Modulation of the activity of amino acid transport system L by phorbol esters and calmodulin antagonists in a human placental choriocarcinoma cell line.

We investigated the regulation of the activity of amino acid transport system L in the JAR human placental choriocarcinoma cell line by agents which are known to modulate the activities of three different classes of protein kinases, A-kinase, C-kinase and CaM-kinase. The system L activity was measured by determining the uptake of leucine in these cells, grown as confluent monolayers. Leucine uptake in these cells was predominantly Na(+)-independent, was stimulated by lowering the extracellular pH and was inhibited by hydrophobic neutral amino acids. These characteristics demonstrate that uptake of leucine in this cell line occurs primarily via system L. Treatment of the cells with cholera toxin and forskolin, agents which are known to elevate intracellular cAMP levels, did not have any effect on the activity of system L. 4 beta-Phorbol 12-myristate 13-acetate, an activator of C-kinase, but not the inactive analogue 4 alpha-phorbol 12,13-didecanoate, caused a significant stimulation of system L. The involvement of C-kinase in the phorbol ester-induced stimulation was supported by the finding that staurosporine, an inhibitor of C-kinase, effectively blocked the stimulation. Calmodulin antagonists, calmidazolium, W-7 and CGS 9343 B stimulated system L activity markedly. The potency of these antagonists was in the following order: calmidazolium greater than CGS 9343 B greater than W-7. This stimulatory effect was specific for system L because systems A and ASC were not stimulated by these agents. The stimulation caused by these agents was primarily due to an increase in the maximal velocity, the apparent Km of the system being only minimally affected. It is concluded that the activity of amino acid transport system L in the JAR human placental choriocarcinoma cell line is stimulated by C-kinase, inhibited by CaM-kinase and unaffected by A-kinase.     

Characterization of system L and system y+ amino acid transport activity in cultured vascular smooth muscle cells

The uptake of L-leucine and L-lysine into vascular smooth muscle cells cultured from the aortas of rats has been investigated. Both amino acids are taken up by saturable systems that are independent of the presence of a ^·Na⁺ gradient and can be stimulated in trans by neutral bulky amino acids for leucine and cationic amino acids for lysine. Leucine uptake is inhibited competitively in cis by several neutral amino acids, whereas lysine uptake is inhibited strongly by other cationic amino acids but also significantly by neutral amino acids such as leucine. The leucine inhibition is noncompetitive. Cells preloaded with leucine and lysine could also export these amino acids and the rate of efflux was stimulated by the presence of appropriate amino acids in trans. These data are all consistent with leucine being transported largely if not entirely by System L and lysine by the System y⁺ transporter. © 1993 Wiley-Liss, Inc.     

Membrane potential and amino acid transport in a mutant Chinese hamster ovary cell line

The bioenergetics of amino acid transport system A was studied in two Chinese hamster ovary (CHO) cell lines, the parent line CHO-PEOT/1 and CHY-1, a mutant of the former exhibiting a low activity of the same transport system. The steady-state transmembrane distribution ratio of the cationic amino acid L-arginine (RARG) was employed as an indicator of membrane potential (delta psi). Evidence for the reliability of RARG to measure delta psi can be summarized as follows: (1) L-arginine transmembrane distribution increased under conditions of cell hyperpolarization and decreased under conditions of cell depolarization; (2) L-arginine distribution conformed closely to that expected for a probe of delta psi in conditions in which delta psi depends largely on the transmembrane potassium gradient; and (3) the value of delta psi obtained through a valinomycin null point experiment (-72.7 mV) was very similar to the value calculated from L-arginine distribution using the Nernst equation (-73.4 mV). The transmembrane gradient of sodium electrochemical potential (delta mu Na), the driving force for the operation of system A, was slightly higher in the mutant cell line CHY-1. In the same line, the intracellular level of the specific system A substrate MeAIB at steady state was also higher. Studies of the rheogenicity of system A in the two lines indicated that the depolarization associated with the entry of substrates of system A was proportional to the amount of amino acid taken up by the cells. Kinetic analysis showed that the low activity of system A in the mutant cell line was referrable to a decrease in transport Vmax. It is concluded that neither a decrease in energy available for the operation of system A nor a decreased efficiency of coupling of the system to delta psi is responsible for the defect observed in the mutant line.     

Characterization of amino acid transport during erythroid cell differentiation

We have studied the changes in amino acid transport in fetal erythroid cells isolated from rat fetal liver at different gestation days. Our results show that System A transport as measured by the Na+-dependent uptake of 2-(methylamino)isobutyric acid (MeAIB) was conspicuous at day 13 but virtually disappeared between days 16 and 18. In contrast, the activity of System ASC measured by the Na+-dependent uptake of MeAIB-insensitive threonine uptake increased after day 14 and was optimal between days 16 and 18. This transport system regressed in activity with further maturation, but remained conspicuously saturable in the matured red blood cell. Interestingly, the newly discovered Na+-independent System asc (Vadgama, J. V., and Christensen, H.N. (1985) J. Biol. Chem. 260, 2912-2921), selective for the uptake of test substrates threonine, serine, and alanine, was present in these erythroid cells. Its activity increased during gestation days 16-18. System L transport was present simultaneously with the Na+-independent System asc. As we had previously demonstrated for the pigeon red blood cell, these two transport systems are kinetically independent as confirmed with inhibition studies and the special selectivity of System L to trans stimulation. Tryptophan uptake could be attributed predominantly to System L, as also observed for the nucleated pigeon red blood cells and certain other cells. Arginine showed its familiar Na+-independent mode of uptake as a cation throughout the interval of study. An exceptional Na+-dependent component of arginine uptake emerged after day 14, peaked at day 18, and then disappeared on further maturation of the erythroid cell.     

Regulation of amino acid transport in L6 muscle cells: I. Stimulation of transport system a by amino acid deprivation

The regulation of amino acid transport in L6 muscle cells by amino acid deprivation was investigated. Proline uptake was Na⁺-dependent, saturable and concentrative, and was predominantly through system A. Proline uptake was inhibited by alanine, α-amino isobutyric acid (AIB), and by α-methylamino isobutyric acid, but not by lysine or valine. At 25°C, Km of proline uptake was 0.5 mM. Amino acid-deprivation resulted in a progressive increase in the rate of proline uptake, reaching up to 6-fold stimulation after 6 hours. The basal and stimulated transport were equally Na⁺-dependent, and both were inhibited by competition with the same amino acids. Kinetic analysis showed that Km decreased by a factor of 2.4 and Vmax increased 1.9-fold in deprived cells. Amino acid-deprivation did not stimulate amino acid uptake through systems other than system A. This suggests that the higher Km in proline-supplemented cells is not due to release of intracellular amino acids into unstirred layers surrounding the cells. The presence of amino acids which are substrates of system A (including AIB) during proline-deprivation, prevented stimulation of proline uptake, whereas those transported by systems Ly⁺ or L exclusively were ineffective. The stimulation of the transport-rate in deprived cells could be reversed by subsequent exposure to proline or other substrates of system A. L6 cells, deprived of proline for 6 hours, retained the stimulation of transport after detachment from the monolayers with trypsin. Uptake rates were comparable in suspended and attached cells in monolayer culture. Thus, amino acid-depreivation of L6 cells results in an adaptive increase in proline uptake, which is not due to unstirred layers but appears to be mediated by other mechanisms of selective transport regulation.     

System A amino acid transport in a rat submandibular ductal cell line

1. Neutral amino acid transport was studied in an established cell line derived from rat submandibular glands, RSMTx. 2. The greatest portion of alpha-amino isobutyrate (AIB) transport is mediated by system A. This component is Na+ dependent, pH sensitive, markedly inhibited by methyl AIB and enhanced 2-5-fold by amino acid depletion. 3. Evidence for the presence of other neutral amino acid transport systems, presumably ASC and L, was also found in these cells.     

Chemical modification of the neutral amino acid transport system L of Chinese hamster ovary cells with p-chloromercuribenzene sulfonate.

Branched-chain and aromatic neutral amino acids enter mammalian cells predominantly through a Na(+)-independent transport agency called System L. The sulfhydryl specific reagent p-chloromercuribenzene sulfonate (pCMBS) has been shown to be a potent inactivator of System L transport activity in Chinese hamster ovary cells, however, inactivation by pCMBS can be prevented by the presence of System L-specific substrate amino acids during the inactivation reaction. In addition, the presence of amino acids that are not substrates for System L have no effect on pCMBS inactivation of System L. Inactivation of System L activity by pCMBS was sensitive to pH and reversible by incubation with dithiothreitol. These findings suggest that there is a sulfhydryl group in, or very near, the amino acid-binding site of the System L transporter of CHO cells. Substrate protection, however, could be explained by conformational changes in the transporter associated with substrate binding. The presence of a substrate protectable sulfhydryl group on the System L transporter would aid in the attempt to identify this transporter using the technique of differential labeling.     

Polarity of neutral amino acid transport and characterization of a broad specificity transport activity in a kidney epithelial cell line, MDCK

The Madin-Darby canine kidney (MDCK) cell line was investigated with respect to the cellular polarity of amino acid transport in early confluent versus late confluent cultures. Early confluent cultures could take up amino acids from the apical and the basolateral sides of the cell layer via amino acid transport Systems A, ASC, and L. However, in late confluent cultures the activities of Systems A and L were clearly localized to the basolateral surface of the cell monolayer. In addition to the presence of systems A, ASC, and L, a novel activity, measurable under conditions used for quantitating System ASC, was found to be active in the apical membrane of these cells. This transporter, termed System G (for general), recognized basic and neutral amino acids with high affinity and acidic amino acids with lower affinity. System G exhibited broad substrate specificity, strict cation specificity, and a broad pH optimum with maximal activity at acidic pH. The activity of System G was relatively low after growth in serum-containing medium but was induced in a defined medium. Induction of System G activity was dependent upon the presence of prostaglandin E1. The broad substrate specificity, low pH optimum, and Na+ dependence suggest that System G may function in apical membranes as an energy-dependent transport route during reabsorption of amino acids from the kidney tubule lumen.     

Characterization of neutral amino acid transport in a marine pseudomonad.

The transport of neutral amino acids in marine pseudomonad B-16 (ATCC 19855) has been investigated. From patterns of competitive inhibition, mutant analysis, and kinetic data, two active transport systems with overlapping substrate specificities were distinguished and characterized. One system (DAG) served glycine, D-alanine, D-serine, and alpha-aminoisobutyric acid (AIB) and, to a lesser extent, L-alanine and possibly other related neutral D- and L-amino acids. The other system (LIV) showed high stereospecificity for neutral amino acids with the L configuration and served primarily to transport L-leucine, L-isoleucine, L-valine, and L-alanine. This system exhibited low affinity for alpha-aminoisobutyric acid. Neither system was able to recognize structural analogues with modified alpha-amino or alpha-carboxyl groups. The kinetic parameters for L-alanine transport by the DAG and LIV systems were determined with appropriate mutants defective in either system. For L-alanine, Kt values of 4.6 X 10(-5) and 1.9 X 10(-4) M and Vmax values of 6.9 and 20.8 nmol/min per mg of cell dry weight were obtained for transport via the DAG and LIV systems respectively. alpha-Aminoisobutyric acid transport heterogeneity was also resolved with the mutants, and Kt values of 2.8 X 10(-5) and 1.4 X 10(-3) M AIB were obtained for transport via the DAG and LIV systems, respectively. Both systems required Na+ for activity (0.3 M Na+ optimal) and in this regard are distinguished from systems of similar substrate specificity reported in nonmarine bacteria.     

Effect of hyperosmolarity on the activity of amino acid transport system L in avian fibroblasts

The transport of selected neutral amino acids known as good substrates of amino acid transport System L has been studied in chick embryo fibroblasts exposed for 4 hours to hyperosmolar culture medium. The activity of the L system, as measured by initial rates of L-phenylalanine uptake, increased in hyperosmolarity treated cells when determined before any cell depletion of intracellular amino acids. This effect was lost after depletion but reappeared after reloading the cells with pertinent substrates of System L. This transport activity appeared to be related to the internal level of amino acids capable of exchange through System L. In hyperosmolarity-treated chick embryo fibroblasts a higher level of System L substrates was obtained during the reloading phase in comparison to control cells. This expanded amino acid pool reflected an increased activity of transport System A, an agency of amino acid mediation known to enlarge its capacity following a hyperosmolar treatment of chick embryo fibroblasts (see Tramacere et al., 1984). L-Methionine, a preferred substrate of both A and L systems, appeared to be involved in the coupling between the activity of amino acid transport Systems A and L in these cells.     

Characterization of an organic anion transport system in a placental cell line

Transporters within the placenta play a crucial role in the distribution of nutrients and xenobiotics across the maternal-fetal interface. An organic anion transport system was identified on the apical membrane of the rat placenta cell line HRP-1, a model for the placenta barrier. The apical uptake of 3H-labeled organic anion estrone sulfate in HRP-1 cells was saturable (Km = 4.67 microM), temperature and Na+ dependent, Li+ tolerant, and pH sensitive. The substrate specificity of the transport system includes various steroid sulfates, such as beta-estradiol 3,17-disulfate, 17 beta-estradiol 3-sulfate, and dehydroepiandrosterone 3-sulfate (DHEAS) but does not include taurocholate, p-aminohippuric acid (PAH), and tetraethylammonium. Preincubation of HRP-1 cells with 8-bromo-cAMP (a cAMP analog) and forskolin (an adenylyl cyclase activator) acutely stimulated the apical transport activity. This stimulation was further enhanced in the presence of IBMX (a phosphodiesterase inhibitor). Together these data show that the apical membrane of HRP-1 cells expresses an organic anion transport system that is regulated by cellular cAMP levels. This transport system appears to be different from the known taurocholate-transporting organic anion-transporting polypeptides and PAH-transporting organic anion transporters, both of which also mediate the transport of estrone sulfate and DHEAS.     

Identification of LAT4, a novel amino acid transporter with system L activity

System L amino acid transporters mediate the movement of bulky neutral amino acids across cell membranes. Until now three proteins that induce system L activity have been identified: LAT1, LAT2, and LAT3. The former two proteins belong to the solute carrier family 7 (SLC7), whereas the latter belongs to SLC43. In the present study we present a new cDNA, designated LAT4, which also mediates system L activity when expressed in Xenopus laevis oocytes. Human LAT4 exhibits 57% identity to human LAT3. Like LAT3, the amino acid transport activity induced by LAT4 is sodium-, chloride- and pH-independent, is not trans-stimulated, and shows two kinetic components. The low affinity component of LAT4 induced activity is sensitive to the sulfhydryl-specific reagent N-ethylmaleimide but not that with high affinity. Mutation in LAT4 of the SLC43 conserved serine 297 to alanine abolishes sensitivity to N-ethylmaleimide. LAT4 activity is detected at the basolateral membrane of PCT kidney cells. In situ hybridization experiments show that LAT4 mRNA is restricted to the epithelial cells of the distal tubule and the collecting duct in the kidney. In the intestine, LAT4 is mainly present in the cells of the crypt.     

Characterization of the methotrexate transport defect in a resistant L1210 lymphoma cell line

L1210/R81 lymphoma cells are resistant to methotrexate (MTX) by virtue of a 35-fold elevation in dihydrofolate reductase and an inability to transport the folate antagonist drug effectively. In a phosphate-containing buffer there was little or no influx into the resistant cells at either 1 or 50 μm MTX. Replacement of this buffer with a 4-(2-hydroxyethyl)-1-piperazine-N′-2-ethanesulfonic acid-Mg²⁺ system resulted in an apparent influx of MTX into the resistant cells. Under these conditions, L1210/R81 cells achieved an apparent steady state at an extracellular MTX concentration of 50 μm. The apparent steady-state level of 5 nmol $\text{[}{}^3\text{H]MTX}\text{10}{}^9$ cells was well below the intracellular level of dihydrofolate reductase (45 nmol/10⁹ cells). Efflux experiments at the apparent steady state indicated that 60% of the MTX was very rapidly removed from the cells by washing. Over the range of the experiment a further 20% of the MTX effluxed more slowly ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 12}\text{min}$). The apparent influx into the resistant cells at 5 μm MTX was inhibited 13% by sodium azide (100 μm) and initially stimulated, then inhibited, by p-chloromercuriphenylsulfonic acid (100 μm). 5-Methyltetrahydrofolate (100 μm) had little effect on the process while aminopterin (100 μm) was inhibitory (68%). K_t and V values of 2 × 10^?5m and 0.31 nmol $\text{[}{}^3\text{H]MTX}\text{10}{}^9$ cells/min, respectively, were determined for the apparent influx in $\text{L}\text{1210}\text{R}\text{81}$ cells. Comparison of apparent MTX influx in the resistant cells with MTX transport in the sensitive cells indicates profound differences in the two processes. The evidence suggests that the apparent influx in the former cell line may consist of MTX binding to the cell membrane together with a small degree of MTX influx into the intracellular compartment.     

Insulin-like growth factor-I stimulates amino acid transport in a glutamine-deprived human neuroblastoma cell line

It is still unknown how insulin-like growth factor-I (IGF-I) regulates cancer cell growth in the condition of the limited availability of key nutrients, such as glutamine. We investigated the effects of IGF-I on cell growth and amino acid transport in a glutamine-deprived human neuroblastoma cell line, SK-N-SH. Cell growth was measured, and ³H-labeled amino acid transport was assayed after treatment with or without IGF-I (50 ng/ml) in 2 mM (control) and 100 μM glutamine concentrations. Cell growth rates were dependent on glutamine concentrations. IGF-I stimulated cell growth in both 2 mM and 100 μM glutamine. IGF-I stimulated glutamine transport in 100 μM glutamine with the mechanism of increasing carrier V_max, but had no effect in 2 mM glutamine. IGF-I also stimulated leucine, glutamate and 2-(methylamino)isobutyric acid transport in 100 μM glutamine. There were significant increases in [³H]thymidine and [³H]leucine incorporation in IGF-I-treated cells in both 2 mM and 100 μM glutamine. These data suggest that IGF-I stimulates cell growth by increasing amino acid transport in the condition of low glutamine levels in a human neuroblastoma cell line. This mechanism may allow to maintain cell growth even in nutrient-deprived tumor tissues.     

Isolation and characterization of Chinese hamster ovary cell mutants defective in amino acid transport System L

The Chinese hamster ovary cell line CHO-tsH1 is a temperature-sensitive leucyl-tRNA synthetase mutant that shows temperature-dependent regulation of the amino acid transport responsible for accumulating leucine, System L. At nonpermissive temperatures, CHO-tsH1 cells are unable to grow because they are unable to incorporate leucine into protein. As a result, System L activity is increased. We have isolated mutants from CHO-tsH1 that have constitutively de-repressed System L activity. These mutants are temperature-resistant as a result of increased intracellular steady-state accumulations of System L-related amino acids, which compensates for the defective synthetase activity. In this study, we have subjected one of these regulatory mutant cell lines (C11B6) to a tritium-suicide selection, in which L-[3H]leucine was used as a toxic substrate. Three mutant cell lines, C4B4, C5D9, and C9D9 that showed reduced System L transport activity were isolated. The decreases in the initial rates of System L transport activity lead to reduced steady-state accumulations of System L-related amino acids. In contrast to the parental cell line, C11B6, the transport-defective mutants are temperature-sensitive because the reduced intracellular pool of leucine can no longer compensate for the defective synthetase activity.     

The acidic amino acid transport system of the baby hamster kidney cell line BHK21-C13

The uptake of L-glutamate into BHK21-C13 cells in culture has been studied. This amino acid appears to be transported via a relatively high affinity, low capacity, Na+-dependent transport system capable of the rapid accumulation of substrate amino acids. Kinetic studies of the inhibition of L-glutamate uptake has provided information as to the substrate and the molecular configuration required for transport via the glutamate transport system. This system exhibited marked substrate specificity and was only capable of transporting L-glutamate and aspartate and certain closely related acidic amino acid analogues.     

Hormonal regulation of the system a amino acid transport adaptive response mechanism in a kidney epithelial cell line (MDCK)

When mamalian cells are starved for amino acids, the activity of the A amino acid transport system increases, a phenomenon called adaptive regulation. We have examined the effects of those factors which support Madin-Darby canine kidney (MDCK) cell growth in a defined medium on the derepression of System A activity. Of the five factors which supported MDCK cell growth, insulin was found to be an absolute requirement for derepression. In contrast, PGE₁ was a negative controlling factor for the transport system. Growth of MDCK cells in the absence of PGE₁ resulted in elevated System A activity which derepressed poorly upon amino acid starvation. Kinetic analysis of α-(methylamino) isobutyric acid (mAIB) uptake as a function of substrate concentration showed that the elevated A activity observed when cells were grown in the absence of PGE₁ was kinetically similar to the activity induced by starvation for amino acids. Transport of mAIB by amino-acid-fed cells grown in the presence of PGE₁ was characterized by a linear Eadie-Hofstee graph and by a relatively low Vmax. Transport by cells starved for amino acids or by cells grown in the absence of PGE₁ was characterized by biphasic kinetics for mAIB transport and by elevated Vmax values. An influence of growth factors on the inactivation of derepressed A activity was also observed. In the presence of cycloheximide the rate of loss of A activity in amino-acid-starved cells was 1/4–1/2 that of amino-acid-fed cells. Insulin slowed inactivation in the absence of most amino acids in a protein-synthesis-independent manner, but insulin did not influence the more rapid inactivation observed in amino-acid-fed cells. These results indicate that the level of System A activity observed in response to regulation by amino acids represents a balance between carrier synthesis and inactivation, which can be positively or negatively influenced by growth factors.     

Identification of the protein responsible for hepatic system N amino acid transport activity.

In the liver, glutamine utilization may be limited by the rate of transport across the plasma membrane by the System N carrier. System N-mediated transport activity has been solubilized from rat liver plasma membrane, partially purified, and then reconstituted into proteoliposomes. To identify the System N carrier protein, monoclonal antibodies were generated against the protein fraction enriched for System N activity. Two antibodies , 3E1-2 and 1E7-3, inhibited System N activity in hepatocytes. These antibodies also immunoprecipitated System N activity from a mixture of solubilized proteins and were specific for antigen recognition in that neither immunoprecipitated System A activity. The antibody recognized a single protein of molecular size 100 kDa by immunoblot analysis. Recognition of this protein by the antibody increased in parallel with the enrichment of System N activity in solubilized membrane fractions. These data suggest that a 100-kDa plasma membrane protein mediates System N transport activity in rat hepatocytes.     

Regulation of amino acid transport in the renal epithelial cell line NBL-1

Summary The activities of the transport systems A, B° and X_AG- are induced by various forms of stress in renal epithelial cells. Amino acid deprivation induces System A and X_AG- in a protein-synthesis dependent process. In the case of System X_AG- evidence is presented that induction of transport does not involve an increase in the amount of mRNA for the transporter or of the amount of transport protein. Preliminary evidence for the existence of a novel glycoprotein which is induced in parallel to the induction of these transport systems is presented. It is suggested that the induction of amino acid transport proteins and of some of the so-called stress proteins may be triggered by a common molecular mechanism.