Short-chain Neutral Amino Acids Research Articles

Inhibitory Effects of Basic Drugs on the Sodium-Dependent Transport of L-Alanine via System B0 in the Small Intestine

To elucidate the mechanism of the interaction of basic drugs with Na(+)-dependent L-alanine absorption from the small intestine, we investigated the effect of imipramine on the transport of L-alanine via system B(0), which is thought to be one of the main Na(+)-dependent systems for intestinal absorption of short-chain neutral amino acids, including L-alanine. The uptake of L-alanine by cells that express hATB(0) (human amino acid transporter B(0)) was inhibited in the presence of imipramine. The Eadie-Hofstee plot showed that the inhibition was not competitive with the substrate (L-alanine) but competitive with Na(+). When rat intestinal brush border membrane vesicles were used, several basic drugs had inhibitory effects. Inhibition was also observed in intestinal absorption evaluated by an in situ single-pass perfusion technique. However, the potencies of inhibition were different. The potency of inhibition was dependent on the lipophilicity of the drugs.

Insulin Promotes the Cell Surface Recruitment of the SAT2/ATA2 System A Amino Acid Transporter from an Endosomal Compartment in Skeletal Muscle Cells

SAT1-3 comprise members of the recently cloned family of System A transporters that mediate the sodium-coupled uptake of short chain neutral amino acids, and their activity is regulated extensively by stimuli such as insulin, growth factors, and amino acid availability. In skeletal muscle, insulin stimulates System A activity rapidly by a presently ill-defined mechanism. Here we demonstrate that insulin induces an increase in the plasma membrane abundance of SAT2 in a phosphatidylinositol 3-kinase-dependent manner and that this increase is derived from an endosomal compartment that is required for the hormonal activation of System A. Chloroquine, an acidotropic weak base that impairs endosomal recycling of membrane proteins, induced a complete inhibition in the insulin-mediated stimulation of System A, which was associated with a loss in SAT2 recruitment to the plasma membrane. The failure to stimulate System A and recruit SAT2 to the cell surface could not be attributed to a block in insulin signaling, as chloroquine had no effect on the insulin-mediated phosphorylation of protein kinase B or glycogen synthase kinase 3 or upon insulin-stimulated GLUT4 translocation and glucose transport. Our data indicate strongly that insulin increases System A transport in L6 cells by stimulating the exocytosis of SAT2 carriers from a chloroquine-sensitive endosomal compartment.

Subcellular localization and adaptive up-regulation of the System A (SAT2) amino acid transporter in skeletal-muscle cells and adipocytes.

The recently cloned amino acid transporter SAT2 is ubiquitously expressed and confers Na(+)-dependent transport of short-chain neutral amino acids, characteristics of the functionally defined System A transporter. Here we report the presence of SAT2 mRNA and protein in both skeletal muscle and adipocytes, and the characterization of polyclonal antibodies directed against this transporter. SAT2 protein was present in both plasma-membrane and internal-membrane fractions derived from rat skeletal muscle and adipose tissue, L6 myotubes and 3T3-L1 adipocytes, having a localization similar to that of the glucose transporter GLUT4. Moreover, consistent with the adaptive up-regulation of System A activity following chronic amino acid deprivation, a time-dependent increase in SAT2 protein abundance was observed in amino-acid-deprived L6 myotubes and 3T3-L1 adipocytes. These studies provide the first evidence regarding the subcellular distribution and adaptive up-regulation of SAT2 protein and the characterization of molecular probes for this physiologically important transporter, the function of which is altered in several disease states.

L-leucine and L-alanine uptake by trout ( Salmo trutta ) cardiomyocytes: the effect of IGF-I and insulin

The uptake of L-leucine and L-alanine by trout cardiomyocytes was analysed to identify the carriers involved in their incorporation into cells. The sodium independent systems L and asc, as well as the sodium dependent system ASC, were found to be present. The Vmax of these carriers was 33.0, 40.1 and 19.4 pmol amino acid mg−1 prot min−1 for L, asc and ASC, respectively, and the Km values obtained for these transport systems were 296, 269 and 349 μM amino acid. The L system had as its main substrates the large apolar branched-chain amino acids and showed stereospecificity. The asc and ASC systems carried the short-chain neutral amino acids and were not inhibited by Me-AIB. The ASC transporter also showed adaptive regulation. The effect of IGF-I and insulin on L-alanine uptake, as well as the incorporation of L-leucine into protein, were also studied. Low IGF-I concentrations (below 2.5 ng ml−1) stimulated the uptake of L-alanine and protein synthesis but this effect reverted at high IGF-I concentrations. Insulin depressed protein synthesis and did not affect L-alanine uptake.

Cloning and Functional Expression of ATA1, a Subtype of Amino Acid Transporter A, from Human Placenta

This report describes the primary structure and functional characteristics of human ATA1, a subtype of the amino acid transport system A. The human ATA1 cDNA was isolated from a placental cDNA library. The cDNA codes for a protein of 487 amino acids with 11 putative transmembrane domains. The transporter mRNA (∼9.0 kb) is expressed most prominently in the placenta and heart, but detectable level of expression is evident in other tissues including the brain. When expressed heterologously in mammalian cells, the cloned transporter mediates Na+-coupled transport of the system A-specific model substrate α-(methylamino)isobutyric acid. The transport process is saturable with a Michaelis-Menten constant of 0.89 ± 0.12 mM. The Na+:amino acid stoichiometry is 1:1 as deduced from the Na+-activation kinetics. The transporter is specific for small short-chain neutral amino acids. The gene for the transporter is located on human chromosome 12.

Lipid, polyamide, and flavonol phagostimulants for adult western corn rootworm from sunflower (Helianthus annuus L.) pollen.

Adult Diabroticites including western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte, consume pollen of corn, squash, sunflower, and other species. Short-chain neutral amino acids in methanol-water extracts of pollen have been previously identified in our laboratory as strong phagostimulants for Diabrotica. Bioassay-driven fractionation was used to characterize the interacting lipid and midpolarity phagostimulants for adult WCR in Giant Gray Stripe sunflower, Helianthus annuus L., pollen. Lipids rich in omega3-linolenic acid including triglycerides, free fatty acids, phosphatidylethanolamines, phosphatidic acids, and phosphatidylcholines were highly phagostimulatory. Other important phagostimulatory components included a hydroxycinnamic acid-polyamine amide, N(1),N(5),N(10)-tri[(E)-p-coumaroyl]spermidine, and a flavonol, quercetin beta-3-O-glucoside. The structural characteristics of these phagoactive compounds and their role in the pollinivory specialization of rootworm beetles are discussed.

Seasonal variation in uptake of short-chain neutral amino acid by red blood cells and hepatocytes in trout (Salmo trutta).

The present study shows that the capacity of trout (Salmo trutta) red blood cells (RBCs) and freshly isolated hepatocytes to take up short-chain neutral amino acids changes according to a seasonal pattern. Maximal amino acid uptake rates in RBCs were obtained in winter and spring, while minima were seen in summer and autumn. In contrast, the maximal rates for the freshly isolated hepatocytes were obtained in autumn and winter, and the minima were seen in spring and summer. In addition, by studying the uptake of glycine, evidence was found that the activities of the amino acids carriers ASC, asc and Gly in RBCs varied according to a seasonal rhythm. The activity of the ASC and asc systems changed in parallel with the global uptake of amino acids. Moreover, the RBC:plasma concentration ratio for certain substrates of these carriers (alanine, serine and glycine) varied accordingly. In contrast, the activity of the Gly system was modified inversely with respect to the overall amino acid uptake. The activity of the ASC system in freshly isolated hepatocytes was also seasonally modified, reaching a maximum in autumn, shortly before the reproductive period.

Regulation of the ASC system and Na+/K + pump activities in brown trout (Salmo trutta) hepatocytes

The present study investigates the regulation of Na+/K+ pump activity and alanine uptake in trout hepatocytes. Pump activity increased when cells were incubated in an amino-acid-free medium, while it was reduced in cells from fasted animals. Short-term exposure (3 h) to glucagon modified the activity of the pump in a complex seasonally dependent pattern: in experiments carried out in autumn and winter there was some inhibition, while in spring the pump was activated by this hormone. Pharmacological modification of levels of two intracellular signal transducers, namely cyclic AMP and Ca2+, always led to a reduction in pump activity. These experiments were conducted in May, when activation of the pump by glucagon exposure occurred. There is no apparent explanation for the mechanism by which this hormone modifies the activity of the pump. Glucagon also regulates the activity of system ASC (a Na+-dependent amino acid carrier with short-chain neutral amino acids as preferred substrates). This regulation also showed a seasonally dependent pattern, although the pattern was opposite to that found for the regulation of Na+/K+ pump activity.

Na(+)-independent L-alanine uptake by trout cells. Evidence for the existence of at least two functionally different acs systems.

The Na(+)-independent uptake of L-alanine has been studied in trout red blood cells, isolated hepatocytes and peripheral blood lymphocytes. The present study shows the existence of two functionally different Na(+)-independent systems for short chain neutral amino acids in these cells. They are designated as asc systems because of their resemblance to systems described in other cell types. Besides their independence of sodium and a rough similarity in substrate preference, the most important property shared by the two carriers is a lack of trans-stimulation, allowing further differentiation from system L. One of them is an unusually stereospecific carrier present in red blood cells, the other is less restrictive and present in hepatocytes and peripheral blood lymphocytes. Extracellular acid pH increases the incorporation to red blood cells, while it slightly depresses the uptake in the other cells. From the data presented, it is not possible, at first, to classify these carriers as asc1 or asc2 systems. Moreover, the system present in red cells resembles that found in the nonerythroid cells, BSC-1, while there is no clear parallelism between the system found in hepatocytes/lymphocytes and any of those described previously.

Effect of Amino Acid Deprivation on L-Alanine Uptake Through the Asc System in Freshly Isolated Trout Hepatocytes

ABSTRACT Fasting in mammals and other vertebrates induces an increase in the ability of liver to extract from blood gluconeogenic substrates, such as plasma amino acids, which can also be used as an energy source (Cowey et al. 1977; Newsholme and Leech, 1983). In mammalian hepatocytes, food deprivation induces the appearance of a high-affinity component for short-chain amino acid transport, with the properties of system A, while there are no changes in the activities of a low-affinity system (the ASC system), system L (Fehlmann et al. 1979) or glutamine uptake (Hayes and McGivan, 1982). ‘A’ is the abbreviation for a Na+-dependent carrier which has L-alanine and other short-chain neutral amino acids as preferred substrates. Its tolerance to N-methylated analogues differentiates it from the ASC system. ‘L’ is the abbreviation for a Na+-independent carrier which has L-leucine as preferred substrate. ‘asc’ is the abbreviation for a carrier similar to the ‘ASC’ carrier with respect to preferred substrates, but it is Na+-independent. The ASC system is a widely distributed, short-chain neutral amino acid carrier that transports alanine, serine, cysteine and threonine in a Na+-dependent mode, although the scope of its substrates is now thought to be wider than when it was first described in Erlich ascites tumour cells (Christensen et al. 1967). Characteristically, it does not accept N-methylated amino acid derivatives (allowing easy distinction from the A system).

Transduction mechanisms for the taste of amino acids

Amino acids are important taste stimuli for a variety of animals. One animal model, the channel catfish, I. punctatus, possesses sensitive taste receptor systems for several amino acids. Neurophysiological and biochemical receptor binding studies suggest the presence of at least three receptor pathways: one is a relatively nonspecific site(s) responsive to short-chain neutral amino acids such as L-alanine (L-ALA); another is responsive to the basic amino acid L-arginine (L-ARG); still another is a low affinity site for L-proline (L-PRO). Several possible transduction pathways are available in the taste system of this animal model for these amino acids. One of these, formation of inositol trisphosphate (IP3) and cyclic AMP (cAMP), is mediated by GTP-binding regulatory proteins, while another involves ion channels directly activated by stimuli. L-ALA is a potent stimulus to cAMP and IP3 accumulation, while L-ARG at low concentrations is without effect. On the other hand, L-ARG and L-PRO, but not L-ALA, are able to activate stimulus-specific and cation-selective channels in taste epithelial membranes reconstituted in phospholipid bilayers at the tips of patch pipettes. Preliminary studies using mouse taste tissue demonstrate that monosodium-L-glutamate (MSG) did not enhance production of IP3 or cAMP. However, in reconstitution experiments using taste epithelium of mouse, conductance changes due to MSG are observed. The specificity of this channel(s) and its uniqueness have yet to be determined.

How many Na +-dependent carriers for l-alanine and l-proline in the eel intestine? Studies with brush-border membrane vesicles

Using brush-border membrane (BBM) vesicles prepared from the intestine of the European eel, the specificity of l-alanine and l-proline Na +-dependent transport was investigated by measuring the uptake of isotopically labelled subsrates. In the presence of Na + ions, cross-inhibition between alanine and proline transports was observed; in addition α-(methylamino)isobutyric acid (MeAIB) inhibited proline but had no effect on alanine uptake. These results can be explained by the presence, in eel intestinal BBM vesicles, of at least two distinct agencies for Na +-dependent proline and alanine translocation. The first system is specific for alanine and short-chain neutral amino-acids; the second system, specific for imino acids and the N-methylated analogues, is regulated by alanine concentration.

Ligand binding specificity of a neutral l-amino acid olfactory receptor

1. 1. The ligand binding specificity of the l-[ 3H]alanine binding site was investigated in isolated cilia preparations from the olfactory epithelium of channel catfish ( Ictahurus punctatus) by competitive binding experiments. 2. 2. Approximately 45 amino acids, derivatives and enantiomers were tested for the ability to compete with radiolabeled l-alanine for common binding sites. 3. 3. Acidic and basic l-amino acids and imino acids did not compete as effectively as l-alanine for the receptor, while long-chain neutral ligands were only partially effective inhibitors of l-alanine binding. 4. 4. d-Alanine and l-alanine derivatives with substituted α-amino or carboxyl groups exhibited decreased ability to compete for the receptor, paralleling their lower neurophysiological potency. 5. 5. In combination, the ligand binding results were consistent with previous electrophysiological data in catfish, and suggest the presence of an olfactory receptor site that selectively recognizes short-chain neutral amino acids.

The amino acid transport systems of the autotrophically grown green alga Chlorella

The kinetic analysis of l-amino acid uptake by the green alga Chlorella revealed at least seven different uptake systems to be present in cells grown autotrophically with nitrate as nitrogen source. There is a ‘general system’ which transports most neutral and acidic amino acids, a system for short-chain neutral amino acids including proline, a system for basic amino acids including histidine, a special system for acidic amino acids, and specific systems for methionine, glutamine and threonine. The ‘general system’ is possibly the same as that which can be stimulated by incubation of cells in glucose plus ammonium (Sauer, N. (1984) Planta 161, 425–431). The incubation of Chlorella in glucose induces the increased synthesis of six amino acid uptake systems, namely the above-mentioned system for short-chain neutral amino acids, a threonine system, a methionine system, and a glutamine system. These results indicate that the uptake of l-amino acids by the green alga Chlorella is as complex as in other free-living organisms such as bacteria or yeast. The small number of amino acid uptake systems found in cells of higher plants, i.e. two or three, seems therefore to be a consequence of integration of the cells in a tissue supplying a relatively constant environment, and not a consequence of autotrophic growth on mineral carbon and mineral nitrogen.

GABA transport by nerve ending fractions of cat brain.

Abstract— Nerve ending and mitochondrial fractions were prepared from cat cerebral cortex by differential and sucrose density gradient centrifugation. The isolated fractions were characterized morphologically and enzymatically and the nerve‐ending fraction was shown to be particularly rich in glutamate decarboxylase activity. The uptake of [3H] γ‐aminobutyric acid (GABA) was studied by trapping the particles on a Millipore filter.GABA uptake was not saturable over the concentration range studied (0.018 to 8000 μM. Tissue/medium ratios were consistently greater than 2·5, and uptake was significantly reduced by ouabain and iodoacetate, or by replacing sodium with lithium or choline chloride. GABA uptake was significantly inhibited by desmethylimipramine (DMI) with an IC50 of approx. 10–20 μM. GABA uptake was reduced by structurally similar amino acids lacking an α‐amino group, and also by methionine and phenylalanine, but not by short chain neutral amino acids.