論文

It is important to address the tissue permeability of drugs, particularly in tissues that have a blood-tissue barrier, in terms of both lipophilicity and the contribution of transporters. Here, we employed umbilical perfusion in rats to evaluate in vivo fetal-to-maternal transfer clearances of various xenobiotics. We measured fetal-to-maternal clearance (CLfm) of 23 compounds, which have a broad range of lipophilicity. Drugs for which CLfm was more than 300 μL/(mL min) belonged exclusively to Biopharmaceutical Drug Disposition Classification System (BDDCS) class 1 (highly permeable) and those for which CLfm was less than 50 μL/(mL min) belonged exclusively to BDDCS class 3 (poorly permeable). For most drugs, CLfm values were broadly consistent with lipophilicity. However, CLfm of digoxin was saturable and was inhibited by verapamil, suggesting that P-glycoprotein (P-gp)-mediated efflux has a substantially effect on measured clearance. CLfm of mitoxantrone continued to increase slightly at high concentrations of mitoxantrone, but placental-to-maternal clearance of mitoxantrone was saturable, implying that Bcrp1 contributes to mitoxantrone efflux across the placenta. Thus, we measured CLfm by umbilical perfusion and examined the relationship between CLfm and lipophilicity of xenobiotics. Fetal-to-maternal transport clearances measured in this study will be helpful to understand the characteristics of the blood-placental barrier. © 2013 Wiley Periodicals, Inc.

We have shown in vitro a hypoxia-induced time-dependent increase in facilitative glucose transporter isoform 3 (GLUT3) expression in N2A murine neuroblasts. This increase in GLUT3 expression is partially reliant on a transcriptional increase noted in actinomycin D and cycloheximide pretreatment experiments. Transient transfection assays in N2A neuroblasts using murine glut3-luciferase reporter constructs mapped the hypoxia-induced enhancer activities to -857- to -573-bp and -203- to -177-bp regions. Hypoxia-exposed N2A nuclear extracts demonstrated an increase in HIF-1α and p-Creb binding to HRE (-828 to -824 bp) and AP-1 (-187 to -180 bp) cis-elements, respectively, in electromobility shift and supershift assays, which was confirmed by chromatin immunoprecipitation assays. In addition, the interaction of CBP with Creb and HIF-1α and CREST with CBP in hypoxia was detected by coimmunoprecipitation. Furthermore, small interference (si)RNA targeting Creb in these cells decreased endogenous Creb concentrations that reduced by twofold hypoxia-induced glut3 gene transcription. Thus, in N2A neuroblasts, phosphorylated HIF-1α and Creb mediated the hypoxia-induced increase in glut3 transcription. Coactivation by the Ca++-dependent CREST and CBP proteins may enhance cross-talk between p-Creb-AP-1 and HIF-1α/HRE of the glut3 gene. Collectively, these processes can facilitate an adaptive response to hypoxic energy depletion targeted at enhancing glucose transport and minimizing injury while fueling the proliferative potential of neuroblasts. © 2013 the American Physiological Society.

A possible approach to improve antiretroviral therapy with nucleoside reverse transcriptase inhibitors is to enhance inhibitor delivery to CD4-positive T cells. We previously showed that dehydroepiandrosterone sulfate (DHEAS) enhances zidovudine (AZT) transfer into syncytiotrophoblast. Here, we investigated whether DHEAS also enhances AZT transfer into a cellular model of human T lymphocytes, and whether AZT is taken up by a specific transport system. The effects of DHEAS and related compounds on the uptake of [3H]AZT and other nucleosides by Molt-4 cells (a model of human CD4-positive T cells) were measured. [3H]AZT uptake by Molt-4 cells was nitrobenzylthioinosine insensitive and pH dependent, and the uptake was significantly inhibited by 1 mM ethylisopropylamiloride. [3H]AZT uptake by Molt-4 cells was increased in the presence of DHEAS, whereas uptake of other nucleosides was reduced. Kinetic study revealed that the maximum uptake velocity (up to 30 min) was increased in the presence of DHEAS. The structural requirements for AZT uptake-enhancing activity were studied using structural analogues of DHEAS. Estrone-3-sulfate and 16α-hydroxy DHEAS also enhanced AZT uptake into Molt-4 cells. The use of uptake enhancers may be a good strategy to improve the efficacy of antiretroviral therapy. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association.

The L-type amino acid transporter 1 (LAT1, SLC7A5) is an Na +-independent neutral amino acid transporter the expression of which is located in retinal endothelial cells. Due to its broad substrate selectivity, LAT1 has been proposed to mediate the transport of amino acid-related drugs across the blood-tissue barriers. Here, we have investigated the transport screening of amino acid-mustards using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) which expresses LAT1. We synthesized 5 amino acid-mustards: tyrosine-mustard, phenylglycine-mustard, alanine-mustard, ornithine-mustard, and lysine-mustard. LAT1-mediated [3H]L- phenylalanine (Phe) uptake by TR-iBRB2 cells was inhibited in a competitive manner by tyrosine-mustard and phenylglycine-mustard as well as melphalan (phenylalanine-mustard). Phenylglycine-mustard was able to induce the efflux of [3H]Phe preloaded into the TR-iBRB2 cells expressing LAT1 through the obligatory exchange mechanism, although tyrosine-mustard, alanine-mustard, ornithine-mustard, lysine-mustard, and melphalan did not induce any significant efflux. These findings suggest that phenylglycine-mustard is a better substrate for LAT1 than melphalan and other amino acid-mustards. © 2008 Pharmaceutical Society of Japan.

Little is known about the cerebral distribution and clearance of guanidinoacetate (GAA), the accumulation of which induces convulsions. The purpose of the present study was to identify creatine transporter (CRT)-mediated GAA transport and to clarify its cerebral expression and role in GAA efflux transport at the blood-cerebrospinal fluid barrier (BCSFB). CRT mediated GAA transport with a Km value of 269 μM/412 μM which was approximately 10-fold greater than that of CRT for creatine. There was wide and distinct cerebral expression of CRT and localization of CRT on the brush-border membrane of choroid plexus epithelial cells. The in vivo elimination clearance of GAA from the CSF was 13-fold greater than that of d-mannitol reflecting bulk flow of the CSF. This process was partially inhibited by creatine. The characteristics of GAA uptake by isolated choroid plexus and an immortalized rat choroid plexus epithelial cell line (TR-CSFB cells) used as an in vitro model of BCSFB are partially consistent with those of CRT. These results suggest that CRT plays a role in the cerebral distribution of GAA and GAA uptake by the choroid plexus. However, in the presence of endogenous creatine in the CSF, CRT may make only a limited contribution to the GAA efflux transport at the BCSFB. © 2008 The Authors.

The purpose of this study was to identify the uptake mechanism of γ-aminobutyric acid (GABA) via taurine transporter (Slc6a6/TauT) and its relationship with GABA transport at the inner BRB. Rat Slc6a6/TauT-transfected HeLa cells exhibited Na+-, Cl--, and concentration-dependent [3H]GABA uptake with a Km of 1.5 mM. Taurine, β-alanine, and GABA markedly inhibited Slc6a6/TauT-mediated uptake of [3H]GABA. The uptake of [3H]GABA by a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) was Na+-, Cl--, and concentration-dependent with a Km of 2.0 mM. This process was more potently inhibited by substrates of Slc6a6/TauT, taurine and β-alanine, than those of GABA transporters, GABA and betaine. In the presence of taurine, there was competitive inhibition with a Ki of 74 μM. [3H]Taurine also exhibited competitive inhibition with a Ki of 1.8 mM in the presence of GABA. In conclusion, rat Slc6a6/TauT has the ability to use GABA as a substrate and Slc6a6/TauT-mediated GABA transport appears to be present at the inner BRB. © 2008 Elsevier B.V. All rights reserved.

In the retina, taurine exerts a number of neuroprotective functions as an osmolyte and antioxidant. The purpose of the present study was to elucidate the taurine transport system(s) at the inner blood-retinal barrier (BRB). [3H]Taurine transport at the inner BRB was characterized using in vivo integration plot analysis and a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells). The expression of the taurine transporter (TauT) was demonstrated by RT-PCR and immunoblot analyses. The apparent influx permeability clearance of [3H]taurine in the rat retina was found to be 259 μL/(min{radical dot}g retina), supporting carrier-mediated influx transport of taurine at the BRB. [3H]Taurine uptake by TR-iBRB2 cells was Na+-, Cl-- and concentration-dependent with a Km of 22.2 μM and inhibited by TauT inhibitors, such as β-alanine and hypotaurine. RT-PCR and immunoblot analyses demonstrated that TauT is expressed in TR-iBRB2 and primary cultured human retinal endothelial cells. The uptake of [3H]taurine and the expression of TauT mRNA in TR-iBRB2 cells increased under hypertonic conditions but decreased following pretreatment with excess taurine. In conclusion, TauT most likely mediates taurine transport and regulate taurine transport at the inner BRB. © 2006 Elsevier Inc. All rights reserved.

Vitamin C is mainly transported across the blood-retinal and -brain barriers as dehydroascorbic acid (DHA) via a facilitative glucose transporter, GLUT1, and accumulates as ascorbic acid in the retina and brain. To investigate whether DHA transport to the retina and brain is changed by hyperglycemia, [14C]DHA transport across the blood-retinal and -brain barriers was examined using in vivo integration plot analysis in streptozotocin-induced diabetic rats with a 3-week duration of diabetes and in normal rats. Blood-to-retina and -brain transport of [14C]DHA was reduced by 65.5% and 84.1%, respectively, in diabetic rats compared with normal rats, whereas there was no major difference in the heart. Therefore, we propose that hyperglycemia reduces the supply of vitamin C to the retina and brain. © 2006 Pharmaceutical Society of Japan.

The purpose of the present study was to characterize the adenosine transport system(s) at the inner blood-retinal barrier (inner BRB). A conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2), used as an in vitro model of the inner BRB, expresses equilibrative nucleoside transporter 1 (ENT1), ENT2, concentrative nucleoside transporter 2 (CNT2), and CNT3 mRNAs. TR-iBRB2 cells exhibited an Na+-independent and concentration-dependent [3H]adenosine uptake with a Michaelis-Menten constant of 28.5 μM and a maximum uptake rate of 814 pmol/(min mg protein). [3H]Adenosine uptake by TR-iBRB2 cells was strongly inhibited by 2 mM adenosine, inosine, uridine, and thymidine. On the other hand, this process was not inhibited by 100 nM nitrobenzylmercaptopurine riboside and dipyridamole. These uptake studies suggest that ENT2 is involved in [3H]adenosine uptake by TR-iBRB2 cells. Quantitative real-time PCR revealed that the expression of ENT2 mRNA is 5.5-fold greater than that of ENT1 mRNA. An in vivo study suggested that [3H]adenosine is transported from the blood to the retina and significantly inhibited by adenosine and thymidine. The results of this study show that ENT2 most likely mediates adenosine transport at the inner BRB and is expected to play an important role in regulating the adenosine concentration in the retina. © 2006 Elsevier B.V. All rights reserved.

© 2018 The Pharmaceutical Society of Japan. Hypotaurine is a precursor of taurine and a physiological antioxidant that circulates in adult and fetal plasma. The purpose of the present study was to clarify whether hypotaurine is a substrate of Slc6a/gammaaminobutyric acid (GABA) transporter family members. Radiolabeled hypotaurine was synthesized from radiolabeled cysteamine and 2-aminoethanethiol dioxygenase. The uptakes of [3H]GABA, [3H]taurine, and [14C]hypotaurine by HEK293 cells expressing mouse GAT1/Slc6a1, TAUT/Slc6a6, GAT3/Slc6a11, BGT1/ Slc6a12, and GAT2/Slc6a13 were measured. TAUT and GAT2 showed strong [14C]hypotaurine uptake activity, while BGT1 showed moderate activity, and GAT1 and GAT3 showed slight but significant activity. Mouse TAUT and GAT2 both showed Michaelis constants of 11 μM for hypotaurine uptake. GAT2-expressing cells pretreated with hypotaurine showed resistance to H2O2-induced oxidative stress. These results suggest that under physiological conditions, TAUT and GAT2 would be major contributors to hypotaurine transfer across the plasma membrane, and that uptake of hypotaurine via GAT2 contributes to the cellular resistance to oxidative stress.

© 2021 John Wiley & Sons Ltd. Equilibrative nucleoside transporters (ENTs) 1 and 2 reportedly accept fluorouracil as a substrate. Here, we evaluated ENT1/2 expression at the messenger RNA (mRNA), protein, and functional levels in a panel of four triple-negative breast cancer (TNBC) cell lines, BT-549, Hs578T, MDA-MB-231, and MDA-MB-435, and we examined the relationship of the observed profiles to fluorouracil sensitivity. Nitrobenzylthioinosine (NBMPR) at 0.1 μM inhibits only ENT1, while dipyridamole at 10 μM or NBMPR at 100 μM inhibits both ENT1 and ENT2. We found that the uptake of [3H]uridine, a typical substrate of ENT1 and ENT2, was decreased to approximately 40% by 0.1 μM NBMPR. At 100 μM, NBMPR almost completely blocked the saturable uptake of [3H]uridine, but this does not imply a functional role of ENT2, because 10 μM dipyridamole showed similar inhibition to 0.1 μM NBMPR. Expression of ENT1 mRNA was almost 1 order of magnitude higher than that of ENT2 in all TNBC cell lines. Liquid chromatography-tandem mass spectrometry(LC-MS/MS) LC-MS/MS-based targeted protein quantification showed that ENT1 protein levels were in the range of 9.3–30 fmol/μg protein in plasma membrane fraction of TNBC cell lines, whereas ENT2 protein was below the detection limit. [3H]Fluorouracil uptake was insensitive to 0.1 μM NBMPR and 10 μM dipyridamole, suggesting a negligible contribution of ENT1 and ENT2 to fluorouracil uptake. The levels of ENT1 mRNA, ENT1 protein, ENT2 mRNA, and ENT1-mediated [3H]uridine uptake in the four TNBC cell lines showed no correlation with fluorouracil sensitivity. These results indicate that neither ENT1 nor ENT2 contributes significantly to the fluorouracil sensitivity of TNBC cell lines.

The purpose of this study was to elucidate the mechanisms of blood-to-retina creatine transport across the blood-retinal barrier (BRB) in vivo and in vitro, and to identify the responsible transporter(s). The creatine transport across the BRB in vivo and creatine uptake in an in vitro model of the inner BRB (TR-iBRB2 cells) were examined using [14C]creatine. Identification and localization of the creatine transporter (CRT) were carried out by RT-PCR, western blot, and immunoperoxidase electron microscopic analyses. An in vivo intravenous administration study suggested that [14C]creatine is transported from the blood to the retina against the creatine concentration gradient that exists between the retina and blood. [14C]Creatine uptake by TR-iBRB2 cells was saturable, Na+- and Cl --dependent and inhibited by CRT inhibitors, suggesting that CRT is involved in creatine transport at the inner BRB. RT-PCR and western blot analyses demonstrated that CRT is expressed in rat retina and TR-iBRB2 cells. Moreover, using an immunoperoxidase electron microscopic analysis, CRT immunoreactivity was found at both the luminal and abluminal membranes of the rat retinal capillary endothelial cells. In conclusion, CRT is expressed at the inner BRB and plays a role in blood-to-retina creatine transport across the inner BRB.

The objective of this study was to establish and characterize a retinal capillary endothelial cell line (TR-iBRB) from a newly developed transgenic rat harboring the temperature-sensitive simian virus 40 (SV 40) large T-antigen gene (Tg rat). Retinal capillary endothelial cells were isolated from a Tg rat and cultured in collagen-coated dishes at 37°C for a period of 48 hr. Cells were subsequently cultured at 33°C to activate the large T-antigen. At the third passage, cells were cloned by colony formation and isolated from other cells. Nine immortalized cell lines of retinal capillary endothelial cells (TR-iBRB1 ∼9) were obtained from a Tg rat. These cell lines had a spindle-fiber shape morphology, expressed the typical endothelial marker, von Willebrand factor, and internalized acetylated-low density lipoprotein. Moreover, vascular endothelial growth factor (VEGF) receptor-2 was expressed in TR-iBRBs. TR-iBRBs expressed a large T-antigen and grew well at 33°C with a doubling time of 19-21 hr. In contrast, cells did not grow at 37 and 39°C due to the reduced expression of large T-antigen. supporting temperature-dependent cell growth, TR-iBRBs expressed GLUT1 and exhibited 3-O-methyl-D-glucose (3-OMG) uptake activity, This 3-OMG uptake was saturable with a Michaelis-Menten constant of 5.56 ± 0.51 mM and a maximum uptake rate of 45.3 ± 2.6 nmol min-1 mg protein-1, P-Glycoprotein, with a molecular weight of ∼180 KDa, was expressed in TR-iBRBs. In addition, mdr 1a, mdr 1b and mdr 2 were detected in TR-iBRB2 using RT-PCR. In conclusion, conditionally immortalized retinal capillary endothelial cell lines were established from a transgenic rat harboring the temperature-sensitive SV 40 large T-antigen gene and these lines were shown to exhibit the properties of retinal capillary endothelial cells. © 2001 Academic Press.

Prenatal nutrient restriction (NR) culminating in intra-uterine growth restriction (IUGR) with postnatal catch up growth leads to diabesity. In contrast, postnatal NR with growth restriction (PNGR) superimposed on IUGR (IPGR) protects young and aging adults from this phenotype. We hypothesized that PNGR/IPGR will compromise the blood-brain metabolic profile impairing neurobehavior and predisposing to Alzheimer's disease (AD). NR (50%) in late gestation followed by cross-fostering of rat pups to either ad lib fed (CON) or NR (50%) lactating mothers generated CON, IUGR, PNGR and IPGR male (M) and female (F) offspring that were examined through the life span. In PNGR/IPGR plasma/CSF glucose and lactate decreased while ketones increased in (M) and (F) (PN21, PN50). In addition increased brain glucose transporters, Glut1 & Glut3, greater brain derived neurotrophic factor (BDNF), reduced Glut4, with unchanged serotonin transporter concentrations were noted in (F) (PN50-60). While (F) displayed more hyperactivity, both (F) and (M) exhibited anxiety although socially and cognitively unimpaired (PN25-28&50). Aging (15-17 m) (F) not (M), expressed low plasma insulin, reduced brain IRS-2, pAkt, and pGSK-3βSer9, unchanged pPDK1, pTau or lipoprotein receptor related protein 1 (LRP1), higher glial fibrillary acidic protein (GFAP) and spinophilin but a 10-fold increased amyloid-β42. We conclude that therapeutically superimposing PNGR on IUGR (IPGR) should be carefully weighed in light of unintended consequences related to perturbed neurobehavior and potential predilection for AD. © 2012 Elsevier B.V.

The purpose of this study was to identify the transporter mediating l-arginine transport at the inner blood-retinal barrier (BRB). The apparent uptake clearance of [3H]l-arginine into the rat retina was found to be 118 μL/(min·g retina), supporting a carrier-mediated influx transport of l-arginine at the BRB. [3H]l-Arginine uptake by a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells), used as an in vitro model of the inner BRB, was primarily an Na +-independent and saturable process with Michaelis-Menten constants of 11.2 μM and 530 μM. This process was inhibited by rat cationic amino acid transporter (CAT) 1-specific small interfering RNA as well as substrates of CATs, l-arginine, l-lysine, and l-ornithine. The expression of cationic amino acid transporter (CAT) 1 mRNA was 25.9- and 796-fold greater than that of CAT3 in TR-iBRB2 and magnetically isolated rat retinal vascular endothelial cells, respectively. The expression of CAT1 protein was detected in TR-iBRB2 cells and immunostaining of CAT1 was observed along the rat retinal capillaries. In conclusion, CAT1 is localized in retinal capillary endothelial cells and at least in part mediates l-arginine transport at the inner BRB. This process seems to be closely involved in visual functions by supplying precursors of biologically important molecules like nitric oxide in the neural retina. © 2009 International Society for Neurochemistry.

Vitamin C is mainly transported across the inner blood-retinal barrier (inner BRB) as dehydroascorbic acid (DHA) via a facilitative glucose transporter (GLUT) 1, and accumulates as ascorbic acid (AA) in the retina. Müller cells, huge glial cells, exhibit passive structural and metabolic functions for retinal neurons and the inner BRB. We characterized DHA transport and its corresponding transporter in a rat Müller cell line (TR-MUL5 cells). [14C]DHA uptake by TR-MUL5 cells took place in a time-dependent and Na+-independent manner. [14C]DHA uptake was inhibited by substrates and inhibitors of GLUTs, suggesting that Müller cells take up DHA via GLUTs. HPLC analysis revealed that most of the DHA taken up by TR-MUL5 cells was converted to AA and accumulated as AA in TR-MUL5 cells. [14C]DHA uptake by TR-MUL5 cells took place in a concentration-dependent manner with a Michaelis-Menten constant of 198 μM and was inhibited by cytochalasin B in a concentration-dependent manner with a 50% inhibition concentration of 0.283 μM. Although GLUT1, 3, and 4 mRNA are expressed in TR-MUL5 cells, quantitative real-time PCR revealed that GLUT1 mRNA expression was 5.85- and 116-fold greater than that of GLUT3 and 4, respectively. Western blot analysis supports the expression of GLUT1 protein with 45 kDa in TR-MUL5 cells. In conclusion, DHA is taken up by facilitative glucose transporters, most likely GLUT1, and converted to AA in TR-MUL5 cells. © 2008 Elsevier Ltd. All rights reserved.

The purpose of the present study was to evaluate vitreous humor/retina-to-blood efflux transport in rats and determine the efflux transport of estradiol 17-β glucuronide (E17βG) across the blood-retinal barrier (BRB) by the use of microdialysis. [3H]E17βG and [14C]d-mannitol, which were used as a model compound for amphipathic organic anions and a bulk flow marker, respectively, were injected into the vitreous humor of rat eye, and a microdialysis probe was placed in the vitreous humor. [3H]E17βG and [14C]d-mannitol were bi-exponentially eliminated from the vitreous humor after vitreous bolus injection. The elimination rate constant of [3H]E17βG during the terminal phase was 1.9-fold greater than that of [14C]d-mannitol and reduced the level of [14C]d-mannitol in the retinal presence of 0.3 mM E17βG, suggesting that [3H]E17βG is transported via a carrier-mediated efflux transport process across the BRB. The efflux transport of [3H]E17βG was significantly inhibited by organic anions, such as probenecid, sulfobromophthalein, digoxin, and dehydroepiandrosterone sulfate, whereas it was not inhibited by p-aminohippuric acid. In conclusion, the efflux transport of [3H]E17βG across the rat BRB was evaluated by microdialysis and its inhibition by organic anions suggests organic anion transporting polypeptide 1a4-mediated E17βG efflux transport at the BRB. © 2006 Elsevier B.V. All rights reserved.

© 2017 American Pharmacists Association® The pharmacokinetics of cetirizine, a nonsedating antihistamine, is profoundly affected by transporter-mediated membrane transport in the kidney. In this study, we aimed to investigate the transport mechanism of levocetirizine, the pharmacologically active enantiomer of cetirizine, via human organic anion transporter 4 (OAT4) expressed in the apical membrane of renal proximal tubules and the basal plasma membrane of placental syncytiotrophoblasts. In cells expressing human OAT4 under the control of tetracycline, levocetirizine uptake was increased by tetracycline treatment. On the other hand, OAT4 expression did not facilitate efflux of preloaded levocetirizine from the cells, either in the presence or absence of extracellular Cl−. The OAT4-mediated levocetirizine uptake was concentration-dependent with a Km of 38 μM. The uptake rate of levocetirizine via OAT4 was approximately twice that of racemic cetirizine, indicating stereoselective uptake of levocetirizine. On the other hand, OAT4-mediated [3H]dehydroepiandrosterone sulfate uptake was inhibited by dextrocetirizine and levocetirizine. Overall, our findings indicate that OAT4 mediates levocetirizine uptake but is unlikely to mediate the efflux.

© 2017, Springer-Verlag Wien. System A consists of three subtypes, sodium-coupled neutral amino acid transporter 1 (SNAT1), SNAT2, and SNAT4, which are all expressed in the placenta. The aim of this study was to evaluate the contributions of each of the three subtypes to total system A-mediated uptake in placental MVM of human and rat, using betaine and l-arginine as subtype-selective inhibitors of SNAT2 and SNAT4, respectively. Appropriate concentrations of betaine and l-arginine for subtype-selective inhibition in SNAT-overexpressing cells were identified. It was found that 10 mM betaine specifically and almost completely inhibited human and rat SNAT2-mediated [14C]α-methylaminoisobutyric acid ([14C]MeAIB) uptake, while 5 mM l-arginine specifically and completely inhibited [3H]glycine uptake via human SNAT4, as well as [14C]MeAIB uptake via rat SNAT4. In both human and rat placental MVM vesicles, sodium-dependent uptake of [14C]MeAIB was almost completely inhibited by 20 mM unlabeled MeAIB. l-Arginine (5 mM) partly inhibited the uptake in humans, but hardly affected that in rats. Betaine (10 mM) partly inhibited the uptake in rats, but hardly affected it in humans. These results suggest that SNAT1 is most likely the major contributor to system A-mediated MeAIB uptake by human and rat MVM vesicles and that the remaining uptake is mainly mediated by SNAT4 in humans and SNAT2 in rats. Thus, inhibition studies using betaine and l-arginine are useful to characterize the molecular mechanisms of system A-mediated transport.

© 2016 The Japanese Society for the Study of Xenobiotics Fluorouracil is used for treatment of breast cancer even in pregnant women, except during fetal organogenesis. The purpose of this study was to clarify the transport mechanism of fluorouracil at the rat placental barrier. Maternal-to-fetal transfer of [3H]fluorouracil in rats at gestational day 19.5 was saturable and much higher than that of [14C]sucrose. The uptake of [3H]fluorouracil was also saturable in rat placental trophoblast TR-TBT 18d-1 cells, which express both equilibrative nucleoside transporter (ENT) 1 and ENT2. Nitrobenzylthioinosine (NBMPR) at 0.1 μM had no effect on [3H]fluorouracil uptake by TR-TBT 18d-1 cells, but 100 μM NBMPR almost completely inhibited the saturable component, suggesting involvement of ENT2, rather than ENT1 in the transport. Rat ENT2 cRNA-injected oocytes showed significantly increased [3H]fluorouracil uptake compared with water-injected oocytes, while rat ENT1 cRNA-injected oocytes did not show an increase of [3H]fluorouracil uptake. The Michaelis–Menten constant for rat ENT2-mediated uptake of [3H]fluorouracil was 4.21 mM. The expression profile of ENT2 mRNA in rat placenta during pregnancy was almost constant from 13.5 to 21.5 days of gestation. In conclusion, ENT2 appears to be the mediator of fluorouracil transport in rat placental trophoblast cells.