Sodium-dependent neutral amino acid transport by human liver plasma membrane vesicles

The activities of several selected Na(+)-dependent amino acid transporters were identified in human liver plasma membrane vesicles by testing for Na(+)-dependent uptake of several naturally occurring neutral amino acids or their analogs. Alanine, 2-(methylamino)isobutyric acid, and 2-aminoisobutyric acid were shown to be almost exclusively transported by the same carrier, system A. Kinetic analysis of 2-(methylamino)isobutyric acid uptake by the human hepatic system A transporter revealed an apparent Km of 0.15 mM and a Vmax of 540 pmol.mg-1 protein.min-1. Human hepatic system A accepts a broad range of neutral amino acids including cysteine, glutamine, and histidine, which have been shown in other species to be transported mainly by disparate carriers. Inhibition analysis of Na(+)-dependent cysteine transport revealed that the portion of uptake not mediated by system A included at least two saturable carriers, system ASC and one other that has yet to be characterized. Most of the glutamine and histidine uptake was Na(+)-dependent, and the component not mediated by system A constituted system N. The largest portion of glycine transport was mediated through system A and the remainder by system ASC with no evidence for system Gly activity. Our examination of Na(+)-dependent amino acid transport documents the presence of several transport systems analogous to those described previously but with some notable differences in their functional activity. Most importantly, the results demonstrate that liver plasma membrane vesicles are a valuable resource for transport analysis of human tissue.     

Homocysteine uptake by human umbilical vein endothelial cells in culture

The characteristics of the uptake of L-homocysteine by cultures of human umbilical vein endothelial cells have been examined. Uptake occurred by Na(+)-dependent and Na(+)-independent systems, but was essentially independent of the pH of the uptake medium. The Na(+)-independent system corresponded to system L, being totally inhibited by the presence of beta-2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH) a system L analogue. It was concluded on the basis of starvation experiments coupled with failure to detect any inhibition in the presence of 2-methylaminoisobutyric acid (MeAIB), a system A analogue, that the Na(+)-dependent uptake was wholly accounted for by system ASC. The kinetic properties of systems L and ASC were determined by omitting Na+ from the uptake medium and incorporating BCH in the medium, respectively. It has been concluded on the basis of the inhibitory effects of a number of amino acids that uptake of homocysteine occurs by those systems which transport cysteine.     

Inhibition of transport system b0,+ in blastocysts by inorganic and organic cations yields insight into the structure of its amino acid receptor site

The most conspicuous, Na(+)-independent amino acid transport process in preimplantation mouse blastocysts was provisionally designated system b0,+ because it accepts some cationic and zwitterionic amino acids about equally well as substrates. Although system b0,+ is not Na(+)-stimulated, it has not been determined if it is inhibited by Na+, or if its activity is affected by most other ions. Therefore, we measured uptake of amino acids by blastocysts in isotonic solutions of different ionic and nonionic osmolites. Na(+)-independent L-leucine uptake was unaffected by the ion concentration, but L-lysine transport was several-fold faster in isotonic solutions of non-electrolytes than in similar solutions of inorganic and organic salts or zwitterions. The Km value for 'Na(+)-independent' L-lysine transport was about 10-fold higher in isotonic salt solutions than in solutions of nonionic osmolites, whereas the Km value for L-leucine transport was about the same in either type of solution. Therefore, inorganic and organic cations and the cationic portions of zwitterions appear to compete with cationic but not zwitterionic amino acids for system b0,+ receptor sites. The cation, harmaline, was a particularly strong competitive inhibitor of 'Na(+)-independent' L-lysine uptake by system b0,+, even in isotonic salt solutions, whereas it inhibited L-leucine uptake noncompetitively. Moreover, harmaline appeared to compete with inorganic cations for the lysine receptor sites of system b0,+. Harmaline also has been found by other investigators to competitively inhibit L-lysine uptake by the Na(+)-independent system asc1 in horse erythrocytes, whereas it noncompetitively inhibits alanine uptake by the same system. Similarly, harmaline noncompetitively inhibits L-alanine uptake by the Na(+)-dependent system ASC in human erythrocytes, but it appears to compete for binding with L-alanine's cosubstrate, Na+. In addition, others have found that the positively-charged side chains of cationic amino acids seem to take the place of Na+ needed near side chains in order for zwitterionic amino acids to be transported by systems ASC and y+. We conclude that system b0,+ may be similar to systems asc1, ASC and y+, and that each of these systems may be a variant of the same ancestral transport process. We speculate that since it appears to accept a broader scope of substrates and to interact with a wider variety of cations than do systems asc1, ASC or y+, system b0,+ may more closely resemble the proposed ancestral transport process than any of the other contemporary systems.     

Cation and harmaline interactions with Na(+)-independent dibasic amino acid transport system y+ in human erythrocytes and in erythrocytes from a primitive vertebrate the pacific hagfish (Eptatretus stouti).

Transport systems y+, asc and ASC exhibit dual interactions with dibasic and neutral amino acids. For conventional Na(+)-dependent neutral amino acid system ASC, side chain amino and guanido groups bind to the Na+ site on the transporter. The topographically equivalent recognition site on related system asc binds harmaline (a Na(+)-site inhibitor) with the same affinity as asc (apparent Ki range 1-4 mM), but exhibits no detectable affinity for Ha. Although also classified as Na(+)-independent, dibasic amino acid transport system y+ accepts neutral amino acids when Na+ or another acceptable cation is also present. This latter observation implies that the y+ translocation site binds Na+ and suggests possible functional and structural similarities with ASC/asc. In the present series of experiments with human erythrocytes, system y(+)-mediated lysine uptake (5 microM, 20 degrees C) was found to be 3-fold higher in isotonic sucrose medium than in normal 150 mM NaCl medium. This difference was not a secondary consequence of changes in membrane potential, but resulted from Na+ functioning as a competitive inhibitor of transport. Apparent Km and Vmax values for lysine transport at 20 degrees C were 15.2 microM and 183 mumol/l cells per h, respectively, in sucrose medium and 59.4 microM and 228 mumol/l cells per h in Na+ medium. Similar results were obtained with y+ in erythrocytes of a primitive vertebrate, the Pacific hagfish (Eptatretus stouti), indicating that Na(+)-inhibition is a general property of this class of amino acid transporter. At a permeant concentration of 5 microM, the IC50 value for Na(+)-inhibition of lysine uptake by human erythrocytes was 27 mM. Other inorganic and organic cations, including K+ and guanidinium+, also inhibited transport. In parallel with its actions on ASC/asc harmaline competitively inhibited lysine uptake by human cells in sucrose medium. As predicted from mutually competitive binding to the y+ translocation site, the presence of 150 mM Na+ increased the harmaline inhibition constant (Ki) from 0.23 mM in sucrose medium to 0.75 mM in NaCl medium. We interpret these observations as further evidence that y+, asc and ASC represent a family of closely related transporters with a common evolutionary origin.     

The Astroglial ASCT2 Amino Acid Transporter as a Mediator of Glutamine Efflux

Glutamine release from astrocytes is an essential part of the glutamate-glutamine cycle in the brain. Uptake of glutamine into cultured rat astrocytes occurs by at least four different routes. In agreement with earlier studies, a significant contribution of amino acid transport systems ASC, A, L, and N was detected. It has not been determined whether these systems are also involved in glutamine efflux or whether specific efflux transporters exist. We show here that ASCT2, a variant of transport system ASC, is strongly expressed in rat astroglia-rich primary cultures but not in neuron-rich primary cultures. The amino acid sequence of rat astroglial ASCT2 is 83% identical to that of mouse ASCT2. In Xenopus laevis oocytes expressing rat ASCT2, we observed high-affinity uptake of [U-14C]glutamine (Km = 70 microM) that was Na(+)-dependent, concentrative, and unaffected by membrane depolarization. When oocytes were preloaded with [U-14C]glutamine, no glutamine efflux was detected in the absence of extracellular amino acids. Neither lowering intracellular pH nor raising the temperature elicited efflux. However, addition of 0.1 mM unlabeled alanine, serine, cysteine, threonine, glutamine, or leucine to the extracellular solution resulted in a rapid release of glutamine from the ASCT2-expressing oocytes. Amino acids that are not recognized as substrates by ASCT2 were ineffective in this role. Extracellular glutamate stimulated glutamine release weakly at pH 7.5 but was more effective on lowering pH to 5.5, consistent with the pH dependence of ASCT2 affinity for glutamate. Our findings suggest a significant role of ASCT2 in glutamine efflux from astrocytes by obligatory exchange with extracellular amino acids. However, the relative contribution of this pathway to glutamine release from cells in vivo or in vitro remains to be determined.     

Neutral amino acid transport in isolated rat pancreatic islets

The neutral amino acid transport systems of freshly isolated rat pancreatic islets have been studied by first examining the transport of L-alanine and the nonmetabolizable analogue 2-(methylamino)isobutyric acid (MeAIB). By comparing the uptake of MeAIB and L-alanine for their pH dependency profile, choline and Li+ substitution for Na+, tolerance to N-methylation, and competition with other amino acids, the existence in pancreatic islets of both A and ASC amino acid transport systems was established. The systems responsible for the inward transport of five natural amino acids was studied using competition analysis and Na+ dependency of uptake. These studies defined three neutral amino acid transport systems: A and ASC (Na+-dependent) and L (Na+-independent). L-Proline entered rat islet cells mainly by system A; L-leucine by the Na+-independent system L. The uptake of L-alanine, L-serine, and L-glutamine was shared by systems ASC and L, the participation of system A being negligible for these three amino acids. An especially broad substrate specificity for systems L and ASC is therefore suggested for the rat pancreatic islet cells. The regulation of amino acid transport was also investigated in two conditions differing as to glucose concentration and/or availability, i.e. islets from fasted rats and islets maintained in tissue culture at high or low glucose concentrations. Neither alanine nor MeAIB transport was altered by fasting of the islet-donor rats. On the other hand, pancreatic islets maintained for 2 days in tissue culture at high (16.7 mM) glucose transported MeAIB at twice the rate of islets maintained at low (2.8 mM) glucose. Amino acid starvation of pancreatic islets during 11 h of tissue culture resulted in a 2-fold increase in MeAIB transport.     

The preferential interaction of L-threonine with transport system ASC in cultured human fibroblasts.

The transport of L-threonine was studied in cultured human fibroblasts. A kinetic analysis of L-threonine transport in a range of extracellular concentrations from 0.01 to 20 mM indicated that this amino acid enters cells through both Na(+)-independent and Na(+)-dependent routes. These routes are: (1) a non-saturable, Na(+)-independent route formally indistinguishable from diffusion; (2) a saturable, Na(+)-independent route inhibitable by the analog BCH and identifiable with system L; (3) a low-affinity, Na(+)-dependent component (Km = 3 mM) which can be attributed to the activity of system A since it is adaptively enhanced by amino acid starvation and suppressed by the characterizing analog MeAIB and (4) a high-affinity, Na(+)-dependent route (Km = 0.05 mM). This latter route is identifiable with system ASC since it is insensitive to adaptive regulation, uninhibited by MeAIB, trans-stimulated by intracellular substrates of system ASC, markedly stereoselective, and relatively insensitive to changes in external pH. At an external concentration of 0.05 mM more than 90% of L-threonine transport is referrable to the activity of system ASC; in these conditions, the transport of the amino acid exhibits typical ASC-features even in the absence of inhibitors of other transport agencies, and, therefore, it can be employed as a reliable indicator of the activity of transport system ASC in cultured human fibroblasts.     

Glycine transport by cultured human fibroblasts

The transport of glycine was studied in cultured human fibroblasts. The amino acid entered the cell by Na+-dependent and Na+-independent mechanisms. Na+-independent glycine (0.1 mM) transport was less than 10% of total uptake and occurred by a mechanism formally indistinguishable from diffusion. Two distinct routes contributed to Na+-dependent glycine transport. The first route was identified with system A because it was inhibited by MeAIB and underwent adaptive regulation. The second route was identified with system ASC as it was inhibited by L-alanine, but not by MeAIB. Kinetic analysis revealed that the two systems operated glycine transport with the same Km of 1.6 mM, a value unusually high for system ASC.     

Developmental changes in glutamine transport by rat jejunal basolateral membrane vesicles

The ontogeny of glutamine uptake by jejunal basolateral membrane vesicles (BLMV) was studied in suckling and weanling rats and the results were compared with adult rats. Glutamine uptake was found to represent a transport into an osmotically active space and not mere binding to the membrane surface. Temperature dependency indicated a carrier-mediated process with optimal pH of 7.0. Transport of glutamine was Na+ (out greater than in) gradient dependent with a distinct "overshoot" phenomenon. The magnitude of the overshoot was higher in suckling compared with weanling rats. The uptake kinetics and inhibition profile indicated the existence of two major transport pathways. A Na(+)-dependent system correlated with System A showed tolerance to System N and System ASC substrates, and a Na(+)-independent system similar to the classical L system that favors leucine and BCH. The Vmax for the Na(+)-dependent system was higher in suckling compared with weanling and adult rats. The Vmax for the Na(+)-dependent system was 0.86 +/- 0.17, 0.64 +/- 0.8, and 0.41 +/- 0.9 nmol.mg protein-1.10 sec-1 for suckling, weanling, and adult rats, respectively. The Vmax for the Na(+)-independent system was 0.68 +/- 0.08, 0.50 +/- 0.03, and 0.24 +/- 0.03 nmol.mg protein-1.10 sec-1 for suckling, weanling, and adult rats, respectively. We conclude that glutamine uptake undergoes developmental changes consistent with more activity and/or number of glutamine transporters during periods of active cellular proliferation and differentiation.     

Primary structure, genomic organization, and functional and electrogenic characteristics of human system N 1, a Na+- and H+-coupled glutamine transporter

We have cloned the human Na(+)- and H(+)-coupled amino acid transport system N (hSN1) from HepG2 liver cells and investigated its functional characteristics. Human SN1 protein consists of 504 amino acids and shows high homology to rat SN1 and rat brain glutamine transporter (GlnT). When expressed in mammalian cells, the transport function of human SN1 could be demonstrated with glutamine as the substrate in the presence of LiCl (instead of NaCl) and cysteine. The transport activity was saturable, pH-sensitive, and specific for glutamine, histidine, asparagine, and alanine. Analysis of Li(+) activation kinetics showed a Li(+):glutamine stoichiometry of 2:1. When expressed in Xenopus laevis oocytes, the transport of glutamine or asparagine via human SN1 was associated with inward currents under voltage-clamped conditions. The transport function, monitored as glutamine- or asparagine-induced currents, was saturable, Na(+)-dependent, Li(+)-tolerant, and pH-sensitive. The transport cycle was associated with the involvement of more than one Na(+) ion. Uptake of asparagine was directly demonstrable in these oocytes by using radiolabeled substrate, and this uptake was inhibited by membrane depolarization. In addition, simultaneous measurement of asparagine influx and charge influx in the same oocyte yielded an asparagine:charge ratio of 1. These data suggest that SN1 mediates the influx of two Na(+) and one amino acid substrate per transport cycle coupled to the efflux of one H(+), rendering the transport process electrogenic.     

System beta and system A amino acid transporters in the feline endotheliochorial placenta

There is no knowledge of the transport mechanisms by which solutes cross the cat placenta or any other endotheliochorial placenta. Here, we investigated whether the amino acid transport systems beta and A are present in the cat placenta using a placental fragment uptake technique. Data were compared with studies in the human placenta, in which the presence of these two transport systems has been well established. A time course of [(3)H]taurine (substrate for system beta) and [(14)C]MeAIB (nonmetabolizable substrate for system A) uptake was determined in the term cat and human placental fragments in the presence and absence (choline substituted) of Na(+), and further studies were carried out over 15 min. Taurine uptake into both cat and human placenta fragments was found to be Na(+) and Cl(-) dependent, and Na(+)-dependent taurine uptake was blocked by excess beta-alanine. MeAIB uptake was found to be Na(+) dependent, and Na(+)-dependent MeAIB uptake was blocked by excess MeAIB or glycine. Western blotting and immunohistochemistry performed on cat and human placenta showed expression of TAUT and ATA2 (SNAT2), proteins associated with system beta and system A activity, respectively. This study therefore provides the first evidence of the presence of amino acid transport systems beta and A in the cat placenta.     

Evidence for two different broad-specificity oligopeptide transporters in intestinal cell line Caco-2 and colonic cell line CCD841

Recently the existence of two different Na(+)-coupled oligopeptide transport systems has been described in mammalian cells. These transport systems are distinct from the previously known H(+)/peptide cotransporters PEPT1 and PEPT2, which transport only dipeptides and tripeptides. To date, the only peptide transport system known to exist in the intestine is PEPT1. Here we investigated the expression of the Na(+)-coupled oligopeptide transporters in intestinal cell lines, using the hydrolysis-resistant synthetic oligopeptides deltorphin II and [d-Ala(2),d-Leu(5)]enkephalin (DADLE) as model substrates. Caco-2 cells and CCD841 cells, both representing epithelial cells from human intestinal tract, were able to take up these oligopeptides. Uptake of deltorphin II was mostly Na(+) dependent, with more than 2 Na(+) involved in the uptake process. In contrast, DADLE uptake was only partially Na(+) dependent. The uptake of both peptides was also influenced by H(+) and Cl(-), although to a varying degree. The processes responsible for the uptake of deltorphin II and DADLE could be differentiated not only by their Na(+) dependence but also by their modulation by small peptides. Several dipeptides and tripeptides stimulated deltorphin II uptake but inhibited DADLE uptake. These modulating small peptides were, however, not transportable substrates for the transport systems that mediate deltorphin II or DADLE uptake. These two oligopeptide transport systems were also able to take up several nonopioid oligopeptides, consisting of 9-17 amino acids. This represents the first report on the existence of transport systems in intestinal cells that are distinct from PEPT1 and capable of transporting oligopeptides consisting of five or more amino acids.     

Differential effects of transmembrane potential on two Na+-dependent transport systems for neutral amino acids

The effects of changes of membrane potential on amino acid transport through systems A, ASC and L was investigated in the Ehrlich cell and the human erythrocyte. Changes of membrane potential were produced by incubating cells whose K+ permeability had been increased, either by valinomycin or by activation of Ca2+-dependent K+ channels, in medium containing different K+ concentrations. The changes in membrane potential were followed by measuring the distribution ratio reached by lipophilic indicators. Transport through Na+-dependent system A was sensitive to the membrane potential, the rate of amino acid uptake increasing 2.2-3.1-times for each 60 mV-hyperpolarization. The Na+-dependent system ASC was insensitive to membrane potential. The Na+-independent system L was not directly affected by membrane potential, but the steady-state accumulation of system L substrates was increased by hyperpolarization.     

The transport of alanine, serine, and cysteine in cultured human fibroblasts

The transport of L-alanine, L-serine, and L-cysteine has been studied in skin-derived diploid human fibroblasts in culture. Competition analysis, mathematical discrimination by nonlinear regression, and conditions varying the relative contribution of the various mediations have been used to characterize the systems engaged in the inward transport of these amino acids. All the adopted criteria yielded results showing that L-alanine, L-serine, and L-cysteine enter the cell by two Na+-dependent systems, System A and System ASC, and by a Na+-independent route, whose major component has been identified as System L. The apparent affinity of L-alanine, L-serine, and L-cysteine for the putative carrier was higher for System ASC than for System A. The transport Vmax for System A increased in response to cell starvation; after 12 h, its values were similar or higher than those exhibited by System ASC. At amino acid concentrations approaching those present in human plasma, System ASC appeared to be the primary mediation for the inward transport of L-alanine, L-serine, and L-cysteine in human fibroblasts. The contribution of System A was negligible in nonstarved cells and became appreciable under conditions of cell starvation. The Na+-independent System L made no substantial contribution to the uptake of L-alanine and L-serine and accounted for approximately one-fourth of the total uptake of L-cysteine.     

Electrogenic Na(+)-dependent L-alanine transport in the lizard duodenum. Involvement of systems A and ASC

L-Alanine transport across the isolated duodenal mucosa of the lizard Gallotia galloti has been studied in Ussing chambers under short-circuit conditions. Net L-alanine fluxes, transepithelial potential difference (PD), and short-circuit current (Isc) showed concentration-dependent relationships. Na(+)-dependent L-alanine transport was substantially inhibited by the analog alpha-methyl aminoisobutyric acid (MeAIB). Likewise, MeAIB fluxes were completely inhibited by L-alanine, indicating the presence of system A for neutral amino acid transport. System A transport activity was electrogenic and exhibited hyperbolic relationships for net MeAIB fluxes, PD, and Isc, which displayed similar apparent K(m) values. Na(+)-dependent L-alanine transport, but not MeAIB transport, was partially inhibited by L-serine and L-cysteine, indicating the participation of system ASC. This transport activity represents the major pathway for L-alanine absorption and seemed to operate in an electroneutral mode with a negligible contribution to the L-alanine-induced electrogenicity. It is concluded from the present study that the active Na(+)-dependent L-alanine transport across the isolated duodenal mucosa of Gallotia galloti results from the independent activity of systems A and ASC for neutral amino acid transport.     

Kinetic analysis of L-glutamine transport into porcine jejunal enterocyte brush-border membrane vesicles

L-Glutamine transport into porcine jejunal enterocyte brush border membrane vesicles was studied. Uptake was mediated by a Na(+)-dependent and a Na(+)-independent pathway as well as by diffusion. The initial rates of glutamine uptake over a range of concentrations is both Na(+)-gradient and Na(+)-free conditions were analyzed and kinetic parameters were obtained. Na(+)-dependent glutamine transport had a K(m) of 0.77 +/- 0.16 mM and a Jmax of 70.7 +/- 5.8 pmol mg protein-1 s-1; Na(+)-independent glutamine transport had a K(m) of 3.55 +/- 0.78 mM and a Jmax of 55.1 +/- 6.6 pmol mg protein-1 s-1. The non-saturable component measured with HgCl2-poisoned brush border membrane vesicles in the Na(+)-free condition contained passive diffusion and non-specific membrane binding and was defined to be apparent glutamine diffusion and the glutamine permeability coefficient (Kdiff) was estimated to be Kdiff = 3.78 +/- 0.06 pmol 1 mg protein-1 mmol-1 s-1. Results of inhibition experiments showed that Na(+)-dependent glutamine uptake occurred primarily through the brush border system-B degree transporters, whereas Na(+)-independent glutamine uptake occurred via the system-L transporters. Furthermore, the kinetics of L-leucine and L-cysteine inhibition of L-glutamine uptake demonstrated that neutral amino acids sharing the same brush border transporters can effectively inhibit each other in their transport.     

Mechanism of polyamine spermidine uptake by Xenopus laevis oocytes

In this study, the mechanisms of polyamine spermidine (Spd) uptake were investigated in Xenopus laevis oocytes. Spd uptake followed a sigmoidal kinetics with [S]90/[S]10 = 3 microM and Hill interaction coefficient (n) = 2. The order of magnitude of uptake and efflux was similar (t1/2 = 45 min). The equilibrium potential for Spd, calculated by Nenrst equation, was 90.78 mV. Free energy change for the uptake (delta G) was found to be 2.31 Kcal/mole of Spd. During efflux, Spd was not converted into putrescine or spermine. It seems that there are two types of Spd uptake pathways: Na(+)-dependent and Na(+)-independent since replacement of Na+ from incubation medium did not completely abolish the Spd uptake. The Na(+)-dependent component of Spd uptake was shared neither by system A nor by system ASC amino acids.     

Glutamine transporter in crypts compensates for loss of villus absorption in bovine cryptosporidiosis

Cryptosporidium parvum infection represents a significant cause of diarrhea in humans and animals. We studied the effect of luminally applied glutamine and the PG synthesis inhibitor indomethacin on NaCl absorption from infected calf ileum in Ussing chambers. Infected ileum displayed a decrease in both mucosal surface area and NaCl absorption. Indomethacin and glutamine or its stable derivative alanyl-glutamine increased the net absorption of Na(+) in infected tissue in an additive manner and to a greater degree than in controls. Immunohistochemical and Western blot studies showed that in control animals neutral amino acid transport system ASC was present in villus and crypts, whereas in infected animals, ASC was strongly present only on the apical border of crypts. These results are consistent with PGs mediating the altered NaCl and water absorption in this infection. Our findings further illustrate that the combined use of a PG synthesis inhibitor and glutamine can fully stimulate Na(+) and Cl(-) absorption despite the severe villous atrophy, an effect associated with increased expression of a Na(+)-dependent amino acid transporter in infected crypts.     

Expression of rat liver glutamine transporters in Xenopus laevis oocytes.

As a first step in attempting to isolate the Na(+)-dependent System N transporter from rat liver we have investigated the use of prophase-arrested oocytes from Xenopus laevis for the functional expression of rat liver glutamine transporters. Individual oocytes, defolliculated by collagenase treatment, were injected with 50 nl of a 1 mg.ml-1 solution of poly(A)+ RNA (mRNA) isolated from rat liver. 50 microM L-[3H]glutamine uptake was measured 1-5 days post-injection: after 48 h, poly(A)+ RNA-injected oocytes showed a 60 +/- 12% increase in Na(+)-dependent glutamine uptake compared to controls. This increased uptake showed characteristic features of hepatic System N: that is, it tolerated Li(+)-for-Na+ substitution and was inhibited by the System N substrate L-histidine (5 mM) in Li medium, unlike endogenous Na(+)-dependent glutamine transport. In subsequent experiments rat liver poly(A)+ RNA, size-fractionated by density gradient fractionation, was injected into oocytes. Injection of poly(A)+ RNA of 1.9-2.8 kilobases (kb) in size resulted in a significant stimulation of Na(+)-dependent glutamine transport to 0.362 +/- 0.080 pmol.min-1/oocyte from 0.178 +/- 0.060 pmol.min-1/oocyte in vehicle-injected oocytes (p less than 0.01). A lighter fraction, with poly(A)+ RNA of less than 1.9 kilobases size resulted in a similar increase in Na(+)-dependent glutamine uptake which was largely Li(+)-tolerant: Li(+)-stimulated glutamine uptake in oocytes injected with this fraction increased to 0.230 +/- 0.070 pmol.min-1/oocyte from 0.098 +/- 0.029 pmol.min-1/oocyte in controls (p less than 0.05). This enhanced rate of Li(+)-stimulated glutamine uptake was inhibited 28 and 70%, respectively, by 1 and 5 mM L-histidine. Na(+)-independent uptake of glutamine rose by 72 +/- 12% in oocytes injected with poly(A)+ RNA of 2.8-3.6 kb (p less than 0.001). These results demonstrate that glutamine transporters, with characteristics associated with hepatic Systems N, L, and A (or ASC), can be expressed in X. laevis oocytes injected with specific size fractions of rat liver mRNA.     

Asymmetry of glutamine transporters in cultured neural cells

Transfer of glutamine between astrocytes and neurons is an essential part of the glutamate-glutamine cycle in the brain. Transport of glutamine was investigated in primary cultures of astrocytes and neurons and compared to glutamine transport in cell lines with glial and neuronal properties. Glutamine uptake in astrocytes was mainly mediated by general amino acid transporters with properties similar to ASCT2, LAT1, LAT2, SN1 and y(+)LAT2. In cultured neurons, transport activities were detected consistent with the presence of LAT1, LAT2 and y(+)LAT2, but the most prominent activity was a novel Na(+)-dependent glutamine transporter that could be inhibited by D-aspartate. The mRNA for system A isoforms ATA1 and ATA2 was detected in both neurons and astrocytes, but system A activity was only detected in neurons. ASCT2 on the other hand appeared to be astrocyte-specific. The cell lines F98 and 108CC-15, having astroglial and neuronal properties, respectively, expressed sets of glutamine transporters that were unrelated to those of the corresponding primary culture and are thus of limited use as models to study transfer of glutamine between astrocytes and neurons.