Basolateral amino acid transport systems in the perfused exocrine pancreas: sodium-dependency and kinetic interactions between influx and efflux mechanisms

Basolateral amino acid transport systems have been characterized in the perfused exocrine pancreas using a high-resolution paired-tracer dilution technique. Significant epithelial uptakes were measured for L-alanine, L-serine, alpha-methylaminoisobutyric acid, glycine, methionine, leucine, phenylalanine, tyrosine and L-arginine, whereas L-tryptophan and L-aspartate had low uptakes. alpha-Methylaminoisobutyric acid transport was highly sodium dependent (81 +/- 3%), while uptake of L-serine, L-leucine and L-phenylalanine was relatively insensitive to perfusion with a sodium-free solution. Cross-inhibition experiments of L-alanine and L-phenylalanine transport by twelve unlabelled amino acids indicated overlapping specificities. Unidirectional L-phenylalanine transport was saturable (Kt = 16 +/- 1 mM, Vmax = 12.3 +/- 0.4 mumol/min per g), and weighted non-linear regression analysis indicated that influx was best described by a single Michaelis-Menten equation. The Vmax/Kt ratio (0.75) for L-phenylalanine remained unchanged in the presence of 10 mM L-serine. Although extremely difficult to fit, L-serine transport appeared to be mediated by two saturable carriers (Kt1 = 5.2 mM, Vmax1 = 7.56 mumol/min per g; Kt2 = 32.8 mM, Vmax2 = 22.9 mumol/min per g). In the presence of 10 mM L-phenylalanine the Vmax/Kt ratio for the two L-serine carriers was reduced, respectively, by 79% and 50%. Efflux of transported L-[3H]phenylalanine or L-[3H]serine was accelerated by increasing perfusate concentrations of, respectively, L-phenylalanine and L-serine, and trans-stimulated by other amino acids. In the pancreas neutral amino acid transport appears to be mediated by Na+-dependent Systems A and ASC, the classical Na+-independent System L and another Na+-independent System asc recently identified in erythrocytes. The interactions in amino acid influx and efflux may provide one of the mechanisms by which the supply of extracellular amino acids for pancreatic protein synthesis is regulated.     

Effect of hyperosmolarity on the activity of amino acid transport system L in avian fibroblasts

The transport of selected neutral amino acids known as good substrates of amino acid transport System L has been studied in chick embryo fibroblasts exposed for 4 hours to hyperosmolar culture medium. The activity of the L system, as measured by initial rates of L-phenylalanine uptake, increased in hyperosmolarity treated cells when determined before any cell depletion of intracellular amino acids. This effect was lost after depletion but reappeared after reloading the cells with pertinent substrates of System L. This transport activity appeared to be related to the internal level of amino acids capable of exchange through System L. In hyperosmolarity-treated chick embryo fibroblasts a higher level of System L substrates was obtained during the reloading phase in comparison to control cells. This expanded amino acid pool reflected an increased activity of transport System A, an agency of amino acid mediation known to enlarge its capacity following a hyperosmolar treatment of chick embryo fibroblasts (see Tramacere et al., 1984). L-Methionine, a preferred substrate of both A and L systems, appeared to be involved in the coupling between the activity of amino acid transport Systems A and L in these cells.     

Characterization of neutral and cationic amino acid transport in Xenopus oocytes

Amino acid transport was characterized in stage 6 Xenopus laevis oocytes. Most amino acids were taken up by the oocytes by way of both Na+-dependent and saturable Na+-independent processes. Na+-dependent transport of 2-aminoisobutyric acid (AIB) was insensitive to cis- or trans-inhibition by the System A-defining substrate 2-(methylamino)-isobutyric acid (MeAIB), although threonine, leucine, and histidine were found to be effective inhibitors, eliminating greater than 80% of Na+-dependent AIB uptake. Lack of inhibition by arginine eliminates possible mediation by System Bo,+ and suggests uptake by System ASC. The Na+-dependent transport of characteristic System ASC substrates such as alanine, serine, cysteine, and threonine was also insensitive to excess MeAIB. Evidence to support the presence of System Bo,+ was obtained through inhibition analysis of Na+-dependent arginine transport as well arginine inhibition of Na+-dependent threonine uptake. The Na+-independent transport of leucine was subject to trans-stimulation and was inhibited by the presence of excess phenylalanine, histidine, and, to a lesser extent, 2-amino-(2,2,1)-bicycloheptane-2-carboxylic acid (BCH). These observations are consistent with mediation by System L. The characteristics of Na+-independent uptake of threonine are not consistent with assignment to System L, and appear to be reflective of Systems asc and bo,+. In its charged state, histidine appears to be transported by a carrier similar in its specificity to System y+, but is taken up by System L when present as a zwitterion.     

Neutral amino acid transport in embryonal carcinoma cells

Neutral amino acid transport was characterized in the pluripotent embryonal carcinoma (EC) cell line, OC15. Ten of the thirteen amino acids tested are transported by all three of the major neutral amino acid transport systems--A, L, and ASC--although one system may make a barely measurable contribution in some cases. The characterization of N-methyl-aminoisobutyric acid (meAIB) transport points to this model amino acid as a definitive substrate for System A transport by OC15 cells. Thus, high concentrations of meAIB can be used selectively to block System A transport, and the transport characteristics of meAIB represent system A transport. Kinetic analysis of System A, with a Km = 0.79mM and Vmax = 14.4 nmol/mg protein/5 min, suggests a single-component transport system, which is sensitive to pH changes. While proline transport in most mammalian cells is largely accomplished through System A, it is about equally divided between Systems A and ASC in OC15 cells, and System A does not contribute at all to proline transport by F9 cells, an EC cell line with limited developmental potential. Kinetic analysis of System L transport, represented by Na+-independent leucine transport, reveals a high-affinity, single-component system. This transport system is relatively insensitive to pH changes and has a Km = 0.0031 mM and Vmax = 0.213 nmol/mg protein/min. The putative System L substrate, 2-aminobicyclo-[2,2,1]heptane-2-carboxylic acid (BCH), inhibits Systems A and ASC as well as System L in OC15 cells. Therefore, BCH cannot be used as a definitive substrate for System L in OC15 cells. Phenylalanine is primarily transported by Na+-dependent Systems A and ASC (83% Na+-dependent; 73% System ASC) in OC15 cells, while it is transported primarily by the Na+-independent System L in most other cell types, including early cleavage stage mouse embryos and F9 cells. We have also found this unusually strong Na+-dependency of phenylalanine transport in mouse uterine blastocysts (82% Na+-dependent). There is no evidence for System N transport by OC15 cells, since histidine is transported primarily by a Na+-independent, BCH-inhibitable mechanism.     

Chemical modification of the neutral amino acid transport system L of Chinese hamster ovary cells with p-chloromercuribenzene sulfonate.

Branched-chain and aromatic neutral amino acids enter mammalian cells predominantly through a Na(+)-independent transport agency called System L. The sulfhydryl specific reagent p-chloromercuribenzene sulfonate (pCMBS) has been shown to be a potent inactivator of System L transport activity in Chinese hamster ovary cells, however, inactivation by pCMBS can be prevented by the presence of System L-specific substrate amino acids during the inactivation reaction. In addition, the presence of amino acids that are not substrates for System L have no effect on pCMBS inactivation of System L. Inactivation of System L activity by pCMBS was sensitive to pH and reversible by incubation with dithiothreitol. These findings suggest that there is a sulfhydryl group in, or very near, the amino acid-binding site of the System L transporter of CHO cells. Substrate protection, however, could be explained by conformational changes in the transporter associated with substrate binding. The presence of a substrate protectable sulfhydryl group on the System L transporter would aid in the attempt to identify this transporter using the technique of differential labeling.     

Expression of the mammalian Na+-independent L system amino acid transporter in Xenopus laevis oocytes

System L is primarily responsible for the Na+-independent transport of neutral amino acids, those with bulky chains such as leucine, isoleucine, phenylalanine, etc., into mammalian cells. mRNA from rat kidney and human lymphoid cells, when microinjected into Xenopus laevis oocytes, induced expression of this transport system. The expressed transport exhibits characteristics similar to those reported for the System L amino acid transporter from a variety of mammalian cells. Injection of size-fractionated mRNA indicates that the System L transporter in both the rat kidney and human lymphoid cells is encoded by mRNA of about 3 to 4 kb.     

L-threonine transport in pig jejunal brush border membrane vesicles. Functional characterization of the unique system B in the intestinal epithelium.

Uptake and inhibitory kinetics of [3H]L-threonine were evaluated in preparations of pig jejunal brush border membrane vesicles. Uptake of [3H]L-threonine under O-trans, Na+ gradient, and O-trans, Na(+)-free conditions was best described by high affinity transport (Km < 0.01 mM) plus a nonsaturable component. The maximal velocity of transport was 3-fold greater under Na+ gradient conditions. 100 mM concentrations of all of the dipolar amino acids and 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid caused complete inhibition of [3H]L-threonine transport under Na+ gradient and Na(+)-free conditions. Imino acids, anionic amino acids, cationic amino acids, and methylamino-isobutyric acid caused significant partial inhibition of L-threonine uptake. Inhibitor concentration profiles for proline and lysine were consistent with low affinity competitive inhibition. The Ki values of alanine and phenylalanine approximated 0.2 and 0.5 mM, respectively, under both Na+ gradient and Na(+)-free conditions. These data indicate that the transport system available for L-threonine in the intestinal brush border membrane (system B) is functionally distinct from other amino acid transport systems. Comparison of kinetics parameters in the presence and absence of a Na+ gradient suggests that both partially and fully loaded forms of the carrier can function to translocate substrate and that Na+ serves to accelerate L-threonine transport by a mechanism that does not involve enhanced substrate binding.     

Polarity of neutral amino acid transport and characterization of a broad specificity transport activity in a kidney epithelial cell line, MDCK

The Madin-Darby canine kidney (MDCK) cell line was investigated with respect to the cellular polarity of amino acid transport in early confluent versus late confluent cultures. Early confluent cultures could take up amino acids from the apical and the basolateral sides of the cell layer via amino acid transport Systems A, ASC, and L. However, in late confluent cultures the activities of Systems A and L were clearly localized to the basolateral surface of the cell monolayer. In addition to the presence of systems A, ASC, and L, a novel activity, measurable under conditions used for quantitating System ASC, was found to be active in the apical membrane of these cells. This transporter, termed System G (for general), recognized basic and neutral amino acids with high affinity and acidic amino acids with lower affinity. System G exhibited broad substrate specificity, strict cation specificity, and a broad pH optimum with maximal activity at acidic pH. The activity of System G was relatively low after growth in serum-containing medium but was induced in a defined medium. Induction of System G activity was dependent upon the presence of prostaglandin E1. The broad substrate specificity, low pH optimum, and Na+ dependence suggest that System G may function in apical membranes as an energy-dependent transport route during reabsorption of amino acids from the kidney tubule lumen.     

Important differences in cationic amino acid transport by lysosomal system c and system y+ of the human fibroblast

Superficial similarities led us to extend our designation for the transport of the plasma membrane for cationic amino acids, y+, to the lysosomal system also serving for such amino acids. Further study on the purified lysosomes of human skin fibroblasts leads us now to redesignate the lysosomal system as c (for cationic), rather than y+, to emphasize important contrasts. Lysosomal uptake of arginine at pH 7.0 was linear during the first 2 min, but attained a steady state in 6 min. This arginine uptake was Na+-independent and was tripled in rate when the lysosomes had first been loaded with the cationic amino acid analog, S-2-aminoethyl-L-cysteine. Uptake was slowed to one-third when 2 mM MgATP was added to the incubation mixture. The following differences in cationic amino acid influx between lysosomal System c and the plasma membrane System y+ became apparent: 1) arginine influx is increased 10-fold by raising the external pH from 5.0 to 7.0. This effect favors net entry of cationic amino acids under the H+ gradient prevailing in vivo. In contrast, arginine uptake across the plasma membrane is insensitive to pH changes in this range. 2) The Km of arginine uptake by lysosomal System c, 0.32 mM, is eight times that for System y+ arginine uptake by the fibroblast. 3) Certain neutral amino acids in the presence of Na+ are accepted as surrogate substrates by System y+, but not by lysosomal system c. 4) Cationic amino acids in which the alpha-amino group is monomethylated or the distal amino group is quaternary, also D-arginine, are recognized by lysosomal System c, whereas System y+ has little affinity for these analogs. This broader substrate specificity of lysosomal system c led us to discover that thiocholine serves to deplete accumulated cystine from cystinotic fibroblasts as effectively as does the therapeutic agent, cysteamine. The quaternary nitrogen of thiocholine renders the mixed disulfide formed when it reacts with cystine unsatisfactory as a substrate for System y+.     

Tryptophan and phenylalanine transport in rat cerebral cortex slices as influenced by sodium ions

Tryptophan and phenylalanine transport in rat cerebral cortex slices was studied in sodium-free media and during influx and efflux of sodium ions. Choline as a substitute for sodium in incubation media increased efflux and decreased influx of tryptophan and phenylalanine. Exchange of intracellular [³H]tryptophan and [³H]phenylalanine with extracellular unlabeled histidine, phenylalanine, and tryptophan was sodium-independent. Efflux of sodium ions from the slices had no immediate effects on phenylalanine and tryptophan efflux, but influx decreased. Influx of sodium into the sodium-depleted slices provoked a transient increase in tryptophan and phenylalanine efflux and also enhanced influx. The results are interpreted to indicate that sodium ions may possibly affect the function of the primary transport sites for aromatic amino acids at cerebral membranes by controlling the orientation of their reactive sites towards the intracellular and extracellular sides, rather than by being directly involved in the binding of amino acids to the carriers.     

Na+-dependent amino acid transport is a major factor determining the rate of (Na+,K+)-ATPase mediated cation transport in intact HeLa cells

Little is known concerning the effects of Na+-coupled solute transport on (Na+,K+)-ATPase mediated cation pumping in the intact cell. We investigated the effect of amino acid transport and growth factor addition on the short term regulation of (Na+,K+)-ATPase cation transport in HeLa cells. The level of pump activity in the presence of amino acids or growth factors was compared to the level measured in phosphate buffered saline. These rates were further related to the maximal pump capacity, operationally defined as ouabain inhibitable 86Rb+ influx in the presence of 15 microM monensin. Of the growth factors tested, only insulin was found to moderately (22%) increase (Na+,K+)-ATPase cation transport. The major determinant of pump activity was found to be the transport of amino acids. Minimal essential medium (MEM) amino acids increased ouabain inhibitable 86Rb+ influx to a level close to that obtained with monensin, indicating that the (Na+,K+)-ATPase is operating near maximal capacity during amino acid transport. This situation may apply to tissue culture conditions and consequently measurements of (Na+,K+)-ATPase activity in buffer solutions alone may yield little information about cation pumping under culture conditions. This finding applies especially to cells having high rates of amino acid transport. Furthermore, rates of amino acid transport may be directly or indirectly involved in the long-term regulation of the number of (Na+,K+)-ATPase molecules in the plasma membrane.     

Comparison of system N in fetal hepatocytes and in related cell lines

In contrast to the changes seen in membrane transport systems for other neutral, anionic, and cationic amino acids, System N for glutamine, histidine, and asparagine in the rat hepatocytes shows nearly constant properties at the fetal, differentiated, and cultured hepatoma stages. These properties were tested by measuring the Na+-dependent transport of glutamine. This approximate constancy applies not only to the transport selectivity of the system among neutral amino acids, but also to its tolerance of Li+ as a substitute for Na+, its characteristic sensitivity to pH lowering, its relative sensitivity to N-ethylmaleimide, its stimulation by amino acid deprivation, and its failure to respond to insulin or glucagon. The properties of histidine as a substrate for System N were also examined. Inhibition studies with different cell types suggest that the Na+-dependent glutamine and histidine uptake is more restricted to System N in the hepatoma line H35 (H4-11-EC,3) and in the fetal hepatocyte than in hepatoma line HTC and the Ehrlich cells. The Na+-independent component of glutamine and histidine uptake was greater in the hepatoma cells in continuous culture than in fetal and adult hepatocytes in primary culture. Trans-stimulation of glutamine and histidine influx into H35 cells occurs predominantly by the Na+-independent route.     

The transport of L-arginine in Chinese hamster ovary cells

The transport of L-arginine has been characterized in Chinese hamster ovary cells (CHO). In the absence of Na+ the influx of the amino acid decreased. Both in the presence and in the absence of Na+ L-arginine influx was trans-stimulated and cis-inhibited by cationic amino acids. The amino acid entered CHO cells through an apparently non saturable mechanism and a single saturable agency whose Km increased in the absence of Na+. These results indicate that the agency devoted to transport cationic amino acids in CHO cells resembles system y+, the Na+-independent route that transports cationic amino acids in a number of mammalian models, although its activity is lowered by the replacement of extracellular sodium.     

Kinetic relations of the Na-amino acid interaction at the mucosal border of intestine

The relation between unidirectional influxes of Na and amino acids across the mucosal border of rabbit ileum was studied under a variety of conditions. At constant Na concentration in the mucosal bathing solution, amino acid influx followed Michaelis-Menten kinetics permitting determination of maximal influx and the apparent Michaelis constant, K_t. Reduction in Na concentration, using choline as substitute cation, caused an increase in K_t for alanine but had no effect on maximal alanine influx. The reciprocal of K_t was a linear function of Na concentration. Similar results were obtained for valine and leucine and these amino acids competitively inhibited alanine influx both in the presence and in the absence of Na. These results lead to a model for the transport system which involves combination of Na and amino acid with a single carrier or site leading to penetration of both solutes. The model predicts that alanine should cause an increase in Na influx and the ratio of this extra Na flux to alanine flux should vary with Na concentration. The observed relation agreed closely with predicted values for Na concentrations from 5 to 140 mM. These results support the hypothesis that interactions between Na and amino acid transport depend in part on a common entry mechanism at the mucosal border of the intestine.     

Transport properties of a system y+L neutral and basic amino acid transporter. Insights into the mechanisms of substrate recognition

The properties of system y(+)L-mediated transport were investigated on rat system y(+)L transporter, ry(+)LAT1, coexpressed with the heavy chain of cell surface antigen 4F2 in Xenopus oocytes. ry(+)LAT1-mediated transport of basic amino acids was Na(+)-independent, whereas that of neutral amino acids, although not completely, was dependent on Na(+), as is typical of system y(+)L-mediated transport. In the absence of Na(+), lowering of pH increased leucine transport, without affecting lysine transport. Therefore, it is proposed that H(+), besides Na(+) and Li(+), is capable of supporting neutral amino acid transport. Na(+) and H(+) augmented leucine transport by decreasing the apparent K(m) values, without affecting the V(max) values. We demonstrate that although ry(+)LAT1-mediated transport of [(14)C]l-leucine was accompanied by the cotransport of (22)Na(+), that of [(14)C]l-lysine was not. The Na(+) to leucine coupling ratio was determined to be 1:1 in the presence of high concentrations of Na(+). ry(+)LAT1-mediated leucine transport, but not lysine transport, induced intracellular acidification in Chinese hamster ovary cells coexpressing ry(+)LAT1 and 4F2 heavy chain in the absence of Na(+), but not in the presence of physiological concentrations of Na(+), indicating that cotransport of H(+) with leucine occurred in the absence of Na(+). Therefore, for the substrate recognition by ry(+)LAT1, the positive charge on basic amino acid side chains or that conferred by inorganic monovalent cations such as Na(+) and H(+), which are cotransported with neutral amino acids, is presumed to be required. We further demonstrate that ry(+)LAT1, due to its peculiar cation dependence, mediates a heteroexchange, wherein the influx of substrate amino acids is accompanied by the efflux of basic amino acids.     

KINETIC ANALYSIS OF PHENYLALANINE-INDUCED INHIBITION IN THE SATURABLE INFLUX OF TYROSINE, TRYPTOPHAN, LEUCINE AND HISTIDINE INTO BRAIN CORTEX SLICES FROM ADULT AND 7-DAY-OLD RATS

—An attempt was made to isolate the saturable uptake from the unidirectional influx of amino acids into tissue slices and to estimate the transport constants and maximal velocities of saturable transport. The method was applied to studies on the inhibition of phenylalanine in the saturable influx of tyrosine, tryptophan, histidine and leucine into brain cortex slices from adult and 7-day-old rats. In both age groups phenylalanine inhibited the influx of the other amino acids, and vice versa. The apparent transport constants of the other amino acids increased in the presence of phenylalanine more noticeably in the slices from 7-day-old rats than in those from adult rats, whereas the concomitant influx of phenylalanine was inhibited less in the slices from 7-day-old rats. In immature animals in vivo competition between amino acids may play a more marked role in the supply of amino acids from plasma to brain, as the transport systems in brain slices from 7-day-old rats become saturated with extracellular amino acids more readily than do the transport systems in brain slices from adult rats.     

Transport of L-methionine in human diploid fibroblast strain WI38

The transport of L-methionine in human diploid fibroblast strain WI38 was investigated. The uptake of L-methionine was measured in sparse cell cultures in a simple balanced salt solution buffered with either Tris.HCl of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). Similar results were obtained with these two buffers. Cultures were allowed to equilibrate with the buffered saline before transport was measured. The presence of glucose in the buffered saline results in a slight reduction in the initial rate of transport for the first 2 h of equilibration in buffered saline. L-Methionine is actively transported in WI38 by saturable, chemicallly specific mechanisms which are temperature, pH and, in part Na+ dependent, and are reactive with both L- and D-stereoisomers. Kinetic analysis of initial rates of transport at substrate concentrations from 0.0005 to 100 mM indicated the presence of two saturable transport systems. System 1 has an apparent KM of 21.7 micrometer and an apparent V of 3.57 nmol/mg per min. System 2 has an apparent KM of 547 micrometer and an apparent V of 22.6 nmol/mg per min. Kinetic analysis of initial rates of transport in Na+-free media or after treatment with ouabain suggested that system 1 is Na+ independent and that system 2 is Na+ dependent. Preloading of cells with unlabeled L-methionine greatly increases the initial rate of uptake. Efflux of transported methionine is temperature dependent, and is greatly increased in the presence of unlabeled L- or D-methionine or L-phenylalanine, but not in the presence of L-arginine. L-Methionine transport is strongly inhibited by other neutral amino acids, and is very weakly inhibited by dibasic amino acids, dicarboxylic amino acids, proline or glycine.     

Coupling of a loop diuretic-sensitive Na+ influx with the net loop diuretic-sensitive K+ efflux in mouse NIH 3T3 cells

Mouse 3T3 fibroblasts have a loop diuretic sensitive Na+ transport system, responsible for more than 50% of the total Na+ influx. This transport system is dependent on the simultaneous presence of all three ions; Na+, K+, (Rb+) and Cl- in the extracellular medium. The same requirement for these three ions was also found for the loop diuretic-sensitive K+ efflux. In addition, the sensitivities of Na+ influx and Rb+ efflux for the two loop diuretics, furosemide and bumetanide were found to be similar. The similar ionic requirement and sensitivity towards loop diuretics of the two fluxes, support the hypothesis, that this loop diuretic-sensitive Na+ influx in mouse 3T3 cells, is accompanied by the net loop diuretic-sensitive K+ efflux.     

Na+-dependent transport of large neutral amino acids occurs at the abluminal membrane of the blood-brain barrier

Several Na+-dependent carriers of amino acids exist on the abluminal membrane of the blood-brain barrier (BBB). These Na+-dependent carriers are in a position to transfer amino acids from the extracellular fluid of brain to the endothelial cells and thence to the circulation. To date, carriers have been found that may remove nonessential, nitrogen-rich, or acidic (excitatory) amino acids, all of which may be detrimental to brain function. We describe here Na+-dependent transport of large neutral amino acids across the abluminal membrane of the BBB that cannot be ascribed to currently known systems. Fresh brains, from cows killed for food, were used. Microvessels were isolated, and contaminating fragments of basement membranes, astrocyte fragments, and pericytes were removed. Abluminal-enriched membrane fractions from these microvessels were prepared. Transport was Na+ dependent, voltage sensitive, and inhibited by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, a particular inhibitor of the facilitative large neutral amino acid transporter 1 (LAT1) system. The carrier has a high affinity for leucine (Km 21 +/- 7 microM) and is inhibited by other neutral amino acids, including glutamine, histidine, methionine, phenylalanine, serine, threonine, tryptophan, and tyrosine. Other established neutral amino acids may enter the brain by way of LAT1-type facilitative transport. The presence of a Na+-dependent carrier on the abluminal membrane capable of removing large neutral amino acids, most of which are essential, from brain indicates a more complex situation that has implications for the control of essential amino acid content of brain.     

Effect of Intracellular Glutamine on the Uptake of Large Neutral Amino Acids in Astrocytes: Concentrative Na+-Independent Transport Exhibits Metastability

To examine whether the concentration gradient of glutamine (Gln) drives concentrative Na(+)-independent uptake of neutral amino acids (NAA) in mouse cerebral astrocytes, uptake was compared in "Gln-depleted" and "Gln-replete" cultures. Uptake (30 min in Na(+)-free buffer) of histidine, kynurenine, leucine, tyrosine, and a model substrate for System L transport was 70-150% greater in Gln-replete cultures. Phenylalanine uptake was not affected. All of these NAA trans-stimulated the export of Gln from astrocytes. However, the increase in NAA uptake was sustained even though the Gln content of Gln-replete cultures declined. Also, uptake of Gln itself was enhanced in Gln-replete cultures. Thus, countertransport of Gln was insufficient to explain the enhancement of NAA uptake. Enhanced uptake was restored, and could be magnified, by reloading Gln-depleted cultures either with Gln or with histidine. It is suggested that substrate-induced asymmetry and molecular hysteresis in the Na(+)-independent carrier could account for the sustained enhancement of NAA uptake. Only histidine and kynurenine were concentrated comparably to Gln (15- to 29-fold at 1 mM in Na(+)-free buffer). The other NAA were four to six times less concentrated. At least two Na(+)-dependent transport systems also supported the concentration gradient of Gln in regular buffer.