Cellular uptake of sepiapterin and push–pull accumulation of tetrahydrobiopterin

doi:10.1016/j.ymgme.2008.04.007

Molecular Genetics and Metabolism

Volume 94, Issue 4, August 2008, Pages 410-416

https://doi.org/10.1016/j.ymgme.2008.04.007 Get rights and content

Abstract

Cellular uptake of sepiapterin resulted in an efficient accumulation of tetrahydrobiopterin. Tetrahydrobiopterin is much less permeable across the cell membrane than sepiapterin or dihydrobiopterin, the precursors of the tetrahydrobiopterin-salvage pathway. The uptake of sepiapterin by the cell was examined under metabolic arrest with N-acetylserotonin, an inhibitor of sepiapterin reductase. The release profile of previously accumulated sepiapterin was also analyzed. Two routes were clearly distinguishable, namely rapid and slow. Both were apparently bi-directional and equilibrating in type. Each route was connected to non-mixable pools somehow separated in the cell. The rapid process was too fast to analyze by the current methods of cell handling. The slower process was associated with conversion of sepiapterin to tetrahydrobiopterin in the absence of N-acetylserotonin, suggesting that this route opens into the cytosolic compartment where use of the salvage pathway was strongly driven by sepiapterin reductase and dihydrofolate reductase with a supply of NADPH which favors tetrahydrobiopterin accumulation. Consequently, sepiapterin was enforcedly taken up by the cell where it accumulated tetrahydrobiopterin in the cytosol in continuous manner.

Introduction

Tetrahydrobiopterin (BH₄) is an essential cofactor for aromatic amino acid hydroxylases of phenylalanine [1], tyrosine [2], and tryptophan [3], [4], fatty acid glycerylether oxygenase [5], and nitric oxide (NO) synthase [6], [7]. BH₄ is synthesized de novo from guanosine triphosphate (GTP), thus it is defined as an endogenous metabolite rather than as a vitamin. The biosynthetic pathway involves at least 4 essential enzymes, GTP-cyclohydrolase I, 6-pyruvoil-tetrahydropterin synthase, 6-pyruvoil-tetrahydropterin 2’-reductase and sepiapterin reductase as reviewed [8], [9], [10]. The immediate precursor of BH₄ in the de novo synthetic pathway is believed to be 6-lactoil-tetrahydropterin, a substrate of sepiapterin reductase. Sepiapterin (SP) is a dihydro form of 6-lactoil-pterin. Since biopterin-deficient mutants of sepia of a fruit fly Drosophila melanogaster and lemon of a silk worm Bombyx mori deposited SP, this pterin was long believed to be the intermediate in biopterin biosynthesis [11]. In normal mammalian tissues, however, neither SP nor 6-lactoil-tetrahydropterin accumulate to any detectable extent.

BH₄ supplementation has been employed in treating patients who are genetically BH₄-deficient. Since BH₄ is an essential cofactor in the production of NO and aromatic monoamines such as dopamine and serotonin, a vast range of vascular and neural disease states has been recognized as potentially benefiting from effective BH₄ supplementation. Further, many classical PKU patients were found to respond to BH₄ supplementation therapy by a reduction in their blood phenylalanine levels [12]. However, due to the extremely low efficiency of the uptake and/or short retention of administered BH₄, its supplementation is not always prescribed except in the case of a malignancy. Cellular homeostasis of BH₄, i.e. mechanisms of uptake and release which maintain a steady endogenous level of BH₄, has not been well understood. To explore methods for effective BH₄ supplementation, the aim of this work was to elucidate the mechanism by which SP is taken up by the cell. The involvement of transporters in SP permeation is described for the first time, and the long known advantage of SP in BH₄ supplementation now stands on a scientific basis.

Section snippets

Materials and methods

l-erythro-(6R)-Tetrahydrobiopterin (6R-BH₄) was donated by Asubio Pharma (Tokyo, Japan), and SP and 7,8-dihydrobiopterin (7,8-BH₂) were purchased from Schirks Laboratories (Jona, Switzerland). N-acetylserotonin (NAS) and methotrexate (MTX) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Stock solutions of the pterin compounds and MTX and NAS were dissolved in 0.1 M HCl (5 mM or 20 mM), 0.2 M NaOH (10 mM) and DMSO (100 mM), respectively. Usually, 100× working solutions

BH₄ accumulation in the cell by feeding SP

Administration of SP to RBL2H3 cells caused a dramatic increase in BH₄ (more than 50-fold within 3 h). In contrast, biopterin accumulation due to BH₄ feeding was only several fold over the endogenous level of BH₄ even after prolonged incubation, as shown in Fig. 1a. Our extended experiments further revealed that the increase in BH₄ noted after BH₄ feeding was mainly due to the uptake and reduction of BH₂, which was secondarily formed from BH₄ during the incubation as a sort of artifact (reviews

Discussion

The conversion of sepiapterin (SP) to enzymically active biopterin was first demonstrated in rat liver extracts [16]. The involvement of SP in the biosynthesis of biopterin, representing the salvage pathway, was evidenced using larvae of the bull frog Rana catesbeiana [11] as well as rat brain [17]. SP is chemically less active than BH₄. It does not function in the cell as the coenzyme, nonetheless, the ready uptake of SP and enzymic conversion to BH₄ were of great advantage in various

Conclusion

Sepiapterin can move across the cell membrane in both an inward and outward direction. BH₄, however, is virtually unable to cross the cell membrane in either direction. The two successive reactions, SP to BH₂, and BH₂ to BH₄, favor production of BH₄ due to cellular redox-homeostasis. Consequently, SP is enforcedly taken up by the cell and BH₄ is accumulated in the cytosol in a continuous fashion.

Acknowledgments

This work was supported by the High-Tech Research Center project for Private Universities: a matching fund subsidy from MEXT (Ministry of Education, Culture, Sports, Science and Technology), 2003–2007. We are grateful for the kind support of Kaneka Corporation. We thank Doctors Naohiko Anzai (Department of Pharmacology and Toxicology, Kyorin University School of Medicine), Hitoshi Endou (Fuji Biomedix Co., Ltd.), Kazuhiko Matsumoto (Torii Pharmaceutical Co., Ltd.), and Michihiro Kasahara

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The extracellular concentration of BH4 in brain is not known but presumably would be no higher than its plasma concentration of around 0.1 μM in rodents (Ohashi et al., 2016). The relatively high intracellular concentration of BH4 is maintained actively by de novo and salvage pathways of BH4 synthesis, both of which are ultimately driven by the redox potential of NADPH (Sawabe et al., 2008). As is the case with tryptophan, it is thought that cellular levels of BH4 are near or slightly below saturation for TPH (Miwa, Watanabe, & Hayaishi, 1985) and increases in its concentration could stimulate 5-HT synthesis.
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Other preclinical data suggest that a substantial proportion of BH4 is degraded in the gut following oral administration [19], and a study in humans showed that exposure to BH4 was 58–76% greater following sublingual, compared with oral, administration [20]. It appears that increases in systemic levels of BH4 following an oral dose of a preparation of BH4 arise mainly from initial conversion of BH4 to BH2 during absorption into the blood, followed by uptake of BH2 into cells, where it is reconverted to BH4 via the BH4salvage pathway (Fig. 1) [18,21,22]. In addition, the incorporation of sepiapterin into cultured cells in vitro proceeded about 10 times faster than incorporation of BH4 [23].
Tetrahydrobiopterin (BH₄) is the natural cofactor of aromatic amino acid hydroxylases and essential for degradation of phenylalanine and synthesis of catecholamines and serotonin. It can be synthesized either de novo from GTP or through the salvage pathway from sepiapterin. Sepiapterin, a natural precursor of BH₄, is a more stable molecule and is transported more efficiently across cellular membranes, thus having potentially significant advantage over BH₄ as a pharmacological agent for diseases associated with BH₄-deficient conditions.
We report the results of a first-in-humans, randomized, double-blind, placebo-controlled, dose-ranging, Phase I clinical trial in 83 healthy volunteers of CNSA-001, a novel formulation of sepiapterin. Single oral doses of 2.5–80 mg/kg CNSA-001 caused dose-related increases in plasma sepiapterin (mean C_max 0.58–2.92 ng/mL) and BH₄ (mean C_max 57–312 ng/mL). Maximum plasma concentrations were achieved in about 1–2 h (sepiapterin) or about 4 h (BH₄) after CNSA-001 oral intake.
Increases in plasma BH₄ were substantially larger in absolute terms and on a dose-for-dose basis following treatment with CNSA-001 vs. sapropterin dihydrochloride, a synthetic form of BH₄. The pharmacokinetics of plasma sepiapterin and BH₄ were similar before and after seven days of repeat daily dosing with CNSA-001 at 5, 20 or 60 mg/kg indicating little or no drug accumulation. Oral administration of CNSA-001 resulted in higher concentrations of sepiapterin in fasted vs. fed subjects, but overall BH₄ plasma exposure following CNSA-001 intake increased by 1.7–1.8-fold in fed subjects. CNSA-001 was well tolerated, with no clear dose-relationship for adverse events (AE), no serious AE and no study discontinuations for AE. These data indicate that CNSA-001 is rapidly and efficiently converted to BH₄ in humans supporting further clinical evaluation of CNSA-001 for the management of PKU, primary BH₄ deficiencies and other diseases associated with deficient BH₄ metabolism.
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Tetrahydrobiopterin (BH4) is an essential cofactor of aromatic amino acid hydroxylases and nitric oxide synthase so that BH4 plays a key role in many biological processes. BH4 deficiency is associated with numerous metabolic syndromes and neuropsychological disorders. BH4 concentration in mammals is maintained through a de novo synthesis pathway and a regeneration pathway. Previous studies showed that the de novo pathway of BH4 is similar between insects and mammals. However, knowledge about the regeneration pathway of BH4 (RPB) is very limited in insects. Several mutants in the silkworm Bombyx mori have been approved to be associated with BH4 deficiency, which are good models to research on the RPB in insects. In this study, homologous genes encoding two enzymes, pterin-4a-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR) involving in RPB have been cloned and identified from B. mori. Enzymatic activity of DHPR was found in the fat body of wild type silkworm larvae. Together with the transcription profiles, it was indicated that BmPcd and BmDhpr might normally act in the RPB of B. mori and the expression of BmDhpr was activated in the brain and sexual glands while BmPcd was expressed in a wider special pattern when the de novo pathway of BH4 was lacked in lemon. Biochemical analyses showed that the recombinant BmDHPR exhibited high enzymatic activity and more suitable parameters to the coenzyme of NADH in vitro. The results in this report give new information about the RPB in B. mori and help in better understanding insect BH4 biosynthetic networks.

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Cellular uptake of sepiapterin and push–pull accumulation of tetrahydrobiopterin

Abstract

Introduction

Section snippets

Materials and methods

BH4 accumulation in the cell by feeding SP

Discussion

Conclusion

Acknowledgments

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

Arch. Biochem. Biophys.

J. Pediatr.

Anal. Biochem.

J. Biol. Chem.

J. Biol. Chem.

J. Pharmacol. Sci.

J. Pharmacol. Sci.

Tryptophan hydroxylation: measurement in pineal gland, brainstem, and carcinoid tumor

Science

Biosynthesis and metabolism of tetrahydrobiopterin and molybdopterin

Annu. Rev. Biochem.

Tetrahydrobiopterin biosynthesis, regeneration and functions

Biochem. J.

Tetrahydrobiopterin biosynthesis, utilization and pharmacological effects

Curr. Drug Metab.

BH₄ accumulation in the cell by feeding SP