Transport of threonine and tryptophan by rat brain slices: relation to other amino acids at concentrations found in plasma

Threonine content of brain decreases in young rats fed a threonine-limiting, low protein diet containing a supplement of small neutral amino acids (serine, glycine and alanine), which are competitors of threonine transport in other systems (Tews et al., 1977). Threonine transport by brain slices was inhibited more by a complex amino acid mixture resembling plasma from rats fed the small neutral amino acid supplement than by mixtures resembling plasma from control rats or from rats fed a supplement of large neutral amino acids. Greater inhibition was seen with mixtures containing only the small neutral amino acids than with mixtures containing only large neutral amino acids. On an equimolar basis, serine and alanine were the most inhibitory; large neutrals were moderately so; and glycine and lysine were without effect. Threonine transport was also strongly inhibited by α-amino-n-butyric acid and homoserine, less so by α-aminoisobutyric acid, and not at all by GABA. The complex amino acid mixtures strongly inhibited α-aminoisobutyric acid transport by brain or liver slices but, in contrast to effects in brain, the extent of the inhibition in liver was not much affected by altering the composition of the mixture. Tryptophan accumulation by brain slices was effectively inhibited by other large neutral amino acids in physiologically occurring concentrations. Threonine, or a mixture of serine, glycine and alanine only slightly inhibited tryptophan uptake; basic amino acids were without effect and histidine stimulated tryptophan transport slightly. These results support the conclusion that a diet-induced decrease in the concentration in brain of a specific amino acid may be related to increased inhibition of its transport into brain by increases in the concentrations of transport-related, plasma amino acids.     

The reduction of cystine during its transport into rat jejunal everted segments and sacs

1. Everted segments and sacs of rat jejunum were incubated in buffer containing [(35)S]cystine. 2. Concentration gradients were achieved by both segments and sacs, and the effects of duration of incubation and of cystine concentration on the isotope distribution ratios were determined. 3. Kinetic constants were determined for the uptake of cystine by both segments and sacs, and the differences between the two systems are discussed. 4. Reduction to cysteine was virtually complete intracellularly and in the sac lumen. Extensive reduction in the medium occurred only when segments were incubated. 5. Anaerobiosis prevented a concentration gradient being obtained between the medium and the tissue, but had little effect on the extent of reduction to cysteine in the tissue and sac lumen. 6. It is concluded that cystine is transported by an active process into rat jejunum, where it is present almost entirely in the reduced form, and that efflux of cysteine occurs through the serosal surface.     

Cytotoxicity as an indicator for transport mechanism. Evidence that melphalan is transported by two leucine-preferring carrier systems in the L1210 murine leukemia cell

Melphalan, l-phenylalanine mustard, is transported by the L1210 cell through carriers of the leucine (L) type. Its initial rate of transport is inhibited by both l-leucine, a naturally occurring L system amino acid and 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH), a synthetic amino acid which is transported by the L system in the Ehrlich ascites tumor cell. Both amino acids inhibited melphalan transport comparably in sodium-free medium. However, BCH, in medium containing sodium, was unable to reduce a component of melphalan transport which was readily inhibited by leucine but not by α-aminoisobutyric acid. Inhibition analysis indicated that leucine competes with BCH for transport but that a portion of leucine transport is not readily inhibited by BCH. These results suggest that in the L1210 cell melphalan is transported equally by a BCH-sensitive, sodium-independent L system and a BCH-insensitive, sodium-dependent L system.     

Transport of methionine and proline by rat liver slices and the effect of certain hormones

1. Transport characteristics of l-methionine and l-proline in rat liver slices in vitro were studied. 2. Intracellular concentration gradients for methionine were obtained. 3. Methionine uptake was inhibited by iodoacetate, dinitrophenol, Na⁺-free media and also by glycine, lysine, cysteine and dithiothreitol but not by α-aminoisobutyrate. 4. The rate of methionine metabolism in the slice was slow. 5. Puromycin inhibited methionine incorporation into protein, but not methionine uptake. 6. Methionine inhibited the transport of α-aminoisobutyrate but not of cystine. 7. Efflux and exchange diffusion of methionine was studied. 8. Amino acid transport in rat liver slices was not affected by thyroidectomy. 9. Addition of insulin, glucagon, adrenaline or cortisol did not affect the transport of methionine. 10. Addition of 6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate increased methionine transport after a 120min incubation period in some experiments. 11. Studies of l-proline transport were invalidated because of the rapid evolution of CO₂ from the substrate.     

Energetics of sodium-dependent α-aminoisobutyric acid transport in the moderate halophile Vibrio costicola

The energetics of α-aminoisobutyric acid transport were examined in Vibrio costicola grown in a medium containing the NaCl content (1 M) optimal for growth. Respiration rate, the membrane potential (Δψ) and α-aminoisobutyric acid transport had similar pH profiles, with optima at 8.5–9.0. Cells specifically required Na⁺ ions to transport α-aminoisobutyric acid and to maintain the highest Δψ (150–160 mV). Sodium was not required to sustain high rates of O₂-uptake. Δψ (and α-aminoisobutyric acid transport) recovered fully upon addition of Na⁺ to Na⁺-deficient cells, showing that Na⁺ is required in formation or maintenance of the transmembrane gradients of ions. Inhibitions by protonophores, monensin, nigericin and respiratory inhibitors revealed a close correlation between the magnitudes of Δψ and α-aminoisobutyric acid transport. Also, dissipation of Δψ with triphenylmethylphosphonium cation abolished α-aminoisobutyric acid transport without affecting respiration greatly. On the other hand, alcohols which stimulated respiration showed corresponding increases in α-aminoisobutyric acid transport, without affecting Δψ. Similarly, N,N′-dicyclohexylcarbodiimide (10 μM) stimulated respiration and α-aminoisobutyric acid transport and did not affect Δψ, but caused a dramatic decline in intracellular ATP content. From these, and results obtained with artificially established energy sources (Δψ and Na⁺ chemical potential), we conclude that Δψ is obligatory for α-aminoisobutyric acid transport, and that for maximum rates of transport an Na⁺ gradient is also required.     

Maturation of membrane function: Transport of amino acid by rat erythroid cells

The membrane changes which occur during cellular maturation of erythroid cells have been investigated. The transport of α-aminoisobutyric acid, alanine, and N-methylated-α-aminoisobutyric acid have been studied in the erythroblastic leukemic cell, the reticulocyte, and the erythrocyte of the Long-Evans rat. The dependence of amino acid transport on extracellular sodium concentration was investigated. Erythrocytes were found to transport these amino acids only by Na-independent systems. The steady state distribution ratio was less than 1. Reticulocytes were found to transport α-aminoisobutyric acid and alanine by Na-dependent systems, but only small amounts of N-methylated-α-aminoisobutyric acid. Small amounts of these amino acids were transported by Na-independent systems. The steady state distribution ratio was greater than one for Na-dependent transport. The erythroblastic leukemia cell, a model immature erythroid cell, showed marked Na-dependence (>90%) for α-aminoisobutyric acid and alanine transport, and >80% for the Na-dependent transport of N-methyl-α-aminoisobutyric acid. The steady state distribution ratio for the Na-dependent transport was >4. In the erythroblastic leukemic cell, at least three Na-dependent systems are present: one includes alanine and α-aminoisobutyric acid, but excludes N-methyl-α-aminoisobutyric acid; one is for α-aminoisobutyric acid, alanine and also N-methyl-α-aminoisobutyric acid; and one is for N-methyl-α-aminoisobutyric acid alone. In the reticulocyte, the number of Na-dependent systems are reduced to two: one for α-aminoisobutyric acid and alanine; one for N-methyl-α-aminoisobutyric acid. In the erythrocytes, no Na-dependent transport was found. Therefore, maturation of the blast cell to the mature erythrocyte is characterized by a systematic loss in the specificity and number of transport systems for amino acids.     

l-Phenylalanine active transport in the midgut of Bombyx mori larva

l-phenylalanine and α-aminoisobutyric acid are actively transported from the lumen to the haemolymph in the isolated midgut of Bombyx mori larva. Kinetics of l-phenylalanine fluxes as a function of concentration have been studied. The influx shows a convex relationship to the aminoacid concentration, while the outflux bears a linear relationship within the range of concentrations considered. The net flux shows a saturation kinetic, is sodium independent and is inhibited by DNP and anoxia. l-phenylalanine pools obtained by luminal or haemolymph loading are similar and no intracellular accumulation of the aminoacid takes place. Conversely the amount of α-aminoisobutyric acid entering the cells through the basolateral membrane exceeds that of luminal origin and it reaches an intracellular concentration twofold higher than that of the bathing medium.     

Mechanism of action of Clostridium perfringens enterotoxin. Effects on membrane permeability and amino acid transport in primary cultures of adult rat hepatocytes

Purified enterotoxin from the bacterium Clostridium perfringens rapidly decreased the hormonally induced uptake of α-aminoisobutyric acid in primary cultures of adult rat hepatocytes. At 5 min after toxin addition the decrease in α-aminoisobutyric acid uptake appeared not due to increased passive permeation (estimated with l-glucose) or to increased α-aminoisobutyric acid efflux. When short uptake assay times were employed a depression of α-aminoisobutyric acid influx was observed in toxin-treated hepatocytes. The depression of α-aminoisobutyric acid influx was correlated with a rapid increase in intracellular Na⁺ (estimated using ²²Na⁺) apparently effected by membrane damage. In contrast, the uptake of cycloleucine in the presence of unlabeled α-aminoisobutyric acid (assay for Na⁺-independent amino acid uptake) by hepatocytes treated with toxin for 5 min was decreased to only a small extent or not at all depending upon experimental design. At later times, C. perfringens enterotoxin increased the exodus of l-glucose, $\text{3-O-}\text{methylglucose}$ and α-aminoisobutyric acid from pre-loaded cells indicating that the toxin effects progressive membrane damage. When enterotoxin was removed by repeated washing after 5–20 min the decay of α-aminoisobutyric acid uptake ceased and appeared to undergo recovery towards the hormonally induced control level. The degree of recovery of α-aminoisobutyric acid uptake was inverse to the length of time of exposure to toxin. Adding at 10 min specific rabbit antiserum against C. perfringens enterotoxin without medium change also reversed the effect of toxin on increased intracellular ²²Na⁺, and on the exodus (from preloaded cells) of α-aminoisobutyric acid, L-glucose, and $\text{3-O-}\text{methylglucose}$.     

Uptake of l-[35S]Cystine by Isolated Rat Brain Capillaries

Adult rat brain capillaries were isolated by a simplified procedure and showed an enrichment of the marker enzyme, γ-glutamyltranspeptidase. The uptake of [³⁵S]cystine at 37°C by this preparation can be divided into two components, a sodium- and energy-dependent transport process for the free amino acid pool, with an apparent K_m of 36 μm , and a binding process, with an apparent K_m of 1.13 mm . Chemical analysis of the amino acid pool indicates that cystine is the major form of intracapillary ³⁵S. Cystine transport was not inhibited by lysine, but glycine, α-methylaminoisobutyric acid and β-2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid were inhibitory to a small extent.     

Transport and metabolic effects of α-aminoisobutyric acid in saccharomyces cerevisiae

α-Aminoisobutyric acid is actively transported into yeast cells by the general amino acid transport system. The system exhibits a K_m for α-aminoisobutyric acid of 270 μM, a V_max of 24 nmol/min per mg cells (dry weight), and a pH optimum of 4.1–4.3. α-Aminoisobutyric acid is also transported by a minor system(s) with a V_max of 1.7 nmol/min per mg cells. Transport occurs against a concentration gradient with the concentration ratio reaching over 1000:1 (in/out). The α-aminoisobutyric acid is not significantly metabolized or incorporated into protein after an 18 h incubation. α-Aminoisobutyric acid inhibits cell growth when a poor nitrogen source such as proline is provided but not with good nitrogen sources such as NH₄⁺. During nitrogen starvation α-aminoisobutric acid strongly inhibits the synthesis of the nitrogen catabolite repression sensitive enzyme, asparaginase II. Studies with a mutant yeast strain (GDH-CR) suggest that α-aminoisobutyric acid inhibition of asparaginase II synthesis occurs because α-aminoisobutyric acid is an effective inhibitor of protein synthesis in nitrogen starved cells.     

Reconstitution and characterization of a sodium-stimulated active aminoisobutyric acid transport system derived from partially purified plasma membranes from mouse fibroblasts transformed by simian virus 40: comparison of reconstituted vesicles with native membrane vesicles

A Na⁺-specific and Na⁺-stimulated active α-aminoisobutyric acid transport system was reconstituted from plasma membranes isolated from mouse fibroblast BALB/c 3T3 cells transformed by simian virus 40. The plasma membranes were treated with dimethylmaleic anhydride and then extracted with 2% cholate. The cholate-solubilized supernatant proteins were combined with exogenous phospholipids and eluted through a Sephadex G-50 column. This yielded reconstituted vesicles which in the presence of Na⁺ could actively transport α-aminoisobutyric acid as shown by the transient accumulation above the equilibrium level (overshoot). The overshoot was not obtained with other monovalent cations such as K⁺, Li⁺, and choline⁺. The electrochemical effect of the lipophilic anion, SCN^?, led to greater α-aminoisobutyric acid uptake as compared to that observed with Cl^? or SO₄^2?. The Na⁺-stimulated transport of a-aminoisobutyric acid was a saturable process with an apparent K_m of 2 mm. Studies of the inhibition of α-aminoisobutyric acid transport by other amino acids showed that methylaminoisobutyric acid [specifically transported by A system (alanine preferring)]had a pronounced inhibitory effect on a-aminoisobutyric acid uptake in contrast to the slight inhibitory effect produced by phenylalanine [primarily transported by L system (leucine preferring)]. The results show that the reconstituted vesicles, prepared from partially purified membrane proteins and exogenous phospholipids, regained the same important transport properties of native membrane vesicles, i.e., Na⁺-specific and Na⁺-stimulated concentrative α-aminoisobutyric acid uptake.     

Uptake of alpha-aminoisobutyric acid in pig spleen lymphocytes stimulated by sodium periodate.

The effect of sodium periodate on the ability of pig spleen lymphocytes to transport the nonmetabolizable amino acid, α-aminoisobutyric acid, was studied. NaIO₄-treated cells exhibited a lowered rate of uptake of α-aminoisobutyric acid in contrast to phytohemagglutinin- and concanavalin A-treated cells. However, when periodate-treated cells were preincubated with untreated cells for 2 h, the mixed cells exhibited twofold stimulation in the uptake of α-aminoisobutyric acid as compared to untreated cells. The increased uptake of α-aminoisobutyric acid in mixed cells was due to a change in the V but not in the K_m. The observed increased uptake of α-aminoisobutyric acid in mixed cells was inhibited (24%) by ouabain, although the level of uptake in untreated and NaIO₄-treated cells was not affected. Na⁺,K⁺-ATPase activity in mixed cells, which was ouabain sensitive, was stimulated 56%. Studies also showed that there was a decrease in the fluorescence polarization (P value) of diphenyl hexatriene in mixed cells (P = 0.21) as compared to untreated cells (P = 0.24). These results demonstrate that NaIO₄ treatment induces a change in the lymphocyte cell membrane and transport of α-aminoisobutyric acid. Incubation of NaIO₄-treated cells with untreated cells is required for the stimulatory effect in the uptake of α-aminoisobutyric acid, and the stimulation appears to be due to changes in Na⁺,K⁺-ATPase activity and membrane fluidity.     

Depression of amino acid transport in cultured rat hepatocytes by purified enterotoxin from Clostridiumperfringens

Rat liver parenchymal cells (hepatocytes) were isolated by a collagenase perfusion technique and maintained as monolayers in serum-free medium in collagen-coated culture dishes. Glucagon, in combination with dexamethasone, induced α-aminoisobutyric acid transport in these cells. Addition of purified $\text{Clostridium}\text{perfringens}$ enterotoxin to hepatocytes preinduced by glucagon and dexamethasone rapidly depressed (but did not abolish) α-aminoisobutyric acid transport. The toxin effect was dose dependent: 1000 or 300 ng/ml produced maximal depression whereas 100 or 40 ng/ml were without effect in 120 minutes. The effect was eliminated by pretreating the toxin with heat or specific antisera. The effect of enterotoxin on α-aminoisobutyric acid transport in two cultured rat hepatoma cell lines (H4-II-E-C3 and McA-RH 7777) was also investigated. Only the McA-RH 7777 cells were sensitive to the toxin suggesting that the enterotoxin may interact with specific membrane components of normal rat liver cells which are also present on some (but not all) cancerous rat liver cells.     

Sodium-dependent neutral amino acid transport by human liver plasma membrane vesicles

The activities of several selected Na(+)-dependent amino acid transporters were identified in human liver plasma membrane vesicles by testing for Na(+)-dependent uptake of several naturally occurring neutral amino acids or their analogs. Alanine, 2-(methylamino)isobutyric acid, and 2-aminoisobutyric acid were shown to be almost exclusively transported by the same carrier, system A. Kinetic analysis of 2-(methylamino)isobutyric acid uptake by the human hepatic system A transporter revealed an apparent Km of 0.15 mM and a Vmax of 540 pmol.mg-1 protein.min-1. Human hepatic system A accepts a broad range of neutral amino acids including cysteine, glutamine, and histidine, which have been shown in other species to be transported mainly by disparate carriers. Inhibition analysis of Na(+)-dependent cysteine transport revealed that the portion of uptake not mediated by system A included at least two saturable carriers, system ASC and one other that has yet to be characterized. Most of the glutamine and histidine uptake was Na(+)-dependent, and the component not mediated by system A constituted system N. The largest portion of glycine transport was mediated through system A and the remainder by system ASC with no evidence for system Gly activity. Our examination of Na(+)-dependent amino acid transport documents the presence of several transport systems analogous to those described previously but with some notable differences in their functional activity. Most importantly, the results demonstrate that liver plasma membrane vesicles are a valuable resource for transport analysis of human tissue.     

A reduction in energy-dependent amino acid transport by microtubular inhibitors in ehrlich ascites tumor cells

Vincristine, other periwinkle alkaloids, and colchicine partially inhibit the energy dependent transport of α-aminoisobutyric acid in Ehrlich ascites tumor cells. The properties of this phenomenon were characterized in detail for vincristine. Maximum depression of the steady-state intracellular α-aminoisobutyric acid level was achieved with a vincristine concentration of > 0.5 m̈M. The inhibitory effect of vincristine increases as the extracellular α-aminoisobutyric acid concentration is increased reaching a maximum, however, of only ∼25% at a level of 5 mM, leaving a large gradient for α-aminoisobutyric acid across the cell membrane. Vincristine produced an asymmetrical effect on the bidirectional fluxes decreasing the initial uptake rate, while increasing the efflux of α-aminoisobutyric acid. Inhibition of net α-aminoisobutyric acid transport by vincristine was partially reversible (∼40%). Colchicine (50 m̈M) reduced the steady-state α-aminoisobutyric acid level by 30%, an effect that was not reversible. Inhibition by vinleurosine and vinrosidine was comparable to that of vincristine. Addition of glucose to the medium resulted in a small, but significant, decrease in the inhibitory effects of both vincristine and colchicine. The data indicate that these agents inhibit a small component of the uphill transport of α-aminoisobutyric acid in Ehrlich ascites tumor cells. The inhibitory effect of vincristine cannot be attributed to an increase in the passive permeability of the cell membrane to this agent. Rather, the data along with other studies from this laboratory suggest that vincristine reduces the energy-dependent transport of α-aminoisobutyric acid by either inhibiting cellular energy metabolism or by inhibiting the coupling of energy-metabolism to the transport of this amino acid and raises the possibility that cellular microtubules play a role in these processes.     

DEVELOPMENTAL AND OTHER ASPECTS OF [35S]CYSTEINE TRANSPORT BY RAT BRAIN SYNAPTOSOMES

Abstract— Cysteine uptake by rat brain synaptosomes occurs by active transport. The uptake by synaptosomes isolated from newborn brain is slower and the concentration gradient achieved is lower than that observed in adult tissue. Synaptosomal fractions from both adult and newborn rat brains accumulate cysteine by two saturable systems. The calculated parameters show that the maximum rates of cysteine uptake in adult synaptosomes are approximately twice that observed in newborn synaptosomes for both the high and low affinity systems. The uptake by the high affinity system is sodium dependent and is inhibited by glycine and dibasic amino acids. Uptake by synaptosomes from 14-day-old animals is close to that observed in adult tissue. The uptake of cysteine differs greatly from that of cystine since the oxidized form, cystine, is taken up more slowly by systems with low affinities which are sodium independent, do not interact with dibasic amino acids and are independent of age.     

Amino Acid Requirements for the Growth of Aedes albopictus Clone C6/36 Cells and for the Production of Dengue and Chikungunya Viruses in the Infected Cells

Amino acid requirements for the growth of Aedes albopictus, clone C6/36, cells and for the production of dengue (DEN) and Chikungunya (CHIK) viruses were examined by growing the cells or the viruses in media which were deprived of one of the 20 amino acids. Cell growth was markedly inhibited when cystine was omitted from the medium, and to a lesser extent by arginine deprivation. On the other hand, omission of alanine, asparagine, aspartic acid, and glutamic acid at the same time did not affect cell growth. Marked accumulation of alanine was observed in the medium when the cells were grown for 8 days in complete medium, with concomitant depletion of aspartic acid and glutamic acid. The production of CHIK virus was inhibited markedly by omission of cystine from the medium after virus infection, while the production of DEN viruses was more affected by glycine deprivation, although cystine deprivation also inhibited virus production to a lesser extent. On the other hand, production of CHIK and DEN viruses was not affected when alanine, asparagine, aspartic acid, and glutamic acid were omitted from the medium at the same time.     

Cystine and dibasic amino acid uptake by opossum kidney cells

The characteristics of the uptake of L-cystine by the continuous opossum kidney cell line, OK, were examined. Uptake of cystine is rapid and, in contrast to other continuous cultured cell lines, these cells retain the cystine/dibasic amino acid transport system which is found in vivo and in freshly isolated kidney tissue. Confluent monolayers of cells also fail to show the presence of the cystine/glutamate transport system present in LLC-PK1 cells, fibroblasts, and cultured hepatocytes. Uptake of cystine occurs via a high-affinity saturable process which is independent of medium sodium concentration. The predominant site of cystine transport is across the apical cell membrane. The intracellular concentration of GSH far exceeds that of cystine with a ratio greater than 100:1 for GSH:cysteine. Incubation of cells for 5 minutes with a physiological level of labelled cystine resulted in the labelling of 66% and 5% of the total intracellular cysteine and glutathione, respectively. The ability of these cells to reflect the shared cystine/dibasic amino acid transport system makes them a suitable model for investigation of the cystine carrier which is altered in human cystinuria.     

Energy requirement for amino acid uptake inSaccharomyces cerevisiae

The uptake of glycine and α-aminoisobutyric acid by baker’s yeast was substantially increased by preincubation withd-glucose,d-fructose, sucrose, and maltose, but much less with ethanol or acetate. The increments in uptake are in rough agreement with the intracellular amount of acid-non-extractable high-energy phosphate (probably polyphosphate). The energy for amino acid transport is thus provided predominantly by the nonmitochondrial catabolic processes.