System A-mediated Amino Acid Transport Research Articles

Effect of high altitude on human placental amino acid transport.

Women residing at high altitudes deliver infants of lower birth weight than at sea level. Birth weight correlates with placental system A-mediated amino acid transport capacity, and severe environmental hypoxia reduces system A activity in isolated trophoblast and the mouse placenta. However, the effect of high altitude on human placental amino acid transport remains unknown. We hypothesized that microvillous membrane (MVM) system A and system L amino acid transporter activity is lower in placentas of women living at high altitude compared with low-altitude controls. Placentas were collected at term from healthy pregnant women residing at high altitude (HA; >2,500 m; n = 14) or low altitude (LA; <1,700 m; n = 14) following planned, unlabored cesarean section. Birth weight, but not placenta weight, was 13% lower in HA pregnancies (2.88 ± 0.11 kg) compared with LA (3.30 ± 0.07 kg, P < 0.01). MVM erythropoietin receptor abundance, determined by immunoblot, was greater in HA than in LA placentas, consistent with lower placental oxygen levels at HA. However, there was no effect of altitude on MVM system A or L activity, determined by Na+-dependent [14C]methylaminoisobutyric acid uptake and [3H]leucine uptake, respectively. MVM abundance of glucose transporters (GLUTs) 1 and 4 and basal membrane GLUT4 were also similar in LA and HA placentas. Low birth weights in the neonates of women residing at high altitude are not a consequence of reduced placental amino acid transport capacity. These observations are in general agreement with studies of IUGR babies at low altitude, in which MVM system A activity is downregulated only in growth-restricted babies with significant compromise.NEW & NOTEWORTHY Babies born at high altitude are smaller than at sea level. Birth weight is dependent on growth in utero and, in turn, placental nutrient transport. We determined amino acid transport capacity in placentas collected from women resident at low and high altitude. Altitude did not affect system A amino acid transport across the syncytiotrophoblast microvillous membrane, suggesting that impaired placental amino acid transport does not contribute to reduced birth weight in this high-altitude population.

The contribution of SNAT1 to system A amino acid transporter activity in human placental trophoblast

System A-mediated amino acid transport across the placenta is important for the supply of neutral amino acids needed for fetal growth. All three system A subtypes (SNAT1, 2, and 4) are expressed in human placental trophoblast suggesting there is an important biological role for each. Placental system A activity increases as pregnancy progresses, coinciding with increased fetal nutrient demands. We have previously shown SNAT4-mediated system A activity is higher in first trimester than at term, suggesting that SNAT1 and/or SNAT2 are responsible for the increased system A activity later in gestation. However, the relative contribution of each subtype to transporter activity in trophoblast at term has yet to be evaluated. The purpose of this study was to identify the predominant subtype of system A in cytotrophoblast cells isolated from term placenta, maintained in culture for 66h, by: (1) measuring mRNA expression of the three subtypes and determining the Michaelis–Menten constants for uptake of the system A-specific substrate, 14C-MeAIB, (2) investigating the contribution of SNAT1 to total system A activity using siRNA. Results: mRNA expression was highest for the SNAT1 subtype of system A. Kinetic analysis of 14C-MeAIB uptake revealed two distinct transport systems; system 1: Km=0.38±0.12mM, Vmax=27.8±9.0pmol/mg protein/20min, which resembles that reported for SNAT1 and SNAT2 in other cell types, and system 2: Km=45.4±25.0mM, Vmax=1190±291pmol/mg protein/20min, which potentially represents SNAT4. Successful knockdown of SNAT1 mRNA using target-specific siRNA significantly reduced system A activity (median 75% knockdown, n=7). Conclusion: These data enhance our limited understanding of the relative importance of the system A subtypes for amino acid transport in human placental trophoblast by demonstrating that SNAT1 is a key contributor to system A activity at term.

Physiological importance of system A-mediated amino acid transport to rat fetal development.

Fetal growth and development are dependent on the delivery of amino acids from maternal amino acid pools to the fetal blood. This is accomplished via transfer across the apical and basal plasma membrane of the placental syncytiotrophoblast. The aim of this study was to determine whether inhibition of system A (amino acid transporter) was associated with a decrease in fetal weight in the rat. System A is a ubiquitous Na(+)-dependent amino acid transporter that actively transports small zwitterionic amino acids. In brief, system A was inhibited by infusing a nonmetabolizable synthetic amino acid analog, 2-(methylamino)isobutyric acid from days 7-20 of gestation. On day 20, the rats were killed and tissues (maternal liver, fetuses, and placentas) were collected for analysis. The degree of system A inhibition was determined, as was the impact of said inhibition on fetal and maternal weights, system A-mediated placental transport, and placental system A-mediated transporter expression. Our results suggest that when system A is inhibited, fetal weight is diminished [control group: -3.55 +/- 0.04 g (n = 113), experimental group: -3.29 +/- 0.04 g (n = 128)], implying an integral role for system A transport in fetal growth and development in the rat.

Posttranscriptional Regulation of ATA2 Transport during Liver Regeneration

The recent cloning of ATA2, a cDNA displaying characteristics identical to the System A transporter, has provided the first molecular tool for study of System A-mediated amino acid transport in liver. Despite the 233 ± 9 and 472 ± 11% increase in System A transport activity following partial hepatectomy at 6 and 12 h, respectively, the steady-state level of ATA2 mRNA did not show a corresponding marked increase. Examination of the kinetic properties of System A following partial hepatectomy revealed a Km of 0.26 ± 0.04 mM which is consistent with the reported Km for ATA2. These results indicate that a System A transporter present in regenerating liver and ATA2 are identical, but that the increase in System A activity following partial hepatectomy does not result from an increase in steady-state levels of ATA2 mRNA. These observations suggest that ATA2-mediated transport of amino acids is regulated at the posttranscriptional level.

Epidermal growth factor regulates amino acid transport in chick embryo hepatocytes via protein kinase C

System A-mediated amino acid transport, activation of different steps of signal transduction and involvement of different isoforms of protein kinase C (PKC) have been investigated in chick embryo hepatocytes after epidermal growth factor (EGF) stimulation. EGF rapidly (10 min) increased the rate of aminoisobutyric acid (AIB) uptake in chick embryo hepatocytes freshly isolated on the 19th day of embryonic life, while no change was detectable at other embryonal stages. The growth factor stimulation was abolished by PKC and tyrosine kinase inhibitors and was mimicked by 4-phorbol-12-myristate-13-acetate, dimethyl-2 (PMA). EGF treatment did not modify the phosphorylation of the γ isoform of phospholipase C (PLC-γ), and inositol trisphosphate (IP3) and intracellular calcium levels, but it induced an increase in PKC activity. Our data show that EGF regulates amino acid uptake, via PKC and without PLC-γ activation, only in the last period of chick embryo hepatocyte development. The effects of growth factor on PKC activity suggest the involvement of PKC-α and -ε isoforms in EGF modulation of amino acid transport.

Epidermal growth factor regulates amino acid transport in chick embryo hepatocytes via protein kinase C.

System A-mediated amino acid transport, activation of different steps of signal transduction and involvement of different isoforms of protein kinase C (PKC) have been investigated in chick embryo hepatocytes after epidermal growth factor (EGF) stimulation. EGF rapidly (10 min) increased the rate of aminoisobutyric acid (AIB) uptake in chick embryo hepatocytes freshly isolated on the 19th day of embryonic life, while no change was detectable at other embryonal stages. The growth factor stimulation was abolished by PKC and tyrosine kinase inhibitors and was mimicked by 4-phorbol-12-myristate-13-acetate, dimethyl-2 (PMA). EGF treatment did not modify the phosphorylation of the isoform of phospholipase C (PLC-), and inositol trisphosphate (IP3) and intracellular calcium levels, but it induced an increase in PKC activity. Our data show that EGF regulates amino acid uptake, via PKC and without PLC- activation, only in the last period of chick embryo hepatocyte development. The effects of growth factor on PKC activity suggest the involvement of PKC- and - isoforms in EGF modulation of amino acid transport.

Inhibition of system A amino acid transport and hepatocyte proliferation following partial hepatectomy in the rat.

System A, the sodium-dependent neutral amino acid transport activity, has a 3-fold increase in its initial uptake velocity into hepatocytes following partial hepatectomy (PH) in the rat. The purpose of this study was to examine the effect of inhibition of System A-mediated amino acid transport on hepatocyte proliferation and liver regeneration. We describe the in vivo competitive inhibition of System A activity following PH by the nonmetabolizable, System A-specific substrate, alpha-(methylamino)isobutyric acid (MeAIB). Administration of MeAIB 60 minutes before PH decreased the incorporation of [(3)H]thymidine into DNA by 45% +/- 5% and 76% +/- 17% at 24 and 36 hours, respectively. The readministration of MeAIB every 12 hours further decreased DNA synthesis by 92% +/- 18% and 82% +/- 11% at 24 and 36 hours. The recovery of liver mass of rats receiving MeAIB was decreased by 46.4% +/- 5.1% at 24 hours after PH. In vitro, 5 mmol/L MeAIB inhibited proliferation of primary hepatocytes by 56% +/- 4% and 61% +/- 12% 48 hours after incubation with 10% fetal calf serum or epidermal growth factor (5 ng/mL), respectively. Thus, MeAIB inhibition of System A transport activity decreased both in vivo and in vitro inducement of hepatocyte proliferation. Treatment with MeAIB did not significantly change the incorporation of [(3)H]leucine into total liver protein, but changes in serum amino acids and hepatocyte cell volume were observed, suggesting System A transport activity during hepatocyte proliferation functions primarily to provide amino acids to fuel liver-specific biochemical pathways and to increase cell volume.

Complex subcellular distribution of sodium-dependent amino acid transport systems in kidney cortex and LLC-PK1/Cl4 cells

To characterize the amino acid transport system in basolateral membranes and to test for possible intracellular loci of amino acid transport activity, we surveyed the distribution of L-alanine transport activity in rabbit proximal tubular cells and LLC-PK1/Cl4 cells. A three-dimensional separation procedure based on differential sedimentation, density gradient centrifugation, and counter-current distribution resolved 21 physically and biochemically distinct membrane populations from rabbit cortex. Inhibition of L-alanine transport by phenylalanine and N-(methylamino)isobutyric acid was used to delineate parallel amino acid transport pathways. Population n was identified as brush border membranes by virtue of its 16-fold maltase enrichment; 94% of its Na(+)-dependent alanine transport activity was mediated by systems previously shown to be characteristic of brush border membranes. Two populations, c' and c", which accounted for 25% of the total Na,K-ATPase activity, were identified as basalateral membranes on the basis of Na,K-ATPase cumulative enrichment factors of 15 and 21; 82% of the total alanine transport in these populations was mediated by a Na(+)-independent system similar to the classical system L. Na,K-ATPase, Na(+)-independent and Na(+)-dependent alanine transport activities were associated with intracellular membrane populations as well as with the plasma membranes. The major intracellular locus of Na,K-ATPase activity, population i accounted for roughly 31% of the Na,K-ATPase, maximally enriched ninefold; it contained 29% of the total system L transport activity. Population l, which was identified as endoplasmic reticulum because it was the major locus of membrane-bound NADPH cytochrome c reductase activity, contained 44% of the total system A transport. Three distinct Golgi-derived populations, m', m", and o, accounted for 39% of the total system A transport. A survey of the amino acid transport systems in LLC-PK1/Cl4 cells showed that the majority of system A-mediated amino acid transport was present in membranes of intracellular and possibly apical origin. The presence of large intracellular pools of amino acid transport activities might reflect newly synthesized transport proteins, ongoing membrane recycling or, perhaps, intracellular reserves available for rapid recruitment to the plasma membrane.

Substrate-dependent adaptive regulation and trans-inhibition of system A-mediated amino acid transport. Studies using rat hepatoma plasma membrane vesicles

Substrate-dependent regulation of amino acid transport by System A occurs by both direct action at the carrier (trans-inhibition) and transcriptional control (adaptive regulation). While experiments with intact cells have led to working models that describe these regulatory phenomena, the use of subcellular will serve to refine the present hypotheses. Adaptive induction of System A transport following amino acid starvation of cells was shown to be dependent on de novo RNA and protein synthesis, and the stimulted activity was shown to be retained in isolated plasma membrane vesicles. This stimulated transport activity was tightly associated with the plasma membrane, but could be solubilized by 4 M urea and 2.5% cholate, and recovered following reconstitution of the protein into artificial proteoliposomes. These data support the working hypothesis that adaptive induction of transport is the result of de novo synthesis and insertion into the plasma membrane of System A carrier protein. In contrast, the activity of System ASC in the vesicles from the amino acid starved cells was actually reduced by 2–5-fold when compared to amino acid-fed cells. A more rapid form of regulation of System A activity is trans-inhibition. The use of isolated plasma membrane vesicles demonstrated that trans-inhibition in whole cells not survive membrane isolation. However, substrate loading of isolated membrane vesicles containing high level of System A activity, produced trans-inhibition in a very specific manner in that System A substrates resulted in decreased transport activity, while those amino acids which are poor substrates for the System A carrier did not. Thus, trans-inhibition is not the result of a recycling process involving an intracellular pool of carrier, but rather can be accounted for by differences in the kinetics for amino acid binding and/or translocation on the two dies of the membrane.

Solubilization and Reconstitution Characteristics of Hepatic System A-mediated Amino Acid Transport

In the liver, System A-mediated uptake of neutral amino acids may play a key role in metabolic control. Knowing the properties of the solubilized and reconstituted System A activity is important for future studies on the purification of the carrier protein. Solubilization of System A activity by the combination of 2.5% cholate and 4 M urea resulted in greater than 85% extraction of the activity. Previous removal of easily extracted plasma membrane proteins with 1% cholate alone followed by solubilization of the transporter with cholate/urea resulted in a 2-fold enrichment in transport activity. Based on the observation that the carrier protein aggregates in the presence of low detergent concentrations, a selective polyethylene glycol precipitation procedure was developed resulting in recovery of more than 70% of the initial transport activity and less than 10% of the total protein. A concomitant 10-fold enrichment in carrier activity was achieved. The precipitated carrier could be resuspended in buffer containing Triton X-100, asolectin, and glycerol. Transporter activity in this buffer was stable for up to 5 days when maintained at -20 degrees C or for 2 days at 4 degrees C. The general applicability of the devised reconstitution is illustrated by the presence of Systems N and Gly in the reconstituted proteoliposomes at specific activities greater than those in the native vesicles.

Solubilization and reconstitution of hepatic System A-mediated amino acid transport. Preparation of proteoliposomes containing glucagon-stimulated transport activity

System A-mediated amino acid transport activity from rat liver plasma membrane vesicles has been solubilized and reconstituted into proteoliposomes using a freeze-thaw-dilution technique. The presence of cholate, at a cholate to protein ratio of 1:1, during the freeze-thaw step resulted in an enhancement in recoverable transport activity. The carrier required both phosphatidylcholine and phosphatidylethanolamine for optimal activity, but the addition of cholesterol to the reconstitution procedure appeared to have no significant effect on the resulting activity. A lipid to protein ratio of 20:1 yielded maximal transport activity. Sonication of the proteoliposomes provided some improvement in the accuracy of replicate assays for a given proteoliposome preparation. Isolated liver plasma membrane vesicles prepared from rats treated in vivo with glucagon in combination with dexamethasone contained stimulated System A activity. This enhanced transport activity could be solubilized and recovered in proteoliposomes generated from these plasma membranes. The data support the proposal that hormone regulation of the hepatic System A gene results in the de novo synthesis and plasma membrane insertion of the carrier protein itself.

Control of growth and amino acid transport of mature rat hepatocytes by human newborn serum.

DNA synthesis and system A-mediated amino acid transport were studied in primary culture of adult rat hepatocytes maintained as a monolayer. Potent growth promoting activity and inducibility of system A-mediated amino acid transport could be found in human newborn sera but not in human adult sera. The maximal activity to promote DNA synthesis was about the same level as insulin (10(-7) M) plus epidermal growth factor (EGF, 20 ng/ml). These results suggest the presence of a hepatocyte-specific growth factor in human newborn sera controlling the amino acid metabolism during the newborn period.

System A-mediated amino acid transport: metabolic control at the plasma membrane

Transport across the plasma membrane can be the rate-limiting step for further catabolism of a nutrient molecule. Regulation of transport via transcriptional control is an important long term adaptational mechanism for the eukaryotic cell. System A-mediated amino acid transport is an example of such a regulatory process.

Cis-inhibition, trans-inhibition, and repression of hepatic amino acid transport mediated by System A. Substrate specificity and other properties.

Substrate regulation of System A-mediated amino acid transport was investigated in primary cultures of rat hepatocytes. Studies on the substrate specificity of trans-inhibition and repression revealed considerable differences between the two processes. Those data along with a difference in temperature sensitivity suggest that the two phenomena are not related or inter-dependent in any direct way. However, kinetic analysis indicates that both trans-inhibition and repression decrease the number of functional carriers within the plasma membrane. Cis-inhibition tests show that the hepatic System A carrier exhibits a wide degree of tolerance with regard to modification of the alpha-amino and alpha-carboxyl groups. In general, the amino acids that cause the greatest degree of trans-inhibition are only moderate cis-inhibitors of System A-mediated transport (40-60% inhibition of Na+-dependent 2-aminoisobutyric acid uptake). The substrate specificity of amino acid-induced repression appears to be similar to that for System A-mediated transport, although an amino acid can exhibit cis-inhibition of System A activity without causing repression (or trans-inhibition). For example, S-methyl-L-cysteine serves as a competitive inhibitor of System A, yet it does not cause trans-inhibition or repression to a considerable degree. However, through its cis-inhibition of transport, S-methyl-L-cysteine blocks asparagine-dependent repression, apparently by suppressing the cytoplasmic accumulation of asparagine.

Control by amino acids of the activity of system A-mediated amino acid transport in isolated rat hepatocytes

The effect of amino acids, in concentrations corresponding to those found in the portal vein of rats given a high-protein diet, was investigated on the activity of system A amino acid transport in hepatocytes from fed rats. Amino acids counteracted the induction of system A by insulin or glucagon. This effect was observed at all concentrations of hormones tested, up to 1 microM. Amino acids did not affect the basal cyclic AMP concentration in hepatocytes, or the large rise in cyclic AMP elicited by glucagon. The reversal of system-A induction was observed at relatively low concentration of amino acids, corresponding to plasma values reported in rats given a basal diet. Amino acids were separately tested: substrates of system A were particularly efficient, but so were glutamine and histidine. Non-metabolizable substrates of system A, such as 2-aminoisobutyrate, were also inhibitory, suggesting that a part of the effect of amino acids is independent of their cellular metabolism. Provision of additional energy substrates such as lactate and oleate did not affect induction of system A or the inhibitory effects of amino acids. Thus amino acids do not act by serving as an energy source and by maintaining the integrity of hepatocytes. Inhibition of mRNA synthesis by actinomycin practically abolished the effect of amino acids on the induction of system A by glucagon. The results suggest that amino acids may promote the synthesis of protein(s) affecting the activity of system A either directly at the carrier unit or at an intermediate stage of its emergence.

Induction and decay of amino acid transport in the liver. Turnover of transport activity in isolated hepatocytes after stimulation by diabetes or glucagon.

System A-mediated amino acid transport in liver tissue is stimulated by diabetes or by exogenous glucagon. The present report describes the decay process for stimulated System A activity in isolated rat hepatocytes. Transport induced by glucagon, insulin, or spontaneous diabetes (BB/G rats) decayed rapidly after initiation of primary cultures; the estimated half-life was about 1.5 h. In contrast, the stimulated activity in cultured hepatocytes from streptozotocin-diabetic rats had a half-life of about 2.5 h. It is not known if the loss of System A activity is the result of proteolysis or of another form of inactivation. The decay was blocked by either actinomycin or cycloheximide, but was unaffected by leupeptin, methylamine, chloroquine, dinitrophenol, rotenone, or tunicamycin. Studies with cycloheximide and actinomycin suggest the following: 1) within 30 min after initiation of cell cultures, synthesis of the corresponding mRNA for the transport-inactivating protein has begun; 2) the mRNA for transport-inactivating protein is relatively long-lived, but the inactivating protein itself has a half-life of less than 1 h; and 3) actinomycin blocks the decay through inhibition of transport-inactivating protein biosynthesis rather than by protection of the mRNA for the protein responsible for System A activity. A working model for the synthesis and decay of System A activity is presented. Cationic amino acid transport, System y+, was also stimulated severalfold after induction of diabetes or glucagon injection of rats. Systems ASC, X-, and N were enhanced to varying degrees in hepatocytes from diabetic or glucagon-injected rats, but the level of stimulation for each was not as great as that found for Systems A or y+.