Energy Metabolism and Amino Acid Transport During Early Development of Antarctic and Temperate Echinoderms.

Among the members of the major facilitator superfamily of Saccharomyces cerevisiae, we identified genes involved in the transport into vacuoles of the basic amino acids histidine, lysine, and arginine. ATP-dependent uptake of histidine and lysine by isolated vacuolar membrane vesicles was impaired in YMR088c, a vacuolar basic amino acid transporter 1 (VBA1)-deleted strain, whereas uptake of tyrosine or calcium was little affected. This defect in histidine and lysine uptake was complemented fully by introducing the VBA1 gene and partially by a gene encoding Vba1p fused with green fluorescent protein, which was determined to localize exclusively to the vacuolar membrane. A defect in the uptake of histidine, lysine, or arginine was also observed in the vacuolar membrane vesicles of mutants YBR293w (VBA2) and YCL069w (VBA3). These three VBA genes are closely related phylogenetically and constitute a new family of basic amino acid transporters in the yeast vacuole.

Some regulatory aspects of neutral amino acid transport were investigated in isolated brain microvessels, an in vitro model of the blood-brain barrier. Preloading of the microvessels with glutamine stimulated the subsequent uptake of other neutral amino acids by way of the Na+-independent L system, but had no effect on the uptake of either basic or acidic amino acids. Moreover, this stimulation was abolished when the loading step was carried out in the absence of Na+ ions or in the presence of a high concentration of alpha-methylaminoisobutyric acid, indicating that the microvessels were able to concentrate glutamine via the A system of amino acid transport. Since the presence of the A system of neutral amino acid transport has not been detected in studies of blood-brain transport performed in vivo, the A system is probably associated with the antiluminal side of brain microvessels. Our results indicate, therefore, that the concentrative Na+-dependent A system and the exchanging Na+-independent L system can cooperate in the uptake of the large neutral hydrophobic amino acids. Such a cooperation may be relevant in the pathogenesis of some neurological disturbances such as hepatic encephalopathy, in which brain glutamine concentration is unusually high.

Perinatal changes in the uptake of amino acids were measured in slices of fetal (15- and 19-day) and newborn (4-, 24-, and 48-hr-old) mouse brain. Uptake increased with age; smaller changes occurred with basic and neutral amino acid transport systems, and the largest changes occurred in fetal brain with amino acids of putative neurotransmitter function (taurine, glycine, GABA, and the acidic amino acids). The pattern of increase in uptake was similar at high and at low external amino acid concentrations. Developmental changes in tissue content of Na(+), K(+), or ATP were small during this period, and so are unlikely to be responsible for the observed changes in uptake. It appears that by the 15th day of fetal life, the transport systems for essential amino acids are fairly well developed in the brain, and the transport systems for neurotransmitter amino acids are not so well developed, but undergo a rapid increase in the 15-19-day period. From birth to adulthood, the concentrative capacity of slices of mouse brain for nonessential (putative neurotransmitter) amino acids is much greater than for essential amino acids.

In this review we discuss the beneficial effects of amino acid transport and metabolism on pre- and peri-implantation embryo development, and we consider how disturbances in these processes lead to undesirable health outcomes in adults. Proline, glutamine, glycine, and methionine transport each foster cleavage-stage development, whereas leucine uptake by blastocysts via transport system B0,+ promotes the development of trophoblast motility and the penetration of the uterine epithelium in mammalian species exhibiting invasive implantation. (Amino acid transport systems and transporters, such as B0,+, are often oddly named. The reader is urged to focus on the transporters’ functions, not their names.) B0,+ also accumulates leucine and other amino acids in oocytes of species with noninvasive implantation, thus helping them to produce proteins to support later development. This difference in the timing of the expression of system B0,+ is termed heterochrony—a process employed in evolution. Disturbances in leucine uptake via system B0,+ in blastocysts appear to alter the subsequent development of embryos, fetuses, and placentae, with undesirable consequences for offspring. These consequences may include greater adiposity, cardiovascular dysfunction, hypertension, neural abnormalities, and altered bone growth in adults. Similarly, alterations in amino acid transport and metabolism in pluripotent cells in the blastocyst inner cell mass likely lead to epigenetic DNA and histone modifications that produce unwanted transgenerational health outcomes. Such outcomes might be avoided if we learn more about the mechanisms of these effects.

Available data indicate little reutilization of 3-methylhistidine (3-MH) in the rat and man. These data led to the use of urinary 3-MH excretion as a measure of muscle protein catabolism in those animal species. However, 3-MH excretion does not accurately measure protein catabolism in the sheep, pig, and rabbit. This is due, at least in part, to the fact that renal amino acid (AA) transport systems reabsorb 3-MH from the glomerular filtrate. The monkey differs from man in that its plasms contains significant quantities of 3-MH, suggesting an active renal transport system for this AA. The present study measured maternal and fetal plasma 3-MH levels in 33 pregnant rhesus monkeys to determine whether the non-human primate placenta contained transport sites concentrating this AA to the fetal plasma. Mean fetal plasma 3-MH concentrations were 16.4 +/- 6.71 micrometers/100 ml, while maternal levels were 9.45 +/- 3.69 micrometers/100 ml. The fetal to maternal gradient was maintained between 1.6 to 1.7 during the course of maternal infusions of various AA. Since placental AA transport systems are similar to those in the kidney and intestine, the data also suggest the presence of AA transport systems for 3-MH in the monkey, indicating that urinary 3-MH excretion would be a poor method for measuring muscle protein catabolism in this species.

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

Amino acid transport during mammalian embryogenesis has been characterized best in preimplantation conceptuses and chorioallantoic placentas. Most of the amino acid transport systems in the placental trophoblast appear to be similar to the well-known systems that are present in many other types of mammalian cells (e.g., Yudilevich and Sweiry, 1985; Ganapathy et al., 1986; Kudo et al., 1987; Johnson and Smith, 1988; Wheeler and Yudilevich, 1989; Moe and Smith, 1989; Karl et al., 1989; Hoeltzli and Smith, 1989; Hoeltzli et al., 1990; Kudo and Boyd, 1990). Although the placental systems probably contribute to net transfer of most amino acids from mother to fetus, the mechanisms of these net transfers remain to be determined. Similarly, the developmental etiologies and fates of the placental systems and their controls are not yet known. For these reasons, we will focus mainly on developmental changes in the activities of several novel amino acid transport systems in preimplantation mouse conceptuses. Since embryogenesis is preceded by oogenesis, we will also discuss available information concerning amino acid transport in mouse oocytes and their associated follicular cells. In addition, we will consider the endogenous amino acid transport systems in Xenopus oocytes since these cells may prove to be useful as a means of helping to isolate and characterize mammalian amino acid transporters and, eventually, their genes.

Amino acid transport by small intestinal, hepatic, and pancreatic epithelia

Alpha-aminoisobutyric acid (AIB), or alpha-methyl alanine, is a nonmetabolized amino acid transported into cells, particularly malignant cells, predominantly by the 'A' amino acid transport system. Since it is not metabolized, [1-11C]-AIB can be used to quantify A-type amino acid transport into cells using a relatively simple compartmental model and quantitative imaging procedures (e.g. positron tomography). The tissue distribution of [1-11C]-AIB was determined in six dogs bearing spontaneous tumors, including lymphosarcoma, osteogenic sarcoma, mammary carcinoma, and adenocarcinoma. Quantitative imaging with tissue radioassay confirmation at necropsy showed poor to excellent tumor localization. However, in all cases the concentrations achieved appear adequate for amino acid transport measurement at known tumor locations. The observed low normal brain (due to blood-brain barrier exclusion) and high (relative to brain) tumor concentrations of [1-11C]-AIB suggest that this agent may prove effective for the early detection of human brain tumors.

Endogenous amino acid transport systems and expression of mammalian amino acid transport proteins in Xenopus oocytes

The relationship between the transport of the thyroid hormone T3 and the transport of neutral amino acids was investigated in JAR human placental choriocarcinoma cells. The uptake of leucine, mediated by the amino acid transport system L, was inhibited by T3 and T4, and the nature of inhibition was competitive. Uptake of T3 into the cells was predominantly Na+ independent, and so was that of leucine. However, although an acidic extracellular pH stimulated leucine uptake, the uptake of T3 remained unaffected. In addition, leucine failed to inhibit T3 uptake. The aromatic amino acids phenylalanine and tryptophan were found to inhibit the uptake of T3, but these two amino acids were transported into the cells predominantly via system L. The amino acid transport system T, which is specific for aromatic amino acids, was not detectable in these cells. Treatment of the cells with the calmodulin antagonist CGS 9343 B stimulated the uptake of leucine and tryptophan, but inhibited the uptake of T3. Kinetic analysis of T3 uptake revealed the presence of a single saturable system for this hormone in these cells, and the Michaelis-Menten constant for this system was 0.77 +/- 0.06 microM. Metabolic poisons that interfere with the cellular generation of ATP had no effect on the uptake of T3. It is concluded that in placental choriocarcinoma cells, 1) T3 and T4 are high affinity competitive inhibitors of the amino acid transport system L, 2) uptake of T3 occurs via a specific Na(+)-independent, energy-independent, and saturable mechanism that is unrelated to the amino acid transport systems L and T, 3) the aromatic amino acids phenylalanine and tryptophan interact, although weakly, with the T3 uptake system, and 4) calmodulin-dependent processes participate in the regulation of the T3 uptake system.

The relationship between the transport of the thyroid hormone T3 and the transport of neutral amino acids was investigated in JAR human placental choriocarcinoma cells. The uptake of leucine, mediated by the amino acid transport system L, was inhibited by T3 and T4, and the nature of inhibition was competitive. Uptake of T3 into the cells was predominantly Na+ independent, and so was that of leucine. However, although an acidic extracellular pH stimulated leucine uptake, the uptake of T3 remained unaffected. In addition, leucine failed to inhibit T3 uptake. The aromatic amino acids phenylalanine and tryptophan were found to inhibit the uptake of T3, but these two amino acids were transported into the cells predominantly via system L. The amino acid transport system T, which is specific for aromatic amino acids, was not detectable in these cells. Treatment of the cells with the calmodulin antagonist CGS 9343 B stimulated the uptake of leucine and tryptophan, but inhibited the uptake of T3. Kinetic analysis of T3 uptake revealed the presence of a single saturable system for this hormone in these cells, and the Michaelis-Menten constant for this system was 0.77 +/- 0.06 microM. Metabolic poisons that interfere with the cellular generation of ATP had no effect on the uptake of T3. It is concluded that in placental choriocarcinoma cells, 1) T3 and T4 are high affinity competitive inhibitors of the amino acid transport system L, 2) uptake of T3 occurs via a specific Na(+)-independent, energy-independent, and saturable mechanism that is unrelated to the amino acid transport systems L and T, 3) the aromatic amino acids phenylalanine and tryptophan interact, although weakly, with the T3 uptake system, and 4) calmodulin-dependent processes participate in the regulation of the T3 uptake system.

The seasonal availability of food for Antarctic zoobenthic consumers affects, among other factors, the levels of excreted metabolites that can serve as a source of nitrogen (N) and phosphorus (P) for autotrophs. This study tested the effects of prolonged starvation on N and P excretion by Nacella concinna, Odontaster validus, and Sterechinus neumayeri, as well as changes in their body chemistry. In all animals starved for 65 days, a significant decrease in body protein content was observed (33% for S. neumayeri and approximately 23% for N. concinna and O. validus). Nitrogen excretion rates were higher than phosphorus, with mean values (in µmol N or P h−1 100 g−1) of 1.351 and 0.094 for N. concinna, 0.779 and 0.037 for O. validus, and 0.538 and 0.075 for S. neumayeri, respectively. Ammonium nitrogen accounted for 50% to 69% of total nitrogen, while total reactive phosphorus represented 35% to 42% of total phosphorus. The study confirmed the natural adaptation of zoobenthos to prolonged food scarcity and demonstrated that (i) protein was the primary energy source during starvation, (ii) excretion rates were negatively correlated with invertebrate body size and were higher for N than P, and (iii) the excreted N and P loads may serve as a source of nutrients for autotrophs and act as chemical signals in trophic chemoreception.

Placental amino acid transport systems and fetal growth restriction – A workshop report

Tyrosine phosphorylation-and epidermal growth factor-dependent regulation of the sodium-coupled amino acid transporter B 0 in the human placental choriocarcinoma cell line JAR