Involvement of TauT/SLC6A6 in Taurine Transport at the Blood-Testis Barrier.

Taurine transport was investigated at the blood-testis barrier (BTB) formed by Sertoli cells. An integration plot analysis of mice showed the apparent influx permeability clearance of [3H]taurine (27.7 µL/(min·g testis)), which was much higher than that of a non-permeable paracellular marker, suggesting blood-to-testis transport of taurine, which may involve a facilitative taurine transport system at the BTB. A mouse Sertoli cell line, TM4 cells, showed temperature- and concentration-dependent [3H]taurine uptake with a Km of 13.5 µM, suggesting that the influx transport of taurine at the BTB involves a carrier-mediated process. [3H]Taurine uptake by TM4 cells was significantly reduced by the substrates of taurine transporter (TauT/SLC6A6), such as ß-alanine, hypotaurine, γ-aminobutyric acid (GABA), and guanidinoacetic acid (GAA), with no significant effect shown by L-alanine, probenecid, and L-leucine. In addition, the concentration-dependent inhibition of [3H]taurine uptake revealed an IC50 of 378 µM for GABA. Protein expression of TauT in the testis, seminiferous tubules, and TM4 cells was confirmed by Western blot analysis and immunohistochemistry by means of anti-TauT antibodies, and knockdown of TauT showed significantly decreased [3H]taurine uptake by TM4 cells. These results suggest the involvement of TauT in the transport of taurine at the BTB.

Involvement of sodium-coupled neutral amino acid transporters (system A) in l-proline transport in the rat retinal pericytes.

The retinal pericytes contribute to the supply of collagen to the basement membrane, and thus, form the structural support of the blood-retinal barrier. Since l-proline (L-Pro) is a major component of collagen, the uptake of L-Pro is an important process for the synthesis of collagen. This study was aimed to elucidate L-Pro transport mechanism(s) in the retinal pericytes. The transport of [3H]L-Pro was evaluated in the conditionally immortalized rat retinal pericyte cell line, TR-rPCT1 cells. The expression of the candidate transporter was examined by qualitative/quantitative reverse transcription-polymerase chain reaction, immunoblot analysis, and immunostaining. The [3H]L-Pro uptake by TR-rPCT1 cells showed Na+-dependence, Cl--independence, and concentration-dependence with a Km of 810 µM. The substrates for system A, such as 2-(methylamino)isobutyric acid (MeAIB), significantly inhibited the L-Pro uptake, suggesting the involvement of system A in the uptake of L-Pro. Among the subtypes of system A, the mRNA expression levels of ATA2 were the highest in TR-rPCT1 cells. Immunostaining analysis of the isolated rat retinal capillaries containing pericytes indicated the protein expression of ATA2 in retinal pericytes. In conclusion, it is suggested that ATA2, at least in part, is involved in the transport of L-Pro in the retinal pericytes.

Function and regulation of taurine transport in Müller cells under osmotic stress.

In the retina, taurine works as an osmolyte to exert a neuroprotective function, and it has been proposed that Müller cells, a major type of retinal glial cells, are involved in the osmolarity regulation of retinal neural cells by controlling the taurine concentration in retinal extracellular fluid (ECF). However, the detailed mechanism of taurine transport in Müller cells has not fully examined, and we investigated this using a conditionally immortalized rat Müller cell line (TR-MUL5 cells). In the uptake study, TR-MUL5 cells exhibited Na(+)-, Cl(-)-dependent [(3)H]taurine uptake with a K(m) of 37.9µM. The [(3)H]taurine uptake by TR-MUL5 cells was strongly inhibited by ß-alanine and hypotaurine, substrates of taurine transporter TAUT (SLC6A6), and RT-PCR and immunoblot analyses demonstrated the expression of TAUT in Müller cells, suggesting the involvement of TAUT in taurine uptake by Müller cells. In the efflux study, [(3)H]taurine efflux by TR-MUL5 cells under hypotonic conditions was significantly greater than that under isotonic conditions, and significantly enhanced by sphingosine 1-phosphate (S1P), suggesting that the volume-sensitive taurine release is enhanced via G protein-coupled receptors (GPCRs) in Müller cells. Furthermore, [(3)H]taurine efflux by TR-MUL5 cells under hypotonic conditions was significantly inhibited in the presence of the volume-sensitive organic osmolyte and anion channel (VSOAC) inhibitor, suggesting a major contribution of VSOAC to the volume-sensitive taurine release by Müller cells. This is the first description of the detailed mechanism of taurine transport in Müller cells, indicating a possible function of Müller cells in retinal neuroprotection by regulating osmolarity of retinal ECF.

Inner blood-retinal barrier mediates l-isomer-predominant transport of serine.

D-serine, a coagonist for N-methyl-D-aspartate-type glutamate receptors, which mediate visual signal transmission, is thought to be generated from L-serine via serine racemase in the retina. However, the source of L-serine and D-serine in the retina are yet to be determined. The purpose of the present study was to investigate the characteristics of the blood-to-retina transport of serine at the inner blood-retinal barrier (BRB). In vivo study revealed the blood-to-retina transport of [(3) H]L-serine with an influx clearance of 49.9 µL/(min·g retina), which is greater than that of [(3) H]D-serine. This was consistent with the L-isomer-predominant uptake of serine by conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells), an in vitro inner BRB model. [(3) H]L-Serine and [(3) H]D-serine uptake by TR-iBRB2 cells took place in an Na(+)-dependent and a concentration-dependent manner with Michaelis constant values of 97.5 µM and 9.63 mM, respectively. The uptake process of [(3) H]L-serine and [(3) H]D-serine was significantly inhibited by system ASC (alanine-serine-cysteine) substrates. Polymerase chain reaction analysis and immunocytochemistry revealed the expression of ASC transporters ASCT1 and ASCT2 in TR-iBRB2 cells. These results suggest that the system ASC at the inner BRB is a potent pathway for supplying serine in the form of the L-isomer from the circulating blood to the retina.

Involvement of system A in the retina-to-blood transport of l-proline across the inner blood-retinal barrier.

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 [(3)H]l-proline across the BRB was evaluated by microdialysis. Transport mechanisms of [(3)H]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. [(3)H]l-Proline and [(14)C]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 [(3)H]l-proline during the terminal phase was 1.6-fold greater than that of [(14)C]d-mannitol. The terminal elimination rate constant difference between [(3)H]l-proline and [(14)C]d-mannitol was reduced in the retinal presence of 3 mM l-proline and 5 mM alpha-methylaminoisobutyric acid, suggesting that l-proline is transported via a carrier-mediated retina-to-blood transport process across the BRB. [(3)H]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 muM. 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.

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 [(3)H]p-aminohippuric acid ([(3)H]PAH), [(3)H]benzylpenicillin ([(3)H]PCG), and [(14)C]6-mercaptopurine ([(14)C]6-MP), substrates of rOat3, from the vitreous humor/retina to the circulating blood across the inner BRB was evaluated using the microdialysis method. [(3)H]PAH, [(3)H]PCG, [(14)C]6-MP, and [(14)C] or [(3)H]d-mannitol, a bulk flow marker, were biexponentially eliminated from the vitreous humor after vitreous bolus injection. The elimination rate constant of [(3)H]PAH, [(3)H]PCG, and [(14)C]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.