Search

Glutamine and alanine are important precursors for the synthesis of glutamate. Provided to neurons by neighboring astrocytes, these amino acids are internalized by classical system A amino acid carriers. In particular, System A transporter (SAT1) is a highly efficient glutamine transporter, whereas SAT2 exhibits broad specificity for neutral amino acids with a preference for alanine. We investigated the localization and the functional relevance of SAT1 and SAT2 in primary cultures of hippocampal neurons. Both carriers have been expressed since early developmental stages and are uniformly distributed throughout all neuronal processes. However, whereas SAT1 is present in axonal growth cones and can be detected at later developmental stages at the sites of synaptic contacts, SAT2 does not appear to be significantly expressed in these compartments. The non-metabolizable amino acid analogue alpha-(methylamino)-isobutyric acid, a competitive inhibitor of system A carriers, significantly reduced miniature excitatory postsynaptic current amplitude in neurons growing on top of astrocytes, being ineffective in pure neuronal cultures. alpha-(Methylamino)-isobutyric acid did not alter neuronal responsitivity to glutamate, thus excluding a postsynaptic effect. These data indicate that system A carriers are expressed with a different subcellular distribution in hippocampal neurons and play a crucial role in controlling the astrocyte-mediated supply of glutamatergic neurons with neurotransmitter precursors.

Primary cultures of bovine adrenomedullary cells actively take up ascorbic acid and alpha-aminoisobutyric acid (AIB). Following a brief incubation with L-[14C] ascorbic acid and alpha-[methyl-3H]aminoisobutyric acid, cells stimulated with the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium iodide or by membrane depolarization with high [K+] or veratridine released newly acquired ascorbic acid (NA-ascorbate) and AIB. NA-ascorbate and endogenous catecholamines are differentially released under a variety of conditions suggesting that release of both substances cannot originate from the same subcellular compartment. In contrast, the release profile for NA-ascorbate and AIB, a putative cytosolic marker, suggest that both of these molecules are released from a cytosolic compartment. Cells permeabilized with the detergent digitonin release catecholamines only in the presence of external Ca2+, whereas release of NA-ascorbate and AIB is Ca2+-independent and time- and detergent concentration-dependent. If the osmolality of the external medium is made either hyper- or hypoosmotic, 1,1-dimethyl-4-phenylpiperazinium iodide-induced release of endogenous catecholamines is inhibited. Release of NA-ascorbate and AIB, however, is progressively inhibited with increasing osmolality and enhanced with decreasing osmolality. Furthermore, differential release of NA-ascorbate and AIB as compared to soluble acetylcholinesterase, which is apparently released form the cisternae of the endoplasmic reticulum, was also observed. To determine the mechanism by which NA-ascorbate and AIB are released from the cell, the requirements for their maximal release were investigated. Release of NA-ascorbate and AIB was sensitive to inhibitors (both metabolic and transport) and to changes in the external ionic environment. The metabolic inhibitors carbonyl cyanide p-trifluoromethoxyphenylhydrazone and KCN (when incubated simultaneously with 2-deoxyglucose) inhibited NA-ascorbate and AIB release by greater than 75%. In contrast, the Na+-K+-ATPase inhibitor ouabain enhanced veratridine-induced release of NA-ascorbate by nearly 100% and had an even greater effect on AIB release. Changes in the external ionic environment (i.e. Na+ and/or Cl- substitution) inhibited both NA-ascorbate and AIB release to varying degrees. Substitution of Cl- by various anions inhibited NA-ascorbate and AIB release to a much greater degree than endogenous catecholamine release. Complete substitution of NaCl with sucrose inhibited release of NA-ascorbate and AIB release by greater than 80%, while Na+ substituted with Li+ inhibited release of all three molecules by about 50%.(ABSTRACT TRUNCATED AT 400 WORDS)

In an effort to clarify the mechanism by which certain plant lectins induce lymphocyte activation, we have studied amino acid (amino[14C]isobutyric acid) uptake in purified human lymphocytes exposed to mitogenic and nonmitogenic lectins. In confirmation of earlier work, mitogenic lectins (concanavalin A and two phytohemagglutinins) produced a dose-related, 2- to 10-fold increase in aminoisobutyric acid transport. Changes occurred as early as 2 hours and reached a maximum after 18 hours. The stimulation by concanavalin A was inhibited by alpha-methyl-D-mannoside but not by other selected monosaccharides, indicating that the effect is modulated through specific carbohydrate receptors. In contrast to the stimulation with concanavalin A and phytohemagglutinin, the nonmitogen wheat germ agglutinin inhibited aminoisobutyric acid transport, both in the presence and absence of the mitogenic lectins. The inhibition was seen over a broad wheat germ agglutinin dose range, was prevented by N-acetylglucosamine, a known inhibitor of wheat germ agglutinin binding, and did not appear to be associated with cytotoxicity. Comparative binding studies with radiolabeled concanavalin A and wheat germ agglutinin demonstrated that differences in transport occurred in cells containing comparable numbers of bound lectin molecules indicating that the failure of wheat germ agglutinin to stimulate a response was not a result of ineffective binding. The lack of stimulation by wheat germ agglutinin was not due to its inability to interact multivalently with membrane receptors since this lectin is divalent, produces capping and agglutination, and continues to inhibit aminoisobutyric acid transport even after the minimum valence was increased to 4 by cross-linking with glutaraldehyde. In contrast both divalent and tetravalent concanavalin A produced stimulation. Competitive binding studies with soluble wheat germ agglutinin or lectin attached to 300 A latex spheres revealed little or no competition for binding sites with radiolabeled concanavalin A, suggesting the two lectins are interacting with different receptors. This was further suggested by kinetic studies of aminoisobutyric acid transport which indicated that wheat germ agglutinin was probably affecting both the Vmax and Km of transport, whereas concanavalin A affected only the Vmax. While the mechanism by which concanavalin A and wheat germ agglutinin produce opposing effects on amino acid transport is not clear, since the two lectins appear to be interacting with different surface receptors we would suggest that they are perturbing microanatomically and functionally different domains on the lymphocyte plasma membrane.

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.

Amino acid transport was studied in C1 cells which contain amplified levels of sodium- and potassium-activated adenosine triphosphatase (Na,K-ATPase), in C4 cells which are ouabain-sensitive revertants, and in parental HeLa S3. Sodium-dependent uptake of aminoisobutyric acid and alanine was increased 2-fold in the amplified C1 cells. After a 6 h amino acid starvation period, the rate of sodium-dependent uptake of methylaminoisobutyric acid was 70-90% greater for C1 than for C4 and HeLa. This uptake was inhibitable by ouabain and the apparent Km values for high affinity uptake were similar in all three lines. Overall, neutral amino acid uptake through Systems A, ASC, and L was 2-fold higher in the Na,K-ATPase amplified C1 cells relative to C4 or HeLa. The induction of System A uptake of methylaminoisobutyric acid after starvation was more rapid in both the amplified C1 cells and the revertant C4 when compared to HeLa, which suggests that the selection for amplification of the Na,K-ATPase produced membrane alterations affecting the adaptive regulation of System A.

When cells of the marine pseudomonad B-16 (ATCC 19855) grown in nutrient broth-salts medium containing 0.01 m KCl, were washed in a complete salts solution consisting of 0.3 m NaCl, 0.05 m MgSO4, and 0.01 m KCl the intracellular K+ concentration was approximately 0.3 m and the cells rapidly accumulated α-aminoisobutyric acid-14C (AIB-14C). Cells washed with 0.05 m MgSO4 solution alone lost some 95 to 98% of the total intracellular K+. Transport studies revealed that K+-depleted cells resuspended in salts solution containing 0.3 m NaCl, 0.05 m MgSO4, and 0.05 m Tris-HCl buffer (pH 7.2) failed to accumulate AIB-14C. Penetration studies under these conditions showed that α-aminoisobutyric acid (AIB) nevertheless entered the cells, and occupied the total available fluid space in a packed cell preparation of K+-depleted cells. Entry of AIB was Na+ dependent, and equivalent concentrations of Li+ or K+ failed to replace this cation in mediating AIB penetration and equilibration. Addition of K+ to the equilibrated system resulted in rapid accumulation of the substrate. The rate of AIB accumulation was maximal at 0.01 m KCl and kinetic analysis revealed that K+ increased Vmax but did not change the Km for transport. Additions of adenine nucleotides further increased the rate of AIB accumulation in the presence but not in the absence of K+. When K+-depleted cells were again loaded with K+ and subsequently transferred to AIB uptake medium lacking K+, there was immediate and rapid accumulation of substrate. Active transport of AIB is apparently mediated by two distinct cation-dependent steps. The first stage involves the entry and equilibration of AIB into the cells by a Na+-dependent facilitated diffusion mechanism. In the K+-dependent second stage, the cation, acting at the intracellular level, brings about the accumulation of AIB against a concentration gradient.

The requirement for Na+ for the uptake of α-aminoisobutyric acid (AIB) by cells of a marine pseudomonad was found to be quantitatively similar to the requirement for Na+ to prevent the release of AIB from previously loaded cells of the organism. At concentrations of Na+ different from the optimum, the viability of cells decreased in proportion to the extent of loss of AIB from previously loaded cells. AIB loss, however, preceded the loss of viability. The uptake of AIB required Na+ specifically while retention could be at least partially effected by other ions, particularly Li+. As the Na+ concentration in the suspending medium increased from 0 to 50 mm, the Km for transport decreased 12-fold while Vmax remained essentially constant. Between 50 and 200 mm Na+, Km remained constant at 8.33 x 10-5 m while Vmax continued to increase. Up to 200 mm Na+ the rate of uptake of AIB and the capacity of the cells for AIB were a direct function of the Na+ concentration or of the Li+ concentration if Na+ was present. Above 200mm Na+ the capacity of the cells for AIB decreased. Na+ could not be replaced by Li+ or K+ in effecting exchange diffusion of unlabeled AIB in the suspending medium with AIB-14C in the cells. Cells suspended at pH 7.2 in a medium containing NaCl or LiCl at a concentration optimum for retention of AIB lost AIB in the presence of NaCl but not LiCl when treated with 2,4-dinitrophenol or KCN. At pH 5.3 dinitrophenol caused the loss of AIB from the cells in the presence of either salt. The results suggest that there are two functions of Na+ in these cells, one concerned with uptake of compounds and the other involved in the prevention of their release.

The impairment by inositol deprivation of the α-aminoisobutyric acid (AIB) transport system of KB cells was studied under various experimental conditions. Kinetic studies under nonsteady state conditions showed that inositol deprivation causes a decrease of the Vmax of AIB influx and efflux without affecting the respective Km values. The Km for AIB influx is 0.4 to 0.5 mm, whereas for AIB efflux it is 6.2 to 6.8 mm, in both normal and inositol-deficient cells. The AIB efflux coefficients are much lower in deficient cells at all levels of intracellular AIB. When AIB exchange fluxes were studied under steady state conditions it was found that inositol deprivation causes a decrease of the influx coefficient and the Vmax. Active AIB transport requires the presence of extracellular Na+. The effect of decreasing the extracellular Na+ concentration is to increase the Km for AIB uptake without affecting the Vmax. This increase is greater in normal (11.0 mm) than in deficient cells (1.5 mm). Active AIB transport and accumulation are inhibited by ouabain. Half-maximal inhibition is obtained at 0.35 µm for AIB uptake and 0.1 µm for AIB accumulation, with both types of cells. The fraction of AIB transport sensitive to ouabain is 62% in normal and 38% in deficient cells. However, ouabain abolishes active transport in both types of cells. It is suggested that inositol deprivation impairs AIB transport because of structural alterations at the carrier sites of the plasma membrane.

When cells of marine pseudomonad B-16 (ATCC 19855) harvested during the logarithmic phase of growth were aerated in a buffered salt solution containing 0.3 m NaCl, 0.01 m KCl, and 0.05 m MgSO4 (Complete Salts Solution) immediately prior to analysis, intracellular Na+ was found to be 0.09 m and intracellular K+ 0.44 m. When the cells were washed with 0.05 m MgSO4 to deplete them of K+ and then were suspended with aeration in the buffered Complete Salts Solution, the intracellular Na+ and K+ concentrations were initially 0.3 and 0.004 m, respectively. On further incubation, the intracellular K+ concentration increased without a lag while the Na+ concentration, after a lag decreased until the ion concentrations prevailing in freshly harvested and aerated cells were obtained. The rate of uptake of K+ by and expulsion of Na+ from the cells was increased by adding ethanol, an oxidizable substrate, to the suspending medium. The Km and Vmax for α-aminoisobutyric acid transport into freshly harvested and aerated cells of the marine pseudomonad were essentially unchanged when the Na+ and K+ concentrations in the suspending medium were changed from those present in the Complete Salts Solution to those prevailing inside the cells. Thus the Na+ + K+ gradients are not required for α-aminoisobutyric acid accumulation, a Na+-dependent process. Cells suspended in the Complete Salts Solution from which KCl was omitted lost K+ at a slow constant rate which was not enhanced by α-aminoisobutyric acid uptake or the addition of NaCN.