Taurine transporter (TauT/SLC6A6) is an "honorary" γ-aminobutyric acid (GABA) transporter because of its low affinity for GABA. The sequence analysis of TauT implied the role of Gly57, Phe58, Leu306 and Glu406 in the substrate recognition of TauT, and amino acid-substitutions were performed. Immunocytochemistry supported no marked effect of mutations on the expression of TauT. TauT H]taurine and [3 H]GABA uptake, suggesting that Gly57 is involved in the determination of substrate pocket volume and in the interaction with substrates. E406C exhibited a decrease and an increase in the affinity for taurine and GABA, respectively, suggesting the involvement of Glu406 in the substrate specificity of TauT. The inhibition study supported the role of Glu406 in the substrate specificity since [ H]taurine and [3 H]GABA uptake by E406C was less sensitive to taurine and β-alanine, and more sensitive to GABA and nipecotic acid than was the case with wild type of TauT. F58I had an increased affinity for GABA, suggesting the involvement of Phe58 in the substrate accessibility. The kinetic parameters showed the decreased and increased affinities of L306Q for taurine and GABA, respectively, supporting that substrate recognition of TauT is conformationally regulated by the branched-side chain of Leu306. In conclusion, the present results suggest that these residues play important roles in the transport function and substrate specificity of TauT.Key words taurine transporter; TauT (SLC6A6); γ-aminobutyric acid (GABA); GABA transporter (GAT); blood-retinal barrier (BRB)In the retina, taurine (2-aminoethanesulfonic acid) play a role in the protection of the retinal neural cells by acting as an osmolyte and antioxidant. [1][2][3][4][5] Recent progress in the research of membrane transporter and the blood-retinal barrier (BRB) has revealed the involvement of taurine transporter (TauT/ SLC6A6) in taurine uptake by the retinal capillary endothelial cells and retinal glial cells (Müller cells) contributing to the regulation of retinal osmolarity.

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We have reported previously that taurine transporter (TauT) mediates γ-aminobutyric acid (GABA) as a substrate in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells). This study investigates how TauT-mediated GABA transport is regulated in TR-iBRB2 cells under hypertonic conditions. [³H]GABA uptake by TR-iBRB2 cells exposed to 12 h- to 24 h-hypertonic culture medium was significantly greater than that of isotonic culture medium. [³H]GABA uptake by TR-iBRB2 cells was Na(+)-, Cl(-)-, and concentration-dependent with a Michaelis-Menten (K(m)) constant of 3.5 mM under isotonic conditions and K(m) of 0.324 and 5.48 mM under hypertonic conditions. Under hypertonic conditions, [³H]GABA uptake by TR-iBRB2 cells was more potently inhibited by substrates of TauT, such as taurine and β-alanine, than those of GABA transporters such as GABA, nipecotic acid, and betaine. These results suggest that an unknown high-affinity GABA transport process and TauT-mediated GABA transport are enhanced under hypertonic conditions. In conclusion, hypertonicity enhances GABA uptake by cultured rat retinal capillary endothelial cells.

Hyperbilirubinemia (HB) is sometimes encountered following bileduct cannulation in rats. It possibly originates from the reduced functioning of multidrug resistance-associated protein 2 (Mrp2) and subsequent adaptive alterations in the expression of Mrp3 and the organic anion transporting polypeptides (Oatps). Our aim was to clarify the importance of excluding bile-duct-cannulated (BDC) rats with HB for proper conduct of drug excretion studies. We detected HB [serum total bilirubin concentration (TBIL) ‡0.20 mg/dl] in 16% of all BDC rats prepared. The serum activities of aspartate aminotransferase, alanine aminotransferase, leucine aminopeptidase, and alkaline phosphatase were within the respective normal ranges in the BDC rats with mild HB (TBIL, 0.20-0.79 mg/dl), indicating the absence of hepatic failure. In the pharmacokinetics of pravastatin, an Oatps/Mrp2 probe drug in the BDC rats, the apparent volume of distribution and the clearance were smaller in the mild HB group as compared with the normal group, suggesting the reduction of apparent hepatic uptake and hepatobiliary elimination. The biliary excretion (percentage of dose) was significantly reduced by 54%, suggesting that the biliary efflux activity via Mrp2 was reduced to a greater extent relative to metabolic activity in hepatocytes. The serum g-glutamyltransferase (GGT) activity correlated with TBIL and inversely correlated with biliary excretion of pravastatin, a finding which could serve as a clue to uncover the regulatory system involving cooperation between GGT and Mrp2. In conclusion, BDC rats with HB, however mild, should be excluded from drug excretion studies to avoid the risk of underestimation of the biliary excretion of drugs.

Uptake and release of reported substrates via organic cation/carnitine transporters (OCT/Ns) was studied in A549 monolayers in an attempt to investigate the role of OCT/Ns in alveolar cell function. The OCT/N probes, ASP+, [14C]‐TEA and [3H]‐acetylcarnitine (Ac‐Car) were used in the experiments. [3H]‐verapamil, generally considered a substrate for P‐gp/MDR1, was also studied. The effect of time, temperature and various pharmacological agents on uptake and release of the compounds was investigated. All compounds exhibited temperature‐sensitive and time‐ and concentration‐dependent uptake and release. The involvement of both non saturable and saturable uptake carriers was observed in the case of the OCT/N probes. Km values were 27.8 μM (ASP+), 744.4 μM (TEA) and 17.7 μM (Ac‐Car), respectively. Uptake was significantly inhibited by MPP+, Decynium 22 and amantadine. Verapamil uptake was found to be saturable, mediated by a single transporter (Km 57.9 μM) and not inhibited by organic cations. The efflux of ASP+ from A549 monolayers was also saturable (Km 32.9 μM). L‐carnitine and amantadine had no significant effect on ASP+ efflux. Our data suggest that OCT/Ns are involved in membrane transport of organic cations in the alveolar epithelium. In addition, OCTs, but not OCTNs contributed to ASP+ efflux from A549 monolayers.