Papers - Tomi, Masatoshi

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

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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

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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

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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

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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

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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

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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.