Evidence in favor of a facilitated transport system for FA uptake in cultured L6 cells (original) (raw)
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Progress in Model Studies of Cellular Uptake
Annals of the New York Academy of Sciences, 1985
Cellular uptake of macromolecules may be regarded either as controlled by specific molecular interactions or affected by some nonspecific factors. Nonspecificity is meant usually in the sense that we are not able to recognize any biological purpose and significance of such interaction. Naturally, both specific and nonspecific interactions depend upon the interplay between structural moieties of the macromolecule and of the cell surface. In order to study these relationships, model synthetic polymers are very useful. Copolymers of poly-a,~-[N(2-hydroxyethyl)-~,~-aspartamide] (PHEA) were used several times as convenient models for biological studies.'-' Sets of polymers with a similar degree of polymerization but differing in the side chain composition may be easily prepared by a stepwise aminolysis of polysuccinimide fractions with various amines6 The monitoring of these polymers in biological experiments was made feasible by either labeling them with I 3 ' I or 12'1 or taking advantage of the fluorescence label N(2-aminoethylcarbonyl)-6-aminofluorescein covalently attached to the polymer.' The latter method of labeling when combined with special fixation of biological samples provides an instructive picture of the histological and cytological deposition of the polymer.'.* The radioactive label, on
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1992
The unidirectional fluxes of palmitate across the liver cell membrane and metabolic uptake rates were m:asured employing the multiple-indicator dilution technique. The following rcsuhs were obtained: (I) Influx and net uptake rates do not vary proportionally to each other when albumin and palmitate conccntratlor,~ arc varlcd. ~2) Effiux ;s significant for albumin concentrations in the range between !.5 and 500 ~M. (3) At 150/tM albumin net uptake rates arc proportional to the total (bound plus free) extracellular palmitate concentration in the range from 10 to 600/~M; the dependence of influx rates on the palmitatc concentration is rather concave up. (4) When albumin and palmitate are both varied at an equimolar ratio, pseudo-saturation appears in the net uptake rates; the influx rates also show pseudo-saturation, but with a declining tendency at the higher concentrations. (5) The intracellular palmitate concentration is strongly influenced by albumin. At very low concentrations of the protein (I.5 /tM) the intracenular concentration is practically equal to the cxtraccllular one; at physiological albumin concentrations, however, the intraccllular paZmitate concentration is less !ban 2% of the extracellular one. (6) Saturation of net uptake with respect to the intraccllular palmitate concentration was not obscrvc~ with conccntratio~ up to 46 p.M.
European Journal of Pharmaceutical Sciences, 2007
Intestinal absorption Transport medium Simulated intestinal fluid a b s t r a c t The purpose of this study was to identify simulated intestinal fluids (SIFs) containing nutrients compatible with the Caco-2 cell culture model and to examine the impact of the identified medium on the transport of a poorly aqueous soluble model compound, estradiol, and a substrate of efflux mechanisms, etoposide. Monolayer integrity was evaluated by transepithelial electrical resistance and cellular viability by release of lactate dehydrogenase to the apical compartment and cellular protein content. It was shown that the viability of Caco-2 cells was enhanced by use of the CO 2 independent nutritional medium, Leibovitz's L-15 compared to Hanks' balanced salt solution. SIF containing 5 mM sodium taurocholate and 1.25 mM phosphatidylcholine or lysophosphatidylcholine in Leibovitz's L-15 induced less release of lactate dehydrogenase than the traditional transport medium, HBSS.
European Journal of Pharmaceutics and Biopharmaceutics, 2007
Membrane transport characteristics of a paracellular permeability marker fluorescein were evaluated using artificial membrane, Caco-2 cell monolayers and rat jejunum, all mounted in side-by-side diffusion cells. Modified Ringer buffers with varied pH values were applied as incubation salines on both sides of artificial membrane, cell culture monolayers or rat jejunum. Passive transport according to pH partition theory was determined using all three permeability models. In addition to that, active transport of fluorescein in the M-S (mucosal-to-serosal) direction through rat jejunum was observed. The highest M-S P app values regarding the active transport through the rat jejunum were observed in incubation saline with pH 6.5. Fluorescein transport through the rat jejunum was inhibited by DIDS (4,4 0 -diisothiocyanatostilbene-2,2 0 -disulfonic acid) and a-CHC (a-cyano-4-hydroxycinnamic acid). Thus, we assume that two pH-dependent influx transporters could be involved in the fluorescein membrane transport through the intestinal (jejunal) epithelium.
H+-coupled α-methylaminoisobutyric acid transport in human intestinal Caco-2 cells
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1995
Transepithelial apical-to-basal transport and cellular uptake of the non-metabolisable amino acid a-methylaminoisobutyric acid (MeAl]B) across confluent raonolayers of the human intestinal epithelial cell line Caco-2 are enhanced by a transepithelial pH gradient (apical pH 6.0, basolateral pH 7.4). In Na+-free conditions (apical pH 7.4, basolateral pH 7.4), net absorption (120 + 58 pmol/cm 2 per h, n = 13) and uptake across the apical membrane (cell/medium ratio 0.56 ___ 0.06, n = 13) are low. However, in Na+-free conditions with apical pH 6.0, net absorpti.on (685_ 95 pmol/cm 2 per h, n = 15) and intracellular accumulation (cell/medium ratio 3.63_ 0.29, n --14) were marked. Continuous monitoring of intracellular pH (pHi) in BCECF (2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein)loaded Caco-2 cell monolayers indicated that apical addition of MeAIB (20 mM) was associated with H+-flow across the apical membrane in both Na + and Na+-free conditions. This transport process is rheogenic in Na+-free media, stimulating an inward short-circuit current in voltage-clamped Caco-2 cell monolayers. On the basis of competition for MeAIB accumulation and pH i experiments, L-proline, glycine, L-alanine and /3-alanine are also substrates for H+-linked transport at the apical membrane of Caco-2 cells but L-valine, L-leucine and L-phenylalanine are not. These data are consistent with the expression, in the apical brush-border membrane of Caco-2 cells, of a H+-coupled, Na+-independent MeAIB carrier.
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.
How fatty acids of different chain length enter and leave cells by free diffusion
Prostaglandins, Leukotrienes and Essential Fatty Acids, 2006
Opposing views exist as to how unesterified fatty acids (FA) enter and leave cells. It is commonly believed that for short- and medium-chain FA free diffusion suffices whereas it is questioned whether proteins are required to facilitate transport of long-chain fatty acid (LCFA). Furthermore, it is unclear whether these proteins facilitate binding to the plasma membrane, trans-membrane movement, dissociation into the cytosol and/or transport in the cytosol. In this mini-review we approach the controversy from a different point of view by focusing on the membrane permeability constant (P) of FA with different chain length. We compare experimentally derived values of the P of short and medium-chain FA with values of apparent permeability coefficients for LCFA calculated from their dissociation rate constant (k(off)), flip-flop rate constant (k(flip)) and partition coefficient (Kp) in phospholipid bilayers. It was found that Overton's rule is valid as long as k(flip)<k(off). With increasing chain length, the permeability increases according to increasing Kp and reaches a maximum for LCFA with chain length of 18 carbons or longer. For fast flip-flop (e.g. k(flip)=15s(-1)), the apparent permeability constant for palmitic acid is very high (P(app)=1.61 cm/s). Even for a slow flip-flop rate constant (e.g. k(flip)=0.3s(-1)), the permeability constant of LCFA is still several orders of magnitude larger than the P of water and other small non-electrolytes. Since polyunsaturated FA have basically the same physico-chemical properties as LCFA, they have similar membrane permeabilities. The implications for theories involving proteins to facilitate uptake of FA are discussed.
Effect of α-ketoglutarate on organic anion transport in single rabbit renal proximal tubules
American Journal of Physiology-renal Physiology, 1998
The effect of exogenous ␣-ketoglutarate (␣KG) and the peritubular Na ϩ-dicarboxylate (Na-DC) cotransporter on organic anion/dicarboxylate (OA/DC) exchange in S2 segments of single, nonperfused rabbit proximal tubules was measured using 1 µM fluorescein (FL), a model OA, and epifluorescence microscopy. The effect of different transmembrane distributions of 10 µM ␣KG on peritubular FL uptake was measured at 37°C using bicarbonate-buffered, nutrientcontaining buffers, which are conditions similar to those found in vivo. Compared with FL uptake in the absence of exogenous ␣KG, preloading tubules with ␣KG (trans-configuration) or acute exposure to ␣KG (cis-configuration) increased FL uptake 62% and 54%, respectively, whereas a cis-transconfiguration of ␣KG increased FL uptake by 76%. The cis-stimulation of FL uptake by ␣KG was rapid, within 5-7 s. This stimulation was blocked 96% by simultaneous exposure to 2 mM Li ϩ , indicating that stimulation of transport was secondary to the uptake of exogenous ␣KG. In the absence of exogenous ␣KG, selective inhibition of Na-DC cotransport using 2 mM Li ϩ or 1 mM methylsuccinate decreased FL uptake by 25% (effects that were reversible but not additive), suggesting that the Na-DC cotransporter recycles endogenous ␣KG that has left the cell in exchange for FL and that this activity supports ϳ25% of baseline activity of the OA/DC exchanger. With recycling of ␣KG accounting for ϳ25% of FL uptake and with accumulation of exogenous ␣KG accounting for another ϳ75% increase in FL uptake, Na-DC cotransport appears to directly support (25% ϩ 75%)/175%, or ϳ57%, of total FL transport.
Journal of nuclear medicine : official publication, Society of Nuclear Medicine, 1999
The aim of the study was to investigate the transport mechanism and uptake kinetics of the new 18F-labeled amino acid O-(2-[18F]fluoroethyl)-L-tyrosine (L-[18F]FET) and D-[18F]FET in human SW 707 colon carcinoma cells and the in vivo biodistribution of this tracer in SW 707 tumor-bearing mice. SW 707 cells were incubated with L- and D-[18F]FET under physiologic amino acid concentrations with and without the competitive transport inhibitors 2-amino-2 norbornane-carboxylic acid and a-(methylamino)isobutyric acid plus serine. For the investigation of the transport capacity, unlabeled L-FET was added to the samples. In addition, xenotransplanted mice were injected intravenously with L-[18F]FET; killed 10, 30, 60 and 120 min after injection; and the radioactivity concentration in different organs was measured in a gamma counter. The in vitro kinetic experiments showed a fast initial uptake of L-[18F]FET into the cells up to 6 min, followed by a nearly constant tracer concentration. The a...