The SLC36 family of proton-coupled amino acid transporters and their potential role in drug transport

Members of the solute carrier (SLC) 36 family are involved in transmembrane movement of amino acids and derivatives. SLC36 consists of four members. SLC36A1 and SLC36A2 both function as H(+) -coupled amino acid symporters. SLC36A1 is expressed at the luminal surface of the small intestine but is also commonly found in lysosomes in many cell types (including neurones), suggesting that it is a multipurpose carrier with distinct roles in different cells including absorption in the small intestine and as an efflux pathway following intralysosomal protein breakdown. SLC36A1 has a relatively low affinity (K(m) 1-10 mM) for its substrates, which include zwitterionic amino and imino acids, heterocyclic amino acids and amino acid-based drugs and derivatives used experimentally and/or clinically to treat epilepsy, schizophrenia, bacterial infections, hyperglycaemia and cancer. SLC36A2 is expressed at the apical surface of the human renal proximal tubule where it functions in the reabsorption of glycine, proline and hydroxyproline. SLC36A2 also transports amino acid derivatives but has a narrower substrate selectivity and higher affinity (K(m) 0.1-0.7 mM) than SLC36A1. Mutations in SLC36A2 lead to hyperglycinuria and iminoglycinuria. SLC36A3 is expressed only in testes and is an orphan transporter with no known function. SLC36A4 is widely distributed at the mRNA level and is a high-affinity (K(m) 2-3 μM) transporter for proline and tryptophan. We have much to learn about this family of transporters, but from current knowledge, it seems likely that their function will influence the pharmacokinetic profiles of amino acid-based drugs by mediating transport in both the small intestine and kidney.     

L-type amino acid transporter-1 expressed in human astrocytomas, U343MGa

LAT1 (L-type amino acid transporter 1), one of the L-type amino acid transporters, transports the branched and aromatic amino acids. LAT1 requires the heavy chain of 4F2 antigen (4F2hc) for the functional expression as an amino acid transporter. The expression of this transporter is up-regulated in tumor cells and rapidly-growing cells to support their proliferation. Here, we studied the expression of LAT1 and 4F2hc in human cultured cells by real-time PCR and Western blot, and found that human brain astrocytomas, U343MGa, highly expressed LAT1 and 4F2hc mRNAs and proteins. The uptake of [14C]leucine by U343MGa cells is Na+-independent and inhibited by BCH (2-amino-2-norbornane carboxylic acid), and branched and aromatic amino acids, indicating that the LAT1 is expressed at the cell surface. Pulse chase labeling and surface labeling experiments of this cell line indicate that the protein synthesis of LAT1 and 4F2hc is slow, however, the heterodimeric complex assembled in the cells is very stable, and that the disulfide bond between the LAT1 and 4F2hc is not directly involved in the stability of the heterodimer.     

Impact of system L amino acid transporter 1 (LAT1) on proliferation of human ovarian cancer cells: A possible target for combination therapy with anti-proliferative aminopeptidase inhibitors

Amino acids activate nutrient signaling via the mammalian target of rapamycin (mTOR), we therefore evaluated the relationship between amino acid transporter gene expression and proliferation in human ovarian cancer cell lines. Expression of three cancer-associated amino acid transporter genes, LAT1, ASCT2 and SN2, was measured by qRT-PCR and Western blot. The effects of silencing the LAT1 gene and its inhibitor BCH on cell growth were evaluated by means of cell proliferation and colony formation assays. The system L amino acid transporter LAT1 was up-regulated in human ovarian cancer SKOV3, IGROV1, A2780, and OVCAR3 cells, compared to normal ovarian epithelial IOSE397 cells, whereas ASCT2 and SN2 were not. BCH reduced phosphorylation of p70S6K, a down-stream effector of mTOR, in SKOV3 and IGROV1 cells, and decreased their proliferation by 30% and 28%, respectively. Although proliferation of SKOV3 (S1) or IGROV1 (I10) cells was unaffected by LAT1-knockdown, plating efficiency in colony formation assays was significantly reduced in SKOV3(S1) and IGROV1(I10) cells to 21% and 52% of the respective plasmid transfected control cells, SKOV3(SC) and IGROV(IC), suggesting that LAT1 affects anchorage-independent cell proliferation. Finally, BCH caused 10.5- and 4.3-fold decrease in the IC₅₀ value of bestatin, an anti-proliferative aminopeptidase inhibitor, in IGROV1 and A2780 cells, respectively, suggesting that the combined therapy is synergistic. Our findings indicate that LAT1 expression is increased in human ovarian cancer cell lines; LAT1 may be a target for combination therapy with anti-proliferative aminopeptidase inhibitors to combat ovarian cancer.     

L-型氨基酸转运体1 在肿瘤诊断和治疗中的研究进展

L-型氨基酸转运体1（LAT1）是L-型氨基酸转运蛋白家族的一个成员,它是由两条多肽链通过二硫键形成的异二聚体,也是异二聚体氨基酸转运体SLC7 亚家族的重要成员。LAT1 的主要功能是介导一些分子中含有苯环或者支链的、分子质量较大的、体液环境中酸碱性呈中性的氨基酸（L-亮氨酸、L-甲硫氨酸和L-苯丙氨酸等）及其类似物（美法仑、多巴和甲状腺素等）的跨膜转运,其跨膜转运方式为不依赖Na+和ATP 的协助扩散。LAT1 转运体在许多肿瘤细胞中有特异性的高表达,且其表达程度与临床上肿瘤的分期情况及治疗预后有密切的联系。LAT1 不仅被证明在肿瘤诊断中具有重要意义,也是一个潜在的肿瘤治疗靶点。本文对LAT1 的结构和转运特点进行了阐述,并总结了LAT1 在肿瘤诊断和治疗中的最新研究进展。     

Pharmacokinetic role of L-type amino acid transporters LAT1 and LAT2

LAT1 and LAT2 are heterodimeric large amino acid transporters that are expressed in various tissues, including the intestinal wall, blood-brain barrier, and kidney. These transporters consist of membrane spanning light chain and heavy chain, and they act as 1:1 exchangers in concert with other amino acid transporters. Only a few drugs (less than 10) are substrates of LAT1 and LAT2, including l-DOPA, alpha-methyldopa, melphalan, and gabapentin. The mechanisms and substrates have been mostly elucidated using mammalian cells and Xenopus oocytes. The in vivo relevance of LAT1 and LAT2 in pharmacokinetics is obscure, because contradictory findings have been reported. It is difficult to make quantitative pharmacokinetic conclusions about LAT1 and LAT2. This is due to the possible involvement of other transporters (including cross-linked heterodimers of light chain with different heavy chains, other overlapping transporters, for example TAT1), competing endogenous amino acids, and saturation phenomena. This review presents the current functional knowledge on LAT1 and LAT2 with emphasis on their potential involvement in pharmacokinetics.     

尿酸转运蛋白研究进展

体内尿酸经肾脏转运时需依赖肾小管上皮细胞上的转运蛋白。现已明确有4种尿酸盐转运蛋白参与了人近曲肾小管对尿酸盐的转运：即负责尿酸重吸收的尿酸盐阴离子转运体1（hURATl）,及负责尿酸分泌的尿酸盐转运体（UAT）和有机阴离子转运体（OATl和OAT3）。最近,又发现了一种负责将尿酸分泌到细胞外,参与肾脏近曲小管对尿酸盐的重吸收的转运蛋白一一葡萄糖转运蛋白9（GLUT9）。本文对尿酸转运蛋白的特点、功能及调节机制进行综述。     

新型抗2型糖尿病药物钠-葡萄糖协同转运蛋白2抑制剂研究进展

钠-葡萄糖协同转运蛋白（SGLT）是一类位于小肠黏膜（SGLT1）和肾近曲小管（SGLT2和SGLT1）中的葡萄糖转运基因家族。其中,SGLT2是一种低亲和力的转运蛋白,在肾脏中特异性表达并且在近曲小管葡萄糖重吸收中发挥非常重要的作用。它可以选择性地抑制SGLT2,即可通过增加尿糖的排出来治疗2型糖尿病,是一种创造性的治疗策略。本文介绍了SGLT2抑制剂在2型糖尿病治疗研究方面的最新进展,重点综述了SGLT2抑制剂的作用机制、部分在研SGLT2抑制剂的生物活性数据及临床试验结果。     

In vitro and in vivo evidence of the importance of organic anion transporters (OATs) in drug therapy

Organic anion transporters 1-10 (OAT1-10) and the urate transporter 1 (URAT1) belong to the SLC22A gene family and accept a huge variety of chemically unrelated endogenous and exogenous organic anions including many frequently described drugs. OAT1 and OAT3 are located in the basolateral membrane of renal proximal tubule cells and are responsible for drug uptake from the blood into the cells. OAT4 in the apical membrane of human proximal tubule cells is related to drug exit into the lumen and to uptake of estrone sulfate and urate from the lumen into the cell. URAT1 is the major urate-absorbing transporter in the apical membrane and is a target for uricosuric drugs. OAT10, also located in the luminal membrane, transports nicotinate with high affinity and interacts with drugs. Major extrarenal locations of OATs include the blood-brain barrier for OAT3, the placenta for OAT4, the nasal epithelium for OAT6, and the liver for OAT2 and OAT7. For all transporters we provide information on cloning, tissue distribution, factors influencing OAT abundance, interaction with endogenous compounds and different drug classes, drug/drug interactions and, if known, single nucleotide polymorphisms.     

LAT1 Is a Central Transporter of Essential Amino Acids in Human Umbilical Vein Endothelial Cells

Endothelial cell proliferation supporting angiogenesis requires sufficient nutrient supply because of facilitated intracellular metabolism. However, little is known about the mechanism for the promotion of nutrient incorporation in proliferating endothelial cells. Here we show that L-type amino acid transporter 1 (LAT1) is a major transporter of essential amino acids in human umbilical vein endothelial cells (HUVECs). Growing HUVECs express a certain level of LAT1. A LAT1-specific inhibitor suppressed leucine uptake, cell proliferation, and tube formation of HUVECs. Therefore, LAT1 acts to support effective uptake of amino acids, which is critical for the optimal function of HUVECs for angiogenesis.     

Amino acid transporter molecule as a drug target

Amino acids, especially essential amino acids are required for protein synthesis and as energy sources in all living cells. Since most amino acids are hydrophilic, special membrane proteins are necessary for their transmembrane transport particularly in transformed cells. We cloned a cDNA encoding L-type amino acid transporter, and named it LAT1. LAT1 is highly expressed in most tumor cells and in a few normal tissues such as the bone marrow, spleen, placenta and brain. As a homolog of LAT1, we also cloned LAT2 which is expressed in various normal tissues. In order to clarify the role of LAT1 in tumor cells, we tested a relatively specific inhibitor to LAT1, BCH (2-aminobicyclo-2(2,2,1)-heptane-2-caboxylic acid), and found that BCH inhibited mortality of mice treated with sarcoma cells. In addition, BCH also suppressed strongly T24 carcinoma cell growth. Moreover, LAT1 transports amino acid mimetic drugs. Thus, these results indicate that amino acid transporter molecule such as LAT1 can be a drug target.     

Establishment of stable cell lines with high expression of heterodimers of human 4F2hc and human amino acid transporter LAT1 or LAT2 and delineation of their differential interaction with α-alkyl moieties

System L is a major transport system for cellular uptake of neutral amino acids. Among system L transporters, L-type amino acid transporter 1 (LAT1) is responsible for the nutrient uptake in cancer cells, whereas L-type amino acid transporter 2 (LAT2) is a transporter for non-cancer cells. In this study, we have established HEK293 cell lines stably expressing high levels of human LAT1 and LAT2 forming heterodimers with native human 4F2hc of the cells. We have found that L-[(14)C]alanine is an appropriate substrate to examine the function of LAT2, whereas L-[(14)C]leucine is used for LAT1. By using L-[(14)C]alanine on LAT2, we have for the first time directly evaluated the function of human LAT2 expressed in mammalian cells and obtained its reliable kinetics. Using α-alkyl amino acids including α-methyl-alanine and α-ethyl-L-alanine, we have demonstrated that α-alkyl groups interfere with the interaction with LAT2. These cell lines with higher practical advantages would be useful for screening and analyzing compounds to develop LAT1-specific drugs that can be used for cancer diagnosis and therapeutics. The strategy that we took to establish the cell lines would also be applicable to the other heterodimeric transporters with important therapeutic implications.     

1,1′-Diethyl-2,2′-cyanine (decynium22) potently inhibits the renal transport of organic cations

Summary The excretion of cationic compounds by renal proximal tubule cells involves at least two distinct transporters: the basolateral type which transports organic cations from the plasma into the proximal tubule cell, and the apical type which secretes the organic cations into the lumen of the tubule. However, potent inhibitors were known for neither type of transporter. Here we introduce a compound, decynium22, that potently, competitively, and selectively inhibits the apical type of the renal organic cation transporter.The transport of the prototypical organic cation ¹⁴C-tetraethylammonium through the apical plasma membrane of clonal proximal tubule cells (LLC-PK₁) was used as experimental system. Initial rates of ¹⁴C-tetraethylammonium transport into LLC-PK₁ cells were saturable, the K_m and V_max being 27 mol/l and 200 pmol/(mg protein · min), respectively. Decynium 22 competitively and potently inhibited ¹⁴C-tetraethylammonium transport (K_i = 5.6 nmol/l). Moreover, the effect of decynium22 on basolateral to apical directed transepithelial transport of ¹⁴C-tetraethylammonium through a confluent monolayer of LLC-PK₁ cells was determined. Decynium22 (30 nmol/l) applied to the apical medium, reduced transepithelial transport by 76% and increased intracellular accumulation of ¹⁴C-tetraethylammonium 1.5-fold. In contrast, application of 30 nmol/l decynium22 to the basolateral medium failed to affect transepithelial transport and intracellular accumulation of ¹⁴C-tetraethylammonium.Decynium22 is the most potent inhibitor of the renal transport of organic cations known so far. With decynium22 it is now possible to distinguish precisely between a decynium22-sensitive apical type and a decynium22-resistant basolateral type of renal organic cation transporter in renal proximal tubule cells.Supported by the Deutsche Forschungsgemeinschaft (SFB176) and the Universitätsbund WürzburgCorrespondence to E. Schömig at the above address     

Role of organic anion and amino acid carriers in transport of inorganic mercury in rat renal basolateral membrane vesicles: influence of compensatory renal growth.

Susceptibility to renal injury induced by inorganic mercury (Hg(2+)) increases significantly as a result of compensatory renal growth (following reductions of renal mass). We hypothesize that this phenomenon is related in part to increased basolateral uptake of Hg(2+) by proximal tubular cells. To determine the mechanistic roles of various transporters, we studied uptake of Hg(2+), in the form of biologically relevant Hg(2+)-thiol conjugates, using basolateral membrane (BLM) vesicles isolated from the kidney(s) of control and uninephrectomized (NPX) rats. Binding of Hg(2+) to membranes, accounted for 52-86% of total Hg(2+) associated with membrane vesicles exposed to HgCl(2), decreased with increasing concentrations of HgCl(2), and decreased slightly in the presence of sodium ions. Conjugation of Hg(2+) with thiols (glutathione, L-cysteine (Cys), N-acetyl-L-cysteine) reduced binding by more than 50%. Under all conditions, BLM vesicles from NPX rats exhibited a markedly lower proportion of binding. Of the Hg(2+)-thiol conjugates studied, transport of Hg-(Cys)(2) was fastest. Selective inhibition of BLM carriers implicated the involvement of organic anion transporter(s) (Oat1 and/or Oat3; Slc22a6 and Slc22a8), amino acid transporter system ASC (Slc7a10), the dibasic amino acid transporter (Slc3a1), and the sodium-dicarboxylate carrier (SDCT2 or NADC3; Slc13a3). Uptake of each mercuric conjugate, when factored by membrane protein content, was higher in BLM vesicles from uninephrectomized (NPX) rats, with specific increases in transport by the carriers noted above. These results support the hypothesis that compensatory renal growth is associated with increased uptake of Hg(2+) in proximal tubular cells and we have identified specific transporters involved in the process.     

Transport characteristics of L‐citrulline in renal apical membrane of proximal tubular cells

L ‐Citrulline has diagnostic potential for renal function, because its plasma concentration increases with the progression of renal failure. Although L ‐citrulline extracted by glomerular filtration in kidney is mostly reabsorbed, the mechanism involved is not clearly understood. The present study was designed to characterize L ‐citrulline transport across the apical membranes of renal epithelial tubular cells, using primary‐cultured rat renal proximal tubular cells, as well as the human kidney proximal tubular cell line HK‐2. L ‐Citrulline was transported in a Na⁺‐dependent manner from the apical side of both cell types cultured on permeable supports with a microporous membrane. Kinetic analysis indicated that the transport involves two distinct Na⁺‐dependent saturable systems and one Na⁺‐independent saturable system in HK‐2 cells. The uptake was competitively inhibited by neutral and cationic, but not anionic amino acids. Relatively large cationic and anionic compounds inhibited the uptake, but smaller ones did not. In HK‐2 cells, mRNA expression of SLC6A19 and SLC7A9, which encode B⁰AT1 and b^0,+AT, respectively, was detected by RT‐PCR. In addition, L ‐citrulline transport was significantly decreased in HK‐2 cells in which either SLC6A19 or SLC7A9 was silenced. Hence, these results suggest that amino acid transporters B⁰AT1 and b^0,+AT are involved in the reabsorption of L ‐citrulline in the kidney, at least in part, by mediating the apical membrane transport of L ‐citrulline in renal tubule cells. Copyright © 2009 John Wiley & Sons, Ltd.     

In vivo antidiabetic activity of nimesulide due to inhibition of amino acid transport

Inhibiting the intestinal and renal neutral amino acid transporter B0AT1 by genetic means has improved insulin sensitivity in mice, but there are no antagonists available for preclinical or clinical use. Since the anti-inflammatory agent nimesulide selectively inhibited B0AT1 in vitro, we hypothesized that nimesulide exhibits in vivo potential to restrict neutral amino acid absorption and, therefore, may improve insulin sensitivity. The dose-related effect of nimesulide (10 to 100 mg/kg, PO) on intestinal absorption of neutral amino acids was estimated in C57 mice. The effect of nimesulide (50 mg/kg, PO) on renal resorption of amino acids was also assessed. The effect of chronic nimesulide (50 mg/kg, PO, BID for 14 days) was assessed in high protein diet-fed C57 mice, diet-induced obese mice and obese and diabetic db/db mice. Acute and chronic nimesulide treatment decreased absorption of neutral amino acids and increased their urinary excretion. Nimesulide treatment improved insulin sensitivity and glycemic control, increased GLP-1, decreased liver lipids and improved FGF-21 in serum. Nimesulide improved insulin sensitivity and glucose tolerance by inhibiting neutral amino acid transport in the intestine and kidneys. Thus, it can serve as a tool compound for in vivo B0AT1 inhibition.     

Targeting L-type amino acid transporter 1 for anticancer therapy: clinical impact from diagnostics to therapeutics

Introduction: L-type amino acid transporter 1 (LAT1) is one of the amino acid transporters. It is overexpressed in various types of cancer cells, while it is produced restrictedly in normal tissues.

Areas covered: We discuss its characteristics in cancer cells compared with normal cells. We also mention the current applications to target LAT1 for anticancer therapy focusing on prognostic biomarkers, radio-labeled tumor imaging reagents, amino acid-stapled prodrugs, LAT1-mediated enhanced transport of anticancer drugs and LAT1 inhibitors.

Expert opinion: LAT1 can be a versatile target to promisingly develop transporter-based drugs with enhanced drug delivery potential for anticancer therapy.     

Transport of methylmercury—cysteine conjugate by system L—tpye amino acid transporters and its transportermediated toxicity

Methylmercury(MeHg) is widely known for its potent neurotoxicity and the causal substance of Minamata disease.Since the conjugates of MeHg with thiol compounds are easily formed in vivo,the metabolism and transport of glutathione,cysteine and their derivatives are important determinants of tissue distribution and elimination of MeHg.It has been proposed that the amino acid transport system L,which transports large neutral amino acids,is one of the major routes for MeHg mobilization.We have identified two isoforms of system L amino acid transporters:L-type amino acid transkporter-1(LAT1) and LAT2 and found that they transport MeHg as a cysteine-conjugate (MeHg-Cys).We have further found that a classical system L inhibitor BCH[2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid] rescued T24 human bladder carcinoma cells expressing LAT1 form the toxicity of MeHg-Cysl.We concluded that the cytotoxicity of MeHg is mediated by system L transporters.The fact that BCH reduced the toxicity of MeHg-Cys suggests that the high-affinity inhibitors of system L transporters could be a new rationable to avoid MeHg-toxicity.     

SGLT2抑制剂研究进展

钠-葡萄糖协同转运蛋白（SGLTs）主要存在于小肠黏膜（SGLTl）和肾近曲小管（SGLT1和SGLT2）的转运基因家族,其表达的膜蛋白负责将葡萄糖、氨基酸、维生素、离子和渗透溶质转运至肾近曲小管的刷状缘细胞及小肠上皮细胞。而SGLT2是一种主要在肾脏特异性表达的高效能．低亲和力转运体。葡萄糖在肾近曲小管的重吸收约有90％由SGLT2完成,因此选择性地阻断SGLT2、减少重吸收、增加尿糖排出这一治疗策略已成为糖尿病领域的又一创新性研究,为糖尿病治疗药物提供了新作用靶点。本文对SGLT2抑制剂在治疗2型糖尿病方面的最新研究进展进行介绍,主要阐述其作用机制、疗效（部分在研SGTL2抑制剂的临床试验结果）和安全性。