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

Expression of amino acid transport systems in cultured human umbilical vein endothelial cells. 1. 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. 3. Endothelial cell transport of L‐leucine was markedly inhibited during perfusion with 1 mM‐BCH (beta‐2‐aminobicyclo‐(2,2,1)‐heptane‐2‐carboxylic acid, system L analogue), L‐leucine, D‐leucine, L‐phenylalanine, L‐methionine and L‐DOPA. 2‐MeAIB, L‐cysteine, glycine, L‐proline, hydroxy‐L‐proline, L‐aspartate and beta‐alanine were poor inhibitors, while L‐serine and the cationic substrates L‐lysine and L‐arginine inhibited uptake by 10‐35%. 4. When the kinetics of L‐leucine transport were examined over a wide range of substrate concentrations (0.025‐1 mM) transport was saturable. A single entry site analysis gave a half‐maximal saturation constant Kt = 0.24 +/‐ 0.08 mM (mean +/‐ S.E.M., n = 5) and a Vmax = 27.8 +/‐ 4.6 nmol/min per column (approximately 3 x 10(6) cells). 5. Removal of sodium from the perfusate inhibited tracer uptake of L‐leucine, L‐serine and L‐arginine by respectively 20 +/‐ 5% (n = 3), 77 +/‐ 5% (n = 3) and 35 +/‐ 4% (n = 3). 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Physiology Wiley

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

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References (45)

Publisher
Wiley
Copyright
© 2014 The Physiological Society
ISSN
0022-3751
eISSN
1469-7793
DOI
10.1113/jphysiol.1989.sp017535
Publisher site
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Abstract

1. 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. 3. Endothelial cell transport of L‐leucine was markedly inhibited during perfusion with 1 mM‐BCH (beta‐2‐aminobicyclo‐(2,2,1)‐heptane‐2‐carboxylic acid, system L analogue), L‐leucine, D‐leucine, L‐phenylalanine, L‐methionine and L‐DOPA. 2‐MeAIB, L‐cysteine, glycine, L‐proline, hydroxy‐L‐proline, L‐aspartate and beta‐alanine were poor inhibitors, while L‐serine and the cationic substrates L‐lysine and L‐arginine inhibited uptake by 10‐35%. 4. When the kinetics of L‐leucine transport were examined over a wide range of substrate concentrations (0.025‐1 mM) transport was saturable. A single entry site analysis gave a half‐maximal saturation constant Kt = 0.24 +/‐ 0.08 mM (mean +/‐ S.E.M., n = 5) and a Vmax = 27.8 +/‐ 4.6 nmol/min per column (approximately 3 x 10(6) cells). 5. Removal of sodium from the perfusate inhibited tracer uptake of L‐leucine, L‐serine and L‐arginine by respectively 20 +/‐ 5% (n = 3), 77 +/‐ 5% (n = 3) and 35 +/‐ 4% (n = 3). 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.

Journal

The Journal of Physiology – Wiley

Published: Mar 1, 1989

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