Postnatal amino acid uptake by the rat small intestine. Energetics of membrane transport systems for amino acids in the developing jejunum

The energetics of amino acid uptake by the developing small intestine was investigated in vitro. L-valine, L-leucine, L-phenylalanine, L-methionine, L-lysine and L-arginine were all actively transported by the newborn rat jejunum. Metabolic inhibitors (e.g. 2,4-dinitrophenol) significantly reduced uptake of all amino acids but uptake against a concentration gradient was not totally abolished. Uptake of all amino acids was reduced at low[Na+]. Inhibition of transport of neutral amino acids by reduced luminal [Na+] was greater than that of basic amino acids, and the tissue was barely able to concentrate the neutral amino acids. [Na+] affected the Michaelis constant (Km) of neutral transport systems for their substrates; for the basic amino acids Km values were unaffected by the presence or absence of Na+. Ouabain significantly inhibited neutral amino acid uptake but had no effect on L-lysine or L-arginine uptake. These results are discussed in terms of the Na+ gradient hypothesis for amino acid transport, and the site of energy input to active transport. The role of glycolysis in providing energy for intestinal transport in the neonatal rat and the efficiency of Na+ dependent and independent transport mechanisms are considered. It is concluded that the energetics of amino acid transport systems in neonatal and adult rats are essentially similar.     

Kinetic analysis of insulin action on amino acid uptake by isolated chick embryo heart cells

1. Isolated chick embryo heart cells were used to investigate the mode of action of insulin on the transport of three naturally occurring amino acids: l-proline, l-serine and glycine. Initial velocities of uptake were measured over a period of 5min with an 80-fold range of amino acid concentration. Corrections for amino acid diffusion, incorporation into protein and conversion into carbon dioxide were introduced. 2. The uptake processes approximated Michaelis-Menten kinetics within definite ranges of amino acid concentrations. A single transport system for proline and at least two transport systems for serine and glycine were detected. 3. The kinetic effects of insulin on transport systems for the amino acids tested were consistent with an acceleration of the maximal velocity of the process, without substantial changes in substrate concentration for half-maximal transport velocity. 4. These hormonal effects were not essentially altered by the corrections for amino acid incorporation into protein and conversion into carbon dioxide.     

Neutral amino acid transport in isolated rat pancreatic islets

The neutral amino acid transport systems of freshly isolated rat pancreatic islets have been studied by first examining the transport of L-alanine and the nonmetabolizable analogue 2-(methylamino)isobutyric acid (MeAIB). By comparing the uptake of MeAIB and L-alanine for their pH dependency profile, choline and Li+ substitution for Na+, tolerance to N-methylation, and competition with other amino acids, the existence in pancreatic islets of both A and ASC amino acid transport systems was established. The systems responsible for the inward transport of five natural amino acids was studied using competition analysis and Na+ dependency of uptake. These studies defined three neutral amino acid transport systems: A and ASC (Na+-dependent) and L (Na+-independent). L-Proline entered rat islet cells mainly by system A; L-leucine by the Na+-independent system L. The uptake of L-alanine, L-serine, and L-glutamine was shared by systems ASC and L, the participation of system A being negligible for these three amino acids. An especially broad substrate specificity for systems L and ASC is therefore suggested for the rat pancreatic islet cells. The regulation of amino acid transport was also investigated in two conditions differing as to glucose concentration and/or availability, i.e. islets from fasted rats and islets maintained in tissue culture at high or low glucose concentrations. Neither alanine nor MeAIB transport was altered by fasting of the islet-donor rats. On the other hand, pancreatic islets maintained for 2 days in tissue culture at high (16.7 mM) glucose transported MeAIB at twice the rate of islets maintained at low (2.8 mM) glucose. Amino acid starvation of pancreatic islets during 11 h of tissue culture resulted in a 2-fold increase in MeAIB transport.     

Acidic amino acid transport in animal cells and tissues

1. The occurrence and characterization of acidic amino acid transport in the plasma membrane of a variety of cells and tissues of a number of organisms is reviewed. 2. Several cell types, especially in brain, possess both high- and low-affinity transport systems for acidic amino acids. 3. High-affinity systems in brain may function to remove neurotransmitter amino acid from the extracellular environment. 4. Many cell systems for acidic amino acid transport are energized by an inwardly directed Na+ gradient. Moreover, certain cell types, such as rat brain neurons, human placental trophoblast and rabbit and rat kidney cortex epithelium, respond to an outwardly directed K+ gradient as an additional source of energization. This simultaneous action may account for the high accumulation ratios seen with acidic amino acids. 5. Rabbit kidney has been found to have a glutamate-H+ co-transport system which is subject to stimulation by protons in the medium. 6. Acidic amino acid transport in rat brain neurons occurs with a stoichiometric coupling of 1 mol of amino acid to 2 mol of Na+. For rabbit intestine, one Na+ is predicted to migrate for each mol of amino acid. 7. Uptake in rat kidney cortex and in high-K+ dog erythrocytes is electrogenic. However, uptake in rabbit and newt kidney and in rat and rabbit intestine is electroneutral. 8. Na+-independent acidic amino acid transport systems have been described in the mouse lymphocyte, the human fibroblast, the mouse Ehrlich cell and in rat hepatoma cells. 9. In a number of cell systems, D-acidic amino acids have substantial affinity for transport; D-glutamate, in a number of systems, however, appears to have little reactivity. 10. Acidic amino acid transport in some cell systems appears to occur via the "classical" routes (Christensen, Adv. Enzymol. Relat. Areas Mol. Biol. 49, 41-101, 1979). For example, uptake in the Ehrlich cell is partitioned between the Na+-dependent A system (which transports a wide spectrum of neutral amino acids), the Na+-dependent ASC system (which transports alanine, serine, threonine, homoserine, etc.), and the Na+-independent L system (which shows reactivity centering around neutral amino acids such as leucine and phenylalanine). Also, a minor component of uptake in mouse lymphocytes occurs by a route resembling the A system. 11. Human fibroblasts possess a Na+-independent adaptive transport system for cystine and glutamate that is enhanced in activity by cystine starvation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Insulin regulation of amino acid transport in mesenchymal cells from avian and mammalian tissues.

Insulin regulation of amino acid transport across the cell membrane was studied in a variety of mesenchymal cell directly isolated from avian and mammalian tissues or collected from confluent cultures. Transport activity of the principal systems of mediation in the presence and absence of insulin was evaluated by measuring the uptake of representative amino acids under conditions approaching initial entry rates. Insulin enhanced the transport rate of substrate amino acids from the A system(alpha-aminoisobutyric acid, L-proline, glycine, L-alanine and L-serine) in fibroblasts and osteoblasts from chick-embryo tissues, in mesenchymal cells (fibroblasts and smooth muscle cells) from immature rat uterus, in thymic lymphocytes from young rats and in chick-embryo fibroblasts from confluent secondary cultures. In these tissues, the uptake of amino acid substrates of transport systems L and Ly+ (L-leucine, L-phenylalanine, L-lysine) was not affected by the presence of the hormone. No insulin control of amino acid transport was detected in chick-embryo chondroblasts and rat peritoneal macrophages. These observations identify the occurrence of hormonal regulatory patterns of amino acid transport for different mesenchymal cells types and indicate that these properties emerge early during cell differentiation.     

Amino acid transport systems of JapaneseParamecium symbiont F36-ZK

Analyses of amino acid transport systems in JapaneseParamecium symbiont F36-ZK were performed using¹⁴C-amino acids. Kinetic analyses of amino acid uptake and competitive experiments revealed three transport systems; a basic amino acid transport system, which catalyzed transport of L-Arg and L-Lys, a general amino acid transport system, which had broad specificity for 19 amino acids (but not L-Arg), and an alanine transport system. These three systems were considered to be capable of active transport. Amino acid-proton symport was indicated by the following data: decreases in pH of the medium observed during L-Ser and L-Ala uptake, and uptake of L-Arg, L-Ser and L-Ala being inhibited by carbonyl cyanide m-chlorophenylhydrazone, sodium azide and vanadate. The optimal pH for uptake of neutral amino acids and L-Arg was around 5 and 5 to 6.5, respectively. Uptake of L-Asp and L-Glu was very sensitive to pH and little uptake of L-Asp was measured above pH 6.0. Amino acid uptake was not inhibited by nitrate or ammonium, and cultured cells with ammonium also possessed constitutive uptake systems.     

Mechanism of amino Acid uptake by sugarcane suspension cells

The amino acid carriers in sugarcane suspension cells were characterized for amino acid specificity and the stoichiometry of proton and potassium flux during amino acid transport.

Amino acid transport by sugarcane cells is dependent upon three distinct transport systems. One system is specific for neutral amino acids and transports all neutral amino acids including glutamine, asparagine, and histidine. The uptake of neutral amino acids is coupled to the uptake of one proton per amino acid; one potassium ion leaves the cells for charge compensation. Histidine is only taken up in the neutral form so that deprotonation of the charged imidazole nitrogen has to occur prior to uptake. The basic amino acids are transported by another system as uniport with charge-compensating efflux of protons and potassium. The acidic amino acids are transported by a third system. Acidic amino acids bind to the transport site only if the distal carboxyl group is in the dissociated form (i.e. if the acidic amino acid is anionic). Two protons are withdrawn from the medium and one potassium leaves the cell for charge compensation during the uptake of acid amino acids. Common to all three uptake systems is a monovalent positively charged amino acidproton carrier complex at the transport site.

     

EVIDENCE FOR SEPARATE SYSTEMS FOR THE TRANSPORT OF NEUTRAL AND BASIC AMINO ACIDS ACROSS THE BLOOD-BRAIN BARRIER

Abstract— The effects of high circulating concentrations of several amino acids on the free amino acids of rat brain were measured, to see whether or not the results followed any consistent pattern. High circulating concentrations of large, neutral amino acids (phenylalanine, valine or isoleucine) caused significantly decreased values only of other large, neutral amino acids in the brains. High circulating concentrations of the basic amino acids lysine or arginine caused significantly decreased values only of each other. The data suggest that there are separate systems for the transport of neutral and basic amino acids across the blood-brain barrier. The effects of valine and lysine on the uptake by brain and the con-vulsant action of allylglycine (a neutral amino acid) were consistent with the concept of separate systems for the transport of amino acids across the blood-brain barrier. Valine inhibited the uptake by brain and the convulsant action of allylglycine in mice, but lysine did not. The data suggest that allylglycine and valine are transported into the brain by a common mechanism that does not transport lysine.     

Amino acid transport in isolated rat hepatocytes

Summary Improvements in the collagenase perfusion techniques have made isolated rat hepatocytes a popular model in which to study hepatic function. Our knowledge of hepatic amino acid transport has been advanced as a result of this methodology. Translocation across the hepatocyte plasma membrane can, in some instances, represent the rate-limiting step in the overall metabolism of certain amino acids. Furthermore, regulation of amino acid uptake by hepatocytes appears to play a role in diabetes, and perhaps in malignant transformation. Comparisons between normal adult hepatocytes and several hepatoma cell lines show basic differences in amino acids transport. There are at least eight distinct systems in normal hepatocytes for transport of the amino acids. One of these, System A, transports the small neutral amino acids most efficiently and responds to a wide variety of hormones. Systems A and N exhibit enhanced uptake rates after the cells have been maintained in the absence of extracellular amino acids, a phenomenon termed adaptive control. Further studies using isolated hepatocytes will increase our basic understanding of membrane transport processes and their regulation.     

Postnatal amino acid uptake by the rat small intestine. Changes in membrane transport systems for amino acids associated with maturation of jejunal morphology.

The uptake of a number of amino acids by the developing small intestine of the rat was investigated in vitro. L-valine, L-leucine, L-methionine, L-phenylalanine, L-arginine and L-lysine were all taken up by active transport and concentrated within the jejunal mucosa. GABA was not actively transported by the jejunum. The kinetics of carrier transport of amino acids was determined from birth to maturity. The Michaelis constant (Km) of the L-leucine, L-methionine, L-arginine and l-lysine transport systems was found to be low postnatally and increased with age, particularly after the time of weaning. The rate of l-leucine, L-methionine, L-phenylalanine and L-lysine transport (Vmax) was high postnatally but decreased after weaning. Neutral amino acids were transported at higher rates than basic amino acids. l-arginine was poorly transported by the jejunum. The specificity of transport systems for amino acids was investigated in inhibition studies. Amino acid transport systems appeared to be polyfunctional in the postnatal period but were more specific in post-weaned animals. The changes in kinetics and specificity of amino acid transport in the small intestine are discussed with reference to their possible functional significance and to the maturational changes in the jejunum, particularly with the appearance of a functionally distinct absorptive cell lining the intestinal villi during the third postnatal week (the time of weaning).     

Inhibition of sodium-independent amino acid transport by dexamethasone in rat hepatoma cells

We studied the uptake of leucine, phenylalanine, and the amino acid analog, 2-aminonorborane-2-carboxylic acid, by rat hepatoma cells in tissue culture. The uptake of these amino acids was partially mediated by a plasma membrane transport system similar to the L agency described in other cell types in that it does not require extracellular sodium and is subject to trans-stimulation. Initial rates of sodium-independent transport of these amino acids were calculated using mathematical transformations of the uptake time course curves. The glucocorticoid dexamethasone inhibits the activity of this transport system; the initial rates of sodium-independent uptake of leucine, phenylalanine, and 2-aminonorborane-2-carboxylic acid are decreased by approximately one-third (average = 30%, n = 19) after incubation of HTC cells with 0.1 microM dexamethasone. This inhibition requires at least 15 h, reaching a maximum at 24 h of exposure of the cells to the hormone. Dexamethasone has an asymmetrical effect on sodium-independent amino acid transport in that exposure of the cells to the hormone does not inhibit the rates of outflow of leucine or phenylalanine from preloaded cells into medium without sodium. Inhibition of uptake is blocked by 0.1 mM cycloheximide and 4 microM actinomycin D, indicating the need for continuous protein synthesis for dexamethasone action. Insulin, which is known to partially reverse the inhibitory effect of dexamethasone on the A amino acid transport system in HTC cells, does not alter the action of dexamethasone on the L system. Previous investigations have demonstrated inhibition by dexamethasone of at least two distinct sodium-dependent amino acid transport activities in HTC cells. The data presented here, showing inhibition by the glucocorticoid of a sodium-independent transport activity, indicate that the effect of the hormone is independent of the energy source of the amino acid transport systems affected.     

Substrate specificity of uptake of diamines in mouse brain slices

Brain slices upon incubation accumulate diamines (cadaverine and putrescine) from the medium against a concentration gradient up to an intracellular-to-medium ratio of 8. The transport system is different from the various systems for amino acids, among which is the transport system for basic amino acids. Diamine uptake, in contrast to amino acid uptake, is independent of Na⁺ and is increased at higher pH. There is some overlap among these transport classes—basic amino acids have a low affinity to the diamine system and some heteroexchange (stimulation of uptake) can be observed at very high concentrations between diamines and some amino acids (glycine, β alanine, γ-aminobutyrate, possibly also with proline and taurine). The diamine system seems also to be separate from the monoamine uptake systems. The results indicate the presence of numerous systems for metabolite transport in the brain with some overlap between systems.     

Cationic and anionic amino acid transport studies in rat red blood cells

The transport of L-proline, L-lysine and L-glutamate in rat red blood cells has been studied. L-proline and L-lysine uptake were Na⁺-independent. When the concentration dependence was studied both showed a non-saturable uptake assimilable to a difussion-like process, with high Kd values (0.718 and 0.191 min^–1 for L-proline and L-lysine respectively). Rat red blood cells showed high impermeability to L-glutamate. No sodium dependence was observed and the Kd value was low (0.067 min^–1). Our results show firstly, that rat red blood cells do not have amino acid transport systems for anionic and cationic amino acids and secondly that erythrocytes show no sodium-dependent L-proline transport, and that these cells are very permeable to this amino acid.Abbreviations MeAIB methyl aminoisobutyric acid     

The 4F2 antigen heavy chain induces uptake of neutral and dibasic amino acids in Xenopus oocytes.

The 4F2 cell surface antigen is a disulfide-linked heterodimer induced during the process of cellular activation and expressed widely in mammalian tissues (Parmacek, M. S., Karpinski, B. A., Gottesdiener, K. M., Thompson, C. B., and Leiden, J. M. (1989) Nucleic Acids Res. 17, 1915-1931). The human heavy chain component, a type II membrane glycoprotein, has 29% identity to the amino acid transport-related protein encoded by the recently cloned rat D2 cDNA. We have demonstrated that Xenopus oocytes injected with in vitro transcribed cRNA from D2 take up cystine and dibasic and neutral amino acids (Wells, R. G., and Hediger, M. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5596-5600). In the present study, we examine the role of the human 4F2 heavy chain in amino acid transport. In vitro transcribed 4F2 cRNA was injected into Xenopus oocytes which were assayed for the uptake of radiolabeled amino acids. Our results show that cRNA from 4F2 stimulates the uptake of dibasic and neutral amino acids into oocytes at levels up to 3-fold higher than for water-injected control oocytes. There is no demonstrable uptake of cystine. Uptake is saturable, with characteristics of high affinity transport, and inhibition data suggest that uptake occurs via a single transporter. Dibasic amino acids are taken up by both 4F2 and D2 cRNA-injected oocytes in a sodium-independent manner. In contrast, 4F2-induced but not D2-induced neutral amino acid uptake has a significant component of sodium dependence. Likewise, neutral amino acids in excess inhibit the 4F2-induced uptake of radiolabeled arginine but not leucine in a sodium-dependent manner. The 4F2-induced uptake we observe most likely represents the activity of a single transport system with some characteristics of systems y+, b0,+, and B0,+. We suggest that 4F2 and D2 represent a new family of proteins which induce amino acid transport with distinct characteristics, possibly functioning as transport activators or regulators.     

Perinatal changes of transport systems for amino acids in slices of mouse brain

Perinatal changes in the uptake of amino acids were measured in slices of fetal (15- and 19-day) and newborn (4-, 24-, and 48-hr-old) mouse brain. Uptake increased with age; smaller changes occurred with basic and neutral amino acid transport systems, and the largest changes occurred in fetal brain with amino acids of putative neurotransmitter function (taurine, glycine, GABA, and the acidic amino acids). The pattern of increase in uptake was similar at high and at low external amino acid concentrations. Developmental changes in tissue content of Na⁺, K⁺, or ATP were small during this period, and so are unlikely to be responsible for the observed changes in uptake. It appears that by the 15th day of fetal life, the transport systems for essential amino acids are fairly well developed in the brain, and the transport systems for neurotransmitter amino acids are not so well developed, but undergo a rapid increase in the 15–19-day period. From birth to adulthood, the concentrative capacity of slices of mouse brain for nonessential (putative neurotransmitter) amino acids is much greater than for essential amino acids.This research was supported in part by NIH Grant No. RR05707.     

Genetic analysis of amino acid transport in the facultatively heterotrophic cyanobacterium Synechocystis sp. strain 6803

The existence of active transport systems (permeases) operating on amino acids in the photoautotrophic cyanobacterium Synechocystis sp. strain 6803 was demonstrated by following the initial rates of uptake with 14C-labeled amino acids, measuring the intracellular pools of amino acids, and isolating mutants resistant to toxic amino acids. One class of mutants (Pfa1) corresponds to a regulatory defect in the biosynthesis of the aromatic amino acids, but two other classes (Can1 and Aza1) are defective in amino acid transport. The Can1 mutants are defective in the active transport of three basic amino acids (arginine, histidine, and lysine) and in one of two transport systems operating on glutamine. The Aza1 mutants are not affected in the transport of the basic amino acids but have lost the capacity to transport all other amino acids except glutamate. The latter amino acid is probably transported by a third permease which could be identical to the Can1-independent transport operating on glutamine. Thus, genetic evidence suggests that strain 6803 has only a small number of amino acid transport systems with fairly broad specificity and that, with the exception of glutamine, each amino acid is accumulated by only one major transport system. Compared with heterotrophic bacteria such as Escherichia coli, these permeases are rather inefficient in terms of affinity (apparent Km ranging from 6 to 60 microM) and of Vmax.     

PERIODIC CHANGES IN RATE OF AMINO ACID UPTAKE DURING YEAST CELL CYCLE

Uptake of amino acids is a complex process but in cells growing with ammonia as sole nitrogen source the initial uptake rate of amino acids is a measure of the transport capacity of the uptake system (permease). In synchronous cultures of Saccharomyces cerevisiae amino acids were transported at all stages of the cell cycle. However, for any one amino acid the initial uptake rate was constant for most of the cycle and doubled during a discrete part of the cycle. Thus, for a variety of amino acids the functioning amino acid transport capacity of the membrane doubles once per cycle at a characteristic stage of the cycle. Arginine, valine, and phenylalanine exhibit periodic doubling of uptake rate at different stages of the cell cycle indicating that the transport of these amino acids is mediated by three different systems. Serine, phenylalanine, and leucine exhibit periodic doubling of the uptake rate at the same stage of the cycle. However, it is unlikely that serine and phenylalanine share the same transport system since the uptake of one is not inhibited by the other amino acid. This phenomenon is analogous to the periodic synthesis of soluble enzymes observed in S. cerevisiae.     

The importance of amino acids as carbon sources for Micromonospora echinospora (ATCC 15837)

AIMS: This study set out to investigate the effect of amino acids on the uptake of glucose by Micromonospora eichinospora (ATCC 15837). METHODS AND RESULTS: The specific rate of glucose uptake was found to be reduced when organic nitrogen components were present in the medium. Radioactive uptake studies revealed that the Km for glucose in this organism was 53 mm, indicating a low affinity for uptake compared with other actinomycete sugar transport systems. Individual amino acids negatively influenced the rate of glucose transport, suggesting a relationship between amino acid metabolism and glucose uptake in this organism. The sugar transport system was found to be an active process being inhibited by ionophores and KCN. CONCLUSIONS: The data suggest a direct link between amino acid metabolism and glucose uptake at the level of sugar transport. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that the uptake of glucose, a major carbon source for many antibiotic fermentations, is significantly reduced in the presence of amino acids. This fact should inform the medium design and feeding regimes of fermentations involving similar actinomycetes.     

Transport of L-4-azaleucine in Escherichia coli.

The uptake of L-4-azaleucine was examined in Escherichia coli K-12 strains to determine the systems that serve for its accumulation. L-4=Azaleucine in radio-labeled form was synthesized and resolved by the action of hog kidney N-acylamino-acid amidohydrolase (EC 3.5.1.B) on the racemic alpha-N-acetyl derivative of DL-[dimethyl-14C]4-azaleucine. L-4-Azaleucine is taken up in E. coli by energy-dependent processes that are sensitive to changes in the pH and to inhibition by leucine and the aromatic amino acids. Although a single set of kinetic parameters was obtained by kinetic experiments, other evidence indicates that transport systems for both the aromatic and the branched-chain amino acids serve for azaleucine. Azaleucine uptake in strain EO317, with a mutation leading to derepression and constitutive expression of branched-chain amino acid (LIV) transport and binding proteins, was not repressed by growth with leucine as it was in parental strain EO300. Lesions in the aromatic amino acid transport system, aroP, also led to changes in the regulation of azaleucine uptake activity when cells were grown on phenylalanine. Experiments on the specificity of azaleucine uptake and exchange experiments with leucine and phenylalanine support the hypothesis that both LIV and aroP systems transport azaleucine. The ability of external azaleucine to exchange rapidly with intracellular leucine may be an important contributor to azaleucine toxicity. We conclude from these and other studies that at least four other process may affect azaleucine sensitivity: the level of branched-chain amino acid biosynthetic enzymes; the level of leucine, isoleucine, and valine transport systems; the level of the aromatic amino acid, aroP, uptake system; and, possibly, the ability of the cell to racemize D and L amino acids. The relative importance of these processes in azaleucine sensitivity under various conditions is not known precisely.