Glutamine Transport in Mouse Cerebral Astrocytes

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

N-System Amino Acid Transport at the Blood-CSF Barrier

Abstract: Despite l -glutamine being the most abundant amino acid in CSF, the mechanisms of its transport at the choroid plexus have not been fully elucidated. This study examines the role of L-, A-, ASC-, and N-system amino acid transporters in l -[¹⁴C]glutamine uptake into isolated rat choroid plexus. In the absence of competing amino acids, approximately half the glutamine uptake was via a Na⁺-dependent mechanism. The Na⁺-independent uptake was inhibited by 2-amino-2-norbornane carboxylic acid, indicating that it is probably via an L-system transporter. Na⁺-dependent uptake was inhibited neither by the A-system substrate α-(methylamino)isobutyric acid nor by the ASC-system substrate cysteine. It was inhibited by histidine, asparagine, and l -glutamate γ-hydroxamate, three N-system substrates. Replacement of Na⁺ with Li⁺ had little effect on uptake, another feature of N-system amino acid transport. These data therefore indicate that N-system amino acid transport is present at the choroid plexus. The V _max and K _max for glutamine transport by this system were 8.1 ± 0.3 nmol/mg/min and 3.3 ± 0.4 m M , respectively. This system may play an important role in the control of CSF glutamine, particularly when the CSF glutamine level is elevated as in hepatic encephalopathy.     

Intracellular Ascorbic Acid Inhibits the Na+-Ca2+ Exchanger in Cultured Rat Astrocytes

Abstract: The effect of ascorbic acid on Ca²⁺ uptake in cultured rat astrocytes was examined in the presence of ouabain and monensin, which are considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Ascorbic acid at 0.1–1 m M inhibited Na⁺-dependent Ca²⁺ uptake significantly but not Na⁺-dependent glutamate uptake in the cells, although the inhibition required pretreatment for more than 30 min. The effect of ascorbic acid on the Ca²⁺ uptake was blocked by simultaneous addition of ascorbate oxidase (10 U/ml). Na⁺-dependent Ca²⁺ uptake was also inhibited by isoascorbate at 1 m M but not by ascorbate 2-sulfate, dehydroascorbate, and sulfhydryl-reducing reagents such as glutathione and 2-mercaptoethanol. The inhibitory effect of ascorbic acid was observed even in the presence of an inhibitor of lipid peroxidation, o -phenanthroline, or a radical scavenger, mannitol, and the degrading enzymes such as catalase and superoxide dismutase. On the other hand, the inhibitory effect was not observed under the Na⁺-free conditions that inhibited the uptake of ascorbic acid in astrocytes. When astrocytes were cultured for 2 weeks in a medium containing ascorbic acid, the content of ascorbic acid in the cells was increased and conversely Na⁺-dependent Ca²⁺ uptake was decreased. These results suggest that an increase in intracellular ascorbic acid results in a decrease of Na⁺-Ca²⁺ exchange activity in cultured astrocytes and the mechanism is not related to lipid peroxidation.     

UPTAKE OF GLUCOSE ANALOGUES BY RAT BRAIN CORTEX SLICES: Na+-INDEPENDENT MEMBRANE TRANSPORT

Abstract— The glucose analogues 3-O-methyl-D-glucose and α-methyl-D-glucoside were not metabolized in brain tissue.
The uptake of these two sugars into the intracellular compartment of brain cortex slices was investigated using media with normal and low Na⁺ concentration (replacement of all NaCl with choline Cl). The cellular transport was not Na⁺-dependent. The transport mechanism clearly distinguished between the two sugars in both normal and low Na⁺ media.     

Early Signals in Serum-Induced Increases in Ouabain-Sensitive Na+-K+ Pump Activity and in Glucose Transport in Rat Skeletal Muscle Are Amiloride-Sensitive

Abstract: The acute effects of serum on sodium-potassium (Na⁺-K⁺) pump activity and glucose uptake in cultured rat skeletal muscle were studied. Addition of serum to myo-tubes in phosphate-buffered saline caused Na⁺-K⁺ pump activity (as measured by changes in the ouabain-sensitive component of both membrane potential and ⁸⁶Rb uptake) to increase, with peak effects obtained after 30 min. The effect was blocked completely by treatment with amiloride, but not by tetrodotoxin, which blocks voltage-dependent Na⁺ channels. On transfer of myotubes to Na⁺-free, choline buffer, resting Na⁺-K⁺ pump activity decreased to about 10% of that in phosphate-buffered saline. Addition of regular serum, but not Na⁺-free serum, caused Na⁺-K⁺ pump activity to increase slightly. Similar results were obtained with serum on glucose uptake, the peak effect being reached within 15 min. Stimulation of glucose uptake by serum was partially reduced by amiloride and was not altered by tetrodotoxin. Removal of external Na⁺ also eliminated serum effects on glucose uptake. The results demonstrate that there are similar signals involving Na⁺-H⁺ exchange for serum-induced increases in Na⁺-K⁺ pump activity and glucose transport. The lack of complete blockade of serum-induced elevation of glucose transport suggests an additional, as yet undefined, intracellular signal for stimulation of this transport system.     

Glutamate Uptake Stimulates Na+,K+-ATPase Activity in Astrocytes via Activation of a Distinct Subunit Highly Sensitive to Ouabain

Abstract: The excitatory amino acid glutamate was previously shown to stimulate aerobic glycolysis in astrocytes by a mechanism involving its uptake through an Na⁺-dependent transporter. Evidence had been provided that Na⁺,K⁺-ATPase might be involved in this process. We have now measured the activity of Na⁺,K⁺-ATPase in cultured astrocytes, using ouabain-sensitive ⁸⁶Rb uptake as an index. l -Glutamate increases glial Na⁺,K⁺-ATPase activity in a concentration-dependent manner with an EC₅₀ = 67 µ M . Both l - and d -aspartate, but not d -glutamate, produce a similar response, an observation that is consistent with an uptake-related effect rather than a receptor-mediated one. Under basal conditions, concentration-dependent inhibition of Na⁺,K⁺-ATPase activity in astrocytes by ouabain indicates the presence of a single catalytic site with a low affinity for ouabain ( K _0.5 = 113 µ M ), compatible with the presence of an α₁ isozyme. On stimulation with glutamate, however, most of the increased activity is inhibited by low concentrations of ouabain ( K _0.5 = 20 n M ), thus revealing a high-affinity site akin to the α₂ isozyme. These results suggest that astrocytes possess a glutamate-sensitive isoform of Na⁺,K⁺-ATPase that can be mobilized in response to increased neuronal activity.     

Nitroprusside and Cyclic GMP Stimulate Na+-Ca2+ Exchange Activity in Neuronal Preparations and Cultured Rat Astrocytes

Abstract: The effects of nitric oxide (NO)-generating agents on ⁴⁵Ca²⁺ uptake in rat brain slices and cultured rat astrocytes were studied in the presence of monensin, which is considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Sodium nitroprusside (SNP) at >10 µ M increased monensin-stimulated Ca²⁺ uptake in the slices, although it did not affect high K⁺-stimulated Ca²⁺ uptake. Another NO donor, 3-morpholinosydnonimine, was effective. The effect of SNP was antagonized by hemoglobin (50 µ M ), a NO scavenger, and mimicked by 8-bromo-cyclic GMP (100 µ M ). In rat brain synaptosomes, SNP increased monensin-stimulated Ca²⁺ uptake, but it did not affect high K⁺-stimulated Ca²⁺ uptake. 8-Bromocyclic GMP, but not SNP, increased Na⁺-dependent Ca²⁺ uptake significantly in synaptic membrane vesicles in the absence of monensin. In cultured rat astrocytes, SNP and 8-bromo-cyclic GMP increased Ca²⁺ uptake in the presence of ouabain and monensin, which were required for the Ca²⁺ uptake in the cells. These findings suggest that NO stimulates the Na⁺-Ca²⁺ exchanger in neuronal preparations and astrocytes in a cyclic GMP-dependent mechanism.     

Alanine metabolism, transport, and cycling in the brain

Brain glutamate/glutamine cycling is incomplete without return of ammonia to glial cells. Previous studies suggest that alanine is an important carrier for ammonia transfer. In this study, we investigated alanine transport and metabolism in Guinea pig brain cortical tissue slices and prisms, in primary cultures of neurons and astrocytes, and in synaptosomes. Alanine uptake into astrocytes was largely mediated by system L isoform LAT2, whereas alanine uptake into neurons was mediated by Na⁺-dependent transporters with properties similar to system B⁰ isoform B⁰AT2. To investigate the role of alanine transport in metabolism, its uptake was inhibited in cortical tissue slices under depolarizing conditions using the system L transport inhibitors 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid and cycloleucine (1-aminocyclopentanecarboxylic acid; cLeu). The results indicated that alanine cycling occurs subsequent to glutamate/glutamine cycling and that a significant proportion of cycling occurs via amino acid transport system L. Our results show that system L isoform LAT2 is critical for alanine uptake into astrocytes. However, alanine does not provide any significant carbon for energy or neurotransmitter metabolism under the conditions studied.     

Tryptophan Transport into Plasma Membrane Vesicles Derived from Rat Brain Synaptosomes

Abstract: Tryptophan uptake by membrane vesicles derived from rat brain was investigated. The uptake is dependent on the Na⁺ gradient [Na⁺] outside > [Na⁺] inside and is maximal when both Na⁺ and Cl⁻ are present. The uptake represents transport into an os-motically active space and not a binding artifact, as indicated by the effect of increasing the medium osmo-larity. The uptake of tryptophan is stimulated by a membrane potential (interior negative) as demonstrated by the effects of the ionophores valinomycin and carbonyl cyanide m-chlorophenylhydrazone and anions with different permeabilities. Kinetic data show that tryptophan is accumulated by two systems with different affinities. Ouabain, an inhibitor of Na⁺, K⁺-activated ATPase, does not affect tryptophan transport. The uptake of tryptophan is inhibited by high concentrations of phenylalanine, tyrosine, leucine and 3, 4-dihydroxyphenylalanine.     

Control of Na+ and K+ transport in Spergularia marina I. Transpiration effects

In this paper we begin our study of factors controlling Na⁺ and K⁺ uptake in the halophyte Spergularia marina (L.) Griseb., with emphasis on plants growing at moderate salinity (0.2x sea water). The involvement of transpiration was considered first because of its potential to account for much or all of the transport of ions, and particularly of Na⁺, to the shoot under these growth conditions. Transpiration was constant with time through most of the light period, quickly dropping to 6% of the day time rate at night. ²²Na⁺ uptake, on the other hand, showed much less day/night variation, and relative transport to the shoot was constant. After establishing that transpiration was linearly related to leaf weight, possible transpiration effects were further considered as correlations between leaf weight and transport to the shoot. Under constant, day-time conditions, with linear effects of time and plant size removed, total transport of ²²Na⁺ to the shoot (per plant) was not correlated to leaf weight. A similar result was found when transport was expressed per gram of root, and when partitioning of total label to the shoot was considered. Finally, the correlation was considered between leaf weight and a Na⁺/K⁺ enrichment factor defined as the Na⁺/K⁺ ratio in the leaves divided by that in the roots. This correlation was also insignificant. The results indicate that analysis of control of Na⁺ and K⁺ uptake and transport in this experimental system need not consider effects of transpiration.     

L-Aspartate Transport into Plasma Membrane Vesicles Derived from Rat Brain Synaptosomes

Abstract: Aspartate uptake by membrane vesicles derived from rat brain was investigated. The uptake is dependent on a Na⁺ gradient ([Na⁺] outside > [Na⁺] inside). Active transport of aspartate is strictly dependent upon the presence of sodium and maximal extent of transport is reached when both Na⁺ and Cl⁻ ions are present. The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. The uptake of aspartate is stimulated by a membrane potential (negative inside), as demonstrated by the effect of the ionophore carbonyl cyanide m -chlorophenylhydrazone and anions with different permeabilities. The presence of ouabain, an inhibitor of (Na⁺+ K⁺)-ATPase, does not affect aspartate transport. The kinetic analysis shows that aspartate is accumulated by two systems with different affinities, showing K _m and V _max values of similar order to those found in slightly "cruder" preparations. Inhibition of the l -aspartate uptake by d -aspartate and d - and l -glutamate indicates that a common carrier is involved in the process, this being stereospecific for the d - and l -glutamate stereoisomers.     

K/Na selectivity at the plasmalemma of cortical root cells and preferential release of sodium to the xylem vessels in roots of Atriplex hortensis

Using excised roots of Atriplex hortensis L., cv. Gelbe Gartenmelde, the uptake, accumulation and xylem transport of K⁺ and Na⁺ have been measured. Influx as well as xylem transport proved to discriminate little between K⁺ and Na⁺, when considered in relation to the external solution. Both K⁺ and Na⁺ inhibited the uptake and xylem transport of each other to about the same degree. Measurements of intracel-lular Na⁺ fluxes by means of compartment analysis indicated that the low degree of K/Na discrimination during uptake was due to low influx selectivity. Moreover, K⁺/Na⁺ exchange at the plasmalemma was not very efficient in Atriplex roots. In order to establish the basis of the low K/Na discrimination in xylem transport, the rates of K⁺ and Na⁺ transport were related to the cytoplasmic K⁺ and Na⁺ concentrations to yield the selectivity ratio of transport, S(transport) = (φ_cx(K) × [Na⁺]_c)/(φ_cx(Na) × [K⁺]_c). Under all conditions this ratio was far below one indicating that Na⁺ was favoured during xylem release in excised roots of Atriplex at low external concentrations. The implications of this discrimination in favour of Na+ are discussed with respect to salt tolerance of A. hortensis .     

Inhibition of Rat Brain Microsomal Na+,K+-ATPase by S-Adenosylmethionine

Abstract: Rat brain microsomes were preincubated with S -adenosylmethionine (SAM), MgCl₂, and CaCl₂, then re-isolated, and the activity of Na⁺,K⁺-ATPase determined. SAM inhibited the Na⁺,K⁺-ATPase activity compared with microsomes subjected to similar treatment in the absence of SAM. A biphasic inhibitory effect was observed with a 50% decrease at a SAM concentration range of 0.4 μ M -3.2 μ M and a 70% reduction at a concentration range above 100 μ M . Inclusion of either S- adenosylhomocysteine or 3-deazaadenosine in the preincubations prevented the SAM inhibition of Na⁺,K⁺-ATPase activity. The inhibition by SAM appeared to be Mg²⁺- or Ca²⁺-dependent.     

Characterization of Na+ exclusion mechanisms of salt-tolerant reed plants in comparison with salt-sensitive rice plants

Salt-tolerant reed plants ( Phragmites communis Trinius) and salt-sensitive rice plants ( Oryza sativa L. cv. Kinmaze) were grown in salinized nutrient solutions up to 50 m M NaCl, and growth, Na⁺ contents and kinetics of ²²Na⁺ uptake and translocation were compared between the species to characterize the salt tolerance mechanisms operating in reed plants. When both plants were grown under the same salinity, Na⁺ contents of the shoots were lower in reed plants, although those of the roots were quite similar. The shoot base region of both species accumulated Na⁺ more than the leaf blades did. Sodium-22 uptake and pulse-chase experiments suggested that the lower Na⁺ transport rate from root to shoot could limit excessive Na⁺ accumulation in the reed shoot. There was a possibility that the apparently lower ²²Na⁺ transport rate to the shoot of reed plants was due to net downward Na⁺ transport from shoot base to root.     

Elevated Levels of Glucose and L-Fucose Reduce 22Na+ Uptake and Whole Cell Na+ Current in Cultured Neuroblastoma Cells

Abstract: Na⁺ flux was studied in cultured neuroblastoma cells grown in medium containing increased glucose or L - fucose concentrations. Chronic exposure of neuroblastoma cells to 30 m M glucose or 30 m M L-fucose caused a decrease in ouabain-sensitive and veratridine-stimulated ²²Na⁺ uptake compared with cells cultured in unsupplemented medium. The Na⁺ current, determined by using whole-cell configuration of the patch clamp, was also decreased in these cells. Tetrodotoxin (3 μ M ), which blocked whole cell Na⁺ currents, also blocked veratridine-stimulated ²²Na⁺ accumulation. Culturing cells in medium containing 30 m M fructose as an osmotic control had no effect on Na⁺ flux. Specific [³H] saxitoxin binding was not affected by 30 m M glucose or 30 m M L-fucose compared with cells grown in unsupplemented medium, suggesting that the number of Na⁺ channels was not decreased. These studies suggest that exposing cultured neuronal cells to conditions that occur in the diabetic milieu alters Na⁺ transport and Na⁺-channel activity.     

Triiodothyronine Is a High-Affinity Inhibitor of Amino Acid Transport System L1 in Cultured Astrocytes

Abstract: The relationship between the transport of thyroid hormones and that of amino acids was examined by measuring the uptake of amino acids that are characteristic substrates of systems L, A, and N, and the effect of 3,3',5-triiodo-L-thyronine (T₃) on this uptake, in cultured astrocytes. Tryptophan and leucine uptakes were rapid, Na⁺-independent, and efficiently inhibited by T₃ (half-inhibition at ∼ 2 μ M ). Two Na⁺-independent L-like systems (L₁ and L₂), common to leucine and aromatic amino acids, were characterized kinetically. System L₂ had a low affinity for leucine and tryptophan ( K _m= 0.3–0.9 m M ). The high-affinity system L₁ ( K _m∼ 10 μ M for both amino acids) was competitively inhibited by T₃ with a K _i of 2–3 μ M (close to the T₃ transport K _m). Several T₃ analogues inhibited system L₁ and the T₃ transport system similarly. Glutamine uptake and α-(methylamino)isobutyric acid uptake were, respectively, two and 200 times lower than tryptophan and leucine uptakes. T₃ had little effect on the uptakes of glutamine and α-(methylamino)isobutyric acid. The results indicate that the T₃ transport system and system L₁ are related.     

Na+/H+ antiport activity in tonoplast vesicles from roots of the salt-tolerant Plantago maritima and the salt-sensitive Plantago media

Plantago species differ in their strategy towards salt stress, a major difference being the uptake and distribution of Na⁺ ions. A salt-sensitive ( Plantago media L.) and a salt-tolerant ( P. maritima L.) species were compared with respect to Na⁺/H⁺ antiport activities at the tonoplast. After exposure of the plants to 50 m M NaCl for 6 days isolated tonoplast vesicles of P. maritima showed Na⁺/H⁺ antiport activity with saturation kinetics and a K_m of 2.4 m M Na⁺, NaCl-grown P. media and the control plants of both species showed no antiport activity. Selectivity of the antiport system for Na⁺ was high and was determined by adding different chloride salts after formation of a Δ pH in the vesicles. Specific tonoplast ATPase activities were similar in the two species and did not alter after exposure to NaCl stress.     

SODIUM AND THE FLUX OF CALCIUM IONS IN ELECTRICALLY-STIMULATED CEREBRAL TISSUE

Abstract— The rate of efflux of ⁴⁵Ca²⁺ from slices of rat cerebral cortex was resolved into two exponential curves which were attributed to an extracellular component and an intracellular or bound component. Electrical stimulation increased efflux of ⁴⁵Ca²⁺ from the more stable pool and the time course for the redistribution of Na⁺ and K⁺ paralleled that for the increased efflux of Ca²⁺. This effect of stimulationwas dependent on the presence of Na⁺ in the incubation medium. Lack of Na⁺ in the medium during loading of the slices with ⁴⁵Ca²⁺ increased uptake but on subsequent transfer to a medium containing Na⁺, electrical pulses failed to increase the rate of efflux of ⁴⁵Ca²⁺. In unstimulated slices, the rate of efflux of ⁴⁵Ca²⁺ was dependent upon the concentration ratio of Na⁺ to Ca²⁺ in the incubation medium. Saxitoxin and tetrodotoxin inhibited the increased efflux of ⁴⁵Ca²⁺ that occurred during electrical stimulation but exerted no effect on Ca²⁺-Ca²⁺ exchange. Our results suggest that there is a Na⁺-dependent turnover of Ca²⁺ in brain slices which may involve changes in affinity at a common binding site. The possible involvement of such a Na⁺-Ca²⁺ interaction in the regulation of neurotransmitter function is discussed.     

Detection of Intracellular Free Na+ Concentration of Synaptosomes by a Fluorescent Indicator, Na+-Binding Benzofuran Isophthalate: The Effect of Veratridine, Ouabain, and α-Latrotoxin

Abstract: A novel fluorescent Na⁺ indicator, Na⁺-binding benzofuran isophthalate (SBFI), was used to follow changes in the intracellular free Na⁺ concentration ([Na⁺]₁) of synaptosomes. The dye, when loaded into synapto- somes in the form of its acetoxymethyl ester, was responsive to changes of [Na⁺]₁. Calibration was made using the 340/380 nm excitation ratio when the cytoplasmic Na⁺ concentration was equilibrated with different concentrations of extracellular Na⁺ in the presence of 2 μ M gramicidin D. The basal value of [Na⁺]₁ in synaptosomes in the presence of 140 m M extracellular Na⁺ was found to be 10.9 ± 1.8 m M. Veratridine, which opens potential-dependent Na⁺ channels, caused a sudden increase in [Na⁺]₁ in a concentration-dependent manner (1 -20 μ M ), whereas the effect of ouabain (20 and 50 μ M ), the inhibitor of the plasma membrane Na⁺,K⁺-ATPase, was more gradual. The rise in the fluorescence intensity upon addition of veratridine was prevented completely by 2 μ M tetrodotoxin. α-Latrotoxin, the black widow spider toxin, caused an increase in the fluorescence intensity, which became evident 1 min after the addition of the toxin. The rate of increase was proportional to the concentration of the toxin (0.19–1.5 n M ). This report confirms our earlier finding demonstrating a Na⁺-dependent component in the action of α-Iatrotoxin, and shows that changes in [Na⁺]₁ in synaptosomes can be followed by SBFI.