TRANSPORT OF LYSINE FROM CEREBROSPINAL FLUID OF THE CAT

—The clearance from cerebrospinal fluid of l-[¹⁴C]lysine and l-[³H]arginine was measured during ventriculo-cisternal perfusions of anaesthetized cats. Increasing in the perfusate the concentration of unlabelled l-lysine produced a gradual reduction in clearance of the labelled amino acids without altering the uptake of l-[¹⁴C]lysine by the choroid plexus. Net transport of l-lysine out of cerebrospinal fluid occurred by saturable and non-saturable components. The saturable component satisfied Michaelis-Menton kinetics, while the behaviour of the non-saturable component was consistent with diffusion. A V_max of 0·017 μmol/min and an affinity constant (k_t) of 0·83 mm were estimated. The clearance of l-lysine was unaffected by the addition to the perfusate of high concentrations of selected neutral amino acids, but was stimulated by the presence of l-cystine. Conversely, a high concentration of l-lysine did not affect the clearance of glycine or cycloleucine. The dibasic amino acids appear to be removed from cerebrospinal fluid by a relatively specific, mediated transport system which may serve to regulate their concentrations in the cerebrospinal fluid.     

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.     

Synergism between different transport systems stimulates the uptake of neutral amino acids by isolated brain microvessels

Summary The polar long-chain amino acids glutamine and methionine can be transported across the endothelial cells of brain microvessels either by an L-system which operates by a facilitated diffusion, exchanging mechanism, or by a concentrating, energy-dependent A-system. The presence of glutamine and/or of methionine can induce a synergism between the two transport systems which results, by a transstimulation mechanism, in a net increased uptake of neutral hydrophobic aminoacids. The methionine analog S-methylthiocysteine, which is the mixed disulfide resulting from the combination of methanethiol with cysteine, behaves similarly to methionine in stimulating the uptake of neutral hydrophobic amino acids. The same transstimulating effect can even be obtained in collagenase-treated, A-system-deprived microvessels by inducing the direct formation of S-methylthiocysteine within the cytoplasmic compartment of the endothelial cells.Abbreviations MeAIB -methylaminoisobutyric acid - BCH DL--aminobicyclo-2,2,1-heptanecarboxylic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid     

Transport of L-tryptophan into slices of rat cerebral cortex

—Slices of rat cerebral cortex, when incubated aerobically at 37°C in Krebs-Ringer-phosphate solution (pH 7.0) containing 10 mm glucose and 1.0 mm l -tryptophan [1-¹⁴C], accumulated tryptophan. Within the first 15 min of incubation the ratio of the concentration of the amino acid in the tissue to that in the medium reached 3.5:1. Uptake of tryptophan was linear for the first 30 min and attained a maximum concentration ratio (tissue:medium) of 6.5:1 within 60 min. The transport mechanism became saturated at 1.0-3.0 mm tryptophan. Entry of the amino acid into the cortical cells was thereafter directly proportional to its initial concentration in the medium. The tissue: medium ratio at 15 min decreased significantly under the following experimental conditions: (1) lowering the incubation temperature to 0°C; (2) incubating under N₂; (3) omitting glucose; (4) decreasing the Na⁺ concentration below 50 mm ; (5) removing K⁺ from the medium; or (6) adding 1.0 mm NaCN or 0.1 mm protoveratrine B to the medium. These results provided evidence that the accumulation of tryptophan against its concentration gradient was an active process. The effects of a number of amino acids on the uptake of tryptophan were studied: of these, l -phenylalanine, dl -p-chlorophenylalanine, l -tyrosine, l -DOPA, the branched chain aliphatic amino acids (l -leucine, l -isoleucine, l -valine) and l -glutamic acid were found to be the most potent inhibitors of tryptophan transport. Several tryptophan metabolites were tested; only l -kynurenine inhibited the uptake of tryptophan.     

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     

Some Characteristics of Threonine Transport Across the Blood-Brain Barrier of the Rat

Threonine entry into brain is altered by diet-induced changes in concentrations of plasma amino acids, especially the small neutrals. To study this finding further, we compared effects of various amino acids (large and small neutrals, analogues, and transport models) on transport of threonine and phenylalanine across the blood-brain barrier. Threonine transport was saturable and was usually depressed more by natural large than small neutrals. Norvaline and 2-amino-n-butyrate (AABA) were stronger competitors than norleucine. 2-Aminobicyclo[2.2.1]heptane-2-carboxylate (BCH), a model in other preparations for the large neutral (L) system, and cysteine, a proposed model for the ASC system only in certain preparations, reduced threonine transport; 2-(methylamino)isobutyrate (MeAIB; a model for the A system for small neutrals) did not. Phenylalanine transport was most depressed by cold phenylalanine and other large neutrals; threonine and other small neutrals had little effect. Norleucine, but not AABA, was a strong competitor; BCH was more competitive than cysteine or MeAIB. Absence of sodium did not affect phenylalanine transport, but decreased threonine uptake by 25% (p less than 0.001). Our results with natural, analogue, and model amino acids, and especially with sodium, suggest that threonine, but not phenylalanine, may enter the brain partly by the sodium-dependent ASC system.     

Evidence for the transport of neutral as well as cationic amino acids by ATA3, a novel and liver-specific subtype of amino acid transport system A

We report here on the cloning and functional characterization of the third subtype of amino acid transport system A, designated ATA3 (amino acid transporter A3), from a human liver cell line. This transporter consists of 547 amino acids and is structurally related to the members of the glutamine transporter family. The human ATA3 (hATA3) exhibits 88% identity in amino acid sequence with rat ATA3. The gene coding for hATA3 contains 16 exons and is located on human chromosome 12q13. It is expressed almost exclusively in the liver. hATA3 mediates the transport of neutral amino acids including α-(methylamino)isobutyric acid (MeAIB), the model substrate for system A, in a Na⁺-coupled manner and the transport of cationic amino acids in a Na⁺-independent manner. The affinity of hATA3 for cationic amino acids is higher than for neutral amino acids. The transport function of hATA3 is thus similar to that of system y⁺L. The ability of hATA3 to transport cationic amino acids with high affinity is unique among the members of the glutamine transporter family. hATA1 and hATA2, the other two known members of the system A subfamily, show little affinity toward cationic amino acids. hATA3 also differs from hATA1 and hATA2 in exhibiting low affinity for MeAIB. Since liver does not express any of the previously known high-affinity cationic amino acid transporters, ATA3 is likely to provide the major route for the uptake of arginine in this tissue.     

Transport ofl-histidine by human diploid fibroblasts in culture

Summary The transport ofl-histidine has been characterized in skin derived diploid human fibroblasts, cultured under strictly controlled conditions. The transport measurements were made on cells grown to subconfluency after 60 to 90 min timed preincubation. The data, at substrate concentrations ranging from 0.050 to 10 mmol/l, were analyzed by a computer program. A saturable transport system (K _m =0.25 mmol/l, V _max =17 nmol/mg protein per min) and a nonsaturable component of influx (K _d =1.6±0.4 nmol/mg protein/min per mmol) were found.l-Histidine displayed no Na⁺ requirement at either low or high concentrations. Inhibition analysis demonstrated thatl-histidine uptake at low concentration was poorly inhibited by amino acids known to be effective inhibitors of system A. The largest fraction ofl-histidine uptake was inhibited by 2-amino-bicyclo (2,2,1)-heptane-2-carboxylic acid (BCH), leucine, and tryptophan. These results indicated thatl-histidine is transported in human fibroblasts, mainly by the Na⁺ independent system L. The differences between this cell type and others studied previously are discussed. This work was supported in part by Grant 773 from UER de Médecine, Université Paris XI (France).     

Characterization of system L and system y+ amino acid transport activity in cultured vascular smooth muscle cells

The uptake of L-leucine and L-lysine into vascular smooth muscle cells cultured from the aortas of rats has been investigated. Both amino acids are taken up by saturable systems that are independent of the presence of a ^·Na⁺ gradient and can be stimulated in trans by neutral bulky amino acids for leucine and cationic amino acids for lysine. Leucine uptake is inhibited competitively in cis by several neutral amino acids, whereas lysine uptake is inhibited strongly by other cationic amino acids but also significantly by neutral amino acids such as leucine. The leucine inhibition is noncompetitive. Cells preloaded with leucine and lysine could also export these amino acids and the rate of efflux was stimulated by the presence of appropriate amino acids in trans. These data are all consistent with leucine being transported largely if not entirely by System L and lysine by the System y⁺ transporter. © 1993 Wiley-Liss, Inc.     

UPTAKE OF 3-O-METHYL-d-GLUCOSE INTO CULTURED HUMAN GLIOMA CELLS

Human glioma cells (138 MG) were found to take up 3-O-methyl-d -glucose (3-OMG) by a saturable low affinity transport system with a K_m of 20 mm and a V_max of 500 nmol/mg protein/min. About 20 per cent of the total uptake was due to passive diffusion. d -Glucose was a competitive inhibitor with a K_i of 10 mm . Follow-up experiments indicated that the same transport mechanism is involved in the uptake of n-glucose and 3-OMG. Phloretin (0·02 mm ) and cytochalasin B (0·002 mm ) strongly inhibited the uptake of 3-OMG, whereas phlorizin (0·02 mm ), ouabain (0·1 mm ), NaCN (0·5 mm ) and iodoacetic acid (1·0 mm ) had no effect. The data suggest that 3-OMG and d -glucose enter 138 MG cells mainly by a Na⁺-independent passive carrier-mediated transport system. Serum-deprivation doubled the population doubling time (T_d) without affecting the total uptake of 3-OMG. An increase in the non-specific (diffusional) uptake was balanced by a decrease in the specific (carrier-medíated) uptake. After addition of dibutyryl cyclic AMP (dbcAMP, 0·25 mm ) the cells attained a morphology characteristic of differentiated glia cells. T_d was maintained unchanged. The non-specific uptake of 3-OMG was not affected in cells grown in serum-containing medium plus dbcAMP, whereas the specific uptake increased by 40 per cent and there-fore also the total uptake. Similar, but more pronounced, changes were observed if serum-deprived cells were treated with dbcAMP.     

ACTIONS OF l- AND d-HOMOCYSTEATE IN RAT CNS: A CORRELATION BETWEEN LOW-AFFINITY UPTAKE AND THE TIME COURSES OF EXCITATION BY MICROELECTROPHORETICALLY APPLIED L-GLUTAMATE ANALOGUES

Abstract— A correlation has been attempted between the uptake characteristics of l - and d -homocysteate and the time courses of neuronal excitation by these and other amino acids related to l -glutamate. The uptake of l - and d -homocysteate and of l -[³⁵S]homocysteate was studied in individual slices of rat cerebral cortex at 37°C. Tissue: medium ratios attained over l0 min for the unlabelled enantiomers at 2.5 mM were 3.7 for l -homocysteate but only 0.8 for the d -isomer. The uptake of l -[³⁵S]homocysteate over the concentration range 0.09 μm -2 mm can be attributed mainly to a low-affinity transport process with K_m approx 3 mm and V_max 1.7 μmol/g/min, but a high-affinity process of low V_max may make a minor contribution at the lower concentrations within this range. In terms of dependence on energy metabolism and [Na⁺], and on inhibition by p-chloromercuriphenylsulphonate, ouabain and structural analogues of the amino acid, the main uptake system for L-[³⁵S]homocysteate appears to be similar to that mediating low-affinity uptake of l -glutamate and other acidic amino acids. d -Homocysteate was but a weak inhibitor of this uptake system compared with other structural analogues. The time courses of excitation by 6 amino acids were determined by microelectrophoretic application to rat spinal neurones. d -Homocysteate induced responses with recovery times considerably longer than those of the other amino acids; this correlates with the absence of rapid uptake systems demonstrated for this amino acid in cortical tissue. d -Glutamate and l -homocysteate, which are only accumulated by low-affinity transport mechanisms, induced responses with recovery periods similar to those of l -glutamate, l -aspartate and d -aspartate, which are accumulated by both high- and low-affinity uptake systems. Although contributions of other factors to the observed time courses, such as rates of association and dissociation of the amino acid-receptor complexes, cannot be excluded, the present results are consistent with the hypothesis that low-affinity uptake systems of high V_max play an important role in the rapid termination of the effects of amino acid excitants.     

Multiple transport pathways for neutral amino acids in rabbit jejunal brush border vesicles

Amino acids enter rabbit jejunal brush border membrane vesicles via three major transport systems: (1) simple passive diffusion; (2) Na-independent carriers; and (3) Na-dependent carriers. The passive permeability sequence of amino acids is very similar to that observed in other studies involving natural and artificial membranes. Based on uptake kinetics and cross-inhibition profiles, at least two Na-independent and three Na-dependent carrier-mediated pathways exist. One Na-independent pathway, similar to the classical L system, favors neutral amino acids, while the other pathway favors dibasic amino acids such as lysine. One Na-dependent pathway primarily serves neutral L-amino acids including 2-amino-2-norbornanecarboxylic acid hemihydrate (BCH), but not beta-alanine or alpha-methylaminoisobutyric acid (MeAIB). Another Na-dependent route favors phenylalanine and methionine, while the third pathway is selective for imino acids and MeAIB. Li is unable to substitute for Na in these systems. Cross-inhibition profiles indicated that none of the Na-dependent systems conform to classical A or ACS paradigms. Other notable features of jejunal brush border vesicles include (1) no beta-alanine carrier, and (2) no major proline/glycine interactions.     

Transport of GABA from the perfused ventricular system of the cat

Abstract— The transport of GABA was studied in anaesthetized cats undergoing ventriculo-cisternal perfusion with radioactive GABA. Steady-state clearance of GABA from the CSF was greater than that of other amino acids previously studied, and was saturated at lower substrate concentrations, with an apparent K_t of 5·4 × 10^-5 M, after correcting for non-saturable transport. GABA clearance was inhibited by the inclusion of taurine or β-alanine in the perfusion fluid, but not by a number of the common neutral and acidic amino acids. Study of punch biopsies of brain tissue taken adjacent to the venticular system, at the completion of perfusions, showed accumulation of radioactive GABA in the tissue to values four times higher than those found in the perfusion fluid. Of the radioactivity which had been removed from the ventricular system, only 11 per cent remained in the brain at the completion of the perfusion. Excised cat choroid plexus showed a saturable uptake of GABA which was inhibited by inclusion of taurine, β-alanine, or β-guanidino propionic acid in the incubation medium.     

KINETICS OF COMPETITIVE INHIBITION OF NEUTRAL AMINO ACID TRANSPORT ACROSS THE BLOOD-BRAIN BARRIER

The transport of tryptophan across the blood-brain barrier is used as a specific example of a general approach by which rates of amino acid influx into brain may be predicted from existing concentrations of amino acids in plasma. The kinetics of inhibition of [¹⁴C]tryptophan transport by four natural neutral amino acids (phenylalanine, leucine, methionine, and valine) and one synthetic amino acid (α-methyl tyrosine) is studied with a tissue-sampling, single injection technique in the barbiturate-anesthetized rat. The equality of the K₁ (determined from cross-inhibition studies) and the K_m (determined from auto-inhibition data) for neutral amino acid transport indicate that these amino acids compete for a single transport site in accordance with the kinetics of competitive inhibition. Based on equations derived for competitive inhibition, apparent K_m values are computed for the essential neutral amino acids from known data on amino acid transport K_m and plasma concentrations. The apparent K_m values make possible predictions of the in vivo rates of amino acid influx into brain based on given plasma amino acid concentrations. Finally, a method is presented for determining transport constants from saturation data obtained with single injection techniques.     

THE EFFECT OF l-DOPA AND AN INHIBITOR OF PERIPHERAL DECARBOXYLATION ON GLUCOSE METABOLISM IN BRAIN1

Abstract– The effect of the administration of l -DOPA plus an inhibitor of peripheral l -aromatic amino acid decarboxylase (aromatic-l -amino-acid carboxy-lyase; EC 4.1.1.28) on the metabolism of glucose in brain was studied by administering [U-^I4C]glucose (20μCi) to three groups of rats: (1) rats that had been injected with l -DOPA (200mg/kg) 28min earlier; (2) rats that had been similarly injected with l -DOPA and also with N-(d,l -seryl)-N′-(2,3,4-trihydroxybenzyl)hydrazine (50 mg/kg), an inhibitor of l -aromatic amino acid decarboxylase, 30min before the l -DOPA; and (3) appropriate controls. The flux of ¹⁴C from glucose in plasma to those amino acids that are in equilibrium with the tricarboxylic acid cycle intermediates was reduced by treatment with l -DOPA and reduced further by treatment with l -DOPA and the decarboxylase inhibitor. Concentrations of glucose in brain and in plasma were increased after treatment with l -DOPA; these increases were attenuated if the inhibitor was given before the l -DOPA. After treatment with l -DOPA, there were decreases in the concentration of aspartate, tryptophan, and tyrosine in brain. After the administration of l -DOPA and the decarboxylase inhibitor, the concentrations in brain of alanine, glutamate, tyrosine, and phenylalanine were greater, and the concentrations of aspartate, leucine, lysine, histidine, arginine, and tryptophan were less than in control rats.     

L-alanine transport in isolated cells of interscapular brown adipose tissue in rat

The pattern of L-alanine uptake in isolated cells of interscapular brown adipose tissue has been determined. The uptake can be divided into the diffusion component (Kd=0.55 min^–1) and a saturable Na⁺-dependent transport (K _M =0.87 mM andV _max=155 nmol/min/10⁶ cells). The saturable component can be subdivided into MeAIB-sensitive (K _M =1.63 mM andV _max=162 nmol/min/10⁶ cells) and MeAIB-insensitive (K _M =3.2 mM andV _max=39.5 nmol/min/10⁶ cells). This kinetic pattern could indicate the presence of transport system (s) that resemble the commonly described transport systems for alanine uptake in several tissues.Abbreviations MeAIB Methyl-aminoisobutyric acid - AIB Aminoisobutyric acid     

Na+-Independent Transporters, LAT-2 and b0,+, Exchange L-DOPA with Neutral and Basic Amino Acids in Two Clonal Renal Cell Lines

The present study examined the functional characteristics of L-DOPA transporters in two functionally different clonal subpopulations of opossum kidney (OKLC and OKHC) cells. The uptake of L-DOPA was largely Na+-independent, though in OKHC cells a minor component (approximately 15%) required extracellular Na+. At least two Na+-independent transporters appear to be involved in L-DOPA uptake. One of these transporters has a broad specificity for small and large neutral amino acids, is stimulated by acid pH and inhibited by 2-aminobicyclo(2,2,l)-heptane-2-carboxylic acid (BCH; OKLC, Ki = 291 mM; OKHC, Ki = 380 mM). The other Na+-independent transporter binds neutral and basic amino acids and also recognizes the di-amino acid cystine. [14C]-L-DOPA efflux from OKLC and OKHC cells over 12 min corresponded to a small amount of intracellular [14C]-L-DOPA. L-Leucine, nonlabelled L-DOPA, BCH and L-arginine, stimulated the efflux of [14C]-L-DOPA in a Na+-independent manner. It is suggested that L-DOPA uses at least two major transporters, systems LAT-2 and b0,+. The transport of L-DOPA by LAT-2 corresponds to a Na+-independent transporter with a broad specificity for small and large neutral amino acids, stimulated by acid pH and inhibited by BCH. The transport of L-DOPA by system b0,+ is a Na+-independent transporter for neutral and basic amino acids that also recognizes cystine. LAT-2 was found equally important at the apical and basolateral membranes, whereas system b0,+ had a predominant distribution in apical membranes.     

Characterization of amino-acid transport in Ricinus communis roots using isolated membrane vesicles

The mechanism and specificity of amino-acid transport at the plasma membrane of Ricinus communis L. roots was investigated using membrane vesicles isolated by phase partitioning. The transport of glutamine, isoleucine, glutamic acid and aspartic acid was driven by both a pH gradient and a membrane potential (internally alkaline and negative), created artificially across the plasma membrane. This is consistent with transport via a proton symport. In contrast, the transport of the basic amino acids, lysine and arginine, was driven by a negative internal membrane potential but not by a pH gradient, suggesting that these amino acids may be taken up via a voltage-driven uniport. The energized uptake of all of the amino acids tested showed a saturable phase, consistent with carrier-mediated transport. In addition, the membrane-potential-driven transport of all the amino acids was greater at pH 5.5 than at pH 7.5, which suggests that there could be a direct pH effect on the carrier. Several amino-acid carriers could be resolved, based on competition studies: a carrier with a high affinity for a range of neutral amino acids (apart from asparagine) but with a low affinity for basic and acidic amino acids; a carrier which has a high affinity for a range of neutral amino acids except isoleucine and valine, but with a low affinity for basic and acidic amino acids; and a carrier which has a higher affinity for basic and some neutral amino acids but has a lower affinity for acidic amino acids. The existence of a separate carrier for acidic amino acids is discussed.Abbreviations PM plasma membrane - TPP⁺ tetraphenylphosphonium ion - pH pH gradient - membrane potential This work was supported by the Agricultural and Food Research Council and The Royal Society. We would like to thank Mrs. Sue Nelson for help with some of the membrane preparations.     

THE UPTAKE OF [3H]GABA BY SLICES OF RAT CEREBRAL CORTEX

—A rapid accumulation of [³H]GABA occurs in slices of rat cerebral cortex incubated at 25° or 37° in a medium containing [³H]GABA. Tissue medium ratios of almost 100:1 are attained after a 60 min incubation at 25°. At the same temperature no labelled metabolites of GABA were found in the tissue or the medium. The process responsible for [³H]GABA uptake has many of the properties of an active transport mechanism: it is temperature sensitive, requires the presence of sodium ions in the external medium, is inhibited by dinitrophenol and ouabain, and shows saturation kinetics. The estimated K_m value for GABA is 2·2 × 10^?5m , and V_max is 0·115 μmoles/min/g cortex. There is only negligible efflux of the accumulated [³H]GABA when cortical slices are exposed to a GABA-free medium. [³H]GABA uptake was not affected by the presence of large molar excesses of glycine, l -glutamic acid, l -aspartic acid, or β-aminobutyrate, but was inhibited in the presence of l -alanine, l -histidine, β-hydroxy-GABA and β-guanidinopropionate. It is suggested that the GABA uptake system may represent a possible mechanism for the inactivation of GABA or some related substance at inhibitory synapses in the cortex.     

L-lysine Transport in Chicken Jejunal Brush Border Membrane Vesicles

The properties of l-lysine transport in chicken jejunum have been studied in brush border membrane vesicles isolated from 6-wk-old birds. l-lysine uptake was found to occur within an osmotically active space with significant binding to the membrane. The vesicles can accumulate l-lysine against a concentration gradient, by a membrane potential-sensitive mechanism. The kinetics of l-lysine transport were described by two saturable processes: first, a high affinity-transport system (K _mA= 2.4 ± 0.7 μmol/L) which recognizes cationic and also neutral amino acids with similar affinity in the presence or absence of Na⁺ (l-methionine inhibition constant K_iA, NaSCN = 21.0 ± 8.7 μmol/L and KSCN = 55.0 ± 8.4 μmol/L); second, a low-affinity transport mechanism (K_mB= 164.0 ± 13.0 μmol/L) which also recognizes neutral amino acids. This latter system shows a higher affinity in the presence of Na⁺ (K_iB for l-methionine, NaSCN = 1.7 ± 0.3 and KSCN = 3.4 ± 0.9 mmol/L). l-lysine influx was significantly reduced with N-ethylmaleimide (0.5 mmol/L) treatment. Accelerative exchange of extravesicular labeled l-lysine was demonstrated in vesicles preloaded with 1 mmol/L l-lysine, l-arginine or l-methionine. Results support the view that l-lysine is transported in the chicken jejunum by two transport systems, A and B, with properties similar to those described for systems b ^0,+ and y⁺, respectively. Received: 14 August 1995/Revised: 2 April 1996