Functional reconstitution of the hepatic system N amino acid transport activity.
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ABSTRACT:
SUBMITTER: Tamarappoo BK
PROVIDER: S-EPMC1149925 | biostudies-other | 1991 Feb
REPOSITORIES: biostudies-other
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Project description:In many cell types, including human erythrocytes, membrane transport of hydrophobic amino acids such as leucine and phenylalanine is mediated primarily by Na(+)-independent system L. In this paper we demonstrate that erythrocytes from the rat have a 400-fold higher system L transport capacity than human erythrocytes. We have exploited this high transport activity to achieve the first successful reconstitution of an erythrocyte amino acid transporter into phospholipid vesicles. Rat erythrocyte membranes were depleted of extrinsic membrane proteins, solubilized in 50 mM n-octyl glucoside and reconstituted into egg-yolk phospholipid vesicles by a gel filtration freeze-thaw protocol. Optimal reconstitution of transport activity occurred at lipid/protein ratios of 25-35:1. At a lipid/protein ratio of 25:1, one-half of the total uptake of L-[14C]leucine (0.2 mM, 25 degrees C) was inhibited by 2 mM phloretin and thus judged to be carrier-mediated. This component of L-leucine uptake was inhibited by non-radioactive L-phenylalanine and L-leucine, and only to a very much weaker extent by glycine and L-alanine. Two other inhibitors of system L in intact cells, MK196 and PCMBS (p-chloromercuriphenylsulphonate), were also effective inhibitors of phloretin-sensitive L-leucine transport in reconstituted proteoliposomes. Phloretin-insensitive uptake of L-leucine in proteoliposomes occurred by simple diffusion across the lipid bilayer.
| S-EPMC1134163 | biostudies-other
Project description:The cystine transport activity of a lens epithelial cell line originated from a canine mature cataract was investigated. The distinct cystine transport activity was observed, which was inhibited to 28% by extracellular 1 mM glutamate. The cDNA sequences of canine cysteine/glutamate exchanger (xCT) and 4F2hc were determined. The predicted amino acid sequences were 527 and 533 amino acid polypeptides, respectively. The amino acid sequences of canine xCT and 4F2hc showed high similarities (>80%) to those of humans. The expression of xCT in lens epithelial cell line was confirmed by western blot analysis. RT-PCR analysis revealed high level expression only in the brain, and it was below the detectable level in other tissues.
| S-EPMC4064136 | biostudies-literature
Project description:A rapid method for the functional reconstruction of amino acid transport from liver plasma-membrane vesicles using the neutral detergent decanoyl-N-glucamide ('MEGA-10') is described. The method is a modification of that previously employed in this laboratory for reconstitution of amino acid transport systems from kidney brush-border membranes [Lynch & McGivan (1987) Biochem. J. 244, 503-508]. The transport activities termed 'System A', 'System N', and 'System L' are all reconstituted. The reconstitution procedure is rapid and efficient and is suitable as an assay for transport activity in studies involving membrane fractionation. By using this reconstitution procedure, System A transport activity was partially purified by lectin-affinity chromatography.
| S-EPMC1135335 | biostudies-other
Project description:BackgroundAmino acids are important nutrients during fetal development, and the activity of placental amino acid transporters is crucial in the regulation of fetal growth. Leptin, an adipocyte- and placenta-derived hormone, has been proposed to act as a peripheral signal in reproduction in humans. Leptin is elevated during pregnancy and elevated further in pathologic pregnancies such as preeclampsia. However, the role of leptin in placental function has not been fully elucidated. We hypothesize that leptin plays a role in the regulation of placental amino acid transport by activation of the JAK-STAT pathway.MethodsPlacental amino acid transport, specifically system A transport was studied in placental villous fragments using the amino acid analog, methylaminoisobutyric acid (MeAIB). Specific inhibitors of the JAK-STAT signal transduction pathway were used to further elucidate their role in leptin-mediated effects on amino acid transport activity. Western blotting was performed to identify STAT3 phosphorylation as a measure of leptin receptor activation.ResultsLeptin significantly increased system A amino acid transporter activity by 22-42% after 1h of incubation. Leptin activated JAK-STAT signaling pathway as evidenced by STAT3 phosphorylation, and inhibition of STAT3 or JAK2 resulted in 36-45% reduction in system A amino acid transporter activity. Furthermore, blocking endogenously produced leptin also decreased system A transport by 45% comparable to STAT3 inhibition.ConclusionsThese data demonstrate that leptin stimulates system A by JAK-STAT dependent pathway in placental villous fragments. Our findings support the autocrine/paracrine role of leptin in regulating amino acid transport in the human placenta.
| S-EPMC2675556 | biostudies-literature
Project description:Classical amino acid transport System A accounts for most of the Na(+)-dependent neutral amino acid uptake by mammalian cells. System A has also provided a paradigm for short- and long-term regulation by physiological stimuli. We now report the isolation of a cDNA encoding System A that shows close similarity to the recently identified System N transporter (SN1). The System A transporter (SA1) and SN1 share many functional characteristics, including a marked sensitivity to low pH, but, unlike SN1, SA1 does not mediate proton exchange. Transport mediated by SA1 is also electrogenic. Amino acid transport Systems A and N thus appear closely related in function as well as structure, but exhibit important differences in ionic coupling.
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Project description:IL-3 mediates human plasmacytoid dendritic cell (pDC) survival in vitro, but its mechanism of action is unclear. We show that, through JAK2/STAT5, IL-3 and the related GM-CSF, induce expression of System L amino acid transporters SLC7A5 and SLC3A2. This primes pDC to allow subsequent activation of mTORC1 in response to TLR9 stimulation. Active mTORC1 facilitates increased metabolic activity, type I IFN and TNF production, and the expression of SLC7A11, a subunit of the cystine/glutamate antiporter, xc—. We found that co-expression of SLC7A5, SLC3A2 and SLC7A11 is a major transcriptional signature shared by cytokine-producing pDC in vitro, and pDC at sites of autoimmune kidney damage in systemic lupus erythematosus (SLE). Targeting the expression or function of these transporters with inhibitors of JAK2 and of xc— synergistically blocked cytokine production by pDC. We propose that such an approach may have value for treating SLE, and other diseases in which pDC are implicated.
2022-04-07 | GSE157305 | GEO
Project description:Cationic amino acid transport activity in a canine lens epithelial cells (LEC) line was investigated. The transporter activity of arginine was 0.424 ± 0.047 nmol/mg protein min, while the presence of N-ethylmaleimide, an inhibitor of the canine cationic amino acid transporter (CAT), reduced transport activity by 30%. A full-length cDNA sequence of canine CAT1 was 2558 bp long and was predicted to encode the 629 amino acid polypeptides. The deduced amino acid sequence of canine CAT1 showed similarities of 92.1% and 88.6% to those of the human and mouse, respectively. Western blot analysis detected a band at 70 kDa in a membrane protein sample of LEC. RT-PCR analysis confirmed that CAT1 was ubiquitously detected in all tissues examined.
| S-EPMC4160958 | biostudies-literature
Project description:Intracellular trafficking of the general amino acid permease, Gap1p, of Saccharomyces cerevisiae is regulated by amino acid abundance. When amino acids are scarce Gap1p is sorted to the plasma membrane, whereas when amino acids are abundant Gap1p is sorted from the trans-Golgi through the multivesicular endosome (MVE) and to the vacuole. Here we test the hypothesis that Gap1p itself is the sensor of amino acid abundance by examining the trafficking of Gap1p mutants with altered substrate specificity and transport activity. We show that trafficking of mutant Gap1p(A297V), which does not transport basic amino acids, is also not regulated by these amino acids. Furthermore, we have identified a catalytically inactive mutant that does not respond to complex amino acid mixtures and constitutively sorts Gap1p to the plasma membrane. Previously we showed that amino acids govern the propensity of Gap1p to recycle from the MVE to the plasma membrane. Here we propose that in the presence of substrate the steady-state conformation of Gap1p shifts to a state that is unable to be recycled from the MVE. These results indicate a parsimonious regulatory mechanism by which Gap1p senses its transport substrates to set an appropriate level of transporter activity at the cell surface.
| S-EPMC3103407 | biostudies-literature
Project description:L-[methyl-11C]Methionine (11C-Met) is useful for estimating the therapeutic efficacy of particle radiotherapy at early stages of the treatment. Given the short half-life of 11C, the development of longer-lived 18F- and 123I-labeled probes that afford diagnostic information similar to 11C-Met, are being sought. Tumor uptake of 11C-Met is involved in many cellular functions such as amino acid transport System-L, protein synthesis, and transmethylation. Among these processes, since the energy-dependent intracellular functions involved with 11C-Met are more reflective of the radiotherapeutic effects, we evaluated the activity of the amino acid transport System-A as an another energy-dependent cellular function in order to estimate radiotherapeutic effects. In this study, using a carbon-ion beam as the radiation source, the activity of System-A was evaluated by a specific System-A substrate, alpha-[1-14C]-methyl-aminoisobutyric acid (14C-MeAIB). Cellular growth and the accumulation of 14C-MeAIB or 14C-Met were evaluated over time in vitro in cultured human salivary gland (HSG) tumor cells (3-Gy) or in vivo in murine xenografts of HSG tumors (6- or 25-Gy) before and after irradiation with the carbon-ion beam. Post 3-Gy irradiation, in vitro accumulation of 14C-Met and 14C-MeAIB decreased over a 5-day period. In xenografts of HSG tumors in mice, tumor re-growth was observed in vivo on day-10 after a 6-Gy irradiation dose, but no re-growth was detected after the 25-Gy irradiation dose. Consistent with the growth results, the in vivo tumor accumulation of 14C-MeAIB did not decrease after the 6-Gy irradiation dose, whereas a significant decrease was observed after the 25-Gy irradiation dose. These results indicate that the activity of energy dependent System-A transporter may reflect the therapeutic efficacy of carbon-ion radiotherapy and suggests that longer half-life radionuclide-labeled probes for System-A may also provide widely available probes to evaluate the effects of particle radiotherapy on tumors at early stage of the treatment.
| S-EPMC5330493 | biostudies-literature
Project description:The effect of amino acids, in concentrations corresponding to those found in the portal vein of rats given a high-protein diet, was investigated on the activity of system A amino acid transport in hepatocytes from fed rats. Amino acids counteracted the induction of system A by insulin or glucagon. This effect was observed at all concentrations of hormones tested, up to 1 microM. Amino acids did not affect the basal cyclic AMP concentration in hepatocytes, or the large rise in cyclic AMP elicited by glucagon. The reversal of system-A induction was observed at relatively low concentration of amino acids, corresponding to plasma values reported in rats given a basal diet. Amino acids were separately tested: substrates of system A were particularly efficient, but so were glutamine and histidine. Non-metabolizable substrates of system A, such as 2-aminoisobutyrate, were also inhibitory, suggesting that a part of the effect of amino acids is independent of their cellular metabolism. Provision of additional energy substrates such as lactate and oleate did not affect induction of system A or the inhibitory effects of amino acids. Thus amino acids do not act by serving as an energy source and by maintaining the integrity of hepatocytes. Inhibition of mRNA synthesis by actinomycin practically abolished the effect of amino acids on the induction of system A by glucagon. The results suggest that amino acids may promote the synthesis of protein(s) affecting the activity of system A either directly at the carrier unit or at an intermediate stage of its emergence.
| S-EPMC1152747 | biostudies-other