Postnatal amino acid uptake by the rat small intestine. Energetics of membrane transport systems for amino acids in the developing jejunum

The energetics of amino acid uptake by the developing small intestine was investigated in vitro. L-valine, L-leucine, L-phenylalanine, L-methionine, L-lysine and L-arginine were all actively transported by the newborn rat jejunum. Metabolic inhibitors (e.g. 2,4-dinitrophenol) significantly reduced uptake of all amino acids but uptake against a concentration gradient was not totally abolished. Uptake of all amino acids was reduced at low[Na+]. Inhibition of transport of neutral amino acids by reduced luminal [Na+] was greater than that of basic amino acids, and the tissue was barely able to concentrate the neutral amino acids. [Na+] affected the Michaelis constant (Km) of neutral transport systems for their substrates; for the basic amino acids Km values were unaffected by the presence or absence of Na+. Ouabain significantly inhibited neutral amino acid uptake but had no effect on L-lysine or L-arginine uptake. These results are discussed in terms of the Na+ gradient hypothesis for amino acid transport, and the site of energy input to active transport. The role of glycolysis in providing energy for intestinal transport in the neonatal rat and the efficiency of Na+ dependent and independent transport mechanisms are considered. It is concluded that the energetics of amino acid transport systems in neonatal and adult rats are essentially similar.     

Active amino acid transport in plasma membrane vesicles from Simian virus 40-transformed mouse fibroblasts. Characteristics of electrochemical Na+ gradient-stimulated uptake.

Selectively permeable membrane vesicles isolated from Simian virus 40-transformed mouse fibroblasts catalyzed Na+ gradient-coupled active transport of several neutral amino acids dissociated from intracellular metabolism. Na+-stimulated alanine transport activity accompanied plasma membrane material during centrifugation in discontinuous dextran 110 gradients. Carrier-mediated transport into the vesicle was demonstrated. When Na+ was equilibrated across the membrane, countertransport stimulation of L-[3H]alanine uptake occurred in the presence of accumulated unlabeled L-alanine, 2-aminoisobutyric acid, or L-methionine. Competitive interactions among neutral amino acids, pH profiles, and apparent Km values for Na+ gradient-stimulated transport into vesicles were similar to those previously described for amino acid uptake in Ehrlich ascites cells, which suggests that the transport activity assayed in vesicles is a component of the corresponding cellular uptake process. Both the initial rate and quasi-steady state of uptake were stimulated as a function of a Na+ gradient (external Na+ greater than internal Na+) applied artificially across the membrane and were independent of endogenous (Na+ + K+)-ATPase activity. Stimulation by Na+ was decreased when the Na+ gradient was dissipated by monensin, gramicidin D or Na+ preincubation. Na+ decreased the apparent Km for alanine, 2-aminoisobutyric acid, and glutamine transport. Na+ gradient-stimulated amino acid transport was electrogenic, stimulated by conditions expected to generate an interior-negative membrane potential, such as the presence of the permeant anions NO3- and SCN-. Na+-stimulated L-alanine transport was also stimulated by an electrogenic potassium diffusion potential (K+ internal greater than K+ external) catalyzed by valinomycin; this stimulation was blocked by nigericin. These observations provide support for a mechanism of active neutral amino acid transport via the "A system" of the plasma membrane in which both a Na+ gradient and membrane potential contribute to the total driving force.     

Na+-Independent Transporters, LAT-2 and b0,+, Exchange L-DOPA with Neutral and Basic Amino Acids in Two Clonal Renal Cell Lines

The present study examined the functional characteristics of L-DOPA transporters in two functionally different clonal subpopulations of opossum kidney (OKLC and OKHC) cells. The uptake of L-DOPA was largely Na+-independent, though in OKHC cells a minor component (approximately 15%) required extracellular Na+. At least two Na+-independent transporters appear to be involved in L-DOPA uptake. One of these transporters has a broad specificity for small and large neutral amino acids, is stimulated by acid pH and inhibited by 2-aminobicyclo(2,2,l)-heptane-2-carboxylic acid (BCH; OKLC, Ki = 291 mM; OKHC, Ki = 380 mM). The other Na+-independent transporter binds neutral and basic amino acids and also recognizes the di-amino acid cystine. [14C]-L-DOPA efflux from OKLC and OKHC cells over 12 min corresponded to a small amount of intracellular [14C]-L-DOPA. L-Leucine, nonlabelled L-DOPA, BCH and L-arginine, stimulated the efflux of [14C]-L-DOPA in a Na+-independent manner. It is suggested that L-DOPA uses at least two major transporters, systems LAT-2 and b0,+. The transport of L-DOPA by LAT-2 corresponds to a Na+-independent transporter with a broad specificity for small and large neutral amino acids, stimulated by acid pH and inhibited by BCH. The transport of L-DOPA by system b0,+ is a Na+-independent transporter for neutral and basic amino acids that also recognizes cystine. LAT-2 was found equally important at the apical and basolateral membranes, whereas system b0,+ had a predominant distribution in apical membranes.     

Increases in transepithelial vectorial Na+ transport facilitates Na+-dependent L-DOPA transport in renal OK cells

The present study evaluated the hypothesis of whether increases in vectorial Na+ transport translate into facilitation of Na+-dependent L-DOPA uptake in cultured renal epithelial tubular cells. Increases in vectorial Na+ transport were obtained in opossum kidney (OK) cells engineered to overexpress Na+-K+-ATPase after transfection of wild type OK cells with the rodent Na+-K+-ATPase alpha1 subunit. The most impressive differences between wild type and transfected OK cells are that the latter overexpressed Na+-K+-ATPase accompanied by an increased activity of the transporter. Non-linear analysis of the saturation curve for l-DOPA uptake revealed a Vmax value (in nmol mg protein/6 min) of 62 and 80 in wild type and transfected cells, respectively. The uptake of a non-saturating concentration (0.25 microM) of [14C]-L-DOPA in OK-WT cells was not affected by Na+ removal, whereas in OK-alpha1 cells accumulation of [14C]-L-DOPA was clearly dependent on the presence of extracellular Na+. When Na+ was replaced by choline, the inhibitory profile of neutral l-amino acids, but not of basic and acidic amino acids, upon [14C]-L-DOPA uptake in both cell types, was significantly greater than that observed in the presence of extracellular Na+. It is concluded that enhanced ability of OK cells overexpressing Na+-K+-ATPase to translocate Na+ from the apical to the basal cell side correlates positively with their ability to accumulate L-DOPA, which is in agreement with the role of Na+ in taking up the precursor of renal dopamine.     

Cation and harmaline interactions with Na(+)-independent dibasic amino acid transport system y+ in human erythrocytes and in erythrocytes from a primitive vertebrate the pacific hagfish (Eptatretus stouti).

Transport systems y+, asc and ASC exhibit dual interactions with dibasic and neutral amino acids. For conventional Na(+)-dependent neutral amino acid system ASC, side chain amino and guanido groups bind to the Na+ site on the transporter. The topographically equivalent recognition site on related system asc binds harmaline (a Na(+)-site inhibitor) with the same affinity as asc (apparent Ki range 1-4 mM), but exhibits no detectable affinity for Ha. Although also classified as Na(+)-independent, dibasic amino acid transport system y+ accepts neutral amino acids when Na+ or another acceptable cation is also present. This latter observation implies that the y+ translocation site binds Na+ and suggests possible functional and structural similarities with ASC/asc. In the present series of experiments with human erythrocytes, system y(+)-mediated lysine uptake (5 microM, 20 degrees C) was found to be 3-fold higher in isotonic sucrose medium than in normal 150 mM NaCl medium. This difference was not a secondary consequence of changes in membrane potential, but resulted from Na+ functioning as a competitive inhibitor of transport. Apparent Km and Vmax values for lysine transport at 20 degrees C were 15.2 microM and 183 mumol/l cells per h, respectively, in sucrose medium and 59.4 microM and 228 mumol/l cells per h in Na+ medium. Similar results were obtained with y+ in erythrocytes of a primitive vertebrate, the Pacific hagfish (Eptatretus stouti), indicating that Na(+)-inhibition is a general property of this class of amino acid transporter. At a permeant concentration of 5 microM, the IC50 value for Na(+)-inhibition of lysine uptake by human erythrocytes was 27 mM. Other inorganic and organic cations, including K+ and guanidinium+, also inhibited transport. In parallel with its actions on ASC/asc harmaline competitively inhibited lysine uptake by human cells in sucrose medium. As predicted from mutually competitive binding to the y+ translocation site, the presence of 150 mM Na+ increased the harmaline inhibition constant (Ki) from 0.23 mM in sucrose medium to 0.75 mM in NaCl medium. We interpret these observations as further evidence that y+, asc and ASC represent a family of closely related transporters with a common evolutionary origin.     

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)     

Comparison of system N in fetal hepatocytes and in related cell lines

In contrast to the changes seen in membrane transport systems for other neutral, anionic, and cationic amino acids, System N for glutamine, histidine, and asparagine in the rat hepatocytes shows nearly constant properties at the fetal, differentiated, and cultured hepatoma stages. These properties were tested by measuring the Na+-dependent transport of glutamine. This approximate constancy applies not only to the transport selectivity of the system among neutral amino acids, but also to its tolerance of Li+ as a substitute for Na+, its characteristic sensitivity to pH lowering, its relative sensitivity to N-ethylmaleimide, its stimulation by amino acid deprivation, and its failure to respond to insulin or glucagon. The properties of histidine as a substrate for System N were also examined. Inhibition studies with different cell types suggest that the Na+-dependent glutamine and histidine uptake is more restricted to System N in the hepatoma line H35 (H4-11-EC,3) and in the fetal hepatocyte than in hepatoma line HTC and the Ehrlich cells. The Na+-independent component of glutamine and histidine uptake was greater in the hepatoma cells in continuous culture than in fetal and adult hepatocytes in primary culture. Trans-stimulation of glutamine and histidine influx into H35 cells occurs predominantly by the Na+-independent route.     

Effect of alkali ions on the active transport of neutral amino acids into Streptomyces hydrogenans

The active transport of neutral amino acids into Streptomyces hydrogenans is inhibited by external Na⁺. There is no indication that in these cells amino acid accumulation is driven by an inward gradient of Na⁺. The extent of transport inhibition by Na⁺ depends on the nature of the amino acid. It decreases with increasing chain length of the amino acid molecules i.e. with increasing non-polar properties of the side chain. Kinetic studies show that Na⁺ competes with the amino acid for a binding site at the amino acid carrier. There is a close relation between the $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ values for Na⁺ and the number of C atoms of the amino acids. Other cations also inhibit neutral amino acid uptake competitively; the effectiveness decreases in the order $\text{Li}{}^{\mathrm{+}}\text{> Na}{}^{\mathrm{+}}\text{> K}{}^{\mathrm{+}}\text{> Rb}{}^{\mathrm{+}}\text{> Cs}{}^{\mathrm{+}}$. Anions do not have a significant effect on the uptake of neutral amino acids. After prolonged incubation of the cells with 150 mM Na⁺, in addition to the competitive inhibition of transport Na⁺ induces an increase in membrane permeability for amino acids.     

Amino acid transport by membrane vesicles of virally transformed and nontransformed cells: effects of sodium gradient and cell density.]

Mixed membrane vesicle populations composed of plasma membrane and endoplasmic reticulum were prepared from Balb/c 3T3 and simian virus 40-transformed Balb/c 3T3 mouse fibroblasts. The initial rates of uptake of L-leucine and alpha-aminoisobutyric acid by these vesicles were stimulated by a NaCl gradient (external greater than internal). Cation specificity for stimulation of L-leucine uptake was Na+ greater than Li+ greater than K+. NaSCN was as effective as NaCl. Stimulation of uptake of both amino acids by a NaCl gradient was twice as great in vesicles from transformed as compared to non-transformed cells. The NaCl gradient produced transient accumulation of both L-leucine and alpha-aminoisobutyric acid to twice the equilibrium level in vesicles from transformed cells. No such "overshoot" was observed in vesicles from nontransformed cells. In vesicles from the contact-inhibitable Balb/c 3T3 cells, transport of alpha-aminoisobutyric acid, but non L-leucine, exhibited a density-dependent decrease in Na+ gradient induced stimulation, from 248% for sub-confluent to 109% with confluent cells. No density-related changes in uptake were noted with vesicles from the transformed cells. These studies suggest that variation in amino acid uptake associated with viral transformation may be related, at least in part, to alterations in Na+ permeability of the surface membrane.     

INHIBITION OF AMINO ACID UPTAKE BY THE ABSENCE OF Na+ IN SLICES OF BRAIN

—The Na+ requirement of amino acid transport was measured in brain slices. The tissue was first washed free of Na+ and then Na+ was replaced by one of the following: choline, Li+, Rb+, or mannose. Amino acid uptake was measured at different times (5–120 min) and at low (10^-7–10^-5m ) and high (10^-3m ) concentrations. Most of the Na+ could be washed out of the tissue; this also decreased K+ levels despite increased K+ in the medium. K+ tissue levels were partially restored when Na+ was added. The absence of Na+ abolished the uptake of Glu, Asp, GABA, Gly, Tau and Pro. Most of the neutral amino acids (Ala, Val, Trp, His) were very strongly inhibited by the absence of Na+ under most experimental conditions. Basic amino acids (Arg, Lys) were not completely inhibited, in that 30 per cent of the equilibrium uptake remained and some of the basic amino acid influx was independent of the Na+ tissue level. The uptake of amines (tyramine, cadaverine, putrescine) did not require Na+, and often was greater in the absence of Na+. We conclude that amino acid uptake in brain slices is Na+ dependent, although the absence of Na+ may affect transport indirectly.     

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.     

Reconstitution into liposomes of the B degrees -like glutamine-neutral amino acid transporter from renal cell plasma membrane

Na+ dependent [3H]glutamine uptake was found in liposomes reconstituted with solubilized rat kidney brush border in the presence of intraliposomal K+. The reconstituted system was optimised with respect to the critical parameters of the cyclic detergent removal procedure, i.e., the detergent used for the solubilization, the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. Time dependent [3H]glutamine accumulation in proteoliposomes occurred only in the presence of external Na+ and internal K+. The transporter showed low if there is any tolerance towards the substitution of Na+ or K+ for other cations. Valinomycin strongly stimulated the transport indicating that it is electrogenic. Intraliposomal glutamine had no effect. From the dependence of the transport rate on the Na+ concentration cooperativity index close to 1 was derived, indicating that 1 Na+ should be involved in the cotransport with glutamine. The electrogenicity of the transport originated from the Na+ transport. Optimal rate of 0.1 mM [3H]glutamine uptake was found in the presence of 50 mM intraliposomal K-gluconate. At higher K-gluconate concentrations the transport rate decreased. The activity of the reconstituted transporter was pH dependent with optimal function in the range pH 6.5-7.0. [3H]glutamine (and [3H]leucine) uptake was inhibited by all the neutral but not by the positively or negatively charged amino acids. The sulfhydryl reagents HgCl2, mersalyl, p-hydroxymercuribenzoate and the substrate analogue 2-aminobicyclo[2,2,1]heptane-2-carboxylate strongly inhibited the transporter, whereas the amino acid analogue alpha-(methylamino)isobutyrate had no effect. The inhibition by mersalyl was protected by the presence of the substrate. On the basis of the Na+ dependence, the electrogenic transport mode and the specificity towards the amino acids, the reconstituted transporter was classified as B degrees-like.     

Identification,functional characterization and expression of a LAT type amino acid transporter from the mosquito Aedes aegypti

We isolated two cDNAs from the mosquito Aedes aegypti, an L-amino acid transporter (AeaLAT) and a CD98 heavy chain (AeaCD98hc). Expression of AeaCD98hc or AeaLAT alone in Xenopus oocyte did not induce amino acid transport activity. However, co-expression of AeaCD98hc and AeaLAT, which are postulated to form a heterodimer protein linked through a disulfide bond, showed significant increase in amino acid transport activity. This heterodimeric protein showed uptake specificity for large neutral and basic amino acids. Small acidic neutral amino acids were poor substrates for this transporter. Neutral amino acid (leucine) uptake activity was partially Na+ dependent, because leucine uptake was approximately 44% lower in the absence of Na+ than in its presence. However, basic amino acid (lysine) uptake activity was completely Na+ independent at pH of 7.4. Extracellular amino acid concentration could be the main factor that determined amino acid transport. These results suggest the heteromeric protein is likely a uniporter mediating diffusion of amino acids in the absence of ions. The AeaLAT showed high level expression in the gastric caeca, Malpighian tubules and hindgut of larvae. In caeca and hindgut expression was in the apical cell membrane. However, in Malpighian tubules and in midgut, the latter showing low level expression, the transporter was detected in the basolateral membrane. This expression profile supports the conclusion that this AeaLAT is a nutrient amino acid transporter.     

Third system for neutral amino acid transport in a marine pseudomonad.

Uptake of leucine by the marine pseudomonad B-16 is an energy-dependent, concentrative process. Respiratory inhibitors, uncouplers, and sulfhydryl reagents block transport. The uptake of leucine is Na+ dependent, although the relationship between the rate of leucine uptake and Na+ concentration depends, to some extent, on the ionic strength of the suspending assay medium and the manner in which cells are washed prior to assay. Leucine transport can be separated into at least two systems: a low-affinity system with an apparent Km of 1.3 X 10(-5) M, and a high-affinity system with an apparent Km of 1.9 X 10(-7) M. The high-affinity system shows a specificity unusual for bacterial systems in that both aromatic and aliphatic amino acids inhibit leucine transport, provided that they have hydrophobic side chains of a length greater than that of two carbon atoms. The system exhibits strict stereospecificity for the L form. Phenylalanine inhibition was investigated in more detail. The Ki for inhibition of leucine transport by phenylalanine is about 1.4 X 10(-7) M. Phenylalanine itself is transported by an energy-dependent process whose specificity is the same as the high-affinity leucine transport system, as is expected if both amino acids share the same transport system. Studies with protoplasts indicate that a periplasmic binding protein is not an essential part of this transport system. Fein and MacLeod (J. Bacteriol. 124:1177-1190, 1975) reported two neutral amino acid transport systems in strain B-16: the DAG system, serving glycine, D-alanine, D-serine, and alpha-aminoisobutyric acid; and the LIV system, serving L-leucine, L-isoleucine, L-valine, and L-alanine. The high-affinity system reported here is a third neutral amino acid transport system in this marine pseudomonad. We propose the name "LIV-II" system.     

PEPT1-mediated uptake of dipeptides enhances the intestinal absorption of amino acids via transport system b(0,+)

Free amino acids and short chain peptides are the main digestion products of dietary proteins in the small intestine. Whether there is a direct interference in transport of both groups of degradation products is not known. We used human intestinal Caco-2 cells to investigate whether the absorption of dipeptides by the peptide transporter PEPT1 alters the apical uptake of free cationic and neutral amino acids. Influx of L-[3H]Arg into Caco-2 cells was Na+-independent and mediated mainly by the b(0,+) system recognizing both cationic and neutral amino acids. Preincubation of cells with 10 mM of selected neutral, mono- or dicationic dipeptides increased the influx of L-Arg up to fourfold. Preloading with equivalent concentrations of the corresponding free amino acids also increased L-Arg influx but dipeptides always proved to be more efficient. The observed trans-stimulation was found to be specific for cationic amino acids since transport of L-[3H]Ala remained unaffected. We here demonstrate for the first time a direct interplay in amino acid and peptide transport in intestinal cells that may selectively alter the kinetics of amino acid absorption.     

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.     

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.     

Adriamycin-liposome interactions. A magnetic resonance study of the differential effects of cardiolipin on drug-induced fusion and permeability

L-Glutamate and L-aspartate transport into osmotically active intestinal brush border membrane vesicles is specifically increased by Na+ gradient (extravesicular greater than intravesicular) which in addition energizes the transient accumulation (overshoot) of the two amino acids against their concentration gradients. The "overshoot" is observed at minimal external Na+ concentration of 100 mM for L-glutamate and 60 mM for L-aspartate; saturation with respect to [Na+] was observed at a concentration near 100 mM for both amino acids. Increasing amino acid concentration, saturation of the uptake rate was observed for L-glutamate and L-aspartate in the concentration range between 1 and 2 mM. Experiments showing mutual inhibition and transtimulation of the two amino acids indicate that the same Na+ -dependent transport system is shared by the two acidic amino acids. The imposition of diffusion potentials across the membrane vesicles artificially induced by addition of valinomycin in the presence of a K+ gradient supports the conclusion that the cotransport Na+/dicarboxylic amino acid in rat brush border membrane vesicles is electroneutral.     

Sodium-ion stimulated amino acid uptake in membrane vesicles of alkalophilic Bacillus no. 8-1

Membrane vesicles were isolated from alkalophilic Bacillus No. 8-1, and the active transport of amino acids was studied. The transport of amino acids was dependent upon substrate oxidation and the presence of Na+. Concentrative uptake of amino acids was stimulated by the addition of an artificial electron donor system, ascorbate-phenazine methosulfate (PMS), and to a lesser extent by NADH, while succinate, L-lactate, and alpha-glycerol-phosphate did not stimulate the uptake. N,N,N',N'-Tetramethyl-p-phenylenediamine (TMPD) and cytochrome c were able to replace PMS, and reduced forms of these compounds were also very efficient electron donors. Amino acid transport was dependent on electron transfer, and inhibition of NADH oxidation by cyanide, 2-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), and sodium azide directly prohibited serine transport. The pH optima for serine transport lay between pH 8 and 9 for all energy sources. Sodium ion stimulated serine transport in the presence of NADH, NADH plus cytochrome c or succinate plus PMS, but had no stimulatory effect on the corresponding dehydrogenase activities. Sodium ion was also required for accumulation of serine in response to an artificial membrane potential where the respiratory chain was not operative. These results indicated that the stimulatory effect of Na+ on amino acid uptake was on the transport process itself.