Details of a Researcher - Tomi, Masatoshi

Papers - Tomi, Masatoshi

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Characterizing Apixaban Pharmacokinetics Through Physiologically-Based Pharmacokinetic Modeling: Critical Role of Biliary Secretion and Enterohepatic Circulation in Humans

Tsuchitani T., Kou W., Tomi M., Sugiyama Y.

Cpt Pharmacometrics and Systems Pharmacology 15 ( 1 ) e70163 2026.01

Research paper (scientific journal), Accepted

Apixaban, a factor Xa inhibitor, is a direct oral anticoagulant with a well-balanced elimination; it is eliminated evenly via feces, urine (with no active secretion), and as metabolites after oral administration. The common understanding is that biliary secretion and enterohepatic circulation (EHC) of apixaban are limited in humans, and that fecal excretion may be attributable to intestinal secretion. However, a decrease in apixaban blood concentration with activated charcoal coadministration in humans suggests possible involvement of EHC. This study aimed to evaluate the contribution of biliary excretion, EHC, and intestinal secretion to apixaban pharmacokinetics (PK) using a physiologically-based pharmacokinetic (PBPK) model. A top-down analysis was performed using blood concentration and mass balance data from healthy volunteers. Model parameters were optimized using the Cluster-Gauss Newton method (CGNM), followed by the bootstrap method. The model accurately described observed data and indicated moderate to high biliary secretion relative to metabolic clearance. Simulated biliary secretion into the duodenum well predicted the biliary secretion data in humans (< 1% of dose at 8 h post-dose). Virtual knockout of EHC resulted in a shortened half-life from 8.7 to 2.9 h, and 17% and 55% decrease in area under the concentration curve (AUC) and fecal excretion after intravenous dosing, respectively, confirming the significant contribution of biliary excretion and EHC. The model also accurately described apixaban PK with activated charcoal coadministration at 2 or 6 h post-dose. Although further experimental validation (e.g., sandwich-cultured hepatocytes) would strengthen these findings, our study demonstrates that biliary secretion and EHC play a substantial role in apixaban elimination and disposition in humans.

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Rat-Specific Expression of Placental MATE1 Contributes to Species Difference in Fetal Transfer of Metformin and 1-Methyl-4-Phenylpyridinium.

Suzuki A, Noguchi S, Tsuchitani T, Sakamoto R, Yamauchi A, Ishinabe T, Hashimoto R, Nakaguchi Y, Sakai M, Imayoshi N, Ishimoto T, Kato Y, Shirasaka Y, Katakura S, Maruyama T, Nishimura T, Tomi M

Molecular pharmaceutics 22 ( 12 ) 7412 - 7422 2025.11

Research paper (scientific journal), ISSN 1543-8384

Metformin readily crosses the human placenta. However, the fetal-to-maternal plasma concentration ratio (F/M ratio) of metformin has been reported to be as low as 0.057 in rats. This study investigated the underlying mechanisms of these interspecies differences by examining the expression and function of candidate transporters, organic cation transporter 3 (OCT3/SLC22A3) and multidrug and toxin extrusion protein 1 (MATE1/SLC47A1), in the placenta across species. Expression analysis at gene and protein levels using RNA-sequencing and absolute quantification by LC-MS/MS revealed minimal species differences in placental OCT3 expression (less than 4-fold). In contrast, MATE1 showed almost exclusive expression in the rat placental labyrinth, but minimal or undetectable levels were observed in samples prepared from human placental villi and mouse placental labyrinth. To evaluate the function of the placental MATE1, we compared the placental transfer of two dual substrates of MATE1 and OCT3, metformin and 1-methyl-4-phenylpyridinium (MPP+), in pregnant rats and mice in the presence and absence of pyrimethamine, a MATE1 inhibitor. The pyrimethamine was dosed to reach the plasma concentration, which specifically inhibits MATE1 based on the measured IC<inf>50</inf> of pyrimethamine to rat MATE1 and OCT3, which were 0.015 and 100 μM, respectively. The F/M ratios of metformin and MPP+ in the absence of pyrimethamine were half or less in rats compared to mice. Notably, preadministration of pyrimethamine increased the F/M ratios of metformin and MPP+ by approximately 50% in rats but not mice. These findings demonstrate that functional placental MATE1 expression is specific to rats, which, in part, contributes to the low fetal transfer of cationic compounds compared with other species. This is of significant concern, as it suggests that nonclinical developmental and reproductive toxicity studies of MATE1 substrate drugs in rats may underestimate fetal exposure and toxicity when extrapolated to humans.

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Fetal ezrin expression affects macrophages and regulatory T cells in mouse placental decidua

Nishimura T., Mizokami R., Yamanaka M., Takahashi M., Yoshida Y., Ogawa Y., Noguchi S., Tomi M.

Biochemical and Biophysical Research Communications 735 150842 2024.11

Research paper (scientific journal), Accepted, ISSN 0006291X

Ezrin is a cross-linker protein between membrane proteins and cytosolic actin, abundantly expressed in the placenta among the ERM protein family. Ezrin gene knockout mice exhibit fetal growth restriction after gestational day (GD) 15.5. This study aimed to clarify the effect of ezrin on immune cells that influence fetal growth and immune tolerance. Ezrin heterozygous knockout (Ez+/−) mice were interbred, and the gene expressions and immune cell distributions in the placentas of wild-type (Ez+/+) and ezrin knockout (Ez−/−) fetuses were analyzed. IL-6 expression in the placenta of Ez−/− fetuses was significantly higher than in Ez+/+ fetuses at GD 15.5. The mRNA expression of IL-6 in the uterine decidua attached to Ez−/− fetuses was higher compared to that attached to Ez+/+ fetuses but not in the junctional zone and labyrinth. Classical M1 and M2 macrophages in the decidua were analyzed by flow cytometry using CD86 and CD206 as markers. M1 macrophages increased in the decidua attached to Ez−/− mice compared to Ez+/+ mice, while M2 macrophages did not increase. CD4-positive T cells showed a reduction in the decidua attached to Ez−/− fetuses. Further analysis involved the subcutaneous administration of tacrolimus in pregnant Ez+/− mice from GD 8.5 to GD 15.5, which prevented the decrease in fetal body weight and decidual CD4-positive T cells in Ez−/− mice at GD 15.5. These results suggest that impaired expression of fetoplacental-derived ezrin induces inflammatory conditions in the uterine decidua through M1 polarization of macrophages, increased IL-6, and decreased CD4-positive T cells, including Treg cells.

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Conversion of Olmesartan to Olmesartan Medoxomil, A Prodrug that Improves Intestinal Absorption, Confers Substrate Recognition by OATP2B1

Fukazawa N., Nishimura T., Orii K., Noguchi S., Tomi M.

Pharmaceutical Research (Pharmaceutical Research) 41 ( 5 ) 849 - 861 2024.05

Research paper (scientific journal), Accepted, ISSN 07248741

Purpose: Olmesartan medoxomil (olmesartan-MX), an ester-type prodrug of the angiotensin II receptor blocker (ARB) olmesartan, is predominantly anionic at intestinal pH. Human organic anion transporting polypeptide 2B1 (OATP2B1) is expressed in the small intestine and is involved in the absorption of various acidic drugs. This study was designed to test the hypothesis that OATP2B1-mediated uptake contributes to the enhanced intestinal absorption of olmesartan-MX, even though olmesartan itself is not a substrate of OATP2B1. Methods: Tetracycline-inducible human OATP2B1- and rat Oatp2b1-overexpressing HEK 293 cell lines (hOATP2B1/T-REx-293 and rOatp2b1/T-REx-293, respectively) were established to characterize OATP2B1-mediated uptake. Rat jejunal permeability was measured using Ussing chambers. ARBs were quantified by liquid chromatography-tandem mass spectrometry. Results: Significant olmesartan-MX uptake was observed in hOATP2B1/T-REx-293 and rOatp2b1/T-REx-293 cells, whereas olmesartan uptake was undetectable or much lower than olmesartan-MX uptake, respectively. Furthermore, olmesartan-MX exhibited several-fold higher uptake in Caco-2 cells and greater permeability in rat jejunum compared to olmesartan. Olmesartan-MX uptake in hOATP2B1/T-REx-293 cells and in Caco-2 cells was significantly decreased by OATP2B1 substrates/inhibitors such as 1 mM estrone-3-sulfate, 100 µM rifamycin SV, and 100 µM fluvastatin. Rat Oatp2b1-mediated uptake and rat jejunal permeability of olmesartan-MX were significantly decreased by 50 µM naringin, an OATP2B1 inhibitor. Oral administration of olmesartan-MX with 50 µM naringin to rats significantly reduced the area under the plasma concentration–time curve of olmesartan to 76.9%. Conclusion: Olmesartan-MX is a substrate for OATP2B1, and the naringin-sensitive transport system contributes to the improved intestinal absorption of olmesartan-MX compared with its parent drug, olmesartan.

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Involvement of GAT2/Slc6a13 in hypotaurine uptake at fetal-facing plasma membrane of syncytiotrophoblasts at mid-to-late gestation in rats and mice

Nishimura T., Araki H., Higuchi K., Noguchi S., Saito K., Hara K., Yagishita H., Akashi R., Obata S., Tomi M.

Placenta (Placenta) 147 59 - 67 2024.03

Research paper (scientific journal), Accepted, ISSN 01434004

Introduction: Hypotaurine, a precursor to taurine, is known for its antioxidant properties and is prominently present in fetal plasma and the placenta. Our previous research revealed that ezrin-knockout mice experience fetal growth retardation, coinciding with reduced hypotaurine levels in fetal plasma. This study aims to elucidate the expression and role of hypotaurine transporters within the placenta. Methods: We employed quantitative RT-PCR to measure mRNA expression of GAT transporter family members in the placenta during mid-to-late gestation. LC/MS/MS was used to analyze the distribution of hypotaurine in different placental subregions. Immunohistochemistry was utilized to examine the localization of GAT2 in mice. Placental hypotaurine uptake from fetal circulation was studied via umbilical perfusion in rats. Results: Among hypotaurine transporters, GAT2 exhibited increased mRNA and protein expression in murine placenta during mid-to-late gestation. Notably, GAT2/Slc6a13 mRNA and hypotaurine were most concentrated in the labyrinth of murine placenta. In contrast, enzymes responsible for hypotaurine synthesis, such as cysteine dioxygenase, cysteine sulfinic acid decarboxylase, and 2-aminoethanethiol dioxygenase, showed minimal expression in the labyrinth. These findings suggest that GAT2 is a key determinant of hypotaurine levels in the placental labyrinth. Immunohistochemical examination unveiled that GAT2 was predominantly localized on the fetal-facing plasma membrane within syncytiotrophoblasts, which co-localized with ezrin. In rat umbilical perfusion experiments, the GAT2/3 and TauT inhibitor, SNAP-5114, significantly reduced hypotaurine extraction from fetal circulation to the placenta. Discussion: The results suggest that GAT2 plays a pivotal role in the concentrative uptake of hypotaurine from fetal plasma within syncytiotrophoblasts of the placenta.

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Breast Cancer Resistance Protein Limits Fetal Transfer of Tadalafil in Mice

Nishimura T., Ishii M., Tanaka H., Noguchi S., Ikeda T., Tomi M.

Journal of Pharmaceutical Sciences (Journal of Pharmaceutical Sciences) 113 ( 2 ) 486 - 492 2024.02

Research paper (scientific journal), Accepted, ISSN 00223549

Tadalafil, a phosphodiesterase 5 (PDE5) inhibitor, is a candidate therapeutic agent for fetal growth restriction and hypertensive disorders of pregnancy. In this study, we elucidated the fetal transfer of tadalafil in comparison with that of sildenafil, the first PDE5 inhibitor to be approved. We also examined the contributions of multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP) to fetal transfer. Tadalafil or sildenafil was administered to wild-type, Mdr1a/b-double-knockout or Bcrp-knockout pregnant mice by continuous infusion from gestational day (GD) 14.5 to 17.5, and the fetal-to-maternal plasma concentration ratio of unbound drug (unbound F/M ratio) was evaluated at GD 17.5. The values of unbound F/M ratio of tadalafil and sildenafil in wild-type mice were 0.80 and 1.6, respectively. The unbound F/M ratio of tadalafil was increased to 1.1 and 1.7 in Mdr1a/b-knockout and Bcrp-knockout mice, respectively, while the corresponding values for sildenafil were equal to or less than that in wild-type mice, respectively. A transcellular transport study revealed that basal-to-apical transport of both tadalafil and sildenafil was significantly higher than transport in the opposite direction in MDCKII-BCRP cells. Our research reveals that tadalafil is a newly identified substrate of human and mouse BCRP, and it appears that the fetal transfer of tadalafil is, at least in part, attributed to the involvement of BCRP within the placental processes in mice. The transfer of sildenafil to the fetus was not significantly constrained by BCRP, even though sildenafil was indeed a substantial substrate for BCRP.

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L-Type Amino Acid Transporter 1 (SLC7A5)-Mediated Transport of Pregabalin at the Rat Blood-Spinal Cord Barrier and its Sensitivity to Plasma Branched-Chain Amino Acids.

Akashi T, Noguchi S, Takahashi Y, Nishimura T, Tomi M

Journal of pharmaceutical sciences (Journal of Pharmaceutical Sciences) 112 ( 4 ) 1137 - 1144 2023.01

Research paper (scientific journal), Accepted, ISSN 0022-3549

Pregabalin is an anti-neuropathic pain drug inhibiting the α2δ subunit of the voltage-dependent calcium channel in the spinal cord. The aim of this study is to characterize the transport mechanism of pregabalin at the blood-spinal cord barrier (BSCB) by means of in vivo experiments in rats and in vitro studies using primary-cultured rat spinal cord endothelial cells. We isolated endothelial cells by culturing rat spinal cord tissue in the presence of puromycin, and confirmed the expression of BSCB markers such as Cd31, Mdr1a, and Claudin-5. The uptake of pregabalin by primary-cultured rat spinal cord endothelial cells was sodium-independent and was significantly inhibited by L-leucine, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, and JPH203. These results suggest the involvement of L-type amino acid transporter (LAT) 1. LAT1 mRNA and protein was expressed in primary-cultured rat spinal cord endothelial cells, which is consistent with LAT1 expression at the BSCB. In the in vivo study, the transfer of pregabalin to rat spinal cord and brain was significantly decreased by the pre-administration of branched chain amino acids (BCAAs), which are endogenous substrates of LAT1. Our results indicate that pregabalin transport across the BSCB is mediated at least in part by LAT1 and is inhibited by plasma BCAAs.

Quantitative Comparison of Breast Cancer Resistance Protein (BCRP/ABCG2) Expression and Function Between Maternal Blood-Brain Barrier and Placental Barrier in Mice at Different Gestational Ages

Arimi Fujita, Saki Noguchi, Rika Hamada, Tsutomu Shimada, Satomi Katakura, Tetsuo Maruyama, Yoshimichi Sai, Tomohiro Nishimura, Masatoshi Tomi

Frontiers in Drug Delivery (Frontiers) 2 932576 2022.07

Research paper (scientific journal), Joint Work, Last author, Corresponding author, Accepted

Breast cancer resistance protein (BCRP) is expressed by brain capillary endothelial cells and at the interface between two placental syncytiotrophoblast layers in rodents and serves to suppress drug distribution to the brain and the fetus. The purpose of the present study is to determine and compare the apparent impact of a single BCRP molecule on drug transfer between the maternal blood-brain barrier and placental barrier in pregnant mice at different gestation ages. BCRP protein was quantified by liquid chromatography-tandem mass spectrometry. Genistein or dantrolene was continuously administered to pregnant Bcrp−/− or wild-type (WT) mice, and the brain-to-plasma concentration ratio in the mother (K<inf>p,brain</inf>) and the fetal-to-maternal ratio of plasma concentrations (K<inf>p,fp</inf>) were determined. At gestational day 15.5 (GD15.5), the protein amount of BCRP at the murine placental barrier was estimated to be approximately three times higher than at the maternal blood-brain barrier, but the levels were approximately the same at GD17.5 due to the decline of placental BCRP expression during gestation. On the other hand, the values of Bcrp−/−/WT ratio of K<inf>p,brain</inf> for genistein and dantrolene were 6.1 and 3.8, respectively, while the K<inf>p,fp</inf> ratios were all less than 2.0. These results indicate that the apparent impact of a single placental BCRP molecule on the restriction of drug distribution is much less than that of a single brain BCRP molecule, probably because the function of placental BCRP is attenuated by bypass transfer through the connexin26 gap junctions between adjacent syncytiotrophoblast layers. The present study also found that the expression amount of BCRP protein at the human placental barrier formed by the monolayer of syncytiotrophoblasts was lower than that in mice, but this species difference appears to be functionally compensated by the murine-specific bypass route through gap junctions, at least in part.

Decellularized Organ-Derived Scaffold Is a Promising Carrier for Human Induced Pluripotent Stem Cells-Derived Hepatocytes

Kojima H., Yagi H., Kushige H., Toda Y., Takayama K., Masuda S., Morisaku T., Tsuchida T., Kuroda K., Hirukawa K., Inui J., Nishi K., Nakano Y., Tanaka M., Hori S., Hasegawa Y., Abe Y., Kitago M., Adachi S., Tomi M., Matsuura K., Mizuguchi H., Kitagawa Y.

Cells (Cells) 11 ( 8 ) 2022.04

Research paper (scientific journal), Accepted

Human induced pluripotent stem cells (hiPSCs) are a promising cell source for elucidating disease pathology and therapy. The mass supply of hiPSC-derived cells is technically feasible. Carriers that can contain a large number of hiPSC-derived cells and evaluate their functions in vivo-like environments will become increasingly important for understanding disease pathogenesis or treating end-stage organ failure. hiPSC-derived hepatocyte-like cells (hiPSC-HLCs; 5 × 108) were seeded into decellularized organ-derived scaffolds under circumfusion culture. The scaffolds were implanted into immunodeficient microminiature pigs to examine their applicability in vivo. The seeded hiPSC-HLCs demonstrated increased albumin secretion and up-regulated cytochrome P450 activities compared with those in standard two-dimensional culture conditions. Moreover, they showed long-term survival accompanied by neovascularization in vivo. The decellularized organ-derived scaffold is a promising carrier for hiPSC-derived cells for ex vivo and in vivo use and is an essential platform for regenerative medicine and research.

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Limited Impact of Murine Placental MDR1 on Fetal Exposure of Certain Drugs Explained by Bypass Transfer Between Adjacent Syncytiotrophoblast Layers.

Fujita A, Noguchi S, Hamada R, Inoue S, Shimada T, Katakura S, Maruyama T, Sai Y, Nishimura T, Tomi M

Pharmaceutical research (Pharmaceutical Research) 39 ( 7 ) 1645 - 1658 2022.01

Research paper (scientific journal), Accepted, ISSN 0724-8741

Purpose: Multidrug resistance protein 1 (MDR1) is located at the interface between two syncytiotrophoblast layers in rodent placenta, and may influence fetal drug distribution. Here, we quantitatively compare the functional impact per single MDR1 molecule of MDR1 at the placental barrier and blood-brain barrier in mice. Methods: MDR1A and MDR1B proteins were quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Paclitaxel or digoxin was continuously administered to pregnant Mdr1a−/−/Mdr1b−/− or wild-type mice, and the drug concentrations in the maternal and fetal plasma and maternal brain were quantified by LC-MS/MS. Results: MDR1A and MDR1B proteins are expressed in the membrane of mouse placental labyrinth, and total MDR1 at the placental barrier amounts to about 30% of that at the blood-brain barrier. The fetal-to-maternal plasma concentration ratio of digoxin was only marginally affected in Mdr1a−/−/Mdr1b−/− mice, while that of paclitaxel showed a several-fold increase. No such difference between the two drugs was found in the maternal brain distribution. The impact per single MDR1 molecule on the fetal distribution of digoxin was calculated to be much lower than that on the brain distribution, but this was not the case for paclitaxel. Our pharmacokinetic model indicates that the impact of placental MDR1 is inversely correlated to the ratio of permeability through gap junctions connecting the two syncytiotrophoblast layers to passive diffusion permeability. Conclusion: Our findings indicate that murine placental MDR1 has a minimal influence on the fetal concentration of certain substrates, such as digoxin, due to bypass transfer, probably via connexin26 gap junctions.

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Transplantation of Bioengineered Liver Capable of Extended Function in a Preclinical Liver Failure Model.

Higashi H, Yagi H, Kuroda K, Tajima K, Kojima H, Nishi K, Morisaku T, Hirukawa K, Fukuda K, Matsubara K, Kitago M, Shinoda M, Obara H, Adachi S, Nishimura K, Natsume T, Tomi M, Soto-Gutierrez A, Kitagawa Y

American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons (American Journal of Transplantation) 22 ( 3 ) 731 - 744 2021.12

Research paper (scientific journal), Accepted, ISSN 1600-6135

Unlimited organ availability would represent a paradigm shift in transplantation. Long-term in vivo engraftment and function of scaled-up bioengineered liver grafts have not been previously reported. In this study, we describe a human-scale transplantable liver graft engineered on a porcine liver-derived scaffold. We repopulated the scaffold parenchyma with primary hepatocytes and the vascular system with endothelial cells. For in vivo functional testing, we performed auxiliary transplantation of the repopulated scaffold in pigs with induced liver failure. It was observed that the auxiliary bioengineered liver graft improved liver function for 28 days and exhibited upregulation of liver-specific genes. This study is the first of its kind to present 28 days of posttransplant evaluation of a bioengineered liver graft using a preclinical large animal model. Furthermore, it provides definitive evidence for the feasibility of engineering human-scale transplantable liver grafts for clinical applications.

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Transplacental pharmacokinetic model of digoxin based on ex vivo human placental perfusion study

Kurosawa K, Noguchi S, Nishimura T, Tomi M, Chiba K

Drug metabolism and disposition: the biological fate of chemicals (Drug Metabolism and Disposition) 50 ( 3 ) 287 - 298 2021.12

Research paper (scientific journal), Accepted, ISSN 0090-9556

Digoxin is used as first-line therapy to treat fetal supraventricular tachycardia; however, because of the narrow therapeutic window, it is essential to estimate digoxin exposure in the fetus. The data from ex vivo human placental perfusion study are used to predict in vivo fetal exposure noninvasively, but the ex vivo fetal-to-maternal concentration (F:M) ratios observed in digoxin perfusion studies were much lower than those in vivo. In the present study, we developed a human transplacental pharmacokinetic model of digoxin using previously reported ex vivo human placental perfusion data. The model consists of maternal intervillous, fetal capillary, non-perfused tissue, and syncytiotrophoblast compartments, with multidrug resistance protein (MDR) 1 and influx transporter at the microvillous membrane (MVM) and influx and efflux transporters at the basal plasma membrane (BM). The model-predicted F:M ratio was 0.66, which is consistent with the mean in vivo value of 0.77 (95% confidence interval: 0.64-0.91). The time to achieve the steady state from the ex vivo perfusion study was estimated as 1,500 minutes, which is considerably longer than the reported ex vivo experimental durations, and this difference is considered to account for the inconsistency between ex vivo and in vivo F:M ratios. Reported digoxin concentrations in a drug-drug interaction study with MDR1 inhibitors quinidine and verapamil were consistent with the profiles simulated by our model incorporating inhibition of efflux transporter at the BM in addition to MVM. Our modeling and simulation approach should be a powerful tool to predict fetal exposure and DDIs in human placenta. SIGNIFICANCE STATEMENT We developed a human transplacental pharmacokinetic model of digoxin based on ex vivo human placental perfusion studies in order to resolve inconsistencies between reported ex vivo and in vivo fetal-to-maternal concentration ratios. The model successfully predicted the in vivo fetal exposure to digoxin and the drug-drug interactions of digoxin and P-glycoprotein/multidrug resistance protein 1 inhibitors in human placenta.

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Role of Uptake Transporters OAT4, OATP2A1, and OATP1A2 in Human Placental Bio-disposition of Pravastatin

Fokina V.M., Patrikeeva S., Wang X.m., Noguchi S., Tomi M., König J., Ahmed M.S., Nanovskaya T.

Journal of Pharmaceutical Sciences (Journal of Pharmaceutical Sciences) 111 ( 2 ) 505 - 516 2021.09

Research paper (scientific journal), Accepted, ISSN 00223549

Pravastatin is currently under evaluation for prevention of preeclampsia. Factors contributing to placental disposition of pravastatin are important in assessment of potential undesirable fetal effects. The purpose of this study was to identify the uptake transporters that contribute to the placental disposition of pravastatin. Our data revealed the expression of organic anion transporting polypeptide 1A2 (OATP1A2) and OATP2A1 in the apical, and OATP2B1 and OATP5A1 in the basolateral membranes of the placenta, while organic anion transporter 4 (OAT4) exhibited higher expression in basolateral membrane but was detected in both membranes. Preloading placental membrane vesicles with glutarate increased the uptake of pravastatin suggesting involvement of glutarate-dependent transporters such as OAT4. In the HEK293 cells overexpressing individual uptake transporters, OATP2A1, OATP1A2 and OAT4 were determined to accept pravastatin as a substrate at physiological pH, while the uptake of pravastatin by OATP2B1 (known to interact with pravastatin at acidic pH) and OATP5A1 was not detected at pH 7.4. These findings led us to propose that OATP1A2 and OATP2A1 are responsible for the placental uptake of pravastatin from the maternal circulation, while OAT4 mediates the passage of the drug across placental basolateral membrane in the fetal-to-maternal direction.

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MicroRNA-126 suppresses the invasion of trophoblast-model JEG-3 cells by targeting LIN28A

Pan X., Noguchi S., Ando M., Nishimura T., Tomi M.

Biochemical and Biophysical Research Communications (Biochemical and Biophysical Research Communications) 545 132 - 137 2021.03

Research paper (scientific journal), Accepted, ISSN 0006291X

© 2021 Elsevier Inc. Inadequate trophoblast invasion and impaired trophoblast-induced vascular remodeling are features of preeclampsia. In this context, an angiogenesis-related microRNA, miR-126, is abnormally expressed in preeclampsia placentas, but its role in trophoblast development remains unclear. The purpose of this study was to investigate the roles of miR-126 in the proliferation, migration, and invasion processes of trophoblast cells using the human choriocarcinoma-derived JEG-3 cell line as a model. The mRNA expression profiling of JEG-3 cells with and without miR-126 overexpression, in combination with bioinformatics analysis, identified LIN28A as a putative target of miR-126. The results of real-time RT-PCR and luciferase assay were consistent with this idea. Overexpression of miR-126 in JEG-3 cells decreased the invasive ability of the cells without affecting proliferation or migration. The invasiveness of JEG-3 cells was significantly reduced to a similar extent by knockdown of LIN28A with siRNA and by miR-126-overexpression-induced downregulation of LIN28A, although the level of LIN28A protein was much lower in the siLIN28A-transfected cells. These results indicate that miR-126 suppresses JEG-3 cell invasion by targeting LIN28A, and suggest that miR-126-mediated downregulation of LIN28A might contribute to the onset/deterioration of preeclampsia.

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Substrate recognition of renally eliminated angiotensin II receptor blockers by organic anion transporter 4

Noguchi S., Okochi M., Atsuta H., Kimura R., Fukumoto A., Takahashi K., Nishimura T., Tomi M.

Drug Metabolism and Pharmacokinetics (Drug Metabolism and Pharmacokinetics) 36 100363 2021.02

Research paper (scientific journal), Accepted, ISSN 13474367

© 2020 The Japanese Society for the Study of Xenobiotics Organic anion transporter (OAT) 4, which is localized at the apical membrane of human renal proximal tubules, transports olmesartan, an angiotensin II receptor blocker (ARB). Many ARBs, including olmesartan, undergo partial tubular secretion as active forms, and inhibit OAT4-mediated uptake activity. Here, we examined the substrate recognition of various ARBs by OAT4 in order to assess whether OAT4 might be involved in the renal handling of ARBs. Concentration-dependent OAT4-mediated uptake of azilsartan, candesartan, carboxylosartan, losartan, and valsartan was observed with Km values of 6.6, 31, 7.2, 13, and 1.7 μM, respectively, in the absence of extracellular Cl−. In the presence of extracellular Cl−, OAT4-mediated uptake of dianionic ARBs (azilsartan, candesartan, carboxylosartan, and valsartan) was lower and reached a steady state faster than in the absence of extracellular Cl−. Thus, OAT4 is proposed to use extracellular Cl− as a counterpart for anion efflux. Our results suggest that OAT4 may play a role in the excretion of azilsartan, candesartan, carboxylosartan, and valsartan, as well as olmesartan. In contrast, OAT4-mediated uptake of losartan, a monoanionic ARB, was little affected by extracellular Cl−, suggesting that only OAT4-mediated dianion transport is Cl−-sensitive.

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Fluorouracil Uptake in Triple-Negative Breast Cancer Cells: Negligible Contribution of Equilibrative Nucleoside Transporters 1 and 2.

Noguchi S, Takagi A, Tanaka T, Takahashi Y, Pan X, Kibayashi Y, Mizokami R, Nishimura T, Tomi M

Biopharmaceutics & drug disposition (Biopharmaceutics and Drug Disposition) 42 ( 2-3 ) 85 - 93 2021.01

Research paper (scientific journal), Accepted, ISSN 0142-2782

© 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.

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Development of a Pharmacokinetic Model of Transplacental Transfer of Metformin to Predict In Vivo Fetal Exposure

Kurosawa K., Chiba K., Noguchi S., Nishimura T., Tomi M.

Drug metabolism and disposition: the biological fate of chemicals (Drug metabolism and disposition: the biological fate of chemicals) 48 ( 12 ) 1293 - 1302 2020.12

Research paper (scientific journal), Accepted, ISSN 0090-9556

Copyright © 2020 The Author(s). Two types of systems are used in ex vivo human placental perfusion studies to predict fetal drug exposures, that is, closed systems with recirculation of the maternal and fetal buffer and open systems using a single-pass mode without recirculation. The in vivo fetal/maternal (F:M) ratio of metformin, a cationic drug that crosses the placenta, is consistent with that reported in an open system ex vivo but not with that in a closed system. In the present study, we aimed to develop a pharmacokinetic (PK) model of transplacental transfer of metformin to predict in vivo fetal exposure to metformin and to resolve the apparent inconsistency between open and closed ex vivo systems. The developed model shows that the difference between open and closed systems is due to the difference in the time required to achieve the steady state. The model-predicted F:M ratio (approx. 0.88) is consistent with reported in vivo values [mean (95% confidence interval): 1.10 (0.69-1.51)]. The model incorporates bidirectional transport via organic cation transporter 3 (OCT3) at the basal plasma membrane, and simulations indicate that the use of trimethoprim (an OCT3 inhibitor) to prevent microbial growth in the placenta ex vivo has a negligible effect on the overall maternal-to-fetal and fetal-to-maternal clearances. The model could successfully predict in vivo fetal exposure using ex vivo human placental perfusion data from both closed and open systems. This transplacental PK modeling approach is expected to be useful for evaluating human fetal exposures to other poorly permeable compounds, besides metformin. SIGNIFICANCE STATEMENT: We developed a pharmacokinetic model of transplacental transfer of metformin, used to treat gestational diabetes mellitus, in order to predict in vivo fetal exposure and resolve the discrepancy between reported findings in open and closed ex vivo perfusion systems. The discrepancy is due to a difference in the time required to reach the steady state. The model can predict in vivo fetal exposure using data from both closed and open systems.

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Contribution of Prostaglandin Transporter OATP2A1/SLCO2A1 to Placenta-to-Maternal Hormone Signaling and Labor Induction

Inagaki M., Nishimura T., Nakanishi T., Shimada H., Noguchi S., Akanuma S.i., Tachikawa M., Hosoya K.i., Tamai I., Nakashima E., Tomi M.

iScience (iScience) 23 ( 5 ) 101098 2020.05

Research paper (scientific journal), Joint Work, Accepted

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Quantification of ENT1 and ENT2 Proteins at the Placental Barrier and Contribution of These Transporters to Ribavirin Uptake

Nishimura T., Sano Y., Takahashi Y., Noguchi S., Uchida Y., Takagi A., Tanaka T., Katakura S., Nakashima E., Tachikawa M., Maruyama T., Terasaki T., Tomi M.

Journal of Pharmaceutical Sciences (Journal of Pharmaceutical Sciences) 108 ( 12 ) 3917 - 3922 2019.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 00223549

© 2019 The Authors The aims of this study are to quantify the protein levels of nucleoside transporters in placental microvillous membranes (MVMs) and to clarify the contributions of these transporters to ribavirin uptake at the placental barrier. Placental MVMs of human and rat expressed equilibrative nucleoside transporter (ENT) 1 protein, whereas the expression of ENT2 protein was obscure. Maternal-to-fetal transfer of [3H]ribavirin in rats was much higher than that of [14C]sucrose. The uptake of [3H]ribavirin by rat placental trophoblast TR-TBT 18 d-1 cells, which functionally express both ENT1 and ENT2 proteins, was saturable, and was significantly inhibited by 0.1 μM nitrobenzylthioinosine, which selectively abolishes ENT1-mediated uptake. Dipyridamole at 10 μM is capable of inhibiting ENT2 as well as ENT1, but a degree of inhibition by 10 μM dipyridamole on [3H]ribavirin uptake was not much different from that by 0.1 μM nitrobenzylthioinosine (ENT1-specific inhibitor). Therefore, ENT2 may contribute little to [3H]ribavirin uptake by these cells. Rat ENT1 cRNA-injected oocytes showed increased [3H]ribavirin uptake compared with water-injected oocytes, while rat ENT2 cRNA-injected oocytes did not. In conclusion, ENT1 protein expressed in placental MVMs appears to play a predominant role in the uptake of ribavirin.

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Transport of Pregabalin Via L-Type Amino Acid Transporter 1 (SLC7A5) in Human Brain Capillary Endothelial Cell Line

Takahashi Y., Nishimura T., Higuchi K., Noguchi S., Tega Y., Kurosawa T., Deguchi Y., Tomi M.

Pharmaceutical Research (Pharmaceutical Research) 35 ( 12 ) 246 2018.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 07248741

© 2018, The Author(s). Purpose: The anti-epileptic drug pregabalin crosses the blood-brain barrier (BBB) in spite of its low lipophilicity. This study was performed to determine whether L-type amino acid transporters (LAT1/SLC7A5 and LAT2/SLC7A8) contribute to the uptake of pregabalin. Methods: Pregabalin uptake by LATs-transfected HEK293 cells or hCMEC/D3 cells, an in vitro human BBB model, was measured by LC-MS/MS analysis. Expression of LAT1 mRNA in hCMEC/D3 cells was determined by quantitative RT-PCR analysis. Results: Overexpression of LAT1, but not LAT2, in HEK293 cells significantly increased the cellular uptake of pregabalin, and the LAT1-mediated uptake was saturable with a Km of 0.288 mM. LAT1-mediated amino acid uptake was inhibited specifically and almost completely in the presence of 1 mM pregabalin. The uptake of pregabalin by hCMEC/D3 cells was sodium-independent, saturable (Km = 0.854 mM), and strongly inhibited by large amino acids at 1 mM, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, a specific system L inhibitor, at 1 mM and by JPH203, a LAT1-selective inhibitor, at 10 μM. Pregabalin uptake in hCMEC/D3 cells was also decreased by 75% by the silencing of LAT1 gene using LAT1 siRNA. Conclusions: Our results indicate that LAT1, but not LAT2, recognizes pregabalin as a substrate. It is suggested that LAT1 mediates pregabalin transport at the BBB.

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Hypotaurine Is a Substrate of GABA Transporter Family Members GAT2/Slc6a13 and TAUT/Slc6a6

Nishimura Tomohiro, Higuchi Kei, Yoshida Yuko, Sugita-Fujisawa Yuki, Kojima Kazuaki, Sugimoto Maiko, Santo Marie, Tomi Masatoshi, Nakashima Emi

Biological and Pharmaceutical Bulletin (The Pharmaceutical Society of Japan) 41 ( 10 ) 1523 - 1529 2018.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 09186158

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/gamma-aminobutyric 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.

Gene expression of A6-like subgroup of ATP-binding cassette transporters in mouse brain parenchyma and microvessels

Tachikawa M., Toki H., Watanabe M., Tomi M., Hosoya K., Terasaki T.

Anatomical Science International (Anatomical Science International) 93 ( 4 ) 456 - 463 2018.03

Research paper (scientific journal), Accepted, ISSN 1447-6959

© 2018, Japanese Association of Anatomists. The A-subclass of ATP-binding cassette (ABC) transporters is a highly conserved superfamily of potent lipid transporters. Although the ABCA1-like subgroup of ABCA1-4, and A7 have been shown to mediate the transport of endogenous lipids, the roles of the ABCA6-like subgroup transporters, which have been identified as a unique gene cluster on human chromosome 17q24 (ABCA5, A6, A8, A9, and A10) and mouse chromosome 11 (Abca5, a6, a8a, a8b, and a9), remains largely unknown. The purpose of the present study was to clarify the spatial and temporal expression profiles of Abca6-like subgroup transporters in embryonic and postnatal mouse brains by a combination of in situ hybridization and quantitative polymerase chain reaction (PCR) using magnetically isolated brain vascular endothelial cells. In embryonic brains, the transcripts of Abca5, a8a and a8b were detected predominantly in the mantle zone, where postmitotic neurons differentiate. At the postnatal stages, they were expressed in various nuclei and neuronal layers. Abca9 mRNA was detected diffusely in the embryonic and postnatal brains and sequential and/or strong spotted signals were detected in the leptomeninges on the brain surface. PCR detected expression of Abca8a and Abca9 mRNAs in isolated vascular endothelial cells. Expression signals for Abca6 mRNA were hardly observed at any stages examined. These distinct spatio-temporal expression patterns of Abca6-like subgroup transporters may reflect their functional significance and diversity to regulate lipid transport, particularly in neurons, leptomeningeal cells, and vascular endothelial cells.

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Effect of serotonin transporter genotype on self-reported efficacy and activity changes of brain prefrontal area in response to placebo

Isawa M., Tashiro R., Naruse C., Yamaguchi Y., Itoh H., Nishimura T., Tomi M., Shimada H., Saito H., Mochizuki M., Nakashima E.

Pharmazie (Pharmazie) 73 ( 1 ) 35 - 41 2018.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 0031-7144

Patients benefit from drug therapy not only through pharmacological mechanisms, but also through non-pharmacological action (placebo effect), which may be mediated in part by the prefrontal area of the brain. We consider that the difference between responders and non-responders to placebo might be related to polymorphisms in the serotonin transporter-linked polymorphic region (5-HTTLPR). To study this idea, we performed a randomized double-blind clinical trial using caffeine and lactose (placebo). Activity in the prefrontal area of the brain was measured in terms of blood flow by means of near-infrared spectroscopy (NIRS) as an objective indicator. Self-reported feelings of drowsiness on established scales were used as subjective indicators. Twenty-one subjects in block A took caffeine on the first day and placebo on the third day, and 21 in block B took placebo on the first day and placebo on the third day. After placebo administration, improvement of sleepiness was significantly enhanced, a similar extent to that after caffeine medication. Among the 42 subjects, 22 showed S/S type polymorphism in the serotonin transporter (52.4 %), 17 showed S/L type (40.5 %) and 3 showed L/L type (7.10 %). Statistical analysis of the results indicate that subjects with L/L genotype showed a significantly greater placebo response in terms of both self-reported feeling of drowsiness and blood flow in the prefrontal area of the brain associated with working memory (46 area). Our results indicate that the L/L genotype of 5-HTTLPR, which is rare in Japanese (3.2 %) but common in Americans (32.2 %), may be associated with a greater placebo effect.

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Cellular Uptake of Levocetirizine by Organic Anion Transporter 4

Noguchi S., Nishimura T., Mukaida S., Benet L., Nakashima E., Tomi M.

Journal of Pharmaceutical Sciences (Journal of Pharmaceutical Sciences) 106 ( 9 ) 2895 - 2898 2017.09

Research paper (scientific journal), Joint Work, Accepted, ISSN 0022-3549

© 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.

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Co-localization of microsomal prostaglandin E synthase-1 with cyclooxygenase-1 in layer II of murine placental syncytiotrophoblasts

Inagaki M., Nishimura T., Akanuma S., Nakanishi T., Tachikawa M., Tamai I., Hosoya K., Nakashima E., Tomi M.

Placenta (Placenta) 53 76 - 82 2017.04

Research paper (scientific journal), Accepted, ISSN 01434004

© 2017 Elsevier Ltd The placenta is an organ that secretes prostaglandin (PG) E2 into the fetal-placental circulation to regulate both vascular tone and remodeling of the fetal ductus arteriosus. Placental PGE2 synthesis might be mediated by microsomal PGE synthase-1 (mPGES-1), in addition to cyclooxygenase (COX) isoforms. Thus, the purpose of this study is to clarify the temporal and spatial expression patterns of mPGES-1, together with COX-1 and COX-2, in murine placenta. We found that mPGES-1 and COX-1 protein levels continuously increased in the placental labyrinth from gestational day (GD) 13.5 to GD19.5, becoming higher than in the decidua or the junctional zone by GD17.5. The PGE2 level at GD17.5 was also highest in the labyrinth. Immunofluorescence stainings for mPGES-1 and COX-1 in the labyrinth at GD17.5 overlapped and were located on the fetal side of the signals for connexin 26, which forms gap junctions between maternal-facing (SynT-I) and fetal-facing (SynT-II) syncytiotrophoblast layers, and on the maternal side of the signals for glucose transporter 1 on the basal plasma membrane of SynT-II. On the other hand, the signals for COX-2 did not overlap with those for mPGES-1. These results indicate that COX-1 and mPGES-1 are co-localized in murine placental SynT-II, facing the fetal-placental circulation. Therefore, SynT-II could contribute to placental synthesis of PGE2 for release into the fetal-placental circulation.

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Contribution of equilibrative nucleoside transporter (ENT) 2 to fluorouracil transport in rat placental trophoblast cells

Takagi A., Nishimura T., Akashi T., Tomi M., Nakashima E.

Drug Metabolism and Pharmacokinetics (Drug Metabolism and Pharmacokinetics) 32 ( 2 ) 151 - 156 2017.04

Research paper (scientific journal), Accepted, ISSN 1347-4367

© 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.

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Sex-specific life course changesin the neuro-metabolic phenotype of glut3 null heterozygous mice: Ketogenic diet ameliorates electroencephalographic seizures and improves sociability

Dai Y., Zhao Y., Tomi M., Shin B.C., Thamotharan S., Mazarati A., Sankar R., Wang E.A., Cepeda C., Levine M.S., Zhang J., Frew A., Alger J.R., Clark P.M., Sondhi M., Kositamongkol S., Leibovitch L., Devaskar S.U.

Endocrinology (Endocrinology) 158 ( 4 ) 936 - 949 2017.04

Research paper (scientific journal), Accepted, ISSN 0013-7227

© 2017 Endocrine Society. We tested the hypothesis that exposure of glut3 +/- mice to a ketogenic diet ameliorates autism-like features, which include aberrant behavior and electrographic seizures. We first investigated the life course sex-specific changes in basal plasma-cerebrospinal fluid (CSF)-brain metabolic profile, brain glucose transport/uptake, glucose and monocarboxylate transporter proteins, and adenosine triphosphate (ATP) in the presence or absence of systemic insulin administration. Glut3 +/- male but not femalemice (5months of age) displayed reduced CSF glucose/lactate concentrations with no change in brain Glut1, Mct2, glucose uptake or ATP. Exogenous insulin-induced hypoglycemia increased brain glucose uptake in glut3 +/- males alone. Higher plasma-CSF ketones (β-hydroxybutyrate) and lower brain Glut3 in females vs males proved protective in the former while enhancing vulnerability in the latter. As a consequence, increased synaptic proteins (neuroligin4 and SAPAP1) with spontaneous excitatory postsynaptic activity subsequently reduced hippocampal glucose content and increased brain amyloid β1-40 deposition in an age-dependent manner in glut3 +/- males but not females (4 to 24 months of age). We then explored the protective effect of a ketogenic diet on ultrasonic vocalization, sociability, spatial learning and memory, and electroencephalogram seizures in male mice (7 days to 6 to 8months of age) alone. A ketogenic diet partially restored sociability without affecting perturbed vocalization, spatial learningandmemory, andreducedseizure events.We concludethat (1) sexspecific and age-dependent perturbations underlie the phenotype of glut3 +/- mice, and (2) a ketogenic diet ameliorates seizures caused by increased cortical excitation and improves sociability, but fails to rescue vocalization and cognitive deficits in glut3 +/- male mice.

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Contributions of system A subtypes to α-methylaminoisobutyric acid uptake by placental microvillous membranes of human and rat

Takahashi Y., Nishimura T., Maruyama T., Tomi M., Nakashima E.

Amino Acids (Amino Acids) 49 ( 4 ) 795 - 803 2017.04

Research paper (scientific journal), Accepted, ISSN 0939-4451

© 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.

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Layer II of placental syncytiotrophoblasts expresses MDR1 and BCRP at the apical membrane in rodents

Akashi T., Nishimura T., Takaki Y., Takahashi M., Shin B.C., Tomi M., Nakashima E.

Reproductive Toxicology (Reproductive Toxicology) 65 375 - 381 2016.10

Research paper (scientific journal), Accepted, ISSN 0890-6238

The purpose of this study is to clarify the subcellular localizations of multidrug resistance protein 1 (MDR1)/ABCB1 and breast cancer resistance protein (BCRP)/ABCG2 in the rodent placental SynT bilayer, i.e., a maternal-facing (SynT-I) layer and a fetal-facing (SynT-II) layer. In double immunofluorescence staining, the signals of MDR1 and BCRP appeared midway between the signals of glucose transporter 1 on the apical membrane of SynT-I and the basal plasma membrane of SynT-II, and mostly overlapped with signals of connexin 26, which forms gap junctions between SynT-I and SynT-II. In detail, median intensities (pixels) of the MDR1 and BCRP signals were significantly closer to the fetal circulation as compared to the location of connexin 26 signals. In double in situ hybridization studies, the signals of Mdr1b mRNA mostly overlapped with those of Syncytin-B, a SynT-II marker. In conclusion, MDR1 and BCRP are expressed on apical membranes of the rodent placental SynT-II layer.

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Influence of Counseling Intervention on the Stress-Reducing Effect of Bergamot Oil and on the Concentration-Improving Effect of Rosemary Oil in Aromatherapy

ISAWA Minae, KOH Risako, ITOH Hiroshi, NISHIMURA Tomohiro, TOMI Masatoshi, SHIMADA Hideyo, SAITO Hidetsugu, NAKASHIMA Emi

Journal of Japanese Cosmetic Science Society (Japanese Cosmetic Science Society) 40 ( 2 ) 79 - 86 2016.06

Research paper (scientific journal), Accepted, ISSN 1880-2532

There is growing evidence that aromatherapy can improve patients' stress and refreshment levels. Also, an explanation to patients of the anticipated pharmacological effects of drugs may serve to improve compliance and to enhance subjective treatment benefit. Here, we aimed to examine the effectiveness of counseling as a means of improving the stress-reducing efficacy of bergamot oil and the concentration-improving effect of rosemary oil in 23 healthy young women. Efficacy was evaluated by using a self-reported questionnaire and multi-dimensional fatigue inventory-20 (MFI-20) as subjective indicators, together with near-infrared spectroscopy (NIRS)-evaluated blood flow in the inferior frontal cortex and amylase concentration in saliva as objective indicators. Counseling combined with bergamot aromatherapy significantly improved physical fatigue and mental fatigue and also improved sleepiness, concentration, and activity (p<0.01, p<0.05). Counseling combined with rosemary aromatherapy significantly improved motivation (p<0.05). Regional blood flow in the inferior frontal cortex was decreased totally. Especially partial right field of the prefrontal cortex area was decreased significantly by exposure to bergamot oil with counseling. By contrast, rosemary oil with counseling was decreased significantly in partial left of the prefrontal cortex area. Interestingly, we found that levels of salivary amylase (a sensitive biological marker of stress) were increased by counseling in subjects with low scores for resilience (capacity of individuals to resist stress). This finding may indicate that counseling was stressful to these subjects. Overall, our results indicate that counseling intervention increased the effectiveness of aromatherapy. Focused counseling taking account of behavioral differences between individuals may be a promising strategy to maximize patients' benefit.

Organic Anion Transporter 4-Mediated Transport of Olmesartan at Basal Plasma Membrane of Human Placental Barrier

Noguchi S., Nishimura T., Fujibayashi A., Maruyama T., Tomi M., Nakashima E.

Journal of Pharmaceutical Sciences (Journal of Pharmaceutical Sciences) 104 ( 9 ) 3128 - 3135 2015.09

Research paper (scientific journal), Accepted, ISSN 0022-3549

Mechanisms regulating fetal transfer of olmesartan, an angiotensin-II receptor type 1 antagonist, are important as potential determinants of life-threatening adverse fetal effects. The purpose of this study was to examine the olmesartan transport mechanism through the basal plasma membrane (BM) of human syncytiotrophoblasts forming the placental barrier. Uptake of olmesartan by human placental BM vesicles was potently inhibited by dehydroepiandrosterone sulfate (DHEAS), estrone 3-sulfate, and bromosulfophthalein, which are all typical substrates of organic anion transporter (OAT) 4 localized at the BM of syncytiotrophoblasts, and was increased in the absence of chloride. In tetracycline-inducible OAT4-expressing cells, [3H]olmesartan uptake was increased by tetracycline treatment. Olmesartan uptake via OAT4 was concentration dependent with a K<inf>m</inf> of 20 μM, and was increased in the absence of chloride. [3H]Olmesartan efflux via OAT4 was also observed and was trans-stimulated by extracellular chloride and DHEAS. Thus, OAT4 mediates bidirectional transport of olmesartan and appears to regulate fetal transfer of olmesartan at the BM of syncytiotrophoblasts. Efflux transport of olmesartan via OAT4 from syncytiotrophoblasts to the fetal circulation might be facilitated in the presence of an inwardly directed physiological chloride gradient and extracellular DHEAS.

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Role of OAT4 in uptake of estriol precursor 16α-hydroxydehydroepiandrosterone sulfate into human placental syncytiotrophoblasts from fetus

Tomi M., Eguchi H., Ozaki M., Tawara T., Nishimura S., Higuchi K., Maruyama T., Nishimura T., Nakashima E.

Endocrinology (Endocrinology) 156 ( 7 ) 2704 - 2712 2015.07

Research paper (scientific journal), Accepted, ISSN 0013-7227

Estriol biosynthesis in human placenta requires the uptake of a fetal liver-derived estriol precursor, 16α-hydroxydehydroepiandrosterone sulfate (16α-OH DHEAS), by placental syncytiotrophoblasts at their basal plasma membrane (BM), which faces the fetal circulation. The aim of this work is to identify the transporter(s) mediating 16α-OH DHEAS uptake at the fetal side of syncytiotrophoblasts by using human placental BM-enriched vesicles and to examine the contribution of the putative transporter to estriol synthesis at the cellular level, using choriocarcinoma JEG-3 cells. Organic anion transporter (OAT)-4 and organic anion transporting polypeptide 2B1 proteins were enriched in human placental BM vesicles compared with crude membrane fraction. Uptake of [3H]16α-OH DHEAS by BM vesicles was partially inhibited in the absence of sodium but was significantly increased in the absence of chloride and after preloading glutarate. Uptake of [3H]16α-OH DHEAS by BM vesicles was significantly inhibited by OAT4 substrates such as dehydroepiandrosterone sulfate, estrone-3-sulfate, and bromosulfophthalein but not by cyclosporin A, tetraethylammonium, p-aminohippuric acid, or cimetidine. These characteristics of vesicular [3H]16α-OH DHEAS uptake are in good agreement with those of human OAT4-transfected COS-7 cells as well as forskolin-differentiated JEG-3 cells. Estriol secretion from differentiated JEG-3 cells was detected when the cells were incubated with 16α-OH DHEAS for 8 hours but was inhibited in the presence of 50 μM bromosulfophthalein. Our results indicate that OAT4 at the BM of human placental syncytiotrophoblasts plays a predominant role in the uptake of 16α-OH DHEAS for placental estriol synthesis.

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Protective effect of hypotaurine against oxidative stress-induced cytotoxicity in rat placental trophoblasts

Nishimura T., Duereh M., Sugita Y., Yoshida Y., Higuchi K., Tomi M., Nakashima E.

Placenta (Placenta) 36 ( 6 ) 693 - 698 2015.06

Research paper (scientific journal), Accepted, ISSN 0143-4004

Introduction Hypotaurine is a precursor of taurine and an antioxidant, and is concentrated in fetal plasma compared to maternal plasma. Hypotaurine is significantly decreased in fetal plasma of ezrin (Vil2) knock-out mice, and fetuses show intrauterine growth retardation. The aim of this study was to characterize the mechanism through which cellular hypotaurine level is maintained in placental trophoblasts, and the effect of hypotaurine on oxidative stress induced by hydrogen peroxide (H<inf>2</inf>O<inf>2</inf>). Methods Hypotaurine transfer from extracellular fluid and antioxidant effect of hypotaurine were analyzed in rat placental trophoblast TR-TBT 18d-1 cells. Results We found that hypotaurine is concentrated into rat placental trophoblast TR-TBT 18d-1 cells, and the level of hypotaurine was markedly reduced by culture in medium supplemented with dialyzed fetal bovine serum (FBS) instead of normal FBS. The hypotaurine level recovered almost completely when hypotaurine was added to the culture medium, indicating that intracellular hypotaurine is predominantly supplied by transport across the plasma membrane from extracellular fluid rather than by biosynthesis. Hypotaurine showed a cytoprotective effect against H<inf>2</inf>O<inf>2</inf>-induced oxidative damage in TR-TBT 18d-1 cells. Hypotaurine treatment of TR-TBT 18d-1 cells increased antioxidant capacity against hydroxyl radical and peroxyl radical. The concentration of intracellular hydroxyl radical induced by H<inf>2</inf>O<inf>2</inf> in TR-TBT 18d-1 cells was significantly reduced by hypotaurine treatment. Discussion These results indicate that intracellular hypotaurine is mainly supplied to placental trophoblasts by transfer from extracellular fluid across the plasma membrane, and may play a role in cell protection by scavenging reactive oxygen species.

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Influence of Counseling Intervention on the Effectiveness of a Revitalizing Medicinal Product in Healthy Volunteers, Using a Brain Monitoring System

Isawa Minae, Shimizu Ayako, Miyashita Eri, Itoh Hiroshi, Nishimura Tomohiro, Tomi Masatoshi, Shimada Hideyo, Saito Hidetsugu, Nakashima Emi

Japanese Journal of Pharmaceutical Health Care and Sciences (Japanese Society of Pharmaceutical Health Care and Sciences) 40 ( 10 ) 558 - 566 2014.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 1346-342X

The purpose of this research was to examine whether the fatigue-relieving efficacy of a revitalizing medicinal product would be increased by counseling (advice to subjects regarding its expected efficacy), using both subjective and objective indicators. Twenty participants took the revitalizing medicinal product (drinkable product: 50 mL, once) on separate occasions, once with and once without counseling, with a 1-week interval. They completed a self-reported questionnaire (covering motivation, feeling of drowsiness, and general fatigue) and multi-dimensional fatigue inventory-20 (MFI-20) as subjective indicators, and blood flow in the inferior frontal cortex was evaluated by means of near-infrared spectroscopy (NIRS) as an objective indicator. The self-reported questionnaire showed that the revitalizing drink with counseling significantly improved general fatigue, as compared to the drink alone. MFI-20 showed no significant difference. Regional blood flow in the inferior frontal cortex was significantly increased by administration of the revitalizing drink with counseling, compared to without counseling (P < 0.05). Overall, the results indicated that counseling intervention increased the effectiveness of the revitalizing medicinal product.

Fetal growth retardation and lack of hypotaurine in ezrin knockout mice

Nishimura T., Higuchi K., Sai Y., Sugita Y., Yoshida Y., Tomi M., Wada M., Wakayama T., Tamura A., Tsukita S., Soga T., Nakashima E.

PLoS ONE (PLoS ONE) 9 ( 8 ) e105423 2014.08

Research paper (scientific journal), Accepted

Ezrin is a membrane-associated cytoplasmic protein that serves to link cell-membrane proteins with the actin-based cytoskeleton, and also plays a role in regulation of the functional activities of some transmembrane proteins. It is expressed in placental trophoblasts. We hypothesized that placental ezrin is involved in the supply of nutrients from mother to fetus, thereby influencing fetal growth. The aim of this study was firstly to clarify the effect of ezrin on fetal growth and secondly to determine whether knockout of ezrin is associated with decreased concentrations of serum and placental nutrients. Ezrin knockout mice (Ez-/-) were confirmed to exhibit fetal growth retardation. Metabolome analysis of fetal serum and placental extract of ezrin knockout mice by means of capillary electrophoresis-time-of-flight mass spectrometry revealed a markedly decreased concentration of hypotaurine, a precursor of taurine. However, placental levels of cysteine and cysteine sulfinic acid (precursors of hypotaurine) and taurine were not affected. Lack of hypotaurine in Ez-/- mice was confirmed by liquid chromatography with tandem mass spectrometry. Administration of hypotaurine to heterogenous dams significantly decreased the placenta-to-maternal plasma ratio of hypotaurine in wild-type fetuses but only slightly decreased it in ezrin knockout fetuses, indicating that the uptake of hypotaurine from mother to placenta is saturable and that disruption of ezrin impairs the uptake of hypotaurine by placental trophoblasts. These results indicate that ezrin is required for uptake of hypotaurine from maternal serum by placental trophoblasts, and plays an important role in fetal growth. © 2014 Nishimura et al.

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Role of protein kinase A in regulating steroid sulfate uptake for estrogen production in human placental choriocarcinoma cells

Tomi M., Miyata Y., Noguchi S., Nishimura S., Nishimura T., Nakashima E.

Placenta (Placenta) 35 ( 8 ) 658 - 660 2014.08

Research paper (scientific journal), Accepted, ISSN 0143-4004

The purpose of this study is to assess the role of the protein kinase A (PKA) in regulating uptake of dehydroepiandrosterone sulfate (DHEAS), an estrogen precursor, by syncytiotrophoblasts. Forskolin, a PKA activator, significantly increased [³H]DHEAS uptake and the mRNA expression levels of organic anion transporter (OAT) 4 and CYP19A1 in choriocarcinoma JEG-3 cells, while other steroid sulfate transporters present in the placenta showed no change in expression level. KT5720, a PKA inhibitor, attenuated these effects of forskolin. Accordingly, the PKA pathway appears to play an important role in estrogen synthesis by cooperatively regulating OAT4 and steroidogenic enzymes in syncytiotrophoblasts. © 2014 Elsevier Ltd. All rights reserved.

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Influence of Counseling Intervention on the Effectiveness of Aromatherapy with Bergamot Oil in Healthy Women Volunteers Measured with a Brain Monitoring System

ISAWA Minae, INOUE Shio, ITOH Hiroshi, NISHIMURA Tomohiro, TOMI Masatoshi, SHIMADA Hideyo, SAITO Hidetsugu, NAKASHIMA Emi

Journal of Japanese Cosmetic Science Society (Japanese Cosmetic Science Society) 38 ( 2 ) 81 - 86 2014.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 1880-2532

There is growing evidence that aromatherapy can improve mood. Since pharmacists may be requested to counsel patients about aromatherapy, this study was designed to evaluate the influence of counseling on the effectiveness of aromatherapy in 14 healthy young women. Participants completed a self-reported questionnaire (motivation, feeling of drowsiness and general fatigue) and multi-dimensional fatigue inventory-20 (MFI-20) in a room filled with the fragrance of bergamot, with or without counseling. Further, as an objective measure of changes in stress, blood flow in the inferior frontal cortex was evaluated by means of near-infrared spectroscopy (NIRS). MFI-20 showed that aromatherapy with counseling significantly improved activity and motivation, as compared to aromatherapy only. The self-reported questionnaire showed no significant difference. Regional blood flow in the inferior frontal cortex was significantly decreased by exposure to aromatherapy with counseling, compared to aromatherapy alone (p<0.01, p<0.05). The value of the laterality index (LI) derived from NIRS measurements, which is considered to be a measure of stress, was decreased by aromatherapy with counseling, indicating that bergamot oil reduces stress. Overall, the results indicated that counseling intervention markedly increased the effectiveness of aromatherapy.

System A amino acid transporter SNAT2 shows subtype-specific affinity for betaine and hyperosmotic inducibility in placental trophoblasts

Nishimura T., Yagi R., Usuda M., Oda K., Yamazaki M., Suda S., Takahashi Y., Okazaki F., Sai Y., Higuchi K., Maruyama T., Tomi M., Nakashima E.

Biochimica et Biophysica Acta - Biomembranes (Biochimica et Biophysica Acta - Biomembranes) 1838 ( 5 ) 1306 - 1312 2014.05

Research paper (scientific journal), Accepted, ISSN 0005-2736

Betaine uptake is induced by hypertonic stress in a placental trophoblast cell line, and involvement of amino acid transport system A was proposed. Here, we aimed to identify the subtype(s) of system A that mediates hypertonicity-induced betaine uptake. Measurement of [¹⁴C]betaine uptake by HEK293 cells transiently transfected with human or rat sodium-coupled neutral amino acid transporters (SNATs), SNAT1, SNAT2 and SNAT4 revealed that only human and rat SNAT2 have betaine uptake activity. The Michaelis constants (K_m) of betaine uptake by human and rat SNAT2 were estimated to be 5.3 mM and 4.6 mM, respectively. Betaine exclusively inhibited the uptake activity of SNAT2 among the rat system A subtypes. We found that rat SNAT1, SNAT2 and SNAT4 were expressed at the mRNA level under isotonic conditions, while expression of SNAT2 and SNAT4 was induced by hypertonicity in TR-TBT 18d-1 cells. Western blot analyses revealed that SNAT2 expression on plasma membrane of TR-TBT 18d-1 cells was more potently induced by hypertonicity than that in total cell lysate. Immunocytochemistry confirmed the induction of SNAT2 expression in TR-TBT 18d-1 cells exposed to hypertonic conditions and indicated that SNAT2 was localized on the plasma membrane in these cells. Our results indicate that SNAT2 transports betaine, and that tonicity-sensitive SNAT2 expression may be involved in regulation of betaine concentration in placental trophoblasts. © 2014 Elsevier B.V.

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Estrogen receptor α induction by mitoxantrone increases Abcg2 expression in placental trophoblast cells

Oda K., Nishimura T., Higuchi K., Ishido N., Ochi K., Iizasa H., Sai Y., Tomi M., Nakashima E.

Journal of Pharmaceutical Sciences (Journal of Pharmaceutical Sciences) 102 ( 9 ) 3364 - 3372 2013.09

Research paper (scientific journal), Accepted, ISSN 0022-3549

Substrate-induced upregulation of ATP-binding cassette subfamily G member 2 (ABCG2) has been well studied in cancer cells, but it is also important to understand whether ABCG2 is upregulated by its substrates in tissues in which it is constitutively expressed. In the present study, we aimed to clarify the regulatory mechanism of Abcg2 expression by its substrate, mitoxantrone, in placental cells. Abcg2 mRNA expression in rat placental TR-TBT 18d-1 cells treated with 10μM mitoxantrone for 24h was increased, compared with that in nontreated cells, whereas 10μM pheophorbide-a had no effect. Methylated CpG level in the promoter region of the Abcg2 gene was low and was not altered by mitoxantrone. On the contrary, mitoxantrone markedly increased the expression of estrogen receptor (ER) α and progesterone receptor (PR) B. Fulvestrant, an ER antagonist, attenuated the mitoxantrone-induced increase of Abcg2 mRNA expression, whereas mifepristone, a PR antagonist, had little effect. 17β-estradiol, an ER ligand, positively regulated the mitoxantrone-induced increase of Abcg2 expression. DNA demethylation by 5-aza-2-deoxycytidine treatment increased ERα expression, but mitoxantrone failed to facilitate the demethylation of ERα promoter in TR-TBT 18d-1 cells. In conclusion, Abcg2 expression is induced by mitoxantrone via the induction of ERα in TR-TBT 18d-1 cells. © 2013 Wiley Periodicals, Inc.

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Evaluation of rat in vivo fetal-to-maternal transfer clearances of various xenobiotics by umbilical perfusion

Nishimura T., Takanohashi T., Tomi M., Horikoshi M., Higuchi K., Sai Y., Nakashima E.

Journal of Pharmaceutical Sciences (Journal of Pharmaceutical Sciences) 102 ( 9 ) 3356 - 3363 2013.09

Research paper (scientific journal), Accepted, ISSN 0022-3549

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 (CL_fm) of 23 compounds, which have a broad range of lipophilicity. Drugs for which CL_fm was more than 300 μL/(mL min) belonged exclusively to Biopharmaceutical Drug Disposition Classification System (BDDCS) class 1 (highly permeable) and those for which CL_fm was less than 50 μL/(mL min) belonged exclusively to BDDCS class 3 (poorly permeable). For most drugs, CL_fm values were broadly consistent with lipophilicity. However, CL_fm of digoxin was saturable and was inhibited by verapamil, suggesting that P-glycoprotein (P-gp)-mediated efflux has a substantially effect on measured clearance. CL_fm 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 CL_fm by umbilical perfusion and examined the relationship between CL_fm 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.

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Hypoxic adaptation engages the CBP/CREST-induced coactivator complex of Creb-HIF-1α in transactivating murine neuroblastic glucose transporter

Thamotharan S., Raychaudhuri N., Tomi M., Shin B., Devaskar S.

American Journal of Physiology - Endocrinology and Metabolism (American Journal of Physiology - Endocrinology and Metabolism) 304 ( 6 ) E583 - E598 2013.03

Research paper (scientific journal), Accepted, ISSN 0193-1849

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.

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Early life nutrient restriction impairs blood-brain metabolic profile and neurobehavior predisposing to Alzheimer's disease with aging

Tomi M., Zhao Y., Thamotharan S., Shin B.C., Devaskar S.U.

Brain Research (Brain Research) 1495 61 - 75 2013.02

Research paper (scientific journal), Accepted, ISSN 0006-8993

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-3β^Ser9, 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.

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Mechanism of Nucleoside Uptake in Rat Placenta and Induction of Placental CNT2 in Experimental Diabetes

NISHIMURA Tomohiro, CHISHU Takuya, TOMI Masatoshi, NAKAMURA Ryo, SATO Kazuko, KOSE Noriko, SAI Yoshimichi, NAKASHIMA Emi

Drug Metabolism and Pharmacokinetics (The Japanese Society for the Study of Xenobiotics) 27 ( 4 ) 439 - 446 2012.08

Research paper (scientific journal), Accepted, ISSN 1347-4367

The purpose of this study was to clarify the transport characteristics of nucleosides in rat placenta and the changes of functional expression of nucleoside transporters in rat placenta with experimental diabetes mellitus. Placental uptake clearances of [³H]adenosine and [³H]zidovudine from maternal blood was much higher than that of [¹⁴C]mannitol. Xenopus oocytes injected with rat ENT1 and ENT2 cRNA took up [³H]adenosine with K_m values of 6.1 and 26 μM, respectively. [³H]Adenosine transport by rat placental brush-border membrane vesicles (BBMV) was saturable and was inhibited by nitrobenzylthioinosine (NBMPR), a specific ENT inhibitor, in a manner consistent with involvement of both rat ENT1 and ENT2. [³H]Didanosine was modestly taken up by placenta, and the inhibitory effect of 100 μM NBMPR on [³H]ddI uptake by BBMV suggested a role of ENT2-mediated transport. Expression of ENT1, ENT2, ENT3, CNT2, and CNT3 mRNAs was detected in placenta of control and streptozotocin (STZ)- induced diabetic pregnant rats, and CNT2 (SLC28A2) expression was significantly increased in STZinduced diabetic rats. Consistently, Na⁺-dependent adenosine uptake by BBMV from STZ-induced diabetic pregnant rats was higher than that from control rats. These results suggest the involvement of placental ENT2 as well as ENT1 in nucleoside uptake from maternal blood, and the induction of CNT2 in experimental diabetes mellitus. © 2012 by the Japanese Society for the Study of Xenobiotics (JSSX).

Up-regulation of L-Type Amino Acid Transporter 1 (LAT1) in Cultured Rat Retinal Capillary Endothelial Cells in Response to Glucose Deprivation

MATSUYAMA Ryo, TOMI Masatoshi, AKANUMA Shin-ichi, TABUCHI Akiko, KUBO Yoshiyuki, TACHIKAWA Masanori, HOSOYA Ken-ichi

Drug Metabolism and Pharmacokinetics (The Japanese Society for the Study of Xenobiotics) 27 ( 3 ) 317 - 324 2012.06

Research paper (scientific journal), Accepted, ISSN 1347-4367

We investigated the regulation of L-type amino acid transporter 1 (LAT1) in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells) in response to glucose deprivation. The amounts of LAT1 and 4F2 heavy chain (4F2hc) mRNA in TR-iBRB2 cells exposed to glucose-free culture medium for 8 to 24 h were significantly elevated compared with those in control medium. Concomitantly, [³H]L-leucine uptake activity was increased, suggesting that LAT1 transport activity is induced under glucose-deprivation. To determine the transcriptional activity of the LAT1 gene under glucose-free conditions, the promoter activity of the LAT1 gene of approximately 2 kbp (-1958 bp to +70 bp) in TR-iBRB2 cells was assayed using a dual-luciferase reporter assay system. The transcriptional activity of the 2 kbp LAT1 promoter under the glucose-free conditions was 1.7-fold greater than that under normal glucose conditions. The presence of an activator site(s) between -162 bp and -155 bp was indicated by the low activities exhibited by the construct spanning this region and mutagenesis. These results suggest that the glucose deprivation sensitivity of LAT1 expression is transcriptionally regulated, and cis-elements within the LAT1 promoter region from -162 bp to -155 bp mediate this regulation. © 2012 by the Japanese Society for the Study of Xenobiotics (JSSX).

Effectiveness of Aromatherapy Evaluated with Subjective and Objective Cognitive Indicators: A Clinical Trial on Young Adults Using Near-infrared Spectroscopy

Shinno Akemi, Isawa Minae, Itoh Hiroshi, Kose Noriko, Ishino Haruyuki, Nishimura Tomohiro, Tomi Masatoshi, Tsujii Takeo, Shimada Hideyo, Saito Hidetsugu, Nakashima Emi

Japanese Journal of Pharmaceutical Health Care and Sciences (Japanese Society of Pharmaceutical Health Care and Sciences) 38 ( 4 ) 265 - 271 2012.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 1346-342X

The purpose of this research was to noninvasively evaluate the influence of aromatherapy on work performance in 14 healthy young adults. Participants in a room filled with the fragrance of lavender completed a self-reported questionnaire and multi-dimensional fatigue inventory-20 (MFI-20). Further, as an objective measure, blood flow in the inferior frontal cortex was evaluated via near-infrared spectroscopy (NIRS) as a parameter of working memory capacity. Compared to the control stage (no aromatherapy), exposure to aromatherapy achieved a significant reduction in general and mental fatigue according to MFI-20. Self-reported questionnaires also indicated improvement, but the differences were not statistically significant. NIRS measurement during the task performance of an N-back program indicated that regional blood flow in the inferior frontal cortex was significantly increased through exposure to aromatherapy, compared to without. The prefrontal area in the brain is involved in working memory, attention concentration and judgment. These results suggest that lavender may improve both cognitive ability and mood. A larger study with more aroma oils seems warranted.

Strategies for therapy of retinal diseases using systemic drug delivery: Relevance of transporters at the blood retinal barrier

Hosoya K., Tomi M., Tachikawa M.

Expert Opinion on Drug Delivery (Expert Opinion on Drug Delivery) 8 ( 12 ) 1571 - 1587 2011.12

Research paper (scientific journal), Joint Work, ISSN 1742-5247

Introduction: There is an increasing need for managing rapidly progressing retinal diseases because of the potential loss of vision. Although systemic drug administration is one possible route for treating retinal diseases, retinal transfer of therapeutic drugs from the circulating blood is strictly regulated by the bloodretinal barrier (BRB). Areas covered: This review discusses the constraints and challenges of drug delivery to the retina. In addition, this article discusses the properties of drugs and the conditions of the BRB that affect drug permeability. The reader will gain insights into the strategies for developing therapeutic drugs that are able to cross the BRB for treating retinal diseases. Further, the reader will gain insights into the role of BRB physiology including barrier functions, and the effect of influx and efflux transporters on retinal drug delivery. Expert opinion: When designing and selecting optimal drug candidates, it's important to consider the fact that they should be recognized by influx transporters and that efflux transporters at the BRB should be avoided. Although lipophilic cationic drugs are known to be transported to the brain across the bloodbrain barrier, verapamil transport to the retina is substantially higher than to the brain. Therefore, lipophilic cationic drugs do have a great ability to increase influx transport across the BRB. © 2011 Informa UK, Ltd.

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Enhancement of zidovudine transfer to molt-4 cells, a human t-cell model, by dehydroepiandrosterone sulfate

Nishimura T., Tanaka J., Tomi M., Seki Y., Kose N., Sai Y., Nakashima E.

Journal of Pharmaceutical Sciences (Journal of Pharmaceutical Sciences) 100 ( 9 ) 3959 - 3967 2011.09

Research paper (scientific journal), Accepted, ISSN 0022-3549

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.

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Mother-to-fetus transfer of antiviral drugs and the involvement of transporters at the placental barrier

Tomi M., Nishimura T., Nakashima E.

Journal of Pharmaceutical Sciences (Journal of Pharmaceutical Sciences) 100 ( 9 ) 3708 - 3718 2011.09

Research paper (scientific journal), Accepted, ISSN 0022-3549

Administration of antiviral drugs to pregnant women with influenza, human immunodeficiency virus (HIV), and herpes simplex virus (HSV) infections is widely accepted as an effective treatment to safeguard the health of the mother and fetus. This review deals with the transfer of antiviral drugs to the fetus across the placental barrier, which is formed by an epithelial layer of syncytiotrophoblasts. First, the structure, function, and developmental change of the placenta and the placental barrier are briefly presented. We then review the transplacental permeability of antiviral drugs, such as oseltamivir for influenza, antiretrovirals (e.g., zidovudine, didanosine, and saquinavir) for HIV, and acyclovir for HSV, focusing on the involvement of ATP-binding cassette, organic anion/cation, and nucleoside transporters. The increasing evidence that is becoming available about transport mechanisms operating at the placental barrier is expected to be useful in the development of techniques to control fetal and placental transfer of antiviral drugs, and thereby to obtain maximum therapeutic benefit while minimizing potential fetal toxicity. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association.

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Effectiveness of Repeated Administration of Energy Drinks Based on Cognitively Subjective and Objective Indicators

Itoh Hiroshi, Isawa Minae, Nishimura Tomohiro, Tomi Masatoshi, Shimada Hideyo, Mochizuki Mayumi, Nakashima Emi

JAPANESE JOURNAL OF PHARMACEUTICAL HEALTH CARE AND SCIENCES (Japanese Society of Pharmaceutical Health Care and Sciences) 36 ( 12 ) 847 - 854 2010.12

Research paper (scientific journal), Joint Work, Accepted, ISSN 1346-342X

The purpose of this research was to clarify the effect of repeated administration of 2 kinds of energy drink,1 containing vitamins,taurine and caffeine (simple-type),and the other containing vitamins,taurine,caffeine and several botanical herb extracts (herb-type),on work performance using cognitively subjective and objective indicators. The simple-type and herb-type drinks were given to 14 healthy young adults in a consecutive double-blind,randomized,2-period cross-over clinical trial.Participants took each energy drink once a day for five days during separate periods.Motivation,feeling of drowsiness,and general fatigue were evaluated on a subjective scale using a self-reported questionnaire both before and after administration.Other subjective evaluations were those based on the fatigue-visual analog scale (Fatigue-VAS) and multi-dimensional fatigue inventory-20 (MFI-20).As an objective measure,work performance was examined using the Kreapelin mental work test. Compared to the simple-type drink,the herb-type drink achieved a significant reduction in drowsiness and significantly increased motivation based on the self-reported questionnaire results for the post-administration assessment.In the Kreapelin mental work test,the mean amount of work was significantly increased by both drinks.MFI-20 revealed significant decreases in 2 categories of physical fatigue and general fatigue due to the 2 drinks.The fatigue-VAS also indicated decreases but they were not statistically significant. Both types of energy drink appeared to be effective in terms of subjective and objective indicators.Though the herb-type drink was found to be superior to the simple-type drink regarding subjective measures,there was no significant difference between the 2 regarding the objective measure.

Roles of TauT and system A in cytoprotection of rat syncytiotrophoblast cell line exposed to hypertonic stress

Nishimura T., Sai Y., Fujii J., Muta M., Iizasa H., Tomi M., Deureh M., Kose N., Nakashima E.

Placenta (Placenta) 31 ( 11 ) 1003-1009 - 1009 2010.11

Research paper (scientific journal), Joint Work, Accepted, ISSN 0143-4004

The purpose of this study was to clarify the cytoprotective mechanism(s) induced in a conditionally immortalized syncytiotrophoblast cell line (TR-TBT 18d-1) exposed to hypertonic conditions. Hypertonicity-induced apoptosis of TR-TBT 18d-1 cells, but this was blocked by addition of 1 mM taurine to the culture medium. TauT-knockdown using siRNA revealed that TauT is a major contributor to taurine uptake by TR-TBT 18d-1 cells, at least under normal conditions. Cellular uptake of [3H]taurine and [14C] betaine by TR-TBT 18d-1 cells cultured under hypertonic conditions was increased compared to that under normal conditions. TauT, BGT-1, ATA2 and HSP70 mRNAs were upregulated by hypertonicity, while OCTN2, ENT1 and CNT1 mRNAs were downregulated. [3H]Taurine uptake was strongly inhibited by TauT inhibitors such as hypotaurine and β-alanine. MeAIB, a system A specific substrate, inhibited hypertonic stress-induced [14C]betaine uptake. These results suggest that TauT and system A play cytoprotective roles in syncytiotrophoblasts exposed to hypertonic stress. © 2010 Elsevier Ltd. All rights reserved.

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Proton-coupled erythromycin antiport at rat blood-placenta barrier

Sai Y., Nishimura T., Ochi K., Tanaka N., Takagi A., Tomi M., Kose N., Kobayashi Y., Miyakoshi N., Kitagaki S., Mukai C., Nakashima E.

Drug Metabolism and Disposition (Drug Metabolism and Disposition) 38 ( 9 ) 1576 - 1581 2010.09

Research paper (scientific journal), Accepted, ISSN 0090-9556

The aim of the present study was to characterize the mechanism of erythromycin transport at the blood-placenta barrier, using TR-TBT 18d-1 cells as a model of rat syncytiotrophoblasts. [14C]Erythromycin was taken up by TR-TBT 18d-1 cells with a Michaelis constant of 466 μM. Although the uptake was not dependent on extracellular Na+ or Cl-, it was increased at weakly alkaline pH. Significant overshoot of [ 14C]erythromycin uptake by placental brush-border membrane vesicles was observed in the presence of an outwardly directed proton gradient. These results indicate that erythromycin is transferred by the H+-coupled transport system in syncytiotrophoblasts. To address the physiological transport of erythromycin in rat placenta, fetal-to-maternal transport clearance was estimated by means of the single placental perfusion technique. Clearance of [14C]erythromycin was higher than that of [14C]inulin, a paracellular pathway marker, and was decreased by the addition of 5 mM erythromycin, indicating that saturable efflux system from fetus to mother is involved. The effect of various transporter inhibitors on [14C] erythromycin efflux from TR-TBT 18d-1 cells was evaluated. cyclosporin A, fumitremorgin C, and probenecid had no effect, whereas ethylisopropylamiloride, a specific inhibitor of Na+/H+ exchangers (NHEs), was significantly inhibitory. These results suggest that erythromycin efflux transport at the rat blood-placenta barrier is mediated by an erythromycin/H+ antiport system, driven by H+ supplied by NHEs. Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics.

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The role of blood-ocular barrier transporters in retinal drug disposition: An overview

TOMI Masatoshi

Expert Opinion on Drug Metabolism and Toxicology (Expert Opinion on Drug Metabolism and Toxicology) 6 ( 9 ) 1111 - 1124 2010.09

Research paper (scientific journal), Accepted, ISSN 1742-5255

Importance of the field: Delivery of drugs to the retina remains a major challenge which needs to be addressed urgently because retinal disorders are leading causes of visual impairment and significantly affect a patient's quality of life. Systemic drug administration is one possible route for treating retinal disorders; however, retinal transfer of drugs from the circulating blood is strictly regulated by two bloodocular barrier systems, the bloodaqueous barrier and the bloodretinal barrier. Areas covered in this review: This review summarizes the latest biological research regarding bloodocular barrier drug transporters. What the reader will gain: The bloodocular barrier sites and their respective roles in aqueous humor dynamics and retinal homeostasis are briefly presented. The potential impact of ATP-binding cassette (ABC) and solute carrier (SLC) drug transporters, such as ABCB, ABCC, ABCG, SLC7, SLC16, SLC19, SLCO/SLC21A, SLC22A and SLC29 transporters, on the permeability of drugs across the bloodocular barriers is then illustrated. Take home message: As more information becomes available regarding the bloodocular barrier transporters, we may be able to design simpler and more effective routes for drug delivery to the retina and, consequently, improve the treatment of retinal diseases. © 2010 Informa UK, Ltd.

Involvement of system A in the retina-to-blood transport of l-proline across the inner blood-retinal barrier

Yoneyama D., Shinozaki Y., Lu W., Tomi M., Tachikawa M., Hosoya K.

Experimental Eye Research (Experimental Eye Research) 90 ( 4 ) 507 - 513 2010.04

Research paper (scientific journal), Accepted, ISSN 0014-4835

The purpose of the present study was to elucidate the mechanisms of retina-to-blood transport of l-proline across the blood-retinal barrier (BRB) in vivo and in vitro, and to identify the responsible transporter(s). The vitreous humor/retina-to-blood transport of [3H]l-proline across the BRB was evaluated by microdialysis. Transport mechanisms of [3H]l-proline were investigated by cellular uptake using an in vitro model of the inner BRB (TR-iBRB2 cells). The mRNA level of system A was determined by quantitative real-time PCR analysis with specific primers. [3H]l-Proline and [14C]d-mannitol, which is a bulk flow marker, were bi-exponentially eliminated from the vitreous humor after vitreous bolus injection. The elimination rate constant of [3H]l-proline during the terminal phase was 1.6-fold greater than that of [14C]d-mannitol. The terminal elimination rate constant difference between [3H]l-proline and [14C]d-mannitol was reduced in the retinal presence of 3 mM l-proline and 5 mM α-methylaminoisobutyric acid, suggesting that l-proline is transported via a carrier-mediated retina-to-blood transport process across the BRB. [3H]l-Proline uptake by TR-iBRB2 cells appeared to be mediated through a saturable and Na+-dependent process. The corresponding Michaelis-Menten constant was 392 μM. This process was reduced by substrates for system A, suggesting that system A is involved in l-proline uptake. Of the isoforms of system A, ATA1, ATA2, and ATA3, ATA2 mRNA is predominantly expressed in TR-iBRB2 cells and isolated rat retinal endothelial cells. In conclusion, system A, most likely ATA2, is responsible for the retina-to-blood transport of l-proline across the inner BRB and may play a role in maintaining the concentration of small neutral amino acids in the retina. © 2010 Elsevier Ltd.

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Involvement of OCTN2 in the transport of acetyl-lcarnitine across the inner blood-retinal barrier

TOMI Masatoshi

Investigative Ophthalmology and Visual Science (Investigative Ophthalmology and Visual Science) 51 ( 1 ) 430 - 436 2010.01

Research paper (scientific journal), Accepted, ISSN 0146-0404

PURPOSE. To elucidate the mechanisms of acetyl-L-carnitine transport across the inner blood-retinal barrier (inner BRB). METHODS. In vivo integration plot and retinal uptake index (RUI) analyses were used to examine acetyl-L-[3H]carnitine transport in the retina across the inner BRB in rats. RUI was determined from the ratio of acetyl-L-[3H]carnitine and [14C]n-butanol, a freely diffusible internal reference, in the retina divided by the same ratio in the solution injected in the carotid artery. The transport mechanism was characterized in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells), as an in vitro inner BRB model. RESULTS. The apparent influx permeability clearance (Kin) per gram retina of acetyl-L-[3H]carnitine was found to be 2.31 μL/(minute·g retina). The Kin of acetyl-L-[3H]carnitine was 3.7-fold greater than that of [3H]D-mannitol, a nonpermeable paracellular marker. Acetyl-L-[3H]carnitine uptake by the retina was found to be significantly inhibited by L-carnitine and acetyl- L-carnitine, supporting a carrier-mediated influx transport of acetyl-L-carnitine at the inner BRB. L-[3H]carnitine and acetyl-L- [3H]carnitine uptake by TR-iBRB2 cells was Na+- and concentration- dependent, with a Km of 29 and 26 μM, respectively. These forms of transport were significantly inhibited by organic cation/carnitine transporter (OCTN) substrates and inhibitors such as L-carnitine and acetyl-L-carnitine, tetraethylammonium, quinidine, and betaine. These transport properties are consistent with those of carnitine transport by OCTN2. OCTN2 was predominantly expressed in TR-iBRB2 cells and isolated rat retinal vascular endothelial cells. CONCLUSIONS. The findings suggest that OCTN2 is involved in the transport of acetyl-L-carnitine from the circulating blood to the retina across the inner BRB. © Association for Research in Vision and Ophthalmology.

Cationic amino acid transporter 1-mediated l-arginine transport at the inner blood-retinal barrier

Tomi M., Kitade N., Hirose S., Yokota N., Akanuma S., Tachikawa M., Hosoya K.

Journal of Neurochemistry (Journal of Neurochemistry) 111 ( 3 ) 716 - 725 2009.11

Research paper (scientific journal), Accepted, ISSN 0022-3042

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.

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Roles of inner blood-retinal barrier organic anion transporter 3 in the vitreous/retina-to-blood efflux transport of p-aminohippuric acid, benzylpenicillin, and 6-mercaptopurine

Hosoya K., Makihara A., Tsujikawa Y., Yoneyama D., Mori S., Terasaki T., Akanuma S., Tomi M., Tachikawa M.

Journal of Pharmacology and Experimental Therapeutics (Journal of Pharmacology and Experimental Therapeutics) 329 ( 1 ) 87 - 93 2009.04

Research paper (scientific journal), Accepted, ISSN 0022-3565

The purpose of the present study was to characterize rat organic anion transporter (Oat) 3 (Oat3, Slc22a8) in the efflux transport at the inner blood-retinal barrier (BRB). Reverse transcription-polymerase chain reaction analysis showed that rat (r) Oat3 mRNA is expressed in retinal vascular endothelial cells (RVECs), but not rOat1 and rOat2 mRNA. The expression of Oat3 in the retina and human cultured retinal endothelial cells was further confirmed by Western blot analysis. Immunohisto-chemical staining in RVECs showed that rOat3 is colocalized with glucose transporter 1, but not P-glycoprotein, suggesting that rOat3 is possibly located at the abluminal membrane of the RVEC. The contribution of rOat3 to the efflux of [3H]p-aminohippuric acid ([3H]PAH), [3H]benzylpenicillin ([3H]PCG), and [14C]6-mercaptopurine ([14C]6-MP), substrates of rOat3, from the vitreous humor/retina to the circulating blood across the inner BRB was evaluated using the microdialysis method. [3H]PAH, [ 3H]PCG, [14C]6-MP, and [14C] or [ 3H]D-mannitol, a bulk flow marker, were biexponentially eliminated from the vitreous humor after vitreous bolus injection. The elimination rate constant of [3H]PAH, [3H]PCG, and [14C]6-MP during the terminal phase was approximately 2-fold greater than that of D-mannitol. This efflux transport was reduced in the retinal presence of probenecid, PAH, and PCG, whereas it was not inhibited by digoxin. In conclusion, rOat3 is expressed at the inner BRB and involved in the vitreous humor/retina-to-blood transport of PAH, PCG, and 6-MP. This transport system is one mechanism to limit the retinal distribution of PAH, PCG, and 6-MP. Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics.

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Modulation of retinal capillary endothelial cells by Müller glial cell-derived factors.

Abukawa H., Tomi M., Kiyokawa J., Hori S., Kondo T., Terasaki T., Hosoya K.

Molecular vision (Molecular vision) 15 451 - 457 2009.02

PURPOSE: The inner blood-retinal barrier (BRB) is a gliovascular unit in which macroglial cells surround capillary endothelial cells and regulate retinal capillaries by paracrine interactions. The purpose of the present study was to identify genes of retinal capillary endothelial cells whose expression is modulated by Müller glial cell-derived factors. METHODS: Conditionally immortalized rat retinal capillary endothelial (TR-iBRB2) and Müller (TR-MUL5) cell lines were chosen as an in vitro model. TR-iBRB2 cells were incubated with conditioned medium of TR-MUL5 (MUL-CM) for 24 h and subjected to microarray and quantitative real-time PCR analysis. RESULTS: TR-MUL5 cell-derived factors increased alkaline phosphatase activity in TR-iBRB2 cells, indicating that paracrine interactions occurred between TR-iBRB2 and TR-MUL5 cells. Microarray analysis demonstrated that MUL-CM treatment leads to a modulation of several genes including an induction of plasminogen activator inhibitor 1 (PAI-1) and a suppression of an inhibitor of DNA binding 2 (Id2) in TR-iBRB2 cells. Treatment with TGF-beta1, which is incorporated in MUL-CM, also resulted in an induction of PAI-1 and a suppression of Id2 in TR-iBRB2 cells. CONCLUSIONS: In vitro inner BRB model study revealed that Müller glial cell-derived factors modulate endothelial cell functions including the induction of anti-angiogenic PAI-1 and the suppression of pro-angiogenic Id2. Therefore, Müller cells appear to be one of the modulators of retinal angiogenesis.

Expression and possible role of creatine transporter in the brain and at the blood-cerebrospinal fluid barrier as a transporting protein of guanidinoacetate, an endogenous convulsant

Tachikawa M., Fujinawa J., Takahashi M., Kasai Y., Fukaya M., Sakai K., Yamazaki M., Tomi M., Watanabe M., Sakimura K., Terasaki T., Hosoya K.I.

Journal of Neurochemistry (Journal of Neurochemistry) 107 ( 3 ) 768 - 778 2008.11

Research paper (scientific journal), Accepted, ISSN 0022-3042

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.

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Evaluation of amino acid-mustard transport as L-type amino acid transporter 1 (LAT1)-mediated alkylating agents

Hosoya K., Kyoko H., Toyooka N., Kato A., Orihashi M., Tomi M., Tachikawa M.

Biological and Pharmaceutical Bulletin (Biological and Pharmaceutical Bulletin) 31 ( 11 ) 2126 - 2130 2008.11

Research paper (scientific journal), Accepted, ISSN 0918-6158

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.

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Function of taurine transporter (Slc6a6/TauT) as a GABA transporting protein and its relevance to GABA transport in rat retinal capillary endothelial cells

Tomi M., Tajima A., Tachikawa M., Hosoya K.

Biochimica et Biophysica Acta - Biomembranes (Biochimica et Biophysica Acta - Biomembranes) 1778 ( 10 ) 2138 - 2142 2008.10

Research paper (scientific journal), Accepted, ISSN 0005-2736

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.

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Dehydroascorbic acid uptake and intracellular ascorbic acid accumulation in cultured Müller glial cells (TR-MUL)

Hosoya K.i., Nakamura G., Akanuma S.i., Tomi M., Tachikawa M.

Neurochemistry International (Neurochemistry International) 52 ( 7 ) 1351 - 1357 2008.06

Research paper (scientific journal), Accepted, ISSN 0197-0186

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.

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Gene expression profiles of ATP-binding cassette transporter A and C subfamilies in mouse retinal vascular endothelial cells

Tachikawa M., Toki H., Tomi M., Hosoya K.

Microvascular Research (Microvascular Research) 75 ( 1 ) 68 - 72 2008.01

Research paper (scientific journal), Accepted, ISSN 0026-2862

The purpose of this study was to quantify gene expression levels of the ATP-binding cassette (ABC) transporter A and C subfamilies ABCA1-A9, and ABCC1-6/Mrp1-6, C10/Mrp7 in mouse retinal vascular endothelial cells (RVEC) using a combination of a magnetic isolation method for mouse RVEC and real-time quantitative PCR analysis. The transcript level of endothelial cell markers, such as CD31, Tie-2, claudin-5, occludin, ABCB1a/mdr1a, and ABCG2, were more than 20-fold higher than those in the non-RVEC fraction, suggesting that RVEC in the RVEC fraction are concentrated at least 20-fold compared with those of the non-RVEC fraction. In the ABCA1 to A9 families, the transcript level of ABCA3 and A9 in the RVEC fraction was 1.2- and 32-fold higher than that in the non-RVEC fraction. Although ABCA3 was expressed in both the RVEC and non-RVEC fractions, A9 is predominantly expressed in the RVEC fraction. In the ABCC1 to C6 and C10 families, the transcript level of ABCC3, C4, and C6 in the RVEC fraction was 27-, 251-, and 242-fold higher, respectively, than that in the non-RVEC fraction, suggesting that ABCC3, C4, and C6 are predominantly expressed in the RVEC. In conclusion, ABCA3, ABCA9, ABCC3, ABCC4, and ABCC6 mRNAs are predominantly expressed at the inner blood-retina barrier (inner BRB) and appear to play a major role in the efflux transport of their substrates at the inner BRB. © 2007 Elsevier Inc. All rights reserved.

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Na+-independent choline transport in rat retinal capillary endothelial cells

Tomi M., Arai K., Tachikawa M., Hosoya K.

Neurochemical Research (Neurochemical Research) 32 ( 11 ) 1833 - 1842 2007.11

Research paper (scientific journal), Accepted, ISSN 0364-3190

The purpose of this study was to clarify the mechanism of the inner blood-retinal barrier (inner BRB) transport of choline and examine the choline uptake ability of rat choline transporter-like protein (CTL) 1. The transcript level of CTL1 in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) is more than 100-fold greater than that of CTL3 and CTL4, and no expression of organic cation transporter (OCT) mRNA was detected. The apparent influx permeability clearance of [3H]choline in the rat retina was found to be 271 μl/(min · g retina). The [ 3H]choline uptake by TR-iBRB2 cells was Na+-independent, potential-dependent, and concentration-dependent with Michaelis-Menten constants of 6.44 μM and 99.7 μM, and inhibited by several organic cations but not tetraethylammonium. The inhibition of CTL1 mRNA by small interfering RNA had little effect on the [3H]choline uptake by TR-iBRB2 cells. Rat CTL1-expressing Xenopus laevis oocytes exhibited an increase in the [ 3H]choline uptake by 45% compared with a control. In conclusion, our findings are consistent with Na+-independent choline transport being the mechanism for blood-to-retina transport of choline at the inner BRB. Although rat CTL1 expression is associated with the choline uptake, CTL1 does not play a major role in the choline uptake at the inner BRB. © 2007 Springer Science+Business Media, LLC.

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Function and regulation of taurine transport at the inner blood-retinal barrier

Tomi M., Terayama T., Isobe T., Egami F., Morito A., Kurachi M., Ohtsuki S., Kang Y., Terasaki T., Hosoya K.

Microvascular Research (Microvascular Research) 73 ( 2 ) 100 - 106 2007.03

Research paper (scientific journal), Accepted, ISSN 0026-2862

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.

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Establishment and characterization of conditionally immortalized endothelial cell lines from the thoracic duct and inferior vena cava of tsA58/EGFP double-transgenic rats

Matsuo M., Koizumi K., Yamada S., Tomi M., Takahashi R., Ueda M., Terasaki T., Obinata M., Hosoya K., Ohtani O., Saiki I.

Cell and Tissue Research (Cell and Tissue Research) 326 ( 3 ) 749 - 758 2006.12

Research paper (scientific journal), Accepted, ISSN 0302-766X

The basic biology of blood vascular endothelial cells has been well documented. However, little is known about that of lymphatic endothelial cells, despite their importance under normal and pathological conditions. The lack of a lymphatic endothelial cell line has hampered progress in this field. The objective of this study has been to establish and characterize lymphatic and venous endothelial cell lines derived from newly developed tsA58/EGFP transgenic rats harboring the temperature-sensitive simian virus 40 (SV40) large T-antigen and enhanced green fluorescent protein (EGFP). Endothelial cells were isolated from the transgenic rats by intraluminal enzymatic digestion. The cloned cell lines were named TR-LE (temperature-sensitive rat lymphatic endothelial cells from thoracic duct) and TR-BE (temperature-sensitive rat blood-vessel endothelial cells from inferior vena cava), respectively, and cultured on fibronectin-coated dishes in HuMedia-EG2 supplemented with 20% fetal bovine serum and Endothelial Mitogen at a permissive temperature, 33°C. A temperature shift to 37°C resulted in a decrease in proliferation with degradation of the large T-antigen and cleavage of poly (ADP-ribose) polymerase. TR-LE cells expressed lymphatic endothelial markers VEGFR-3 (vascular endothelial growth factor receptor), LYVE-1 (a lymphatic endothelial receptor), Prox-1 (a homeobox gene product), and podoplanin (a glomerular podocyte membrane mucoprotein), together with endothelial markers CD31, Tie-2, and VEGFR-2, whereas TR-BE cells expressed CD31, Tie-2, and VEGFR-2, but no lymphatic endothelial markers. Thus, these conditionally immortalized and EGFP-expressing lymphatic and vascular endothelial cell lines might represent an important tool for the study of endothelial cell functions in vitro. © 2006 Springer-Verlag.

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Inhibition of dehydroascorbic acid transport across the rat blood-retinal and -brain barriers in experimental diabetes

Minamizono A., Tomi M., Hosoya K.

Biological and Pharmaceutical Bulletin (Biological and Pharmaceutical Bulletin) 29 ( 10 ) 2148 - 2150 2006.10

Research paper (scientific journal), Accepted, ISSN 0918-6158

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.

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Application of microdialysis to evaluate the efflux transport of estradiol 17-β glucuronide across the rat blood-retinal barrier

Katayama K., Ohshima Y., Tomi M., Hosoya K.

Journal of Neuroscience Methods (Journal of Neuroscience Methods) 156 ( 1-2 ) 249 - 256 2006.09

Research paper (scientific journal), Accepted, ISSN 0165-0270

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.

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Functional and molecular characterization of adenosine transport at the rat inner blood-retinal barrier

Nagase K., Tomi M., Tachikawa M., Hosoya K.i.

Biochimica et Biophysica Acta - Biomembranes (Biochimica et Biophysica Acta - Biomembranes) 1758 ( 1 ) 13 - 19 2006.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 0005-2736

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.

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Evidence for creatine biosynthesis in Müller glia

Nakashima T., Tomi M., Tachikawa M., Watanabe M., Terasaki T., Hosoya K.

GLIA (GLIA) 52 ( 1 ) 47 - 52 2005.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 0894-1491

In high-energy metabolic tissues like the retina, creatine may play an important role in energy storage and in transmission of phosphate-bound energy substrates. To prove this, we investigated creatine synthesis in Müller glia. We also characterized the localization of the creatine synthetic enzyme, S-adenosyl-L-methionine:N-guanidinoacetate methyltransferase (GAMT) in the retina. Reverse transcription-polymerase chain reaction analysis revealed that L-arginine:glycine amidinotransferase and GAMT mRNAs were expressed in the retina and the Müller cell line, TR-MUL5. [14C]Creatine was detected after incubation of isolated rat retina or TR-MUL5 cells with [ 14C]glycine, L-arginine and L-methionine, suggesting creatine synthesis in Müller glia. Western blot analysis also revealed expression of GAMT protein in the rat retina and TR-MUL5 cells. Furthermore, confocal immunofluorescent microscopy of dual-labeled rat retinal sections demonstrated co-localization of GAMT with glutamine synthetase. Taken together, the results of the present study indicate creatine synthesis in Müller glia, implying an important role of creatine in energy metabolism in the retina. ©2005 Wiley-Liss, Inc.

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Dominant expression of androgen receptors and their functional regulation of organic anion transporter 3 in rat brain capillary endothelial cells; comparison of gene expression between the blood-brain and -retinal barriers

Ohtsuki S., Tomi M., Hata T., Nagai Y., Hori S., Mori S., Hosoya K., Terasaki T.

Journal of Cellular Physiology (Journal of Cellular Physiology) 204 ( 3 ) 896 - 900 2005.09

Research paper (scientific journal), Joint Work, Accepted, ISSN 0021-9541

Brain and retinal capillary endothelial cells (BCECs and RCECs, respectively) exhibit a barrier structure and function. Comparison of gene expression in these cells could clarify the selective function of each barrier. The purpose of this study was to identify the genes selecti vely expressed at the blood-brain barrier (BBB) and to clarify the function of the selective gene, androgen receptor (AR). Gene expression was compared by a differential display using conditionally immortalized rat BCECs and RCECs (TR-BBB and TR-iBRB, respectively). A total of 12 gene fragments were identified as the selective genes dominantly expressed in TR-BBB cells. The most selective fragment in TR-BBB cells had the highest homology with the 3′-UTR of human and mouse AR. Rat AR mRNA was detected in TR-BBB cells and the brain capillary rich fraction, but not in TR-iBRB cells. Expression of organic anion transporter 3 (OAT3) mRNA in TR-BBB cells was induced by treatment with dihydrotestosterone (DHT), an AR ligand, and this induction was suppressed by flutamide. Moreover, uptake of benzylpenicillin by TR-BBB cells was also induced by DHT treatment. In contrast, OAT3 mRNA expression in TR-iBRB cells was not affected by DHT treatment. The brain-to-blood efflux rate of benzylpenicillin was not affected by gender. These results suggest that AR is involved in the functional regulation of OAT3 at the BBB, but not at the inner blood-retinal barrier (iBRB), and this regulation is not affected by gender. The BBB function will be affected by the androgen levels in the brain and/or plasma via AR. © 2005 Wiley-Liss, Inc.

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L-type amino acid transporter 1-mediated L-leucine transport at the inner blood-retinal barrier

Tomi M., Mori M., Tachikawa M., Katayama K., Terasaki T., Hosoya K.

Investigative Ophthalmology and Visual Science (Investigative Ophthalmology and Visual Science) 46 ( 7 ) 2522 - 2530 2005.07

Research paper (scientific journal), Joint Work, Accepted, ISSN 0146-0404

PURPOSE. L-type amino acid transporters (LATs) prefer branched-chain and aromatic amino acids, including neurotransmitter precursors. The objective of this study was to clarify the expression and function of LAT at the inner blood-retinal barrier (BRB). METHODS. [3H]L-Leucine transport at the inner BRB was characterized by using in vivo integration plot analysis and a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2). The expression of the LAT1 was demonstrated by quantitative real-time RT-PCR, immunoblot, and immunohistochemical analyses. RESULTS. The apparent influx permeability clearance of [3H]L-leucine in the rat retina was found to be 203 μL/(min · g retina), supporting a carrier-mediated influx transport of L-leucine at the BRB. [3H]L- Leucine uptake by TR-iBRB2 cells was an Na+-independent and concentration-dependent process with a Km of 14.1 μM. This process was more potently cis inhibited by substrates of LAT1, D-leucine, D-phenylalanine, and D-methionine, than those of LAT2, L-alanine, and L-glutamine. [3H]L-Leucine efflux from TR-iBRB2 cells was trans-stimulated by substrates of LAT1. The expression of LAT1 mRNA was 100- and 15-fold greater than that of LAT2 in TR-iBRB2 and magnetically isolated rat retinal vascular endothelial cells, respectively. The expression of LAT1 protein was observed in TR-iBRB2 and primary cultured human retinal endothelial cells and immunostaining of LAT1 was observed along the rat retinal capillaries. CONCLUSIONS. LAT1 is expressed at the inner BRB and mediates blood-to-retina L-leucine transport. This transport system plays a key role in maintaining large neutral amino acids as well as neurotransmitters in the neural retina. Copyright © Association for Research in Vision and Ophthalmology.

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PKC/MAPK signaling suppression by retinal pericyte conditioned medium prevents retinal endothelial cell proliferation

Kondo T., Hosoya K., Hori S., Tomi M., Ohtsuki S., Terasaki T.

Journal of Cellular Physiology (Journal of Cellular Physiology) 203 ( 2 ) 378 - 386 2005.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 0021-9541

Little is known about the regulation mechanism of endothelial cell proliferation by retinal pericytes. The purpose of this study was to elucidate the suppression mechanism of retinal capillary endothelial cell growth by soluble factors derived from retinal pericytes. Conditioned medium of retinal pericytes (rPCT1-CM) suppressed ischemia-induced retinal neovascularization. The growth and DNA synthesis of TR-iBRB2 cells, a conditionally immortalized rat retinal capillary endothelial cell line, were suppressed in a concentration-dependent manner by concentrated rPCT1-CM. The number of human cultured endothelial cells was also reduced by rPCT1-CM. These results provide the first evidence that CM from the cultivation of pericytes alone can inhibit retinal neovascularization in vivo and in vitro. Although the growth reduction of TR-iBRB2 cells was only partly reversed by treatment of rPCT1-CM with antibodies to transforming growth factor-β1, it was completely lost by heat-treatment of rPCT1-CM, suggesting that anti-angiogenic factors are soluble proteins. The levels of expression of G1/S-phase-related proteins, such as cyclin D1, cyclin-dependent kinase (cdk)4, cdk6, and proliferating cell nuclear antigen, were reduced and a cdk inhibitor, p21Cip1, was induced in rPCT1-CM-treated TR-iBRB2 cells. Moreover, phosphorylated p44/42 mitogen-activated protein kinase (p44/42 MAPK) in TR-iBRB2 cells was reduced by rPCT1-CM treatment and phosphorylated protein kinase C (PKC)α/βII, which is upstream of p44/42 MAPK, was also suppressed. In conclusion, CM from retinal pericytes suppresses PKC-p44/42 MAPK signaling, inhibits endothelial cell growth, and prevents retinal neovascularization. Anti-angiogenic factors derived from retinal pericytes are likely to play a critical role in the regulation of retinal endothelial cell growth. © 2004 Wiley-Liss, Inc.

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Advances in the cell biology of transport via the inner blood-retinal barrier: Establishment of cell lines and transport functions

Hosoya K., Tomi M.

Biological and Pharmaceutical Bulletin (Biological and Pharmaceutical Bulletin) 28 ( 1 ) 1 - 8 2005.01

Research paper (scientific journal), Joint Work, Accepted, ISSN 0918-6158

The retinal capillary endothelial cells are connected to each other by tight junctions that play a key role in permeability as the inner blood-retinal barrier (inner BRB). Thus, understanding the inner BRB transport mechanism is an important step towards drug targeting of the retina. Nevertheless, inner BRB transport studies have been very limited in number since it is not easy to use the retinal capillaries, which are very small in size, for in vitro transport studies. Conditionally immortalized rat retinal capillary endothelial cells (TR-iBRB), pericytes (TR-rPCT) and Müller cell lines (TR-MUL) have been established from transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene. These cell lines posses respective cell type markers and maintain certain in vivo functions. Using a combination of newly developed cell lines and in vivo studies, we have elucidated the mechanism whereby vitamin C, L-cystine, and creatine are supplied to the retina. TR-iBRB cells are also able to identify transporters and apply to study regulation of transporters under pathophysiological conditions. Furthermore, these cell lines permit the investigation of cell-to-cell interactions and the expression of inner BRB-specific genes between TR-iBRB and other cell lines. © 2005 Pharmaceutical Society of Japan.

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Application of magnetically isolated rat retinal vascular endothelial cells for the determination of transporter gene expression levels at the inner blood-retinal barrier

Tomi M., Hosoya K.

Journal of Neurochemistry (Journal of Neurochemistry) 91 ( 5 ) 1244 - 1248 2004.12

Research paper (scientific journal), Accepted, ISSN 0022-3042

The purpose of the present study was to quantify transporter gene levels at the inner blood-retinal barrier (inner BRB) using a combination of magnetic isolation method for rat retinal vascular endothelial cells (RVEC) and real-time quantitative PCR analysis. The transcript levels of CD31, Tie-2, claudin-5, occludin, Jam-1, mdr1a, oatp2, and oatp14 in the RVEC fraction were more than 100-fold greater than those in the non-RVEC fraction, suggesting that these genes are predominantly expressed at the inner BRB. The transcript levels of GLUT1 and MCT1 in the RVEC fraction were the most abundant in the respective transporter family, suggesting that GLUT1 and MCT1 play a predominant role in D-glucose and monocarboxylate transport, respectively, at the inner BRB. In conclusion, application of magnetically isolated RVEC is able to determine transporter gene levels at the inner BRB thereby increasing our understanding of inner BRB functions at a molecular level.

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Transporter mRNA expression in a conditionally immortalized rat small intestine epithelial cell line (TR-SIE).

Hosoya K., Tomi M., Takayama M., Komokata Y., Nakai D., Tokui T., Nishimura K., Ueda M., Obinata M., Hori S., Ohtsuki S., Amidon G., Terasaki T.

Drug metabolism and pharmacokinetics (Drug metabolism and pharmacokinetics) 19 ( 4 ) 264 - 269 2004.08

Research paper (scientific journal), Accepted, ISSN 1347-4367

Small intestine epithelial cell lines (TR-SIE), which are established from the small intestine of transgenic rats harboring temperature-sensitive simian virus 40 large T-antigen gene (tsA58 Tg rat), were used to characterize the mRNA expression of small intestine transporters. TR-SIE cells had a polygonal morphology and expressed cytokeratin protein and villin mRNA. Although the large T-antigen was strongly expressed at 33 degrees C, this was reduced at 37 and 39 degrees C. Concomitantly, the cell growth was arrested at 37 and 39 degrees C compared with that at 33 degrees C, suggesting that TR-SIE cells are conditionally immortalized cell lines. RT-PCR analysis revealed that TR-SIE cells expressed ABCB1 (mdr1a and mdr1b), ABCB4 (mdr2), ABCC2 (mrp2), ABCC6 (mrp6), ABCG1, ABCG2 (bcrp/mxr), Slc21a7 (Oatp3), Slc15a1 (PepT1), and Slc16a1 (Mct1). Conditionally immortalized rat small intestine epithelial cell lines were established from tsA58 Tg rats and expressed the mRNA of intestinal transporters.

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Retinal selectivity of gene expression in rat retinal versus brain capillary endothelial cell lines by differential display analysis

Tomi M., Abukawa H., Nagai Y., Hata T., Takanaga H., Ohtsuki S., Terasaki T., Hosoya K.

Molecular Vision (Molecular Vision) 10 ( 66 ) 537 - 543 2004.08

Research paper (scientific journal), Accepted, ISSN 1090-0535

Purpose: The retina is a neural tissue especially differentiated for vision and, thus, the inner blood-retinal barrier (inner BRB) specific molecules may play an essential role in maintaining neural functions in the retina. The purpose of the present study was to identify selectively expressed genes at the inner blood-retinal barrier compared with the blood-brain barrier (BBB). Methods: A comparison of expressed genes between conditionally immortalized rat retinal (TR-iBRB) cell lines and brain capillary endothelial (TR-BBB) cell lines was performed using mRNA differential display analysis and quantitative real time PCR analysis. The rat M-cadherin gene was cloned by performing 5' RACE, and its protein expression was detected by immunoblot analysis. Results: Eight clones were identified as highly expressed genes in TR-iBRB cells including GATA-binding protein-3 (GATA-3), cytosolic branched chain amino transferase (BCATc), and M-cadherin (cadherin-15). The rat M-cadherin gene was cloned from TR-iBRB cells, for the first time, and has >86% amino acid sequence identity to the previously cloned mammalian M-cadherins. Rat M-cadherin expression in TR-iBRB cells was much greater than that in TR-BBB cells as far as mRNA and protein levels were concerned. Conclusions: M-cadherin, GATA-3, and BCATc are highly expressed in TR-iBRB cells compared with TR-BBB cells and may indeed be involved in unique functions at the inner BRB. © 2004 Molecular Vision.

Blood-to-retina transport of creatine via creatine transporter (CRT) at the rat inner blood-retinal barrier

Nakashima T., Tomi M., Katayama K., Tachikawa M., Watanabe M., Terasaki T., Hosoya K.

Journal of Neurochemistry (Journal of Neurochemistry) 89 ( 6 ) 1454 - 1461 2004.06

Research paper (scientific journal), Accepted, ISSN 0022-3042

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.

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Vitamin C transport in oxidized form across the rat blood-retinal barrier

Hosoya K., Minamizono A., Katayama K., Terasaki T., Tomi M.

Investigative Ophthalmology and Visual Science (Investigative Ophthalmology and Visual Science) 45 ( 4 ) 1232 - 1239 2004.04

Research paper (scientific journal), Accepted, ISSN 0146-0404

PURPOSE. To elucidate the mechanisms of vitamin C transport across the blood-retinal barrier (BRB) in vivo and in vitro. METHODS. [14C] Dehydroascorbic acid (DHA) and [14C]ascorbic acid (AA) transport in the retina across the BRB were examined using in vivo integration plot analysis in rats, and the transport mechanism was characterized using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) as an in vitro model of the inner BRB. RESULTS. The apparent influx permeability clearance (Kin) per gram of retina of [14C]DHA and [ 14C]AA was found to be 2.44 × 103 μL/(min · g retina) and 65.4 μL/(min · g retina), respectively. In the retina and brain, the Kin of [14C]DHA was approximately 38 times greater than that of [14C]AA. whereas there was no major difference in the heart. The Kin of [14C]DHA in the retina was eight times greater than that in the brain. HPLC analysis revealed that most of the vitamin C accumulated in AA form in the retina. These results suggest that vitamin C is mainly transported in DHA form across the BRB and accumulates in AA form in the rat retina. In an in vitro uptake study in TR-iBRB2 cells, the initial uptake rate of [14C]DHA was 37 times greater than that of [14C]AA, which is in agreement with the results of the in vivo study. [14C]DHA uptake by TR-iBRB2 cells took place in an Na +-independent and concentration-dependent manner with a Km of 93.4 μM. This process was inhibited by substrates and inhibitors of glucose transporters. [14C]DHA uptake was inhibited by D-glucose in a concentration-dependent manner with a 50% inhibition concentration of 5.56 mM. Quantitative real-time PCR and immunostaining analyses revealed that expression of GLUT1 and -3 was greater than that of the Na+-dependent L-ascorbic acid transporter (SVCT)-2 in TR-iBRB2 cells. CONCLUSIONS. Vitamin C is mainly transported across the BRB as DHA mediated through facilitative glucose transporters and accumulates as AA in the rat retina.

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Expression and regulation of L-cystine transporter, system X <inf>c</inf>-, in the newly developed rat retinal Müller cell line (TR-MUL)

Tomi M., Funaki T., Abukawa H., Katayama K., Kondo T., Ohtsuki S., Ueda M., Obinata M., Terasaki T., Hosoya K.

GLIA (GLIA) 43 ( 3 ) 208 - 217 2003.09

Research paper (scientific journal), Accepted, ISSN 0894-1491

The purpose of the present study was to elucidate the expression and regulation of the L-cystine transporter, system xc-, in Müller cells. In this study, newly developed conditionally immortalized rat Müller cell lines (TR-MUL) from transgenic rats harboring the temperature-sensitive SV 40 large T-antigen gene were used as an in vitro model. TR-MUL cells express large T-antigen and grow well at 33°C with a doubling time of 30 h, but do not grow at 39°C. TR-MUL cells express typical Müller cell markers such as S-100, glutamine synthetase, and EAAT1/GLAST, whereas EAAT2/GLT-1 and EAAT5 are not detected. TR-MUL cells also exhibit little or no expression of glial fibrillary acidic protein. We found that TR-MUL5 cells exhibited [14C]L-cystine uptake activity and expressed xCT and 4F2hc, which involve system xc-. The uptake of [14C]L-cystine was significantly inhibited by L-glutamic acid and L-aspartic acid, whereas L-leucine had no effect. Following diethyl maleate (DEM) treatment, the glutathione concentration in TR-MUL5 cells was reduced in the first 24 h, then gradually recovered for more than 24 h. The L-cystine uptake rate and the xCT expression level in TR-MUL5 cells were enhanced by DEM treatment. In contrast, the 4F2hc expression level was unchanged. In conclusion, TR-MUL cells have the properties of Müller cells and exhibit system x c--mediated L-cystine uptake activity. The oxidative stress conditions following DEM treatment activate L-cystine transport in TR-MUL cells due to the enhanced transcription of the xCT gene. © 2003 Wiley-Liss, Inc.

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Establishment of conditionally immortalized rat retinal pericyte cell lines (TR-rPCT) and their application in a Co-culture system using retinal capillary endothelial cell line (TR-iBRB2)

Kondo T., Hosoya K., Hori S., Tomi M., Ohtsuki S., Takanaga H., Nakashima E., Iizasa H., Asashima T., Ueda M., Obinata M., Terasaki T.

Cell Structure and Function (Cell Structure and Function) 28 ( 3 ) 145 - 153 2003.06

Research paper (scientific journal), Accepted, ISSN 0386-7196

The purpose of this study was to establish and characterize a retinal pericyte cell line from retinal capillaries of transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene (tsA58 Tg rat), and to apply this to the co-culture with a retinal capillary endothelial cell line. The conditionally immortalized rat retinal pericyte cell lines (TR-rPCTs), which express a temperature-sensitive large T-antigen, were obtained from two tsA58 Tg rats. These cell lines had a multicellular nodule morphology and reacted positively with von Kossa staining, a marker of calcification. TR-rPCTs cells expressed mRNA of pericyte markers such as rat intercellular adhesion molecule-1, platelet-derived growth factor-receptor β, angiopoietin-1, and osteopontin. Western blot analysis indicated that α-smooth muscle actin (α-SMA) was expressed in TR-rPCT3 and 4 cells. In contrast, α-SMA was induced by transforming growth factor-β1 and its enhancement was reduced by basic fibroblast growth factor in TR-rPCT1 and 2 cells. When TR-rPCT1 cells were cultured with a rat retinal endothelial cell line (TR-iBRB2) in a contact co-culture system, the number of TR-iBRB2 cells were significantly reduced in comparison with that of a single culture of TR-iBRB2 cells, suggesting that physical contact between pericytes and retinal endothelial cells is important for the growth of retinal endothelial cells. In conclusion, conditionally immortalized retinal pericyte cell lines were established from tsA58 Tg rats. These cell lines exhibited the properties of retinal pericytes and can be applied in co-culture systems with a retinal capillary endothelial cell line.

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Regulation of taurine transport at the blood-brain barrier by tumor necrosis factor-α, taurine and hypertonicity

Kang Y., Ohtsuki S., Takanaga H., Tomi M., Hosoya K., Terasaki T.

Journal of Neurochemistry (Journal of Neurochemistry) 83 ( 5 ) 1188 - 1195 2002.12

Research paper (scientific journal), Accepted, ISSN 0022-3042

Taurine is the abundant sulfur-containing β-amino acid in brain where it exerts a neuroprotective effect. Although it is known that the blood-brain barrier (BBB) mediates taurine transport, the regulation of taurine transport have not been clarified yet. A conditionally immortalized rat brain capillary endothelial cells (TR-BBB13), an in vitro model of the BBB, exhibited [3H]taurine uptake, which was dependent on both Na+ and Cl-, and inhibited by β-alanine. Taurine transporter (TAUT) mRNA was detected in TR-BBB13 cells, and TAUT protein was also expressed at 70 kDa. TR-BBB13 cells exposed to 20 ng/mL TNF-α and under hypertonic conditions showed a 1.7-fold and 3.2-fold increase in [3H]taurine uptake, respectively. In contrast, lipopolysaccharide and diethyl maleate did not significantly affect taurine uptake. The taurine uptake was reduced by pre-treatment with excess taurine (50 mM). The mRNA level of the TAUT in TNF-α and following hypertonic treatment was greater than that in control cells, whereas that under excess taurine conditions was lower than in controls. Therefore, taurine transport activity at the BBB appears to be regulated at the transcriptional level by cell damage, osmolality and taurine in the brain.

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Enhancement of L-cystine transport activity and its relation to xCT gene induction at the blood-brain barrier by diethyl maleate treatment

Hosoya K., Tomi M., Ohtsuki S., Takanaga H., Saeki S., Kanai Y., Endou H., Naito M., Tsuruo T., Terasaki T.

Journal of Pharmacology and Experimental Therapeutics (Journal of Pharmacology and Experimental Therapeutics) 302 ( 1 ) 225 - 231 2002.07

Research paper (scientific journal), Accepted, ISSN 0022-3565

The purpose of the present study was to elucidate the mechanism of enhancement of L-cystine uptake at the blood-brain barrier (BBB). The uptake of [ 14C]L-cystine and [ 3H]L-glutamic acid (L-Glu) was determined using a mouse brain endothelial cell line (MBEC4) as an in vitro BBB model. The mRNA levels of L-cystine/L-Glu exchanger, system X c-, which consists of xCT and 4F2hc, were determined by quantitative real-time reverse transcription-polymerase chain reaction analysis. The [ 14C]L-cystine uptake by MBEC4 cells appeared to be mediated via an Na +-independent saturable process. The corresponding Michaelis-Menten constant (K m) was 63.7 μM. In the presence of L-Glu, there was competitive inhibition with an inhibition constant (K i) of 83.5 μM. [ 3H]L-Glu uptake in the absence of Na + was saturable with a K m of 48.1 μM, and it exhibited competitive inhibition with a K i of 24.9 μM in the presence of L-cystine. The mutual inhibition between L-cystine and L-Glu and the type of inhibition suggest that system X c- operates in MBEC4 cells. The xCT and 4F2hc mRNAs were expressed in MBEC4 cells and, following diethyl maleate (DEM) treatment, the xCT mRNA level and L-cystine uptake in MBEC4 cells were enhanced in parallel with an increase in DEM concentration (up to 500 μM). Concomitantly, the glutathione concentration in MBEC4 cells was increased. In conclusion, system X c--mediated L-cystine uptake takes place in MBEC4 cells. L-Cystine transport via system X c- at the BBB is likely to be induced under oxidative stress conditions following DEM treatment due to enhanced transcription of the xCT gene.

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Induction of xCT gene expression and L-cystine transport activity by diethyl maleate at the inner blood-retinal barrier

Tomi M., Hosoya K., Takanaga H., Ohtsuki S., Terasaki T.

Investigative Ophthalmology and Visual Science (Investigative Ophthalmology and Visual Science) 43 ( 3 ) 774 - 779 2002.03

Research paper (scientific journal), Accepted, ISSN 0146-0404

Purpose. In this study, the expression and regulation of the L-cystine transporter, system xc-, at the inner blood-retinal barrier (inner BRB) was investigated using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) as an in vitro model. Methods. For the uptake study, TR-iBRB2 cells were cultured at 33°C in the presence or absence of diethyl maleate (DEM), and the uptake rate of [14C]L-cystine was measured at 37°C. The mRNA levels of system xc-, which consists of xCT and 4F2hc, were determined by quantitative real-time RT-PCR analysis with specific primers. Results. The xCT and 4F2hc mRNAs were expressed in TR-iBRB2 cells. The [14C]L-cystine uptake by TR-iBRB2 cells appeared to be mediated through a saturable Na+-independent process. The corresponding Michaelis-Menten constant was 9.18 μM. At 100 μM DEM, the xCT mRNA level and L-cystine uptake activity in TR-iBRB2 cells were enhanced in a time-dependent manner. Concomitantly, the glutathione concentration in TR-iBRB2 cells was increased. In contrast, the 4F2hc mRNA level was unchanged up to 24 hours and was induced for more than 24 hours by DEM treatment. Under both normal and DEM treatment conditions, the uptake of [14C]L-cystine was strongly inhibited by L-glutamic acid, L-α-aminoadipic acid, L-homocysteic acid, and L-quisqualic acid, whereas L-aspartic acid and L-arginine had no effect, which is evidence of the induction of system xc-. Conclusions. System xc--mediated L-cystine uptake appears to be present at the inner BRB. DEM induces L-cystine transport through system xc- at the inner BRB by enhanced transcription of the xCT gene.

MCT1-mediated transport of L-lactic acid at the inner blood-retinal barrier: A possible route for delivery of monocarboxylic acid drugs to the retina

Hosoya K., Kondo T., Tomi M., Takanaga H., Ohtsuki S., Terasaki T.

Pharmaceutical Research (Pharmaceutical Research) 18 ( 12 ) 1669 - 1676 2001.12

Research paper (scientific journal), Accepted, ISSN 0724-8741

Purpose. The aim of this study was to characterize L-lactic acid transport using a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) as a model of in vitro inner blood-retinal barrier (iBRB) to obtain a better understanding of the transport mechanism at the iBRB. Methods. TR-iBRB2 cells were cultured at 33°C, and L-lactic acid uptake was monitored by measuring [ 14C]L-lactic acid at 37°C. The expression and mRNA level of monocarboxylate transporter (MCT)1 and MCT2 were determined by reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR with specific primers, respectively. Results. The [ 14C]L-lactic acid uptake by TR-iBRB2 cells increased up to a pH of 5.0 and was inhibited in the presence of 10 mM L-lactic acid. The [ 14C]L-lactic acid uptake at pH 6.0 was both temperature-and concentration-dependent with a Michaelis-Menten constant of 1.7 mM and a maximum uptake rate of 15 nmol/(30 s·mg of protein). This process was reduced by carbonylcyanide p-trifluoromethoxy-phenylhydrazone (protonophore), α-cyano-4-hydroxycinnamate, and p-chloromercuribenzenesulfonate (typical inhibitors for H +-coupled monocarboxylic acid transport), suggesting that L-lactic acid uptake by TR-iBRB2 cells is a carrier-mediated transport process coupled with an H + gradient. [ 14C]L-Lactic acid uptake was markedly inhibited by monocarboxylic acids but not dicarboxylic acids and amino acids. Moreover, salicylic and valproic acids competitively inhibited this process with an inhibition constant of 4.7 mM and 5.4 mM, respectively. Although MCT1 and MCT2 mRNA were found to be expressed in TR-iBRB2 cells, MCT1 mRNA was found to be present at a concentration 33-fold greater than that of MCT2 mRNA using quantitative real-time PCR. [ 14C]L-Lactic acid was significantly reduced by 5-(N,N-hexamethylene)-amiloride at pH 7.4 and Na +/H + exchanger 1 mRNA was expressed in TR-iBRB2 cells. Conclusion. L-Lactic acid transport at the iBRB is an H +-coupled and carrier-mediated mechanism via MCT1 that is competitively inhibited by monocarboxylate drugs.

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Conditionally immortalized retinal capillary endothelial cell lines (TR-iBRB) expressing differentiated endothelial cell functions derived from a transgenic rat

Hosoya K., Tomi M., Ohtsuki S., Takanaga H., Ueda M., Yanai N., Obinata M., Terasaki T.

Experimental Eye Research (Experimental Eye Research) 72 ( 2 ) 163 - 172 2001.02

Research paper (scientific journal), Accepted, ISSN 0014-4835

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.

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Conditionally immortalized brain capillary endothelial cell lines established from a transgenic mouse harboring temperature-sensitive simian virus 40 large T-antigen gene

Hosoya K., Tetsuka K., Nagase K., Tomi M., Saeki S., Ohtsuki S., Terasaki T., Yanai N., Obinata M., Kikuchi A., Okano T., Takanaga H.

AAPS PharmSci (AAPS PharmSci) 2 ( 3 ) 1 - 11 2000.12

ISSN 15221059

Five immortalized brain capillary endothelial cell lines (TM-BBB1-5) were established from 3 transgenic mice harboring temperature-sensitive simian virus 40 large T-antigen gene (Tg mouse). These cell lines expressed active large Tantigen and grew well at 33°C with a doubling time of about 20 to 30 hours. TM-BBBs also grew at 37°C but not at 39°C. However, growth was restored when the temperature of the culture was lowered to 33°C. Although significant amounts of large T-antigen were shown to be present in the cell culture at 33°C, there was less of this complex at 37°C and 39°C. TMBBBs expressed the typical endothelial marker, von Willebrand factor, and exhibited acetylated lowdensity lipoprotein uptake activity. The alkaline phosphatase and γ-glutamyltranspeptidase activity in TM-BBBs were -10% and 50% to 80% of brain capillary fraction of normal mice, respectively. DMannitol transport in the both apical-to-basal and basal-to-apical directions across the TM-BBB was 2-fold greater than for inulin. TM-BBBs were found to express GLUT-1 but not GLUT-3, and exhibited concentration-dependent 3-O-methyl-D-glucose (3-OMG) uptake activity with a Michaelis-Menten constant of 6.59 ± 1.16 mmol/1. Moreover, Pglycoprotein (P-gp) with a molecular weight of-170 kDa was expressed in all TM-BBBs. Both mdr la and mdr 1b mRNA were detected in TM-BBB4 using reverse transcription-polymerase chain reaction (RT- 7 PCR) analysis. [3H]-Cyclosporin A uptake by TMBBB was significantly increased in the presence of 100 μmol/1 verapamil and vincristine, suggesting that TM-BBB exhibits efflux transport activity via P-gp. In conclusion, conditional brain capillary endothelial cell lines were established from Tg mice. This cell line expresses endothelial markers and transporters at the BBB and is able to regulate cell growth, due to the amount of active large T-antigen in the cell, by changing the culture temperature.

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