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Tetrahydrobiopterin (BH4) is a common coenzyme of phenylalanine-, tyrosine-, and tryptophan hydroxylases, alkylglycerol monooxygenase, and NO synthases (NOS). Synthetic BH4 is used medicinally for BH4-responsive phenylketonuria and inherited BH4 deficiency. BH4 supplementation has also drawn attention as a therapy for various NOS-related cardio-vascular diseases, but its use has met with limited success in decreasing BH2, the oxidized form of BH4. An increase in the BH2/BH4 ratio leads to NOS dysfunction. Previous studies revealed that BH4 supplementation caused a rapid urinary loss of BH4 accompanied by an increase in the blood BH2/BH4 ratio and an involvement of probenecid-sensitive but unknown transporters was strongly suggested in these processes. Here we show that OAT1 and OAT3 enabled cells to take up BP (BH4 and/or BH2) in a probenecid-sensitive manner using rat kidney slices and transporter-expressing cell systems, LLC-PK1 cells and Xenopus oocytes. Both OAT1 and OAT3 preferred BH2 and sepiapterin as their substrate roughly 5- to 10-fold more than BH4. Administration of probenecid acutely reduced the urinary exclusion of endogenous BP accompanied by a rise in blood BP in vivo. These results indicated that OAT1 and OAT3 played crucial roles: (1) in determining baseline levels of blood BP by excluding endogenous BP through the urine, (2) in the rapid distribution to organs of exogenous BH4 and the exclusion to urine of a BH4 excess, particularly when BH4 was administered, and (3) in scavenging blood BH2 by cellular uptake as the gateway to the salvage pathway of BH4, which reduces BH2 back to BH4.

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.

In treating hereditary deficiency of tetrahydrobiopterin (BH(4)), supplementation with BH(4) might be the ultimate choice of therapy. Oral administration of BH(4) has been believed to be inefficient owing to poor absorption of BH(4) in the intestine. In this study, we found a considerable amount of BH(4) as well as its oxidized pterins in the ingredients of intestinal lumen of mice when they were served food that did not contain significant amounts of biopterin. Ligation of the biliary duct led to significant decrease in luminal biopterin. Supplementation of BH(4) either by intraperitoneal administration of sepiapterin or of 6RBH(4) ((6R)-L-erythro-5,6,7,8-tetrahydrobiopterin) increased the BH(4) content in the intestinal lumen with a slight delay after the rise of blood BH(4). In these mice, biopterin appeared in the large intestine, caecum and colon, 2 h after the administration. The appearance of BH(4) in the large intestine was accompanied by a large amount of pterin (2-amino-4-hydroxypteridine). The amounts of biopterin + pterin that appeared in the large intestine after intraperitoneal administration of BH(4) were not greater than those found after oral administration at the same dose. When the mice were treated with a large dose of antibiotics prior to the BH(4) administration, the amount of biopterin increased in the caecum but the amount of pterin decreased greatly. These results suggested that a large proportion of BH(4) administered moved to the large intestine, where most biopterin was decomposed presumably by enteric bacteria. Nonetheless, most of the orally administered biopterin was taken up by the small intestine and the amount of biopterin reaching the large intestine was almost the same as that which appeared after direct injection of 6RBH(4) into the peritoneal cavity.

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.

Oral administration of tetrahydrobiopterin (BH(4)) has been known to be effective in treating BH(4)-deficient patients. It has long been established that BH(4) is absorbed by the intestinal mucosa. However, the mechanism for translocation of BH(4) across epithelial cells has not been elucidated. In order to study BH(4) transport mechanisms, Caco-2 cells were employed in this study as an epithelial cell model. Caco-2 cells were cultured (2 x 10(4) cells/0.3 cm(2) well) for 21 days in a 24-well format using Transwell, a porous membrane-based culture dish, at which point they had established themselves as a tight sheet with a definite polarity. When BH(4) (100 micromol/L) was given to cells from the apical side, a considerable translocation toward their basolateral side was noted. The rate of BH(4) movement was around 150 pmol/h per well. This was comparable to the highest rate of BH(4) uptake or its release so far obtained using a monolayer culture of Caco-2 cells on an ordinary plastic plate. The transcellular movement of BH(4) across the polar culture on the porous membrane was effectively prevented by benzbromarone (10 micromol/L), a well known inhibitor of a group of transporters including urate transporter (URAT1), organic anion transporters (OATs), and multidrug-resistance-associated proteins (MRPs). It was thus concluded that in Caco-2 cells, BH(4) moved across the cell interior in a rapid ligand-specific manner that was driven by a transporter.