L-alanine Uptake Research Articles

Na(+)-independent L-alanine uptake by trout cells. Evidence for the existence of at least two functionally different acs systems.

The Na(+)-independent uptake of L-alanine has been studied in trout red blood cells, isolated hepatocytes and peripheral blood lymphocytes. The present study shows the existence of two functionally different Na(+)-independent systems for short chain neutral amino acids in these cells. They are designated as asc systems because of their resemblance to systems described in other cell types. Besides their independence of sodium and a rough similarity in substrate preference, the most important property shared by the two carriers is a lack of trans-stimulation, allowing further differentiation from system L. One of them is an unusually stereospecific carrier present in red blood cells, the other is less restrictive and present in hepatocytes and peripheral blood lymphocytes. Extracellular acid pH increases the incorporation to red blood cells, while it slightly depresses the uptake in the other cells. From the data presented, it is not possible, at first, to classify these carriers as asc1 or asc2 systems. Moreover, the system present in red cells resembles that found in the nonerythroid cells, BSC-1, while there is no clear parallelism between the system found in hepatocytes/lymphocytes and any of those described previously.

Up-regulation of liver system A for neutral amino acid transport in euglycemic hyperinsulinemic rats

To determine the role of insulin on the in vivo modulation of liver system A activity, we used the euglycemic hyperinsulinemic clamp coupled to the measurement of solute uptakes into plasma membrane vesicles partially purified from livers of hyperinsulinemic rats and their saline-infused controls. The clamp was performed in chronically catheterized rats, either in the fasted state, 24 h after surgery (Group I), or after 3 days of recovery (Group II). System A activity, measured as the MeAIB-inhibitable l-alanine uptake, was selectively induced by hyperinsulinemia, although the effect was much greater in Group II than in Group I rats (137% vs. 24% over the basal values, respectively). This might be explained by the higher basal levels found in those liver plasma membrane vesicles from Group I fasted animals. Hyperinsulinemia also decreased blood amino acids but to a similar extent in both experimental groups. This suggests that amino acid depletion by itself may not cause up-regulation of system A. Other transport activities involved in neutral amino acid transport (Systems ASC, N and L) were not modified by the clamp. The induction of system A cannot be explained by changes in the dissipation rate of the Na + transmembrane gradient, because the differences between insulin- and saline-infused rats remained even when the electrochemical Na + gradient was disrupted in the presence of monensin. Thus, hyperinsulinemia might induce an increase in the number of transporters inserted into the plasma membrane.

Tripeptide transport in rat lung

Transport of L-alanyl- D-phenylalanyl- L-alanine was investigated with an in situ vascular perfusion preparation of rat lung and brush border membrane vesicles prepared from type II pneumocytes. In the perfused lung 1 mM tripeptide was transported intact from the alveolar lumen to the vascular perfusate at a mean rate of 25.1 ± 1.29 (3) nmol/min per g dry weight. D-Phenylalanine also appeared in the vascular perfusate at a rate of 21.9 ± 1.74 (3) nmol/min per g dry weight indicating that 47% of the absorbed tripeptide was split during passage across the epithelial layer. No dipeptide could he detected in the vascular effluent during perfusions with tripeptide. Rapid L-alanyl- D-phenylalanyl- L-alanine uptake occurred with fresh apical membrane vesicles prepared from type II pneumocytes and this was abolished by treatment with 0.1% triton. The related tripeptide, D-alanyl- L-phenylalanyl- D-alanine, was taken up significantly more slowly by the vesicles, D-phenylalanyl- L-alanine and D-phenylalanyl- D-alanine, were also studied with the vascularly perfused preparation; the mixed dipeptide appeared in the vascular perfusate significantly faster than L-alanyl- D-phenylalanyl- L-alanine whereas D-phenylalanyl- D-alanine appeared more slowly and was not hydrolysed.

Redistribution of hepatocyte chloride during L-alanine uptake.

We used ion-sensitive, double-barrel microelectrodes to measure changes in hepatocyte transmembrane potential (Vm), intracellular K+, Cl-, and Na+ activities (aiK, aiCl and aiNa), and water volume during L-alanine uptake. Mouse liver slices were superfused with control and experimental Krebs physiological salt solutions. The experimental solution contained 20 mM L-alanine, and the control solution was adjusted to the same osmolality (305 mOsm) with added sucrose. Hepatocytes also were loaded with 50 mM tetramethylammonium ion (TMA+) for 10 min. Changes in cell water volume during L-alanine uptake were determined by changes in intracellular, steady-state TMA+ activity measured with the K+ electrode. Hepatocyte control Vm was -33 +/- 1 mV. L-alanine uptake first depolarized Vm by 2 +/- 0.2 mV and then hyperpolarized Vm by 5 mV to -38 +/- 1 mV (n = 16) over 6 to 13 min. During this hyperpolarization, aiNa increased by 30% from 19 +/- 2 to 25 +/- 3 mM (P < 0.01), and aiK did not change significantly from 83 +/- 3 mM. However, with added ouabain (1 mM) L-alanine caused only a 2-mV increase in Vm, but now aiK decreased from 61 +/- 3 to 54 +/- 5 mM (P < 0.05). Hyperpolarization of Vm by L-alanine uptake also resulted in a 38% decrease of aiCl from 20 +/- 2 to 12 +/- 3 mM (P < 0.001). Changes in Vm and VCl-Vm voltage traces were parallel during the time of L-alanine hyperpolarization, which is consistent with passive distribution of intracellular Cl- with the Vm in hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine uptake by liver of mid-lactating rats

l-Alanine transport in liver plasma membrane vesicle preparations from led virgin and 15-day-lactating rats was studied. Lactation was found to induce a decrease of the maximal rate (Vmax) of a high-capacity-low-affinity component of the Na +-dependent l-alanine uptake. However, a high-affinity-low-capacity agency was significantly induced in lactating-rat livers. l-Alanine uptake was differentially inhibited by other amino acids in those preparations from lactating rats, and showed different sensitivity to Li + as a cosubstrate instead of Na + and to inhibition by sulfhydryl modifying reagents ( N-ethylmaleimide [NEM] and p-chloromercuribenzosulfonate [PCMBS]). All of these observations taken together suggest that system A is upregulated in lactating-rat livers, thus resulting in a different contribution of both agencies A and ASC to the total Na +-dependent alanine transport into liver plasma membrane vesicles. This was demonstrated using the analogue α-methyl-aminoisobutyric acid (MeAIB), a specific system A substrate. l-Alanine uptake rates, as calculated from plasma membrane enzyme marker recoveries, were also enhanced in the physiologic range of alanine concentrations in blood. Our results prove that the physiologic adaptation to lactation involves modulation of system A activity in the liver.

Up-regulation of system A activity in the regenerating rat liver

System A activity for neutral amino acid transport, measured as the MeAIB-sensitive Na +-dependent l-alanine uptake, is induced 6 h after partial hepatectomy in plasma membrane vesicles from rat livers. Other Na +-dependent transporters, like system ASC (MeAIB-insensitive Na +-dependent l-alanine transport) and the nucleoside carrier show similar inductions. Up-regulation of system A is not explained by changes in the dissipation rate of the Na + transmembrane gradient, as deduced from uptake measurements performed in the presence of monensin. To determine whether induced system A shared any similarity with the activity found in hepatoma cell lines, we analyzed the N-ethylmaleimide (NEM) sensitivity of system A in both regenerating and control rat liver plasma membrane vesicles. NEM treatment was equally effective in inhibiting system A in both experimental groups. Thus, during the prereplicative phase of liver growth, a transport activity similar to basal system A is up-regulated in liver parenchymal cells, by a stable mechanism that does not involve changes in the Na + transmembrane gradient.

L-alanine uptake by rat liver parenchymal and haematopoietic cells during the perinatal period.

Alanine disposal by liver parenchymal and haematopoietic cells from 21-day fetuses, newborns and adult rats was studied. Preparations selectively enriched in either haematopoietic cells or hepatocytes were obtained by direct perfusion of fetal- and neonatal-rat livers. L-Alanine transport into liver parenchymal cells was best fitted to two Na(+)-dependent saturable systems. The high-affinity system showed a much higher activity (Vmax.) in hepatocytes from fetuses and newborns than in those from adult rats (2.4, 4.3 and 0.3 nmol/8 min per 10(6) cells for fetuses, newborns and adults respectively). Vmax. for the low-affinity component was slightly lower during the perinatal period than in the adult (about 30 nmol/8 min per 10(6) cells for hepatocytes from fetuses and newborns, versus 48 nmol/8 min per 10(6) cells for adult rat parenchymal cells). Haematopoietic cells from fetal-rat livers showed significant Na(+)-dependent L-alanine uptake which was completely abolished after birth. These results show that the transport systems involved in L-alanine uptake by liver parenchymal cells are fully developed before birth. This probably contributes to fulfilling the high requirement for neutral amino acids for protein synthesis during development. Haematopoietic cells may play an important role in liver amino acid metabolism during fetal life.

Cloning and expression of a novel Na(+)-dependent neutral amino acid transporter structurally related to mammalian Na+/glutamate cotransporters

A cDNA has been isolated from human hippocampus that appears to encode a novel Na(+)-dependent, Cl(-)-independent, neutral amino acid transporter. The putative protein, designated SATT, is 529 amino acids long and exhibits significant amino acid sequence identity (39-44%) with mammalian L-glutamate transporters. Expression of SATT cDNA in HeLa cells induced stereospecific uptake of L-serine, L-alanine, and L-threonine that was not inhibited by excess (3 mM) 2-(methylamino)-isobutyric acid, a specific substrate for the System A amino acid transporter. SATT expression in HeLa cells did not induce the transport of radiolabeled L-cysteine, L-glutamate, or related dicarboxylates. Northern blot hybridization revealed high levels of SATT mRNA in human skeletal muscle, pancreas, and brain, intermediate levels in heart, and low levels in liver, placenta, lung, and kidney. SATT transport characteristics are similar to the Na(+)-dependent neutral amino acid transport activity designated System ASC, but important differences are noted. These include: 1) SATT's apparent low expression in ASC-containing tissues such as liver or placenta; 2) the lack of mutual inhibition between serine and cysteine; and 3) the lack of trans-stimulation. SATT may represent one of multiple activities that exhibit System ASC-like transport characteristics in diverse tissues and cell lines.

Effect of Amino Acid Deprivation on L-Alanine Uptake Through the Asc System in Freshly Isolated Trout Hepatocytes

ABSTRACT Fasting in mammals and other vertebrates induces an increase in the ability of liver to extract from blood gluconeogenic substrates, such as plasma amino acids, which can also be used as an energy source (Cowey et al. 1977; Newsholme and Leech, 1983). In mammalian hepatocytes, food deprivation induces the appearance of a high-affinity component for short-chain amino acid transport, with the properties of system A, while there are no changes in the activities of a low-affinity system (the ASC system), system L (Fehlmann et al. 1979) or glutamine uptake (Hayes and McGivan, 1982). ‘A’ is the abbreviation for a Na+-dependent carrier which has L-alanine and other short-chain neutral amino acids as preferred substrates. Its tolerance to N-methylated analogues differentiates it from the ASC system. ‘L’ is the abbreviation for a Na+-independent carrier which has L-leucine as preferred substrate. ‘asc’ is the abbreviation for a carrier similar to the ‘ASC’ carrier with respect to preferred substrates, but it is Na+-independent. The ASC system is a widely distributed, short-chain neutral amino acid carrier that transports alanine, serine, cysteine and threonine in a Na+-dependent mode, although the scope of its substrates is now thought to be wider than when it was first described in Erlich ascites tumour cells (Christensen et al. 1967). Characteristically, it does not accept N-methylated amino acid derivatives (allowing easy distinction from the A system).

Effect of starvation on neutral amino acid transport in isolated small-intestinal cells from guinea pigs

The effects of starvation on neutral amino acid transport were examined in isolated enterocytes. Starvation stimulated L-alanine transport by the Na(+)-dependent system A and the Na(+)-independent system L without producing any changes in either the Na(+)-dependent systems ASC or the passive non-mediated uptake. Starvation produces a twofold increase in Vmax of system A without any change in Kt. Starvation produces an increase in Vmax of system L of 1.7 times without any change in Kt. Activation of systems A and L by starvation was reversible with subsequent refeeding. The effects of a series of amino acids on systems A and L were evaluated. A different inhibition pattern was found in starved animals as compared to controls. Starvation increases Na(+)-dependent L-alanine uptake and Na(+)-independent cycloleucine uptake by small-intestinal brush-border membrane vesicles. These results suggest that starvation stimulates amino acid transport across the apical plasma membrane of the enterocytes by inducing specific carrier units.

Hyperosmolarity leads to an increase in derepressed system A activity in the renal epithelial cell line NBL-1

Hyperosmolarity induced an increase in Na(+)-dependent L-alanine uptake in confluent monolayers of the established renal epithelial cell line NBL-1. This induction was attributable to system A and was only seen when the cells had been previously deprived of amino acids in the culture medium to derepress system A activity. It was additive to the adaptive regulation induction, and both were inhibited by cycloheximide. However, the hyperosmolarity effect was inhibited by colcemid (an inhibitor of microtubular function), but adaptive regulation was not. Otherwise, when cell monolayers were incubated in a control medium, basal Na(+)-dependent L-alanine uptake mediated by system B0 decreased. The results of this study show that: (i) system A activity was not induced by cell shrinkage and subsequent swelling due to extracellular hyperosmolarity when cells were incubated in control medium; (ii) previous expression of system A activity induced by amino acid starvation seems to be a prerequisite for further induction due to hyperosmolarity; and (iii) the effects of adaptive regulation and hyperosmotic stress are mediated by different mechanisms.

Transport of amino-acids across renal brush border membrane vesicles in Mycobacterium leprae infected Swiss albino mice--effect of Convit vaccine.

Brush border membrane vesicles prepared from kidneys of Mycobacterium leprae infected (non-vaccinated) and vaccinated-infected Swiss albino mice were used to assess the effect of Convit's combined vaccine (BCG + M. leprae) on amino acid transport activity across the tubular basement membrane. The protective effect of Convit's vaccine was more pronounced with respect to the uptake of L-alanine than L-aspartate. Uptake of L-lysine showed no significant difference in the different groups. Footpad counts followed characteristic growth curves in the non-vaccinated infected group but showed a lag in the development of peak levels in the vaccinated group. Further Convit's vaccine appeared to have a protective effect on renal impairment in the mouse model of leprosy in the initial stages of infection only, as indicated by the transient reversal of amino acid uptake and a diminution in the footpad counts induced by M. leprae infection. No significant (P > 0.05) protective effect of the vaccine was found in the advanced disease state.

Heterogeneity of L-alanine transport systems in brush-border membrane vesicles from rat placenta during late gestation.

The placental uptake of L-alanine was studied by using purified brush-border membrane vesicles from rat trophoblasts. Saturation curves were carried out at 37 degrees C in buffers containing 100 mM (zero-trans)-NaSCN, -NaCl, -KSCN, -KCl, or -N-methyl-D-glucamine gluconate. The uncorrected uptake results were fitted by non-linear regression analysis to an equation involving one diffusional component either one or two saturable Michaelian transport terms. In the presence of NaCl, two distinct L-alanine transport systems were distinguished, named respectively System 1 (S-1; Vm1 about 760 pmol/s per mg of protein; KT1 = 0.5 mM) and System 2 (S-2; Vm2 about 1700 pmol/s per mg; KT2 = 9 mM). In contrast, in the presence of K+ (KCl = KSCN) or in the absence of any alkali-metal ions (N-methyl-D-glucamine gluconate), only one saturable system was apparent, which we identify as S-2. When Na+ is present, S-1, but not S-2, appears to be rheogenic, since its maximal transport capacity significantly increases in the presence of an inside-negative membrane potential, created either by replacing Cl- with the permeant anion thiocyanate (NaSCN > NaCl) or by applying an appropriate K+ gradient and valinomycin. alpha-(Methylamino)isobutyrate (methyl-AIB) appears to be a substrate of S-1, but not of S-2. For reasons that remain to be explained, however, methyl-AIB inhibits S-2. We conclude that S-1 represents a truly Na(+)-dependent mechanism, where Na+ behaves as an obligatory activator, whereas S-2 cannot discriminate between Na+ and K+, although its activity is higher in the presence of alkali-metal ions than in their absence (Na+ = K+ > N-methyl-D-glucammonium ion). S-2 appears to be fully developed 2 days before birth, whereas S-1 undergoes a capacity-type activation between days 19.5 and 21.5 of gestation, i.e. its apparent Vmax. nearly doubles, whereas its KT remains constant.

Amino Acid Uptake and Metabolism by Embryos of the Blenny Zoarces Viviparus

ABSTRACT The ability of blenny Zoarces viviparus (L.) embryos in early and late development to assimilate and metabolize ambient L-alanine was investigated in vitro and in vivo by means of autoradiographic and radiochemical methods. Autoradiograms showed that after 24 h of exposure to L-[14C]alanine, label was distributed in the tissues of the embryos. Uptake rates for l4C-labelled L-alanine in vitro were estimated by measuring the disappearance of radioactivity from the medium. Net uptake rates were measured by high performance liquid chromatography of samples taken simultaneously from the medium. Uptake rates, based on uptake of the tracer (0.60 μmol g−1 h−1), were similar to net uptake rates (0.54 μmol g−1 h−1) in embryos in late development in the in vitro incubations in 60 μmol l−1 alanine. In vivo, the injected tracer was completely cleared from the ovarian fluid over 24h. After intraovarian and in vitro incubation of the embryos with labelled alanine, there was evidence for both catabolic and anabolic metabolism of the amino acid by production of l4CO2 and by incorporation of radioactivity into molecules insoluble in trichloroacetic acid, respectively. The data provide evidence for uptake and metabolism of amino acids by embryos in vitro and in vivo. An increase in the capacity for uptake and metabolism of L-alanine may occur during development of the embryos in the ovary.

Uptake and Metabolism of L-Alanine by Freshly Isolated Trout (Salmo Trutta) Hepatocytes: The Effect of Fasting

ABSTRACT Uptake and metabolism of L-alanine by freshly isolated trout hepatocytes has been analyzed. This amino acid is incorporated ‘uphill’ by different carriers, either Na+-dependent or Na+-independent. Na+-dependent uptake shows the characteristics of an ASC system on the basis of cation dependence and substrate preferences. The Na+-independent uptake is split between an L-cysteine-sensitive system and a non-saturable component. No uptake through system A has been found, suggesting that this carrier is lacking in trout hepatocytes. On the basis of inhibition by several preferred amino acids, L-alanine is not taken up through either the L or N systems. Fasting induced changes in both the uptake of L-alanine and its metabolism to CO2 and glucose. There was an increase in uptake that showed an inverse relationship with L-alanine plasma levels. Glucose production from L-alanine rose during food deprivation, while CO2 production showed an initial increase, similar to that of glucose. At the end of the fasting time considered, however, there was a drop in CO2 production, indicating a different kind of regulation for alanine oxidation and gluconeogenesis.

Secondary transport of amino acids in Nitrosomonas europaea

Nitrosomonas europaea is capable of incorporating exogenously supplied amino acids. Studies in whole cells revealed that at least eight amino acids are actively accumulated, probably by the action of three different transport systems, each with high affinity (μ molar range) for several amino acids. Evidence for the action of secondary mechanisms of transport was obtained from efflux, counterflow and exchange experiments. More detailed information was obtained from studies in liposomes in which solubilized integral membrane proteins of N. europaea were incorporated. Uptake of l-alanine in these liposomes could be driven by artificially imposed pH gradients and electrical potentials, but not by chemical sodium-ion gradients. These observations indicate that l-alanine is transported by a H+/alanine symport system. The ecological significance of secondary amino acid transport systems in autotrophic ammonium-oxidizing bacteria is discussed.

Membrane vesicles from brown adipose tissue: a tool for the study of amino acid transport. The case of L-alanine.

A density gradient method is used to isolate membrane vesicles from brown adipose tissue. These respond to changes in osmolarity and show the classical overshoot pattern when L-alanine uptake is assayed. Transport is shown to be effected by two components: a linear (Kd = 0.498 min-1) and Na(+)-dependent saturable component (Km = 2.3 mM) and a Vmax = 19.9 pmol/micrograms protein.min). This pattern is similar to that shown by cells isolated from brown adipose tissue.

Expression of Na(+)-independent amino acid transport in Xenopus laevis oocytes by injection of rabbit kidney cortex mRNA.

Poly(A)+ mRNA was isolated from rabbit kidney cortex and injected into Xenopus laevis oocytes. Injection of mRNA resulted in a time- and dose-dependent increase in Na(+)-independent uptake of L-[3H]alanine and L-[3H]arginine. L-Alanine uptake was stimulated about 3-fold and L-arginine uptake was stimulated about 8-fold after injection of mRNA (25-50 ng, after 3-6 days) as compared with water-injected oocytes. T.I.C. of oocyte extracts suggested that the increased uptake actually represented an increase in the oocyte content of labelled L-alanine and L-arginine. The expressed L-alanine uptake, obtained by subtracting the uptake in water-injected oocytes from that in mRNA-injected oocytes, showed saturability and was inhibited completely by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) and L-arginine. The expressed L-arginine uptake in mRNA-injected oocytes also showed saturability, being completely inhibited by L-dibasic amino acids) and partially inhibited by BCH. Expression of both L-alanine and L-arginine uptake showed clear cis-inhibition by cationic (e.g. L-arginine) and neutral (e.g. L-leucine) amino acids. In all, this points to the expression of a Na(+)-independent transport system with broad specificity (i.e. b degree, (+)-like). In addition, part of the expressed uptake of L-arginine could be due to a system y(+)-like transporter. After size fractionation through a sucrose density gradient, the mRNA species encoding these increased transport activities (Na(+)-independent transport of L-alanine and of L-arginine) were found in fractions of an average mRNA chain-length of 1.8-2.4 kb. On the basis of these results, we conclude that Na(+)-independent transport system(s) for L-alanine and L-arginine from rabbit renal cortical tissues, most likely proximal tubules, are expressed in Xenopus laevis oocytes. These observations may represent the first steps towards expression and cloning of these transport pathways.

Amino acid uptake by liver of genetically obese Zucker rats

Alanine and glutamine uptake by the liver of 50-52-day-old genetically obese Zucker rats and their lean littermates has been studied. The net uptake in vivo of L-alanine is 2-fold higher in the obese animals. No significant change in L-glutamine net balance was found. We also studied the Na(+)-dependent uptake of L-alanine and L-glutamine into plasma-membrane vesicles isolated from either obese- or lean-rat livers. Vmax. values of both L-alanine and L-glutamine transport were 2-fold higher in those preparations from obese rats. No change in Km was observed. As suggested by inhibition studies, this seemed to be mediated by an enhancement of the activities of systems A, ASC and N. We conclude that the liver of the obese Zucker rat is extremely efficient in taking up neutral amino acids from the afferent blood, which results in an enhanced net uptake of L-alanine in vivo. The changes in transport activities at the plasma-membrane level might contribute to increase amino acid disposal by liver, probably for lipogenic purposes, as recently reported by Terrettaz & Jeanrenaud [Biochem. J. (1990) 270, 803-807].

Neutral amino acid transport by isolated small intestinal cells from guinea pigs

Neutral amino acid transport was examined by using isolated enterocytes. Cells transport L-alanine by at least three different mechanisms: two Na(+)-dependent systems (A and ASC) and one Na(+)-independent mechanism (system L), in addition to passive entry. System A was characterized acterized by measuring the Na(+)-dependent alpha-(methylamino)isobutyric acid (MeAIB) uptake. Na(+)-dependent MeAIB uptake was concentrative and saturable. Vmax was obtained at 80mM Na+ in the incubation medium and Kt app for Na+ was 21.5 mM. Kt app for MeAIB was 6.75 +/- 0.37 mM and the Vmax was 14.2 +/- 0.3 nmol.mg-1.min-1. System ASC was studied by evaluating the Na(+)-dependent L-alanine uptake, insensitive to MeAIB and inhibitable by L-serine and L-cysteine. Uptake by this mechanism was also concentrative and saturable. Maximal uptake was obtained with 80 mM Na+ in the incubation medium and Kt app for Na+ was 29.7 mM. Kt app for L-alanine was 7.02 +/- 0.61 mM and Vmax was 5.44 +/- 0.19 nmol.mg-1.min-1. The Na(+)-independent system L was studied by measuring cycloleucine uptake in Na(+)-free medium. It had a saturable and a nonsaturable component. Only the saturable component was concentrative; it was inhibited by 2-amino-2-norbornanecarboxylic acid and was capable of mediating exchange diffusion. Kt app for cycloleucine was 4.05 +/- 0.72 mM and the Vmax was 31.9 +/- 1.3 nmol.mg-1.min-1. These results confirm the existence of Na(+)-dependent systems A and ASC and Na(+)-independent system L in isolated enterocytes.