L-type amino acid transporters in two intestinal epithelial cell lines function as exchangers with neutral amino acids (original) (raw)
Related papers
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1993
High-and low-affinity Na+-dependent neutral L-a-amino acid transporters were solubilized with 0.25% octaethylene glycol dodecyl ether (C12E8) after removal of the proteins from the brush-border membrane vesicles with 2% CHAPS and 4 M urea. When the CHAPS-insoluble protein was treated with papain before its solubilization with C12E8, a substantial amount of protein was removed without any decrease of the transport activities. The solubilized transporters were reconstituted into proteoliposomes after removal of C12E a with Bio-Beads SM2. Several parameters proved to be important for optimal reconstitution efficiency: (a) the type of detergent, and (b) the phospholipid/protein and detergent/protein ratio during reconstitution, and (c) the salt concentration during reconstitution. Reconstituted proteoliposomes showed rapid uptake of neutral L-a-amino acids but not imino acid, basic or acidic amino acids driven by an electrochemical potential of Na ÷ (out > in). The uptakes under low-and high-substrate condition were further augmented by an artificial membrane potential introduced by K ÷ diffusion via valinomycin (negative interior). Kinetic analysis revealed that both the brush-border membranes and the solubilized fraction involved two carrier-mediated pathways for alanine transport. The kinetic parameters were determined by curve fitting with a computer to be Ka-0.28 mM (0.21 mM) and Kt2 = 43.2 mM (28.4 mM), respectively (those with brush-border membrane vesicles in parentheses). Studies on the specific activities for transport of individual amino acids under low or high substrate concentration and the cross-inhibitory effects of various amino acids on alanine uptake (low concentration) revealed that these transporters possess broad specificity for neutral L-a-amino acids.
Amino Acid Transport Across Mammalian Intestinal and Renal Epithelia
Physiological Reviews, 2008
The transport of amino acids in kidney and intestine is critical for the supply of amino acids to all tissues and the homeostasis of plasma amino acid levels. This is illustrated by a number of inherited disorders affecting amino acid transport in epithelial cells, such as cystinuria, lysinuric protein intolerance, Hartnup disorder, iminoglycinuria, dicarboxylic aminoaciduria, and some other less well-described disturbances of amino acid transport. The identification of most epithelial amino acid transporters over the past 15 years allows the definition of these disorders at the molecular level and provides a clear picture of the functional cooperation between transporters in the apical and basolateral membranes of mammalian epithelial cells. Transport of amino acids across the apical membrane not only makes use of sodium-dependent symporters, but also uses the proton-motive force and the gradient of other amino acids to efficiently absorb amino acids from the lumen. In the basolate...
The role of sodium ion in the transport of amino acids by the intestine
Biochimica et biophysica acta, 1965
I. Decreasing the incubation medium Na+ concentration below physiologic levels resulted in decreased transport of Na + and water in the isolated rabbit jejunum as well as diminution of amino acid transport. 2. Ouabain inhibited the transport of Na+ and amino acids in everted jejunal sacs. 3. Reduction of medium Na+ was associated with decreased tissue Na + and K+ content while ouabain produced elevated tissue Na+ and reduced tissue K+ concentrations. 4. Kinetic analysis of amino acid-uptake data showed that ouabain and reduction of medium Na + caused a slowing of both influx and, to a lesser degree, effiux coefficients. 5. The Na+ ion was shown to affect energy-independent (diffusion) as well as energy-dependent (active) transport of amino acids.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1990
We have characterized the transcellular transport of a large neutral amino acid (LNAA) in Caco-2 cell monolayers. The apical (AP) to basolateral (BL) transport of phenylalanine (Phe) was approximately 10-times faster than BL-to-AP transport. The evidence for the carrier-mediated AP-to-BL transport of Pbe include: (a) temperature dependence and saturability, (b) Phe transport was not affected by a reverse gradient, (c) the activation energy for transport was 12.0 kcal/tool, and (d) an excess amount of unlabeled Phe caused a 75% reduction in transport rate and a delay (lag time) in the appearance of Phe in the BL side. The V m and K m for Phe transport were 572.4 pmol • mg protein-t. min-t and 0.56 raM, respectively. Phe transport was decreased in the absence of glucose and in the presence of sodium azide or ouahain. The carrier interacted with LNAAs and with cationic amino acids but not with small neutral or anionic amino acids.
Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2000
Amino acid influx across the brush border membrane of the intact pig ileal epithelium was studied. It was examine whether in addition to system B, systems ASC and b o, + were involved in transport of bipolar amino acids. The kinetics of interactions between lysine and leucine demonstrates that system b o, + is present and accessible also to L-glutamine. D-aspartate (K 1/2 0.3 mM) and L-glutamate (K i 0.5 mM) share a high affinity transporter with a maximum rate of 1.3 mmol cm − 2 h − 1 , while only L-glutamate with a K 1/2 of 14.4 mM uses a low affinity transporter with a maximum rate of 2.7 mmol cm − 2 h − 1 , system ASC, against which serine has a K i of 1.6 mM. In the presence of 100 mM lysine, L-glutamine (A), leucine (B), and methionine (C) fulfilled the criteria of the ABC test for transport by one and the same transporter. However, serine inhibits not only transport of L-glutamate but also of glutamine (K i 0.5 mM), and L-glutamate inhibits part of the transport of glutamine. The test does, therefore, only indicate that the three bipolar amino acids have similar affinities for transport by systems B and ASC. Further study of the function of system B must be carried out under full inhibition by lysine and glutamate.
Amino acid transport by small intestinal, hepatic, and pancreatic epithelia
Gastroenterology, 1995
Abbreviations used in this paper: AIB, 2-aminoisobutyric acid; GGT, y-glutamyltranspeptidase; MeAIB, 2-(methylamino) isobutyric acid; NAA, neutral amino acid; NBAT, neutral and basic amino acid transporter; ORT, oral rehydration therapy; rBAT, related to b °'+ amino acid transporter; SGLT, sodium-dependent glucose transporter; Vm~x, maximum velocity.
The Journal of Membrane Biology, 1995
We determined the extent of Na+-independent, proton-driven amino acid transport in human intestinal epithelia (Caco-2). In Na+-free conditions, acidification of the apical medium (apical pH 6.0, basolateral pH 7.4) is associated with a saturable net absorption of glycine. With Na+-free media and apical pH set at 6.0, (basolateral pH 7.4), competition studies with glycine indicate that proline, hydroxyproline, sarcosine, betaine, taurine, [3-alanine, r acid (AIB), (x-methylaminoisobutyric acid (MeAIB), x-amino-nbutyric acid and L-alanine are likely substrates for pHdependent transport in the brush border of Caco-2 cells. Both D-serine and D-alanine were also substrates. In contrast leucine, isoleucine, valine, phenylalanine, methionine, threonine, cysteine, asparagine, glutamine, histidine, arginine, lysine, glutamate and D-aspartate were not effective substrates. Perfusion of those amino acids capable of inhibition of acid-stimulated net glycine transport at the brush-border surface of Caeo-2 cell monolayers loaded with the pH-sensitive dye 2',7'-bis(2carboxyethyl-5(6)-carboxyfluorescein) (BCECF) caused cytosolic acidification consistent with proton/amino acid symport. In addition, these amino acids stimulate an inward short-circuit current (Iso) in voltage-clamped Caco-2 cell monolayers in Na+-free media (pH 6.0). Other amino acids such as leucine, isoleucine, phenylalanine, tryptophan, methionine, valine, sefine, glutamine, asparagine, D-aspartic acid, glutamic acid, cysteine, lysine, arginine and histidine were without effect on both pH i and inward Iso. In conclusion, Caco-2 cells express a Na § H+-coupled, rheogenic amino acid transporter at the apical brush-border membrane which plays an important role in the transepithelial Correspondence to: N.L. Simmons transport of a range of amino acids across this human intestinal epithelium.
Journal of Nutrition
The efflux of the nutritionally essential amino acid, L-lysine from the basolateral (BL) membrane was characterized in human cultured intestinal cells (Caco-2) grown and differentiated on permeable filter supports. Cells were loaded by incubating with 3H-lysine from the apical (AP) side in the absence of sodium (substituted with choline) in the BL medium; under these conditions, cells accumulated lysine in the intracellular soluble pool to 10- to 20-fold the extracellular concentration. L-Lysine efflux in the BL medium was then followed, and initial rates of efflux were calculated under different experimental conditions. L-Lysine efflux exhibited a strong energy dependence. The presence of an inwardly directed gradient of sodium or lithium stimulated lysine efflux; ouabain reduced efflux in both sodium- and lithium-containing medium. When zwitterionic or cationic amino acids were added to the BL medium, L-lysine efflux was strongly stimulated. The most efficient trans-stimulating am...
Deciphering the mechanisms of intestinal imino (and amino) acid transport: The redemption of SLC36A1
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2007
The absorption of zwitterionic imino and amino acids, and related drugs, is an essential function of the small intestinal epithelium. This review focuses on the physiological roles of transporters recently identified at the molecular level, in particular SLC36A1, by identifying how they relate to the classical epithelial imino and amino acid transporters characterised in mammalian small intestine in the 1960s-1990s. SLC36A1 transports a number of D-and L-imino and amino acids, βand γ-amino acids and orally-active neuromodulatory and antibacterial agents. SLC36A1 (or PAT1) functions as a proton-coupled imino and amino acid symporter in cooperation with the Na + /H + exchanger NHE3 (SLC9A3) to produce the imino acid carrier identified in rat small intestine in the 1960s but subsequently ignored because of confusion with the IMINO transporter. However, it is the sodium/imino and amino acid cotransporter SLC6A20 which corresponds to the betaine carrier (identified in hamster, 1960s) and IMINO transporter (identified in rabbit and guinea pig, 1980s). This review summarises evidence for expression of SLC36A1 and SLC6A20 in human small intestine, highlights the differences in functional characteristics of the imino acid carrier and IMINO transporter, and explains the confusion surrounding these two distinct transport systems.