Involvement of γ-aminobutyric acid transporter 2 in the hepatic uptake of taurine in rats.

Taurine is essential for the hepatic synthesis of bile salts and, although taurine is synthesized mainly in pericentral hepatocytes, taurine and taurine-conjugated bile acids are abundant in periportal hepatocytes. One possible explanation for this discrepancy is that the active supply of taurine to hepatocytes from the blood stream is a key regulatory factor. The purpose of the present study is to investigate and identify the transporter responsible for taurine uptake by periportal hepatocytes. An in vivo bolus injection of [(3)H]taurine into the rat portal vein demonstrated that 25% of the injected [(3)H]taurine was taken up by the liver on a single pass. The in vivo uptake was significantly inhibited by GABA, taurine, ß-alanine, and nipecotic acid, a GABA transporter (GAT) inhibitor, each at a concentration of 10 mM. The characteristics of Na(+)- and Cl(-)-dependent [(3)H]taurine uptake by freshly isolated rat hepatocytes were consistent with those of GAT2 (solute carrier SLC6A13). Indeed, the K(m) value of the saturable uptake (594 µM) was close to that of mouse SLC6A13-mediated taurine transport. Although GABA, taurine, and ß-alanine inhibited the [(3)H]taurine uptake by > 50%, each at a concentration of 10 mM, GABA caused a marked inhibition with an IC(50) value of 95 µM. The [(3)H]taurine uptake exhibited a significant reduction when the GAT2 gene was silenced. Immunohistochemical analysis showed that GAT2 was localized on the sinusoidal membrane of the hepatocytes predominantly in the periportal region. These results suggest that GAT2 is responsible for taurine transport from the circulating blood to hepatocytes predominantly in the periportal region.

The blood-brain barrier transport and cerebral distribution of guanidinoacetate in rats: involvement of creatine and taurine transporters.

Although the cerebral accumulation of guanidinoacetate (GAA) contributes to neurological complications in S-adenosylmethionine:guanidinoacetate N-methyltransferase (GAMT) deficiency, how GAA is abnormally distributed in the brain remains unknown. The purpose of this study was to investigate the transport of GAA across the blood-brain barrier (BBB) and in brain parenchymal cells in rats. [(14)C]GAA microinjected into the rat cerebrum was not eliminated from the brain, implying the negligible contribution of GAA efflux transport across the BBB. In contrast, in vivo analysis and an uptake study by TR-BBB cells, a rat in vitro BBB model, revealed that GAA was transported from the circulating blood across the BBB most likely via a creatine transporter (CRT). Although CRT at the BBB is almost saturated by endogenous creatine under physiological conditions, the creatine level in the blood significantly decreases in GAMT deficiency. This might lead to the increase of CRT-mediated blood-to-brain transport of GAA at the BBB. Furthermore, [(14)C]GAA was taken up by brain parenchymal cells in a concentrative manner most likely via taurine transporter and CRT. These characteristics of GAA transport across the BBB and in the brain parenchymal cells could be the key factors that facilitate GAA accumulation in the brains of patients with GAMT deficiency.

The blood-cerebrospinal fluid barrier is a major pathway of cerebral creatinine clearance: involvement of transporter-mediated process.

There is still incomplete evidence for the cerebral clearance of creatinine (CTN) which is an endogenous convulsant and accumulates in the brain and CSF of patients with renal failure. The purpose of this study was to clarify the transporter-mediated CTN efflux transport from the brain/CSF. In vivo data demonstrated that CTN after intracerebral administration was not significantly eliminated from the brain across the blood-brain barrier. In contrast, the elimination clearance of CTN from the CSF was 60-fold greater than that of inulin, reflecting CSF bulk flow. Even in renal failure model rats, the increasing ratio of the CTN concentration in the CSF was lower than that in the plasma, suggesting a significant role for the CSF-to-blood efflux process. The inhibitory effects of inhibitors and antisense oligonucleotides on CTN uptake by isolated choroid plexus indicated the involvement of rat organic cation transporter 3 (rOCT3) and creatine transporter (CRT) in CTN transport. rOCT3- and CRT-mediated low-affinity CTN transport with K(m) values of 47.7 and 52.0 mM, respectively. Our findings suggest that CTN is eliminated from the CSF across the blood-CSF barrier as a major pathway of cerebral CTN clearance and transporter-mediated processes are involved in the CTN transport in the choroid plexus.

Expression and possible role of creatine transporter in the brain and at the blood-cerebrospinal fluid barrier as a transporting protein of guanidinoacetate, an endogenous convulsant.

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 K(m) value of 269 microM/412 microM 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.

γ-Aminobutyric acid transporter 2 mediates the hepatic uptake of guanidinoacetate, the creatine biosynthetic precursor, in rats.

Guanidinoacetic acid (GAA) is the biosynthetic precursor of creatine which is involved in storage and transmission of phosphate-bound energy. Hepatocytes readily convert GAA to creatine, raising the possibility that the active uptake of GAA by hepatocytes is a regulatory factor. The purpose of this study is to investigate and identify the transporter responsible for GAA uptake by hepatocytes. The characteristics of [(14)C]GAA uptake by hepatocytes were elucidated using the in vivo liver uptake method, freshly isolated rat hepatocytes, an expression system of Xenopus laevis oocytes, gene knockdown, and an immunohistochemical technique. In vivo injection of [(14)C]GAA into the rat femoral vein and portal vein results in the rapid uptake of [(14)C]GAA by the liver. The uptake was markedly inhibited by γ-aminobutyric acid (GABA) and nipecotinic acid, an inhibitor of GABA transporters (GATs). The characteristics of Na(+)- and Cl(-)-dependent [(14)C]GAA uptake by freshly isolated rat hepatocytes were consistent with those of GAT2. The Km value of the GAA uptake (134 µM) was close to that of GAT2-mediated GAA transport (78.9 µM). GABA caused a marked inhibition with an IC(50) value of 8.81 µM. The [(14)C]GAA uptake exhibited a significant reduction corresponding to the reduction in GAT2 protein expression. GAT2 was localized on the sinusoidal membrane of the hepatocytes predominantly in the periportal region. This distribution pattern was consistent with that of the creatine biosynthetic enzyme, S-adenosylmethionine:guanidinoacetate N-methyltransferase. GAT2 makes a major contribution to the sinusoidal GAA uptake by periportal hepatocytes, thus regulating creatine biosynthesis in the liver.