Biomedical subjects

P D Prasad

Publications and source records attributed to P D Prasad.

At least 19 recordsLinked to original sources

PubMed2006

Molecular mechanism involved in the transport of a prodrug dopamine glycosyl conjugate.

We have previously demonstrated that dopamine conjugation to glucose allows it to induce therapeutic effects against Parkinson's disease after intravenous administration. In this paper we demonstrate that, unlike dopamine, the prodrug glu-dopamine is a transportable substrate of glucose transporters. Towards this, the effect of glucose-conjugation on the affinity and uptake of dopamine have been assessed in vitro, using human retinal pigment epithelium (HRPE) cells. Glucose transporter-mediated uptake was measured using [(3)H]3-O-methylglucose ([(3)H]3-O-MG) as the tracer. The uptake was found to be rapid and hyperbolically related to its concentrations (K(t)=7.8+/-1.2mM and V(max)=54+/-2 nmol/min mg protein). Inhibition experiments showed that dopamine was able to interact with glucose carriers only when conjugated to glucose (IC(50)=2.6+/-0.6mM). HPLC analysis of HRPE cell extracts showed that both dopamine and the prodrug permeate the cell, but only the uptake of the prodrug is inhibitable by glucose. This confirms that glucose transporters mediate the transport of the prodrug glu-dopamine, but not of dopamine. HRPE cells is therefore proposed as a promising model for in vitro studies involving the glucose transporter-mediated transport of drugs and their conjugates.

3-O-Methylglucose↗

PubMed2005

Functional characteristics of NaS2, a placenta-specific Na+-coupled transporter for sulfate and oxyanions of the micronutrients selenium and chromium.

NaS2 is a Na+-coupled transporter for sulfate that belongs to the SLC13 gene family. This transporter was originally cloned from high endothelial venule endothelial cells, but nothing is known about the functional characteristics of this transporter except that it transports sulfate in a Na+-coupled manner. Northern blot analysis indicates that NaS2 is expressed most robustly in placenta. In the present study, we cloned NaS2 from rat placenta and characterized its transport function in detail using the Xenopus laevis oocyte expression system. Rat NaS2 consists of 629 amino acids and is highly similar to human NaS2. In situ hybridization studies with mouse placental sections show that NaS2 transcripts are expressed primarily in trophoblasts of the labyrinth zone. The expression of the transporter is confirmed in primary cultures of trophoblasts isolated from human placenta. When expressed in X. laevis oocytes, rat NaS2 mediates Na+-coupled transport of sulfate. The transport of sulfate is inhibited by oxyanions of selenium, chromium, arsenic, molybdenum, and phosphorous, suggesting that the transporter may mediate the transport of these oxyanions in addition to sulfate. The Kt for sulfate is 153+/-30 microM and the Na+:sulfate stoichiometry is 3:1. The transport process is electrogenic as evidenced from the inhibition of the uptake process by K+-induced depolarization. We conclude that NaS2 is a placenta-specific Na+-coupled, electrogenic, transporter for sulfate expressed in trophoblasts and that it is also responsible for the transport of oxyanions of the micronutrients selenium and chromium.

Amino Acid Sequence↗

PubMed2005

Biological functions of SLC5A8, a candidate tumour suppressor.

SLC5A8 is a candidate tumour suppressor gene that is silenced in colon cancer, gastric cancer and possibly other cancers in humans. This gene codes for a transporter belonging to the Na(+)/glucose co-transporter gene family (SLC5). The cancer-associated silencing of the gene involves hypermethylation of CpG islands present in exon 1 of the gene. SLC5A8 is expressed in colon, ileum, kidney and thyroid gland. The protein coded by the gene mediates the Na(+)-coupled and electrogenic transport of a variety of monocarboxylates, including short-chain fatty acids, lactate and nicotinate. It may also transport iodide. The normal physiological function of this transporter in the intestinal tract and kidney is likely to facilitate the active absorption of short-chain fatty acids, lactate and nicotinate. One of the short-chain fatty acids that serves as a substrate for SLC5A8 is butyrate. This fatty acid is an inhibitor of histone deacetylases and is known to induce apoptosis in a variety of tumours including colonic tumour. Since butyrate is produced in the colonic lumen at high concentrations by bacterial fermentation of dietary fibre, we speculate that the ability of SLC5A8 to mediate the entry of this short-chain fatty acid into colonic epithelial cells underlies the potential tumour suppressor function of this transporter.

Cation Transport Proteins↗

PubMed2001

Transport of choline and its relationship to the expression of the organic cation transporters in a rat brain microvessel endothelial cell line (RBE4).

The present study was undertaken to elucidate the functional characteristics of choline uptake and deduce the relationship between choline uptake and the expression of organic cation transporters in the rat brain microvessel endothelial cell line RBE4. Confluent RBE4 cells were found to express a high affinity choline uptake system. The system is Na(+)-independent and shows a Michaelis-Menten constant of approx. 20 microM for choline. The choline analogue hemicholinium-3 inhibits choline uptake in these cells with an inhibition constant of approx. 50 microM. The uptake system is also susceptible for inhibition by various organic cations, including 1-methyl-4-phenylpyridinium, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, clonidine, procainamide, and tetramethylammonium. The prototypical organic cation tetraethylammonium shows very little affinity for the choline uptake system in these cells. The inhibition of choline uptake by hemicholinium-3 is competitive. Northern analysis and RT-PCR show that these cells do not express the organic cation transporters OCT2 and OCT3. These cells do express, however, low levels of OCT1, but the functional characteristics of choline uptake in these cells are very different from the known properties of choline uptake via OCT1. The Na(+)-coupled high affinity choline transporter CHT1 is not expressed in these cells as evidenced by RT-PCR. This corroborates the Na(+)-independent nature of choline uptake in these cells. It is concluded that RBE4 cells express an organic cation transporter that is responsible for choline uptake in these cells and that this transporter is not identical to any of the organic cation transporters thus far identified at the molecular level in mammalian cells.

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

PubMed2001

Involvement of transporter recruitment as well as gene expression in the substrate-induced adaptive regulation of amino acid transport system A.

We investigated the molecular mechanism involved in the adaptive regulation of the amino acid transport system A, a process in which amino acid starvation induces the transport activity. These studies were done with rat C6 glioma cells. System A activity in these cells is mediated exclusively by the system A subtype, amino acid transporter A2 (ATA2). The other two known system A subtypes, ATA1 and ATA3, are not expressed in these cells. Exposure of these cells to an amino acid-free medium induces system A activity. This process consists of an acute phase and a chronic phase. Laser-scanning confocal microscopic immunolocalization of ATA2 reveals that the acute phase is associated with recruitment of preformed ATA2 from an intracellular pool to the plasma membrane. In contrast, the chronic phase is associated with an induction of ata2 gene expression as evidenced from the increase in the steady-state levels of ATA2 mRNA, restoration of the intracellular pool of ATA2 protein, and blockade of the induction by cycloheximide and actinomycin D. The increase in system A activity induced by amino acid starvation is blocked specifically by system A substrates, including the non-metabolizable alpha-(methylamino)isobutyric acid.

Adaptation, Physiological↗

PubMed2001

Na+- and Cl--coupled active transport of carnitine by the amino acid transporter ATB(0,+) from mouse colon expressed in HRPE cells and Xenopus oocytes.

1. ATB(0,+) is an amino acid transporter energized by transmembrane gradients of Na+ and Cl(-) and membrane potential. We cloned this transporter from mouse colon and expressed the clone functionally in mammalian (human retinal pigment epithelial, HRPE) cells and Xenopus laevis oocytes to investigate the interaction of carnitine and its acyl esters with the transporter. 2. When expressed in mammalian cells, the cloned ATB(0,+) was able to transport carnitine, propionylcarnitine and acetylcarnitine. The transport process was Na(+) and Cl(-) dependent and inhibitable by the amino acid substrates of the transporter. The Michaelis constant for carnitine was 0.83 +/- 0.08 mM and the Hill coefficient for Na(+) activation was 1.6 +/- 0.1. 3. When expressed in Xenopus laevis oocytes, the cloned ATB(0,+) was able to induce inward currents in the presence of carnitine and propionylcarnitine under voltage-clamped conditions. There was no detectable current in the presence of acetylcarnitine. Carnitine-induced currents were obligatorily dependent on the presence of Na(+) and Cl(-). The currents were saturable with carnitine and the Michaelis constant was 1.8 +/- 0.4 mM. The analysis of Na(+)- and Cl(-)-activation kinetics revealed that 2 Na(+) and 1 Cl(-) were involved in the transport of carnitine via the transporter. 4. These studies describe the identification of a novel function for the amino acid transporter ATB(0,+). Since this transporter is expressed in the intestinal tract, lung and mammary gland, it is likely to play a significant role in the handling of carnitine in these tissues. 5. A Na(+)-dependent transport system for carnitine has already been described. This transporter, known as OCTN2 (novel organic cation transporter 2), is expressed in most tissues and transports carnitine with high affinity. It is energized, however, only by a Na(+) gradient and membrane potential. In contrast, ATB(0,+) is a low-affinity transporter for carnitine, but exhibits much higher concentrative capacity than OCTN2 because of its energization by transmembrane gradients of Na(+) and Cl(-) as well as by membrane potential.

Amino Acid Sequence↗

PubMed2001

Evidence for the transport of neutral as well as cationic amino acids by ATA3, a novel and liver-specific subtype of amino acid transport system A.

We report here on the cloning and functional characterization of the third subtype of amino acid transport system A, designated ATA3 (amino acid transporter A3), from a human liver cell line. This transporter consists of 547 amino acids and is structurally related to the members of the glutamine transporter family. The human ATA3 (hATA3) exhibits 88% identity in amino acid sequence with rat ATA3. The gene coding for hATA3 contains 16 exons and is located on human chromosome 12q13. It is expressed almost exclusively in the liver. hATA3 mediates the transport of neutral amino acids including alpha-(methylamino)isobutyric acid (MeAIB), the model substrate for system A, in a Na(+)-coupled manner and the transport of cationic amino acids in a Na(+)-independent manner. The affinity of hATA3 for cationic amino acids is higher than for neutral amino acids. The transport function of hATA3 is thus similar to that of system y(+)L. The ability of hATA3 to transport cationic amino acids with high affinity is unique among the members of the glutamine transporter family. hATA1 and hATA2, the other two known members of the system A subfamily, show little affinity toward cationic amino acids. hATA3 also differs from hATA1 and hATA2 in exhibiting low affinity for MeAIB. Since liver does not express any of the previously known high-affinity cationic amino acid transporters, ATA3 is likely to provide the major route for the uptake of arginine in this tissue.

Amino Acid Transport Systems↗

PubMed2001

Structure, function, and tissue expression pattern of human SN2, a subtype of the amino acid transport system N.

We have cloned a new subtype of the amino acid transport system N from a human liver cell line. This transporter, designated SN2, consists of 472 amino acids and exhibits 62% identity with human SN1 at the level of amino acid sequence. SN2-specific transcripts are expressed predominantly in the stomach, brain, liver, lung, and intestinal tract. The sizes of the transcripts vary in different tissues, indicating tissue-specific alternative splicing of the SN2 mRNA. In contrast, SN1 is expressed primarily in the brain and liver and there is no evidence for the presence of multiple transcripts of varying size for SN1. When expressed in mammalian cells, the cloned human SN2 mediates Na(+)-coupled transport of system N-specific amino acid substrates (glutamine, asparagine, and histidine). In addition, SN2 also transports serine, alanine, and glycine. Anionic amino acids, cationic amino acids, imino acids, and N-alkylated amino acids are not recognized as substrates by human SN2. The SN2-mediated transport process is Li(+)-tolerant and highly pH-dependent. The Michaelis-Menten constant for histidine uptake via human SN2 is 0.6 +/- 0.1 mM. The gene coding for SN2 is located on human chromosome Xp11.23. Successful cloning of SN2 provides the first molecular evidence for the existence of subtypes within the amino acid transport system N in mammalian tissues.

Amino Acid Sequence↗

PubMed2001

Vitamin C transport in human lens epithelial cells: evidence for the presence of SVCT2.

Vitamin C [ascorbic acid (AA)] is an important antioxidant present in m M amounts in the aqueous humor. Recently, two specific transporters for vitamin C (SVCT1, SVCT2) have been cloned in the rat and the human. The aim of the present study was to characterize vitamin C transport in an immortalized human lens epithelial cell line (HLE-B3). AA uptake was linear for 120 min in experiments conducted with 14C AA + 40 microM unlabelled AA. Uptake was measured at varying AA concentrations (0.04-1 m M) in Na+-containing and Na+-free buffers for 30 min at 37 degrees C. Effect of potential inhibitors of AA transport was also examined. Presence (or absence) of SVCT1 and SVCT2 was studied by RT-PCR of HLE-B3 poly (A)+ RNA using gene specific primers. Uptake studies revealed that AA uptake was highly Na+-dependent and exhibited saturation. Na+-dependent 14C-AA uptake was strongly inhibited (85-90%) by 10 m M unlabelled AA. Incubation of HLE-B3 cells with cAMP (0.1 m M), cytocholasin B (0.1 m M) and phorbol dibutyrate (1 microM) resulted in partial inhibition (36-51%) of AA uptake. Under similar conditions, D -glucose (10 m M) and staurosporine (0.1 microM) had no effect. RT-PCR showed the presence of SVCT2 while SVCT1 could not be amplified. Exposure to the chemical oxidant tert-butylhydroperoxide (TBH) up-regulated SVCT2 gene expression in HLE-B3 cells. Our data suggest that Na+-dependent transport of AA in normal lens epithelium is most likely mediated by SVCT2 rather than by SVCT1. This transport system may be subject to regulation by oxidant stress and by various second messenger signals.

Antioxidants↗

PubMed2001

Targeting the sodium-dependent multivitamin transporter (SMVT) for improving the oral absorption properties of a retro-inverso Tat nonapeptide.

PURPOSE: To investigate the potential for delivering large peptides orally by altering their absorptive transport pathways and improving intestinal permeability. The absorptive transport of retro-inverso (R.I.-) K-Tat9 and R.I.-K(biotin)-Tat9, novel peptidic inhibitors of the Tat protein of HIV-1, and their interactions with human SMVT (hSMVT), a high affinity, low capacity transporter, were investigated using Caco-2 and transfected CHO cells. METHODS: Following synthesis on a PAL resin using Fmoc chemistry, the transport of R.I.-K-Tat9 (0.01-25 microM) and R.I.-K(biotin)-Tat9 (0.1-25 microM) was evaluated across Caco-2 cells. The transport and kinetics of biotin, biocytin and desthiobiotin (positive controls for SMVT) were also determined. Uptake of R.I.-K-Tat9 and R.I.K(biotin)-Tat9 (both 0.1-10 microM) was determined in CHO/hSMVT and CHO/pSPORT (control) cells. RESULTS: The absorptive transport of R.I.-K-Tat9 was passive, low (Pm approximately 1 x 10(-6) cm/sec) and not concentration dependent. R.I.K(biotin)-Tat9 permeability was 3.2-fold higher than R.I.-K-Tat9 demonstrating active (Ea = 9.1 kcal/mole), concentration dependent and saturable transport (Km = 3.3 microM). R.I.-K(biotin)-Tat9 uptake in CHO/hSMVT cells (Km = 1.0 microM) was - 500-fold greater than R.I.-K-Tat9 (at 10 microM). R.I.-K(biotin)-Tat9 transport in Caco-2 and CHO/hSMVT cells was significantly inhibited by known substrates of SMVT including biotin, biocytin, and desthiobiotin. Passive uptake of R.I.-K(biotin)-Tat9 was significantly greater than R.I.-K-Tat9 uptake in CHO/pSPORT cells. CONCLUSIONS: These results demonstrate that the structural modification of R.I.-K-Tat9 to R.I.-K(biotin)-Tat9 altered its intestinal transport pathway resulting in a significant improvement in its absorptive permeability by enhancing nonspecific passive and carrier-mediated uptake by means of SMVT. The specific interactions between R.I.-K(biotin)-Tat9 and SMVT suggest that targeting approaches utilizing transporters such as SMVT may substantially improve the oral delivery of large peptides.

Administration, Oral↗

PubMed2001

Cloning and functional characterization of a new subtype of the amino acid transport system N.

We have cloned a new subtype of the amino acid transport system N2 (SN2 or second subtype of system N) from rat brain. Rat SN2 consists of 471 amino acids and belongs to the recently identified glutamine transporter gene family that consists of system N and system A. Rat SN2 exhibits 63% identity with rat SN1. It also shows considerable sequence identity (50-56%) with the members of the amino acid transporter A subfamily. In the rat, SN2 mRNA is most abundant in the liver but is detectable in the brain, lung, stomach, kidney, testis, and spleen. When expressed in Xenopus laevis oocytes and in mammalian cells, rat SN2 mediates Na(+)-dependent transport of several neutral amino acids, including glycine, asparagine, alanine, serine, glutamine, and histidine. The transport process is electrogenic, Li(+) tolerant, and pH sensitive. The transport mechanism involves the influx of Na(+) and amino acids coupled to the efflux of H(+), resulting in intracellular alkalization. Proline, alpha-(methylamino)isobutyric acid, and anionic and cationic amino acids are not recognized by rat SN2.

Amino Acid Sequence↗

PubMed2001

Regulation of taurine transporter expression by NO in cultured human retinal pigment epithelial cells.

Taurine is actively transported at the retinal pigment epithelial (RPE) apical membrane in an Na(+)- and Cl(-)-dependent manner. Diabetes may alter the function of the taurine transporter. Because nitric oxide (NO) is a molecule implicated in the pathogenesis of diabetes, we asked whether NO would alter the activity of the taurine transporter in cultured ARPE-19 cells. The activity of the transporter was stimulated in the presence of the NO donor 3-morpholinosydnonimine. The stimulatory effects of 3-morpholinosydnonimine were not observed during the initial 16-h treatment; however, stimulation of taurine uptake was elevated dramatically above control values with 20- and 24-h treatments. Kinetic analysis revealed that the stimulation was associated with an increase in the maximal velocity of the transporter with no significant change in the substrate affinity. The NO-induced increase in taurine uptake was inhibited by actinomycin D and cycloheximide. RT-PCR analysis and nuclear run-on assays provided evidence for upregulation of the transporter gene. This study provides the first evidence of an increase in taurine transporter gene expression in human RPE cells cultured under conditions of elevated levels of NO.

Animals↗

PubMed2001

Na+ - and Cl- -coupled active transport of nitric oxide synthase inhibitors via amino acid transport system B(0,+).

Nitric oxide synthase (NOS) inhibitors have therapeutic potential in the management of numerous conditions in which NO overproduction plays a critical role. Identification of transport systems in the intestine that can mediate the uptake of NOS inhibitors is important to assess the oral bioavailability and therapeutic efficacy of these potential drugs. Here, we have cloned the Na+ - and Cl- -coupled amino acid transport system B(0,+) (ATB(0,+)) from the mouse colon and investigated its ability to transport NOS inhibitors. When expressed in mammalian cells, ATB(0,+) can transport a variety of zwitterionic and cationic amino acids in a Na+ - and Cl- -coupled manner. Each of the NOS inhibitors tested compete with glycine for uptake through this transport system. Furthermore, using a tritiated analog of the NOS inhibitor N(G)-nitro-L-arginine, we showed that Na+ - and Cl- -coupled transport occurs via ATB(0,+). We then studied transport of a wide variety of NOS inhibitors in Xenopus laevis oocytes expressing the cloned ATB(0,+) and found that ATB(0,+) can transport a broad range of zwitterionic or cationic NOS inhibitors. These data represent the first identification of an ion gradient-driven transport system for NOS inhibitors in the intestinal tract.

Amino Acid Transport System ASC↗

PubMed2001

Structure, function, and regulation of human cystine/glutamate transporter in retinal pigment epithelial cells.

PURPOSE: The purpose of this investigation was to provide evidence for the expression of the cystine/glutamate transporter (x(c)(-)) in the human retinal pigment epithelial cell line ARPE-19, clone the light chain of the transporter from an ARPE-19 cell cDNA library and study its function, and investigate the regulation of this transporter by nitric oxide (NO) in ARPE-19 cells. METHODS: Uptake of radiolabeled cystine and glutamate was measured in ARPE-19 cells. The functional identity of x(c)(-) in these cells was established by substrate specificity and Na(+)-independence of the uptake process. The human x(c)(-) light chain (human xCT) was cloned from an ARPE-19 cell cDNA library. The functional identity of the cloned human xCT was investigated by heterologous coexpression of the light chain with the heavy chain (human 4F2hc) in HeLa cells. ARPE-19 cells were treated with or without the NO donor 3-nitroso-N:-acetylpenicillamine (SNAP) and the expression of x(c)(-) was studied at the functional and molecular levels. RESULTS: ARPE-19 cells take up cystine as well as glutamate in the absence of Na(+). Substrate specificity studies indicate that although the uptake of cystine in the absence of Na(+) is mediated by multiple amino acid transport systems including x(c)(-), the uptake of glutamate in the absence of Na(+) occurs exclusively via x(c)(-). The human xCT cloned from ARPE-19 cells is a protein of 501 amino acids. These cells express the heavy chain 4F2hc as evidenced from RT-PCR analysis. Coexpression of human xCT with 4F2hc in HeLa cells leads to the induction of cystine and glutamate uptake with characteristics similar to that of x(c)(-). The activity of x(c)(-) in ARPE-19 cells is upregulated by SNAP, and the process is associated with an increase in the expression of xCT with no detectable change in the expression of 4F2hc. CONCLUSIONS: ARPE-19 cells express the cystine/glutamate transporter x(c)(-) (the light chain xCT and the heavy chain 4F2hc) as is evident from functional and molecular studies. NO upregulates this transport system and the process is associated with an increase in xCT mRNA but with no change in 4F2hc mRNA.

Amino Acid Sequence↗

PubMed2000

Primary structure, genomic organization, and functional and electrogenic characteristics of human system N 1, a Na+- and H+-coupled glutamine transporter.

We have cloned the human Na(+)- and H(+)-coupled amino acid transport system N (hSN1) from HepG2 liver cells and investigated its functional characteristics. Human SN1 protein consists of 504 amino acids and shows high homology to rat SN1 and rat brain glutamine transporter (GlnT). When expressed in mammalian cells, the transport function of human SN1 could be demonstrated with glutamine as the substrate in the presence of LiCl (instead of NaCl) and cysteine. The transport activity was saturable, pH-sensitive, and specific for glutamine, histidine, asparagine, and alanine. Analysis of Li(+) activation kinetics showed a Li(+):glutamine stoichiometry of 2:1. When expressed in Xenopus laevis oocytes, the transport of glutamine or asparagine via human SN1 was associated with inward currents under voltage-clamped conditions. The transport function, monitored as glutamine- or asparagine-induced currents, was saturable, Na(+)-dependent, Li(+)-tolerant, and pH-sensitive. The transport cycle was associated with the involvement of more than one Na(+) ion. Uptake of asparagine was directly demonstrable in these oocytes by using radiolabeled substrate, and this uptake was inhibited by membrane depolarization. In addition, simultaneous measurement of asparagine influx and charge influx in the same oocyte yielded an asparagine:charge ratio of 1. These data suggest that SN1 mediates the influx of two Na(+) and one amino acid substrate per transport cycle coupled to the efflux of one H(+), rendering the transport process electrogenic.

Amino Acid Transport Systems, Neutral↗

PubMed2000

Primary structure, functional characteristics and tissue expression pattern of human ATA2, a subtype of amino acid transport system A.

We report here on the primary structure and functional characteristics of the protein responsible for the system A amino acid transport activity that is known to be expressed in most human tissues. This transporter, designated ATA2 for amino acid transporter A2, was cloned from the human hepatoma cell line HepG2. Human ATA2 (hATA2) consists of 506 amino acids and exhibits a high degree of homology to rat ATA2. hATA2-specific mRNA is ubiquitously expressed in human tissues. When expressed in mammalian cells, hATA2 mediates Na+-dependent transport of alpha-(methylamino)isobutyric acid, a specific model substrate for system A. The transporter is specific for neutral amino acids. It is pH-sensitive and Li+-intolerant. The Na+:amino acid stoichiometry is 1:1.

Amino Acid Sequence↗

PubMed2000

Cloning and functional expression of ATA1, a subtype of amino acid transporter A, from human placenta.

This report describes the primary structure and functional characteristics of human ATA1, a subtype of the amino acid transport system A. The human ATA1 cDNA was isolated from a placental cDNA library. The cDNA codes for a protein of 487 amino acids with 11 putative transmembrane domains. The transporter mRNA ( approximately 9.0 kb) is expressed most prominently in the placenta and heart, but detectable level of expression is evident in other tissues including the brain. When expressed heterologously in mammalian cells, the cloned transporter mediates Na(+)-coupled transport of the system A-specific model substrate alpha-(methylamino)isobutyric acid. The transport process is saturable with a Michaelis-Menten constant of 0. 89 +/- 0.12 mM. The Na(+):amino acid stoichiometry is 1:1 as deduced from the Na(+)-activation kinetics. The transporter is specific for small short-chain neutral amino acids. The gene for the transporter is located on human chromosome 12.

Amino Acid Sequence↗

PubMed2000

Expression and differential polarization of the reduced-folate transporter-1 and the folate receptor alpha in mammalian retinal pigment epithelium.

The differential polarized distribution of the reduced- folate transporter (RFT-1) and folate receptor alpha (FRalpha), the two proteins involved in the transport of folate, has been characterized in normal mouse retinal pigment epithelium (RPE) and in cultured human RPE cells. RPE cells mediate the vectorial transfer of nutrients from choroidal blood to neural retina. Whereas FRalpha is known to be present in many cell types of the neural retina, in situ hybridization analysis in the present study demonstrated that RFT-1 is present only in RPE. Laser-scanning confocal microscopy using antibodies specific for RFT-1 demonstrated an apical distribution of this protein in cultured human and intact mouse RPE, which contrasts with the basolateral distribution of FRalpha in these cells. The expression of RFT-1 in the RPE cell apical membrane was confirmed by functional studies with purified apical membrane vesicles from bovine RPE. These studies, done with N(5)-methyltetrahydrofolate (the predominant folate derivative in blood) and folate as substrates, have shown that RFT-1 functions in a Na(+)- and C1(-)-independent manner. The transporter is specific for folate and its analogs. A transmembrane H(+) gradient influences the transport function of this protein markedly; the transport mechanism is likely to be either folate/H(+) co-transport or folate/OH(-) exchange. Based on the differential polarization of FRalpha and RFT-1 in RPE, we suggest that these two proteins work in a concerted manner to bring about the vectorial transfer of folate across the RPE cell layer from the choroidal blood to the neural retina. This constitutes the first report of the differential polarization of the two folate transport proteins in any polarized epithelium.

Animals↗