Ohashi A et al. (2017), Organic anion transporters, OAT1 and OAT3, are ...

XB-ART-54204

Mol Cell Biochem 2017 Nov 01;4351-2:97-108. doi: 10.1007/s11010-017-3060-7.

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Organic anion transporters, OAT1 and OAT3, are crucial biopterin transporters involved in bodily distribution of tetrahydrobiopterin and exclusion of its excess.

Ohashi A , Mamada K , Harada T , Naito M , Takahashi T , Aizawa S , Hasegawa H .

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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.

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Species referenced: Xenopus
Genes referenced: kcnk3 nos1 pklr prok1 slc22a6

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	Fig. 1. Uptake of 6RBH4 by kidney slices in the presence or absence of typical ligands of organic anion transporters. The kidney slices were prepared as described in “Materials and methods” section. The slices took up 6RBH4 and the uptake was inhibited by OAT ligands. The reagents used were 6RBH4 (10 µM and 3 mM), penicillin G (PCG, 1 mM), probenecid (PBC, 1 mM) and p-aminohippuric acid (PAH, 1 mM). The uptake of BH4 for 15 min was expressed as a portion of the clearance. P < 0.05, *P < 0.01 (Holm’s test); each point represents the mean ± S.D. (n = 3–7)
	Fig. 2. Biopterin uptake by rOat1- and rOat3-expressing LLC-PK1 cells and naïve LLC-PK1 cells. Tetrahydrobiopterin uptake by OAT-expressing or naïve LLC-PK1 cells was examined under a monolayer culture (4 × 104 cells/well, 96-well analytical culture plate). a LLC-PK1 cells transfected with rOat1 (left) or with rOat3 (right) were used. The cells were exposed for 1 h to 50 µM 6RBH4 in the absence (gray bars, control labeled “none”) or in the presence (open bars) of OAT ligands (1 mM each except for methotrexate (MTX) at 80 µM). The rOat1- and rOat3-expressing LLC-PK1 cells took up 6RBH4 and the uptake was inhibited by the ligands of OAT1 and OAT3. The OAT ligands used were probenecid (PBC), estronesulfate (ES), p-aminohippuric acid (PAH), penicillin G (PCG), methotrexate (MTX) and cimetidine (CIM). b rOat1-LLC-PK1 cells (left) or rOat3- LLC-PK1 cells (right) were given 50 µM each of 6RBH4, dihydrobiopterin (BH2) or sepiapterin (SP) in the absence (gray bars) or presence (open bars) of 1 mM PBC for 1 h. The resultant biopterin accumulations of BH2 + BH4 were then compared between those in the absence of PBC vs the presence of PBC, and levels of BH4 vs. BH2 and of BH2 vs SP. c Uptakes of 6RBH4 and BH2 (50 µM each) by naïve LLC-PK1 cells were also compared (left panel). The uptake of 6RBH4 (50 µM) for 1 h (right panel) was analyzed in the absence (gray bar, labeled “none”) or presence (open bars) of 200 µM nitrobenzylthioinosine (NBMPR) or 1 mM PBC. The uptake of the pterins was expressed as a portion of the clearance. P < 0.05, *P < 0.01 (Holm’s test); †† P < 0.01 (Student’s t-test); each point represents the mean ± S.D. (n = 5–6)
	Fig. 3. Uptake of 6RBH4, BH2, and sepiapterin by hOAT1- and hOAT3-expressing Xenopus oocytes. Xenopus oocytes were individually injected (gray bars) with 50 nL of hOAT1 (a) or hOAT3 (b) cRNA (1 ng/nL). As the control (hatched bars), oocytes were injected with 50 nL of distilled water. The oocytes were then allowed to express the respective transporters at 19 °C for 2 days. In the uptake experiment, all pterins were used at 50 µM. All oocytes injected with either cRNA took up significantly more pterins than the control and the uptake was inhibited by OAT ligands (1 mM each). The OAT ligands used were probenecid (PBC), p-aminohippuric acid (PAH), estronesulfate (ES) and penicillin G (PCG). a 6RBH4, BH2, and sepiapterin (SP) were taken up by hOAT1-expressing oocytes (main panel) for 1 h, and the BH2 uptake was analyzed in the absence (gray bar, labeled “none”) or presence (open bars) of OAT1 ligands (upper panel). (b) Uptake of 6RBH4, BH2, and SP by hOAT3-expressing oocytes (main panel) and inhibition of BH4 uptake by OAT3 ligands in the absence (gray bar, labeled “none”) or presence (open bars) of OAT3 ligands (upper panel). The uptake of the pterins was expressed as a portion of the clearance. P < 0.05, *P < 0.01 (Holm’s test); each point represents the mean ± S.D. (n = 4–9)
	Fig. 4. Elevation of blood BP accompanied by a decrease in the urinary loss of endogenous BP with a single dose of probenecid. Rats were given probenecid (PBC, 200 mg/kg, i.p.), and the blood (a) and urine (b) were collected sequentially from individual rats at the indicated times under sustained anesthesia for 6 h, then the kidney and liver (c) were dissected from the same rats at 6 h after the PBC dosing. The 0-time samples were taken from the rats without PBC treatment. The 0-time amounts of BH2 + BH4 (BP, open symbols) were compared with those of PBC-treated rat samples (grey symbols). In a and b, P < 0.05, P < 0.01 (“0-time” vs. “PBC-treated”, paired Student’s test), and in c, *P < 0.01, or n.s., no significant difference (“before” vs. “PBC-treated”, Williams’ test). Data are mean ± S.E. (n = 4)

References [+] :

Baek, Macrophage nitric oxide synthase subunits. Purification, characterization, and role of prosthetic groups and substrate in regulating their association into a dimeric enzyme. 1993, Pubmed

Baek, Macrophage nitric oxide synthase subunits. Purification, characterization, and role of prosthetic groups and substrate in regulating their association into a dimeric enzyme. 1993, Pubmed
Bartholomé, Atypical phenylketonuria with normal phenylalanine hydroxylase and dihydropteridine reductase activity in vitro. 1977, Pubmed
Bianchi, Heterologous expression of C. elegans ion channels in Xenopus oocytes. 2006, Pubmed , Xenbase
Blau, Alternative therapies to address the unmet medical needs of patients with phenylketonuria. 2015, Pubmed
Burckhardt, Drug transport by Organic Anion Transporters (OATs). 2012, Pubmed
Chalupsky, Endothelial dihydrofolate reductase: critical for nitric oxide bioavailability and role in angiotensin II uncoupling of endothelial nitric oxide synthase. 2005, Pubmed
Crabtree, Ratio of 5,6,7,8-tetrahydrobiopterin to 7,8-dihydrobiopterin in endothelial cells determines glucose-elicited changes in NO vs. superoxide production by eNOS. 2008, Pubmed
Crabtree, Dihydrofolate reductase protects endothelial nitric oxide synthase from uncoupling in tetrahydrobiopterin deficiency. 2011, Pubmed
Cunnington, Systemic and vascular oxidation limits the efficacy of oral tetrahydrobiopterin treatment in patients with coronary artery disease. 2012, Pubmed
El-Sheikh, Mechanisms of renal anionic drug transport. 2008, Pubmed
Fukushima, Analysis of reduced forms of biopterin in biological tissues and fluids. 1980, Pubmed
Fukushima, Pterins in human urine. 1972, Pubmed
Hasegawa, Delivery of exogenous tetrahydrobiopterin (BH4) to cells of target organs: role of salvage pathway and uptake of its precursor in effective elevation of tissue BH4. 2005, Pubmed
Hasegawa, Functional involvement of rat organic anion transporter 3 (rOat3; Slc22a8) in the renal uptake of organic anions. 2002, Pubmed
Hasegawa, Contribution of organic anion transporters to the renal uptake of anionic compounds and nucleoside derivatives in rat. 2003, Pubmed
Heintz, Tetrahydrobiopterin, its mode of action on phenylalanine hydroxylase, and importance of genotypes for pharmacological therapy of phenylketonuria. 2013, Pubmed
Hull, The origin and characteristics of a pig kidney cell strain, LLC-PK. 1976, Pubmed
KAUFMAN, THE STRUCTURE OF THE PHENYLALANINE-HYDROXYLATION COFACTOR. 1963, Pubmed
Keil, Long-term follow-up and outcome of phenylketonuria patients on sapropterin: a retrospective study. 2013, Pubmed
Klatt, The pteridine binding site of brain nitric oxide synthase. Tetrahydrobiopterin binding kinetics, specificity, and allosteric interaction with the substrate domain. 1994, Pubmed
Kure, Tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency. 1999, Pubmed
Kusuhara, Molecular cloning and characterization of a new multispecific organic anion transporter from rat brain. 1999, Pubmed , Xenbase
Kwon, Reduced biopterin as a cofactor in the generation of nitrogen oxides by murine macrophages. 1989, Pubmed
Longo, Long-term safety and efficacy of sapropterin: the PKUDOS registry experience. 2015, Pubmed
Lovenberg, Tryptophan hydroxylation: measurement in pineal gland, brainstem, and carcinoid tumor. 1967, Pubmed
Moens, Targeting endothelial and myocardial dysfunction with tetrahydrobiopterin. 2011, Pubmed
NAGATSU, TYROSINE HYDROXYLASE. THE INITIAL STEP IN NOREPINEPHRINE BIOSYNTHESIS. 1964, Pubmed
Ohashi, Rapid clearance of supplemented tetrahydrobiopterin is driven by high-capacity transporters in the kidney. 2012, Pubmed
Ohashi, Asymmetric uptake of sepiapterin and 7,8-dihydrobiopterin as a gateway of the salvage pathway of tetrahydrobiopterin biosynthesis from the lumenal surface of rat endothelial cells. 2011, Pubmed
Ohashi, Tetrahydrobiopterin Supplementation: Elevation of Tissue Biopterin Levels Accompanied by a Relative Increase in Dihydrobiopterin in the Blood and the Role of Probenecid-Sensitive Uptake in Scavenging Dihydrobiopterin in the Liver and Kidney of Rats. 2016, Pubmed
Ohashi, Membrane transport of sepiapterin and dihydrobiopterin by equilibrative nucleoside transporters: a plausible gateway for the salvage pathway of tetrahydrobiopterin biosynthesis. 2011, Pubmed , Xenbase
Reverter, Effects of Sapropterin on Portal and Systemic Hemodynamics in Patients With Cirrhosis and Portal Hypertension: A Bicentric Double-Blind Placebo-Controlled Study. 2015, Pubmed
Sawabe, Tetrahydrobiopterin uptake in supplemental administration: elevation of tissue tetrahydrobiopterin in mice following uptake of the exogenously oxidized product 7,8-dihydrobiopterin and subsequent reduction by an anti-folate-sensitive process. 2004, Pubmed
Sawabe, Accumulated BH4 in mouse liver caused by administration of either 6R- or 6SBH4 consisted solely of the 6R-diastereomer: evidence of oxidation to BH2 and enzymic reduction. 2005, Pubmed
Sawabe, Cellular uptake of sepiapterin and push-pull accumulation of tetrahydrobiopterin. 2008, Pubmed
Sawabe, Cellular accumulation of tetrahydrobiopterin following its administration is mediated by two different processes; direct uptake and indirect uptake mediated by a methotrexate-sensitive process. 2005, Pubmed
Schaub, Tetrahydrobiopterin therapy of atypical phenylketonuria due to defective dihydrobiopterin biosynthesis. 1978, Pubmed
Schmidt, Cell type-specific recycling of tetrahydrobiopterin by dihydrofolate reductase explains differential effects of 7,8-dihydrobiopterin on endothelial nitric oxide synthase uncoupling. 2014, Pubmed
Sekine, Molecular physiology of renal organic anion transporters. 2006, Pubmed
Stuehr, Oxygen reduction by nitric-oxide synthases. 2001, Pubmed
Sugiyama, Characterization of the efflux transport of 17beta-estradiol-D-17beta-glucuronide from the brain across the blood-brain barrier. 2001, Pubmed
TIETZ, A NEW PTERIDINE-REQUIRING ENZYME SYSTEM FOR THE OXIDATION OF GLYCERYL ETHERS. 1964, Pubmed
Takeda, Characterization of organic anion transport inhibitors using cells stably expressing human organic anion transporters. 2001, Pubmed
Thiele, The challenge of long-term tetrahydrobiopterin (BH4) therapy in phenylketonuria: Effects on metabolic control, nutritional habits and nutrient supply. 2015, Pubmed
Watschinger, Alkylglycerol monooxygenase. 2013, Pubmed
Watschinger, Tetrahydrobiopterin and alkylglycerol monooxygenase substantially alter the murine macrophage lipidome. 2015, Pubmed
Wedeen, The distribution of p-aminohippuric acid in rat kidney slices. I. Tubular localization. 1973, Pubmed
Wettstein, Linking genotypes database with locus-specific database and genotype-phenotype correlation in phenylketonuria. 2015, Pubmed
Whitsett, Human endothelial dihydrofolate reductase low activity limits vascular tetrahydrobiopterin recycling. 2013, Pubmed

	Fig. 1. Uptake of 6RBH4 by kidney slices in the presence or absence of typical ligands of organic anion transporters. The kidney slices were prepared as described in “Materials and methods” section. The slices took up 6RBH4 and the uptake was inhibited by OAT ligands. The reagents used were 6RBH4 (10 µM and 3 mM), penicillin G (PCG, 1 mM), probenecid (PBC, 1 mM) and p-aminohippuric acid (PAH, 1 mM). The uptake of BH4 for 15 min was expressed as a portion of the clearance. P < 0.05, *P < 0.01 (Holm’s test); each point represents the mean ± S.D. (n = 3–7)
	Fig. 2. Biopterin uptake by rOat1- and rOat3-expressing LLC-PK1 cells and naïve LLC-PK1 cells. Tetrahydrobiopterin uptake by OAT-expressing or naïve LLC-PK1 cells was examined under a monolayer culture (4 × 104 cells/well, 96-well analytical culture plate). a LLC-PK1 cells transfected with rOat1 (left) or with rOat3 (right) were used. The cells were exposed for 1 h to 50 µM 6RBH4 in the absence (gray bars, control labeled “none”) or in the presence (open bars) of OAT ligands (1 mM each except for methotrexate (MTX) at 80 µM). The rOat1- and rOat3-expressing LLC-PK1 cells took up 6RBH4 and the uptake was inhibited by the ligands of OAT1 and OAT3. The OAT ligands used were probenecid (PBC), estronesulfate (ES), p-aminohippuric acid (PAH), penicillin G (PCG), methotrexate (MTX) and cimetidine (CIM). b rOat1-LLC-PK1 cells (left) or rOat3- LLC-PK1 cells (right) were given 50 µM each of 6RBH4, dihydrobiopterin (BH2) or sepiapterin (SP) in the absence (gray bars) or presence (open bars) of 1 mM PBC for 1 h. The resultant biopterin accumulations of BH2 + BH4 were then compared between those in the absence of PBC vs the presence of PBC, and levels of BH4 vs. BH2 and of BH2 vs SP. c Uptakes of 6RBH4 and BH2 (50 µM each) by naïve LLC-PK1 cells were also compared (left panel). The uptake of 6RBH4 (50 µM) for 1 h (right panel) was analyzed in the absence (gray bar, labeled “none”) or presence (open bars) of 200 µM nitrobenzylthioinosine (NBMPR) or 1 mM PBC. The uptake of the pterins was expressed as a portion of the clearance. P < 0.05, *P < 0.01 (Holm’s test); †† P < 0.01 (Student’s t-test); each point represents the mean ± S.D. (n = 5–6)
	Fig. 3. Uptake of 6RBH4, BH2, and sepiapterin by hOAT1- and hOAT3-expressing Xenopus oocytes. Xenopus oocytes were individually injected (gray bars) with 50 nL of hOAT1 (a) or hOAT3 (b) cRNA (1 ng/nL). As the control (hatched bars), oocytes were injected with 50 nL of distilled water. The oocytes were then allowed to express the respective transporters at 19 °C for 2 days. In the uptake experiment, all pterins were used at 50 µM. All oocytes injected with either cRNA took up significantly more pterins than the control and the uptake was inhibited by OAT ligands (1 mM each). The OAT ligands used were probenecid (PBC), p-aminohippuric acid (PAH), estronesulfate (ES) and penicillin G (PCG). a 6RBH4, BH2, and sepiapterin (SP) were taken up by hOAT1-expressing oocytes (main panel) for 1 h, and the BH2 uptake was analyzed in the absence (gray bar, labeled “none”) or presence (open bars) of OAT1 ligands (upper panel). (b) Uptake of 6RBH4, BH2, and SP by hOAT3-expressing oocytes (main panel) and inhibition of BH4 uptake by OAT3 ligands in the absence (gray bar, labeled “none”) or presence (open bars) of OAT3 ligands (upper panel). The uptake of the pterins was expressed as a portion of the clearance. P < 0.05, *P < 0.01 (Holm’s test); each point represents the mean ± S.D. (n = 4–9)
	Fig. 4. Elevation of blood BP accompanied by a decrease in the urinary loss of endogenous BP with a single dose of probenecid. Rats were given probenecid (PBC, 200 mg/kg, i.p.), and the blood (a) and urine (b) were collected sequentially from individual rats at the indicated times under sustained anesthesia for 6 h, then the kidney and liver (c) were dissected from the same rats at 6 h after the PBC dosing. The 0-time samples were taken from the rats without PBC treatment. The 0-time amounts of BH2 + BH4 (BP, open symbols) were compared with those of PBC-treated rat samples (grey symbols). In a and b, P < 0.05, P < 0.01 (“0-time” vs. “PBC-treated”, paired Student’s test), and in c, *P < 0.01, or n.s., no significant difference (“before” vs. “PBC-treated”, Williams’ test). Data are mean ± S.E. (n = 4)