Basic Amino Acid Transport Systems Research Articles

Several transport systems contribute to the intestinal uptake of Paraquat, modulating its cytotoxic effects

Paraquat (PQ) is an extremely toxic herbicide upon oral ingestion that lacks a specific antidote. In case of intoxication, treatment primarily relies on limiting its intestinal absorption. In this study, we elucidate the intestinal transport mechanisms of PQ uptake using Caco-2 cells as a model of the human intestinal epithelium. The cells were incubated with a wide range of PQ concentrations (0–5000μM) for 24h with or without simultaneous exposure to different transporters substrates/inhibitors including, choline or hemicolinium-3 (for choline carrier-mediated transport system inhibition) and putrescine, trifluoperazine, valine, lysine, arginine or N-ethylmaleimide (for basic amino acid transport systems inhibition). PQ cytotoxicity was evaluated by the MTT reduction assay and correlated with PQ intracellular levels quantified by gas chromatography-ion trap-mass spectrometry (GC–IT/MS). Potential interactions of PQ with the substrates/inhibitors of the transport systems were investigated and discarded by infrared spectroscopy.Our results showed a significant reduction in PQ intracellular accumulation and, consequently, in PQ cytotoxicity, in the presence of both choline and hemicolinium-3, demonstrating that the choline carrier-mediated transport system is partially involved in PQ intestinal uptake. Likewise, PQ cytotoxicity and intracellular accumulation were significantly attenuated by simultaneous exposure to putrescine, trifluoperazine, valine, lysine, arginine and N-ethylmaleimide. These data suggested the involvement of more than one of the basic amino acids transport systems, including the y+, b0,+ or y+L systems.In conclusion, this study demonstrated that several transport systems mediate PQ intestinal absorption and, therefore, their modulation may provide alternative efficient pathways for limiting PQ toxicity in intoxication scenarios.

RBE4 cells are highly resistant to paraquat‐induced cytotoxicity: studies on uptake and efflux mechanisms

Paraquat (PQ) is a widely used, highly toxic and non-selective contact herbicide, which has been associated with central neurotoxic effects, namely the development of Parkinson's disease, but whose effects to the blood-brain barrier (BBB) itself have rarely been studied. This work studied the mechanisms of PQ uptake and efflux in a rat's BBB cell model, the RBE4 cells. PQ is believed to enter cells using the basic or neutral amino acid or polyamine transport systems or through the choline-uptake system. In contrast, PQ efflux from cells is reported to be mediated by P-glycoprotein. Therefore, we evaluated PQ-induced cytotoxicity and the effect of some substrates/blockers of these transporters (such as arginine, L-valine, putrescine, hemicholinium-3 and GF120918) on such cytotoxicity. RBE4 cells were shown to be extremely resistant to PQ after 24 h of exposure; even at concentrations as high as 50 mM approximately 45% of the cells remained viable. Prolonging exposure until 48 h elicited significant cytotoxicity only for PQ concentrations above 5 mM. Although hemicholinium-3, a choline-uptake system inhibitor, significantly protected cells against PQ-induced toxicity, none of the effects were observed for arginine, L-valine or putrescine. Meanwhile, inhibiting the efflux pump P-glycoprotein using GF120918 significantly enhanced PQ-induced cytotoxicity. In conclusion, PQ used the choline-uptake system, instead of the transporters for the basic or neutral amino acids or for the polyamines, to enter RBE4 cells. P-glycoprotein extrudes PQ back to the extracellular medium. However, this efflux mechanism only partially explains the observed RBE4 resistance to PQ.

Amino acid transport systems of JapaneseParamecium symbiont F36-ZK

Analyses of amino acid transport systems in JapaneseParamecium symbiont F36-ZK were performed using14C-amino acids. Kinetic analyses of amino acid uptake and competitive experiments revealed three transport systems; a basic amino acid transport system, which catalyzed transport of L-Arg and L-Lys, a general amino acid transport system, which had broad specificity for 19 amino acids (but not L-Arg), and an alanine transport system. These three systems were considered to be capable of active transport. Amino acid-proton symport was indicated by the following data: decreases in pH of the medium observed during L-Ser and L-Ala uptake, and uptake of L-Arg, L-Ser and L-Ala being inhibited by carbonyl cyanide m-chlorophenylhydrazone, sodium azide and vanadate. The optimal pH for uptake of neutral amino acids and L-Arg was around 5 and 5 to 6.5, respectively. Uptake of L-Asp and L-Glu was very sensitive to pH and little uptake of L-Asp was measured above pH 6.0. Amino acid uptake was not inhibited by nitrate or ammonium, and cultured cells with ammonium also possessed constitutive uptake systems.

Pralidoxime iodide (2-pAM) penetrates across the blood-brain barrier.

The in vivo rat brain microdialysis technique with HPLC/UV was used to determine the blood-brain barrier (BBB) penetration of pralidoxime iodide (2-PAM), which is a component of the current nerve agent antidote therapy. After intravenous dosage of 2-PAM (10, 50, 100 mg/kg), 2-PAM appeared dose-dependently in the dialysate; the striatal extracellular/blood concentration ratio at 1 h after 50 mg/kg dosage was 0.093 +/- 0.053 (mean +/- SEM). This finding offered conclusive evidence of the BBB penetration of 2-PAM. We also examined whether the BBB penetration of 2-PAM was mediated by a certain specific transporter, such as a neutral or basic amino acid transport system. Although it was unclear, the neural uptake of 2-PAM was Na+ dependent. The mean BBB penetration by 2-PAM was approximately 10%, indicating the intravenous administration of 2-PAM might be to a degree effective to reactivation of the blocked cholinesterase in the brain.

RBAT is an amino acid exchanger with variable stoichiometry.

The rBAT protein, when expressed in Xenopus oocytes, was previously shown to reproduce the selectivity of the Na(+)-independent neutral and basic amino acid transport system called bo,+. More recently, the capacity of rBAT to generate a transmembrane current was demonstrated when addition of neutral amino acids stimulated the efflux of cations (presumably basic amino acids) in rBAT-injected oocytes. In the present paper, aminoisobutyric acid (AIB), a neutral amino acid analogue, was shown to induce outward currents (efflux of basic amino acids) through rBAT similar to those caused by alanine in terms of affinity, maximal currents and I-V curves. Despite generating similar currents, the AIB transport rate was more than 30 times lower than that of alanine, thus challenging the assumption that rBAT functions as a classical exchanger. Experiments using a cut-open oocyte voltage clamp demonstrated that AIB was capable of stimulating rBAT-mediated currents from either side of the membrane. AIB, like alanine, was able to stimulate the efflux of radiolabeled alanine and arginine while no rBAT-mediated efflux was measurable in the absence of external rBAT substrates. These results demonstrate that (i) the presence of amino acids is required on both sides of the membrane for rBAT to mediate amino acid flux and thus rBAT must be some type of exchanger but (ii) rBAT-mediated amino acid influx is not stoichiometrically related to the efflux. A model of a "double gated pore" is proposed to account for these properties of rBAT, which contravene standard models of exchangers and other transporters.

Phenylmethylsulfonyl fluoride protects l-lysine transport in Schizosaccharomyces pombe against inactivation by ammonium ions

Ammonium ions inactivate the basic amino acid transport system in Schizosaccharomyces pombe in an irreversible manner. The inactivation is accompanied by a 4-fold decrease of K T of l-lysine transport, leaving its J max unchanged; phenylmethylsulfonyl fluoride protects the system against inactivation. In contrast, two basic amino acid transport systems in a gap1 mutant of Saccharomyces cerevisiae are influenced by NH 4 + ions in such a way that only the J max decreases while the K T of l-lysine transport is unchanged. Phenylmethylsulfonyl fluoride does not act here as a protective agent.

Characteristics of gentamicin uptake in the isolated crista ampullaris of the inner EAR of the guinea pig

The characteristics of aminoglycoside uptake in the inner ear were investigated in the isolated crista ampullaris. The organ was incubated with radiolabeled gentamicin, and the stable, nonexchangeable radioactivity was considered the intracellular pool of the drug. Gentamicin was transported against a concentration gradient, and the resulting tissue to medium ratios ranged from 1.4 to 18.6. Transport was inhibited by reduction in temperature or by the addition of metabolic blockers, indicative of an energy-dependent component. The uptake system showed at least two sites, the first a high-affinity site with a dissociation constant K d = 39 nM and a capacity of n = 0.2 pmole/crista; the second had a K d = 16 μ M and a capacity of n = 11 pmoles/crista. Aminoglycosides competed with uptake of gentamicin in the order netilmicin ≥ neomycin > tobramycin, and polyamines competed in the order spermine > spermidine > putrescine; glucosamine and the basic amino acids lysine and asparagine were ineffective, excluding the participation of glucose and basic amino acid transport systems in gentamicin uptake. These results along with direct comparisons with some characteristics of putrescine uptake suggest that aminoglycoside and polyamine transport have common features in the crista ampullaris.

Ornithine accumulation and metabolism in rat lens

The non-protein amino acid ornithine is readily accumulated into rat lenses cultured in TC-199 bicarbonate medium. This accumulation, measured by the lens water/lens medium ratios of radiolabeled ornithine, appears to be the result of an apparent energy-dependent basic amino acid transport system. Moreover, competition studies suggest that increased levels of ornithine can depress the concomitant lenticular accumulation of arginine and lysine. Examination of the trichloroacetic acid protein precipitates of these cultured lenses indicates that the radiolabel from ornithine can also be incorporated into lens proteins as proline. This results from the conversion by ornithine aminotransferase (EC 2. 6. 1. 13) of radiolabeled ornithine to delta 1-pyrroline-5-carboxylic acid (P5C), which is subsequently converted to proline by P5C reductase (EC 1. 5. 1. 2). This incorporation of radiolabel into cultured lens proteins is reduced upon addition of either 1 m m proline or P5C to the culture medium or inhibited by the addition of the protein synthesis inhibitor, puromycin.

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.

Physiological and regulatory properties of the general amino acid transport system of Neurospora crassa.

The fundamental properties of the general amino acid transport system of Neurospora crassa were investigated in the conidial stage of the life cycle. The transport activity was found to be under genetic control, and an isogenic set of mutants deficient for the neutral, basic, or general amino acid transport systems and combinations thereof was constructed and used for analyzing the properties specific to the general permease. Amino acid transport by this system was found to be a carrier-mediated active process with broad specificity for the neutral and basic amino acids. Kinetic analysis revealed that a common binding site functioned to transport both neutral and basic amino acids and that the permease had a high affinity for its substrates. The kinetic parameters Km, Vmax, and Ki were defined for several substrates. Two modes of regulation were detected: substrate inhibition and ammonium repression. Activity of the general system was enhanced by the removal of ammonium ions from the incubation medium with a concomitant decline in either neutral or basic permease activity, suggesting that a common component exists between the neutral and the general systems and between the basic and the general systems.

Escherichia coli regulatory mutation affecting lysine transport and lysine decarboxylase.

A spontaneous thiosine-resistant mutant of Escherichia coli was shown to have the following characteristics: lowered initial rate of lysine uptake and lowered plateau level of accumulation of exogenous lysine by both the lysine-specific and the general basic amino acid transport systems; altered repressibility of these two lysine transport systems; a derepressed level of lysine decarboxylase; normal growth rate; parental levels of lysyl-transfer ribonucleic acid synthetase and the inducible and constitutive arginine and ornithine decarboxylases. Both the mutant (lysP) and its parent (lysP+) feed a lysine auxotroph when they are plated in proximity on solid medium. However, the feeding response was observable after 1 day less of incubation when the mutant was the feeding strain. Despite the derepressed level of lysine decarboxylase in exponential cultures of the mutant extracts of these cultures had no detectable cadaverine pool. Conjugation experiments established the following gene order: gyrA (formerly nalA) lysP metG his. All thiosine-resistant recombinants assayed showed reduced lysine transport. In many of these recombinants the derepression of lysine decarboxylase was not expressed.

Inhibition of amino acid transport in rat small intestine by N.EPSILON.-substituted L-lysine derivatives.

Effect of Ne-dansyl-L-lysine [Ne-(5-dimethylaminonaphthalenesulfonyl)-L-lysine] on amino acid transport across the brush border of isolated rat small intestine was investigated. Ne-Dansy-L-lysine was not actively accumulated nor transmurally transported by the intestinal tissue; nonetheless, it competitively inhibited the basic amino acid transport with a Ki of about 0.4mM for L-lysine, but entirely not the neutral amino acid transport. The inhibition was relatively independent of extracellular Na+ The minimal dependence on Na+ parallels with the least Na+ dependence of lysine transport among various amino acid transport systems, suggesting the inhibition competes for a step involved in a heteroexchange process mediating the greater portion of lysine transport. The results provided a basis for a potential use of Ne-dansyl-L-lysine as a fluorescent probe in delineating its interaction with basic amino acid transport system in the small intestine.

Perinatal changes of transport systems for amino acids in slices of mouse brain

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.

Basic amino acid transport in Escherichia coli: properties of canavanine-resistant mutants.

A mutant of Escherichia coli strain CanR 22 has been isolated which is resistant to growth inhibition by canavanine, an analogue of arginine. The properties of this strain and of another canavanine-resistant mutant, JC182-5 (isolated by Celis et al. [5]), were studied. The mutation is pleiotropic in that it results in a reduction in the activity of two distinct permeases, the arginine-specific and lysine-arginine-ornithine transport systems. The lesion maps at min 56 of the E. coli linkage map, at or near the argP locus. Although strain CanR 22 excretes arginine, this excretion appears to result from reduced ability to concentrate arginine, rather than the loss of transport ability being the result of excretion. This conclusion is based on findings with a canavanine-resistant strain auxotrophic for arginine, which exhibits transport properties similar to those of the prototrophic strains. Additionally, growth in the presence of arginine or ornithine results in a repression of the activity of the two basic amino acid transport systems. Neither the arginine-specific nor the lysine-arginine-ornithine binding proteins of the mutant cells show significant alterations in terms of amount, physical properties, or kinetic parameters. These observations lead to the proposal of a model for the two basic amino acid transport systems in which two carrier proteins with different specificities interact with a common energy coupling mechanism. A lesion in the gene (or one of the genes) for this coupling mechanism can confer canavanine resistance.

Mechanisms and specificity of amino acid transport in Taenia crassiceps larvae (Cestoda)

The absorption of lysine, arginine, phenylalanine and methionine by Taenia crassiceps larvae is linear with respect to time for at least 2 min. Arginine uptake occurs by a mediated system and diffusion, and arginine, lysine and ornithine (in order of decreasing affinity) are completely competitive inhibitors of arginine uptake. The basic amino acid transport system has a higher affinity for l-amino acids than d-amino acids, and blocking the α-amino group of an amino acid destroys its inhibitory action. Phenylalanine uptake by T. crassiceps larvae is inhibited in a completely competitive fashion by serine, leucine, alanine, methionine, histidine, phenylalanine, tyrosine and tryptophan (in order of increasing affinity). Methionine apparently binds non-productively to the phenylalanine (aromatic amino acid-preferring) transport system. l-methionine uptake by larvae is inhibited more by d-alanine and d-valine than by their respective l-isomers, while d- and l-methionine inhibit l-methionine uptake equally well. The presence of an unsubstituted α-amino group is essential for an inhibitor to have a high affinity for the methionine transport system. Uptake of arginine, phenylalanine and methionine is Na +-insensitive, and both phenylalanine and methionine are accumulated by larvae against a concentration difference in the presence or absence of Na +. Arginine accumulation is precluded by its rapid metabolism to proline, ornithine and an unidentified compound.

Amino acid transport pathways in the small intestine of the neonatal rat.

Extract: The activity of amino acid transport pathways in the small intestine of the 2-day-old rat was investigated. Transport was determined by measuring the uptake of 1 mM concentrations of various amino acids by intestinal segments after a 5- or 10-min incubation and it was expressed as intracellular accumulation. The neutral amino acid transport pathway was well developed with intracellular accumulation values for leucine, isoleucine, valine, methionine, tryptophan, phenylalanine, tyrosine, and alanine ranging from 3.9–5.6 mM/5 min. The intracellular accumulation of the hydroxy-containing neutral amino acids threonine (essential) and serine (nonessential) were 2.7 mM/5 min, a value significantly lower than those of the other neutral amino acids. The accumulation of histidine was also well below the level for the other neutral amino acids (1.9 mM/5 min). The basic amino acid transport pathway was also operational with accumulation values for lysine, arginine and ornithine ranging from 1.7–2.0 mM/5 min. Accumulation of the essential amino acid lysine was not statistically different from that of nonessential ornithine. Accumulation of aspartic and glutamic acid was only 0.24–0.28 mM/5 min indicating a very low activity of the acidic amino acid transport pathway. Accumulation of sarcosine and betaine was about 0.5 mM/5 min, which indicates a low activity for the imino acid-glycine pathway. The presence of 1 mM alanine significantly (P < 0.01) increased the accumulation of lysine by 28.5%, which suggests the presence of an exchange transport system. These results show that by operation of the neutral and basic amino acid transport systems, the neonatal rat can effectively absorb all the essential amino acids; however, accumulation of individual amino acids appears to be governed by structural rather than nutritional considerations. Speculation: The data presented demonstrate the differential ability of the neonatal rat to absorb various ammo acids. This finding is of potential importance in the formulation and evaluation of synthetic protein diets.

Amino Acid Transport Pathways in the Small Intestine of the Neonatal Rat

Extract: The activity of amino acid transport pathways in the small intestine of the 2-day-old rat was investigated. Transport was determined by measuring the uptake of 1 mM concentrations of various amino acids by intestinal segments after a 5− or 10-min incubation and it was expressed as intracellular accumulation.The neutral amino acid transport pathway was well developed with intracellular accumulation values for leucine, isoleucine, valine, methionine, tryptophan, phenylalanine, tyrosine, and alanine ranging from 3.9–5.6 mM/5 min. The intracellular accumulation of the hydroxy-containing neutral amino acids threonine (essential) and serine (nonessential) were 2.7 mM/5 min, a value significantly lower than those of the other neutral amino acids. The accumulation of histidine was also well below the level for the other neutral amino acids (1.9 mM/5 min). The basic amino acid transport pathway was also operational with accumulation values for lysine, arginine and ornithine ranging from 1.7–2.0 mM/5 min. Accumulation of the essential amino acid lysine was not statistically different from that of nonessential ornithine. Accumulation of aspartic and glutamic acid was only 0.24–0.28 mM/5 min indicating a very low activity of the acidic amino acid transport pathway. Accumulation of sarcosine and betaine was about 0.5 mM/5 min, which indicates a low activity for the imino acid-glycine pathway. The presence of 1 mM alanine significantly (P < 0.01) increased the accumulation of lysine by 28.5%, which suggests the presence of an exchange transport system.These results show that by operation of the neutral and basic amino acid transport systems, the neonatal rat can effectively absorb all the essential amino acids; however, accumulation of individual amino acids appears to be governed by structural rather than nutritional considerations.

Histidine Uptake in Strains of Neurospora crassa with Normal and Mutant Transport Systems

Kinetic parameters for three systems of active histidine uptake by germinated conidia of Neurospora crassa have been measured. Each system appears to follow typical Michaelis-Menten kinetics when studied separately from the other systems. Under the conditions studied, the general amino acid transport system was found to account for the major portion of histidine uptake from low concentrations. Three types of transport mutants with altered growth inhibition patterns were selected in a histidine auxotroph. Growth of one mutant, type bas(a), could be inhibited by the addition of methionine to a histidine-supplemented medium, and another type, neu(a), could be inhibited by the addition of arginine. These mutants were shown to be lacking active histidine uptake by the basic amino acid and neutral amino acid transport systems, respectively. Another type of double mutant (his-3, neu(r)) could be inhibited only by the addition of very high concentrations of methionine in the presence of arginine and histidine, and the mutation appeared to have altered the specificity of the neutral amino acid permease.

Acidic and basic amino acid transport systems of Penicillium chrysogenum

Penicillium chrysogenum possesses a relatively specific constitutive transport system for the basic amino acids l-lysine and l-arginine (K m = 6 × 10 −6 m, V max = 1 μmole/g-min). This system has a low affinity for 2,4-diaminobutryate, l-ornithine, and l-histidine. The basic amino acid transport system can be assayed readily in nutrient-sufficient mycelia where a nonspecific amino acid transport system (Benko, P. V., Wood, T. C., and Segel, I. H., Arch. Biochem. Biophys. 129, 498, 1969), which also transports basic amino acids, is extremely low in activity. Nutrient-sufficient mycelia also contain a low-level lysine-insensitive arginine transport system and a low-level arginine-insensitive lysine transport system. The basic amino acid transport system can be assayed in nitrogen-starved and in carbon-starved mycelia if a large excess of, e.g., l-leucine is added to the assay mixture in order to suppress transport of the labeled substrate by the nonspecific amino acid transport system. The acidic amino acids, l-glutamate and l-aspartate, are also substrates of the general amino acid transport system. However, the preferred ionic form is the uncharged species. l-Glutamate and l-aspartate are also transported by a distinct stereospecific acidic amino acid transport system that develops along with the nonspecific system during nitrogen starvation or carbon starvation. The acidic amino acid system can be assayed at pH 6.0 in the presence of a large excess of, e.g., l-leucine. These conditions minimize the transport of glutamate and aspartate by the nonspecific system. A distinct proline transport system has also been detected in nitrogen-starved and in carbon-starved mycelia. No distinct or quantitatively significant specific systems for aromatic amino acids or histidine were detected. The general and acidic amino acid transport systems are subject to feedback inhibition (“transinhibition”) by their intracellular substrates. The transinhibition is most evident under conditions in which the amino acid is transported much faster than it is metabolized. In this respect, l-α-aminoadipate is a better transinhibitor of the acidic system than the preferred substrates, aspartate and glutamate. The feedback inhibitor of the nonspecific system has been shown to be the accumulated substrate rather than NH 4 +.