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.     

Growth regulation and amino acid transport in epithelial cells: Influence of culture conditions and transformation on A,ASC, and l transport activities

Amino acid transport in Madin-Darby canine kidney (MDCK) cells, grown in a defined medium, was investigated as a function of cell density, exposure to specific growth factors, and transformation. MDCK cells were found to transport neutral amino acids by systems similar to the A, ASC, L, and N systems which have been characterized using other cell lines. Experimental conditions were developed for MDCK cells which allowed independent measurement of A, ASC, and L transport activities. The activity of the L system was measured as Na⁺-independent leucine or methionine uptake at pH 7.4. The activity of the A system was measured as Na⁺-dependent α(methylamino)isobutyric acid (mAIB) uptake at pH 7.4, the activity of the ASC system was measured as Na⁺-dependent alanine uptake in the presence of 0.1 mM mAIB at pH 6.0, and the activity of system N was observed by measuring Na⁺-dependent glutamine uptake at pH 7.4 in the presence of high concentrations of A and ASC system substrates. The L transport system responded minimally to changes in growth state, but Na⁺-dependent amino add transport responded to regulation by growth factors, cell density, and transformation. The activities of the A and ASC systems both decreased at high cell density, but these activities responded dissimilarly under other conditions. The activity of the A system was stimulated by insulin, was inhibited by PGE₁, and was elevated 3–7 fold in the transformed cell line, MDCK-T₁. The activity of the ASC system was slightly stimulated by insulin and by PGE₁, but was unchanged after chemical transformation. Changes in cellular growth were monitored and were found to correlate best with the activity of the A system. These results suggested that MDCK cell growth may be more closely related to the activity of the A than of the ASC system.     

Hormonal regulation of amino acid transport and cAMP production in monolayer cultures of rat hepatocytes

The effects of insulin, glucagon or Dexamethasone (DEX) and of glucagon with insulin or DEX were examined on the uptake of 2-amino [1-¹⁴C]isobutyric acid (AIB) and N-Methyl-2-amino [1-¹⁴C]isobutyric acid (NMe AIB) in monolayer cultures of rat hepatocytes. Insulin and glucagon stimulated the uptake of both the amino acids and DEX inhibited it, showing that all three of these hormones regulate the A system (the sodium-dependent system that permits the transport of NMe AIB) for amino acid transport in these cultures. Experiments investigating the transport of aminocyclopentane-1-carboxylic acid, 1- [carboxyl-¹⁴C] in the presence of excess AIB or in the absence of sodium showed that insulin had no effect on the activity of the L system (the sodium-independent system that prefers leucine). Experiments on the uptake of AIB in the presence of excess NMe AIB showed insulin had no effect on the transport activity of the ASC system (the sodium-dependent system that does not transport NEe AIB). Insulin concentrations ranging from 0.1 nM to 100 nM did not antagonize the stimulatory effect of optimum or suboptimum concentrations of glucagon on the uptake of either AIB or NMe AIB. Similarly, glucagon did not antagonize the stimulatory effect of optimum or suboptimum concentrations of insulin on the uptake of both the amino acids. The combined effect of insulin and glucagon was additive on the rate as well as the cumulative uptake of both AIB and NMe AIB. DEX alone inhibited the transport of both AIB and NMe AIB by about 25%, while glucagon caused a 2–3-fold increase; however, the addition of glucagon to cultures containing DEX caused a 7–8-fold increase in the uptake of both AIB and NMe AIB when compared to cultures containing DEX alone. The effect of insulin on the levels of cAMP was also investigated. Insulin had no effect on the cAMP levels in cultures treated or untreated with optimum or suboptimum concentrations of glucagon.     

Hormonal regulation of hepatic amino acid transport

The transport of 2-aminoisobutyric acid (AIB) into liver tissue was increased by both insulin and glucagon. We have now shown that these hormones do not stimulate the same transport system. Glucagon, possibly via cAMP, increased the hepatic uptake of AIB by a mechanism which resembled system A. This glucagon-sensitive system could be monitored by the use of the model amino acid MeAIB. In contrast, the insulin-stimulated system exhibited little or no affinity for MeAIB and will be referred to as system B. On the basis of other reports that the hepatic transport of AIB is almost entirely Na⁺ dependent and the present finding that the uptake of 2-aminobicyclo [2,2,1] heptane-2-carboxylic acid (BCH) was not stimulated by either hormone, we conclude that system B is Na⁺ dependent. Furthermore, insulin added to the perfusate of livers from glucagon-pretreated donors suppressed the increase in AIB or MeAIB uptake. Depending upon the specificities of systems A and B, both of which are unknown for liver tissue, the insulin/glucagon ratio may alter the composition of the intracellular pool of amino acids.     

Transport system a is not responsive to hormonal stimulation in primary cultures of fetal rat hepatocytes

Hepatocytes isolated from rat fetuses have been shown to contain Systems A, ASC, and N for the Na⁺-dependent transport of neutral amino acids and the activity of each of these systems is significantly higher in the fetal cells than those of an adult (J. Vadgama and H.N. Christensen, personal communication). In contrast to the hepatocytes isolated from adult or newborn animals, the fetal cells do not respond to insulin, glucagon, or dexamethasone with an increase in System A-mediated transport. The System A activity present in the fetal hepatocytes does undergo adaptive regulation in a manner similar to that seen for adult cells, however, the same is not true for System N. Like the cells isolated from the adult rats, the hepatocytes isolated from fetal liver tissue exhibit an increase in Na⁺-independent transport with time in culture.     

The characterisation of two partially purified systems for Na+-dependent amino acid transport

Two systems mediating the transport of amino acids were studied in vesicles derived from protein-depleted membranes of pigeon erythrocytes. One system (ASC system) catalysed the Na⁺-dependent exchange of small neutral amino acids, such as alanine, serine and cysteine. The other system, also Na⁺-dependent, mediated the active transport of glycine. The ASC and glycine systems were distinguished by the sensitivity of the latter to the anion present, by the former's requirement for an exchangeable amino acid and by the inability of alanine to inhibit the transport of glycine. Preliminary results indicated that the influx of glycine was electrically silent. The only major integral protein retained in the vesicles was the band 3 protein, but that could not be unequivocally identified as the transporter.     

Maintenance of glucagon-stimulated system A amino acid transport activity in rat liver plasma membrane vesicles

Plasma membrane vesicles prepared from intact rat liver or isolated hepatocytes retain transport activity by systems A, ASC, N, and Gly. Selective substrates for these systems showed a Na+-dependent overshoot indicative of energy-dependent transport, in this instance, driven by an artificially-imposed Na+ gradient. Greater than 85% of Na+-dependent 2-aminoisobutyric acid (AIB) uptake was blocked by an excess of 2-(methylamino)isobutyric acid (MeAIB) with an apparent Ki of 0.6 mM. Intact hepatocytes obtained from glucagon-treated rats exhibited a stimulation of system A activity and plasma membrane vesicles isolated from those same cells partially retained the elevated activity. Transport activity induced by substrate starvation of cultured hepatocytes was also evident in membrane vesicles prepared from those cells. The membrane-bound glucagon-stimulated system A activity decays rapidly during incubation of vesicles at 4 degrees C (t1/2 = 13 h), but not at -75 degrees C. Several different inhibitors of proteolysis were ineffective in blocking the decay of transport activity. Hepatic system N transport activity was also elevated in plasma membrane vesicles from glucagon-treated rats, whereas system ASC was essentially unchanged. The results indicate that both glucagon and adaptive regulation cause an induction of amino acid transport through a plasma membrane-associated protein.     

Amiloride inhibits protein synthesis in isolated rat hepatocytes

The effects of amiloride on Na⁺ ion influx, amino acid transport, protein synthesis and RNA synthesis have been studied in isolated rat hepatocytes. The initial rate of ²²Na⁺ uptake and the amount of ²²Na⁺ taken up at later time points were decreased in hepatocytes incubated in the presence of amiloride. Amiloride inhibited by about 25% the influx of α-methylamino[1?¹⁴C]isobutyric acid, a specific substrate for the A (Alanine preferring) system of neutral amino acid transport. By contrast, the activity of system L (Leucine preferring) was not affected by amiloride. Rates of protein synthesis were determined by using high extracellular concentrations of [¹⁴C]valine in order to maintain a constant amino acid precursor pool. Amiloride inhibited protein synthesis by 85% and had no effect on RNA synthesis. Half-maximal inhibition of protein synthesis occurred with amiloride at about 150 μM. In the absence of Na⁺ in the incubation medium, the rate of protein synthesis was reduced by about 35% and no further inhibition was observed with amiloride. These results suggest that in isolated rat hepatocytes protein synthesis is partially dependent on Na⁺, and that amiloride is an efficient inhibitor of protein synthesis.     

Insulin and glucagon stimulation of amino acid transport in isolated rat hepatocytes. Synthesis of a high affinity component of transport.

Insulin and glucagon stimulate amino acid transport in freshly prepared suspensions of isolated rat hepatocytes. The kinetic properties of alpha-amino[1-14C]isobutyric acid (AIB) transport were investigated in isolated hepatocytes following stimulation by either hormone in vitro. In nonhormonally treated cells (i.e. basal state), saturable transport occurred mainly through a low affinity (Km approximately equal to 40 mM) component. In insulin or glucagon-treated hepatocytes, saturable transport occurred through both a low affinity component (similar to that observed in the basal state) and a high affinity (Km approximately equal to 1 mM) component. At low AIB concentrations (less than 0.5 mM), insulin and glucagon at maximally stimulating doses increased AIB uptake about 2-fold and 5-fold, respectively. The high affinity component induced by either hormone exhibited the properties of the A (alanine preferring) mediation of amino acid transport. This component required 2 to 3 h for maximal expression, and its emergence was completely prevented by cycloheximide. Half-maximal stimulation was elicited by insulin at about 3 nM and by glucagon at about 1 nM. Dibutyryl cyclic AMP mimicked the glucagon effect and was not additive to it at maximal stimulation. Maximal effects of insulin and glucagon, or insulin and dibutyryl cyclic AMP, were additive. We conclude that insulin and glucagon can modulate amino acid entry in hepatocytes through the synthesis of a high affinity transport component.     

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

The 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 chicken embryo fibroblasts by purified multiplication-stimulating activity (MSA)

Enhanced amino acid transport is observed when quiescent cultures of chicken embryo fibroblasts are stimulated to proliferate by the addition of purified multiplication-stimulating activity (MSA). This increase in amino acid transport is an early event occuring prior to the onset of DNA synthesis in stimulated cells. Results indicate that the changes in transport activity, as measured by α-aminoisobutyric acid (AIB) uptake, are due to stimulation of only the Na⁺-dependent A transport system. There is little or no change in the activities of transport systems ASC, L, or Ly⁺ upon exposure to MSA. A kinetic analysis shows this increased activity is due to a change in Vmax while K_m remains unaltered. Continuous exposure to the stimulus is required to maintain the increased level of transport activity and the presence of inhibitors of RNA and protein synthesis significantly inhibits the response. Results also indicate that a similar specific increase in the A transport system is initiated when RSV tsNY68 infected cells are shifted to the permissive temperature. It appears that the A system of mediation is emerging as a strategic regulatory site for cell function.     

Glucagon resistance of hepatoma cells. Evidence for receptor and post-receptor defects.

Of all available liver cells in culture, only primary cultured hepatocytes are known to respond to glucagon in vitro. In the present study we investigated whether glucagon could stimulate amino acid transport and tyrosine aminotransferase (TAT;EC 2.6.1.5) activity (two well-characterized glucagon effects in the liver) in Fao cells, a highly differentiated rat hepatoma cell line. We found that glucagon had no effect on transport of alpha-aminoisobutyric acid (AIB; a non-metabolizable alanine analogue) nor on TAT activity, even though both activities could be fully induced by insulin [2-fold and 3-fold effects for AIB transport and TAT activity, respectively, after 6h; EC50 (median effective concentration) = 0.3 nM], or by dexamethasone (5-8-fold effects after 20 h; EC50 = 2 nM). Analysis of [125I]iodoglucagon binding revealed that Fao cells bind less than 1% as much glucagon as do hepatocytes, whereas insulin binding in Fao cells was 50% higher than in hepatocytes. The addition of dibutyryl cyclic AMP, which fully mimics the glucagon stimulation of both AIB transport and TAT activity in hepatocytes, induced TAT activity in Fao cells (a 2-fold effect at 0.1 mM-dibutyryl cyclic AMP) but had no effect on AIB transport. Cholera toxin stimulated TAT activity to the same extent as did dibutyryl cyclic AMP. These results indicate that the lack of glucagon responsiveness in cultured hepatoma cells results from both a receptor defect and, for amino acid transport, an additional post-receptor defect. Moreover, the results show that amino acid transport and TAT activity, which appeared to be co-induced by insulin or by dexamethasone in these cells, respond differently to cyclic AMP. This suggests that different mechanisms are involved in the induction of these activities by glucagon in liver.     

Cell density and amino acid transport in 3T3, SV3T3, and SV3T3 revertant cells

The transport of selected neutral and cationic amino acids has been studied in Balb/c 3T3, SV3T3, and SV3T3 revertant cell lines. After properly timed preincubations to control the size of internal amino acid pools, the activity of systems A, ASC, L, and Ly⁺ has been discriminated by measurements of amino acid uptake (initial entry rate) in the presence and absence of sodium and of transportspecific model substrates. L-Proline, 2-aminoisobutyric acid, and glycine were primarily taken up by system A; L-alanine and L-serine by system ASC; L-phenylalanine by system L; and L-lysine by system Ly⁺ in SV3T3 cells. L-Proline and L-serine were also preferential substrates of systems A and ASC, respectively, in 3T3 and SV3T3 revertant cells. Transport activity of the Na⁺-dependent systems A and ASC decreased markedly with the increase of cell density, whereas the activity of the Na⁺-independent systems L and Ly⁺remained substantially unchanged. The density-dependent change in activity of system A occurred through a mechanism affecting transport maximum (V_max) rather than substrate concentration for half-maximal velocity (K_m). Transport activity of systems A and ASC was severalfold higher in transformed SV3T3 cells than in 3T3 parental cells at all the culture densities that could be compared. In SV3T3 revertant cells, transport activity by these systems remained substantially similar to that observed in transformed SV3T3 cells. The results presented here add cell density as a regulatory factor of the activity of systems A and ASC, and show that this control mechanism of amino acid transport is maintained in SV40 virus-transformed 3T3 cells that have lost density-dependent inhibition of growth, as well as in SV3T3 revertant cells that have resumed it.     

Antigen-specific stimulation of amino acid transport in bovine lymphocytes

Treatment of bovine lymphocytes isolated from animals which were either infected with Mycobacterium bovis or sensitized to a purified protein derivative (PPD-B) from this organism induced an increase in the transport of α-aminoisobutyric acid (AIB) and α-methylaminoisobutyric acid (MeAIB). PPD-B did not stimulate these transport activities in lymphocytes from nonsensitized animals. The transport stimulation was first measurable after about 7 hours of treatment, reached about a two-fold enhancement after 20 hours, and continued to increase to 30- to 40-fold after 6 days. The stimulation of AIB transport was inhibited by both ouabain and cycloheximide. Experiments to determine transport system specificities in nonstimulated lymphocytes showed that MeAIB transport was primarily by the Na⁺-dependent, A-system, and leucine transport was mostly by Na⁺-independent system(s). In contrast, AIB transport was about 25% by the A-system, 25% by at least one Na⁺-dependent, non-A-system, and 50% by one or more Na⁺-independent system(s). Analysis of the three components of AIB transport after treatment with PPD-B showed that: (1) transport by both the A-system and the Na⁺-independent system(s) was stimulated; (2) A-system transport was stimulated to a larger extent than Na⁺-independent transport; and (3) Na⁺-dependent, non-A-system transport was not stimulated significantly.     

Alanine and leucine transport in unfertilized pig oocytes and early blastocysts

Amino acid transport is facilitated by specific transporters within the plasma membrane of the cell. In mouse oocytes and cleavage-stage conceptus Na⁺-dependent L-alanine and L-leucine transport are nearly undetectable. Sodium-dependent transport via system B^O,+ in the mouse conceptus increases greatly between the 8-cell and blastocyst stages. By contrast, data presented here for the pig show that L-alanine and L-leucine transport is mainly Na⁺-dependent in the oocyte; this Na⁺-dependent component of transport becomes undetectable by the blastocyst stage. The Na⁺-dependent component of transport in oocytes is inhibited by BCH (2-aminoendo-bicyclo[2.2.1] hexane-2-carboxylic acid) and L-lysine and thus could be a form of system B^O,+. In both oocytes and blastocysts Na⁺-independent L-leucine transport is inhibited by BCH, which is consistent with the presence of system L. The dramatic decrease in Na⁺-dependent amino acid transport activity could occur in pig conceptuses in association with the onset of RNA synthesis during the 4-cell stage. Regardless of the precise time during development at which it occurs, however, this dramatic, developmentally regulated decrease in Na⁺-dependent alanine and leucine transport activity contrasts sharply with the large increase in Na⁺-dependent system B^O,+ activity that occurs during preimplantation development of murine conceptuses. Elucidation of the molecular mechanisms by which these changes occur should contribute to an understanding of regulation of gene expression during early development. © 1993 Wiley-Liss, Inc.     

Regulation of neutral amino acid transport in hepatocytes isolated from adrenalectomized rats

The present report shows that System A-mediated 2-aminoisobutyric acid (AIB) uptake is elevated in hepatocytes isolated from adrenalectomized rats when they are compared to control cells. Although System ASC activity also shows this perturbation, Systems N, beta, L1, and L2 are unaffected. Transport of AIB in both cell types is stimulated by dexamethasone, insulin, and glucagon, yet the hepatocytes from the adrenalectomized rats are much less responsive to these hormones. This apparent decrease in competence is seen for adaptive regulation of System A as well. The in vitro addition of dexamethasone to the hepatocytes from the adrenalectomized animals does not restore fully their ability to respond to hormones or amino acid deprivation. These effects are observed even after the cells have been held in primary culture for 24 hr. The simultaneous addition of glucagon and dexamethasone to either cell type resulted in stimulation of transport to rates significantly greater than the sum of the increases produced by the two hormones when added separately. In contrast, insulin and dexamethasone were additive in their effects rather than synergistic. These results suggest that hepatocytes from adrenalectomized rats are less competent than control cells with respect to regulation of neutral amino acid transport, including stimulation by insulin or amino acid starvation, two processes which appear not to depend on glucocorticoid for maximal response.     

Neutral amino acid transport in human synovial cells: substrate specificity of adaptative regulation and transinhibition

Neutral amino acid transport was characterized in human synovial cells. The amino acids tested are transported by all three major neutral amino acid transport systems, that is, A, L, and ASC. The model amino acid 2-aminoisobutyric acid (AIB) was found to be a strong specific substrate for system A in synovial cells. When cells were starved of amino acids, the activity of AIB transport increased, reaching a maximum within 1 h. The stimulation of transport activity was not blocked by cycloheximide and would thus appear to be related to a release from transinhibition. Similarly, the decrease in the activity of AIB transport observed after the addition of alpha-methyl-aminoisobutyric acid (meAIB) appeared to be related to transinhibition. However, using a different approach, that is, amino acid starvation followed by incubation with 10 mM meAIB and transfer to an amino acid-free medium with or without cycloheximide supplementation, a clear increase in AIB uptake, due both to derepression and a release from transinhibition, was observed. Unlike human fibroblasts, the depression of system A in these synovial cells was not serum-dependent. The process of derepression was observed only after preloading with meAIB. Neither AIB nor alanine produced this phenomenon. Moreover, alanine preloading led to a large increase in AIB transport activity due to a release from transinhibition. These observations indicate that the process of derepression and release from transinhibition are specific to the substrates present in the culture medium prior to amino acid starvation.     

Characterisation and cloning of a Na-dependent broad-specificity neutral amino acid transporter from NBL-1 cells: a novel member of the ASC/B transporter family

Na⁺-dependent neutral amino acid transport into the bovine renal epithelial cell line NBL-1 is catalysed by a broad-specificity transporter originally termed System B⁰. This transporter is shown to differ in specificity from the B⁰ transporter cloned from JAR cells [J. Biol. Chem. 271 (1996) 18657] in that it interacts much more strongly with phenylalanine. Using probes designed to conserved transmembrane regions of the ASC/B⁰ transporter family we have isolated a cDNA encoding the NBL-1 cell System B⁰ transporter. When expressed in Xenopus oocytes the clone catalysed Na⁺-dependent alanine uptake which was inhibited by glutamine, leucine and phenylalanine. However, the clone did not catalyse Na⁺-dependent phenylalanine transport, again as in NBL-1 cells. The clone encoded a protein of 539 amino acids; the predicted transmembrane domains were almost identical in sequence to those of the other members of the B⁰/ASC transporter family. Comparison of the sequences of NBL-1 and JAR cell transporters showed some differences near the N-terminus, C-terminus and in the loop between helices 3 and 4. The NBL-1 B⁰ transporter is not the same as the renal brush border membrane transporter since it does not transport phenylalanine. Differences in specificity in this protein family arise from relatively small differences in amino acid sequence.     

Neutral amino acid transport by marine fish intestine: role of the side chain

This work was devoted to the study of the structure-affinity relationships in neutral amino acid transport by intestinal brush border of marine fish (Dicentrarchus labrax). The effects of the length of the side chain on kinetics of glycine, alanine, methionine and amino isobutyric acid were investigated. In the presence of K⁺ two components were characterized: one is saturable by increased substrate concentrations, whereas the other can be described by simple diffusion mechanism. Simple diffusion, a passive, non-saturable, Na⁺-independent route, contributes largely to the transport of methionine and to a much lesser extend to alanine, glycine or alphaaminoisobutyric acid uptakes. If a branched chain is present, as in the case of amino isobutyric acid, diffusion is low. A Na⁺-independent, saturable system has been fully characterized for methionine, but not for branched amino acids such as amino isobutyric acid. In the presence of Na⁺ saturable components were shown. Two distinct Na⁺-dependent pathways have been characterized for glycine uptake, with low and high affinities. For alanine and methionine only one Na⁺-dependent high affinity system exists with the same half-saturation concentration and the same maximum uptake at saturable concentrations. Glycine high affinity system has the same half-saturation concentration as methionine or alanine uptake, whereas maximum uptake is lower. The substitution of the hydrogen by a methyl group results in a severe decrease of uptake (aminoisobutyric acid). Mutual inhibition experiments indicate that the same carriers could be responsible for methionine and alanine uptakes and probably glycine Na⁺-dependent uptake. The influence of Na⁺ concentrations (100^-1 mol·l^-1) on amino acid uptake was examined. Glycine, alanine, methionine and amino isobutyric acid transport can be described by a hyperbolic function, with a saturation uptake which is highly increased for methionine. However, the half-saturation concentration does not seem to be strongly affected by the amino acid structure. The effect of Na⁺ concentration (25 and 100 mmol·l^-1) on the kinetics of methionine uptake have been also examined. The maximum uptake of the saturable system clearly shows a typical relationship with concentration.Abbreviations [AA] amino acid concentration - AIB aminoisobutyric acid - [I] Inhibitor amino acid concentration - J _i uptake in the presence of inhibitor - J _o uptake without inhibitor - K _d passive diffusion constant - K _i inhibitor constant - K _t concentration of test amino acid for half-maximal flux - MES 2[N-morpholino]ethanesulphonic acid - V _max maximum uptake at saturable amino acid concentrations - V _tot total amino acid uptake     

Stimulatory effect of glucagon and dibutyryl-cAMP specifically on the Na+-independent amino acid transport of chang liver cell

Pretreatment(2 h) of Chang liver cells with glucagon and dibutyryl-cAMP has been shown to stimulate specifically the Na⁺-independent transport system for amino acids. The stimulation of Na⁺-independent leucine uptake was completely or largely inhibited by prior incubation(30 min) of the cells with colchicine or cytochalasin B. Glucagon treatment colud only change the Vmax value of the high-affinity component of Na⁺-independent leucine transport and the apparent Km value of the low-affinity one, whereas dibutyryl-cAMP treatment more or less affected all of the kinetic constants of both of the two components in favor of promotion of leucine tansport. These results of kinetic analysis indicate that dibutyryl-cAMP may not perfectly play the role of glucagon in activation of the Na⁺-independent transport system for leucine.