Dependent Amino Acid Transport Research Articles

Identification of a regulatory pathway of L-phenylalanine-induced GLP-1 secretion in the enteroendocrine L cells

Glucagon like peptide-1 (GLP-1) is one of incretin hormone and is secreted when enteroendocrine L cells sense saccharides, amino acids, and fatty acids. Some amino acids have been shown to promote GLP-1 secretion from small intestinal enteroendocrine L cells. However, the molecular mechanisms that L-phenylalanine, a potent trigger of GLP-1 secretion, causes GLP-1 secretion from enteroendocrine L cells has not been elucidated. In this study, we used live-cell imaging to clarify the pathway by which L-phenylalanine activates enteroendocrine L cells. The results showed that L-phenylalanine was sensed by Gq-coupled receptor GPR142 and caused an increase in intracellular Ca2+ concentration. In addition, L-phenylalanine was taken up directly into the cell via Na+-dependent amino acid transporter, causing membrane depolarization and enhancing GLP-1 secretion. In summary, enteroendocrine L cells may regulate blood glucose levels in the body by detecting L-phenylalanine in the lumen and secreting GLP-1 via GPR142 and Na+-dependent amino acid transporters.

Age-related Regulation of Active Amino Acid Transport in the Ileum of Broiler Chickens.

Broiler chickens grow rapidly within a short period; in this regard, our group had previously reported a decrease in the active transport of glucose in the intestines of broiler chickens with their growth. Therefore, in this study, we compared the active transport process of amino acids in the intestines between 1- and 5-week-old broilers using everted sac, Ussing chamber techniques, and real-time quantitative polymerase chain reaction (RT-PCR). The everted sac experiment showed that amino acids were absorbed from all segments of the small intestine in both age groups. There were no significant differences in the serosal to mucosal ratio between 1- and 5-week-old broilers. The Ussing chamber experiment showed that amino acid-induced short-circuit current (ΔIsc) in the ileal epithelium was significantly greater in the 5-week-old chickens than in the 1-week-old chicks (P=0.035). Membrane conductance, an indicator of ion permeability, showed no significant difference between the two groups. Moreover, the mRNA expression levels of amino acid transporters (ASCT1, EAAT3, B0AT1, and y+LAT1) were significantly elevated in the distal ileum of the 5-week-old broilers compared to those in the 1-week-old broilers (P<0.05), while no significant differences were observed in the mRNA levels of ATB0'+, B0/+AT, rBAT, CAT1, and CAT2 in both groups. Our study provides clear evidence that age-dependent increase in the active transport of amino acid across the ileal epithelium is caused by the high expression of Na+-dependent amino acid transporters in broiler chickens.

SLC6 neurotransmitter transporter family (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

Members of the solute carrier family 6 (SLC6) of sodium- and (sometimes chloride-) dependent neurotransmitter transporters [29, 22, 70] are primarily plasma membrane located and may be divided into four subfamilies that transport monoamines, GABA, glycine and neutral amino acids, plus the related bacterial NSS transporters [99]. The members of this superfamily share a structural motif of 10 TM segments that has been observed in crystal structures of the NSS bacterial homolog LeuTAa, a Na+-dependent amino acid transporter from Aquiflex aeolicus [126] and in several other transporter families structurally related to LeuT [45].

Rho family of GTP proteins Cell Division Cycle 42 (Cdc42) and Rac Family Small GTPase 1 (Rac1) regulates F-actin dependent amino acid transporter trafficking in primary human trophoblast cells (PHT)

Rho family of GTP proteins Cell Division Cycle 42 (Cdc42) and Rac Family Small GTPase 1 (Rac1) regulates F-actin dependent amino acid transporter trafficking in primary human trophoblast cells (PHT)

Abstract 3585: Amino acid transporter SLC6A14: A novel drug target for colorectal cancer

Abstract SLC6A14 is a Na+/Cl− -dependent amino acid transporter capable of transporting 18 of the 20 proteinogenic amino acids, including arginine, leucine (mTOR activators) and glutamine (necessary for nucleotide biosynthesis). This transporter is expressed at basal levels in normal colon but significantly upregulated in colorectal cancer (CRC). However, the relevance of this upregulation to disease progression remains unknown. We postulated that deletion of SLC6A14 or pharmacological blockade of its function would suppress CRC by depleting amino acids and interfering with mTOR signaling selectively in tumor cells. To test this postulate, we first used Slc6a14-null mice. With two different models of spontaneous CRC (Apcmin/- and DSS/AOM), we found the tumor incidence and tumor growth were much lower in mice with Slc6a14-null background than in mice with Slc6a14. To evaluate the impact of pharmacologic blockade of the transporter on tumor growth we used a syngeneic tumor mouse model with MC-38 cells (a mouse CRC cell line); blockade of Slc6a14 with alpha-methyl tryptophan (α-MT) markedly reduced tumor growth. We then determined the transcriptome profiles of colonic epithelial cells and colonic non-epithelial cells from wild type mice and Slc6a14-null mice by RNAseq. There were hundreds of genes that were either upregulated or downregulated in the null mice. Ingenuity pathway analysis (IPA) of these data revealed predictive activation of canonical AMPK signaling pathway in colonic epithelial of Slc6a14-null mice. AMPK has anticancer activities by modulating multiple pathways including negatively regulating mTOR signaling. Moreover, upstream analysis showed predictive inactivation of two important tumor growth and survival signaling pathways ERK and EGF in the colon of the null mice. Likewise, significant upregulation of APC downregulated 1 (APCDD1) in epithelial cells in null mice implies predictive suppression of canonical Wnt signaling pathway. We also found marked differences in fecal microbiota as a result of Slc6a14 deletion. There was an increase in the relative abundance of beneficial microbiota including those capable of generating short chain fatty acids and lactic acid in null mice. We conclude that deletion of Slc6a14 or its pharmacological blockade protects against CRC by inducing amino acid starvation in tumor cells, thereby causing changes in multiple signaling pathways and in colonic microbiome. These studies identify SLC6A14 as a novel drug target for the treatment of colorectal cancer. Citation Format: Mohd Omar Faruk Sikder, Sathish Sivaprakasam, Vadivel Ganapathy. Amino acid transporter SLC6A14: A novel drug target for colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3585.

Abstract 5735: Amino acid transporter SLC6A14: A novel drug target for colorectal cancer and colitis and its transcriptional regulation by TCF4/beta-catenin pathway

Abstract Amino Acid Transporter SLC6A14: A Novel Drug Target for Colorectal Cancer &amp; Colitis and Its Transcriptional Regulation by TCF4/β-catenin Pathway Mohd Omar Faruk Sikder, Sathish Sivaprakasam, Vadivel Ganapathy Texas Tech University Health Sciences Center SLC6A14 is a Na+/Cl− -dependent amino acid transporter capable of transporting 18 of the 20 proteinogenic amino acids, including leucine (mTOR activator), glutamine (necessary for nucleotide biosynthesis), and arginine (substrate for iNOS). This transporter is expressed at basal levels in normal colon but significantly upregulated in colorectal cancer (CRC) and colitis. However, the relevance of this upregulation to disease progression and the mechanisms involved in the upregulation remain unknown. We postulated that deletion of SLC6A14 or pharmacological blockade of its function may suppress CRC by depleting amino acids and interfering with mTOR signaling selectively in tumor cells. It may also ameliorate the severity of colitis by diminishing NO synthesis by iNOS in colonic epithelial cells. Since TCF4/β-catenin/Wnt signaling is activated in CRC and colitis, we postulated that TCF4/β-catenin might control the expression of SLC6A14. We tested these hypotheses in the present study. CRC cells treated in vitro with α-methyl tryptophan, a selective blocker of SLC6A14, showed evidence of amino acid deprivation, decreased mTOR activity, and increased autophagy and apoptosis. In nude mouse xenografts with LS174T cells (a CRC cell line), silencing of SLC6A14 by shRNA markedly reduced tumor growth. Accordingly, Slc6a14-/- mice showed reduced tumor incident and tumor size compared to the wild type mice in AOM/DSS-induced colitis-associated colon cancer mouse model. Furthermore, Slc6a14-null mice showed decreased disease progression in an experimental colitis model (Dextran sulfate sodium-induced colitis) compared to wild type mice. In vitro treatment of LS174T cells with calphostin-C, a potent inhibitor of TCF4/β-catenin, dramatically reduced the expression of SLC6A14 mRNA and protein levels, whereas treatment of KM12L4 and KM12C cells with Wnt agonist AMBMP showed the opposite trend. Finally, ChIP assay demonstrated that TCF4/β-catenin complex directly regulates the expression of SLC6A14 in human CRC cells by binding with its promoter. We conclude that the increased β-catenin/Wnt signaling in CRC and colitis is responsible for the upregulation of SLC6A14 under these pathological conditions and that deletion of the transporter or its pharmacological blockade protects against colitis and CRC. These studies identify SLC6A14 as a novel drug target for the treatment of colonic inflammation and CRC. Citation Format: Mohd Omar Sikder, Sathish Sivaprakasam, Ganapathy Vadivel. Amino acid transporter SLC6A14: A novel drug target for colorectal cancer and colitis and its transcriptional regulation by TCF4/beta-catenin pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5735.

Abstract LB-295: MEDI7247, a novel pyrrolobenzodiazepine ADC targeting ASCT2 with potent in vivo activity across a spectrum of hematological malignancies

Abstract MEDI7247 is a first in class ADC consisting of a human anti-ASCT2 monoclonal antibody site specifically conjugated to DNA cross-linking pyrrolobenzodiazepine (PBD) dimers. ASCT2 (SLC1A5) is a multi-pass, Na+-dependent neutral amino acid transporter that mediates the uptake of amino acids required for tumor growth and progression. ASCT2 is highly overexpressed in many hematologic cancers, most notably Multiple Myeloma (MM - 100% positive), Acute Myeloid Leukemia (AML - 100% positive) and Diffuse Large B cell lymphoma (DLBCL - 95% positive). ASCT2 expression is low in normal tissues. MEDI7247 (Q1Wx4) demonstrated a significant survival advantage in 3 disseminated AML cell line models, TF1α(ASCT2-High), MOLM-13(ASCT2-low) and M.V.411(ASCT2-High), when compared to the untreated control at the lowest dose levels examined: 0.05, 0.1 and 0.1 mg/kg, respectively. Further exemplifying the activity of MEDI7247, both the TF1α and MOLM-13 models did not reach 50% survival by the end of the study, with 80% survival at &amp;gt;200 days for TF1α and 70% survival at &amp;gt;180 days for MOLM-13. Similarly, a single dose of MEDI7247 in the TF1α model resulted in a 60% survival at &amp;gt;200 days at 0.05 mg/kg. MEDI7247 was also tested in a disseminated AML PDX(ASCT2-low) model at 0.05, 0.1 and 0.4 mg/kg. A significant improvement in survival was observed at both 0.1 and 0.4 mg/kg with the higher dose level extending survival by &amp;gt;80 days. MEDI7247 activity was further confirmed by monitoring peripheral blood CD33+ve cells, which initially receded, with the timing of reappearance preempting survival. Multiple Myeloma is another indication that exhibits a high level of ASCT2 expression. MEDI7247 (Q1Wx4) efficacy was examined in 3 disseminated MM cell line models, NCI-H929(ASCT2-High), MM.1S(ASCT2-medium) and OPM2(ASCT2-Medium), with a significant improvement in survival from control at the lowest dose levels examined: 0.1, 0.1 and 0.05 mg/kg, respectively. The activity of MEDI7247 (Q1Wx4) was also examined in the subcutaneous DLBCL model KARPAS 422(ASCT2-High). Tumor regressions were observed at all dose levels tested (0.1, 0.2, 0.3 and 0.4 mg/kg), with the higher two dose levels resulting in complete tumor regression without regrowth beyond 150 days. Additionally, MEDI7247 (Q1Wx4) is efficacious against the disseminated 697(ASCT2-Low) (Acute Lymphoblastic Leukemia - ALL) and RAJI(ASCT2-High) (Burkitt's lymphoma) models. A significant survival advantage was seen in both tumor models at the lowest dose examined of 0.05 mg/kg. In conclusion, MEDI7247 demonstrates antitumor efficacy across all tumor indications tested and varying levels of ASCT2 expression. These data support the use of MEDI7247 in ASCT2 positive hematological malignancies. MEDI7247 is currently in Phase 1 clinical trials. Citation Format: Noel R. Monks, Kevin P. Schifferli, Ravinder Tammali, M. Jack Borrok, Steven R. Coats, Ronald Herbst, David A. Tice, Nabendu Pore. MEDI7247, a novel pyrrolobenzodiazepine ADC targeting ASCT2 with potent in vivo activity across a spectrum of hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-295.

Abstract LB-296: Discovery and development of MEDI7247, a novel Pyrrolobenzodiazepine (PBD)-based antibody drug conjugate targeting ASCT2, for treating hematological and solid cancers

Abstract The sodium dependent neutral amino acid transporter, ASCT2/SLC1A5 is frequently overexpressed in cancer to sustain “glutamine addiction” of cancer cells and to meet their increased demand for energy and nutrients. High expression of ASCT2 is often associated with poor disease prognosis. Immuno-histochemistry (IHC) data from 600+ tumor samples suggest ASCT2 expression in hematologic and solid cancers, including MM, AML, and DLBCL (≥ 95% positive in respective tumor samples), as well as squamous cell carcinoma of the head and neck (SCCHN), non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), prostate, pancreatic, and colorectal cancer (66% to 95% positivity in respective tumors). Broad expression across various cancers and restricted expression in normal tissues warrant ASCT2 as an attractive candidate for an antibody drug conjugate. MEDI7247 is a novel investigational antibody-drug conjugate (ADC) comprising an anti-ASCT2 human monoclonal antibody site-specifically conjugated to pyrrolobenzodiazepine (PBD) dimer via a protease-cleavable linker, with a drug to antibody ratio (DAR) of 2. MEDI7247 specifically binds to cell surface ASCT2 while exhibiting no affinity to the other members of the family, including ASCT1. Following binding, MEDI7247 is internalized and trafficked to the lysosomes to subsequently release the PBD warhead. PBD enters the nucleus where it induces DNA damage and results in tumor cell death. MEDI7247 shows potent in vitro cytotoxicity in several human cancer cell lines expressing variable levels of ASCT2. Dose dependent in vivo efficacy of MEDI7247 was determined in solid tumor xenografts and disseminated hematological malignancy models. MEDI7247 is cross-reactive with cynomolgous monkey ASCT2 and shows similar binding and killing with cyno ASCT2 expressing cells. MEDI7247 demonstrated acceptable safety profile in toxicity studies with non-human primates to support first in human trials. Based on its combined efficacy and safety, MEDI7247 is in clinical development for the treatment of ASCT2 positive cancers. Citation Format: Nabendu Pore, M Jack Borrok, Marlon Rebellato, MaryJane Hinrichs, Kevin P. Schifferli, Noel R. Monks, Ravinder Tammali, Ronald Herbst, Steven R. Coats, David A. Tice. Discovery and development of MEDI7247, a novel Pyrrolobenzodiazepine (PBD)-based antibody drug conjugate targeting ASCT2, for treating hematological and solid cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-296.

The amino acid transporter SLC36A4 regulates the amino acid pool in retinal pigmented epithelial cells and mediates the mechanistic target of rapamycin, complex 1 signaling.

SummaryThe dry (nonneovascular) form of age‐related macular degeneration (AMD), a leading cause of blindness in the elderly, has few, if any, treatment options at present. It is characterized by early accumulation of cellular waste products in the retinal pigmented epithelium (RPE); rejuvenating impaired lysosome function in RPE is a well‐justified target for treatment. It is now clear that amino acids and vacuolar‐type H+‐ATPase (V‐ATPase) regulate the mechanistic target of rapamycin, complex 1 (mTORC1) signaling in lysosomes. Here, we provide evidence for the first time that the amino acid transporter SLC36A4/proton‐dependent amino acid transporter (PAT4) regulates the amino acid pool in the lysosomes of RPE. In Cryba1 (gene encoding βA3/A1‐crystallin) KO (knockout) mice, where PAT4 and amino acid levels are increased in the RPE, the transcription factors EB (TFEB) and E3 (TFE3) are retained in the cytoplasm, even after 24 h of fasting. Consequently, genes in the coordinated lysosomal expression and regulation (CLEAR) network are not activated, and lysosomal function remains low. As these mice age, expression of RPE65 and lecithin retinol acyltransferase (LRAT), two vital visual cycle proteins, decreases in the RPE. A defective visual cycle would possibly slow down the regeneration of new photoreceptor outer segments (POS). Further, photoreceptor degeneration also becomes obvious during aging, reminiscent of human dry AMD disease. Electron microscopy shows basal laminar deposits in Bruch's membrane, a hallmark of development of AMD. For dry AMD patients, targeting PAT4/V‐ATPase in the lysosomes of RPE cells may be an effective means of preventing or delaying disease progression.

14C]Fluciclovine (alias anti-[14C]FACBC) uptake and ASCT2 expression in castration-resistant prostate cancer cells

Introductiontrans-1-Amino-3-[18F]fluorocyclobutanecarboxylic acid ([18F]fluciclovine, also known as anti-[18F]FACBC), is a tracer for positron emission tomography (PET) imaging for detection of tumors such as prostate cancer (PCa). Our previous study showed that ASCT2 (Na+-dependent amino acid transporter (AAT)) mediates fluciclovine uptake in androgen-dependent PCa cells; its expression is influenced by androgen, a key hormone in the progression of primary PCa and castration-resistant prostate cancer (CRPC). In this study, we investigated the uptake mechanisms and feasibility of [18F]fluciclovine for CRPC in the androgen-dependent PCa cell line LNCaP and LNCaP-derivatives LNCaP-SF and LN-REC4. MethodsLNCaP-SF was established after long-term cultivation of LNCaP in steroid-free conditions, and LN-Pre and LN-REC4 were established from LNCaP inoculated in intact and castrated severe combined immunodeficient mice, respectively. Uptake and competitive inhibition experiments were performed with trans-1-amino-3-fluoro[1-14C]cyclobutanecarboxylic acid ([14C]fluciclovine) to characterize the involvement of AATs in androgen-dependent PCa (LNCaP and LN-Pre) and CRPC-like (LNCaP-SF and LN-REC4) cell lines. AAT expression was analyzed by Western blotting, and [14C]fluciclovine uptake in androgen-dependent PCa and CRPC-like cell lines were investigated in the presence or absence of dihydrotestosterone (DHT). ResultsThe contribution of Na+-dependent AATs to [14C]fluciclovine uptake in all cell lines was 88−98%, and [14C]fluciclovine uptake was strongly inhibited by l-glutamine and l-serine, the substrates for Na+-dependent alanine-serine-cysteine (system ASC) AATs, in the presence of Na+. DHT enhanced ASCT2 expression in LNCaP, LN-Pre, and LN-REC4, but not in LNCaP-SF, and the responses of ASCT2 expression to DHT correlated with [14C]fluciclovine uptake. ConclusionsSystem ASC, especially ASCT2, could play a major role in [14C]fluciclovine uptake into CRPC-like and androgen-dependent PCa cells, suggesting [18F]fluciclovine-PET is applicable to the detection of CRPC as well as androgen-dependent PCa. Advance in knowledge[18F]fluciclovine-PET may be applied for the detection of CRPC. Implication for patient care[18F]fluciclovine-PET may permit early intervention for CRPC treatment.

Cardenolide aglycones inhibit tumor necrosis factor α-induced expression of intercellular adhesion molecule-1 at the translation step by blocking Na⁺/K⁺-ATPase.

Cardiac glycosides, which are inhibitors of Na(+)/K(+)-ATPase, are classified into cardenolides and bufadienolides. We have recently shown that two cardenolide glycosides, ouabain and odoroside A, inhibit Na(+)/K(+)-ATPase, thereby preventing nuclear factor κB-inducible protein expression by blocking Na(+)-dependent amino acid transport. In this study, we investigated the mechanism of action of cardenolide aglycones in tumor necrosis factor α (TNF-α)-induced gene expression. Ouabagenin, digitoxigenin, and digoxigenin were found to inhibit the TNF-α-induced cell-surface expression of intercellular adhesion molecule-1 (ICAM-1) in human lung carcinoma A549 cells. Those cardenolide aglycones did not inhibit the TNF-α-induced expression of ICAM-1 mRNA, but strongly inhibited the TNF-α-induced expression of ICAM-1 as translation product. The inhibition of the TNF-α-induced ICAM-1 expression by ouabagenin, digitoxigenin, and digoxigenin was significantly reversed by the ectopic expression of ouabain-resistant rat Na(+)/K(+)-ATPase α1 isoform. Moreover, knockdown of Na(+)/K(+)-ATPase α1 isoform augmented the inhibition of the TNF-α-induced ICAM-1 expression by ouabagenin or ouabain. These results clearly indicate that cardenolide aglycones inhibit the TNF-α-induced ICAM-1 expression at the translation step by blocking Na(+)/K(+)-ATPase.

PAT4 is abundantly expressed in excitatory and inhibitory neurons as well as epithelial cells

PAT4, the fourth member of the SLC36/proton dependent amino acid transporter (PAT) family, is a high-affinity, low capacity electroneutral transporter of neutral amino acids like proline and tryptophan. It has also been associated with the function of mTORC1, a complex in the mammalian target of rapamycin (mTOR) pathway. We performed in situ hybridization and immunohistological analysis to determine the expression profile of PAT4, as well as an RT-PCR study on tissue from mice exposed to leucine. We performed a phylogenetic analysis to determine the evolutionary origin of PAT4. The in situ hybridization and the immunohistochemistry on mouse brain sections and hypothalamic cells showed abundant PAT4 expression in the mouse brain intracellularly in both inhibitory and excitatory neurons, partially co-localizing with lysosomal markers and epithelial cells lining the ventricles. Its location in epithelial cells around the ventricles indicates a transport of substrates across the blood brain barrier. Phylogenetic analysis showed that PAT4 belongs to an evolutionary old family most likely predating animals, and PAT4 is the oldest member of that family.

Differences in Transport Mechanisms of trans-1-Amino-3-[18F]Fluorocyclobutanecarboxylic Acid in Inflammation, Prostate Cancer, and Glioma Cells: Comparison with l-[Methyl-11C]Methionine and 2-Deoxy-2-[18F]Fluoro-d-Glucose

We aimed to elucidate trans-1-amino-3-[(18)F]fluorocyclobutanecarboxylic acid (anti-[(18)F]FACBC) uptake mechanisms in inflammatory and tumor cells, in comparison with those of L-[methyl-(11)C]methionine ([(11)C]Met) and 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F]FDG). Using carbon-14-labeled tracers, in vitro time-course, pH dependence, and competitive inhibition uptake experiments were performed in rat inflammatory (T cells, B cells, granulocytes, macrophages), prostate cancer (MLLB2), and glioma (C6) cells. Anti-[(14)C]FACBC uptake ratios of T/B cells to tumor cells were comparable, while those of granulocytes/macrophages to tumor cells were lower than those for [(14)C]FDG. Over half of anti-[(14)C]FACBC uptake by T/B and tumor cells was mediated by Na(+)-dependent amino acid transporters (system ASC), whereas most [(14)C]Met transport in all cells was mediated by Na(+)-independent carriers (system L). The low anti-[(18)F]FACBC accumulation in granulocytes/macrophages may be advantageous in discriminating inflamed regions from tumors. The significant anti-[(18)F]FACBC uptake in T/B cells may cause false-positives in some cancer patients who undergo FACBC-positron emission tomography (PET).

Leucine and methionine share a Na/K‐dependent amino acid transporter in shrimp hepatopancreas

Hepatopancreatic brush border membrane vesicles (BBMV), from Atlantic White shrimp (Litopeneaus setiferus), were used to characterize 3H‐L‐leucine influx and how other essential amino acids and the cations, Na and K, interact with this transport system. L‐leucine uptake occurred against transmembrane concentration gradients in both Na and K media, suggesting that either cation could drive amino acid accumulation. L‐methionine at 20 mM, significantly inhibited 2 min uptakes of 1 mM L‐leucine in both Na‐ and K‐containing incubation media. L‐leucine influxes in both NaCl and KCl media were hyperbolic functions of [L‐leucine], following the Michaelis‐Menten equation. In NaCl, L‐leucine influx displayed a low Km (high affinity) and low Jmax, while in KCl the transport exhibited a high Km (low affinity) and high Jmax. L‐methionine was a competitive inhibitor of L‐leucine influxes in both NaCl and KCl media, (increase in L‐leucine influx Km, but no response in L‐leucine influx Jmax). Potassium was a competitive inhibitor of Na co‐transport with L‐leucine, increasing L‐leucine influx Km in the presence of Na, but having no effect on L‐leucine influx Jmax. Shrimp BBMV transport L‐leucine by a methionine‐shared carrier system stimulated by either Naor Kacting as co‐transport drivers binding to shared activator sites. Agriculture and Food Research Initiative Competitive USDA Grant no. 2010–65206‐20617.

Putative Transport Mechanism and Intracellular Fate of Trans-1-Amino-3-18F-Fluorocyclobutanecarboxylic Acid in Human Prostate Cancer

Trans-1-amino-3-(18)F-fluorocyclobutanecarboxylic acid (anti-(18)F-FACBC) is an amino acid PET tracer that has shown promise for visualizing prostate cancer. Therefore, we aimed to clarify the anti-(18)F-FACBC transport mechanism in prostate cancer cells. We also studied the fate of anti-(18)F-FACBC after it is transported into cells. For convenience, because of their longer half-lives, (14)C compounds were used instead of (18)F-labeled tracers. Trans-1-amino-3-fluoro-1-(14)C-cyclobutanecarboxylic acid ((14)C-FACBC) uptake was examined in human prostate cancer DU145 cells with the following substrates of amino acid transporters: α-(methylamino) isobutyric acid (a system A-specific substrate) and 2-amino-2-norbornanecarboxylic acid (a system L-specific substrate). The messenger RNA expression of amino acid transporters in human prostate cancer specimens was analyzed by complementary DNA microarray and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Gene expression in DU145 cells was analyzed by qRT-PCR. We also examined the knockdown effect of the amino acid transporters system ASC transporter 2 (ASCT2) and sodium-coupled neutral amino acid transporter 2 (SNAT2) on (14)C-FACBC uptake. In addition, the possibility of (14)C-FACBC incorporation into proteins was examined. (14)C-FACBC uptake by DU145 cells was markedly decreased to approximately 20% in the absence of Na(+), compared with that in its presence, indicating that Na(+)-dependent transporters are mainly responsible for the uptake of this tracer. Moreover, 2-amino-2-norbornanecarboxylic acid inhibited the transport of (14)C-FACBC to the basal level in Na(+)-free buffer. In contrast, α-(methylamino) isobutyric acid did not inhibit (14)C-FACBC accumulation in DU145 cells. Human prostate tumor specimens and DU145 cells had similar messenger RNA expression patterns of amino acid transporter genes. Although SNAT2 and ASCT2 are 2 major amino acid transporters expressed in prostate tumor tissues and DU145 cells, ASCT2 knockdown using small interfering RNA was more effective in lowering (14)C-FACBC transport than SNAT2. Almost all intracellular (14)C-FACBC was recovered from the nonprotein fraction. ASCT2, which is a Na(+)-dependent amino acid transporter, and to a lesser extent Na(+)-independent transporters play a role in the uptake of (14)C-FACBC by DU145 cells. Among the Na(+)-independent transporters, system L transporters are also involved in the transport of (14)C-FACBC. Moreover, (14)C-FACBC is not incorporated into proteins in cells. These findings suggest a possible mechanism of anti-(18)F-FACBC PET for prostate cancer.

Molecular Mechanisms of Monovalent Cation Selectivity in Na+-Coupled Secondary Transporters from Free Energy Simulations with Implicit and Explicit Treatment of Electronic Effects

The ability to efficiently discriminate different cations is essential for the proper physiological functioning of channels and transporters. Strict monovalent selectivity of Na+-coupled secondary transporters is essential to realize the strong coupling between the transport of the substrate and Na+ flux. Loss of selectivity would result in the substrate being pumped outward. The x-ray structures of Na+-dependent amino-acid transporters (LeuT and Glt)[1,2], provides a great opportunity to better understand the molecular basis of monovalent cation selectivity.Extensive all-atom free energy molecular dynamics simulations with hamiltonian construted with additive, polarizable force-fields and QM/MM descriptions of the binding pocket are performed at various occupancy states of the binding sites. In this work, we demonstrate that there is a collective effect of multiple binding sites on a total selectivity for Na+ over K+ (and Tl+) in LeuT and Glt transporters. The polarizable models of metal ions and protein (for sites Na1 and Na2 identified in structural studies) employed to provide better understanding for ion selectivity [3] as well as to mechanism of ion-substrate coupling in Glt transporter. The role of local connectivity, site rigidity and atomic polarization in monovalent cation selectivity is discussed. We will also present results from decomposition analysis.[1] Boudker, O., Ryan, R.M., Yernool, D., Shimamoto, K., Gouaux, E., 2007, Nature 445: 387-393.[2] Yamashita, A., Singh, S.K., Kawate, T., Jin, Y., Gouaux, E., 2005, Nature 437: 215-223.[3] Lev B., Salohub D., Noskov S., Computational Life Sciences,Vol. 2, No. 1, pp. 12-20.

Pertussis toxin nullifies the depolarization of the membrane potential and the stimulation of the rapid phase of 45Ca2+ entry through L-type calcium channels that are produced by follicle stimulating hormone in 10- to 12-day-old rat Sertoli cells

The aim of this study was to evaluate the effect of pertussis toxin (PTX) on the depolarizing component of the action of follicle stimulating hormone (FSH) on the membrane potential (MP) of Sertoli cells, which is linked to the rapid entry of Ca2+ into cells and to the Ca2+-dependent transport of neutral amino acids by the A system. This model allowed us to analyze the involvement of Gi proteins in the action of FSH in these phenomena. In parallel, using an inactive analog of insulin-like growth factor type I (IGF-1), JB1, and an anti-IGF-I antibody we investigated the possible mediating role of IGF-I on these effects of FSH because IGF-I is produced and released by testicular cells in response to stimulation by FSH and shows depolarization effects on MP similar to those from FSH. Our results have the following implications: (a) the rapid membrane actions of FSH, which occur in a time-frame of seconds to minutes and include the depolarization of the MP, and stimulation of 45Ca2+ uptake and [14C]-methyl aminoisobutyric acid ([14C]-MeAIB) transport, are nullified by the action of PTX and, therefore, are probably mediated by GiPCR activation; (b) the effects of FSH were also nullified by verapamil, an L-type voltage-dependent Ca2+ channel blocker; (c) wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K), prevented FSH stimulation of 45Ca2+ entry and [14C]-MeAIB transport; and (d) these FSH actions are independent of the IGF-I effects. In conclusion, these results strongly suggest that the rapid action of FSH on L-type Ca2+ channel activity in Sertoli cells from 10- to 12-day-old rats is mediated by the Gi/βγ/PI3Kγ pathway, independent of the effects of IGF-I.

Regulation of Renal LAT2 and 4F2hc Expression by Aldosterone

In the spontaneous hypertensive rat, overexpression of the renal Na + -independent L-amino acid transporter LAT2 is organ specific, precedes the onset of hypertension, correlates negatively with plasma aldosterone, and parallels the enhanced ability to take up L-DOPA and form renal dopamine. The present study evaluated the role of aldosterone on transcript and protein abundance of Na + -independent and Na + -dependent amino acid transporters. Na + -independent het- erodimeric amino acid transporters LAT1/4F2hc, LAT2/4F2hc and a Na + -dependent transporter ASCT2 transcript and protein abundance was determined in the renal cortex of normotensive Wistar rats chronically treated with aldosterone (1.5 mg), spironolactone (200 mg) or aldosterone plus spironolactone. Aldosterone significantly increased renal cortical LAT2 mRNA levels (45 % increase), with no changes in LAT1, 4F2hc and ASCT2 transcript levels. The effect of aldos- terone upon LAT2 mRNA levels was completely prevented by spironolactone. At the protein level, aldosterone treatment did not significantly affect LAT1 and LAT2 expression, but markedly reduced (51 % decrease) the abundance of 4F2hc and the urinary excretion of dopamine and DOPAC. The effect of aldosterone upon 4F2hc protein abundance was not re- versed by spironolactone. Increases in renal LAT2 transcript during chronic treatment with aldosterone occur through a spironolactone-sensitive genomic mechanism. This effect parallels with a decrease in LAT2 functionality, resulting from decreases in 4F2hc protein abundance, which appears to be either a non-genomic effect or an indirect effect of aldoster- one. The decrease in LAT2 functionality by aldosterone correlates well with the reduction in urinary dopamine.

A Conserved Na+ Binding Site of the Sodium-coupled Neutral Amino Acid Transporter 2 (SNAT2)

The SLC38 family of solute transporters mediates the coupled transport of amino acids and Na(+) into or out of cells. The structural basis for this coupled transport process is not known. Here, a profile-based sequence analysis approach was used, predicting a distant relationship with the SLC5/6 transporter families. Homology models using the LeuT(Aa) and Mhp1 transporters of known structure as templates were established, predicting the location of a conserved Na(+) binding site in the center of membrane helices 1 and 8. This homology model was tested experimentally in the SLC38 member SNAT2 by analyzing the effect of a mutation to Thr-384, which is predicted to be part of this Na(+) binding site. The results show that the T384A mutation not only inhibits the anion leak current, which requires Na(+) binding to SNAT2, but also dramatically lowers the Na(+) affinity of the transporter. This result is consistent with a previous analysis of the N82A mutant transporter, which has a similar effect on anion leak current and Na(+) binding and which is also expected to form part of the Na(+) binding site. In contrast, random mutations to other sites in the transporter had little or no effect on Na(+) affinity. Our results are consistent with a cation binding site formed by transmembrane helices 1 and 8 that is conserved among the SLC38 transporters as well as among many other bacterial and plant transporter families of unknown structure, which are homologous to SLC38.

Odoroside A and ouabain inhibit Na +/K +-ATPase and prevent NF-κB-inducible protein expression by blocking Na +-dependent amino acid transport

Inflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin-1 (IL-1), trigger the activation of transcription factor NF-κB that induces the expression of a variety of genes, including intercellular adhesion molecule (ICAM)-1. Odoroside A [3 β-O-( β- d-diginosyl)-14-hydroxy-5 β,14 β-card-20(22)-enolide] was found to inhibit the cell-surface expression of ICAM-1 induced by TNF-α and IL-1 at comparable concentrations in human lung carcinoma A549 cells. In this study, the molecular mechanism underlying the inhibition of TNF-α-induced cell-surface ICAM-1 expression by odoroside A together with the specific Na +/K +-ATPase inhibitor ouabain was further investigated. Odoroside A and ouabain neither prevented IκBα degradation nor NF-κB translocation to the nucleus upon TNF-α stimulation. While odoroside A and ouabain had no inhibitory effect on the induction of ICAM-1 mRNA, they inhibited the TNF-α-induced ICAM-1 expression at the protein level. Consistent with these results, odoroside A and ouabain potently reduced de novo protein synthesis, largely due to its ability to block Na +-dependent transport of amino acids across the plasma membrane, but not to interfering with the translation machinery. As a direct molecular target, odoroside A was found to inhibit the ATP-hydrolyzing activity of Na +/K +-ATPase as potently as ouabain. These results clearly demonstrate that odoroside A and ouabain prevent NF-κB-inducible protein expression by blocking the Na +-dependent amino acid transport.