L-glutamine Uptake Research Articles

Fluoride Exposure Affects Glutamine Uptake in Müller Glia Cells.

Fluoride, a pollutant present in contaminated ground water, oral care products, food, and pesticides, has deleterious effects in the structure and function of the central nervous system. Among the established neurological defects described in the exposed population, a reduced score in intelligence quotient tests in children of contaminated areas has gained attention over the past years. Maternal fluoride exposure during gestation decreases learning and memory abilities that correlate with a significant diminution of glutamate receptors expression. Since the involvement of glia cells in the maintenance and regulation of glutamatergic synapses is well-documented, in this contribution, we characterized the effect of fluoride exposure in the regulation of glia glutamine transporters. To this end, we used the Müller glia cell line, Mio-M1, and through the use of [3H]L-Glutamine uptake experiments and a Western blot approach, we demonstrate here the functional expression of system N of glutamine transporters, SNAT3 and SNAT5, in this model of human retina radial glia cells. Furthermore, these transporters interact with the glutamate transporter excitatory amino acid transporter 1, in an activity-dependent manner. Fluoride treatment reduces glutamine uptake and cell membrane [3H]glutamine surface binding, in good correlation with a decrease in SNAT3 and 5 protein levels. These results demonstrate that glia cells respond to the presence of fluoride reducing glutamine mobilization and by these means decreases glutamate turnover suggesting a disruption of glutamatergic transmission.

L-Glutamine uptake is developmentally regulated and is involved in metacyclogenesis in Trypanosoma cruzi

Trypanosoma cruzi, the aetiological agent of Chagas disease, can obtain L-glutamine (Gln) through the enzyme glutamine synthetase (GS) using glutamate (Glu) and ammonia as substrates. In this work, we show additional non-canonical roles for this amino acid: its involvement in ATP maintenance and parasite survival under severe metabolic stress conditions and its participation in the differentiation process occurring in the insect vector (metacyclogenesis). These roles are dependent on the supply of Gln from an extracellular source. We show that T. cruzi incorporates Gln through a saturable and specific transport system, which results in unusual stability at elevated temperatures. The activity was moderately higher at pH values between 6 and 7 and was sensitive to the dissipation of the H+ gradient at the plasma membrane. When analysed in the different life cycle stages, we found that Gln transport is developmentally regulated. In fact, Gln uptake and GS activity seem to be finely regulated at most stages: when GS activity is increased, transport is decreased and vice versa, with the exception of trypomastigotes, where both sources of Gln are diminished. This metabolic adaptation reflects the relevance of Gln in T. cruzi biology and the plasticity of these parasites to adjust their metabolism to changing environments.

L-glutamine Induces Expression of Listeria monocytogenes Virulence Genes.

The high environmental adaptability of bacteria is contingent upon their ability to sense changes in their surroundings. Bacterial pathogen entry into host poses an abrupt and dramatic environmental change, during which successful pathogens gauge multiple parameters that signal host localization. The facultative human pathogen Listeria monocytogenes flourishes in soil, water and food, and in ~50 different animals, and serves as a model for intracellular infection. L. monocytogenes identifies host entry by sensing both physical (e.g., temperature) and chemical (e.g., metabolite concentrations) factors. We report here that L-glutamine, an abundant nitrogen source in host serum and cells, serves as an environmental indicator and inducer of virulence gene expression. In contrast, ammonia, which is the most abundant nitrogen source in soil and water, fully supports growth, but fails to activate virulence gene transcription. We demonstrate that induction of virulence genes only occurs when the Listerial intracellular concentration of L-glutamine crosses a certain threshold, acting as an on/off switch: off when L-glutamine concentrations are below the threshold, and fully on when the threshold is crossed. To turn on the switch, L-glutamine must be present, and the L-glutamine high affinity ABC transporter, GlnPQ, must be active. Inactivation of GlnPQ led to complete arrest of L-glutamine uptake, reduced type I interferon response in infected macrophages, dramatic reduction in expression of virulence genes, and attenuated virulence in a mouse infection model. These results may explain observations made with other pathogens correlating nitrogen metabolism and virulence, and suggest that gauging of L-glutamine as a means of ascertaining host localization may be a general mechanism.

The regulation and role of L-glutamine in B-lymphocyte activation (LYM7P.618)

Abstract B lymphocyte activation is a highly energetic event that promotes cell survival and growth. Aberrant activation of B lymphocytes can lead to disease states such as lymphomas or autoimmune diseases. To meet the metabolic demands of activation, B cells increase nutrient uptake to provide energy and intermediates for macromolecule synthesis. We have investigated the role of L-glutamine, which is emerging as a critical immunomodulatory nutrient; however, the precise role of L-glutamine in B cell growth responses is poorly understood. Depletion of extracellular L-glutamine results in impaired cell growth and proliferation in response to B-cell antigen receptor (BCR) ligation, although, early activation events such as induction of CD86 are unaffected. We show that L-glutamine uptake is induced in response to BCR ligation. This corresponds to an increase in the surface expression of the high affinity L-glutamine transporter, ASCT2, indicating its involvement in L-glutamine uptake. Further, inhibition of ASCT2 by the small molecule N-glutaryl-L-phenylalanine p-nitroanilide (GPNA) caused impaired B lymphocyte activation similar to that observed in response to L-glutamine depletion. The addition of L-leucine abated some of the effects of GPNA treatment on B lymphocyte growth responses, indicating mTORC involvement. These data provide new insight on the requirement of L-glutamine in B lymphocyte activation and suggest a possible underlying mechanism of growth regulation by mTORC.

SLC1A5 Mediates Glutamine Transport Required for Lung Cancer Cell Growth and Survival

We have previously identified solute-linked carrier family A1 member 5 (SLC1A5) as an overexpressed protein in a shotgun proteomic analysis of stage I non-small cell lung cancer (NSCLC) when compared with matched controls. We hypothesized that overexpression of SLC1A5 occurs to meet the metabolic demand for lung cancer cell growth and survival. To test our hypothesis, we first analyzed the protein expression of SLC1A5 in archival lung cancer tissues by immunohistochemistry and immunoblotting (N = 98) and in cell lines (N = 36). To examine SLC1A5 involvement in amino acid transportation, we conducted kinetic analysis of l-glutamine (Gln) uptake in lung cancer cell lines in the presence and absence of a pharmacologic inhibitor of SLC1A5, gamma-l-Glutamyl-p-Nitroanilide (GPNA). Finally, we examined the effect of Gln deprivation and uptake inhibition on cell growth, cell-cycle progression, and growth signaling pathways of five lung cancer cell lines. Our results show that (i) SLC1A5 protein is expressed in 95% of squamous cell carcinomas (SCC), 74% of adenocarcinomas (ADC), and 50% of neuroendocrine tumors; (ii) SLC1A5 is located at the cytoplasmic membrane and is significantly associated with SCC histology and male gender; (iii) 68% of Gln is transported in a Na(+)-dependent manner, 50% of which is attributed to SLC1A5 activity; and (iv) pharmacologic and genetic targeting of SLC1A5 decreased cell growth and viability in lung cancer cells, an effect mediated in part by mTOR signaling. These results suggest that SLC1A5 plays a key role in Gln transport controlling lung cancer cells' metabolism, growth, and survival.

Immunosuppressive activity enhances central carbon metabolism and bioenergetics in myeloid-derived suppressor cells in vitro models

BackgroundThe tumor microenvironment contains a vast array of pro- and anti-inflammatory cytokines that alter myelopoiesis and lead to the maturation of immunosuppressive cells known as myeloid-derived suppressor cells (MDSCs). Incubating bone marrow (BM) precursors with a combination of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-6 (IL-6) generated a tumor-infiltrating MDSC-like population that impaired anti-tumor specific T-cell functions. This in vitro experimental approach was used to simulate MDSC maturation, and the cellular metabolic response was then monitored. A complementary experimental model that inhibited L-arginine (L-Arg) metabolizing enzymes in MSC-1 cells, an immortalized cell line derived from primary MDSCs, was used to study the metabolic events related to immunosuppression.ResultsExposure of BM cells to GM-CSF and IL-6 activated, within 24 h, L-Arg metabolizing enzymes which are responsible for the MDSCs immunosuppressive potential. This was accompanied by an increased uptake of L-glutamine (L-Gln) and glucose, the latter being metabolized by anaerobic glycolysis. The up-regulation of nutrient uptake lead to the accumulation of TCA cycle intermediates and lactate as well as the endogenous synthesis of L-Arg and the production of energy-rich nucleotides. Moreover, inhibition of L-Arg metabolism in MSC-1 cells down-regulated central carbon metabolism activity, including glycolysis, glutaminolysis and TCA cycle activity, and led to a deterioration of cell bioenergetic status. The simultaneous increase of cell specific concentrations of ATP and a decrease in ATP-to-ADP ratio in BM-derived MDSCs suggested cells were metabolically active during maturation. Moreover, AMP-activated protein kinase (AMPK) was activated during MDSC maturation in GM-CSF and IL-6–treated cultures, as revealed by the continuous increase of AMP-to-ATP ratios and the phosphorylation of AMPK. Likewise, AMPK activity was decreased in MSC-1 cells when L-Arg metabolizing enzymes were inhibited. Finally, inhibition of AMPK activity by the specific inhibitor Compound C (Comp-C) resulted in the inhibition of L-Arg metabolizing enzyme activity and abolished MDSCs immunosuppressive activity.ConclusionsWe anticipate that the inhibition of AMPK and the control of metabolic fluxes may be considered as a novel therapeutic target for the recovery of the immunosurveillance process in cancer-bearing hosts.

Bidirectional Transport of Amino Acids Regulates mTOR and Autophagy

Amino acids are required for activation of the mammalian target of rapamycin (mTOR) kinase which regulates protein translation, cell growth, and autophagy. Cell surface transporters that allow amino acids to enter the cell and signal to mTOR are unknown. We show that cellular uptake of L-glutamine and its subsequent rapid efflux in the presence of essential amino acids (EAA) is the rate-limiting step that activates mTOR. L-glutamine uptake is regulated by SLC1A5 and loss of SLC1A5 function inhibits cell growth and activates autophagy. The molecular basis for L-glutamine sensitivity is due to SLC7A5/SLC3A2, a bidirectional transporter that regulates the simultaneous efflux of L-glutamine out of cells and transport of L-leucine/EAA into cells. Certain tumor cell lines with high basal cellular levels of L-glutamine bypass the need for L-glutamine uptake and are primed for mTOR activation. Thus, L-glutamine flux regulates mTOR, translation and autophagy to coordinate cell growth and proliferation.

Cellular distribution of the neutral amino acid transporter subtype ASCT2 in mouse brain

ASCT2 is an ASC (alanine-, serine-, cysteine-preferring) neutral amino acid exchanger that may regulate CNS function by transporting amino acid substrates including L-serine, L-cysteine, L-glutamine, L-glutamate and D-serine. Despite the potentially important role of ASCT2 in influencing metabolic and signaling functions of these amino acids in brain, there has been little description of its distribution in brain tissue. We employed a commercially available human ASCT2 antibody in immunohistochemistry studies in adult mouse brain and found a wide regional distribution for ASCT2 that was limited to dendrites labeled by anti-microtubule-associated protein-2 in cortex, hippocampus and striatum. No ASCT2 immunoreactivity was observed in areas labeled by antibodies against a neuronal cell body marker (NeuN), or either of the astrocyte markers, glial fibrillary acidic protein or S100beta. In cerebellum both Purkinje cell bodies and dendrites were positive for ASCT2 immunoreactivity. In support of a dendritic localization for ASCT2 in cortex, low affinity (K(T) > 1 mM), Na(+)-dependent D-serine and L-glutamine uptake characteristic of ASCT2-mediated transport was observed in P2 synaptosomal preparations. These results suggest that ASCT2 may be an important neuronal neutral amino acid transporter and highlight a discrepancy between findings of astrocyte ASCT2 function in tissue culture and brain in situ.

Design, synthesis, and biological activity of novel triazole amino acids used to probe binding interactions between ligand and neutral amino acid transport protein SN1

Novel triazole amino acids were synthesized as probes to investigate ligand–protein binding interactions of the neutral amino acid transporter SN1. The bonding hypothesis to be tested was that the side chains of endogenous substrates are acting as H-bond acceptors. Although limited inhibition of 3H- l-glutamine uptake by SN1 expressing oocytes was observed, the synthetic compounds show a trend that suggests a hydrogen bond interaction just outside the endogenous ligand binding pocket.

Effect of melatonin on the retinal glutamate/glutamine cycle in the golden hamster retina.

Glutamate is the main excitatory neurotransmitter in the retina, but it is neurotoxic when present in excessive amounts. The metabolic dependence of glutamatergic neurons upon glia via the glutamate/glutamine cycle to provide the precursor for neurotransmitter glutamate is well established. Since melatonin has been shown to be neuroprotective in several systems, in the present report, its effect on the glutamate/glutamine cycle activity was examined in the golden hamster retina. Melatonin (0.1-10 nM) significantly increased retinal glutamine synthetase activity but it did not affect L-glutamine release. A characterization of the hamster retinal L-glutamine uptake mechanism was performed. This mechanism was partly Na+-dependent, and it was significantly inhibited by 2-aminobicyclo (2, 2, 1) heptane 2-carboxylic acid (BCH, a selective antagonists for the L-type system) and by alpha-(methylamino)-isobutyric acid (MeAIB, substrate characteristic for the A -type transporter) suggesting the coexistence of these transport systems in the hamster retina. Melatonin (0.1-10 nM) significantly increased total glutamine uptake as well as the BCH and the MeAIB-insensitive transporters activity. On the other hand, melatonin significantly decreased retinal glutaminase activity. On the basis of these results, it might be presumed that hamster retinal glutamate/glutamine cycle activity is regulated by physiological concentrations of melatonin. Furthermore, these findings suggest that a treatment with melatonin could be considered as a new approach to handling glutamate-mediated neuronal degeneration.

Phase III study: AES-14 in patients at risk for mucositis secondary to anthracycline-based chemotherapy

8008 Background: Oral mucositis (OM) limits chemotherapy dosing and increases health care costs. AES-14 (Saforis) enhances direct oral mucosal cell uptake of L-glutamine by the Aesgen UpTec system. Prior AES-14 studies in chemotherapy patients suggested safe, effective reduction (20%) of clinically significant OM. Methods: A randomized, double-blind, placebo-controlled crossover multicenter trial was conducted in breast carcinoma patients receiving at least 3 cycles of an anthracycline regimen (AC, CAF or FAC). Patients developing WHO Grade (Gr) 2–4 OM during screening cycle were randomized for crossover in 2 subsequent cycles. Study drug began 1st day of chemotherapy and continued 14 days after last chemotherapy dose or 5 days after OM resolved. Safety was assessed by Adverse Event (AE)/Serious AE evaluations. Primary endpoint analysis compared proportion of patients with ≥ Gr 2 OM while on AES-14 versus placebo. Secondary endpoints included OM duration and OM Assessment Scale outcomes. Results: 2,064 female patients were screened for OM ≥ WHO Gr 2. 326 patients (mean age 51 years) were randomized (15.7% OM rate). Peak OM was primarily WHO Gr 2. No significant randomization imbalances occurred. Comparison of treatment versus placebo outcomes in crossover design requires that no significant drug treatment carryover effect exists. AES-14 administered during 1st treatment cycle reduced ≥ Gr 2 OM incidence in the placebo 2nd treatment cycle (p=0.0269). This carryover effect invalidated crossover efficacy comparison. Primary endpoint analysis thus compared parallel group treatment during 1st cycle only. Incidence of clinically significant OM (WHO Gr 2–4) was 22% less with AES-14 versus placebo (p=0.0261). No AES-14-related SAEs and 2 placebo-associated SAEs were reported. Conclusions: AES-14 reduced incidence of 1st cycle Gr 2–4 OM by 22% in these chemotherapy patients. The significant AES-14 carryover effect suggests that optimizing mucosal repair during one cycle may protect patients from OM during subsequent chemotherapy. Supported by Aesgen Inc. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Aesgen Inc. Aesgen Inc.

Phase III study: AES-14 in patients at risk for mucositis secondary to anthracycline-based chemotherapy

8008 Background: Oral mucositis (OM) limits chemotherapy dosing and increases health care costs. AES-14 (Saforis) enhances direct oral mucosal cell uptake of L-glutamine by the Aesgen UpTec system. Prior AES-14 studies in chemotherapy patients suggested safe, effective reduction (20%) of clinically significant OM. Methods: A randomized, double-blind, placebo-controlled crossover multicenter trial was conducted in breast carcinoma patients receiving at least 3 cycles of an anthracycline regimen (AC, CAF or FAC). Patients developing WHO Grade (Gr) 2–4 OM during screening cycle were randomized for crossover in 2 subsequent cycles. Study drug began 1st day of chemotherapy and continued 14 days after last chemotherapy dose or 5 days after OM resolved. Safety was assessed by Adverse Event (AE)/Serious AE evaluations. Primary endpoint analysis compared proportion of patients with ≥ Gr 2 OM while on AES-14 versus placebo. Secondary endpoints included OM duration and OM Assessment Scale outcomes. Results: 2,064 female patients were screened for OM ≥ WHO Gr 2. 326 patients (mean age 51 years) were randomized (15.7% OM rate). Peak OM was primarily WHO Gr 2. No significant randomization imbalances occurred. Comparison of treatment versus placebo outcomes in crossover design requires that no significant drug treatment carryover effect exists. AES-14 administered during 1st treatment cycle reduced ≥ Gr 2 OM incidence in the placebo 2nd treatment cycle (p=0.0269). This carryover effect invalidated crossover efficacy comparison. Primary endpoint analysis thus compared parallel group treatment during 1st cycle only. Incidence of clinically significant OM (WHO Gr 2–4) was 22% less with AES-14 versus placebo (p=0.0261). No AES-14-related SAEs and 2 placebo-associated SAEs were reported. Conclusions: AES-14 reduced incidence of 1st cycle Gr 2–4 OM by 22% in these chemotherapy patients. The significant AES-14 carryover effect suggests that optimizing mucosal repair during one cycle may protect patients from OM during subsequent chemotherapy. Supported by Aesgen Inc. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Aesgen Inc. Aesgen Inc.

Glutamine transport in isolated epithelial intestinal cells. Identification of a Na+-dependent transport mechanism, highly specific for glutamine.

L-glutamine transport was evaluated in isolated cells from the guinea-pig small intestine by measuring [(3)H]- L-glutamine uptake. Villous and crypt cells expressed Na(+)-dependent and Na(+)-independent transport mechanisms. Glutamine transport systems were identified using various amino acids and analogues as inhibitors. In both villous and crypt cells, 2-(methylamino)-isobutyrate (MeAIB), a system A inhibitor, did not inhibit Na(+)-dependent glutamine influx. 2-Aminobicyclo(2,2,1)heptane-2-carboxylate (BCH), a system B(0) and B(0,+) substrate, had no effect on Na(+)-dependent influx. Serine, cysteine and threonine, system ASC inhibitors, reduced Na(+)-dependent influx by 50%. Asparagine, but not histidine, system N inhibitors, reduced Na(+)-dependent glutamine influx by 50%, however the effect of asparagine was not additive to that of threonine. The remaining Na(+)-dependent glutamine influx (50%) was only inhibited by glutamine itself, by Na(+) substitution ( N-methyl-glucamine, K(+), Li(+)) or by external pH reduction. Phenyl-acetyl-glutamine (PAG), a synthetic amino acid analogue, also inhibited this Na(+)-dependent, threonine-insensitive glutamine influx (IC(50) 2.45 mM). The Na(+)-independent uptake was partially inhibited by BCH, a system L inhibitor, and other neutral amino acids, but was not affect by PAG. Our results suggest that glutamine is transported in both villous and crypt cells by the Na(+)-independent system L, by the Na(+)-dependent system ASC and by an as yet undescribed Na(+)-dependent transport mechanism, highly specific for glutamine.

Insulin-like growth factor-I stimulates Na+-dependent glutamine absorption in piglet enterocytes.

Agents that enhance intestinal glutamine transport may be useful under conditions in which intestinal mucosal function is compromised. Here we examined whether oral administration of insulin-like growth factor (IGF-I) stimulates absorption of L-glutamine in piglet intestine. Colostrum-deprived piglets received human recombinant IGF-I (3.5 mg/kg/day) or vehicle orogastrically every 8 hr for four days after birth. Piglets were killed on day 5 and the proximal jejunum was removed. Basal electrical parameters and L-glutamine-stimulated changes in short-circuit current were measured in muscle-stripped tissues, and rates of L-glutamine uptake were measured in everted jejunal sleeves. Oral IGF-I had no effect on jejunal mucosal mass. Short-circuit current responses to mucosal addition of 10 mM L-glutamine were increased by oral IGF-I. Total and carrier-mediated uptakes of L-glutamine per milligram were greater in tissues from IGF-I-treated piglets due to a significantly greater maximal rate of uptake (Jmax). Thus, oral administration of IGF-I stimulates Na+-dependent glutamine absorption in piglet small intestine, an effect that is independent of changes in intestinal mucosal mass.

Characteristics of L-glutamine transport during Caco-2 cell differentiation

Glutamine is the main fuel of intestinal epithelial cells, as well as a precursor for the intense nucleotide biosynthesis which arises with the rapid turnover of enterocytes. In order to determine whether glutamine uptake may vary as a function of metabolic demand, glutamine transport across the brush-border membrane of differentiating Caco-2 cells has been investigated. The uptake of L-[3H]glutamine was measured between day 7 and day 21 post-seeding. Kinetic analysis with glutamine concentrations ranging from 6.25 μM to 12.8 mM revealed the involvement of high affinity Na+-dependent (Kt=110 μM) and low affinity Na+-independent (Kt=900 μM) transport components at day 7. Both components were partially inhibited by L-lysine in a competitive fashion, suggesting that four different systems were responsible for glutamine uptake: B0, B0,+, b0,+ and L. All four systems were present during the differentiation process, with systems L and B0 being responsible for up to 80% of glutamine uptake. Caco-2 cell differentiation was associated with a marked decrease in L-glutamine uptake, which affected both the Na+-dependent and the Na+-independent components. In contrast to glucose uptake, the development of L-glutamine uptake across the brush-border membrane of Caco-2 cells may reflect an adjustment to cell metabolic demand rather than the progressive appearance of a vectorial transport process.

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 in 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 J max 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 J max of 55.1±6.6 pmol mg protein −1 s −1. The non-saturable component measured with HgCl 2-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 ( K diff) was estimated to be K diff=3.78±0.06 pmol l 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° 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.

Characteristics of L-glutamine transport by lactating mammary tissue.

The transport of L-glutamine by the lactating rat mammary gland has been investigated using rat mammary tissue explants and the in situ perfused rat mammary gland. L-glutamine uptake by both explants and the perfused mammary gland was via both Na(+)-dependent and Na(+)-independent pathways. It appeared that these pathways are situated on the blood-facing aspect of the mammary gland. L-glutamine uptake by both mammary preparations was markedly inhibited by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid in the absence of external Na+. This is consistent with L-glutamine uptake via system L. The Na(+)-dependent component(s) of L-glutamine uptake remains to be precisely identified. However, system A can be ruled out on the basis that L-glutamine was not inhibited by (methylamino)isobutyric acid. Mammary tissue concentrates L-glutamine with respect to both milk and plasma: we suggest that the Na(+)-dependent component(s) of L-glutamine uptake is responsible for generating the intracellular to extracellular concentration gradient.

Choroid plexus histidine transport

System-N transport plays an important role in l-glutamine uptake into isolated rat choroid plexus but its role in the transport of another System-N substrate, l-histidine, has yet to be determined. Similarly, the possible effects on System-N mediated l-histidine transport of changes in pH and extracellular l-glutamine, such as occur in cerebral ischemia and hepatic encephalopathy, have yet to be examined. In the absence of competing amino acids, l-[ 3H]histidine uptake in isolated rat choroid plexus was mediated by both Na +-independent and Na +-dependent transport. The former was inhibited by 2-amino-2-norbornane carboxlic acid, indicating System-L transport, while the latter appears System-N mediated as it was inhibited by three System-N substrates but not substrates for System-A and -ASC. The Na +-dependent uptake had a K m of 0.2 mM and a V max of 1.4 nmol/mg/min. It accounted for 30% of l-histidine uptake in the presence of physiological concentrations of amino acids. Reductions in pH markedly inhibited Na +-dependent but not Na +-independent transport indicating that, as in liver but not neurons, System-N mediated transport at the choroid plexus is pH sensitive. Increases in l-glutamine concentration in the pathophysiological range reduced l-histidine uptake via both System-L and -N.

Factors affecting amino acid uptake by the ectomycorrhizal fungus Paxillus involutus

Factors affecting amino acid uptake by the ectomycorrhizal fungus Paxillus involutus were investigated using tracer kinetic experiments. Amino acid uptake by P. involutus was dependent on the pH of the incubation medium. Uptake of L-glutamate and L-glutamine had a distinct pH optimum around pH 4.0 and declined sharply between pH 4.5 and 6.4. Uptake of alanine and aspartate was also highest at low pH values (3-4) but declined more slowly with increasing pH. Total uptake of labelled amino acids was unaffected by 0.05 or 0.5 mM ammonium while 5 mM ammonium decreased amino acid uptake. Nitrate or glucose did not significantly affect amino acid uptake but glucose greatly increased the proportion of carbon that was respired. Uptake of the different amino acids decreased by a factor of 4 to 10 with increasing age (1-5 wk) and the proportion of absorbed C-14 that was respired increased with age. Changes in the size of the total amino acid pool were inversely related to those in the rate of uptake and the pool increased steadily with age. The implications of the ability of P. involutus to take up amino acids are discussed. (Less)

Glutamine transport by vesicles isolated from tumour-cell mitochondrial inner membrane.

Mitochondrial-inner-membrane vesicles, isolated from Ehrlich ascites carcinoma cells by titration with detergents, accumulated L-glutamine by a very efficient transport system. The vesicles lack any phosphate-activated glutaminase activity, allowing measurement of transport rates without interference by L-glutamine metabolism. The time course of the transport was linear for the first 60 s, reaching a steady state after 120 min. L-Glutamine transport showed co-operativity, with a Hill coefficient of 2.2; the kinetic parameters S0.5 and Vmax had values of 5 mM and 26 nmol/30 s per mg of protein respectively. The pH-dependence curve showed a bell shape, with a pH optimum about 8.0. The uptake of L-glutamine was not affected by the presence of a 50-fold molar excess of D-glutamine, L-cysteine, L-histidine, L-alanine, L-serine and L-leucine, whereas L-glutamate behaved as a poor inhibitor. The structural analogue L-glutamate gamma-hydroxamate (5mM) inhibited the net uptake by 68%; interestingly, other analogues (6-diazo-5-oxo-L-norleucine, acivicin and L-glutamate gamma-hydrazide) were ineffective. The impermeant thiol reagent p-chloromercuriphenylsulphonic acid (0.5mM) completely abolished the mitochondrial L-glutamine uptake; in contrast, other thiol reagents (mersalyl and N-ethylmaleimide) did not significantly affect the transport. These data confirm the existence of a specific transport system with high capacity for L-glutamine in the mitochondrial inner membrane, a step preceding the highly operative glutaminolysis in tumour cells.