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Masanori Tachikawa - One of the best experts on this subject based on the ideXlab platform.

  • astrocytic γ aminobutyric acid gaba transporters mediate guanidinoacetate transport in rat brain
    Neurochemistry International, 2018
    Co-Authors: Masanori Tachikawa, Ayane Yashiki, Shinichi Akanuma, Haruka Matsukawa, Masabumi Minami, Kenichi Hosoya
    Abstract:

    Abstract Guanidinoacetate (GAA) is a biosynthetic precursor of creatine, which plays a critical role in homeostasis of high-energy phosphates in the brain, but cerebral accumulation of GAA leads to neurological complications, such as epilepsy and seizures. The purpose of the present study was to clarify the contribution of the γ-aminobutyric acid (GABA) transport systems to GAA transport in astrocytes by means of uptake studies in rat brain slices, primary astrocyte cultures and Chinese hamster ovary (CHO) cells expressing human GABA transporters (GATS). GAA uptake by rat brain slices was Na+- and Cl−-dependent, and GABA-sensitive. The inhibitory effect of GABA, a common substrate of GATS, on GAA uptake by the brain slices was similar to that of β-alanine, a selective substrate of GAT2/Slc6a13, GAT3/Slc6a11, and taurine transporter (TauT)/Slc6a6. Taurine, a high-affinity substrate of TauT/Slc6a6, exhibited a lesser inhibitory effect. In contrast, betaine, a substrate of betaine-GABA transporter 1 (BGT1)/Slc6a12, and creatine, a substrate of creatine transporter (CRT)/Slc6a8, had little inhibitory effect. A similar inhibition profile was observed in primary-cultured astrocytes. CHO cells expressing human GAT2/SLC6A13, GAT3/SLC6A11 and BGT1/SLC6A12 exhibited GAA transport, whereas CHO cells expressing GAT1/SLC6A1 did not. The Michaelis-Menten values in CHO cells expressing GAT2/SLC6A13 and GAT3/SLC6A11 were similar to those in primary-cultured astrocytes. Overall, our results suggest that astrocytic GAT2/Slc6a13 and GAT3/Slc6a11 play major roles in GAA uptake as regulatory mechanisms of GAA in rat brain, while TauT/Slc6a6, BGT1/Slc6a12, and CRT/Slc6a8 make relatively small contributions.

  • γ aminobutyric acid transporter 2 mediates the hepatic uptake of guanidinoacetate the creatine biosynthetic precursor in rats
    PLOS ONE, 2012
    Co-Authors: Masanori Tachikawa, Shinichi Akanuma, Saori Ikeda, Jun Fujinawa, Shirou Hirose, Kenichi Hosoya
    Abstract:

    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 [14C]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 [14C]GAA into the rat femoral vein and portal vein results in the rapid uptake of [14C]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 [14C]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 IC50 value of 8.81 µM. The [14C]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.

Kenichi Hosoya - One of the best experts on this subject based on the ideXlab platform.

  • astrocytic γ aminobutyric acid gaba transporters mediate guanidinoacetate transport in rat brain
    Neurochemistry International, 2018
    Co-Authors: Masanori Tachikawa, Ayane Yashiki, Shinichi Akanuma, Haruka Matsukawa, Masabumi Minami, Kenichi Hosoya
    Abstract:

    Abstract Guanidinoacetate (GAA) is a biosynthetic precursor of creatine, which plays a critical role in homeostasis of high-energy phosphates in the brain, but cerebral accumulation of GAA leads to neurological complications, such as epilepsy and seizures. The purpose of the present study was to clarify the contribution of the γ-aminobutyric acid (GABA) transport systems to GAA transport in astrocytes by means of uptake studies in rat brain slices, primary astrocyte cultures and Chinese hamster ovary (CHO) cells expressing human GABA transporters (GATS). GAA uptake by rat brain slices was Na+- and Cl−-dependent, and GABA-sensitive. The inhibitory effect of GABA, a common substrate of GATS, on GAA uptake by the brain slices was similar to that of β-alanine, a selective substrate of GAT2/Slc6a13, GAT3/Slc6a11, and taurine transporter (TauT)/Slc6a6. Taurine, a high-affinity substrate of TauT/Slc6a6, exhibited a lesser inhibitory effect. In contrast, betaine, a substrate of betaine-GABA transporter 1 (BGT1)/Slc6a12, and creatine, a substrate of creatine transporter (CRT)/Slc6a8, had little inhibitory effect. A similar inhibition profile was observed in primary-cultured astrocytes. CHO cells expressing human GAT2/SLC6A13, GAT3/SLC6A11 and BGT1/SLC6A12 exhibited GAA transport, whereas CHO cells expressing GAT1/SLC6A1 did not. The Michaelis-Menten values in CHO cells expressing GAT2/SLC6A13 and GAT3/SLC6A11 were similar to those in primary-cultured astrocytes. Overall, our results suggest that astrocytic GAT2/Slc6a13 and GAT3/Slc6a11 play major roles in GAA uptake as regulatory mechanisms of GAA in rat brain, while TauT/Slc6a6, BGT1/Slc6a12, and CRT/Slc6a8 make relatively small contributions.

  • γ aminobutyric acid transporter 2 mediates the hepatic uptake of guanidinoacetate the creatine biosynthetic precursor in rats
    PLOS ONE, 2012
    Co-Authors: Masanori Tachikawa, Shinichi Akanuma, Saori Ikeda, Jun Fujinawa, Shirou Hirose, Kenichi Hosoya
    Abstract:

    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 [14C]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 [14C]GAA into the rat femoral vein and portal vein results in the rapid uptake of [14C]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 [14C]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 IC50 value of 8.81 µM. The [14C]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.

Shinichi Akanuma - One of the best experts on this subject based on the ideXlab platform.

  • astrocytic γ aminobutyric acid gaba transporters mediate guanidinoacetate transport in rat brain
    Neurochemistry International, 2018
    Co-Authors: Masanori Tachikawa, Ayane Yashiki, Shinichi Akanuma, Haruka Matsukawa, Masabumi Minami, Kenichi Hosoya
    Abstract:

    Abstract Guanidinoacetate (GAA) is a biosynthetic precursor of creatine, which plays a critical role in homeostasis of high-energy phosphates in the brain, but cerebral accumulation of GAA leads to neurological complications, such as epilepsy and seizures. The purpose of the present study was to clarify the contribution of the γ-aminobutyric acid (GABA) transport systems to GAA transport in astrocytes by means of uptake studies in rat brain slices, primary astrocyte cultures and Chinese hamster ovary (CHO) cells expressing human GABA transporters (GATS). GAA uptake by rat brain slices was Na+- and Cl−-dependent, and GABA-sensitive. The inhibitory effect of GABA, a common substrate of GATS, on GAA uptake by the brain slices was similar to that of β-alanine, a selective substrate of GAT2/Slc6a13, GAT3/Slc6a11, and taurine transporter (TauT)/Slc6a6. Taurine, a high-affinity substrate of TauT/Slc6a6, exhibited a lesser inhibitory effect. In contrast, betaine, a substrate of betaine-GABA transporter 1 (BGT1)/Slc6a12, and creatine, a substrate of creatine transporter (CRT)/Slc6a8, had little inhibitory effect. A similar inhibition profile was observed in primary-cultured astrocytes. CHO cells expressing human GAT2/SLC6A13, GAT3/SLC6A11 and BGT1/SLC6A12 exhibited GAA transport, whereas CHO cells expressing GAT1/SLC6A1 did not. The Michaelis-Menten values in CHO cells expressing GAT2/SLC6A13 and GAT3/SLC6A11 were similar to those in primary-cultured astrocytes. Overall, our results suggest that astrocytic GAT2/Slc6a13 and GAT3/Slc6a11 play major roles in GAA uptake as regulatory mechanisms of GAA in rat brain, while TauT/Slc6a6, BGT1/Slc6a12, and CRT/Slc6a8 make relatively small contributions.

  • γ aminobutyric acid transporter 2 mediates the hepatic uptake of guanidinoacetate the creatine biosynthetic precursor in rats
    PLOS ONE, 2012
    Co-Authors: Masanori Tachikawa, Shinichi Akanuma, Saori Ikeda, Jun Fujinawa, Shirou Hirose, Kenichi Hosoya
    Abstract:

    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 [14C]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 [14C]GAA into the rat femoral vein and portal vein results in the rapid uptake of [14C]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 [14C]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 IC50 value of 8.81 µM. The [14C]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.

Klaus T. Wanner - One of the best experts on this subject based on the ideXlab platform.

  • Synthesis and biological evaluation of 4-hydroxy-4-(4-methoxyphenyl)-substituted proline and pyrrolidin-2-ylacetic acid derivatives as GABA uptake inhibitors.
    Bioorganic & Medicinal Chemistry, 2012
    Co-Authors: Xueqing Zhao, Georg Höfner, Jörg Pabel, Klaus T. Wanner
    Abstract:

    Abstract A series of enantiomerically pure 4-hydroxy-4-(4-methoxyphenyl)-substituted proline and pyrrolidin-2-ylacetic acid derivatives have been synthesized starting from the respective N-protected 4-hydroxy derivatives via oxidation to the corresponding 4-oxo compounds, subsequent addition of organometallic reagents, final hydrolysis and deprotection. The major diastereoisomers obtained by the addition of the Grignard reagents were found to have opposite stereoconfigurations depending on whether cerium trichloride was present or absent as an additive. The final compounds were evaluated for their capability to inhibit the GABA transport proteins GAT1 and GAT3. 4-Hydroxyproline derivatives substituted with a tris(4-methoxyphenyl)methyloxyethyl residue at the nitrogen and a 4-methoxyphenyl group in 4-position showed, with the exception of the (2 R ,4 R )-diastereomer, an improved inhibition at GAT3 compared to the derivatives missing the 4-methoxyphenyl group in 4-position. This may imply that an appropriate lipophilic group at the C-4 position of the proline moiety is beneficial for potent inhibition at GAT3.

  • deletion of the γ aminobutyric acid transporter 2 gat2 and slc6a13 gene in mice leads to changes in liver and brain taurine contents
    Journal of Biological Chemistry, 2012
    Co-Authors: Yun Zhou, Georg Höfner, Klaus T. Wanner, Silvia Holmseth, Bjornar Hassel, Henrik S Huitfeldt, Niels C Danbolt
    Abstract:

    Abstract The GABA transporters (GAT1, GAT2, GAT3, and BGT1) have mostly been discussed in relation to their potential roles in controlling the action of transmitter GABA in the nervous system. We have generated the first mice lacking the GAT2 (slc6a13) gene. Deletion of GAT2 (both mRNA and protein) neither affected growth, fertility, nor life span under nonchallenging rearing conditions. Immunocytochemistry showed that the GAT2 protein was predominantly expressed in the plasma membranes of periportal hepatocytes and in the basolateral membranes of proximal tubules in the renal cortex. This was validated by processing tissue from wild-type and knockout mice in parallel. Deletion of GAT2 reduced liver taurine levels by 50%, without affecting the expression of the taurine transporter TAUT. These results suggest an important role for GAT2 in taurine uptake from portal blood into liver. In support of this notion, GAT2-transfected HEK293 cells transported [3H]taurine. Furthermore, most of the uptake of [3H]GABA by cultured rat hepatocytes was due to GAT2, and this uptake was inhibited by taurine. GAT2 was not detected in brain parenchyma proper, excluding a role in GABA inactivation. It was, however, expressed in the leptomeninges and in a subpopulation of brain blood vessels. Deletion of GAT2 increased brain taurine levels by 20%, suggesting a taurine-exporting role for GAT2 in the brain.

  • Synthesis and biological evaluation of new derivatives of 2-substituted 4-hydroxybutanamides as GABA uptake inhibitors
    European Journal of Medicinal Chemistry, 2010
    Co-Authors: Katarzyna Kulig, Georg Höfner, Klaus T. Wanner, Krzysztof Więckowski, Anna Więckowska, Justyna Gajda, Bartłomiej Pochwat, Barbara Malawska
    Abstract:

    Abstract This study presents the synthesis of novel substituted 4-hydroxybutanamides and their influence on the activity of murine GABA transport proteins GAT1–GAT4. The active compounds, derivatives of N-arylalkyl-2-(4-diphenylmethylpiperazin-1-yl)-4-hydroxybutyramide, are characterized by pIC50 values in range of 3.92–5.06 and by slight subtype-selectivity. Among them N-4-chlorobenzylamide was the most potent GAT inhibitor (mGAT3), while N-benzylamide was the most active in GAT1-binding assay (pKi = 4.96). The results pointed out that benzhydryl and benzylamide moieties are crucial for the activity of this class of compounds as murine GAT inhibitors.

Georg Höfner - One of the best experts on this subject based on the ideXlab platform.

  • Synthesis and biological evaluation of 4-hydroxy-4-(4-methoxyphenyl)-substituted proline and pyrrolidin-2-ylacetic acid derivatives as GABA uptake inhibitors.
    Bioorganic & Medicinal Chemistry, 2012
    Co-Authors: Xueqing Zhao, Georg Höfner, Jörg Pabel, Klaus T. Wanner
    Abstract:

    Abstract A series of enantiomerically pure 4-hydroxy-4-(4-methoxyphenyl)-substituted proline and pyrrolidin-2-ylacetic acid derivatives have been synthesized starting from the respective N-protected 4-hydroxy derivatives via oxidation to the corresponding 4-oxo compounds, subsequent addition of organometallic reagents, final hydrolysis and deprotection. The major diastereoisomers obtained by the addition of the Grignard reagents were found to have opposite stereoconfigurations depending on whether cerium trichloride was present or absent as an additive. The final compounds were evaluated for their capability to inhibit the GABA transport proteins GAT1 and GAT3. 4-Hydroxyproline derivatives substituted with a tris(4-methoxyphenyl)methyloxyethyl residue at the nitrogen and a 4-methoxyphenyl group in 4-position showed, with the exception of the (2 R ,4 R )-diastereomer, an improved inhibition at GAT3 compared to the derivatives missing the 4-methoxyphenyl group in 4-position. This may imply that an appropriate lipophilic group at the C-4 position of the proline moiety is beneficial for potent inhibition at GAT3.

  • deletion of the γ aminobutyric acid transporter 2 gat2 and slc6a13 gene in mice leads to changes in liver and brain taurine contents
    Journal of Biological Chemistry, 2012
    Co-Authors: Yun Zhou, Georg Höfner, Klaus T. Wanner, Silvia Holmseth, Bjornar Hassel, Henrik S Huitfeldt, Niels C Danbolt
    Abstract:

    Abstract The GABA transporters (GAT1, GAT2, GAT3, and BGT1) have mostly been discussed in relation to their potential roles in controlling the action of transmitter GABA in the nervous system. We have generated the first mice lacking the GAT2 (slc6a13) gene. Deletion of GAT2 (both mRNA and protein) neither affected growth, fertility, nor life span under nonchallenging rearing conditions. Immunocytochemistry showed that the GAT2 protein was predominantly expressed in the plasma membranes of periportal hepatocytes and in the basolateral membranes of proximal tubules in the renal cortex. This was validated by processing tissue from wild-type and knockout mice in parallel. Deletion of GAT2 reduced liver taurine levels by 50%, without affecting the expression of the taurine transporter TAUT. These results suggest an important role for GAT2 in taurine uptake from portal blood into liver. In support of this notion, GAT2-transfected HEK293 cells transported [3H]taurine. Furthermore, most of the uptake of [3H]GABA by cultured rat hepatocytes was due to GAT2, and this uptake was inhibited by taurine. GAT2 was not detected in brain parenchyma proper, excluding a role in GABA inactivation. It was, however, expressed in the leptomeninges and in a subpopulation of brain blood vessels. Deletion of GAT2 increased brain taurine levels by 20%, suggesting a taurine-exporting role for GAT2 in the brain.

  • Synthesis and biological evaluation of new derivatives of 2-substituted 4-hydroxybutanamides as GABA uptake inhibitors
    European Journal of Medicinal Chemistry, 2010
    Co-Authors: Katarzyna Kulig, Georg Höfner, Klaus T. Wanner, Krzysztof Więckowski, Anna Więckowska, Justyna Gajda, Bartłomiej Pochwat, Barbara Malawska
    Abstract:

    Abstract This study presents the synthesis of novel substituted 4-hydroxybutanamides and their influence on the activity of murine GABA transport proteins GAT1–GAT4. The active compounds, derivatives of N-arylalkyl-2-(4-diphenylmethylpiperazin-1-yl)-4-hydroxybutyramide, are characterized by pIC50 values in range of 3.92–5.06 and by slight subtype-selectivity. Among them N-4-chlorobenzylamide was the most potent GAT inhibitor (mGAT3), while N-benzylamide was the most active in GAT1-binding assay (pKi = 4.96). The results pointed out that benzhydryl and benzylamide moieties are crucial for the activity of this class of compounds as murine GAT inhibitors.