Specificity of transinhibition of amino acid transport in neurospora

Amino acid transport systems I and III in Neurospora are inhibited by amino acids in the intracellular pool (transinhibition). The transinhibition is system specific. The ability of an amino acid to transinhibit a transport system is highly correlated with its affinity for the system. The significance of the system specificity of transinhibition is discussed.     

Neutral amino acid transport in embryonal carcinoma cells

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

Neutral amino acid transport at the human blood-brain barrier

The kinetics of human blood-brain barrier neutral amino acid transport sites are described using isolated human brain capillaries as an in vitro model of the human blood-brain barrier. Kinetic parameters of transport (Km, Vmax, and KD) were determined for eight large neutral amino acids. Km values ranged from 0.30 +/- 0.08 microM for phenylalanine to 8.8 +/- 4.6 microM for valine. The amino acid analogs N-methylaminoisobutyric acid and 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid were used as model substrates of the alanine- and leucine-preferring transport systems, respectively. Phenylalanine is transported solely by the L-system (which is sensitive to 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid), and leucine is transported equally by the L- and ASC-system (which is sodium-dependent and N-methylaminoisobutyric acid-independent). Dose-dependent inhibition of the high affinity transport system by p-chloromercuribenzenesulfonic acid is demonstrated for phenylalanine, similar to the known sensitivity of blood-brain barrier transport in vivo. The Km values for the human brain capillary in vitro correlate significantly (r = 0.83, p less than 0.01) with the Km values for the rat brain capillary in vivo. The results show that the affinity of human blood-brain barrier neutral amino acid transport is very high, i.e. very low Km compared to plasma amino acid concentrations. This provides a physical basis for the selective vulnerability of the human brain to derangements in amino acid availability caused by a selective hyperaminoacidemia, e.g. hyperphenylalaninemia.     

Neutral amino acid transport at the human blood-brain barrier

Transport regulates nutrient availability in the brain, and many pathways of brain amino acid metabolism are influenced by precursor supply. Therefore, amino acid transport through the blood-brain barrier (BBB) plays an important rate-affecting role in brain metabolism. Information on the Km of BBB amino acid transport provides the quantitative basis for understanding the physiological importance of BBB transport competition effects. For example, the uniquely low Km values of BBB amino acid transport as compared to other organs in the rat provides the basis for the selective vulnerability of the rat brain to changes in amino acid supply caused by nutritional factors. The development of amino acid imbalances in the human brain in parallel with amino acid imbalances in blood is likely to occur if the Km of BBB neutral amino acid transport in humans is low, e.g., 25-100 microM, as is the case for the rat. A new model system of the human BBB, the isolated human brain capillary, has been developed. Recent studies with this system indicate that the Km of phenylalanine transport into human brain microvessels is approximately the same as that found during in vivo studies with laboratory rats. These results support the emerging hypothesis that the human brain, like the rat brain, is subject to acute regulation by dietary-related amino acid imbalances, and that the major site of this regulation is the amino acid transport system at the BBB.     

Neutral amino acid transport in Pseudomonas fluorescens

Membrane transport of beta-alanine, l-alanine, and l-proline was studied in a beta-alanine transaminaseless mutant (strain 67) of Pseudomonas fluorescens. In this mutant beta-alanine is metabolically inert, and it was therefore possible to demonstrate active transport of this substrate in the absence of intracellular catabolism. The permease which catalyzes the uptake of beta-alanine also transports l-proline and l-alanine. This common transport system was distinguished from permeases which transport only l-alanine and only l-proline by competition studies in strain 67 and by studies of transport specificity in a permeaseless mutant (strain 67/4MTR).     

Neutral amino acid transport in an androgen-dependent epithelium

• 1.1. Transport of neutral amino acids by the isolated seminal vesicle epithelium of normal and gonadectomized guinea pigs has been investigated by measurement of the uptake of 2-amino[1-¹⁴C]-isobutyric acid and 2-methylamino[1-¹⁴C]isobutyric acid.
• 2.2. The V_max for Na-dependent and -independent transport of both amino acids was reduced by gonadectomy but the general transport characteristics appeared to be unchanged by this treatment.
• 3.3. The most likely explanation of the decreased transport is the loss of transporter molecules associated with the tissue regression that follows rapidly on gonadectomy.

     

Neutral amino acid transport in Leishmania promastigotes

Neutral amino acid transport was investigated in Leishmania promastigotes. Proline and alanine transport occur against their concentration gradient although there is a very rapid (40% at 30 min) conversion of proline to alanine. Uptake of these amino acids occurs by a sodium-independent route which is completely eliminated by addition of CCCP or KCN. $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for proline and alanine are 80 μM and 63 μM with $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ values of 6.4 and 7.2 nmol/min per mg dry weight, respectively. Countertransport of proline, alanine and phenylalanine was measured by loading the cells with a variety of neutral amino acids and proline analogs, followed by CCCP addition. The effect of aminooxyacetic acid, an inhibitor of alanine aminotransferase (EC 2.6.1.2), on proline and alanine countertransport was also examined. The results obtained are consistent with the presence of at least two systems for neutral amino acid transport in Leishmania promastigotes.     

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.     

Neutral amino acid transport in surface membrane vesicles isolated from mouse fibroblasts: Intrinsic and extrinsic models of regulation

Membrane transport carrier function, its regulation and coupling to metabolism, can be selectively investigated dissociated from metabolism and in the presence of a defined electrochemical ion gradient driving force, using the single internal compartment system provided by vesiculated surface membranes. Vesicles isolated from nontransformed and Simian virus 40-transformed mouse fibroblast cultures catalyzed carrier-mediated transport of several neutral amino acids into an osmotically-sensitive intravesicular space without detectable metabolic conversion of substrate. When a Na⁺ gradient, external Na⁺ > internal Na⁺, was artifically imposed across vesicle membranes, accumulation of several neutral amino acids achieved apparent intravesicular concentrations 6- to 9-fold above their external concentrations. Na⁺-stimulated alanine transport activity accompanied plasma membrane material during subcellular fractionation procedures. Competitive interactions among several neutral amino acids for Na⁺-stimulated transport into vesicles and inactivation studies indicated that at least 3 separate transport systems with specificity properties previously defined for neutral amino acid transport in Ehrlich ascites cells were functional in vesicles from mouse fibroblasts: the A system, the L system and a glycine transport system. The pH profiles and apparent K_m values for alanine and 2-aminoisobutyric acid transport into vesicles were those expected of components of the corresponding cellular uptake system. Several observations indicated that both a Na⁺ chemical concentration gradient and an electrical membrane potential contribute to the total driving force for active amino acid transport via the A system and the glycine system. Both the initial rate and quasi-steady-state of accumulation were stimulated as a function of increasing concentrations of Na⁺ applied as a gradient (external > internal) across the membrane. This stimulation was independent of endogenous Na⁺, K⁺-ATPase activity in vesicles and was diminished by monensin or by preincubation of vesicles with Na⁺. The apparent K_m for transport of alanine and 2-aminoisobutyric acid was decreased as a function of Na⁺ concentration. Similarly, in the presence of a standard initial Na⁺ gradient, quasi-steady-state alanine accumulation in vesicles increased as a function of increasing magnitudes of interior-negative membrane potential imposed across the membrane by means of K⁺ diffusion potentials (internal > external) in the presence of valinomycin; the magnitude of this electrical component was estimated by the apparent distributions of the freely permeant lipophilic cation triphenylme thylphosphonium ion. Alanine transport stimulation by charge asymmetry required Na⁺ and was blocked by the further addition of either nigericin or external K⁺. As a corollary, Na⁺-stimulated alanine transport was associated with an apparent depolarization, detectable as an increased labeled thiocyanate accumulation. Permeant anions stimulated Na⁺-coupled active transport of these amino acids but did not affect Na⁺-independent transport. Translocation of K⁺, H⁺, or anions did not appear to be directly involved in this transport mechanism. These characteristics support an electrogenic mechanism in which amino acid translocation is coupled t o an electrochemical Na⁺ gradient by formation of a positively charged complex, stoichiometry unspecified, of Na⁺, amino acid, and membrane component. Functional changes expressed in isolated membranes were observed t o accompany a change in cellular proliferative state or viral transformation. Vesicles from Simian virus 40-transformed cells exhibited an increased Vmax of Na⁺-stimulated 2-aminoisobutyric acid transport, as well as an increased capacity for steady-state accumulation of amino acids in response t o a standard Na⁺ gradient, relative t o vesicles from nontransformed cells. Density-inhibition of nontransformed cells was associated with a marked decrease in these parameters assayed in vesicles. Several possibilities for regulatory interactions involving gradient-coupled transport systems are discussed.     

Hormonal regulation of hepatic amino acid transport

The transport of 2-aminoisobutyric acid (AIB) into liver tissue was increased by both insulin and glucagon. We have now shown that these hormones do not stimulate the same transport system. Glucagon, possibly via cAMP, increased the hepatic uptake of AIB by a mechanism which resembled system A. This glucagon-sensitive system could be monitored by the use of the model amino acid MeAIB. In contrast, the insulin-stimulated system exhibited little or no affinity for MeAIB and will be referred to as system B. On the basis of other reports that the hepatic transport of AIB is almost entirely Na⁺ dependent and the present finding that the uptake of 2-aminobicyclo [2,2,1] heptane-2-carboxylic acid (BCH) was not stimulated by either hormone, we conclude that system B is Na⁺ dependent. Furthermore, insulin added to the perfusate of livers from glucagon-pretreated donors suppressed the increase in AIB or MeAIB uptake. Depending upon the specificities of systems A and B, both of which are unknown for liver tissue, the insulin/glucagon ratio may alter the composition of the intracellular pool of amino acids.     

Dependence of adaptative regulation for IL-1β action on system A activity in human synovial cells

Human synovial cells are a suitable model for estimating the physiopathological effects of IL-1β (IL-1) in joint. Given the importance of this cytokine in the modulation of cell metabolic activities, we set out to study the action of IL-1 on the neutral amino acid transport A system, using the methyl (aminoisobutyric) acid (MeAIB), the most highly specific and nonmetabolizable substrate for the A system. Stimulation of system A activity by adaptative regulation is a prerequisite to obtain an increase of MeAIB uptake in IL-1-treated cells, since cells which had been grown in a normal medium did not express stimulation of system A activity when IL-1 was added. The IL-1-mediated MeAIB uptake is independent of protein synthesis, since cycloheximide (CHX) did not inhibit MeAIB uptake, and characterized by a decrease in the Michaelis constant (K_m) (0.147 vs. 0.270 mmol/l, IL-1 vs. control) and a slight increase in maximal velocity (V_max) (4.59 vs. 3.89 nmol/mg prot/10 min, IL-1 vs. control). These observations indicate that IL-1 induces modifications in both system A transporter affinity and number. Moreover, we indicate that system A should be responsive in vivo to IL-1 in the same way since derepression and IL-1 action occurred in the presence of human synovial fluid. © 1996 Wiley-Liss, Inc.