Inhibition Of Amino Acid Transport Research Articles

Na + influx and cell growth in cultured human fibroblasts : Effect of indomethacin

The effect of indomethacin on Na + influx and cell growth in human diploid fibroblasts (HSWP) has been investigated. It was found that both indomethacin and aspirin block serum-stimulated Na + influx in a dose-dependent manner (Ki = 0.34 ± 0.04 mM and 11 ± 1 mM respectively) while having no effect on influx of Na + in the absence of serum. The Ki for inhibition of [ 3H]thymidine incorporation into HSWP cells (0.28 ± 0.02 mM) closely correlated with the Ki for inhibition of Na + influx. The onset of action of indomethacin is rapid (within 2 min) and inhibition of Na + flux is readily reversed (within 5 min). Other workers have reported that indomethacin is cytostatic for human fibroblasts presumably via a slowly developing inhibition of “A” system amino acid transport [6]; however, present results indicate that inhibition of Na + influx in HSWP cells occurs much more rapidly than the inhibition of amino acid transport observed in other human foreskin fibroblasts and therefore may be more closely related to the primary cellular locus of indomethacin action.

Inhibition of amino acid transport and enhancement of tryptophan binding in a water mold by a range of natural and synthetic cytokinins

Approximately 30 purine compounds were examined for their influence on the energy-linked amino acid transport systems of Achlya (a freshwater mold). Most of them were inhibitory, and their effectiveness was related to (a) the presence of a substituent group at position 6 of the purine ring, (b) the length of hydrocarbon chain as substituent, and (c) the type of linkage between the substituent group and the heterocyclic ring. The most effective linkage group was SH followed by an NH2 group, and an oxygen bridge which was the least effective. Inhibition was immediate (suggesting an interaction with the cell membrane) and pH and temperature dependent. Only tryptophan uptake was anomalous, for while its transport was inhibited, its binding to a cell wall – membrane proteoglycan was stimulated severalfold. From kinetic studies, we deduced that the analogues and tryptophan were interacting with the same cell entity. Nucleoside forms of the analogues did not inhibit amino acid transport; this was understandable, since nucleosides are transported by an energy-linked system whereas nucleic acid bases are not. The purine analogues themselves were taken up very poorly by this organism.

Steroid specificity of the glucocorticoid inhibition of amino acid transport in rat hepatoma cells.

Dexamethasone, a synthetic glucocorticoid, inhibits the initial rate of transport of the nonmetabolizable amino acid, alpha-aminoisobutyric acid, in rat hepatoma tissue culture (HTC) cells. To determine whether this inhibition is mediated by the same proximal steps as is the steroidal induction of tyrosine aminotransferase, we have examined the hormonal specificity of these two responses for various steroids previously characterized with respect to transaminase induction as agonists, partial agonists, antagonists, or inactive steroids. We conclude that the steroidal inhibition of amino acid transport, at steroid concentrations of 10(-5) M or less is mediated by the same glucocorticoid receptor mechanisms as the induction of tyrosine aminotransferase. First, the concentrations at which the full agonists, dexamethasone and cortisol, produce their half-maximal effects on phenomena are the same. Second, tetrahydrocortisol, which does not interact with the glucocorticoid receptor, neither inhibits transport nor induces transaminase. Third, the competitive interactions between partial agonists or antagonists and the full agonist dexamethasone with respect to both transport inhibition and enzyme induction are virtually identical. At concentrations greater than 10(-5)M, however, both partial agonist and antagonist steroids are capable of fully inhibiting amino acid transport. The effects of these steroids on transport, like those of dexamethasone, are reversible and are blocked by cycloheximide and actinomycin D. Furthermore, these steroids, like dexamethasone, slow the rate of efflux of alpha-aminoisobutyric acid from preloaded cells. Thus, the effects of high concentrations of partial agonist and antagonist steroids on amino acid transport do not appear to reflect a generalized toxic effect on membrane function.

Growth inhibitory protein(s) in the 3T3 cell plasma membrane. Partial purification and dissociation of growth inhibitory events from inhibition of amino acid transport.

It has been reported previously that a plasma membrane-enriched fraction from 3T3 cells arrests the growth of sparse 3T3 cells early in the G1 phase of growth (Whittenberger et al., '78, '79). Addition of membranes to sparse 3T3 cells also results in a decrease of the rate of transport of alpha-aminoisobutyric acid (Lieberman et al., '79). We have partially purified the growth-inhibitory proteins from plasma membrane of 3T3 cells. These growth-inhibitory proteins behave as intrinsic membrane proteins and do not require membrane lipids for activity. The partially purified proteins arrest cell growth without affecting the rate of alpha-aminoisobutyric transport; thus, inhibition of both transport and cell growth are not obligatorily coupled events.

A colchicine-sensitive uptake system in Morris hepatomas.

The interference of microtubular disruptors with the uptake of amino acids and other low molecular weight substrates has been studied in Morris hepatomas, host liver, and regenerating liver. Colchicine inhibits amino acid transport (alpha-aminoisobutyric acid, L-methionine, and L-leucine) in hepatomas by 59-98% whereas transport in host and regenerating liver is not impeded but increased. In hepatomas, treatment urea, and carbonate. Vinblastine, but not lumicolchicine or cytochalasin B, is an effective inhibitor. The inhibition of uptake is not linked to a decrease of cellular ATP and UTP. The data suggest that the transport of low molecular weight substrates in hepatomas is related to microtubules or other colchicine-binding structures, e.g., of the plasma membrane. This colchicine-sensitive uptake system in hepatomas may be due to the malignant transformation of hepatocytes.

Uncoupling Action of Amytal in Membrane Vesicles from Escherichia coli

The barbiturate amytal (5-ethyl-5-isopentylbarbituric acid) has been shown to inhibit amino acid transport in membrane vesicles from anaerobically grown Escherichia coli. Amytal has no effect on the activity of the enzymes of the nitrate respiration system, nor on electron transfer in this system. However, addition of amytal to the membrane vesicles results in a decrease of the membrane potential from -90 mV to -72 mV, and to a decrease of the pH-gradient of -61 mV to undetectable values. Furthermore, amytal causes an increase in the rate of ferricyanide reduction in liposomes, indicating that amytal increases the proton permeability of phospholipid membranes. These results demonstrate that amytal acts as an uncoupler in membrane vesicles from anaerobically grown E. coli.

Inhibition of amino acid transport into lymphoid cells by the glutamine analog L-2-amino-4-oxo-5-chloropentanoate.

Transport of L-glutamine and of the chloroketone glutamine analog L-2-amino-4-oxo-5-chloropentanoate into lymphoid cells is mediated by the same system. Arginine and a number of other amino acids (e.g., glutamate, aspartate, and lysine) are transported to a much lesser extent by this system. However, after uptake of the chloroketone into the cells, the transport of glutamine, arginine, and other amino acids is markedly inhibited, due evidently to reaction of the chloroketone with intracellular components that are involved in amino acid transport. The chloroketone acts more effectively on growing than on resting cells. Treatment of lymphoid cells with the chloroketone or with L-chloro-2,4-dinitrobenzene leads to rapid and complete depletion of intracellular glutathione without affecting cell viability. These reagents appear to be useful experimental tools for studies of glutathione function and metabolism.

Effect of xanthines and some derivatives on protein metabolism in isolated parenchymal rat liver cells

(1) Isolated parenchymal rat liver cells were incubated in Krebs-Henseleit buffer containing 0.5% gelatine. (2) Xanthines (caffeine, theophylline, theobromine, paraxanthine) and some derivatives (IBMX, aminophylline, proxiphylline) all inhibited the incorporation of [ 14C]valine into proteins in the medium and albumin secretion. (3) The incorporation into liver cell proteins was only significantly inhibited by IBMX, caffeine and theophylline. (4) Xanthine and metabolites of xanthines had no or only slight effect on the variables mentioned above. (5) The inhibitory effect of xanthines and their derivatives on protein metabolism could not be explained by an increased level of cyclic AMP, a decreased secretion of proteins from the liver cells, an inhibition of amino acid transport, an increased degradation of proteins or an effect at the transcription level. (6) The results are compatible with an effect on protein synthesis on the translation level.

Inhibition of bacterial transport by uncouplers of oxidative phosphorylation. Effects of pentachlorophenol and analogues in Bacillus subtilis.

Analogues of the potent uncoupler of oxidative phosphorylation pentachlorophenol were tested as inhibitors of proline and glycine transport by Bacillus subtilis. These analogues included less highly substituted chlorophenols and pentachlorothiophenol. Like pentachlorophenol, they are non-competitive inhibitors of proline transport and uncompetitive inhibitors of glycine transport. However, the less highly substituted chlorophenols are weaker acids than pentachlorophenol and also weaker inhibitors. Analysis indicated that the anionic form of the uncouplers is the inhibiting species. Pentachlorothiophenol, a water-insoluble anion, is also a potent inhibitor. These results support previous studies that concluded that uncouplers of oxidative phosphorylation inhibit amino acid transport by binding at specific sites on proteins, the free energy of interaction stabilizing 'unproductive' conformations. Such specific interactions of uncoupler with protein are probably commonplace.

Mechanism of inhibition of amino acid transport by naricinoleate

Mechanism of inhibition of amino acid transport by naricinoleate

Inhibition by colchicine of changes in amino acid transport and initiation of DNA synthesis in regenerating rat liver.

The uptake of amino acids (measured with alpha-aminoisobutyric acid) by hepatocytes stimulated to proliferate by partial hepatectomy followed a biphasic pattern, with an initial peak 8-10 hr after surgery followed by a release of amino acids between 10 and 16 hr and a further increase between 16 and 20 hr after the operation. The second period of increased uptake coincided with the increase in DNA synthesis by the hepatocytes. Colchicine, and other microtubule disrupters administered at the time of partial hepatectomy, abolished the first period of increased amino acid uptake and prevented the cells from initiating DNA synthesis. Pulse-labeling experiments with alpha-aminoisobutyric acid revealed that colchicine did not inhibit amino acid transport per se, but prevented the increased capacity for amino acid transport induced by partial hepatectomy. An injection of colchicine 14 hr after hepatectomy prevented the release of amino acids by the liver and also substantially reduced the initiation of DNA synthesis. The data suggest that the microtubules may play a role in proliferative activation and also in the initiation of DNA synthesis of hepatocytes in vivo and that changes in amino acid transport are linked to these microtubule-requiring processes.

Glucocorticoid inhibition of amino acid transport in rat hepatoma cells

Dexamethasone rapidly and reversibly inhibits the initial rate of transport of α-aminoisobutyric acid in rat hepatoma cells in tissue culture. Colcemid and cytochalasin B neither inhibit transport nor interfere with its inhibition by dexamethasone, arguing that microtubules and microfilaments are not involved in this hormonal effect. Continuous protein synthesis is required both for the dexamethasone inhibition of transport and for its reversal, although cycloheximide alone inhibits transport in control cells by less than 25%. A model for the dexamethasone inhibition of amino acid transport is presented suggesting that glucocorticoids either block the synthesis or enhance the degradation of a rate-limiting protein in the transport system.

Negative interactions between amino acid and methylamine/ammonia transport systems of Saccharomyces cerevisiae.

The transport of methylamine (methylammonium ion) and ammonia (ammonium ion) is accomplished in Saccharomyces cerevisiae by means of a specific active transport system. L-Amino acids are noncompetitive inhibitors of methylamine transport. This inhibition is relieved or eliminated in mutant strains that have a reduced ability to transport amino acids. The inhibition of methylamine transport occurs immediately upon the addition of amino acids to the assay system and persists until the external amino acid pool is depleted. The degree of inhibition observed is a direct function of the rate of amino acid transport. Both methylamine and ammonia are capable of inhibiting amino acid transport. The inhibition of amino acid transport is eliminated in mutant strains that cannot transport methylamine and ammonia.

Inhibition of Amino Acid Transport in Escherichia coli by Some Beta-Lactam Antibiotics

Among 10 antibiotics tested, cephaloridine and cephalothin showed the greatest inhibition of proline active transport. Ethylenediaminetetraacetate pretreatment of the cells did not enhance inhibition by any of these 10 antibiotics. The inhibition of active transport of 10 additional amino acids by cephaloridine and cephalothin was studied. Both antibiotics inhibited transport of three amino acids which, like proline, have transport systems resistant to osmotic shock; neither antibiotic inhibited transport of the remaining amino acids, including three with shock-resistant and four with shock-sensitive systems.

Amino acid transport in the rat exocrine pancreas. II. Inhibition by lanthanum and tetracaine.

The two calcium antagonistic agents lanthanum and tetracaine cause severe disturbances in the secretory process of the exocrine pancreas, including inhibition of the rate of protein synthesis and exocytosis. The former effect resulted mainly from the inhibition of amino acid transport. Lanthanum in a concentration up to 1 mM inhibited transport of different species of amino acids in an unspecific way whereas tetracaine interfered specifically with the Na+ -dependent transport system for neutral amino acids (14C-alpha-amino-isobutyric acid). Na+ -dependent transport of neutral amino acids (3H-leucine) was not affected. Transport inhibition was correlated to the acitvity of the Na+, K+ -ATPase system which was measured in isolated plasma membrane fractions. At higher concentrations (5-10 mM) some uptake of lanthanum into the cells by limited endocytosis was observed. At lower concentrations lanthanum seemed to bind exclusively to certain components of the plasms membrane, mainly at the lateral and basal cell surface. Even at a concentration of 5-10 mM no binding to the apical surface occurred. Similarly no binding of lanthanum was observed to the limiting membrane of isolated zymogen granules, while mitochondria, contained in the same fraction showed considerable binding affinity. The action of lanthanum and tetracaine on membrane carrier systems did not affect the interior organization of the plasma membrane. Particle density and distribution in freeze-fracture replicas as well as the submembrane microfilamentous=microtubular system and the junctional elements remained unaffected.

ANTIMICROBIAL ACTION OF ALIPHATIC DIOLS AND THEIR ESTERS

ABSTRACTThe antimicrobial activity of a variety of aliphatic diols and their esters has been investigated. Inhibition of growth of Bacillus subtilis 60015 was found to depend on chain length and position of the hydroxyl groups. 1,2‐diols were more effective than 1,3diols. Esterification also affected antimicrobial activity. Studies with membrane vesicles prepared from B. subtilis indicated that inhibition of amino acid transport is a primary antimicrobial effect of diols and their esters.

Inhibition of amino acid transport in rat small intestine by N.EPSILON.-substituted L-lysine derivatives.

Effect of Ne-dansyl-L-lysine [Ne-(5-dimethylaminonaphthalenesulfonyl)-L-lysine] on amino acid transport across the brush border of isolated rat small intestine was investigated. Ne-Dansy-L-lysine was not actively accumulated nor transmurally transported by the intestinal tissue; nonetheless, it competitively inhibited the basic amino acid transport with a Ki of about 0.4mM for L-lysine, but entirely not the neutral amino acid transport. The inhibition was relatively independent of extracellular Na+ The minimal dependence on Na+ parallels with the least Na+ dependence of lysine transport among various amino acid transport systems, suggesting the inhibition competes for a step involved in a heteroexchange process mediating the greater portion of lysine transport. The results provided a basis for a potential use of Ne-dansyl-L-lysine as a fluorescent probe in delineating its interaction with basic amino acid transport system in the small intestine.

Inhibition of amino acid transport in Bacillus subtilis by uncouplers of oxidative phosphorylation

The effect of three uncouplers of oxidative phosphorylation, trifluoromethoxycarbon-ylcyanidephenylhydrazone (FCCP), 3,3′,4′,5-tetrachlorosalicylanilide (TCSA), and pentachlorophenol (PCP), on transport of glycine and proline by Bacillus subtilis were examined. FCCP inhibited proline uptake uncompetitively, but glycine uptake competitively. TCSA inhibited proline uptake noncompetitively, but glycine uptake competitively. PCP inhibited proline uptake noncompetitively, but glycine uptake uncompetitively. The results indicate that these uncouplers inhibit amino acid transport by interacting at specific sites rather than by reducing any central supply of energy used to fuel metabolic processes.

Studies on the stimulation of cAMP metabolism in human lymphocytes by latex polymers

The purpose of this investigation was to further characterize the marked increase in intracellular cAMP which follows the interaction of human lymphocytes and latex polymers. Six distinct cell types, each of which either bind or ingest these latex particles, were studied; however, only lymphocytes responded with increases in intracellular cAMP. The initial attachment of the latex particles to the lymphocyte surface was independent of temperature, cyclic nucleotides and divalent cations in the external milieu. The subsequent cAMP response was maximal at physiologic temperatures and modulated by agents thought to alter microfilament and microtubule function. Four different types of polymers produced increases in intralymphocytic cAMP and the maximal increases were confined to particles having a mean diameter of 0.4–2.02 μm. Within this latter size range, there was a close correlation between the number of membrane-associated particles and the magnitude of the cAMP response. Similarities to the lymphocyte-lectin activation system included: 1. 1. A requirement for binding of the latex polymers to the external plasma membrane. 2. 2. A biphasic cAMP response characterized by an early rise followed by a later fall. 3. 3. Modulation of this response by pharmacologic agents which compromise microtubule and microfilament function. In contrast to the lectin-induced activation, latex beads inhibited amino acid transport and phosphatidylinositol turnover and did not lead to later increases in DNA synthesis. These data suggest that latex polymers attach to receptors on the plasma membrane different from those responsible for lymphocyte activation, and through cAMP induce metabolic responses dissimilar to those associated with lectin activation.

Inhibition of amino acid transport by ammonium ion in Saccharomyces cerevisiae

The rate of transport of L-amino acids by Saccharomyces cerevisiae epsilon 1278b increased with time in response to nitrogen starvation. This increase could be prevented by the addition of ammonium sulfate or cycloheximide. A slow time-dependent loss of transport activity was observed when ammonium sulfate (or ammonium sulfate plus cycloheximide) was added to cells after 3 h of nitrogen starvation. This loss of activity was not observed in the presence of cycloheximide alone. In a mutant yeast strain which lacks the nicotinamide adenine dinucleotide phosphate-dependent (anabolic) glutamate dehydrogenase, no significant decrease in amino acid transport was observed when ammonium sulfate was added to nitrogen-starved cells. A double mutant, which lacks the nicotinamide adenine dinucleotide phosphate-dependent enzyme and in addition has a depressed level of the nicotinamide adenine dinucleotide-dependent (catabolic) glutamate dehydrogenase, shows the same sensitivity to ammonium ion as the wild-type strain. These data suggest that the inhibition of amino acid transport by ammonium ion results from the uptake of this metabolite into the cell and its subsequent incorporation into the alpha-amino groups of glutamate and other amino acids.