Published papers

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 (Km) 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.

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 Km 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).

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.

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.

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.

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.

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.

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.

© 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.

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 Km 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.

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.

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 (H2O2). 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 H2O2-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 H2O2 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.

© 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.

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.

© 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.

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.

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.

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.

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.

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.