Yamamoto et al.: Tetrahydrobiopterin uptake Pteridines Vol. 7, 1996, pp. 154~ 156 A Comparison of Sepiapterin and Tetrahydrobiopterin Uptake by BRL2H3 Cells 2 Kazumasa Yamamoto!, Mutsumi Nagano!, Nobuo Nakanishi , and Hiroyuki Hasegawa! § !Department of Bioscience, Teikyo University of Science, Uenohara, Yamanashi 409-01 2Department of Biochemistry, Meikai University School of Dentistry, Sakato, Saitama 350-02, Japan (Received October 4, 1996) Summary Cellular uptake of sepiapterin and tetrahydrobiopterin was compared quantitatively with RBL2H3 cells in culture. RBL2H3 is a mast cell line and has the ability to synthesize serotonin. Sepiapterin was in­ corporated and converted into tetrahydrobiopterin in the cell. The incorporation of sepiapterin was about 10 times faster than tetrahydrobiopterin. A low concentration of tetrahydrobiopterin was in­ corporated in a saturable manner up to about 10 J..LM, and it was superimposed upon a linearly con­ centration-dependent process. Overnight treatment with DAHP depleted endogenous tetrahy­ drobiopterin. The DAHP treated cells took up tetrahydrobiopterin faster than untreated cells. Introduction In this report, incorporation of sepiapterin and tetrahydrobiopterin was compared using RBL2H3 Tetrahydrobiopterin plays an essential role as cells in culture. The cells can synthesize. tetrahy­ cofactor of variety of hydroxylases or oxygen-linked drobiopterin and utilize it in its biosynthesis of enzymic reactions such as phenylalanine hydroxy­ serotonin as a cofactor of tryptophan hydroxylase. lase (1), tyrosine hydroxylase (2), tryptophan hy­ droxylase (3), glyceryl-ether mono-oxygenase (4), Materials and Methods and NO-synthetase (5). In case of newborn error in tetrahydrobiopterin metabolism which leads to Tetrahydrobiopterin was donated by Suntory Ltd. insufficient supply of this compound, tetrahydro­ (Tokyo, Japan), sepiapterin was purchased from biopterin therapy is unavoidable. It is well known, Schirks Laboratories (Jona, Switzerland). RBL2H3, however, that incorporation of tetrahydrobiopterin a mast cell line derived from rat basophilic leu­ into cells is not efficient when the compound must kemia cells, was obtained from The Japanese Can­ be supplied especially in the brain. cer Research Resources Bank (Tokyo, Japan). Tetrahydrobiopterin is synthesized from GTP via RBL2H3 cells were kept as a monolayer culture in neopterin-triphosphate through tetrahydropterin Dulbecco's Modified Eagle's Medium (DMEM) compounds following of de novo pathway. Sepia­ containing 10% fetal calf serum. All cultures were pterin has been known to be incorporated and be maintained at 37"C under 5% CO2/95% air. For converted to tetrahydrobiopterin, this pathway is biopterin depletion experiments, cells were given 5 now called a salvage pathway (6). mM 2,4-diamino-6-hydroxypyrimidine (DAHP) overnight and through the experiment. Although § Author to whom correspondence should be addressed. tetrahydrobiopterin is rather stable under acid con- Pteridines/ Vol. 7 / No. 4 Yamamoto et at.: Tetrahydrobiopterin uptake 155 ditions, it is labile in neutral pH conditions. In the 6000 culture medium, the half-life was about 2 hours. Hence tetrahydrobiopterin was added in the cul­ ~ Q) ture medium with 1 mM DTI which allowed u 4000 <D tetrahydrobiopterin to be stable for 2 hr at least. 0 Determination of tetrahydrobiopterin in the cell was conducted essentially according to the method "0 E 2000 of Fukushin1a and Nixon (7). For routine work, ..9: Q.. however, acid oxidation was employed for measur­ cc ing total biopterin. In brief, either tetrahydro­ 0 biopterin or sepiapterin was given to the culture 0 50 100 150 200 and after the incubation, the cells were washed Incubation Period (min) three times with Dulbecco's phosphate buffered sa­ Figure 1. Time dependent incorporation of sepiapterin line (PBS), then HCI was added to the cells to and tetrahydrobiopterin by RBL2H3 cells in culture: bring it to 0.1 N, containing 1.2 ~M of pterin (a RBL2H3 cells were given 50 ~M sepiapterin (circles) or far larger amount than endogenous pterin) as an tetrahydrobiopterin (dots). At the indicated times, cells internal standard. I2/KI solution was added and were washed 3 times with PBS then subjected to acid the excessive iodine was reduced with ascorbic acid. oxidation with iodine for measuring biopterin (n=5). In­ For determination of tetrahydrobiopterin, pterin set: the same data for tetrahydrobiopterin were replotted was measured after alkaline oxidation with I2/KI in in expanded scale. (n=5) 0.1 N NaOH without the internal standard. Bio­ pterin and pterin were determined using an HPLC equipped with a fluorescence monitor (JASCO, Tokyo, Japan, FP-920) set 350 nm and 450 nm for excitation and emission, respectively. The solid phase used FineSIL C18-5T and the mobile phase used 7% methanol. "0 Results ~ Q.. In order to compare the incorporation of sepia­ CC pterin with that of tetrahydrobiopterin, 50 /J.M concentrations of these compounds were added o 10 20 30 40 so to RBL2H3 cells in monolayer culture. The initial Tetrahydrobiopterin (J.L M) amount of cellular biopterin was, in a typical case, 108.8± 17.5 pmoljl06 cells. As shown in Fig. 1, Figure 2. Concentration-dependent incorporation of sepiapterin was incorporated and converted to tetrahydrobiopterin with DAHP-treated (dots) or non­ treated (circles) RBL2H3 cells: RBL2H3 cells were treat­ biopterin linearly for at least 1 hours. The cellular ed with or without 5 mM DAHP overnight. Cells were biopterin amount after 1 hr incubation with 50 given tetrahydrobiopterin at concentrations indicated in 6 /J.M sepiapterin reached 4,125±287 pmoljl0 the figure. Two hours later, cells were washed and acid­ cells. A significant amount of biopterin was not oxidized, then the cellular biopterin was measured. (n=5) detected when the samples were subjected to al­ kaline oxidation, indicating that the redox-state of cellular biopterin was mainly (>98%) in the its efflux because the incorporated sepiapterin is tetrahydro- or quinonoid dihydro-form. On the converted to dihydrobiopterin and hence the other hand, the increase in biopterin in the pre­ equilibrium between sepiapterin inside and out­ sence of 50 /J.M tetrahydrobiopterin was only side is continuously advanced by the influx. In 475.3±46.6 pmoljl06 cells; this rise was about contrast, tetrahydrobiopterin uptake may be en­ one tenth that of sepiapterin. countered to the efflux of tetrahydrobiopterin. In Sepiapterin is known to be converted into 7,8- the latter case, intracellular concentration may re­ dihydrobiopterin by the enzymatic action of sepia­ flect downward to the efficiency of the biopterin pterin reductase and then to tetrahydrobiopterin uptake. Thus we established biopterin-depleting by diliydrofolate reductase (salvage pathway). The conditions. Cells were exposed to 5 mM DAHP sepiapterin influx might not be encountered with overnight and the intracellular biopterin con- Pteridines/Vol. 7/No. 4 156 Yamamoto et at.: Tetrahydrobiopterin uptake centration decreased below the detection limit, corporated by diffusion. The fact that DAHP treat­ whereas control cells (not-treated with DAHP) ed cells took up tetrahydrobiopterin to a greater ex­ contained 41.4± 5 pmol of biopterin per 106 cells. tent may suggest an ability of the cell to attenuate When various concentrations of tetrahydrobio­ the incorporation, depending on the intracellular pterin were given to the DAHP-treated cells, the concentration of the pterin. This experiment was incorporation of tetrahydrobiopterin was signi­ performed after overnight exposure of the cells to 5 ficantly more efficient than that observed with the mM DAHP. Although no indication of cellular control cells at concentrations higher than 2-3 damage was observed, this treatment could have im­ ~ (Fig. 2). In both cases, with or without DAHP posed certain modifications upon the cells causing treatment, the concentration dependence seems to increased membrane permeability. be related to the saturable process up to about 10 In conclusion, sepiapterin incorporation by ~M and a process of linear-concentration depen­ RBL2H3 cells was efficient, whereas tetrahy­ dence. drobiopterin uptake was inefficient. Biopterin-de­ ficient cells may take up tetrahydrobiopterin more Discussion efficiently than cells that already have an ordinary level of the pterin. In the early work by Fukushima (8) on biosyn­ thetic pathways of biopterin from GTP, sepiapterin Acknowledgment administered to tadpoles of Rana catesviana was de­ monstrated to dilute the radioactivity of biopterin We are grateful to Emiko Asakawa and Naomi derived from GTP-(U)-14C efficiently. This was in­ Nakazawa for their collaboration in the Student terpreted to indicate that sepiapterin was an in­ Research Program 1996. This research was sup­ termediate in biopterin biosynthesis and suggested ported by the Japan Private School Promotion that sepiapterin was efficiently taken up and merged Foundation. into the metabolic pathway of biopterin. Based on the current knowledge regarding the bio­ References synthesis of tetrahydrobiopterin, the above phe­ nomenon was new evidence that the salvage path­ 1. Kaufinan S. A new cofactor required for the enzy­ way was functioning. Efficient uptake of sepia­ matic conversion of phenylalanine to tyrosine. J. BioI. pterin by primary neuron culture of rat superior Chern. 1958; 234: 931-939. 2 . Nagatsu T ., Levitt M., Vdenfriend S. Tyrosine hy­ cervical ganglion and conversion to tetrahydro­ droxylase. The initial