)2, restored drosopterins in purple to normal levels. The sepia mutant is also deficient in the levels of both quench spot and drosopterins. In this report we propose that quench spot is a precursor of drosopterins, but not sepiapterin, and that it is formed from the sepiapterin synthase intermediate mentioned above. An additional precursor that is formed independently of the sepiapterin synthase pathway is also proposed that would react with quench spot to form drosopterins. These proposals are based on the following: (1) quench spot biosynthesis is observed in extracts of Drosophila heads in which [U-14C]dihydroneopterin triphosphate is the substrate; (2) Mg2+ is required for the synthesis of quench spot but either NADH or NADPH causes diminished incorporation of the label; (3) extracts from heads of a purple mutant (prbwcn) contain only 30% of the quench spot biosynthetic activity as compared to heads from wild type (Oregon-R); (4) quench spot has been purified from heads of wild-type Drosophila; (5) addition of quench spot stimulates the biosynthesis of drosopterins in an enzyme preparation from Oregon-R.

Animals↗
PubMed1983

Biosynthesis of tetrahydrobiopterin by de novo and salvage pathways in adrenal medulla extracts, mammalian cell cultures, and rat brain in vivo.

Mammalian cells and tissues were found to have two pathways for the biosynthesis of tetrahydrobiopterin (BH4): (i) the conversion of GTP to BH4 by a methotrexate-insensitive de novo pathway, and (ii) the conversion of sepiapterin to BH4 by a pterin salvage pathway dependent on dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) activity. In a Chinese hamster ovary cell mutant lacking dihydrofolate reductase (DUKX-B11), endogenous formation of BH4 proceeds normally but, unlike the parent cells, these cells or extracts of them do not convert sepiapterin or 7,8-dihydrobiopterin to BH4. KB cells, which do not contain detectable levels of GTP cyclohydrolase or BH4 but do contain dihydrofolate reductase, readily convert sepiapterin to BH4 and this conversion is completely prevented by methotrexate. In supernatant fractions of bovine adrenal medulla, the conversion of sepiapterin to BH4 is completely inhibited by methotrexate. Similarly, this conversion in rat brain in vivo is methotrexate-sensitive. Sepiapterin and 7,8-dihydrobiopterin apparently do not enter the de novo pathway of BH4 biosynthesis and may be derived from labile intermediates which have not yet been characterized.

Adrenal Medulla↗
PubMed2000

Development of the pteridine pathway in the zebrafish, Danio rerio.

In the zebrafish, the peripheral neurons and the pigment cells are derived from the neural crest and share the pteridine pathway, which leads either to the cofactor tetrahydrobiopterin or to xanthophore pigments. The components of the pteridine pattern were identified as tetrahydrobiopterin, sepiapterin, 7-oxobiopterin, isoxanthopterin, and 2,4,7-trioxopteridine. The expression of GTP cyclohydrolase I activity during the first 24-h postfertilization, followed by 6-pyruvoyl-5,6,7,8-tetrahydropterin synthase and sepiapterin reductase, suggest an early supply of tetrahydrobiopterin for neurotransmitter synthesis in the neurons and for tyrosine supply in the melanophores. At 48-h postfertilization, sepiapterin formation branches off the de novo pathway of tetrahydrobiopterin synthesis. Sepiapterin, via 7,8-dihydrobiopterin and biopterin, serves as a precursor for the formation of 7-oxobiopterin, which may be further catabolized to isoxanthopterin and 2,4,7-trioxopteridine. Neither 7, 8-dihydrobiopterin nor biopterin is a substrate for xanthine oxidoreductase. In contrast, both of these compounds are oxidized at C-7 by a xanthine oxidase variant form, which is inactivated by KCN, but is insensitive to allopurinol. The oxidase and the dehydrogenase form of xanthine oxidoreductase as well as the xanthine oxidase variant have specific developmental patterns. It follows that GTP cyclohydrolase I, the formation of sepiapterin, and the xanthine oxidoreductase family control the pteridine pathway in the zebrafish.

Alcohol Oxidoreductases↗
PubMed1985

Tetrahydrobiopterin biosynthesis. Studies with specifically labeled (2H)NAD(P)H and 2H2O and of the enzymes involved.

The biosynthesis of tetrahydrobiopterin from either dihydroneopterin triphosphate, sepiapterin, dihydrosepiapterin or dihydrobiopterin was investigated using extracts from human liver, dihydrofolate reductase and purified sepiapterin reductase from human liver and rat erythrocytes. The incorporation of hydrogen in tetrahydrobiopterin was studied in either 2H2O or in H2O using unlabeled NAD(P)H or (R)-(4-2H)NAD(P)H or (S)-(4-2H)NAD(P)H. Dihydrofolate reductase catalyzed the transfer of the pro-R hydrogen of NAD(P)H during the reduction of 7,8-dihydrobiopterin to tetrahydrobiopterin. Sepiapterin reductase catalyzed the transfer of the pro-S hydrogen of NADPH during the reduction of sepiapterin to 7,8-dihydrobiopterin. In the presence of partially purified human liver extracts one hydrogen from the solvent is introduced at position C(6) and the 4-pro-S hydrogen from NADPH is incorporated at each of the C(1') and C(2') position of BH4. Label from the solvent is also introduced into position C(3'). These results suggest that dihydrofolate reductase is not involved in the biosynthesis of tetrahydrobiopterin from dihydroneopterin triphosphate. They are consistent with the assumption of the occurrence of a 6-pyruvoyl-tetrahydropterin intermediate, which is proposed to be formed upon triphosphate elimination from dihyroneopterin triphosphate, and via an intramolecular redox reaction. Our results suggest that the reduction of 6-pyruvoyl-tetrahydropterin might be catalyzed by sepiapterin reductase.

Affinity Labels↗
PubMed1982

Biosynthesis of biopterin by rat brain.

A method for the determination of [14C]biopterin biosynthesis from [14C]guanosine-5'-triphosphate by a desalted preparation from rat striatum, based on sequential reverse-phase and cation-exchange high performance liquid chromatography, is described. Synthesis of reduced forms of biopterin by this striatal extract was found to be dependent on enzymatic activity, guanosine-5'-triphosphate, magnesium ions, and a reduced pyridine nucleotide. As demonstrated by the technique of isotope dilution, isotope trapping, 6-lactyl-7,8-dihydropterin (sepiapterin) was found to be an intermediate in biopterin biosynthesis that is catalyzed by the striatal extract. Rat brain was also shown to synthesize biopterin in vivo from intraventricularly administered [14C]guanosine or sepiapterin. Intraventricular injection of sepiapterin increased dihydro- and 5,6,7,8-tetrahydrobiopterin levels in rat brain by more than eightfold. The temporal relationship between the appearance of dihydro- and 5,6,7,8-tetrahydrobiopterin following intraventricular injection of sepiapterin suggests that dihydrobiopterin is the immediate product of sepiapterin reduction which is then reduced further to the functional cofactor 5,6,7,8-tetrahydrobiopterin. Therefore, in contrast to previous reports, the biosynthesis of biopterin by rat brain does not appear to differ from that occurring in other, nonneural tissues.

Animals↗