Hypoxia Enhances Ecto-5′-Nucleotidase Activity and Cell Surface Expression in Endothelial Cells: Role of Membrane Lipids

• Published in: Circulation research 2003-05, Vol.92 (8), p.848-855
• Main Authors: Ledoux, S, Runembert, I, Koumanov, K, Michel, J B, Trugnan, G, Friedlander, G
• Format: Article
• Language: English
• Subjects: 5'-Nucleotidase - genetics; 5'-Nucleotidase - metabolism; Adenosine Monophosphate - pharmacology; Animals; Biological and medical sciences; Blood vessels and receptors; Blotting, Western; Cell Membrane - chemistry; Cell Membrane - drug effects; Cell Membrane - enzymology; Cells, Cultured; Dose-Response Relationship, Drug; Endocytosis; Endothelium, Vascular - cytology; Endothelium, Vascular - drug effects; Endothelium, Vascular - enzymology; Fundamental and applied biological sciences. Psychology; Gas Chromatography-Mass Spectrometry; Gene Expression Regulation, Enzymologic - drug effects; Hypoxia - physiopathology; Membrane Lipids - chemistry; Oxygen - pharmacology; Palmitic Acid - metabolism; Rats; RNA, Messenger - drug effects; RNA, Messenger - genetics; RNA, Messenger - metabolism; Vertebrates: cardiovascular system
• ISSN: 0009-7330; 1524-4571
• DOI: 10.1161/01.RES.0000069022.95401.FE

ABSTRACT—Extracellular adenosine production by the glycosyl-phosphatidyl-inositol-anchored Ecto-5′-Nucleotidase plays an important role in the defense against hypoxia, particularly in the intravascular space. The present study was designed in order to elucidate the mechanisms underlying hypoxia-induced stimulation of Ecto-5′-Nucleotidase in endothelial cells. For this purpose, aortic endothelial cells (SVARECs) were submitted to hypoxic gas mixture. Hypoxia (0% O2 for 18 hours) induced a 2-fold increase of Ecto-5′-Nucleotidase activity (Vmax 19.78±0.53 versus 8.82±1.12 nmol/mg protein per min), whereas mRNA abundance and total amount of the protein were unmodified. By contrast, hypoxia enhanced cell surface expression of Ecto-5′-Nucleotidase, as evidenced both by biotinylation and immunostaining. This effect was accompanied by a decrease of Ecto-5′-Nucleotidase endocytosis, without modification of Ecto-5′-Nucleotidase association with detergent-resistant membranes. Finally, whereas cholesterol content was unmodified, hypoxia induced a time-dependent increase of saturated fatty acids in SVARECs, which was reversed by reoxygenation, in parallel to Ecto-5′-Nucleotidase stimulation. Incubation of normoxic cells with palmitic acid enhanced Ecto-5′-Nucleotidase activity and cell surface expression. In conclusion, hypoxia enhances cell surface expression of Ecto-5′-Nucleotidase in endothelial cells. This effect could be supported by a decrease of Ecto-5′-Nucleotidase endocytosis through modification of plasma membrane fatty acid composition.