Published online before print March 27, 2003, doi: 10.1161/01.RES.0000069022.95401.FE
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© 2003 American Heart Association, Inc.
Molecular Medicine |
Hypoxia Enhances Ecto-5'-Nucleotidase Activity and Cell Surface Expression in Endothelial Cells
Role of Membrane Lipids
From INSERM U426 (S.L., I.R., G.F.), Faculté de Médecine Xavier Bichat, Université Paris 7; INSERM U538 (K.K., G.T.), Faculté de Médecine Saint-Antoine; INSERM U460 (J.B.M.), Faculté de Médecine Xavier Bichat, Université Paris 7, Paris, France.
Correspondence to Séverine Ledoux, INSERM U426, Faculté Xavier Bichat, BP416, 16 rue Henri Huchard, 75870 Paris, Cedex 18, France. E-mail ledoux{at}bichat.inserm.fr
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.
Key Words: hypoxia • Ecto-5'-Nucleotidase • membrane lipids
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