Biomedical subjects

V Ganapathy

Publications and source records attributed to V Ganapathy.

At least 73 records · Page 4Linked to original sources

PubMed2000

Transport of N-acetylaspartate by the Na(+)-dependent high-affinity dicarboxylate transporter NaDC3 and its relevance to the expression of the transporter in the brain.

N-Acetylaspartate is a highly specific marker for neurons and is present at high concentrations in the central nervous system. It is not present at detectable levels anywhere else in the body other than brain. Glial cells express a high-affinity transporter for N-acetylaspartate, but the molecular identity of the transporter has not been established. The transport of N-acetylaspartate into glial cells is obligatory for its intracellular hydrolysis, a process intimately involved in myelination. N-Acetylaspartate is a dicarboxylate structurally related to succinate. We investigated in the present study the ability of NaDC3, a Na(+)-coupled high-affinity dicarboxylate transporter, to transport N-acetylaspartate. The cloned rat and human NaDC3s were found to transport N-acetylaspartate in a Na(+)-coupled manner in two different heterologous expression systems. The Michaelis-Menten constant for N-acetylaspartate was approximately 60 microM for rat NaDC3 and approximately 250 microM for human NaDC3. The transport process was electrogenic and the Na(+):N-acetylaspartate stoichiometry was 3:1. The functional expression of NaDC3 in the brain was demonstrated by in situ hybridization and reverse transcription-polymerase chain reaction as well as by isolation of a full-length functional NaDC3 from a rat brain cDNA library. In addition, the expression of a Na(+)-coupled high-affinity dicarboxylate transporter and the interaction of the transporter with N-acetylaspartate were demonstrable in rat primary astrocyte cultures. These studies establish NaDC3 as the transporter responsible for the Na(+)-coupled transport of N-acetylaspartate in the brain. This transporter is likely to be an essential component in the metabolic role of N-acetylaspartate in the process of myelination.

Animals↗

PubMed1999

Mutations in novel organic cation transporter (OCTN2), an organic cation/carnitine transporter, with differential effects on the organic cation transport function and the carnitine transport function.

Novel organic cation transporter (OCTN2) is an organic cation/carnitine transporter, and two missense mutations, L352R and P478L, in OCTN2 have been identified as the cause for primary carnitine deficiency. In the present study, we assessed the influence of these two mutations on the carnitine transport function and the organic cation transport function of OCTN2. The L352R mutation resulted in a complete loss of both transport functions. In contrast, the P478L mutation resulted in a complete loss of only the carnitine transport function but significantly stimulated the organic cation transport function. Studies with human OCTN2/rat OCTN2 chimeric transporters indicated that the carnitine transport site and the organic cation transport site were not identical. Because carnitine transport is Na(+)-dependent whereas organic cation transport is Na(+)-independent, we investigated the possibility that the P478L mutation affected Na(+) binding. The Na(+) activation kinetics were found to be similar for the P478L mutant and wild type OCTN2. We then mutated nine different tyrosine residues located in or near transmembrane domains and assessed the transport function of these mutants. One of these mutations, Y211F, was found to have differential influence on the two transport activities of OCTN2 as did the P478L mutation. However, the Na(+) activation kinetics were not affected. These findings are of clinical relevance to patients with primary carnitine deficiency because whereas each and every mutation in these patients is expected to result in the loss of the carnitine transport function, all of these mutations may not interfere with the organic cation transport function.

Animals↗

PubMed1999

Human Na(+)-dependent vitamin C transporter 1 (hSVCT1): primary structure, functional characteristics and evidence for a non-functional splice variant.

We report here on the cloning and functional characterization of human Na(+)-dependent vitamin C transporter 1 (SVCT1). The human SVCT1 cDNA, obtained from a Caco2 cell cDNA library, encodes a protein of 598 amino acids with 12 putative transmembrane domains. The SVCT1-specific transcript, 2.4 kb in size, is expressed in kidney, liver, small intestine, thymus and prostate. When expressed heterologously in HRPE cells, SVCT1 mediates the transport of ascorbate, the reduced form of vitamin C, in a Na(+)-dependent manner. The transporter is specific for ascorbate with a K(t) of approximately 75 microM. The relationship between the cDNA-specific uptake rate of ascorbate and Na(+) concentration is sigmoidal with a Na(+):ascorbate stoichiometry of 2:1, indicating that the transport process is electrogenic. In Caco2 cells and in normal human intestine, SVCT1 also exists as a non-functional splice variant with a four amino acid sequence inserted between E-155 and V-156. The splice variant results from the use of a donor site 12 bp downstream of the normal donor site.

Amino Acid Sequence↗

PubMed1999

Cloning of the human thiamine transporter, a member of the folate transporter family.

We have isolated a cDNA from human placenta, which, when expressed heterologously in mammalian cells, mediates the transport of the water-soluble vitamin thiamine. The cDNA codes for a protein of 497 amino acids containing 12 putative transmembrane domains. Northern blot analysis indicates that this transporter is widely expressed in human tissues. When expressed in HeLa cells, the cDNA induces the transport of thiamine (K(t) = 2.5 +/- 0.6 microM) in a Na(+)-independent manner. The cDNA-mediated transport of thiamine is stimulated by an outwardly directed H(+) gradient. Substrate specificity assays indicate that the transporter is specific to thiamine. Even though thiamine is an organic cation, the cDNA-induced thiamine transport is not inhibited by other organic cations. Similarly, thiamine is not a substrate for the known members of mammalian organic cation transporter family. The thiamine transporter gene, located on human chromosome 1q24, consists of 6 exons and is most likely the gene defective in the metabolic disorder, thiamine-responsive megaloblastic anemia. At the level of amino acid sequence, the thiamine transporter is most closely related to the reduced-folate transporter and thus represents the second member of the folate transporter family.

Amino Acid Sequence↗

PubMed1999

Carnitine transport and its inhibition by sulfonylureas in human kidney proximal tubular epithelial cells.

The kidney plays an important role in the homeostasis of carnitine by its ability to reabsorb carnitine almost completely from the glomerular filtrate. The transport process responsible for this reabsorption has been investigated thus far only in laboratory animals. Here we report on the characteristics of carnitine uptake in a proximal tubular epithelial cell line derived from human kidney. The uptake process was found to be obligatorily dependent on Na+ with no involvement of anions. The process was saturable, with a Michaelis-Menten constant of 14 +/- 1 microM. The Na+:carnitine stoichiometry was 1:1. The same process also was found to be responsible for the uptake of acetylcarnitine and propionylcarnitine, two acyl esters of carnitine with potential for therapeutic use in humans. The uptake process was specific for carnitine and its acyl esters. Betaine, a structural analog of carnitine, interacted with the uptake process to a significant extent. The present studies also showed that sulfonylureas, oral hypoglycemic agents currently used in the management of type 2 diabetes, inhibited the carnitine uptake system. Among the sulfonylureas tested, glibenclamide was the most potent inhibitor. The inhibition was competitive. Glibenclamide inhibited the uptake not only of carnitine but also of acetylcarnitine and propionylcarnitine. The inhibition most likely was the result of direct interaction of the compound with the carnitine transporter because the inhibition could be demonstrated in purified rat kidney brush border membrane vesicles.

Administration, Oral↗

PubMed1999

Cloning and expression of a b(0,+)-like amino acid transporter functioning as a heterodimer with 4F2hc instead of rBAT. A new candidate gene for cystinuria.

We have cloned a transporter protein from rabbit small intestine, which, when coexpressed with the 4F2 heavy chain (4F2hc) in mammalian cells, induces a b(0,+)-like amino acid transport activity. This protein (4F2-lc6 for the sixth member of the 4F2 light chain family) consists of 487 amino acids and has 12 putative transmembrane domains. At the level of amino acid sequence, 4F2-lc6 shows significant homology (44% identity) to the other five known members of the 4F2 light chain family, namely LAT1 (4F2-lc1), y(+)LAT1 (4F2-lc2), y(+)LAT2 (4F2-lc3), xCT (4F2-lc4), and LAT2 (4F2-lc5). The 4F2hc/4F2-lc6 complex-mediated transport process is Na(+)-independent and exhibits high affinity for neutral and cationic amino acids and cystine. These characteristics are similar to those of the b(0,+)-like amino acid transport activity previously shown to be associated with rBAT (protein related to b(0,+) amino acid transport system). However, the newly cloned 4F2-lc6 does not interact with rBAT. This is the first report of the existence of a b(0,+)-like amino acid transport process that is independent of rBAT. 4F2-lc6 is expressed predominantly in the small intestine and kidney. Based on the characteristics of the transport process mediated by the 4F2hc/4F2-lc6 complex and the expression pattern of 4F2-lc6 in mammalian tissues, we suggest that 4F2-lc6 is a new candidate gene for cystinuria.

Alanine↗

PubMed1999

Human placental sodium-dependent vitamin C transporter (SVCT2): molecular cloning and transport function.

We report here on the cloning and functional characterization of human SVCT2, a sodium-dependent vitamin C (ascorbate) transporter. The hSVCT2 cDNA obtained from a human placental choriocarcinoma cell cDNA library, codes for a protein of 650 amino acids with a predicted molecular mass of 70 kDa. At the level of amino acid sequence, the human SVCT2 exhibits 95% identity to its rat homolog. When functionally expressed in mammalian cells, hSVCT2 induces the transport of ascorbic acid. The transport process induced by hSVCT2 is Na(+)-dependent and is specific for ascorbate. The Michaelis-Menton constant (K(t)) for the transport of ascorbate in cDNA-transfected cells is 69 +/- 5 microM. The relationship between the cDNA-specific uptake rate of ascorbate and Na(+) concentration is sigmoidal with a Na(+):ascorbate stoichiometry of 2:1. Northern blot analysis shows that SVCT2-specific transcripts are present in heart, brain, placenta, and liver and is absent in lung and skeletal muscle. The size of the principal transcript is approximately 7.5 kb.

Amino Acid Sequence↗

PubMed1999

Interaction of kyotorphin and brain peptide transporter in synaptosomes prepared from rat cerebellum: implication of high affinity type H+/peptide transporter PEPT2 mediated transport system.

High-affinity type H+/peptide cotransporter PEPT2 is preferentially expressed in the kidney, and is responsible for reabsorption of di- and tripeptides in epithelial tubules. Interestingly, PEPT2 has been recently cloned from rat brain. However, there is very little information available on the peptide transporter activity in the brain. In the present study, we investigated the interaction of kyotorphin (L-tyrosyl-L-arginine) with the peptide transporter using synaptosomes prepared from rat cerebellum. The activity of the peptide transporter was assessed by measuring the uptake of radiolabeled glycyl-sarcosine (Gly-Sar), which is a prototypical substrate for the peptide transporter, in the presence of H+-gradient. Kyotorphin competitively inhibited the uptake of Gly-Sar with an inhibitory constant (Ki) of 30 +/- 4 microM in rat cerebellum synaptosomes. This uptake property is very close to that of PEPT2. Carnosine (beta-alanyl-L-histidine) also inhibited the uptake of Gly-Sar, on the other hand, TRH did not interact with the peptide transporter. RT-PCR using specific primers revealed that PEPT2 mRNA exists in cerebellum in rat. Taken collectively, these results indicate that the functional peptide transport system in rat cerebellum might be the high affinity transporter PEPT2.

Animals↗

PubMed1999

sigma Receptor ligand-induced up-regulation of the H(+)/peptide transporter PEPT1 in the human intestinal cell line Caco-2.

We determined the effects of (+)pentazocine, a selective sigma(1) ligand, on the uptake of glycylsarcosine (Gly-Sar) in the human intestinal cell line Caco-2 which expresses the low affinity/high capacity peptide transporter PEPT1. Confluent Caco-2 cells were treated with various concentrations of (+)pentazocine for desired time (mostly 24 hr). The activity of PEPT1 was assessed by measuring the uptake of [(14)C]Gly-Sar in the presence of a H(+) gradient. (+)Pentazocine increased the uptake of [(14)C]Gly-Sar mediated by PEPT1 in a concentration- and time-dependent manner. Kinetic analyses have indicated that (+)pentazocine increased the maximal velocity (V(max)) for Gly-Sar uptake in Caco-2 cells without affecting the Michaelis-Menten constant (K(t)). In addition, semi-quantitative RT-PCR revealed that treatment of (+)pentazocine increased PEPT1 mRNA in Caco-2 cells in a concentration-dependent manner. These data suggest that sigma(1) receptor ligand (+)pentazocine up-regulates PEPT1 in Caco-2 cells at the level of increased mRNA, causing an increase in the density of the transporter protein in the cell membrane.

Base Sequence↗

PubMed1999

Drugs of abuse and placental transport.

The placenta provides the only link between the mother and the developing fetus. The function of the placenta as a transport organ is obligatory for fetal development because this process, mediated by a variety of transport systems, is responsible for the delivery of nutrients from the mother to the fetus. Some of the transport systems in the placenta also play a role in the clearance of vasoactive compounds, thus maintaining optimal blood flow to this organ. There is strong supporting evidence to indicate that several of these placental transport systems are either direct or indirect targets for the abusable drugs cocaine, amphetamines, nicotine, and cannabinoids. These drugs of abuse compromise the placental transport function and consequently produce detrimental effects on the developing fetus.

Journal Article↗

PubMed1999

Molecular and functional characterization of the intestinal Na+-dependent multivitamin transporter.

We have cloned a Na+-dependent multivitamin transporter from rabbit intestine (riSMVT). The cDNA codes for a protein of 636 amino acids with 12 putative transmembrane domains. When expressed in mammalian cells, the cDNA induces Na+-dependent uptake of the vitamins pantothenate and biotin. Lipoate is also a substrate for the cDNA-induced uptake process. The affinity constant for the cDNA-specific transport of pantothenate and biotin is approximately 2 and approximately 8 microM, respectively. The Na+:vitamin stoichiometry is greater than 1, indicating that the transport process is electrogenic. The SMVT-specific transcripts of 3.2 kbp are equally distributed throughout the small intestine. We have also cloned SMVT from the human intestinal cell line Caco-2. The Caco-2 SMVT cDNA codes for a protein of 635 amino acids which is homologous to riSMVT and is identical to the SMVT expressed in the human choriocarcinoma cell line JAR. Caco-2 SMVT also catalyzes Na+-dependent uptake of pantothenate, biotin, and lipoate. In oocytes expressing Caco-2 SMVT, all three vitamins evoke inward currents, confirming the electrogenicity of the transport process.

Amino Acid Sequence↗

PubMed1999

Human placental Na+-dependent multivitamin transporter. Cloning, functional expression, gene structure, and chromosomal localization.

We have cloned the human Na+-dependent multivitamin transporter (SMVT), which transports the water-soluble vitamins pantothenate, biotin, and lipoate, from a placental choriocarcinoma cell line (JAR). The cDNA codes for a protein of 635 amino acids with 12 transmembrane domains and 4 putative sites for N-linked glycosylation. The human SMVT exhibits a high degree of homology (84% identity and 89% similarity) to the rat counterpart. When expressed in HRPE cells, the cDNA-induced transport process is obligatorily dependent on Na+ and accepts pantothenate, biotin, and lipoate as substrates. The relationship between the cDNA-specific uptake rate of pantothenate or biotin and Na+ concentration is sigmoidal with a Na+:vitamin stoichiometry of 2:1. The human SMVT, when expressed in Xenopus laevis oocytes, induces inward currents in the presence of pantothenate, biotin, and lipoate in a Na+-, concentration-, and potential-dependent manner. We also report here on the structural organization and chromosomal localization of the human SMVT gene. The SMVT gene is approximately 14 kilobase pairs in length and consists of 17 exons. The SMVT gene is located on chromosome 2p23 as evidenced by somatic cell hybrid analysis and fluorescence in situ hybridization.

Amino Acid Sequence↗

PubMed1999

Preferential recognition of zwitterionic dipeptides as transportable substrates by the high-affinity peptide transporter PEPT2.

We investigated the interaction of rat PEPT2, a high-affinity peptide transporter, with neutral, anionic, and cationic dipeptides using electrophysiological approaches as well as tracer uptake methods. D-Phe-L-Gln (neutral), D-Phe-L-Glu (anionic), and D-Phe-L-Lys (cationic) were used as representative, non-hydrolyzable, dipeptides. All three dipeptides induced H+-dependent inward currents in Xenopus laevis oocytes heterologously expressing rat PEPT2. The H+:peptide stoichiometry was 1:1 in each case. A simultaneous measurement of radiolabeled dipeptide influx and charge transfer in the same oocyte indicated a transfer of one net positive charge into the oocyte per transfer of one peptide molecule irrespective of the charged nature of the peptide. We conclude that the zwitterionic peptides are preferentially recognized by PEPT2 as transportable substrates and that the proton/peptide stoichiometry is 1 for the transport process.

Animals↗

PubMed1999

Identity of the F52F12.1 gene product in Caenorhabditis elegans as an organic cation transporter.

We describe here the cloning and functional characterization of an organic cation transporter from Caenorhabditis elegans (CeOCT1). The CeOCT1 cDNA is 1826 bp long and codes for a protein of 568 amino acids. The oct1 gene is approximately 3.2 kb in size and consists of 12 exons. The location of this gene corresponds to the F52F12.1 gene locus on chromosome I. The predicted protein contains 12 putative transmembrane domains. It exhibits significant homology to mammalian OCTs. When expressed in mammalian cells, CeOCT1 induces the transport of the prototypical organic cation tetraethylammonium. The Michaelis-Menten constant for this substrate is 80+/-16 microM. The substrate specificity of CeOCT1 is broad. This represents the first report on the cloning and functional characteristics of an organic cation transporter from C. elegans.

Amino Acid Sequence↗

PubMed1999

Regulation of the reduced-folate transporter by nitric oxide in cultured human retinal pigment epithelial cells.

The regulation of the reduced-folate transporter (RFT) by nitric oxide (NO) was analyzed in human retinal pigment epithelial (HRPE) cells. NO inhibited specifically and reversibly the uptake of N5-methyltetrahydrofolate by a cGMP-independent mechanism. The inhibition was associated with a decrease in substrate affinity. The NO-induced inhibition was prevented by antioxidants and NO scavengers. Agents capable of modifying thiol groups in proteins inhibited RFT, indicating that the likely mechanism of NO-induced inhibition is via modification of essential thiol groups in this protein. These studies suggest that NO produced during retinal disease may affect the function of RFT in adjacent RPE cells.

4-Chloro-7-nitrobenzofurazan↗

PubMed1999

Mutations in the organic cation/carnitine transporter OCTN2 in primary carnitine deficiency.

Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation caused by defective carnitine transport. This disease presents early in life with hypoketotic hypoglycemia or later in life with skeletal myopathy or cardiomyopathy. The gene for this condition maps to 5q31.2-32 and OCTN2, an organic cation/carnitine transporter, also maps to the same chromosomal region. Here we test the causative role of OCTN2 in primary carnitine deficiency by searching for mutations in this gene in affected patients. Fibroblasts from patients with primary carnitine deficiency lacked mediated carnitine transport. Transfection of patient's fibroblasts with the OCTN2 cDNA partially restored carnitine transport. Sequencing of the OCTN2 gene revealed different mutations in two unrelated patients. The first patient was homozygous (and both parents heterozygous) for a single base pair substitution converting the codon for Arg-282 to a STOP codon (R282X). The second patient was a compound heterozygote for a paternal 1-bp insertion producing a STOP codon (Y401X) and a maternal 1-bp deletion that produced a frameshift creating a subsequent STOP codon (458X). These mutations decreased the levels of mature OCTN2 mRNA and resulted in nonfunctional transporters, confirming that defects in the organic cation/carnitine transporter OCTN2 are responsible for primary carnitine deficiency.

Amino Acid Sequence↗

PubMed1999

Human LAT1, a subunit of system L amino acid transporter: molecular cloning and transport function.

We report here on the cloning and functional characterization of human LAT1, a subunit of the amino acid transport system L. The hLAT1 cDNA, obtained from a human placental cDNA library, codes for a protein of 507 amino acids. When functionally expressed in mammalian cells together with the heavy chain of the rat 4F2 antigen (r4F2hc), hLAT1 induces the transport of neutral amino acids. When expressed independently, neither hLAT1 nor r4F2hc was capable of amino acid transport to any significant extent. Thus, the hLAT1-r4F2hc heterodimeric complex is responsible for the observed amino acid transport. The transport process induced by the heterodimer is Na+ independent and is not influenced by pH. It recognizes exclusively neutral amino acids with high affinity. LAT1-specific mRNA is expressed in most human tissues with the notable exception of the intestine.

Amino Acid Sequence↗

PubMed1999

Primary structure and functional characteristics of a mammalian sodium-coupled high affinity dicarboxylate transporter.

We have cloned a Na+-dependent, high affinity dicarboxylate transporter (NaDC3) from rat placenta. NaDC3 exhibits 48% identity in amino acid sequence with rat NaDC1, a Na+-dependent, low affinity dicarboxylate transporter. NaDC3-specific mRNA is detectable in kidney, brain, liver, and placenta. When expressed in mammalian cells, NaDC3 mediates Na+-dependent transport of succinate with a Kt of 2 microM. The transport function of NaDC3 shows a sigmoidal relationship with regard to Na+ concentration, with a Hill coefficient of 2.7. NaDC3 accepts a number of dicarboxylates including dimethylsuccinate as substrates and excludes monocarboxylates. Li+ inhibits NaDC3 in the presence of Na+. Transport of succinate by NaDC3 is markedly influenced by pH, the transport function gradually decreasing when pH is acidified from 8. 0 to 5.5. In contrast, the influence of pH on NaDC3-mediated transport of citrate is biphasic in which a pH change from 8.0 to 6. 5 stimulates the transport and any further acidification inhibits the transport. In addition, the potency of citrate to compete with NaDC3-mediated transport of succinate increases 25-fold when pH is changed from 7.5 to 5.5. These data show that NaDC3 interacts preferentially with the divalent anionic species of citrate. This represents the first report on the cloning and functional characterization of a mammalian Na+-dependent, high affinity dicarboxylate transporter.

Amino Acid Sequence↗