Characterisation of <Emphasis Type="SmallCaps">l</Emphasis>-alanine and glycine absorption across the gut of an ancient vertebrate

This study utilised an in vitro technique to characterise absorption of two amino acids across the intestinal epithelium of Pacific hagfish, Eptatretus stoutii. Uptake of l-alanine and glycine conformed to Michaelis–Menten kinetics. An uptake affinity (K _m; substrate concentration required to attain a 50% uptake saturation) of 7.0 mM and an uptake capacity (J _max) of 83 nmol cm⁻² h⁻¹ were described for l-alanine. The K _m and J _max for glycine were 2.2 mM and 11.9 nmol cm⁻² h⁻¹, respectively. Evidence suggested that the pathways of l-alanine and glycine absorption were shared, and sodium dependent. Further analysis indicated that glycine uptake was independent of luminal pH and proline, but a component of uptake was significantly impaired by 100-fold excesses of threonine or asparagine. The presence of a short-term (24 h) exposure to waterborne glycine, similar in nature to that which may be expected to occur when feeding inside an animal carcass, had no significant impact on gastrointestinal glycine uptake. This may indicate a lack of cross talk between absorptive epithelia. These results are the first published data to describe gastrointestinal uptake of an organic nutrient in the oldest extant vertebrate and may provide potential insight into the evolution of nutrient transport systems.     

Histidine Absorption across Apical Surfaces of Freshwater Rainbow Trout Intestine: Mechanistic Characterization and the Influence of Copper

The essential amino acid histidine performs critical roles in health and disease. These functions are generally attributed to the amino acid itself, but could also be mediated by a positive effect on trace element bioavailability. Mechanistic information regarding the absorption of histidine across the gastrointestinal tract is essential for understanding the interplay between amino acid and mineral nutrients and the implications of these interactions for nutrition and toxicology. Using intestinal brush-border membrane vesicles obtained from freshwater rainbow trout, absorption of histidine over the range 0.78–780 μm was found to be saturable, with a maximal transport rate (J _max) of 9.1 ± 0.8 nmol mg protein⁻¹ min⁻¹ and a K _m (histidine concentration required to reach 50% of this level) of 339 ± 68 μm. Histidine uptake was highly specific as 10-fold elevated levels of a variety of amino acids with putative shared transporters failed to significantly inhibit uptake. Elevated levels of d-histidine, however, impaired uptake of the natural l-isomer. The presence of “luminal” copper (8.3 μm) significantly increased both the J _max and K _m of histidine transport. This suggests that chelated copper–histidine species cross the brush-border epithelium through transport pathways distinct from those used by histidine alone.     

Blood-endothelial cell and blood-brain transport ofl-proline, α-aminoisobutyric acid,andl-alanine

We report the application of multiple time regression analysis with the in situ brain perfusion technique to measure the rates of passage between blood and brain for [¹⁴C]l-proline, [¹⁴C]l-alanine, and [¹⁴C] α-aminoisobutyric acid (AIB) and their rapidly reversible volumes following perfusion of these amino acids from 10 to 60 seconds. We also report on their mechanism of transport. Proline diffused through the blood-brain barrier with a transfer coefficient (K_in) of 0.55 ± 0.15 × 10⁻⁴ ml/s/g and had no reversible compartment. AIB had a low K_in of 0.68±0.14×10⁻⁴ ml/s/g and a significant reversible volume of 4.34±0.51×10⁻³ ml/g in parietal cortex.l-alanine had the highest transfer coefficient, 3.11±0.26 × 10⁻⁴ ml/s/g, and a reversible volume of 10.03±0.93×10⁻³ ml/g in the same cerebral region. Postwash procedures which remove any radiotracer in the vasculature and capillary depletion were performed for alanine and AIB, as they had significant reversible compartments, to test the possibility of rapid efflux from the endothelial cells. Results obtained from wash and capillary depletion procedures suggest that a rapid efflux could occur from endothelial cells after entry of alanine and AIB. Mechanisms of transport forl-alanine and AIB were investigated using amino acids (5 mM) as substrates and inhibitors of different amino acid transport systems. AIB transport was reduced by plasma andl-leucine and unchanged by sodium-free buffer, confirming its passage by the L₁ system.l-alanine uptake was sodium-independent and not reduced by plasma.l-serine,l-cysteine,l-leucine andl-phenylalanine produced similar inhibition (66%) whilel-alanine produced a lower inhibition (41%).l-arginine increased alanine uptake in cortex and thalamus. Addingl-serine tol-phenylalanine reduced the uptake only in cortex and hippocampus. These data suggest thatl-alanine is transported by another L transport system different from the L₁ system at the luminal membrane.     

The gills as an important uptake route for the essential nutrient iron in freshwater rainbow trout Oncorhynchus mykiss

Short-term (3h) acquisition of iron (16 nmol ⁵⁹FeCl₃ l⁻¹) from oxic, alkaline fresh water was assessed in rainbow trout Oncorhynchus mykiss in the presence or absence of a range of iron chelators, all of which had differing binding affinities for ferric iron [100 μmol l⁻¹ of desferrioxamine (DFO), Log₁₀K₁ 32·5; citric acid Log₁₀K₁ 11·9; nitrilotriacetic acid (NTA) Log₁₀K₁ 15·9, CP20 and CP94 (Log₁₀K₁ > 30), as well as humic acid (HA), Log₁₀K₁ 5·04, 5 mg l⁻¹]. In the absence of chelators (control conditions) O. mykiss acquired iron from the water under laboratory lights (wavelength range of the lights 440–650 nm, peak intensity 548–626 nm) via the gill. In these conditions iron uptake onto the gill had a maximum transport capacity (J_max) of 11·2 pmol Fe g⁻¹ h⁻¹ (gill organ mass) and a K_m of 21·3 nmol Fe l⁻¹ h⁻¹. Furthermore, there were two components to iron accumulation into the carcass of these fish, a slow rate of aqueous iron uptake at low concentrations (6–24 nmol Fe l⁻¹), followed by a faster rate of uptake at higher iron concentrations (48–96 nmol Fe l⁻¹), suggesting that the rate-limiting step of iron uptake at low iron concentrations is the apical entry step. O. mykiss also acquired iron in the presence of HA, although the majority of the other chelators prevented iron uptake. Ultraviolet light (354 nm) treatment of Fe-DFO increased iron bioavailability. Results suggest that rainbow trout are able to access either the predicted very low concentrations (picomolar) of ferrous iron present in fresh water or the ferric oxide complexes present in oxic environments. The iron uptake rate measured (0·75 pmol g⁻¹ h⁻¹) would be sufficient to provide a substantial proportion (c. 85%) of the daily iron requirements of growing salmonid fry.     

Redistribution of hepatocyte chloride during l-alanine uptake

We used ion-sensitive, double-barrel microelectrodes to measure changes in hepatocyte transmembrane potential (V _m), intracellular K⁺, Cl^-, and Na⁺ activities (a ⁱ _k, a _Cl ⁱ and a _Na ⁱ ), and water volume during l-alanine uptake. Mouse liver slices were superfused with control and experimental Krebs physiological salt solutions. The experimental solution contained 20 m l-alanine, and the control solution was adjusted to the same osmolality (305 mOsm) with added sucrose. Hepatocytes also were loaded with 50 mm tetramethylammonium ion (TMA⁺) for 10 min. Changes in cell water volume during l-alanine uptake were determined by changes in intracellular, steady-state TMA⁺ activity measured with the K⁺ electrode. Hepatocyte control V _m was -33±1 mV. l-alanine uptake first depolarized V _m by 2±0.2 mV and then hyperpolarized V _m by 5 mV to-38±1 mV (n = 16) over 6 to 13 min. During this hyperpolarization, a _Na ⁱ increased by 30% from 19±2 to 25±3 mm (P < 0.01), and a _K ⁱ did not change significantly from 83±3 mm. However, with added ouabain (1 mm) l-alanine caused only a 2-mV increase in V _m, but now a _K ⁱ decreased from 61±3 to 54±5 mm (P < 0.05). Hyperpolarization of V _m by l-alanine uptake also resulted in a 38% decrease of a _Cl ⁱ from 20±2 to 12±3 mm (P < 0.001). Changes in V _m and V _Cl — V _m voltage traces were parallel during the time of l-alanine hyperpolarization, which is consistent with passive distribution of intracellular Cl^– with the V _m in hepatocytes. Added Ba²⁺ abolished the l-alanineinduced hyperpolarization, and a _Cl ⁱ remained unchanged. Hepatocyte water volume during l-alanine uptake increased by 12±3%. This swelling did not account for any changes in ion activities following l-alanine uptake. We conclude that hepatocyte a _K ⁱ is regulated by increased Na⁺-K⁺ pump activity during l-alanine uptake in spite of cell swelling and increased V _m due to increased K⁺ conductance. The hyperpolarization of V _m during l-alanine uptake provides electromotive force to decrease a _Cl ⁱ . The latter may contribute to hepatocyte volume regulation during organic solute transport.This work was supported by grant AA-08867 from the Alcohol, Drug Abuse, and Mental Health Association.     

MRP2 and the Transport Kinetics of Cysteine Conjugates of Inorganic Mercury

Human exposure to mercuric species occurs regularly throughout the world. Mercuric ions may accumulate in target cells and subsequently lead to cellular intoxication and death. Therefore, it is important to have a thorough understanding of how transportable species of mercury are handled by specific membrane transporters. The purpose of the current study was to characterize the transport kinetics of cysteine (Cys)-S-conjugates of inorganic mercury (Cys-S-Hg-S-Cys) at the site of the multidrug resistance-associated transporter 2 (MRP2). In order to estimate the maximum velocity (V _max) and Michaelis constant (K _m) for the uptake of Cys-S-Hg-S-Cys mediated by MRP2, in vitro studies were carried out using radioactive Cys-S-Hg-S-Cys (5 μM) and inside-out membrane vesicles made from Sf9 cells transfected with MRP2. The V _max was estimated to be 74.3 ± 10.1 nmol mg protein⁻¹ 30 s⁻¹ while the K _m was calculated to be 63.4 ± 27.3 μM. In addition, in vivo studies were utilized to measure the disposition of inorganic mercury (administered dose 0.5 μmol kg⁻¹ in 2 mL normal saline) over time in Wistar and TR¯ (Mrp2-deficient) rats. These studies measured the disposition of mercuric ions in the kidney, liver, and blood. In general, the data suggest that the initial uptake of mercuric conjugates into select target cells is rapid followed by a period of slower uptake and accumulation. Overall, the data indicate that MRP2 transports Cys-S-Hg-S-Cys in a manner that is similar to that of other MRP2 substrates.

     

Facilitated Transport of Lactate by Rat Jejunal Enterocyte

L-lactate transport mechanism across rat jejunal enterocyte was investigated using isolated membrane vesicles. In basolateral membrane vesicles l-lactate uptake is stimulated by an inwardly directed H⁺ gradient; the effect of the pH difference is drastically reduced by FCCP, pCMBS and phloretin, while furosemide is ineffective. The pH gradient effect is strongly temperature dependent. The initial rate of the proton gradient-induced lactate uptake is saturable with respect to external lactate with a K _m of 39.2 ± 4.8 mm and a J _max of 8.9 ± 0.7 nmoles mg protein⁻¹ sec⁻¹. A very small conductive pathway for l-lactate is present in basolateral membranes. In brush border membrane vesicles both Na⁺ and H⁺ gradients exert a small stimulatory effect on lactate uptake. We conclude that rat jejunal basolateral membrane contains a H⁺-lactate cotransporter, whereas in the apical membrane both H⁺-lactate and Na⁺-lactate cotransporters are present, even if they exhibit a low transport rate. Received: 22 October 1996/Revised: 11 March 1997     

Sulphate transport in the cyanobacterium Synechococcus R-2 (Anacystis nidulans, S. leopoliensis) PCC 7942

Synechococcus R-2 (PCC 1942) actively accumulates sulphate in the light and dark. Intracellular sulphate was 1.35 ± 0.23 mol m^?3 (light) and 0.894 ± 0.152 mol m^?3 (dark) under control conditions (BG-11 media: pH_o, 7.5; [SO₄^2?]_o, 0.304 mol m^?3). The sulphate transporter is different from that found in higher plants: it appears to be an ATP-driven pump transporting one SO₄^2?/ATP [ΔμSO₄^2?_i,o=+ 27.7 ± 0.24 kJ mol^?1 (light) and + 24 ± 0.34 kj mol^?1 (dark)]. The rate of metabolism of SO₄^2?at pH_o, 7.5 was 150 ± 28 pmol m^?2 s^?1 (n = 185) in the light but only 12.8 ± 3.6 pmol m^?2 s^?1 (n = 61) in the dark. Light-driven sulphate uptake is partially inhibited by DCMU and chloramphenicol. Sulphate uptake is not linked to potassium, proton, sodium or chloride transport. The alga has a constitutive over-capacity for sulphate uptake [light (n= 105): K_m= 0.3 ± 0.1 mmol m^?3, V_max, = 1.8 ± 0.6 nmol m^?2 s^?1; dark (n= 56): K_m= 1.4 ± 0.4 mmol m^?3, V_max= 41 ± 22 pmol m^?2 s^?1]. Sulphite (SO₃^2?) was a competitive inhibitor of sulphate uptake. Selenate (SeO₄^2?) was an uncompetitive inhibitor.     

CARRIER MEDIATED BLOOD-BRAIN BARRIER TRANSPORT OF CHOLINE AND CERTAIN CHOLINE ANALOGS

Blood-brain barrier (BBB) transport of choline and certain choline analogs was studied in adult and suckling rats, and additionally compared in the paleocortex and neocortex of adult rats. Saturable uptake was characterized by a single kinetic system in all cases examined, and in adult rat forebrains we determined a K_m= 442 ± 60 μM and V_max= 10.0 ± 0.6 nmol min^-1 g^-1. In 14–15-day-old suckling forebrains a similar K_m (= 404 ± 88 μM) but higher V_max (= 12.5 ± 1.5 nmol min^-1 g^-1) was determined. When choline uptake was compared in two regions of the forebrain, similar Michaelis-Menten constants were determined but a higher uptake velocity was found in the neocortex (i.e. neocortex K_m= 310 ± 103 μM and V_max= 12.6 ± 2.8 nmol min^-1g^-1; paleocortex K_m= 217 ± 76 μM and V_max= 7.2 ± 1.5 nmol min^-1 g^-1). Administration of radiolabelled choline at low (5 μM) and high (100 μM) concentrations, followed by microwave fixation 60 s later and chloroform-methanol-water separations of the homogenized brain did not suggest a relationship between concentration and the appearance of label in lipid or aqueous fractions as observed in another in-vitro study elaborating two-component kinetics of choline uptake. It was observed that 60s after carotid injection 12–14% of the radiolabel in the ipsilateral cortex was found in the chloroform-soluble fraction. Hemicholinium-3 (K_i= 111 μM), dimethylaminoethanol (K_i= 42 μM), tetraethyl ammonium chloride, tetramethyl ammonium chloride, 2-hydroxyethyl triethylammonium iodide, carnitine, normal rat serum, and to a lesser extent lithium and spermidine all inhibited choline uptake in the BBB. Unsubstituted ammonium chloride and imipramine did not inhibit choline uptake. No difference was observed in blood-brain barrier choline uptake of unanesthetised, carotid artery-catheterized animals, and comparable sodium pentobarbital-anesthetized controls.     

Kinetics of Transport and Phosphorylation of 2-Fluoro-2-Deoxy-d-Glucose in Rat Brain

Abstract: The kinetics of transport across the blood-brain barrier and metabolism in brain (hemisphere) of [¹⁴C]2-fluoro-2-deoxy-d -glucose (FDG) were compared to that of [³H]2-deoxy-d -glucose (DG) and d -glucose in the pentobarbital-anesthetized adult rat. Saturation kinetics of transport were measured with the brain uptake index (BUI) method. The BUI for FDG was 54.3 ± 5.6. Nonlinear regression analysis gave a K_m of 6.9 ± 1.1 mM and a V_max of 1.70 ± 0.32 μmol/min/g. The K₁ for glucose inhibition of FDG transport was 10.7 ± 4.4 mM. The kinetic constants of influx (k₁) and efflux (K₂) for FDG were calculated from the K_m, V_max, and glucose concentrations of the hemisphere and plasma (2.3 ± 0.2 μmol/g and 9.9 ± 0.4 mM, respectively). The transport coefficient (k_{1 FDG}/k₁glucose) was 1.67 ± 0.07 and the phosphorylation constant was 0.55 ± 0.16. The predicted lumped constant for FDG was 0.89, whereas the measured hexose utilization index for FDG was 0.85 ± 0.16. Conclusion: The value for the lumped constant can be predicted on the basis of the known kinetic constants of FDG and glucose transport and metabolism, as well as brain and plasma glucose levels. Knowledge of the lumped constant is crucial in interpreting data obtained from ¹⁸FDG analysis of regional glucose utilization in human brain in pathological states. We propose that the lumped constant will rise to a maximum equal to the transport coefficient for FDG under conditions of transport limitation (hypoglycemia) or elevated glycolysis (ischemia, seizures), and will fall to a minimum equal to the phosphorylation coefficient during phosphorylation limitation (extreme hyperglycemia).     

l- andd-alanine transport in brush border membrane vesicles from lepidopteran midgut: Evidence for two transport systems

Summary In brush border membrane vesicles from the midgut ofPhilosamia cynthia larvae (Lepidoptera) thel- andd-alanine uptake is dependent on a potassium gradient and on transmembrane electrical potential difference. Each isomer inhibits the uptake of the other form: inhibition ofl-alanine uptake byd-alanine is competitive, whereas inhibition ofd-alanine uptake byl-alanine is noncompetitive. Transstimulation experiments as well as the different pattern of specificity to cations suggest the existence of two transport systems. Kinetic parameters for the two transporters have been calculated both when K_out>K_in and K_out=K_in.d-alanine is actively transported also by the whole midgut, but it is not metabolized by the intestinal tissue.     

Effect of beta-alanine supplementation on muscle carnosine concentrations and exercise performance

High-intensity exercise results in reduced substrate levels and accumulation of metabolites in the skeletal muscle. The accumulation of these metabolites (e.g. ADP, Pi and H⁺) can have deleterious effects on skeletal muscle function and force generation, thus contributing to fatigue. Clearly this is a challenge to sport and exercise performance and, as such, any intervention capable of reducing the negative impact of these metabolites would be of use. Carnosine (β-alanyl-l-histidine) is a cytoplasmic dipeptide found in high concentrations in the skeletal muscle of both vertebrates and non-vertebrates and is formed by bonding histidine and β-alanine in a reaction catalysed by carnosine synthase. Due to the pKa of its imidazole ring (6.83) and its location within skeletal muscle, carnosine has a key role to play in intracellular pH buffering over the physiological pH range, although other physiological roles for carnosine have also been suggested. The concentration of histidine in muscle and plasma is high relative to its K _m with muscle carnosine synthase, whereas β-alanine exists in low concentration in muscle and has a higher K _m with muscle carnosine synthase, which indicates that it is the availability of β-alanine that is limiting to the synthesis of carnosine in skeletal muscle. Thus, the elevation of muscle carnosine concentrations through the dietary intake of carnosine, or chemically related dipeptides that release β-alanine on absorption, or supplementation with β-alanine directly could provide a method of increasing intracellular buffering capacity during exercise, which could provide a means of increasing high-intensity exercise capacity and performance. This paper reviews the available evidence relating to the effects of β-alanine supplementation on muscle carnosine synthesis and the subsequent effects on exercise performance. In addition, the effects of training, with or without β-alanine supplementation, on muscle carnosine concentrations are also reviewed.     

Uptake by Yeast: Interaction of Rb+, Na+ and Phosphate

It has been shown that addition of phosphate to phosphate deficient yeast gives rise to an immediate increase in the rate of Na⁺ uptake and an immediate decrease in the rate of Rb⁺ uptake. In addition, phosphate uptake is enhanced specifically by Na ions presumably by a process with a very high affinity for phosphate with a K_m of about 2 × 10⁻⁶M at pH 7.2, whereas the K_m for phosphate uptake of the Na⁺ independent process amounts to 1.3 × 10⁻⁴M.     

Regulation of intestinal hPepT1 (SLC15A1) activity by phosphodiesterase inhibitors is via inhibition of NHE3 (SLC9A3)

The H⁺-coupled transporter hPepT1 (SLC15A1) mediates the transport of di/tripeptides and many orally-active drugs across the brush-border membrane of the small intestinal epithelium. Incubation of Caco-2 cell monolayers (15 min) with the dietary phosphodiesterase inhibitors caffeine and theophylline inhibited Gly-Sar uptake across the apical membrane. Pentoxifylline, a phosphodiesterase inhibitor given orally to treat intermittent claudication, also decreased Gly-Sar uptake through a reduction in capacity (V_max) without any effect on affinity (K_m). The reduction in dipeptide transport was dependent upon both extracellular Na⁺ and apical pH but was not observed in the presence of the selective Na⁺/H⁺ exchanger NHE3 (SLC9A3) inhibitor S1611. Measurement of intracellular pH confirmed that caffeine was not directly inhibiting hPepT1 but rather having an indirect effect through inhibition of NHE3 activity. NHE3 maintains the H⁺-electrochemical gradient which, in turn, acts as the driving force for H⁺-coupled solute transport. Uptake of β-alanine, a substrate for the H⁺-coupled amino acid transporter hPAT1 (SLC36A1), was also inhibited by caffeine. The regulation of NHE3 by non-nutrient components of diet or orally-delivered drugs may alter the function of any solute carrier dependent upon the H⁺-electrochemical gradient and may, therefore, be a site for both nutrient-drug and drug-drug interactions in the small intestine.     

Isolation and function of the amino acid transporter PAT1 (slc36a1) from rabbit and discrimination between transport via PAT1 and system IMINO in renal brush-border membrane vesicles

Reabsorption of amino acids is an important function of the renal proximal tubule. pH-dependent amino acid transport has been measured previously using rabbit renal brush-border membrane vesicles (BBMV). The purpose of this investigation was to determine whether this pH-dependent uptake represents H⁺/amino acid cotransport via a PAT1-like transport system. The rabbit PAT1 cDNA was isolated (2296bp including both 5′ and 3′ untranslated regions and poly(A) tail) and the open reading frame codes for a protein of 475 amino acids (92% identity to human PAT1). Rabbit PAT1 mRNA was found in all tissues investigated including kidney. When expressed heterologously in a mammalian cell line, rabbit PAT1 mediates pH-dependent, Na⁺-independent uptake of proline, glycine, l-alanine and α-(methylamino)isobutyric acid. Proline uptake was maximal at pH?5.0 (K_m?2.2±0.7?mM). A transport system with identical characteristics (ion dependency, substrate specificity) was detected in rabbit renal BBMV where an overshoot was observed in the absence of Na⁺ but in the presence of an inwardly directed H⁺ gradient. In the presence of Na⁺ and under conditions in which PAT1 transport function was suppressed, a second proline uptake system was detected that exhibited functional characteristics similar to those of the IMINO system. The functional characteristics of rabbit PAT1 in either mammalian cells or renal BBMV suggest that PAT1 is the low-affinity transporter of proline, glycine and hydroxyproline believed to be defective in patients with iminoglycinuria.     

Characteristics of dipeptide transport in pig jejunum in vitro

 Characteristics of dipeptide transport in pig jejunum were investigated in vitro by applying the Ussing-chamber technique and mucosal uptake studies. Addition of both glycyl-l-glutamine and glycyl-l-sarcosine (20 mmol · l⁻¹) to the mucosal buffer solution significantly increased the short-circuit current by 2.60 ± 0.15 and 1.57 ± 0.20 μeq · cm⁻² · h⁻¹, respectively. Concentration-dependent changes in short-circuit current followed Michaelis-Menten kinetics with similar affinity constants for both dipeptides. From unidirectional flux rates for radiolabelled glycyl-l-sarcosine, a net flux rate for glycyl-l-sarcosine of 49.8 ± 6.7 nmol · cm⁻² · h⁻¹ was calculated. In mucosal uptake experiments, the apical influx of ¹⁴C-labelled glycyl-l-sarcosine into isolated porcine mucosa was pH dependent and significantly inhibited by glycyl-l-glutamine. Moreover, RT-PCR studies with primers derived from rabbit PepT1 identified two PCR fragments of identical size to rabbit PepT1 from pig intestinal mRNA preparations. In conclusion, our studies revealed key features of mammalian intestinal peptide transporters and give evidence for a PepT1-like transporter in the pig jejunum that could significantly contribute to the overall amino acid absorption from the gut. Accepted: 30 June 1999     

Function of taurine transporter (Slc6a6/TauT) as a GABA transporting protein and its relevance to GABA transport in rat retinal capillary endothelial cells

The purpose of this study was to identify the uptake mechanism of γ-aminobutyric acid (GABA) via taurine transporter (Slc6a6/TauT) and its relationship with GABA transport at the inner BRB. Rat Slc6a6/TauT-transfected HeLa cells exhibited Na⁺-, Cl⁻-, and concentration-dependent [³H]GABA uptake with a K_m of 1.5 mM. Taurine, β-alanine, and GABA markedly inhibited Slc6a6/TauT-mediated uptake of [³H]GABA. The uptake of [³H]GABA by a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) was Na⁺-, Cl⁻-, and concentration-dependent with a K_m of 2.0 mM. This process was more potently inhibited by substrates of Slc6a6/TauT, taurine and β-alanine, than those of GABA transporters, GABA and betaine. In the presence of taurine, there was competitive inhibition with a K_i of 74 μM. [³H]Taurine also exhibited competitive inhibition with a K_i of 1.8 mM in the presence of GABA. In conclusion, rat Slc6a6/TauT has the ability to use GABA as a substrate and Slc6a6/TauT-mediated GABA transport appears to be present at the inner BRB.     

Interaction of Synthetic Dipeptide Bestim with Mouse Thymocytes and Macrophages

Tritium-labeled dipeptide bestim (γ-D-Glu-L-Trp) with a specific activity of 45 Ci/mmol was obtained by the high-temperature solid-state catalytic isotope exchange (HSCIE) reaction. [³H]bestim was found to bind with high affinity to mouse peritoneal macrophages (K _d 2.1 ± 0.1 nM) and thymocytes (K _d 3.1 ± 0.2 nM) and also plasma membranes isolated from these cells (K _d 18.6 ± 0.2 and 16.7 ± 0.3 nM respectively). The specific bonding of [³H]bestim with macrophages and thymocytes was inhibited by unlabeled dipeptide thymogen (L-Glu-L-Trp) (K _i 0.9 ± 0.1 and 1.1 ± 0.1 nM respectively). Treatment of the macrophages and thymocytes with trypsin led to their loss of capacity to bind [³H]bestim. Bestim at concentrations range of 0.1–1000 nМ reduced the adenylate cyclase activity in macrophage and thymocyte membranes.     

Mechanism and Putative Structure of B0-like Neutral Amino Acid Transporters

The Na⁺-dependent transport of neutral amino acids in epithelial cells and neurons is mediated by B⁰-type neutral amino acid transporters. Two B⁰-type amino acid transporters have been identified in the neurotransmitter transporter family SLC6, namely B⁰AT1 (SLC6A19) and B⁰AT2 (SLC6A15). In contrast to other members of this family, B⁰-like transporters are chloride-independent. B⁰AT1 and B⁰AT2 preferentially bind the substrate prior to the Na⁺-ion. The Na⁺-concentration affects the K _m of the substrate and vice versa. A kinetic scheme is proposed that is consistent with the experimental data. An overlapping binding site of substrate and cosubstrate has been demonstrated in the bacterial orthologue LeuT _Aa from Aquifex aeolicus, which elegantly explains the mutual effect of substrate and cosubstrate on each other’s K _m -value. LeuT _Aa is sequence-related to transporters of the SLC6 family, allowing homology modeling of B⁰-like transporters along its structure.     

Active Transport of Glutamate in Leishmania (Leishmania) amazonensis

ABSTRACT. Leishmania spp. are the causative agents of leishmaniasis, a complex of diseases with a broad spectrum of clinical manifestations. Leishmania (Leishmania) amazonensis is a main etiological agent of diffuse cutaneous leishmaniasis. Leishmania spp., as other trypanosomatids, possess a metabolism based significantly on the consumption of amino acids. However, the transport of amino acids in these organisms remains poorly understood with few exceptions. Glutamate transport is an important biological process in many organisms. In the present work, the transport of glutamate is characterized. This process is performed by a single kinetic system (K_m=0.59±0.04 mM, V_max=0.123±0.003 nmol/min per 20 × 10⁶ cells) showing an energy of activation of 52.38±4.7 kJ/mol and was shown to be partially inhibited by analogues, such as glutamine, aspartate, α‐ketoglutarate and oxaloacetate, methionine, and alanine. The transport activity was sensitive to the extracellular concentration of H⁺ but not to Na⁺ or K⁺. However, unlike other amino acid transporters presently characterized, the treatment with specific ionophores confirmed the participation of a K⁺, and not H⁺ membrane gradient in the transport process.