Glycine transport in mouse eggs and preimplantation conceptuses

At least two Na+-dependent systems for glycine transport became detectable, while another became undetectable during preimplantation development of mouse conceptuses. Glycine was taken up by a process in eggs and cleavage-stage conceptuses which closely resembles system Gly. Mediated transport at these stages was more rapid at higher Cl- concentrations, sigmoidally related to the exogenous Na+ concentration, and strongly inhibited by sarcosine but not by amino acids with larger side chains. Moreover, neither Li+ nor choline could substitute for Na+ in stimulating glycine transport. System Gly was the only mediated process detected for glycine uptake in unfertilized and fertilized eggs and two-cell conceptuses, but two, less conspicuous, sarcosine-resistant, Na+-dependent components of transport also appeared to be present in eight-cell conceptuses. One of the latter components seemed to remain relatively inconspicuous when conceptuses formed blastocysts, while system Gly became undetectable. In contrast, the other less conspicuous component in eight-cell conceptuses appeared to become the most conspicuous transport process in blastocysts. The latter process, previously designated system B0,+, was shown here also to interact strongly with a broad scope of zwitterionic and cationic amino acid structures. Moreover, transport of glycine via system B0,+ was more rapid at higher Cl- concentrations, and this Na+-dependent process as well as Na+-independent leucine uptake were inhibited by choline. Furthermore, Na+-dependent amino acid transport in two-cell conceptuses and blastocysts was inhibited by 1.0 or 10 mM ouabain, but the inhibition was incomplete at both concentrations. Since Na+/K+-ATPase has not been detected in two-cell conceptuses, inhibition of amino acid transport by ouabain may not have been due solely to an effect on this enzyme. The level of system Gly activity decreased during the development of eight-cell conceptuses from eggs, and this decrease could contribute to an associated decline in intracellular glycine. Since other amino acids begin to compete strongly with glycine for transport when system B0,+ replaces system Gly in conceptuses, this qualitative change in transport activity may help account for a further decrease in the glycine content of conceptuses, reported elsewhere to occur after they form blastocysts.     

Transport of cationic and zwitterionic amino acids in preimplantation rat conceptuses

The ability of preimplantation rat conceptuses to take up several amino acids was examined under a variety of conditions, and the characteristics of uptake were compared to those determined previously for mouse conceptuses. Mediated leucine transport in two-cell rat conceptuses is Na(+)-independent and inhibited almost completely by 2-amino-endobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), so it resembles system L which predominates in two-cell mouse conceptuses. System L becomes less conspicuous than homoarginine-sensitive, Na(+)-independent leucine transport (provisionally designated system bo,+) by the time rat conceptuses develop into blastocysts, as is also the case for mouse conceptuses. In contrast to leucine transport, system bo,+ appears to be the most conspicuous transporter of cationic amino acids throughout preimplantation development of both species. A Na(+)-independent cation-preferring amino acid transport process also appears to be present in rat as well as in mouse conceptuses. Moreover, rat conceptuses resemble mouse conceptuses because Na(+)-dependent transport system Gly activity virtually disappears from them by the time they form blastocysts. Unlike mouse conceptuses, however, Na(+)-dependent system Bo,+ activity appears to be present throughout preimplantation development of rat conceptuses, whereas it has not been detected until at least the two-cell stage in the mouse. Although system Bo,+ becomes more conspicuous in mouse than in rat conceptuses by the time they form blastocysts, system Bo,+ activity appears to increase when blastocysts of both species are removed from the uterus just prior to implantation. The latter observation is consistent with the possibility that system Bo,+ activity is controlled, in part, by the uterus near the time of implantation, although further studies are needed to verify this possibility. Similarities as well as differences in the amino acid transport processes present in conceptuses of rats and mice may eventually be understood best in relation to the environments in which they develop in vitro and in situ.     

Heterogeneity of transport systems for L-glutamine in mouse mammary gland

The characteristics of the transport systems of L-glutamine in lactating mouse mammary gland have been studied. L-glutamine uptake was mediated by three Na+-dependent and one Na+-independent systems. The 2-(methylamino)isobutyric acid-sensitive component of Na+-dependent uptake exhibited the usual characteristics of system A. The other two Na+-dependent systems, which we have named BCI(-)-dependent and BCl(-)-independent, are the new systems identified. These are broad specificity systems and were discriminated on the basis of inhibition analysis, Cl- dependency and the effect of preloading mammary tissue with amino acids. While L-aspargine inhibited the uptake of L-glutamine via both these broad specificity systems, L-homoserine inhibited the uptake of L-glutamine via only BCl(-)-dependent system. The uptake of L-glutamine via the BCl(-)-independent system was upregulated by preloading mammary tissue with L-serine, while BCl(-)-dependent system was unaffected. The Na+-independent uptake of L-glutamine was inhibited by 2-aminobicyclo-(2,2,1)heptane carboxylic acid and other neutral amino acids, and identified as the system L.     

Changes in neutral amino acid transport activity in myeloid leukemia cells differentiated by lipopolysaccharide

M1 cells derived from mouse myeloid leukemia have been reported to differentiate to macrophage-like cells upon treatment with substances such as lipopolysaccharide. Previously we found that in mouse peritoneal macrophages most of the neutral amino acids were taken up through a unique Na+-independent system. In this paper we have investigated the neutral amino acid transport in M1 cells and in those treated with lipopolysaccharide. In M1 cells serine, alanine and proline were taken up mainly by Na+-dependent transport systems, and leucine was largely transported by a Na+-independent system. By treating the cells with lipopolysaccharide, the activities of the Na+-dependent systems markedly decreased, whereas the activity of the Na+-independent system was little affected. The amino acid concentrations in the cells and the culture medium were measured. As a whole, the intracellular to extracellular distribution ratios for neutral amino acids that are preferred substrates for Na+-dependent systems were decreased on lipopolysaccharide treatment, whereas those for amino acids that are mainly transported by a Na+-independent system were slightly increased. From these results we conclude that M1 cells treated with lipopolysaccharide tend to differentiate to macrophage-like cells with respect to the neutral amino acid transport.     

Neutral amino acid transport in embryonal carcinoma cells

Neutral amino acid transport was characterized in the pluripotent embryonal carcinoma (EC) cell line, OC15. Ten of the thirteen amino acids tested are transported by all three of the major neutral amino acid transport systems--A, L, and ASC--although one system may make a barely measurable contribution in some cases. The characterization of N-methyl-aminoisobutyric acid (meAIB) transport points to this model amino acid as a definitive substrate for System A transport by OC15 cells. Thus, high concentrations of meAIB can be used selectively to block System A transport, and the transport characteristics of meAIB represent system A transport. Kinetic analysis of System A, with a Km = 0.79mM and Vmax = 14.4 nmol/mg protein/5 min, suggests a single-component transport system, which is sensitive to pH changes. While proline transport in most mammalian cells is largely accomplished through System A, it is about equally divided between Systems A and ASC in OC15 cells, and System A does not contribute at all to proline transport by F9 cells, an EC cell line with limited developmental potential. Kinetic analysis of System L transport, represented by Na+-independent leucine transport, reveals a high-affinity, single-component system. This transport system is relatively insensitive to pH changes and has a Km = 0.0031 mM and Vmax = 0.213 nmol/mg protein/min. The putative System L substrate, 2-aminobicyclo-[2,2,1]heptane-2-carboxylic acid (BCH), inhibits Systems A and ASC as well as System L in OC15 cells. Therefore, BCH cannot be used as a definitive substrate for System L in OC15 cells. Phenylalanine is primarily transported by Na+-dependent Systems A and ASC (83% Na+-dependent; 73% System ASC) in OC15 cells, while it is transported primarily by the Na+-independent System L in most other cell types, including early cleavage stage mouse embryos and F9 cells. We have also found this unusually strong Na+-dependency of phenylalanine transport in mouse uterine blastocysts (82% Na+-dependent). There is no evidence for System N transport by OC15 cells, since histidine is transported primarily by a Na+-independent, BCH-inhibitable mechanism.     

Acidic amino acid transport in animal cells and tissues

1. The occurrence and characterization of acidic amino acid transport in the plasma membrane of a variety of cells and tissues of a number of organisms is reviewed. 2. Several cell types, especially in brain, possess both high- and low-affinity transport systems for acidic amino acids. 3. High-affinity systems in brain may function to remove neurotransmitter amino acid from the extracellular environment. 4. Many cell systems for acidic amino acid transport are energized by an inwardly directed Na+ gradient. Moreover, certain cell types, such as rat brain neurons, human placental trophoblast and rabbit and rat kidney cortex epithelium, respond to an outwardly directed K+ gradient as an additional source of energization. This simultaneous action may account for the high accumulation ratios seen with acidic amino acids. 5. Rabbit kidney has been found to have a glutamate-H+ co-transport system which is subject to stimulation by protons in the medium. 6. Acidic amino acid transport in rat brain neurons occurs with a stoichiometric coupling of 1 mol of amino acid to 2 mol of Na+. For rabbit intestine, one Na+ is predicted to migrate for each mol of amino acid. 7. Uptake in rat kidney cortex and in high-K+ dog erythrocytes is electrogenic. However, uptake in rabbit and newt kidney and in rat and rabbit intestine is electroneutral. 8. Na+-independent acidic amino acid transport systems have been described in the mouse lymphocyte, the human fibroblast, the mouse Ehrlich cell and in rat hepatoma cells. 9. In a number of cell systems, D-acidic amino acids have substantial affinity for transport; D-glutamate, in a number of systems, however, appears to have little reactivity. 10. Acidic amino acid transport in some cell systems appears to occur via the "classical" routes (Christensen, Adv. Enzymol. Relat. Areas Mol. Biol. 49, 41-101, 1979). For example, uptake in the Ehrlich cell is partitioned between the Na+-dependent A system (which transports a wide spectrum of neutral amino acids), the Na+-dependent ASC system (which transports alanine, serine, threonine, homoserine, etc.), and the Na+-independent L system (which shows reactivity centering around neutral amino acids such as leucine and phenylalanine). Also, a minor component of uptake in mouse lymphocytes occurs by a route resembling the A system. 11. Human fibroblasts possess a Na+-independent adaptive transport system for cystine and glutamate that is enhanced in activity by cystine starvation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Na+-independent transport of basic and zwitterionic amino acids in mouse blastocysts by a shared system and by processes which distinguish between these substrates

Preimplantation mouse blastocysts were found to contain at least three mediated components of Na+-independent amino acid transport. The two less conspicuous components seemed to be selective for either cationic or zwitterionic substrates but were not characterized further or examined for multiple transport activities. L-Leucine and L-lysine competed strongly for uptake by the most conspicuous Na+-independent transport process detected in these conceptuses (referred to as component b0,+), and no further heterogeneity of transport activities was found within this component. A series of inhibitors of various strengths had about the same effect on component b0,+ when either leucine or lysine was the substrate, and uptake of each substrate was not affected significantly by changes in the pH between 6.3 and 8.0. Furthermore, the Ki values for mutually competitive inhibition of transport between leucine and lysine and their Km values for transport via component b0,+ were all on the order of about 100 microM. In addition, the Ki values for competitive inhibition of leucine or lysine uptake by valine were approximately 5 mM in both cases, and alanine appeared to be a similarly weak competitive inhibitor of leucine transport. Based on these results, component b0,+ prefers to interact with bulky amino acids that do not branch at the beta-carbon. Moreover, amino acids that branch at the alpha-carbon, such as the leucine analog 3-amino-endo-bicyclo[3.2.1]octane-3-carboxylic acid, were virtually excluded by this component. The substrate reactivity of component b0,+ is more limited than the Na+-dependent transport system B0,+ in blastocysts which accepts both these branched species and less bulky amino acids relatively well as substrates. Thus, mediated amino acid transport in the mouse trophoblast is clearly distinguishable from that in most other mammalian tissues that have been studied. Not only do component b0,+ and system B0,+ and system B0,+ fail to discriminate strongly between basic and zwitterionic substrates, but their relative reactivity with bicyclic amino acids, such as 3-amino-endo-bicyclo[3.2.1]octane-3-carboxylic acid, is the reverse of transport processes in other cell types where these amino acids react strongly with Na+-independent, but not Na+-dependent, systems.     

Neutral Amino Acid Transport in Astrocytes: Characterization of Na+-Dependent and Na+-Independent Components of α-Aminoisobutyric Acid Uptake

Neutral amino acid transport is largely unexplored in astrocytes, although a role for these cells in blood-brain barrier function is suggested by their close apposition to cerebrovascular endothelium. This study examined the uptake into mouse astrocyte cultures of alpha-aminoisobutyric acid (AIB), a synthetic model substrate for Na+-dependent system A transport. Na+-dependent uptake of AIB was characteristic of system A in its pH sensitivity, kinetic properties, regulatory control, and pattern of analog inhibition. The rate of system A transport declined markedly with increasing age of the astrocyte cultures. There was an unexpectedly active Na+-independent component of AIB uptake that declined less markedly than system A transport as culture age increased. Although the saturability of the Na+-independent component and its pattern of analog inhibition were consistent with system L transport, the following properties deviated: (1) virtually complete inhibition of Na+-independent AIB uptake by characteristic L system substrates, suggesting unusually high affinity of the transporter; (2) apparent absence of trans-stimulation of AIB influx; (3) unusually concentrative uptake at steady state (the estimated distribution ratio for 0.2 mM AIB was 55); and (4) susceptibility to inhibition by N-ethylmaleimide. Direct study of the uptake of system L substrates in astrocytes is needed to confirm the present indications of high affinity and concentrative Na+-independent transport.     

Acidic amino acid transport characteristics of a newly developed conditionally immortalized rat type 2 astrocyte cell line (TR-AST).

To characterize acidic amino acid transport in type 2 astrocytes, we established conditionally immortalized rat astrocyte cell lines (TR-AST) from newly developed transgenic rats harboring temperature-sensitive SV40 large T-antigen gene. TR-AST exhibited positive immunostaining for anti-GFAP antibody and A2B5 antibody, characteristics associated with type 2 astrocytes, and expressed glutamine synthetase. Acidic amino acid transporters, GLT-1 and system xc-, which consists of xCT and 4F2hc, were expressed in all TR-ASTs by RT-PCR. On the other hand, GLAST expression was found in TR-AST3 and 5. The characteristics of [3H]L-glutamic acid (L-Glu) uptake by TR-AST5 include an Na+-dependent and Na+-independent manner, concentration-dependence, and inhibition by L-aspartic acid (L-Asp) and D-aspartic acid (D-Asp). The corresponding Michaelis-Menten constants for the Na+-dependent and Na+-independent process were 36.3 microM and 155 microM, respectively. [3H]L-Asp and [3H]D-Asp uptake by TR-AST5 had an Na+-dependent and Na+-independent manner. This study demonstrated that GLT-1, system xc-, and GLAST were expressed in TR-AST, which has the characteristics of type 2 astrocytes and is able to transport acidic amino acids.     

Glycine transport by cultured human fibroblasts

The transport of glycine was studied in cultured human fibroblasts. The amino acid entered the cell by Na+-dependent and Na+-independent mechanisms. Na+-independent glycine (0.1 mM) transport was less than 10% of total uptake and occurred by a mechanism formally indistinguishable from diffusion. Two distinct routes contributed to Na+-dependent glycine transport. The first route was identified with system A because it was inhibited by MeAIB and underwent adaptive regulation. The second route was identified with system ASC as it was inhibited by L-alanine, but not by MeAIB. Kinetic analysis revealed that the two systems operated glycine transport with the same Km of 1.6 mM, a value unusually high for system ASC.     

Plasma membrane transport of 2-ketoisocaproate by rat hepatocytes in primary culture

Others have shown that the branched chain 2-keto acids are generated in muscle, released into the bloodstream, and then removed by the liver where further catabolism occurs. The present investigation describes the plasma membrane transport systems for these metabolites in cultured rat hepatocytes. One of these systems in Na+-dependent, concentrates the 2-keto acids against a gradient, and is inhibited by pyruvate. The second process is Na+-independent, is less concentrative, and may be composed of two distinct systems as suggested by pyruvate inhibition studies. None of these systems accept neutral amino acids. For the transport of 2-ketoisocaproate, the Na+-dependent system exhibits a Km value of about 5 mM, whereas the corresponding value for the Na+-independent agency is 60 microM. The activity of the Na+-dependent system is moderately increased by insulin treatment of the cells, while neither agency is stimulated by glucagon, dexamethasone, or the combination of these two hormones. Hepatocytes from diabetic rats show enhanced transport by the Na+-dependent system and incubation of cultured hepatocytes for 24 h in the absence of 2-keto acids results in a 3-fold stimulation of the Na+-dependent system, but has no effect on the rate of Na+-independent transport. These results demonstrate the existence of at least two saturable transport systems for the branched chain 2-keto acids in the rat hepatocyte and the ability of the Na+-dependent system to respond to the extracellular environment.     

Homocysteine uptake by human umbilical vein endothelial cells in culture

The characteristics of the uptake of L-homocysteine by cultures of human umbilical vein endothelial cells have been examined. Uptake occurred by Na(+)-dependent and Na(+)-independent systems, but was essentially independent of the pH of the uptake medium. The Na(+)-independent system corresponded to system L, being totally inhibited by the presence of beta-2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH) a system L analogue. It was concluded on the basis of starvation experiments coupled with failure to detect any inhibition in the presence of 2-methylaminoisobutyric acid (MeAIB), a system A analogue, that the Na(+)-dependent uptake was wholly accounted for by system ASC. The kinetic properties of systems L and ASC were determined by omitting Na+ from the uptake medium and incorporating BCH in the medium, respectively. It has been concluded on the basis of the inhibitory effects of a number of amino acids that uptake of homocysteine occurs by those systems which transport cysteine.     

Characterization of neutral and cationic amino acid transport in Xenopus oocytes

Amino acid transport was characterized in stage 6 Xenopus laevis oocytes. Most amino acids were taken up by the oocytes by way of both Na+-dependent and saturable Na+-independent processes. Na+-dependent transport of 2-aminoisobutyric acid (AIB) was insensitive to cis- or trans-inhibition by the System A-defining substrate 2-(methylamino)-isobutyric acid (MeAIB), although threonine, leucine, and histidine were found to be effective inhibitors, eliminating greater than 80% of Na+-dependent AIB uptake. Lack of inhibition by arginine eliminates possible mediation by System Bo,+ and suggests uptake by System ASC. The Na+-dependent transport of characteristic System ASC substrates such as alanine, serine, cysteine, and threonine was also insensitive to excess MeAIB. Evidence to support the presence of System Bo,+ was obtained through inhibition analysis of Na+-dependent arginine transport as well arginine inhibition of Na+-dependent threonine uptake. The Na+-independent transport of leucine was subject to trans-stimulation and was inhibited by the presence of excess phenylalanine, histidine, and, to a lesser extent, 2-amino-(2,2,1)-bicycloheptane-2-carboxylic acid (BCH). These observations are consistent with mediation by System L. The characteristics of Na+-independent uptake of threonine are not consistent with assignment to System L, and appear to be reflective of Systems asc and bo,+. In its charged state, histidine appears to be transported by a carrier similar in its specificity to System y+, but is taken up by System L when present as a zwitterion.     

Differential effects of transmembrane potential on two Na+-dependent transport systems for neutral amino acids

The effects of changes of membrane potential on amino acid transport through systems A, ASC and L was investigated in the Ehrlich cell and the human erythrocyte. Changes of membrane potential were produced by incubating cells whose K+ permeability had been increased, either by valinomycin or by activation of Ca2+-dependent K+ channels, in medium containing different K+ concentrations. The changes in membrane potential were followed by measuring the distribution ratio reached by lipophilic indicators. Transport through Na+-dependent system A was sensitive to the membrane potential, the rate of amino acid uptake increasing 2.2-3.1-times for each 60 mV-hyperpolarization. The Na+-dependent system ASC was insensitive to membrane potential. The Na+-independent system L was not directly affected by membrane potential, but the steady-state accumulation of system L substrates was increased by hyperpolarization.     

Characterization of L-arginine transport in adrenal cells: effect of ACTH

Nitric oxide synthesis depends on the availability of its precursor L-arginine, which could be regulated by the presence of a specific uptake system. In the present report, the characterization of the L-arginine transport system in mouse adrenal Y1 cells was performed. L-arginine transport was mediated by the cationic/neutral amino acid transport system y+L and the cationic amino acid transporter (CAT) y+ in Y1 cells. These Na+-independent transporters were identified by their selectivity for neutral amino acids in both the presence and absence of Na+ and by the effect of N-ethylmaleimide. Transport data correlated to expression of genes encoding for CAT-1, CAT-2, CD-98, and y+LAT-2. A similar expression profile was detected in rat adrenal zona fasciculata. In addition, cationic amino acid uptake in Y1 cells was upregulated by ACTH and/or cAMP with a concomitant increase in nitric oxide (NO) production.     

Na+-dependent transport of basic, zwitterionic, and bicyclic amino acids by a broad-scope system in mouse blastocysts

Mouse blastocysts which had been activated from diapause in utero appeared to take up amino acids via a Na+-dependent transport system with novel characteristics. In contrast to other cell types, uptake of 3-aminoendobicyclo [3,2,1]octane-3-carboxylic acid (BCO) by blastocysts was largely Na+ dependent. Moreover, L-alanine and BCO met standard criteria for mutual competitive inhibition of the Na+-dependent transport of each other. The Ki for each of these amino acids as an inhibitor of transport of the other had a value similar to the value of its Km for transport. In addition, both 2-aminoendobicyclo [2,2,1]heptane-2-carboxylic acid (Ki approximately 1.0 mM) and L-valine (Ki approximately 0.10 mM) appeared to inhibit Na+-dependent transport of alanine and BCO competitively. Finally, alanine and L-lysine appeared to compete for the same Na+-dependent transport sites in blastocysts. For these reasons, we conclude that lysine, alanine, and BCO are transported by a common Na+-dependent system in blastocysts. In addition, the apparent interaction of the system with other basic amino acids, such as 1-dimethylpiperidine-4-amino-4-carboxylic acid, which has a nondissociable positive charge on its side chain, and L-arginine and L-homoarginine, whose cationic forms are highly predominant at neutral pH, suggests that the cationic forms of basic amino acids are transported by the wide-scope system.     

Amino acid transport of y+L-type by heterodimers of 4F2hc/CD98 and members of the glycoprotein-associated amino acid transporter family.

Amino acid transport across cellular membranes is mediated by multiple transporters with overlapping specificities. We recently have identified the vertebrate proteins which mediate Na+-independent exchange of large neutral amino acids corresponding to transport system L. This transporter consists of a novel amino acid permease-related protein (LAT1 or AmAT-L-lc) which for surface expression and function requires formation of disulfide-linked heterodimers with the glycosylated heavy chain of the h4F2/CD98 surface antigen. We show that h4F2hc also associates with other mammalian light chains, e.g. y+LAT1 from mouse and human which are approximately 48% identical with LAT1 and thus belong to the same family of glycoprotein-associated amino acid transporters. The novel heterodimers form exchangers which mediate the cellular efflux of cationic amino acids and the Na+-dependent uptake of large neutral amino acids. These transport characteristics and kinetic and pharmacological fingerprints identify them as y+L-type transport systems. The mRNA encoding my+LAT1 is detectable in most adult tissues and expressed at high levels in kidney cortex and intestine. This suggests that the y+LAT1-4F2hc heterodimer, besides participating in amino acid uptake/secretion in many cell types, is the basolateral amino acid exchanger involved in transepithelial reabsorption of cationic amino acids; hence, its defect might be the cause of the human genetic disease lysinuric protein intolerance.     

Neutral amino acid transport in isolated rat pancreatic islets

The neutral amino acid transport systems of freshly isolated rat pancreatic islets have been studied by first examining the transport of L-alanine and the nonmetabolizable analogue 2-(methylamino)isobutyric acid (MeAIB). By comparing the uptake of MeAIB and L-alanine for their pH dependency profile, choline and Li+ substitution for Na+, tolerance to N-methylation, and competition with other amino acids, the existence in pancreatic islets of both A and ASC amino acid transport systems was established. The systems responsible for the inward transport of five natural amino acids was studied using competition analysis and Na+ dependency of uptake. These studies defined three neutral amino acid transport systems: A and ASC (Na+-dependent) and L (Na+-independent). L-Proline entered rat islet cells mainly by system A; L-leucine by the Na+-independent system L. The uptake of L-alanine, L-serine, and L-glutamine was shared by systems ASC and L, the participation of system A being negligible for these three amino acids. An especially broad substrate specificity for systems L and ASC is therefore suggested for the rat pancreatic islet cells. The regulation of amino acid transport was also investigated in two conditions differing as to glucose concentration and/or availability, i.e. islets from fasted rats and islets maintained in tissue culture at high or low glucose concentrations. Neither alanine nor MeAIB transport was altered by fasting of the islet-donor rats. On the other hand, pancreatic islets maintained for 2 days in tissue culture at high (16.7 mM) glucose transported MeAIB at twice the rate of islets maintained at low (2.8 mM) glucose. Amino acid starvation of pancreatic islets during 11 h of tissue culture resulted in a 2-fold increase in MeAIB transport.     

Aspartate and glutamate transport in unfertilized pig oocytes and blastocysts

Amino acid transport is facilitated by specific transporters within the plasma membrane of the cell. Mediated Na⁺-independent transport of L-glutamate can be easily detected in mouse oocytes, but it is nearly undetectable in blastocyst-stage embryos. In contrast, the Na⁺-dependent transport of L-aspartate is not detectable in oocytes, but it is detectable in eight-cell embryos and reaches relatively high levels by the blastocyst stage. It is believed that the amino acid transporters responsible are systems x^?_c and X^?_AG, respectively. Here we report the detection of Na⁺-dependent L-aspartate transport, which increased as pig blastocysts developed, although Na⁺-dependent aspartate transport was not detected in pig oocytes. Mediated Na⁺-independent L-glutamate transport was not detected in pig oocytes, in contrast to the mouse, nor in early or hatched pig blastocysts. Thus, while the developmental regulation of system X^?_AG is similar in both the pig and the mouse, system x^?_c was not detectable in pig oocytes or blastocysts. Elucidation of the molecular mechanisms controlling amino acid transport and other gene expression in early embryos should contribute to an understanding of whether and even why some aspects of developmental regulation of gene expression may need to differ among species. © 1993 Wiley-Liss, Inc.     

Inhibition of transport system b0,+ in blastocysts by inorganic and organic cations yields insight into the structure of its amino acid receptor site

The most conspicuous, Na(+)-independent amino acid transport process in preimplantation mouse blastocysts was provisionally designated system b0,+ because it accepts some cationic and zwitterionic amino acids about equally well as substrates. Although system b0,+ is not Na(+)-stimulated, it has not been determined if it is inhibited by Na+, or if its activity is affected by most other ions. Therefore, we measured uptake of amino acids by blastocysts in isotonic solutions of different ionic and nonionic osmolites. Na(+)-independent L-leucine uptake was unaffected by the ion concentration, but L-lysine transport was several-fold faster in isotonic solutions of non-electrolytes than in similar solutions of inorganic and organic salts or zwitterions. The Km value for 'Na(+)-independent' L-lysine transport was about 10-fold higher in isotonic salt solutions than in solutions of nonionic osmolites, whereas the Km value for L-leucine transport was about the same in either type of solution. Therefore, inorganic and organic cations and the cationic portions of zwitterions appear to compete with cationic but not zwitterionic amino acids for system b0,+ receptor sites. The cation, harmaline, was a particularly strong competitive inhibitor of 'Na(+)-independent' L-lysine uptake by system b0,+, even in isotonic salt solutions, whereas it inhibited L-leucine uptake noncompetitively. Moreover, harmaline appeared to compete with inorganic cations for the lysine receptor sites of system b0,+. Harmaline also has been found by other investigators to competitively inhibit L-lysine uptake by the Na(+)-independent system asc1 in horse erythrocytes, whereas it noncompetitively inhibits alanine uptake by the same system. Similarly, harmaline noncompetitively inhibits L-alanine uptake by the Na(+)-dependent system ASC in human erythrocytes, but it appears to compete for binding with L-alanine's cosubstrate, Na+. In addition, others have found that the positively-charged side chains of cationic amino acids seem to take the place of Na+ needed near side chains in order for zwitterionic amino acids to be transported by systems ASC and y+. We conclude that system b0,+ may be similar to systems asc1, ASC and y+, and that each of these systems may be a variant of the same ancestral transport process. We speculate that since it appears to accept a broader scope of substrates and to interact with a wider variety of cations than do systems asc1, ASC or y+, system b0,+ may more closely resemble the proposed ancestral transport process than any of the other contemporary systems.