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Cloning of neutral amino acid transporter B0 from a human placental choriocarcinoma cell line
A.24
Affect of Diabetes in Human Trophoblast and Umbilical Endothehum: Transport and Metabolism of Adenosine (ADO) and L-Arginine (NO Synthesis). ‘L. Sobrevia, ‘G.E. Mann and 3D. Yudilevich. ‘Vascular Biology Research Centre, Physiology, King’s College London, U.K.; ‘Physiology Dept. Fat. Biol. Sci., Univ. Conception, Chile; ‘Dept. Physiol. Biophysics, Fat. Medicine, Univ. Chile, Santiago, Chile. Gestational diabetes may result in fetal embryopathy. We prepared placental brush border membrane vesicles (BBMV) and cultured human umbilical vein endothelial cells (HUVEC). We found that diabetesaltered HUVEC in culture, whereas we did not find changes in BBMV ADO transport or metabolism. In HUVEC diabetes reduced i. cell proliferation, protein synthesis and DNA turnover; ii. ADO transport and metabolism; iii. prostacyclin release. On the contrary, HUVEC showed i. increasedL-Arginine (ARG) transport and nitric oxide (NO) synthesis; ii. hyperpolarization and increased intracelluar calcium. Human insulin stimulated ARG transport and NO synthesis in non-diabetic HUVEC, but insulin reduced the diabeteselevated ARG transport and restore diabetes-impaired protein and DNA synthesis, These results suggest that diabetes induces phenotypic changes in fetal endothelial cells. Supported by FONDECYT (Chile), Wellcome Trust and British Council.
Placenta
Cloning of Neutral Amino Acid Transporter Bo from a Human Placental Choriocarcinoma Cell Line. R. Kekuda P.D. Prasad, Y.J. Fei, V. Torres-Zamorano, -, F.H. Leibach and V. Ganapathy. Dept. of Biochem. & Mol. Biol., Medical College of Georgia, USA. A cDNA library was constructed from the human placental choriocarcinoma cell line JAR using Superscript Plasmid System. The 1.6 kb cDNA comprising the coding region of the amino acid transport system ASC was used as the probe to screen this library. Functional characterization of the positive clones was done using the vaccinia virus expression system in HeLa cells. One of the positive clones, 2.9 kb in size, was found to encode a Na’-dependent amino acid transport system with preference for zwitterionic amino acids, Anionic, cationic and N-methylated amino acids and imino acids are excluded by this system. These characteristicsare identical to those describedfor the amino acid transporter B” . The cDNA codes for a polypeptide containing 541 amino acids with ten putative transmembranedomains. Amino acid sequence homology predicts this transporter to be a member of a superfamily consisting of the glutamate transporters, the neutral amino acid transport system ASCT, and the insulin-activatable neutral/anionic amino acid transporter. Chromosomal assignment studies with somatic cell hybrid analysis and fluorescent in situ hybridization have located this gene to human chromosome 19q13.3.
Tyrosine Phosphorylation-Dependent Regulation of the Amino Acid Transponer Bo in JAR Human Placental Choriocarcinoma Cells. V.Torres-Zamorano, R.Kekuda, F.H.Leibach & V.Ganapathy, Dept. of Biochem. & Mol. Biol., Medical College of Georgia, Augusta, GA, USA.
Direct Evidencefor Functional Coupling between Human Placental Folate Transporter and Folate Receptor in a Heterologous Expression System.P. D. Prasad, Y. J. Fei, F. H. Leibach, and V. Ganapathy. Dept. of Biochemistry & Molecular Biology, Medical College of Georgia, USA.
The human placental choriocarcinoma cell line JAR expresses a highly active Na+-dependent amino acid transport system whose substratespecificity identifies it as System B”. The substrates of the system include small aliphatic amino acids, bulky branched chain amino acids, aromatic amino acids and amidated amino acids. Treatment of the cells with aurintricarboxylic acid (ATA) for 16 h leads to a significant increase (= 25 %) in the activity of this transporter. This increase is effectively blocked by genistein, an inhibitor of tyrosine kinases. The increasein the transporter activity is associatedwith a parallel increase in the steady state levels of B”-specific mRNA. Kinetic analysisreveals that the increasein the activity is due to an increase in the maximal velocity of the transporter. Treatment of the cells with ATA results in enhanced phosphotyrosine content of specific proteins in these cells, indicating that tyrosine phosphorylation is involved in ATA effect. Treatment of the cells with epidermal growth factor, which acts through a receptor associated with tyrosine kinase, for 24 h leads to a similar increase in B” activity which is associated with an increase in Vmax and in B” mRNA levels. It is concluded that tyrosine phosphorylation plays a role in the regulation of the amino acid transporter B”.
Folate efficiently crossesthe placenta from mother to fetus, but the cellular mechanism responsible for this transfer remains unknown. Recent findings from our laboratory implicate a novel transport mechanism called potocytosis in placental folate uptake. This mechanismwas hypothesized to involve functional coordination between folate transporter (FOLT) and folate receptor (FR). The present study provides, for the first time, direct evidence for functional coupling between the human FR and human FOLT using a heterologous expression system. We have functionally expressedthe cloned human FR (cw-isoform) and the FOLT either independently or together and measured the resultant uptake of r3H]-5-methyltetrahydrofolate. This was done using vacciniavirus expression system in MTXR-ZR-75-1 cells which lack functional FR and FOLT. Expression of FR alone led to a 17-fold increase in folate uptake and expression of FOLT alone led to 37-fold increasecompared to control cells. However when the receptor and the transporter were co-expressed, the resultant folate uptake was 114-fold higher, This increasewhich is much greater than the increaseexpected from a simple additive effect provides strong evidence for functional coordination between the FR and FOLT in effective folate uptake in the placenta,
(1996),
Vol. 17
Affect of Diabetes in Human Trophoblast and Umbilical Endothehum: Transport and Metabolism of Adenosine (ADO) and L-Arginine (NO Synthesis). ‘L. Sobrevia, ‘G.E. Mann and 3D. Yudilevich. ‘Vascular Biology Research Centre, Physiology, King’s College London, U.K.; ‘Physiology Dept. Fat. Biol. Sci., Univ. Conception, Chile; ‘Dept. Physiol. Biophysics, Fat. Medicine, Univ. Chile, Santiago, Chile. Gestational diabetes may result in fetal embryopathy. We prepared placental brush border membrane vesicles (BBMV) and cultured human umbilical vein endothelial cells (HUVEC). We found that diabetesaltered HUVEC in culture, whereas we did not find changes in BBMV ADO transport or metabolism. In HUVEC diabetes reduced i. cell proliferation, protein synthesis and DNA turnover; ii. ADO transport and metabolism; iii. prostacyclin release. On the contrary, HUVEC showed i. increasedL-Arginine (ARG) transport and nitric oxide (NO) synthesis; ii. hyperpolarization and increased intracelluar calcium. Human insulin stimulated ARG transport and NO synthesis in non-diabetic HUVEC, but insulin reduced the diabeteselevated ARG transport and restore diabetes-impaired protein and DNA synthesis, These results suggest that diabetes induces phenotypic changes in fetal endothelial cells. Supported by FONDECYT (Chile), Wellcome Trust and British Council.
Placenta
Cloning of Neutral Amino Acid Transporter Bo from a Human Placental Choriocarcinoma Cell Line. R. Kekuda P.D. Prasad, Y.J. Fei, V. Torres-Zamorano, -, F.H. Leibach and V. Ganapathy. Dept. of Biochem. & Mol. Biol., Medical College of Georgia, USA. A cDNA library was constructed from the human placental choriocarcinoma cell line JAR using Superscript Plasmid System. The 1.6 kb cDNA comprising the coding region of the amino acid transport system ASC was used as the probe to screen this library. Functional characterization of the positive clones was done using the vaccinia virus expression system in HeLa cells. One of the positive clones, 2.9 kb in size, was found to encode a Na’-dependent amino acid transport system with preference for zwitterionic amino acids, Anionic, cationic and N-methylated amino acids and imino acids are excluded by this system. These characteristicsare identical to those describedfor the amino acid transporter B” . The cDNA codes for a polypeptide containing 541 amino acids with ten putative transmembranedomains. Amino acid sequence homology predicts this transporter to be a member of a superfamily consisting of the glutamate transporters, the neutral amino acid transport system ASCT, and the insulin-activatable neutral/anionic amino acid transporter. Chromosomal assignment studies with somatic cell hybrid analysis and fluorescent in situ hybridization have located this gene to human chromosome 19q13.3.
Tyrosine Phosphorylation-Dependent Regulation of the Amino Acid Transponer Bo in JAR Human Placental Choriocarcinoma Cells. V.Torres-Zamorano, R.Kekuda, F.H.Leibach & V.Ganapathy, Dept. of Biochem. & Mol. Biol., Medical College of Georgia, Augusta, GA, USA.
Direct Evidencefor Functional Coupling between Human Placental Folate Transporter and Folate Receptor in a Heterologous Expression System.P. D. Prasad, Y. J. Fei, F. H. Leibach, and V. Ganapathy. Dept. of Biochemistry & Molecular Biology, Medical College of Georgia, USA.
The human placental choriocarcinoma cell line JAR expresses a highly active Na+-dependent amino acid transport system whose substratespecificity identifies it as System B”. The substrates of the system include small aliphatic amino acids, bulky branched chain amino acids, aromatic amino acids and amidated amino acids. Treatment of the cells with aurintricarboxylic acid (ATA) for 16 h leads to a significant increase (= 25 %) in the activity of this transporter. This increase is effectively blocked by genistein, an inhibitor of tyrosine kinases. The increasein the transporter activity is associatedwith a parallel increase in the steady state levels of B”-specific mRNA. Kinetic analysisreveals that the increasein the activity is due to an increase in the maximal velocity of the transporter. Treatment of the cells with ATA results in enhanced phosphotyrosine content of specific proteins in these cells, indicating that tyrosine phosphorylation is involved in ATA effect. Treatment of the cells with epidermal growth factor, which acts through a receptor associated with tyrosine kinase, for 24 h leads to a similar increase in B” activity which is associated with an increase in Vmax and in B” mRNA levels. It is concluded that tyrosine phosphorylation plays a role in the regulation of the amino acid transporter B”.
Folate efficiently crossesthe placenta from mother to fetus, but the cellular mechanism responsible for this transfer remains unknown. Recent findings from our laboratory implicate a novel transport mechanism called potocytosis in placental folate uptake. This mechanismwas hypothesized to involve functional coordination between folate transporter (FOLT) and folate receptor (FR). The present study provides, for the first time, direct evidence for functional coupling between the human FR and human FOLT using a heterologous expression system. We have functionally expressedthe cloned human FR (cw-isoform) and the FOLT either independently or together and measured the resultant uptake of r3H]-5-methyltetrahydrofolate. This was done using vacciniavirus expression system in MTXR-ZR-75-1 cells which lack functional FR and FOLT. Expression of FR alone led to a 17-fold increase in folate uptake and expression of FOLT alone led to 37-fold increasecompared to control cells. However when the receptor and the transporter were co-expressed, the resultant folate uptake was 114-fold higher, This increasewhich is much greater than the increaseexpected from a simple additive effect provides strong evidence for functional coordination between the FR and FOLT in effective folate uptake in the placenta,
(1996),
Vol. 17