Characterization of tryptophan transport in human placental brush-border membrane vesicles.

The characteristics of tryptophan uptake in isolated human placental brush-border membrane vesicles were investigated. Tryptophan uptake in these vesicles was predominantly Na+-independent. Uptake of tryptophan as measured with short incubations occurred exclusively by a carrier-mediated process, but significant binding of this amino acid to the membrane vesicles was observed with longer incubations. The carrier-mediated system obeyed Michaelis-Menten kinetics, with an apparent affinity constant of 12.7 +/- 1.0 microM and a maximal velocity of 91 +/- 5 pmol/15 s per mg of protein. The kinetic constants were similar in the presence and absence of a Na+ gradient. Competition experiments showed that tryptophan uptake was effectively inhibited by many neutral amino acids except proline, hydroxyproline and 2-(methylamino)isobutyric acid. The inhibitory amino acids included aromatic amino acids as well as other system-1-specific amino acids (system 1 refers to the classical L system, according to the most recent nomenclature of amino acid transport systems). The transport system showed very low affinity for D-isomers, was not affected by phloretin or glucose but was inhibited by p-azidophenylalanine and N-ethylmaleimide. The uptake rates were only minimally affected by change in pH over the range 4.5-8.0. Tryptophan uptake markedly responded to trans-stimulation, and the amino acids capable of causing trans-stimulation included all amino acids with system-1-specificity. The patterns of inhibition of uptake of tryptophan and leucine by various amino acids were very similar. We conclude that system t, which is specific for aromatic amino acids, is absent from human placenta and that tryptophan transport in this tissue occurs via system 1, which has very broad specificity.     

High affinity insulin binding and insulin receptor-effector coupling: modulation by Ca2+.

Insulin binding and insulin stimulated amino acid and glucose uptake were determined in cultured HTC hepatoma cells in the presence of Ca2+ and ruthenium red (RR) in order to further characterise the putative calcium binding site on the receptor. These ions increased insulin receptor high affinity binding and the sensitivity of these responses to insulin. The insulin concentration required to half-maximally stimulate amino acid uptake decreased significantly from 26.9 +/- 5.8 ng/ml to 6.0 +/- 1.3 ng/ml in the presence of 10 mM Ca2+ and to 1.3 +/- 0.5 ng/ml in the presence of RR. The effect of Ca2+ and RR was more pronounced on insulin stimulated glucose uptake. These agents also increased receptor-effector coupling, reducing the percentage of occupied receptors required for maximal insulin stimulation of amino acid uptake from 10.8% in control cells to 3.4 and 1.4% in the presence of Ca2+ and RR respectively. The receptor occupancy required to produce maximal insulin responses on glucose uptake decreased from 20% (control) to 3.8% (Ca2+ and RR). We hypothesize that since Ca2+ and RR have similar effects, that occupation of Ca2+ binding sites on the receptor produces a conformational change in the insulin receptor which increases insulin receptor affinity, insulin sensitivity and acts on an early post-receptor event responsible for coupling binding to insulin action.     

Amino acid and putative neurotransmitter transport in human Y79 retinoblastoma cells. Effect of insulin and insulin-like growth factor

The binding of insulin and insulin-like growth factor I (IGF-I) and their effect on amino acid and neurotransmitter transport was studied in cultured human Y79 retinoblastoma cells. Y79 cells possess specific receptors for both insulin and IGF-I. Insulin binding to Y79 cells is characterized by a curvilinear Scatchard plot suggesting a two-site or two-affinity binding system. In contrast, IGF-I binding has a linear plot indicative of a one-site, one-affinity binding system. The uptake of glycine, a putative neurotransmitter in the retina occurs by a specific transport system in Y79 cells, independent of the uptake of other neutral amino acids. The uptake of glycine was increased 25-50% by either insulin or IGF-I. The response to insulin or IGF-I on glycine uptake is gradual and concentration dependent. The accumulation of other amino acids and putative retinal neurotransmitters by Y79 cells was not significantly affected by insulin of IGF-I. In addition, the activity of Na+/K+-ATPase was not influenced. The analysis of high affinity glycine uptake indicates that insulin and IGF-I are stimulating glycine transport by increasing the V'max without significantly affecting the K'm. Further analysis suggests that insulin and IGF-I are causing a recruitment of additional glycine transporters at the cell surface or activating otherwise nonfunctional transporters by an unexplained mechanism. Because of the implication that glycine responds as a neuroactive amino acid in Y79 cells these studies suggest that insulin and IGF-I may influence neuroactivity in the human retina by regulating the transport of glycine.     

Stimulation of synaptosomal uptake of neurotransmitter amino acids by insulin: possible role of insulin as a neuromodulator

Insulin stimulates in a dose-dependent manner (concentration range of 0.1 - 10 microM) the synaptosomal uptake of amino acids characterized by high-affinity, Na+-dependent, veratridine-sensitive transport systems. This stimulation is observed in synaptosomes prepared from each of several regions of the adult rat brain. Both the initial rate of amino acid uptake and the overall capacity for amino acid accumulation are increased. Since these transport systems have been associated with the neurotransmitter role of the amino acids, we postulate that insulin can modulate neurotransmission in the rat central nervous system by increasing the efficiency of neuroactive amino acid reuptake.     

Insulin and glucagon stimulation of (Na+-K+)-ATPase transport activity in isolated rat hepatocytes

The effects of insulin and glucagon on the (Na+-K+)-ATPase transport activity in freshly isolated rat hepatocytes were investigated by measuring the ouabain-sensitive, active uptake of 86Rb+. The active uptake of 86Rb+ was increased by 18% (p less than 0.05) in the presence of 100 nM insulin, and by 28% (p less than 0.005) in the presence of nM glucagon. These effects were detected as early as 2 min after hepatocyte exposure to either hormone. Half-maximal stimulation was observed with about 0.5 nm insulin and 0.3 nM glucagon. The stimulation of 86Rb+ uptake by insulin occurred in direct proportion to the steady state occupancy of a high affinity receptor by the hormone (the predominant insulin-binding species in hepatocytes at 37 degrees C. For glucagon, half-maximal response was obtained with about 5% of the total receptors occupied by the hormone. Amiloride (a specific inhibitor of Na+ influx) abolished the insulin stimulation of 86Rb+ uptake while inhibiting that of glucagon only partially. Accordingly, insulin was found to rapidly enhance the initial rate of 22Na+ uptake, whereas glucagon had no detectable effect on 22Na+ influx. These results indicate that monovalent cation transport is influenced by insulin and glucagon in isolated rat hepatocytes. In contrast to glucagon, which appears to enhance 86Rb+ influx through the (Na+-K+)-ATPase without affecting Na+ influx, insulin stimulates Na+ entry which in turn may increase the pump activity by increasing the availability of Na+ ions to internal Na+ transport sites of the (Na+-K+)-ATPase.     

Induction of amino acid transport in primary cultures of adult rat liver parenchymal cells by insulin.

Amino acid transport was studied in primary cultures of parenchymal cells isolated from adult rat liver by a collagenase perfusion technique and maintained as a monolayer in a serum-free culture medium. Amino acid transport was assayed by measuring the uptake of the nonmetabolizable amino acid, alpha-aminoisobutyric acid. Rat liver parenchymal cells transported alpha-aminoisobutyric acid by an energy-dependent Na+-requiring system which displayed Michaelis-Menten kinetics. Addition of insulin to cultured rat liver parenchymal cells resulted in an increased influx of alpha-aminoisobutyric acid which was reflected in a higher initial rate of alpha-aminoisobutyric acid transport as well as an increased accumulation of alpha-aminoisobutyric acid at later time points. Cycloheximide effectively blocked the increase while results with actinomycin D were equivocal. Insulin at concentrations as low as 50 pM was effective in stimulating alpha-aminoisobutyric acid transport while the maximal response was observed at 80 nM.     

Effects of different oxidizing agents on neutral amino acid transport systems in isolated bovine brain microvessels

Using isolated bovine brain microvessels as an in vitro model of the blood-brain barrier (BBB) we have evaluated the role of free radical generating solutions on some amino acid transport systems operating on the endothelial cell membrane. Fe(2+)/ascorbate, phenylhydrazine and CuSO(4) did not affect any of the transport system tested, while exposure of bovine brain microvessels to tert-butylhydroperoxide (t-BHP) caused a reduced capacity to take up small neutral amino acids via the Na(+)-dependent A-system. The presence of glucose during t-BHP treatment did not prevent this inhibition, which was partially counteracted when the isolated microvessels were incubated with 5mM inosine before the oxidative stress. Incubation of the isolated capillaries with 5mM dithiothreitol, after exposure to t-BHP, resulted in a 50% recovery of the alpha-methylaminoisobutyrate (MeAIB) uptake by the A-system. Treatment with t-BHP, which had no effect on the L-system of neutral amino acid transport, caused a significant decrease of the intracellular levels of ATP, of glutathione (GSH), and of gamma-glutamyltranspeptidase (GGT) activity, while no significant modification of hexokinase (HK) or of alkaline phosphatase (ALKP) activities were observed. Oxidative damage of the BBB appears therefore to impair essentially the metabolic pathways which ensure the energy requirement for the endothelial cells, thus inhibiting the energy-dependent amino acid transport system "A".     

Insulin-stimulated alpha-(methyl)aminoisobutyric acid uptake in skeletal muscle. Evidence for a short-term activation of uptake independent of Na+ electrochemical gradient and protein synthesis.

1. The present study was designed to explore the mechanisms by which insulin stimulates system A of amino acid transport in extensor digitorum longus (EDL) muscles, by using a system A analogue, alpha-(methyl)aminoisobutyric acid (MeAIB). 2. Insulin stimulation of MeAIB uptake was noted after only 30 min of incubation and was maximal at 60 min. Kinetics of the insulin effect on MeAIB uptake were characterized by an increased Vmax. without modification of Km for MeAIB. 3. Incubation of EDL muscles with cycloheximide for 90 min did not modify MeAIB uptake in either the presence or the absence of insulin, indicating the independence of insulin action from protein synthesis de novo. Incubations for 180 min with cycloheximide caused a decrease in basal MeAIB uptake; however, the percentage stimulation of amino acid transport by insulin was unaltered. Basal MeAIB uptake was increased by incubation for 180 min, but under these conditions no change in the percentage effect of insulin was found. 4. Ouabain, gramicidin D, or both, markedly decreased basal MeAIB uptake by EDL muscle, but the percentage effect of insulin was unaltered. 5. We conclude that insulin action on amino acid transport through system A in muscle is rapid, is characterized by an increased Vmax., and is independent of protein synthesis de novo and the Na+ electrochemical gradient. Our data are compatible with insulin acting directly on the system A transporter.     

Stimulation of Na+-Ca2+ exchange in heart sarcolemma by insulin

Insulin was found to stimulate Na+-dependent Ca2+ uptake in dog heart sarcolemma in a concentration dependent manner (0.001 to 1 milliunits/ml). Maximal stimulation (160 to 170%) was seen at 0.1 to 1 milliunits/ml of insulin. Unlike Na+-dependent Ca2+ uptake, ATP-dependent Ca2+ uptake was unaltered by 1 microunit/ml of insulin. However, high concentrations of insulin (0.01 to 1 milliunits/ml) significantly increased the ATP-dependent Ca2+ uptake activity of heart sarcolemma; maximal increase (60%) was observed at 1 milliunit/ml of insulin. The Na+ K+-ATPase activity did not change upon incubating sarcolemma with insulin. The membrane preparation exhibited specific insulin binding characteristics. The Scatchard plot analysis of the data indicated two binding sites for insulin; the association constants for the high and low affinity sites were 2 X 10(9) M-1 and 4.4 X 10(8) M-1, respectively. These results support the view regarding the presence of insulin receptors in the heart cell membrane and indicate a dramatic effect of insulin on the sarcolemmal Ca2+ transport systems.     

Insulin stimulation of (Na+,K+)-adenosine triphosphatase-dependent 86Rb+ uptake in rat adipocytes

Insulin stimulated the uptake of 86Rb+ (a K+ analog) in rat adipocytes and increased the steady state concentration of intracellular potassium. Half-maximal stimulation occurred at an insulin concentration of 200 pM. Both basal- and insulin-stimulated 86Rb+ transport rates depended on the concentration of external K+, external Na+, and were 90% inhibited by 10(-3) M ouabain and 10(-3) M KCN, indicating that the hormone was activating the (Na+,K+)-ATPase. Insulin had no effect on the entry of 22Na+ or exit of 86Rb+. Kinetic analysis demonstrated that insulin acted by increasing the maximum velocity, Vmax, of 86Rb+ entry. Inhibition of the rate of Rb+ uptake by ouabain was best described by a biphasic inhibition curve. Scatchard analysis of ouabain binding to intact cells indicated binding sites with multiple affinities. Only the rubidium transport sites which exhibited a high affinity for ouabain were stimulated by insulin. Stimulation required insulin binding to an intact cell surface receptor, as it was reversible by trypsinization. We conclude that the uptake of 86Rb+ by the (Na+,K+)-ATPase is an insulin-sensitive membrane transport process in the fat cell.     

Kinetics of Neutral Amino Acid Transport Through the Blood-Brain Barrier of the Newborn Rabbit

Abstract: Since protein synthesis in the developing brain may, under certain conditions, be limited by amino acid availability, the present studies were undertaken to characterize the kinetics of large neutral amino acid transport through the blood-brain barrier (BBB) of the newborn rabbit. The K_m, V_max, and K_D of the transport of eight amino acids were determined by a nonlinear regression analysis of data obtained with the carotid injection technique. Compared with kinetic parameters observed for the adult rat, the K_m, V_max, and K_D of amino acid transport were all two- to threefold higher in the newborn. Albumin was found to bind tryptophan actively in vitro , but had no inhibitory effect on tryptophan transport through the newborn BBB. Glutamine was transported through the BBB of the newborn at rates severalfold higher than are seen in the adult rat. However, glutamine transport was not inhibited by high concentrations of N -methylaminoisobutyric acid (NMAIB), a model amino acid that is specific for the alanine-preferring or A-system present in peripheral tissues. In conclusion, these studies show that the BBB neutral amino acid transport system of the newborn rabbit has a lower affinity and higher capacity than does the BBB of the adult rat. Under conditions of high plasma amino acids, the increased capacity of the newborn transport system allows for a higher rate of amino acid transport into brain than would occur via the lower capacity system present in the adult rat brain.     

Enhancement of Tryptophan Uptake by Divalent Cations in the Absence of Sodium Ions

Abstract: Nations were found to inhibit the uptake of L-tryptophan into synaptosomes with a shallow dose-response curve. Almost maximal inhibition was obtained with 10 mM-Na⁺. The divalent cations Ca²⁺ and Mg²⁺ were shown to be responsible for the increased uptake of L-tryptophan in the absence of Na⁺ ions. Other divalent cations also promoted tryptophan uptake under this condition (Ca²⁺ < Mg²⁺ < Mn²⁺ < Fe²⁺ < Zn²⁺ < Cu²⁺). It was concluded that monovalent chelate complexes were responsible for this enhancing effect. The measured L-tryptophan uptake was the net product of membrane bound and unbound tryptophan. Both bound and unbound tryptophan were increased in the presence of divalent cations. If no divalent cations were added to the incubation medium, Na⁺ ions decreased the unbound tryptophan but were without effect on bound tryptophan. Under these circumstances D-tryptophan had no effect on binding of the L-isomer and affected the transport of 1.-tryptophan only at very high does (100 x conc. L-tryptophan). These results suggest that I -tryptophan binds to a stereospecific transport carrier located in the synaptosomal membrane and that Na⁺ ions prevent the translocation of this carrier amino acid complex from the outer to the inner site of the neuronal membrane.     

Developmental control of 2-aminoisobutyric acid transport by 7-and 14-day chick heart cell aggregates. Roles of insulin and amino acids.

Cell aggregates cultured from 7-day embryonic avian heart showed a spontaneous increase in A-system 2-aminoisobutyric acid transport when placed in protein-free and amino acid-free buffer for 3 hr. The apparent V_max increased from 4.0 to 9.9 nmoles/μl of intracellular fluid volume/10 min in 3 hr. l-Proline (5 mM), an amino acid transported primarily by the A system, prevented this rise, but l-phenylalanine, primarily an L-system substrate, had no effect. Actinomycin, puromycin, and cycloheximide (55 μM) also prevented the time-dependent increase in transport. In contrast, cell aggregates cultured from 14-day embryonic heart exhibited a decrease in apparent V_max during the 3-hr incubation, from 8.3 to 3.3 nmoles/μl of intracellular volume/10 min. l-Proline, but not l-phenylalanine, enhanced this decrease in A-system transport. The percentage proline inhibition of transport was reduced by actinomycin or cycloheximide (55 μM) at both ages. Insulin stimulated A-system transport at identical half-maximal concentrations of 18 nM at 7 and 14 days of embryonic development. In the presence of cycloheximide at 7 days of age, insulin prolonged the half-life of transport activity twofold. However, at 14 days, cycloheximide reduced the insulin response by 88% [Elsas, L. J., Wheeler, F. B., Danner, D. J., and DeHaan, R. L. (1975). J. Biol. Chem.250, 9381–9390]. l-Proline or actinomycin reduced both basal and insulin-stimulated transport by 7-day cell aggregates, but neither reduced the percentage insulin stimulation. We conclude that inherent developmental control(s), A-system amino acids, and insulin regulated the maximal velocity of A-system transport by controlling the biological turnover of transport protein(s). l-Proline decreased the existing synthesis of transport protein(s) at both ages. The predominant effect of insulin shifted from a posttranslational level at 7 days to a synthetic level by 14 days of embryonic development. Seven-day cell aggregates spontaneously increased synthesis in the absence of A-system amino acids, but 14-day cell aggregates required hormonal stimulation to shift the balance from degradation to synthesis of transport protein(s).     

Evidence for modulation by ATP of amino acid transport in human placenta

The effect of ATP on placental amino acid transport was studied by measuring the uptake of alpha-(methylamino)-isobutyrate in brush border microvillous plasma membrane vesicles prepared from human full-term placental syncytiotrophoblasts which were incubated with or without ATP. The presence of a Na+ gradient from the outside to the inside of the vesicles prepared after incubation with ATP resulted in a higher initial rate and an increased transport of alpha-(methylamino)-isobutyrate, while Na+ gradient-independent alpha-(methylamino)-isobutyrate uptake was not different in either type of membrane vesicle. The increase in transport activity was not inhibited by cycloheximide. Kinetic analysis showed that ATP enhanced transport activity by increasing the maximal velocity (Vmax) of transport, without significant changes in the affinity (Km) of the carrier for the substrate, suggesting an increase in carrier number in placental syncytiotrophoblasts incubated with ATP.     

Involvement of membrane sulfhydryls in the activation and maintenance of nutrient transport in chick embryo fibroblasts

At 5 μg/ml, insulin stimulates hexose, A-system amino acid, and nucleoside transport by serum-starved chick embryo fibroblasts (CEF). This stimulation, although variable, is comparable to that induced by 4% serum. The sulfhydryl oxidants diamide (1–20 μM). hydrogen peroxide (500 μM), and methylene blue (50 μM) mimic the effect of insulin in CEF. PCMB-S,¹ a sulfhydryl-reacting compound which penetrates the membrane slowly, has a complex effect on nutrient transport in serum- and glucose-starved CEF. Hexose uptake is inhibited by 0.1–1 mM PCMB-S in a time- and concentration-dependent manner, whereas A-system amino acid transport is inhibited maximally within 10 min of incubation and approaches control rates after 60 min. A differential sensitivity of CEF transport systems is also seen in cells exposed to membrane-impermeant glutathione-maleimide I, designated GS-Mal. At 2 mM GS-Mal reduces the rate of hexose uptake 80–100% in serum- and glucose-starved CEF; in contrast A-system amino acid uptake is unaffected. D-glucose, but not L-glucose or cytochalasin B, protects against GS-Mal inhibition. These results are consistent with the hypothesis that sulfhydryl groups are involved in nutrient transport and that those sulfhydryls associated with the hexose transport system and essential for its function are located near the exofacial surface of the membrane in CEF.     

Insulin regulates ionic metabolism in a fresh water teleost, anabas testudineus (bloch)

Short term effects of insulin on total brain and branchial Na+K+ ATPase, Ca2+ ATPase and Na+, K+ and Ca2+ ions were investigated in A. testudineus. The increase in brain Ca2+ ATPase after alloxan treatment may account for an increased amount of intracellular calcium required for biochemical events taking place inside the cells. Branchial Na+K+ATPase was significantly stimulated while Ca2+ ATPase significantly inhibited after alloxan treatment. This suggests that alloxan exerts its inhibitory effect on the ATP-driven Ca2+ transport via; its action on the Ca2+ pump protein rather than the membrane permeability to Ca2+. The increased activity of brain Na+K+ ATPase at 3 and 24 hr by insulin to alloxan pretreated fish may account for the stimulated co-transport of glucose and its utilization for energy requirements and the excitatory action on neurons in the brain. The elevated brain Ca2+ ATPase may be due to the role of calcium as a second messenger in hormone action. At 24 hr, the activity of branchial Na+K+ ATPase and Ca2+ ATPase in alloxan pretreated specimens was significantly stimulated by insulin. This may be due to increased synthesis of these enzyme units. Administration of insulin (lU/fish) in normal fish significantly inhibited the activity of brain and branchial Na+K+ ATPase while brain Ca2+ ATPase showed a stimulatory effect at 3 and 24 hr compared to control. Inhibition of total branchial Ca2+ ATPase activity by insulin may be due to increased Ca2+ concentration. Higher plasma glucose level in alloxan treated groups confirms the diabetic effect of alloxan. Insulin reverses this effect. The possible mechanism by which insulin controls Na+K+ ATPase activity appears to be tissue specific. The results seem to be the first report on the effect of insulin on ATPase activity in a teleost. These data are consistent with the hypothesis that insulin performs a role in hydro mineral regulation in freshwater teleosts.     

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.     

Characterization of amino acid transport in membrane vesicles from the thermophilic fermentative bacterium Clostridium fervidus.

Amino acid transport was studied in membrane vesicles of the thermophilic anaerobic bacterium Clostridium fervidus. Neutral, acidic, and basic as well as aromatic amino acids were transported at 40 degrees C upon the imposition of an artificial membrane potential (delta psi) and a chemical gradient of sodium ions (delta microNa+). The presence of sodium ions was essential for the uptake of amino acids, and imposition of a chemical gradient of sodium ions alone was sufficient to drive amino acid uptake, indicating that amino acids are symported with sodium ions instead of with protons. Lithium ions, but no other cations tested, could replace sodium ions in serine transport. The transient character of artificial membrane potentials, especially at higher temperatures, severely limits their applicability for more detailed studies of a specific transport system. To obtain a constant proton motive force, the thermostable and thermoactive primary proton pump cytochrome c oxidase from Bacillus stearothermophilus was incorporated into membrane vesicles of C. fervidus. Serine transport could be driven by a membrane potential generated by the proton pump. Interconversion of the pH gradient into a sodium gradient by the ionophore monensin stimulated serine uptake. The serine carrier had a high affinity for serine (Kt = 10 microM) and a low affinity for sodium ions (apparent Kt = 2.5 mM). The mechanistic Na+-serine stoichiometry was determined to be 1:1 from the steady-state levels of the proton motive force, sodium gradient, and serine uptake. A 1:1 stoichiometry was also found for Na+-glutamate transport, and uptake of glutamate appeared to be an electroneutral process.     

Sodium ion-dependent amino acid transport in membrane vesicles of Bacillus stearothermophilus.

Amino acid transport in membrane vesicles of Bacillus stearothermophilus was studied. A relatively high concentration of sodium ions is needed for uptake of L-alanine (Kt = 1.0 mM) and L-leucine (Kt = 0.4 mM). In contrast, the Na(+)-H(+)-L-glutamate transport system has a high affinity for sodium ions (Kt less than 5.5 microM). Lithium ions, but no other cations tested, can replace sodium ions in neutral amino acid transport. The stimulatory effect of monensin on the steady-state accumulation level of these amino acids and the absence of transport in the presence of nonactin indicate that these amino acids are translocated by a Na+ symport mechanism. This is confirmed by the observation that an artificial delta psi and delta mu Na+/F but not a delta pH can act as a driving force for uptake. The transport system for L-alanine is rather specific. L-Serine, but not L-glycine or other amino acids tested, was found to be a competitive inhibitor of L-alanine uptake. On the other hand, the transport carrier for L-leucine also translocates the amino acids L-isoleucine and L-valine. The initial rates of L-glutamate and L-alanine uptake are strongly dependent on the medium pH. The uptake rates of both amino acids are highest at low external pH (5.5 to 6.0) and decline with increasing pH. The pH allosterically affects the L-glutamate and L-alanine transport systems. The maximal rate of L-glutamate uptake (Vmax) is independent of the external pH between pH 5.5 and 8.5, whereas the affinity constant (Kt) increases with increasing pH. A specific transport system for the basic amino acids L-lysine and L-arginine in the membrane vesicles has also been observed. Transport of these amino acids occurs most likely by a uniport mechanism.     

CORRELATION OF PLASMA AND BRAIN AMINO ACID AND PUTATIVE NEUROTRANSMITTER ALTERATIONS DURING ACUTE HEPATIC COMA IN THE RAT

During acute hepatic coma following two-stage hepatic devascularization in the rat, profound changes occurred in plasma and whole-brain amino acids and putative neurotransmitters. Brain ammonia, glutamine and GABA were increased, aspartate was decreased, while glutamate was unchanged. An increase in brain tryptophan was accompanied by a similar increase in plasma unbound tryptophan but decreased plasma total tryptophan. These changes occurred in the presence of high plasma levels of the other neutral amino acids, including the branched chain amino acids. Plasma insulin was unchanged while glucagon levels rose, resulting in a decreased insulin to glucagon ratio. These results suggest that while plasma unbound tryptophan may influence brain tryptophan levels, altered plasma concentrations of neutral amino acids which compete with tryptophan for transport into the brain do not contribute to the increase in brain tryptophan observed during acute hepatic coma.