Sepiapterin Research Articles

Role of tetrahydrobiopterin in resistance to myocardial ischemia in Brown Norway and Dahl S 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.

Role of a Serotonin Precursor in Development of Gut Microvilli

Monoamines exert diverse functions in various cells in peripheral organs as well as in the central nervous system. 5-Hydroxy-l-tryptophan (5-HTP) has been simply regarded as a precursor of serotonin, and it is believed that the biological significance of 5-HTP is essentially ascribable to the production of serotonin. Systemic treatment with 5-HTP is often applied to patients with low serotonin levels in the brain. Here we show that endogenous and exogenous 5-HTP but not serotonin induced the development of microvilli in the gut villi epithelium. In contrast, serotonin but not 5-HTP regulated phagocytosis by macrophages. 5-HTP specifically induced actin remodeling and decreased phosphorylation of extracellular signal-regulated kinase (ERK) in the gut, whereas serotonin stimulated actin remodeling and increased ERK phosphorylation in macrophages. Functionally, inhibition of ERK activity promoted the development of microvilli in the gut and ameliorated phagocytosis by macrophages. Thus, 5-HTP and serotonin contribute to distinct cell-type-specific functions via common mediators. Our study might create an opportunity to explore the effects of exogenously applied 5-HTP in humans.

Singlet Oxygen (O2(1Δg)) Quenching by Dihydropterins

Pterins belong to a class of heterocyclic compounds present in a wide range of living systems. They participate in relevant biological functions and are involved in different photobiological processes. Dihydropterins are one of the biologically active forms of pterins. The photoinduced production and quenching of singlet oxygen (1O2) by a series of dihydropterins (7,8-dihydrobiopterin (DHBPT), 7,8-dihydroneopterin (DHNPT), 6-formyl-7,8-dihydropterin (FDHPT), sepiapterin (SPT), 7,8-dihydrofolic acid (DHFA), and 7,8-dihydroxanthopterin (DHXPT)) in aqueous solution at physiological pH ( approximately 7) were investigated, and the quantum yields of 1O2 production (PhiDelta) and rate constants of total quenching (kt) of 1O2 were determined. Studied compounds do not produce 1O2 under UV-A irradiation and are very efficient 1O2 quenchers. The chemical reactions between 1O2 and dihydropterin derivatives were investigated, and the corresponding rate constants (kr) were found to be particularly high. The oxidized pterin derivatives, biopterin (BPT), neopterin (NPT), 6-formylpterin (FPT), and folic acid (FA), were identified and quantified during the reaction of 1O2 with DHBPT, DHNPT, FDHPT, and DHFA, respectively. Besides the oxidation of the dihydropyrazine ring to yield the corresponding oxidized pterins, a second oxidation pathway, leading to fragmentation of the dihydropterin and formation of non-pterinic products, was identified. Mechanisms and biological implications are discussed.

Exogenous biopterins requirement for iNOS function in vascular smooth muscle cells.

The authors evaluated whether supplementation of tetrahydrobiopterin (BH4) or the BH4 precursor in vascular smooth muscle cells (VSMC) that are deficient in de novo BH4 production affected the ability of these cells to synthesize nitric oxide (NO). GTP cyclohydrolase I (GTPCH) is the rate-limiting enzyme for the de novo synthetic pathway of BH4. The selective GTPCH inhibitor 2,4-diamino-6-hydroxypyrimidine (DAHP) restricts the de novo synthesis of BH4 in VSMC. Thus, treatment with DAHP and cytokines should lead to the intracellular accumulation of inactive inducible nitric oxide synthase (iNOS) apoenzyme (relative to BH4), which can be reactivated upon the repletion of BH4. Using such VSMC deficient in de novo BH4, the authors evaluated their ability to synthesize NO by supplementing with BH4 or the BH4 precursor sepiapterin or dihydrobiopterin. Adding BH4 to VSMC increased NO production in a concentration-dependent manner. Sepiapterin (SEP) or 7,8-dihydrobiopterin (BH2) also dose-dependently induced NO generation. Nitric oxide was produced in the order SEP >BH2>> BH4 at half-maximal concentrations for stimulation of 0.05, 0.1, and 1 micromol/L, respectively. Addition of BH4 or SEP substantially induced iNOS enzyme activity, which was quantified as the formation of [3H]L-citrulline from [3H]L-arginine. SEP or BH2 in the presence of methotrexate, a selective inhibitor of dihydrofolate reductase, no longer induced NO production. In contrast, although the amount of NO induced by supplemented BH4 was substantially depressed, higher concentrations of BH4, but not SEP or BH2, significantly caused NO production even in the presence of methotrexate. Thus, BH4 produced from BH2 is largely responsible for NO production in VSMC that are deficient in de novo BH4. NOS would preferentially use BH4 that is regenerated from BH2 via dihydrofolate reductase, rather than BH4 or BH2 obtained from the cytosolic milieu.

Cytokine-activated endothelial cells express an isotype of nitric oxide synthase which is tetrahydrobiopterin-dependent, calmodulin-independent and inhibited by arginine analogs with a rank-order of potency characteristic of activated macrophages

We have previously reported that cultured murine brain endothelial cells (MBE) produce large quantities of nitric oxide (NO •) after activation with interferon-gamma in combination with any of several immunoactivators including: bacterial endotoxin, tumor necrosis factor and interleukin-1. Since endothelial cells are the first example of a cell-type which may possess both a constitutive and an inducible type of NO • synthase, it was of interest to compare the requirements of these two enzyme activities. Induction of NO • synthesis in MBE by cytokines was abolished by the protein synthesis inhibitor, cycloheximide, and by 2,4-diamino-6-hydroxypyridine (DAHP), a selective inhibitor of GTP cyclohydrolase I, the rate-limiting enzyme for de novo synthesis of tetrahydrobiopterin (THB). In the presence of DAHP, NO • synthesis was restored by sepiapterin (SEP), a substrate for the alternative pathway of THB synthesis occuring via pterin salvage. Moreover, SEP increased NO • synthesis to >150% of control values, suggesting that THB availability is rate-limiting for NO • synthesis by cytokine-induced MBE. Methotrexate, an inhibitor of the pterin salvage pathway of THB synthesis, completely reversed the stimulation of NO • synthesis by sepiapterin. Thus, cytokine-induced MBE NO • synthase appears to have an absolute requirement for THB as cofactor. In additional studies we found that NO • synthesis by cytokine-activated MBE was inhibited by N G-monosubstituted arginine analogs with a rank-order of potency NH 2 > CH 3 > NO 2, in contrast with the rank-order of NO 2 > NH 2 > CH 3 previously described for inhibition of the constitutive endothelial cell enzyme. Using a kinetic assay for NO • synthase activity, based on oxidation of myoglobin heme-iron, we have found that these rank orders of potency also apply to cytosol preparations of cytokine-induced and untreated endothelial cells, respectively. Further differences between constitutive and cytokine-induced NO • synthase were observed with regard to calmodulin requirements. Whereas constitutive NO • synthase was potently inhibited by the calmodulin antagonists mellitin and trifluoperazine, cytokine-induced NO • synthase was unaffected. In summary, NO • synthesis by cytokine-activated MBE is THB-dependent, calmodulin-independent and inhibited by NG-substituted arginine analogs with a rank-order profile distinct from that for untreated endothelial cells but identical to that for cytokine-activated macrophages.

On the presence of sepiapterin reductase different from folate and dihydrofolate reductase in chicken liver.

Nous avons demontre dans le foie de poussin l'existence d'un ferment qui reduit specifiquement la sepiapterine. Il est capable de separer le ferment de reductase d'acide folique et d'acide dihydrofolique apres precipitation fractionnaire de sulfate d'ammonium. Le ferment, reductase de sepiapterine, se trouve largement dans les foies des mammiferes, tels que rat, cochon, lapin, cheval et boeuf.

キイロショウジョウバエのヘンナ座位における対立遺伝子作用の生化学的研究

The allelism at Henna locus in Drosophila melanogaster was iochemically investigated. From the experlmental, results Henna-recessive alleles seem to be pseudoallelic.Red and yellow eye pigments extracted from male heads with 30 per cent acidic ethanol were estimated photometrically and compared with each other. Various genotypes resulting from allele combinations, regarding the eye pigment formation are in the following order:+/+>+/Hnr-1=+/Hnr-3>Hnr-1/Hnr-1>Hnr-1/Hnr-3=Hnr-3/Hnr-1>Hnr-3/Hnr-3.The separation of pteridines extracted from male heads and bodies was conducted by means of two-dimensional paper chromatography. Relative amounts of the separated pteridines were measured fluorometrically. A positive correlation between the amounts of sepiapterin (SP) and biopterin (BP) was recognized. A striking difference between the alleles was detected in amounts of SP and isoxanthopterin (IXP); the Hnr-3 homozygotes had less IXP and more SP than the other genotypes.The sepiapterin reductase, closely related to the conversion of SP to 2-amino-4-hydroxypteridine (AHP) was remarkably lower activity in Hnr-3 than in the others. Hence, the accumulation of SP in the Hnr-3 flies, especially in testes, was considered to depend upon the low activity of that enzyme.A synthetic pathway of Drosophila pteridines was proposed from the experimental results. The Hnr-3 gene apparently inhibits the process between dihydropteridine (YPH2) and tetrahydropteridine (YPH4), but the Hnr-1 gene does not inhibit this step.