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(Circulation Research. 2003;93:813.)
© 2003 American Heart Association, Inc.

Molecular Medicine

Role of Neutral Amino Acid Transport and Protein Breakdown for Substrate Supply of Nitric Oxide Synthase in Human Endothelial Cells

Alexandra Simon, Lars Plies, Alice Habermeier, Ursula Martiné, Marco Reining, Ellen I. Closs

From the Department of Pharmacology, Johannes Gutenberg University, Mainz, Germany.

Correspondence to Ellen Ildicho Closs, PhD, Department of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Str. 67, 55101 Mainz, Germany. E-mail Closs{at}mail.uni-mainz.de

Endothelial dysfunction is often associated with a relative substrate deficiency of the endothelial nitric oxide synthase (eNOS) in spite of apparently high intracellular arginine concentrations. For a better understanding of the underlying pathophysiological mechanisms, we aimed to characterize the intracellular arginine sources of eNOS. Our previous studies in human endothelial EA.hy926 cells suggested the existence of two arginine pools: pool I can be depleted by extracellular lysine, whereas pool II is not freely exchangeable with the extracellular space, but accessible to eNOS. In this study, we demonstrate that the eNOS accessible pool II is also present in human umbilical vein endothelial cells (HUVECs), but not in ECV bladder carcinoma cells transfected with an expression plasmid for eNOS. In the endothelial cells, one part of pool II (referred to as pool IIA) consisted of recycling of citrulline to arginine. This part could be depleted by neutral amino acids that match the substrate profile of system N transporter 1 (SN1), presumably by the removal of intracellular citrulline. SN1 was expressed in EA.hy926 cells and HUVECs as shown by real-time RT-PCR. The second part of pool II (referred to as pool IIB) could not be depleted by any of the cationic or neutral amino acids tested. Our data demonstrate that pool IIB is nourished by protein breakdown and thus represents a substrate pool likely to accumulate protein-derived endogenous inhibitors of eNOS. Preferential use of the arginine pool IIB under pathophysiological conditions might therefore explain the arginine paradox.

Key Words: endothelial nitric oxide synthase • neutral amino acid transport • system N • intracellular arginine pool • proteasome

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