Biomedical subjects

V Ganapathy

Publications and source records attributed to V Ganapathy.

At least 127 records · Page 7Linked to original sources

PubMed1996

The human intestinal H+/oligopeptide cotransporter hPEPT1 transports differently-charged dipeptides with identical electrogenic properties.

The human intestinal H+/oligopeptide cotransporter hPEPT1, expressed in Xenopus oocytes, transported neutral, anionic and cationic dipeptides with identical electrogenic properties and maximal evoked currents. Currents were activated by 1 H+ regardless of the net charge on the driven substrate, and were independent of Na+o, K+i and Clo-, calling into question the familiar concept of the origin of the transporter-mediated current.

Animals↗

PubMed1996

Sodium-dependent carnitine transport in human placental choriocarcinoma cells.

The JAR human placental choriocarcinoma cells were found to transport carnitine into the intracellular space by a Na(+)-dependent process. The transport showed no requirement for anions. The Na+-dependent process was saturable and the apparent Michaelis-Menten constant for carnitine was 12.3 +/- 0.5 microM. Na+ activated the transport by increasing the affinity of the transport system for carnitine. The transport system specifically interacted with L-carnitine, D-carnitine, acetyl-DL-carnitine and betaine. 6-N-Trimethyllysine and choline had little or no effect on carnitine transport. Of the total transport measured, transport into the intracellular space represented 90%. Plasma membrane vesicles prepared from JAR cells were found to bind carnitine in a Na(+)-dependent manner. The binding was saturable with an apparent dissociation constant of 0.66 +/- 0.08 microM. The binding process was specific for L-carnitine, D-carnitine, acetyl-DL-carnitine, and betaine. 6-N-Trimethyllysine and choline showed little or no affinity. It is concluded that the JAR cells express a Na(+)-dependent high-affinity system for carnitine transport and that the Na(+)-dependent high-affinity carnitine binding detected in purified JAR cell plasma membrane vesicles is possibly related to the transmembrane transport process.

Binding Sites↗

PubMed1996

Cloning of the sodium-dependent, broad-scope, neutral amino acid transporter Bo from a human placental choriocarcinoma cell line.

We have isolated a cDNA from a human placental choriocarcinoma cell cDNA library which, when expressed in HeLa cells, induces a Na+-dependent amino acid transport system with preference for zwitterionic amino acids. Anionic amino acids, cationic amino acids, imino acids, and N-methylated amino acids are excluded by this system. These characteristics are identical to those described for the amino acid transporter Bo. When expressed in Xenopus laevis oocytes that do not have detectable endogenous activity of the amino acid transporter Bo, the cloned transporter increases alanine transport in the oocytes severalfold and induces alanine-evoked inward currents in the presence of Na+. The cDNA codes for a polypeptide containing 541 amino acids with 10 putative transmembrane domains. Amino acid sequence homology predicts this transporter (hATBo) to be a member of a superfamily consisting of the glutamate transporters, the neutral amino acid transport system ASCT, and the insulin-activable neutral/anionic amino acid transporter. Chromosomal assignment studies with somatic cell hybrid analysis and fluorescent in situ hybridization have located the ATBo gene to human chromosome 19q13.3.

Alanine↗

PubMed1996

Sodium-dependent high-affinity binding of carnitine to human placental brush border membranes.

The interaction of carnitine with human placental brush-border membrane vesicles was investigated. Carnitine was found to associate with the membrane vesicles in a Na(+)-dependent manner. The time course of this association did not exhibit an overshoot, which is typical of a Na+ gradient-driven transport process. The absolute requirement for Na+ was noticeable whether the association of carnitine with the vesicles was measured with a short time incubation or under equilibrium conditions, indicating Na(+)-dependent binding of carnitine to the human placental brush-border membranes. The binding was saturable and was of a high-affinity type with a dissociation constant of 1.37 +/- 0.03 microM. Anions had little or no influence on the binding process. The binding process was specific for carnitine and its acyl derivatives. Betaine also competed for the binding process, but other structurally related compounds did not. Kinetic analyses revealed that Na+ increased the affinity of the binding process for carnitine and the Na+/carnitine coupling ratio for the binding process was 1. The dissociation constant for the interaction of Na+ with the binding of carnitine was 24 +/- 4 mM. This constitutes the first report on the identification of Na(+)-dependent high-affinity carnitine binding in the plasma membrane of a mammalian cell. Studies with purified rat renal brush-border membrane vesicles demonstrated the presence of Na+ gradient-driven carnitine transport but no Na(+)-dependent carnitine binding in these membrane vesicles. In contrast, purified intestinal brush-border membrane vesicles posses neither Na+ gradient-driven carnitine transport nor Na(+)-dependent carnitine binding.

Anions↗

PubMed1996

Mechanisms of the human intestinal H+-coupled oligopeptide transporter hPEPT1.

The hPEPT1 cDNA cloned from human intestine (Liang, R., Fei, Y.-J., Prasad, P. D., Ramamoorthy, S., Han, H., Yang-Feng, T. L., Hediger, M. A., Ganapathy, V., and Leibach, F. H. (1995) J. Biol. Chem. 270, 6456-6463) encodes a H+/oligopeptide cotransporter. Using two-microelectrode voltage-clamp in Xenopus oocytes expressing hPEPT1, we have investigated the transport mechanisms of hPEPT1 with regard to voltage dependence, steady-state kinetics, and transient charge movements. The currents evoked by 20 mM glycyl-sarcosine (Gly-Sar) at pH 5.0 were dependent upon membrane potential (Vm) between -150 mV and +50 mV. Gly-Sar-evoked currents increased hyperbolically with increasing extracellular [H+], with Hill coefficient approximately 1, and the apparent affinity constant (K0.5H) for H+ was in the range of 0.05 1 microM. K0.5 for Gly-Sar (K0.5GS) was dependent upon Vm and pH; at -50 mV, K0.5H was minimal (approximately 0.7 mM) at pH 6.0. Following step-changes in Vm, in the absence of Gly-Sar, hPEPT1 exhibited H+-dependent transient currents with characteristics similar to those of Na+-coupled transporters. These charge movements (which relaxed with time constants of 2-10 ms) were fitted to Boltzmann relations with maximal charge (Qmax) of up to 12 nC; the apparent valence was determined to be approximately 1. Qmax is an index of the level of transporter expression which for hPEPT1 was in the order of 1011/oocyte. In general our data are consistent with an ordered, simultaneous transport model for hPEPT1 in which H+ binds first.

Animals↗

PubMed1996

Inhibition of the H+/peptide cotransporter in the human intestinal cell line Caco-2 by cyclic AMP.

Treatment of Caco-2 cells with cholera toxin inhibits the activity of the H+/peptide cotransporter. The effect of cholera toxin is mimicked by E. coli heat-labile enterotoxin, forskolin and isobutylmethylxanthine and is associated with an increase in cAMP levels in the cells. The inhibition is due to a decrease in the maximal velocity of the transport system. Inhibitors of protein kinase A and protein kinase C block the effect of cholera toxin. Interestingly, the H+/peptide cotransporter in Caco-2 cells does not possess any putative site for phosphorylation by protein kinase A but does possess sites for phosphorylation by protein kinase C. It appears that the cAMP-dependent inhibition of the H+/peptide cotransporter in Caco-2 cells is mediated through activation of protein kinase C.

Base Sequence↗

PubMed1996

Functional expression of the plasma membrane serotonin transporter but not the vesicular monoamine transporter in human placental trophoblasts and choriocarcinoma cells.

We investigated the functional expression of the plasma membrane serotonin transporter and the vesicular monoamine transporter in choriocarcinoma cells and normal trophoblasts. The RBL 2H3 cells, a rat basophilic leukaemia cell line, which express both transporters were used for comparison. The choriocarcinoma cells JAr and BeWo were found to possess the plasma membrane serotonin transporter as assessed by the presence of serotonin transport activity in intact cells that was Na(+)-dependent and was sensitive to inhibition by tricyclic and non-tricyclic antidepressants. The activity of the vesicular monoamine transporter in these cells was determined by measuring serotonin transport in digitonin-permeabilized cells. The transport in permeabilized cells was very slow, was not stimulated by ATP and was insensitive to inhibition by reserpine. Under similar conditions, the vesicular monoamine transporter activity was demonstrable in RBL cells, which was stimulated by ATP and was inhibitable by reserpine, bafilomycin A1 (an inhibitor of the V-type H(+)-pump) and carbonyl cyanide p-trifluoromethoxy phenylhydrazone (a protonophore which dissipates transmembrane H+ gradients). In corroboration with these findings, mRNA transcripts hybridizable to the vesicular monoamine transporter cDNA probe were detectable in RBL cells but not in JAr choriocarcinoma cells. Similarly, there was no evidence for the expression of the vesicular monoamine transporter as assessed by Northern blot analysis in normal trophoblasts which were maintained in culture to differentiate to form multinucleated syncytial cells. It is concluded that the trophoblasts and choriocarcinoma cells express the plasma membrane serotonin transporter but not the vesicular monoamine transporter.

Adenosine Triphosphate↗

PubMed1996

Peptide transporters.

Proton-coupled peptide transporters found in animal cells are unique because of their energetics and their ability to accept a broad spectrum of chemically diverse peptides as substrates. The abundant expression of these transporters in the small intestine and the kidney indicates their importance in the maintenance of protein nutrition. There is also evidence for additional functions of the peptide transporters which are of physiological, pharmacological and clinical relevance.

Animals↗

PubMed1996

Peptide transporters in the intestine and the kidney.

Even though the existence of a transport process for intact peptides in the brush border membrane of intestinal and renal absorptive epithelial cells has been known for almost three decades, it is only recently that the molecular nature of the proteins responsible for the transport process has been elucidated. Two peptide transporters, PEPT 1 and PEPT 2, have been cloned. The cloned transporters catalyze active transport of intact di- and tripeptides and utilize a transmembrane electrochemical H+ gradient as the driving force. The characteristic of H+ coupling makes PEPT 1 and PEPT 2 unique among the transporters thus far identified in mammalian cells. In addition, the peptide transporters have immediate pharmacologic relevance because a number of peptide-like drugs are recognized as substrates by these transporters. Recently, cultured cell lines of intestinal and renal origin that express PEPT 1 and PEPT 2 have been identified. These cell lines are likely to facilitate studies on the regulatory aspects of the peptide transporters.

Animals↗

PubMed1995

Proton/peptide cotransporter (PEPT 2) from human kidney: functional characterization and chromosomal localization.

We report here on the functional characterization of the H+/peptide cotransporter PEPT 2 cloned from human kidney and on the chromosomal localization of the PEPT 2 gene. PEPT 2, when functionally expressed in HeLa cells, induces the transport of the neutral dipeptide glycylsarcosine. The induced transport activity is markedly influenced by extracellular pH. The optimum pH for the transport process is 6.0-7.0. Kinetic analysis has revealed that PEPT 2 is a high-affinity transporter, the Michaelis-Menten constant for glycylsarcosine being 74 +/- 14 microM. The human intestinal H+/peptide cotransporter PEPT 1 has 4-fold less affinity for the dipeptide under identical experimental conditions. Studies with other chemically diverse dipeptides have established that PEPT 2 possesses higher affinity than PEPT 1 not only for neutral peptides but also for peptides consisting of anionic and/or cationic amino acids. Somatic cell hybrid analysis and in situ hybridization have shown that the gene encoding PEPT 2 maps to human chromosome 3q13.3-q21.

Carrier Proteins↗

PubMed1995

Regulation of the human serotonin transporter by interleukin-1 beta.

The serotonin transporter expressed in brain, platelets, and placenta is a primary target for antidepressants, cocaine, and amphetamines. Here we report that interleukin-1 beta is a potent regulator of the expression of this transporter. The activity of the serotonin transporter in human JAR choriocarcinoma cells is stimulated by this cytokine and the stimulation is accompanied by an increase in the steady state levels of the transporter mRNAs and in the transporter density. The increase in mRNA levels is effectively blocked by actinomycin D. The stimulatory effect of interleukin-1 beta is not associated with any change in the cellular levels of cAMP, indicating involvement of a cAMP-independent pathway. Even though cholera toxin, which is known to increase cAMP levels, also upregulates the serotonin transporter in JAR cells, the effects of interleukin-1 beta and cholera toxin on the transporter activity are additive. This constitutes the first report on the identification of a physiologically occurring hormone/cytokine which upregulates the expression of the human serotonin transporter gene.

Biological Transport↗

PubMed1995

Differential recognition of beta -lactam antibiotics by intestinal and renal peptide transporters, PEPT 1 and PEPT 2.

This study was initiated to determine if there are differences in the recognition of beta -lactam antibiotics as substrates between intestinal and renal peptide transporters, PEPT 1 and PEPT 2. Reverse transcription-coupled polymerase chain reaction and/or Northern blot analysis have established that the human intestinal cell line Caco-2 expresses PEPT 1 but not PEPT 2, whereas the rat proximal tubule cell line SKPT expresses PEPT 2 but not PEPT 1. Detailed kinetic analysis has provided unequivocal evidence for participation of PEPT 2 in SKPT cells in the transport of the dipeptide glycylsarcosine and the aminocephalosporin cephalexin. The substrate recognition pattern of PEPT 1 and PEPT 2 was studied with cefadroxil (a cephalosporin) and cyclacillin (a penicillin) as model substrates for the peptide transporters constitutively expressed in Caco-2 cells (PEPT 1) and SKPT cells (PEPT 2). Cyclacillin was 9-fold more potent than cefadroxil in competing with glycylsacosine for uptake via PEPT 1. In contrast, cefadroxil was 13-fold more potent than cyclacillin in competing with the dipeptide for uptake via PEPT 2. The substrate recognition pattern of PEPT 1 and PEPT 2 was also investigated using cloned human peptide transporters functionally expressed in HeLa cells. Expression of PEPT 1 or PEPT 2 in HeLa cells was found to induce H(+)-coupled cephalexin uptake in these cells. As was the case with Caco-2 cells and SKPT cells, the uptake of glycylsarcosine induced in HeLa cells by PEPT 1 cDNA and PEPT 2 cDNA was inhibitable by cyclacillin and cefadroxil. Again, the PEPT 1 cDNA-induced dipeptide uptake was inhibited more potently by cyclacillin than by cefadroxil, and the PEPT 2 cDNA-induced dipeptide uptake was inhibited more potently by cefadroxil than by cyclacillin. It is concluded that there are marked differences between the intestinal and renal peptide transporters in the recognition of beta -lactam antibiotics as substrates.

Animals↗

PubMed1995

Cyclic AMP-independent up-regulation of the human serotonin transporter by staurosporine in choriocarcinoma cells.

Treatment of confluent cultures of JAR human placental choriocarcinoma cells with staurosporine caused a marked stimulation of serotonin transport activity in these cells. The stimulatory effect was noticeable at nanomolar concentrations of staurosporine, and a treatment time of > 4 h was required for staurosporine to elicit the effect. At 40 nM and with a treatment time of 16 h, the stimulation of the transport activity was 3.5-6.0-fold. None of the several other protein kinase inhibitors tested had similar effect except KT 5720, a protein kinase A inhibitor, which showed a small but significant (approximately 1.4-fold) stimulatory effect at a concentration of 5 microM. Blockade of RNA synthesis and protein synthesis in the cells prevented completely the stimulation of the transport activity induced by staurosporine. The stimulation was observed not only in intact cells but also in plasma membrane vesicles prepared from staurosporine-treated cells. The stimulation was accompanied by a 5-7-fold increase in the steady state levels of the transporter-specific mRNAs, by a 7-fold increase in the maximal velocity of the transport process, and by a 6-fold increase in the transporter density in the plasma membrane. Even though both staurosporine and cholera toxin had similar effects on the serotonin transport activity in these cells, the effect was not additive when the cells were treated with both reagents together. While treatment of the cells with cholera toxin markedly elevated intracellular levels of cAMP, staurosporine did not have any effect on the cellular levels of this cyclic nucleotide. It is concluded that staurosporine up-regulates the serotonin transport activity in JAR cells by increasing the steady state levels of the serotonin transporter mRNA and by the consequent increase in the transporter density in the plasma membrane and that the process involves a cAMP-independent signaling pathway.

Alkaloids↗

PubMed1995

The amino acid transport system y+L induced in Xenopus laevis oocytes by human choriocarcinoma cell (JAR) mRNA is functionally related to the heavy chain of the 4F2 cell surface antigen.

Injection of mRNA isolated from human placental choriocarcinoma cells (JAR) into Xenopus laevis oocytes induced the transport of the neutral amino acid leucine as well as the transport of the cationic amino acid arginine. The induced transport of leucine was predominantly Na(+)-dependent, whereas that of arginine was Na(+)-independent. The ratio of transport activity for these amino acids in mRNA-injected oocytes versus water-injected oocytes was much greater if the transport activity was measured at pH 5.5 instead of at pH 7.5. Leucine transport in mRNA-injected oocytes was inhibited to a marked extent by arginine and lysine. The bicyclic amino acid BCH and the N-methyl amino acid MeAIB had no effect. Arginine transport in mRNA-injected oocytes was insensitive to N-ethylmaleimide and inhibited markedly by micromolar concentrations of leucine in the presence of Na+. The inhibitory potency of leucine was reduced severalfold in the absence of Na+. These results indicated that the arginine transport activity induced in Xenopus oocytes by JAR cell mRNA was due to an amino acid transport system which is identical with or highly similar to y+L and that this system was responsible for a major portion of leucine transport activity measured in these oocytes. Northern blot analysis showed that normal placenta and JAR cells do not possess detectable levels of mRNA transcripts for D2, a protein closely related to the function of another transport system, namely bo.+, which is also involved in the cellular uptake of neutral and cationic amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Transport Systems↗

PubMed1995

Assignment of the human folate transporter gene to chromosome 21q22.3 by somatic cell hybrid analysis and in situ hybridization.

We have used the human folate transporter cDNA and a human genomic clone hybridizing to the cDNA to perform chromosomal mapping of the folate transporter gene. Human-rodent somatic cell hybrid analysis using the cDNA as the probe revealed perfect segregation with human chromosome 21. In situ hybridization of the cDNA probe to human metaphase chromosome spreads mapped the folate transporter gene to the distal long arm of chromosome 21, band q22.3. This chromosomal location was confirmed by fluorescence in situ hybridization using the genomic clone.

Animals↗

PubMed1995

Substrate-specific regulation of the taurine transporter in human placental choriocarcinoma cells (JAR).

Exposure of the JAR human placental choriocarcinoma cells to taurine leads to a marked decrease in the activity of the taurine transporter in these cells. The ability to induce this adaptive response is not unique to taurine but is shared by other substrates of the transporter as well. Compounds such as betaine and alpha-aminoisobutyric acid which are not substrates for the transporter do not produce this effect. The change in the taurine transporter activity induced by taurine exposure is however unique to the taurine transporter because the activities of many other transport systems remain unaffected under these conditions. The adaptive regulation is not associated with any change in the dependence of the transporter activity on Na+ and Cl-, in the Na+/Cl-/taurine stoichiometry and in the affinities of the transporter for Na+ and Cl-. The decrease in the transporter activity caused by taurine exposure is due to a decrease in the maximal velocity of the transporter, and to a lesser extent, in the substrate affinity of the transporter. The decrease in the transporter activity observed in intact cells is demonstrable in plasma membrane vesicles after isolation from control and taurine-exposed cells. Cycloheximide and actinomycin D block the adaptive response in intact cells to a significant extent, but not completely. Northern blot analysis of mRNA from control and taurine-exposed cells shows that taurine exposure causes a significant decrease in the steady state levels of the taurine transporter mRNA. It is concluded that the activity of the taurine transporter in JAR cells is subject to substrate-specific adaptive regulation and that transcriptional as well as posttranscriptional events are involved in this regulatory process.

Carrier Proteins↗

PubMed1995

Molecular cloning of PEPT 2, a new member of the H+/peptide cotransporter family, from human kidney.

Mammalian kidney is known to express a transport system specific for small peptides and pharmacologically active aminocephalosporins. This system is energized by a transmembrane electrochemical H+ gradient. Recently, a H(+)-coupled peptide transporter has been cloned from rabbit and human intestine (Fei et al. (1994) Nature 368, 563-566; Liang et al., J. Biol. Chem., in press). Functional studies have established that the renal peptide transport system is similar but not identical to its intestinal counterpart. Therefore, in an attempt to isolate the renal H+/peptide cotransporter cDNA, we screened a human kidney cDNA library with a probe derived from the rabbit intestinal H+/peptide cotransporter cDNA. This has resulted in the isolation of a positive clone with a 2190 bp long open reading frame. The predicted protein consists of 729 amino acids. Hydropathy analysis of the amino acid sequence indicates the presence of twelve putative transmembrane domains. The primary structure of this protein exhibits 50% identity and 70% similarity to the human intestinal H+/peptide cotransporter. Functional expression of the kidney cDNA in HeLa cells results in the induction of a H(+)-coupled transport system specific for small peptides and aminocephalosporins. Reverse transcription-coupled polymerase chain reaction demonstrates that the cloned transporter is expressed in human kidney but not in human intestine. This transporter, henceforth called PEPT 2, represents a new member in the growing family of H(+)-coupled transport systems in the mammalian plasma membrane.

Amino Acid Sequence↗

PubMed1995

Human intestinal H+/peptide cotransporter. Cloning, functional expression, and chromosomal localization.

In mammalian small intestine, a H(+)-coupled peptide transporter is responsible for the absorption of small peptides arising from digestion of dietary proteins. Recently a cDNA clone encoding a H+/peptide cotransporter has been isolated from a rabbit intestinal cDNA library (Fei, Y.J., Kanai, Y., Nussberger, S., Ganapathy, V., Leibach, F.H., Romero, M.F., Singh, S.K., Boron, W. F., and Hediger, M. A. (1994) Nature 368, 563-566). Screening of a human intestinal cDNA library with a probe derived from the rabbit H+/peptide cotransporter cDNA resulted in the identification of a cDNA which when expressed in HeLa cells or in Xenopus laevis oocytes induced H(+)-dependent peptide transport activity. The predicted protein consists of 708 amino acids with 12 membrane-spanning domains and two putative sites for protein kinase C-dependent phosphorylation. The cDNA-induced transport process accepts dipeptides, tripeptides, and amino beta-lactam antibiotics but not free amino acids as substrates. The human H+/peptide cotransporter exhibits a high degree of homology (81% identity and 92% similarity) to the rabbit H+/peptide cotransporter. But surprisingly these transporters show only a weak homology to the H(+)-coupled peptide transport proteins present in bacteria and yeast. Chromosomal assignment studies with somatic cell hybrid analysis and in situ hybridization have located the gene encoding the cloned human H+/peptide cotransporter to chromosome 13 q33-->q34.

Amino Acid Sequence↗