Placental Amino Acid Transfer Research Articles

Reduction of In Vivo Placental Amino Acid Transport Precedes the Development of Intrauterine Growth Restriction in the Non-Human Primate.

Intrauterine growth restriction (IUGR) is associated with reduced placental amino acid transport (AAT). However, it remains to be established if changes in AAT contribute to restricted fetal growth. We hypothesized that reduced in vivo placental AAT precedes the development of IUGR in baboons with maternal nutrient restriction (MNR). Baboons were fed either a control (ad libitum) or MNR diet (70% of control diet) from gestational day (GD) 30. At GD 140, in vivo transplacental AA transport was measured by infusing nine (13)C- or (2)H-labeled essential amino acids (EAAs) as a bolus into the maternal circulation at cesarean section. A fetal vein-to-maternal artery mole percent excess ratio for each EAA was measured. Microvillous plasma membrane (MVM) system A and system L transport activity were determined. Fetal and placental weights were not significantly different between MNR and control. In vivo, the fetal vein-to-maternal artery mole percent excess ratio was significantly decreased for tryptophan in MNR. MVM system A and system L activity was markedly reduced in MNR. Reduction of in vivo placental amino acid transport precedes fetal growth restriction in the non-human primate, suggesting that reduced placental amino acid transfer may contribute to IUGR.

Down-regulation of placental Cdc42 and Rac1 links mTORC2 inhibition to decreased trophoblast amino acid transport in human intrauterine growth restriction.

Intrauterine growth restriction (IUGR) increases the risk for perinatal complications and metabolic and cardiovascular disease later in life. The syncytiotrophoblast (ST) is the transporting epithelium of the human placenta, and decreased expression of amino acid transporter isoforms in the ST plasma membranes is believed to contribute to IUGR. Placental mechanistic target of rapamycin Complex 2 (mTORC2) signaling is inhibited in IUGR and regulates the trafficking of key amino acid transporter (AAT) isoforms to the ST plasma membrane; however, the molecular mechanisms are unknown. Cdc42 and Rac1 are Rho-GTPases that regulate actin-binding proteins, thereby modulating the structure and dynamics of the actin cytoskeleton. We hypothesized that inhibition of mTORC2 decreases AAT expression in the plasma membrane and amino acid uptake in primary human trophoblast (PHT) cells mediated by down-regulation of Cdc42 and Rac1. mTORC2, but not mTORC1, inhibition decreased the Cdc42 and Rac1 expression. Silencing of Cdc42 and Rac1 inhibited the activity of the System L and A transporters and markedly decreased the trafficking of LAT1 (System L isoform) and SNAT2 (System A isoform) to the plasma membrane. mTORC2 inhibition by silencing of rictor failed to decrease AAT following activation of Cdc42/Rac1. Placental Cdc42 and Rac1 protein expression was down-regulated in human IUGR and was positively correlated with placental mTORC2 signaling. In conclusion, mTORC2 regulates AAT trafficking in PHT cells by modulating Cdc42 and Rac1. Placental mTORC2 inhibition in human IUGR may contribute to decreased placental amino acid transfer and reduced fetal growth mediated by down-regulation of Cdc42 and Rac1.

A systems perspective on placental amino acid transport.

Placental amino acid transfer is a complex process that is essential for fetal development. Impaired amino acid transfer causes fetal growth restriction, which may have lifelong health consequences. Transepithelial transfer of amino acids across the placental syncytiotrophoblast requires accumulative, exchange and facilitated transporters on the apical and basal membranes to work in concert. However, transporters alone do not determine amino acid transfer and factors that affect substrate availability, such as blood flow and metabolism, may also become rate‐limiting for transfer. In order to determine the rate‐limiting processes, it is necessary to take a systems approach which recognises the interdependence of these processes. New technologies have the potential to deliver targeted interventions to the placenta and help poorly growing fetuses. While many factors are necessary for amino acid transfer, novel therapies need to target the rate‐limiting factors if they are going to be effective. This review will outline the factors which determine amino acid transfer and describe how they become interdependent. It will also highlight the role of computational modelling as a tool to understand this process.

Computational modelling of placental amino acid transfer as an integrated system

Placental amino acid transfer is essential for fetal development and its impairment is associated with poor fetal growth. Amino acid transfer is mediated by a broad array of specific plasma membrane transporters with overlapping substrate specificity. However, it is not fully understood how these different transporters work together to mediate net flux across the placenta. Therefore the aim of this study was to develop a new computational model to describe how human placental amino acid transfer functions as an integrated system. Amino acid transfer from mother to fetus requires transport across the two plasma membranes of the placental syncytiotrophoblast, each of which contains a distinct complement of transporter proteins. A compartmental modelling approach was combined with a carrier based modelling framework to represent the kinetics of the individual accumulative, exchange and facilitative classes of transporters on each plasma membrane. The model successfully captured the principal features of transplacental transfer. Modelling results clearly demonstrate how modulating transporter activity and conditions such as phenylketonuria, can increase the transfer of certain groups of amino acids, but that this comes at the cost of decreasing the transfer of others, which has implications for developing clinical treatment options in the placenta and other transporting epithelia.

Regulation of amino acid transporter trafficking by mTORC1 in primary human trophoblast cells is mediated by the ubiquitin ligase Nedd4-2.

Changes in placental amino acid transfer directly contribute to altered fetal growth, which increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. Placental amino acid transfer is critically dependent on the expression of specific transporters in the plasma membrane of the trophoblast, the transporting epithelium of the human placenta. However, the molecular mechanisms regulating this process are largely unknown. Nedd4-2 is an ubiquitin ligase that catalyses the ubiquitination of proteins, resulting in proteasomal degradation. We hypothesized that inhibition of mechanistic target of rapamycin complex 1 (mTORC1) decreases amino acid uptake in primary human trophoblast (PHT) cells by activation of Nedd4-2, which increases transporter ubiquitination resulting in decreased transporter expression in the plasma membrane. mTORC 1 inhibition increased the expression of Nedd4-2, promoted ubiquitination and decreased the plasma membrane expression of SNAT2 (an isoform of the System A amino acid transporter) and LAT1 (a System L amino acid transporter isoform), resulting in decreased cellular amino acid uptake. Nedd4-2 silencing markedly increased the trafficking of SNAT2 and LAT1 to the plasma membrane, which stimulated cellular amino acid uptake. mTORC1 inhibition by silencing of raptor failed to decrease amino acid transport following Nedd4-2 silencing. In conclusion, we have identified a novel link between mTORC1 signalling and ubiquitination, a common posttranslational modification. Because placental mTORC1 is inhibited in fetal growth restriction and activated in fetal overgrowth, we propose that regulation of placental amino acid transporter ubiquitination by mTORC1 and Nedd4-2 constitutes a molecular mechanisms underlying abnormal fetal growth.

Phenylalanine transfer across the isolated perfused human placenta: an experimental and modeling investigation.

Membrane transporters are considered essential for placental amino acid transfer, but the contribution of other factors, such as blood flow and metabolism, is poorly defined. In this study we combine experimental and modeling approaches to understand the determinants of [14C]phenylalanine transfer across the isolated perfused human placenta. Transfer of [14C]phenylalanine across the isolated perfused human placenta was determined at different maternal and fetal flow rates. Maternal flow rate was set at 10, 14, and 18 ml/min for 1 h each. At each maternal flow rate, fetal flow rates were set at 3, 6, and 9 ml/min for 20 min each. Appearance of [14C]phenylalanine was measured in the maternal and fetal venous exudates. Computational modeling of phenylalanine transfer was undertaken to allow comparison of the experimental data with predicted phenylalanine uptake and transfer under different initial assumptions. Placental uptake (mol/min) of [14C]phenylalanine increased with maternal, but not fetal, flow. Delivery (mol/min) of [14C]phenylalanine to the fetal circulation was not associated with fetal or maternal flow. The absence of a relationship between placental phenylalanine uptake and net flux of phenylalanine to the fetal circulation suggests that factors other than flow or transporter-mediated uptake are important determinants of phenylalanine transfer. These observations could be explained by tight regulation of free amino acid levels within the placenta or properties of the facilitated transporters mediating phenylalanine transport. We suggest that amino acid metabolism, primarily incorporation into protein, is controlling free amino acid levels and, thus, placental transfer.

UPLC-MS metabolic profiling of second trimester amniotic fluid and maternal urine and comparison with NMR spectral profiling for the identification of pregnancy disorder biomarkers

We report on the first untargeted UPLC-MS study of 2nd trimester maternal urine and amniotic fluid (AF), to investigate the possible metabolic effects of fetal malformations (FM), gestational diabetes mellitus (GDM) and preterm delivery (PTD). For fetal malformations, considerable metabolite variations were identified in AF and, to a lesser extent, in urine. Using validated PLS-DA models and statistical correlations between UPLC-MS data and previously acquired NMR data, a metabolic picture of fetal hypoxia, enhanced gluconeogenesis, TCA activity and hindered kidney development affecting FM pregnancies was reinforced. Moreover, changes in carnitine, pyroglutamate and polyols were newly noted, respectively, reflecting lipid oxidation, altered placental amino acid transfer and alterations in polyol pathways. Higher excretion of conjugated products in maternal urine was seen suggesting alterations in conjugation reactions. For the pre-diagnostic GDM group, no significant changes were observed, either considering amniotic fluid or maternal urine, whereas, for the pre-PTD group, some newly observed changes were noted, namely, the decrease of particular amino acids and the increase of an hexose (possibly glucose), suggesting alteration in placental amino acid fluxes and a possible tendency for hyperglycemia. This work shows the potential of UPLC-MS for the study of fetal and maternal biofluids, particularly when used in tandem with comparable NMR data. The important roles played by sampling characteristics (e.g. group dimensions) and the specific experimental conditions chosen for MS methods are discussed.

Facilitated transporters mediate net efflux of amino acids to the fetus across the basal membrane of the placental syncytiotrophoblast

Fetal growth depends on placental transfer of amino acids from maternal to fetal blood. The mechanisms of net amino acid efflux across the basal membrane (BM) of the placental syncytiotrophoblast to the fetus, although vital for amino acid transport, are poorly understood. We examined the hypothesis that facilitated diffusion by the amino acid transporters TAT1, LAT3 and LAT4 plays an important role in this process, with possible effects on fetal growth. Amino acid transfer was measured in isolated perfused human placental cotyledons (n = 5 per experiment) using techniques which distinguish between different transport processes. Placental TAT1, LAT3 and LAT4 proteins were measured, and mRNA expression levels (measured using real-time quantitative-PCR) were related to fetal and neonatal anthropometry and dual-energy X-ray absorptiometry measurements of neonatal lean mass in 102 Southampton Women's Survey (SWS) infants. Under conditions preventing transport by amino acid exchangers, all amino acids appearing in the fetal circulation were substrates of TAT1, LAT3 or LAT4. Western blots demonstrated the presence of TAT1, LAT3 and LAT4 in placental BM preparations. Placental TAT1 and LAT3 mRNA expression were positively associated with measures of fetal growth in SWS infants (P < 0.05). We provide evidence that the efflux transporters TAT1, LAT3 and LAT4 are present in the human placental BM, and may play an important role in the net efflux of amino acids to the fetus. Unlike other transporters they can increase fetal amino acid concentrations. Consistent with a role in placental amino acid transfer capacity and fetal growth TAT1 and LAT3 mRNA expression showed positive associations with infant size at birth.

Computational modelling of amino acid transfer interactions in the placenta

Amino acid transfer from mother to fetus via the placenta plays a critical role in normal development, and restricted transfer is associated with fetal growth restriction. Placental amino acid transfer involves the interaction of 15 or more transporters and 20 amino acids. This complexity means that knowing which transporters are present is not sufficient to predict how they operate together as a system. Therefore, in order to investigate how placental amino acid transfer occurs as a system, an integrated mathematical/computational modelling framework was developed to represent the simultaneous transport of multiple amino acids. The approach was based on a compartmental model, in which separate maternal, syncytiotrophoblast and fetal volumes were distinguished, and transporters were modelled on the maternal- and fetal-facing membranes of the syncytiotrophoblast using Michaelis-Menten-type kinetics. The model was tested in comparison with placental perfusion experiments studying serine-alanine exchange and found to correspond well. The results demonstrated how the different transporters can work together as an integrated system and allowed their relative importance to be assessed. Placental-fetal serine exchange was found to be most sensitive to basal membrane transporter characteristics, but a range of secondary, less intuitive effects were also revealed. While this work only addressed a relatively simple three amino acid system, it demonstrates the feasibility of the approach and could be extended to incorporate additional experimental parameters. Ultimately, this approach will allow physiological simulations of amino acid transfer. This will enhance our understanding of these complex systems and placental function in health and disease.

Effects of branched-chain amino acids on placental amino acid transfer and insulin and glucagon release in the ovine fetus

Objective: Competition for placental amino acid transporters can affect the fetal supply of amino acids. Specifically, the branched-chain amino acids—isoleucine, leucine, and valine—may inhibit the transfer of other amino acids. This study was undertaken to determine the effect of branched-chain amino acids on the umbilical uptake of amino acids. Study Design: Six late-gestation ewes were infused sequentially for 2 hours with 3 different mixtures of amino acids: (1) one that was comparable to commercial parenteral nutrition preparations, (2) the same solution without branched-chain amino acids, and (3) branched-chain amino acids alone. Maternal and fetal blood samples were collected simultaneously for the determination of uterine and umbilical uptake values of amino acids, and for concentrations of arterial insulin, glucagon, glucose, and lactate before (control) and during (experimental) infusion. Results: Umbilical uptake of branched-chain amino acids increased significantly when they were present in the infusates. The fetal uptake of several other amino acids could be increased by increasing their maternal concentrations. Inhibition of umbilical uptake by branched-chain amino acids could be shown for threonine and methionine. The infusion of branched-chain amino acids alone did not affect maternal and fetal insulin or glucagon concentrations. Conclusions: In late-gestation sheep, an increase in maternal plasma concentration of branched-chain amino acids led to increased branched-chain amino acid umbilical uptake, but branched-chain amino acids can also inhibit the transport of some amino acids to the fetus. Changes in fetal plasma concentration and uptake of branched-chain amino acid appear to have no significant effect on fetal insulin or glucagon. (Am J Obstet Gynecol 2001;185:487-95.)

Administration of tumor necrosis factor-alpha results in a decreased placental transfer of amino acids in the rat.

The administration of an acute tumor necrosis factor-alpha (TNF) dose (100 micrograms/kg BW) to 20-day pregnant rats resulted in a substantial decrease in the fetal availability of maternally administered amino acids, as measured by the accumulation of alpha-amino-[1-14C]isobutyrate ([14C]AIB) and [1-14C]cycloleucine ([14C]CLEU), nonmetabolizable analogs of the amino acids alanine and leucine, respectively. Thus, TNF treatment resulted in a decreased accumulation of the tracers in the whole fetus as well as in fetal liver. The cytokine also caused important changes on the maternal liver, where it increased both [14C]AIB and [14C]CLEU accumulation. In skeletal muscle and heart, TNF treatment resulted in decreased [14C]AIB accumulation, but increased [14C]CLEU. These changes in tissue amino acid uptake were accompanied by changes in circulating amino acids. TNF treatment promoted an increase in the concentrations of both alanine and leucine in the maternal circulation, whereas no changes in the circulating concentrations of these amino acids were observed in the fetuses. The decreased fetal accumulation of maternally derived amino acid analogs is partially explained by a decrease in fetal blood flow [as measured by the accumulation of [14C]1,1,1-trichloro-2,2- bis-(p-chlorophenyl)ethane] induced by the cytokine). It is suggested that the cytokine may be involved in fetal growth impairment during pathological states (such as tumor growth or chronic infection) by promoting a decreased transplacental passage of amino acids, essential compounds for both protein accretion and oxidation in fetal metabolism.

Administration of tumor necrosis factor-alpha results in a decreased placental transfer of amino acids in the rat

Administration of tumor necrosis factor-alpha results in a decreased placental transfer of amino acids in the rat

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.

THE ROLE OF THE INSULIN-LIKE GROWTH FACTORS IN NORMAL AND ABNORMAL FETAL GROWTH

Fetal growth (FG) in late gestation (LG) is primarily determined by uteroplacental transfer of nutrients. FG is normally constrained by the capacity of this transfer. In LG, fetal IGF-1 is acutely regulated by glucose, but not by amino acid availability, acting via enhanced insulin release; this provides the mechanism of fetal overgrowth induced by hyperinsulinism. Fetal IGF-1 levels correlate with fetal nutritional status and thus FG. Direct evidence for the role of IGF-1 in FG is provided by embryo transfer experiments in mice selected for high or low IGF-1 levels. The infusion of IGF-1 (60μg/kg/hr) to LG fetal sheep increased IGF-1 4 fold without a measurable change in fetal or placental glucose uptake. However placental lactate production fell (p<0.03). The amino-nitrogen concentration fell (p<0.01) in both mother and fetus as did fetal urea production (p <0.05). These observations suggest that IGF-1 inhibits fetal protein catabolism, promotes fetal anabolism, enhances placental amino acid transfer and alters placental metabolism favourably. It is suggested that glucose availability affects fetal IGF-1 production via altered insulin secretion and this in turn has anabolic and anticatabolic consequences. IGF binding proteins are also acutely modulated by nutrient availability. Fetal IGF-2 in late gestation is under lesser nutritional regulation compatible with a more constitutive role in the regulation of FG. Maternal IGF-1 administration throughout pregnancy in rodents abolishes the physiological constraints on FG without affecting placental growth. Maternal IGF-1 administration reduces fetal urea production in sheep. It is suggested that maternal IGF-1, under nutritional and hormonal control, determines nutrient availability and transfer across the placenta. Thus optimal FG depends on coordinate increases in both fetal and maternal IGF-1.

Effect of inorganic mercury on in vitro placental nutrient transfer and oxygen consumption

The effect of mercury (HgCl 2) on placental amino acid and glucose transfer as determined by the use of their nonmetabolizable radioactive analogues, aminoisobutyric acid (AIB) and 3- o-metyhl glucose (3MG), respectively, was studied in an in vitro perfusion model of a term human placenta. Hg 2+ was found to decrease the transfer and accumulation of AIB without affecting 3MG transfer. It was also found to decrease the placental oxygen consumption rate. Placental circulation and tissue morphology remained intact, as demonstrated by the antipyrine transfer rate, and by electron microscopy, respectively. The mechanism by which Hg 2+ may interfere with placental amino acid transfer and accumulation is discussed. Although much higher concentrations than those found in the ordinary polluted environment were used, this is the first report showing that Hg 2+ interferes with an essential human placental function in a system employing a whole human placental cotyledon. This finding may indicate the possible involvement of Hg 2+ in impaired organogenesis in early pregnancy or deranged fetal growth during the last trimester.

Investigations of protein metabolism in human pregnancy: the term foetus and placenta studied using stable isotope labelled amino-acids

Because the placenta functions as a very efficient provider of ‘total parenteral nutrition’, the foetus enjoys a rapid growth rate in relation to its body weight (14). The growth rate exceeds that of any other time during life, including the adolescent growth spurt (12). In human pregnancy, maternal plasma total a-amino nitrogen is reduced from about 3.0 to 2.3mM, however, the total a-amino nitrogen in foetal plasma is 3.8mM, giving a F:M ratio of 1.7 (31). Thus, in fact, all the free amino-acid concentrations in the umbilical artery are higher than in the maternal ante-cubital vein (30), so the placental transfer of amino-acids to the foetus must occur against a concentration gradient, i.e. be due to some process of active transport (22), probably Na-linked. Widdowson et al (29) measured the amino-acid composition of the body and organs of a few human foetuses of accurately known gestational ages (79 days-term), and although such values included the small amount of amino-acids in the free pool as well as the larger amount of aminoacids incorporated into the protein, they clearly showed that nitrogen does not begin to increase rapidly until about 160 days of gestation. Thereafter, the foetus gains weight quickly with rapid nitrogen (mainly protein) deposition: from midgestation to term, the human foetus grows at an average rate of 1.5% per day (19); for a birth weight at term gestation of 3.5 kg, this equals a final growth velocity of 15 g/kg birth weight/day. Amino-acids are the obligatory precursors for body protein, and naturally they are utilised by the foetus for growth but, despite their obvious importance, it is only relatively recently that we have information concerning their metabolism in utero. In sheep, it has been found that the rate of amino-acid provision to the foetus exceeded its rate of accretion in tissue proteins (2, 3). This suggested that amino-acids might also be used as fuel for oxidation or as substrates for lipid, neurotransmitter and nucleic acid synthesis and possibly for gluconeogenesis in the kidney but not in the liver, which is poorly developed. Studies in fasting pregnant sheep using 14C labelled leucine have shown that the ratio of the foetal leucine oxidation rate to the leucine disposal rate was doubled when compared to the fed state (27). Foetal oxidation of alanine, glycine and lysine has also been observed (19). Results of experiments using 14C lysine and 14C leucine in foetal lambs have also shown that the fractional protein synthetic rate in late gestation is twoor three-fold higher than the fractional growth rate, confirming the hypothesis that foetal protein turnover is likely to be high (19). Starting from a low base, the last 10 years have seen an increasing amount of information being gathered on the rates of growth and protein turnover in human neonates, almost exclusively for mature infants. In studies using the 15N glycine/ NH3-urea end product method on very low birth weight premature infants (< 1500g) receiving 483 kJ/kg/day and 3g protein/kg/day as breast or formula milk, the total body protein accretion rate was found to be 1.8 g/kg/day and the observed average weight gain was found to be 15 g/kg/day (8). This agrees closely with the value of 1.9g of protein gained/kg/day obtained from studies using L-[l-13C] leucine (4) in a continuous infusion plasma kinetics protocol. Studies to measure whole blood umbilical V-A concentration differences and umbilical blood flow in chronic unstressed foetal lamb have been performed by the group of Battaglia and Meschia:

Human placental acetylcholine

The human placenta contains both acetylcholine (ACh) and choline acetyltransferase, and in vitro bilaterally perfused placental lobules release ACh. The function of this placental cholinergic system has not yet been clearly defined, although changes occur in it during parturition and it may be linked to placental prostaglandin generation at this time. It has also been suggested that ACh may regulate placental amino-acid transport and/or blood flow. It has been found that ACh release from fetal vessels of bilaterally perfused placental lobules is reduced during preeclampsia but is not necessarily correlated with any change in perfusion pressure or materno-fetal transfer of the nonmetabolizable amino acid alpha-aminoisobutyric acid. However, a correlation has been found between releases from human placental explants of ACh (when inhibited by (2-benzoylethyl)trimethylammonium or vesamicol) and of prostaglandins E2 and F2 alpha. Thus, although the evidence for a role of ACh in the control of placental amino-acid transfer or vascular tone is not conclusive, inhibition of the human placental cholinergic system has been shown to be associated with reduced output of prostaglandins from this tissue.

Lysine and alanine transport in the perfused guinea-pig placenta

The characteristics of l-lysine transport were investigated at brush-border (maternal) and basal (fetal) sides of the syncytiotrophoblast in the term guinea-pig placenta artificially perfused either through the umbilical vessels in situ or through both circulations simultaneously. Cellular uptake, efflux and transplacental transfer were determined using a single-circulation paired-tracer dilution technique. Unidirectional l-[ 3H]lysine uptake (%) (perfusate lysine 50 μM) was high on maternal ( M = 87 ± 1) and fetal ( F = 73 ± 2) sides. l-[ 3H]Lysine efflux back into the ipsilateral circulation was asymmetrical (F/M ratio = 2.3) and transplacental flux occurred in favour of the fetal circulation. Unidirectional lysine influx kinetics (0.05–8.00 mM) gave K m values of 1.75 ± 0.70 mM and 0.90 ± 0.25 mM at maternal and fetal sides, respectively; corresponding V max values were 1.95 ± 0.38 and 0.87 ± 0.10 μmol·min −1·g −1. At both sides, lysine influx (50 μM) could be inhibited (about 60–80%) by 4 mM l-lysine and l-ornithine and less effectively (about 10–40%) by l-citrulline, l-arginine, d-lysine and l-histidine. At the basal side: (i) lysine influx kinetics were greatly modified in the presence of 10 mM l-alanine ( K m = 6.25 ± 3.27 mM; V max = 2.62 ± 0.94 μmol·min −1 · g −1), but unchanged by equimolar l-phenylalanine or l-tryptophan; (ii) in the converse experiments, lysine (10 mM) did not affect the kinetic characteristics for either l-alanine or l-phenylalanine; (iii) l-lysine and l-alaninie influx kinetics were not dependent on the sodium gradient; (iv) the inhibition of l-[ 3H]lysine uptake by 4 mM l-homoserine was partially (60%) Na +-dependent. At the maternal side the kinetic characteristics for alanine influx were highly Na +-dependent, while lysine influx was partially Na +-dependent only at low concentrations (0.05–0.5 mM). Bilateral perfusion with 2,4-dinitrophenol (1 mM) reduced l-[ 3H]lysine uptake into the trophoblast and abolished transplacental transfer. It is suggested that lysine transport in the guinea-pig placenta is mediated by a specific transport system (y +) for cationic amino-acids. The asymmetry in the degree of sodium-dependency at both trophoblast membranes may in part explain the maternal-to-foetal polarity of placental amino-acid transfer in vivo.

Some aspects of amino acid metabolism in the rat fetus

1. 1. In spite of an eventual catabolic phase during the last third of pregnancy, nitrogen retention seems to increase in pregnant rats. Furthermore, the high uterine blood flow and the high placental transfer of amino acids maintains an adequate nutrient supply to the fetuses. 2. 2. The terminal rat fetus has a high circulating plasma amino acid level, as well as an increased free amino acid tissue pool when compared to its mother's. 3. 3. In the rat fetus the development of enzymatic capabilities shows a sudden emergence (also denomined clustering) in late fetal life. In a general trend, the activities of enzymes related with amino acid metabolism are not well developed during rat fetal life. 4. 4. The rate of amino nitrogen excertion in rat fetus is low, mainly due to the low development of urea cycle enzyme activities. 5. 5. The rates of protein synthesis in many tissues are high in the rat fetus and they show a progressive decrease until delivery. On the other hand, the rates of protein breakdown are also higher during fetal life than in the adult.

Inhibition of placental γ-glutamyltransferase results in a decreased placental transfer of alanine

The possibility that the y-glutamyl cycle functions as an amino acid transport system (Meister, 1973) is still an open question. Evidence against a direct role for yglutamyltransferase (GGT) (EC 2.3.2.2.) in amino acid transport in renal brush-border membrane vesicles has been offered (Hsu et al., 1984). However, we reported that GGT is involved in amino acid uptake by mammary gland in vivo (Viiia et al., 1981, 1983). The aim of this work was to study the possible role of GGT in placental transfer of amino acids. We recently developed a method to measure placental transfer of metabolites (Lasuncion et al., 1983) and have used it to study the effect of starvation (Palacin et al, 1985a) and diabetes (Herrera et al., 1985) on the placental transfer of substrates. It basically consists of the infusion through the left uterine artery of tracer amounts of I4C-labelled metabolites dissolved in physiological saline. Thus, the left uterine horn receives the tracer directly, whereas it reaches the right horn after dilution and metabolism in the mother. Twenty-day-pregnant Wistar rats were anaesthetized with pentobarbital and then 1 ml of 0.9% NaCl (controls) or 1 ml of 0.9% NaCl containing 5 mg of acivicin (AT. 125, Upjohn, Kalamazoo, U.S.A.) was injected. After 30 min we opened the abdominal cavity and infused 10 pCi of ~-[U-'~C]alanine through the left uterine artery. Foetuses from both uterine horns were processed separately. GGT activity was measured as described by Tate & Meister (1974) and alanine as described by Williamson (1974). E. Herrera, J. Lorenzo, M. A. Lasuncion & M. Palacin (personal communication) observed that the blood flow to rat uterine horns is proportional to the number of foetuses contained in the