Dependent Alanine Uptake Research Articles

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.

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.

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.

Differentiation- and Protein Kinase C-dependent Regulation of Alanine Transport via System B

The regulation of sodium-dependent L-alanine transport is described for the first time in intestinal cells. Substrate analogue inhibition patterns and Dixon analyses indicated that uptake occurred via transport system B, an epithelial cell variant of systems B0,+ and ASC. System B served > 95% of the Na(+)-dependent alanine uptake in both undifferentiated (2 days postpassaging) and differentiated (> 9 days postpassaging) states of the human Caco-2 cultured intestinal cell line. (Methylamino)isobutyric acid-inhibitable system A transport accounted for < 5% of total alanine uptake. System B activity was greater in undifferentiated cells compared with the differentiated state, and activity at any differentiation state was stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA). The maximal stimulation, determined by TPA dose-response/exposure time data, was attributable to a change in cell transport capacity (Vmax), with Km unaffected. The Vmax of system B was greater in 2-day-old cells (2.79 +/- 0.21 nmol min-1 mg of protein-1; Km = 164 +/- 26 microM alanine), decreasing to Vmax = 0.51 +/- 0.03 nmol min-1 mg of protein-1 (Km = 159 +/- 14 microM) in 9-day-old cells. Regardless of differentiation status, the sodium-activation Hill coefficient was 1.06 +/- 0.10, and the alanine passive diffusion permeability coefficient was 0.53 +/- 0.08 microliter min-1 mg of protein-1. Phorbol ester up-regulated the Vmax of system B in 2-day-old cells to Vmax = 6.32 +/- 0.37 nmol min-1 mg of protein-1 (Km = 169 +/- 18 microM), and in 9-day-old cells to Vmax = 1.42 +/- 0.05 nmole min-1 mg of protein-1 (Km = 180 +/- 10 microM). Phorbol ester stimulation of transport occurred after at least 6 h of continual exposure, and was blocked by the protein kinase C inhibitors chelerythrine or photoactivated calphostin C. Protein synthesis inhibitors cycloheximide and actinomycin D each blocked the phorbol ester up-regulation of system B activity. It is concluded that Caco-2 cells regulate carrier-mediated sodium-dependent transport of L-alanine by changing the membrane capacity to transport alanine via system B and that this regulation involves de novo protein synthesis under the control of protein kinase C.

Adaptation to phosphate deprivation in osteoblast‐like cells

The rat osteosarcoma cell line UMR-106-01 has an osteoblast-like phenotype. When grown in monolayer culture these cells transport inorganic phosphate and L-alanine via Na(+)-dependent transport systems. Exposure of these cells to a low phosphate medium for 4 h produced a 60-70 per cent increase in Na(+)-dependent phosphate uptake compared to control cells maintained in medium with a normal phosphate concentration. In contrast, Na(+)-dependent alanine uptake and Na(+)-independent phosphate uptake were not changed during phosphate deprivation. The increased phosphate uptake was due, in part, to an increased Vmax and was blocked completely by pretreatment with cycloheximide (70 microM). In these cells recovery of intracellular pH after acidification with NH4Cl is due primarily to the Na+/H+ exchange system. The rate of this recovery process, monitored with a pH sensitive indicator (BCECF), was decreased by more than 50 per cent in phosphate-deprived cells compared to controls indicating that Na+/H+ exchange was inhibited during phosphate deprivation.

Impaired alanine uptake by basolateral liver plasma membrane vesicles from rats consuming ethanol

Chronic ethanol consumption reduces alanine transport by rat basolateral liver plasma membrane (blLPM) vesicles; however, the mechanism for this effect remains uncertain. It may be related to the ethanol-induced changes in blLPM fluidity and lipid composition; alternatively ethanol might reduce the number of transporters in the blLPM. To investigate the effect of blLPM fluidity and lipid composition on Na(+)-dependent alanine uptake these parameters were altered in vitro. Increasing the blLPM fluidity had no effect on Na(+)-dependent alanine uptake by blLPM vesicles or the activity of amino acid transport systems, A and ASC. Because ethanol is known to reduce the blLPM cholesterol content, the influence of altering blLPM cholesterol on alanine transport by these membranes was investigated next. Neither an increase nor a decrease of the cholesterol content of the blLPM altered Na(+)-dependent alanine uptake or the activity of system A or ASC. Finally, the influence of chronic ethanol consumption on the specific binding of [3H]alanine to blLPM was studied. The dissociation constant for alanine binding to blLPM from ethanol-fed rats and their pair-fed controls was similar (1.9 +/- 0.2 vs. 2.0 +/- 0.3 mM); however, the maximal binding capacity for alanine was significantly (P less than 0.05) lower in the blLPM from ethanol-fed rats (316 +/- 53 pmol/mg protein) compared with their pair-fed controls (527 +/- 79 pmol/mg protein). These studies do not support the hypothesis that ethanol-induced changes in blLPM fluidity are responsible for the impaired alanine transport; they do suggest that ethanol may reduce the

Mechanism of mercurial inhibition of sodium-coupled alanine uptake in liver plasma membrane vesicles from Raja erinacea

In mammalian hepatocytes the l-alanine carrier contains a sulfhydryl group that is essential for its activity and is inhibited by mercurials. In hepatocytes of the evolutionarily primitive little skate ( Raja erinacea), HgCl 2 inhibits Na +-dependent alanine uptake and Na + K + -ATPase and increases K + permeability. To distinguish between direct effects of HgCl 2 on the Na +-alanine cotransporter and indirect effects on membrane permeability, [ 3H]alanine transport was studied in plasma membrane vesicles. [ 3H]Alanine uptake was stimulated by an “out-to-in” Na + but not K + gradient and was saturable confirming the presence of Na +-alanine cotransport in liver plasma membranes from this species. Preincubation of the vesicles with HgCl 2 for 5 min reduced initial rates of Na +-dependent but not Na +-independent alanine uptake in a dose-dependent manner (10–200 μ m). In the presence of equal concentrations of NaCl or KCl inside and outside of the vesicles, 75 μ m HgCl 2 directly inhibited sodium-dependent alanine-[ 3H]alanine exchange, demonstrating that HgCl 2 directly affected the alanine cotransporter. Inhibition of Na +-dependent alanine uptake by 30 μ m HgCl 2 was reversed by dithiothreitol (1 m m). HgCl 2 (10–30 μ m) also increased initial rates of 22Na uptake (at 5 sec), whereas 22Na uptake rates were decreased at HgCl 2 concentrations> 50μ m. Higher concentrations of HgCl 2 (100–200 μ m) produced nonspecific effects on vesicle integrity. These studies indicate that HgCl 2 inhibits Na +-dependent alanine uptake in skate hepatocytes by three different concentration-dependent mechanisms: direct interaction with the transporters, dissipation of the driving force (Na + gradient), and loss of membrane integrity. Inactivation of the Na +-coupled alanine carrier by mercury in hepatocytes of this evolutionarily primitive vertebrate, as in mammals, suggests that the sulfhydryl groups on this transport protein are highly conserved.

Effects of ethanol on amino acid transport in basolateral liver plasma membrane vesicles.

Ethanol has been reported to inhibit hepatocellular processes such as gluconeogenesis and protein synthesis that depend, in part, on amino acid uptake. Since previous studies in cultured hepatocytes indicate that ethanol may have a direct and selective inhibitory effect on amino acid transport, the effects of ethanol on amino acid uptake into basolateral (sinusoidal) rat liver plasma membrane (blLPM) vesicles were examined. Uptake of [3H]alanine, [3H]leucine, and [35S]cysteine was measured by a rapid Millipore filtration technique in the presence of inwardly directed Na+ and K+ gradients and under tetramethylammonium (TMA+)- and Na+-equilibrated conditions. Ethanol preincubation produced a concentration-dependent inhibition of Na+-dependent alanine and cysteine uptake; no effect was observed on either Na+-independent alanine and cysteine uptake or Na+-independent leucine transport. Ethanol had no effect on L-alanine transport under Na+-equilibrated conditions; however, initial rates of 22Na flux were enhanced in the presence of ethanol. On the basis of differences in 2-(methylamino)isobutyrate and L-cysteine sensitivity, ethanol inhibition of Na+-dependent alanine transport in blLPM vesicles largely but not exclusively corresponded to the hormone-responsive system A for amino acid transport described in isolated hepatocytes. Kinetic analysis showed that ethanol treatment resulted in an alteration in the apparent maximum velocity of reaction (Vmax) of Na+-dependent alanine transport without affecting the apparent Km for alanine. The inhibitory effects of ethanol on the time course of Na+-dependent alanine uptake were reversible.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular potassium as a possible inducer of amino acid transport across hamster jejunal enterocytes.

Brush border membrane potentials (Vm), intracellular K+ activity (aiK) and alanine uptake were measured in different parts of villi in mid-jejunal tissue taken from hamsters fed different amounts of food at high and low environmental temperatures. Basal villus enterocytes (Y cells) were found to have lower values for Vm and aiK than upper villus enterocytes (O cells). Alanine uptake was confined to O cells. The position on the villus where values for Vm and aiK changed, and where alanine uptake could first be seen to take place, depended on the energy intake and environmental temperature at which hamsters were kept. Na+-dependent alanine uptake and Vm were both higher in O cells of hamsters fed 10 kcal day-1 at 30 degrees C (10 k/30 degrees C) compared with animals fed 30 kcal day-1 at an environmental temperature of 12 degrees C (30 k/12 degrees C hamsters). The rates at which enterocytes migrated along the crypt-villus axis, measured separately in thymidine-labelling experiments, were 6.9 and 16.1 micron h-1 for 10 k/30 degrees C and 30 k/12 degrees C hamsters respectively. Both Vm and aiK were estimated, from these measurements, to have increased significantly by the time enterocytes became 30 h old. Alanine uptake began 15 h later. Neither of these parameters were influenced by previous adaptation conditions. The close temporal and variable positional relationship found between changes in aiK and onset of transport suggests that early changes in electrolyte composition might initiate a second phase of development enabling the enterocyte to absorb nutrients. The possibility that other ions besides K+ might also be involved in this aspect of regulation is also discussed.

Inhibition of Na-alanine cotransport in oocytes of Xenopus laevis during meiotic maturation is voltage-regulated.

During progesterone-induced maturation of oocytes of Xenopus laevis the fast decrease of the rate of Na+-alanine cotransport is accompanied by a depolarization of the membrane potential and an increase in the intracellular Na+ activity. It is demonstrated that the decrease of the Na+-dependent alanine uptake is the result of the depolarization while the simultaneous increase in intracellular Na+ activity has no significant effect.

Growth regulation and amino acid transport in epithelial cells: Influence of culture conditions and transformation on A, ASC, and l transport activities

AbstractAmino acid transport in Madin‐Darby canine kidney (MDCK) cells, grown in a defined medium, was investigated as a function of cell density, exposure to specific growth factors, and transformation. MDCK cells were found to transport neutral amino acids by systems similar to the A, ASC, L, and N systems which have been characterized using other cell lines. Experimental conditions were developed for MDCK cells which allowed independent measurement of A, ASC, and L transport activities. The activity of the L system was measured as Na+‐independent leucine or methionine uptake at pH 7.4. The activity of the A system was measured as Na+‐dependent α(methylamino)isobutyric acid (mAIB) uptake at pH 7.4, the activity of the ASC system was measured as Na+‐dependent alanine uptake in the presence of 0.1 mM mAIB at pH 6.0, and the activity of system N was observed by measuring Na+‐dependent glutamine uptake at pH 7.4 in the presence of high concentrations of A and ASC system substrates. The L transport system responded minimally to changes in growth state, but Na+‐dependent amino add transport responded to regulation by growth factors, cell density, and transformation. The activities of the A and ASC systems both decreased at high cell density, but these activities responded dissimilarly under other conditions. The activity of the A system was stimulated by insulin, was inhibited by PGE1, and was elevated 3–7 fold in the transformed cell line, MDCK‐T1. The activity of the ASC system was slightly stimulated by insulin and by PGE1, but was unchanged after chemical transformation. Changes in cellular growth were monitored and were found to correlate best with the activity of the A system. These results suggested that MDCK cell growth may be more closely related to the activity of the A than of the ASC system.