Transport of butyryl-L-carnitine, a potential prodrug, via the carnitine transporter OCTN2 and the amino acid transporter ATB(0,+)

L-carnitine is absorbed in the intestinal tract via the carnitine transporter OCTN2 and the amino acid transporter ATB(0,+). Loss-of-function mutations in OCTN2 may be associated with inflammatory bowel disease (IBD), suggesting a role for carnitine in intestinal/colonic health. In contrast, ATB(0,+) is upregulated in bowel inflammation. Butyrate, a bacterial fermentation product, is beneficial for prevention/treatment of ulcerative colitis. Butyryl-L-carnitine (BC), a butyrate ester of carnitine, may have potential for treatment of gut inflammation, since BC would supply both butyrate and carnitine. We examined the transport of BC via ATB(0,+) to determine if this transporter could serve as a delivery system for BC. We also examined the transport of BC via OCTN2. Studies were done with cloned ATB(0,+) and OCTN2 in heterologous expression systems. BC inhibited ATB(0,+)-mediated glycine transport in mammalian cells (IC(50), 4.6 +/- 0.7 mM). In Xenopus laevis oocytes expressing human ATB(0,+), BC induced Na(+) -dependent inward currents under voltage-clamp conditions. The currents were saturable with a K(0.5) of 1.4 +/- 0.1 mM. Na(+) activation kinetics of BC-induced currents suggested involvement of two Na(+) per transport cycle. BC also inhibited OCTN2-mediated carnitine uptake (IC(50), 1.5 +/- 0.3 microM). Transport of BC via OCTN2 is electrogenic, as evidenced from BC-induced inward currents. These currents were Na(+) dependent and saturable (K(0.5), 0.40 +/- 0.02 microM). We conclude that ATB(0,+) is a low-affinity/high-capacity transporter for BC, whereas OCTN2 is a high-affinity/low-capacity transporter. ATB(0,+) may mediate intestinal absorption of BC when OCTN2 is defective.     

Transport of D-serine via the amino acid transporter ATB(0,+) expressed in the colon

D-Serine, synthesized endogenously in the brain, is an important modulator of glutamatergic neurotransmission. Since colonic bacteria produce D-serine, we asked the question whether there are transport mechanisms in the colon that might make this exogenously produced D-serine available to the host. Here we identify for the first time an amino acid transporter in the intestine for high-affinity active transport of D-serine. This transporter, called ATB(0,+), is a Na(+)- and Cl(-)-coupled transporter for L-enantiomers of neutral and cationic amino acids. Here we demonstrate that ATB(0,+) is also capable of mediating the Na(+)- and Cl(-)-coupled transport of D-serine. The affinity of ATB(0,+) for L-serine and D-serine is similar, the K(t) value for the two enantiomers being approximately 150 microM. In addition to D-serine, ATB(0,+) transports D-alanine, D-methionine, D-leucine, and D-tryptophan. However, several other neutral and cationic amino acids that are transportable substrates for ATB(0,+) as L-enantiomers are not transported when presented as D-enantiomers. ATB(0,+) is expressed in the intestinal tract, interestingly not in the proximal intestine but in the distal intestine. Expression is most predominant in the colon where the transporter is localized to the luminal membrane of colonocytes, making this transporter uniquely suitable for absorption of bacteria-derived D-serine.     

Upregulation of the amino acid transporter ATB0,+ (SLC6A14) in colorectal cancer and metastasis in humans

ATB(0,+) (SLC6A14) is a Na(+)/Cl(-)-coupled arginine transporter expressed at low levels in normal colon. Arginine is an essential amino acid for tumor cells. Arginine is also the substrate for nitric oxide synthases (NOSs). Since arginine and arginine-derived nitric oxide (NO) play a critical role in cancer, we examined the expression of ATB(0,+) in colorectal cancer. Paired normal and cancer tissues from colectomy specimens of 10 patients with colorectal cancer and from the liver tissue of one patient with hepatic metastasis from a colonic primary were used for the analysis of the levels of ATB(0,+) mRNA, inducible NOS (iNOS) mRNA and the corresponding proteins. Tissues samples from the colon, liver, and lymph nodes of an additional patient with metastatic colon cancer were analyzed for ATB(0,+) protein alone. We also examined the levels of nitrotyrosylated proteins. The ATB(0,+) mRNA increased 22.9+/-3.0-fold in colorectal cancer compared to normal tissue and the increase was evident in each of the 10 cases examined. iNOS mRNA increased 5.2+/-1.1-fold in cancer specimens. The changes in mRNA levels were associated with an increase in ATB(0,+), iNOS, and nitrotyrosylated proteins. The increased expression of ATB(0,+) and iNOS was also demonstrated in liver and lymph node specimens with metastases from colonic primaries. This study strongly suggests that the upregulation of ATB(0,+) may have a pathogenic role in colorectal cancer. Since ATB(0,+) is a versatile transporter not only for arginine but also for several drugs including NOS inhibitors, these findings have significant clinical and therapeutic relevance.     

Functional characterization and cloning of amino acid transporter B(0,+) (ATB(0,+)) in primary cultured rat pneumocytes

Cationic amino acid transport in primary cultured rat pneumocytes exhibiting characteristics of alveolar epithelial type I-like cells are described. Asymmetry and activator ion dependency of (3)H-L-arginine uptake were characterized from the apical or basolateral fluid of pneumocytes grown on permeable support. Substrate specificity of transport was evaluated as a function of (3)H-L-arginine uptake inhibition in the presence of other amino acids. Transepithelial transport studies estimated (3)H-L-arginine flux in the apical-to-basolateral and basolateral-to-apical directions. Full length cDNA of rat amino acid transporter B(0,+) (rATB(0,+)) was cloned and its relative expression level studied. Results indicate that uptake of (3)H-L-arginine from apical fluid is dependent on Na(+) and Cl(-). Zwitterionic and cationic amino acids (excluding L-proline and anionic amino acids) inhibited uptake of (3)H-L-arginine from apical, but not basolateral incubation fluid. Apical-to-basolateral transepithelial flux of (3)H-L-arginine was 20x higher than basolateral-to-apical transport. Kinetic studies of (3)H-L-arginine uptake from apical fluid revealed maximal velocity (V(max)) and Michaelis-Menten constants (K(t)) of 33.32 +/- 2.12 pmol/mg protein/15 min and 0.50 +/- 0.11 mM, respectively, in a cooperative process having a coupling ratio of 1.18 +/- 0.16 with Na(+) and 1.11 +/- 0.13 with Cl(-). Expression of rATB(0,+) mRNA was identified by RT-PCR and Northern analysis. Corresponding cloned 3.2 kb rATB(0,+) cDNA sequence exhibits pronounced homology in deduced amino acid sequence to mouse (95% identity and 97% similarity) and human (89% identity and 95% similarity) ATB(0,+) homologues. We conclude that rat pneumocytes express ATB(0,+), which may partly contribute towards recovering cationic and neutral amino acids from alveolar luminal fluid.     

Regulation of amino acid/carnitine transporter B 0,+ (ATB 0,+) in astrocytes by protein kinase C: independent effects on raft and non-raft transporter subpopulations

Neutral and basic amino acid transporter B(0,+) belongs to a Na,Cl-dependent superfamily of proteins transporting neurotransmitters, amino acids and osmolytes, known to be regulated by protein kinase C (PKC). The present study demonstrates an increased phosphorylation of B(0,+) on serine moiety after treatment of rat astrocytes with phorbol 12-myristate 13-acetate, a process correlated with an augmented activity of l-leucine transport and an enhanced presence of the transporter at the cell surface. After solubilization with Triton X-100 and sucrose gradient centrifugation, B(0,+) was detected in non-raft as well as in detergent-resistant raft fractions under control conditions, while phorbol 12-myristate 13-acetate treatment resulted in a complete disappearance of the transporter from the raft fraction. B(0,+) was observed to interact with caveolin-1 and flotillin-1 (reggie-2) proteins, the markers of detergent-resistant microdomains of plasma membrane. As verified in immunocytochemistry and immunoprecipitation experiments, modification of PKC activity did not affect these interactions. It is proposed that PKC reveals different effects on raft and non-raft subpopulations of B(0,+). Phorbol ester treatment results in trafficking of the transporter from the intracellular pool to non-raft microdomains and increased activity, while B(0,+) present in raft microdomains undergoes either internalization or is transferred laterally to non-raft domains.     

Identification of an amino acid transporter associated with the cystinuria-related type II membrane glycoprotein.

We identified an amino acid transporter that is associated with the cystinuria-related type II membrane glycoprotein, rBAT (related to b(0,+) amino acid transporter). The transporter designated BAT1 (b(0, +)-type amino acid transporter 1) from rat kidney was found to be structurally related to recently identified amino acid transporters for system L, system y(+)L, and system x(-)C, which are linked, via a disulfide bond, to the other type II membrane glycoprotein, 4F2hc (4F2 heavy chain). In the nonreducing condition, a 125-kDa band, which seems to correspond to the heterodimeric complex of BAT1 and rBAT, was detected in rat kidney with anti-BAT1 antibody. The band was shifted to 41 kDa in the reducing condition, confirming that BAT1 and rBAT are linked via a disulfide bond. The BAT1 and rBAT proteins were shown to be colocalized in the apical membrane of the renal proximal tubules where massive cystine transport had been proposed. When expressed in COS-7 cells with rBAT, but not with 4F2hc, BAT1 exhibited a Na(+)-independent transport of cystine as well as basic and neutral amino acids with the properties of system b(0,+). The results from the present investigation were used to establish a family of amino acid transporters associated with type II membrane glycoproteins.     

Functional regulation of Na+-dependent neutral amino acid transporter ASCT2 by S-nitrosothiols and nitric oxide in Caco-2 cells

We describe the regulation mechanisms of the Na(+)-dependent neutral amino acid transporter ASCT2 via nitric oxide (NO) in the human intestinal cell line, Caco-2. Exposure of Caco-2 cells to S-nitrosothiol, such as S-nitroso-N-acetyl-DL-penicillamine (SNAP) and S-nitrosoglutathione, and the NO-donor, NOC12, concentration- and time-dependently increased Na(+)-dependent alanine uptake. Kinetic analyses indicated that SNAP increases the maximal velocity (V(max)) of Na(+)-dependent alanine uptake in Caco-2 cells without affecting the Michaelis-Menten constant (K(t)). The stimulatory effect was partially eliminated by actinomycin D and cycloheximide. Increased Na(+)-dependent alanine uptake by SNAP was partially abolished by the NO scavengers, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide sodium salt (carboxy-PTIO) and N-(dithiocarboxy)sarcosine disodium salts (DTCS), as well as the NADPH oxidase inhibitor, diphenyleneiodonium. RT-PCR revealed that Caco-2 cells expressed the Na(+)-dependent neutral amino acid transporter ASCT2, but not the other Na(+)-dependent neutral amino acid transporters ATB(0,+) and B(0)AT1. These results suggested that functional up-regulation of ASCT2 by SNAP might be partially associated with an increase in the density of transporter protein via de novo synthesis.     

Expression of the amino acid transporter ATB 0+ in lung: possible role in luminal protein removal

Normal lung function requires transepithelial clearance of luminal proteins; however, little is known about the molecular mechanisms of protein transport. Protein degradation followed by transport of peptides and amino acids may play an important role in this process. We previously cloned and functionally characterized the neutral and cationic amino acid transporter ATB(0+) and showed expression in the lung by mRNA analysis. In this study, the tissue distribution, subcellular localization, and function of the transporter in native tissue were investigated. Western blots showed expression of the ATB(0+) protein in mouse lung, stomach, colon, testis, blastocysts, and human lung. Immunohistochemistry revealed that ATB(0+) is predominantly expressed on the apical membrane of ciliated epithelial cells throughout mouse airways from trachea to bronchioles and in alveolar type I cells. Electrical measurements from mouse trachea preparations showed Na(+)- and Cl(-)-dependent, amino acid-induced short-circuit current consistent with the properties of ATB(0+). We hypothesize that, by removing amino acids from the airway lumen, the transporter contributes to protein clearance and, by maintaining a low nutrient environment, plays a role in lung defense.     

Molecular and functional analysis of glutamine uptake in human hepatoma and liver-derived cells

Human hepatoma cells take up glutamine at rates severalfold faster than the system N-mediated transport rates observed in normal human hepatocytes. Amino acid inhibition, kinetic, Northern blotting, RT-PCR, and restriction enzyme analyses collectively identified the transporter responsible in six human hepatoma cell lines as amino acid transporter B(0) (ATB(0)), the human ortholog of rodent ASCT2. The majority of glutamine uptake in liver fibroblasts and an immortalized human liver epithelial cell line (THLE-5B) was also mediated by ATB(0). The 2.9-kb ATB(0) mRNA was equally expressed in all cell lines, whereas expression of the system A transporters ATA2 and ATA3 was variable. In contrast, the system N isoforms (SN1 and SN2) were expressed only in well-differentiated hepatomas. ATB(0) mRNA was also expressed in cirrhotic liver and adult and pediatric liver cancer biopsies but was not detectable in isolated human hepatocytes or fetal liver. Although the growth of all hepatomas was glutamine dependent, competitive inhibition of ATB(0)-mediated glutamine uptake blocked proliferation only in poorly differentiated cells lacking SN1 or SN2 expression and exhibiting low glutamine synthetase mRNA levels.     

Identification and characterization of a Na(+)-independent neutral amino acid transporter that associates with the 4F2 heavy chain and exhibits substrate selectivity for small neutral D- and L-amino acids

A cDNA was isolated from the mouse brain that encodes a novel Na(+)-independent neutral amino acid transporter. The encoded protein, designated as Asc-1 (asc-type amino acid transporter 1), was found to be structurally related to recently identified mammalian amino acid transporters for the transport systems L, y(+)L, x(C)(-), and b(0,+), which are linked, via a disulfide bond, to the type II membrane glycoproteins, 4F2 heavy chain (4F2hc), or rBAT (related to b(0,+) amino acid transporter). Asc-1 required 4F2hc for its functional expression. In Western blot analysis in the nonreducing condition, a 118-kDa band, which seems to correspond to the heterodimeric complex of Asc-1 and 4F2hc, was detected in the mouse brain. The band shifted to 33 kDa in the reducing condition, confirming that Asc-1 and 4F2hc are linked via a disulfide bond. Asc-1-mediated transport was not dependent on the presence of Na(+) or Cl(-). Although Asc-1 showed a high sequence homology (66% identity at the amino acid level) to the Na(+)-independent broad scope neutral amino acid transporter LAT2 (Segawa, H., Fukasawa, Y., Miyamoto, K., Takeda, E., Endou, H., and Kanai, Y. (1999) J. Biol. Chem. 274, 19745-19751), Asc-1 also exhibited distinctive substrate selectivity and transport properties. Asc-1 preferred small neutral amino acids such as Gly, L-Ala, L-Ser, L-Thr, and L-Cys, and alpha-aminoisobutyric acid as substrates. Asc-1 also transported D-isomers of the small neutral amino acids, in particular D-Ser, a putative endogenous modulator of N-methyl-D-aspartate-type glutamate receptors, with high affinity. Asc-1 operated preferentially, although not exclusively, in an exchange mode. Asc-1 mRNA was detected in the brain, lung, small intestine, and placenta. The functional properties of Asc-1 seem to be consistent with those of a transporter subserving the Na(+)-independent small neutral amino acid transport system asc.     

A hyperosmotic stress-induced mRNA of carp cell encodes Na(+)- and Cl(-)-dependent high affinity taurine transporter

A cDNA clone encoding a Na(+)- and Cl(-)-dependent high affinity taurine transporter was isolated from a common carp cell line, Epithelioma papulosum cyprini (EPC), as a hyperosmotic stress-inducible gene by RNA arbitrarily primed PCR. The clone contained a 2.5-kb cDNA fragment including an open reading frame of 1878 bp encoding a protein of 625 amino acids. The deduced amino acid sequence of carp taurine transporter shows 78-80% identity to those of cloned mammalian taurine transporters. The functional characteristics of the cloned transporter were analyzed by expression in COS-7 cells. Transfection with the cDNA induced Na(+)- and Cl(-)-dependent taurine transport activity with an apparent K(m) of 56 microM. The Na(+)/Cl(-)hepatopancreas. Taurine transporter mRNA level increased up to 7.5-fold on raising the ambient osmolality from 300 to 450 mosmol/kgH(2)O. These data suggest the significant role of taurine as an osmolyte in carp cells.     

Differential influence of the 4F2 heavy chain and the protein related to b(0,+) amino acid transport on substrate affinity of the heteromeric b(0,+) amino acid transporter

We provide evidence here that b(0,+) amino acid transporter (b(0, +)AT) interacts with 4F2 heavy chain (4F2hc) as well as with the protein related to b(0,+) amino acid transporter (rBAT) to constitute functionally competent b(0,+)-like amino acid transport systems. This evidence has been obtained by co-expression of b(0, +)AT and 4F2hc or b(0,+)AT and rBAT in human retinal pigment epithelial cells and in COS-1 cells. The ability to interact with 4F2hc and rBAT is demonstrable with mouse b(0,+)AT as well as with human b(0,+)AT. Even though both the 4F2hc x b(0,+)AT complex and the rBAT x b(0,+)AT complex exhibit substrate specificity that is characteristic of system b(0,+), these two complexes differ significantly in substrate affinity. The 4F2hc x b(0,+)AT complex has higher substrate affinity than the rBAT x b(0,+)AT complex. In situ hybridization studies demonstrate that the regional distribution pattern of mRNA in the kidney is identical for b(0,+)AT and 4F2hc. The pattern of rBAT mRNA expression is different from that of b(0,+)AT mRNA and 4F2hc mRNA, but there are regions in the kidney where b(0,+)AT mRNA expression overlaps with rBAT mRNA expression as well as with 4F2hc mRNA expression.     

Carnitine/xenobiotics transporters in the human mammary gland epithelia, MCF12A

The barrier function of the human mammary gland collapses if challenged with cationic drugs, causing their accumulation in milk. However, underlying molecular mechanisms are not well understood. To gain insight into the mechanism, we characterized transport of organic cations in the MCF12A human mammary gland epithelial cells, using carnitine and tetraethylammonium (TEA) as representative nutrient and xenobiotics probes, respectively. Our results show that the mammary gland cells express mRNA and proteins of human (h) novel organic cation transporters (OCTN) 1 and hOCTN2 (a Na+-dependent carnitine carrier with Na+-independent xenobiotics transport function), which belong to the solute carrier superfamily (SLC) of transporters. Other SLC OCTs such as hOCT1 and extraneuronal monoamine transporter (EMT)/hOCT3 are also expressed at mRNA levels, but hOCT2 was undetectable. We further showed mRNA expression of ATB0+ (an amino acid transporter with a Na+/Cl(-)-dependent carnitine transport activity), and Fly-like putative transporter 2/OCT6 (a splice variant of carnitine transporter 2: a testis-specific Na+-dependent carnitine transporter). TEA uptake was pH dependent. Carnitine uptake was dependent on Na+, and partly on Cl-, compatible with hOCTN2 and ATB0+ function. Modeling analyses predicted multiplicity of the uptake mechanisms with the high-affinity systems characterized by K(m) of 5.1 microM for carnitine and 1.6 mM for TEA, apparently similar to the reported hOCTN2 parameter for carnitine, and that of EMT/hOCT3 for TEA. Verapamil, cimetidine, carbamazepine, quinidine, and desipramine inhibited the carnitine uptake but required supratherapeutic concentrations, suggesting robustness of the carnitine uptake systems against xenobiotic challenge. Our findings suggest functional roles of a network of multiple SLC organic cation/nutrient transporters in human mammary gland drug transfer.     

SLC6A14 (ATB0,+) protein, a highly concentrative and broad specific amino acid transporter, is a novel and effective drug target for treatment of estrogen receptor-positive breast cancer

SLC6A14, also known as ATB(0,+), is an amino acid transporter with unique characteristics. It transports 18 of the 20 proteinogenic amino acids. However, this transporter is expressed only at low levels in normal tissues. Here, we show that the transporter is up-regulated specifically in estrogen receptor (ER)-positive breast cancer, demonstrable with primary human breast cancer tissues and human breast cancer cell lines. SLC6A14 is an estrogen/ER target. The transport features of SLC6A14 include concentrative transport of leucine (an activator of mTOR), glutamine (an essential amino acid for nucleotide biosynthesis and substrate for glutaminolysis), and arginine (an essential amino acid for tumor cells), suggesting that ER-positive breast cancer cells up-regulate SLC6A14 to meet their increased demand for these amino acids. Consequently, treatment of ER-positive breast cancer cells in vitro with α-methyl-DL-tryptophan (α-MT), a selective blocker of SLC6A14, induces amino acid deprivation, inhibits mTOR, and activates autophagy. Prolongation of the treatment with α-MT causes apoptosis. Addition of an autophagy inhibitor (3-methyladenine) during α-MT treatment also induces apoptosis. These effects of α-MT are specific to ER-positive breast cancer cells, which express the transporter. The ability of α-MT to cause amino acid deprivation is significantly attenuated in MCF-7 cells, an ER-positive breast cancer cell line, when SLC6A14 is silenced with shRNA. In mouse xenograft studies, α-MT by itself is able to reduce the growth of the ER-positive ZR-75-1 breast cancer cells. These studies identify SLC6A14 as a novel and effective drug target for the treatment of ER-positive breast cancer.     

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.     

Luminal heterodimeric amino acid transporter defective in cystinuria

Mutations of the glycoprotein rBAT cause cystinuria type I, an autosomal recessive failure of dibasic amino acid transport (b(0,+) type) across luminal membranes of intestine and kidney cells. Here we identify the permease-like protein b(0,+)AT as the catalytic subunit that associates by a disulfide bond with rBAT to form a hetero-oligomeric b(0,+) amino acid transporter complex. We demonstrate its b(0,+)-type amino acid transport kinetics using a heterodimeric fusion construct and show its luminal brush border localization in kidney proximal tubule. These biochemical, transport, and localization characteristics as well as the chromosomal localization on 19q support the notion that the b(0,+)AT protein is the product of the gene defective in non-type I cystinuria.     

Involvement of OCTN2 and B0,+ in the transport of carnitine through an in vitro model of the blood-brain barrier

Carnitine is known to accumulate in brain, therefore transport of carnitine through the blood-brain barrier was studied in an in vitro system using bovine brain capillary endothelial cells (BBCEC) grown on filter inserts in a co-culture system with glial cells. Long-term exposure of BBCEC to carnitine resulted in a high accumulation of long-chain acyl carnitines, which decreased dramatically upon removal of carnitine. Kinetic analysis of carnitine accumulation indicated a possibility of functioning of more than one transporter. BBCEC were incubated in the presence of substrates and inhibitors of known carnitine transporters added from either apical or basolateral side. Inhibition by replacement of sodium and expression of OCTN2 (RT-PCR) were in agreement with earlier reports on the functioning of OCTN2 in apical membrane. For the first time, functioning of OCTN2 was demonstrated in the basolateral membrane, as well as functioning in both membranes of a low affinity carnitine transporter B(0,+). Expression of B(0,+) in BBCEC was confirmed by RT-PCR. These results suggest that OCTN2 and B(0,+) could be involved in carnitine transport in both the apical and basolateral membrane.