Alanine Uptake Research Articles

Underexpression of the Na+-dependent neutral amino acid transporter ASCT2 in the spontaneously hypertensive rat kidney

This study examined the inward transport of l-[(14)C]alanine, an ASCT2 preferential substrate, in monolayers of immortalized renal proximal tubular epithelial (PTE) cells from Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. The expression of ASCT2 in WKY and SHR PTE cells and kidney cortices from WKY and SHR was also evaluated. l-[(14)C]alanine uptake was highly dependent on extracellular Na(+). Replacement of NaCl by LiCl or choline chloride abolished transport activity in SHR and WKY PTE cells. In the presence of the system L inhibitor BCH, Na(+)-dependent l-alanine uptake in WKY and SHR PTE cells was inhibited by alanine, serine, and cysteine, which is consistent with amino acid transport through ASCT2. The saturable component of Na(+)-dependent l-alanine transport under V(max) conditions in SHR PTE cells was one-half of that in WKY PTE cells, with similar K(m) values. Differences in magnitude of Na(+)-dependent l-alanine uptake through ASCT2 between WKY and SHR PTE cells correlated positively with differences in ASCT2 protein expression, this being more abundant in WKY PTE cells. Abundance of ASCT2 transcript and protein in kidney cortices of SHR rats was also lower than that in normotensive WKY rats. In conclusion, immortalized SHR and WKY PTE cells take up l-alanine mainly through a high-affinity Na(+)-dependent amino acid transporter, with functional features of ASCT2 transport. The activity and expression of the ASCT2 transporter were considerably lower in the SHR cells.

Solute-Inhibitor Interactions in the Plasmodial Surface Anion Channel Reveal Complexities in the Transport Process

Human red blood cells infected with the malaria parasite Plasmodium falciparum have markedly increased permeabilities to diverse organic and inorganic solutes. The plasmodial surface anion channel (PSAC), recently identified with electrophysiological methods, contributes to the uptake of many small solutes. In this study, we explored the effects of known PSAC antagonists on transport of different solutes. We were surprised to find that the transport of two solutes, phenyltrimethylammonium and isoleucine, was only partially inhibited by concentrations of three inhibitors that abolish sorbitol or alanine uptake. Residual uptake via endogenous transporters could not account for this finding because uninfected red blood cells (RBCs) do not have adequate permeability for these solutes. In infected RBCs, the residual uptake of these solutes could be abolished by higher concentrations of specific and nonspecific PSAC antagonists. Adding sorbitol or alanine, permeant solutes that do not exhibit residual uptake, could also abolish it. The residual uptake did not exhibit anomalous mole fraction behavior and had a steep activation energy. These observations exclude uptake via unrelated pathways and instead point to differences in how PSAC recognizes and transports various solutes. We propose a possible model that also may help explain the unique selectivity properties of PSAC.

Liver Failure and Liver Disease

Liver Failure and Liver Disease

Brain oxygen utilization is unchanged by hypoglycemia in normal humans: lactate, alanine, and leucine uptake are not sufficient to offset energy deficit

During hypoglycemia, substrates other than glucose have been suggested to serve as alternate neural fuels. We evaluated brain uptake of endogenously produced lactate, alanine, and leucine at euglycemia and during insulin-induced hypoglycemia in 17 normal subjects. Cross-brain arteriovenous differences for plasma glucose, lactate, alanine, leucine, and oxygen content were quantitated. Cerebral blood flow (CBF) was measured by Fick methodology using N(2)O as the dilution indicator gas. Substrate uptake was measured as the product of CBF and the arteriovenous concentration difference. As arterial glucose concentration fell, cerebral oxygen utilization and CBF remained unchanged. Brain glucose uptake (BGU) decreased from 36.3+/-2.6 to 26.6+/-2.1 micromol.100 g of brain(-1).min(-1) (P<0.001), equivalent to a drop in ATP of 291 micromol.100 g(-1).min(-1). Arterial lactate rose (P<0.001), whereas arterial alanine and leucine fell (P<0.009 and P<0.001, respectively). Brain lactate uptake (BLU) increased from a net release of -1.8+/- 0.6 to a net uptake of 2.5+/-1.2 micromol.100 g(-1).min(-1) (P<0.001), equivalent to an increase in ATP of 74 micromol.100 g(-1).min(-1). Brain leucine uptake decreased from 7.1+/-1.2 to 2.5 +/- 0.5 micromol.100 g(-1).min(-1) (P<0.001), and brain alanine uptake trended downward (P<0.08). We conclude that the ATP generated from the physiological increase in BLU during hypoglycemia accounts for no more than 25% of the brain glucose energy deficit.

Na-glucose and Na-neutral amino acid cotransport are uniquely regulated by constitutive nitric oxide in rabbit small intestinal villus cells

Na-nutrient cotransport processes are not only important for the assimilation of essential nutrients but also for the absorption of Na in the mammalian small intestine. The effect of constitutive nitric oxide (cNO) on Na-glucose (SGLT-1) and Na-amino acid cotransport (NAcT) in the mammalian small intestine is unknown. Inhibition of cNO synthase with N(G)-nitro-l-arginine methyl ester (L-NAME) resulted in the inhibition of Na-stimulated (3)H-O-methyl-D-glucose uptake in villus cells. However, Na-stimulated alanine uptake was not affected in these cells. The L-NAME-induced reduction in SGLT-1 in villus cells was not secondary to an alteration in basolateral membrane Na-K-ATPase activity, which provides the favorable Na gradient for this cotransport process. In fact, SGLT-1 was inhibited in villus cell brush-border membrane (BBM) vesicles prepared from animals treated with L-NAME. Kinetic studies demonstrated that the mechanism of inhibition of SGLT-1 was secondary to a decrease in the affinity for glucose without a change in the maximal rate of uptake of glucose. Northern blot studies demonstrated no change in the mRNA levels of SGLT-1. Western blot studies demonstrated no significant change in the immunoreactive protein levels of SGLT-1 in ileal villus cell BBM from L-NAME-treated rabbits. These studies indicate that inhibition of cNO production inhibits SGLT-1 but not NAcT in the rabbit small intestine. Therefore, whereas cNO promotes Na-glucose cotransport, it does not affect NAcT in the mammalian small intestine.

Effect of individual amino acids and glucose on activation and germination of Rhizopus oligosporus sporangiospores in tempe starter

To understand the conditions promoting activation and germination of spores, and to contribute to the control of tempe starters. Using microscopic counts of fluorescent labelled spores, the following results were obtained: (1) L-alanine plays an important role (of the same order as that of peptone) in stimulation of germination of dormant spores. Alanine can satisfy the requirements of carbon as well as nitrogen for spore germination; (2) L-proline, on the other hand, inhibits alanine uptake presumably by blocking/congesting transporters of spore cells, resulting in apparent low viability on agar media; (3) L-leucine and L-isoleucine slightly favour spore germination while L-arginine and L-lysine do not have any stimulating effect; (4) The stimulatory role of glucose was only evident in the presence of phosphate (in minimal medium); when glucose is used in the absence of phosphate, either alone or in combination with single amino acids its role is hardly distinguishable; (5) Phosphate plays a facilitating role in spore germination. Glucose and amino acids play important roles in activation and germination of sporangiospores of Rhizopus oligosporus in tempe starter (stored for 12 months). The ability and rate of germination of dormant/old sporangiospores of R. oligosporus, depend on their ability for uptake of individual amino acids and/or glucose. New light was shed on the counteractive role of proline and the stimulating effect of phosphate. Soybeans subjected to traditional preparation for tempe making are heavily leached; germination of starter spores on such beans is sub-optimal, and bean processing could be optimized.

Impact of glutamine supplementation on glucose homeostasis during and after exercise

The interaction of glutamine availability and glucose homeostasis during and after exercise was investigated, measuring whole body glucose kinetics with [3-3H]glucose and net organ balances of glucose and amino acids (AA) during basal, exercise, and postexercise hyperinsulinemic-euglycemic clamp periods in six multicatheterized dogs. Dogs were studied twice in random treatment order: once with glutamine (12 micromol.kg(-1).min(-1); Gln) and once with saline (Con) infused intravenously during and after exercise. Plasma glucose fell by 7 mg/dl with exercise in Con (P < 0.05), but it did not fall with Gln. Gln further stimulated whole body glucose production and utilization an additional 24% above a normal exercise response (P < 0.05). Net hepatic uptake of glutamine and alanine was greater with Gln than Con during exercise (P < 0.05). Net hepatic glucose output was increased sevenfold during exercise with Gln (P < 0.05) but not with Con. Net hindlimb glucose uptake was increased similarly during exercise in both groups (P < 0.05). During the postexercise hyperinsulinemic-euglycemic period, glucose production decreased to near zero with Con, but it did not decrease below basal levels with Gln. Gln increased glucose utilization by 16% compared with Con after exercise (P < 0.05). Furthermore, net hindlimb glucose uptake in the postexercise period was increased approximately twofold vs. basal with Gln (P < 0.05) but not with Con. Net hepatic uptake of glutamine during the postexercise period was threefold greater for Gln than Con (P < 0.05). In conclusion, glutamine availability modulates glucose homeostasis during and after exercise, which may have implications for postexercise recovery.

Functional regulation of Na+-dependent neutral amino acid transporter ASCT2 by S-nitrosothiols and nitric oxide in Caco-2 cells

We describe the regulation mechanisms of the Na+-dependent neutral amino acid transporter ASCT2 via nitric oxide (NO) in the human intestinal cell line, Caco-2. Exposure of Caco-2 cells to S-nitrosothiol, such as S-nitroso-N-acetyl-dl-penicillamine (SNAP) and S-nitrosoglutathione, and the NO-donor, NOC12, concentration- and time-dependently increased Na+-dependent alanine uptake. Kinetic analyses indicated that SNAP increases the maximal velocity (Vmax) of Na+-dependent alanine uptake in Caco-2 cells without affecting the Michaelis–Menten constant (Kt). The stimulatory effect was partially eliminated by actinomycin D and cycloheximide. Increased Na+-dependent alanine uptake by SNAP was partially abolished by the NO scavengers, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide sodium salt (carboxy-PTIO) and N-(dithiocarboxy)sarcosine disodium salts (DTCS), as well as the NADPH oxidase inhibitor, diphenyleneiodonium. RT-PCR revealed that Caco-2 cells expressed the Na+-dependent neutral amino acid transporter ASCT2, but not the other Na+-dependent neutral amino acid transporters ATB0,+ and B0AT1. These results suggested that functional up-regulation of ASCT2 by SNAP might be partially associated with an increase in the density of transporter protein via de novo synthesis.

Identification and characterization of the main beta-alanine uptake system in Escherichia coli.

In Escherichia coli, beta-alanine is a direct precursor in the biosynthesis of pantothenic acid (vitamin B5). Although a sufficient beta-alanine supply is crucial for biotechnological vitamin B5 production, nothing was known about beta-alanine transport in E. coli until now. The aim of this work was the characterization of beta-alanine transport by E. coli and the identification and overexpression of the corresponding carrier-encoding gene for the rational improvement of pantothenic acid-producing strains. beta-Alanine uptake was found to be an active process catalyzed by the amino acid carrier CycA. The corresponding gene was cloned and overexpressed, resulting in an increase in the uptake rate, compared with the wild type. In all tested strains, this overexpression led to a strong sensitivity to beta-alanine, but not to the other CycA substrates, such as L-alanine, D-alanine, and glycine. This prevented a direct application for the improvement of pantothenic acid-producing strains by an enhanced precursor supply.

Metabolic and mitogenic effects of IGF-I and insulin on muscle cells of rainbow trout.

The relative function of IGF-I and insulin on fish muscle metabolism and growth has been investigated by the isolation and culture at different stages (myoblasts at day 1, myocytes at day 4, and myotubes at day 10) of rainbow trout muscle cells. This in vitro model avoids interactions with endogenous peptides, which could interfere with the muscle response. In these cells, the effects of IGF-I and insulin on cell proliferation, 2-deoxyglucose (2-DG), and l-alanine uptake at different development stages, and the use of inhibitors were studied and quantified. Insulin (10-1,000 nM) and IGF-I (10-100 nM) stimulated 2-DG uptake in trout myocytes at day 4 in a similar manner (maximum of 124% for insulin and of 142% for IGF-I), and this stimulation increased when cells differentiated to myotubes (maximum for IGF-I of 193%). When incubating the cells with PD-98059 and especially cytochalasin B, a reduction in 2-DG uptake was observed, suggesting that glucose transport takes place through specific facilitative transporters. IGF-I (1-100 nM) stimulated the l-alanine uptake in myocytes at day 4 (maximum of 239%), reaching higher values of stimulation than insulin (100-1,000 nM) (maximum of 160%). This stimulation decreased when cells developed to myotubes at day 10 (118% for IGF-I and 114% for insulin). IGF-I (0.125-25 nM) had a significant effect on myoblast proliferation, measured by thymidine incorporation (maximum of 170%), and required the presence of 2-5% fetal serum (FBS) to promote thymidine uptake. On the other hand, insulin was totally ineffective in stimulating thymidine uptake. We conclude that IGF-I is more effective than insulin in stimulating glucose and alanine uptake in rainbow trout myosatellite cells and that the degree of stimulation changes when cells differentiate to myotubes. IGF-I stimulates cell proliferation in this model of muscle in vitro and insulin does not. These results indicate the important role of IGF-I on growth and metabolism of fish muscle.

Ryegrass-based diet and barley supplementation: partition of nitrogenous nutrients among splanchnic tissues and hind limb in finishing lambs.

Splanchnic metabolism of nitrogenous nutrients and their uptake by the hind limb were studied in finishing lambs receiving ryegrass harvested at grazing stage with or without barley supplementation. Six multicatheterized lambs (40.2 +/- 1.5 kg) were fed with frozen ryegrass (RG) at 690 kJ of ME intake (MEI) x d(-1) x BW(-0.75) and 20.8 g of N intake (NI)/d successively without and with barley supplementation (RG + B), according to a crossover design. Barley supplementation represented 21% of DM intake and increased the MEI and the NI by 32 and 24% respectively, (P < 0.01). In the ruminal fluid, barley increased acetate and butyrate concentrations by 21.2 and 49.6%, respectively (P < 0.04), without any effect on the ammonia concentration. Consequently, the net portal appearance (NPA) of ammonia was not modified, but the NPA of total amino acids (TAA; +38%) and nonessential amino acids (NEAA; +45%) was increased (P < 0.05) by barley supplementation. Taken individually, the NPA of the essential amino acids (EAA) was increased for isoleucine (+32%; P < 0.05), threonine (+151%; P < 0.03), and lysine (+26%; P < 0.06), with no effect for the other EAA. In contrast to what was observed at the PDV level, no significant alteration in the net hepatic amino acid flux was observed for TAA, EAA, NEAA, branched-chain amino acids (BCAA), urea, and ammonia after barley supplementation, showing a relatively minor role of the liver in the regulation of the supply of amino acids to the peripheral tissues. However, taken individually, the net hepatic uptake of some NEAA involved in gluconeogenesis and/or ureagenesis was altered with barley supplementation: the alanine uptake was increased by 44% (P < 0.05), aspartate + asparagine (asx) uptake was decreased by 18% (P < 0.01), and glutamate + glutamine (glx) release tended (P < 0.10) to be increased by 208%. With barley supplementation, NI increased by 5 g of N/d, and net splanchnic release increased by 4.63 g of N/d. Consequently, the additional dietary N supply (together with energy supply) was nearly exclusively available to peripheral tissues as AA-N (N as amino acids), but no strong effect of this additional supply of AA to the hind limb could be demonstrated in terms of net AA hind limb fluxes. Consequently, barley supplementation of a ryegrass-based diet increased the net AA release by the splanchnic tissues, with little effect on the AA net uptake by the peripheral tissues.

Dietary protein reduction in sheep and goats: different effects on L-alanine and L-leucine transport across the brush-border membrane of jejunal enterocytes.

It was the aim of this study to examine the potential regulatory effects of a long-term low dietary protein supply on the transport capacity of the jejunal brush-border membrane for amino acids. For this purpose, we used the neutral amino acids L-alanine (representative for nonessential amino acids) and L-leucine (representative for essential amino acids) as model substances. Ten sheep lambs, 8 weeks of age and 19-27 kg body weight, were allotted to two dietary regimes with either adequate or reduced protein supply which was achieved by 17.9% and 9.7% of crude protein in the concentrated feed, respectively. The feeding periods were 4-6 weeks in length. Similarly, eight goat kids of 5-7 weeks of age and 8-14 kg body weight were allotted to either adequate (crude protein 20.1%, feeding period 9-12 weeks) or reduced protein supply (10.1%, feeding period 17-18 weeks). Dietary protein reduction in lambs caused a significant body weight loss of 0.6 +/- 0.7 kg, whereas the body weight in control animals increased by 1.9 +/- 0.7 kg (P<0.05). Plasma urea concentrations decreased significantly by 60% (low protein 2.3 +/- 0.1 versus control 5.7 +/- 0.2 mmol l(-1), P<0.001). In kids, reduction of dietary protein intake led to significant decreases of the daily weight gain by 48% from 181 +/- 8 g to 94 +/- 3 g (P<0.001) and daily dry matter intake by 27% from 568 +/- 13 g to 417 +/- 6 g (P<0.01). Respective urea concentrations in plasma were reduced by 77% from 5.2 +/- 0.4 to 1.2 +/- 0.2 mmol l(-1) (P<0.01). Kinetic analyses of the initial rates of alanine uptake into isolated jejunal brush-border membrane vesicles from sheep and goats as affected by low dietary protein supply yielded that the apparent Km was neither significantly different between the species nor significantly affected by the feeding regime thus ranging between 0.12 and 0.16 mmol.l(-1). Reduction of dietary protein, however, resulted in significantly decreased Vmax values of the transport system by 25-30%, irrespective of the species. Kinetic analyses of the initial rates of leucine uptake into jejunal brush-border membrane vesicles from sheep and goats yielded that leucine uptake was mediated by Na+-dependent as well as Na+-independent processes. Similar to alanine, apparent Km values of leucine uptake were neither different between the species nor affected due to low dietary protein and ranged between 0.08 and 0.15 mmol l(-1). In contrast to the alanine transport mechanism, dietary protein reduction resulted in increased Vmax values of Na+-dependent leucine transport by 53% in sheep and 230% in goats. Similarly, Na+-independent leucine uptake was stimulated by 85% and 200% in sheep and in goats, respectively. This study shows adaptation of amino acid absorption at the brush-border membrane level of jejunal enterocytes of small ruminants due to dietary protein reduction. Whereas the transport capacity for the nonessential amino acid alanine was reduced due to low dietary protein, the transport capacity for the essential amino acid leucine was markedly stimulated. From this, the involvement of rather different feedback mechanisms in adaptation of intestinal amino acid transport mechanisms has to be discussed.

Disruption of F-actin stimulates hypertonic activation of the BGT1 transporter in MDCK cells.

Many membrane transport systems are altered by changes in the state of the actin cytoskeleton. Although an intact microtubule network is required for hypertonic activation of the betaine transporter (BGT1), the possible role of the actin cytoskeleton is unknown. BGT1 function in Madin-Darby canine kidney cell monolayers was assessed as Na(+)-dependent uptake of GABA, following disassembly of F-actin by cytochalasin D (1.0 microM) or latrunculin A (0.6 microM). Both drugs significantly increased (P < 0.001) the activation of BGT1 transport by 24-h hypertonicity (500 mosmol/kgH(2)O). In contrast, the hypertonic upregulation of Na(+)-dependent alanine uptake remained unaltered by cytochalasin D. Disruption of F-actin did not interfere with downregulation of BGT1 transport when cells were transferred from hypertonic to isotonic medium. Immunofluorescence staining revealed colocalization of BGT1 and F-actin at the plasma membrane of hypertonic cells. Surface biotinylation revealed no major change in BGT1 protein abundance after cytochalasin D action, suggesting that stimulation of hypertonic activation of BGT1 transport is due to increased activity of existing BGT1 transporters.

A phosphorylated phloretin derivative. Synthesis and effect on intestinal Na(+)-dependent phosphate absorption.

2'-Phosphophloretin (2'-PP), a phosphorylated derivative of the plant chalcone, was synthesized. The effect of 2'-PP, on Na(+)-dependent phosphate uptake into intestinal brush-border membrane vesicles (BBMV) isolated from rabbit and rat duodenum and jejunum was examined. 2'-PP decreased Na(+)-dependent phosphate uptake into rabbit BBMV with an IC(50) of 55 nM and into rat BBMV with an IC(50) of 58 nM. 2'-PP did not affect Na(+)-dependent glucose, Na(+)-dependent sulfate, or Na(+)-dependent alanine uptake by rabbit intestinal BBMVs. 2'-PP inhibition of rabbit intestinal BBMV Na(+)-dependent phosphate uptake was sensitive to external phosphate concentration, suggesting that 2'-PP inhibition of Na(+)-dependent phosphate uptake was competitive with respect to phosphate. Binding of [(3)H]2'-PP to rabbit intestinal BBMV was examined. Binding of [(3)H]2'-PP was Na(+)-dependent with a K(0.5) for Na(+)(Na(+) concentration for 50% 2'-PP binding) of 30 mM. The apparent K(s) for Na(+)-dependent [(3)H]2'-PP binding to rabbit BBMVs was 58 nM in agreement with the IC(50) for 2'-PP inhibition of Na(+)-dependent phosphate uptake. These results indicate that 2'-PP bound to rabbit or rat intestinal BBMV Na(+)-phosphate cotransporter and inhibited Na(+)-dependent phosphate uptake. In rats treated with 2'-PP by daily gavage, the effect of 2'-PP on serum phosphate, serum glucose, and serum calcium was examined. In a concentration-dependent manner, 2'-PP reduced serum phosphate by 45% 1 wk after starting treatment. 2'-PP did not alter serum calcium or serum glucose. The apparent IC(50) for 2'-PP in vivo was 3 microM.

Characteristics of bacterial populations responsible for uptake of amino acids in activated sludge acclimated to peptone

Amino acids (AAs) are produced from the hydrolysis of proteins, which are the major biodegradable organic compounds in municipal sewage. The characteristics of bacterial populations responsible for the assimilation of thirteen AAs into activated sludge (AS) acclimated to peptone are investigated. The results are as follows. (1) The bacterial populations responsible for the uptake of AAs were partly aggregated in AS flocs. (2) The amounts of the bacterial populations responsible for the uptake of leucine, valine, isoleucine, histidine, threonine, lysine and glycine are limited in AS acclimated to peptone. (3) The bacterial populations responsible for the uptake of phenylalanine, leucine and lysine were different. (4) The amounts of bacterial populations responsible for the uptake of aspartate, arginine, alanine, glutamate and phenylalanine are not limited. (5) The functions of the assimilation of these AAs were induced in many bacterial cells as a result of the BOD determination methods applied to these pure AAs.

Glutamine concentration and tissue exchange with intravenously administered α-ketoglutaric acid and ammonium: a dose-response study in the pig

OBJECTIVE: We investigated the effects of an intravenous load of α-ketoglutaric acid, ammonium (NH 4 +), and metabolic acidosis on plasma concentration and splanchnic and hindleg tissue exchange of glutamine, glutamate, alanine, and arginine in postabsorptive, anesthetized pigs. METHODS: Sixteen anesthetized piglets received a constant infusion of NH 4Cl for 4 h and α-ketoglutaric acid in incremental dosages for 3 h (group 1, n = 8) or a constant infusion of α-ketoglutaric acid for 4 h and NH 4Cl in incremental dosages for 3 h (group 2, n = 8). Plasma amino acids were analyzed and splanchnic blood flow was calculated according to the indocyanine green dye infusion technique. Femoral artery blood flow was measured with ultrasound flowmetry. Statistical evaluation of within-group differences was made with the Wilcoxon signed rank test. RESULTS: Plasma glutamine levels increased dose-dependently in group 2 ( P < 0.05) but not in group 1. Glutamate concentration increased, mainly in group 2 ( P < 0.05), whereas the plasma concentration of alanine decreased in both groups ( P < 0.05). Plasma concentration of arginine increased in both groups ( P < 0.05). Splanchnic uptake and skeletal muscle release of glutamine did not change in either group compared with baseline values. Splanchnic glutamate release decreased ( P < 0.05) in group 1 at 240 min; muscular uptake was unaffected in both groups. Splanchnic uptake and muscular release of alanine were unaffected in both groups. The significance level was set at 0.05. CONCLUSION: Our findings indicate that the splanchnic bed or hindleg skeletal muscle was not the source of the increased plasma concentration of glutamine in this study.

Posttranslational Alanine Trans-Stimulation of Zwitterionic Amino Acid Transport Systems in Human Intestinal Caco-2 Cells

Background. Neutral dietary amino acids, such as alanine, are transported across the gut lumen by both Na+-dependent (System B) and Na+-independent (System L) carriers, but the nature of the acute phase of substrate-induced uptake is unknown. This study examined the effects of acute amino acid substrate exposure on the rapid modulation of apical membrane alanine transport in cultured human intestinal cells.Methods. System B and System L transport activity kinetics, as well as ATB0 mRNA levels, were measured in confluent Caco-2 monolayers treated with various metabolic agents during short-term and extended time periods.Results. Depleting the incubation medium of alanine attenuated both System B and System L uptake activities within 30 mins, with a complete return to baseline values within 3 h. Extracellular alanine added to depleted Caco-2 cells rapidly (within 5 min) increased alanine transport activities. Kinetic analysis showed that acute alanine exposure increased both Km and Vmax of each transport system, indicative of a trans-stimulation effect. Augmenting intracellular alanine levels using the cytosolic alanine aminotransferase inhibitor, aminooxyacetic acid, increased alanine uptake activity. Acute exposure to other substrates of Systems B and L also increased the uptake of alanine, while nonsubstrates did not affect alanine uptake. Cycloheximide or actinomycin did not affect substrate acute activation of System B, and the steady-state level of ATB0 mRNA was not altered by amino acid exposure.Conclusion. Increasing alanine availability to intestinal cells, by either exogenous substrate exposure or inhibition of intracellular catabolism, acutely and reversibly increases apical membrane alanine transport activity via a posttranslation trans-stimulation mechanism.

Characterisation and cloning of a Na +-dependent broad-specificity neutral amino acid transporter from NBL-1 cells: a novel member of the ASC/B 0 transporter family

Na +-dependent neutral amino acid transport into the bovine renal epithelial cell line NBL-1 is catalysed by a broad-specificity transporter originally termed System B 0. This transporter is shown to differ in specificity from the B 0 transporter cloned from JAR cells [J. Biol. Chem. 271 (1996) 18657] in that it interacts much more strongly with phenylalanine. Using probes designed to conserved transmembrane regions of the ASC/B 0 transporter family we have isolated a cDNA encoding the NBL-1 cell System B 0 transporter. When expressed in Xenopus oocytes the clone catalysed Na +-dependent alanine uptake which was inhibited by glutamine, leucine and phenylalanine. However, the clone did not catalyse Na +-dependent phenylalanine transport, again as in NBL-1 cells. The clone encoded a protein of 539 amino acids; the predicted transmembrane domains were almost identical in sequence to those of the other members of the B 0/ASC transporter family. Comparison of the sequences of NBL-1 and JAR cell transporters showed some differences near the N-terminus, C-terminus and in the loop between helices 3 and 4. The NBL-1 B 0 transporter is not the same as the renal brush border membrane transporter since it does not transport phenylalanine. Differences in specificity in this protein family arise from relatively small differences in amino acid sequence.

Fetal hepatic and umbilical uptakes of glucogenic substrates during a glucagon-somatostatin infusion.

To test the hypothesis that fetal hepatic glutamate output diverts the products of hepatic amino acid metabolism from hepatic gluconeogenesis, ovine fetal hepatic and umbilical uptakes of glucose and glucogenic substrates were measured before and during fetal glucagon-somatostatin (GS) infusion and during the combined infusion of GS, alanine, glutamine, and arginine. Before the infusions, hepatic uptake of lactate, alanine, glutamine, arginine, and other substrates was accompanied by hepatic output of pyruvate, aspartate, serine, glutamate, and ornithine. The GS infusion induced hepatic output of 1.00 +/- 0.07 mol glucose carbon/mol O(2) uptake, an equivalent reduction in hepatic output of pyruvate and glutamate carbon, a decrease in umbilical glucose uptake and placental uptake of fetal glutamate, an increase in hepatic alanine and arginine clearances, and a decrease in umbilical alanine, glutamine, and arginine uptakes. The latter result suggests that glucagon inhibits umbilical amino acid uptake. We conclude that fetal hepatic pyruvate and glutamate output is part of an adaptation to placental function that requires the fetal liver to maintain both a high rate of catabolism of glucogenic substrates and a low rate of gluconeogenesis.

Active transport of alanine by the neutral amino-acid exchanger ASCT1

ASCT1 protein is a member of the glutamate transporter superfamily, which shows system ASC selectivity and properties and has been characterized as a Na+-dependent neutral amino-acid exchanger. Here, by using ASCT1-expressing oocytes, the uptake of alanine and glutamate was measured to investigate ASCT1's ability to mediate a concentrative transport of alanine, ASCT1's sodium dependence, and the influence of pH on the mutual inhibition between alanine and glutamate. Alanine uptake was measured after 30 min incubation. Kinetic analysis of the Na+ dependence of alanine uptake showed an apparent K0.5 (affinity constant) value for Na+ of 23.1 +/- 4.3 mM (mean +/- SE). Concentration dependence of alanine uptake was tested at 100 and 1 mM Na+, with apparent K0.5 values of 0.16 +/- 0.04 and 1.8 +/- 0.4 mM, respectively, at pH 7.5, and 0.21 +/- 0.06 and 1.9 +/- 0.3 mM at pH 6. Vmax was not modified between 100 and 1 mM Na+ at either pH. ASCT1 actively transports alanine and accumulates it in the cytosol even when the Na+ concentration in the medium was as low as 1-3 mM. 22Na uptake studies revealed that Na+ transport was stimulated by the presence of alanine in the medium. Our results demonstrate that ASCT1 is able to mediate a concentrative transport of alanine, which is Na+-dependent but not coupled to the Na+ gradient.