Ontogeny of glutamine transport by rat liver plasma membrane vesicles.

Glutamine metabolism in the liver is essential for gluconeogenesis and ureagenesis. During the suckling period there is high hepatic protein accretion and the portal vein glutamine concentration is twice that in the adult, whereas hepatic vein glutamine concentration is similar between adult and suckling rats. Therefore, we hypothesized that glutamine uptake by the liver could be greater in the suckling period compared to the adult period. The present studies were, therefore, designed to investigate the transport of glutamine by plasma membranes of rat liver during maturation (suckling--2-week old, weanling--3-week old and adult--12-week old). Glutamine uptake by the plasma membranes of the liver represented transport into an osmotically sensitive space in all age groups. Inwardly directed Na+ gradient resulted in an "overshoot" phenomenon compared to K+ gradient. The magnitude of the overshoot was greater in suckling rats plasma membranes compared to adult membranes. Glutamine uptake under Na+ gradient was electrogenic and maximal at pH 7.5, whereas uptake under K+ gradient was electroneutral. Glutamine uptake with various concentrations of glutamine under Na+ gradient was saturable in all age groups with a Vmax of 1.5 +/- 0.1, 0.7 +/- 0.1 and 0.5 +/- 0.06 nmoles/mg protein/10 seconds in suckling, weanling and adult rats, respectively (P < 0.01). Km values were 0.6 +/- 0.1, 0.5 +/- 0.1 and 0.5 +/- 0.1 mM respectively. Vmax for Na(+)-independent glutamine uptake were 0.6 +/- 0.1, 0.55 +/- 0.07 and 0.54 +/- 0.06 nmoles/mg protein with Km values of 0.54 +/- 0.2, 0. +/- 0.1 and 0.5 +/- 0.2 mM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of the sodium-dependent glutamine transporter in human intestinal cell confluent monolayers.

The intestinal epithelium metabolism of glutamine plays a critical role in inter-organ nitrogen flow. Although it is known that glutamine is the primary oxidative energy source and nucleotide precursor in intestinal cells, the luminal uptake of glutamine by the apical surface of enterocytes is poorly understood. In this study we have uncovered the sodium-dependent transporter system responsible for L-glutamine uptake by the apical membrane of a human intestinal epithelial cell line. The sodium-dependent Michaelis constant (Km) = 247 +/- 45 microM glutamine, and Jmax = 4.44 +/- 0.65 x 10(-9) mole min-1(mg protein)-1 (37 degrees C). Glutamine shares the transporter with alanine, as demonstrated by unlabeled glutamine inhibition of [3H]alanine uptake kinetics with a purely competitive-type inhibition pattern, and glutamine inhibition Ki = 205 +/- 18 microM by Dixon analysis. The inhibition pattern for a series of amino acid analogs indicated that this intestinal apical membrane sodium-dependent transporter for glutamine is distinct from any other transport system found in membranes of non-intestinal cells.     

Glutamine transport in C6 glioma cells

Glutamine transport across the cell membranes of a variety of mammalian tissues is mediated by at least four transport systems: a sodium-independent system L, and sodium-dependent systems A, ASC and N, the latter occurring in different tissue-specific variants. In this study we assessed the contribution of these systems to the uptake of [(3)H]glutamine in C6 rat glioma cells. The sodium-dependent uptake, which accounted for more than 80% of the total uptake, was not inhibited by 2-methylaminoisobutyric acid (MeAIB), indicating that system A was inactive, possibly being depressed by glutamine present in the culture medium. About 80% of the sodium-dependent uptake was mediated by system ASC, which differed from system ASC common to other CNS- and non-CNS tissues by its pH-dependence and partial lithium tolerance. The residual 20% of sodium-dependent uptake appeared to be mediated by system N, which was identified as a component resistant to inhibition by MeAIB+threonine. The system N in C6 cells appeared to be neither fully compatible with the neuronal system Nb, nor with the N system described in astrocytes: it differed from the former in being strongly inhibited by histidine and showing fair tolerance for lithium, and from the latter in its pH-insensitivity and strong inhibition by glutamate. The sodium-independent glutamine uptake differed from the astrocytic or neuronal uptake in its relatively weak inhibition by system L substrates and a strong inhibition by system ASC substrates, indicating a possible contribution of a variant of the ASC system.     

Cyclooxygenase blockade and exogenous glutamine enhance sodium absorption in infected bovine ileum

We have previously shown that prostanoids inhibit electroneutral sodium absorption in Cryptosporidium parvum-infected porcine ileum, whereas glutamine stimulates electroneutral sodium absorption. We postulated that glutamine would stimulate sodium absorption via a cyclooxygenase (COX)-dependent pathway. We tested this hypothesis in C. parvum-infected calves, which are the natural hosts of cryptosporidiosis. Tissues from healthy and infected calves were studied in Ussing chambers and analyzed via immunohistochemistry and Western blots. Treatment of infected tissue with selective COX inhibitors revealed that COX-1 and -2 must be blocked to restore electroneutral sodium absorption, although the transporter involved did not appear to be the expected Na(+)/H(+) exchanger 3 isoform. Glutamine addition also stimulated sodium absorption in calf tissue, but although this transport was electroneutral in healthy tissue, sodium absorption was electrogenic in infected tissue and was additive to sodium transport uncovered by COX inhibition. Blockade of both COX isoforms is necessary to release the prostaglandin-mediated inhibition of electroneutral sodium uptake in C. parvum-infected calf ileal tissue, whereas glutamine increases sodium uptake by an electrogenic mechanism in this same tissue.     

Uptake of Kynurenine into Rat Brain Slices

The transport of [3H]kynurenine ([3H]KYN) into slices from rat tissue was examined in vitro. Brain accumulated KYN seven to eight times more effectively than any of several peripheral organs. Of all the organs tested, only the brain exhibited a sodium-dependent component of the uptake process. After an incubation period of 1 h, sodium-dependent transport amounted to 60% of total uptake. Both processes were abolished by prior sonication of the tissue and significantly inhibited by inclusion of metabolic blockers in the incubation medium. Time resolution showed that the sodium-independent uptake occurred rapidly and reached saturation within 30 min. In contrast, sodium-dependent transport was linear for at least 2 h of incubation. Brain regional analysis revealed a sevenfold difference between the areas of highest (cortex) and lowest (cerebellum) uptake. With the exception of cerebellar tissue, the ratio between sodium-dependent and sodium-independent processes was consistent among brain regions. Kinetic analyses were performed on striatal slices and revealed a Km of 927 microM and a Vmax of 18 nmol/h/mg of protein for the sodium-dependent process, and a Km of 3.8 mM and a Vmax of 38 nmol/10 min/mg of protein for the sodium-independent transport. The transporters were equally amenable to inhibition by KYN and tryptophan, indicating that KYN entry into the cell may be mediated by neutral amino acid uptake sites. No strict stereoselectivity existed, but L enantiomers were clearly more active than the D forms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methodological aspects of measuring amino acid uptake in studies with porcine jejunal brush border membrane vesicles

With L-glutamine, as a representative amino acid this study was undertaken to examine the effects of substrate concentrations on initial and equilibrium amino acid uptake and intravesicular volume determined with porcine jejunal brush border membrane vesicles prepared by Mg2+-aggregation and differential centrifugation. Transport measurements (24 degrees C) were conducted by the rapid filtration manual procedure. Glutamine uptake was shown to occur into an osmotically-active space ranging between 1.09-1.58 microl/mg protein with little non-specific membrane binding. At different concentrations (in parentheses), the duration of initial glutamine uptake in both Na+ gradient and Na+-free conditions was 10 s (0.01 mM), 15 s (0.17 mM), and 20 s (1.9 and 9.4 mM), respectively. Substrate concentrations affected the duration of initial uptake, with lower substrate concentrations giving shorter duration for initial amino acid uptake. At different substrate concentrations (in parentheses), the time required to reach equilibrium glutamine uptake was 5 min (0.01 mM), 10 min (0.17 mM), and 60 min (1.9 and 9.4 mM), respectively. Thus, substrate concentrations also affected the time required to reach equilibrium uptake. The higher the substrate concentration, the longer the incubation time needed to reach equilibrium amino acid uptake. At the glutamine concentrations of 0.01, 0.17, 1.9, and 9.4 mM, the average intravesicular volume was estimated to be 1.58+/-0.21, 1.09+/-0.28, 1.24+/-0.18, and 1.36+/-0.21 microl/mg protein, respectively. Substrate concentrations had no effect (p>0.05) on the intravesicular volume of membrane vesicles. In conclusion, in the experiments on amino acid transport kinetics measured with the rapid filtration manual procedure, the incubation time used for measuring the initial uptake rate should be determined from the time course experiments conducted at the lowest substrate concentration used, whereas the intravesicular volume can be obtained from equilibrium uptake measured at any substrate concentrations.     

Uptake of L-leucine and L-phenylalanine across the basolateral cell surface in isolated oxyntic glands.

The time course, kinetic, specificity and sodium-dependence of L-leucine and L-phenylalanine uptake by rabbit isolated oxyntic glands were studied in order to identify the systems involved in the transport of branched-chain and aromatic neutral amino acids through the basolateral cell membrane. The uptake was measured directly in the disrupted cells after incubation of the glands with the 3H-labelled amino acid both in a sodium-containing and a sodium-free medium. The uptake of L-leucine was largely carrier-mediated whilst L-phenylalanine was taken up by either carrier-mediated and nonsaturable processes. Both amino acids were taken up by a Na(+)-independent process. The kinetic parameters of L-leucine and L-phenylalanine carrier-mediated influx were, respectively: Kt = 2.71 mM and Jmax = 1390 nmol mg-1 s-1, Kt = 1.03 mM and Jmax = 176 nmol mg-1 s-1. From cross-inhibition studies it can be inferred that L-leucine is primarily transported by a Na(+)-independent system which shows specificity for bulky side chains dipolar amino acids. The system displays similar affinities for L-phenylalanine (Ki = 2.81 mM) and L-isoleucine (Ki = 2.62 mM). Similar results were obtained from self-inhibition experiments: the Ki of the carrier-mediated uptake of L-leucine and L-phenylalanine were 2.12 and 2.40 mM (from a Hanes plot) or 3.2 and 0.8 mM (from a Dixon plot), respectively. It is concluded that a sodium-independent transport system, like Christensen's 'L' type, is shared by branched-chain and aromatic dipolar amino acids, which only shows slight differences in their affinities for the carrier.     

Chloride transport across rat ileal basolateral membrane vesicles.

The present study was designed to investigate Cl- transport across rat ileal basolateral membranes. Basolateral membrane vesicles were prepared by a well-validated technique. The purity of the basolateral membrane vesicles was verified by marker enzyme studies and by studies of d-glucose and calcium uptake. Cl- uptake was studied by a rapid filtration technique. Neither an outwardly directed pH gradient, nor a HCO3- gradient, or their combination could elicit any stimulation of Cl- transport when compared with no gradient. 4,4-Diisothiocyanostilbene-2,2-disulfonic acid at 5 mM concentration did not inhibit Cl- uptake under gradient condition. Similarly, the presence of the combination of outwardly directed Na+ and HCO3- gradients did not stimulate Cl- uptake compared with the combination of K+ and HCO3- gradients or no HCO3- gradient. This is in contrast to our results in the brush border membranes, where an outwardly directed pH gradient caused an increase in Cl- uptake. Cl- uptake was stimulated in the presence of combined Na+ and K+ gradient. Bumetanide at 0.1 mM concentration inhibited the initial rate of Cl- uptake in the presence of combined Na+ and K+ gradients. Kinetic studies of bumetanide-sensitive Cl- uptake showed a Vmax of 5.6 +/- 0.7 nmol/mg protein/5 sec and a Km of 30 +/- 8.7 mM. Cl- uptake was stimulated by an inside positive membrane potential induced by the ionophore valinomycin in the setting of inwardly directed K+ gradient compared with voltage clamp condition. These studies demonstrate two processes for Cl- transport across the rat ileal basolateral membrane: one is driven by an electrogenic diffusive process and the second is a bumetanide-sensitive Na+/K+/2 Cl- process. Cl- uptake is not enhanced by pH gradient, HCO3- gradient, their combination, or outwardly directed HCO3- and Na+ gradients.     

Glutamine uptake by rat renal basolateral membrane vesicles

The presence of a sodium-dependent, saturable uptake process is described in basolateral membranes of rat renal cortex for L-glutamine. Concentration-dependence studies indicate the presence of multiple transport systems withK _{m 1} of 0.032 mM and V₁ of 0.028 nmol/mg of protein per min, andK _{m 2} of 17.6 mM and V₂ of 17.6 nmol/mg of protein per min. Lysine completely inhibits the high-affinity, low-capacityK _m system and partially inhibits the low-affinity, high-capacity system. Cystine and other dibasic amino acids also affect glutamine uptake.     

Glutamine-stimulated amino acid and peptide incorporation in Bacteroides melaninogenicus.

The uptake of a number of amino acids and dipeptides by cells and spheroplasts of Bacteroides melaninogenicus was stimulated by the presence of glutamine; 50 mM glutamine induced maximum uptake of glycine or alanine, and glutamine stimulated the uptake of glycine over a wide concentration range (0.17 to 170 mM). Glutamine stimulated the uptake of the dipeptides glycylleucine and glycylproline at significantly faster rates compared with glycine and leucine. The amino acids whose uptake was stimulated by glutamine were incorporated into trichloroacetic acid-precipitable material, and the inclusion of chloramphenicol or puromycin did not affect this incorporation. The uptake of glutamine by cells was concentration dependent. In contrast, in the absence of chloramphenicol 79% of the glutamine taken up by cells supplied with a high external concentration (4.4 mM) was trichloroacetic acid soluble. Glutamate and alpha-ketoglutarate were identified in the intracellular pool of glutamine-incubated spheroplasts. The amino acids and peptides were incorporated into cell envelope material, and a portion (30 to 50%) of the incorporated amino acids could be removed by trypsinization or treatment with papain. The effect of glutamine was depressed by inhibitors of energy metabolism, suggesting that glutamine-stimulated incorporation is an energy-mediated effect.     

Transport ofl-cysteine by rat renal brush border membrane vesicles

Brush border membranes were isolated from rat renal cortex by a divalent cation precipitation method. L-35S-cysteine uptake into the vesicles was measured by a rapid filtration method. Only minimal binding of the amino acid to the vesicles was observed. Sodium stimulates L-cysteine uptake specifically. Anion replacement experiments, experiments in the presence of potassium/valinomycin-induced diffusion potential as well as experiments with a potential-sensitive fluorescent dye document an electrogenic sodium-dependent uptake mechanism for L-cysteine. Tracer replacement experiments as well as the fluorescence experiments indicate a preferential transport of L-cysteine. Transport of L-cysteine is inhibited by L-alanine and L-phenylalanine but not by L-glutamic acid and the L-basic amino acids. Initial, linear influx kinetics provide evidence for the existence of two transport sites. The results suggest (a) sodium-dependent mechanism(s) for L-cysteine shared by other neutral amino acids.     

Effect of parathyroid hormone and dietary phosphate on phosphate transport in renal outer cortical and outer medullary brush-border membrane vesicles

Two distinctive sodium-dependent phosphate transport systems have been identified in early and late proximal tubules; a high-capacity process located only in outer cortical tissue, and a high affinity present in both outer cortical and outer medullary brush-border membranes (Km 0.1-0.25 mM). A third, sodium-independent, pH gradient-stimulated system (Vmax 4.7 +/- 0.3 nmol.mg-1.min-1, Km 0.15 +/- 0.002 mM) is present in the outer medulla, but absent in outer cortex. Brush-border vesicles were prepared from outer cortical and outer medullary tissue of pigs maintained on low (less than 0.05%), normal (0.4%), or high (4%) phosphate diets. Sodium-dependent phosphate uptake of the high-capacity system decreased (Vmax, 9.4 to 2.2 nmol.mg-1.min-1) from low to high phosphate diet, whereas uptake rates decreased about 50% in the high-affinity system. There were no changes in the respective Km values. The pH gradient-stimulated uptake also decreased (Vmax, 6.9 to 3.0 nmol.mg-1.min-1) with no change in mean Km value (0.15 +/- 0.001 mM) with dietary manipulation. Administration of 1 U parathyroid hormone prior to study resulted in a decrease in sodium-dependent uptake by 40-50% and in pH-dependent uptake (36%) with no change in the respective Km values. In conclusion, the antecedent dietary phosphate intake and parathyroid hormone administration appropriately alters phosphate uptake across the brush-border membrane of all three systems, sodium-dependent and pH gradient-stimulated phosphate transport.     

Reactivating tammar wallaby blastocysts oxidize fatty acids and amino acids.

The tammar wallaby, Macropus eugenii, has a ruminant-like digestive system which may make a significant concentration of amino acids and fatty acids available to the blastocyst via uterine fluids. Fluorescent and radioisotope analyses were performed to determine the rate of glutamine and palmitate use by blastocysts recovered on day 0, 3, 4, 5 and 10 after reactivation induced by removal of pouch young (RPY). Between day 0 and 4 glutamine uptake increased from 15.6 +/- 6.6 to 36.1 +/- 2.7 pmol per embryo h-1 (P < 0.01) and ammonium production increased from 8.2 +/- 4.3 to 26.6 +/- 3.0 pmol per embryo h-1 (P < 0.01). Glutamine oxidation did not increase until day 10 after RPY (P < 0.01), but the percentage of glutamine oxidized increased from 4.5 +/- 3.1% during diapause to 31.2 +/- 12.6% (P < 0.01) by day 5 after RPY and increased further to 51.0 +/- 15.8% (P < 0.01) by day 10 after RPY. Palmitate oxidation also increased from 0.3 +/- 0.1 by day 0 blastocysts to 3.8 +/- 1.7 pmol per embryo h-1 (P < 0.01) by day 4 blastocysts. This increase provides a greater potential for ATP production, possibly to supply increased demand due to the coincident resumption of mitoses. The ATP:ADP ratio within blastocysts had reduced by the time of the first measurement at day 3 (0.5 +/- 0.2 pmol per embryo h-1; P < 0.01) compared with day 0 blastocysts (1.4 +/- 0.3 pmol per embryo h-1). It is likely that metabolism of amino acids and fatty acids contributes to the energy supply during reactivation of tammar wallaby blastocysts after embryonic diapause.     

The transport of glutamine and alanine into rat colonocytes.

The transport of glutamine and alanine into isolated rat colonocytes was studied. The transport of both amino acids appears to be dependent on a Na+ gradient. The apparent Km values for the transport of glutamine and alanine were 2.56 +/- 0.84 and 5.35 +/- 1.20 mM respectively, but with similar Vmax. values. Glutamine and alanine transport were mutually competitive, and the transport of both amino acids was competitively inhibited by 2-methylaminoisobutyrate. In contrast, histidine inhibited the transport of both glutamine and alanine non-competitively. It is concluded that glutamine and alanine are transported into rat colonocytes by a common carrier system similar to System A of other cells. It is suggested that the metabolic function of this transport system in rat colonocytes might be the partial exchange of extracellular glutamine for intracellular alanine.     

SLC36A4 (hPAT4) is a high affinity amino acid transporter when expressed in Xenopus laevis oocytes

The SLC36 family of transporters consists of four genes, two of which, SLC36A1 and SLC36A2, have been demonstrated to code for human proton-coupled amino acid transporters or hPATs. Here we report the characterization of the fourth member of the family, SLC36A4 or hPAT4, which when expressed in Xenopus laevis oocytes also encodes a plasma membrane amino acid transporter, but one that is not proton-coupled and has a very high substrate affinity for the amino acids proline and tryptophan. hPAT4 in Xenopus oocytes mediated sodium-independent, electroneutral uptake of [(3)H]proline, with the highest rate of uptake when the uptake medium pH was 7.4 and an affinity of 3.13 μM. Tryptophan was also an excellently transported substrate with a similarly high affinity (1.72 μM). Other amino acids that inhibited [(3)H]proline were isoleucine (K(i) 0.23 mM), glutamine (0.43 mM), methionine (0.44 mM), and alanine (1.48 mM), and with lower affinity, glycine, threonine, and cysteine (K(i) >5 mM for all). Of the amino acids directly tested for transport, only proline, tryptophan, and alanine showed significant uptake, whereas glycine and cysteine did not. Of the non-proteogenic amino acids and drugs tested, only sarcosine produced inhibition (K(i) 1.09 mM), whereas γ-aminobutyric acid (GABA), β-alanine, L-Dopa, D-serine, and δ-aminolevulinic acid were without effect on [(3)H]proline uptake. This characterization of hPAT4 as a very high affinity/low capacity non-proton-coupled amino acid transporter raises questions about its physiological role, especially as the transport characteristics of hPAT4 are very similar to the Drosophila orthologue PATH, an amino acid "transceptor" that plays a role in nutrient sensing.     

Uterine uptake of amino acids and placental glutamine--glutamate balance in the pregnant ewe

The uterine uptake of amino acids was studied in 10 pregnant sheep with gestational ages of 114-146 days. After recovery from surgery, arterial and uterine venous samples were drawn simultaneously via indwelling catheters and analysed for amino acid and oxygen content. In seven ewes, amino acid concentrations were measured by a chromatographic technique. In four ewes, glutamate and glutamine arterio-venous differences across the uterine and umbilical circulations were measured by an enzymatic method. The uptake of neutral and basic amino acids was 66 mumol/mmol O2 and 17.3 mumol/mmol O2, respectively. Comparison of uterine and umbilical uptake shows that the bulk of the neutral and basic amino acids taken up by the pregnant uterus are transferred to the fetus. there was no significant uptake of acidic amino acids (i.e. glutamate, aspartate and taurine). glutamate was delivered from the fetus to the placenta but excretion of glutamate into the uterine circulation was negligible. Glutamine and asparagine were delivered to the fetus in amount which were two to three times larger than the placental uptake of glutamate and aspartate. Therefore placental conversion of exogenous glutamate and aspartate to glutamine and asparagine cannot account entirely for the fetal uptake of these amino acids.     

Glutamine and glutamic acid uptake by rat renal brushborder membrane vesicles

Summary Glutamine uptake by rat renal brushborder vesicles occurred via two distinct saturable processes withK _m values of 0.145 and 8.5 mM which were stimulated by both ionic and sodium gradients with a pH optimum of 6.8–7.1 Glutamic acid uptake also occurred by a two-component system withK _m values of 0.016 and 3.60 mM. Both components were stimulated specifically by a sodium gradient. The lowK _m system for glutamic acid had a pH optimum of 7.2–7.4. Glutamine entry at 0.06 mM was inhibited by a variety of amino acids at 3 mM, including dibasic amino acids, glycine, valine, and phenylalanine. Glutamic acid entry at 0.06 mM was inhibited 20–30% by 3 mM phenylalanine, valine, -aminoisobutyric acid, and glutamine. No metabolic alteration of glutamic acid was observed on incubation with membrane vesicles, but glutamine was significantly hydrolyzed to glutamic acid upon prolonged incubation. Hydrolysis of glutamine was negligible at 15 sec incubation which was employed for measurement of initial rate of entry. These studies provide support for the existence of an uptake system in the brushborder of the renal proximal tubule cell capable of handling the reabsorption of glutamine normally present in glomerular filtrate.     

Reconstitution into liposomes of the B degrees -like glutamine-neutral amino acid transporter from renal cell plasma membrane

Na+ dependent [3H]glutamine uptake was found in liposomes reconstituted with solubilized rat kidney brush border in the presence of intraliposomal K+. The reconstituted system was optimised with respect to the critical parameters of the cyclic detergent removal procedure, i.e., the detergent used for the solubilization, the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. Time dependent [3H]glutamine accumulation in proteoliposomes occurred only in the presence of external Na+ and internal K+. The transporter showed low if there is any tolerance towards the substitution of Na+ or K+ for other cations. Valinomycin strongly stimulated the transport indicating that it is electrogenic. Intraliposomal glutamine had no effect. From the dependence of the transport rate on the Na+ concentration cooperativity index close to 1 was derived, indicating that 1 Na+ should be involved in the cotransport with glutamine. The electrogenicity of the transport originated from the Na+ transport. Optimal rate of 0.1 mM [3H]glutamine uptake was found in the presence of 50 mM intraliposomal K-gluconate. At higher K-gluconate concentrations the transport rate decreased. The activity of the reconstituted transporter was pH dependent with optimal function in the range pH 6.5-7.0. [3H]glutamine (and [3H]leucine) uptake was inhibited by all the neutral but not by the positively or negatively charged amino acids. The sulfhydryl reagents HgCl2, mersalyl, p-hydroxymercuribenzoate and the substrate analogue 2-aminobicyclo[2,2,1]heptane-2-carboxylate strongly inhibited the transporter, whereas the amino acid analogue alpha-(methylamino)isobutyrate had no effect. The inhibition by mersalyl was protected by the presence of the substrate. On the basis of the Na+ dependence, the electrogenic transport mode and the specificity towards the amino acids, the reconstituted transporter was classified as B degrees-like.     

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.     

Regulation of L-leucine transport in rat kidney by dexamethasone and triiodothyronine

We have investigated the transport mechanisms involved in the stimulation of renal tubular reabsorption of large amino acids by glucocorticoids in vivo through the examination of activity and expression of specific transport systems L and y(+)L for L-leucine in membrane preparations of rat kidneys. Kidneys were removed from adult female Wistar rats treated with dexamethasone or triiodothyronine, and the fractions of brush-border and basolateral membranes were isolated by density gradient centrifugation. Functional analysis of L-leucine uptake using rapid filtration technique revealed induction of a sodium-dependent, arginine-inhibitable system y(+)L transport component in the basolateral membrane in the dexamethasone-treated group. A minor sodium-independent, BCH-inhibitable, system L transport component was unaffected by glucocorticoids. L-leucine uptake remained unaffected in the triiodothyronine-treated group. Expression of both subunits of the system y(+)L transporter was increased in dexamethasone-treated rat kidneys: Western blot analysis showed a significant (46%) increase of 4F2hc protein abundance in the basolateral membrane fraction and competitive RT-PCR revealed an almost 4-times induced expression of y(+)LAT1 mRNA. Our results indicate that system y(+)L in rat kidney is regulated by glucocorticoids. We suggest that enhancement of both 4F2 heavy chain and y(+)LAT1 light chain is necessary for induction of this transport system in the kidney.