Stimulation Of Amino Acid Uptake Research Articles

Regulation of enzyme activity by glucagon: Increased hormonal activity of iodinated glucagon

Iodination of glucagon enhanced its ability to activate adenyl cyclase in vitro, to elevate cyclic AMP levels in plasma in vivo, to induce two aminotransferases (PPT and TAT) in rat liver, and to stimulate amino acid uptake into rat liver in vivo. The degree of enhanced hormonal activity was proportional to the degree of iodination. Presumably the iodination of glucagon either confers on it greater intrinsic activity on the adenyl cyclase receptor or renders it less susceptible to degradation so that more glucagon molecules are available to act on the receptor.

Effects of gonadotrophins, dibutyryl cyclic AMP and prostaglandin E2 on amino acid transport and glycolysis in isolated rat corpora lutea.

AbstractCorpora lutea (CL) were isolated by free hand dissection from ovaries of 34–39 day‐old Sprague‐Dawley rats injected with 10 IU Pregnant Mare's Serum gonadotropin (PMSG) when 30 days old leacling to ovulation in the morning of day 33. Injection of luteinizing hormone (LH) to rats 2 h before removal of the ovaries stimulated the in vitro uptake of the non‐utilizable amino acids, a‐aminoisobutyric acid (AIB‐14C) and 1‐amino‐cyclopentane‐carboxylic acid (cycloleucine‐3H) by 3‐day‐old CL during a subsequent incubation in Krebs bicarbonate buffer. Prolactin and FSH were without effects. Addition of LH directly to the incubation medium did not stimulate amino acid uptake of the isolated CL, while addition of dibutyryl cyclic AMP (dbcAMP) enhanced this uptake. Glycolysis was estimated by determination of lactic acid accumulation in die incubation medium. When LH was injected to the animals 2 h before sacrifice, lactic acid production of 3‐day‐old CL was stimulated during a subsequent incubation period. Addition of LH in vitro caused a slight increase in the rate of glycolysis, and dbcAMP and prostaglanclin E2 (PGE2) also exhibited stimulatory effects on lactic acid production by the incubated CL. In a few experiments rats with 7‐day‐oId CL were given injections of LH. While amino acid uptake was not stimulated in these old CL, LH did enhance the rate of glycolysis in these CL.

Stimulation of Amino Acid Uptake by Euglena gracilis and the Effect of Cycloheximide

Summary Euglena gracilis cells of the logarithmic growth phase show a much lower rate of amino acid uptake than the cells of the stationary phase. This ‘repressed’ uptake can be stimulated several-fold by preincubation with those L-amino acids which have been taken up. D-amino acids and the basic L-amino acids are ineffective. The stimulation effect is reversible and decreases after removal of the stimulating amino acid. Cycloheximide in low concentrations strongly inhibits amino incorporation into bulk protein and some minutes later also the uptake process, but it does not prevent the stimulation effect.

Transport of amino acids into the estrogen-primed uterus IV. Effects of sulfhydryl agents on the uptake in vitro

1. 1. Sulfhydryl agents have been found to stimulate uptakes of some amino acids by the immature rat uterus in vitro, and to inhibit the uptakes of others. Those whose uptakes were stimulated can enter the cells well by the L transport system for amino acids, while those inhibited use the A system. Uptake of 1-aminocyclopentanecarboxylic acid (cycloleucine), which probably involves both systems, could be stimulated or inhibited, depending on the conditions. 2. 2. The results suggest that the uterus contains at least two kinds of groups that react with SH agents and are involved in amino acid transport. N- Ethylmalcimide combines relatively rapidly with one type, apparently involving the L system to produce stimulation of amino acid transport; but it combines more slowly with the other, the A system, to inhibit transport. 3. 3. The stimulation of amino acid uptake by estradiol, which probably involves the A system, can be inhibited by N- ethylmaleimide . This inhibition, however, is apparently not due to an action of N- ethylmaleimide to prevent a possible binding of the hormone to necessary uterine sites. 4. 4. The stimulation of amino acid uptake by N- ethylmaleimide appears to be independent of the stimulation by estradiol.

Studies on contact inhibition of growth in the mouse fibroblast, 3T3. II. Effects of amino acid deprivation and serum on growth rate.

ABSTRACT The effect of amino acid concentration on the initial growth rate of the contact-inhibited mouse fibroblasts 3T3 and its SV40-transformed derivative 3T3T has been studied. In medium in which the serum had been passed through a Sephadex column, and the concentration of amino acids lowered 1000-fold, one acid at a time, the growth rate constants were computed. For most of the 16 amino acids studied, the initial growth rate of 3T3 decreased more profoundly than that of 3T3T. The presence in serum of a factor which stimulates amino acid uptake and growth rate of 3T3T is described.

Effects of chelating agents on the initial interaction of phytohemagglutinin with lymphocytes and the subsequent stimulation of amino acid uptake

The chelating agents, ethylene glycol bis-(β-aminoethyl ether)- N, N′-tetraacetic acid (EGTA) and EDTA, had no effect on the initial interaction of phytohemagglutinin with lymphocytes at concentrations which have been shown previously to inhibit the development of the phytohemagglutinin response completely. However, they had a marked inhibitory effect on uptake of the amino acid analog, α-aminoisobutyric acid in both unstimulated and phytohemagglutinin-stimulated cells. The inhibition of amino acid uptake by EGTA could be reversed by adding Ca 2+ but not Mg 2+. These results demonstrated that Ca 2+ is not essential to the initial interaction of phytohemagglutinin with the cell, but does influence amino acid transport which may be a critical preparatory event for later increased protein synthesis.

Mechanisms of Active Transport in Isolated Bacterial Membrane Vesicles: VIII. THE TRANSPORT OF AMINO ACIDS BY MEMBRANES PREPARED FROM ESCHERICHIA COLI

Abstract The studies presented in this paper demonstrate that the transport of 16 amino acids by membrane vesicles prepared from Escherichia coli ML 308–225 exhibits properties similar to those found previously for respiration-linked β-galactoside transport. Concentrative uptake of all 16 amino acids is stimulated maximally by d-lactate and by the artificial electron donor system ascorbate-phenazine methosulfate, and to a lesser extent by succinate, l-lactate, dl-α-hydroxybutyrate, and NADH. Competitive uptake and displacement studies indicate that the vesicles possess at least nine distinct amino acid uptake systems with the following substrate specificities: proline, lysine, glycine-alanine, serine-threonine, glutamic acid-aspartic acid, phenylalanine-tyrosine-tryptophan, histidine, leucine-isoleucine-valine, cysteine. In the presence of ascorbate-phenazine methosulfate, the initial rates of lysine, alanine, serine, glutamic acid, tyrosine, leucine, cysteine, and lactose transport by membrane vesicles are 42 to 102% of those exhibited by appropriately treated whole cells. Initial rates of d-lactate-dependent transport as a function of external amino acid concentration yield simple hyperbolic kinetics for all but four amino acids: histidine, leucine, isoleucine, and valine yield biphasic velocity curves. Apparent Km values determined for the amino acids are in general agreement with results reported for intact cells. Steady state levels of d-lactate-dependent amino acid accumulation vary with temperature. These levels represent a balance between rates of influx and efflux, which can be shifted readily from one steady state level to another by raising or lowering the temperature. Different amino acid transport systems exhibit different steady state temperature optima, ranging from 20°–35°. In contrast, temperature optima for the initial rate of uptake occur at the same temperature (45°) with nearly all of the transport systems, and are similar to the temperature optimum for d-lactic dehydrogenase. There is no relationship between rates of oxidation of electron donors by the membranes (NADH g d-lactate g succinate g dl-α-hydroxybutyrate g l-lactate) and the ability of these compounds to stimulate amino acid uptake. These results indicate that the site of energy coupling between respiration and amino acid transport lies between the primary dehydrogenase and cytochrome b1. Furthermore, the relative effectiveness of these electron donors in supporting uptake varies widely among the various transport systems. The d-lactate-coupled concentrative uptake of proline, lysine, alanine, threonine, aspartic acid, tyrosine, histidine, leucine, and cysteine is inhibited by anaerobiosis, by the electron transfer inhibitors cyanide, 2-heptyl-4-hydroxyquinoline-N-oxide, amytal, and oxamate, and by p-chloromercuribenzoate; inhibition by the latter compound is reversed by dithiothreitol. Furthermore, anaerobiosis, cyanide, 2-heptyl-4-hydroxyquinoline-N-oxide, and amytal cause rapid efflux of the same amino acids from the intravesicular pool. Oxamate and p-chloromercuribenzoate, however, produce little or no efflux of accumulated amino acids. Moreover, p-chloromercuribenzoate blocks tyrosine exchange and cyanide-induced tyrosine efflux; in each case inhibition is reversed by dithiothreitol. Finally, cyanide-induced efflux of proline exhibits a Km for intramembranal proline which is 350 times higher than the Km determined for d-lactate-dependent uptake of external proline, whereas the Vmax values are the same for both processes. The findings presented are consistent with the conceptual model suggested for d-lactic dehydrogenase-coupled β-galactoside transport.

Differentiation on the basis of glucose requirements between the effects of somatomedin on protein synthesis and sulphate incorporation in embryonic chick cartilage

Extracellular glucose stimulates sulphate incorporation by embryonic chick or embryonic rat cartilage but not by 21-day old rat cartilage. In contrast glucose has little or no effect on amino acid transport or proline incorporation in any of these cartilages. Time course studies, in the presence or absence of glucose, suggest that the effect of extracellular glucose on sulphate incorporation in embryonic cartilage is due to the requirement for substrate for polysaccharide chain formation and not to a lack of an endogenous energy source. Stimulation by somatomedin of amino acid transport and incorporation in embryonic chick cartilage was independent of extracellular glucose. Glucose was required, however, for stimulation by somatomedin of sulphate incorporation. Stimulation of protein synthesis by somatomedin in the absence of glucose will result in a subsequent stimulation of sulphate incorporation if the tissue is transferred to a second medium containing glucose but no somatomedin. These results are interpreted as suggesting that in embryonic cartilage the limiting step in sulphated proteoglycan synthesis is protein synthesis in the presence of extracellular glucose but polysaccharide synthesis in the absence of glucose. They indicate that while the primary action of somatomedin is to stimulate amino acid uptake and hence incorporation, in the absence of glucose this stimulation cannot be potentiated as a stimulation of sulphate incorporation until adequate extracellular glucose is provided.

Role of serum, insulin and amino acid concentration in contact inhibition of growth of human cells in culture

When cultures of diploid human cells were confluent or approaching confluency, a change of medium produced a balanced stimulation of macromolecular synthesis and cell division. The essential components of the medium were to be found in serum, which was required for DNA synthesis, and in the increased concentration of amino acids which were necessary to promote protein synthesis when the capacity of cells in crowded cultures to take up amino acids is greatly reduced. There were indications that serum had the secondary function of stimulating amino acid uptake and this resulted in a limited amount of cell division when the amino acid level in the medium was growth-limiting. The role of insulin in promoting cell division was also studied, especially in relation to serum concentration. Insulin facilitated amino acid uptake, thus possibly having some serum sparing action, and there was also circumstantial evidence that insulin facilitated serum factor uptake by the cells and thereby promoted cell division in serum-limited media.

In vitro work load and rat heart metabolism. II. Effect on amino acid transport.

Abstract In vitro pressure overload was found to accelerate protein synthesis in the isolated working rat heart (Hjalmarson and Isaksson 1972 a). Since membrane transport of amino acids is considered to be one rate‐limiting step in protein synthesis, the amino acid transport in the isolated rat heart was investigated using the non‐utilizable amino acids α‐aminoisobutyric acid (AIB) and 1‐aminocyclopentane carboxylic acid (cycloleucine). Increased pressure load (afterload) accelerated amino acid uptake after a perfusion period of 15 ruin, and a 50—100% increase in the intracellular to extracellular distribution ratio of the amino acids was seen after 60 min of perfusion. This accelerated uptake was not inhibited by cycloheximide, suggesting that the work load effect was not dependent upon a continuous synthesis of proteins. The accumulation rate continued to be stimulated for some time after normalization of the work load and coronary flow, indicating that the work load effect was not directly linked to coronary flow. Increased preload did not stimulate amino acid uptake. Hearts from hypophysectomized rats showed a decreased concentrative uptake but the amino acid uptake was still accelerated by pressure overload. It is suggested that an increased uptake of amino acids could be of physiological significance in relation to the increased protein synthesis under these conditions.

Amino Acid Transport in Membrane Vesicles of Bacillus subtilis

Abstract Membrane vesicles of Bacillus subtilis actively transport amino acids in the presence of electron donors. The effectiveness of each physiological electron donor depends on the presence of the necessary membrane proteins that allow substrate oxidation and couple it to amino acid transport. Some of these proteins are present in all membranes while others (e.g. oxidizing glycerol phosphate) appear only in certain growth media (containing glycerol). Physiological electron donors are NADH, NADPH, l-α-glycerol phosphate, l-lactate, and succinate. NADH stimulates amino acid uptake at the highest initial rate whereas glycerol phosphate is the most effective electron donor since it energizes the transport of the largest number of amino acid molecules per nmole of substrate oxidized per min. Reduced phenazine methosulfate (PMS) is a very efficient nonphysiological electron donor which energizes amino acid transport at a high initial rate. At the membrane concentrations usually employed (1 to 5 mg of membrane protein per ml), NADH, NADPH, and reduced PMS consume oxygen so rapidly that special oxygenation is required to maintain the uptake of amino acids at a high rate. This uptake stops when the energy source has been oxidized; the accumulated amino acid then leaks out at a rate of 8% per min. With reduced (by ascorbate) PMS as electron donor, the Km and Vmax values of the uptake of 18 amino acids were determined. The Km values range from 10-5 to 10-6 m whereas the Vmax values vary widely for the different amino acids. Competition experiments have shown the presence of at least nine different amino acid transport carriers in the vesicles. The energy for active transport appears to be produced in the cytochrome-linked electron transport chain: all electron donors effective in amino acid transport cause the reduction of the membrane-bound cytochromes. The electron transport inhibitors antimycin, 2-heptyl-4-hydroxyquinoline-N-oxide, cyanide, and azide also inhibit amino acid transport, for all energy sources. Rotenone and oligomycin inhibit only the lactate-energized transport. Under conditions under which amino acids are transported no net ATP synthesis has been observed.

Action of insulin and growth hormone on protein synthesis in muscle from non-hypophysectomized rabbits.

The separate effects of insulin and growth hormone on the uptake and incorporation of five amino acids into diaphragm muscle from non-hypophysectomized rabbits has been examined. Both growth hormone and insulin, when present in the medium separately, stimulated the incorporation into protein of the amino acids, leucine, arginine, valine, lysine and histidine. Insulin also stimulated amino acid uptake, but growth hormone did not. When insulin and growth hormone were present in the incubation medium together, the uptake and incorporation of valine, the only amino acid studied under these conditions, tended to be greater than the sum of the separate effects of the two hormones.

Effect of isoproterenol on amino acid transport into rat salivary glands.

The effect of isoproterenol on the transport of a non-metabolizable amino acid, 14 C-α-aminoisobutyric acid ( 14 C-AIB), into salivary glands in vitro and in vivo was studied. Treatment with isoproterenol (IPR) in vivo increased the capacity of submandibular gland of rat to concentrate 14 C-AIB in vitro. Glands that were enlarged by the administration of isoproterenol for several days also had an increased intracellular uptake of 14 C-AIB. A single injection of isoproterenol stimulated 14 C-AIB uptake in vivo transiently. The submandibular glands of rats concentrated more 14 C-AIB 2–3 h after the injection of isoproterenol than the glands of control rats. At the same time the drug significantly increased the distribution ratio of 14 C-AIB/g wet tissue/ 14 C-AIB/ml blood measured during steady state distribution of 14 C-AIB. It is suggested that stimulation of amino acid uptake is probably one of the important mechanisms by which isoproterenol affects the growth of the salivary glands.

Hormonal control of protein synthesis.

Most hormones stimulate protein biosynthesis either of specific proteins, or in specific tissues, or in a more generalized fashion. In general protein anabolic hormones can be shown to stimulate amino acid uptake by tissues; to increase protein biosynthesis by a mechanism independent of the increased uptake of amino acids; to stimulate the labelling of ribonucleic acid (RNA) by tissues again in addition to stimulatory effects on precursor entry ; to stimulate RNA polymerase activity; to enhance the activity of ribosomes assayed in cell-free systems and to alter the patterns of polysomes analysed by sucrose density gradient centrifugation; to stimulate the synthesis of endoplasmic reticulum and of ribosomes and, in some instances, to stimulate deoxyribonucleic acid (DNA) synthesis. In general these effects are seen only after the hormone is injected into a suitable experimental animal or are added to the medium in which a cell culture or a tissue is incubated or with which an organ is perfused. There are a few, well-authenticated cases where hormones stimulate protein synthesis when added to cell-free systems. This fact means that certain difficulties are inherent in work with hormones. It is difficult to ensure that the effect observed after injection is not a secondary effect of the hormone through its influence on some other endocrine gland, or other tissue, or some other metabolic event.

Cyclic AMP stimulation of amino acid uptake in bone and kidney

Cyclic AMP stimulation of amino acid uptake in bone and kidney

Effect of Thyroid States on Amino Acid Uptake and Incorporation in the Chicken Anterior Pituitary

RECENTLY Tonoue and Yamamoto (1967a, b, 1968) demonstrated that amino acid metabolism in the rat pituitary gland is regulated by the feedback relationship between the thyroid and pituitary. For example, thyroidectomy stimulated amino acid uptake by the pituitary and the stimulation was depressed by injection of thyroxine. Thyroidectomy enhanced amino acid incorporation into pituitary proteins, and thyroxine injection was effective in suppression of this increased incorporation. Furthermore, they showed evidence that these changes in amino acid metabolism are closely associated with those of thyrotroph cells.Mikami (1958) has reached the conclusion the thyrotroph cells are confined largely to the cephalic lobe of the pars distalis of fowl hypophysis, because after thyroidectomy a marked hypertrophy and thyroidectomy cells are observed within this lobe, and thyrotrophic potency of the cephalic lobe is much higher than that of the caudal lobe.In view of this specialty in the fowl pituitary it seemed of…

Biochemical Studies of Synapses in Vitro: III. IONIC ACTIVATION OF PROTEIN SYNTHESIS

Abstract In synaptosomes isolated from rat brain cortex, sodium- and potassium-activated adenosine triphosphatase is present in high concentration (70 µmoles of Pi per mg of protein per 30 min). The maximal sodium-potassium-ATPase activity occurred with sodium and potassium concentrations similar to those necessary to achieve maximal protein synthesis, oxygen uptake, and potassium accumulation (50 mm sodium and 10 mm potassium). Furthermore, varying the concentrations of sodium or potassium yielded parallel alterations in sodium-potassium-ATPase activity, protein synthesis, and potassium transport. Ouabain (10-4 m) inhibited sodium-potassium-ATPase activity by 98%; protein synthesis, 40%; and active transport of potassium, 33%. Diphenylhydantoin, which may stimulate or inhibit ATPase activity depending upon the sodium to potassium ratio present, exerted parallel effects on protein synthesis. On the other hand, the ionic stimulation of amino acid uptake did not parallel the ionic stimulation of protein synthesis and potassium transport. Amino acid uptake was stimulated by sodium and inhibited by potassium. The apparent Km for 14C-leucine uptake was 3.0 mm in the absence of sodium and 0.7 mm in 50 mm sodium. Although potassium was inhibitory, both sodium and potassium were necessary for the optimal inhibitory action of ouabain. The results suggest a tight coupling of membrane ATPase activity, potassium transport, and the synthesis of synaptosome proteins. They further suggest that changes in ionic pump activity may modulate the synaptic metabolism of macromolecules.

Energy-limitation of a sodium-independent amino acid transport in ovarian oocytes of the frog.

AbstractThe frog, Rana pipiens, hibernates through the winter with ovaries containing oocytes which, in size and appearance, are ready for ovulation and maturation. From November through April, the normal time of egg laying, ovulation and maturation can be induced by interrupting hibernation and administering gonadotropic hormones. In the studies reported here, it has been found that oocytes taken from hibernating animals in early winter take up amino acids from a saline medium at a relatively rapid rate. Respiratory inhibition produced by such agents as dinitrophenol (DNP) and anaerobiosis, does not completely stop uptake but slows it down markedly.In late winter, amino acid uptake tends to be slower in normal cells and when DNP or cyanide is added, a marked acceleration of the rate of uptake is observed. The uptake in poisoned cells is accumulative, producing internal concentrations higher than that of the medium. At this concentration of DNP, amino acid incorporation is almost completely stopped. Fluoride abolishes the DNP stimulation of amino acid uptake. Removal of sodium ion from the incubating medium has no effect on either uptake or incorporation.These data are interpreted to mean that a capacity for oxidative phosphorylation in oocytes diminishes during winter hibernation. In the spring either an anaerobic capacity comes into being or becomes capable of being switched on. During this time the rate of amino acid uptake, even in the downhill phase, is limited by energy availability and is not dependent on a sodium gradient.

Effect of insulin in vivo on diaphragm and adipose tissue of obese mice.

Effect of insulin in vivo on diaphragm and adipose tissue of obese mice.

ACTION OF ESTROGEN SULFATES ON ACCUMULATION OF AMINO ACIDS BY EHRLICH ASCITES TUMOR CELLS IN VITRO.

Two water-soluble estrogen sulfates, estradiol disulfate and estrone sulfate, have been found to elevate the equilibrium distribution ratio for a number of free amino acids between the interior and the exterior of the Ehrlich mouse ascites carcinoma cells in vitro. The optimal effect is given when either agent is present at a level of around 1 mM. When estrone sulfate is added to levels above about 2 mM, it causes exchange of cellular K+ for external Na+, and amino acid uptake comes to be inhibited. In contrast, levels of estradiol disulfate as high as 10 mM do not affect cellular cation composition and they still stimulate amino acid uptake. Estrone sulfate therefore shows effects on the tumor cell more like those produced by equimolar amounts of pyridoxal. These and other experiments show that estradiol disulfate and estrone sulfate act somewhat differently on amino acid uptake. Both agents, however, lead to increased equilibrium distribution ratios by inhibiting efflux without an appreciable action on ...