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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.

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.

Conference Article| August 01 1996 Rat placental amino acid transport after protein-deprivation-induced intrauterine growth retardation M. S. Malandro; M. S. Malandro *Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Box 100245, Gainesville, FL 32610-0245, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar M. J. Beveridge; M. J. Beveridge †Department of Pediatrics, University of Florida College of Medicine, Box 100245, Gainesville, FL 32610-0245, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar D. A. Novak; D. A. Novak †Department of Pediatrics, University of Florida College of Medicine, Box 100245, Gainesville, FL 32610-0245, U.S.A. Search for other works by this author on: This Site PubMed Google Scholar M. S. Kilberg M. S. Kilberg ‡ *Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Box 100245, Gainesville, FL 32610-0245, U.S.A. †To whom correspondence should be addressed. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1996) 24 (3): 839–843. https://doi.org/10.1042/bst0240839 Article history Received: March 29 1996 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation M. S. Malandro, M. J. Beveridge, D. A. Novak, M. S. Kilberg; Rat placental amino acid transport after protein-deprivation-induced intrauterine growth retardation. Biochem Soc Trans 1 August 1996; 24 (3): 839–843. doi: https://doi.org/10.1042/bst0240839 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: IUGR, intrauterine growth retardation, AIB, 2-aminoisobutyric acid This content is only available as a PDF. © 1996 Biochemical Society1996 Article PDF first page preview Close Modal You do not currently have access to this content.

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.

The extracellular concentration of glutamate and other related excitatory amino acids (EAA) is regulated by the action of transporter proteins located on either presynaptic terminals or adjacent astroglial processes. Recent molecular advances have led to the cloning of three separate cDNAs encoding for Na(+)-dependent glutamate transporters; two are thought to be primarily glial in origin (GLAST and GLT-1) and the third (EAAC1) is localized to neurons in the brain and other nonneural tissues. An EAAC1 cDNA was initially cloned from rabbit small intestine (13). In this study, we report isolation and characterization of the homologous clone from rat brain. Northern blot hybridization revealed high levels of EAAC1 mRNA in rat brain and kidney and low levels in heart, lung, and skeletal muscle. Transient expression of EAAC1 in HeLa cells resulted in an increase in Na(+)-dependent high-affinity L-[3H]glutamate and D-[3H]aspartate transport. The pharmacological profile of EAAC1 was very similar to that reported for the rabbit and human EAAC1 homologues. Transport activity was potently inhibited by D- and L-threo-beta-hydroxyaspartate and L-trans-pyrrolodine-2,4-dicarboxylate. Dihydrokainate and L-alpha-aminoadipate did not inhibit transport at concentrations below 1 mM. Oligonucleotide cDNA probes (45-mer) were constructed and labeled with 35S-ATP for film- and emulsion-based in situ hybridization of rat brain. EAAC1 mRNA had the highest density in the cerebellar granule cell layer, hippocampus, superior colliculus, and neocortex. Sections that were emulsion-dipped and counterstained with cresyl violet revealed EAAC1 labeling localized exclusively over neuronal cell bodies, including some nonglutamatergic neurons such as spinal cord ventral horn cells.

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.