Improved method for the measurement of large neutral amino acids in biological matrices

A high-performance liquid chromatographic method for measuring neutral amino acids in rat sera, brain tissues, and perfusates was developed by using o-phthalaldehyde sulfite as a pre-column derivatization reagent. With the present method, it was possible to separate the neutral amino acids within a single run in 25 min, while the acidic amino acids were eluted near or at the solvent front. The recovery was above 88.8% with a relative standard deviation (RSD) below 4.2%. The within- and between-day assay reproducibility for the determination of rat serum amino acids showed RSDs below 1.35 and 7.61%, respectively. In the present study, the neutral amino acids were assayed with high sensitivity, accuracy and good reproducibility in a relatively short time and on a small sample size.     

Na-independent and Na-dependent transport of neutral amino acids in the human red blood cell

Experiments performed at 25°C, pH 7.4, confirmed that the human red blood cell possesses both Na-independent and Na-dependent transport systems for neutral amino acids. Further evidence for the existence of a major Na-independent pathway for the large neutral amino acids (L-leucine, L-isoleucine, L-phenylalanine, L-valine and L-methionine), designated the ‘L-system’, was provided from trans-acceleration experiments of L-leucine efflux. Transport via the L-system with respect to kinetics of substrates and stereospecificity was studied. Experiments on inhibition of transport and on kinetics of Na-dependence of uptake of L-alanine and glycine were consistent with Na-dependent amino acid transport being mediated by two different systems, i. e. an ASC-system for L-alanine, L-serine and L-cysteine and a Gly-system for glycine transport. When compared with a ‘high capacity/low affinity’ pattern of kinetics of the L-system, Na-dependent uptake of L-alanine and glycine was found to exhibit ‘low capacity/high affinity’ kinetics. The Na-dependence of L-alanine uptake conformed to first order interaction, that of glycine uptake to second order. An effect predominantly on the maximum transport capacity (V₁₂) of the saturable Nadependent uptake route of L-alanine and glycine, respectively, by a 50% reduction of the extracellular Na⁺ concentration, was suggested.     

Glycoprotein-associated amino acid exchangers: broadening the range of transport specificity

Members of the newly discovered glycoprotein-associated amino acid transporter family (gpaAT-family) share a similar primary structure with >40% identity, a predicted 12-transmembrane segment topology and the requirement for association with a glycoprotein (heavy chain) for functional surface expression. Five of the six identified gpaATs (light chains) associate with the surface antigen 4F2 heavy chain (4F2hc = CD98), a ubiquitous plasma membrane protein induced in cell proliferation, and which is also highly expressed at the basolateral surface of amino acid transporting epithelia. The differing tissue localizations of the 4F2hc-associated gpaATs appear to complement each other. As yet, a single gpaAT (b(0,+)AT) has been shown to associate with rBAT, a 4F2hc-related glycoprotein mainly localized in intestine and kidney luminal brush-border membranes. The transport characteristics of gpaATs have been shown, by expression in heterologous systems, to correspond to the previously described transport systems L, y+L, xc- and b(o,+). These (obligatory) exchangers of broad substrate specificity (with the exception of xCT) are expected to equilibrate the concentrations of their substrate amino acids across membranes. Thus, the driving force provided by a transmembrane gradient of one substrate amino acid, such as that generated by a parallel functioning unidirectional transporter, can be used by a gpaAT to fuel the secondary active vectorial transport of other exchangeable species. Vectorial transport of specific amino acids is also promoted by the intrinsic asymmetry of these exchangers. The fact that genetic defects of the epithelial gpaATs b(0,+)AT and y+LAT1 cause non-type I cystinuria and lysinuric protein intolerance, respectively, demonstrates that these gpaATs perform vectorial secondary and/or tertiary active transport of specific amino acids in vivo.     

Renal transport of amino acids

Summary According to recent experimental data the renal transport of amino acids (AA) is characterized as follows. 1.Kinetics: Several reabsorption systems remove AA from the tubular fluid by active transport with Michaelis-Menten type kinetics. Passive diffusion does play only a relatively small role in reabsorption, but determines the pump leak steady state concentration at the end of the tubule. 2.Stereospecificity: Except for aspartate the naturally occurring L-analogs show a much larger affinity to the transport carriers than the D-isomers do. 3.Specificity: Separate transport mechanisms exist for a) the acidic AA (Glu and Asp); b) the dibasic AA (Arg, Lys, Orn); c) cystine/cysteine; d) the imino acids (Pro, OH-Pro and other N-substituted AA); e) the - and -AA (-Ala, GABA, Taurine); f) all other neutral AA. For the group (d) and maybe also for (b) and glycine additional low capacity/high affinity systems exist. 4.Localization: Except for glycine and taurine under normal conditions more than 80% of the filtered load are reabsorbed within the first third of the proximal tubule. At an elevated load the rest of the proximal tubule (including pars recta) but not the distal nephron is included into the reabsorptive process. AA are also taken up from the peritubular blood. 5.Energy sources: At least the main part of AA uptake at the brushborder membrane is dependent from a transmembranal Na⁺-gradient which in turn is established by the ATP driven Na⁺-pumps at the basolateral side of the cell (Secondary active transport or co-transport of AA). 6.Biochemistry: The biochemical nature of the AA-carriers is unknown. The recent hypothesis that a -glutamyl cycle plays a major role in this context has been disproved to great extent. 7.Peptides: Oligopeptides (Angiotensin, Glutathion) filtered at the glomerulum are hydrolyzed by brushborder peptidases within the tubule lumen. The splitting products, the free constituent amino acids, are reabsorbed subsequently by their respective transport systems.

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Zusammenfassung Der renale Transport von Aminosäuren (AS) läßt sich heute folgendermaßen charakterisieren:1. Kinetik: AS werden durch aktive Transportmechanismen aus dem Tubulus resorbiert. Passive Diffusion spielt dabei nur insofern eine Rolle, daß sie entscheidend die Gleichgewichtskonzentration, bei der Resorption gleich Rückdiffusion ist, am Ende des proximalen Tubulus bestimmt.2. Stereospezifität: Ausgenommen die Asparaginsäure haben die natürlich vorkommenden L-AS eine höhere Affinität zum Transportsystem als die jeweiligen D-Formen.3. Spezifität: Getrennte Transportsysteme existieren für a) die sauren AS (Glu, Asp), b) die dibasischen AS (Arg, Lys, Orn), c) Cystin/Cystein, d) die sog. Iminosäuren (Pro, OH-Pro und andere, N-substituierte AS), e) die - und -AS (-Ala, GABA, Taurin), f) alle anderen neutralen AS. Für die Gruppe (d) und evtl. auch für (b) und Glyzin gibt es zusätzliche Systeme mit niedriger Kapazität, aber hoher Affinität.4. Lokalisation: Normalerweise werden mehr als 80% der filtrierten AS-Menge innerhalb des ersten Drittels des proximalen Tubulus resorbiert (Ausnahme: Glyzin, Taurin). Erhöht sich die filtrierte AS-Menge, kann der Rest des proximalen Tubulus (incl. pars recta), nicht jedoch das distale Nephron, zur Resorption herangezogen werden. Die Tubuluszelle nimmt AS auch aus dem peritubulären Kapillarblut auf.5. Energiebereitstellung: Der Bergauf-Transport der AS in die Tubuluszelle wird durch das einströmende Na⁺ getrieben (Co-Transport), dessen Gradient wiederum durch die ATP-getriebenen, peritubulären Na⁺-Pumpen aufrechterhalten wird (Sekundär-aktiver Transport).6. Biochemie: Die chemische Natur der AS-Transportsysteme ist unbekannt. Die Hypothese, daß ein sog. -Glutamyl-Zyklus dabei eine wesentliche Rolle spielt, ist nicht mehr haltbar.7. Peptide: Oligopeptide (Angiotensin, Glutathion u.a.) werden nach der glomerulären Filtration innerhalb des Tubuluslumens durch Bürstensaum-Peptidasen hydrolysiert. Die dabei freigesetzten AS werden gleich anschließend durch ihre jeweiligen Transportsysteme resorbiert.

     

Lysine transport in lactating rat mammary tissue: evidence for an interaction between cationic and neutral amino acids

The transport of lysine by the lactating rat mammary gland has been examined to determine whether there is an interaction between cationic and neutral amino acids. Lysine uptake was time dependent and unaffected by replacing Na⁺ with choline. In the presence of Na⁺, lysine influx was inhibited by cationic amino acids (arginine, homoarginine, ornithine and lysine) and by a range of neutral amino acids (methionine, glutamine, leucine, phenylalanine, alanine, asparagine, α-aminoisobutyric acid (AIB), 2-aminobicyclo [2,2,1] heptane-2-carboxylic acid (BCH), proline and tryptophan). Leucine and glutamine also inhibited lysine influx in the absence of Na⁺ but phenylalanine and proline did not. Lysine efflux from mammary tissue was trans-accelerated by various cationic amino acids (lysine, arginine, homoarginine and ornithine). In addition, leucine and glutamine were capable of trans-stimulating lysine efflux in the presence and absence of Na⁺. It appears that cationic and neutral amino acids stimulated lysine efflux at a single locus.     

Electrogenic transport of neutral and dibasic amino acids in a cultured opossum kidney cell line (OK)

A study has been made of electrogenic cellular uptake of amino acids resulting in the depolarization of cell membrane potential (PD_m) in confluent monolayers of an established opossum kidney (OK) cell line using conventional and pH-selective microelectrodes. Apical superfusion of neutral and dibasic amino acids rapidly depolarized the cell membrane, while application of acidic amino acids had no effect on PD_m. The depolarization in response toL-phenylalanine andL-arginine was stereoselective, dose-dependent and saturable. 10 mmol/l ofL-phenylalanine reduced PD_m by 4.8±0.4 mV (n=51) in a completely sodium-dependent way and the concentration necessary for halfmaximal depolarization (C_1/2) was about 1.5 mmol/l. On the other hand, the C_1/2 forL-arginine was about 0.02 mmol/l. The maximal depolarization produced byL-arginine (measured at 10 mmol/l) amounted to 6.8±1.2 mV (n=10) and this was not affected when extracellular sodium was replaced by choline (6.3±1.2 mV;n=10). The depolarizations induced byL-phenylalanine andL-arginine were significantly additive (p<0.001). The intracellular pH of OK cells was 7.09±0.03 (n=11) and did not change duringL-arginine application. We conclude that (1) carrier-mediated uptake of neutral and dibasic amino acids into OK cells is at least partially electrogenic. (2)L-Phenylalanine is transported by a Na⁺-symport. (3) In contrast,L-arginine depolarizes PD_m independently of extracellular sodium. (4) Electrogenic uptake of acidic amino acids is not detectable in OK cells.Parts of this study were presented at the 65th meeting of the Deutsche Physiologische Gesellschaft, March 1988, Würzburg (Pflügers Arch 411, Suppl. 1, 1988, R96) and the 6th European Colloquium on Renal Physiology, May 1988, Varna (Acta Physiol Pharmacol Bulg 14, Suppl. 1, 1988, 77)     

Transport of neutral amino acids by adult articular cartilage

The transport of three neutral amino acids: alanine, serine and leucine and the non-metabolizable analogue aminoisobutyric acid (AIB) was characterized in bovine articular cartilage slices. Alanine and serine were strongly concentrated intracellularly by a factor of 4.5 and 5 respectively and were transported in a sodium dependent and pH sensitive manner. AIB was concentrated by a factor of 1.8 with respect to the extracellular medium and was also transported in a sodium dependent and pH sensitive manner. Leucine was concentrated weakly across the chondrocyte membrane (X1.1) and its transport was independent of sodium ion concentration and the lowering of the extracellular pH. The apparent Km of transport of alanine, serine, leucine and AIB was determined to be 0.46 mM, 0.69 mM, 0.93 mM and 0.66 mM respectively and the values of Vmax were 35.8, 52.1, 14.8 and 6.8 pmol/min per mg respectively. It was shown that both alanine and serine were transported predominantly by system ASC (63.8% and 67.4% respectively) with a small percentage of transport occurring via system A. AIB was transported mainly by system A (76.0%) and leucine was transported equally via systems L (48.4%) and ASC (45.0%).     

Interorgan transport of amino acids in hemorrhagic shock.

Arterial concentrations and net organ metabolism of amino acids (AA), O2, CO2, H+, and glucose (Glc) were measured in two dogs before and during hemorrhage and after blood replacement. Shock caused increased splanchnic and decreased peripheral blood flow and O2 consumption. Po2 decreased more in hepatic venous than in mixed venous blood. pH fell in hemorrhage and increased with retransfusion. Increased liver output and arterial concentration of Glc were observed during hemorrhage. Differences between animals correlated with nutritional status. Blood concentrations of most AA showed little change during hemorrhage but increased after retransfusion. In contrast, arginine concentrations declined sharply. Peripheral output and hepatic uptake of most AA occurred during the control period. During shock, peripheral output and hepatic uptake of total AA and most individual AA declined progressively; after retransfusion peripheral uptake and hepatic output of many AA occurred. By contrast, peripheral output and hepatic uptake increased for alanine, glutamine, serine, phenylalanine, and tyrosine. After retransfusion net transport of some compounds occurred from periphery to liver; others, from liver to periphery. During shock, hepatic protein catabolism increased. and this catabolism, accompanied by decreased hepatic uptake (increased hepatic output), seemed the main cause of increased blood AA concentrations. Protein catabolism in peripheral tissue was not a cause of increased blood concentrations. Pathological changes in pH, Po2, and blood flow, occurred early in hemorrhage. In contrast, changes in AA movements and concentrations were within normal limits until late in shock.