Regulation Of Amino Acid Transport Research Articles

Regulation of amino acid transport in hepatocytes isolated from adult and old rats

The 2-aminoisobutyric acid transport in hepatocytes isolated from 3- and 24- month-old rats studied and some age-related differences were observed. The basal uptake appeared to be almost constant in cells from old animals during the incubation time, while, in the cells from adults, it showed a progressive increase, interpreted as being due to a depression mechanism. Epinephrine and glucagon increased the transport in hepatocytes from animals of both ages, even if with a slightly different pattern; the hormones increased the V max , while the K m was unchanged at each age tested. However, the glucagon-induced increase in V max was lower in the older animals. The mechanism of hormonal action appeared to be similar in adult and old rats. In fact the uptake stimulation by glucagon and epinephrine showed a dependence on protein synthesis. The epinephrine effect was mediated by α-adrenergic receptors. No effect was exerted by extracellular amino acids on hepatocytes rom 24-month-old animals, suggesting a loss of adaptative regulation mechanism with aging. This behaviour was reflected in the kinetic parameters; in the fact the V max was not modified by extracellular amino acids at 24 months of age, while it appeared to be strongly decreased in the adult.

Regulation of amino acid transport in isolated rat hepatocytes during development.

The effect of amino acid depletion or supplementation and the effect of glucagon and insulin on the amino acid transport mediated by system A were investigated by determining the uptake of either 2-amino [1-14C]isobutyric acid (AIB) or N-methyl 2-amino [1-14C]isobutyric acid (MeAIB) in rat hepatocytes, freshly isolated at different stages of pre- and postnatal development. The data obtained show that the Na+-dependent uptake was higher at the earliest developmental stages, and steadily decreased until the adult level. The hormones increased AlB and MeAIB uptake enhancing the Vmax, while the Km was unchanged. This effect was evident in cells from adult and 18-20-day-old fetuses, while no response was present before the 18th day of fetal life and in the perinatal period. Actinomycin D or cycloheximide abolished this hormone-dependent increase. A decrease in AlB and MeAIB transport after incubation in an amino acid-rich medium was demonstrated at all ages tested, but was particularly evident in the prenatal life. The increase in the activity of the system following amino acid starvation was shown to be mostly dependent from de novo protein synthesis in the fetal life; on the contrary in the adult the increase appeared to be more linked to the release from transinhibition of the transport.

Regulation of amino acid transport in rat hepatocytes

Regulation of amino acid transport in rat hepatocytes

Short-term hormonal regulation of amino acid transport in isolated hepatocytes

Short-term hormonal regulation of amino acid transport in isolated hepatocytes

Nonspecific adaptation of jejunal amino acid uptake in the rat

Luminal nutrients are a major effector of intestinal adaptation. Amino acids are trophic to the intestine, but their role in regulating amino acid transport is not well documented. The presence of several distinct amino acid transport systems raises the question of whether adaptation is class-specific. Studies were carried out in parenterally nourished rats receiving a 7-day jejunal infusion of a 3% solution of either aminoisobutyric acid, aspartic acid, glutamine, histidine, lysine, or valine. While all amino acids were trophic to the intestine, their effects on the in vitro uptake of 0.1, 1.0 and 10.0 mM aspartic acid, lysine, and valine (representative acid, basic, and neutral amino acids) were variable and nonspecific. Compared to controls receiving either total parenteral nutrition alone or total parenteral nutrition plus luminal saline, prior lysine and aspartic acid infusion significantly increased in vitro uptake of all three amino acids tested, whereas valine had little effect on trarisport. No effect on transport was seen with glutamine (actively metabolized by the intestine as is aspartic acid), aminoisobutyric acid (a nonmetabolizable amino acid congener), or histidine (the most trophic amino acid). In conclusion, while individual amino acids cause an adaptation of amino acid Uptake, the effects are nonspecific and independent of their metabolic or trophic potential.

Effect of insulin on the activity of amino acid transport systems in cultured human fibroblasts

The regulation of amino acid transport by insulin has been studied in cultured human fibroblasts. Among the six amino acid transport systems operating in cultured human fibroblasts, two systems (A and x − C) are strongly stimulated by insulin and four (ASC, X − AG, y + and L) are essentially not sensitive to the presence of the hormone in the incubation medium. The hormonal stimulation of system A and system x − C became significant after 3 h of incubation and increased up to 12 h. The stimulatory effect was related to insulin concentration, with a half-maximal stimulation at 10 −9 M hormone concentration. Insulin enhanced transport activity by increasing the maximal velocity ( V max) of transport, without significant changes in K m values.

Regulatory effects of insulin and experimental diabetes on neutral amino acid transport in the perfused rat exocrine pancreas. Kinetics of unidirectional l-serine influx and efflux at the basolateral plasma membrane

Relatively little is known about the hormonal regulation of amino acid transport in the normal and diabetic exocrine pancreas. In this study unidirectional influx and tracer efflux of l-serine at the basolateral interface of the rat pancreatic epithelium was investigated in the perfused exocrine pancreas using a rapid (< 30 s) paired-tracer dilution technique. In the non-diabetic pancreas l-serine influx was saturable and stimulated by perfusion with exogenous bovine insulin (100 μU/ml). Transport of l-serine and methylaminoisobutyric acid was markedly elevated in pancreata isolated from streptozotocin diabetic rats and insulin partially reversed the stimulation of l-serine transport induced by experimental diabetes. These results suggest that insulin and diabetes modulate the epithelial transport activity for small neutral amino acids in the intact exocrine pancreas.

Inhibition by Peptides of Amino Acid Uptake by Bacterial Populations in Natural Waters: Implications for the Regulation of Amino Acid Transport and Incorporation

To investigate the regulatory interactions of amino acid transport and incorporation, we determined the effects of dipeptides on amino acid uptake by bacteria in an estuary and a freshwater lake. Dipeptides noncompetitively inhibited net transport and incorporation of amino acids into macromolecules but had no effect on the ratio of respiration to incorporation. Nearly maximum inhibition occurred at peptide concentrations of <10 nM. In contrast, the initial uptake rate of glycyl-[C]phenylalanine was not affected by glycine or phenylalanine. Net amino acid transport appeared to be inhibited by the increased flux into the intracellular pools, whereas the incorporation of labeled monomers into macromolecules was isotopically diluted by the unlabeled amino acids resulting from intracellular hydrolysis of the dipeptide. Chloramphenicol, sodium azide, and dinitrophenol all inhibited the initial uptake rate of leucine and phenylalanine. These results suggest that in aquatic environments amino acids are taken up by active transport which is coupled closely to protein synthesis.

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.

Effects of amino acids, glucagon, insulin and acetylcholine on cyclic nucleotide metabolism and amylase secretion in isolated mouse pancreatic fragments

The effects of amino acids, exogenous islet hormones and acetylcholine on cyclic nucleotide metabolism and amylase secretion in the isolated mouse pancreas have been investigated. The changes in levels of adenosine 3',5'-cyclic monophosphate (cyclic AMP) and guanosine 3',5'-cyclic monophosphate (cyclic GMP) were measured at different times during exposure of pancreatic fragments to amino acids ( l-alanine and l-arginine), islet hormones (insulin and glucagon) or acetylcholine (ACh). lAlanine (1–20 mM) evoked a transient increase in cyclic AMP concentration accompanied by an initial decrease and subsequent increase in the tissue concentration of cyclic GMP. l-Arginine (1–20mM) induced a complex triphasic change in cyclic AMP concentrations involving an initial rise and a delayed sustained elevation. The changes in levels of cyclic GMP increased only transiently. The effects of insulin (10 −6M) and to some extent glucagon (5 × 10 −7M) resembled those seen with l-arginine. The effects of amino acids and islet hormones were all dose-dependent. ACh (10 −7M) elicited a marked reduction in cyclic AMP concentration and this was accompanied by a concomitant increase in the level of cyclic GMP. The amino acids and the islet hormones had no significant effect on amylase secretion whereas ACh, of course, evoked a large increase in amylase output. The results with the amino acids and islet hormones reveal a clear dissociation between cyclic nucleotide changes and amylase secretion and further suggest that the marked reciprocal changes in cyclic AMP and cyclic GMP concentrations may constitute an important physiological role for the cyclic nucleotides to regulate amino acid transport in the pancreas.

Glucagon regulation of amino acid transport in hepatocytes: effect of cell enucleation.

Glucagon and cAMP analogs stimulate amino acid transport in freshly isolated hepatocytes by inducing the synthesis of new transport proteins. The role of the cell nucleus in the glucagon regulation of amino acid transport has been studied in rat hepatocytes enucleated by centrifugation through a discontinuous Ficoll gradient in the presence of cytochalasin B. Enucleated hepatocytes take up alpha-aminoisobutyric acid (AIB) through a Na+-dependent transport component with kinetic properties similar to those found in intact hepatocytes. Cytoplasts prepared from glucagon-stimulated cells retain the increase AIB transport induced by the hormone in the intact cells. The direct addition of glucagon to cytoplasts has no effect on AIB transport, in spite of the fact that the cytoplasts exhibit a higher capacity to bind glucagon than their nucleated counterparts. These data indicate that the nucleus is required for the glucagon stimulation of amino acid transport in isolated hepatocytes.

小腸におけるアミノ酸輸送とその調節

Amino acid transport in small intestine has been briefly reviewed. The specificity of the transport systems has not been conclusively classified yet. Simple diffusion, sodium-dependent and -independent transports are generally involved in amino acid transport. Experimental materials and methods used are very important to deduce any definitive conclusion regarding the specificity. Membrane vesicles prepared either from brush border or basal lateral membrane are assumed to be best to analyze the specificity, energy coupling and regulation of the transport. Regulation of amino acid transport has been noticed in semistarved state, diabetic condition and developing process. In these cases, changes in kinetic parameters conflict each other. Therefore, amino acid transport in small intestine requires much more detailed studies on the kinetics.

Regulation of amino acid transport in L6 myoblasts. II. Different chemical properties of transport after amino acid deprivation.

The mechanism of stimulation of amino acid transport system A caused by amino acid deprivation in L6 cells was investigated. In cells loaded with alpha-aminoisobutyric acid (AIB), amino acid deprivation increased the rate of proline uptake only after the intracellular [AIB] dropped below 7 mM. Efflux of proline was not sensitive to the presence of proline in the outer medium (with or without external Na+), suggesting that efflux through system A (and possibly uptake) is not susceptible to transinhibition. Transport (stimulated uptake) into amino acid-deprived cells and that into amino acid-supplemented cells differed in several chemical properties: 1) In the former group, transport was higher at lower pH values than in the latter, and the optimum pH values were 7.5 and 7.8, respectively. 2) Unlike proline uptake in supplemented cells, uptake in deprived cells was inhibited by 50% with N-ethylmaleimide (1 mM) or by 50 microM p-chloromercuribenzoate (PCMBS). Inhibition by PCMBS was not due to collapse of the Na+ gradient. The mercurial inhibited only the deprivation-induced stimulation of transport, bringing the rate of proline uptake to the "basal" uptake level observed in amino acid-supplemented cells. Proline uptake was not stimulated by a second deprivation following treatment with PCMBS and a supplementation-deprivation cycle. However, in untreated cells, or by reversing mercaptide formation with dithiotreitol, the second deprivation stimulated transport. Deprivation at 4 degrees C did not elicit stimulation of proline uptake. Cycloheximide prevented the stimulation and decreased the rate of proline uptake in deprived cells more efficiently than in supplemented cells. Actinomycin D prevented stimulation when added at the onset of deprivation. The above data indicate that stimulation of transport by deprivation is protein synthesis-dependent and that the stimulated transport had chemical properties distinct from the "basal" transport in supplemented cells. The evidence presented is consistent with a model of activation of a finite pool of transporters upon deprivation, the chemical characteristics of which differ from those of the "basal" transport system.

Regulation of amino acid transport in L6 muscle cells: I. Stimulation of transport system A by amino acid deprivation.

AbstractThe regulation of amino acid transport in L6 muscle cells by amino acid deprivation was investigated. Proline uptake was Na+‐dependent, saturable and concentrative, and was predominantly through system A. Proline uptake was inhibited by alanine, α‐amino isobutyric acid (AIB), and by α‐methylamino isobutyric acid, but not by lysine or valine. At 25°C, Km of proline uptake was 0.5 mM. Amino acid‐deprivation resulted in a progressive increase in the rate of proline uptake, reaching up to 6‐fold stimulation after 6 hours. The basal and stimulated transport were equally Na+‐dependent, and both were inhibited by competition with the same amino acids. Kinetic analysis showed that Km decreased by a factor of 2.4 and Vmax increased 1.9‐fold in deprived cells. Amino acid‐deprivation did not stimulate amino acid uptake through systems other than system A. This suggests that the higher Km in proline‐supplemented cells is not due to release of intracellular amino acids into unstirred layers surrounding the cells. The presence of amino acids which are substrates of system A (including AIB) during proline‐deprivation, prevented stimulation of proline uptake, whereas those transported by systems Ly+ or L exclusively were ineffective. The stimulation of the transport‐rate in deprived cells could be reversed by subsequent exposure to proline or other substrates of system A. L6 cells, deprived of proline for 6 hours, retained the stimulation of transport after detachment from the monolayers with trypsin. Uptake rates were comparable in suspended and attached cells in monolayer culture. Thus, amino acid‐depreivation of L6 cells results in an adaptive increase in proline uptake, which is not due to unstirred layers but appears to be mediated by other mechanisms of selective transport regulation.

Regulation of amino acid transport in Thiobacillus thioparus

Amino acid transport in amino acid auxotrophs of Thiobacillus thioparus was enhanced during growth on rate-limiting amino acid concentration. A pleiotropic mutation enhanced general amino acid transport as manifested by higher values of Vmax of amino acid transport. Affinity constants remained unaltered. Mutants with enhanced transport properties did not show changes in oxidation of thiosulfate, did not oxidize various organic compounds, and did not increase the heterotrophic potential of T. thioparus. The mutations for enhanced transport caused increased synthesis of amino acid transport system components. A method for genetic transformation of T. thioparus is described.

Compartmentation in amino acid transport across the blood brain barrier.

Under steady-state conditions, the transport rates for amino acids from blood to brain have been found to be about half that seen using the intraarterial injection technique. Using a method that mathematically mimics the constant infusion procedure, we were able to reconcile this apparent discrepancy. At less than 1 min after subcutaneous injection of [14C]tyrosine in mice, we have observed a rate of entry into brain of 19.7 nmol/g/min, while from 1-15 min we have measured the rate at 6.4 nmol/g/min. Using methionine sulfoximine as an inhibitor of the gamma-glutamyl cycle, the early rate was reduced to 10.0 nmol/g/min and the later rate to 3.7 nmol/g/min. These data are consistent with a two-compartment system regulating amino acid transport into the neurons. A mathematical model fit to these data indicates that the first compartment contains 8.3 nanomoles of tyrosine per gram brain or about 6.7% of the brain total. It is speculated that the first compartment consists primarily of the astrocytes.

Adaptive regulation of amino acid transport in cultured human fibroblasts. Sites and mechanism of action.

The regulation of the transport of neutral amino acids across the cell membrane by adaptive mechanisms has been studied in cultured human fibroblasts. Among the three transport systems (A, ASC, and L) individually discriminated, only the Na+-dependent System A was subject to adaptive regulation, showing enhancement of its activity when the cells were incubated under conditions of amino acid shortage (derepression phase) and decrease of its activity when the cells were exposed to a medium supplied with Site A-reactive amino acids (repression phase). Starvation-induced derepression of transport activity and its reversal by amino acid refeeding required active RNA and protein synthesis. Derepression involved an early mRNA synthesis which started within 30 min from the abrupt change in extracellular amino acid concentration and apparently lasted 90 min. The transcribed mRNA was rather stable and translatable for a few hours (presumably into transport proteins) in cells maintained in the absence of amino acids. Repression by amino acid refeeding also involved an early mRNA synthesis, the product of its translation being presumably a protein capable of causing degradation or inactivation of transport proteins. The rate of decay in transport activity of previously derepressed cells was somewhat faster in the presence of added Site A-reactive amino acids than in their absence. A model is proposed in which the concentration of Site A-reactive amino acids affects transport activity of System A by modulating transcription of mRNA species coding for transport proteins and their putative inactivators and by regulating the efficiency of transport protein inactivation at the cell membrane.

Glucocorticoid regulation of amino acid transport in anucleate rat hepatoma (HTC) cells.

The transport of alpha-aminoisobutyric acid (AIB) by rat hepatoma tissue culture (HTC) cells is rapidly and reversibly inhibited by dexamethasone and other glucocorticoids. To investigate the role of the nucleus in the regulation of transport and to determine whether steroid hormones or steroid-receptor complexes may have direct effects on cytoplasmic or membrane functions, we have examined the regulation of transport by dexamethasone in anucleate HTC cells. Cytoplasts prepared from suspension cultures of HTC cells fully retain active transport of AIB with the same kinetic properties as intact cells. However, the uptake of AIB is not inhibited by dexamethasone or other corticosteroids. Neither is the inhibited rate of transport, manifested by cytoplasts prepared from dexamethasone-treated cells, restored to normal upon removal of the hormone. Anucleate cells exhibit specific, saturable binding of [3H]dexamethasone; however, the binding is reduced compared with that of intact cells. The nucleus is thus required for the glucocorticoid regulation of amino acid transport in HTC cells.

The effect of amiloride on hormonal regulation of amino acid transport in isolated and cultured adult rat hepatocytes

Insulin and glucagon stimulate amino acid transport in isolated rat hepatocytes. Amiloride, a specific Na +-influx inhibitor, completely inhibited the hormonal (glucagon or insulin) stimulation of α-aminoisobutyric acid influx by preventing the emergence of a high-affinity transport component. The drug also inhibited [ 14C]valine incorporation into hepatocyte protein. The half-maximal concentration of amiloride for inhibition of protein synthesis was similar to that required for inhibition of hormone-stimulated amino acid transport (approx. 0.1 mM). In primary cultured rat hepatocytes, amiloride markedly depressed the stimulation of α-aminoisobutyric acid transport by glucagon, or a mixture of glucagon, insulin and epidermal growth factor. These results suggest that amiloride inhibits the hormonal stimulation of hepatocyte amino acid transport by preventing the synthesis of high-affinity transport proteins. They also suggest that the hormonal stimulation of hepatocyte amino acid transport is dependent, at least partly, on Na + influx.

Calcium-dependent hormonal regulation of amino acid transport and cyclic AMP accumulation in rat hepatocyte monolayer cultures.

The effect of glucagon, epinephrine, norepinephrine, dexamethasone, insulin, and dexamethasone plus glucagon on the transport of 2-aminoisobutyric acid (AIB) and that of glucagon on the production of cyclic AMP were examined in rat hepatocyte monolayer cultures under three different culture conditions involving calcium. The hepatocytes were studied in calcium-contaning medium after treatment with or without 0.033% dimethyl sulfoxide, the solvent for the calcium ionophore A23187 (calcium controls); calcium-free medium after treatment with A23187 (calcium-depleted); and calcium-containing medium after treatment with ionophore (calcium-restored). The basal and hormonally regulated rates of AIB transport for hepatocytes in calcium control and calcium-depleted cultures were comparable. The restoration of calcium in calcium-restored cultures increased the basal and the hormonally stimulated transport of AIB when compared to the other conditions. Calcium markedly enhanced the stimulation of AIB transport in cultures treated with glucagon, catecholamines, and dexamethasone plus glucagon. The level of cyclic AMP production in response to glucagon in calcium control and calcium-depleted cultures was the same and it was conspicuously higher than the level in calcium-restored cultures. Varying the concentration of calcium in the medium used to maintain the hepatocytes in calcium control cultures did not affect the stimulation of AIB transport or cyclic AMP production by glucagon. However, in calcium-restored cultures, increasing the calcium concentration of the medium resulted in increased stimulation of AIB transport and decreased production of cyclic AMP by glucagon. In the calcium-restored cultures, calcium in the absence of glucagon enhanced AIB transport but had no effect on cyclic AMP production. Cultures maintained for 6 hr in calcium-free medium after the depletion of calcium showed a 6- to 7-fold increase in the production of cyclic AMP in response to glucagon, but no stimulation of AIB transport. We suggest that mobilization of cellular calcium by glucagon either directly or through cyclic AMP mediates its stimulation of amino acid transport.