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F H Leibach

Publications and source records attributed to F H Leibach.

At least 37 records · Page 2Linked to original sources
PubMed2000

Electrogenic nature of rat sodium-dependent multivitamin transport.

We report on the electrogenic nature of the transport process mediated by the rat sodium-dependent multivitamin transporter. In Cos-7 cells, the relationship of Na(+) concentration versus biotin and pantothenate uptake rate was sigmoidal with a Na(+):substrate stoichiometry of 2:1. In Cos-7 cells expressing rat SMVT biotin transport was significantly higher when the membrane was hyperpolarized and considerably reduced when the membrane was depolarized. Similarly, biotin uptake in X. laevis oocytes expressing rat SMVT was inhibited with depolarized oocyte membrane by altering the K(+) permeability across the membrane. It is concluded that the transport of biotin and pantothenate mediated by rat SMVT is electrogenic with a Na(+):substrate coupling ratio of 2:1 and that the transport process is associated with the transfer of one net positive charge across the membrane per transport cycle.

Animals↗
PubMed2000

A novel H(+)-coupled oligopeptide transporter (OPT3) from Caenorhabditis elegans with a predominant function as a H(+) channel and an exclusive expression in neurons.

We have cloned and functionally characterized a novel, neuron-specific, H(+)-coupled oligopeptide transporter (OPT3) from Caenorhabditis elegans that functions predominantly as a H(+) channel. The opt3 gene is approximately 4.4 kilobases long and consists of 13 exons. The cDNA codes for a protein of 701 amino acids with 11 putative transmembrane domains. When expressed in mammalian cells and in Xenopus laevis oocytes, OPT3 cDNA induces H(+)-coupled transport of the dipeptide glycylsarcosine. Electrophysiological studies of the transport function of OPT3 in Xenopus oocytes show that this transporter, although capable of mediating H(+)-coupled peptide transport, functions predominantly as a H(+) channel. The H(+) channel activity of OPT3 is approximately 3-4-fold greater than the H(+)/peptide cotransport activity as determined by measurements of H(+) gradient-induced inward currents in the absence and presence of the dipeptide using the two-microelectrode voltage clamp technique. A downhill influx of H(+) was accompanied by a large intracellular acidification as evidenced from the changes in intracellular pH using an ion-selective microelectrode. The H(+) channel activity exhibits a K(0.5)(H) of 1.0 microM at a membrane potential of -50 mV. At the level of primary structure, OPT3 has moderate homology with OPT1 and OPT2, two other H(+)-coupled oligopeptide transporters previously cloned from C. elegans. Expression studies using the opt3::gfp fusion constructs in transgenic C. elegans demonstrate that opt3 gene is exclusively expressed in neurons. OPT3 may play an important physiological role as a pH balancer in the maintenance of H(+) homeostasis in C. elegans.

Acids↗
PubMed2000

beta-lactam antibiotics as substrates for OCTN2, an organic cation/carnitine transporter.

Therapeutic use of cephaloridine, a beta-lactam antibiotic, in humans is associated with carnitine deficiency. A potential mechanism for the development of carnitine deficiency is competition between cephaloridine and carnitine for the renal reabsorptive process. OCTN2 is an organic cation/carnitine transporter that is responsible for Na(+)-coupled transport of carnitine in the kidney and other tissues. We investigated the interaction of several beta-lactam antibiotics with OCTN2 using human cell lines that express the transporter constitutively as well as using cloned human and rat OCTN2s expressed heterologously in human cell lines. The beta-lactam antibiotics cephaloridine, cefoselis, cefepime, and cefluprenam were found to inhibit OCTN2-mediated carnitine transport. These antibiotics possess a quaternary nitrogen as does carnitine. Several other beta-lactam antibiotics that do not possess this structural feature did not interact with OCTN2. The interaction of cephaloridine with OCTN2 is competitive with respect to carnitine. Interestingly, many of the beta-lactam antibiotics that were not recognized by OCTN2 were good substrates for the H(+)-coupled peptide transporters PEPT1 and PEPT2. In contrast, cephaloridine, cefoselis, cefepime, and cefluprenam, which were recognized by OCTN2, did not interact with PEPT1 and PEPT2. The interaction of cephaloridine with OCTN2 was Na(+)-dependent, whereas the interaction of cefoselis and cefepime with OCTN2 was largely Na(+)-independent. Furthermore, the Na(+)-dependent, OCTN2-mediated cellular uptake of cephaloridine could be demonstrated by direct uptake measurements. These studies show that OCTN2 plays a crucial role in the pharmacokinetics and therapeutic efficacy of certain beta-lactam antibiotics such as cephaloridine and that cephaloridine-induced carnitine deficiency is likely to be due to inhibition of carnitine reabsorption in the kidney.

Acetylcarnitine↗
PubMed2000

Cloning and functional characterization of a Na(+)-independent, broad-specific neutral amino acid transporter from mammalian intestine.

We have isolated a cDNA from a rabbit intestinal cDNA library which, when co-expressed with the heavy chain of the human 4F2 antigen (4F2hc) in mammalian cells, induces system L-like amino acid transport activity. This protein, called LAT2, consists of 535 amino acids and is distinct from LAT1 which also interacts with 4F2hc to induce system L-like amino acid transport activity. LAT2 does not interact with rBAT, a protein with a significant structural similarity to 4F2hc. The 4F2hc/LAT2-mediated transport process differs from the 4F2hc/LAT1-mediated transport in substrate specificity, substrate affinity, tissue distribution, interaction with D-amino acids, and pH-dependence. The 4F2hc/LAT2-associated transport process has a broad specificity towards neutral amino acids with K(t) values in the range of 100-1000 microM, does not interact with D-amino acids to any significant extent, and is stimulated by acidic pH. In contrast, the 4F2hc/LAT1-associated transport process has a narrower specificity towards neutral amino acids, but with comparatively higher affinity (K(t) values in the range of 10-20 microM), interacts with some D-amino acids with high affinity, and is not influenced by pH. LAT2 is expressed primarily in the small intestine and kidney, whereas LAT1 exhibits a much broader tissue distribution.

Amino Acid Sequence↗
PubMed2000

Transport of valganciclovir, a ganciclovir prodrug, via peptide transporters PEPT1 and PEPT2.

In clinical trials, valganciclovir, the valyl ester of ganciclovir, has been shown to enhance the bioavailability of ganciclovir when taken orally by patients with cytomegalovirus infection. We investigated the role of the intestinal peptide transporter PEPT1 in this process by comparing the interaction of ganciclovir and valganciclovir with the transporter in different experimental systems. We also studied the interaction of these two compounds with the renal peptide transporter PEPT2. In cell culture model systems using Caco-2 cells for PEPT1 and SKPT cells for PEPT2, valganciclovir inhibited glycylsarcosine transport mediated by PEPT1 and PEPT2 with K(i) values (inhibition constant) of 1.68+/-0.30 and 0.043+/- 0.005 mM, respectively. The inhibition by valganciclovir was competitive in both cases. Ganciclovir did not interact with either transporter. Similar studies done with cloned PEPT1 and PEPT2 in heterologous expression systems yielded comparable results. The transport of valganciclovir via PEPT1 was investigated directly in PEPT1-expressing Xenopus laevis oocytes with an electrophysiological approach. Valganciclovir, but not ganciclovir, induced inward currents in PEPT1-expressing oocytes. These results demonstrate that the increased bioavailability of valganciclovir is related to its recognition as a substrate by the intestinal peptide transporter PEPT1. This prodrug is also recognized by the renal peptide transporter PEPT2 with high affinity.

Animals↗
PubMed2000

Effects of NO donors and synthase agonists on endothelial cell uptake of L-Arg and superoxide production.

It is commonly believed that the activity of NO synthase (NOS) solely controls NO production from its substrates, L-Arg and O(2). The Michaelis-Menten constant (K(m)) of NOS for L-Arg is in the micromolar range; cellular levels of L-Arg are much higher. However, evidence strongly suggests that cellular supply of L-Arg may become limiting and lead to reduced NO and increased superoxide anion (O(-)(2)*) formation, promoting cardiovascular dysfunction. Uptake of L-Arg into cells occurs primarily (approximately 85%) through the actions of a Na(+)-independent, carrier-mediated transporter (system y(+)). We have examined the effects of NOS agonists (substance P, bradykinin, and ACh) and NO donors (S-nitroso-N-acetyl-penicillamine and dipropylenetriamine NONOate) on transport of L-Arg into bovine aortic endothelial cells (BAEC). Our results demonstrate that NOS agonists increase y(+) transporter activity. A rapidly acting NO donor initially increases L-Arg uptake; however, after longer exposure, L-Arg uptake is suppressed. Exposure of BAEC without L-Arg to substance P and a Ca(2+) ionophore (A-23187) increased O(-)(2)* formation, which was blocked with concurrent presence of L-Arg or the NOS antagonist N(omega)-nitro-L-arginine methyl ester. We conclude that factors including NO itself control y(+) transport function and the production of NO and O(-)(2)*.

Animals↗
PubMed2000

Structure, function, and genomic organization of human Na(+)-dependent high-affinity dicarboxylate transporter.

We have cloned and functionally characterized the human Na(+)-dependent high-affinity dicarboxylate transporter (hNaDC3) from placenta. The hNaDC3 cDNA codes for a protein of 602 amino acids with 12 transmembrane domains. When expressed in mammalian cells, the cloned transporter mediates the transport of succinate in the presence of Na(+) [concentration of substrate necessary for half-maximal transport (K(t)) for succinate = 20+/-1 microM]. Dimethylsuccinate also interacts with hNaDC3. The Na(+)-to-succinate stoichiometry is 3:1 and concentration of Na(+) necessary for half-maximal transport (K(Na(+))(0.5)) is 49+/-1 mM as determined by uptake studies with radiolabeled succinate. When expressed in Xenopus laevis oocytes, hNaDC3 induces Na(+)-dependent inward currents in the presence of succinate and dimethylsuccinate. At a membrane potential of -50 mV, K(Suc)(0.5) is 102+/-20 microM and K(Na(+))(0.5) is 22+/-4 mM as determined by the electrophysiological approach. Simultaneous measurements of succinate-evoked charge transfer and radiolabeled succinate uptake in hNaDC3-expressing oocytes indicate a charge-to-succinate ratio of 1:1 for the transport process, suggesting a Na(+)-to-succinate stoichiometry of 3:1. pH titration of citrate-induced currents shows that hNaDC3 accepts preferentially the divalent anionic form of citrate as a substrate. Li(+) inhibits succinate-induced currents in the presence of Na(+). Functional analysis of rat-human and human-rat NaDC3 chimeric transporters indicates that the catalytic domain of the transporter lies in the carboxy-terminal half of the protein. The human NaDC3 gene is located on chromosome 20q12-13.1, as evidenced by fluorescent in situ hybridization. The gene is >80 kbp long and consists of 13 exons and 12 introns.

Amino Acid Sequence↗
PubMed2000

Structure, function, and regional distribution of the organic cation transporter OCT3 in the kidney.

We examined in this study the expression of the potential-sensitive organic cation transporter OCT3 in the kidney. A functionally active OCT3 was cloned from a mouse kidney cDNA library. The cloned transporter was found to be capable of mediating potential-dependent transport of a variety of organic cations including tetraethylammonium. This function was confirmed in two different heterologous expression systems involving mammalian cells and Xenopus laevis oocytes. We have also isolated the mouse OCT3 gene and deduced its structure and organization. The OCT3 gene consists of 11 exons and 10 introns. In situ hybridization studies in the mouse kidney have shown that OCT3 mRNA is expressed primarily in the cortex. The expression is evident in the proximal and distal convoluted tubules. The expression of OCT3 in human kidney was confirmed by RT-PCR. We have also cloned OCT3 from human placenta and human kidney. Human OCT3 exhibits 86% identity with mouse OCT3 in amino acid sequence. Human OCT3 was found to transport tetraethylammonium and a variety of other organic cations. The transport process was electrogenic. We conclude that OCT3 is expressed in mammalian kidney and that it plays an important role in the renal clearance of cationic drugs.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed2000

Polarized distribution of interleukin-1 receptors and their role in regulation of serotonin transporter in placenta.

We investigated the expression of interleukin-1 (IL-1) receptors and their involvement in the regulation of the serotonin transporter gene expression in human placenta. IL-1beta is an activator of the serotonin transporter gene expression in JAR human placental choriocarcinoma cells as demonstrated by an increase in the steady-state levels of the transporter mRNA and in serotonin transport activity. This activation is blocked by IL-1 receptor antagonist. Genistein also blocks the effect of IL-1beta, indicating involvement of tyrosine phosphorylation in the process. Treatment of JAR cells with IL-1beta activates mitogen-activated protein kinases and nuclear factor-kappaB. The nuclear factor-kappaB that is responsive to IL-1beta in these cells is the p65 homodimer. Northern blot analysis and reverse transcription-polymerase chain reaction revealed that JAR cells and human placenta express type I and type II IL-1 receptors. The binding sites for (125)I-IL-1beta are localized predominantly in the maternal-facing brush border membrane of the syncytiotrophoblast. These results show that IL-1 in the maternal circulation is likely to play a critical role in the regulation of the serotonin transporter gene expression in the placenta.

Carrier Proteins↗
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↗