No abstract

The retina, which is a highly differentiated tissue playing a key role in vision, has a blood-retinal barrier (BRB) to maintain a constant milieu and shield the neural retina from the circulating blood. The BRB forms complex tight junctions of retinal capillary endothelial cells (inner BRB) and retinal pigment epithelial cells (RPE; outer BRB).1,2) The inner two thirds of the human retina is nourished by retinal capillaries and the remainder is covered by choriocapillaris via the outer BRB.3) In addition to the BRB, the blood-aqueous barrier, which is formed by epithelial barriers of the cilliary body and by the iridial endothelial cells, is present in the anterior segment of the eye to maintain aqueous humor conditions. Both barriers form the so-called blood-ocular barrier (Fig. 1). 4)The concept of the BRB was first proposed by Schnaudigel in 1913 5) following the classical work of Ehrlich and Goldman who discovered the blood-brain barrier (BBB). 6,7)The inner BRB is structurally similar to the BBB and the retinal capillary endothelial cells are covered with pericytes and glial cells.2) Glial Müller cells predominantly support retinal endothelial cells, although glial astrocytes play a major role in supporting endothelial functions at the BBB and also, partly, at the inner BRB (Fig. 1). 8) Many groups have carried out detailed investigations of the transport functions at the BBB and Cornford postulated that the BBB acts as a dynamic regulatory interface. [9][10][11] Since then, many influx and efflux transporters have been identified and characterized at the BBB.12,13) It was believed that the transport functions at the inner BRB are the same as those at the BBB. Nevertheless, information about transport functions and transporters at the inner BRB is very limited. Until 1999, only three transporters, i.e. facilitative D-glucose transporter (GLUT)1, 14) monocarboxylate transporter (MCT)1, 15) and Pglycoprotein (P-gp), 16) had been identified immunohistochemically at the inner BRB. This lack of interest in this aspect of vision research is somewhat surprising, given that the inner BRB plays important roles in supplying nutrients to the neural retina and is responsible for the efflux of neurotransmitter metabolites from the retina to maintain neural functions. In vivo transport studies using the Retinal Uptake Index (RUI) method have been performed to investigate solute transport into the retina. [17][18][19] Although these have the advantage of being able to estimate the ability to transport solutes from the circulating blood to the retina under physiological conditions, it is difficult to distinguish between substrates that are taken up by the inner BRB and the outer BRB. In order to successfully identify the transporters and transport mechanisms at the inner BRB, we need to develop a good in vitro system, which accurately reflects in vivo transport functions. The techniques of isolation 20) and primary culture of bovine retinal capillaries 21) have been applied to studies of the inner BRB. However, it is no...

Little is known about the cerebral distribution and clearance of guanidinoacetate (GAA), the accumulation of which induces convulsions. The purpose of the present study was to identify creatine transporter (CRT)‐mediated GAA transport and to clarify its cerebral expression and role in GAA efflux transport at the blood‐cerebrospinal fluid barrier (BCSFB). CRT mediated GAA transport with a Km value of 269 μM/412 μM which was approximately 10‐fold greater than that of CRT for creatine. There was wide and distinct cerebral expression of CRT and localization of CRT on the brush‐border membrane of choroid plexus epithelial cells. The in vivo elimination clearance of GAA from the CSF was 13‐fold greater than that of d‐mannitol reflecting bulk flow of the CSF. This process was partially inhibited by creatine. The characteristics of GAA uptake by isolated choroid plexus and an immortalized rat choroid plexus epithelial cell line (TR‐CSFB cells) used as an in vitro model of BCSFB are partially consistent with those of CRT. These results suggest that CRT plays a role in the cerebral distribution of GAA and GAA uptake by the choroid plexus. However, in the presence of endogenous creatine in the CSF, CRT may make only a limited contribution to the GAA efflux transport at the BCSFB.

Taurine is the abundant sulfur-containing b-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 [3 H]taurine uptake, which was dependent on both Na + and Cl -, and inhibited by b-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-a and under hypertonic conditions showed a 1.7-fold and 3.2-fold increase in [ 3 H]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-a 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. Keywords: blood-brain barrier, immortalized brain capillary endothelial cell line, mRNA expression, osmoregulation, taurine transport, tumor necrosis factor-a. Taurine (2-aminoethanesulfonic acid) is one of the abundant free sulfur-containing b-amino acids in the CNS and is thought to play a role as an osmoregulator (Tuz et al. 2001) and a neuromodulator (Oja and Saransaari 1996). Taurine is also known to exert a neuroprotective effect against excitotoxic agents (French et al. 1986) and oxidative stress (Boldyrev et al. 1999). The taurine level in brain interstitial fluid is elevated in ischemia (Uchiyama-Tsuyuki et al. 1994;Matsumoto et al. 1996;Nakane et al. 1998), indicating that the brain controls its taurine level in response to cell damage for protecting neurons. The release of taurine from neuronal cells could be one of the regulating mechanisms. The release of taurine from hippocampal slices is increased under hypoglycemic and ischemic conditions, and in the presence of 2,4-dinitrophenol or media inducing free radical production by H 2 O 2 (Saransaari and Oja 2000). Another regulatory mechanism could involve the transport system at the blood-brain barrier (BBB). The BBB, which is formed by a complex of tight junctions of brain capillary endothelial cells, possesses a transport system for amino acids including taurine. The blood-to-brain influx Received July 16, 2002; revised manuscript received August 27, 2002; accepted September 11, 2002. Address correspondence and reprint requests to Professor Tetsuya Terasaki, Department of Molecular Biopharmacy and Genetics, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan. E-mail: terasaki@mail.pharm.tohoku.ac.jp Abbreviations used: BBB, blood-brain barrier; DEM, diethyl maleat...

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.

The purpose of the present study was to elucidate the expression and regulation of the L-cystine transporter, system x(c) (-), 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 degrees C with a doubling time of 30 h, but do not grow at 39 degrees 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 [(14)C]L-cystine uptake activity and expressed xCT and 4F2hc, which involve system x(c) (-). The uptake of [(14)C]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.

Five immortalized brain capillary endothelial cell lines (TM-BBB1-5) were established from 3 transgenic mice harboring temperaturesensitive 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 Ȗ-glutamyltranspeptidase activity in

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.