Activity and protein localization of multiple glutamate transporters in gestation day 14 vs. day 20 rat placenta.

Concentrative absorption of glutamate by the developing placenta is critical for proper fetal development. The expression of GLAST1, GLT1, EAAC1, and EAAT4, known to be capable of D-aspartate-inhibitable and Na(+)-coupled glutamate transport (system X-AG), was evaluated in day 14 vs. day 20 rat chorioallantoic placenta. Steady-state mRNA levels were greater at day 20 for all transporters. Immunohistochemistry determined that the expression of GLAST1, GLT1, and EAAC1 was greater throughout the day 20 placenta and was asymmetric with respect to cellular localization. EAAT4 protein was not detected. System X-AG activity was responsible for most of the Na(+)-dependent glutamate uptake and was greater in day 20 than in day 14 apical and basal membrane subdomains of the labyrinth syncytiotrophoblast. Greater quantities of EAAC1 and GLAST1 protein were identified on day 20, and quantities were greater in basal than in apical membranes. GLT1 expression, unchanged in apical membranes, was decreased in basal membranes. These data correlate transporter mRNA and protein content with transport activity and demonstrate an increasing capacity for glutamate absorption by the developing placenta.

Adaptive regulation of the cationic amino acid transporter-1 (Cat-1) in Fao cells.

The regulation of the high affinity cationic amino acid transporter Cat-1 in Fao rat hepatoma cells by amino acid availability has been studied. Cat-1 mRNA level increased (3-fold) in 4 h in response to amino acid starvation and remained high for at least 24 h. This induction was independent of the presence of serum in the media and transcription and protein synthesis were required for induction to occur. When Fao cells were shifted from amino acid-depleted media to amino acid-fed media, the levels of the induced cat-1 mRNA returned to the basal level. In amino acid-fed cells, accumulation of cat-1 mRNA was dependent on protein synthesis, indicating that a labile protein is required to sustain cat-1 mRNA level. No change in the transcription rate of the cat-1 gene during amino acid starvation was observed, indicating that cat-1 is regulated at a post-transcriptional step. System y+ mediated transport of arginine was reduced by 50% in 1 h and by 70% in 24 h after amino acid starvation. However, when 24-h amino acid-starved Fao cells were preloaded with 2 mM lysine or arginine for 1 h prior to the transport assays, arginine uptake was trans-stimulated by 5-fold. This stimulation was specific for cationic amino acids, since alanine, proline, or leucine had no effect. These data lead to the hypothesis that amino acid starvation results in an increased cat-1 mRNA level to support synthesis of additional Cat-1 protein. The following lines of evidence support the hypothesis: (i) the use of inhibitors of protein synthesis in starved cells inhibits the trans-zero transport of arginine; (ii) cells starved for 1-24 h exhibited an increase of trans-stimulated arginine transport activity for the first 6 h and had no loss of activity at 24 h, suggesting that constant replenishment of the transporter protein occurs; (iii) immunofluorescent staining of 24-h fed and starved cells for cat-1 showed similar cell surface distribution; (iv) new protein synthesis is not required for trans-stimulation of arginine transport upon refeeding of 24-h starved cells. We conclude that the increased level of cat-1 mRNA in response to amino acid starvation support the synthesis of Cat-1 protein during starvation and increased amino acid transport upon substrate presentation. Therefore, the cat-1 mRNA content is regulated by a derepression/repression mechanism in response to amino acid availability. We propose that the amino acid-signal transduction pathway consists of a series of steps which include the post-transcriptional regulation of amino acid transporter genes.

An expression system for mammalian amino acid transporters using a stably maintained episomal vector.

Despite its versatility and effectiveness in numerous studies, the vaccinia/HeLa cell expression model may not be optimal for the study of all transport proteins. To evaluate an alternative expression model for amino acid transport Systems ASC and X-AG, the mRNA content and transport activity encoded by human hippocampal ASCT1 cDNA and rat hippocampal EAAC1 cDNA, respectively, were measured in pDR2-cDNA-transfected human embryonic kidney 293 cells made competent by stable transfection with the Epstein-Barr neutral antigen-1 (EBNA-1) cDNA (293c18 cells) to evaluate the EBNA-1/293c18 expression system. The results show that (i) the EBNA-1/293c18 expression system results in a larger increase over background of Systems ASCT1 (6.4x) and EAAC1 (39x) transport activity than does the vaccinia/HeLa expression system (2.6x and 22x, respectively); (ii) transfection and hygromycin B selection for the pDR2 vector do not affect the endogenous transport velocities of Systems ASC, X-AG, or A; and (iii) the endogenous transport velocities of Systems ASC and X-AG in 293c18 cells were not affected by the expression of exogenous EAAC1 or ASCT1. We conclude that the EBNA-1/293c18 cell expression model represents a useful transient expression regimen to characterize mammalian amino acid transport proteins, especially for transporters that may exhibit relatively low activity in transient expression systems lacking a selection mechanism.

Ontogeny of amino acid transport system A in rat placenta.

Amino acid transport System A has previously been demonstrated in apical membranes derived from rat placenta, as well as in apical and basal membranes derived from human placenta. We have studied Na(+)-dependent alpha-(methylamino)isobutyric acid (MeAIB) transport in apical and basal predominant membrane fractions prepared from 14 and 20 day gestation rat placenta. Marker enzyme recoveries did not differ significantly between age groups. Markers for intracellular organelles were also found to be comparable. Na(+)-dependent MeAIB transport was not sensitive to freezing and could be found in all membrane components tested. Kinetic parameters were studied--Km = 852 +/- 215 microM, Vmax = 718 +/- 126 pmol/5 sec/mg protein--20 day apical; Km = 748 +/- 269 microM, Vmax = 610 +/- 176 pmol/5 sec/mg protein--20 day basal-predominant; Km 614 +/- 261 microM, Vmax = 123 +/- 45 pmol/5 sec/mg protein-14 day apical. Kinetic parameters could not be determined in the 14 day gestation basal-predominant fraction because of the small amount of uptake present. We conclude that System A like activity is found in both apical and basal predominant membrane fractions derived from rat placenta, and that this activity increases over the last one third of gestation.

Effect of low-protein diet-induced intrauterine growth retardation on rat placental amino acid transport.

Given the central role of the placenta in nutrient transport to the fetus, one might propose that maternal nutrition would have a regulatory effect on this nutrient delivery. We have examined the effect of a low-protein adequate-calorie diet on specific amino acid transport processes by the rat placenta. Maternal weight, fetal weight, and placental weight were all significantly reduced in dams fed a low-protein (5% casein), isocaloric diet when compared with dams pair-fed a control (20% casein) diet. Even though maternal serum amino acid levels were maintained in the low-protein animals, fetomaternal serum amino acid ratios were significantly reduced, suggesting a reduction in nutrient transfer to the fetus. Apical and basal membrane vesicles were isolated from the placental trophoblast and were used to examine the amino acid transport capacity of both maternal-facing and fetal-facing membranes, respectively. Na+-dependent neutral amino acid transport mediated by system A was decreased in both membrane preparations, while transport mediated by system ASC was unaffected. The Na+-dependent anionic amino acid uptake by system X(-)AG (EAAC1) was reduced on the basal membrane, while the Na+-independent component was similar between the low-protein and control diet-fed dams. Cationic amino acid uptake was also reduced on both membrane surfaces. A decreased steady-state mRNA content for EAAC1 and CAT1 (system y+) suggests that reduced synthesis of the transporter proteins is responsible for the decrease in transport activity. Taken together, these data support the hypothesis that maternal protein malnutrition affects nutrient delivery to the fetus by downregulation of specific amino acid transport proteins.

Molecular biology of mammalian amino acid transporters.

Recently a number of alpha-amino acid transport proteins and corresponding cDNA clones have been isolated and categorized into gene families. The "CAT family" contains two members that mediate high-affinity Na(+)-independent transport of cationic amino acids in many tissues, and a third member that encodes a liver-specific low-affinity activity. The "glutamate transporter family" contains at least four members that mediate Na(+)-dependent glutamate/aspartate uptake and two members that are selective for neutral amino acids. The glutamate transporters are expressed at high levels in both glia and neurons of the central nervous system. The Na+/Cl(-)-dependent proline transporter (PROT) belongs to a large superfamily of neurotransmitter transporters and is expressed in regions of the brain that contain glutamanergic neurons. All four glycine transporters of the "GLYT family" also belong to the neurotransmitter superfamily and exhibit the greatest expression in the central nervous system. The "rBAT/4F2hc family" of proteins induce both neutral and cationic amino acid uptake when expressed in Xenopus oocytes. Cystinuria is linked to specific mutations in the rBAT sequence.

Regulation of system y+ arginine transport capacity in differentiating human intestinal Caco-2 cells.

This study describes the ability of passaged human intestinal Caco-2 cells to regulate transport of L-arginine via system y+. Subconfluent and confluent cells possessed system y+ activity, as determined by the sodium independence of uptake and the pattern of inhibition by amino acid analogues or N-ethylmaleimide. Initial rates of arginine uptake via system y+ decreased as the cells advanced from the undifferentiated to the differentiated state following culture passaging. Furthermore, kinetic analysis of the leucine-insensitive portion of uptake indicated that the Caco-2 system y+ transport capacity decreased with cell age, dropping from a maximal velocity (Vmax) = 1,094 pmol.mg-1.min-1 [Michaelis constant (Km) = 41 microM] in undifferentiated cells 2 days postseeding to Vmax = 320 pmol.mg-1.min-1 (Km = 37 microM) in confluent cells 9 days postseeding (from cells of the same passage). Northern analysis indicated that the levels of a single 7.9-kb mCAT-1 mRNA species were relatively constant over the course of Caco-2 differentiation and therefore were unsynchronized with the system y+ relative transport activities. It is concluded that the Caco-2 capacity to transport arginine via system y+ may be downregulated by posttransitional modifications in confluent cells compared with newly passaged undifferentiated cells. These data serve as a well-defined in vitro model for further studies regarding regulation of arginine transport in epithelial cells.

Ontogeny of cationic amino acid transport systems in rat placenta.

Gestational regulation of the placental transfer of amino acids from maternal to fetal circulations is essential for the proper development of the fetus. The cationic amino acid transport systems of the microvillous (maternal facing) and basal (fetal facing) membranes of the rat placental syncytiotrophoblast were examined. Inhibition analysis documented the presence of three kinetically distinct cationic amino acid transport mechanisms: a single Na(+)-dependent mechanism in the microvillous membrane, which increased in activity from 14 to 20 days gestation but was absent from the basal membrane throughout the entire gestational period (system Bo,+), and two Na(+)-independent transport systems in both membrane domains, one that is completely inhibited by leucine, which increased in activity in both the microvillous and basal membrane domains, and the other that is leucine insensitive, which remained fairly constant in the basal membrane and increased throughout gestation in the microvillous membrane (system y1+). Northern analysis with the system y1+ cDNA revealed a specific band of approximately 7.4-7.9 kb, which increased with increasing gestational age.

Plasma membrane clustering of system y+ (CAT-1) amino acid transporter as detected by immunohistochemistry.

Transport of cationic amino acids in fully differentiated mammalian cells is mediated primarily by system y1+ [cationic amino acid transporter (CAT)-1 gene product]. Antibodies, prepared against synthetic peptide sequences predicted to be extracellular loops of the CAT-1 transporter protein, detected the transporter on the surface of cultured cells. In human fibroblasts, porcine pulmonary artery endothelial cells, and cultured rat hepatoma cells, the CAT-1 transporter protein was clustered in an apparent random pattern throughout the plasma membrane. In contrast, labeling of the fibroblasts with antibodies against the epidermal growth factor receptor or the GLUT-1 glucose transporter demonstrated a uniform staining pattern covering the entire cell surface. The CAT-1 antibody labeling was specific, as demonstrated by peptide inhibition and the lack of staining by preimmune serum. Furthermore, hepatocytes did not exhibit specific antibody binding consistent with the lack of system y1+ activity. Disruption of the microtubule assembly resulted in a reversible loss of the CAT-1 transporter clusters and a more generalized labeling of the cell body. The data demonstrate the existence of microdomains within the plasma membrane that contain the CAT-1 transporter protein.