Characterization of Na(+)-independent glutamine transport in rat liver

In hepatic plasma membrane vesicles (HPMVs) from rat liver, we observed that approximately 40-45% of Na(+)-independent glutamine uptake occurs by a saturable carrier-mediated process. This component of glutamine uptake is mediated by a transport agency distinct from that of previously described systems for the Na(+)-independent transport of amino acids. Transport of glutamine was electroneutral and occurred into an osmotically active space with negligible membrane binding. The model system L substrate 2-amino-2-norbornane-carboxylic acid (BCH) showed no appreciable inhibition of Na(+)-independent glutamine uptake by HPMVs but effectively inhibited the uptake of leucine, a classic system L substrate, in identical vesicle preparations. Further evidence against system L-mediated glutamine transport was provided by the pH dependence and the lack of trans-stimulation of saturable uptake. Competition experiments with selected amino acids revealed a pattern of inhibition of glutamine transport that was inconsistent with assignment of glutamine entry to systems asc, T, or systems for the Na(+)-independent transport of the charged amino acids. This BCH-noninhibitable transport system in HPMVs was highly selective for glutamine, histidine, and, to a lesser extent, asparagine. Inhibition of Na(+)-independent glutamine transport by leucine was noncompetitive in nature. On the basis of Na+ independence, pH sensitivity, absence of trans-stimulation, and an amino acid selectivity similar to that of the previously described hepatic Na(+)-dependent system N, we have provisionally designated the glutamine transport agency described in this article as system "n."

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Glutamine transport by basolateral plasma-membrane vesicles prepared from rabbit intestine

Biochemical Journal ◽

10.1042/bj2770687 ◽

1991 ◽

Vol 277 (3) ◽

pp. 687-691 ◽

Cited By ~ 11

Author(s):

S W Wilde ◽

M S Kilberg

Keyword(s):

Epithelial Cells ◽

Basolateral Membrane ◽

Membrane Vesicles ◽

Reciprocal Inhibition ◽

Isobutyric Acid ◽

Plasma Membrane Vesicles ◽

Arterial Blood ◽

System L ◽

Glutamine Transport ◽

Glutamine Uptake

L-Glutamine, a major energy substrate for intestinal epithelial cells, can be extracted from intraluminal contents across the brush-border membrane and from arterial blood via the basolateral membrane. The purpose of the present study was to characterize glutamine transport by the basolateral membrane of rabbit epithelial cells. Transport of glutamine by isolated basolateral-membrane vesicles was mediated by both Na(+)-dependent and Na(+)-independent carriers. Tests were performed to distinguish glutamine uptake by likely transport agencies, including Systems A, ASC, N, IMINO, NBB, L and asc. The Na(+)-dependent glutamine uptake was strongly inhibited by an excess of 2-(methylamino)isobutyric acid (MeAIB), and glutamine was equally effective in inhibiting MeAIB transport. The reciprocal inhibition analysis, as well as a sensitivity to increased H+ concentration, indicates that Na(+)-dependent glutamine transport across the basolateral membrane is mediated by System A. The saturable Na(+)-independent glutamine transport was markedly inhibited by 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid (‘BCH’) and insensitive to changes in assay pH, suggesting uptake via System L rather than System asc. The presence of a Na(+)-dependent carrier to mediate active transport of glutamine across the basolateral membrane is probably essential to ensure a continuous supply of this vital substrate to the enterocyte in the post-absorptive state.

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Characteristics of glutamine transport in dog jejunal brush-border membrane vesicles

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1989.257.1.g80 ◽

1989 ◽

Vol 257 (1) ◽

pp. G80-G85 ◽

Cited By ~ 2

Author(s):

N. M. Bulus ◽

N. N. Abumrad ◽

F. K. Ghishan

Keyword(s):

Brush Border ◽

Brush Border Membrane ◽

Ph Dependence ◽

Membrane Vesicles ◽

Brush Border Membrane Vesicles ◽

Transport Systems ◽

System A ◽

System L ◽

Glutamine Transport ◽

Glutamine Uptake

The present study characterizes glutamine transport across brush-border membrane vesicles (BBMV) prepared from dog jejunum. The purity of these vesicles was demonstrated by a 20-fold enrichment of leucine aminopeptidase, a marker for BBM. Glutamine uptake was found to occur into an osmotically active space with no membrane binding and to exhibit temperature and pH dependence (optimal uptake at pH 7-7.5). Glutamine uptake was driven by an inwardly directed Na+ gradient with a distinct overshoot not observed under K+ gradient. Lithium could not substitute for Na+ as a stimulator of glutamine uptake. Na+-dependent glutamine uptake was not inhibited by methylaminoisobutyric acid, a typical substrate for system A, and was found to be electrogenic and saturable with a Km of 0.97 +/- 0.58 mM and a Vmax of 3.93 +/- 0.99 nmol.mg protein-1.10 s-1. A Na+-glutamine coupling ratio of 1:1 could be demonstrated by a plot of Hill transformation. Na+-independent glutamine uptake was found to be electroneutral and saturable with a Km of 3.70 +/- 0.66 mM and a Vmax of 2.70 +/- 1.55 nmol.mg protein-1.10 s-1. Inhibition studies confirmed the presence of a Na+-dependent as well as a Na+-independent carrier for glutamine uptake. We conclude that glutamine uptake across dog BBMV occurs via two transport systems: a Na+-dependent high-affinity system similar to the neutral brush-border system and a Na+-independent lower-affinity system similar to system L.

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Substrate-specificity of glutamine transporters in membrane vesicles from rat liver and skeletal muscle investigated using amino acid analogues

Biochemical Journal ◽

10.1042/bj2780105 ◽

1991 ◽

Vol 278 (1) ◽

pp. 105-111 ◽

Cited By ~ 13

Author(s):

S Y Low ◽

P M Taylor ◽

A Ahmed ◽

C I Pogson ◽

M J Rennie

Keyword(s):

Skeletal Muscle ◽

Amino Acid ◽

Rat Liver ◽

Membrane Vesicles ◽

Transport Processes ◽

System A ◽

Liver Membrane ◽

Glutamine Transport ◽

Fold Excess ◽

Glutamine Uptake

We investigated the effects of glutamine and histidine analogues on glutamine transport processes in membrane vesicles prepared from rat liver (sinusoidal membrane) and skeletal muscle (sarcolemma). L-[14C]Glutamine is transported in these membranes predominantly by Systems N/Nm (liver and muscle respectively), and to a lesser extent by Systems A and L (e.g. about 60, 20 and 20% of total flux respectively via Systems N, A and L at 0.05 mM-glutamine in liver membrane vesicles). The glutamine anti-metabolites 6-diazo-5-oxo-L-norleucine and acivicin were relatively poor inhibitors of glutamine uptake into liver membrane vesicles (less than 25% inhibition at 20-fold excess) and appeared primarily to inhibit System A activity (i.e. N-methylaminoisobutyric acid-inhibitable glutamine uptake). In similar experiments azaserine (also a glutamine anti-metabolite) inhibited approx. 50% of glutamine uptake, apparently by inhibition of System A and also of System L (i.e. 2-amino-2-carboxybicyclo[2,2,1]heptane-inhibitable glutamine uptake). Glutamate gamma-hydroxamate, aspartate beta-hydroxamate, histidine and N'-methylhistidine were all strong inhibitors of glutamine uptake into liver membrane vesicles (greater than 65% inhibition at 20-fold excess), but neither homoglutamine nor N'-methylhistidine produced inhibition. L-Glutamate-gamma-hydroxamate was shown to be a competitive inhibitor of glutamine transport via System N (Ki approximately 0.6 mM). Glutamine uptake in sarcolemmal vesicles showed a similar general pattern of inhibition as in liver membrane vesicles. The results highlight limits on the substrate tolerance of System N; we suggest that the presence of both an L-alpha-amino acid group and a nitrogen group with a delocalized lone-pair of electrons (amide or pyrrole type), separated by a specific intramolecular distance (C2-C4 chain equivalent), is important for substrate recognition by this transporter.

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Activation by intravesicular amino acids of system L-mediated serine uptake into human placental microvillous plasma membrane vesicles

Placenta ◽

10.1016/j.placenta.2014.06.321 ◽

2014 ◽

Vol 35 (9) ◽

pp. A99

Author(s):

Kate Widdows ◽

Nont Panitchob ◽

Emma Lofthouse ◽

Ian Crocker ◽

Colin Sibley ◽

...

Keyword(s):

Amino Acids ◽

Plasma Membrane ◽

Membrane Vesicles ◽

Plasma Membrane Vesicles ◽

System L

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A new method for the rapid isolation of basolateral plasma membrane vesicles from rat liver. Characterization, validation, and bile acid transport studies.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)47298-x ◽

1984 ◽

Vol 259 (14) ◽

pp. 9295-9301 ◽

Cited By ~ 5

Author(s):

B L Blitzer ◽

C B Donovan

Keyword(s):

Plasma Membrane ◽

Bile Acid ◽

Rat Liver ◽

Membrane Vesicles ◽

New Method ◽

Acid Transport ◽

Plasma Membrane Vesicles ◽

Rapid Isolation ◽

Transport Studies ◽

Basolateral Plasma Membrane

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Albumin binding of unconjugated [3H]bilirubin and its uptake by rat liver basolateral plasma membrane vesicles

Biochemical Journal ◽

10.1042/bj3160999 ◽

1996 ◽

Vol 316 (3) ◽

pp. 999-1004 ◽

Cited By ~ 19

Author(s):

Lorella PASCOLO ◽

Savino DEL VECCHIO ◽

Ronald K. KOEHLER ◽

J. Enrique BAYON ◽

Cecile C. WEBSTER ◽

...

Keyword(s):

Plasma Membrane ◽

Rat Liver ◽

Basolateral Membrane ◽

Membrane Vesicles ◽

Plasma Membrane Vesicles ◽

Experimental Conditions ◽

Saturation Kinetics ◽

Basolateral Plasma Membrane ◽

Component C ◽

Uptake Studies

Using highly purified unconjugated [3H]bilirubin (UCB), we measured UCB binding to delipidated human serum albumin (HSA) and its uptake by basolateral rat liver plasma membrane vesicles, in both the absence and presence of an inside-positive membrane potential. Free UCB concentrations ([Bf]) were calculated from UCB–HSA affinity constants (K´f), determined by five cycles of ultrafiltration through a Centricon-10 device (Amicon) of the same solutions used in the uptake studies. At HSA concentrations from 12 to 380 μM, K´f (litre/mol) was inversely related to [HSA], irrespective of the [Bt]/[HSA] ratio. K´f was 2.066×106+(3.258×108/[HSA]). When 50 mM KCl was iso-osmotically substituted for sucrose, the K´f value was significantly lower {2.077×106+(1.099×108/[HSA])}. The transport occurred into an osmotic-sensitive space. Below saturation ([Bf] ⩽ 65 nM), both electroneutral and electrogenic components followed saturation kinetics with respect to [Bf], with Km values of 28±7 and 57±8 nM respectively (mean±S.D., n = 3, P < 0.001). The Vmax was greater for the electrogenic than for the electroneutral component (112±12 versus 45±4 pmol of UCB·mg-1 of protein·15 s-1, P < 0.001). Sulphobromophthalein trans-stimulated both electrogenic (61%) and electroneutral (72%) UCB uptake. These data indicate that: (a) as [HSA] increases, K´f decreases, thus increasing the concentration of free UCB. This may account for much of the enhanced hepatocytic uptake of organic anions observed with increasing [HSA]. (b) UCB is taken up at the basolateral membrane of the hepatocyte by two systems with Km values within the range of physiological free UCB levels in plasma. The electrogenic component shows a lower affinity and a higher capacity than the electroneutral component. (c) It is important to calculate the actual [Bf] using a K´f value determined under the same experimental conditions (medium and [HSA]) used for the uptake studies.

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Carrier-mediated uptake of L-(+)-lactate in plasma membrane vesicles from rat liver

FEBS Letters ◽

10.1016/0014-5793(88)81267-5 ◽

1988 ◽

Vol 235 (1-2) ◽

pp. 224-228 ◽

Cited By ~ 12

Author(s):

Irene Quintana ◽

Antonio Felipe ◽

Xavier Remesar ◽

Marçal Pastor-Anglada

Keyword(s):

Plasma Membrane ◽

Rat Liver ◽

Membrane Vesicles ◽

Plasma Membrane Vesicles

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Characterization of two Mg2+transporters in sealed plasma membrane vesicles from rat liver

AJP Cell Physiology ◽

10.1152/ajpcell.1998.275.4.c995 ◽

1998 ◽

Vol 275 (4) ◽

pp. C995-C1008 ◽

Cited By ~ 49

Author(s):

Christie Cefaratti ◽

Andrea Romani ◽

Antonio Scarpa

Keyword(s):

Plasma Membrane ◽

Rat Liver ◽

Intracellular Signaling ◽

Mammalian Cells ◽

Plasma Membranes ◽

Membrane Vesicles ◽

Channel Blockers ◽

Transport Mechanisms ◽

Plasma Membrane Vesicles ◽

New Information

The plasma membrane of mammalian cells possesses rapid Mg2+ transport mechanisms. The identity of Mg2+ transporters is unknown, and so are their properties. In this study, Mg2+ transporters were characterized using a biochemically and morphologically standardized preparation of sealed rat liver plasma membranes (LPM) whose intravesicular content could be set and controlled. The system has the advantages that it is not regulated by intracellular signaling machinery and that the intravesicular ion milieu can be designed. The results indicate that 1) LPM retain trapped intravesicular total Mg2+with negligible leak; 2) the addition of Na+ or Ca2+ induces a concentration- and temperature-dependent efflux corresponding to 30–50% of the intravesicular Mg2+; 3) the rate of flux is very rapid (137.6 and 86.8 nmol total Mg2+ ⋅ μm−2 ⋅ min−1after Na+ and Ca2+ addition, respectively); 4) coaddition of maximal concentrations of Na+ and Ca2+ induces an additive Mg2+ efflux; 5) both Na+- and Ca2+-stimulated Mg2+ effluxes are inhibited by amiloride, imipramine, or quinidine but not by vanadate or Ca2+ channel blockers; 6) extracellular Na+ or Ca2+ can stimulate Mg2+ efflux in the absence of Mg2+ gradients; and 7) Mg2+ uptake occurs in LPM loaded with Na+ but not with Ca2+, thus indicating that Na+/Mg2+but not Ca2+/Mg2+exchange is reversible. These data are consistent with the operation of two distinct Mg2+ transport mechanisms and provide new information on rates of Mg2+ transport, specificity of the cotransported ions, and reversibility of the transport.

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Glutamine transport by the blood-brain barrier: a possible mechanism for nitrogen removal

AJP Cell Physiology ◽

10.1152/ajpcell.1998.274.4.c1101 ◽

1998 ◽

Vol 274 (4) ◽

pp. C1101-C1107 ◽

Cited By ~ 127

Author(s):

Wha-Joon Lee ◽

Richard A. Hawkins ◽

Juan R. Viña ◽

Darryl R. Peterson

Keyword(s):

Endothelial Cells ◽

Amino Acid ◽

Membrane Vesicles ◽

Bovine Brain ◽

Glutamate Transport ◽

Plasma Membrane Vesicles ◽

Amino Acid Transport System ◽

Luminal Membrane ◽

Glutamine Transport ◽

Glutamine Transporters

Glutamine and glutamate transport activities were measured in isolated luminal and abluminal plasma membrane vesicles derived from bovine brain endothelial cells. Facilitative systems for glutamine and glutamate were almost exclusively located in luminal-enriched membranes. The facilitative glutamine carrier was neither sensitive to 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid inhibition nor did it participate in accelerated amino acid exchange; it therefore appeared to be distinct from the neutral amino acid transport system L1. Two Na-dependent glutamine transporters were found in abluminal-enriched membranes: systems A and N. System N accounted for ∼80% of Na-dependent glutamine transport at 100 μM. Abluminal-enriched membranes showed Na-dependent glutamate transport activity. The presence of 1) Na-dependent carriers capable of pumping glutamine and glutamate from brain into endothelial cells, 2) glutaminase within endothelial cells to hydrolyze glutamine to glutamate and ammonia, and 3) facilitative carriers for glutamine and glutamate at the luminal membrane may provide a mechanism for removing nitrogen and nitrogen-rich amino acids from brain.

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