Unidirectional flux of phenylalanine into Vero cells. Measurement using paired tracers in perfused cultures

Peran, Suzana; McGee, Maria P.

doi:10.1016/0005-2736(86)90032-5

Cited by 7 publications

(7 citation statements)

References 8 publications

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“…Cell viability and metabolic integrity, as demonstrated by amino acid uptake, were maintained for periods exceeding the kinetic measurements described here (34). The cells expressed surface-TF constitutively and interacted functionally with human fVIIa and fX with apparent steady-state kinetic parameters similar to other procoagulant cells, as reported previously (29,35).…”

Section: Methodssupporting

confidence: 83%

“…Cell Culture and Reaction Chambers-Cells and cell cultures have been described and characterized in detail elsewhere (29,34). Briefly, Vero cells (American Tissue Type Collection) were grown to confluency on microcarrier beads (Cytodex 2, Amersham Pharmacia Biotech) of 150-m average diameter.…”

Section: Methodsmentioning

confidence: 99%

“…Several studies using lipid-coated capillaries also indicate that flow rates influence the activity of coagulation proteases (30,31). In the present study, we use high resolution tracerdilution analyses (32)(33)(34)(35) along with numerical modeling to identify the surface and flow-dependent kinetics of fX activation via the TF pathway. We show that the generation of fXa from plasma fX proceeds via an intermediate step within the membrane.…”

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confidence: 99%

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Surface-dependent Coagulation Enzymes

McGee

Chou²

2001

Journal of Biological Chemistry

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The initial surface reactions of the extrinsic coagulation pathway on live cell membranes were examined under flow conditions. Generation of activated coagulation factor X (fXa) was measured on spherical monolayers of epithelial cells with a total surface area of 41-47 cm 2 expressing tissue factor (TF) at >25 fmol/cm 2 . Concentrations of reactants and product were monitored as a function of time with radiolabeled proteins and a chromogenic substrate at resolutions of 2-8 s. At physiological concentrations of fVIIa and fX, the reaction rate was 3.05 ؎ 0.75 fmol fXa/s/cm 2 , independent of flux, and 10 times slower than that expected for collision-limited reactions. Rates were also independent of surface fVIIa concentrations within the range 0.6 -25 fmol/cm 2 . The transit time of fX activated on the reaction chamber was prolonged relative to transit times of nonreacting tracers or preformed fXa. Membrane reactions were modeled using a set of nonlinear kinetic equations and a lagged normal density curve to track the expected surface concentration of reactants for various hypothetical reaction mechanisms. The experimental results were theoretically predicted only when the models used a slow intermediate reaction step, consistent with surface diffusion. These results provide evidence that the transfer of substrate within the membrane is rate-limiting in the kinetic mechanisms leading to initiation of blood coagulation by the TF pathway.Blood coagulation reactions mediate fibrin deposition in hemostasis and many pathological processes. Blood clots are directly implicated in the lethal complications of cardiovascular disease and contribute significantly to the pathogenesis of infectious, autoimmune, and neoplastic diseases (1-6).The blood coagulation process is initiated by an assembly of complexes comprised of an essential cofactor, TF 1 (tissue factor) and a protease component, fVIIa. The functional complex, TF⅐fVIIa, cleaves the natural substrates, fVII, fIX (factor IX), and fX at specific sites, generating fVIIa, fIXa, and fXa, respectively (4 -6). Factors VII, IX, and X circulate in the blood and extravascular fluids (7-10), whereas TF is expressed on the membranes of many extravascular tissues (11). The anatomic distribution of cells expressing TF is consistent with its role as the initiator of hemostatic reactions. Cell surfaces in contact with blood do not appear to express functional TF constitutively. However, inflammatory stimuli induce expression of functional TF on endothelial cell membranes and blood monocytes (12-14).Factors VII, IX, and X are vitamin K-dependent proteins, and their functional interaction with negatively charged procoagulant membranes has a calcium-dependent, electrostatic component (15-19). The interaction sites are located in highly homologous ␥-carboxyglutamic acid (Gla)-rich regions near the N terminus of all vitamin K-dependent coagulation proteins (4, 19). The specific binding and functional kinetics of interaction between coagulation proteins and biological membranes have been ...

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Section: Methodssupporting

confidence: 83%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Surface-dependent Coagulation Enzymes

McGee

Chou²

2001

Journal of Biological Chemistry

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“…We have adapted this experimental system to examine the transport properties of endothelial cells using a dual-isotope tracer dilution technique (Yudilevich & Mann, 1982). We are aware of only a single related publication on the kinetics and inhibition of phenylalanine transport in cultured Vero cells (Peran & McGee, 1986).…”

Section: Discussionmentioning

confidence: 99%

Expression of amino acid transport systems in cultured human umbilical vein endothelial cells.

Mann

Pearson

Sheriff

et al. 1989

The Journal of Physiology

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SUMMARY1. Nutrient transport in cultured human umbilical vein endothelial cells was characterized using a rapid dual-isotope dilution technique. Microcarrier beads with confluent endothelial cells were perfused in small columns, and uptake and efflux were assessed relative to D-mannitol (extracellular tracer) during a single transit through the column.2. At tracer concentrations significant unidirectional uptakes were measured for L-leucine (53 + 2 %), L-phenylalanine (73 + 2 %), L-serine (40 +4%), L-arginine (42 + 3 %) and L-ornithine (26 + 3 %). Uptake for L-proline, D-glucose, dopamine and serotonin was lower (6-10 %), whereas uptake for the system A analogue 2-methylaminoisobutyric acid (2-MeAIB) was negligible. Uptakes rapidly decreased with time due to tracer efflux. 6. Our results provide the first evidence that cultured human endothelial cells of venous origin express a saturable transport system for large neutral amino acids resembling system L described in brain microvascular endothelium. Detection of Na+-dependent and Na+-independent L-arginine uptake is of interest in view of recent reports that this cationic amino acid may be the physiological precursor for nitric oxide released by endothelium.

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“…Amino acid uptake was measured using a rapid paired-tracer dilution technique [5]. Briefly, cells were packed in a dialysis column (Minifiltro™ Hemofiltro Amicon Corporation, SA) and perfused with Hanks balanced salt solution (Sigma) at 37˚C and pH 7.…”

Section: Amino Acid Uptakementioning

confidence: 99%

Modifications of L-Glutamine and L-Leucine Transport in Proliferating Lymphocytes

Sanchez¹,

Gil²,

Perán³

2015

JBM

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Lymphocytes respond to mitogens that stimulate proliferation by increasing theirs metabolic activity. In this study we investigate L-Glutamine and L-Leucine uptake as markers of cell response to Concavalina A (ConcaA) stimulation, using a high-resolution flow technique. We found that lymphocytes induced to blast transformation enhanced rate and efficiency of amino acid uptake during cell proliferation. Considering that increases in transport is the first quantifiable response of cells during malignant transformation, amino acid uptake could also be useful as an early marker of malignancy.

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Unidirectional flux of phenylalanine into Vero cells. Measurement using paired tracers in perfused cultures

Cited by 7 publications

References 8 publications

Surface-dependent Coagulation Enzymes

Surface-dependent Coagulation Enzymes

Expression of amino acid transport systems in cultured human umbilical vein endothelial cells.

Modifications of L-Glutamine and L-Leucine Transport in Proliferating Lymphocytes

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