Masatoshi Tomi

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.

AbstractPurpose. 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.

Regulation of taurine transport at the blood–brain barrier by tumor necrosis factor-α, taurine and hypertonicity

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.

Expression and regulation of L-cystine transporter, system xc−, in the newly developed rat retinal Müller cell line (TR-MUL)

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 xc−‐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. GLIA 9999:000–000, 2003.

Blood-to-retina transport of creatine via creatine transporter (CRT) at the rat inner blood-retinal barrier

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.

Roles of Inner Blood-Retinal Barrier Organic Anion Transporter 3 in the Vitreous/Retina-to-Blood Efflux Transport of p-Aminohippuric Acid, Benzylpenicillin, and 6-Mercaptopurine

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. Immunohistochemical 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.

Function of taurine transporter (Slc6a6/TauT) as a GABA transporting protein and its relevance to GABA transport in rat retinal capillary endothelial cells.

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.

The role of blood–ocular barrier transporters in retinal drug disposition: an overview

Importance of the field: Delivery of drugs to the retina remains a major challenge which needs to be addressed urgently because retinal disorders are leading causes of visual impairment and significantly affect a patients quality of life. Systemic drug administration is one possible route for treating retinal disorders; however, retinal transfer of drugs from the circulating blood is strictly regulated by two blood–ocular barrier systems, the blood–aqueous barrier and the blood–retinal barrier. Areas covered in this review: This review summarizes the latest biological research regarding blood–ocular barrier drug transporters. What the reader will gain: The blood–ocular barrier sites and their respective roles in aqueous humor dynamics and retinal homeostasis are briefly presented. The potential impact of ATP-binding cassette (ABC) and solute carrier (SLC) drug transporters, such as ABCB, ABCC, ABCG, SLC7, SLC16, SLC19, SLCO/SLC21A, SLC22A and SLC29 transporters, on the permeability of drugs across the blood–ocular barriers is then illustrated. Take home message: As more information becomes available regarding the blood–ocular barrier transporters, we may be able to design simpler and more effective routes for drug delivery to the retina and, consequently, improve the treatment of retinal diseases.

Application of magnetically isolated rat retinal vascular endothelial cells for the determination of transporter gene expression levels at the inner blood–retinal barrier

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.

Evidence for creatine biosynthesis in Müller glia

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.

Involvement of OCTN2 in the transport of acetyl-L-carnitine across the inner blood-retinal barrier.

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-[(3)H]carnitine transport in the retina across the inner BRB in rats. RUI was determined from the ratio of acetyl-L-[(3)H]carnitine and [(14)C]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 (K(in)) per gram retina of acetyl-L-[(3)H]carnitine was found to be 2.31 microL/(minute . g retina). The K(in) of acetyl-L-[(3)H]carnitine was 3.7-fold greater than that of [(3)H]D-mannitol, a nonpermeable paracellular marker. Acetyl-L-[(3)H]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-[(3)H]carnitine and acetyl-L-[(3)H]carnitine uptake by TR-iBRB2 cells was Na(+)- and concentration-dependent, with a K(m) of 29 and 26 microM, 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.