The Experts below are selected from a list of 306 Experts worldwide ranked by ideXlab platform
Tetsuya Terasaki - One of the best experts on this subject based on the ideXlab platform.
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functional expression of a proton coupled organic cation h oc antiporter in human brain Capillary Endothelial Cell line hcmec d3 a human blood brain barrier model
Fluids and Barriers of the CNS, 2013Co-Authors: Keita Shimomura, Tetsuya Terasaki, Pierre-olivier Couraud, Takashi Okura, Sayaka Kato, Jeanmichel Schermann, Yoshiharu DeguchiAbstract:Background Knowledge of the molecular basis and transport function of the human blood–brain barrier (BBB) is important for not only understanding human cerebral physiology, but also development of new central nervous system (CNS)-acting drugs. However, few studies have been done using human brain Capillary Endothelial Cells, because human brain materials are difficult to obtain. The purpose of this study is to clarify the functional expression of a proton-coupled organic cation (H+/OC) antiporter in human brain Capillary Endothelial Cell line hCMEC/D3, which has been recently developed as an in vitro human BBB model.
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functional expression of a proton coupled organic cation h oc antiporter in human brain Capillary Endothelial Cell line hcmec d3 a human blood brain barrier model
Fluids and Barriers of the CNS, 2013Co-Authors: Keita Shimomura, Tetsuya Terasaki, Pierre-olivier Couraud, Takashi Okura, Sayaka Kato, Jeanmichel Schermann, Yoshiharu DeguchiAbstract:Knowledge of the molecular basis and transport function of the human blood–brain barrier (BBB) is important for not only understanding human cerebral physiology, but also development of new central nervous system (CNS)-acting drugs. However, few studies have been done using human brain Capillary Endothelial Cells, because human brain materials are difficult to obtain. The purpose of this study is to clarify the functional expression of a proton-coupled organic cation (H+/OC) antiporter in human brain Capillary Endothelial Cell line hCMEC/D3, which has been recently developed as an in vitro human BBB model. Diphenhydramine, [3H]pyrilamine and oxycodone were used as cationic drugs that proved to be H+/OC antiporter substrates. The in vitro uptake experiments by hCMEC/D3 Cells were carried out under several conditions. Diphenhydramine and [3H]pyrilamine were both transported into hCMEC/D3 Cells in a time- and concentration-dependent manner with Km values of 59 μM and 19 μM, respectively. Each inhibited uptake of the other in a competitive manner, suggesting that a common mechanism is involved in their transport. The diphenhydramine uptake was significantly inhibited by amantadine and quinidine, but not tetraethylammonium and 1-methyl-4-phenylpyridinium (substrates for well-known organic cation transporters). The uptake was inhibited by metabolic inhibitors, but was insensitive to extraCellular sodium and membrane potential. Further, the uptake was increased by extraCellular alkalization and intraCellular acidification. These transport properties are completely consistent with those of previously characterized H+/OC antiporter in rat BBB. The present results suggest that H+/OC antiporter is functionally expressed in hCMEC/D3 Cells.
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expression of nuclear receptor mrna and liver x receptor mediated regulation of abc transporter a1 at rat blood brain barrier
Neurochemistry International, 2008Co-Authors: Shin-ichi Akanuma, Masachika Fujiyoshi, Sumio Ohtsuki, Satoko Hori, Tetsuya TerasakiAbstract:Abstract The aim of the present study was to investigate the expression of nuclear receptor mRNA and regulation of the expression of ATP-binding cassette (ABC) transporters by nuclear receptor agonists in rat brain Capillary Endothelial Cells, which form the blood–brain barrier, by using rat brain Capillary fraction from 8-week-old rats and a conditionally immortalized brain Capillary Endothelial Cell line (TR-BBB13). RT-PCR analysis revealed that liver X receptor α and β, retinoid X receptor α and β and peroxisome proliferator-activating receptor α and β mRNAs were expressed in the rat brain Capillary Endothelial Cells and TR-BBB Cells. In contrast, pregnane X receptor, farnesoid X receptor and constitutive androstane receptor were not detected. Furthermore, treatment with a liver X receptor agonist increased the ABCA1 mRNA level in TR-BBB13 Cells, while ABCG2 mRNA expression was not affected. Treatment with a rat pregnane X receptor agonist did not affect the ABCB1 mRNA level in TR-BBB13 Cells. These results demonstrate that the rat blood–brain barrier has an expressional regulation mechanism via sterol-related nuclear receptor, and indicate that the blood–brain barrier in 8-week-old rats lacks ABCB1 regulation via pregnane X receptor.
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donepezil tacrine and α phenyl n tert butyl nitrone pbn inhibit choline transport by conditionally immortalized rat brain Capillary Endothelial Cell lines tr bbb
Archives of Pharmacal Research, 2005Co-Authors: Young Sook Kang, Tetsuya Terasaki, Kyeong Eun Lee, Nayoung LeeAbstract:In the present study, we have characterized the choline transport system and examined the influence of various amine drugs on the choline transporter using a conditionally immortalized rat brain Capillary Endothelial Cell line (TR-BBB) in vitro. The Cell-to-medium (C/M) ratio of [3H]choline in TR-BBB Cells increased time-dependently. The initial uptake rate of [3H]choline was concentration-dependent with a Michaelis-Menten value, Km, of 26.2 +/- 2.7 microM. The [3H]choline uptake into TR-BBB was Na+-independent, but was membrane potential-dependent. The [3H]choline uptake was susceptible to inhibition by hemicholinium-3, and tetraethylammonium (TEA), which are organic cation transporter substrates. Also, the uptake of [3H]choline was competitively inhibited with Ki values of 274 microM, 251 microM and 180 microM in the presence of donepezil hydrochloride, tacrine and alpha-phenyl-n-tert-butyl nitrone (PBN), respectively. These characteristics of choline transport are consistent with those of the organic cation transporter (OCT). OCT2 mRNA was expressed in TR-BBB Cells, while the expression of OCT3 or choline transporter (CHT) was not detected. Accordingly, these results suggest that OCT2 is a candidate for choline transport at the BBB and may influence the BBB permeability of amine drugs.
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new approaches to in vitro models of blood brain barrier drug transport
Drug Discovery Today, 2003Co-Authors: Tetsuya Terasaki, Sumio Ohtsuki, Satoko Hori, Hitomi Takanaga, Emi Nakashima, Ken-ichi HosoyaAbstract:The pharmaceutical industry has been searching for an in vitro blood-brain barrier (BBB) model that preserves in vivo transporter functions in CNS drug discovery and development. The application of conditionally immortalized Cell lines derived from transgenic animals harboring temperature-sensitive SV40 large T-antigen gene, is a rational and promising approach to such a workable in vitro BBB model. The established brain Capillary Endothelial Cell lines retain the in vivo transport rate of several compounds and various forms of gene expression. Furthermore, this new approach has enabled the development of stable and reproducible co-culture models with a pericyte Cell line and/or an astrocyte Cell line.
Ken-ichi Hosoya - One of the best experts on this subject based on the ideXlab platform.
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carrier mediated transport of nicotine across the inner blood retinal barrier involvement of a novel organic cation transporter driven by an outward h gradient
Journal of Pharmaceutical Sciences, 2015Co-Authors: Yuma Tega, Shin-ichi Akanuma, Yoshiyuki Kubo, Chihiro Yuzurihara, Ken-ichi HosoyaAbstract:The present study was carried out to investigate the blood-to-retina transport of nicotine across the inner blood-retinal barrier (BRB). Using the in vivo vascular injection method, the blood-to-retina influx clearance of nicotine across the BRB was determined as 131 μL/(min?g retina), which is much higher than that of a nonpermeable paraCellular marker, and blood-to-retina transport of nicotine was inhibited by organic cations such as pyrilamine and verapamil. The nicotine uptake by a conditionally immortalized rat retinal Capillary Endothelial Cell line (TR-iBRB2 Cells), an in vitro model of the inner BRB, exhibited time, temperature, and concentration dependence with a Km of 492 μM. These results suggest the involvement of a carrier-mediated transport process in nicotine transport in the inner BRB. The nicotine uptake by TR-iBRB2 Cells was stimulated by an outwardly directed H(+) gradient, and the uptake was significantly inhibited by bulky and hydrophobic cationic drugs, whereas inhibitors of organic cation transporters did not show inhibitory effect. These results suggest that the novel organic cation transport system driven by an outwardly directed H(+) gradient is involved in the blood-to-retina transport of nicotine across the inner BRB.
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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
Biochimica et Biophysica Acta, 2008Co-Authors: Masatoshi Tomi, Ayumi Tajima, Masanori Tachikawa, Ken-ichi HosoyaAbstract:The purpose of this study was to identify the uptake mechanism of gamma-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, beta-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 beta-alanine, than those of GABA transporters, GABA and betaine. In the presence of taurine, there was competitive inhibition with a Ki of 74 microM. [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.
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new approaches to in vitro models of blood brain barrier drug transport
Drug Discovery Today, 2003Co-Authors: Tetsuya Terasaki, Sumio Ohtsuki, Satoko Hori, Hitomi Takanaga, Emi Nakashima, Ken-ichi HosoyaAbstract:The pharmaceutical industry has been searching for an in vitro blood-brain barrier (BBB) model that preserves in vivo transporter functions in CNS drug discovery and development. The application of conditionally immortalized Cell lines derived from transgenic animals harboring temperature-sensitive SV40 large T-antigen gene, is a rational and promising approach to such a workable in vitro BBB model. The established brain Capillary Endothelial Cell lines retain the in vivo transport rate of several compounds and various forms of gene expression. Furthermore, this new approach has enabled the development of stable and reproducible co-culture models with a pericyte Cell line and/or an astrocyte Cell line.
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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
Cell Structure and Function, 2003Co-Authors: Tetsu Kondo, Sumio Ohtsuki, Satoko Hori, Ken-ichi Hosoya, Masatoshi Tomi, Hitomi Takanaga, Emi Nakashima, Hisashi Iizasa, Tomoko Asashima, Masatsugu UedaAbstract: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 beta, angiopoietin-1, and osteopontin. Western blot analysis indicated that alpha-smooth muscle actin (alpha-SMA) was expressed in TR-rPCT3 and 4 Cells. In contrast, alpha-SMA was induced by transforming growth factor-beta1 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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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
Pharmaceutical Research, 2001Co-Authors: Ken-ichi Hosoya, Sumio Ohtsuki, Masatoshi Tomi, Hitomi Takanaga, Tetsu Kondo, Tetsuya TerasakiAbstract: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.
Pierre-olivier Couraud - One of the best experts on this subject based on the ideXlab platform.
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functional expression of a proton coupled organic cation h oc antiporter in human brain Capillary Endothelial Cell line hcmec d3 a human blood brain barrier model
Fluids and Barriers of the CNS, 2013Co-Authors: Keita Shimomura, Tetsuya Terasaki, Pierre-olivier Couraud, Takashi Okura, Sayaka Kato, Jeanmichel Schermann, Yoshiharu DeguchiAbstract:Background Knowledge of the molecular basis and transport function of the human blood–brain barrier (BBB) is important for not only understanding human cerebral physiology, but also development of new central nervous system (CNS)-acting drugs. However, few studies have been done using human brain Capillary Endothelial Cells, because human brain materials are difficult to obtain. The purpose of this study is to clarify the functional expression of a proton-coupled organic cation (H+/OC) antiporter in human brain Capillary Endothelial Cell line hCMEC/D3, which has been recently developed as an in vitro human BBB model.
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functional expression of a proton coupled organic cation h oc antiporter in human brain Capillary Endothelial Cell line hcmec d3 a human blood brain barrier model
Fluids and Barriers of the CNS, 2013Co-Authors: Keita Shimomura, Tetsuya Terasaki, Pierre-olivier Couraud, Takashi Okura, Sayaka Kato, Jeanmichel Schermann, Yoshiharu DeguchiAbstract:Knowledge of the molecular basis and transport function of the human blood–brain barrier (BBB) is important for not only understanding human cerebral physiology, but also development of new central nervous system (CNS)-acting drugs. However, few studies have been done using human brain Capillary Endothelial Cells, because human brain materials are difficult to obtain. The purpose of this study is to clarify the functional expression of a proton-coupled organic cation (H+/OC) antiporter in human brain Capillary Endothelial Cell line hCMEC/D3, which has been recently developed as an in vitro human BBB model. Diphenhydramine, [3H]pyrilamine and oxycodone were used as cationic drugs that proved to be H+/OC antiporter substrates. The in vitro uptake experiments by hCMEC/D3 Cells were carried out under several conditions. Diphenhydramine and [3H]pyrilamine were both transported into hCMEC/D3 Cells in a time- and concentration-dependent manner with Km values of 59 μM and 19 μM, respectively. Each inhibited uptake of the other in a competitive manner, suggesting that a common mechanism is involved in their transport. The diphenhydramine uptake was significantly inhibited by amantadine and quinidine, but not tetraethylammonium and 1-methyl-4-phenylpyridinium (substrates for well-known organic cation transporters). The uptake was inhibited by metabolic inhibitors, but was insensitive to extraCellular sodium and membrane potential. Further, the uptake was increased by extraCellular alkalization and intraCellular acidification. These transport properties are completely consistent with those of previously characterized H+/OC antiporter in rat BBB. The present results suggest that H+/OC antiporter is functionally expressed in hCMEC/D3 Cells.
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puromycin based purification of rat brain Capillary Endothelial Cell cultures effect on the expression of blood brain barrier specific properties
Journal of Neurochemistry, 2005Co-Authors: N Perriere, Anthony Regina, P H Demeuse, E Garcia, Marcel Debray, Jean P Andreux, Patrick Couvreur, J M Scherrmann, Jamal Temsamani, Pierre-olivier CouraudAbstract:One of the main difficulties with primary rat brain Endothelial Cell (RBEC) cultures is obtaining pure cultures. The variation in purity limits the achievement of in vitro models of the rat blood-brain barrier. As P-glycoprotein expression is known to be much higher in RBECs than in any contaminating Cells, we have tested the effect of five P-glycoprotein substrates (vincristine, vinblastine, colchicine, puromycin and doxorubicin) on RBEC cultures, assuming that RBECs would resist the treatment with these toxic compounds whereas contaminating Cells would not. Treatment with either 4 mu g/mL puromycin for the first 2 days of culture or 3 mu g/mL puromycin for the first 3 days showed the best results without causing toxicity to the Cells. TransEndothelial electrical resistance was significantly increased in Cell monolayers treated with puromycin compared with untreated Cell monolayers. When cocultured with astrocytes in the presence of cAMP, the puromycin-treated RBEC monolayer showed a highly reduced permeability to sodium fluorescein (down to 0.75 x 10(-6) cm/s) and a high electrical resistance (up to 500 Omega x cm(2)). In conclusion, this method of RBEC purification will allow the production of in vitro models of the rat blood-brain barrier for Cellular and molecular biology studies as well as pharmacological investigations.
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interactions of racemic mefloquine and its enantiomers with p glycoprotein in an immortalised rat brain Capillary Endothelial Cell line gpnt
Biochimica et Biophysica Acta, 2000Co-Authors: Yenthu Pham, Pierre-olivier Couraud, Anthony Regina, Robert Farinotti, Irving W Wainer, Francoise Roux, Francois GimenezAbstract:The brain distribution of the enantiomers of the antimalarial drug mefloquine is stereoselective according to the species. This stereoselectivity may be related to species-specific differences in the properties of some membrane-bound transport proteins, such as P-glycoprotein (P-gp). The interactions of racemic mefloquine and its individual enantiomers with the P-glycoprotein efflux transport system have been analysed in immortalised rat brain Capillary Endothelial GPNT Cells. Parallel studies were carried out for comparison in human colon carcinoma Caco-2 Cells. The Cellular accumulation of the P-glycoprotein substrate, [(3)H]vinblastine, was significantly increased both in GPNT Cells and in Caco-2 Cells when treated with racemic mefloquine and the individual enantiomers. In GPNT Cells, the (+)-stereoisomer of mefloquine was up to 8-fold more effective than its antipode in increasing Cellular accumulation of [(3)H]vinblastine, while in Caco-2 Cells, both enantiomers were equally effective. These results suggest that racemic mefloquine and its enantiomers are effective inhibitors of P-gp. Furthermore, a stereoselective P-glycoprotein inhibition is observed in rat Cells but not in human Cells. The efflux of [(14)C]mefloquine from GPNT Cells was decreased when the Cells were incubated with the P-gp modulators, verapamil, cyclosporin A or chlorpromazine, suggesting that MQ could be a P-gp substrate.
Sumio Ohtsuki - One of the best experts on this subject based on the ideXlab platform.
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expression of nuclear receptor mrna and liver x receptor mediated regulation of abc transporter a1 at rat blood brain barrier
Neurochemistry International, 2008Co-Authors: Shin-ichi Akanuma, Masachika Fujiyoshi, Sumio Ohtsuki, Satoko Hori, Tetsuya TerasakiAbstract:Abstract The aim of the present study was to investigate the expression of nuclear receptor mRNA and regulation of the expression of ATP-binding cassette (ABC) transporters by nuclear receptor agonists in rat brain Capillary Endothelial Cells, which form the blood–brain barrier, by using rat brain Capillary fraction from 8-week-old rats and a conditionally immortalized brain Capillary Endothelial Cell line (TR-BBB13). RT-PCR analysis revealed that liver X receptor α and β, retinoid X receptor α and β and peroxisome proliferator-activating receptor α and β mRNAs were expressed in the rat brain Capillary Endothelial Cells and TR-BBB Cells. In contrast, pregnane X receptor, farnesoid X receptor and constitutive androstane receptor were not detected. Furthermore, treatment with a liver X receptor agonist increased the ABCA1 mRNA level in TR-BBB13 Cells, while ABCG2 mRNA expression was not affected. Treatment with a rat pregnane X receptor agonist did not affect the ABCB1 mRNA level in TR-BBB13 Cells. These results demonstrate that the rat blood–brain barrier has an expressional regulation mechanism via sterol-related nuclear receptor, and indicate that the blood–brain barrier in 8-week-old rats lacks ABCB1 regulation via pregnane X receptor.
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new approaches to in vitro models of blood brain barrier drug transport
Drug Discovery Today, 2003Co-Authors: Tetsuya Terasaki, Sumio Ohtsuki, Satoko Hori, Hitomi Takanaga, Emi Nakashima, Ken-ichi HosoyaAbstract:The pharmaceutical industry has been searching for an in vitro blood-brain barrier (BBB) model that preserves in vivo transporter functions in CNS drug discovery and development. The application of conditionally immortalized Cell lines derived from transgenic animals harboring temperature-sensitive SV40 large T-antigen gene, is a rational and promising approach to such a workable in vitro BBB model. The established brain Capillary Endothelial Cell lines retain the in vivo transport rate of several compounds and various forms of gene expression. Furthermore, this new approach has enabled the development of stable and reproducible co-culture models with a pericyte Cell line and/or an astrocyte Cell line.
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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
Cell Structure and Function, 2003Co-Authors: Tetsu Kondo, Sumio Ohtsuki, Satoko Hori, Ken-ichi Hosoya, Masatoshi Tomi, Hitomi Takanaga, Emi Nakashima, Hisashi Iizasa, Tomoko Asashima, Masatsugu UedaAbstract: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 beta, angiopoietin-1, and osteopontin. Western blot analysis indicated that alpha-smooth muscle actin (alpha-SMA) was expressed in TR-rPCT3 and 4 Cells. In contrast, alpha-SMA was induced by transforming growth factor-beta1 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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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
Pharmaceutical Research, 2001Co-Authors: Ken-ichi Hosoya, Sumio Ohtsuki, Masatoshi Tomi, Hitomi Takanaga, Tetsu Kondo, Tetsuya TerasakiAbstract: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.
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conditionally immortalized retinal Capillary Endothelial Cell lines tr ibrb expressing differentiated Endothelial Cell functions derived from a transgenic rat
Experimental Eye Research, 2001Co-Authors: Ken-ichi Hosoya, Sumio Ohtsuki, Masatsugu Ueda, Masatoshi Tomi, Hitomi Takanaga, Nobuaki Yanai, Masuo Obinata, Tetsuya TerasakiAbstract: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 degrees C for a period of 48 hr. Cells were subsequently cultured at 33 degrees 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 approximately 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 degrees C with a doubling time of 19-21 hr. In contrast, Cells did not grow at 37 and 39 degrees 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 m M and a maximum uptake rate of 45.3 +/- 2.6 nmol min(-1) mg protein(-1). P-Glycoprotein, with a molecular weight of approximately 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.
Hitomi Takanaga - One of the best experts on this subject based on the ideXlab platform.
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new approaches to in vitro models of blood brain barrier drug transport
Drug Discovery Today, 2003Co-Authors: Tetsuya Terasaki, Sumio Ohtsuki, Satoko Hori, Hitomi Takanaga, Emi Nakashima, Ken-ichi HosoyaAbstract:The pharmaceutical industry has been searching for an in vitro blood-brain barrier (BBB) model that preserves in vivo transporter functions in CNS drug discovery and development. The application of conditionally immortalized Cell lines derived from transgenic animals harboring temperature-sensitive SV40 large T-antigen gene, is a rational and promising approach to such a workable in vitro BBB model. The established brain Capillary Endothelial Cell lines retain the in vivo transport rate of several compounds and various forms of gene expression. Furthermore, this new approach has enabled the development of stable and reproducible co-culture models with a pericyte Cell line and/or an astrocyte Cell line.
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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
Cell Structure and Function, 2003Co-Authors: Tetsu Kondo, Sumio Ohtsuki, Satoko Hori, Ken-ichi Hosoya, Masatoshi Tomi, Hitomi Takanaga, Emi Nakashima, Hisashi Iizasa, Tomoko Asashima, Masatsugu UedaAbstract: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 beta, angiopoietin-1, and osteopontin. Western blot analysis indicated that alpha-smooth muscle actin (alpha-SMA) was expressed in TR-rPCT3 and 4 Cells. In contrast, alpha-SMA was induced by transforming growth factor-beta1 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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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
Pharmaceutical Research, 2001Co-Authors: Ken-ichi Hosoya, Sumio Ohtsuki, Masatoshi Tomi, Hitomi Takanaga, Tetsu Kondo, Tetsuya TerasakiAbstract: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.
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conditionally immortalized retinal Capillary Endothelial Cell lines tr ibrb expressing differentiated Endothelial Cell functions derived from a transgenic rat
Experimental Eye Research, 2001Co-Authors: Ken-ichi Hosoya, Sumio Ohtsuki, Masatsugu Ueda, Masatoshi Tomi, Hitomi Takanaga, Nobuaki Yanai, Masuo Obinata, Tetsuya TerasakiAbstract: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 degrees C for a period of 48 hr. Cells were subsequently cultured at 33 degrees 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 approximately 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 degrees C with a doubling time of 19-21 hr. In contrast, Cells did not grow at 37 and 39 degrees 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 m M and a maximum uptake rate of 45.3 +/- 2.6 nmol min(-1) mg protein(-1). P-Glycoprotein, with a molecular weight of approximately 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.
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Conditionally immortalized retinal Capillary Endothelial Cell lines (TR-iBRB) expressing differentiated Endothelial Cell functions derived from a transgenic rat.
Experimental eye research, 2001Co-Authors: Ken-ichi Hosoya, Sumio Ohtsuki, Masatsugu Ueda, Masatoshi Tomi, Hitomi Takanaga, Nobuaki Yanai, Masuo Obinata, Tetsuya TerasakiAbstract:Abstract 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 m M 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 andmdr 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.
