Details of a Researcher - Tomi, Masatoshi

Papers - Tomi, Masatoshi

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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<sup>+</sup>-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><sup>-</sup>, 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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