Membrane function in differentiating skeletal muscle cells: I. Kinetic analysis of amino acid transport

Primary cultures of mononucleated myoblasts from 12-day-old chick embryos have a twofold higher rate of α-aminoisobutyric acid (AIB) transport before fusion occurs to form multinucleated myotubes. Several lines of evidence indicate that the uptake of AIB observed in both myoblasts and myotubes is primarily carrier-mediated by a membrane transport system. Increasing the temperature from 24 to 37°C results in a threefold increase in the rate of AIB uptake; both methionine and glycine inhibit AIB uptake by more than 85%; and 2,4-dinitrophenol inhibits AIB uptake by approximately 50%. In addition, the energies of activation (14.5 and 14.0 kcal/mole for myoblasts and myotubes, respectively) are characteristic of carrier-mediated transport. Resolution of AIB uptake into a saturable, carrier-mediated component and a nonsaturable, diffusion component shows that at concentrations of AIB≤1.5 mM over 97% of total AIB uptake is carriermediated in both myoblasts and myotubes. Kinetic analysis of carrier-mediated AIB uptake indicates that myoblasts and myotube membrane carriers have the same affinity for AIB (K_m values = 1.73 and 1.31 mM, respectively). However, the V_max for myoblasts is 23.7 nmole/mg/min while myotubes have a V_max of 12.6 nmole/mg/min. The twofold difference in V_max is shown to be due to a twofold difference in the quantity of membrane transport sites per milligram of protein.     

Characteristics of an Uptake System for α-Aminoisobutyric Acid in Leishmania tropica Promastigotes1

ABSTRACT. Leishmania tropica promastigotes transport α-aminoisobutyric acid (AIB), the nonmetabolizable analog of neutral amino acids, against a substantial concentration gradient. AIB is not incorporated into cellular material but accumulates within the cells in an unaltered form. Intracellular AIB exchanges with external AIB. Various energy inhibitors (amytal, HOQNO, KCN, DNP, CCCP, and arsenate) and sulfhydryl reagents (NEM, pCMB, and iodoacetate) severely inhibit uptake. The uptake system is saturable with reference to AIB-and the Lineweaver-Burk plots show biphasic kinetics suggesting the involvement of two transport systems. AIB shares a common transport system with alanine, cysteine, glycine, methionine, serine, and proline. Uptake is regulated by feedback inhibition and transinhibition.     

Sucrose uptake by developing soybean cotyledons

Sucrose uptake by excised developing soybean cotyledons shows a biphasic dependence on sucrose concentration. At concentrations less than about 50 millimolar external sucrose, uptake can be described as a carrier-mediated process, with a K_m of 8 millimolar. At higher external sucrose concentrations, a linear dependence becomes apparent, which suggests the participation of a nonsaturable component in total uptake. Sucrose absorption is dependent on the presence of an electrochemical potential gradient for protons since agents interfering with the generation or maintenance of this gradient (NaN₃ or carbonylcyanide-m-chlorophenyl hydrazone) decrease sucrose transport to a level at or below that predicted from the operation of the noncarrier-mediated process alone. The saturable component of sucrose uptake is also sensitive to the sulfhydryl-modifying compounds N-ethylmaleimide and p-chloro-mercuribenzenesulfonate. The thiol-reducing agent diethioerythritol reverses fully the p-chloro-mercuri-benzenesulfonate inhibition, but not that of N-ethyl maleim de. Sucrose transport is sensitive to external pH, being decreased at high pH₀. Since sucrose-induced depolarization of the membrane potential and carrier-mediated sucrose influx show similar pH-dependence, inhibitor sensitivity, and values of K_m for sucrose, a sucrose/proton contransport process appears to operate in developing soybean cotyledon cells. Measurement of free space and intracellular sucrose concentrations in vivo suggests that the carrier-mediated process is fully saturated and that sucrose transport may be limiting for sucrose accumulation by the developing seed.     

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     

Glutamine Transport in Mouse Cerebral Astrocytes

Abstract: We measured initial influx and exchange of [¹⁴C]glutamine in primary astrocyte cultures in the presence and absence of Na⁺. Kinetic analysis of transport in Na⁺-free solution indicated two saturable Na⁺-independent components, one of which was identifiable functionally as system L₁ transport. In the presence of Na⁺, multiple hyperbolic components were not resolvable from the kinetic data. Nevertheless, other evidence supported participation by at least three Na⁺-dependent neutral amino acid transporters (systems A, ASC, and N). System A transport of glutamine was usually absent or minimal, based on lack of inhibition by α-(methylamino)isobutyric acid. However, vigorous system A-mediated transport emerged after derepression by substrate deprivation. Participation by system ASC was indicated by trans-acceleration of Na⁺-dependent uptake, preferential inhibition of an Li⁺-intolerant component of uptake by cysteine, and inhibition by cysteine of a component resistant to inhibition by histidine and α-(methylamino)isobutyric acid. Because nonsaturable transport of glutamine appeared negligible, and system L transport of glutamine was suppressed in the presence of Na⁺, low-affinity system ASC transport may be the major route of export of glutamine from astrocytes. At 700 µ M glutamine, the primary uptake route was system N transport, identified on the basis of selective inhibition by histidine and asparagine, pH sensitivity, and tolerance of Li⁺ in place of Na⁺.     

Factors involved in the uptake of corticosterone by rat liver cells

Isolated rat liver cells take up corticosterone rapidly; the initial rates increase with increasing temperature. A plot of the initial rates against the concentration of corticosterone indicated the presence of saturable and nonsaturable uptake systems. The Eadie-Hofstee plot showed the presence of two saturable and one nonsaturable uptake components. The apparent K_t values of the saturable systems were 64 ± 40 nM (n =3) and 1085 ± 313 nM (n =12). The nonsaturable system, probably diffusion, contributed 12% to the total uptake between 15 and 72 nM corticosterone, the physiological concentration of the free corticosterone in rat serum. Metabolic inhibitors did not influence the uptake of corticosterone. N-Ethylmaleimide, 1-fluor-2,4-dinitrobenzene and sodium ethyl mercurithiosalicylate (1 mM each) decreased the uptake by 40%. Iodoacetate did not have any influence. Treatment of cells with phospholipase A inhibited the uptake 35–45%. In the presence of cortisone, cortisol, dexamethasone, aldosteron, testosterone, estradiol-17β and estrone (2 μM each) the uptake decreased 30–50%. The presence of serum proteins in the external medium inhibits the uptake of corticosterone. These results suggest that corticosterone is transported into the cell and is accumulated. Only the free hormone is available for uptake which in turn may be regulated by protein and lipid components in the plasma membrane of the liver cell.     

Saturable uptake of indol-3yl-acetic Acid by maize roots

The uptake of 5-[³H]indol-3yl-acetic acid (IAA^*) by segments of Zea mays L. roots was measured in the presence of nonradioactive indol-3yl-acetic acid (IAA°) at different concentrations. IAA uptake was found to have a nonsaturable component and a saturable part with (at pH 5.0) an apparent K_m of 0.285 micromolar and apparent V_max 55.0 picomoles per gram fresh mass per minute. These results are consistent with those which might be expected for a saturable carrier capable of regulating IAA levels. High performance liquid chromatography analyses showed that very little metabolism of IAA^* took place during 4 minute uptake experiments. Whereas nonsaturable uptake was similar for all 2 millimeter long segments prepared within the 2 to 10 millimeter region, saturable uptake was greatest for the 2 to 4 millimeter region. High levels of uptake by stelar (as compared with cortical) segments are partly attributable to the saturable carrier, and also to a high level of uptake by nonsaturable processes. The carrier may play an essential role in controlling IAA levels in maize roots, especially the accumulation of IAA in the apical region. The increase in saturable uptake toward the root tip may also contribute to the acropetal polarity of auxin transport.     

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.     

Amino Acid transport in protoplasts isolated from soybean leaves

We isolated large quantities of mesophyll protoplasts from source and sink leaves of soybean plants and examined them for amino acid uptake. Accumulation of amino acids in isolated protoplasts was linear for at least 40 minutes. Uptake kinetics revealed the presence of both saturable and linear components. Increasing external pH decreases the uptake. The uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone at 15 micromolar inhibited and fusicoccin at 10 micromolar stimulated amino acid uptake. Our data are consistent with a proton-cotransport mechanism for the uptake of l-glutamine and α-amino isobutyric acid into soybean mesophyll cells.     

Differential effects of glucocorticoids on insulin-like growth factor I action in cultured human fibroblasts

Glucocorticoids act synergistically with insulin-like growth factor I (IGF-I) to stimulate DNA synthesis and replication of cultured human fibroblasts. In the present study, we further define glucocorticoid and IGF-I interactive effects on human fibroblast metabolism and growth. IGF-I stimulated dose-dependent increases in early metabolic events. Half-maximal effectiveness was seen at 5–8 ng/ml IGF-I, with mean maximal responses of 1.5-, 2-, and 6-fold for [³H]2-deoxyglucose uptake, [¹⁴C]glucose incorporation, and [¹⁴C]aminoisobutyric acid (AIB) uptake, respectively. A 48-hour preincubation with 10^?7 M dexamethasone markedly enhanced both the sensitivity and maximal effectiveness of IGF-I stimulation of AIB uptake. In contrast, dexamethasone had no effect on IGF-I-stimulated glucose uptake and utilization. Maximum specific binding of [125I]IGF-I to fibroblast monolayers was identical in ethanol control and glucocorticoid-treated cells, with 50% displacement at ～5 ng/ml IGF-I. In addition to its synergism with IGF-I, preincubation with dexamethasone augmented insulin and epidermal growth factor (EGF) stimulation of [³H]thymidine incorporation; dexamethasone had no effect on platelet-derived growth factor or fibroblast growth factor action. Two-dimensional gel electrophoresis identified two specific glucocorticoid-induced proteins in human fibroblast cell extracts with molecular weights of 45K and 53K and pls of 6.8 and 6.3, respectively. These data indicate that IGF-I receptor-mediated actions in human fibroblasts are differentially modulated by glucocorticoids. Glucocorticoids are synergistic with IGF-I in stimulating mitogenesis and amino acid uptake, without having any apparent effect on IGF-I-stimulated glucose metabolism. Glucocorticoid enhancement of growth factor bioactivity may involve modulation of a regulatory event in the mitogenic signaling pathway subsequent to cell surface receptor activation. © 1995 Wiley-Liss, Inc.     

Characterization of the active sucrose transport system of immature soybean embryos

Immature soybean embryos were isolated from soybean [Glycine max (L.) Merr.] seeds at various stages of development to study their accumulation of [¹⁴C]sucrose in vitro. Isolated embryos accumulate sucrose at a constant rate over several hours, the label entering large, endogenous pools of sucrose from which starch, protein, and lipid storage products are formed. Accumulation is without extracellular sucrose hydrolysis and occurs predominantly by active transport at physiological sucrose concentrations. A nonsaturable diffusion component, apparently superimposed upon the active saturable component, dominates overall uptake at exogenous concentrations greater than approximately 50 millimolar sucrose. Active transport is sensitive to uncoupling agents and the sulfhydryl-modifying reagent p-chloromecuribenzene sulfonate, is dependent on more than one energy source, and exhibits well-defined requirements for incubation temperature, pH, and oxygen availability. Under optimal incubation conditions of 35°C, saturating illumination (pH 6), and 21% oxygen, the apparent K_m for sucrose is approximately 8 millimolar and V_max is approximately 0.6 micromoles per hour per 100 milligrams fresh weight. Embryos readily accumulate sucrose from dilute exogenous solutions and, when preloaded with large amounts of sucrose, maintain the internal sucrose pool against steep outward gradients. These and other observations indicate that, although perhaps fully saturated in vivo, active sucrose transport is a significant component of photosynthate uptake in developing soybean embryos, enhancing uptake at physiological sucrose concentrations 2- to 5-fold over diffusion alone.     

Effect of pH on IAA Uptake by Maize Root Segments

The uptake of [5-³H]indoleacetic acid (IAA) by Zea mays L. root segments involves nonsaturable and saturable processes. The pH optimum of the saturable component was found to be 5.0. The proton ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone inhibited at 100 micromolar the saturable component of IAA uptake but had no effect on non-saturable uptake. This indicates that the saturable component of IAA uptake is dependent on the proton gradient across the plasmalemma. The high level of proton extrusion in the elongation zone of the root will stimulate nonsaturable and saturable uptake of IAA in that zone.     

Amino acid transport inRhodotorula glutinis

The yeastRhodotorula glutinis was found to transport amino acids against a concentration gradient (100∶1 for 10⁻⁶ m l-lysine and 1500∶1 for 10⁻⁶ m α-aminoisobutyric acid). Anaerobically, the concentration gradients of free amino acids were occasionally higher than aerobically. The influx is saturable with an apparentK _m of 1mm forl-lysine and 2mm for α-aminoisobutyric acid. The pH optimum for AIB uptake was 5.0, the apparent activation energy between 5° and 30° was 13,200 cal/mole. Competition of an asymmetric nature among various amino acids for uptake was observed. Intracellular amino acids did not leave the cell under any conditions of incubation, short of breaking up the plasma membrane, but they showed a powerful “trans” inhibitory effect on the uptake of amino acids.     

Effect of insulin on folic-acid transport in cultured fibroblasts

Uptake of folic acid was measured in secondary cultures of skin fibroblasts from fetal rats. The cultures were made quiescent by 24 hours preincubation in medium containing 1% serum and subsequent 3 hours preincubation in phosphate buffered saline. The uptake of ³H-folic acid was linear with time during 15 seconds and reached a plateau level at 2–3 minutes. There was no further increase in the intracellular radioactivity until the end of the experiments at 10 minutes. The uptake of folic acid in fibroblasts was not concentrative and proceeded until equilibration with the extracellular concentration. Intracellular metabolic conversion of folic acid was not significant during the time of the experiments (up to 10 minutes). Insulin caused a two-fold increase in the initial rate of folate uptake as determined from the 15 second uptake values. The dose response curves for the insulin effect showed that 85% of the maximal effect was exerted by 1 m?M insulin. A lag period of 7–10 minutes was observed after the addition of insulin and before the effect on folic acid uptake was manifested. Thereafter the effect increased with the time of preincubation with insulin. The concentration dependence of folate uptake yielded non homogeneous curves. At low concentrations of substrate, saturable components were observed while at high concentration (above 5 × 10^?6 M) a linear component was observed. Insulin increased the slope of the linear component and the V_max of the saturable component while the K_m remained unaltered.     

The uptake of gibberellin A1 by suspension-cultured Spinacia oleracea cells has a carrier-mediated component

The kinetics of the uptake of [³H]gibberellin A₁ (GA₁) by light- and dark-grown suspension-cultured cells of Spinacia oleracea (spinach) have been studied. Use of nonradioactive GA₁ and gibberellic acid (GA₃) show that the uptake has a saturable and a nonsaturable component. The nonsaturable component increases as the pH is lowered at a fixed concentration of [³H]GA₁ and is probably caused by non-mediated diffusion of the uncharged protonated species of GA₁. The saturable component is not the result of metabolic transformation or to GA₁ binding to the cell wall and is suggested to represent the operation of a transport carrier for which GA₁ and GA₃ are substrates. Auxin, abscisic acid and a cytokinin did not alter the GA₁ uptake. The K_m is approx. 0.3 mol dm^-3 at pH 4.4 in light- and dark-grown cells. The V_max of the carrier is higher in the light-grown cells. The optimum pH for the carrier at a physiological GA₁ concentration (3 nmol dm^-3) was pH 4.0, with no activity detectable at pH 7.0. Both saturable and nonsaturable components were decreased by protonophores indicating that the pH gradient between the cells and the medium may be a component of the driving forces for both types of transport. Both the permeability coefficient for the undissociated GA₁ and the ratio V _max/K _m for the carrier are lower than the corresponding values for the indole-3-acetic acid and abscisic acid carriers studied in other species.Abbreviations and symbols ABA abscisic acid - DMO 5,5-dimethyloxazolidine-2,4-dione - GA gibberellin - GA₃ gibberellic acid - IAA indole-3-acetic acid - P permeability coefficient     

SYNTHETIC AMINO ACIDS AND THE NATURE OF l-DOPA TRANSPORT AT THE BLOOD-BRAIN BARRIER

—The blood-brain barrier transport of amino acids has been measured using the carotid injection technique in the rat. The synthetic amino acids, 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH) and α-(methylamino)isobutyric acid (MeAIB), were model substrates in the Ehrlich cell for the leucine (L) and alanine (A) neutral amino acid transport mechanisms, respectively. The uptake (±)b-[carboxyl-¹⁴C]BCH at the same rate for the five brain regions tested suggested a similarity between regions for the L transport mechanism. At injectant concentrations of 0·1 mm (similar to naturally occurring aromatic neutral amino acids), BCH was mainly taken up by a saturable mediated transport mechanism (K₁, 0·16 mm and V_max, 0·03/μmol/g per min). At higher concentrations, uptake by a nonsaturable or diffusional mechanism could be demonstrated. When BCH was added as a second amino acid to l -[3-¹⁴C]DOPA, the saturable component of l -DOPA transport was significantly inhibited. MeAIB had no measurable effect on the rate of l -DOPA transport. These results suggested that the mediated transport mechanism for l -DOPA at the cerebral capillaries is similar to the l -neutral amino acid transport system.     

Stimulation of Amino Acid Uptake and Na+, K+-ATPase Activity by Norepinephrine in Superior Cervical Sympathetic Ganglia Excised from Adult Rats

Active uptake of a labelled nonmetabolizable amino acid, alpha-aminoisobutyric acid (AIB), into isolated superior cervical sympathetic ganglia (SCG) excised from adult rats was considerably stimulated by the addition of either norepinephrine (NE, 50 microM) or 3,4-dihydroxyphenylethylamine (dopamine, DA, 100 microM) to the medium during aerobic incubation for 2 h at 37 degrees C. The NE-induced increase in AIB uptake was significantly antagonized by the addition of an alpha 1-adrenoceptor antagonist (prazosin, 10 microM) in SCG axotomized 1 week prior to the examination, in which most of the ganglionic neurons had degenerated and reactive proliferation of the satellite glial components was in progress. The addition of neither acetylcholine (ACh, 1 mM) plus eserine (0.1 mM) nor cyclic nucleotides (1 mM) changed the AIB uptake by the SCG. In the axotomized SCG, the NE-evoked increase in AIB uptake was much more pronounced than that of intact or denervated SCG. A kinetic study of the active AIB uptake in the SCG showed that NE produced a decrease of the Km value and an increase in the Vmax, especially in the axotomized SCG. Ganglionic Na+, K+-ATPase activity was greatly stimulated in the presence of NE, but not by ACh. These results strongly suggest that the NE-induced enhancement of active AIB uptake in the isolated SCG is occurring in glial cells rather than in neuronal cells, with a possible alteration of membrane properties for amino acid uptake and with an apparent regulation by the stimulated transport enzyme Na+, K+-ATPase.     

Effect of oxygen on ascorbic acid uptake and concentration in embryonic chick brain

The effects of oxygen on ascorbic acid concentration and transport were studied in chick embryo (Gallus gallus domesticus). During normoxic incubations, plasma ascorbic acid concentration peaked on fetal day 12 and then fell, before increasing again on day 20 when pulmonary respiration began. In contrast, cerebral ascorbic acid concentration rose after day 6, was maintained at a relatively high level during days 8–18, and then fell significantly by day 20. Exposure of day 16 embryos for 48 h to 42% ambient O₂ concentration decreased ascorbic acid concentration by four-fifths in plasma and by one-half in brain, compared to values in normoxic (21% O₂) or hypoxic (15% O₂) controls. Hyperoxic preincubation of embryos also inhibited ascorbic acid transport, as evidenced by decreased initial rates of saturable and Na⁺-dependent [¹⁴C]ascorbic acid uptake into isolated brain cells. It may be concluded that changes in ascorbic acid concentration occur in response to oxidative stress, consistent with a role for the vitamin in the detoxification of oxygen radicals in fetal tissues. However, changing O₂ levels have less effect on ascorbic acid concentration in brain than in plasma, indicating regulation of the vitamin by brain cells. Furthermore, the effect of hyperoxia on cerebral vitamin C may result, in part, from inhibition of cellular ascorbic acid transport.     

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.     

Aminoisobutyric acid transport in primary cultures of normal adult rat hepatocytes.

In contrast to suspensions of freshly isolated hepatic parenchymal cells (HPC), short-term monolayer cultures of HPC displayed properties of active transport for the amino acid analog aminoisobutyric acid (AIB). The uptake of AIB was inhibited by KCN and iodoacetate, failed to occur at 4 degrees, and was stimulated by glucagon. The apparent Km for AIB uptake by cultured HPC was approximately 19 mM. Glucagon did not alter the apparent Km but did increase V.