Amino Acid Transport Function Research Articles

Transport Kinetics of Alpha-Aminoisobutyric Acid and Water in Pre-Eclamptic Pregnancies: An in vitro Study

Objective: To investigate the transport kinetics of a model amino acid, alpha-aminoisobutyric acid (AIB), in the maternal-fetal direction in placentae from pre-eclamptic pregnancies. Methods: Transport kinetics of the amino acid was assessed in vitro using perfusion of human placental lobules. Control placental lobules were perfused for comparison. National Culture and Tissue Collection-135 medium diluted with Earle’s buffered salt solution was used as the perfusate, and tritiated water served as the internal reference marker. AIB along with reference marker was injected as a single bolus into the maternal arterial perfusate, and serial perfusate samples were collected from maternal and fetal venous circuits for a study period of 5 min. Results: The differential transport rate of the amino acid for various efflux fractions differed significantly between control and experimental groups when compared to that of the reference marker. The transport rate (corresponding to 50% of efflux into the fetal vein), transport fraction, absorption and elimination rates of the amino acid differed significantly compared to the reference marker values in study and control groups. Conclusion: The results indicate that amino acid transport function is compromised in placentae of pre-eclamptic pregnancies.

Function and structure of heterodimeric amino acid transporters.

Heterodimeric amino acid transporters are comprised of two subunits, a polytopic membrane protein (light chain) and an associated type II membrane protein (heavy chain). The heavy chain rbAT (related to b(0,+) amino acid transporter) associates with the light chain b(0,+)AT (b(0,+) amino acid transporter) to form the amino acid transport system b(0,+), whereas the homologous heavy chain 4F2hc interacts with several light chains to form system L (with LAT1 and LAT2), system y(+)L (with y(+)LAT1 and y(+)LAT2), system x (with xAT), or system asc (with asc1). The association of light chains with the two heavy chains is not unambiguous. rbAT may interact with LAT2 and y(+)LAT1 and vice versa; 4F2hc may interact with b(0,+)AT when overexpressed. 4F2hc is necessary for trafficking of the light chain to the plasma membrane, whereas the light chains are thought to determine the transport characteristics of the respective heterodimer. In contrast to 4F2hc, mutations in rbAT suggest that rbAT itself takes part in the transport besides serving for the trafficking of the light chain to the cell surface. Heavy and light subunits are linked together by a disulfide bridge. The disulfide bridge, however, is not necessary for the trafficking of rbAT or 4F2 heterodimers to the membrane or for the functioning of the transporter. However, there is experimental evidence that the disulfide bridge in the 4F2hc/LAT1 heterodimer plays a role in the regulation of a cation channel. These results highlight complex interactions between the different subunits of heterodimeric amino acid transporters and suggest that despite high grades of homology, the interactions between rbAT and 4F2hc and their respective partners may be different.

Regulation of amino acid transporters by amino acid availability.

There is growing recognition that amino acid availability has profound effects on many aspects of cell function, including the control of membrane transport mechanisms, cell signalling, and gene expression. The precise mechanisms by which amino acids are able to elicit control over such diverse processes have become the focus of intense investigation recently. One particular area that has seen considerable advances is the molecular characterization of amino acid transporters, including members of the System A family, which are known to be regulated by amino acid supply. Recent developments concerning how cells sense and signal amino acid availability, and how this process influences the expression and function of amino acid transporters, are reviewed here. Elucidating the molecular mechanisms of these events will be important in clarifying how amino acid transporters might be regulated during altered nutritional states, and will be crucial for the design of new strategies aimed at improving nutritional support.

Structure and function of plasma membrane amino acid, oligopeptide and sucrose transporters from higher plants.

Structure and function of plasma membrane amino acid, oligopeptide and sucrose transporters from higher plants.

Erythroid Progenitor Proliferation is Stimulated by Phenoxyacetic and Phenylalkyl Acids

ABSTRACT Short-chain fatty acids, such as butyrate and propionate, are under investigation as therapeutic stimulants of fetal hemoglobin production in the β-hemoglobin disorders. Significant limitations to these fatty acids and derivatives as optimal therapeutics are their rapid metabolism in vivo and their induction of cell growth arrest in the G 1 phase of the cell cycle. This antiproliferative activity is related to their inhibition of metabolic transport pumps which are essential for cell proliferation. Other small carbon compounds, the phenylalkyl acids, phenoxyacetic acids, and phenylacetic acids, which are structurally resistant to oxidative metabolism, are shown here to induce fetal globin production in human erythroid cultures at concentrations of 0.2 mM, lower than those required for most other fatty acids. Certain of these compounds were found not to inhibit cellular neutral amino acid transport function in erythroid cells, nor to inhibit erythroid colony (Bfu-e) growth. Certain of these compounds even stimulated human Bfu-e proliferation in vitro beyond that induced by optimal concentrations of hematopoietic growth factors. The combination of increased fetal globin chain production by these compounds and their stimulatory effects on erythropoiesis result in an increase in Hb F-expressing erythroid cells in culture several-fold greater than that achieved by the butyrates. These new compounds thus have the potential to provide superior therapy for the β-hemoglobinopathies and other anemias.

Placental amino acid transport

Normal fetal growth and development depend on a continuous supply of amino acids from the mother to the fetus. The placenta is responsible for the transfer of amino acids between the two circulations. The human placenta is hemomonochorial, meaning that the maternal and fetal circulations are separated by a single layer of polarized epithelium called the syncytiotrophoblast, which is in direct contact with maternal blood. Transport proteins located in the microvillous and basal membranes of the syncytiotrophoblast are the principal mechanism for transfer from maternal blood to fetal blood. Knowledge of the function and regulation of syncytiotrophoblast amino acid transporters is of great importance in understanding the mechanism of placental transport and potentially improving fetal and newborn outcomes. The development of methods for the isolation of microvillous and basal membrane vesicles from human placenta over the past two decades has contributed greatly to this understanding. Now a primary cultured trophoblast model is available to study amino acid transport and regulation as the cells differentiate. The types of amino acid transporters and their distribution between the syncytiotrophoblast microvillous and basal membranes are somewhat unique compared with other polarized epithelia. These differences may reflect the unusual circumstance of this epithelium that is exposed to blood on both sides. The current state of knowledge as to the types of transport systems present in syncytiotrophoblast, their regulation, and the effects of maternal consumption of drugs on transport are discussed.

Amino acid transporters of lower eukaryotes: regulation, structure and topogenesis

Lower eukaryotes such as the yeast Saccharomyces cerevisiae and the filamentous fungus Aspergillus nidulans possess a multiplicity of amino acid transporters or permeases which exhibit different properties with respect to substrate affinity, specificity, capacity and regulation. Regulation of amino acid uptake in response to physiological conditions of growth is achieved principally by a dual mechanism; control of gene expression, mediated by a complex interplay of pathway-specific and wide-domain transcription regulatory proteins, and control of transport activities, mediated by a series of protein factors, including a kinase, and possibly, by amino acids. All fungal and a number of bacterial amino acid permeases show significant sequence similarities (33-62% identity scores in binary comparisons), revealing a unique transporter family conserved across the prokaryotic-eukaryotic boundary. Prediction of the topology of this transporter family utilizing a multiple sequence alignment strongly suggests the presence of a common structural motif consisting of 12 alpha-helical putative transmembrane segments and cytoplasmically located N- and C-terminal hydrophilic regions. Interestingly, recent genetic and molecular results strongly suggest that yeast amino acid permeases are integrated into the plasma membrane through a specific intracellular translocation system. Finally, speculating on their predicted structure and on amino acid sequence similarities conserved within this family of permeases reveals regions of putative importance in amino acid transporter structure, function, post-translational regulation or biogenesis.

Amino acid (system A) transporter activity in microvillous membrane vesicles from the placentas of appropriate and small for gestational age babies.

Although a number of causes of poor fetal growth are known, the involvement of placental transport proteins in the etiology of growth retardation is not understood. The aim of this study was to investigate the activity of the system A amino acid transporter and the Na+/H+ exchanger in vesicles isolated from the microvillous membrane of the syncytiotrophoblast of placentas of appropriate and small for gestational age babies. There were no biochemical differences between the membranes from the two groups of placentas, and there was no difference in the activity of the Na+/H+ exchanger. The initial rate of uptake of methylaminoisobutyric acid (a nonmetabolizable amino acid analogue) was 63% lower in vesicles from placentas of small for gestational age babies. Kinetic analysis of the system A transporter (utilized by methylaminoisobutyric acid) showed that the Vmax in the vesicles from placentas of small for gestational age babies (0.24 +/- 0.03 nmol/mg protein/30 s, n = 5) was significantly lower than that in vesicles from placentas of appropriate for gestational age babies (0.64 +/- 0.09 nmol/mg protein/30 s, n = 4, p < 0.001), whereas the Km was not different between the two groups. It is concluded that there is an abnormality of system A amino acid transporter function in placentas of small for gestational age babies.

Cellular distribution of neutral and basic amino acid transport systems in rabbit ileal mucosa.

1. An autoradiographic technique is described whereby the cellular location of tritiated amino acids can be determined following uptake by rabbit ileal mucosa. 2. Stirring solutions in contact with the intestinal mucosa during measurement of rapid influx changes the quantity, but not the distribution, of alanine taken up by the tissue. 3. Conditions predicted to favour either a high affinity system (Ly1) or a low affinity system (Ly2) were used to measure lysine distribution following uptake. Maximal uptake for both transport systems occurred in fully differentiated enterocytes at the tips of villi. Initial maturation of the Ly1 system, which was slow, was followed by a rapid phase of development. The Ly2 system lacked this rapid phase of late development. 4. The cellular distribution of alanine entering on a low affinity Na-independent neutral amino acid carrier closely resembles that determine for Ly1 system for lysine entry. 5. Arginine is a potent inhibitor of lysine uptake through the Ly2 system. Little or no diffusion of lysine appears to take place into rabbit ileal enterocytes. 6. The different distribution of the high and low affinity systems for lysine transport provides further support for their independent existence. It also suggests that more than one message exists fo the switching on of amino acid transport function in differentiating enterocytes.

Transport characteristics of papain-treated brush-border membrane vesicles Non-involvement of γ-glutamyltransferase in leucine transport

Papain treatment of isolated brush border membrane vesicles was carried out to correlate directly the solubilization of γ-glutamyltransferase with the uptake of leucine. Digestion of membrane vesicles with either soluble or gel-complexed papain resulted in nearly complete removal of γ-glutamyltransferase. However, the treated vesicles exhibited increased specific activity of leucine and glucose uptake, indicating the non-involvement of the transferase in leucine transport. The partial purification of amino acid and sugar transport function was better controlled with gel-complexed papain. In contrast to the digestion with soluble papain, the treatment with gel-complexed papain did not modify the diffusional components for solutes and ions and did not alter the intravesicular volume. It appears that controlled papain-digestion, resulting in nearly a 2-fold purification of the transport function with high reproducibility and quantitative recovery of uptake, should be useful in future attempts to purify the ‘carrier’ proteins.